The Role of Bioactive Lipids in Cancer, Inflammation and Related Diseases. Advances in Experimental Medicine and Biology

Advances in Experimental Medicine and Biology 1161
Kenneth V. Honn
Darryl C. Zeldin Editors
The Role of Bioactive

Lipids in Cancer,
Inflammation and
Related Diseases
Advances in Experimental Medicine
and Biology
Volume 1161
Editorial Board
IRUN R. COHEN, The Weizmann Institute of Science, Rehovot, Israel
ABEL LAJTHA, N.S. Kline Institute for Psychiatric Research,
Orangeburg, NY, USA
JOHN D. LAMBRIS, University of Pennsylvania, Philadelphia, PA, USA
RODOLFO PAOLETTI, University of Milan, Milan, Italy
NIMA REZAEI, Children’s Medical Center Hospital, Tehran University
of Medical Sciences, Tehran, Iran
More information about this series at http://www.springer.com/series/5584
Kenneth V. Honn • Darryl C. Zeldin
Editors
The Role of Bioactive

Lipids in Cancer,
Inflammation
and Related Diseases
Editors
Kenneth V. Honn Darryl C. Zeldin
431 Chemistry Building National Institutes of Health
Wayne State University Durham, NC, USA
Detroit, MI, USA
ISSN 0065-2598 ISSN 2214-8019 (electronic)

Advances in Experimental Medicine and Biology
ISBN 978-3-030-21636-8 ISBN 978-3-030-21735-8 (eBook)
https://doi.org/10.1007/978-3-030-21735-8
© Springer Nature Switzerland AG 2019

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Contents
1 Introduction................................................................................... 1
Kenneth V. Honn and Darryl C. Zeldin
2 Understanding the Role of Pro-resolving Lipid Mediators
in Infectious Keratitis................................................................... 3
Elizabeth A. Berger
3 Immunoresolvent Resolvin D1 Maintains the Health
of the Ocular Surface.................................................................... 13
Darlene A. Dartt, Robin R. Hodges, and Charles N. Serhan
4 The Evolving Role of Specialized Pro-resolving Mediators
in Modulating Neuroinflammation in Perioperative
Neurocognitive Disorders............................................................. 27
Ting Yang and Niccolò Terrando
5 Relationship Between Specialized Pro-resolving
Mediators and Inflammatory Markers
in Chronic Cardiac Disorders...................................................... 37
M. Brianza-Padilla and R. Bojalil
6 Specialized Pro-resolving Mediators Directs Cardiac
Healing and Repair with Activation of Inflammation
and Resolution Program in Heart Failure.................................. 45
Ganesh V. Halade and Bochra Tourki
7 Novel n-3 Docosapentaneoic Acid-Derived
Pro-resolving Mediators Are Vasculoprotective
and Mediate the Actions of Statins in Controlling
Inflammation................................................................................. 65
Jesmond Dalli, Kimberly Pistorius, and Mary E. Walker
8 Aspects of Prostaglandin Glycerol Ester Biology....................... 77
Philip J. Kingsley, Carol A. Rouzer, Amanda J. Morgan,
Sachin Patel, and Lawrence J. Marnett
9 Targeting the COX/mPGES-1/PGE2 Pathway
in Neuroblastoma.......................................................................... 89
Karin Larsson, Anna Kock, Per Kogner,
and Per-Johan Jakobsson
v
vi Contents
10 Metabolomics Biomarkers for Precision Psychiatry................. 101

Pei-an (Betty) Shih
11 Cytochrome P450 Eicosanoid Signaling Pathway
in Colorectal Tumorigenesis......................................................... 115
Weicang Wang, Katherine Z. Sanidad, and Guodong Zhang
12 Contributions of 12/15-Lipoxygenase to Bleeding
in the Brain Following Ischemic Stroke...................................... 125
Yi Zheng, Yu Liu, Hulya Karatas, Kazim Yigitkanli,
Theodore R. Holman, and Klaus van Leyen
13 Systematic Understanding of Bioactive Lipids
in Neuro-Immune Interactions: Lessons from
an Animal Model of Multiple Sclerosis....................................... 133
Yasuyuki Kihara
14 Role of Bioactive Sphingolipids in Inflammation
and Eye Diseases........................................................................... 149
Koushik Mondal and Nawajes Mandal
15 Roles of Ceramides and Other Sphingolipids
in Immune Cell Function and Inflammation............................. 169
Sabrin Albeituni and Johnny Stiban
16 Bioactive Lipids in Cancer, Inflammation
and Related Diseases..................................................................... 193
Emma Leishman, Phillip E. Kunkler, Joyce H. Hurley,
Sally Miller, and Heather B. Bradshaw
17 Novel Anti-inflammatory and Vasodilatory
ω-3 Endocannabinoid Epoxide Regioisomers............................ 219
Lauren N. Carnevale and Aditi Das
18 Overview of Lipid Biomarkers in Amyotrophic
Lateral Sclerosis (ALS)................................................................. 233
Andres Trostchansky
19 Flavonoids Ability to Disrupt Inflammation
Mediated by Lipid and Cholesterol Oxidation........................... 243
Carlo Barnaba and Ilce G. Medina-Meza
Index....................................................................................................... 255
About the Editors
Kenneth V. Honn, Ph.D., Distinguished

Professor and Director, Bioactive Lipids
Research Program, Wayne State University
Kenneth Honn, Ph.D., is a distinguished profes-
sor and an adjunct professor in the Wayne State
University School of Medicine Departments of
Pathology and Oncology and Department of
Chemistry, respectively. He is director of the
Bioactive Lipids Research Program and serves
as a member of the Cancer Biology Graduate
Program and of the Barbara Ann Karmanos
Cancer Institute. He is the founding member and
president of the Eicosanoid Research Foundation
and chairman of the International Conference on
Bioactive Lipids in Cancer, Inflammation and
Related Diseases, a biennial international confer-
ence he initiated in 1989. He received his Ph.D.
in Endocrinology from Wayne State University
in 1977. With more than 30 years of experience
in the fields of cancer, inflammation, and bioac-
tive lipids, his laboratory focuses on bioactive
lipids and integrin receptors and the role they
play in various aspects of tumor progression,
namely, cell growth and apoptosis, angiogenesis,
and tumor cell matrix interactions. His lab con-
centrates on lipoxygenases, in particular
12-lipoxygenase and its metabolic product
12(S)-HETE. In addition to his research on bio-
active lipids in tumor progression, he has col-
laborated with scientists at the Perinatology
Research Branch of the National Institutes of
Health for the past 4 years, studying the role of
lipids in human parturition, in particular their
role in preterm labor and term labor. Research
efforts in his laboratory have directly led to six
vii
viii About the Editors
clinical trials, and he holds 17 US patents, 7 of

which are based on the generation of novel che-
motherapeutic/radiation sensitizing compounds.
He is the author of more than 300 published
works. He has had continuous external funding
with more than 50 grants totaling in excess of
$25 million. He also has and continues to serve
on numerous study sections, reviewing grants for
the National Cancer Institute and the Department
of Defense, and provides consultation to phar-
maceutical companies. In addition, he is a mem-
ber of the editorial boards of 12 scientific journals
and is coeditor in chief of Cancer and Metastasis
Reviews.
Darryl C. Zeldin, M.D., Scientific Director,

National Institute of Environmental Health
Sciences (NIEHS) National Institutes of Health
Darryl C. Zeldin, M.D., is the scientific director
at the National Institute of Environmental Health
Sciences (NIEHS), National Institutes of Health
(NIH). He is an internationally recognized expert
on eicosanoids (lipid mediators) and their role in
regulating respiratory and cardiovascular func-
tion. He received his medical degree from
Indiana University in 1986 and completed his
Internal Medicine Residency at Duke University
Medical Center in 1989 and a Fellowship in
Pulmonary/Critical Care Medicine at Vanderbilt
University in 1993. He was recruited to the NIH
in 1994 and promoted to senior investigator with
tenure in 2001. He served as the NIEHS clinical
director from 2007 to 2011 prior to becoming
scientific director in 2011. He is board certified
in Internal Medicine and Pulmonary Medicine, is
a fellow in the American College of Chest
Physicians and in the American Heart
Association, and is an elected member of the
American Society for Clinical Investigation and
Association of American Physicians. He has
coauthored over 340 primary peer-reviewed
manuscripts, and his work has been cited over
25,000 times (h-index 86). He has mentored over
75 postbaccalaureate fellows, graduate students,
medical students, postdoctoral research fellows,
About the Editors ix
and clinical research fellows. As the NIEHS sci-

entific director, he is responsible for one of the
largest intramural research programs at the NIH
with over 1000 scientists in 10 departments and
15 core facilities and an annual budget of over
$130M. His own research has also been featured
on NPR, Good Morning America, USA Today,
US News & World Report, PBS, and on other
internationally recognized media venues.
Introduction
1
Kenneth V. Honn and Darryl C. Zeldin
In addition to this introduction, this book con- presented a lecture on “Decoding New Lipid
tains 18 outstanding chapters based on compre- Mediators and Mechanisms of Resolution of
hensive and detailed reviews of timely topics Inflammation, Infections and Tissue
presented at the 15th International Conference on Regeneration.” Drs. Bruce D. Hammock, Jorge
Bioactive Lipids in Cancer, Inflammation and H. Capdevila and Darryl C. Zeldin shared the
Related Diseases held in Puerto Vallarta, Mexico Outstanding Achievement Award for their work
on October 22–25, 2017. on the P450 epoxygenase pathway of arachidonic
Held biennially since 1989, this ongoing con- acid metabolism and delivered lectures on the
ference series provides a scientific program that role of fatty acids in neuropathic pain, cardiovas-
is comprehensive and structured to highlight cular disease and lung inflammation. There were
research at the cutting edge of science on the role three Eicosanoid Research Foundation Young
of lipid mediators in various physiological and Investigator awards presented to Drs. Karin
pathological processes in a wide range of thera- Larsson (cancer), Jesmond Dalli (inflammation)
peutic areas. Over 315 basic researchers and cli- and Scott Hansen (structure-function). Dr. Shu
nicians representing 32 countries attended this Xu from the University of Toledo was presented
most recent meeting. Dr. Jeffrey M. Drazen, with the Santosh Nigam Memorial Outstanding
Editor-in-Chief of the New England Journal of Young Scientist award for his presentation on
Medicine, received the Exceptional Contributions “The Structure of the Catalytic Domain of
to Human Physiology and Translational Medicine 12-Lipoxygenase.” There were 50 lectures orga-
Award and presented an opening lecture on “Data nized into 3 Plenary Sessions, 14 scientific ses-
Sharing in Clinical Trials.” Dr. Charles N. Serhan sions and 140 poster presentations.
received the Lifetime Achievement Award and The specialized pro-resolving lipid mediators
or SPMs (e.g., resolvins, lipoxins, maresins) have
received wide-spread attention as to their role in
K. V. Honn (*) inflammatory processes. Reviews presented in
Departments of Pathology, Oncology and Chemistry, this book detail recent advances in understanding
Wayne State University, Detroit, MI, USA
e-mail: [email protected] the role of SPMs in inflammatory conditions of
the eye, brain and cardiovascular system. Dr.
D. C. Zeldin (*)
Division of Intramural Research, National Institutes Elizabeth Berger (Chap. 2) summarized recent
of Health/National Institute of Environmental Health literature on infectious keratitis (bacterial, viral,
Sciences, Research Triangle Park, NC, USA fungal) and endophthalmitis with an emphasis on
e-mail: [email protected]
© Springer Nature Switzerland AG 2019 1

K. V. Honn, D. C. Zeldin (eds.), The Role of Bioactive Lipids in Cancer, Inflammation
and Related Diseases, Advances in Experimental Medicine and Biology 1161,
https://doi.org/10.1007/978-3-030-21735-8_1
2 K. V. Honn and D. C. Zeldin
the host response and the role of SPMs in resolu- inflammation and colorectal cancer. Dr. Klaus
tion of the eye inflammation. Drs. Darlene Dartt, Van Leyen and co-workers (Chap. 12) summa-
Robin R. Hodges, and Charles N. Serhan (Chap. rized the biological relevance of lipoxygenase-
3) provided a comprehensive summary of the derived eicosanoids in ischemic stroke and
role of resolvin D1 (RvD1), its cognate receptor neuronal cell death in the brain.
(ALX/FPR2 or GPR32) and downstream signal- Sphingolipids are a unique class of bioactive
ing pathways (PLC, PLD, PLA2, EGFR, and lipids with significant chemical diversity. Recent
ERK) in preserving ocular surface homeostasis advances in the field of sphingolipids and their
and regulating conjunctival goblet cell mucin role in the eye, brain, intestine and immune sys-
secretion. Dr. Niccolo Terrando (Chap. 4) tem are discussed in this book. Dr. Yasuyuki
reported on the role of SPMs in the brain with an Kihara (Chap. 13) provided a review of eico-
emphasis on postoperative neuroinflammation sanoid and other bioactive lipid signaling involve-
and cognitive function. Dr. Rafael Bojalil ment in the neuroinflammatory mechanisms
(Chap. 5) reviewed the role of pro-resolving underlying multiple sclerosis. Drs. Koushik
mediators in cardiovascular diseases, notably in Mondal and Nawajes Mandal (Chap. 14) reported
regulating inflammation. Dr. Ganesh Halade on the role of bioactive sphingolipids including
(Chap. 6) summarized recent literature on the ceramide and sphingosine in ocular inflammation
role of SPMs in resolution of cardiac inflamma- and eye diseases such as uveitis, diabetic retinop-
tion, tissue repair and remodeling in heart failure athy and glaucoma. Drs. Sabrin Albeituni and
models. Dr. Jesmond Dalli (Chap. 7) reported on Johnny Stiban (Chap. 15) reported on the role of
the biological actions of resolvins in the cardio- ceramides and other sphingolipids in immune
vascular system and their role in mediating the cell function and inflammation in cancer, multi-
actions of statins. ple sclerosis and inflammatory bowel disease.
The book contains two chapters that overview The endocannabinoids and their receptors
recent advances in cyclooxygenase-derived eico- affect both central nervous system and peripheral
sanoids in the brain. Dr. Philip Kingsley (Chap. 8) processes. There are two chapters on recent
reported on the identification and role of prosta- advances in endocannabinoids in the brain. Dr.
glandin glycerol esters (PG-Gs) in the brain, Heather Bradshaw and colleagues (Chap. 16)
detailing their production, metabolism and bioac- reported on the role of endogenous cannabinoids
tivity. Dr. Karin Larsson (Chap. 9) summarized and their receptors in the pathogenesis of trau-
recent data on the role of prostaglandin E2 matic brain injury. Dr. Aditi Das (Chap. 17) sum-
(PGE2) in neuroblastoma, in particular its actions marized recent information on the
in regulating angiogenesis, immunosuppression anti-inflammatory and vasoactive role of omega-3
in the tumor microenvironment, and tumor endocannabinoid epoxide metabolites in the
growth. There are three chapters that provide an brain. In addition, there is a chapter by Dr. Andres
overview on the biological relevance of cyto- Trostchansky (Chap. 18) on the relationship of
chrome P450 (CYP) and lipoxygenase-derived lipid metabolism and lipid derived metabolites
eicosanoids. Dr. Pei-an Shih (Chap. 10) reported with the onset and progression of amylotrophic
that CYP-derived eicosanoids are prognostic bio- latereral sclerosis in humans. This book is capped
markers for certain neuropsychiatric disorders off with a concise, scholarly review by Drs. Carlo
such as depression. Dr. Guodong Zhang and co- Barnaba and Ilce G. Medina-Meza on the role of
workers (Chap. 11) discussed recent studies on flavonoids in the reduction of inflammation and
the roles of CYP-derived eicosanoids and soluble lipid oxidation in the pathogenesis of cardiovas-
epoxide hydrolase in the pathogenesis of colonic cular disease (Chap. 19).
Understanding the Role of
Pro-resolving Lipid Mediators 2
in Infectious Keratitis
Elizabeth A. Berger
2.1 Infectious Keratitis ally characterized by rapid progression, focal

white infiltrates with underlying stromal inflam-
Keratitis is a sight-threatening inflammatory con- mation, corneal thinning, stromal edema, muco-
dition of the cornea that can be caused by both purulent discharge and hypopyon, which can lead
infectious and non-infectious agents. Physical or to corneal scarring, endophthalmitis, and perfora-
chemical trauma are typically related to non- tion. In fact, corneal opacity is not only a compli-
infectious keratitis, which may then become sec- cation of keratitis, but among the leading causes
ondarily infected or remain non- infected. of legal blindness worldwide. Despite that empir-
Etiology of infectious keratitis is most often asso- ical treatment effectively controls most of the
ciated with bacteria; but viruses, fungi, and para- pathogens implicated in infectious keratitis,
sites are common causative pathogens as well. As improved clinical outcomes are not guaranteed.
a global concern, common risk factors include: Further, if treatment is not initiated in a timely
systemic immunosuppression (secondary to mal- manner, good visual outcome is reduced to
nutrition, alcoholism, diabetes, steroid use), pre- approximately 50% of keratitis patients [9].
vious corneal surgery (refractive corneal surgery, Moreover, resultant structural alterations, loss of
penetrating keratoplasty), extended wear contact tissue and an unresolved host response remain
lens use, pre-existing ocular surface diseases (dry unaddressed through current clinical manage-
eye, epithelial defect) and ocular trauma (agricul- ment of this condition.
ture- or farm-related) [1–8]. Annual rates of inci-
dence include nearly one million clinical visits
due to keratitis in the United States, while it has 2.2 ost Response to Infectious
H
been reported that roughly two million people Keratitis
develop corneal ulcers in India. Clinically,
patients may show signs of eye pain (ranging A large body of work has been carried out there-
from mild to severe), blurred vision, photopho- fore elucidating the keratitis-induced host innate
bia, chemosis and redness. Pathogenesis is gener- immune response. During infection, corneal
ulceration progresses due to inflammatory medi-
E. A. Berger (*) ators released by the pathogen, as well as from
Department of Ophthalmology, Visual & Anatomical corneal epithelial and stromal cells and infiltrated
Sciences, Wayne State University School of
inflammatory cells [10]. Though infiltrating leu-
Medicine, Detroit, MI, USA
e-mail: [email protected] kocytes are a requisite feature of an effective

4 E. A. Berger
inflammatory response, persistence and chronic PGD2) initiate lipid mediator class switching
activation of macrophages, neutrophils/PMN and [11]. Biosynthesized during the active phase of
T cells contribute to disease pathogenesis through resolution, SPMs play a pivotal role as
sustained production of inflammatory mediators, endogenous agonists stimulating the resolution
proteolytic enzymes and resultant tissue necrosis. of inflammation – a key feature of a healthy,
Similarly, cytokines and chemokines such as productive inflammatory response [12].
IL-1α/β, TNF-α, IFN-γ and MIP-2 promote anti- Proresolving actions include limiting PMN
microbial effector mechanisms, activate phago- infiltration, regulating the cytokine profile [13,
cytic killing and recruit additional inflammatory 14], reducing pain [15], and efferocytosis [16,
cells. However, the continued production of such 17]. SPMs best associated with the eye are
pro-inflammatory cytokines/chemokines contrib- derived from two major polyunsaturated fatty
utes to the destruction of the corneal microenvi- acids; arachidonic acid (AA) and docosahexaenoic
ronment. In addition to an upregulation of acid (DHA) and are targeted by lipoxygenase
cytokines, lipid mediators are also released, (LOX) enzymes, which comprise the main path-
including prostaglandins, thromboxanes and ways for SPM biosynthesis (Fig. 2.1). Studies
platelet-activating factor. It is well-known that have demonstrated LOX activity in the cornea
these arachidonic acid-derived mediators play with 15-LOX appearing to be largely protective
key roles in the initiation of inflammation, as a key enzyme for the generation of SPMs [12,
including the recruitment of leukocytes 18–20] . Alox15 is the most abundant lipoxygen-
(particularly neutrophils). Although necessary, it ase in the healthy murine cornea [21–23]. Gronert
is imperative that this biological cascade is both et al. have established the presence of functional
inhibited and dissolved. These initial pro- 5-LOX and 12/15-LOX in mouse corneal epithe-
inflammatory pathways are balanced by lium and corneas [19]. Furthermore, both healthy
proresolving lipid signals known as specialized and inflamed corneas express 12/15- LOX as
pro-resolving lipid mediators (SPMs). In fact, it detected from epithelial cells and infiltrated neu-
has been shown that prostaglandins (PGE2 and trophils [19]. AA-derived lipoxin A4 (LXA4) can
Fig. 2.1 Schematic of major SPM pathways involved in the cornea

2 Understanding the Role of Pro-resolving Lipid Mediators in Infectious Keratitis 5
be transcellularly biosynthesized during leuko- events. Yet, other studies have indicated delayed
cyte interactions with mucosal cells, such as epithelial healing and worsened disease outcome
PMN interactions with epithelial cells [24]. [35–37]. Additionally, a comprehensive review
Studies have revealed a protective role for LXA4, evaluating the effects of corticosteroids from
whereby it has been shown to accelerate corneal 1950 to 2000 concluded that the role of
re-epithelization and limit neovascularization corticosteroids in the adjunctive treatment of
using a thermal injury model [25]. In addition to infectious keratitis remains unsubstantiated [38].
AA-derived lipoxins, DHA is targeted by phos- Further, a review by McGhee et al. states that
pholipase A2 and subsequently LOX and COX-2 corticosteroid-induced ocular morbidity includes:
produce di- and trihydroxylated DHA derivatives ocular hypertension, glaucoma, cataract
including D series resolvins, protectins and formation, tear film instability, and crystalline
maresins [26]. These SPMs are particularly rele- keratopathy [39]. To this end, this review will
vant to the eye as studies have shown that RvD1 highlight the compelling evidence for the
reduces the inflammatory response during endo- development of pro-resolving molecules as an
toxin-induced uveitis [25], allergic eye disease adjuvant therapy for the treatment of infectious
[27], and dry eye [28]. keratitis – with focus on bacterial, viral and
fungal infections.
2.3 Current Treatments

for Infectious Keratitis 2.4 Bacterial Keratitis
This collective work illuminates host factors that Bacteria remain the most common cause of infec-
exacerbate the progression of keratitis, indicating tious keratitis with studies indicating that bacte-
that the pathogenicity of disease is a combination rial keratitis comprises approximately 90% of
of microbe-induced damage and an unresolved infectious keratitis cases. Epidemiological stud-
host response – the latter of which may be due to ies have revealed that between 54–94% of all cor-
ocular immune privilege. Regardless, a number neal infections are due to bacteria. Pseudomonas
of adjunctive treatments have been considered, aeruginosa and Staphylococcus aureus have
including cross-linking, anti-collagenases, anti- been reported as the more prevalent bacteria
inflammatories, and corticosteroids. Collagen associated with infectious keratitis [40–42].
cross-linking was found to improve corneal Staphylococcus aureus as the most common ocu-
re-epithelization and resistance to enzymatic lar isolate [43]; while Pseudomonas aeruginosa
degradation, reduce corneal melting, and exert is the most frequently isolated of Gram negative
bactericidal effects [29–31]. However, ocular infections [22]. Moreover, Pseudomonas
complications include endothelial cell loss and aeruginosa ulcers have been shown to result in a
reactivation of HSV infections, thus warranting more progressive disease with large infiltrate and
further investigation. Non-steroidal anti-scarring [43, 56] and not benefit from the addi-
inflammatory treatments have been investigated tion of corticosteroids [36]. Current treatment of
in an attempt to prevent excessive inflammation; microbial keratitis consists primarily of fourth
but shown to exacerbate the disease response in generation ophthalmic fluoroquinolones (moxi-
the absence of concomitant antibiotic therapy or floxcin and gatifloxacin), which are effective
exhibit toxicity issues complicated by corneal against both Gram-positive and Gram-negative
melting [32–34]. Indecision continues to exist bacteria. This class of antibiotics also exhibits
regarding the benefits and risks associated with better mutant prevention characteristics and pen-
use of corticosteroids. The Steroids for Corneal etrates well into ocular tissues. However, antibi-
Ulcers Trial (SCUT) provides evidence for otic-resistance is of major concern; in the U.S.,
improved visual acuity in bacterial keratitis roughly 80% of methicillin-resistant S. aureus
patients with no reported increase in adverse ocular isolates have been reported as resistant to
6 E. A. Berger
fluoroquinolones [44–46]. Multi-drug resistance the importance of SPM circuit activation in the
rates for Pseudomonas are on the rise, as well resolution of inflammation following corneal
[47, 48]. infection. Further, it highlights the significant
Work using a mouse model of Pseudomonas- potential of combination treatments – where
induced keratitis has revealed the importance of antibiotics alone cannot activate the resolution
SPM circuits in the resolution of inflammation arm of inflammation; yet without the antibiotic,
[23]. Lipid mediator profiles were characterized Tβ4 (and other activators of SPM pathways) may
in susceptible (corneal thinning, perforation) and not be able to overcome the bacterial burden and
resistant (resolution) phenotypes and indicated subsequently move toward active resolution.
that activation of SPM pathways are requisite for Work carried out by the Pearlman group
a balanced inflammatory response characterized investigated the role of RvE1 in the corneal
by the resolution of disease. Furthermore, this inflammatory response induced by LPS and
study indicated a critical role for 15-LOX in antibiotic-killed Pseudomonas aeruginosa and
influencing inflammatory cell function. Without Staphylococcus aureus [56]. Cytokine production
15-LOX enzymatic activity, essential innate was significantly reduced in human corneal
inflammatory functions (phagocytosis, epithelial cells and neutrophils, as well as mouse
efferocytosis) were impaired, leading to an cornea and macrophages. In addition, a decrease
exacerbated disease response. This supports in neutrophil infiltrate and reduced corneal haze
previous work that showed 15-LOX−/− mice was observed after RvE1 treatment following
resulted in impaired wound healing and amplified bacteria- and LPS-induced corneal inflammation.
inflammatory angiogenesis [19, 49, 50]. In
addition, potential experimental treatments that
have been shown to improve disease response to 2.5 Viral Keratitis
Pseudomonas-induced keratitis appear to have a
common theme – the activation of SPM circuitry. Herpes simplex virus (HSV) keratitis affects an
Vasoactive intestinal peptide or VIP has been estimated 500,000 people in the US and an
demonstrated to exert its immunoregulatory estimated 1.5 million globally per annum [57,
effects, in part, through upregulation of 58]. Though it begins as a subclinical infection of
lipoxygenase enzymes in corneas of the cornea, HSV establishes latency within the
Pseudomonas-infected mice [51, 52]. Additional trigeminal ganglia and when reactivated, it causes
work in the mouse model has revealed that VIP recurrent infections that result in decreased
increases RvD1 and modulates the ALX/FPR2 corneal sensation, neurotrophic keratitis, stromal
receptor axis toward inflammation resolution opacity, scarring, and blindness. In fact, HSV
[52]. Ligands for this receptor include both keratitis is the predominant cause of unilateral
LXA4 and RvD1 [53]; the latter of which has infectious corneal blindness in most developed
been shown to reduce corneal neovascularization nations [59]. Though rare, bilateral infection
[54]. Evidence also indicates that thymosin does occur in approximately 1.3–12% of patients
beta-4 (Tβ4), another therapeutic molecule, may [60]. Primary HSV infections are typically
carry out its profound effects through activation overlooked in the clinic as adenoviral
of SPM circuits [55]. Adjunct Tβ4 treatment with conjunctivitis. Epithelial infection is typically
antibiotic resulted in increased 12- and 12/15- caused by actively replicating virus and presents
LOX mRNA levels, while COX-2 and 5-LOX itself as the classic herpetic corneal lesion – a
were decreased after infection [55]. This is dendritic ulcer – with minimal stromal
further supported by increased RvD1 and its inflammation [61]. Once resolved, a dendritic
receptor, ALX/FPR2. In contrast, pro-scar or ghost dendrite may persist in the
inflammatory lipid mediator pathways were superficial stroma. A more progressive geographic
affected, as evidenced by lower levels of LTB4 ulcer (also caused by live replicating virus) can
and its receptor, BLT1. This work underscores occur in immunocompromised patients and
results in a larger epithelial defect. Unfortunately, of infection. The effect on infiltrating

insufficient corneal wound healing secondary to inflammatory leukocytes is particularly relevant
antiviral toxicity, loss of innervation, and chronic given that HSK-induced lesions are primarily
stromal inflammation can result in trophic ulcers. driven by neutrophils and CD4+ T cells [63].
The greatest morbidity is associated with Work in a corneal transplant model suggests that
recurrent stromal keratitis and HSV endothelitis the observed effect on T cells may be due to an
due to excessive corneal scarring and inhibition of dendritic cell maturation and
neovascularization commonly in response to activation [64]. While in vitro studies revealed
non-replicating viral particles. Herpes stromal that AT-RvD1 decreased IFN-γ and IL-12 – the
keratitis or HSK can range from focal, multifocal, latter of which is a requisite signal for Th1
or diffuse stromal opacities. Disciform keratitis polarization [63]. Another study by the Rouse
results from endothelial dysfunction in response group found that RvE1 similarly improved HSV-
to viral antigens. Necrotizing keratitis is most induced keratitis through reduced
commonly observed in patients with multiple neovascularization, reduced corneal infiltrate of
recurrent HSV infections and can result in corneal neutrophils and T cells (Th1/Th17),
melting and perforation. Less common viral downregulation of pro-inflammatory cytokines
pathogens include varicella-zoster virus (VZV) and chemokines, along with less severe lesions
and cytomegalovirus (CMV). Though treatment [65]. Neuroprotectin D1(NPD1), another DHA-
varies based on the severity of disease, trifluridine derived pro-resolving mediator [66], was shown
is the most commonly prescribed topical antiviral to modulate corneal inflammation through similar
medication for HSV keratitis in the US [62]. mechanisms following HSV-induced stromal
However, due to low bioavailability and ocular keratitis in the mouse, as well [67]. These results
surface toxicity, its use is more limited. While in are in line with a number of studies that have
the rest of the world, acyclovir is the preferred reported NPD1 pro-resolving bioactivities,
treatment as it is equally effective and has lower including reduced T cell migration [68] and
toxicity issues. Topical corticosteroids, under neutrophil influx [69], enhanced efferocytosis of
antiviral cover, are used for the treatment of apoptotic neutrophils [13], and decreased
HSK. Ganciclovir has been found to be just as production of proinflammatory mediators
effective as acyclovir with less toxicity, less (cytokines, chemokines, ROS) [70, 71]. Taken
likely to promote resistance, and effective against together, this body of work indicates promise for
HSV, VZV and CMV. Despite prompt treatment DHA-derived pro-resolving mediators in the
however, scarring is a major complication treatment of viral keratitis.
associated with viral keratitis. Furthermore, since
all of the available antivirals for HSV keratitis are
nucleoside analogues that inhibit viral replication, 2.6 Fungal Keratitis
there remains the toxicity issue due to host DNA
synthesis interference. Fungal keratitis tends to be more prevalent in
Similar to bacterial keratitis, the therapeutic tropical and subtropical climates, where it has
potential of SPMs regarding viral keratitis has been reported that up to 50% of infectious ulcers
been investigated. Work by Rajasagi et al. are caused by fungi – predominately Fusarium
revealed that topical application of aspirin- and Aspergillus [72–74]. However, fungal kerati-
triggered (AT)-RvD1 reduced lesion severity, tis can occur in more temperate regions as demon-
corneal neovascularization, pro-inflammatory strated by the Fusarium outbreak related to
mediators, and infiltration of effector CD4+ T contact lens solution contamination [75–77]. The
cells and neutrophils using a mouse model of prognosis of fungal keratitis is worse than that of
HSK [63]. Further, they found that AT-RvD1 was bacterial keratitis. Surprisingly, no new treat-
equally efficacious regardless of whether ments have been introduced since natamycin in
treatment was started before or after the induction the 1960s [36]. This topical polyene remains the
8 E. A. Berger
first choice of treatment; though amphotericin B mediators in the treatment of this disease.
is preferred for Aspergillus and Candida keratitis. Intravitreal injections of RvD1 was shown to
Limitations exist for both however; natamycin attenuate the progression of disease while
exhibits poor penetration into the corneal stroma preserving retinal function [85]. This was
and topical amphotericin B has toxicity issues indicated by reduced levels of proinflammatory
[78]. Although voriconazole, a newer-generation mediators, reduced bacterial burden, fewer
triazole, demonstrates excellent ocular penetra- infiltrated PMN, and induction of antimicrobial
tion [79] and high susceptibility among common molecules. Further, it was suggested that this
fungal isolates [80], natamycin remains superior effect may involve TLR signaling pathways as
to voriconazole in the topical treatment of fungal TLR2 deficient mice were not protected by RvD1
keratitis [36, 81]. However, there are no reports treatment. Similar to fungal keratitis though,
examining the effects of SPMs in the treatment of therapeutic potential of pro-resolving mediators
this disease either as a monotherapy or as an have yet to be examined in fungal endopthalmitis.
adjunct to available antifungals. In vitro antifun-
gal susceptibility testing and in vivo fungal kerati-
tis models could provide valuable insight into the 2.8 Conclusion
potential use of SPMs for fungal keratitis given
the diversity of fungal aetiology and emergence of Although currently available antibiotics, antivi-
drug resistance. rals, and antifungals are efficacious in success-
fully controlling most of the pathogens implicated
in infectious keratitis, clinical outcomes remain
2.7 Endophthalmitis poor. A common limitation among these thera-
pies is evident – a sustained host response that
In rare cases, infectious keratitis can extend from compromises corneal integrity. The cornea dem-
the cornea into the vitreous and/or aqueous onstrates a deficiency in switching from active
humor and is termed endophthalmitis. Though inflammation to active resolution – perhaps due
uncommon, it is among the most devastating eye to ocular immune privilege – resulting in struc-
infections that bears the worst possible outcome – tural alterations and loss of tissue. There exists a
irreversible blindness that can occur within even clear need for treatments that modify the immune
hours of symptom onset [82]. Incidence of response toward resolution and activate tissue
disease ranges from 0.03–1.3% with cataract restoration. In fact, a review on the clinical man-
surgery to as high as 30–40% following open agement of infectious keratitis suggested that the
globe injuries [83]. The most prevalent pathogens development of future adjuvant therapies with
related to endogenous endophthalmitis are the greatest potential are likely to be multidimen-
bacteria (Staphylococcus sp., Streptococcus sp.) sional that are aimed at modifying the immune
or fungi (Candida, Aspergillus). With more than response to infection [36]. The current review
50% of keratitis-related endophthalmitis cases provides evidentiary support for using pro-
due to mold, Fusarium and Aspergillus are the resolving molecules to treat infectious keratitis.
most common etiologies [84]. Moreover, fungal SPMs are being targeted as a beneficial mono-
endophthalmitis is particularly difficult to treat therapy and adjuvant for infectious keratitis. The
due to delayed diagnosis and poor ocular concept of combining SPMs with other drugs
penetration of antifungals compounded by has been demonstrated to be highly effective in
toxicity issues. Despite this, treatment typically resolving inflammation [86]. This class of pro-
consists of intravitreal antibiotics. In addition, resolving lipid mediators holds promise in the
vitrectomy is often carried out due to the rapidity treatment of drug-resistant infections while
and severity of disease progression. avoiding ocular surface toxicity issues that cur-
Initial studies in a S. aureus-induced model of rently impede proper treatment of infectious
endophthalmitis show promise for pro-resolving keratitis. As reviewed, SPMs function to dis-
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Immunoresolvent Resolvin D1
Maintains the Health of the Ocular 3
Surface
Darlene A. Dartt, Robin R. Hodges,
and Charles N. Serhan
Abstract in human tears and induces goblet cell mucin

The present review focuses on the role of one secretion. RvD1 interacts with its receptors
of the D-series resolvins (Rv) RvD1 in the ALX/FPR2 and GPR32, activates phospholi-
regulation of conjunctival goblet cell secretion pases C, D, and A2, as well as the EGFR. This
and its role in ocular surface health. RvD1 is stimulation increases the intracellular [Ca2+]
the most thoroughly studied of the specialized and activates extracellular regulated kinase
proresolution mediators in the goblet cells. (ERK) 1/2 to cause mucin secretion into the
The anterior surface of the eye consists of the tear film. This mucin secretion protects the
cornea (the transparent central area) and the ocular surface from the challenges in the
conjunctiva (opaque tissue that surrounds the external milieu thus maintaining a healthy
cornea and lines the eyelids). The secretory interface between the eye and the environ-
mucin MUC5AC produced by the conjuncti- ment. RvD1 forms a second important mecha-
val goblet cells is protective of the ocular sur- nism along with activation of a neural reflex
face and especially helps to maintain clear pathway to regulate goblet cell mucin secre-
vision through the cornea. In health, a com- tion and protect the ocular surface in health.
plex neural reflex stimulates goblet cell secre-
tion to maintain an optimum amount of mucin Keywords
in the tear film. The specialized pro-resolution Conjunctiva · Goblet cells · Allergic eye
mediator, D-series resolvin (RvD1) is present disease · Dry eye disease · Ocular surface ·
Mucins · Tear film · Signaling pathways ·
D. A. Dartt (*) · R. R. Hodges Resolvin D1 · Inflammation · Secretion
Schepens Eye Research Institute/Massachusetts Eye
and Ear, Boston, MA, USA
Department of Ophthalmology, Harvard Medical
School, Boston, MA, USA 3.1 ear Film, Cornea,
T
and Conjunctiva
C. N. Serhan
Center for Experimental Therapeutics and
Reperfusion Injury, Department of Anesthesiology, 3.1.1 Introduction
Perioperative and Pain Medicine, Brigham and
Women’s Hospital, Boston, MA, USA The present review focuses on the role of one of
Harvard Medical School, Boston, MA, USA the D-series resolvins (Rv) RvD1 in the r egulation

14 D. A. Dartt et al.
of conjunctival goblet cell secretion. RvD1 is the film and ocular surface tissues (cornea and con-
most thoroughly studied of the specialized pro- junctiva) form the outermost protective layer that
resolution mediators in the goblet cells. This functions to maintain a transparent, healthy and
review is also focused on the role of RvD1 in impermeable cornea [1]. These components each
maintenance of ocular surface health, but not its have unique properties that contribute to the pro-
role in disease. tection of the eye from mechanical, thermal, and
chemical injury; desiccation; allergens and pol-
lutants; and pathogens from the external
3.1.2 F
unction of Tear Film environment.
and Ocular Surface At the basal aspect, the cornea consists of a
single layered endothelium, overlaid by stroma
The eye is a unique, specialized organ whose containing keratocytes, and on the apical side a
function is to provide a clear optical path for multi-layered epithelium. One of the major pro-
image presentation on the retina and its analysis tective properties of the cornea is that it is a very
by the brain (Fig. 3.1). The first optical surface in tight epithelium with limited permeability [1]. It
the eye is the tear film that overspreads the cor- is difficult to permeate the corneal epithelium
nea, a transparent avascular tissue. Together the because of the extensive tight junctions and other
tear film and cornea provide most of the refrac- types of junctions between the topmost layers of
tive power of the eye [1]. The tear film and the cells. For example, bacteria cannot penetrate the
cornea are among the first components of the corneal epithelium unless it is damaged [2].
anterior eye aided by the conjunctiva to interact Furthermore, the cornea is an immune privileged
with the external environment. The conjunctiva is site and maintains an immunosuppressive and
a vascular, optically dense tissue that surrounds anti-inflammatory environment preventing
the cornea and lines the eyelids. Together the tear immune cells from migrating into the cornea or
Fig. 3.1 Scehmatic diagram of the eye and ocular surface

3 Immunoresolvent Resolvin D1 Maintains the Health of the Ocular Surface 15
aqueous humor in response to an immune or The stratified squamous cells and the goblet cells
inflammatory challenge [3]. When injured, the both produce the mucous layer of the tear film but
cornea normally heals rapidly with minimal scar- release different types of mucins. Both cells
ring and no blood vessel ingrowth. The number secrete electrolytes and water.
of mechanisms that the cornea can use to respond Secretion of mucins is one of the major pro-
to the external environment is limited and often tective mechanisms of the conjunctiva. The
constrained, as this tissue needs to preserve trans- mucins produce an extensive glycocalyx and the
parency and remain avascular to ensure clear soluble mucous layer that overspreads both the
vision. cornea and conjunctiva. Stratified squamous cells
The conjunctiva, the focus of this review, sur- produce the membrane spanning mucins MUC1,
rounds the cornea and lines the lids. One of its MUC4, MUC16, and MUC20 (for a review see
major functions is to secrete electrolytes, water, [6]). These mucins help form the glycocalyx, a
and multiple types of mucins that together form thick coat of carbohydrates that emerges from the
the glycocalyx and the inner layer of the tear film apical membranes of epithelial cells and protects
[4]. The conjunctiva is a stratified squamous epi- the corneal and conjunctival surface. The glyco-
thelium that overlies a loose, disorganized stroma calyx interfaces with the tear film. The glycoca-
containing plentiful blood vessels and nerves. lyx is critical to maintaining the barrier function
This epithelium consists of two major cell types, of the cornea, preventing access of microbes to
stratified squamous cells and goblet cells the plasma membrane, and preventing apical
(Fig. 3.2) [4]. Stratified squamous cells are multi- adhesion [7]. In contrast, the goblet cells secrete
layered, have complex interdigitated lateral the large soluble, gel-forming mucin MUC5AC
membranes, and contain a plethora of small clear [8]. This mucin is the major component of the
vesicles. The second cell type is the goblet cells inner mucous layer of the tear film and is essen-
[5]. These cells occur singly or in clusters and tial for ocular surface health. MUC5AC upon
span the entire epithelium in rodents, but only secretion by the goblet cells overspreads the
occupy the mid portion of the epithelium reach- entire ocular surface including the cornea. It pro-
ing to the surface in humans [5]. The goblet cells tects the underlying epithelia as a mucus gel by
are polarized with a basal nucleus, substantial preventing attachment of bacteria and keeping
Golgi apparatus, and a plethora of secretory gran- them suspended in the gel. The water and electro-
ules that occupy most of the cell volume and lytes in the gel keep the ocular surface hydrated.
reach to the apical membrane and the tear film. The movement of the mucus gel across the ocular
Fig. 3.2 An electron

micrograph of rat
conjunctival goblet cells
(CG). Numerous
secretory vesicles can be
seen in the apical
portion of the cells,
while nuclei can be seen
in the basal portion.
SCC stratified squamous
cells. Magnification
×6000. Reprinted from
Dartt [4]
surface into the lacrimal drainage ducts removes depletion of MUC5AC in the tear film lead to
particles, pollutants, and other environmental serious damaging, painful ocular surface diseases
components from the ocular surface. Goblet cells such as dry eye and vitamin A deficiency [14].
and their secretion of MUC5AC are regulated by Increase in goblet cell mucin in the tear film is
nerves. Afferent sensory nerves and efferent also characteristic of specific diseases, such as
parasympathetic and sympathetic nerves extend ocular allergy, that upset ocular surface homeo-
into the conjunctival epithelium interdigitating stasis and can be damaging to the ocular surface.
between the stratified squamous cells. Efferent The finding that both a decrease and an increase
parasympathetic and sympathetic nerves sur- in goblet cell mucin secretion leads to disease
round the goblet cells. The localization of the suggests that goblet cell mucin secretion must be
nerves in the epithelium provides the basis for tightly regulated to maintain an optimal amount
these epithelial cells to respond quickly to of mucin in the tear film [6, 8, 15].
changes in the external environment by secretion In health neural regulation of goblet cell
of MUC5AC into the tears. mucin secretion provides this regulation [16, 17].
The conjunctiva has multiple protective mech- Using a complex neural reflex, goblet cell mucin
anisms in addition to the secretion of secretion can be exquisitely regulated to respond
MUC5AC. As the conjunctival epithelium is to changes in the external environment to secrete
opaque, well vascularized, and very permeable, mucins as needed to protect the occur surface
unlike the cornea, it is not constrained in its [18]. Corneal and conjunctival afferent sensory
responses to the environment. The conjunctiva in nerves are activated by changes in temperature,
fact can respond very robustly. The conjunctiva acid or bases, or mechanical stimuli (including
contains conjunctival associated lymphoid tissue trauma and particulates that occur in pollution),
that is part of the mucosal immune system [9]. In for examples. The stimulated afferent nerves then
addition the conjunctival stroma contains numer- activate the trigeminal ganglion that by a com-
ous different cells for innate defense (macro- plex neural reflex within the brain activates effer-
phages, neutrophils, and mast cells) and for ent parasympathetic nerves In the conjunctiva
immune protection (lymphocytes, plasma cells, parasympathetic nerve endings surround the gob-
and dendritic cells). In addition for the innate let cells and stimulate them to secrete mucins into
defense system the conjunctiva has most of the the tear film [17]. The goblet cells also likely
Toll-like receptors [10], multiple NOD-like secrete electrolytes and water, but the evidence
receptors, and a constitutively assembled NLRP3 for this is indirect [19, 20].
inflammasome poised for activation [11, 12]. The Parasympathetic nerves are the major stimulus
conjunctiva also has goblet cell associated pas- of goblet cell mucin secretion in health [16, 21,
sages (GAPs) that are openings between goblet 22] (Fig. 3.3). There is no published evidence for
cells that allow the stroma to sample antigens and the role of sympathetic nerves in this secretion.
other immune activating material in the external Parasympathetic nerves release the neurotrans-
environment [13]. These GAPs are under para- mitters acetylcholine that activate muscarinic
sympathetic muscarinic control. receptors (MAchR) type 1, 2, and 3 and vasoac-
tive intestinal peptide (VIP) that uses the VPAC1
and 2 receptors [21]. Both acetylcholine (carba-
3.1.3 R
ole Goblet Cells in Ocular chol is used experimentally) and VIP stimulate
Surface Health secretion by increasing intracellular [Ca2+]
([Ca2+]i) and activating extracellular regulated
As goblet cells and their secretion of MUC5AC kinase (ERK)1/2 also known as p44/p42 mitogen
are critical for ocular surface health, both a activated protein kinase (MAPK) [13, 23]. In
decrease and an increase in goblet cell mucin addition VIP activates adenylyl cyclase to
secretion leads to ocular surface disease. Loss of increase the cellular cAMP level (For a review
goblet cells from the conjunctival epithelium and see [24]). Acetylcholine (carbachol), but not VIP,
Fig. 3.3 Schematic representation of cholinergic path- mitogen activated kinase kinase (MEK), and ERK 1/2.
way leading to goblet cell mucin secretion. Muscarinic Vasoactive intestinal peptide (VIP) binds to its receptors
receptors activate phospholipase C (PLC) to generate the VPAC1 or 2 to activate adenylyl cyclase (AC) to generate
production of inositol trisphosphate (IP3), which releases cAMP from ATP. cAMP then activates protein kinase A
intracellular Ca2+ and diacylglycerol (DAG), which acti- (PKA) to stimulate secretion. Reprinted from Hodges
vates protein kinase C (PKC). The EGF receptor (EGFR) et al. Encyclopedia of the Eye, Dartt D, Dana R, Besharse
is transactivated leading to the activation of Ras, Raf, J eds. Elsevier, 2010
also works by activating a matrix metalloprotein- 3.2 Specialized Pro-resolving

ase to cause ectodomain shedding of EGF to Mediators
stimulate the EGFR that in turn increases [Ca2+]i
and activates ERK1/2 to stimulate goblet cell The SPMs comprise four families, lipoxins,
secretion [22]. resolvins, protectins, and maresins. The SPMs
Thus under normal conditions, the parasym- function in the resolution phase of acute inflam-
pathetic neurotransmitters use a common intra- matory diseases and each family possess unique
cellular signaling pathway, an increase in [Ca2+]i bioactions to resolve inflammation [26]. Each
and activation of ERK1/2 to stimulate mucin family of mediators has very potent actions as
secretion by inducing exocytosis and release of well as being structurally distinct, displaying ste-
all the secretory granules within a given goblet reospecific actions, and utilizing different bio-
cell [25]. The molecular mechanism by which the synthetic pathways. Lipoxins are biosynthesized
exocytosis occurs is unstudied in the goblet cell. from the omega 6 fatty acid arachidonic acid
This pathway is tightly regulated in response to after class switching from production of pro-
neural activation of the cornea and conjunctiva to inflammatory mediators. The omega 3 fatty acid
maintain an optimal mucin layer that is critical docosahexaenoic acid (DHA) is precursor for the
for a healthy ocular surface. biosynthesis the D-series resolvins, the protec-
As there is far less research on the conjunctiva tins, and the maresins. The omega 3 fatty acid,
than the cornea, the mechanisms used by the con- eicosapentaenoic acid (EPA) is precursor for the
junctiva to respond to the environment are only biosynthesis of the potent E-series resolvins.
beginning to be described. The role of the spe- Each member of the RvD (RvD1-6) and RvE
cialized pro-resolving mediator (SPM) resolvin (RvE1 and 2) families possesses unique struc-
D1 (RvD1) is one of these mechanisms and is the tures and has distinct functions in the treatment
topic of the present review. of disease in animal models and in cells.
The enzymes responsible for this biosynthesis found that the normal, uninflamed conjunctiva
are the lipoxygenases (LOX) 5-LOX and 12/15 from various species of animals has the capacity
LOX. The location and specificity of the LOXs to synthesize both cyclooxygenases and lipoxy-
are cell and tissue specific. This determines the genases [27, 28]. The cycloxygenase was higher
type and amount of SPM produced. In general than the lipoxygenase activity. The major lipoxy-
15-LOX and 5-LOX are needed to produce lipox- genases produced by the conjunctiva were 12-
ins from arachidonic acid. 5-LOX is required to HETE, 5-HETE, and 5,12-diHETE suggesting
biosynthesize E-series resolvins. 15-LOX and production of hepoxilin and LTA4. Subsequently
5-LOX biosynthesize D-series resolvins and pro- conjunctiva and eyelids were demonstrated to
tectin (Fig. 3.4). 12-LOX biosynthesizes mares- possess EPA lipoxygenase products of the
ins. There is little published information on LOX 5-series suggesting the capability of producing
enzymes in the conjunctiva and especially the E-series resolvins [29].
goblet cells. Several articles from the 1980s
Docosahexaenoic Acid
15-LO
COX-2/
ASA
17S-H(p)-DHA 17R-H(p)-DHA
5-LO 5-LO
Enzymatic Enzymatic
epoxidation epoxidation
7,8-epoxy-17S-HDHA
7,8-epoxy-17R-HDHA
Enzymatic
Enzymatic
Hydrolysis
Hydrolysis
RvD1 AT-RvD1
7S,8R,17S-trihydroxy- 7S,8R,17R-trihydroxy-
4Z,9E,11E,13Z,15E,19Z-DHA 4Z,9E,11E,13Z,15E,19Z-DHA
Fig. 3.4 Synthetic Pathways of the D-series specialized pro-resolving mediators. Each is derived from DHA. 5-LO: 5
lipoxygenase Modified from Sun Y, Oh SF et al. J Biol Chem 282:9323–9234, 2007
There is substantial work on the LOX enzymes we will review the evidence for the role of the
in the cornea and the draining lymph nodes of the D-series resolvin RvD1 in conjunctival health.
eye that contribute to ocular surface disease espe-
cially [30–32], 5- and 15-LOX are the rate limit-
ing enzymes responsible for the generating SPMs 3.3 vD1 Is Present in Tears
R
[33]. The corneal epithelium has high expression from Healthy Individuals
of 15-LOX and functions as part of a LXA4-ALX/
FPR2 circuit that controls wound healing and A number of eicosanoids as well as SPMs are
immune response in the cornea. In particular there found in human tears. Human emotional tears
is a population of tissue specific polymorphonu- were collected from six male and six female sub-
clear leukocytes (PMNs) that contain especially jects and the lipid profile analyzed using an
high amounts of 15-LOX and that in a sex-spe- LC-MS-MS based metabololipidomics along
cific manner play a role in exacerbating autoim- with deuterium-labeled SPM as internal stan-
mune dry eye disease in females. These PMNs dards for quantitation [41]. We documented the
were detected in the lacrimal gland, draining presence of pro-inflammatory prostaglandins and
lymph nodes, and limbus of the cornea. As the the leukotriene B4. For the SPMs, the D-series
cornea and its limbus is adjacent to the conjunc- resolvins (RvD1, RvD2, RvD5), protectin D1,
tiva, it is possible that conjunctival epithelial cells and lipoxin A4, but neither the maresins nor
also have elevated levels of 15-LOX. Furthermore, E-series resolvins, were identified in these sam-
conjunctival goblet cells are a target of disease in ples from healthy human subjects. The SPM bio-
autoimmune dry eye. Study of ocular surface dis- synthesis pathway markers 17-HDHA,
ease has shown the presence of high levels of the 14-HDHA, and 18-HEPE were also identified.
biosynthetic enzymes for the SPMs and the cir- These compounds could be bioactive themselves
cuits for their regulation. These enzymes could [42] or could suggest that both D- and E-series
also function in health especially as during eye resolvins may be present in higher amounts
closure in sleep, PMNs migrate into the tear film locally than measured in the present study. The
and could interact with the conjunctival epithelial presence of pathway markers would also be con-
cells to set up a transient, low level activation of a sistent with the further metabolism of these bio-
LXA4-ALX/FPR2 like circuit or other circuits active resolvins to their oxo- and dehydro-resolvin
potentially in the conjunctiva. The same enzymes products that were not profiled in the present
produce RvD1 and could be critical for regulating study and are usually less or devoid of bioactiv-
RvD1 biosynthesis in conjunctival goblet cells. ity. Thus, RvD1 was present in human tears and
In addition to the production of SPMs in dis- available to regulate the function of the conjunc-
ease, SPMs are also endogenously produced in tival goblet cells. There are two other studies on
human tissues rich in omega 3 fatty acids in the SPMs in tears that are in agreement with that of
absence of disease [34]. Thus SPMs are produced English et al. [41]. Walter et al. [43] found DHA,
in human milk [35, 36], blood [37, 38], brain [39], the ω 3 fatty acid from which RvD1 is biosynthe-
and retina [40]. SPMs are major players in the sized, in tears. RvD1 was not measured directly
regulation of ocular surface health as SPMs are in this study. Masoudi S et al. [44] found RvD1 in
detected in the tear film and stimulate conjuncti- tears, but this was the only SPM analyzed.
val goblet cell mucin secretion under physiologic Surprisingly the lipid profile of male and female
conditions. They function to maintain an opti- tears differed substantially [41]. Use of a principal
mum mucin layer of the tear film in response to component analysis demonstrated a gender differ-
the normal changes in the cornea and conjunctiva ence in the SPMs in tears (Fig. 3.5). The loading
and to the extracellular environment. Multiple plot calculated from LC-MS-MS identified RvD1,
SPM family members are present within emo- RvD2, RvD5 and protectin D1 in male tears. In
tional human tears and stimulate goblet cell secre- contrast LXA4 and aspirin-triggered LXA4 were
tion to maintain the healthy tear film [41] . Herein detected in female tears. When the ratio of SPMs,
b Human Emotional Tears 2D Loading Plot
AT-LXA,
LXA,
RvD1
5-HETE
a
Principal component 1
5S, 15S-diHETE 12-HETE
LTB,
Human Emotional Tears 2D Score Plot 18-HEPE 5S,12S-diHETE
PGD,
14-HDHA
RvD5
15-HETE
RvD2
Female Donors (n=6) 17-HDHA
PGE,
PGF,a
PD1 20-OH-LTB,
c Total SPMs and PG Gender

Comparison
SPM & PG Levels (pg/100 mL) 1800 #
Male Donors (n=6) 1600
1400
1200
Principal component 2 1000
800 SPM
600 #
PG
400 *
200 *
0
Males Females
Gender
Fig. 3.5 PCA and quantitative ratio by gender for male donors & green circles are associated with female
LM-SPMs identified in human emotional tears. (a) donors. (c) Bar graph depicting the ratio of total SPMs
2-dimensional score plot of human emotional tear donors; including RvD1, RvD2, RvD5, PD1, LXA4, AT-LXA4,
blue circles (n = 6) are representative of males, while 17-HDHA, and 18-HEPE compared to PGE2 and PGF2α
green circles (n = 6) are representative of females. Gray (pg/100 μl), in males compared females (n = 6 for each
ellipse denotes 95% confidence interval. (b) 2-dimensional gender; #P < 0.05 for male donors vs. female donors;
loading plot of LM-SPMs identified in human emotional ∗P < 0.05, females vs. males). Reprinted from English
tears; blue circles are those mediators associated with et al. [41]
17-HDHA, and 18-HEPE versus the pro-inflam- was our focus. RvD1 stimulated human and rat
matory compounds PGE2 and PGF2a was com- conjunctival goblet cell high molecular weight
pared between males and females, the ratio of glycoconjugate secretion (that includes mucin)
SPMs to prostaglandins was much higher in males. [45, 46]. Secretion peaked at 1 h and then declined
over time for the next 3 h. RvD1 stimulated
secretion by increasing the [Ca2+]i and activating
3.4 xogenous RvD1 Stimulates
E ERK1/2 [45], similarly to the effect of choliner-
Conjunctival Goblet Cell gic agonists and VIP [21] (Fig. 3.6). RvD1 was
Mucin Secretion Using effective in the range of 10−10–10−8 M, a much
Multiple Intracellular lower concentration range than for carbachol
Signaling Pathways (10−6–10−4 M), but similar to VIP (10−11–10−7 M)
[21, 23]. The RvD1 stimulation of secretion was
3.4.1 R
vD1 Stimulates Goblet Cell blocked by chelating the intracellular Ca2+ with
Mucin Secretion BAPTA and by inhibiting ERK1/2 activation
with U0126 [47] substantiating the role of these
As the major function of goblet cells is to secrete signaling molecules in RvD1 stimulation of gob-
mucins, the action of RvD1 on mucin secretion let cell secretion.
Fig. 3.6 Schematic RvD1

diagram of signaling
pathways of RvD1 in
conjunctival goblet cells.
RvD1 binds to the ALX/
FPR2 receptor and
activates the signaling
pathways of PLA2, PLC,
and PLD. PLA2 and
PLD are believed to
activate ERK 1/2 to
increase [Ca2+]i and Aris Acid U73122
Ca2+/CamK. PLC 1-but
increases IP3 and

DAG. IP3 releases Ca2+
PLA2 PLD PLC
while DAG activates
PKC. Activation of these
pathways leads to mucin
UO126 UO126
IP3 DAG
secretion. PLA2-
phospholipase A2; 2-APB
PLD- phospholipase D; Ro 31-8220
PLC- phospholipase C;
ERK 1/2 Ca2+
ERK 1/2- extracellular
regulated kinase 1/2;
IP3- inositol KN93 Ca2+ PKC
trisphosphate; DAG-
diacylglycerol; Ca2+/ Ca2+/CamK
CamK- calcium/ X
calmodulin dependent
kinase; PKC- protein Mucins
kinase C. Inhibitors to
pathways are shown in
red. Reprinted from
Lippestad et al. [47]
Tears
To induce goblet cell function the appropri- blocked RvD1 stimulated increase in [Ca2+]i
ate receptors must be present on conjunctival [45, 47]. Functional evidence also demon-
goblet cells. RvD1 uses the ALX/FPR2 recep- strates that RvD1 uses the GPR32/DRV1
tor in rat and the GPR32/DRV1 receptor in receptor in human goblet cells. In human cells
human goblet cells. PCR, western blot and inhibition of ALX/FPR2 with its inhibitor
immunofluorescence microscopy demon- BOC-2 does not block RvD1 stimulated
strated that the ALX/FPR2 receptor was pres- increase in [Ca2+]i [49]. Furthermore, in desen-
ent on rat conjunctival goblet cells in vivo and sitization experiments in human cells in which
in culture [48, 49]. There is as yet no published LXA4 and RvD1 were used sequentially, RvD1
report of GPR32/DRV1 on human goblet cells. does not desensitize LXA4 response [49]. This
There is also functional evidence for the pres- suggests use of separate receptors in human
ence of ALX/FPR2 receptor in rat conjunctival goblet cells. Thus RvD1 activates ALX/
goblet cells. To address whether RvD1 uses FPR2 in rat goblet cells and GPR32/DRV1 in
the ALX/FPR2 receptor in the rat, both the human goblet cells. Further experiments using
ALX/FPR2 inhibitor N-Boc−Phe-Leu-Phe- GPR32 siRNA in human goblet cells is
Leu-Phe (BOC-2) and siRNA for ALX/FPR2 warranted.
3.4.2 C
ellular Signaling Pathways [Ca2+]i supported the activation of Ca2+ influx by
Activated by RvD1 RvD1 [45].
The DAG arm of the PLC pathway was inves-
3.4.2.1 Phospholipase C Pathway tigated by determining the role of PKC using the
RvD1 activates a several specific intracellular PKC inhibitor Ro 31-8220. The PKC inhibitor
signaling pathways to stimulate mucin secretion blocked the RvD1-stimulated increase in [Ca2+]i,
from rat goblet cells. One pathway is activation but not in secretion. Activation of PKC may be
of phospholipase (PL) C that produces water important for the increase in [Ca2+]i, but not for
soluble 1,4,5-inositol trisphosphate (IP3) and secretion. There are multiple isoforms of PKC
lipid soluble (membrane-bound) diacylglycerol that are differentially activated by Ca2+ and diac-
(DAG) (Fig. 3.6). IP3 binds to its receptors ylglycerol and can have opposing effects on a
(IP3R) on the endoplasmic reticulum that given process [50]. Thus investigation of the
releases Ca2+ from this intracellular store to types of PKC isoforms present in goblet cells and
increase the cytosolic [Ca2+] that stimulates exo- inhibition of single isoforms would demonstrate
cytosis and mucin secretion. The DAG produced more accurately whether PKC isoforms are
activates protein kinase (PK)C that phosphory- involved in goblet cell secretion.
lates as yet unidentified substrates to stimulate We concluded that RvD1 uses a PLC pathway
secretion. We used multiple techniques and to increase IP3 that releases Ca2+ from intracel-
inhibitors to determine if a PLC-dependent path- lular stores and causes an increase in the influx of
way plays a role in RvD1 stimulated goblet cell extracellular Ca2+ (Fig. 3.6). The resultant
secretion [47]. First the active PLC inhibitor increase in the cytosolic [Ca2+]i stimulates secre-
U73122 blocked RvD1 stimulated increase in tion. The second arm of the PLC pathway pro-
[Ca2+]i and secretion as well as the increase duction of DAG and activation of PKC is used to
caused by the positive control the cholinergic increase the [Ca2+]i, but whether PKC plays a role
agonist carbachol. As expected, the inactive in secretion awaits investigation of the different
inhibitor U73343 did not block either the RvD1 PKC isoforms.
or cholinergic agonist stimulation of secretion PLCγ is activated by EGF as PLCγ is an
nor the cholinergic agonist induced increase in adapter molecule attached to the EGFR. PLCγ
[Ca2+]i. Unfortunately the inactive inhibitor can be phosphorylated and activated upon stimu-
blocked the RvD1 induced increase in [Ca2+]i but lation and dimerization of the EGFR. We recently
was not as effective as the active analog. As the found that RvD1 uses a matrix metalloproteinase
inactive inhibitor did not block three out of four ADAM17 to release EGF by ectodomain shed-
responses, we concluded that RvD1 activates ding and activate the EGFR to increase [Ca2+]i
PLC in conjunctival goblet cells. To investigate and activate ERK1/2 to stimulate secretion [51].
IP3 interaction with its receptor to release Ca2+ Thus RvD1 could also activate PLCγ in addition
from intracellular stores, an inhibitor of the IP3 to PLCβ to increase [Ca2+]i and stimulate secre-
receptor 2-aminoethyl diphenylborate (2-APB) tion. Thus two different types of PLC are used by
was used. 2-APB blocked both the RvD1 stimu- RvD1 to stimulate goblet cell mucin secretion.
lated increase in [Ca2+]i and secretion. A second
method of determining the role of intracellular 3.4.2.2 Phospholipase D
Ca2+ stores is the use of thapsigargin that blocks and A2 Pathways
the re-uptake of Ca2+ into the stores, thereby The next two pathways investigated were activa-
depleting them of Ca2+. If RvD1 used intracellu- tion of PLD and PLA2 [47] (Fig. 3.6). These path-
lar Ca2+ stores, the addition of thapsigargin ways were not investigated in as much detail as
before RvD1 should prevent the increase in PLC. The role of PLD in RVD1 stimulated increase
[Ca2+]i by RvD1. In goblet cells this did not occur in [Ca2+]i and secretion was investigated using
[45]. However, that depletion of extracellular 1-butanol the active inhibitor of PLD and tert-buta-
Ca2+ blocked the RvD1 stimulated elevation in nol, its inactive control. The RvD1- induced
increase in [Ca2+]i and stimulation of secretion was gens, particulate matter, and pathogens in the
almost completely blocked by 1-butanol. The inac- external environment. RvD1 is present in the
tive control only partially blocked the increase in tear film where it can access its receptors on the
[Ca2+]i and did not block the stimulation of secre- basolateral membranes of the goblet cells. RvD1
tion. To study PLA2 aristolochic acid was used. uses the ALX/FPR2 receptor in rat goblet cells
Aristolochic acid blocked both the RvD1 caused and the GPR32 in human goblet cells. Activation
increase in [Ca2+]i and stimulation of secretion. of these receptors employs multiple intracellular
These results are consistent with RvD1 using both pathways including PLC, PLD, PLA2 and the
PLD and PLA2 to stimulate goblet cell secretion. EGFR to increase [Ca2+]i and activate ERK1/2 to
Additional experiments should identify the com- stimulate secretion. The main secretory product
ponents of these pathways. of the goblet cells is the large, gel-forming
mucin MU5AC, which is released into the inner-
3.4.2.3 Extracellular Regulated Kinase most layer of the tear film where it is protective
1/2 Pathway of the ocular surface. This mucin can trap bacte-
RvD1 activates ERK1/2 to increase [Ca2+]i and ria and particulate matter and remove them from
stimulate secretion as shown by inhibition of the ocular surface via the nasal lacrimal drain-
both functions by the MEK inhibitor U0126 [45] age. RvD1 thus preserves ocular surface homeo-
(Fig. 3.6). ERK1/2 could function as a compo- stasis and maintains this surface in a
nent in several of the pathways studied. ERK1/2 non-inflamed, normal physiologic state of an
could be activated by induction of the EGFR optimum amount of mucin secretion. RvD1
using the adapter proteins Ras, Raf, and forms a second important mechanism along with
MEK. ERK1/2 could also be downstream of PLD activation of a neural reflex pathway to regulate
or PLA2. goblet cell mucin secretion and protect the ocu-
lar surface in health.
3.4.3 Summary of Pathways Acknowledgements Dr. Dartt is supported by the

Activated by RvD1 National Institutes of Health RO1 EY019470 and Dr.
Serhan also thanks the NIH for support from
1P01GM095467 (CNS).
RvD1 stimulates mucin secretion from conjuncti-
val goblet cells by a receptor specific mechanism
using ALX/FPR2 in rats and GPR32 in humans.
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The Evolving Role of Specialized
Pro-resolving Mediators 4
in Modulating Neuroinflammation
in Perioperative Neurocognitive
Disorders
Ting Yang and Niccolò Terrando
Abstract postoperative neuroinflammation and neuro-

Surgery can be a life-saving procedure; how- logical complications after surgery.
ever, significant complications may occur
after routine procedures especially in older Keywords
and more frail patients. Perioperative neuro- Astrocytes · Blood-brain barrier · Bone ·
cognitive disorders (PNDs), including delir- Brain · Cytokines · Delirium · Healing ·
ium and postoperative cognitive dysfunction, Macrophages · Maresins · Memory ·
are the most common complications in older Microglia · Neuroinflammation · Resolvins ·
adults following common procedures such as Surgery
orthopedic or cardiac surgery. The conse-
quences of PNDs can be devastating, with lon-
ger in-hospital stay, poorer prognosis, and
higher mortality rates. Inflammation is gain- Perioperative neurocognitive disorders (PNDs),
ing considerable interest as a critical driver of previously referred to as postoperative cognitive
cognitive deficits. In this regard, resolution of dysfunction (POCD), include acute changes in
inflammation, once thought to be a passive cognitive function (i.e. delirium) and longer-
process, may provide novel approaches to lasting memory impairments [1]. PNDs have
treat neuroinflammation and PNDs. Herein we become the most common complications in older
review the role for impaired resolution after adults after common surgical procedures such as
surgery and the growing role of specialized cardiac and orthopedic surgery, and negatively
pro-resolving mediators (SPMs) in regulating affect post-operative outcomes and recovery in
at-risk subjects [2]. Indeed, patients who suffer
from PNDs require intensive nursing care, report
higher mortality rates, and become at greater risk
T. Yang for further complications, including permanent
Division of Nephrology, Department of Medicine, dementia [3, 4]. Although advanced age has been
Duke University and Durham VA Medical Centers,
Durham, NC, USA recognized as a prominent risk factor for PNDs,
the pathophysiology of these complications is
N. Terrando (*)
Center for Translational Pain Medicine, Department still poorly understood. Recent studies showed
of Anesthesiology, Duke University Medical Center, pro-inflammatory signaling molecules can be
Durham, NC, USA identified in the central nervous system (CNS) of

28 T. Yang and N. Terrando
both PND patients and animal models, implicat- nuclear factor (NF)-κB signaling pathway [9]
ing surgery-induced neuroinflammation as a key [10]. BBB homeostasis plays fundamental role in
contributor in the pathophysiology of PNDs. The the communication between peripheral inflam-
aim of this review is to discuss the role of dys- mation and neuroinflammation [11]. Using
regulated immunity after surgery and the thera- fibrinogen as a classical marker of BBB disrup-
peutic potential for specialized pro-resolving tion, we reported a significant deposition of this
lipid mediators (SPMs) in the perioperative neu- blood-derived molecule in the hippocampal
rocognitive arena. parenchyma [12]. Similar results were reported
We and others have been interested in clarify- in other surgical models, with evident BBB open-
ing the pathogenesis of PNDs and developed pre- ing and disruption of tight junctions after anes-
clinical translational models to study the impact thesia and surgery [13]. More recently we
of anesthesia and surgery on the CNS. During described a role for neuronal processing, includ-
surgery, aside from direct effects of anesthetic ing pain signaling at the level of the spinal cord,
agents on the CNS (reviewed in [5]), acute in disrupting acute neurogenesis after orthopedic
inflammation triggered by trauma and sterile surgery [14]. These factors, together with the
injury can negatively affect brain function, con- overall activation of innate immune pathways,
tributing to sickness behavior and PND-like have been highlighted as pathogenic mechanisms
pathology [6]. If poorly controlled, dysregulated underlying cognitive decline in preclinical mod-
inflammation leads to more extensive organ dys- els and in early clinical studies. Preclinical mod-
function and tissue injury. Release of pro- els have evaluated multiple therapeutic strategies
inflammatory cytokines, alarmins, and to modulate neuroinflammation and PND-like
leukotrienes appear to associate with distant behavior. Selective targeting of key pro-
long-term effects on the brain, contributing inflammatory cytokines (such as anti-TNFα and
to neuroinflammation, neurotoxicity, and subse- IL-1 receptor agonist) prevent neuroinflamma-
quent memory impairments. The mechanisms tion and improve cognitive decline in animal
whereby systemic inflammation affects CNS models [6, 15]. Macrophage-specific deletion of
function remain partly defined and are object of IKKβ, a central coordinator of TNFα activation
active investigations. Systemic cytokines have of NF-κB, prevents BBB disruption and macro-
been shown to enter the brain through different phage infiltration in the hippocampus following
immune-to-brain signaling pathways, which surgery. Moreover, harnessing the cholinergic
include humoral, neuronal, and cellular routes reflex by stimulating the α7 subtype of nicotinic
[7]. Using a well-established PND model of acetylcholine receptors (α7 nAChR) in macro-
orthopedic surgery, which frequently leads to phages inhibited NF-κB activity and BBB dis-
cognitive dysfunctions in humans, we first ruption, thus preventing neuroinflammation and
reported a key role for systemic pro-inflammatory cognitive decline following surgery [12].
cytokines, including interleukin (IL)-1β and Although these and several other therapies may
tumor necrosis factor alpha (TNFα), in mediating successfully reduce postoperative neuroinflam-
surgery-induced neuroinflammation and cogni- mation and limit PNDs, they often contribute to
tive decline following surgery [6, 8]. These initial side effects by over suppressing the immune sys-
results as well as work from other models of tem, increasing the risk of infection and delaying
surgery-induced cognitive dysfunction, prompt wound healing, which are all crucial in the con-
for a prominent role of the systemic and humoral text of postoperative recovery. Thus, it is pivotal
response in PND pathogenesis. However, periph- to identify safer therapeutic strategies to prevent
eral cytokines in response to non-CNS surgery neuroinflammation without suppressing systemic
were also shown to disrupt the blood-brain bar- immune functions.
rier (BBB), thus facilitating the migration of Is now well appreciated that resolution of
peripheral cells, including macrophages, into the inflammation is an active process [16–18].
brain parenchyma through activation of TNFα/ Synthesis and release of anti-inflammatory
4 The Evolving Role of Specialized Pro-resolving Mediators in Modulating Neuroinflammation… 29
olecules is required to balance and orchestrate

m SPMs in AD is provided in [29]. Here we will
the overall immune response [19]. The acute review the growing role of SPMs in the periop-
inflammatory response can be divided into two erative space, focusing on neuroinflammation in
stages: initiation and resolution. Resolution PNDs.
launches shortly after the initiation of inflamma- Microglia are the primary active immune
tory response [20]. Seminal work pioneered by defense in the CNS responsible to maintain brain
Dr. Serhan and colleagues has demonstrated how homeostasis. However, activated microglia can
different mediators actively participate in resolv- cause overproduction of pro-inflammatory cyto-
ing acute inflammation in a highly regulated kines and reactive oxidative species that can lead
manner (recently reviewed in [21]). Fundamental to persistent inflammation and exacerbate patho-
to the resolution process are SPMs, endogenous logical changes in CNS [30, 31]. In PND microg-
mediators generated by polyunsaturated fatty lia respond to surgical trauma following BBB
acids (PUFA) with signaling abilities to limit opening and monocytes infiltration. Modulation
inflammation [22]. Omega-3 PUFAs are impor- of microglial activity is an attractive therapeutic
tant catalysts for the synthesis of potent lipid targeted for multiple conditions ranging from
mediators that can exert pro-resolving and anti- neurodegeneration to chronic pain [32]. In vitro
inflammatory actions. Oxygenated metabolites studies using immortalised murine microglial
derived from eicosapentaenoic acid (20:5(n-3) or cell line BV-2 showed pre-incubation with resol-
EPA) and docosahexaenoic acid (22:6(n-3) or vin D1 (RvD1) and E1 (RvE1) inhibit lipopoly-
DHA), enriched in fish oils, lead to structurally saccharide (LPS)-induced TNF-α, IL-6 and IL-1β
distinct families of “resolution phase interaction gene expression via regulating of miRNAs
products” [23], which include resolvins, mares- expression and NF-κB signaling pathway [33].
ins, protectins (as well as sulfide-conjugates in Similar effects of RvD1 were demonstrated in
tissue regeneration), and lipoxins (from arachi- separate studies using human glioma cells or iso-
donic acid). All SPMs display potent anti-inflam- lated primary rat microglia [34, 35]. RvD1 and
matory actions and immunoregulatory properties maresin-1 (MaR1) also down-regulate β-amyloid
now tested in several pre-clinical models of acute (Aβ)42-induced CD11b and CD40 expression in
and chronic inflammation. Indeed, SPMs inhibit human microglial cells. Moreover, MaR1 and
the excessive swarming of neutrophils infiltration aspirin triggered lipoxin A4 (ATL) both exert
and enhance the microbial clearance function of stimulatory effect on microglial cells to uptake
innate immune cells [24]. SPMs also operate as Aβ42 [36, 37]. In addition to reducing the expres-
“resolution agonists” to regulate the acute inflam- sion of M1-like markers activation of microglia,
mation and limit the development of chronic RvD1 also promotes the expression of Arg1 and
inflammation; importantly they do not show Ym1 in IL-4 activated BV2 cells [38]. These in
immunosuppressive effects [23, 25]. The exag- vitro findings demonstrate that SPMs are directly
gerated inflammation and non-resolution of pro- involved in regulating both classical and alterna-
inflammatory processes are characteristic of tive microglia activation, thus may display potent
several neurological conditions and disease states therapeutic effects in inflammatory diseases of
like stroke, neurodegeneration, and chronic pain. the CNS. The regulatory effects of SPMs on
Evidence from clinical studies using dietary fish microglial cells have also been also verified in
oil supplementation suggest promising effects of vivo. A study using Tg2576 mice, which overex-
omega 3 fatty acid in modulating inflammation presses a mutant form of amyloid precursor pro-
both in acute and chronic conditions [26, 27]. Yet tein (APP) and develops early AD onset, showed
the role of SPMs in resolution of neuroinflamma- neuroprotective effects of ATL by shifting
tion and neuroprotection is just emerging. In microglial phenotype to a more anti-inflammatory
Alzheimer’s disease (AD), circulating SPMs and state [37]. In models of surgery-induced microg-
receptor expression are diminished in the brain lial activation, both lipoxins and resolvins have
[28]. A comprehensive review on the role of been shown to improve neuroinflammation by
attenuating release of pro-inflammatory cyto- cytes actions may be determined by the danger
kines [39–41]. Importantly, other classes of signals in the local environment and regulated in
inflammatory-stop signals can modulate neuroin- time-specific manner [56]. The controversial
flammation in PND-models. Using a rat model of roles of astrocytes indicate that these cells may
cardiopulmonary bypass with deep hypothermic play key roles in cognitive function, both in
circulatory arrest treatment with annexin A1 health and diseases. The specific SPMs actions
(ANXA1) was able to improve cognitive out- on astrocytes have not been thoroughly investi-
comes and modulate microglial activation by gated. In pain models, Lipoxin A1 exerts anti-
inhibiting NF-κB p65 transcriptional activity and nociceptive effect by modulation of astrocytic
subsequent cytokine production [42]. Recently, activation [57]. Similarly, AT-RvD1 was shown
we demonstrated that prophylaxis with MaR1 to attenuate TNF-α release from spinal astro-
can rescue microglial activation after orthopedic cytes and improve mechanical hypersensitivity
surgery, as detected by morphological changes- in a rat model of carrageenan-induced peripheral
such as shifting to a more ramified homeo- inflammation [58]. In PND models we described
static morphology [43]. Thus, resolution agonists changes in astrocytes morphology at 24 h after
including SPMs may diminish neuroinflamma- orthopedic surgery [41, 43]. The astrogliosis is
tion in part by regulating microglial phenotype, associated with increased basal glutamatergic
leading to improved cognitive outcomes. To date synaptic transmission, reduced short term plas-
many of the SPMs have not been evaluated in ticity and long-term potentiation in hippocam-
models of PND and surgical recovery, which pus [41]. These pathological changes in
warrants future investigations. astrocytes can be eliminated by prophylaxis with
Aside from microglia, astrocytes also contrib- aspirin triggered resolvin-D1 (AT-RvD1) as low
ute a key role in maintaining CNS homeostasis, as ~0.1 μg/kg [41] or MaR1 at ~4 μg/kg [43].
including regulating brain blood flow [44, 45], The precise mechanisms underlying the effects
synapse function [46], extracellular ion concen- of AT-RvD1 and MaR1 on postoperative astro-
tration [47], and interacting with endothelial gliosis still require further exploration.
cells to support the BBB [48]. Notably, a role for Accumulated evidences suggest the pro-inflam-
immune-regulation has emerged from these cells matory activity of astrocytes may be regulated
[49]. As an active immune regulator in CNS, by microglia [59] via cytokines, chemokine,
astrocytes also sense stimulation and danger sig- complement activation [60], growth factors, and
nals, responding by releasing glia-transmitters other signaling molecules [61]. After surgery the
and communicating to neurons [50]. Together CCL2/CCR2 axis has been implicated in the
with microglia they also contribute to further neuroinflammatory response; Xu et al. showed
activating adaptive immune defense [51] [49]. that increased CCL2 expression in astrocytes is
Following brain injury or in neurodegenerative sufficient to activate microglia and cause learn-
diseases, astrocytes undergo pronounced trans- ing impairments [62]. Therefore, regulation of
formation called “astrogliosis”. Astrogliosis astrogliosis may indirectly regulate microglia
changes the cellular expression and morphology activity and targeting cell-specific interactions
of astrocytes and contribute to the repair and may result into effective therapies for different
scarring process in CNS [52]. However, this neurological conditions, including PNDs
reactive phenotype has also been suggested to (Figs. 4.1 and 4.2).
become detrimental by upregulating the expres- Intrinsic to the postoperative neuroinflamma-
sion of IL-17 receptor and sphingosine 1-phos- tion is the activation of the peripheral innate
phate (S1P) [53, 54]. This further triggers the immune system. Sterile injury, as during surgery,
production of pro-inflammatory cytokines and rapidly triggers the release of TNFα and alarmins
chemokines, which can lead to exacerbated neu- into the circulation. TNFα can initiate a pro-
roinflammation and neurodegeneration [55, 56]. inflammatory cytokine cascade that eventually
During neuroinflammation, the status of astro- impairs the BBB homeostasis. The BBB dys-
function in turn facilitates the migration of LPS-induced TNFα release, NF-κB nuclear
macrophages into the hippocampus [6, 12, 41, translocation, superoxide generation and M1-like
43]. Inhibition of the TNFα signaling by anti- phenotype surface markers expression in primary
TNFα antibody or genetic abrogation of bone marrow derived macrophages [43]. These
macrophage-specific IKKβ prevent postoperative modulatory effects on macrophage function/phe-
BBB disruption and macrophage infiltration in notype may be a key mechanism for SPMs to pre-
the hippocampus [6, 12]. This work demon- vent surgery induced BBB disruption, ensuing
strated the primary impact of innate immune sys- neuroinflammation, and cognitive decline [43].
tem and systemic inflammation on the Importantly, because SPMs exert both anti-
development of neuroinflammation and cognitive inflammatory and pro-resolving effects, they are
decline. During resolution of inflammation, crucial to terminate inflammation but also stimu-
SPMs stimulate the cessation of PMN influx [22, late tissue repair [65], which is fundamental in
34] and macrophage clearance of cellular and the context of perioperative recovery. In fact,
toxic debris [16, 20, 63]. The regulation of mac- RvD1 and maresins have shown to accelerate
rophage phagocytosis function by SPMs may be wound healing in diabetic patients [66, 67].
mediated by the switch of macrophage pheno- AT-RvD1 delivered through nanoparticles also
type [16]. For example, Dalli et al. demonstrated enhance wound healing in a mice model of peri-
that 10 nM of MaR1 or RvD1 lead to significant tonitis [68]. We recently demonstrated that MaR1
reductions in CD54 and CD80 expression and a pretreatment boosts systemic levels of IL-10 with
concomitant upregulation of CD163 and a long- lasting trend up to 14 days after sur-
CD206 in human macrophage [64]. Moreover, gery [43]. Further, we found no difference in cal-
M2-like macrophages are more efficient in con- lus formation in mice treated with MaR1
verting DHA into MaR1 [64]. Similarly, our in compared to vehicle, suggesting MaR1 may be a
vitro work indicated 10 nM MaR1 can inhibit safe option to be tested in future clinical trials.
BBB CNS
TNF
IL-1 GFAP+
IL-6 cld-5
IL-12
CXCL1
Iba-1+
MΦ
SPMs
pro-inflammatory pro-resolving
Fig. 4.1 Putative mechanisms of surgery-induced neuro- dependent memory function. Treatment with SPMs, such
inflammation and cognitive dysfunction. Surgery triggers as resolvins and maresins, actively promote resolution of
systemic pro-inflammatory cytokines that can impair the inflammation after surgical trauma and prevent central
blood-brain barrier, thus allowing peripheral cells into the nervous system dysfunction through the modulation of
brain parenchyma. Macrophage infiltration activates glia, systemic and central cell types, such as monocytes,
including resident microglia and astrocytes, that acutely microglia, and neurons
affect processes of synaptic plasticity and hippocampal-
Fig. 4.2 Proposed mechanisms for neuroinflammation microglia but also by infiltration of peripheral cells into
and surgery-induced cognitive dysfunction. Surgery has the brain parenchyma via a disrupted blood-brain barrier.
been shown to engage the innate immune system and acti- This pro-inflammatory milieu and glia dysfunction impair
vate a cascade of pro-inflammatory mediators, including neuronal activity and synaptic plasticity, impinging on
alarmins, cytokines and eicosanoids. These molecules processes of long-term potentiation, neurotransmission,
exert effects on the humoral and neuronal signaling over- and receptor function at the synapse. In combination,
all contributing to the neuroinflammatory response. These these pathological hallmarks contribute to learning and
processes are mediated not only by activation of resident memory impairments following surgical trauma
Overall, we are starting to uncover the poten- intervene before an elective surgical procedure.
tial for resolution agonists in the context of peri- SPMs biomarkers may take us a step closer to
operative disorders and surgical recovery. Further personalized approaches and targeted interven-
research is needed to evaluate the exact mecha- tions to treat common complications, like post-
nisms of action of different SPMs on different operative pain and PNDs. As coined by Serhan
CNS cell types, and whether specific mediators and Savill ‘alpha’ programs ‘omega’ (i.e. the
may better target the immune response triggered beginning programs the end) [20] could not bet-
by surgical trauma. SPMs have been detected ter define the importance of the temporal events
in human cerebrospinal fluid, establishing proof- that orchestrate the acute inflammatory
of-principle that may serve as biomarkers for response in the perioperative space. Further char-
disease progression in neurological conditions acterizing these complex molecular events may
like multiple sclerosis [69] and clinical PND provide novel and urgently needed approaches to
[43]. These mediators can also be modified by safely treat PND and overall improve brain
dietary interventions, for example omega-3 health for the millions of patients that undergo
PUFA [70], thus providing attractive strategies to surgery every year.
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Relationship Between Specialized
Pro-resolving Mediators 5
and Inflammatory Markers
in Chronic Cardiac Disorders
M. Brianza-Padilla and R. Bojalil
Keywords We can consider acute inflammation as an

Pro-resolving mediators · Inflammation · extreme cellular response to stress that occurs
Cardiac disorders when a stimulus perceived as harmful triggers
mechanisms of the innate immune system [58].
Plasmatic and cellular molecules, leukocytes,
and endothelial cells take part in the inflamma-
5.1 Introduction tory process which induces an increase of vascu-
lar permeability, cell-cell interactions, and the
The term cardiovascular diseases (CVD) refers to massive recruitment of leukocytes and molecules
disorders of heart and blood vessels, and include to the tissues [8]. Since cellular dysfunction can
coronary heart disease, cerebrovascular disease, be an inflammatory trigger, and inflammation
peripheral vascular disease, and heart failure, itself can induce dysfunction or damage, a lack of
among others. Atherosclerosis is a common control of the process can set up a perpetuating
background of these diseases. It is not infrequent cycle [58]. Thus, if the homeostatic mechanisms
that some acute diseases, such as coronary syn- are incapable of returning to the earlier limits,
dromes, appear superimposed on a chronic arte- chronic inflammation takes place, eventually
rial disease. Acute coronary syndromes (ACS), leading to systemic diseases. Various cardiovas-
found worldwide among the leading causes of cular and metabolic diseases are examples of this
death, can be the origin of disabling chronic CVD phenomenon. Indeed, current knowledge permits
such as heart failure [46]. Clinical and experi- association of metabolic disorders which show
mental evidence associates this group of altera- chronic inflammation, such as obesity and its
tions with an inflammatory process that takes part comorbidities (accumulation of visceral fat,
in its pathophysiology. In fact, inflammation is hypertension, dyslipidemia, and glucose resis-
one of the most important factors for its initia- tance) with CVD [42]. Hence, evidence points to
tion, progression, and consolidation [6]. inflammation as an important player in CVD. The
alterations in the concentrations of inflammatory
M. Brianza-Padilla markers reflect such a response. Their serum
Department of Immunology, Instituto Nacional de levels predict cardiovascular risk [43], and the
Cardiología Ignacio Chávez, Mexico City, Mexico outcome and mortality due to acute complica-
R. Bojalil (*) tions of ACS or to heart failure [10, 25].
Department of Health Care, Universidad Autónoma
Metropolitana-Xochimilco, Mexico City, Mexico

38 M. Brianza-Padilla and R. Bojalil
5.2 Cells and Mediators in CVD Thromboxane B2 (TXB2) and leukotriene B4

(LTB4) [12].
As with other inflammatory-related conditions, Tissue repair seems to need the lipid profile of
cells of the immune and inflammatory responses the M2 subtype. The involved mediators include
crucially take part in the progression of CVD. The derivatives of the omega-3 (n-3) polyunsaturated
active and coordinated inflammatory resolution fatty acids (PUFAs) eicosapentaenoic acid (EPA)
process, involves changes in subpopulations of and docosahexaenoic acid (DHA). We call their
inflammatory cells and their proportions, and derivates specialized pro-resolving lipid media-
changes in pro- and anti-inflammatory cytokines tors (SPMs): resolvins (Rv), protectins (PD) and
and lipid mediators, among other factors [51]. maresins (MaR). Lipoxins (LX) are also SPMs
Macrophages are key cells that take part in mul- but their precursor is AA [32]. Serhan and his
tiple roles in the immune response, inflammation, group have identified two classes of resolvins:
and in different pathologies. They respond to that of the E series correspond to the EPA deriva-
various stimuli and express and secrete a wide tives; and the class of the D series corresponds to
range of soluble molecules and receptors which the DHA derivatives; the latter being also the pre-
include lipid mediators, scavenger receptors, pro- cursor of protectins and maresins. M2 produce
and anti-inflammatory cytokines and chemo- more SPMs than the M1 macrophages, such as
kines; macrophages also present autophagic and RvD5, MaR1, PD1, LXA4, LXB4, LXB5, and
anti-apoptotic properties [37]. We find these cells RvE2, [12]. Apoptotic neutrophils stimulate M2
of the innate immune system in varying propor- macrophages: they produce lipid mediators that
tions in healthy and inflamed tissues or tissues activate the SPM profile characteristic of M2
being repaired [41]. For example, in a healthy [12]. One of the main features of M2 macro-
murine heart, they represent approximately 8% phages is clearing apoptotic cells, a process
of all cardiac cells [28] whilst after a myocardial known as epherocytosis, essential for restoring
infarction, the proportion of macrophages damaged tissue.
increases significantly [17].
We can distinguish two main types of macro-
phages: the inflammatory phenotype M1 (also 5.3 SPMs and Other Mediators
known as activated by the classical route), and
the anti-inflammatory phenotype M2 or acti- SPMs play important roles in resolving inflam-
vated by the alternative route; stimuli such as mation, and thus in a physiologic repair of tissues
gamma interferon (IFNγ) or interleukin-4 (IL- [4]. The membrane’s phospholipids of activated
4), and the lymphocyte subpopulation responsi- cells are the precursors of SPMs and other lipid
ble for those stimuli, differentially regulate their mediators of the inflammatory process [50].
activation pathways [22, 54]. For example, some Inflammatory stimuli such as lesions, microor-
authors have observed that regulatory T cells ganisms, and IL-8 activate phospholipase (FLP)
induce the differentiation of macrophages to A2 that produces free AA [8, 49]. When metabo-
M2 in myocardial repair. Both subtypes of mac- lized, this fatty acid forms a family of oxygenated
rophages have different functions and capacities, products called eicosanoids, because of their
partly related to the different profiles of inflam- 20-carbon structure [34]. Eicosanoids include
matory and pro-resolving molecules they syn- prostaglandins, prostacyclins, thromboxanes,
thesize. The pathogen- associated molecular leukotrienes, lipoxins, and hydroxy and hydro-
patterns (PAMPs) activate the M1 macrophages, peroxy fatty acids. While the enzymatic activity
a fact that induces the release of pro-inflamma- of cyclooxygenase (COX) synthesizes
tory cytokines [21]. These macrophages synthe- prostaglandins and thromboxanes, that of lipoxy-
size a pro- inflammatory lipid profile mainly genase (LOX) synthesizes leukotrienes and
derived from the omega-6 (n-6) arachidonic acid lipoxins. In a later stage of inflammation, and
(AA) such as prostaglandin E2 (PGE2), PGF2, depending on the composition of the cell mem-
5 Relationship Between Specialized Pro-resolving Mediators and Inflammatory Markers in Chronic… 39
brane, EPA and DHA substitute AA as the sub- erosclerotic plaques represents a risk to develop
strates of lipid mediators. The SPMs derived myocardial infarction and cerebrovascular events
from those n-3 fatty acids, exert powerful anti- [57]. In atherosclerosis, vascular smooth muscle
inflammatory and pro-resolving actions mainly cells have increased proliferative and chemotactic
by decreasing the influx of neutrophils to tissues, features, reduced expression of contractile pro-
by enhancing non-phlogistic recruitment of teins and increased production of proinflamma-
monocytes into tissues, and by decreasing the tory cytokines [4]; they show increased
production of pro-inflammatory mediators [11]. inflammation and oxidative stress, and extensive
Cell membrane surface receptors mediate their areas of necrosis composed of non-clarified apop-
signaling [29, 33]. Annexin A1, a stress response totic cells [19]. Some authors suggest that poor
endogenous protein, also mediates inflammatory resolution of inflammation merges the above [19],
resolution and can decrease the production of hence various researchers have tested diverse
leukotrienes and prostaglandins by an inhibitory experimental approaches to prove this hypothesis.
action on FLPA2 [15]. SPMs delay the progression of atherosclerosis
Annexin
PLP A2 Regulation
PUFA’s Omega 6 Inflammation
Resolution
Intake AA EPA DHA
Omega 3
Prostaglandins Resolvins E Resolvins D
Thromboxanes Protectins
Maresins
Leukotrienes
Lipoxins
5.4 Resolution and CVD and promote greater plaque stability [18]. A nota-
ble observation is that mouse atherosclerotic
Atherosclerosis is a disease that is a convincing plaque treated with SPMs increases its thickness
example of the consequences of an inefficient and the synthesis of collagen in the fibrous layer,
inflammatory resolution. In humans, having ath- thus gaining stability [18, 19].
Today there is a great interest in studying the proliferation, migration, and production of
resolvins in cardiac alterations because they seem superoxide anion, and the expression of proin-
to be crucial players in restoring lesions since flammatory genes [3, 59]. The phosphoinositide
they reduce the response of vascular smooth 3-kinase (PI3K)/serine/threonine kinase Akt
muscle cells and decrease the damage through (PI3K/Akt) signaling interactive pathway medi-
the local activation of resolution mechanisms [8]. ates the protective effects of RvD1 (and of lipox-
There are reports that resolvins can reduce the ins), as treating with its inhibitor can block the
risk of developing atherosclerosis and its compli- cardioprotective effect of RvD1 [23]. In a model
cations by preventing platelet aggregation, induc- of damage caused by hepatic ischemia, RvD1
ing vasodilation [13], inducing neutrophil inhibits inflammatory pathways, decreasing the
apoptosis and limiting the size of the infarct [8]. levels of IL-6, TNF, and myeloperoxidase, and
In humans, however, there is still a long way to increases phosphorylation of Akt, which favors
fully assess their therapeutic efficiency and pos- its protective effect. In addition, RvD1 reduces
sible toxic doses. the expression of pro-fibrotic genes and the depo-
Resolvin E1 was the first metabolite of EPA sition of collagen, which reduces post-infarction
identified, its biosynthesis depends on the inter- fibrosis [30]. In ischemic lesions, neutrophils
action between endothelial cells and leukocytes infiltrate and release reactive oxygen species
in the inflammatory process [48]. Several studies (ROS) and other mediators that damage tissue
suggest that RvE1 plays an important role in the integrity [56]. Exogenous administration of
restoration and repair of tissue damage, favoring RvD1 after myocardial infarction in mice reduces
resolution by decreasing the production of pro- both the accumulation of neutrophils and fibro-
inflammatory proteins and platelet aggregation. sis, which induces an improvement in cardiac
Administering RvE1 reduces the size of the function [30].
lesion and decreases aortic expression of tumor RvD2 has effects on cellular recruitment and
necrosis factor (TNF) and IFNγ in a murine proliferation. In an animal model, RvD2 limits
model of atherosclerosis [47]. In a model of peri- the recruitment of neutrophils to the left ventricle
odontitis and atherogenesis in mice, it decreases and reduces the density of macrophages in the
the levels of C-reactive protein (CRP) and the infarcted area [8]. This lipid reduces cell prolif-
infiltration of macrophages to the intima [27]. eration and lymphocyte recruitment in a model of
RvE1 takes part in reducing platelet aggregation arterial angioplasty in rabbits [38]. Similarly, in
induced by thromboxane [20] and, in a model of an intimal arterial neoformation model in mice
ischemia-reperfusion of the myocardium in rats, induced by carotid ligation, RvD2 reduced the
its intravenous administration reduces the size of proliferation of smooth muscle cells and the
the infarct in a dose-dependent manner [31]. recruitment of neutrophils and macrophages. It
The mechanisms of action of resolvins of the also induced lower levels of TNF and of granulo-
D series in the inflammatory resolution of cardiac cyte and macrophage colony-stimulating factor
alterations seem to be different. In a model of (GM-CSF) [3]. Acting in a similar way of RvD2,
arterial damage in rats, treatment with RvD1 the administration of LXA4 in rats with myocar-
attenuates both the levels of oxidative stress and ditis reduces the infiltration of inflammatory cells
the activation of the NFκB signaling pathway [9]. [53].
Intact human arteries incubated with DHA Not being an SPM, but because of its role in
ex vivo, produce precursors of resolvins of the D inhibiting FLPA2, annexin A1 has been studied
series, which inhibit the adhesion of monocytes in atherosclerosis. Asymptomatic plaques of the
induced by stimulation of endothelial cells with carotid artery have elevated levels of the protein,
TNF [9], suggesting that the local production of and its plasmatic concentrations correlate nega-
SPMs is necessary to promote inflammatory res- tively with the total areas of the plaques in ath-
olution. The in vitro treatment with RvD1 and erosclerosis models, which suggests an important
RvD2 of vascular smooth muscle cells decreases role of annexin in resolving cardiac inflammation
[15]. Its action on regulating the production of tion (few months to more than 6 years), the great
leukotrienes and prostaglandins may account for variability in doses (<1–4 g/d), the diseases stud-
its capacity to inhibit the migration of leukocytes ied, comorbidities (diabetes, hypertension and
into the plaques and its early protective effects stroke), and the interaction with drugs. [1, 2].
[19]. To reach effective blood levels to reduce the
risk of CVD or prevent secondary complications,
various studies have concluded that the daily con-
5.5 iet PUFAs as Precursors
D sumption of EPA plus DHA must be of at least
of SPMs and CVD 1 g [5, 36, 44]. Consistently, in studies with lower
doses of PUFAs, no protection against a possible
Independent of the use of pure SPMs in the cur- cardiovascular event is achieved [14]. Some
rent approaches, there is a long history of use of authors suggest higher doses to prevent compli-
their precursors n-6 and n-3 PUFAs derived from cations in patients with cardiac disease, for
the diet, specifically by consuming seafoods or example, consuming 3.36 g of EPA plus DHA in
their purified compounds. Being essential in the patients with stable coronary disease, helps to
development and function of the organism [8], restore the levels of SPMs and promotes the
the human body can synthesize only small phagocytosis of clots. [16]. Administering high
amounts of EPA and DHA [60]. Some studies doses of n-3 PUFAs in patients with atherosclero-
observe that consuming fatty acids changes the sis and type 2 diabetes, accompanied by adequate
profile of the membrane phospholipids, and the medical therapy, increases the serum concentra-
composition of the fatty acids of different cells tions of EPA and DHA, although it does not
and tissues, affecting their availability [32]. Other induce changes in the concentrations of RvD1
studies have shown beneficial effects of n-3 on [45]. In rats, a diet enriched with DHA decreases
various pathological conditions [52]. The quality pro-inflammatory proteins such as CRP, IL-6,
of life of people with chronic inflammatory dis- TNF, and IL-1β, and favors an increase in the
eases such as arthritis and asthma [35], the sever- concentrations of RvD2 and RvD3 [39]. Fish oil
ity of chronic inflammation [7] and the risk for treatment favors protection against thrombosis
cardiovascular diseases [26] improve with a diet and damage induced by vascular remodeling in
with n-3 PUFAs. The incidence of heart failure in mice, reducing local inflammation and increasing
older adults associates negatively with the total resolution by increasing RvE1 [24].
plasma concentration of n-3 [40]. Coincidently, a
meta-analysis shows that supplementing with
EPA and DHA has positive effects on risk factors 5.6 Conclusions
for cardiovascular diseases because they have
hypolipemic, hypotensive, antiarrhythmic and Many studies suggest that uncontrolled inflam-
anti-inflammatory actions [1]. mation and failure in the resolution response are
Until today it is not fully clear the role of diets the basis of a myriad of human diseases. SPMs
that include DHA and EPA to protect against car- seem to play a crucial role in restoring damaged
diovascular diseases. There is not a consensus tissue and in the recovery of function. In fact, it is
despite a wide range of studies that show their possible that the success of the inflammatory
beneficial effects, in part because many factors resolution largely depends on the capacity of pro-
are involved and influence the outcome. For ducing SPMs and how these mediators can con-
example, a diet enriched with n-6 and n-3 in a 4:1 tain chronic inflammation. The study of
ratio (as opposed to the 15:1 obtained with a typi- inflammatory resolution in cardiovascular
cal occidental diet) decreases the mortality rate diseases still represents a challenge. It is still nec-
for cardiovascular diseases [55]. Other factors to essary to find out if administering SPMs or their
consider include the different EPA/DHA ratios precursors will have the beneficial therapeutic
used (0.5:1–1.4:1), different lengths of consump- use they promise. Indeed, finding successful
ways of resolving inflammation can provide 12. Dalli J, Serhan CN (2012) Specific lipid media-

tor signatures of human phagocytes: microparticles
important advances in preventing cardiometa- stimulate macrophage efferocytosis and pro-resolving
bolic diseases. mediators. Blood 120(15):e60–e72. https://doi.
org/10.1182/blood-2012-04-423525
13. Das UN (2008) Can endogenous lipid molecules

serve as predictors and prognostic markers of coro-
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Specialized Pro-resolving
Mediators Directs Cardiac Healing 6
and Repair with Activation
of Inflammation and Resolution
Program in Heart Failure
Ganesh V. Halade and Bochra Tourki
Abstract Keywords
After myocardial infarction, splenic leukocytes Cardiac repair · Heart failure · Leukocytes ·
direct biosynthesis of specialized pro-resolving Myocardial infarction · Resolution of
mediators (SPMs) that are essential for the res- inflammation · Specialized pro-resolving
olution of inflammation and tissue repair. In a mediators
laboratory environment, after coronary ligation
of healthy risk free rodents (young adult mice)
leukocytes biosynthesize SPMs with induced
activity of lipoxygenases and cyclooxygenases,
which facilitate cardiac repair. Activated mono-
cytes/macrophages drive the biosynthesis of Abbreviations
SPMs following experimental myocardial
infarction in mice during the acute heart failure. AA Arachidonic acid
In the presented review, we provided the recent AT-LXA4 Aspirin-triggered LXA4
updates on SPMs (resolvins, lipoxins and DHA docosahexaenoic acid
maresins) in cardiac repair that may serve as EPA eicosapentaenoic acid
novel therapeutics for future heart failure ther- H and E hematoxylin and eosin
apy/management. We incorporated the under- HF heart failure
lying causes of non-resolving inflammation LV left ventricle
following cardiac injury if superimposed with LX4 lipoxin A4
obesity, hypertension, diabetes, disrupted circa- LXB4 lipoxin B4
dian rhythm, co-medication (painkillers or MaR1 maresin 1
oncological therapeutics), and/or aging that MaR2 maresin 2
may delay or impair the biosynthesis of SPMs, MI myocardial infarction
intensifying pathological remodeling in heart RvD1 resolvin D1
failure. RvD4 resolvin D4
G. V. Halade (*) · B. Tourki

Department of Medicine, Division of Cardiovascular
Disease, The University of Alabama at Birmingham,
Birmingham, AL, USA

46 G. V. Halade and B. Tourki
6.1 Introduction Currently, 5.7 million people are diagnosed

with HF in the US, but the projections of this dis-
Heart failure (HF) is the chronic, end stage, and ease are expected to increase to 46% [6], with
irreversible pathology secondary to ischemic cor- more than 8 million affected by 2030. As of
onary artery disease, predominantly superim- today, a total of 26 million people are suffering
posed on aging. HF is also common with from advanced HF worldwide [7]. During recent
non-ischemic cardiovascular diseases with pre- years, the discovery of advanced surgery tools,
served ejection fraction (HFpEF) [1, 2]. After novel medical device technology, therapeutics,
myocardial infarction (MI; heart attack), inflam- and team-based symptom management
matory HF is categorized by maladaptive changes approaches have advanced in order to minimize
in size, shape, and function of the left ventricle, patient discomfort. The combination of medical
advancing to cardiac remodeling (Fig. 6.1) [3]. In devices and therapeutic procedures have either
a clinical setting, the size/area of a patient’s helped or interfered with the majority of initial
infarct does not determine the likelihood of inflammatory and reparative responses to MI, but
adverse remodeling, with or without reperfusion have not slowed the trajectory for disease pro-
[4]. Therefore, the high rate of hospital admission gression to HF. Every year there are still 915,000
or re-admission, as well as cardiovascular mor- new cases of HF, accounting for an incidence
bidity and mortality, is primarily due to HF asso- approaching 10 in 1000 individuals over 65 years
ciated with aging [5]. of age [7]. The continuous growth of HF patients
reveals a major gap in our knowledge in how to
Fig. 6.1 Heart attack induced left ventricle (LV) 1 to day 56 compared to day 0 naïve control. Hematoxylin
structural and fibrotic remodeling from acute (day 1 and Eosin (H and E; 40X) staining of LV showing the
and 5) to chronic (day 28–56) heart failure in mice organized myocardium structure before MI compared to
model of permanent coronary ligation. From the top to necrotic and apoptotic disorganized myocardium from
the bottom: difference in ECG from normal mice to day 5 to day 56; Picrosirius red (PSR) staining confirmed
MI-induced heart after coronary ligation; Periodic acid– the deposition of collagen and compact fibrotic remodel-
Schiff (PAS; 1.25X) staining of the LV after MI from day ing in the infarcted area initiated at day 5 and progressive
to day 56
6 SPMs and Cardiac Repair 47
properly treat and identify novel target signaling out signal’). The synchronicity of leukocyte
pathways to resolve inflammation without the entry, coordinated phagocyte activity and an on
adverse effects of medication or therapies, which time exit prevents undesirable consequences of
frequently occurs with current anti-inflammatory an excessive inflammatory process through the
treatments [8, 9]. Aspirin, often considered the resolution process, which enhances the healing
safest anti-inflammatory treatment, has failed to and tissue repair mechanism [15].
limit the event of coronary heart disease and Although HF is a multifactorial and hetero-
stroke in elderly individuals and is often associ- geneous end stage disease, the mechanisms of
ated with high incidences of intracranial and initial inflammation and subsequent initiation
extracranial bleeding within the same risk group of tissue repair are relatively undefined in the
[10]. acute setting of MI and the role of these critical
In this review, we summarized the leukocyte- pathways in the chronic setting remains largely
directed biosynthesis of SPMs in myocardial unexplored [16]. HF is predominantly superim-
healing and the beginning of future applications posed on other risk factors, like aging and renal
of novel SPMs to limit chronic inflammation in failure; however, it is not only risk factors that
the setting of HF with potential enhancement to amplify the complexity of this disease. There is
the reparative program [11]. SPMs are biosynthe- an inter-organ crosstalk between the heart and
sized by leukocytes triggering endogenous car- peripheral organs in HF that delays the cardiac
diac healing as part of the acute innate response healing program (Fig. 6.1). We defined a clear
involved in the inflammation-resolution process. interaction between the spleen and heart, as ‘the
The successful biosynthesis of SPMs terminates splenocardiac axis’ as well as the cardio-renal
inflammation and facilitates repair by stimulating axis [17]. In this context, the spleen is not act-
distinct resolution processes necessary for tissue ing as a leukocyte reservoir but instead as a site
repair and regeneration from MI to advanced HF for the continuous activation of SPMs in car-
[12–14]. diac injury. The spleen is considered an integra-
tive organ in cardiac healing through the
production of multiple families of SPMs to acti-
6.2 Immune-Responsive vate the cardiac resolution program because
Systems in Myocardium splenic leukocytes define the resolution of
Wound Healing inflammation in HF [18, 19]. In order to prevent
the progression of HF pathology from acute to
Pathological inflammation and physiological chronic inflammation, the inflammatory
inflammation are two distinct and primary chal- response must be actively resolved (Fig. 6.2). In
lenges in identifying and treating injury, infec- this review, we highlighted the role of leuko-
tion, or stress (physical or neuro-hormonal cytes, particularly macrophages, that biosynthe-
mental exhaustion). Physiological inflammation, size SPMs in cardiac repair and remodeling
although necessary, needs to be both activated after injury.
and inactivated within a specified time in order to
avoid damage caused by sustained pathological
inflammation. Distinct differences include the 6.3 Immune-Responsive
phenotype and/or biomarker of injured cells and and Leukocytes Directed
tissues and the nature and timing of immune sig- Cardiac Repair
nals. Successful physiological inflammation
depends on the infiltration of neutrophils and Cardiac repair is based on the timely activation
monocytes (‘get-in signal’), phagocytic activity of cellular and molecular pathways that delay
of leukocytes (‘eat-me signal’), and the on time reactive fibrosis signaling, resulting in the forma-
departure, or efferocytosis, of neutrophils (‘get tion of scar tissue [20]. Both innate and adaptive
Fig. 6.2 SPMs balance or imbalance defines the enzymes (Jadapalli JK et al., AJP-HC 2018). Likewise,
inflammation and resolution outcome. Resolution of pain-killer caprofen, when administrated before MI for
post-MI inflammation is an active process that requires a 2 weeks to mice, impairs the splenocardiac axis, dysregu-
balance between proresolving mediators (SPMs) and lated resolving mediators, over activates neutrophils, and
clearance of leukocytes after acute inflammatory response. decrease the reparative macrophages at the site of the
In case of heart failure associated to co-medication, this injury therefore non-resolving inflammation (Halade GV
balance is impaired. For example doxorubicin, an onco- et al., J Leukoc Biol 2018). LV Left Ventricle, MI
logical drug induced a spleen atrophy with a decrease of Myocardial Infarction, MØ macrophages, Neu
splenic macrophages that impairs SPMs biosynthesizing neutrophils
immune mechanisms are involved in this process of HF [24]. Following cardiac or other tissue
[21]. Recently, the belief that activated splenic injury, the resolution of inflammation is a prime
leukocytes play a critical role in both repair and indicator and precursor of tissue repair [25]. In a
remodeling has been validated. Specialists in clinical setting, the hematological profile of a HF
adaptive immune responses demonstrated that patient shows elevated levels of leukocytes, par-
the depletion of mature B lymphocytes impeded ticularly neutrophils and inflammation of the
monocyte mobilization, limited myocardial arterial wall in addition to the infarcted myocar-
injury, and improved heart function post-MI and dium [26, 27]. In mice, the density of leukocytes
Treg function in cardiac repair [22, 23]. However, (neutrophils/monocytes) increase in the infarcted
cardiac repair alone is insufficient due to various LV in response to myocardial injury [28]. This
additional risk factors, such as obesity, insulin spike is due to the depletion of splenic leukocytes
resistance, diabetes, dyslipidemia, and hyperten- that occurs while a steady flow of leukocytes are
sion. These risk factors can hinder healing, pro- provided to the infarcted LV after MI [18]. In
moting defective tissue repair or unresolved fact, splenic leukocytes mobilize to injured myo-
inflammation that can progress to adverse ven- cardium enriched with fatty acids, such as ara-
tricular remodeling thereby leading to end stage chidonic acid (AA), docosahexaenoic acid
(DHA), eicosapentaenoic acid (EPA), and acti- 6.3.1 Neutrophils – Digest

vated lipoxygenases (LOXs). These activated Deceased Cardiomyocyte
cells, primarily monocytes, then further differen-
tiate into macrophages and biosynthesize SPMs After cardiac injury, the clearance of deceased
during the acute inflammatory response, suggest- myocytes is the primary objective of the
ing that the acute inflammatory response coin- leukocytes-based phagocytic system. Neutrophils
cides with the resolving response in cardiac are important effector cells to activate resolution
healing [18, 29]. and cardiac healing post-MI and neutrophil
The spleen, as the immune reservoir, coordi- depletion in early healing can lead to early
nates the time-dependent healing of an infarcted remodeling and HF [15]. Neutrophils dismantle
LV and determines resolving and non-resolving the extracellular matrix and enter the site of
inflammation. First, immune cells are recruited to injury in order to access the deceased and the
stimulate the repair of damaged myocardium necrotic tissue, accelerating pro-inflammatory
[30]. The fatty acid composition in the spleen and cytokine production [37]. Neutrophils are highly
the enrichment of the immune system may indi- phagocytic cells containing granules filled with
rectly affect the cellular events of both of these various defensins, elastases, and proteases
processes. The depletion of leukocytes (macro- (matrix matelloproteinase, MMP2, cathepsins,
phages/neutrophils) is correlated with the lower cathelicidins, phosphatases, oxidases, and colla-
levels of pro-resolving mediators. Macrophages genases) and their primary function is to dissolve
and the interaction of macrophages with innate matrix and phagocytose; however, this can
leukocyte players are essential for the biosynthe- amplify the inflammatory response [38].
sis of bioactive mediators and cardiac healing Neutrophils are classically known as immune-
[31]. The magnitude of cardiac injury, the activa- responsive inflammatory cells (termed as N1
tion, and initiation of the inflammatory response, neutrophils) but recent reports indicated that, like
and its counter regulation triggers the recruitment macrophages, neutrophils undergo polarization
of reparative cells that protect against adverse and show anti-inflammatory properties (termed
remodeling [32, 33]. Physiological importance of as N2). These N2 neutrophils account for 20% of
spleen is recorded in humans, since asplenic the neutrophils present 7 days post-MI [39]. The
patients develop ischemic heart disease and phenotypes of N2 neutrophils are distinguishable
excess risk of infection [34]. by the high expression of the macrophage man-
Recently, the technological advancements in nose receptor CD206 and interlukin-10 (IL-10).
molecular and cellular biology have yielded Neutrophils orchestrate post-MI healing by
progress in biomarker discovery and functional polarizing macrophages towards a reparative
characterization that improve our understanding phenotype but can also play an active role in the
of LV remodeling mechanisms. In the context of resolution of inflammation by depleting the che-
cardiac healing, the resolution process has moattractants that initially drew them to the site
become a frontline focus of inflammation- of injury [40, 41]. Therefore, these cells contrib-
resolution research and SPMs have become a ute directly to the inflammation-resolution pro-
new strategy for chronic diseases. Many pre- cess and indirectly to cardiac repair after MI.
clinical studies and some clinical reports have
shown benefits of SPMs, such as limiting the sec-
ond wave of tissue infiltration of neutrophils, 6.3.2 Macrophage – Programs
reducing collateral tissue damage, shortening the Neutrophils Clearance
resolution interval, enhancing macrophage and SPMs Biosynthesis
phagocytosis, amplifying efferocytosis, and
counter regulating pro-inflammatory chemical Recent findings imply a potential role of the
mediators [35, 36]. mononuclear phagocytic system includes macro-
phage cells in the repair of infarcted myocardium
[42, 43]. According to macrophage biology found in resolving exudates referred to as ‘reso-
research, many studies are devoted to the descrip- lution phase macrophages’. These macrophages
tion of mouse cardiac monocyte and macrophage possess characteristics of both M1 and M2 cells
subsets and the refinement of macrophage clas- and we believe that they represent cardiac repara-
sifications are ongoing [44]. With respect to tive macrophages, especially with the recent clin-
recent data, the composition of macrophage sub- ical strategies focused on improving macrophage
sets in the myocardium undergo dynamic changes function to improve healing outcomes [55–57].
throughout the course of a lifetime [45]. Fate Macrophage biology and phagocytic systems
mapping studies confirmed that the monocyte/ likely vary with the magnitude of injury and
macrophage systems placed in the heart prior to therefore the monocyte and macrophage
birth maintain self-renewal properties indepen- responses to injury can differ depending on the
dently of a circulation feedback system [46]. experimental model. For example, in a model of
Unfortunately, this discovery has limited transla- neonatal mice, the depletion of macrophages
tional potential since it is still unclear whether after MI impaired cardiac function and angiogen-
generation and differentiation of macrophages esis. The heart can fully regenerate without scar-
from precursors are impaired with age. However, ring following MI in neonatal mice; however, the
it is well proven that macrophages become either regenerative capacity of macrophages is lost
partially or totally dysfunctional in wound heal- 7 days after birth [28]. Under other conditions,
ing within the context of obesity and diabetes like in a model of adult mice, after MI, Ly6Chi
superimposed on aging in both humans and macrophages (M1 macrophages), and Ly6Clow
rodents [47]. (M2 macrophages) are the main effectors of car-
We know that macrophages have polarization diac remodeling.
profiles similar to neutrophils; within 1 day post- The kinetic and detailed macrophage and neu-
MI, macrophage exhibit a pro-inflammatory M1 trophil profiling studies demonstrated that after
profile [48–50]. At the post-MI “healing stage” cardiac injury, splenic monocyte-enriched leuko-
(up to 7 days post-MI), there is a transition from cytes mobilized to the infarct area and biosynthe-
predominately M1 macrophages to primarily sized SPMs. Macrophages are a major contributor
anti-inflammatory macrophages, and then to in SPMs biosynthesis [58]. In addition, lipid
reparative M2 cells [51, 52]. Cardiac reparative mediator profiling of the spleen indicates that the
M2-like macrophages are a complicated mixture spleen also receives feedback signal for biosyn-
of heterogeneous subsets that have strengthened thesis of lipid mediators, which promote self-
their tissue-repairing abilities by upregulating resolution of inflammation at the left ventricular
various anti-inflammatory and repair-associated site [59]. In cardiac injury, SPMs are biosynthe-
genes after MI [42, 53]. These two types of cells sized immediately by primarily activated macro-
(M1 and M2) are derived from Ly6Chi monocytes phages with the initiation of inflammation [60].
and Ly6Clow monocytes, respectively. Reparative In response to cardiac injury or stress, the
M2 macrophages are recruited simultaneously resolution-phase activated macrophages also
with inflammatory leukocytes in the post-infarct express ALOX-15, indicating that they may also
LV to mediate myocardial healing through the contribute to SPMs production during resolution
secretion of anti-inflammatory cytokines and [61, 62]. M2 macrophages produce more pro-
growth factors, such as VEGF and TGF-ß, that resolving lipid mediators than M1 macrophages
contribute to myofibroblast activation and neoan- [63]. SPMs initiate the switch from inflammation
giogenesis [54]. Deceased cardiac fibroblasts to resolution by reducing neutrophil recruitment
emit signals causing the activation of monocytes and T cell cytokine production and increasing
via MCP-1-mediated chemotaxis and adhesion to recruitment of phagocytic monocytes [64].
ICAM-1/VCAM-1, and induce their differentia- Resident macrophages express TGF-β1 and
tion to M1 or M2 macrophages [44]. Some IL-10, presumably because of phagocytizing
researches include a third macrophage subtype apoptotic neutrophils; these cells also expressed
ALOX-15 and TIMD4 (T-cell immunoglobulin phocytes [72]. Although data on SPMs primarily
domain and mucin domain 4) to facilitate the rec- focuses on innate immune cells involved in the
ognition and uptake of apoptotic cells [65, 66]. resolution of acute inflammation, resolution cre-
Recent results indicate that treatment with DHA ates a microenvironment conducive for the opti-
derived pro-resolving mediators, like exogenous mal development, tissue repair, and modulation
RvD1, stimulate a switch in macrophage pheno- of the adaptive immunity [19, 58, 73]. We con-
type from pro-inflammatory to pro-resolving, a clude that a better understanding of SPMs bio-
M2-like phenotype [67, 68]. Studies found that synthesis and the pharmacology of SPMs
human M2 macrophages are associated with receptors, especially in chronic inflammatory set-
increased MaR1 levels [61]. This increase is due tings like HF, could minimize the gap between
to the ability of this macrophage subtype to con- the initiation of inflammation and initiation of
vert the 13S, 14S epoxide intermediate to MaR1 resolution, capitalizing the fundamental actions
[60]. As critical regulators of the resolution proof these effectors of resolution.
gram, targeting the actions of macrophages could
be an effective strategy to control inflammation
and cardiac remodeling. 6.4 SPMs Biosynthesis
and Resolution
of Inflammation in Cardiac
6.3.3 Knowledge Gap Repair
of Inflammation
and Resolution as Part Splenic leukocytes mobilize to the site of
of Cardiac Reparative infarcted injury and biosynthesize SPMs to limit
Program the excessive inflammation that can be detrimen-
tal to healthy tissues, particularly in sterile
After cardiac injury, the resolution phase is an inflammation. In addition to the stop signal in
active biosynthetic process that overlaps or coin- acute inflammatory response, SPMs also stimu-
cides with the first response of the innate immune late distinct processes necessary for tissue repair
system. This was confirmed using structural elu- and regeneration [74]. SPMs have been associ-
cidation of the SPMs in a variety of wound heal- ated with positive feedback loops, where one
ing models [19]. Inflammation-resolution pro-resolving mediator induces the biosynthesis
process-derived bioactive metabolites implicate of another [15]. The administration of RvD1 3 h
an array of receptors that transduce pro-resolving after MI in mice induces lipoxin A4 and MaR1 to
action. Among them, the most studied receptors facilitate the resolution of inflammation and car-
are GPCRs as effectors of resolution shown indi- diac regeneration [75]. SPMs are produced
rectly through use of the receptor knockout cells mainly by the inter-organ interactions of
and mice. These GPCR receptors (ChemR23, monocyte-derived macrophages and neutrophils
GPR32, and FPR2) activate signals and trans- via distinct enzymatic pathways from essential
duce the pro-resolving signals of chemerin pep- fatty acids, such as omega-3 and omega 6, poly-
tides, resolvin E1 (RvE1) and resolvin D1 unsaturated fatty acids (PUFAs), eicosapentae-
(RvD1) and lipoxins [69–71]. noic acid (EPA), and docosahexaenoic acid
Recent, emerging data added confirmation to (DHA) [76]. Several endogenous SPMs have
previous thoughts that resolution is a primarily, been discovered, including lipoxins, resolvins,
active stage of the immune response to cardiac protectins, and maresins [36], which are directly
injury and not an inactive, secondary stage. This and indirectly involved in driving the initiation
stage acts as an overlap with the innate response, of inflammation-resolution and successfully ter-
adding a third phase after the initiation of inflam- minating inflammation. Healthy and risk
mation and resolution, creating a post-resolution free animals, such as young or adult mice, effec-
response dominated by macrophages and lym- tively mobilize circulating neutrophils, platelets,
and monocytes/macrophages from the spleen changes of ECM in acute HF suggest that RvD1
after cardiac injury to promote left ventricle orchestrates mature scar formation and stabilizes
healing, which is essential to delay HF [59]. the ECM by resolving inflammation. An experi-
After cardiac injury, monocytes/macrophages mental model of hind limb ischemia (HLI) in
serve as a major contributor for the biosynthesis mice, RvD2 stimulated arteriogenic revascular-
of SPMs that have multiple cellular targets in the ization suggesting that resolvins may be a novel
inflammatory response, including immune cells, class of mediators that both resolve inflammation
platelets, and vascular cells [74]. Leukocyte- and promote arteriogenesis [84] (Table 6.1).
directed SPMs actively counter-regulate and Presented experimental studies in rodent models
control the production of pro-inflammatory strongly indicate that SPMs offer pharmacologi-
mediators, including cytokines, leukotrienes, and cal action in the range of ng or pg doses sugges-
eicosanoids, and regulate leukocyte trafficking tive of outstanding potency [85], in contrast to
and phenotype after both sterile and infectious nonsteroidal anti-inflammatory drugs (NSAID)
challenges [77]. which effective in a range of mg/kg doses that are
immune suppressive and impact the cardiovascu-
lar physiology and pathobiology [86].
6.4.1 Resolvins SPMs are produced by the coordinated activ-
ity of lipoxygenases (LOXs) in the heart and
These are novel bioactive autacoids termed cyclooxygenases (COXs) in the spleen; these are
“resolvins” because they are endogenously gen- the key enzymes for generating potent bioactive
erated by resolution phase interaction products lipid mediators, which primarily synchronizes
classified as either E-series resolvins, if leuko- the resolution of inflammation in pathophysio-
cyte directed biosynthesis is initiated from EPA, logical settings [87]. Fredman et al. reported that
or D-series resolvins, when resolvins are biosyn- RvD1 favors synthesis of the pro-resolving lipid
thesized from DHA [78, 79]. Subcutaneous mediator, LXA4, via limiting nuclear transloca-
administration of RvD1 limits the activation of tion of 5-LOX in atherosclerotic inflammation
neutrophils in the spleen and promotes the clear- [88]. RvD1 critically balanced LOX enzymes by
ance of macrophages and neutrophils from the stimulating 5-LOX expression, which is essen-
infarcted site to facilitate cardiac healing. After tial for wound healing post-MI [89]. RvD1 has
ischemic injury, treatment of RvD1 primarily the potential to delay HF by limiting cytoplas-
activates the ALX/FPR2 receptor in both the mic to nucleus translocation to delay atheroscle-
spleen and left ventricle to promote effective res- rotic inflammation and facilitate neutrophil
olution of inflammation and limit residual time of clearance. Future long-term studies focused on
neutrophils at the site of infarction (‘get-out’ sig- RvD1 are warranted in order to prove its utiliza-
nal) [30, 80]. tion in chronic HF management in patients with
A reperfusion study in rats confirmed that heterogeneous cardiovascular pathology [75].
intraventricular administration of RvD1 reduces Another compound of the D-series resolvins
myocardium infarct size; however, presence of family, RvD3, was used as a model of self-
excess linoleic acid attenuates the myocardial resolving peritonitis in aging mice. The study,
protection offered by RvD1 [81, 82]. In another performed by Arnardottir and colleagues,
experimental study, after cardiac injury using reported that increased levels of RvD1 and
permanent coronary ligation model, RvD1 RvD3 treatment stimulated increases in mono-
administration in mice not only resolves the cyte-accelerated resolution of acute inflamma-
inflammatory response, but also reduced the tion via efferocytosis [90]. RvD3 has been
expression of fibrotic genes, such as colla1, argued to be one of the most potent of the
coll2a1 and tnc, in the early phase of cardiac D-series resolvins [91].
healing. Thus, RvD1 limits fibrotic signaling and Regarding the E-series resolvins, Arita et al.
facilitate organ homeostasis [75, 83]. These reported leukocyte-directed actions of RvE1 as
Table 6.1 Role of SPMs in cardiac repair in different experimental models

Molecules Experimental Model Mechanisms Effect References
Resolvins RvD1 In vivo, mice model of ALX/FPR2 ↑ clearance of neutrophils [75]
heart failure receptor from the spleen
↑ clearance of macrophages
from healing site
↓ expression of fibrotic genes
RvE1 In vivo, mice model of ChemR23 Protected cardiomyocytes [107]
acute heart failure receptor against apoptosis
BLT1 receptor ↓ protected cardiomyocytes
against apoptosis.
↓infiltration of dominant
Ly6Chi Mos/Mps
Lipoxins 15-epi- In vivo, mice model of ALX/FPR2 ↑ clearance of neutrophils [103]
LXA4 heart failure receptor Improve ventricular function
Activate GPR120 receptor
Inhibit GPR40 receptor
Maresins MaR1 In vitro, human GPR32 ↑ Efferocytosis and [60, 108]
macrophages phagocytosis
In vivo, model of BLT1 receptor ↓PMN infiltration
atherosclerosis ↑reparative macrophages
one of the local mediators of tissue homeostasis nanomolar concentrations, inhibiting chemokine-
during inflammation-resolution [92, 93]. The driven recruitment of both granulocytes and
physiological functions of RvE1, and RvE2, both monocytes [98, 99]. At the site of inflammation,
biosynthesized by neutrophils via the 5LOX lipoxins also stimulate macrophages to ingest
pathway and RvE3 biosynthesized by 12/15- and clear apoptotic neutrophils, exerting pro-
LOXs in eosinophils, have been studied in the resolving actions through the activation of ALX/
last few years [94, 95]. The myocardial protective FPR2 [100, 101]. In correlation with lipoxins
action of RvE1 action is quite different from generation, LTB4 was proved higher in the spleen
other resolvins and protectins. RvE1 specifically at a naïve state and increases along with lipoxins
binds to ChemR23 and BLT1 to offer proin the LV at day 1 post-MI [18]. Recent studies
resolving responses in human polymorphonu- demonstrated that 15-epi-LXA4 coordinates the
clear (PMN) cells [96]. This binding enhances upregulation of IL-6 and IL-1β at day 1 post-MI
macrophage phagocytosis via phosphoprotein- to facilitate an unaltered, active inflammatory
mediated signaling [97]. It blocks LTB4 binding phase post-MI without altering the acute inflam-
and signals via BLT1 to promote apoptosis of matory response. Liposomal delivery of 15-epi-
PMN for their clearance by macrophages, while LXA4 showed a similar protective function as
LTB4-BLT1 signals PMN survival. free 15-epi-LXA4, improving ventricular func-
tion through the activation of neutrophil clear-
ance in the LV during the resolving phase with an
6.4.2 Lipoxins increase of ALX/FPR2 and Ccl2 during the acute
inflammatory phase in a murine model of HF
Lipoxins A4 and B4 are lipoxygenase-interaction [102]. Additionally, 15-epi-LXA4 promotes the
products of arachidonic acid and are biosynthe- resolution of inflammation by activating GPR120
sized at the site of injury to facilitate resolution of and inhibiting GPR40 in a translational HF model
inflammation. Lipoxins are endogenous that (Table 6.1) [3, 102, 103].
offers anti-inflammation and pro-resolution at
6.4.3 Maresins dual roles as counter-regulators of inflammation,

as anti-inflammatory without being immunosup-
Maresins (macrophage mediators in resolving pressive, and activators of resolution [109]. For
inflammation) are derived from the marine example, RvD1 reduces nuclear localization of
omega-3 fatty acid DHA [104]. 5-LOX in macrophages and diverts arachidonic
Leukocyte-
directed maresins (MaR) are pro- acid metabolism from pro-inflammatory lipid
duced by macrophages via initial lipoxygenation mediator LTB4, to SPM LXA4 [62]. RvD1, acti-
at the carbon-14 position created by the insertion vated through ALX/FPR2, protects macrophages
of a molecular oxygen, producing a 13S,14S- from oxidative stress–induced apoptosis during
epoxide-maresin intermediate that is enzymati- efferocytosis, in part, by regulating nicotinamide
cally converted to maresin family members (MaR adenine dinucleotide phosphate oxidase activa-
1, MaR2, and MaR conjugate in tissue regeneration and expression of apoptotic proteins,
tion (MCTR) that regulate phagocytosis, and the Bcl-XL, and Bcl-2 [110]. In the other hand, the
repair and regeneration of damaged tissue [36, downregulation of NF-κβ and TNF-α by RvD5
102]. An recent study by Halade et al. showed could be beneficial in the HF model, as both of
that the quantification of MaR1 at day 1 post-MI these factors are increased in the heart post-
in mice is significantly higher in LV than spleen. cardiac injury [111]. Therefore, SPM emerge
However, at the naïve state without any induced both as potent regulators of macrophage
injury, MaR1 is higher in the spleen than in the responses of interest during the resolution phase
LV. These findings confirmed that splenic of acute inflammatory responses and effectors in
monocyte- enriched leukocytes mobilize to the macrophage mediated responses [112]. Of inter-
infarct area and biosynthesize SPMs [18]. In an est, receptors of SPMs, including ALX/FPR2 and
in vivo study, MaR1 reduced neutrophil and mac- ChemR23, are expressed in the human saphenous
rophage recruitment and increased polarization vein SMC, and administration of RvE1 and
of M2 macrophages in the arterial wall [105]. 15-epi-LXA4 counter-regulate platelet-derived
MaR1 have the ability to skew macrophage phe- growth factor–stimulated VSMC migration in a
notypes toward resolution-like, with increased dose-dependent manner [113]. These results
TGF-β production and secretion [106]. After an build on previous studies showing that lipoxins
infection, activated macrophages biosynthesize counter-regulate leukocyte-mediated microvas-
MaRs that increases phagocytosis and efferocy- cular permeability in vivo during acute inflamma-
tosis, resulting in the clearance of microbes tion, stimulated by pro-inflammatory lipid
(Table 6.1) [80]. mediators [114]. The actions of SPMs were
recently reported in human settings where an
RvE1 analog entered human clinical trials for dry
6.5 SPMs Regulation eye inflammation [115]. In light of the role of
and Dysregulation SPMs in the resolution, these compounds display
potent actions in the adaptive immune system
Fatty acids-derived drug (FADD) discovery including the regulation of T-cell phenotypes and
exemplified the generation of SPMs from essen- responses [58, 116]. Because Treg cells are an
tial fatty acids [59]. This discovery is unique and important cell subset involved in modulating and
novel for endogenous, bioactive pro-resolving maintaining self-regulation of the immune sys-
mediators, represents a paradigm shift in our tem, SPMs potentially induced Treg (iTreg) dif-
understanding of the dynamic regulation of acute ferentiation, with the lipids significantly
inflammation, and has led to a new era of resolu- enhancing Foxp3 expression compared to control
tion physiology and resolution pharmacology. iTreg cells. They affect not only Treg induction,
Within a single cell type, lipid mediator class but also specific functional properties, like TH
switching can occur and pro-resolving lipid cell polarization [58]. The actions of SPMs on T
mediators can regulate each other. SPMs have cells are mediated by GPR32 and ALX/FPR2
receptors. It has been proven that RvD1, RvD2, local adipokine production and monocyte accu-
and MaR1 exert a non-cytotoxic regulatory role mulation in obesity-induced adipose inflamma-
on cells; it represents a promising beginning for a tion [90]. However, it has been demonstrated,
new avenue of resolution physiology research. that the formation of SPMs is severely dysregu-
Biosynthesized maresins counter-regulate the lated in inflamed, obese adipose tissue [120]. A
pro-inflammatory cytokines, such as IL-1β, IL-6, deficiency in pro-resolving mediators in obese
and TNF-α. They also regulate nuclear factor adipose tissue within the setting of obesity could
kappa B (NF-κ B) gene products, increase the be the consequence of a structural deficiency in
regulation of T cell de novo synthesis and intra- the tissue content of omega-3-PUFAs, which are
cellular levels of cyclic adenosine monophos- established substrates for SPM biosynthesis
phate, regenerate tissue, and play a role in [122]. Alternatively, the loss of SPMs in obesity
anti-nociceptive action [36, 117]. SPMs might may reflect accelerated tissue SPMs conversion
possibly act on the balance between pathogenic and clearance to inactive further metabolites
TH1/TH17 and tolerogenic Treg cells, which are because 15-PG-dehydrogenase/eicosanoid oxi-
typically altered during chronic inflammation doreductase, the key enzyme in SPM inactiva-
[58]. However, these endogenous pathways of tion, is markedly up-regulated in obese adipose
resolution could be disrupted or dysregulated tissue [123]. Moreover, not only is activated
with co-medication. In fact, treatment with pain- lipoxygenase essential for SPM biosynthesis, but
killer caprofen or oncologic drug doxorubicin also the substrate product of these enzymes is
facilitate splenocardiac resolution deficiency in essential for precise immune responses. A study
mice with cardiac injury, creating non-resolving involving mice found lipidomic analysis showed
inflammation (Fig. 6.2) [118, 119]. Other factors, higher levels of arachidonic acid and
like metabolic dysfunction, are major contribu- 12(S)-hydroxyeicosatetraenoic acid (12-HETE)
tors of SPMs dysregulation. For example, obe- at day 1 post-MI an obese group compared with
sity, hypertension, diabetes, and aging are the non-obese group (Fig. 6.3) [124]. However,
associated with HF and could give an extra rise to studies involving the obesity paradox recently
unresolved chronic inflammation [120]. Next, we provided evidence that both mouse and human
discussed the impact of SPMs dysregulation in adipose tissue have the capacity to generate
cardiometabolic disorder thereby impact on reso- LXA4 [111].
lution physiology.
6.5.2 Diabetes
6.5.1 Obesity
Uncontrolled inflammation plays an essential
The unprecedented, continuous rise in the preva- role in the pathogenesis of diabetes and its asso-
lence of obesity and obesity-related disorders is ciated pathologies, like HF [125]. Also, the key
causally linked to a chronic state of low-grade sequela of adipose tissue inflammation is insulin
inflammation in adipose tissue and many other resistance leading to type 2 diabetes (T2D) [126–
organs [120, 121]. In the case of obesity, the mol- 128]. In this setting, a known complication of
ecules and signaling pathways have dual roles as T2D is impaired wound healing; the administra-
inflammatory mediators as well as regulators of tion of RvD1 to diabetic mice enhances wound
energy storage and metabolism [109]. The on healing, compared with control or vehicle treat-
time resolution of inflammation and the return of ment [129]. Therefore, SPMs may limit factors
this tissue to homeostasis are key components to associated with T2D via its ability to quell
reducing obesity-induced metabolic dysfunc- inflammation and promote adipose and liver tis-
tions. Results indicate that in inflammatory adi- sue homeostasis. RvD1 improved insulin sensi-
pose tissue, RvD1, and RvD2 are potent tivity, reduced adipose inflammation, and reduced
pro-resolving mediators that counteract both steatosis in mouse models of T2D [130, 131]. In
Fig. 6.3 Physiological and pathological healing in like obesity and aging that induce a low-grade chronic
mice delineates the effective and defective resolution in state of inflammation with a high levels of COXs and
heart failure. Physiological or pathological healing LOXs-derived pro-inflammatory mediators with low lev-
depends on resolution of inflammation process. els of SPMs and reparative macrophages along with the
Physiological cardiac remodeling is a consequence of an increase of VCAM-1 (vascular cell adhesion molecule 1),
effective resolution, with the production of necessary MPO (myeloperoxidase), CD40 (cluster of differentiation
SPMs at the right time by reparative macrophages. 40), PGs (prostaglandins), LTs (leukotrienes), and TxA2
However, many factors can lead to a defective resolution (thromboxane A2)
addition to resolvins, lipoxins also showed prom- levels of pro-inflammatory mediators, 5-HETE,
inent results in both mice and human and may 12-HETE and 15-HETE, were observed in pre-
have therapeutic potential in the context of diabe- eclamptic placentae, a disorder of a multifactorial
tes associated vascular complications [132]. etiology that compromise maternal and fetal
However, it is unknown if T2D affects the resolu- well-being as well as cardiovascular health later
tion of inflammation and whether treatment with in life [135]. Endothelial and vascular inflamma-
pro-resolving mediators would stimulate resolution also contribute in sustaining “low-grade
tion and ameliorate clinical complications of inflammation” and have shown to be more asso-
T2D such as impaired wound healing. ciated with pulmonary hypertension (PH) [136].
Recent data demonstrated that in vitro RvD1 pre-
vents arterial wall over-activity in human (HPH).
6.5.3 Hypertension This effect appears to be related to a decrease in
Ca2+ sensitivity in HPA smooth muscle cells.
Hypertension remains a significant risk factor for RvD1 also decreases the expression of trans-
the development of HF with various mechanisms membrane protein member 16A (TMEM16A), a
contributing to both systolic and diastolic dys- specific marker of PH [137]. In the same context,
functions [133]. Much less is known about the Jannaway et al. demonstrated that low nanomolar
role of anti-inflammatory molecules in the regu- concentrations of RvE1, RvD1, and RvD2 can
lation of blood pressure in general or in the devel- prevent constriction in rat and human arteries
opment of hypertension [134]. In contrast, higher induced by a thromboxane mimetic [138].
6.5.4 Disruption of Circadian Clock One study measured SPMs in human blood dur-
ing post-exercise recovery. It was demonstrated
Many physiological functions, including leuko- that lipoxins (LXA4 and LXB4), resolvins (RvE1
cyte and platelet responses, are responsive to day and RvD1), and the protectin D1 isomer increased
and light schedules in order to regulate cell and in human serum during the early hours of post-
organ specific circadian clocks. Results from exercise recovery (0–3 h post-exercise). However,
several recent studies demonstrate that many
ibuprofen treatment with exercise reduced the
metabolic functions are controlled by circadian response of pro-resolving lipid mediators to exer-
rhythm genes, enzymes, and proteins [139]. cise [146]. These results are consistent with those
Disturbances to various aspects of these funda- reported earlier by Gangemi et al. in which LXA4
mental mechanisms are thought to be responsible in human urine was found to increase with tread-
for many of the diseases that affect modern soci- mill exercise [147]. In contrast to the former
eties, including cardiovascular and metabolic dis- belief, exercise training in HF patients has proven
orders [140, 141]. In context to cardiac biology, to be safe and has no adverse effect on left ven-
the direct influence of circadian rhythms on tricular remodeling [148]. However, an exercise
SPMs and endogenous mechanisms in the resolu- paradox exists, and recently a study showed that
tion of inflammation remains largely unexplored. exercise alters β-alanine, augments histidyl
A study demonstrated that cardiomyocte-specific dipeptide levels, and scavenges lipid peroxida-
deletion of the circadian clock component, tion products in human skeletal muscle [149].
Bmal1, leads to age-dependent dilated cardiomy-
opathy and decreased lifespan in mice [142, 143].
Additionally, the circadian release of glucocorti- 6.5.6 Aging and Microbiome
coids and their link to downstream anti-
inflammatory and pro-resolution mediator Aging and age-associated chronic and unre-
annexin A1 facilitates the return to homeostasis solved low-grade inflammation, “inflammaging,”
[140]. A recent study suggests that patients with are progressive and all-time predominant factors
risk of coronary vascular disease (CVD) have for CVD [150, 151]. The underlying mechanisms
defective diurnal regulation of vascular n-3 doc- of inflammaging remain of interest, but a plausi-
osapentaenoic acid (DPA) derived resolvins. ble hypothesis for the non-resolving inflamma-
RvDn-3 DPA is involved in regulating peripheral tion in the elderly is a defect in the
blood cell responses and tissue protection. The inflammation–resolution process. The baseline
marked reductions in plasma RvDn-3 DPA dur- pre- and post-MI environments are also altered
ing the early morning hours indicate that altera- with age [94, 152, 153]. A recent study in a con-
tions in biosynthesis due to dysregulation of text of muscle generation showed a correlation
lipoxygenase and may contribute to CVD onset between aging and CCR2. The data explained
and propagation. This finding can be explained that in young WT CCR2−/−, both dysfunctional
by the increase of adenosine, a regulator of macrophages and a pro-inflammatory environ-
5-LOX activity in plasma from patients with ment were observed. Moreover, this study con-
CVD [144]. cludes that CCR2−/− in mice have a positive
feedback loop that promotes inflammaging in
young mice when compared to aging WT mice
6.5.5 Exercise [154]. In humans, urinary lipoxins (LXs) were
decreased in the elderly, resulting in a profound
Exercise limitation is one of the hallmarks of imbalance between pro-resolving LXs and LTs
heart failure, and an increasing degree of intoler- [147]. In line with these results, a study demon-
ance is associated with a poor prognosis [145]. strated that aging mice dysregulate the formation
of pro-inflammatory and pro- resolving mole- of pro-resolving mediators and therefore non-
cules with a decrease in LOXs expression, resolving inflammation in cardiovascular dis-
depending on the influx or availability of sub- eases. Future studies are warranted to determine
strates that temper acute inflammatory-resolving the mechanism of action, additional receptors for
phases, leading to the promotion or repression of SPMs, and interactions with lifestyles or medica-
inflammation post-MI (Fig. 6.3) [155]. Aged tions in the cardiac repair program.
mice have increased inflammation, LTs, and
decreased SPMs compared to young mice [91]. A Acknowledgements Authors acknowledge the support
recent, aging, metabolome human study from National Institutes of Health (NIH)-NCCIH (for-
merly known as NCCAM) AT006704, HL132989 and
described that RvD6 levels were decreased in an UAB Pittman Scholar Award to GVH. The authors would
aging individuals, indicating the role of lipid like to thank Servier Medical Art images bank that used to
metabolism in progressive aging [156]. Thus, create the illustrations in Figs. 6.1, 6.2 and 6.3.
there is evidence of defective SPMs in aging but
the mechanisms remain of interest. Compared to
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Novel n-3 Docosapentaneoic Acid-
Derived Pro-resolving Mediators 7
Are Vasculoprotective and Mediate
the Actions of Statins
in Controlling Inflammation
Jesmond Dalli, Kimberly Pistorius,
and Mary E. Walker
Abstract biological actions, actively reprogramming

Inflammation is a fundamentally protective the inflammatory reaction to promote its ter-
process that guards the host from invading mination by counter-regulating the production
pathogens and is central in the repair and of pro-inflammatory mediators and regulate
regeneration of damaged tissue. However, leukocyte trafficking as well as phenotype.
when uncontrolled, the overzealous response Recently we found that n-3 docosapentaenoic
leads to tissue damage and malaise. Indeed, acid (DPA), which was until then only
this process is now appreciated to be at the regarded as a biosynthetic intermediate in the
center of many chronic inflammatory diseases formation of DHA from EPA, is also con-
including vascular disease and arthritis. verted to structurally distinct bioactive media-
Studies investigating the mechanisms through tors that reprogram the host immune response.
which acute inflammation is actively turned In the present review we will discuss the evi-
off allowing tissues to regain function demon- dence underpinning the biological actions of
strated that the essential fatty acids, arachi- these novel n-3 DPA-derived autacoids in par-
donic acid (AA), eicosapentaenoic acid (EPA) ticular as they pertain to the vascular system.
and docosahexaenoic acid (DHA) are enzy-
matically converted to bioactive mediators. Keywords
These autacoids carry distinct structures and n-3 docosapentaenoic acid · Resolution ·
Inflammation · Lipid mediators · Vascular
inflammation · Leukocytes · Statins ·
J. Dalli (*) Biomarkers
William Harvey Research Institute, Barts and The
London School of Medicine and Dentistry, Queen
Mary University of London, London, UK
Centre for Inflammation and Therapeutic Innovation,
Queen Mary University of London, London, UK 7.1 Introduction
K. Pistorius · M. E. Walker Success in the evolution of multicellular organ-
William Harvey Research Institute, Barts and The isms was, at least in part, reliant on the develop-
London School of Medicine and Dentistry, Queen ment of a system to repair and regenerate
Mary University of London, London, UK

66 J. Dalli et al.
damaged tissues as well as to defend the organ- (5S-hydroxy-6R-(S-cysteinylglycinyl)-7E,9E,

ism from invading microbial pathogens [1]. This 11Z,14Z-eicosatetraenoic acid) [16]. Studies
defense system is embodied in the inflammatory investigating mechanisms involved in the onset
process which when self-limited is fundamen- and propagation of inflammatory disorders indi-
tally protective and coordinates both the killing cate defects in the production of the pro-resolving
and disposal of invading pathogens as well as the mediators, their enhanced further metabolism or
repair and regeneration of damaged tissues [2]. an impairment in their ability to activate down-
However, when this process becomes dysregu- stream signaling via their cognate receptors are
lated it leads to disease [3–7]. Pioneering studies linked with disease onset and/or progression [3–
investigating mechanisms that regulate the termi- 6, 8].
nation of inflammation uncovered a new genus of In mammals, the essential fatty acid alpha-
mediators produced via the stereoselective con- linolenic acid (9Z, 12Z, 15Z-octadecatrienoic
version of essential fatty acids, termed as special- acid; ALA) is enzymatically elongated and
ized pro-resolving mediators (SPM) [8]. This desaturated to produce EPA and subsequently
superfamily includes the arachidonic acid (AA)- to DHA with n-3 docosapentaenoic acid
derived lipoxins, the eicosapentaenoic acid (EPA) (7Z,10Z,13Z,16Z,19Z-docosapentaenoic acid;
and docosahexaenoic acid (DHA) resolvins [9], n-3 DPA) being the biosynthetic intermediate in
and the DHA-derived protectins [10] and mares- this process [17–19]. n-3 DPA contains a
ins [11]. These mediators share select biological 22-carbon chain with 5 double bonds and differs
actions that include i) limiting neutrophil recruit- from DHA as it lacks a cis-double bond at carbon
ment to the site of inflammation, ii) they counter- 4, which, although a small change, provides its
regulate the production of pro-inflammatory own specific biologically relevant actions [19,
mediators including prostaglandins, leukotrienes, 20]. These actions have been identified in a vari-
cytokines and chemokines (e.g. Tumor necrosis ety of mammalian tissues, including plasma,
factor alpha), iii) promote the uptake and killing brain, retina and heart. Genome-wide association
of bacteria and iv) increase the uptake and clear- studies in humans uncovered a correlation
ance of apoptotic cells [3–6, 8]. In addition, each between increases in peripheral blood n-3 DPA
of the mediators exerts unique biological actions concentrations and single nucleotide polymor-
for example, the EPA-derived resolvin E1 regu- phisms in the gene encoding for the fatty acid
lates platelet activation [12], the DHA-derived elongase 2 (ELOVL2, 21]. We recently found
protectins regulate viral replication [13] and the that, in addition to EPA and DHA, n-3 DPA is
DHA-derived maresins promote tissue regenera- substrate for conversion to novel families of bio-
tion [3, 14]. active mediators [20]. The aim of the present
SPM exert their potent biological actions via review is to discuss the actions of these novel
the activation of specific G-protein coupled families of mediators in regulating key leukocyte
receptors which include the Lipoxin A4 receptor responses.
(ALX/FPR2), GPR32/DRV1, GPR18/DRV2 and
the chemerin receptor ChemR23/ERV1 [15]. In
addition to activating cognate receptors, these 7.2 -3 DPA-Derived SPM Are
n
protective mediators also regulate the onset Novel Resolution Agonists
and propagation of inflammatory responses by
acting as partial agonists or antagonists to Events that occur in the early phases of an inflam-
receptors of inflammatory mediators including matory reaction are suggested to determine
BLT-1, the Leukotriene (LT)B4 (5S,12R- whether the response is self-limited or perpetu-
dihydroxy- 6Z,8E,10E,14Z- eicosatetraenoic ates and becomes chronic [22]. Assessment of
acid) receptor [16], and cysLT1 the receptor events occurring within the circulation during
for LTC4 (5S-hydroxy-6R-(S-glutathionyl)- acute self-limited inflammation demonstrated
7E,9E,11Z,14Z-eicosatetraenoic acid) and LTD4 that the concentrations of n-3 DPA were rapidly
7 Novel n-3 Docosapentaneoic Acid-Derived Pro-resolving Mediators Are Vasculoprotective… 67
upregulated during acute inflammation, to an cells and chemotaxis towards IL-8, to a similar
extent that was comparable to essential fatty extent as the DHA-derived RvD2 (7S,16R,17S-
acids that are involved in the biosynthesis of lipid trihydroxy- 4Z,8E,10Z,12E,14E,19Z- docosa-
mediators, including arachidonic acid and hexaenoic acid) [20, 23, 24]. RvD2n-3 DPA and
DHA. Using a systematic approach coupling RvD5n-3 DPA also regulate the expression of adhe-
structure elucidation with functional readouts we sion molecules on peripheral blood leukocytes
found that endogenous n-3 DPA is converted to and platelets including the expression of CD11b
bioactive mediators in both mice and human leu- on neutrophils and monocytes as well as CD62P
kocytes that carried pro-resolving properties and CD63 on platelets [25]. Furthermore, they
[20]. These mediators are congenerous with also regulate the formation of leukocyte-platelet
DHA products, namely D-series resolvins heterotypic aggregates in both human and mouse
(RvDn-3 DPA), protectins (PDn-3 DPA) and maresins peripheral blood. The biological actions of these
(MaRn-3 DPA), with unique stereochemistries [20]. molecules extend beyond the regulation of mech-
In self resolving exudates, the production of these anisms in leukocyte trafficking. Indeed, n-3
molecules was temporally regulated where for DPA-derived SPMs also regulate the uptake of
example RvD1n-3 DPA (7S,8R,17S-trihydroxy- apoptotic cells by macrophages, a key biological
9E,11E,13Z,15E,19Z -docosapentaenoic acid) action in the resolution of inflammation, with
and PD2n-3 DPA (16,17-dihydroxy-7Z,10,13, increases in macrophage efferocytosis of up to
14,19Z- docosapentaenoic acid) displayed a 70% at doses as low as 1 nM [20, 23]. These
bi-phasic profile, reaching a maximum during mediators also display endothelial directed
peak neutrophil infiltration and late into actions, counteracting the TNF-α- mediated
resolution. PD1n-3 DPA (10R,17S-dihydroxy-upregulation of adhesion molecules, such as
7Z,11E,13E,15Z,19Z-docosapentaenoic acid), Intercellular Adhesion Molecule 1 (ICAM-1/
MaR2n-3 DPA (13,14-dihydroxy-7Z,9,11, 16Z, CD54), on endothelial cells [20].
19Z- docosapentaenoic acid) and MaR3n-3 DPA
(4, 21-dihydroxy-7Z,10Z,12E,16Z,19Z-docosa
pentaenoic acid) levels were each found to 7.3 Diurnal Regulation of RvDn-3
reach a maximum at the 4 h interval and DPA Controls Vascular
gradually decreased over the next 20 h. The peak Leukocyte and Platelet
in exudate RvD2n-3 DPA (7S,16,17S-trihydroxy- Activation
8,10Z,12,14,19Z-docosapentaenoic acid) levels
coincided with the onset of resolution (the point Circadian mechanisms are at the heart of a num-
where PMN levels reach ~50% of maximim neu- ber of physiological functions, including leuko-
trophil counts). RvD5n-3 DPA (7S,17S-dihydroxy- cyte and platelet responses [26, 27]. Disturbances
4Z,8,10Z,13Z,15,19Z-docosahexaenoic acid) to various aspects of these fundamental mecha-
levels were found to gradually increase over the nisms are thought to be responsible for many of
course of inflammation-resolution, with a maxi- the diseases that afflict modern societies, includ-
mum being reached late in the resolution phase. ing cardiovascular and metabolic disorders [26–
The n-3 DPA product corresponding to MaR1n-3 28]. These conditions are characterized by a
DPA (7S,14S-dihydroxy-8E, 10E, 12Z, 16Z, dysregulated inflammatory response, although
19Z-docosapentaenoic acid) gave levels that the exact mechanisms that underlie this inflam-
were elevated in the peritoneum of naive mice, matory state remain of interest. Recent studies
where upon challenge with zymosan these levels demonstrate that the production of RvDn-3 DPA are
drastically decreased [20]. Of note, each of these diurnally regulated in the peripheral blood of
molecules displayed leukocyte directed actions healthy volunteers [25] (Fig. 7.1). Multivariate
whereby incubation of human neutrophils with analysis of plasma lipid mediator profiles demon-
RvD5n-3 DPA or PD1n-3 DPA markedly reduced neu- strated a diurnal shift in plasma LM-SPM con-
trophil adhesion to TNF-α activated endothelial centrations. This shift was associated with an
68 J. Dalli et al.
Fig. 7.1 Diurnal changes in RvDn-3 DPA regulates monocytes, neutrophils and platelets during the early
peripheral blood leukocyte and platelet activation pro- morning hours. Increases in circulating adenosine concen-
tecting from cardiovascular disease. In peripheral blood trations in patients with cardiovascular disease inhibit
diurnal changes in acetylcholine (ACh) upregulates ALOX5 activity disrupting the diurnal changes in plasma
15-lipoxygenase (ALOX15) activity promoting RvDn-3 DPA RvDn-3 DPA and increasing peripheral blood leukocyte and
biosynthesis that limit the physiological activation of platelet activation
increase in the amounts of n-3 DPA derived tion of whole blood with ACh increased RvDn-3
mediators, including RvD1n-3 DPA and RvD5n-3 DPA DPA concentrations, including RvD2n-3 DPA, under
from the evening (18:00 h) to morning intervals both static and flow conditions.
(7:00 and 9:00 h). These diurnal changes in Assessment of the production of these media-
peripheral blood RvDn-3 DPA concentrations were tors in patients with cardiovascular disease
abrogated in mice lacking the main orchestrator (CVD) demonstrated significant decreases in
of the molecular clock, Aryl hydrocarbon recep- plasma RvDn-3 DPA concentrations and a marked
tor nuclear translocator-like protein 1, in myeloid impairment in their diurnal regulation when
cells. Of note, the fluctuations in plasma RvDn-3 compared with healthy volunteers. Flow cyto-
DPA were associated with a regulation of leukocyte metric analysis of peripheral blood leukocyte
and platelet activation that reaches a maximum from patients with CVD demonstrated increases
between 7:00 and 9:00 h coincident with an in the expression of CD11b on both neutrophils
increase in RvDn-3 DPA concentrations. The pro- and monocytes when compared with healthy vol-
duction of these mediators was found to be under unteers. This was coupled with increases in
the control of acetylcholine (ACh), with periph- platelet–neutrophil and platelet–monocyte aggre-
eral blood concentrations of this neurotransmitter gates in peripheral blood from patients with CVD
also reaching a maximum during the early hours [25]. In addition, we found a significant relation-
of the morning (i.e. 7:00 h). Furthermore, incuba- ship between peripheral blood RvDn-3 DPA
c oncentrations and leukocyte and platelet activa- 7.4 PDn-3 DPA Regulates
tion, as demonstrated by a negative correlation Macrophage Phenotype
between RvDn-3 DPA and neutrophil CD41, mono- and Function
cyte CD41, and platelet CD63 and CD42b During Monocyte
expression. Investigations into mechanisms that to Macrophage
lead to the downregulation of peripheral blood Differentiation
RvDn-3 DPA in patients with CVD demonstrated a
link between peripheral blood adenosine concen- It is now well appreciated that in chronic inflam-
trations and the activity of one of the RvDn-3 DPA matory conditions, such as atherosclerosis and
biosynthetic enzymes, ALOX5. Adenosine, rheumatoid arthritis, monocytes play a central
which via the activation of the A2a receptor, role in the initiation, propagation and termination
downregulates the activity of ALOX5 [29], was of inflammation [32, 33]. Upon recruitment to
increased in peripheral blood from patients with the site of inflammation, these cells either differ-
CVD. The role of adenosine in downregulating entiate to inflammatory macrophages that propa-
RvDn-3 DPA concentrations was further under- gate the inflammatory response or to resolution
scored by experiments where peripheral blood phase macrophages that promote the termination
from CVD patients was incubated with adenos- of inflammation and restitution of tissue func-
ine deaminase, leding to the restoration of tion. Studies investigating mechanism that regu-
peripheral blood RvDn-3 DPA concentrations [25]. late the differentiation of macrophages
These findings led us to propose RvDn-3 DPA as demonstrate a role for the PDn-3 DPA pathway in
endogenous protective signals that control phys- regulating the phenotype and function of
iological platelet and leukocyte activation. This monocyte-derived macrophages [34] (Fig. 7.2).
is further supported by observations made in Using a total organic synthetic approach coupled
Apolipoprotein E deficient mice (ApoE−/−) mice with lipid mediator profiling, human ALOX15
fed a western diet. Treatment of these mice with and ALOX15B were identified as the enzymes
RvD5n-3 DPA reduced platelet-leukocytes aggre- that catalyse the first two steps in the PDn-3 DPA
gates in vivo and modulated vascular lipid medi- biosynthetic pathway yielding an allylic epoxide.
ator profiles reducing concentrations of the Using total organic synthesis we established the
pro-thrombogenic mediator Thromboxane (Tx) absolute stereochemistry of this epoxide as
A2 (measured as its metabolite TxB2) and upreg- 1 6 S , 1 7 S - e p o x y - 7 Z , 1 0 Z , 1 2 E , 1 4 E , 1 9 Z -
ulating the formation of pro-resolving mediators docosapentaenoic acid [34, 35]. This intermedi-
including MaR1 (7S,14S-dihydroxy-4Z, 8E, ate was in turn converted to PD1n-3 DPA and PD2n-3
10E, 12Z, 16Z, 19Z-docosahexaenoic acid) and DPA by distinct epoxide hydrolase enzymes, where
aspirin triggered (AT)-LXA4 (5S,6R,15R- in human cells epoxide hydrolase 2 (EPHX2)
trihydroxy-7 E,9E,11Z,13E-eicosatetraenoic was found to catalyse the conversion of 16S,17S-
acid). Furthermore RvD5n-3 DPA also decreased ePDn-3 DPA to PD2n-3 DPA. Experiments investigat-
early aortic lesions in ApoE−/− mice. The present ing the expression of the PDn-3 DPA biosynthetic
findings are in line with published findings dem- pathway during monocyte to macrophage differ-
onstrating an altered production of vascular entiation found the expression of all three
DHA-derived SPMs including RvD2 and MaR1 enzymes was upregulated during monocyte to
and impaired resolution responses in the patho- macrophage differentiation. Of note, ALOX15
genesis of atherosclerosis [30, 31]. Together expression was higher in M2 differentiated cells,
these findings demonstrate that alterations in the whereas the expression of ALOX15B was higher
diurnal regulation of vascular RvDn-3 DPA may in M1 cells [34].
occur early in the pathogenesis of cardiovascu- Pharmacological inhibition and genetic dele-
lar diseases that results in vascular inflammation tion of ALOX15 enzymes in monocytes led to
and impaired biosynthesis of DHA derived phenotypic and functional changes in monocyte-
SPM. derived macrophages. In cells where ALOX15
70 J. Dalli et al.
Fig. 7.2 PDn-3 DPA

biosynthetic pathway
and its regulation of
monocyte-derived
macrophage phenotype
and function. In the
PDn-3 DPA pathway n-3
DPA is converted to
17-HpDHA and then to
16S, 17S-epoxy-PDn-3
DPA by either ALOX15 or
ALOX15B. This is then
hydrolyzed by epoxide
hydrolase activity to
PD1n-3 DPA and PD2n-3 DPA.
This pathway regulates
macrophage phenotype
and function during
monocyte-to-
macrophage
differentiation
was inhibited there was a downregulation of sev- ALOX15 deficient mice also restored the pheno-
eral lineage markers including CD206, CD163 type and efferocytic activity of macrophages in
and CD64 and a shift in macrophage phenotype vivo [34]. Thus, these findings identify the PDn-3
[34]. This downregulation in phagocytic recep- DPA pathway as a component in the monocyte-to-
tors was of functional consequence since inhibi- macrophage differentiation program that regu-
tion of the PDn-3 DPA biosynthetic pathway also lates their phenotype and function.
significantly downregulated the ability of human
macrophages to uptake apoptotic cells, a key pro-
resolving action [36, 37]. Of note, this alteration 7.5 vTs Are Produced
R
in macrophage phenotype and function was During the Early Stages
recovered with the reconstitution of components of Acute Inflammation
within the PDn-3 DPA pathway. Incubation of and Temper Host Immune
human monocyte-derived macrophages with Responses
either PD1n-3 DPA or 16S, 17S-epoxy-PDn-3 DPA led
to a restoration of several phagocytic receptors Recent studies have described and characterized
and increased macrophage efferocytosis. a new family of bioactive mediators produced
Furthermore, administration of PD1n-3 DPA to from n-3 DPA. This new family of four resolvins
is termed the 13-series resolvins (RvT) given that when compared to healthy volunteers. RvTs are
all four molecules display potent host protective protective in mice during acute inflammation
actions and carry a hydroxyl group on carbon 13 where a mixture of RvT1, RvT2, RvT3 and
[38]. RvTs are biosynthesized from n-3 DPA in a RvT4 immediately before or 2 h after intraperi-
process that requires both endothelial cell COX-2 toneal Escherichia coli inoculation resulted in
and neutrophil lipoxygenase (ALOX) activity. host- protection, increasing survival by >60%.
The role for ALOX enzymes in the biosynthesis Indeed, RvT limited neutrophil recruitment to
of RvT was established using heavy oxygen the site of inflammation, increased phagocytosis
incorporation [38] with the identity of the ALOX and intracellular ROS levels and upregulated
enzymes catalyzing this reaction remaining of macrophage efferocytosis. Of note, the protec-
interest. tive actions of RvTs resulted from the repro-
During acute inflammation, cytokines such as gramming of the innate host response since RvT
TNF-α and IL-1β are released, activating did not display direct bactericidal actions at bio-
endothelial cells that upregulate the expression logically-relevant concentrations. Additionally,
of COX-2. Endothelial COX-2 converts n-3 DPA RvT reduced monocyte and macrophage expres-
to 13R-hydro(peroxy)-docosa-7Z,10Z,14E,16Z, sion of inflammasome components, decreasing
19Z-pentaenoic acid (13R-HpDPA); 13R-HpDPA caspase-1 and IL-1β expression and lactate
or its reduced alcohol form 13R-hydroxy- dehydrogenase activity, a marker of pyroptosis.
docosa-7Z,10Z,14E,16Z,19Z-pentaenoic acid RvT also reduced peripheral blood platelet-leu-
(13R-HDPA) is then donated to neutrophils dur- kocyte aggregates, an observation associated
ing neutrophil-endothelial cell interactions, with reduced systemic inflammation. Exudate
whereby it is first converted to 7-hydro(peroxy)- macrophage efferocytosis was also increased
13R-hydroxy-docosa-8,10Z,14E,16Z,19Z- and a significant reduction in local and systemic
pentaenoic acid [38]. This molecule can eicosanoid levels was found in mice given RvT
then undergo a second lipoxygenation [38].
reaction to yield 7,13R,20-trihydroxy-docosa- Using a total organic synthetic approach, we
8,10,14E,16Z,18-pentaenoic acid that was coined recently established the complete stereochemis-
as RvT1. The hydroperoxide can also, in a try and biosynthetic role of 13R-HDPA in the
lipoxygenase- dependent manner, undergo an RvT pathway [39]. Chirally pure precursors were
epoxidation reaction to yield the allylic used in conjunction with stereoselective reac-
epoxide 7,8-epoxy-13-hydroxy-docosa-9,11, tions that installed the configuration at the carbon
14E,16Z,19Z-pentaenoic acid. This is then enzy- 13 atom as R and formed geometrically pure
matically hydrolysed to 7,12,13-trihydroxy- double-bond moieties.
docosa-8,10,14E,16Z,19Z-pentaenoic acid and Synthetic 13R-HDPA was matched with bio-
7,8,13-trihydroxy-docosa-9,11,14E,16Z,19Z- genic 13-HDPA, obtained by incubating n-3 DPA
pentaenoic acid, coined RvT2 and RvT3, respec- with human recombinant COX-2. Structural eval-
tively. Finally, 7-hydro(peroxy)-13R-hydroxy- uation of synthetic 13R-HDPA and biogenic
docosa- 8,10Z,14E,16Z,19Z-pentaenoic acid is 13-HDPA was carried out using liquid chroma-
also reduced to 7,13R-dihydroxy-docosa-tography tandem mass spectrometry (LC-MS/
8,10Z,14E,16Z,19Z-pentaenoic acid that is MS) to attain MRM chromatograms to match
coined RvT4 (Fig. 7.3). retention times and MS-MS spectra to identify
RvTs are produced in humans during exer- matching daughter ions. Chiral LC-MS/MS was
cise, that is now seen as a self-resolving inflam- used to confirm the stereochemistry around car-
matory state, marked by an increase in bon 13, and UV-Vis spectrophotometry was used
neutrophil-endothelial interactions with upregu- to match the double bond conjugation system.
lation of plasma RvT [38]. These mediators are Incubation of the synthetic material with human
also produced during infection when RvT levels leukocytes demonstrated that this was rapidly
are upregulated in plasma from septic patients converted to all four RvTs [39]. These findings
72 J. Dalli et al.
Fig. 7.3 RvT biosynthesis and actions in mediating expression yielding 13-HpDHA, that can then be con-
the protective actions of statins in infections and verted to 13-HDPA and donated to neutrophils where via
inflammatory arthritis. Production of RvT is initiated ALOX activity this is converted to RvT1–4. Statin medi-
via the conversion of n-3 DPA by the endothelial COX-2 ated S-nitrosylation of COX-2 upregulates 13-HDPA pro-
duction contributing to the upregulation of RvTs
confirmed that the stereochemistry around the ple, upregulates the biosynthesis of 15-epi-LXA4
hydroxyl group on carbon 13 was in the R [41]. In addition, atorvastatin was recently found
orientation and the double bond geometry to increase RvT formation during human
around the conjugated double bond system neutrophil-endothelial cell interactions, as well
was found to be in E, Z with the complete as in mice during infections. This increase in RvT
stereochemistry established as 13(R)-hydroxy- production resulted from the S-nitrosylation of
7Z,10Z,13R,14E,16Z,19Z-docosapentaenoic COX-2 leading to increased 13R-HDPA levels,
acid as well as the role of this intermediate in the suggesting that the S-nitrosylation of COX-2
RvT biosynthetic pathway. increased catalytic activity of the enzyme. This
finding was in concordance with the
S-nitrosylation of COX-2 cysteine residues in the
7.6 vT Mediate the Biological
R presence of atorvastatin [38]. Inhibition of induc-
Actions of Statins ible nitric oxide synthase (iNOS) by L-NG-
in Infectious-Inflammation nitroarginine (L-NAME) reduced the
and Inflammatory Arthritis atorvastatin-mediated increases in plasma RvT
levels after E. coli inoculation. A similar reduc-
SPMs are implicated in mediating the protective tion was observed when mice were given cele-
actions of a number of clinically relevant thera- coxib, a COX-2 specific inhibitor. This highlights
peutics including aspirin and statins, whereby the complex regulatory axis of COX-2, as post-
aspirin initiates the biosynthesis of epimeric translational modification of the enzyme may
forms of SPMs [40], while lovastatin, for exam-
yield mediators with distinct biological activities ~34% respectively in mice given atorvastatin
to the classic eicosanoids. compared to mice given vehicle. In mice given
This mechanism was recently also found to be pravastatin, CD11b expression was decreased by
protective in arthritic inflammation where admin- ~40% and platelet-monocyte aggregation reduced
istration of atorvastatin upregulated RvT concen- by ~35%. Compared to vehicle, neutrophil acti-
trations during inflammatory arthritis in both vation markers were significantly reduced by
peripheral blood and joints [42]. Of note, this atorvastatin and pravastatin, reducing CD11b
protective mechanism was not unique to atorvas- expression by ~30% and platelet-neutrophil
tatin and was shared with pravastatin. Atorvastatin aggregation by ~24%. Of note, administration of
administration during ongoing arthritis led to a celecoxib that inhibits the upregulation of RvT
43% increase in total RvT amounts in arthritic by pravastatin and atorvastatin reverses the pro-
paws compared to vehicle-treated mice. tective actions of these statins on both disease
Pravastatin also increased paw RvT by ~20% severity and leukocyte responses [42].
with increases in RvT1 and RvT2.
These increases in joint RvT concentrations
were also linked with decreases in tissue pros- 7.7 Conclusion
tanoids and LTB4 concentrations. Prostaglandins
were reduced by 20–40% by all statins tested The identification of n-3 DPA as a substrate to
when compared to vehicle. Exceptionally, PGF2α novel, structurally distinct, mediators that display
was reduced by ~75% in mice given pravastatin. potent host protective activities demonstrates that
LTB4 concentrations were reduced ~50% by ator- complex mediator networks become activated
vastatin and ~15% by pravastatin. Additionally, during acute inflammation to ensure tissue
TxB2 was reduced 20–50% both statins [42]. The homeostasis. This is further underscored by the
upregulation in RvT concentrations were linked observation of a selective regulation of distinct
with a reduction in disease severity where in mice lipid mediator pathways in a tissue and cell spe-
administered atorvastatin, disease progression cific manner. In addition, mounting evidence
was dampened at day 4 post disease initiation, suggests that some of the beneficial actions of a
with disease scores reaching a maximum of number of widely used drugs, including statins,
9.1 ± 1.2 at day 5 which was sustained until day is mediated via the regulation of these protective
7. When mice were administered pravastatin, dis- pathways. Given the potent actions of n-3 DPA-
ease activity at day 5 was lower compared with derived SPMs in regulating systemic and periph-
mice administered vehicle, with a reduction in eral inflammatory responses, utilizing analogues
disease activity maintained until day 7 measured and mimetics may be useful therapeutics in the
both as reduction in of clinical score and edema. prevention and treatment of chronic inflamma-
In addition, both statins also lead to a reduction in tory diseases. In addition, strategies to boost their
joint damage at a histological level. endogenous production potentially via supple-
In inflammatory arthritis atorvastatin and mentation with n-3 DPA may also be useful in
pravastatin administration also regulates both cir- controlling inflammation. While the clinical evi-
culating and tissue resident leukocyte responses. dence for this approach is currently limited,
In non-classical monocytes, atorvastatin reduced recent studies in healthy volunteers provide evi-
the expression of CD11b by ~18% and platelet- dence for its effectiveness, whereby administra-
monocyte aggregation (measured by a decrease tion of n-3 DPA upregulates peripheral blood
in CD62P) was reduced by ~24% compared to concentrations of RvD5n-3 DPA [43]. Future studies
mice given vehicle [42]. Pravastatin significantly will need to identify the patient populations that
reduced platelet-monocyte aggregation by ~35%, will be responsive to this approach and the sup-
and decreased CD11b expression by ~10%. In plement forms that will be effective in regulating
classical monocytes, expression of CD11b and n-3 DPA-derived SPM concentrations.
CD62P were significantly reduced by ~42% and Furthermore, changes in the observation that
74 J. Dalli et al.
tissue concentrations of these SPM are altered in macrophage mediators with potent antiinflammatory
and proresolving actions. J Exp Med 206(1):15–23.
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https://doi.org/10.1038/ncomms12859 https://doi.org/10.1096/fj.201600360R
Aspects of Prostaglandin Glycerol
Ester Biology 8
Philip J. Kingsley, Carol A. Rouzer,
Amanda J. Morgan, Sachin Patel,
and Lawrence J. Marnett
Abstract
analogous to canonical prostaglandins. This
The Cyclooxygenase enzymes (COX-1 and chapter reviews the literature detailing the
COX-2) incorporate 2 molecules of O2 into production, metabolism, and bioactivity of
arachidonic acid (AA), resulting in an array of these compounds, as well as their detection in
bioactive prostaglandins. However, much intact animals.
work has been done showing that COX-2 will
perform this reaction on several different Keywords
AA-containing molecules, most importantly, Endocannabinoid · Cannabinoid ·
the endocannabinoid 2-arachidonoylglycerol Cyclooxygenase · COX-2 · Inflammation ·
(2-AG). The products of 2-AG oxygenation, Prostaglandin · Prostaglandin glyceryl ester ·
prostaglandin glycerol esters (PG-Gs), are Transgenic mouse · Anandamide ·
2-Arachidonoyl glycerol
P. J. Kingsley · L. J. Marnett (*)

A. B. Hancock Memorial Laboratory for Cancer
Research, Departments of Biochemistry, Chemistry,
and Pharmacology, Vanderbilt Institute of Chemical
8.1 Introduction
Biology, Vanderbilt University, Nashville, TN, USA
e-mail: [email protected]; The cyclooxygenase (COX) enzymes, which
[email protected] exist as two isoforms (COX-1 and COX-2),
C. A. Rouzer · A. J. Morgan incorporate two molecules of O2 into arachi-
A. B. Hancock Memorial Laboratory for Cancer donic acid (AA), generating the intermediate
Research, Departments of Biochemistry, Chemistry,
prostaglandin H2 (PGH 2). PGH 2 is further
and Pharmacology, Vanderbilt Institute of Chemical
Biology, Vanderbilt University School of Medicine, transformed by various prostaglandin syn-
Nashville, TN, USA thases to produce the array of canonical
e-mail: [email protected] prostaglandins familiar to many researchers
S. Patel (Scheme 8.1). Yu [1] and Kozak [2]
Department of Psychiatry and Behavioral Sciences, reported that the arachidonoyl-containing
Department of Molecular Physiology & Biophysics,
endogenous lipids anandamide (AEA) and
and the Vanderbilt Brain Institute, Vanderbilt
University Medical Center, Nashville, TN, USA 2-
a rachidonoylglycerol (2-AG) are efficient
e-mail: [email protected] substrates for COX-2, but not COX-1. These

78 P. J. Kingsley et al.
COR
COR
R = OH OH
OH COR
COX-2
N
H
OH HO
O
15- & 11-HETE O
OH PGF2a COR
Thromboxane
N
OH HO
Synthase O
X Synthasea
H OH
O COR O
COOH
OH O
O OH HO
N OH PGD2 OH
O P O +
Synthase PGI2
O
Synthase COR
OH
+ PGE2
O OH HO
NH3 Synthase
O P O
O COR
O
O
OH
O
COR HO OH
HO OH
Scheme 8.1 Schematic of COX-2-mediated oxygen- (a – PGH2 derived from AEA or 2-AG is not a substrate
ation of various arachidonoyl-containing lipid species. for thromboxane synthase. It is unknown if PGH2-LPC or
PGH2-LPE are acted upon by this enzyme)
compounds – referred to as endocannabinoids lipids is mainly based on three observations:

because they are ligands for the cannabinoid (1) 2-AG is present at high levels in many
receptors CB1 and CB2 – are processed by mammalian tissues (i.e., nmol/g in brain tis-
COX-2 analogously to AA, generating the sue); (2) kinetic studies indicate 2-AG and AA
corresponding PGH2 ethanolamide (PG-EA) are utilized by COX-2 with similar efficiencies
or glyceryl ester (PG-G). These intermediates in vitro [2]; and (3) PG-Gs have been shown to
are acted upon by most of the prostaglandin possess significant biological activity. This
synthases, leading to an array of prostaglan- chapter will review research on (i) production
din derivatives (Scheme 8.1). Curiously, of PG-Gs in enzymatic and cellular systems,
thromboxane synthase does not accept PGH2 (ii) biological activity of PG-Gs, (iii) analyti-
derivatives; thus, there are no thromboxane cal methodology for detection and measure-
analogs produced from endocannabinoids (2). ment of PG-Gs, (iv) the presence of PG-Gs in
Subsequent to these reports, other researchers intact animals, and (v) recent work from the
identified additional arachidonic acid analogs Patel and Marnett laboratories involving
that are selective substrates for COX-2. These PG-Gs in the murine central nervous system
compounds include N-arachidonylglycine [3], (CNS). It should be noted that the biochemis-
2-O-arachidonoyl ether [4], arachidonoyl- try, enzymology, and pharmacology of the
lysophosphatidylethanolamine (AA-LPE), PG-EAs, formed from the oxygenation of AEA
and arachidonyl-lysophosphatidylcholine by COX-2, are distinct from those of the PG-Gs
(AA-LPC) [5]. but are beyond the scope of this review. Several
Our laboratory has a long-standing interest groups have published interesting work eluci-
in exploring the biology of these putative dating the biochemistry of PG-EAs, and recent
COX-2 products, especially those of 2-AG. The reviews and original research in this area can
hypothesis that PG-Gs are relevant bioactive be found here [6–9].
8 Aspects of Prostaglandin Glycerol Ester Biology 79
8.2 PG-Gs in in Vitro and Cellular Rouzer and Marnett [12] further investigated
Systems the cellular production of PG-Gs in a more physi-
ologically relevant setting by isolating murine
The majority of reports regarding non-AA- resident peritoneal macrophages (RPMs). These
derived products of COX-2 oxygenation used cells are known to have a relatively high level of
both reconstituted enzymatic and cellular sys- AA in their phospholipid pool and are able to
tems to characterize the oxygenated products as synthesize COX-2 and generate large amounts of
well as the mechanism of their production. In the PGs in response to a variety of inflammatory
initial report of PG-G formation, Kozak et al. [2] stimuli. Here, RPMs were pretreated with LPS to
used purified murine and human COX-2 for induce COX-2 expression and then challenged
assessment of the kinetics (via O2 uptake) of with zymosan, which stimulated the release of
2-AG oxygenation and the identification of 2-AG. The authors showed that PGE2-G and
COX-2 reaction products. These studies estab- PGI2-G were produced by RPMs without the
lished that, when assayed individually, COX-2 addition of exogenous 2-AG, and these PG-Gs
utilizes AA and 2-AG with similar efficiencies were released from the cells. As in the case of
(kcat/Km = 2.4 s−1 μM−1 and 4.0 s−1 μM−1 for AA RAW264.7 cells, the primary PG-G products in
and 2-AG, respectively, for human COX-2 and RPMs were consistent with the primary PG prod-
2.5 s−1 μM−1 and 2.3 s−1 μM−1 for AA and 2-AG, ucts, which were PGE2 and PGI2.
respectively, for murine COX-2). The authors Based on kinetic analyses of COX-2-mediated
used the RAW264.7 murine macrophage-like cell oxygenation of AA and 2-AG in vitro, which had
line to confirm that the observed enzymatic reac- shown similar enzymatic efficiency for the two
tions could also take place in a cellular setting, substrates, one would predict that the ratio of
where 2-AG would be subject to metabolism by PG-Gs to PGs produced in cells should be simi-
several different pathways. Since RAW264.7 lar to the ratio of available 2-AG to AA. However,
cells do not express COX-2 under basal condi- in zymosan-stimulated RPMs, the ratio of total
tions, they were pretreated with interferon-γ PG to PG-G production was roughly 1000:1,
(INF-γ) and bacterial lipopolysaccharide (LPS) whereas the ratio of free AA to 2-AG in these
to induce the COX-2 enzyme. The investigators cells was 10:1. The much lower than expected
established that exogenous 2-AG can be taken up production of PG-Gs relative to PGs based on
by cells, processed to PGD2-G via COX-2, and substrate availability could not be explained on
secreted into the extracellular medium. They fur- the basis of PG-G hydrolysis [12]. These obser-
ther demonstrated the formation of PGD2-G from vations led to more extensive kinetic analyses of
endogenous 2-AG following stimulation of LPS- 2-AG and AA oxygenation by COX-2 via exper-
and IFN-γ-pretreated RAW264.7 cells with iono- iments in which both substrates were present.
mycin. The formation of PGD2-G by the cells Results of these kinetic studies were consistent
was consistent with the fact that they produce with previously published data suggesting that
predominantly PGD2 from AA. In subsequent the homodimeric COX-2 enzyme behaves as a
studies, Kozak et al. demonstrated the formation heterodimer with one subunit acting as the cata-
of PGE2-G and PGF2α-G from exogenous 2-AG lytic site and the other serving an allosteric func-
by human HCA-7 colon adenocarcinoma cells, tion. Specifically, the results supported the
which constitutively express COX-2 [10]. In hypothesis that AA and 2-AG compete with each
addition, Siegmund et al. reported the synthesis other for both the catalytic and allosteric sites
of PGD2-G from exogenously provided 2-AG by and that binding of either substrate in the alloste-
hepatic stellate cells isolated from mice pre- ric site suppresses 2-AG oxygenation and pro-
treated with bile duct ligation to induce liver motes AA oxygenation. Consequently, when
fibrosis. The investigators confirmed that bile both substrates are present, oxygenation of AA is
duct ligation led to induction of COX-2 expres- favored over that of 2-AG [13]. It is not clear
sion in the hepatic stellate cells [11]. whether this phenomenon fully explains the
observed relatively low yield of PG-Gs produced PG-G-associated biological activity immediately
by zymosan-stimulated RPMs. In fact, COX-2 led to the question of whether or not the effects
requires peroxide-dependent activation before were mediated by known endocannabinoid or
oxygenation of any substrate can occur, and a prostanoid receptors as opposed to distinct recep-
report from Musee and Marnett indicates that tors. An initial pharmacologic study ruled out sig-
higher levels of peroxide are required to activate nificant activity of PGE2-G at the four EP receptors
2-AG oxygenation compared to AA oxygenation that modulate the effects of PGE2, as well as the
[14]. Other factors, such as the actual availability FP, DP, TP, and IP receptors [16]. More recently,
of free 2-AG and AA at the COX-2 active site Woodward et al. used the more metabolically and
within the cell, may also play a role. chemically stable amide derivatives of the 1(3)-
While investigating the phenomenon of and 2-glyceryl esters of PGE2 and PGF2α to show
substrate-selective inhibition, in which an that neither compound has significant activity at
inhibitor blocks COX-2-dependent oxygen- any of the prostanoid receptors. The highest activ-
ation of 2-AG more strongly than that of AA, ity was noted in the case of PGE2-serinolamide
Duggan et al. [15] employed a model utilizing (the stable analog of the 2-glyceryl ester of PGE2)
dorsal root ganglia (DRG). DRGs were har- at the EP3 receptor, for which an EC50 of 500 nM
vested, plated, and treated with a cocktail of was observed. EC50 values were > 3000 nM for all
compounds that included LPS and IFN-γ to tested analogs at all other receptors [17].
induce COX-2. Ionomycin was subsequently The earliest report of PG-G-mediated biologi-
added to stimulate release of 2-AG, resulting in cal activity came from Nirodi et al., who found
the production of PG-Gs. The predominant that PGE2-G, but not PGF2α-G or PGD2-G, caused
products of 2-AG oxygenation were PGE2-G Ca2+ mobilization in the murine RAW264.7
and PGF2α-G. Interestingly, ethanolamide macrophage-like cell line at picomolar concen-
derivatives of PGs were also produced by the trations. Ca2+ mobilization was secondary to
cells, in addition to free acid PGs. The absolute increases in intracellular inositol
quantities of the various products were not 1,4,5-trisphospate, and it resulted in the activa-
reported. With this model, the authors showed tion of protein kinase C and, subsequently, extra-
that within the physiological system of DRGs, cellular signal regulated kinases (ERKs) 1 and 2.
the R-profen class of inhibitors acted as sub- Importantly, the authors also showed that PGE2
strate-selective inhibitors. did not elucidate these effects [16]. In follow-up
It should be noted that other reports have dem- studies, Richie-Jannetta et al. demonstrated
onstrated COX-2-dependent effects of 2-AG in a PGE2-G-mediated Ca2+ mobilization in the
variety of cell systems, suggesting that these H1819 human non-small-cell lung cancer cell
effects may be due to conversion of the endocan- line. In these studies, similar activity was
nabinoid to a PG-G. However, in many of these observed regardless of whether the PGE2 moiety
studies, the actual synthesis of a PG-G in the tis- was attached at the sn-1 or sn-2 position of the
sue or cells of interest was not confirmed. We glycerol, and PGF2α-G exhibited activity similar
have limited this discussion to cases in which to that of PGE2-G. In contrast, the corresponding
PG-G formation was demonstrated analytically. free acid prostanoids were inactive [18].
A number of studies have suggested a potential
role for PG-Gs in the nervous system. Sang et al.
8.3 Biological Activity of PG-Gs [19] demonstrated that PGE2-G, PGF2α-G, and
PGD2-G increased miniature inhibitory postsyn-
Concurrent with the elucidation of the production aptic currents (mIPSCs) in murine hippocampal
and control of PG-Gs, several investigators have neurons. Shortly thereafter, the same group
reported interesting and potent biological activi- reported that these three PG-Gs enhanced minia-
ties of PG-Gs, both in cellular systems and in ture excitatory postsynaptic currents (mEPSCs) in
intact mammals. However, the discovery of any hippocampal neurons, an indication of enhanced
glutamatergic synaptic transmission [20]. In both tis model; the related compounds PGD2 and
cases, 2-AG had the opposite effect, a role for the PGD2-EA did not have similar effects [25].
CB1 receptor was ruled out, and the involvement Raman and colleagues demonstrated that the
of both IP3 and ERKs was implicated. The PGE2- nonenzymatic breakdown product of PGD2-G,
G-mediated enhancement of mEPSCs was also 15-deoxy-Δ12,14-PGJ2-G (15d-PGJ2-G), activates
found to induce neuronal injury and/or death. Yang the PPARγ receptor leading to decreased tran-
et al. also reported an effect of PGE2-G, PGF2α-G, scriptional activity of nuclear factor of activated
and PGD2-G on hippocampal neurotransmission. T cells (NFAT), and ultimately, reduced IL-2
In their case, an increase in basal synaptic trans- expression in the human Jurkat T lymphoblas-
mission and long-term potentiation in hippocam- toid cell line. The researchers suggest that
pal slices was observed that, consistent with prior 15d-PGJ2-G mediates the 2-AG-dependent sup-
findings, was dependent on IP3-mediated Ca2+ pression of IL-2 production in these cells [26,
mobilization and ERK signaling [21]. 27]. In one of the few studies in intact animals,
Commensurate with the neurotoxic effects noted Hu et al. reported that intraplantar administra-
by Sang et al., Valdeolivas and colleagues reported tion of PGE2-G in rats produced hyperalgesia
that PGE2-G was neurotoxic to the M-213-2O fetal and allodynia (8). In this study, PGE2 induced
rat striatum-derived cell line. Their studies sug- hyperalgesia in a manner similar to
gested that PGE2-G is a mediator of malonate- PGE2-G. However, while EP1–4 receptor antago-
dependent neurotoxicity, which in M-213-2O nists blocked the hyperalgesic effect of PGE2,
cells, serves as a model of Huntington’s disease they did not block the hyperalgesic effects of
[22]. In studies involving tissues other than the PGE2-G, suggesting that the PGE2-G response is
hippocampus, Lindgren and colleagues reported mediated by a pathway distinct from that of
that PGE2-G induces an enhancement of stimulus- PGE2. Nevertheless, this study highlights one of
evoked neurotransmitter release by the presynap- the major challenges of elucidating the role of
tic neuron at the neuromuscular junction. They PG-Gs in vivo. The compounds are subject to
further demonstrated that the effect is dependent hydrolysis by multiple esterases such that the
on production of nitric oxide and does not involve half-life of PG-Gs in biological tissues is short
the EP1 or EP2 receptors for PGE2 [23]. Woodward (see below). Consequently, some reports of
et al. demonstrated that the amide derivatives of PG-G-mediated biological effects are actually
the 1(3)- and 2-glycerol esters of PGE2 lower due to free PGs obtained following hydrolysis.
intraocular pressure in canine eyes, whereas the We have limited this discussion to cases in which
comparable derivatives of PGF2α are much less the observed pharmacology appears to be due
effective. In the monkey eye, the derivative of the directly to the action of PG-Gs.
1(3)-glycerol ester of PGE2 was more effective A common finding in these studies of the bio-
than the 2-glycerol ester [17]. logical activities of PG-Gs is that neither 2-AG nor
Most other actions of PG-Gs have been iden- the free acid prostaglandins mediate the same
tified in various models of immune or inflamma- effects, and in many cases, a role for CB or pros-
tory responses. Alhouayek et al. [24] tanoid receptors was specifically ruled out. This
demonstrated that PGD2-G suppresses while strongly implies that there is one (or several)
PGE2-G and PGF2α-G increase IL-1β secretion receptors specific for PG-Gs. In support of this
by murine J774 macrophage-like cells. Their hypothesis, Bruser et al. recently reported that
accompanying exploration of LPS-stimulated PGE2-G is a potent ligand of the P2Y6 receptor
2-AG metabolism by the cells suggested that [28]. In this study, the authors identified G protein-
2-AG-dependent decreases in macrophage acti- coupled receptors (GPCRs) in PGE2-G response-
vation in response to LPS is mediated by positive cells via RNA sequence analysis. From a
PGD2-G. In later work, Alhouayek et al. reported pool of these candidate GPCRs, the authors estab-
that PGD2-G reduced symptoms of inflammation lished that the UDP receptor P2Y6 is also a recep-
in a murine dextran sodium sulfate-induced coli- tor for PGE2-G. In fact, they report that PGE2-G
activates P2Y6 with an EC50 of 1.2 pM, compared Our most recent report [31] employed homog-
to an EC50 of 78 nM for the canonical ligand, UDP. enization of brain tissue via sonication in aceto-
nitrile. No secondary purification was employed.
Phospholipids are known to be less soluble than
8.4 Analytical Methodology eicosanoids in acetonitrile, and it has been our
for PG-G Quantitation experience that little interference is experienced
in the analysis of PG-Gs and related lipids with
While several types of analytical methodologies this method. PG-Gs were chromatographed on a
have been applied to lipid detection and quantita- C18 column with ammonium acetate in the
tion in the last several decades, reports of PG-G mobile phase to promote the generation of the
quantitation have employed almost exclusively [M + NH4]+ ion. For unknown reasons, PG-Gs
LC-MS techniques. Previously, our group pro- preferentially co-ordinate with the ammonium
duced protocols [29] and reviews [30] regarding cation, and this complex provides several candi-
this type of analysis, and other researchers have date fragment ions (Fig. 8.1), giving the
employed similar methodologies. The typical researcher several options for SRM analysis.
work flow involves homogenization of cells or PG-Gs will also coordinate with Na+, but typi-
animal tissue followed by a secondary purifica- cally sodium complexes give rise to relatively
tion step (often solid phase extraction (SPE)). weak product spectra compared to those of H+
Finally, an aliquot of the dried and reconstituted and NH4+ complexes, rendering this ionization
sample is injected onto an LC-MS system where method not useful for SRM detection.
the analytes are chromatographed on a reverse- In one of the few studies of PG-G detection in
phase column and detected by mass spectrometry samples from an intact animal, Hu et al. (8) mea-
via selected reaction monitoring (SRM) (Note: sured PGE2-G in rat hind paws by homogenizing
sometimes researchers use the term “multiple the tissues in methanol, then subjecting the
reaction monitoring” (MRM), which is equiva- diluted homogenates to purification via C18
lent to SRM). SPE. Samples were finally analyzed on a LC-MS/
MS system, where PGE2-G was ionized via
Fig. 8.1 Product ion spectrum of PGE2-G complexed to 444 and the Q3 m/z to any of the several product masses
with an ammonium cation (m/z of [M + NH4]+ = 444.2). observed
SRM analysis may be accomplished by setting the Q1 m/z
co-ordination with the ammonium ion PGF2α-ethanolamide (PGF2α-EA) in the spinal

([M + NH4]+) and detected on a triple quadrupole cords of mice subjected to kaolin/λ-carrageenan
mass spectrometer operating in SRM mode. For inflammation of the knee [6]. More recently, Liu
analyte confirmation, the authors used an LC sys- reported the presence of several COX-2 deriva-
tem coupled to a quadrupole/time-of-flight mass tives in human myocardium and murine liver [5].
spectrometer and compared the product ion spec- Here, the authors detail the discovery of PGE2-
tra of the putative, endogenous PGE2-G and an LPE and -LPC, as well as the 11-HETE- and
authentic standard. 15-HETE- derivatives of AA LPE and AA LPC,
Similarly, Alhouayek et al. [25] homogenized which are also COX-2 products.
tissues of interest in chloroform and subsequently One potential factor for the relative lack of
performed a modified Folch-type extraction. detectable levels of PG-Gs in vivo is a high sus-
Samples were further purified via reverse-phase ceptibility of PG-Gs to enzymatic hydrolysis,
SPE, then subjected to LC-MS/MS analysis on a resulting in the generation of the corresponding
triple quadrupole instrument, again operating the free PG and glycerol. Kozak et al. reported that
mass spectrometer in SRM mode. the half-life of PGE2-G in rat plasma is 14 s, and
Researchers have quantitated PG-EAs using the half-life for appearance of PGE2 is 16 s [34].
similar techniques. Gatta et al. [6] quantified Additionally, these authors reported that no start-
PGF2α-EA in murine spinal cord by homogenizing material was detectable in rat plasma 5 min
ing the tissue and extracting with acetone. The after dosing the animal with 2 mg/kg of
samples were further purified by open-bed chro- PGE2-G. Vila reported that monoacylglycerol
matography on silica gel. The eluted fraction lipase (MAGL) and fatty acid amide hydrolase
containing PG-EAs was analyzed on a reverse- (FAAH) both hydrolyze PG-Gs [35], whereas
phase LC-MS system (using a C18 column). Manna reported that LYPLA2 does this as well
Interestingly, the analytes were detected as [36]. Indeed, several other enzymes have been
[M + Na]+ ions by a high-resolution mass spec- reported to carry out this reaction, among them
trometer (an IT-TOF instrument manufactured by ABHD6, ABHD12, CES-1, and PPT1 [37, 38].
Shimadzu). Uraquhart et al. [32] examined rabbit As noted above, there also appear to be kinetic
cornea and cornea homogenates for PG-EAs. A limitations on the generation of PG-Gs via COX-
Folch-type extraction was used to purify the ana- 2. AA is typically present at higher concentrations
lytes from the homogenized tissue, and resultant in mammalian tissue, with the excess of up to an
samples were quantified via LC-MS/MS, where order of magnitude. This, combined with the
several SRM transitions were employed for AA-dependent allosteric suppression of 2-AG
PGF2α-EA, PGE2-EA, and PGD2-EA detection. oxygenation by COX-2 [13] and the lower perox-
The authors utilized precursor ion [M + H-H2O]+ ide tone required for AA oxygenation as opposed
for all PG-EA species. to 2-AG oxygenation [14] suggests that AA will
likely be the preferred substrate of the enzyme
under most in vivo conditions.
8.5 PG-Gs in in Vivo Systems
Despite the relative abundance of reports of 8.5.1 P

G-Gs in Transgenic Mouse
PG-G generation in both in vitro and cellular Brain Tissue
experimental models, non-traditional COX-2
products (both PG-Gs and other classes) have Historically, and possibly for the reasons detailed
proven difficult to detect in vivo, and few such above, in analyses of murine CNS tissue, PG-Gs
reports exist in the literature. As previously men- have proven to be below the limit of detection for
tioned, Hu reported the presence of PGE2-G in both our methods and those of other researchers
the hindpaw and brain of rats treated with carra- [33, 39]. We hypothesized that increased levels of
geenan [33]. Gatta reported the presence of the COX-2 enzyme would generate PG-Gs in a
mouse brain setting. Towards this end, we acquired concurrently with the highly selective COX-2
the Thy-1 hCOX-2 mouse. These mice overexpress inhibitor lumiracoxib (LMX) [42]. The LMX-
human COX-2 (hCOX-2) primarily in the neurons treated animals showed LMX levels in brain tissue
of the striatum, amygdala, cerebral cortex, and hip- of 200–400 pmol/g and an almost complete elimi-
pocampus [40]. Litters resulting from the breeding nation of PG-G species (Fig. 8.4). Interestingly,
of these mice contain both wild-type and transgenic PGE2 and PGD2 were also markedly reduced.
(Tg) overexpressor animals. The experimental data
shown below were generated with 60–90 day-old
mice that were obtained in this way [31]. 8.5.2 PG-Gs in Response
Western blotting analysis verified the presence of to Systemic Inflammation
hCOX-2 in the brain tissue (prefontal cortex and hip-
pocampus) of the Tg animals (Fig. 8.2) [31]. Having established a model system in which
However, PG-Gs were not detected in analyses of Tg robust PG-Gs can be observed with Tg mice, we
or WT brain tissue (Fig. 8.3). We hypothesized that wanted to explore the possibility of observing
an increase in substrate levels (i.e., 2-AG) may afford PG-Gs without genetic manipulation. As previ-
detectable PG-G generation. Thus, WT and Tg mice ous reports of COX-2 products in vivo involved
were dosed with the monoacylglycerol lipase the administration of inflammatory stimuli, we
(MAGL) inhibitor, JZL184 [41]. In agreement with treated WT mice with LPS (3 mg/kg once per day
previous literature reports, 2-AG was increased in the for 2 days) with and without concurrent adminis-
JZL184-dosed subjects (data not shown), and PG-Gs tration of JZL184. Some of the LPS-treated mice
were detected (Fig. 8.3). Levels of PGF2α-G, PGE2-G, produced PG-Gs, albeit at levels 1–2 orders of
and PGD2-G were found to be in the range of magnitude lower than those of the Tg + JZL184
5–50 pmol per g of tissue (wet weight). subjects (Fig. 8.5). Note that tissues from animals
The COX-2 dependence of PG-G production treated with LPS alone exhibited a trend toward
was tested by dosing the Tg + JZL184 animals increased PG-G levels, though this did not reach
Fig. 8.2 Western blot of Tg WT

selected brain regions of
Tg and WT mice (ref
-2
17). (Figure reproduced Cerebellum PFC Pfc
OX
by permission from mC
Morgan et al. [31].
Copyright 2018, hCOX-2
American Chemical
Society)
Actin
Fig. 8.3 Detection and quantitation of PG-G species in permission from Morgan et al. [31]. Copyright 2018,
Tg mice. PG-Gs are observed only in the Tg + JZL184 American Chemical Society)
subjects (∗∗ indicated p < 0.01). (Figure reproduced by
Fig. 8.4 Effect of LMX on lipid species in brain tissue of Y-axis. (Figure reproduced by permission from Morgan
Tg + JZL184 animals. (∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ et al. [31]. Copyright 2018, American Chemical Society)
p < 0.001, ∗ p < 0.0001). Note: Figure d has a non-zero
Fig. 8.5 Effect of systemic LPS treatment on brain lipid levels of Tg and Tg + JZL184 subjects (∗p < 0.05, ∗∗
p < 0.01). (Figure reproduced by permission from Morgan et al. [31]. Copyright 2018, American Chemical Society)
statistical significance when compared to con- synthesis can be detected and monitored under
trols. It is interesting to note, however, that there conditions that will allow us to assess their role in
was no statistical significance between PG-G lev- physiological or pathophysiological processes.
els in the brains of animals treated with LPS Such models will also allow further study of
alone and those treated with LPS and JZL184, additional aspects of COX-2 biology, such as the
although levels in the latter animals were statisti- in vivo relevance of substrate selective
cally different from those in the controls. These inhibition.
findings suggest that LPS treatment increased
PG-G levels in the brains of the mice even in the Acknowledgments This work was supported by
absence of JZL184; however, a larger study will CA89450, GM15431 and (cite Sachin’s grant(s)).
be necessary to test this hypothesis.
References
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Targeting the COX/mPGES-1/PGE2
Pathway in Neuroblastoma 9
Karin Larsson, Anna Kock, Per Kogner,
and Per-Johan Jakobsson
Abstract geted therapies. Cancer-associated fibroblasts

The importance of prostaglandin E2 in cancer is the main source of prostaglandin E2 in neu-
progression is well established, but research roblastoma contributing to angiogenesis,
on its role in cancer has so far mostly been immunosuppression and tumor growth.
focused on epithelial cancer in adults while Prostaglandin E2 is formed from its precursor
the knowledge about the contribution of pros- arachidonic acid in a two-step enzymatic reac-
taglandin E2 to childhood malignancies is lim- tion. Arachidonic acid is first converted by
ited. Neuroblastoma, an extracranial solid cyclooxygenases into prostaglandin H2 and
tumor of the sympathetic nervous system, then further converted by microsomal prosta-
mainly affects young children. Patients with glandin E synthase-1 into prostaglandin E2.
tumors classified as high-risk have poor sur- We believe targeting of microsomal prosta-
vival despite receiving intensive treatment, glandin E synthase-1 in cancer-associated
illustrating a need for new treatments compli- fibroblasts will be an effective future thera-
menting existing ones. The basis of neuroblas- peutic strategy in fighting neuroblastoma.
toma treatment e.g. chemotherapy and
radiation therapy, target the proliferating Keywords
genetically unstable tumor cells leading to Microsomal prostaglandin E synthase-1 ·
treatment resistance and relapses. The tumor Prostaglandin E2 · mPGES-1 inhibition ·
microenvironment is an avenue, still to a great COX-inhibition · Neuroblastoma · Cancer-
extent, unexplored and lacking effective tar- associated fibroblasts · Tumor microenviron-
ment · Targeted therapy · Tumor-promoting
Per Kogner and Per-Johan Jakobsson contributed equally inflammation · Cancer
to this work.
K. Larsson (*) · P.-J. Jakobsson

Rheumatology Unit, Department of Medicine, 9.1 Introduction
Karolinska University Hospital, Solna, Karolinska
Institutet, Stockholm, Sweden
e-mail: [email protected] Prostaglandin E2 (PGE2) is a pro-inflammatory
A. Kock · P. Kogner mediator belonging to the eicosanoid family of
Childhood Cancer Research Unit, Department of bioactive lipids and the most abundant lipid
Women’s and Children’s Health, Karolinska mediator in tumor tissue. Numerous studies
Institutet, Stockholm, Sweden

90 K. Larsson et al.
describe upregulation of PGE2 in different tumors cells that are not targeted by chemotherapy and
[1–5] and the contribution of PGE2 to tumor pro- radiation, continue to produce important media-
gression is linked to several of the hallmarks of tors such as cytokines, chemokines, growth fac-
cancer including increased proliferation of tumor tors and PGE2 that sustains inflammation and
cells [6, 7], withstanding apoptosis signals [8], promote tumor repopulation [20]. It has therefore
increased angiogenesis [9], invasion and metasta- lately been suggested that therapies targeting
sis [10], and evasion of host immune response genetically stable cells in the stromal compart-
[11, 12]. ment will be a promising alternative or an addi-
Neuroblastoma is a neural crest derived tional therapeutic strategy [21, 22]. This review
childhood malignancy of the sympathetic ner- will address PGE2-driven inflammation, its con-
vous system. Neuroblastoma accounts for tribution to neuroblastoma progression and inhi-
approximately 6–10% of the childhood cancer bition of PGE2 as a new therapeutic strategy in
cases and 9–15% of the pediatric cancer deaths neuroblastoma treatment.
making it the single most common and deadly
tumor of childhood [13, 14]. The disease ranges
from localized tumors with favorable biology 9.2 Prostaglandin E2
and high survival to metastatic tumors with Biosynthesis and Signaling
aggressive biology and poor prognosis.
Neuroblastoma is stratified into three risk- Arachidonic acid (AA), the initial precursor for
groups, low-risk, intermediate-risk and high- prostanoid (prostaglandins and thromboxane)
risk groups, depending on clinical age, biosynthesis, is an omega-6 fatty acid and
metastatic stage and histologic appearance with/ released from cellular membranes by phospholi-
without differentiation and biology including pases A2 (PLA2). PGE2 is synthesized from AA
genetic aberrations [15]. Two of the most com- in a two-step enzymatic reaction. In the first step
mon genetic aberration in the unfavorable high- AA is converted by cyclooxygenases (COX)-1
risk neuroblastoma is amplification of the and − 2 into a reactive intermediate, prostaglan-
MYCN oncogene (MYCN-amplified) and loss of din (PG)H2 via a transition molecule PGG. PGH2
the long arm of chromosome 11 (11q-deletion) is then converted into PGE2, PGD2, PGF2α, pros-
[16]. Children with high-risk tumors only have a tacyclin (PGI2) and thromboxane (TXA2) by spe-
survival rate of 40–50% despite intensive multi- cific terminal synthases i.e. PGE synthases, PGD
modal treatment including intense induction synthases, PGF synthase, PGI synthase and TX
chemotherapy, ablative therapy with stem cell synthase respectively. The key PGE2 producing
rescue, radiation therapy, immune therapy and synthase is microsomal prostaglandin E syn-
attempts to complete surgical removal [17]. The thase-1 (mPGES-1) [23]. During normal condi-
remarkable progression in pediatric oncology tions mPGES-1 is expressed at low levels but
with increasing survival of most childhood then rapidly induced upon stimuli with pro-
malignancies during the second half of the inflammatory cytokines (IL-1β, TNF-α) [24, 25],
twentieth century has stagnated in the last endotoxins (LPS) [26], growth factors (EGF)
decade highlighting the need for new therapeu- [27] and hypoxia [28]. There is two additional
tic targets and strategies to further increase sur- PGE2 synthases reported, namely cytosolic pros-
vival. Intensification of current treatments is not taglandin E synthase (cPGES) and microsomal
an option since life long side effects and second- prostaglandin E synthase-2 (mPGES-2), with no
ary malignancies already pose an increasing structural relationship to mPGES-1 [29, 30].
problem in surviving children [18]. While the function of mPGES-2 as a PGE2 syn-
Chemotherapy and radiation therapy hit the thase has been shown both for recombinantly
fast proliferating tumor cells that are genetically expressed enzyme [31] and in mammalian cells
unstable resulting in clonal evolution and the [29], the function of mPGES-2 as a PGE2 syn-
development of therapy resistance [19]. Stromal thase in physiological and pathological condi-
9 Targeting the COX/mPGES-1/PGE2 Pathway in Neuroblastoma 91
EP receptors
PLA2
Leukotrienes LOX
HETEs Arachidonic acid
Lipoxins
Cyt P450
COX-1 PGE
COX-2 PGE 2
synthase
EETs
PGH 2 PGF synthase PGF2α

PGI
synthase PGD synthase
TX
synthase
PGI2 PGD2
TXA 2
Fig. 9.1 Prostaglandin E2 biosynthesis. Arachidonic acid converts PGH2 into PGE2. PGH2 can also be converted
(AA) released from cellular membranes is converted to into other prostanoids (PGD2, PGF2α, prostacyclin (PGI2)
prostaglandin (PG)E2 in a two-step enzymatic reaction. In or thromboxane (TXA2)) by respective terminal syn-
the first step AA is oxidized by cyclooxygenase (COX) -1 thases. PLA2, phospholipase A2; EP, E prostanoid;
or -2 into the short lived intermediate PGH2. In the second HETEs, hydroxyeicosatetraenoic acids; EETs, epoxye-
step microsomal prostaglandin E synthase-1 (mPGES-1) icosatrienoic acids; LOX, lipoxygenase; Cyt, Cytochrome
tions remains to be proven [32, 33]. PGE 9.3 PGE2 in the Tumor

synthase activity was found in cPGES purified Microenvironment
from rat brain and in mammalian cells trans-
fected with cPGES cDNA [30]. cPGES (also Solid tumors are not only composed of malignant
known as p23 or cPGES/p23) is a co-chaperone cells but a complex mix of cellular components
to Hsp90 and has been linked to cancer but not including cancer cells, immune cells, endothelial
coupled to any PGE synthase activity in vivo [34, cells, cancer-associated fibroblasts (CAFs) and
35]. PGE2 can also be metabolized non-enzy- the non-cellular extracellular matrix (ECM) con-
matically from PGH2 but at a much slower rate tributing to the production of growth factors and
than enzymatic conversion by mPGES-1 [36]. pro-inflammatory mediators, thus providing the
The biosynthesis of prostanoids is summarized prerequisites for tumor progression. PGE2 play
in Fig. 9.1. an important role in the orchestration of the many
PGE2 elicit its response by four G-protein processes involved in the developing tumor
coupled receptors, the E prostanoid receptors microenvironment [38]. By downregulating Th1
EP1, EP2, EP3 and EP4. The EP receptors are cytokines (IFNγ, TNFα and IL-2) and upregulat-
coupled to Gα proteins with stimulatory or inhib- ing Th2 cytokines (IL-10, IL-4 and IL-6) PGE2
itory subunits leading to the activation of a shift the balance from the anti-tumor Th1
diverse panel of downstream signaling pathways response towards immunosuppressive Th2
(Fig. 9.2). EP2 and EP4 are coupled with Gαs response [39–41]. PGE2 both directly and via the
that activates adenylyl cyclase (AC) that in turn shift in cytokine production suppress the cyto-
leads to cAMP production. EP3 is coupled to Gαi toxic activity of cytotoxic T lymphocytes and
that instead inhibits AC. EP1 is coupled to Gαq natural killer cells, inhibits maturation of den-
that activates phospholipase C leading to dritic cells and promotes immunosuppressive
increased Ca2+ influx [37]. cells like T regulatory cells and myeloid derived
EP1-4
EP2 EP3
EP1 EP4
PLA2 β γ β γ β γ
Gαq Gαs Gα i
AA PGH 2 PGE 2
COX-1 mPGES-1
COX-2
PLC AC AC
mPGES-1
NSAIDs
inhibitor
Coxibs
Ca2+ cAMP cAMP
Proliferation, survival, migration, invasion,

angiogenesis, immune suppression and evation
Fig. 9.2 PGE2 signaling. PGE2 formed via COXs and results in increased Ca2+ flux mediated through phospholi-
mPGES-1 is released from cells and confer downstream pase C (PLC), EP2 and EP4 leads to increased cAMP lev-
signaling via four E prostanoid (EP) receptors, EP1-EP4, els and EP3 in decrease levels of cAMP via activation or
either on the same cell (autocrine) or neighboring cells inhibition of adenylyl cyclase (AC) respectively. To
(paracrine). The EP receptors are G-protein coupled inhibit PGE2 signaling, either COXs or mPGES-1 can be
receptors that leads to changes in intracellular Ca2+ or inhibited or EP receptor antagonists can be used.
cyclic adenosine monophosphate (cAMP) levels. EP1 NSAIDs, nonsteroidal anti-inflammatory drugs
suppressor cells [42–46]. PGE2 also promotes the PGE2 produced by CAFs, TAMs and tumor cells
polarization of tumor-associated macrophages can stimulate vascular endothelial growth factor
(TAMs) towards a phenotype close to M2 polar- (VEGF) production in both CAFs and tumor
ized macrophages with surface expression of cells and thereby promote angiogenesis [50, 51].
CD163 and CD206 [47]. The M2-like TAMs in
turn contribute to tumor progression and immune
suppression by the production of cytokines and 9.4 Tumor-Promoting
chemokines e.g. IL-6 and IL-10 [48]. CAFs, one Inflammation
of the most abundant cell type within the tumor in Neuroblastoma
microenvironment, are recruited and activated by
tumor cells and influence several important pro- Lately, several investigations about the stromal
cesses in tumor progression including ECM compartment of neuroblastoma tumors have
remodeling, migration and metastasis, immune improved our knowledge about the role of non-
modulation and angiogenesis via secretion of a malignant cells in the tumor microenvironment to
plethora of soluble factors, including PGE2 [49]. neuroblastoma progression [21]. In a study by
Carlson and coworkers, a shift was found during 9.5 Prostaglandin E2

tumor progression in an experimental MYCN- in Neuroblastoma
driven neuroblastoma model, from an adaptive
immune response in early lesions to a state of 9.5.1 Expression of PGE2 Producing
immature innate immune response in larger Enzymes and Receptors
tumors. Also, a change in the population of infil- in Neuroblastoma
trating macrophages, the TAMs, was detected
from an M1 macrophage phenotype in early neo- As reported by us previously, mPGES-1 is
plastic lesions to an M2-like macrophage pheno- expressed in human neuroblastoma tumors [2] as
type in the established tumors leading to a well as in experimental neuroblastoma tumors
gradually increasing immunosuppressive micro- [58]. The highest levels of mPGES-1 was found
environment [52]. Asgharzadeh and colleagues in a subset of high-risk tumors with 11q-deletion
also illuminated the importance of TAMs in met- but without MYCN-amplification, compared to
astatic spread of neuroblastoma. Metastatic high-risk tumors with MYCN-amplification and
MYCN non-amplified tumors had a higher degree low-risk tumors without poor prognosis biologi-
of TAM infiltration than non-metastatic tumors cal features. Colocalization of mPGES-1 with
and by calculating an inflammation-related gene markers such as vimentin, fibroblast specific pro-
score, survival rate could be predicted [53]. We tein (FSP-1), fibroblast activating protein (FAP)
also found an increased infiltration of and platelet derived growth factor receptor β
M2-like TAMs (CD163 and CD206 expres- (PDGFRβ) in the tumors suggested that the major
sion) in high-risk neuroblastoma compared to source of mPGES-1 expression was cancer-
low-risk neuroblastoma [2]. In addition to TAMs, associated fibroblasts [2]. In experimental tumors
other cell types were also infiltrating the neuro- we also found colocalization of mPGES-1 with
blastoma tumors e.g. dendritic cells, T cells, B PDGFRβ indicating the validity of these models
cells and CAFs. The CAFs were found in prox- to study mPGES-1 inhibition in neuroblastoma.
imity to the dendritic cells and T-cells indicating Even though we did not find mPGES-1 expres-
an interaction between these cell types [2]. The sion in tumor cells in primary human or experi-
numbers of TAMs and area of CAFs were found mental neuroblastoma tumors there are reports
to correlate with clinical stage, in a study by about mPGES-1 expression in IL-1β stimulated
Hashimoto et al. They also observed that TAMs SK-N-SH cells [59, 60], and PGE2 production in
resided close to the CAFs, suggesting an interac- SK-N-SH and SK-N-BE(2) cells when treated
tion also between CAFs and macrophages in the with IL-1β and AA [61].
neuroblastoma tumor microenvironment and that The source of PGH2, the substrate for PGE2
these cell together promote neuroblastoma pro- biosynthesis, in neuroblastoma tumors is not
gression [54]. These results were recently con- yet fully understood. In a study by Johnsen
fimed in a study by Borriello et al., where they et al., extensive COX-2 expression was found
demonstrated a correlation between CD163 both in neuroblastoma tumors and cell lines that
expressing cells and CAFs in neuroblastoma further responded to NSAID therapy in vitro
tumors and also in this study it was found that the and in vivo [62]. In a more recent study, we on
cell types resided in proximity to each other indi- the other hand primarily found COX-1 expres-
cating an interaction [55]. In addition to promot- sion in the neuroblastoma tumors both on
ing cancer cell growth and interacting with mRNA level and protein level, with the excep-
immune cells, CAFs also promote angiogenesis tion of MYCN-amplified tumors where COX-2
[56]. Zeine et al. found an association between was the dominant isoform. Interestingly, there
high levels of α-SMA positive CAFs and micro- were not many cells co-expressing COX-1 and
vascular proliferation in neuroblastoma tumors mPGES-1, suggesting transcellular transfer of
suggesting a role of CAFs in promoting angio- PGH2 [2].
genesis also in neuroblastoma [57].
The levels of PGE2 not only depend on COX 9.5.2 COX-Inhibitors

and mPGES-1 expression but also on the PGE2 in Experimental
degrading enzyme 15-hydroxyprostaglandin Neuroblastoma Models in vivo
dehydrogenase (15-PGDH). In the subset of high
mPGES-1 expressing neuroblastoma tumors with The benefits of COX-inhibition to reduced tumor
11q-deletion only low levels of 15-PGDH was growth in neuroblastoma experimental models
detected whereas in the low mPGES-1 express- have been demonstrated in several studies, sum-
ing tumors (MYCN-amplified tumors and low- marized in Table 9.1. Johnsen et al. showed a sig-
risk tumors) high expression of 15-PGDH was nificantly reduced tumor growth and tumor
detected. The low expression of 15-PGDH prob- weight at sacrifice, in nude rats with SK-SY5Y
ably contributed to the increased levels of PGE2 neuroblastoma xenografts, treated with either
found in the 11q-deleted tumors [2]. celecoxib (10 mg/day for 10 days) or diclofenac
In a comprehensive study by Rasmuson et al., (250 g/l drinking water for 11 days). They also
an abundant expression of all EP receptors in the demonstrated increased apoptosis in the tumors
tumor tissue irrespective of tumor subset was from diclofenac treated rats [62]. In another
detected. Expression of the EP receptors was study, transgenic MYCN+/+ mice were treated
found in tumor cells and in the stromal vascula- with low-dose aspirin for 10 days (10 mg/kg/
ture [61]. In a following study, we also found day), leading to reduced tumor growth and a
expression of all EP receptors in neuroblastoma decrease in tumor-promoting inflammation [52].
tumors with MYCN-amplification and Additionally, a COX-2 specific inhibitor NS-398
11q-deletion. EP1, EP2 and EP3 was found in (15 mg/kg/day for 4.5 weeks) was found to
both tumor cells and stromal cells whereas EP4 reduce growth of subcutaneous SK-N-AS xeno-
showed a clear stromal localization [58]. The grafts as well as bone metastasis in nude mice.
reason for this discrepancy could be that in the Treatment with NS-398 also reduced expression
previous study paraffin embedded material was of VEGF-A and microvessel density [63]. We
used while in the latter frozen tumor material also showed that diclofenac (250 g/l drinking
was used. water for 9 days) significantly reduced tumor
growth in mice carrying SK-N-AS neuroblas-
Table 9.1 Overview of neuroblastoma in vivo studies targeting PGE2 biosynthesis

Target In vivo model Treatment Effects
Inhibitor Dose Time References
Celecoxib COX-2 rat xeno, SK-SY5Y 10 mg, p.o. 10 days Tumor growth↓ [62]
Diclofenaca COX-1/2 rat xeno, SK-SY5Y 250 g/l 11 days Inhibit growth
Apoptosis↑
Aspirinb COX-1 mice, tg-MYCN+/+ 10 mg/kg, p.o. 10 days Tumor growth↓ [52]
Inflammation↓
NS-398 COX-2 mice xeno, SK-N-AS 15 mg/kg, i.p. 4.5 weeks Tumor growth↓ [63]
Bone metastasis↓
Angiogenesis↓
Diclofenaca COX-1/2 mice xeno, SK-N-AS 250 g/l 9 days Tumor growth↓ [2]
Compound III mPGES-1 mice xeno, SK-N-AS 50 mg/kg, i.p. 8 days Tumor growth↓ [58]
mice, tg-MYCN+/+ 50 mg/kg, i.p. 10 days Tumor growth↓
Angiogenesis↓
CAF infiltration↓
M1/M2↑
a
Mice had free access to drinking water supplemented with diclofenac at the indicated concentration
b
Dose of aspirin administered equivalent to low-dose aspirin in humans. Xeno, xenograft; tg, transgenic; p.o., oral
gavage, i.p., intraperitoneal. In all experiments, inhibitors were administered daily during the indicated time
toma xenografts, and that the reduction of tumor decreased presence of PDGFRβ expressing
growth was associated with a reduction of PGE2 CAFs, thus not only inhibiting mPGES-1 enzyme
in the tumor tissue [2]. activity in CAFs already infiltrating the tumor
but, consistent with the in vitro data, reducing fur-
ther recruitment of CAFs. These data together
9.5.3 Selective Inhibition provide strong evidence that mPGES-1/PGE2
of mPGES-1 in Experimental play an important role in the developing tumor
Neuroblastoma Models in vivo microenvironment in neuroblastoma creating an
immunosuppressive milieu and contributing to
In a recently published paper, we investigated the CAF infiltration and angiogenesis.
effect of selective mPGES-1 inhibition on neuro-
blastoma growth [58]. We used two experimental
neuroblastoma mouse models, a xenograft model 9.6 linical Benefit of PGE2
C
reflecting the 11q-deleted subset of high-risk neu- Blocking Therapy in Cancer
roblastoma and an inflammatory MYCN-driven Treatment
transgenic model [52, 64]. In both models, daily
intraperitoneal injections of the cross- species All anti-tumor treatments strive to induce apopto-
human/murine mPGES-1 inhibitor Compound III sis or cell death, either by directly targeting the
(CIII, 50 mg/kg) for 8–10 days, resulted in tumor cells or via the immune system. The cel-
reduced tumor growth. We also found interesting lular stress that follow massive cell death, induced
changes in the tumor microenvironment upon by cytotoxic treatment or irradiation of cells, also
pharmacological mPGES-1 inhibition with CIII, leads to induction and release of PGE2, stimulat-
highlighting the importance of PGE2 in several ing tumor repopulation [65–67]. Proposed mech-
aspects of the tumor microenvironment all of anisms how PGE2 support tumor cell survival and
which probably contributed to the resulting repopulation include changing the apoptotic
decrease in tumor growth. Firstly, tumors from threshold [68], suppression of inflammatory
CIII treated mice were less vascularized com- responses [69] and by promotion of cancer stem
pared to controls, assessed by immunohistochem- cells [70, 71]. Several studies have illuminated
ical (IHC) analysis with an antibody towards the the benefits of PGE2 inhibition in combination
endothelial marker CD31. Secondly, IHC analy- with chemotherapy. Recently, Kurtova et al.
sis of macrophage polarization markers revealed showed that the cytotoxic treatment-induced
less pro-tumorigenic M2 phenotype of the TAMs apoptosis and the accompanied PGE2 release pro-
(CD206) in tumors from CIII treated mice com- moted cancer stem cells repopulation and chemo-
pared to controls. A concomitant increase in anti- resistance. Inhibition of PGE2 with celecoxib
tumorigenic M1 phenotype of the TAMs, detected (5 mg/kg/day, starting 2 days before cytotoxic
with flow cytometry, confirmed a shift towards a treatment and then continuing throughout
less immunosuppressive tumor microenviron- the experiment) in combination with gem-
ment in CIII treated mice compared to controls. citabine/cisplatin (GC) administered in several
Thirdly, host-derived PGE2 producing CAFs treatment cycles reduced chemotherapy resis-
infiltrating the tumors were positive for PDGFRβ, tance compared to GC treatment alone in a blad-
EP4 and the IL-1 receptor type I as well as phos- der cancer xenograft model [20]. Also in
phorylated STAT3, describing a pro-inflammatory neuroblastoma in vivo studies, there are reports
pro-tumorigenic CAF phenotype. In an in vitro about beneficial combination effects with cele-
migration assay, IL-1β treatment induced migra- coxib and cytotoxic drugs. Celecoxib (10 mg/day
tion of fibroblasts towards tumor cells, a migra- for 12 days) was found to potentiate the anti-
tion that was inhibited either by CIII or an EP4 tumor effect of irinotecan and doxorubicin in
antagonist, suggesting a role of PGE2 in recruit- neuroblastoma (SH-SY5Y) xenografted rats
ing stromal cells to the tumor microenvironment. [72]. Additional studies confirmed an enhanced
Inhibition of mPGES-1 in vivo also resulted in anti-tumor effect of irinotecan when adminis-
tered in combination with low-dose celecoxib cardiovascular system. (ii) A three amino acid
(5 mg/kg/day for 20 consecutive days) in three phylogenetic difference between human
patient-derived neuroblastoma xenografts [73]. mPGES-1 and murine mPGES-1 in the catalytic
Celecoxib (250 mg/kg/day for 20 days) and a cleft renders most candidate drugs developed to
PGE2 neutralizing antibody was also found to human mPGES-1 inefficient towards murine
enhance the effect of radiation in a head and neck mPGES-1 and thus obstructing pre-clinical test-
squamous carcinoma xenograft model [74]. ing [81]. (iii) Lack of differentiation to NSAIDs.
Several recent studies also indicate that inhibition Researchers have failed to provide compelling
of PGE2 could be a strategy to enhance the clini- evidence of the potential benefits of mPGES-1
cal outcome of immune-based therapies [75–77]. inhibition compared to COX-inhibition.
Weather mPGES-1 inhibitors will have the same In contrast to COX-2 inhibitors, mPGES-1
beneficial combinatorial effects as celecoxib inhibitors would not affect the thromboxane/
remains to be proven. prostacyclin balance in favor of thromboxane
production and would thereby not have the same
effect on the cardiovascular system. Indeed, phar-
9.7 A Niche for mPGES-1 macological inhibition of mPGES-1 with CIII
Inhibition in Cancer [82], a cross-species human/murine mPGES-1
Treatment inhibitor, was even shown to increase production
of prostacyclin and reduce vasoconstriction of
Although NSAIDs and Coxibs have proved anti- blood vessels [83]. There are also studies show-
tumor efficiency in mouse models and epidemi- ing chemoprotective effects of prostacyclin and
ology data supports the benefits of regular PGD2, effects that would be lost upon COX inhi-
NSAID intake to prevent cancer, the severe side bition but would be spared or even enhanced by
effects on the gastrointestinal tract and cardiovas- mPGES-1 inhibition and the potential shunting to
cular system has hampered their use as chemo- prostacyclin and PGD2. For comprehensive
preventing and chemopotentiating agents. In reviews of pre-clinical mPGES-1 inhibitors see
children, there is also a fear of Reye’s syndrome references [84, 85]. For a summary of the advan-
that has been associated with viral induced tages and disadvantages with mPGES-1 inhibi-
fever and intake of aspirin, which is why the use tion versus COX inhibition see Table 9.2.
of aspirin in children below 16 years of age is
usually not recommended [78, 79]. In addition, Table 9.2 Advantages and disadvantages with mPGES-1
some cytostatic drugs like the anthracyclines are inhibition vs COX inhibition in cancer treatment
cardiotoxic and therefore may increase the risk of mPGES-1 inhibition
cardiovascular adverse effects seen with Coxibs + Anti-tumor efficiency in vivo.
in adults, also in children [80]. + Selective PGE2 inhibition.
An mPGES-1 inhibitor would potentially have + Cardiovascular safe.
the same anti-tumor properties seen with COX − No available inhibitor in the clinic.
inhibitors without the severe side effects on the − Unknown shunting effects.
gastrointestinal tract and the cardiovascular sys- − Poor efficiency of human inhibitors in mouse
models.
tem, resulting from the inhibition of all prosta-
COX inhibition
glandins. Still, there are no mPGES-1 inhibitors + Anti-tumor efficiency in vivo.
available in the clinic to date. There are three + Supportive epidemiologic data.
main reasons why the development of mPGES-1 − Inhibit chemoprotective prostacyclin and PGD2.
inhibitors has failed to reach the clinic. (i) ‘Guilt − Gastrointestinal side effects.
of association’ to COX-2 inhibitors. Since − Cardiovascular side effects.
mPGES-1 preferentially couples with COX-2 − Fear that aspirin causes Reye’s syndrome in
there are concerns that an mPGES-1 inhibitor children.
would have the same deleterious effect on the − Could increase cardiotoxicity of cytotoxic drugs.
9.8 Conclusions (www.nnbcr.se), Märta and Gunnar V Philipson

Foundation and Karolinska Institutet Foundation.
A link between tumor-promoting inflammation

and prostaglandin E2 is well established in adult
tumors [41]. There are recent studies highlighting
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Metabolomics Biomarkers
for Precision Psychiatry 10
Pei-an (Betty) Shih
Abstract chiatric disorders such as anorexia nervosa

The treatment of psychiatric disorders remains and depression. This review (1) provides
a significant challenge in part due to imprecise background on the current clinical challenges
diagnostic criteria and incomplete understand- of psychiatric disorders, (2) gives an overview
ing of the molecular pathology involved. of metabolomics application as a tool to
Current diagnostic and pharmacological treat- develop improved biomarkers for precision
ment guidelines use a uniform approach to psychiatry, and (3) summarizes current knowl-
address each disorder even though psychiatric edge on metabolomics and lipidomic findings
clinical presentation and prognosis within a in common psychiatric disorders, with a focus
disorder are known to be heterogeneous. on eicosanoids. Metabolomics is a promising
Limited therapeutic success highlights the tool for precision psychiatry. This research has
need for a precision medicine approach in great potential for both discovering biomark-
psychiatry, termed precision psychiatry. To ers and elucidating molecular mechanisms
practice precision psychiatry, it is essential to underlying psychiatric disorders.
research and develop multiple omics-based
biomarkers that consider environmental fac- Keywords
tors and careful phenotype determination. Systematic review · Psychiatric disorders ·
Metabolomics, which lies at the endpoint of Metabolomics · Eicosanoids ·
the “omics cascade,” allows for detection of Polyunsaturated fatty acids · Biomarkers
alterations in systems-level metabolites within
biological pathways, thereby providing
insights into the mechanisms that underlie
various physiological conditions and patholo- 10.1 Challenges in Clinical
gies. The eicosanoids, a family of metabolites Psychiatry
derived from oxygenated polyunsaturated
fatty acids, play a key role in inflammatory Psychiatric disorders can impair one’s thinking,
mechanisms and have been implicated in psy- perceptions, emotions, and behaviors, resulting
in significant distress or impairment of personal
Pei-an (Betty) Shih (*) functioning [1]. The five most common catego-
Department of Psychiatry, University of California, ries of psychiatric disorders are anxiety disor-
San Diego, San Diego, CA, USA ders, including generalized anxiety disorder and

102 Pei-an (Betty) Shih
post-traumatic stress disorder; mood disorders, 10.2 Omics-Based Strategies

such as depression and bipolar disorder; schizo- in Precision Psychiatry
phrenia and psychotic disorders; dementia; and
eating disorders, including anorexia nervosa and A promising strategy to overcome obstacles in
binge-eating disorder. Psychiatric disorders are clinical psychiatry is “precision medicine,” an
prevalent, with an astonishing 46.4% lifetime emerging approach that aims to improve health
prevalence of having at least one major psychiat- and advance individualized care by taking into
ric disorders in the United States [2]. Psychiatric account “each person’s variability in genes, envi-
conditions represent a major public health prob- ronment, and lifestyle” [12]. This ambitious ini-
lem due to their associated disabilities [3] and tiative requires collecting dense data from a large
mortality [4]. The estimated global burden of number of cohort studies, including studies of
psychiatric disorders accounts for up to 32% of psychiatric disorders [13]. The rise of biotech-
years lived with disability, and more than 13% of nologies that simultaneously measure thousands
disability-adjusted life-years [5]. Moreover, psy- of data points has been timely in meeting the
chiatric disorders are significant predictors of the needs of precision medicine. These high-
onset and severity of subsequent serious medical throughput technologies yield multifaceted data,
illnesses such as heart disease [6]. including genomics, epigenomics, transcrip-
Diagnosis in psychiatry is based on a classifi- tomics, proteomics, metabolomics and are col-
cation system that includes clinical nosologies lectively referred to as “multi-omics” [14]. Used
such as the International Classification of effectively, multi-omics investigation enables
Diseases [7] and the Diagnostic and Statistical exploration of complex interactions in biological
Manual of Mental Disorders [8]. The current systems and their roles in health and disorders.
diagnostic system is universally applied, not only Among individual “omics” disciplines, the
clinically, but also in research and policy settings most frequently published in psychiatry are
such as drug-approval and insurance-reimburse- genome-wide association studies (GWAS) [15,
ment systems. Although these diagnostic criteria 16]. GWAS have revealed evidence of substantial
are regularly revised to improve validity, more pleiotropy or shared genetic etiology among sev-
disagreements about diagnosis fundamentals are eral psychiatric disorders [17]. Due to the poly-
found in psychiatry than in any other branch of genic, multi-factorial nature of psychiatric
medicine [9, 10]. The heterogeneous presentation disorders and the inherent limitations of GWAS
of psychiatric disorders is a result not only of design, the genetic loci identified are typically
phenotypic, biological, and genetic heterogene- small in effect size and of questionable clinical
ity, but also the outcome of complex interactions significance [18]. By itself, GWAS likely remain
between environmental and biological factors. A limited in yielding significant translational
lack of clear understanding about the complex advances to improve diagnostic accuracy and
psychopathology contributing to each disorder treatment effectiveness [19].
leads to inadequate or ineffective treatment strat- A multidisciplinary approach that combines
egy. Taking depression as an example, although multiple omics data– integrated multi-dimensional
antidepressants provide substantial benefits for omics– is much more likely to offer complemen-
many, issues including lack of efficacy, intoler- tary vantage points to enrich our knowledge of
ance, delayed therapeutic onset, and risk of expression and functions of genomic factors asso-
relapse are frequently reported. In fact, results ciated with a disorder, thus improving diagnosis,
from one of the largest randomized trials involv- prognosis, and treatment development [20]. For
ing 4041 patients from 41 clinical sites around example, a recent GWAS meta-analysis revealed
the country showed that the remission rate from a high degree of correlation [average genetic cor-
the first line of treatment was only 28% [11]. relation (rg) = 0.40] among bipolar disorder,
Clearly, there is a lot of room for improvement in major depressive disorder (MDD), and schizo-
clinical psychiatry. phrenia [21]. On the other hand, a molecular pro-
10 Metabolomics Biomarkers for Precision Psychiatry 103
filing approach characterizing 181 proteins and 10.4 Overview of Metabolomics

small molecules in serum showed excellent poten- Analytical Techniques
tial to distinguish schizophrenia from healthy and Methodologies
controls, as well as from subjects with MDD,
bipolar disorder, and Asperger syndrome [22]. The likelihood of success for precision psychia-
Studies incorporating both investigation methods try lies in the accuracy and comprehensive
in the same study cohort likely will lead to signifi- dimensionality of the data. Analytical techniques
cant improvement in diagnostic accuracy. for metabolomics have come a long way. Nuclear
Biomarkers are objective surrogates of magnetic resonance (NMR), mass spectrometry
genetic, tissue-specific, and environmental fac- (MS), and electrochemical detection are com-
tors, as well as their interactions [23]. An effec- monly used techniques to identify and quantify
tive biomarker system such as integrated metabolites [27]. NMR is less sensitive than
multi-dimensional omics will thus serve as one of MS-based methods, yet it is favorable due to the
the most informative research and clinical tools absence of detection bias and is useful in identi-
and move the practice of psychiatry closer to the fying novel metabolite structures [28]. Compared
goal of precision psychiatry [24]. to NMR, MS is superior in mass analysis capa-
bilities and is usually used together with other
separation instruments such as gas chromatogra-
10.3 Unique Role phy (GC), liquid chromatography (LC), or capil-
of Metabolomics Biomarkers lary electrophoresis (CE). GC and LC have
traditionally been used in metabolomics studies;
While GWAS provide information on genomic CE has gained popularity in recent years [27]. In
risk factors that are often unmodifiable, metabo- clinical laboratories, liquid chromatography-
lomics studies measure our metabolic state, tandem mass spectrometry (LC-MS/MS) is
determined not only by genomic factors but also known for high specificity and sensitivity [29].
modified by diet, environmental factors, and host LC-MS/MS can detect compounds with low
factors such as the childhood experiences and gut molecular weight (such as eicosanoids) with bet-
microbiome. The metabolic profile serves as a ter sensitivity, selectivity, and higher throughput
quantifiable, dynamic readout of biochemical than high performance liquid chromatography or
state that can inform underlying molecular mech- GC-MS [29].
anisms of the disorder or phenotype. As such, Equally as important as choosing the right
metabolomics data have higher relevance to the instrument for metabolomics measurement are
“disordered state” and may serve well as predic- the design and analysis aspects of metabolo-
tive, prognostic, diagnostic biomarkers [25] for mics. Metabolomics analysis methods can
psychiatric disorders. The remainder of this broadly be categorized in two ways. “Untargeted
chapter provides a brief summary of the analyti- metabolomics” (global) analysis captures a
cal techniques most commonly used in metabolo- wide array of detectable metabolites, including
mics studies, and reports on and discusses a those with unknown functions or that have not
selection of psychiatric metabolomics and lipido- been seen previously [30]. Untargeted metabo-
mic studies. In particular, it highlights a specific lomics offers the unique advantage of discover-
class of metabolites called eicosanoids and their ing novel perturbations and detecting the
unique role in unraveling how disorders are influ- relationship between interconnected metabo-
enced by the interactive relationship between lites from multiple pathways in an unbiased
genes and diet [26]. fashion [30]. In contrast, a “targeted metabolo-
mics” approach focuses on a narrower, pre-
specified cluster of metabolites that have been
hypothesized to play a role in the disorder stud-
ied. Internal standards allow for quantification
of analytes in targeted metabolomics, offering brain-specific alterations in glucoregulatory

better control of sensitivity, stability, and repro- processes were intrinsic to disease because
ducibility of each targeted metabolite. Having these dysregulations normalized after treatment
some prior knowledge of these metabolites and with atypical antipsychotic medications in half
biochemical pathways means associations iden- of schizophrenia patients [35]. Patients with
tified in targeted analysis can move more schizophrenia and other psychiatric disorders
quickly to other molecular or translational stud- often experience significant weight gain during
ies to further define mechanisms underlying the their course of treatment [36]. Lipidomic and
phenotype associations. metabolomic analyses have identified lipids
Recent advances in these complementary associated with medication-associated weight
approaches have helped elucidate informative gain [37] and metabolic predictors of future
metabolomics biomarkers relevant in psychiat- weight gain [38]. These results emphasize the
ric disorders such as eicosanoids with inflam- added usefulness of a metabolomics approach in
mation regulatory functions. These biomarkers identifying psychiatric patients at risk of devel-
show promise for capturing early biochemical oping metabolic comorbidities [38]. To begin to
changes in the disease state [31] and enabling address the variability in treatment response
early diagnosis of psychiatric disorders. While commonly found in psychiatric disorders, serum
analytical considerations for generating metab- metabolites were investigated in 8 schizophre-
olomics data are beyond the scope of this chap- nia patients before and after risperidone mono-
ter, it is important to note that metabolites are therapy together with healthy controls. Although
volatile, with a short half-life, making rigorous the sample size was small, partial least squares
quality control of biological samples and assay discriminant analysis model derived from GC-
preparation necessary to ensure validity of the MS spectra revealed clear separations not only
findings. between schizophrenia and controls, but also
between risperidone responders and non-
responders [39]. These data suggest a global
10.5 Metabolomics Studies change of metabolites after risperidone treat-
of Common Psychiatric ment, and disturbances of energy metabolism,
Disorders antioxidant defense systems, neurotransmitter
metabolism, fatty acid biosynthesis, and phos-
Using an untargeted metabolomics approach and pholipid metabolism in schizophrenia, which
proton NMR (1H-NMR) system, a serum metab- could be partially normalized by risperidone
olite profile was effective in separating schizo- therapy [39].
phrenia from healthy controls [32]. Moreover, In major depressive disorder, at least 17
the results seem to reinforce the importance of peripheral blood mononuclear cell -derived
the glycolysis pathway and a “hyperglutamate metabolites identified in the GC-MC platform
hypothesis” previously proposed [33] in schizo- were significantly altered when compared with
phrenia. Examining both serum and urinary controls, indicating disturbances of energy and
metabolites, 1H-NMR and gas chromatography neurotransmitter metabolism [40]. In a urinary
with two-dimensional gas chromatography metabolomics study, the NMR- and GC-MS–
(GC-TOFMS) platforms revealed several path- based methods identified two sets of metabolites
ways implicated in schizophrenia including fatty that effectively discriminate “moderate” and
acid metabolism, carbohydrate metabolism, and “severe” patients from healthy controls, respec-
amino acid metabolism [34]. tively [41]. These metabolites implicate involve-
Metabolic profiles of cerebrospinal fluid ment of gut microbial metabolites, glycine
samples from drug-naïve (or minimally treated) biosynthesis, and cell death and survival in MDD
first-onset schizophrenia and controls suggest [41].
Depression is heterogeneous in its presenta- 10.6 Polyunsaturated Fatty Acids

tion and pathophysiology, affecting people of in Psychiatric Disorders
all ages, including those with medical diseases.
Metabolomic analysis of plasma from older While advances in mass spectrometry have
adults with and without depression revealed expanded our knowledge of the patterns of metab-
lower levels of several neurotransmitters and olomic perturbation in psychiatric disorders, non-
medium chain fatty acids in depression. Also, genetic risk factors such as diet play a major role
the profile of those with remission from depres- in neuronal fitness [50–52]. Essential fatty acids
sion was more similar to non-depressed con- represent a modifiable risk factor for neuropatho-
trols than to the depressed individuals [42]. In a physiological processes [53, 54]. While a number
medical cohort of patients with heart failure of hypotheses exist for etiology of psychiatric dis-
with and without depression, GC-MS and orders, inflammation has recently been shown to
LC-MS platforms revealed higher concentra- play a role in common mental disorders such as
tions of several amino acids and dicarboxylic depression [55] and schizophrenia [56].
fatty acids [43], consistent with prior findings Bioactive lipid mediators are a class of under-
in neurotransmitter systems and fatty acid appreciated, under-utilized molecules in studies
metabolism dysregulation. These results sug- of inflammation. Specifically, the bioactive
gest that metabolomics biomarkers might be metabolites derived from fatty acids, termed
useful as objective diagnostic tests for depres- eicosanoids, participate in modulation of inflam-
sive disorder. Based on findings in several mation [57, 58] and pain [59], and have been
untargeted metabolomics studies of depression shown to affect risks of hypertension [60], car-
and pharmacometabolomic studies [44, 45], a diovascular diseases [61], cancer [62], anorexia
new study investigating whether these metabo- nervosa [63], and schizophrenia [64]. To more
lites (sphingomyelins, lysophosphatidylcho- comprehensively assess how dietary-based inter-
lines, phosphatidylcholines, and acylcarnitines) vention [65] may affect inflammation and psychi-
could act as predictors of depression recovery atric outcomes [66–68], MS technology has been
found that the addition of metabolites in all pre- extended to lipidomics analysis for polyunsatu-
dictive models outperformed models without rated fatty acids (PUFA) [69, 70]. These lipids
these metabolites [46]. include the 18-carbon “essential” PUFA such as
In bipolar disorder, 1H-NMR-based analysis linoleic acid (LA; 18:2n-6) and alpha-linolenic
revealed lipids, lipid metabolism-related mole- acid (ALA; 18:3n-3), 20-carbon PUFA arachi-
cules, and some amino acids that distinguished donic acid (ARA; 20:4n-6) and eicosapentaenoic
bipolar subjects, with 7 specific markers as “key acid (EPA; 20:5n-3), and 22-carbon PUFA such
metabolites” [47]. A study using dual platform as docosapentaenoic acid (DPA; 22:5) and doco-
(NMR spectroscopy and GC-MS) revealed 5 uri- sahexaenoic acid (DHA; 22:6n-3).
nary metabolite biomarkers with higher accuracy The clinical benefits observed when supple-
than single-platform derived markers in discrimi- menting n-3 PUFA in inflammation-driven dis-
nating bipolar disorder from healthy controls eases were the initial clues that n-3 PUFA may
[48]. In another study, an increased proportion of similarly yield symptom relief in psychiatric
serum sphingolipids and glycerolipids and a disorders.
decreased proportion of glycerophospholipids Rheumatoid arthritis patients who took n-3
were found in bipolar disorder patients when fatty acids supplementation showed significant
UltraPerformance LC coupled with high- clinical benefits compared with those who did
resolution MS was used [49]. However, of the top not receive n-3 supplementation [71]. Patients
5 most differential lipids identified, 3 had with colon cancer who received fish oil supple-
unknown biology and could not be identified in mentation had a significant reduction in IL-6 and
any databases [49]. TNF-alpha levels and an increase in the
percentages of CD3+ and CD+ lymphocytes Schizophrenia is a serious psychiatric disorder

when compared with the control group [72]. that has been proposed as “neurodevelopmental”
These data suggest that n-3 PUFA might also based on early subclinical brain imaging charac-
benefit symptoms in psychiatric disorders [73], teristics [83]. Decreased levels of AA and DHA
which are now recognized as disorders of neuro- were observed in never-medicated first-episode
nal inflammation [74–76]. schizophrenia patients compared with medicated
Peripheral levels of PUFA and n-6:n-3 PUFA patients and healthy controls [84]. In another
ratios have been investigated in depressive disor- study using established schizophrenia cases and
der and bipolar disorder, the two most common carefully selected matched controls, DHA, DPA,
mood disorders. The erythrocyte membrane ARA and DHA:ARA ratio were reduced in patients
and DHA were significantly reduced in Taiwanese compared to controls [85].
patients with bipolar disorder patients when com- For another psychiatric disorder, anorexia ner-
pared with healthy controls, while no differences vosa, we found the ARA:EPA ratio to be lowered
in total PUFAs were observed [77]. Similarly, in in anorexia nervosa compared with healthy con-
a sample of Italian patients with bipolar disorder, trols [63]. To determine if PUFA levels were
plasma DHA was significantly lower than that of “risk factors” or “consequences” of schizophre-
healthy controls; however, EPA, AA, ALA, and nia, ultra-high risk individuals (for developing
ARA appeared to be elevated. Based on this schizophrenia) were followed and dietary intake
result, the authors suggest that DHA may be a was assessed. Those who later developed psycho-
useful adjuvant for bipolar disorder [78]. sis were found to consume more dietary n-6
To account for the competitive biology PUFAs (LA, AA) and had higher AA:EPA + DHA
between the n-3 and n-6 classes of PUFA, another ratios than those who did not develop psychosis
group analyzed a broad panel of serum lipids [86]. Similarly, n-3 PUFA supplementation was
including PUFA in individuals with euthymic associated with beneficial effects for anorexia
bipolar, depressive bipolar, major depressive dis- nervosa in several studies [87, 88]. Together,
order, and non-psychiatric controls. They found these data support a possible protective role n-3
that higher AA:EPA and AA:EPA + DHA ratios PUFAs play in preventing onset or worsening of
were consistently found in the group with bipolar psychotic symptoms. Several mechanisms have
depression. Moreover, the AA:EPA + DHA ratio been proposed to explain n-3 PUFAs’ favorable
was positively correlated with depression sever- effect in psychiatric disorders, including anti-
ity in all groups, despite a lack of control on the neuronal inflammation [89] and neuronal protec-
fasting status of the subjects [79]. In another tion [90].
study, a high n-6:n-3 ratio and low DHA were Although the data in psychiatric literature
found to be predictive of suicide risk among largely paint a favorable view for supplementa-
depressed patients [80], highlighting the poten- tion of n-3 PUFA, the verdict on the supplements’
tial prognostic role PUFA markers may play in therapeutic role in psychiatric disorders cannot
depression. Lastly, several eloquent meta- be established without randomized clinical trials
analyses have examined evidence of the efficacy with well-characterized longitudinal data and
of treating depression or depressive symptoms large sample size. While many independent stud-
with n-3 PUFA. Some took a conservative posi- ies in the past have supported the benefits of n-3
tion, stating that the antidepressant efficacy of PUFAs for serious medical disorders such as car-
n-3 PUFAs in unipolar and bipolar depression diovascular disease and cancer, the most recent
cannot be confirmed until further replication with meta-analysis showed that n-3 fatty acids did not
more “homogeneous” and larger samples [81]. reduce the incidence of cancer and cardiovascu-
Two independent meta-analyses reported n-3 lar events (e.g., stroke, myocardial infarction, and
PUFA supplementation, especially EPA, to be cardiovascular event-related death) [91]. In a
effective in treating major depression disorder large randomized trial including 15,480 diabetic
and depressive symptoms [54, 82]. patients without cardiovascular disease, no
significant difference was found in the risk of mation biology include prostaglandin E2 (PGE2),
serious vascular events between those who were a pro-inflammatory molecule stimulated by
assigned to n-3 PUFA supplementation and those COX, and 5-LOX-produced leukotrienes, which
who were assigned to a placebo [92]. It is thus contribute to potent inflammation in asthma and
imperative to conduct further research not only to other allergic diseases [96]. CYP regulates
confirm the effectiveness of n-3 PUFAs in psy- inflammation by oxidizing ARA with its active
chiatric disorders but also to explore molecular heme iron to form anti-inflammatory HETE or
mechanisms driving clinical benefits in patients epoxy-eicosatrienoic acid (EETs), which is then
and develop biomarkers to classify individuals hydrolyzed into pro-inflammatory dihydroxy-
with high likelihood of benefiting from PUFA eicosatrienoic acids (DHET) by soluble epoxide
supplementation. To achieve these goals, the log- hydrolase (sEH) [97].
ical next step is to take advantage of targeted We have demonstrated the effectiveness of a
metabolomics technology to focus on a specific combined use of lipidomics and targeted metabo-
class of metabolites, termed eicosanoids [93, 94]. lomics in investigating anorexia nervosa, an ill-
ness characterized by rapid weight loss and
reduction in food consumption [63]. Higher
10.7 Eicosanoids as Biomarkers ratios of dihydroxy to epoxy fatty acids were
for Psychiatric Disorders found in anorexia nervosa patients than in con-
trols, suggesting an upregulation of sEH activity,
While the pattern of association between PUFA an elevation in pro-inflammatory eicosanoid pro-
and psychiatric disorders may at first seem file, and a reduction in anti-inflammatory epoxy
straightforward, one must understand the func- fatty acids [63]. Additionally, recovered anorexia
tions and mechanisms underlying beneficial nervosa patients showed a partial normalization
effects of any compound to use such benefits in PUFA and eicosanoids, implying the resolu-
clinically. The bioactive lipids family represents tion of inflammation and that it may be achieved
the next logical class of molecules to study to by dietary intervention [26]. This is clinically rel-
elucidate PUFA mechanisms, and to establish a evant as well for patients with other types of psy-
biomarker system that is biologically and clini- chiatric disorders because medication
cally informative to guide precision psychiatry. non-adherence rate is notoriously high, up to
Major n-6 and n-3 PUFA can be oxygenated 80% in schizophrenia [98]. Dietary intervention
by at least 3 different enzymes to synthesize over may be an important alternative treatment modal-
120 heterogeneous and pleiotropic bioactive ity for patients refusing medications.
molecules termed eicosanoids [59, 95]. While the The results of the anorexia nervosa study sug-
word eicosanoid was derived from the Greek gest that psychopathology and inflammatory pro-
word “eikosa,” meaning “20,” based on the deriv- cesses in eating disorders are affected by
atives of the 20-carbon ARA, here “eicosanoid” interactions between dietary PUFA and geneti-
is applied to also include the oxygenated prod- cally driven metabolism. With additional empiri-
ucts of other PUFA including LA, ALA, DHA, cal research, food-based treatment or a
and DPA. The 3 enzymatic families that affect nutraceutical strategy may be employed to
PUFA are cyclooxygenases 1 and 2 (COX-1/2); improve outcomes in clinical psychiatry.
5-, 12-, and 15-lypooxygenases (5/12/15-LOX); Furthermore, as eicosanoid variation reflects in
and P450 epoxygenase. The COX-1/2 are known vivo cellular inflammation, targeted metabolo-
to drive the synthesis of prostanoids such as pros- mics can be applied to develop improved progno-
taglandins and thromboxanes, while 5/12/15- sis biomarkers.
LOX produce leukotrienes, lipoxins, and Untargeted metabolomics emerged as a use-
hydroxyeicosatetraenoids, and P450 synthesize ful tool to uncover unsuspected pathways
HETEs and epoxyeicosatrienoids [95]. The eico- involved in psychiatric disorders, and a targeted
sanoids most well-studied for their link to inflam- metabolomics approach is particularly helpful in
characterizing the specificity, direction and mag- The discovery of an association between cyto-
nitude of disease-associated metabolites, which chrome P450-associated bioactive lipid media-
provide molecular insight helpful to develop tors and psychiatric disorders is made possible in
new treatments. In a pilot study of adolescent part because of advances in technology, but the
major depressive disorder, we characterized involvement of eicosanoids in psychiatric disor-
eicosanoids in fasting plasma at the baseline ders was reported as early as the 1980s. Using
visit and final visit after a 2 year follow-up low throughput techniques such as radioimmuno-
period. While all subjects displayed no differ- assay, elevated levels of PGE and PGE2 were
ence in depression severity or profile of depres- identified in schizophrenia [104, 105], whereas
sion risk factors at the baseline visit, half of the PGD2, PGE2, and PGF2α and TXB2 were found
subjects had progressed to significantly worse to be elevated in major depressive disorder [106–
depression (refractory group) while the other 109]. Almost 40 years later, the field can now
half remitted. Strikingly, the eicosanoids profile take advantage of both untargeted and targeted
in the refractory group revealed a pattern very liquid chromatography-mass spectrometry-based
similar to that found in patients with anorexia methods to monitor a much larger number of
nervosa [99], implicating an epoxy fatty acid potential markers. A recent schizophrenia study
catalyzing enzyme, soluble epoxide hydrolase that investigated 158 markers including PUFA,
(sEH), as a common risk factor for depression eicosanoids, and related mediators from enzyme-
and anorexia nervosa.In a study of seasonal dependent or independent pathways uncovered
major depression [31], quantitative changes of 23 metabolites that were significantly altered in
CYP450 pathway eicosanoids during the winter patients compared with healthy controls [64].
season (when subjects experienced severe While some abnormal markers were reversed
depression symptoms) were similar in pattern to after antipsychotic treatment, anandamide,
the eicosanoids profile we found in the refrac- oleoylethanolamine, and ARA were identified as
tory adolescent depression group [99], suggest- having the best potential for differentiating
ing that sEH-mediated metabolism of PUFA patients from controls [64].
eicosanoids underlies the psychopathology of Leveraging what is already known about the
depressive disorders [31]. biology of bioactive lipids and the plethora of
sEH is known as a regulator of inflammatory physiological and homeostatic processes they
resolution due to its potent and complex mecha- participate in, several drugs have already been
nisms in the formation/catabolism of epoxy- and developed to inhibit the production of pro-
diol eicosanoids [100], but its involvement with inflammatory mediators, including nonsteroidal
psychiatric phenotypes was only recently uncov- anti-inflammatory drugs (NSAIDS) that reduce
ered through MS-based discovery [31, 99] and the activity of both COX-1 and COX-2 [110],
sequencing [101]. Another group has since dem- cysteinyl leukotriene (cysLT) receptor antago-
onstrated that sEH inhibition showed antidepres- nists that reduce bronchoconstriction caused by
sant effects in both inflammation and social cycLT and pro-inflammatory cytokines in the
defeat stress models of depression [102] and pulmonary system [111], and COX-2 inhibitors
attenuated behavioral abnormalities (i.e., hyper- [112]. In fact, administration of COX-2 inhibitor
locomotion and prepulse inhibition deficits) in an celecoxib has been shown to improve symptoms
animal model of schizophrenia [103]. Moreover, in schizophrenia [113], possibly through inhibit-
a higher level of sEH was found in postmortem ing conversion of ARA into prostanoids.
brain samples from patients with depression, Additionally, COX-2 inhibitors may be effective
schizophrenia, and bipolar disorder compared as an adjunctive treatment by accelerating the
with control samples [102], strengthening the onset of antidepressant effects for bipolar depres-
role sEH plays in psychiatric pathology. sion and refractory major depression [114, 115].
10.8 Conclusions psychopathology of psychiatric disorders, metab-

olomics coupled with other multi-omics
While an untargeted metabolomics strategy has approaches can (1) provide deeper insights into
gained popularity for its ability to screen new and the biological underpinnings of psychiatric disor-
unsuspected pathways involved in psychiatric ders, (2) be used as powerful diagnostic, disease-
disorders, evidence of a role for eicosanoids in monitoring, and treatment response biomarkers,
psychiatry is accumulating. Eicosanoids partici- and (3) bring precision psychiatry closer to real-
pate in the modulation of inflammatory processes ity by enabling improved drug discovery and
and affect the risk of a number of neuropsychiat- development processes, thereby advancing phar-
ric disorders. Characterizing the eicosanoid sig- macometabolomics, nutrigenomics, and metabo-
nature in major psychiatric disorders and lomic engineering technologies.
subtypes within can lay the foundation for indi-
vidualized treatment approaches. Much work is
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Cytochrome P450 Eicosanoid
Signaling Pathway in Colorectal
11
Tumorigenesis
Weicang Wang, Katherine Z. Sanidad,
and Guodong Zhang
Abstract Keywords
Colorectal cancer (CRC) is the third most Colorectal cancer · Colonic inflammation ·
common cancer and the second leading cause Cytochrome P450 · Soluble epoxide hydro-
of cancer-related death in the United States. It lase · Eicosanoids
is important to discover novel cellular targets
which are crucial in the pathogenesis of CRC,
which could facilitate development of
mechanism- based strategies to reduce the 11.1 Introduction
risks of CRC. Emerging studies support that
the cytochrome P450 (CYP) monooxygenase/ Colorectal cancer (CRC) is a serious health prob-
soluble epoxide hydrolase (sEH) pathway and lem: there were ~140,250 new cases and ~50,630
their eicosanoid metabolites play critical roles deaths in the United States in 2018 [1], empha-
in colonic inflammation and CRC, and could sizing the need for discovering novel cellular tar-
be therapeutically explored for treating or pre- gets which are crucial in the pathogenesis of
venting CRC. Here in this review, we discuss CRC. Colonic inflammation is a major risk factor
recent studies about the roles of the CYP/sEH for developing CRC, therefore, targeting the
eicosanoid pathway in the pathogenesis of pathological components involved in colonic
colonic inflammation and CRC. inflammation is a promising strategy to reduce
the risks of CRC [2]. Eicosanoids, which are
W. Wang endogenous lipid signaling molecules produced
Department of Food Science, University of from enzymatic metabolism of polyunsaturated
Massachusetts, Amherst, MA, USA fatty acids (PUFAs), play essential roles in
K. Z. Sanidad inflammatory responses and were recently impli-
Molecular and Cellular Biology Graduate Program, cated in cancer [3, 4]. The most prominent CRC-
University of Massachusetts,
Amherst, MA, USA
associated eicosanoids are prostaglandins, which
are produced by the cyclooxygenase-2 (COX-2)
G. Zhang (*)
Department of Food Science, University of
enzyme that is overexpressed in most human
Massachusetts, Amherst, MA, USA CRC samples [4]. Genetic knockout of Cox-2
Molecular and Cellular Biology Graduate Program,
reduces polyp formation in azoxymethane
University of Massachusetts, Amherst, MA, USA (AOM)- or Apc mutation-induced CRC models
e-mail: [email protected] [5, 6]. Furthermore, clinical and epidemiological

116 W. Wang et al.
studies support that pharmacological inhibitors this previously unappreciated pathway in the
of COX-2, such as nonsteroidal anti-inflammatory pathogenesis of CRC could help to develop new
drugs (NSAIDs), are effective in reducing the strategies for cancer treatment or prevention. In
risk of CRC [4]. These results support the critical this review, we will discuss the roles of the CYP/
importance of eicosanoid signaling in sEH eicosanoid pathway in the pathogenesis of
CRC. However, the gastrointestinal and cardio- colonic inflammation and CRC, as well as
vascular toxicities induced by the COX-2 inhibi- obesity-associated colonic inflammation and
tors have limited their clinical applications [7]. CRC.
Besides COX-2, the roles of other eicosanoid
pathways in colonic inflammation and CRC are
not well understood [8]. It is therefore important 11.2 Expression of CYP
to discover novel eicosanoid signaling pathways Monooxygenases and sEH
involved in the pathogenesis of CRC. in Colonic Inflammation
Besides being substrates of COX-2, PUFAs and CRC
are also substrates of cytochrome P450 (CYP)
monooxygenases (predominately the CYP2C Our recent study has shown that CYP monooxy-
and CYP2J isoforms), which convert them to genases are overexpressed in colon tumor tissues
epoxygenated fatty acids (EpFAs). EpFAs include and colon cancer cells [21]. Compared with con-
epoxyeicosatrienoic acids (EETs) produced from trol healthy mice, the expressions of mouse CYP
arachidonic acid (ARA, 20:4ω-6), monooxygenases, such as Cyp2c38, Cyp2c39,
epoxyoctadecenoic acids (EpOMEs) from lin-
Cyp2c55, Cyp2c65, Cyp2c70, Cyp2j6, Cyp2j9,
oleic acid (LA, 18:2ω-6), epoxyoctadecadienoic and Cyp2j13, are increased in colon tissues of
acids (EpODEs) from α-linolenic acid (ALA, AOM/dextran sulfate sodium (DSS)-induced
18:3ω-3), and epoxydocosapentaenoic acids CRC mice [21]. In addition, the concentrations of
(EDPs) from docosahexaenoic acid (DHA, CYP-produced EpFAs are increased in both the
22:6ω-3) [9]. EpFAs are metabolically unstable plasma and colon tissues of AOM/DSS-induced
with a half-life of several seconds in vivo, in part CRC mice [21]. Furthermore, we find that com-
because they could be rapidly metabolized by pared with normal human colon cells (CCD-
soluble epoxide hydrolase (sEH) to generate the 18co), the expression of CYP2C8, CYP2C9,
corresponding fatty acid diols [10]. Currently, the CYP2C19, and CYP2J2 is increased in human
CYP/sEH eicosanoid pathway is being explored CRC cells (HCT116 and Caco-2) [21]. These
by academic laboratories and pharmaceutical results support that the CYP monooxygenase
companies for clinical applications. For example, pathway is upregulated in mouse and cell culture
GlaxoSmithKline is conducting human clinical models.
trials to test an sEH inhibitor GSK2256294; the The expressions of CYP monooxygenases in
company has found that this drug candidate is human CRC are more complicated. We analyzed
well-tolerated and causes sustained inhibition of gene expression of CYP monooxygenases
sEH in humans [11]. Other novel classes of sEH (CYP2C8, CYP2C9, CYP2C19, and CYP2J2) in
inhibitors are also being considered for human the Cancer Genome Atlas (TCGA) database, and
trials [12]. In addition, recent studies have shown found that their expressions were not increased in
that some FDA-approved drugs are potent inhibi- colorectal adenocarcinoma [21]. However, we
tors of CYP monooxygenases [13]. must note that the expression and activity of the
Emerging research supports that the CYP/sEH CYP enzymes are regulated by multiple mecha-
pathway plays critical roles in regulating inflam- nisms, including transcription, translation, and
mation, angiogenesis, tumor growth, and tumor post-translational modification, not only the
metastasis [14–17], and could be involved in the mRNA expression levels [22]. Indeed,
pathogenesis of colonic inflammation and CRC Enayetallah et al. report that CYP2C9 is detected
[18–21]. A better understanding of the roles of in 13 out of 17 human colon tumor samples,
11 Cytochrome P450 Eicosanoid Signaling Pathway in Colorectal Tumorigenesis 117
while it is not detected in matched benign sam- mice, pharmacological inhibition of sEH reduces
ples [23]. In addition to CRC, previous studies the infiltration of macrophages and expression of
have shown that CYP monooxygenases are over- pro-inflammatory cytokines in epididymal fat
expressed in other tumor tissues, such as breast, and liver [25, 26]. Deletion of sEH also leads to
liver, and stomach tumors [16, 23]. There could reduced infiltration of neutrophils, decreased lev-
be many mechanisms by which CYP monooxy- els of pro-inflammatory cytokines and less neu-
genases are overexpressed in tumor tissues. The ronal damage in an intracerebral hemorrhage
expression of CYP monooxygenases has been mouse model [27]. Together, these studies dem-
shown to be elevated by hypoxia [10], which is a onstrate sEH is involved in many inflammation-
common feature of tumor tissues [24]. It is fea- associated diseases.
sible that the hypoxic tumor microenvironment More studies are needed to better characterize
could contribute to the increased expression of the roles of sEH in cancer. Panigrahy et al. have
CYP monooxygenases in tumor tissues. shown that pharmacological inhibition or genetic
In humans, the expression of sEH is increased deletion of sEH increases tumor growth and
in colonic dysplasia and adenocarcinomas in metastasis in xenograft models by stimulating
ulcerative colitis (UC) patients [18]. sEH has tumor angiogenesis [15]. However, this finding is
been detected in ~40% of human colon adenocar- different from the results observed in the DSS
cinomas (7 out of 17 samples) with relatively and IL-10−/− mouse models [18–20]. The differ-
high expression levels, but is not detected in ent results could be, at least in part, due to the
matched benign samples (0 out of 4 samples) differences in mouse models. The phenotypes in
[23]. More studies are needed to characterize the the DSS and IL-10−/− mouse models are strongly
expression of sEH and the concentrations of associated with inflammation, therefore, inhibi-
CYP/sEH-produced eicosanoid metabolites in tion of sEH would reduce inflammatory response
colonic inflammation and CRC. and attenuate inflammation-associated CRC [18–
20]. In other models such as xenograft models,
inhibition of sEH could upregulate angiogenesis
11.3 R
oles of CYP/sEH Eicosanoid and increase tumorigenesis [15]. However, the
Pathway in Colonic pro-angiogenic effects of sEH inhibition were
Inflammation and CRC mainly observed in mouse models [15]. A recent
human clinical trial has shown that even at 100%
Recent research has shown that sEH plays a criti- inhibition of sEH, there is no change of the
cal role in colonic inflammation and CRC. In the plasma concentration of vascular endothelial
DSS-induced mouse model, sEH−/− mice have growth factor (VEGF), which is an important
reduced colonic inflammation (as assessed by biomarker of angiogenesis [11]. Since sEH inhib-
mucosal erosion and lymphoplasmocytosis) and itors are currently being evaluated in human clin-
carcinogenesis (tumor incidence and volume) ical trials [11, 12], it is of critical importance to
compared with wild-type mice [18]. Similarly, in better understand the roles of sEH and sEH inhib-
an interleukin 10 (IL-10) deficiency-induced itors in tumorigenesis.
CRC model, sEH−/− IL-10−/− mice have reduced Recent studies also support the critical roles of
colonic expression of pro-inflammatory cyto- CYP monooxygenases in tumorigenesis. In a
kines and formation of ulcers and carcinomas xenograft tumor model, overexpression of human
compared with sEH+/+ IL-10−/− mice [19, 20]. CYP2C8 or CYP2J2 in endothelial cells led to
Together, these results support that sEH could increased xenograft tumor growth of B16F10
contribute to colonic inflammation and melanoma and T241 fibrosarcoma [15]. Using a
inflammation-associated CRC. This is largely in Lewis lung carcinoma (LLC) resection-induced
agreement with previous studies which show that tumor metastasis model, endothelial expression
inhibition of sEH reduces inflammatory responses of human CYP2C8 or CYP2J2 increases lung
in various disease models. For example, in obese metastases [15]. Together, these results support
118 W. Wang et al.
the pro-tumorigenic and pro-metastatic effect of further metabolized by sEH to form the corre-
CYP monooxygenases. sponding fatty acid diols termed dihydroxyocta-
Our recent studies also support a potential role decenoic acids (DiHOMEs) [10]. Similar to
of CYP monooxygenases in the tumorigenesis of EpOMEs, DiHOMEs have also been shown to
CRC [21]. Compared with AOM/DSS-induced induce chemotaxis, tissue injury, and cause mor-
Cyp2c+/+ mice, the AOM/DSS-induced Cyp2c+/− tality in animal models [34, 35].
mice have lower tumor numbers and total tumor The biological actions of EETs are more
burden, as well as reduced expression of CYP complicated, as they have been shown to have
monooxygenases and concentrations of CYP- anti-inflammatory effects (negatively associated
derived fatty acid epoxides in colon tissues, sup- with tumorigenesis) and pro-angiogenic effects
porting the roles of Cyp2c monooxygenases in (positively associated with tumorigenesis).
colon tumorigenesis [21]. Consistent with the Indeed, many studies have shown that EETs
results observed in transgenic mouse models, we have potent anti-inflammatory effects. In a
also find that pharmacological inhibition of CYP murine carotid artery model, treatment with
monooxygenases suppresses AOM/DSS-induced 11,12-EET decreases tumor necrosis factor-α
colon tumorigenesis in mice [21]. Together, these (TNF-α)–induced mononuclear cell adhesion to
results support that CYP monooxygenases con- the arterial endothelium [36]. Treatment with
tribute to the tumorigenesis of CRC. 14,15-EET inhibits TNF-induced degradation
of IκBα in primary human lung tissue [37] and
reduces LPS-activated IL-1β and TNF-α expres-
11.4 Effects of CYP-Produced sion in mouse macrophages [18]. Together,
Eicosanoids on Inflammation these results support the anti-inflammatory
and CRC effects of EETs. The effects of EETs on tumor
inflammation are not well characterized and
The metabolism of PUFAs by CYP monooxy- require more studies. Previous studies have
genases leads to the formation of EpFAs [9]. The shown that inhibition or deletion of sEH attenu-
major EpFAs in tissues and plasma include ates DSS- or IL-10 knockout-induced CRC [18–
EpOMEs produced from LA, EETs from ARA, 20]; these results could be, at least in part, due
and EDPs from DHA. Emerging research sup- to the anti-inflammatory effects of EETs. On the
ports that these eicosanoid metabolites have other hand, EETs have pro-angiogenic effects,
potent effects on inflammation and tumorigenesis and therefore could promote tumor growth and
with the details discussed below. metastasis. In an orthotopic cancer model, treat-
Previous studies show that EpOMEs have a ment of 14,15-EET at a dose as low as 15 μg/kg/
series of detrimental actions such as inducing day increases orthotopic PC3M-LN4 prostate
chemotaxis, inflammation, cardiovascular dis- tumor growth in SCID mice [15]. In transgenic
eases, and pulmonary injury [28–33]. In human adenocarcinoma of the mouse prostate
studies, EpOMEs, which are termed “leukotox- (TRAMP) mice, administration of 14,15-EET at
ins,” are associated with multiple organ failures a dose of 15 μg/kg/day promotes prostate tumor
and adult respiratory distress syndrome in severe growth, suggesting that EETs increase tumor
burn patients [29, 31, 34]. Notably, at a concen- growth in vivo [15]. In addition, treatment with
tration as low as 10 nM, EpOMEs enhance neu- 14,15-EET increases tumor metastasis in a LLC
trophil chemotaxis, suggesting potent resection model [15]. More studies are needed
pro-inflammatory effects of EpOMEs [33]. to further characterize the role of EETs in
Regarding CRC, our recent study shows that tumorigenesis.
treatment with 12,13-EpOME exaggerates AOM/ Opposite to the effects of EpOMEs and EETs,
DSS-induced CRC in mice [21], supporting the our studies have shown that ω-3 PUFA-produced
pro-CRC actions of EpOMEs. EpOMEs can be EDPs inhibit angiogenesis, tumor growth, and
tumor metastasis [38]. 19,20-EDP inhibits tube been shown to be biologically active and could
formation, migration, and production of matrix contribute to the biological actions of CYP
metalloproteinases (MMPs) in endothelial cells, monooxygenases and sEH. Recent studies have
and suppresses VEGF- and basic fibroblast shown that dihydroxyeicosatrienoic acids
growth factor (bFGF)-induced angiogenesis in a (DHETs), which are metabolites of EETs pro-
Matrigel plug assay in mice, demonstrating its duced by sEH, have pro-inflammatory effects
anti-angiogenic effect [38]. Treatment with stabi- [10, 47]. Treatment of 5,6-DHET, 8,9-DHET,
lized 19,20-EDP also suppresses primary tumor 11,12-DHET, or 14,15-DHET at a dose of 3 μM
growth and tumor metastasis in mice [38]. stimulates primary human monocyte migration
Furthermore, our recent study showed that in vitro [50]. Similar to the pro-inflammatory
treatment with EDPs suppresses growth of MC38 effects of ARA-produced DHETs, recent studies
xenograft colon tumors, supporting the anti-CRC show the DHA-produced dihydroxydocosapen-
effects of EDPs [39]. Consistent with our results, taenoic acids (DHDPs) promote progression of
a recent study by Yanai et al. has shown that sys- retinopathy in mice [48, 49]. Treatment of
tematic treatment with EDPs inhibits pathologi- 19,20-DHDP induces retinal angiogenesis by
cal angiogenesis in a mouse model of macular increasing tip cell, sprouting, and filopodia
degeneration [14]. numbers in mice, all of which contributes to
To date, the molecular mechanisms for the proliferative retinopathy [48]. Moreover, in an
biological actions of EpFAs are not well ex vivo whole mount retina model, treatment
understood. Many eicosanoids act by binding to with 19,20-DHDP increases vascular endothe-
specific cellular targets. For example, COX-2- lial cell permeability and pericyte migration
produced prostaglandin E2 (PGE2) and lipoxy- into extravascular space, both of which are dis-
genase (LOX)-produced leukotriene B4 (LTB4) ease markers for diabetic retinopathy [49]. In
act by binding to their G-protein coupled recep- cultured murine brain microvascular endothelial
tors [40]. The direct cellular target(s) of CYP cells, treatment of 19,20-DHDP decreases junc-
monooxygenase metabolites are not well under- tion formation between cells and reduces
stood, hampering our understanding of their expression of N-cadherin, which could contrib-
mechanisms of action. Previous studies show that ute to effect of 19,20-DHDP on vascular perme-
EETs bind to cell membrane-bound proteins in a ability during diabetic retinopathy [49]. Overall,
high-affinity, specific, and saturable manner [41– these studies demonstrate that fatty acid diols
43], and some of the biological actions of EETs are bioactive eicosanoids which could contrib-
are G protein-dependent [43–46]. These results ute to the pathogenesis of many diseases.
support that the CYP monooxygenase metabo- Together, these results support that CYP-
lites also act via interactions with specific cellu- produced eicosanoid metabolites, including
lar proteins; however, the identities of the cellular EpOMEs, EETs, and EDPs, have potent effects
targets are not well understood. Beyond EETs, no on tumorigenesis. These results could help to
study has investigated the cellular targets of other establish a novel mechanistic linkage between
CYP monooxygenase metabolites (such as fatty acid intake and cancer risks. For example,
EpOMEs). Identification of their direct cellular animal experiments have shown that a high
targets could greatly enhance our understanding dietary intake of LA increases AOM-induced
for the molecular mechanisms of the CYP mono- colon tumorigenesis, suggesting its potential
oxygenase pathway. In addition, the identified adverse effect on CRC [50–54]. Here our study
cellular targets could also serve as novel molecu- about the promoting effects of LA-produced
lar targets for preventing or treating cancer and EpOMEs on CRC suggests that the formation of
other human diseases. EpOMEs could contribute to the promoting
Besides the EpFAs, their down-stream effects of LA on the risks of CRC [21].
metabolites, termed fatty acid diols, have also
120 W. Wang et al.
11.5 R
oles of CYP/sEH Eicosanoid sues of obese mice [60, 61]. In addition, pharma-
Pathway in Obesity-Induced cological inhibition or genetic ablation of sEH
Colonic Inflammation attenuated various adverse consequences of obe-
sity, including endoplasmic reticulum stress,
More than one-third of US adults (34.9% or metabolic syndrome, fatty liver, hepatic steatosis,
78.6 million) are obese [55], and obese individu- inflammation, endothelial dysfunction, and dia-
als have a 30–60% higher risk of developing betes [25, 26, 60, 62–68]. Together, these results
CRC [56, 57]. Considering the obesity epidemic support that targeting sEH could be a promising
and the potential lethal consequence of CRC, strategy to reduce the risks of CRC in obese indi-
obesity-enhanced CRC is a serious health prob- viduals. Further studies are needed to character-
lem in the US. However, the mechanism by ize the roles of sEH in obesity-enhanced CRC.
which obesity increases the risks of CRC is not In our studies, we find that the colonic expres-
well understood, and there are few effective strat- sions of CYP2C and CYP2J monooxygenases
egies to prevent obesity-enhanced CRC [58]. are not altered in HFD-treated mice [59].
Using LC-MS/MS-based metabolomics, our However, considering that the fatty acid diols are
recent research suggests that sEH could be a down-stream metabolites of CYP monooxygen-
novel therapeutic target of obesity-induced ases, it is feasible that inhibition of CYP mono-
colonic inflammation [59]. In a high fat diet oxygenases could also reduce obesity-induced
(HFD)-induced obesity model in C57BL/6 mice, colonic inflammation and associated tumorigen-
we find that the expression of sEH and the con- esis. Targeting CYP monooxygenases could be
centrations of sEH-produced fatty acid diols are an alternative approach to reduce the risks of
significantly increased in the colon tissues of obesity-enhanced CRC. This approach could
HFD-induced obese mice [59]. Furthermore, minimize potential adverse effects of sEH inhibi-
pharmacological inhibition or genetic ablation of tion on angiogenesis [15], and further studies are
sEH abolishes HFD-induced colonic inflamma- needed to better characterize the roles of the
tion in mice, with reduced expression of pro- CYP/sEH eicosanoid pathway in obesity-
inflammatory cytokines (Il-1β and Tnf-α) and/or enhanced CRC.
decreased infiltration of immune cells in colon
tissues [59]. Furthermore, we find that the inhibi-
tion or ablation of sEH attenuates HFD-induced 11.6 Conclusion
activation of Wnt signaling pathway in colon tis-
sues [59]. CRC is the third most common cancer and the
Considering the critical roles of colonic second leading cause of cancer-related death in
inflammation and Wnt signaling in the pathogen- the US [1], emphasizing the need for discovery of
esis of CRC, these results support that sEH could novel cellular targets which are crucial in the
be a potential therapeutic target of obesity- pathogenesis of colon cancer. Recent research by
enhanced CRC. This notion is supported by pre- us and others support that CYP monooxygenases,
vious studies, which show that inhibition of sEH sEH, and associated eicosanoid metabolites
has beneficial effects on colonic inflammation, (EpFAs and fatty acid diols) play critical roles in
CRC, and obesity, supporting that targeting sEH regulating angiogenesis, tumor growth, and tumor
is a promising strategy to reduce the risks of metastasis [14–17], and could be involved in the
obesity-enhanced CRC. Previous studies have pathogenesis of colonic inflammation and CRC
shown that: [1] compared with normal colon tis- [18–21] (Fig. 11.1). Currently pharmaceutical
sues, the expression of sEH is increased in human companies and academic laboratories are t argeting
CRC samples [18, 23]; [2] pharmacological inhi- the CYP/sEH eicosanoid pathway to develop
bition or genetic ablation of sEH attenuates therapeutic drugs. Further understanding about
colonic inflammation and CRC [18–20]; and [3] the roles of the CYP/sEH eicosanoid pathway in
sEH is overexpressed in the liver and adipose tis- colonic inflammation and CRC could facilitate
Fig. 11.1 Biochemistry
of the CYP/sEH
eicosanoid pathway. The PLA2
metabolism of PUFAs
by cytochrome P450 COOH
COOH COOH
monooxygenases
(largely CYP2C and
CYP2J isoforms) leads LA ARA DHA
to formation of
epoxygenated fatty acids Cytochrome P450
(EpFAs), which are
further metabolized by COOH
COOH COOH
soluble epoxide
hydrolase (sEH) to form
O O O
the corresponding fatty
acid diols. Recent EpOMEs EETs EDPs
research shows that the
CYP and sEH enzymes
Soluble epoxide hydrolase
contribute to the
pathogenesis of colonic
inflammation and CRC COOH COOH COOH
HO OH HO OH
HO OH
DiHOMEs DHETs DiHDPEs
5. Chulada PC et al (2000) Genetic disruption of Ptgs-1,

the development of novel therapeutic strategies in
as well as Ptgs-2, reduces intestinal tumorigenesis in
the prevention or treatment of CRC. In addition, min mice. Cancer Res 60:4705–4708
our research supports that the omega-6-series 6. Ishikawa TO, Herschman HR (2010) Tumor forma-
EpFAs (e.g. EpOMEs) are associated with tion in a mouse model of colitis-associated colon
cancer does not require COX-1 or COX-2 expression.
increased inflammation and CRC [21], while the
Carcinogenesis 31:729–736
omega-3-series EpFAs (e.g. EDPs) are associated 7. Grosser T, Fries S, FitzGerald GA (2006) Biological
with decreased CRC [39] (Fig. 11.1), providing a basis for the cardiovascular consequences of COX-2
potential mechanism-based nutritional strategy to inhibition: therapeutic challenges and opportunities.
J Clin Invest 116:4–15
reduce the risks of CRC.
8. Wang Y et al (2018) Eicosanoid signaling in carcino-
genesis of colorectal cancer. Cancer Metastasis Rev
Acknowledgement This research is supported by USDA 37:257–267
NIFA grant 2016-67017-24423, USDA/Hatch grant 9. Zeldin DC (2001) Epoxygenase pathways of
MAS00492, and NIH/NCI R03CA218520 (to G.Z.). arachidonic acid metabolism. J Biol Chem
276:36059–36062
10. Zhang G, Kodani S, Hammock BD (2014) Stabilized
epoxygenated fatty acids regulate inflammation,
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Contributions
of 12/15-Lipoxygenase to Bleeding 12
in the Brain Following Ischemic
Stroke
Yi Zheng, Yu Liu, Hulya Karatas, Kazim Yigitkanli,

Theodore R. Holman, and Klaus van Leyen
Abstract implications for the use of 12/15-LOX inhibi-

Ischemic strokes are caused by one or more tors in the treatment of stroke.
blood clots that typically obstruct one of the
major arteries in the brain, but frequently also Keywords
result in leakage of the blood-brain barrier and Lipoxygenase · Eicosanoid · 12-HETE ·
subsequent hemorrhage. While it has long 15-HETE · Ischemic stroke · Hemorrhage ·
been known that the enzyme Hemorrhagic transformation · Ischemia ·
12/15- lipoxygenase (12/15-LOX) is up- Blood-brain barrier · Tissue plasminogen
regulated following ischemic strokes and con- activator · tPA · Neuroprotection · Warfarin ·
tributes to neuronal cell death, recent research STAT6
has shown an additional major role for 12/15-
LOX in causing this hemorrhagic transforma-
tion. These findings have important
Strokes are typically classified into two major
subtypes. Of these, ischemic strokes caused by
Y. Zheng · K. van Leyen (*) blockage of a major artery account for around
Neuroprotection Research Laboratories,
Massachusetts General Hospital, Harvard Medical 85% of cases, while the remainder are caused by
School, Charlestown, MA, USA hemorrhage which can be either intracerebral or
e-mail: [email protected] subarachnoid, depending on the location of the
Y. Liu vessel rupture. However, even among the isch-
Zhuhai Interventional Medical Center, Zhuhai emic strokes a substantial number go on to
Precision Medical Center, Zhuhai People’s Hospital include subsequent bleeding, leading to increased
of Jinan University, Zhuhai, Guangdong, China
brain injury. This hemorrhagic transformation
H. Karatas frequently occurs when tissue plasminogen acti-
Institute of Neurological Sciences and Psychiatry,
Hacettepe University, Ankara, Turkey vator (tPA) is used to lyse the obstructive blood
clot, contributing to catastrophically low usage of
K. Yigitkanli
Medicana Bursa Hospital, Neurosurgery Clinic, this potentially lifesaving therapy - only a minor
Bursa, Turkey percentage of ischemic stroke patients receive
T. R. Holman thrombolytic treatment. Mechanisms involving
Department of Chemistry and Biochemistry, the enzyme 12/15-lipoxygenase (12/15-LOX)
University of California at Santa Cruz, contribute to the hemorrhagic transformation of
Santa Cruz, CA, USA

126 Y. Zheng et al.
ischemic strokes both in the presence and absence infarct sizes (Sen group 90% reduction 72 h after
of tPA, as will be discussed in this mini-review. stroke onset, van Leyen group 40% infarct size
The liberation of polyunsaturated fatty acids reduction measured 24 h after initiation of exper-
including arachidonic acid in the brain following imental stroke). Moreover, subsequent studies
a stroke has been recognized since the early from our lab found that the mRNA encoding
1970s [1]. These give rise to a dizzying spectrum 12/15-LOX was up-regulated 2.2 fold in mice
of eicosanoids and related compounds produced 24 h after an experimental stroke [7].
by lipoxygenases, cyclooxygenases, and cyto- Immunohistochemistry showed the increased
chromes P450, including prostaglandins, leukot- 12/15-LOX signal both in neurons, and in endo-
rienes, and hydroxyeicosatetraenoic acids thelial cells [8]. Early work focused on injury to
(HETEs). Increased levels of 12-HETE were neurons, initially with the discovery that 12/15-
found along with leukotrienes C4 and D4 in ger- LOX contributes to a form of cell death termed
bil brains following an experimental stroke [2]. oxidative glutamate toxicity or oxytosis in neuro-
We have similarly seen massively increased lev- nal cells [9]. More recently, a related redox path-
els of 12-HETE specifically on the infarcted side way termed ferroptosis was introduced, which is
of the brain in mice both 12 and 24 h after onset characterized by the loss of glutathione peroxi-
of ischemia (Fig. 12.1). Therapeutically, initially dase 4 (Gpx-4) activity [10] and in which 12/15-
5-LOX was seen as the most promising target, LOX is also involved. The commonalities and
mostly due to a much better understanding of differences between these two pathways have yet
leukotriene biology compared to the much less to be clearly defined [10–12]. Glutathione as the
studied 12/15-LOX. However, two independent major intracellular antioxidant in neurons is
findings changed this perception: In 2004, a clearly important for both pathways, and gluta-
Japanese study found that Alox5 knockout mice thione levels also drop on the ischemic side of
developed the same level of ischemic injury fol- the brain following stroke, which presumably
lowing experimental stroke as matched wild type contributes to the activation of 12/15-LOX. The
mice [3]. Around the same time, the group of function of 12/15-LOX in this neuronal cell
Chandan Sen at Ohio State University [4] and death pathway is to damage mitochondria and
our group [5, 6] found that Alox15 knockout other organelles, for which the 12/15-LOX is
mice were protected against the consequences of uniquely qualified: in stress reticulocytes pro-
an experimental stroke, developing smaller duced during severe anemia, as well as when
Fig. 12.1 (a) 12-HETE was significantly increased in the identity of 12-HETE was confirmed by high-performance
infarcted ipsilateral hemisphere of mice both 12 and liquid chromatography (HPLC)/mass spectrometry analy-
24 hours after transient focal ischemia, compared to sis. The smaller peak for 15-HETE in the HPLC profile
sham-operated mice (∗p < 0.05, ∗∗∗p < 0.001; sham, n = 6 (top panel) is also a 12/15-LOX product. (Reprinted with
brains; 12 h, n = 3 brains; 24 h, n = 5 brains). (b) The permission from reference Rai et al. [24])
12 Contributions of 12/15-Lipoxygenase to Bleeding in the Brain Following Ischemic Stroke 127
incubated in vitro, the enzyme attacks mitochon- anticoagulant warfarin [22, 23]. Warfarin is a
drial membranes [13–16], priming the mitochon- vitamin K inhibitor that is frequently given to
dria for further degradation via the ubiquitin/ patients with atrial fibrillation to reduce their risk
proteasome system. This is one of three path- of blood clot formation and subsequent stroke.
ways by which reticulocytes lose their organelles While warfarin reduces the risk of stroke in these
during maturation to become functioning eryth- patients, this anticoagulant can cause excessive
rocytes, the others being degradation via autoph- bleeding leading to increased injury when a
agic vacuoles and exosome formation [17, 18]. stroke does occur. Moreover, thrombolysis with
The redundancy of these pathways may be the the clotbuster tissue plasminogen activator (tPA)
reason why no outright defects in erythropoiesis is contraindicated in effectively anticoagulated
were found in Alox15 knockout mice [19]. patients on warfarin (international normalized
Beyond causing cell death in neurons, how- ratio of coagulation time (INR) > 1.7) because
ever, it has in recent years become clear that tPA itself has bleeding as a side effect, thus elimi-
12/15-LOX also contributes to vessel injury in nating the only drug currently approved by the
rodent models of stroke. Alox15 knockout mice FDA as a treatment option. Mice pretreated with
develop 51% less edema following experimental warfarin via their drinking water for 24 h prior to
stroke than their wild type counterparts, and 30% experimental stroke develop severe hemorrhage
less immunoglobulin G (representative of blood both when the mice receive a tPA infusion fol-
proteins) extravasates into the brain parenchyma lowing removal of the filament, or in the absence
[8]. Several years later, we made the striking of tPA when the stroke is severe enough (3 h of
observation in a mouse model of thrombotic filament occlusion; Fig. 12.2) [23]. Along with
stroke that tPA infusion intended to lyse the the increased hemorrhage, we also found 25%
occluding thrombus led to massive brain hemor- higher levels of 12/15-LOX in the brains of the
rhage in these mice, which was reduced by 82% warfarin-treated mice. The increase was seen
through simultaneous administration of a 12/15- mostly in the vasculature, consistent with the
LOX inhibitor, LOXBlock-1 [20] (Fig. 12.2). We idea that the increased vessel leakage following
have expanded on these results by investigating warfarin pretreatment is due to 12/15-LOX.
the effects of 12/15-LOX inhibition on both Consistent with the idea of 12/15-LOX as con-
bleeding and infarct size in this thrombosis tributor to hemorrhage, 41% less bleeding was
model, and found that LOXBlock-1 also seen following warfarin pretreatment in Alox15
improved behavioral scores in the mice [21]. knockout mice [23]. In wild type mice, a simi-
This finding led us to systematically study larly drastic reduction in hemorrhage by 38%
mouse models of stroke where reperfusion fol- was detected when the mice were treated with the
lowing the ischemic event is associated with second generation 12/15-LOX inhibitor ML351
increased bleeding. In the classical filament [24], administered intraperitoneally at the time of
model of transient focal ischemia, a filament is reperfusion, 3 hours after onset of ischemia. The
inserted into the internal carotid artery to par- reduction in bleeding remained significant even
tially block the middle cerebral artery on one side when the results were adjusted to account for the
of the brain, which leads to a reduction of blood reduced infarct size in the ML351-treated mice,
flow and ischemia in the striatum and cortex. The confirming that there is a specific effect on hem-
filament is then removed after a pre-specified orrhage. When ML351 was administered along
time to allow for reperfusion. When the mouse is with tPA in warfarin pretreated mice, hemorrhage
sacrificed after 24 h an infarct is detected, the size was similarly reduced by 59%. Taken together,
of which is determined by the duration of the these results demonstrated that increased 12/15-
ischemia. Typically, this model does not lead to LOX in the brain vasculature can contribute to
significant bleeding, but so called hemorrhagic excessive bleeding in the brain, which is reduced
transformation of the ischemic stroke can be by treatment with a 12/15-LOX inhibitor.
induced, for example when mice are fed with the
128 Y. Zheng et al.
Fig. 12.2 Examples of stroke models associated with 24 h with the anticoagulant warfarin added to the drinking
increased hemorrhage. (a) Thrombosis was induced when water causes massive hemorrhage following a severe form
10% ferric chloride solution was topically applied to the of experimental ischemic stroke with 3 h of occlusion of
brain. An infusion of tissue plasminogen activator (tPA) the middle cerebral artery. This is again visible both on the
2 h later led to distinct hemorrhage in vehicle-treated mice surface, as well as in brain sections. Mice treated with the
after 24 h, visible both on the surface of the brain (top) 12/15-LOX inhibitor ML351 (50 mg/kg) develop far less
and in sections (below). In contrast, when mice were hemorrhage (bottom right). Quantitation graphs represent
intraperitoneally injected with the 12/15-LOX inhibitor hemorrhage area measured in brain sections and are
LOXBlock-1 (50 mg/kg), significantly less hemorrhage reprinted with permission from references [24] (top) and
was detected in the brain. (b) Pretreatment of mice for [23] (bottom)
Much work remains to be done to elucidate STAT6 are involved in up-regulating 12/15-LOX
the mechanism by which 12/15-LOX contributes under ischemic conditions [7]. Are the same
to increased hemorrhage after an ischemic stroke. STATs active here, or is a different form of regu-
Important open questions include the selective lation relevant? Finally, what happens after
up-regulation of 12/15-LOX in brain vascular 12/15-LOX is up-regulated in the endothelial
endothelial cells following warfarin administra- cells and how does this lead to vessel rupture? In
tion, both with and without subsequent tPA infu- addition to destroying endothelial cells of the
sion. Is this a direct effect of warfarin and/or tPA, brain vasculature by damaging mitochondria,
or are intermediate factors involved? Also, in there may be a second injury mechanism induced
neurons signal transducers and activators of tran- by the signaling function of 12/15-LOX via
scription (STATs), specifically STAT1 and metabolites of arachidonic acid. Both 12-HETE
and its immediate precursor 12-HPETE are we found increased 12/15-LOX in the brains of
known as second messengers [25], activated mice 24 h after hemorrhage induction (92 ± 60
along the semaphorin pathway [26, 27]. In neu- 12/15-LOX positive cells/field vs. 2 ± 2 in sham-
rons, this can lead to axon retraction [28, 29], but operated controls, p < 0.05) [38]. In this case,
under some conditions also to cell death [30]. 12/15-LOX expression was detected mostly in
Semaphorin 3A has also been reported to increase macrophages however, rather than in neurons and
vascular permeability in experimental stroke endothelial cells; the injury mechanism may thus
models [31]. Downstream of 12-HETE and differ from that in ischemic stroke. Regardless,
12-HPETE, secretion of destabilizing matrix Alox15 knockout mice developed 72% less
metalloproteinases (MMPs) may play a role [32]. injury than wild type mice, and 12/15-LOX inhi-
Both the molecular details of this signaling path- bition also reduced injury by 55% compared to
way, and the relative contributions of both path- vehicle-treated mice in this model of hemor-
ways to vascular injury require further study. rhagic stroke.
Because 12/15-LOX contributes to both neu- In conclusion, despite different triggers – in
ronal cell death and to vessel leakage following a the presence or absence of anticoagulant, with or
stroke, 12/15-LOX inhibition appears to be a par- without tPA treatment – 12/15-LOX is activated
ticularly promising approach to stroke therapy by in various models of stroke-related hemorrhage.
targeting two separate modes of injury, killing In addition to its benefits in infarct size reduction,
two birds with one stone. Both our group [20, 23, 12/15-LOX inhibition may thus independently
24, 33, 34] and others [35, 36] have in recent reduce hemorrhagic conversion of ischemic
years focused on developing improved inhibitors strokes by protecting the vasculature.
of 12/15-LOX, and it will be exciting to see if
these novel molecules can turn the tide in the Acknowledgments Funding from the National Institutes
seemingly endless war to combat stroke. The of Health (R01 NS049430, UG3 NS106854-01 and R21
NS087165 to KvL) and the American Heart Association
finding that we can reduce bleeding subsequent (17GRNT33460100 to KvL) is gratefully acknowledged.
to an ischemic stroke in the rodent model broad-
ens the spectrum of patients that could be treated
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Systematic Understanding
of Bioactive Lipids 13
in Neuro-Immune Interactions:
Lessons from an Animal Model
of Multiple Sclerosis
Yasuyuki Kihara
Abstract lysophosphatidic acid, and S1P) in MS and

Bioactive lipids, or lipid mediators, are uti- the animal model, experimental autoimmune
lized for intercellular communications. They encephalomyelitis (EAE). Genetic ablation,
are rapidly produced in response to various along with pharmacological inhibition, of
stimuli and exported to extracellular spaces lipid metabolic enzymes and lipid GPCRs
followed by binding to cell surface G protein- revealed that each bioactive lipid has a
coupled receptors (GPCRs) or nuclear recep- unique role in regulating immune and neural
tors. Many drugs targeting lipid signaling functions, including helper T cell (TH1 and
such as non-steroidal anti-inflammatory TH17) differentiation and proliferation,
drugs (NSAIDs), prostaglandins, and antago- immune cell migration, astrocyte responses,
nists for lipid GPCRs are in use. For exam- endothelium function, and microglial phago-
ple, the sphingolipid analog, fingolimod cytosis. A systematic understanding of bio-
(also known as FTY720), was the first oral active lipids in MS and EAE dredges up
disease-modifying therapy (DMT) for information about understudied lipid signal-
relapsing-remitting multiple sclerosis (MS), ing pathways, which should be clarified in
whose mechanisms of action (MOA) includes the near future to better understand MS
sequestration of pathogenic lymphocytes pathology and to develop novel DMTs.
into secondary lymphoid organs, as well as
astrocytic modulation, via down-regulation Keywords
of the sphingosine 1-phosphate (S1P) recep- Neuroimmunology · Autoimmunity ·
tor, S1P1, by in vivo-phosphorylated fingoli- Neuroinflammation · Neurodegeneration ·
mod. Though the cause of MS is still under Demyelination · Inflammation · Microglia ·
debate, MS is considered to be an autoim- Astrocyte · Blood-brain barrier · Eicosanoid ·
mune demyelinating and neurodegenerative PLA2 · PGE2 · LTB4 · PAF · LPA
disease. This review summarizes the involve-
ment of bioactive lipids (prostaglandins, leu-
kotrienes, platelet-
activating factors,
Y. Kihara (*)
Sanford Burnham Prebys Medical Discovery
Institute, La Jolla, CA, USA
e-mail: kihara-[email protected]

134 Y. Kihara
13.1 Neuro-immune Interactions ing difficulties, numbness/tingling, vision prob-

and Their Failures lems, and dizziness/vertigo. Surprisingly, many
of these symptoms were recorded in documents
The central nervous system (CNS) is separated about St. Lidwina of Schiedam (1380–1433),
from the peripheral immune system by the which is thought to be the oldest depiction of MS
blood-brain barrier (BBB) and it is also immu- [14].
nologically inactive which provides immune Based on the disease course, MS is classified
privilege [1] despite the existence of autoim- into four types: (1) clinically isolated syndrome
mune demyelinating diseases such as multiple (CIS), (2) relapsing-remitting MS (RRMS), (3)
sclerosis (MS). Recent studies clearly show the secondary progressive MS (SPMS), and (4) pri-
communication pathways between the CNS vs. mary progressive MS (PPMS) (nationalmssoci-
peripheral immune system through a lymphatic ety.org). Currently, about 2.3 million people live
system in the meninges [2] and tunnels connect- with MS around the world, with a higher preva-
ing the brain and the skull [3]. Furthermore, an lence in North America and Europe (>100/100,000
animal model of MS proposed that pathogenic T inhabitants) compared to Asia and Africa
cells penetrate into the CNS via the fifth lumber (2/100,000 population) [15]. MS affects more
spinal cord [4]. Failure of homeostatic neuro- women than men (3~4:1 ratio) (nationalmssoci-
immune interactions exerts pernicious effects on ety.org). Although involvement of genetic and
the CNS, resulting in the development of several environmental factors in MS pathogenesis have
diseases including MS [4], Alzheimer’s disease been proposed, the cause of MS is still under
[5], and social behavior impairment [6]. In this debate. By 2018, the Food and Drug
section, MS and its animal model, experimental Administration (FDA) has approved 16 medica-
autoimmune encephalomyelitis (EAE), is tions as disease-modifying therapies (DMTs).
introduced. However, daclizumab, an anti-CD25 monoclonal
antibody (Ab), was withdrawn from the market
worldwide in March 2018 [16]. Additional details
13.1.1 Multiple Sclerosis and updated information about MS and its treat-
ment can be found on the National Multiple
Publications in the 1830s described a disease Sclerosis Society’s website (nationalmssociety.
exhibiting multiple, irregular foci of discolor- org). Since MS is traditionally considered to be
ation and shrinkage throughout neuroaxis [7]. In an autoimmune disorder, immunomodulation is
1868, a French neurologist, Jean-Martin Charcot, the mechanism of action (MOA) of most
gathered prior clinical depictions and framed the MS-drugs such as natalizumab (anti-VLA4 Ab)
disease as sclérose en plaques (multiple sclerosis [17], alemtzumab (anti-CD52 Ab) [18], ocreli-
in French) [8]. The pathologic hallmarks of MS zumab (anti-CD20 Ab) [19], interferon β (IFN-β)
are myelin loss (demyelination), accompanied by [20], fingolimod [21, 22], and dimethyl fumarate
inflammation within the CNS, which are called (DMF) [23, 24]. However, a growing body of lit-
plaques or lesions. Introduction of magnetic reso- erature supports that some MS drugs, such as fin-
nance imaging (MRI) in 1981 [9] conquered the golimod [25], IFN-β [26], and DMF [27], exhibit
chaos in diagnosing MS, understanding MS direct effects in the CNS. Exploring and develop-
pathogenesis, and evaluation of treatment. The ing CNS-targeted MS-drugs is meaningful for
current standard of MS diagnosis, the McDonald finding a cure for progressive forms of MS that
criteria [10, 11], uses MRI to identify CNS dam- show more neurodegenerative features than
age as seen over time and location. In 1983, John RRMS. Neuro-immune interactions underlying
F. Kurtzke developed the Expanded Disability MS are far more complex than previously under-
Status Scale (EDSS) [12, 13] that quantify the stood, and thus further exploration of the com-
disabilities in MS patients whose symptoms are munications between the immune and nervous
variable and numerous including fatigue, walk- systems is highly desirable.
13 Systematic Understanding of Bioactive Lipids in Neuro-Immune Interactions: Lessons from an Animal… 135
13.1.2 Experimental Autoimmune since NOD mice develop spontaneous diabetes

Encephalomyelitis at a higher rate.
Failure of neuro-immune communication
A breakthrough in understanding MS pathogen- results in T cells attacking CNS elements, par-
esis and MS drug development was the establish- ticularly oligodendrocytes, which causes demye-
ment of an animal model of MS, now called lination and neurodegeneration. Although
experimental autoimmune (or allergic) encepha- immunological aspects of these diseases have
lomyelitis (EAE), by Thomas Rivers in 1933 been well-studied, it remained unknown whether
[28]. The study provided the first evidence of CNS cells are actively involved in the disease. By
immune cell-mediated CNS damage. using genetically engineered mice harboring a
Recapitulation of EAE by transfer of lymph node tetracycline-controlled c-Fos reporter (a green
cells [29] guided several important findings that fluorescent protein-histone H2B fusion protein
include EAE induction with T cells reactive under a tetO promoter controlled by a c-Fos
against myelin basic protein (MBP) [30] and def- inducible tetracycline transactivator), we identi-
inition of encephalitogenic epitopes of MBP fied that astrocytes were activated by EAE insults
[31]. Further investigation revealed CD4+ T at the initial phase of disease course [41]. We
helper type 1 (TH1) cells recognize named the astrocytes that experienced c-Fos acti-
oligodendrocyte- specific proteins or peptide vation during EAE as immediate-early astrocytes
fragments (such as MBP; proteolipid protein, (ieAstrocytes) [41]. The number of ieAstrocytes
PLP; myelin oligodendrocyte glycoprotein, increased linearly with EAE progression, indicat-
MOG) [32, 33] that are responsible for EAE ing that astrocytes are the key CNS cell type in
induction and progression. In 2005, interleukin- EAE pathogenesis [41]. Although there are sev-
23 (IL-23)-derived IL-17 producing encephalito- eral differences between MS and EAE, both dis-
genic T cells, which are now known as TH17 eases share the pathological, clinical features,
cells, were found to play key roles in CNS auto- which justify EAE as an MS model to study
immunity [34]. neuro-immune interactions. In the following sec-
EAE susceptibility varies in mouse strains tions, the roles of bioactive lipids in neuro-
and may be explained by the major histocom- immune interactions from EAE studies that used
patibility complex H2 haplotypes [35]. The knockout (KO) mice or pharmacological tools
most commonly used mouse strains in the sci- are summarized.
entific field are TH1-prone C57BL/6 (H2b) and
CD4+ T helper type 2 (TH2)-prone Balb/c (H2d)
mice. Both active immunization with MOG 13.2 A
rachidonic Acid Cascade
peptide (MOG35-55) and passive immunization in EAE and MS
by adoptive transfer of encephalitogenic T cells
in C57BL/6 mice display monophasic disease Arachidonic acid (AA) cascade is a pathway that
course [23, 36–39]. All the knockout studies produces eicosanoids from ω-6 polyunsaturated
introduced below used the C57BL/6 genetic fatty acid (PUFA), AA (5Z,8Z,11Z,14Z-
background unless otherwise stated. Balb/c eicosatetraenoic acid) [42]. Eicosanoid is the
mice are not susceptible to EAE, but some con- general term used for potent bioactive lipids that
troversial results are reported [40]. SJL mice have 20 (eicosa in Greek) carbon units in their
harboring H2s present a relapsing-remitting structure, which include prostaglandins (PGs),
disease course when immunized with PLP pep- thromboxanes (TXs), leukotrienes (LTs),
tide (PLP139-151) [35]. Non-obese diabetic hydroxyl eicosatetraenoic acids (HETEs), and
(NOD) mice harboring H2g7 immunized with epoxyeicosatrienoic acids (EETs). Figure 13.1
MOG35-55 display progressive MS-like disease summarizes the relationship between AA cascade
signs [35]. This needs careful consideration and EAE/MS pathology.
136 Y. Kihara
Fig. 13.1 EAE phenotypes in AA cascade-KO mice
13.2.1 Phospholipase A2s (PLA2s) progression [44, 47–50]. These results suggest
that eicosanoids, which are produced down-
PLA2s including cytosolic PLA2s (cPLA2), stream of PLA2, should have unique roles in EAE
secreted PLA2s (sPLA2) and calcium-independent and MS.
PLA2s (iPLA2), hydrolyze the sn-2 position of
membrane glycerophospholipids to liberate fatty
acyls including AA [43]. Among them, group 13.2.2 Cyclooxygenases (COXs)
IVA cPLA2 (cPLA2α; gene name, Pla2g4a) is a
key enzyme to produce eicosanoids, since its COXs (COX-1 and COX-2; gene names, Ptgs1
deficiency lacks the ability of producing eico- and Ptgs2, respectively) insert two molecular
sanoids, resulting in resistance to various disease oxygen into AA to produce an unstable endoper-
models [43]. Several lines of evidence, such as oxide intermediate, PGH2, which in turn metabo-
(1) up-regulation of Ca2+-dependent PLA2 activ- lizes to PGD2, PGE2, PGF2α, PGI2, and TXA2 by
ity and PLA2 mRNAs (cPLA2α, cPLA2β, cPLA2ζ, isomerases and terminal synthases [51].
and sPLA2-V) in EAE spinal cords (SCs) [38], Acetylsalicylic acid (Aspirin®) is the first non-
(2) cPLA2α expression in brain endothelium and steroidal anti-inflammatory drug (NSAID) syn-
infiltrated immune cells in EAE mice [44], and thesized by Bayer in 1897 [52]. The MOA of
(3) increased sPLA2 activity in both MS and EAE aspirin is suppression of prostaglandin synthesis
urine [45], suggest active involvement of these by inhibiting COXs, which was revealed by John
enzymes in EAE. Using cPLA2α-KO mice [46], Vane in 1971 [53].
Marusic et al. revealed the requirement of both There were no beneficial effects of aspirin in
peripheral and neuronal cPLA2α in EAE develop- guinea pig EAE [54, 55], while a high dose
ment and progression [47]. No other PLA2-KO sodium salicylate, a major metabolite of aspirin,
mice have been tested for EAE. Pharmacological showed efficacy in guinea pig EAE [56]. Two
inhibition of PLA2s (Pan inhibitors, AACOCF3 clinical trials in the early 1960’s examined aspi-
and FKGK2; cPLA2α inhibitor, WAY-196025; rin’s efficacy in MS failed [57, 58]. However,
sPLA2 inhibitor, CHEC-9; and iPLA2 inhibitor, mouse EAE studies tested other NSAIDs includ-
FKGK11) also blocked EAE development and ing indomethacin and naproxen (non-selective
COX inhibitors) that clearly demonstrated the lesions [37]. mPGES-1 deficiency almost com-
involvement of COXs in EAE [50, 59]. Miyamoto pletely inhibited PGE2 production in EAE SCs,
et al. reported that COX-2-KO mice showed while doubling PGI2 production. mPEGS-1-KO
equivalent disease course with controls [59]. The mice showed EAE amelioration and impairment
study also demonstrated that a COX-2 selective of TH1/TH17 differentiation [37]. Later, another
inhibitor, celecoxib, prevented EAE in mice, group reported essentially the same results using
while COX-2-KO mice were not rescued by cele- the same mPGES-1-KO mice. They proposed
coxib. Thus, the MOA of celecoxib in EAE sup- that mPGES-1 in vascular endothelial cells con-
pression is independent from COX-2 inhibition trols IL-1β signaling in both CD4+ T cells and
[59]. Recently, the efficacy of low-dose aspirin (1endothelial cells, resulting in EAE exacerbation
~ 3 mg/kg), which is commonly used for lower- [65–67]. EAE phenomics using 8 PG receptor
ing cardiovascular risks [60], on the EAE pheno- KO mice was reported [68], which identified a
type was reported [61]. The study revealed that significant suppression of EAE only in PGE2
low-dose aspirin prevented loss of regulatory T receptor 4 (EP4; gene name, Ptger4)-KO mice on
cells (Treg) by stimulating IL-11 production, and the mixed background of C57BL/6 and 129/Ola.
also suppressed TH1/TH17 d ifferentiation, result- EAE phenotypes in other PGE2 receptors (EP1,
ing in EAE amelioration [61]. However, since EP2, and EP3) were equivalent to their matching
this MOA of low-dose aspirin is not tested in controls [68]. Prophylactic administration of the
COX-1/2-KO mice and eicosanoid levels were EP4 antagonist (ONO-AE3-208) prevented mice
not quantified, it remains unclear if the efficacy from developing EAE, while the therapeutic par-
of low-dose aspirin is related to COX-1/2 inhibi- adigm showed little effect on EAE development
tion. Considering the results from cPLA2α-KO and severity [68]. The efficacy of ONO-AE3-208
and COX-2-KO studies, COX-1 may play some was greater in EP2-KO mice than wild-type (WT)
role in EAE, but EAE has not been tested in mice, suggesting a redundancy of EP4 and EP2
COX-1-KO mice to date. [68]. Indeed, PGE2 facilitated TH1 differentiation
and IL-23-induced TH17 expansion through both
EP2 and EP4 [69]. These results clearly demon-
13.2.3 Prostaglandins (PGs) strate that the mPGES-1-PGE2-EP2/4 axis is an
exacerbating pathway in EAE and MS.
PGs are produced downstream of COX-1/2 and Although the EAE phenomics study con-
are functionally coupled with terminal PG syn- cluded no aggravation or facilitation of EAE in
thases to produce each PG efficiently [62–64], any PG receptor KO mice except for EP4-KO
followed by binding to their specific receptors (a mice, the clinical score of PGI2 receptor (IP; gene
member of the superfamily of G-protein coupled name, Ptgir)-KO mice appeared to show a right
receptors (GPCRs)), to elicit a variety of biologi- shift (delayed onset and/or milder EAE) as com-
cal responses [42]. pared to controls [68]. Indeed, another group
We applied targeted lipidomics using liquid reported a similar trend with significant differ-
chromatography tandem mass spectrometry (LC- ences between IP-KO mice vs. controls [70].
MS/MS) in combination with transcriptomics to They also demonstrated that PGI2-IP signaling
identify a key eicosanoid pathway that is involved induces IL-17A production in naïve CD4+ T cells
in EAE [37]. Lipidomics identified a metabolic and promotes TH17 differentiation [70]. The
shift from the PGD2 pathway in naïve spinal decreased PGI2 in EAE SCs could be explained
cords (SCs) to the PGE2 pathway in EAE SCs by down-regulation of PGI synthase (PGIS; gene
[37]. Among the three PGE2 synthases, micro- name, Ptgis) mRNA that is abundant in endothe-
somal PGE synthase 1 (mPGES-1; gene name, lial cells [37], suggesting a direct neuroprotective
Ptges) mRNA was uniquely up-regulated in EAE role of PGI2 in the CNS. Muramatsu et al. reported
SCs. mPGES-1 protein was expressed in that knockdown of PGIS and IP by siRNA deliv-
microglia/macrophages in both EAE and MS ered directly into the CNS, delayed recovery
138 Y. Kihara
from motor dysfunction associated with EAE 13.2.4 Lipoxygenases (LOs)

[71]. Furthermore, PGI2 promoted neurite elon-
gation in vitro. Systemic administration of an IP LOs (5-LO, 8-LO, and 12/15-LO; gene names,
agonist, iloprost, improved motor deficits in EAE, Alox5, Alox8, and Alox12, respectively) insert
indicating that vascular endothelial cell-derived one molecule of molecular oxygen into the 5, 8,
PGI2 promotes neuronal rewiring in EAE via the 12, and 15 positions of AA, to produce hydroper-
PGIS-PGI2-IP pathway [71]. Collectively, PGI2 oxy eicosatetraenoic acids (HpETE) [80, 81].
shows anti-immune and neuroprotective effects. 5-LO catalyzes the two-step conversion of AA to
PGD2 is the most abundant eicosanoid in the LTA4 that are metabolized to LTs and HETEs by
CNS [72]. The decreased PGD2 in EAE SCs enzymatic and non-enzymatic reactions, respec-
might be associated with reductions in lipocalin- tively [80]. LTs elicit their biological actions by
type PGD synthase (L-PGDS; gene name, Ptgds) binding to their specific GPCRs [42]. In addition
mRNA that is highly expressed in oligodendro- to LTs, LOs also produce specialized pro-
cytes (OLs) [37]. L-PGDS deficiency in a genetic resolving mediators (SPMs) including
demyelinating mouse model (twicher mice AA-derived lipoxins, docosahexaenoic acid
harboring a homozygote for a nonsense point (DHA) and eicosapentaenoic acid (EPA)-drived
mutation of the galactosylceramidase) showed resolvins (RvDs and RvEs, respectively), and
increases of apoptotic neurons and OLs [73]. DHA-derived neuroprotectin (NPD1) [82–84].
Moreover, increased L-PGDS expression was Gene expression profiling in MS lesions and
reported in remyelinated lesions of MS brains EAE brains identified an up-regulation of the
[76], indicating an anti-apoptotic and pro- 5-LO gene [85]. 5-LO-KO mice on a 129
remyelinating property of the L-PGDS-PGD2 genetic background showed severe disease as
pathway in OLs. No difference in EAE signs compared to controls on the 129S1/SvImJ back-
between PGD2 receptor (DP1; gene name, ground [86]. 12/15-LO-KO mice on a mixed
Ptgdr1)-KO mice vs. controls was reported [68], B6:129S2 background also showed severe dis-
while DP1-KO mice appeared to show a slightly ease as compared to controls on a C57BL/6 J
severer disease course than controls. This requires background [86]. These severe phenotypes may
a more careful evaluation of the EAE phenotype be accounted for by a metabolic shift towards
in DP1-KO mice. Moreover, little is known about the COX-PG pathway in 5-LO-KO mice, and/or
the roles of L-PGDS, hematopoietic-type PGD by a reduction of SPMs in 12/15-LO-KO mice.
synthase (H-PGDS; gene name, Ptgds2), and However, the mismatched genetic backgrounds
another PGD2 receptor (DP2/CRTH2; gene name, between KO vs. controls make it difficult to
Ptgdr2) in EAE. conclude roles of these LOs. On the other hand,
Although PGD2 levels decreased in EAE SCs, the FDA-approved anti-asthma drug, zileuton,
one PGD2 metabolite, 15-deoxy-Δ12,14-PGJ2 which is an 5-LO selective inhibitor, effectively
(15d-PGJ2), was substantially increased in EAE delayed EAE onset and ameliorated EAE [49],
SCs [37]. 15d-PGJ2 binds selectively to one of suggesting that LTs produced by 5-LO may
three peroxisome proliferator-activated recep- worsen EAE.
tors, PPARγ (gene name, Pparg), which inhibits
pro-inflammatory cytokine production [75]. Both
PPARγ-hetero [76, 77] and PPARγ antagonist 13.2.5 Leukotrienes (LTs)
treated mice developed severe EAE [77].
Furthermore, prophylactic and therapeutic LTs are produced downstream of 5-LO. LTA4
administration of 15d-PGJ2 [78], as well as pro- hydrolase (LTA4H; gene name, Lta4h) metabo-
phylactic treatment of a PPARγ agonist (trogli- lizes LTA4 to LTB4 [87]. Cysteinyl LTs (cys-LTs)
tazone) [79], ameliorated EAE [78], indicating are produced by LTC4 synthase (LTC4S; gene
that 15d-PGJ2 production in EAE SCs might be a name, Ltc4s) that conjugates reduced glutathione
defensive reaction as an innate response. to LTA4, resulting in LTC4 formation that is
metabolized to LTD4 followed by LTE4 [88, 89]. trial using ginkgolide B, a PAFR antagonist
Levels of these 5-LO metabolites in naïve SCs extracted from Ginkgo biloba, showed no effi-
were very low as compared to COX metabolites, cacy for the treatment of acute exacerbations of
which were further declined in EAE SCs [37]. MS [95], several lines of evidence support an
Therefore, LTs may not play pivotal roles in involvement of PAF in MS/EAE to certain
EAE. However, mRNAs of the LTB4 receptor 1 extent, including (1) elevated PAF levels in cere-
(BLT1; gene name, Ltb4r1) and the cys-LT recep- brospinal fluids (CSF) and plasma of RRMS
tor 1 (CysLT1; gene name, Cysltr1) were highly patients [96], (2) up-regulation of PAFR mRNA
up-regulated in EAE SCs from disease onset in chronic MS plaques [97], (3) a relationship
through the acute phase [37, 39], implying accu- between PAFR gene missense mutation vs. the
mulation of immune cells expressing these recep- susceptibility for MS [98], and (4) an efficacy of
tors into the CNS. The BLT1 antagonist, PAFR antagonist (BN52021) on EAE [99]. We
CP-105,696, prevented EAE and suppressed found increased activity of PAF producing
eosinophil infiltration into the CNS in a enzymes (PLA2 and LysoPAFAT) and unchanged
dose-dependent manner [90]. We reported that PAFAH activity in EAE SCs as compared to
BLT1-KO mice showed delayed onset, less severe controls, resulting in a significant elevation of
EAE, and reduced TH1/TH17 responses than con- PAF levels [36, 38]. We also tested EAE in
trols [39]. Moreover, BLT1-KO SCs from asymp- PAFR-KO mice that showed a lower incidence
tomatic EAE mice showed no T cell, neutrophil, and less severe disease than controls [36]. PAF-
or macrophage infiltrations, whereas those cells PAFR signaling promoted cytokine and chemo-
were found in asymptomatic WT EAE controls kine production in SCs before disease onset [36].
[39]. These results suggest that BLT1 is respon- In addition, PAF accelerated phagocytotic activ-
sible for immune cell recruitment into the CNS ity and its associated TNF-α production in mac-
in the early phase of the disease. EAE studies rophages via PAFR [36]. Another group
using KO mice of LTA4H, LTC4S, BLT2 (gene independently confirmed these results using the
name, Ltb4r2), CysLT1, 2, 3 (gene name, Cysltr1, same PAFR-KO mice [100]. These results sug-
Cysltr2, Cyltr3/Oxgr1/Gpr99) are not available gest a dual role of PAF-PAFR signaling in EAE
at this time. development and progression.
13.3 Platelet-Activating Factor 13.4 Lysophospholipid (LP)

(PAF) in EAE and MS Mediators in EAE and MS
PAF (1-O-alkyl-2-acetyl-sn-glycero-3-phospho LP is a derivative of a phospholipid in which one

choline) is a potent lipid mediator that activates acyl chain is missing from the glycerol backbone
immune cells and induces vascular hyperperme- combined with a phosphate head group, such as
ability, hypotension, and bronchoconstriction lysophosphatidic acid (LPA, 1 or 2-acyl-sn-
via its specific GPCR, PAF receptor (PAFR; glycero-3-phosphate), lysophosphatidylserine
gene name, Ptafr) [91, 92]. PAF production (LysoPS), lysophosphatidylinositol (LPI), and
through a remodeling pathway requires mem- lysophosphatidylcholine (LPC) [101]. Since
brane phospholipid hydrolysis by PLA2s that LIPID MAPS® classifies sphingosine 1-phosphate
supply lyso-PAF (1-O-alkyl-sn-glycero-3- (S1P) in the sphingolipid group [102], it is tech-
phosphocholine), which in turn metabolizes to nically not a lysophospholipid. However, S1P is
PAF by acetyl-CoA:lyso-PAF acetyltransferase included here based on the IUPAR (International
(LysoPAFAT; gene name, Lpcat2) [93]. PAF is Union of Basic and Clinical Pharmacology)
rapidly degraded to lyso-PAF by PAF acetylhy- GPCR classification [103]. Figure 13.2 summa-
drolase (PAF-AH; gene name, Pafah1b1, rizes the relationship between LPs and EAE/MS
Pafah1b2, Pafah1b3) [94]. Although a clinical pathology.
140 Y. Kihara
Fig. 13.2 EAE phenotypes in LP signaling-KO mice. (a) LPA pathway. (b) S1P pathway
13.4.1 Lysophosphatidic Acid (LPA) carrying TCR specific for MOG92-106 in the con-
text of I-As in SJL/J genetic background; TCR1640
LPA is produced by three distinct pathways: (1) mice [115]), unsaturated LPAs in plasma and SCs
de novo synthesis from glycerol-3-phosphate were highly elevated during the first remission,
(G3P) by G3P acyltransferase (GPAT; gene followed by suppression in the second relapse
names, Gpam, Gpat2, Gpat3 and Gpat4) [93, [114]. Receptor expression profiles in spleen
104], (2) head group removal from LPs by auto- cells of EAE mice identified a suppression of
taxin (ATX; gene name, Enpp2) [105, 106], and LPA2 protein expression on CD4+ T cells and
(3) hydrolysis of phosphatidic acid (PA) by myeloid cells [114]. LPA1 mRNA in spleen, LPA2
PA-specific PLA1 (PA-PLA1α; gene name, Liph) and LPA3 mRNA in white blood cells, and LPA2,
[105, 107]. LPA levels in blood (0.1 μM in plasma LPA3, LPA4 and LPA5 mRNA in SCs were up-
~ 10 μM in serum) are of sufficiently high con- regulated in EAE mice [114]. In LPA2-KO mice
centration to activate six cognate GPCRs (LPA1-6; on a C57BL6 x Sv129 mixed background, about
gene names, Lpar1-6) [101, 108–111], in which 25% developed EAE with increased T cell and
LPA1 dissociation constants (Kd) for LPA approx- myeloid cell infiltration into the SCs, while their
imated subnanomolar range [112]. Although LPA matching WT controls showed very mild disease
was isolated and identified from soy beans in course because Sv129 mice are resistant to EAE
1978 [113], it has not been well-studied in MS [114]. LPA2 agonist, GRI-977,143, effectively
and EAE until recently. In 2017, Schmitz et al. ameliorated EAE in SJL mice [114].
reported a meticulous investigation of the rela- Controversial, small cohort studies were also
tionship between LPA and MS and EAE [114]. reported that LPA levels increased in serum of
LPA concentrations quantified by LC-MS/MS RRMS patients, while they were determined by
were significantly reduced in the serum of MS ELISA or phosphate quantification [116, 117].
patients, regardless of disease type, and EAE ATX activity was elevated in the CSF and serum
mice on an SJL/J background as compared to of RRMS patients [118]. A naturally occurring
their controls [114]. Moreover, unsaturated LPA LPA analog, cyclic phosphatidic acid (cPA,
levels in serum were responsive to some DMTs 1-acyl-sn-glycero-2,3-cyclic phosphate), is
(fingolimod and natalizumab), which were also reported to activate LPA receptors [119].
observed in EAE mice on an SJL/J background Treatment using a metabolically stable analog of
[114]. In a spontaneous relapsing-remitting EAE cPA (2ccPA) in both a prophylactic and therapeu-
model (T cell receptor (TCR) transgenic mice tic manner ameliorated EAE and reduced inflam-
mation in the SCs [120], which may be mediated administration of fingolimod. Moreover, fingoli-
via LPA2. The EAE course of LPA4-KO mice was mod-P was identified as a high affinity agonist to
equivalent to controls (unpublished observation). four out of the five known S1P receptors (S1P1,
The EAE disease course in LPA1, LPA3, LPA5, S1P3, S1P4, and S1P5). Brinkman et al. also
and LPA6 deficiency, as well as LPA-producing reported the efficacy of fingolimod in EAE by a
and degrading enzyme deficiency, need to be prophylactic treatment paradigm [127]. In 2003,
studied to characterize their roles in EAE. Sphk2 was reported to show a greater effect of
fingolimod phosphorylation as compared to
Sphk1 [129]. In 2004, Jason Cyster’s group
13.4.2 Sphingosine 1-phosphate revised the MOA of fingolimod from agonism to
(S1P) functional antagonism based upon findings that
(1) S1P1 was required for lymphocyte egress
S1P is produced from sphingosine (2-amino-4- from lymphoid organs to circulation, and (2) fin-
octadecene-1,3-diol) by the action of sphingosine golimod treatment down-regulated S1P1 cell sur-
kinases (Sphks; gene name, Sphk1, Sphk2) [121]. face expression [130, 131]. These studies
S1P is enriched in blood plasma (low micromolar provided a basis for the biological significance of
range), where S1P binds to albumin (gene name, the S1P gradient in lymphocyte trafficking
Alb) and apolipoprotein M (ApoM; gene name, between circulatory and lymph systems, which
Apom) tethered to high-density lipoprotein are supported by other studies using S1P-
(HDL) at a ratio of approximately three to seven generating (Sphks) and -degrading (S1P lyase;
[122]. S1P levels in lymph and tissues are lower gene name, Sgpl1) enzyme KO mice [132, 133].
than in blood (nanomolar range), which gener- Additional studies provided more evidence of
ates an S1P gradient between the circulatory sys- S1P1 involvement in MS and EAE. Proteomics
tem and tissues [122]. The biological significance analysis on MS brain tissue identified S1P1 phos-
of this gradient was recognized about 10 years phorylation on a C-terminal serine residue that is
after the discovery of fingolimod (trade name, crucial for receptor internalization [134]. A
Gilenya™), originally called FTY720. knock-in mouse harboring alanine mutations on
In 1995, fingolimod was first synthesized S1P1 C-terminal serine residues (S1P1-S5A mice)
from an immunosuppressive natural product, [135] developed more severe EAE than controls
myriocin (ISP-I), which was isolated from cul- with an enhanced TH17 immunity. S1P1 floxed
ture broths of Isaria sinclairii [123–125]. Chiba mice (S1P1flox/flox) were used to study cell-specific
et al. reported that fingolimod strikingly reduced roles of S1P1 in EAE, since global S1P1-KO mice
the number of circulating lymphocytes [125], were embryonically lethal due to impairment of
while the molecular basis of fingolimod activity vascular maturation [136]. TH17-specific
remained unclear at that time. This immunosup- S1P1-KO mice (IL-17A-Cre: S1P1flox/flox mice)
pressive effect of fingolimod was clinically tested were completely resistant to EAE development
for preventing organ graft rejection, while phase [137]. On the other hand, Treg-specific S1P1-KO
III clinical trials did not support a superior effi- mice (Foxp3-Cre: S1P1flox/flox mice) developed
cacy of fingolimod in renal transplantation as systemic autoimmunity, since Treg cells were
compared to the prior standard treatment [126]. retained in lymphoid organs and reduced in
During 1998–2000, five S1P receptors (S1P1-5: peripheral tissues. Also, S1P1 deletion, as well as
gene names, S1pr1-5) that belong to rhodopsin fingolimod treatment, promoted to effector Treg
family GPCRs were identified [101, 108]. In cell conversion from central Treg cell. Tamoxifen-
2002, the MOA of fingolimod was independently inducible Treg-specific S1P1-KO mice (Foxp3-
proposed by two pharmaceutical companies, CreERT2: S1P1flox/flox mice) developed severe EAE
Merck and Novartis [127, 128]. Both groups [137]. The immunological roles of ApoM-bound
clearly demonstrated that phosphorylated fingoli- S1P were investigated using ApoM-KO mice,
mod (fingolimod-P) was generated in vivo after which revealed that ApoM-S1P suppresses
142 Y. Kihara
expansion of Lin−Sac-1+cKit+ hematopoietic pro- Knockout and antagonist studies targeting

genitor cells (LSKs) and common lymphoid pro- S1P3, S1P4, and S1P5 have not been reported, but
genitors (CLPs) through S1P1 [138]. This the EAE phenotype in S1P2-KO mice was
immune stimulation led to exacerbation of EAE reported [142, 143]. Microarray analysis search-
in mice lacking ApoM, while less severe EAE ing for sexually dimorphic genes in the CNS of
was observed in ApoM transgenic mice (driving naïve SJL mice identified higher S1P2 expression
with endogenous promoter). Although endothe- levels in female vs. male [142]. S1P2 expression
lium-specific S1P1-KO mice (Cdh5-Cre-ERT2: increased in brain endothelial cells of EAE-
S1P1flox/flox mice) showed pulmonary vascular induced female mice and female MS patients,
leakage, EAE disease course in these mice were which enhanced BBB permeability to
equivalent to their matching controls [138]. Na-Fluorescein [142]. Therapeutic treatment
Collectively, S1P1 signaling is responsible for with the S1P2 antagonist (JTE-013) ameliorated
homeostatic lymphocyte trafficking and lympho- EAE in SJL mice [142]. Furthermore, S1P2-KO
poiesis, whose functional antagonism by fingoli- mice on a C57BL/6 × 129S mixed background
mod reduces circulating pathogenic T cells, showed milder EAE course than controls [142].
resulting in EAE/MS amelioration. Using the same S1P2-KO mice, another group
Given the fact that fingolimod penetrated and also reported that S1P2 deficiency ameliorated
accumulated in the CNS [139] where S1P recep- EAE, increased the number of oligodendrocytes
tors are abundant (S1P1 in astrocytes and S1P5 in in EAE lesions, and decreased fibrinogen extrav-
oligodendrocytes) [36], direct CNS effects of fin- asation and macrophage infiltration [143].
golimod were expected. In 2011, Jerold Chun’s
group reported EAE amelioration and no fingoli-
mod efficacy in CNS-specific S1P1-KO mice 13.5 Conclusions
(Nestin-Cre: S1P1flox/flox mice) and astrocyte-
specific S1P1-KO mice (GFAP-Cre: S1P1flox/flox Collectively, this review provides an overview of
mice), while neuron-specific S1P1-KO mice lipid signaling pathways in MS and EAE. Each
(Synapsin-Cre: S1P1flox/flox mice) did not show bioactive lipid, lipid metabolic enzyme, and lipid
such phenotypes [140]. Most recently, we identi- GPCR play key roles in regulating immune and
fied a novel functional astrocyte, ieAstrocyte, in neural functions. One of the most important con-
EAE SCs, whose formation was significantly tributions to the lipid biology field in neurode-
blocked by fingolimod administration and S1P1 generative diseases was the development of
deficiency [41]. These results suggest that S1P1 fingolimod for the treatment of RRMS patients.
inhibition on astrocytes appears to be another Although most DMTs are immunomodulatory,
MOA of fingolimod, which seems to be desirable fingolimod shows dual regulatory roles in the
for the treatment of progressive type of MS immune and nervous systems. Considering the
(PPMS and SPMS). Development of S1P recep- neurodegenerative features of progressive forms
tor modulators in next generation compounds are of MS, not only immunomodulation, but also
designed to have a receptor subtype selectivity, neuroprotection and neuroregeneration, should
such as the S1P1/S1P5 specificity in siponimod, be explored. Unfortunately, we still have a lot to
ponesimod, and ozanimod, and S1P1 specificity learn about the roles of lipid mediators including
in KRP-203 and GSK2018682 [108]. Among (EETs, HETEs, SPMs, cannabinoids, and lyso-
these, the phase III clinical trial (EXPAND) dem- phospholipids), lipid metabolic enzymes (PLA2s,
onstrated that siponimod reduced the risk of dis- COX-1, PGDS, LysoPAFAT and other acyltrans-
ability progression in SPMS patients [141], ferases, diacylglycerol lipase, Sphks, and ATX),
which might be mediated through direct CNS lipid GPCRs (DP2, BLT2, CysLT1, 2, 3, cannabi-
action. Several reviews have been published that noid receptors, fatty acid receptors, LPA1, 3, 5, 6,
summarize direct CNS effect of fingolimod and S1P3-5, and other LP receptors), and more in EAE
S1P receptor modulators [21, 25, 108, 140]. and MS. Among them, several proteins (L-PGDS,
LPA1, S1P5, and GPR17) that are highly expressed 6. Filiano AJ, Xu Y, Tustison NJ, Marsh RL, Baker
W, Smirnov I, Overall CC, Gadani SP, Turner SD,
in oligodendrocytes and anti-inflammatory/pro- Weng Z, Peerzade SN, Chen H, Lee KS, Scott
resolving lipid mediators (EETs and SPMs), are MM, Beenhakker MP, Litvak V, Kipnis J (2016)
attractive neuroprotective targets to study in Unexpected role of interferon-gamma in regulating
EAE/MS. neuronal connectivity and social behaviour. Nature
535:425–429
7. Hickey WF (1999) The pathology of multiple scle-
Acknowledgements Thanks to Prof. Takao Shimizu and rosis: a historical perspective. J Neuroimmunol
all the members in his laboratory (The University of 98:37–44
Tokyo), and Prof. Takehiko Yokomizo (Juntendo 8. Murray TJ (2009) The history of multiple sclerosis:
University) and Prof. Satoshi Ishii (Akita University) for the changing frame of the disease over the centuries.
their support to complete my Ph.D. work, Profs. K. Frank J Neurol Sci 277(Suppl 1):S3–S8
Austen and Yoshihide Kanaoka (Harvard University) for 9. Young IR, Hall AS, Pallis CA, Legg NJ, Bydder
providing me an opportunity to study in the U.S.A., Profs. GM, Steiner RE (1981) Nuclear magnetic resonance
Edward A. Dennis and Shankar Subramaniam for accep- imaging of the brain in multiple sclerosis. Lancet
tance to join LIPID MAPS®, Prof. Jerold Chun (Sanford 2:1063–1066
Burnham Prebys Medical Discovery Institute (SBP)) for 10. Mcdonald WI, Compston A, Edan G, Goodkin D,
his continuous support and encouragement, Dr. Deepa Hartung HP, Lublin FD, Mcfarland HF, Paty DW,
Jonnalagadda (SBP) for helpful discussions, and Ms. Polman CH, Reingold SC, Sandberg-Wollheim
Danielle Jones (SBP) for editorial assistance. This work M, Sibley W, Thompson A, Van Den Noort S,
was supported by a grant from NIH/NINDS R01NS103940 Weinshenker BY, Wolinsky JS (2001) Recommended
and fellowships from Japan Society for the Promotion of diagnostic criteria for multiple sclerosis: guidelines
Science and Human Frontier Science Program. from the International Panel on the diagnosis of mul-
tiple sclerosis. Ann Neurol 50:121–127
Conflict of Interest Y.K. declares no competing financial 11. Thompson AJ, Banwell BL, Barkhof F, Carroll WM,
interests. Coetzee T, Comi G, Correale J, Fazekas F, Filippi
M, Freedman MS, Fujihara K, Galetta SL, Hartung
HP, Kappos L, Lublin FD, Marrie RA, Miller AE,
Miller DH, Montalban X, Mowry EM, Sorensen PS,
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Role of Bioactive Sphingolipids
in Inflammation and Eye Diseases 14
Koushik Mondal and Nawajes Mandal
Abstract retinal pigment epithelial (RPE) and photore-

Inflammation is a common underlying factor ceptor cells. Inflammatory stress by reactive
in a diversity of ocular diseases, ranging from oxygen species leads to LacCer accumulation
macular degeneration, autoimmune uveitis, and S1P secretion and induces proliferation of
glaucoma, diabetic retinopathy and microbial retinal endothelial cells and eventual forma-
infection. In addition to the variety of known tion of new vessels. In sphingolipid/lysosomal
cellular mediators of inflammation, such as storage disorders, sphingolipid metabolites
cytokines, chemokines and lipid mediators, accumulate in lysosomes and can cause ocular
there is now considerable evidence that sphin- disorders that have an inflammatory etiology.
golipid metabolites also play a central role in Sphingolipid metabolites activate comple-
the regulation of inflammatory pathways. ment factors in the immune-response medi-
Various sphingolipid metabolites, such as ated pathogenesis of macular degeneration.
ceramide (Cer), ceramide-1-phosphate (C1P), These examples highlight the integral associa-
sphingosine-1-phosphate (S1P), and lactosyl- tion between sphingolipids and inflammation
ceramide (LacCer) can contribute to ocular in ocular diseases.
inflammatory diseases through multiple path-
ways. For example, inflammation generates Keywords
Cer from sphingomyelins (SM) in the plasma Inflammation · Sphingolipid · Ceramide ·
membrane, which induces death receptor Ceramide-1-phosphate · Sphingosine-1-
ligand formation and leads to apoptosis of phosphate · Lactosylceramide · AMD ·
Uveitis · Diabetic retinopathy · Glaucoma
K. Mondal
Department of Ophthalmology, University of
Tennessee Health Science Center, UTHSC,
Memphis, TN, USA 14.1 Introduction
N. Mandal (*)
Department of Ophthalmology, University of Inflammation is a defensive mechanism of a host
Tennessee Health Science Center, UTHSC, organism against infectious agents and injury.
Memphis, TN, USA Inflammatory mechanisms represent a network
Anatomy and Neurobiology, University of Tennessee of complex processes requiring the involvement
Health Science Center, UTHSC, Memphis, TN, USA of different metabolic and signaling pathways to

150 K. Mondal and N. Mandal
resolve damage to tissue or to fight against infec- cant roles in immune surveillance and
tion. However, inflammation may also be detri- inflammatory processes [10]. During
mental if it progresses out of control. Host complement-mediated targeted cell lysis, there is
organisms have evolved different signaling an initiation of strong opsonization of the foreign
mechanisms to respond appropriately against a pathogen or apoptotic cell/cellular compartment,
range of threats by utilizing specialized immune followed by induction of proinflammatory sig-
cells such as neutrophils, resident and recruited naling by anaphylatoxins, which lead to recruit-
macrophages, dendritic cells, and lymphocytes ment of macrophages and eventually phagocytosis
[57]. Host immune machinery is activated against of the pathogen through the formation of the
microbial pathogens and recognizes molecular membrane attack complex (MAC) [129]. Thus,
structures found in pathogens, known as the inflammasome-complement pathway elimi-
Pathogen-Associated Molecular Patterns nates the pathogen and clears and eliminates
(PAMPs) [80], whereas signals released from potential mediators of damage and injury. Acute
stressed and damaged host cells are known as and chronic inflammation can influence vascular
Damage Associated Molecular Patterns (DAMPs) permeability of the cell. Activation of proinflam-
[175]. Both PAMPs and DAMPs are recognized matory cytokines up-regulates selectins (e.g.,
by molecular structures on immune cells, known P-selectin) and integrin ligands, e.g., Vascular
as Pattern recognition receptors (PRR). The Toll- cell adhesion molecule 1 (VCAM-1) and
like receptors (TLR) are important class of PRR Intercellular adhesion molecule 1 (ICAM-1), on
[82] that recognize bacterial (pathogen) mem- the lumen of endothelial cells. These are sensed
brane lipopolysaccharides and viral RNA as well by selectin ligands, e.g., P-selectin glycoprotein
as endogenous molecules that are secreted from ligand 1 (PSGL1) and integrins, e.g., Lymphocyte
damaged or dying cells [126]. After activation, function-associated antigen 1 (LFA1) on the sur-
TLRs recruit downstream signal adaptor pro- face of leukocytes, and promote loosening of
teins, including Myeloid differentiation primary tight junctions between endothelial cells while
response 88 (MyD88) and TIR-domain contain- permitting transfer of solutes to peripheral tissues
ing adapter inducing interferon β (TRIF), which and leukocyte infiltration through the blood-brain
leads to activation of kinases, such as Inhibitor of barrier [127]. Inflammasome activation can also
kappa B (IkB) and Mitogen activated protein regulate synthesis of various eicosanoids, such as
kinase (MAPK), downstream transcription fac- prostaglandins (PGs), thromboxane, hydroxye-
tors, such as Nuclear factor kappa B (NF-kB), icosatetraenoic acid (HETEs) and leukotrienes
Activator protein-1 (AP-1), and interferon regu- [128]. In addition to inflammasome-mediated
latory factor family proteins. These factors can canonical activation of caspase-1-dependent mat-
stimulate transcription of several amplifiers and uration of proinflammatory cytokines IL-1β and
effectors [155]. Different types of cytokines, IL-18, caspase-1 independently activates cyto-
such as Tumor necrosis factor (TNF)-α, solic phospholipase A2 (cPLA2) that stimulates
Interleukin (IL)-1β and IL-6, and chemokines, eicosanoid synthesis. In this mechanism, there is
e.g., C-C motif chemokine ligand 2 (CCL2), formation of a membrane pore, which drives
C-X-C motif chemokine ligand 1 (CXCL1), and rapid Ca2+ influx. The influx of Ca2+ then acti-
C-X-C motif chemokine ligand 10 (CXCL10) act vates cPLA2 and generates arachidonic acid
as amplifiers and effector molecules in this mech- (AA) from membrane phospholipids. This ara-
anism. Another member of the innate immune chidonic acid is further converted to prostaglan-
sensor is the NOD-like receptor (NLR), which is dins and thromboxanes by cyclooxygenases- 1
a component of the inflammasome multiprotein (COX-1) and COX-2, and leukotrienes and
complex [125]. The third type of pattern recogni- HETEs are converted by lipoxygenases [163].
tion receptor is Retinoic acid inducible gene 1 The generation of eicosanoids is responsible for
(RIG1) [87]. Cooperative interactions of inflam- increasing vascular permeability and leucocyte
masomes and complement cascades play signifi- recruitment during diverse homeostatic and path-
14 Role of Bioactive Sphingolipids in Inflammation and Eye Diseases 151
ological processes [30]. Thus, during injury or research that reveals involvement of sphingolipid
disease, the immune cells become reactive and metabolites in inflammation and their role in ocu-
their PRRs are activated, which leads to genera- lar diseases.
tion of innate inflammatory mediators including
complement pathway, chemokines and cytokines,
and inflammatory enzymes. These proinflamma- 14.2.1 Ceramides
tory mediators stimulate immune cells to prolif-
erate, migrate and induce expression of adhesion Sphingolipid metabolism is regulated by cas-
molecules on endothelial cells, which promote cades of enzyme activation within different cel-
loosening of tight junctions and eventually infil- lular compartments wherein Cer occupies a
tration of immune cells leading to recovery from central position. [56, 109] Cer plays a structural
injury or infection from pathogens or otherwise role by regulating membrane properties and its
pathological changes in diseased state. permeability [161] leading to promotion of raft
fusion [46]. Cer- enriched platforms facilitate
clustering of receptor molecules and their ligands
14.2 Sphingolipid Metabolites [65]. This in turn helps the induction of apoptosis
and Inflammation by clustering of CD95/Fas death receptor ligand
[47]. In addition to its structural role, Cer acts as
Sphingolipids serve both structural and regula- a second messenger by activating a diverse set of
tory roles in eukaryotic cells [31, 56]. The sphin- kinases and phosphatases [132]. The de novo Cer
golipid metabolites Ceramide (Cer), biosynthesis pathway is an anabolic pathway,
Sphingosine-1-phosphate (S1P), Ceramide-1 which begins with condensation of serine and
phosphate (C1P), and Lactosylceramide (LacCer) palmitoyl-CoA catalyzed by Serine palmitoyl-
are the major signaling molecules regulating key transferase (SPT) in the endoplasmic reticulum
physiological functions and a variety of patho- (ER). The catabolic pathway of Cer generation
logical processes, mainly related to inflammatory occurs in the plasma membrane and lysosome via
responses or inflammation-associated diseases degradation of sphingomyelin (SM) to Cer and
[55]. Cer acts as a potent pro-inflammatory agent, phosphorylcholine by sphingomyelinase. The
whereas C1P and S1P can regulate either inflam- third pathway is the lysosomal salvage pathway
mation or participate in anti-inflammatory func- involving complex sphingolipids. Ceramide syn-
tions. LacCer plays a role as a signaling molecule thases play a significant role in the salvage path-
in inflammation-induced proliferation or angio- way, thereby bypassing the formation of
genesis. Immune cell mediated secretion of pro- dihydroceramide. The fourth pathway of Cer bio-
inflammatory cytokines stimulate inflammatory synthesis occurs in liver mitochondria. The first
sphingolipid metabolic enzymes to convert report of the involvement of Cer in the inflamma-
sphingomyelin (SM) into Cer. Cer is then con- tory process demonstrated intracellular induction
verted into either S1P or C1P or glycosphingo- of a proinflammatory cytokine, TNF-α, which
lipid. The schematic diagram of sphingolipid induced sphingomyelinase and, in turn, elevated
metabolism and the biosynthetic enzymes Cer [75, 94]. Sphingolipidomics and transcrip-
involved in this process has been presented in tomics studies revealed that lipopolysaccharide
Fig. 14.1. The conversion of different sphingo- (LPS) induces inflammation through TLR4 in
lipid metabolites varies with cell type. These macrophage cell lines by inducing an increase in
inflammatory sphingolipid mediators then induce Cer [29]. In macrophages, the LPS-induced
different types of inflammatory transcription fac- TLR4-mediated increase in de novo Cer biosyn-
tors (e.g., NFkB) or they may activate cyclooxy- thesis is necessary for autophagosome formation,
genase -2, leading to production of which could play a role in innate immunity [140].
pro-inflammatory prostaglandins. In this review, Production of Cer subsequently activates a
we provide an overview of the significant body of proinflammatory transcription factor, NFkB, the
Fig. 14.1 Sphingolipid Metabolites in Ocular Diseases. Sphingosine kinase isoenzymes (SPHKs) and for reverse
The name of the disorders are presented in red and reaction Sphingosine-1-phosphate phosphatase (S1PP)
enzymes responsible for these metabolites are presented plays its role. Addition of glucose moiety with Cer is
in blue. The de-novo Ceramide (Cer) biosynthesis is mediated by Glucosylceramide synthase (GCS).
mediated by Serine palmitoyltransferase (SPT). In sal- Lactosylceramide synthase (LCS) converts
vage pathway Ceramide synthase isoenzymes (CerSs) Glucosylceramide (GlcCer) to Lactosylceramide (LacCer)
plays role in formation of Cer from Sphingosine. and Lactosylceramidase (LCdase) converts LacCer to
Sphingomyelin phosphodiesterase isoenzymes (SMPDs) GlcCer.GM3 ganglioside formation is mediated by GM3
converts Sphingomyelin (SM) to Cer. Sphingomyelin syn- synthase. Complex Gangliosides are generated by other
thase (SmS) is the enzyme that converts Cer to carbohydrate moiety adding enzymes, which we dis-
SM. Ceramide kinase (CerK) and Ceramide-1-phosphate cussed in our earlier review. AMD Age-related macular
phosphatase (C1PP), respectively converts Cer to degeneration, DR Diabetic Retinopathy, DTS
Ceramide-1-phosphate or vice versa. Conversion of Dysfunctional Tear Syndrome, MS Multiple Sclerosis, RP
Sphingosine-1-phosphate (S1P) is mediated by Retinitis Pigmentosa
ubiquitously expressed transcription factor in modulating inflammatory pathways. The Nlrp3

mammalian cells [139]. The induction of NFkB dependent inflammasome pathway increased
encodes different cytokines, such as IL-1β, IL-6, caspase-1 activity through lipotoxicity-associated
IL-8, as well as chemokines, Monocyte chemoat- ceramide production in macrophage and adipose
tractant protein-1 (MCP-1) including proinflam- tissue of obese mice [162]. In mouse models, it
matory enzymes Cyclooxygenase -2 (COX-2), has been reported that increases in Cer produc-
which is involved in the production of prosta- tion leads to TLR-4 dependent insulin resistance
glandins [169]. All these factors play important by inhibiting Akt [60]. Stimulation of protein
roles in inflammation. Another family of tran- kinase C ζ (PKCζ) by Cer is another pathway
scription factors, CCAAT/enhancer binding pro- which inhibits Akt activity [39]. In summary,
teins (cEBP), is also activated by Cer in these studies support a strong link between Cer
hepatocytes and macrophages. In hepatocytes, and inflammation-related disorders.
proinflammatory cytokine IL-1β induced CCAAT
activation through Cer-mediated Extracellular
signal regulated kinase 1/2 (ERK-1/2) pathway 14.2.2 Ceramide-1-P
[42]. Cer can lead to induction of COX-2 via
cEBP activation in macrophages stimulated with Ceramide-1-phosphate (C1P) is mitogenic and
LPS [25]. Cer also plays a role in obesity by anti-apoptotic for different cell types [44].
Interestingly, C1P can have a dual regulatory role peripheral blood mononuclear cells [54]. The
by serving as an intracellular second messenger bimodal behavior of C1P in pro- and anti-
to regulate cell survival and/or as an extracellular inflammation could provide a novel therapeutic
receptor ligand to stimulate chemotaxis [43]. In strategy for modulating inflammation associated
inflammation, CIP also behaves in a promiscuous with different diseases.
manner, either as a pro-inflammatory or anti-
inflammatory agent. The biosynthesis of C1P
takes place in the trans-Golgi network through 14.2.3 Sphingosine-1-P
phosphorylation of Cer by Ceramide kinase
(CerK). The role of C1P in inflammation was first To date, Sphingosine-1-phosphate (S1P) is the
reported in A549 lung adenocarcinoma cells, best-described mediator within the sphingolipid
where it stimulated release of arachidonic acid pathway. Unlike Cer, which promotes apoptosis,
(AA) and subsequent production of proinflam- S1P is responsible for suppressing apoptosis
matory eicosanoids [120]. C1P-mediated inflam- [146]. As an extracellular and intracellular mes-
mation is directly regulated by activation of senger, S1P plays diversified roles in inflamma-
cytosolic phospholipase-A2α (cPLA2α), an tion, cancer, atherosclerosis, autoimmunity, and
enzyme that releases AA from membrane phos- angiogenesis [153]. The diversified actions of
pholipids [121]. Further studies demonstrated S1P are mainly regulated by five widely expressed
that C1P-mediated activation of group IV cPLA2 S1P G-protein-coupled receptors, S1PR(1–5)
proinflammatory enzyme is chain length- [147]. S1P is produced by phosphorylation of
specific; C1P bearing an acyl chain of 6 carbon or free sphingosine by Sphingosine kinase isoen-
higher, activated cPLA2α in vitro, whereas CIP zymes 1 and 2 (SphK1 and SphK2). Topologically,
with a shorter acyl chain length (C2-C1P) was SphK1 is a cytosolic protein and may also be
unable to activate this proinflammatory enzyme localized at the plasma membrane and endocytic
[150, 166]. Interestingly, prostaglandin produc- membrane trafficking network, whereas SphK2
tion is a coordinated function of C1P and S1P, mainly resides in the nucleus, mitochondria or in
where S1P stimulates COX-2 activity and then the ER [53]. It has been well documented that
cPLA2α functions in synthesis of AA to produce S1P gradient in circulation plays major role in
prostaglandins [122]. The interaction of C1P in lymphocyte migration and trafficking [12].
proinflammation has also been proposed in some Usually, in the blood and lymph S1P level is
studies. It has been shown that, compared to wild higher than in tissues, which is important for
type mice, CerK knock-out mice generate maintaining vascular integrity. The maintenance
decreased level of proinflammatory cytokines, of circulating lymphocytes is balanced through
IL-6 and TNF-α in high fat diets and show nor- S1P; lymphocytes are attached through S1P with
mal insulin signaling [98]. These knock out ani- their receptors in the lymphoid organ to prevent
mals show higher expression of insulin receptors their egression into blood. In inflamed tissues,
and glucose transporter GLUT4 and decreased there is production of S1P by SphK1 in endothe-
signaling of MCP-1 in infiltrating macrophages lial cells. Simultaneously, S1PR1 is also acti-
of adipose tissue. However, the role of C1P in vated by inflammatory signals, which in turn lead
proinflammation is complex, as other studies to disbalance in circulating lymphocytes [133].
have demonstrated that C1P may also reduce The synthetic S1P analog FTY720 (Fingolimod)
inflammation. C1P was shown to inhibit tumor prevents egression of lymphocytes in circulation
necrosis factor (TNF)-converting enzyme or and accumulation in the lymph nodes [131].
TACE, which is the major metalloprotease for Thus, FTY720 acts as an immunosuppressive
cleaving pro-TNF to its active form that plays a drug in inflammation and autoimmune diseases.
role in inflammation [79]. Also, exogenous use of The role of S1P in inflammation varies with cell
C1P was shown to suppress production of IL-6, type. It plays an important role during activation
IL-8, TNF, and IL-1β in LPS treated human of mast cells and the subsequent development of
inflammatory responses [111]. The involvement 14.2.4 Lactosylceramide

of S1P in inflammation is supported by the fact
that loss of generation of S1P leads to decreased Lactosylceramide (LacCer) acts as a common
levels of proinflammatory cytokines (TNF-α, precursor of all types of the lactose series of
IL-6) and proinflammatory factor, arachidonic complex glycosphingolipids (GSLs) (e.g. the
acid, in SphK2 deficient fetal liver mast cells gangliosides and globotriosylceramide). It acts
[112]. TNF-α induced proinflammatory enzyme, as a bioactive lipid in various physiological pro-
COX2 and production of prostaglandin E2 are cesses ranging from inflammation, proliferation,
regulated by activation of the Sphk1/S1P axis expression of adhesion molecules, angiogenesis
[120]. In addition, in an animal model of inflam- and endocytosis [17]. LacCer is synthesized
mation, S1P levels were decreased in dextran- from ceramide generated by the de novo path-
sulfate induced colitis of Sphk1-lacking mice way and from other sphingolipids.
[143]. TNF-α mediated activation of Sphk1 is Glucosylceramide (GlcCer) is the first glycosyl-
responsible for activation of inflammatory tran- ation product of ceramide generated at the cyto-
scription factor NFkB. S1P binds and stimulates solic surface of the Golgi. GlcCer is then
ubiquitin E3 ligase TRAF2 activity, resulting in translocated to the lumen of the Golgi and
Lys-63 linked polyubiquitination of Receptor LacCer synthase [UDP-Galactose: glucosylce-
interacting protein-1 (RIP-1), leading to phos- ramide β1,4 galactosyl transferase (β4GalT)]
phorylation of IKK complex and activation of converts GlcCer to LacCer. LacCer is an impor-
NFkB [6]. Similarly, in Rheumatoid arthritis, tant signaling component in astrogliosis and
S1P/S1P receptor 1 signaling upregulates pro- induction of inflammatory mediators in neuroin-
inflammatory receptor activator of NFkB ligand flammatory diseases. The proinflammatory fac-
(RANKL) [154]. Contrary to its role in inflam- tor arachidonic acid is generated by
mation, S1P has also been reported to have anti- LacCer-mediated activation of cytosolic phos-
inflammatory effects by causing a switch from pholipase A2α [103]. The inflammatory media-
pro-inflammatory macrophage subtype M1 to tors TNF α [116] and LPS/IFN-γ [117] activate
anti-inflammatory subtype M2. [130]. In mice, it LacCer, which in turn induces inflammatory fac-
has been reported that lymphopoiesis and neuro- tors, namely inducible nitric oxide synthase
inflammation has been restrained by HDL-bound enzyme (iNOS) that eventually generates nitric
S1P [18]. Although lots of information supports oxide(NO) and causes neuroinflammation. In
the role of the S1P/Sphk1 axis in activation of this LacCer-mediated inflammatory mechanism,
NFkB, nevertheless it has also been reported that Ras-NFkB-MAPK [117] and Ras-ERK1/2 [116]
downregulation of SphK1 enhances chemokine pathways play significant roles. LacCer also
CCL5, which plays an active role in recruiting induces inflammatory mediator, NADPH oxi-
leukocytes in inflammatory sites. Downregulation dase and generates reactive oxygen species
of Sphk2 reduced CCL5 expression. In this (ROS) from endothelial cells [16] and from neu-
mechanism p38MAPK may play a significant trophils [9]. Inflammation stimulates prolifera-
role without affecting NFkB [2]. Another com- tion and migration of immune cells and induces
plexity in S1P biology is that not all tissues expression of adhesion molecules on endothelial
respond in a similar fashion to regulation of SIP cells. LacCer induces expression of Platelet
production. For example, in a mouse model, endothelial cell adhesion molecule-1 (PECAM1)
deletion of Sphk1 decreases S1P in blood, on endothelial cells in the microenvironment of
whereas deletion of Sphk2 increases it [86, 143]. monocyte accumulated cells at the site of inflam-
Although we have progressed significantly in mation [45]. This LacCer-mediated expression
various aspects of S1P research, we are still lack- of PECAM1 is regulated by cross-talk between
ing in a comprehensive understanding of the activation of PKC-α/ζ and stimulation of phos-
involvement of SIP mechanistic pathways in pholipase A2. The inflammation induced LacCer
inflammation related diseases. activation plays a role in PECAM1 expression
and induces angiogenesis. LacCer mediated 14.3.2 Sphingolipidoses and Ocular

induction of angiogenesis has been shown in Inflammation
Human umbilical vein endothelial cells
(HUVEC). Loss of the LacCer synthase gene in There are similar reports on ocular inflammation
HUVEC cells leads to reduction of angiogenesis from metabolic diseases, which often cause neu-
by reduction of PECAM1 expression when rodegeneration and visual impairment. Many of
induced with Vascular endothelial growth factor these diseases are lysosomal storage disorders
(VEGF) [124]. LacCer serves as a lipid second resulting in functional impairment of lysosomal
messenger in VEGF induced angiogenesis, sepa- enzymes or co-factors responsible for accumula-
rate from S1P mediated pathway [77]. LacCer tion of sphingolipid metabolites in the cell [23].
also contributes to mitochondrial dysfunction Sphingolipids are fatty amino alcohols, which
and generation of ROS in murine model of dia- regulate cell survival, growth, inflammation,
betes [107]. This information suggests that senescence and apoptosis [89]. In the mouse
LacCer acts as a connecting modulator between model of GM1 and GM2 gangliosidoses, there is
inflammation and angiogenesis by expression of microglial activation leading to elevation of pro-
cell adhesion molecules and eventually, inflammatory cytokines TNF-α, IL-1β [68]. The
angiogenesis. GM1 and GM2 Gangliosides are sialic acid-
containing glycosphingolipids, which plays
important role in cell-cell recognition, adhesion
14.3 Sphingolipids in Ocular and signal transduction. The loss of beta hexosa-
Disease minidase A and hexosaminidase B causes accu-
mulation of gangliosides in the brain and nerve
14.3.1 Ocular Inflammation cells during Tay Sachs and Sandhoff disease, the
lysosomal storage disorders [171]. The accumu-
Interestingly, ocular tissues prevent and resolve lation of gangliosides in patients’ retinas has
inflammation by different mechanisms. The bar- been reported in Tay Sachs [104] and Sandhoff
rier between circulating blood and the retina, disease [135]. Accumulation of glucocerebroside
lack of a direct lymphatic drainage pathway, and has been observed in Gaucher patients leading to
the intraocular microenvironment limit local pathological abnormalities in the eye ranging
immune and inflammatory responses and make from ocular motor apraxia [35] to corneal opacity
the eye an immune-privileged tissue [148]. A [51] followed by infiltration of monocytes/mac-
major population of innate inflammatory cells rophages [40]. In Krabbe’s disease (globoid cell
involved in ocular inflammation are macro- leukodystrophy or galactosyl ceramide lipidosis),
phages [27], whereas adaptive immune elements, neuroinflammation is the major component of
CD4+ and CD8+ T cells, express effector func- pathogenesis. A reduction of retinal ganglion
tion in the eye [119]. In the most common ocular cells and nerve fiber layers of the retina are
inflammatory disease, uveitis, inflammation is observed in Krabbe’s disease [34]. In murine
associated with Th1 and Th17 cells [21] along models, there is a generation of numerous proin-
with different innate effectors, such as mono- flammatory molecules, including Major histo-
cytes and neutrophils. In Age-related macular compatibility complexes (MHC) [95], TNF-α,
degeneration (AMD), innate/complement IL-6 [84], and Monocyte chemoattractant pro-
inflammatory responses play a significant role tein-
1 (MCP 1), and IL-10 [167]. Activated
[32] along with adaptive immune responses microglia produce an inflammatory signaling
[108]. Diabetic retinopathy (DR) is another mediator, Prostaglandin D2 (PGD2), which acti-
inflammatory and a classical microvascular dis- vates astrocytes in mouse models of Krabbe’s
ease, where presence of microglia has been disease [101]. Monoglycosylsphingolipid
reported in the outer nuclear layer and photore- (Psychosine) plays a significant role as an inflam-
ceptor layer [173]. matory component in the pathogenesis of
Krabbe’s disease [152]. Niemann-Pick is a lyso- Uveitis. In Intermediate Uveitis there is chronic
somal storage disease caused by a mutation in the inflammation, which affects the vitreous. It
acid sphingomyelinase gene leading to dysfunc- includes Pars planitis, Posterior cyclitis and
tion of sphingolipid signaling [81]. In Neimann- Hyalitis. During Posterior Uveitis inflammation
Pick Type A, ocular abnormalities range from affects retina, choroid and optic nerve. It could be
corneal opacification to retinal opacification with chronic and recurrent in nature. The disease
a macular cherry red spot [164]. Whereas in the named Chorioretinitis, Retinochoroiditis,
case of Neimann-Pick Type B, the ocular mani- Retinitis and Neuroretinitis are under the cate-
festation is mainly retinal with pathological fea- gory of Posterior Uveitis. Pathologically,
tures including macular halos and cherry-red Posterior Uveitis involves breakdown of blood-
maculae [96]. Optic nerve pallor and perimacular retinal barrier (BRB), whereas in other forms of
gray discoloration in Neimann-Pick Type C have uveitis do not [37]. Like Posterior uveitis pathol-
been observed, both clinically and histologically. ogy, in Experimental Autoimmune Uveitis
In knock-out mouse models of Neimann-Pick (EAU), there is an extensive breakdown of BRB
disease, there is enhancement of microglial activ- and release of retinal autoantigen [49]. The EAU
ity and upregulation of IL-1β from astrocytes follows classical example of organ-specific auto-
[15]. In Farber disease, there is accumulation of immune disease that resembles Posterior Uveitis
ceramide in the joints, liver, throat, CNS and retin humans [22]. In case of Panuveitis, the inflam-
ina due to deficiency of ceramidase [41]. The mation affects entire uvea. The inflammation
pathological changes are observed in retinal gan- associated with uveitis is due to infiltration of
glion cells with gross distention and inclusions both innate and adaptive immune cells [61].
[172]. Fabry disease is a deficiency of Using a murine model of uveitis, it has been con-
α-galactosidase A which leads to accumulation firmed that T cells are involved and that Th17 and
of globotriosylceramide. The most common ocu- Th1 play a significant role in the inflammatory
lar condition arising from this is cornea verticil- mechanism. Th17 and Th1 recruit different innate
late [144]. Overall, these observations suggest a effector molecules: Th17 recruits neutrophils and
role for sphingolipids in ocular inflammatory Th1 recruits monocytes; both cause tissue
diseases. destruction with independent mechanisms of
pathology, with proinflammatory cytokines play-
ing a major role [119]. Interestingly, FTY720, a
14.3.3 Sphingolipids structural analog of Sphingosine (Sph) and an
and Autoimmune Eye FDA-approved therapeutic drug for Multiple
Diseases sclerosis (MS), has been found to be effective in
a rat model of experimental autoimmune uveitis
Uveitis is an autoimmune eye disease where the [26]. The exact mechanism of FTY720 is still
uvea is pathologically affected. There is inflam- unknown, but it acts in a complex way on sphin-
mation of the uvea, which composes the middle golipid metabolism. Sphk2 phosphorylates
layer of eye including the iris, ciliary body and FTY720 to FTY720-P, which is a mimetic of S1P
choroid. There are different types of uveitis, and inactivates S1P receptor mediated signaling
which is classified according to International [91]. It also inhibits de novo ceramide synthesis
Uveitis Study Group (IUSG) Classification, and also acts to inhibit ceramide synthase
which includes Anterior Uveitis, Intermediate enzymes [23]. This same drug was earlier used in
Uveitis, Posterior Uveitis and Panuveitis [66]. experimental treatment of Vogt-Koyanagi-Harada
Anterior Uveitis is acute type and it is most com- (VKH) uveitis patients to suppress production of
mon kind of uveitis where anterior chamber is granulocyte monocyte colony stimulating factor
inflamed. It mainly affects the iris and is often by T cells [134]. The T cell clones (TCC) from
called as Iritis. Iridocyclitis and Anterior cyclitis aqueous humor (AH) or peripheral blood mono-
are also included in this category of Anterior nuclear cells (PBMCs) from VKH patients pro-
duced significantly higher level of duction of ceramide has been linked with types of
proinflammatory cytokines IL-6, IL-8 and IFN-γ neuroinflammation other than MS, including
in comparison with healthy donors. This finding those that are connected to NFkB regulated path-
suggests a role for sphingolipid in inflammation ways that cause blood brain barrier disruption,
and lymphocyte migration in uveitis. Recently, in vascular leakage, and lymphocyte migration with
a Wister rat model of endotoxin-induced uveitis upregulation of ICAM1, VCAM1 and selectin
(EIV), increased levels of proinflammatory cyto- [67]. Although Fingolimod is currently used in
kines IL-6 and TNF-α were noted in the aqueous the treatment of MS, one of the common side
humor [165]. Increased levels of ceramides effects of this drug is Fingolimod-associated
C24:0 and C24:1, and sphingomyelin C24:0 were macular edema (FAME) [90]. Retinal hemor-
also reported in the aqueous humor. In the retina, rhages and retinal vein occlusion can also occur
similar length carbon chain species of ceramide in Fingolimod treated patients. Although infor-
also have been noticed in EIV rats. Increased lev- mation pertaining to molecular mechanisms
els of proinflammatory transcription factor NFkB associated with FAME is still lacking, a possible
were also observed in the retina of EIV rats. mechanism could be disruption of cell-to-cell
These observations suggest that infiltration of and cell-to-matrix adhesion complexes in retinal
innate and adaptive immune cells induces inflam- vessels resulting in stress in vascular permeabil-
mation, which could be mediated through modu- ity and subsequent macular edema [97, 113].
lation of sphingolipid metabolites. Thus, While our current understanding of MS is incom-
sphingolipids may play a major role in uveitis plete, there appears to be a strong correlation
pathology. between MS related retinal degeneration and
Multiple Sclerosis is an autoimmune disease. ceramide-related inflammatory pathways.
Inflammation-related retinal atrophy is one of the
pathological features related to MS. Significant
loss of retinal ganglion cells and the presence of 14.3.4 Sphingolipids
human leukocyte antigen-DR positive cells in the and Degenerative Retinal
retina, with activation of microglia are character- Diseases
istic abnormalities associated with MS [48, 160].
In the central nervous system (CNS), oligoden- Progressive damage to the retina and death of
drocytes are the myelin forming cells, which are photoreceptors is a hallmark for degenerative
affected during MS by activation of glial cells retinal diseases, including Age-related macular
and infiltration of lymphocytes and macrophages, degeneration (AMD) and Retinitis Pigmentosa
leading to apoptosis of oligodendrocytes. (RP). AMD is associated with several pathologi-
Sphingolipids are the major component of myelin cal disorders, ranging from inflammation, mal-
sheath and there are multiple pathophysiological functioning of autophagy and chronic oxidative
roles of sphingolipids in MS. In MS patients, stress leading to degeneration of retinal pigment
increased levels of ceramide have been reported epithelium (RPE) and ultimately photoreceptor
in oligodendrocytes [141] in association with an death with vision loss [99, 123]. RPE is the pig-
increase in sphingosine in white matter [102]. In mented cell layer, which is attached to underlying
addition to NSMase- Ceramide upregulation in choroid and provides nourishment to overlying
MS, sphingosine kinase 1- S1P receptor signal- retinal visual cells. It also functions in phagocy-
ing regulates astroglial proliferation and gliosis tosis, secretion and immune modulation.
[168]. As S1P- S1P receptor1 is a main pathway Photoreceptor cells function in visual phototrans-
of lymphocyte egression in MS, application of duction and visual signal generation. There are
Fingolimod or FTY720, an immunosuppressive two types of photoreceptor cells in mammalian
S1P receptor agonist drug reduces lesion forma- retinas, rods and cones, along with second and
tion in MS patients [73]. Neutral third order neurons, bipolar and ganglion cells,
Sphingomyelinase (NSMase) activation and pro- respectively. During early stage of AMD there is
accumulation of extracellular deposits called dru- [151]. On the other hand, overexpression of
sen in the retina, between RPE and Bruch’s mem- Sphingomyelin phosphodiesterase 3 (SMPD3)
brane. Drusen formation is linked to chronic enhances Cer production, which in turn leads to
low-level inflammation and complement activa- enhancement of RPE cell death by increasing
tion during initial stages in the pathogenesis of inflammatory factors and stress [174]. In mouse
AMD [7, 69]. Activation and secretion of various models, it has been reported that cholesterol
cytokines and chemokines, e.g., IL-1β, IL-6, mediates activation of acid sphingomyelinase,
TNF-α, CXCL8 play a significant role in initia- which disrupts autophagy in RPE and leads to
tion of inflammation [83]. The later stage of dis- early onset macular degeneration [159]. Increases
ease progression is classified as either ‘Dry in Cer eventually promote inflammatory factors
AMD’ or ‘Wet AMD’. Dry AMD is limited to and oxidative stress, which prevent proper endo-
damage of the macula region of the retina caused somal recycling of complement regulatory pro-
by atrophy whereas wet AMD also includes both teins after complement attack and disrupt
macular atrophy as well as choroidal neovascu- endosomal biogenesis [156]. Aberrant endosomal
larization (CNV). Inflammation mediated by biogenesis mediates complement activation in
complement factor plays an important role in the RPE cells in murine model of macular degen-
AMD. In addition to this, genetic mutations asso- eration [74]. In Rd10 mouse models, inhibition
ciated with complement factor gene is among the of de novo Cer biosynthesis by myriocin lowers
major risk factors in AMD pathogenesis. One retinal Cer levels and restricts photoreceptor
such factor in AMD is the inheritable genetic death in RP [149]. Accumulation of POS
mutation, Y402H in complement factor H (CFH) increases oxidative stress and activates CFB,
[52, 76]. Other variants are present in C3, CFB, leading to AMD associated neovascularization
C2 genes, associated with susceptibility to AMD [157]. The inflammatory factor also activates
[8]. These mutations are associated with a reduc- complement factor C3 and aggravates AMD
tion of anti-inflammatory iC3b component and pathogenesis [105]. Ceramide induces retinal
an increase of proinflammatory cytokines TNF-α degeneration, whereas choroidal neovasculariza-
and IFN-γ [24]. RPE also plays role in autophagy tion (CNV) is promoted by administration of
by autophagic degradation of photoreceptor outer alpha-galactosylceramide into the vitreous cavity
segments (POS) in the process called heteroph- of C57BL/6 mice [58]. Similarly, S1P2 receptor
agy [70]. In aging, the function of RPE declines deficient mice do not develop neovascularization
and results in accumulation of POS, which even- in the murine model of ischemia driven retinopa-
tually forms lipofuscin in lysosomes leading to thy [142]. The blockage of S1P by sonepcizumab,
malfunctioning of lysosomes, generation of oxi- a humanized monoclonal antibody, also signifi-
dative stress and retinal inflammation [36]. cantly reduces CNV in mouse models [170]. In
Oxidative stress-induced Cer biosynthesis genes summary, sphingolipids appear to play a signifi-
are involved in photoreceptor cell death [13]. cant role in retinal degenerative diseases by
Increased Cer levels in RPE cells raises the level increasing inflammation, generating oxidative
of inflammatory factor and ROS, which leads to stress and deregulating lysosomal function in
mitochondrial permeabilization and activation of RPE and triggering photoreceptor cell death and/
caspase-3, followed by apoptosis [72]. Use of or neovascularization.
desipramine protects photoreceptor death by
reducing inflammatory factors and oxidative
stress augmented by Cer, as desipramine inhibits 14.3.5 Sphingolipids and Diabetic
sphingomyelinase’s ability to convert sphingo- Retinopathy
myelin to Cer [136]. Similarly, overexpression of
Acid ceramidase (ASAH1) in ARPE19 cells Diabetic retinopathy is a microvascular disease
(Human retinal pigment epithelial cell line) pro- affecting retinal vascular degeneration and defec-
tects from oxidative stress by reducing Cer level tive repair of retinal endothelial cells with persis-
tent low-grade inflammation. It has been reported 14.3.6 Sphingolipids and Glaucoma

that activated retinal glial cells and pigment epi-
thelial cells express proinflammatory cytokines Glaucoma is a neurodegenerative disease where
and VEGF in diabetes, which contributes to dam- retinal ganglion cells (RGC) and their axons in
age of retinal vasculature [1, 20, 100]. In addi- the optic nerve are affected. The major risk factor
tion to this, activation of circulating of glaucoma is elevation of intraocular pressure
myelomonocytic cells from bone marrow (IOP). The neuroinflammatory responses during
increases leukocyte adhesion and contributes to early stages of glaucoma are mediated by astro-
retinal inflammation [85, 138]. There is also cytes, resident microglia, and other monocyte-
myeloid derived monocyte infiltration in diabetic derived cells in the optic nerve head (ONH).
retinas and exacerbation of inflammation by Proteomic analysis of human glaucomatous reti-
secreting proinflammatory cytokines, which fur- nas revealed upregulation of TLR signaling,
ther activates resident microglia, astrocytes and where TLR2, TLR3 and TLR4 was observed in
Muller glia in the retina [1, 59, 145]. The proin- microglia and astrocytes from glaucomatous reti-
flammatory cytokines secreted from these cells nas [88]. In DBA/2J (Dilute Brown Non Agouti,
cause endothelial cells to produce Acid sphingo- which develops progressive eye abnormalities
myelinase (ASMase). Endothelial cells produce that closely mimic hereditary glaucoma) mice in
up to 20-fold more secretory sphingomyelinase early stages of glaucoma, 11 of the 13 TLRs were
than macrophages in response to cytokine stimu- upregulated in the optic nerve head (ONH) [62,
lation [92]. The increase in ASMase regulates 64]. As there was upregulation of TLR, the down-
cytokine-mediated inflammation by generation stream factors such as MyD88, MAPK and NFkB
of Cer in diabetic human and animal models all were activated, which in turn lead to activation
[115]. Using different inhibitors, it has been of proinflammatory cytokines. In RGC degenera-
observed that ASMase plays a major role in dia- tion, Fas ligand also is a major effector in DBA/2J
betic retinopathy. Increases in ASMase by TNF-α mice models [50]. In disease pathology apart
and IL-1β induce VEGF and ICAM-1 in Human from monocytes derived cells [63], the comple-
retinal endothelial cells (HREC) and regulate ment cascade system plays a significant part in
retinal microangiopathy [114]. Retinal vascular inflammation in DBA/2J animal models.
permeability is mediated by very long chain Upregulation of C1 complex was observed in
ceramide, which is decreased in diabetic condi- microglial cells in ONH in DBA/2J glaucoma-
tions due to decreases of biosynthetic enzyme, tous mice [62]. The second component of the
an Elongation of very long-chain fatty acids pro- complement cascade that plays a damaging role
tein 4 (ELOVL4) [71, 158]. The streptozocin- is complement component C5, a necessary com-
induced rat models exhibit decreased levels of ponent for MAC generation. Although there is
Cer and a concomitant increase of incomplete information on role of sphingolipids
Glucosylceramide (GlcCer). The inhibition of in glaucoma development, it has been reported
glucosylceramide synthase increases the viabil- that in the aqueous humor there are native sphin-
ity of retinal neuronal cells and insulin sensitiv- golipid species. The levels of sphingomyelin and
ity in retinal neurons [38]. In addition to this it sphingoid base were reduced in hypertensive
has also been observed that the pharmacological state from normotensive conditions, whereas S1P
inhibition of glucosylceramide synthase and ceramide levels increased in a hypertensive
increases insulin sensitivity in Zucker diabetic state [33] in DBA/2J mouse models. Data per-
fatty (ZDF) rat [3]. Thus sphingolipid, more spe- taining to sphingolipid composition of human
cifically Cer and GlcCer, play significant role in aqueous humor [5] and trabecular meshwork [4]
inflammation and retinal neovascularization in have also been generated. There is still a signifi-
diabetic retinopathy. cant lack of information regarding the sphingo-
lipid biology in glaucoma.
14.3.7 Sphingolipids and Dry Eye bomian gland tear film as compare to the good
Syndrome quality individuals [118]. The sphingolipid
metabolites in meibomian gland tear film could
Dysfunctional tear syndrome (DTS), commonly serve as clinical signature of different types of
known as dry eye disease, is caused by tear defi- eye diseases.
ciency or excessive evaporation [19]. In addition
to this, ocular surface inflammation due to
increase in tear osmolarity plays a major role in 14.4 Summary and Conclusion
DTS [78]. The tear film performs diversified
functions, ranging from maintaining light refrac- This chapter summarizes our current understand-
tion, supplying the cornea with nutrients and ing of inflammation and its correlation with
oxygen, lubrication of the cornea and conjunc- sphingolipid metabolites in eye diseases.
tiva, and ocular surface protection against foreign Although ocular immune privilege protects the
materials [110]. There are three different layers eye and retina from inflammation, the modula-
in tear film composition: the carbohydrate-rich tion and accumulation of different sphingolipid
glycocalyx layer, the intermediate aqueous layer, metabolites can perturb the ocular anti-
and the superficial tear film lipid layer (TFLL) inflammatory environment and lead to ocular
[28]. Meibomian glands are the major source of pathology in different lysosomal storage disor-
TFLL lipids. Meibomian Gland Dysfunction ders, autoimmune diseases, age related macular
(MGD) is a complex multifactorial disorder arise degeneration and diabetic retinopathy, suggest-
from combination of five different pathophysio- ing an involvement of sphingolipid metabolites
logical mechanisms; these are eyelid inflamma- in maintaining homeostasis of the eye. Fig. 14.1
tion, conjunctival inflammation, corneal damage, shows a schematic diagram of sphingolipid
microbiological changes and dry eye disease metabolism and involvement of different sphin-
resulting from tear film instability [14]. Mass golipid metabolites in ocular diseases. The bioac-
spectrometry (MS) data from dry eye disease tive sphingolipid ceramide acts as a
patient reveals the role of sphingolipid in main- proinflammatory lipid, whereas C1P and S1P
taining TFLL integrity [78]. In patient meibo- have both pro- and anti-inflammatory functions.
mian samples, significant increases of SM levels LacCer, on the other hand, acts as an angiogenic
were observed compared to normal subjects. The sphingolipid and induces neovascularization. In
individual short chain GlcCer species were sig- ocular diseases, ceramides are found to be inflam-
nificantly increased in patient meibomian sam- matory factors for stimulating proinflammatory
ples. Whereas in case of meibomian cytokines and in some cases, proinflammatory
keratoconjunctivitis (MKC), increased levels of cytokines induce the formation of ceramide that
Cer were reported due to abnormal hyperkerati- may be ultimately responsible for retinal cell
nization [93]. Similarly, increased Cer levels dis- death. GlcCer, LacCer, and S1P have been found
rupt stability and elasticity of TFLL [11]. to be associated with the cross talk between
However, patients with chronic blepharitis had immune cells and endothelial cells that eventu-
been reported with decreased amount of cerebro- ally develop neovascularization in ‘wet AMD’
sides in their meibomian gland [106]. In animal and diabetic retinopathy. The loss of homeostasis
models, very long chain ceramides (acylce- in diseased conditions leads to stress in the endo-
ramide) in TFLL prevent dry eye disease, as plasmic reticulum, mitochondria, lysosomes and
transgene ELOVL1 mice developed corneal ultimately to activation of different proinflamma-
opacity with vascular invasion, accompanied by tory factors. A more complete understanding of
epidermalization of the cornea due to lower level sphingolipid metabolites and their role in inflam-
of acylceramide in epidermis and in the meibo- mation will help in our understanding of the eti-
mian gland [137]. Recently, our lab has reported ology and pathobiology of various eye diseases
increase in Cer/S1P ratio from poor quality mei- that have inflammatory links.
Acknowledgements This work was supported by 11. Arciniega JC, Uchiyama E, Butovich IA (2013)
National Eye Institute grants [EY022071, EY025256, Disruption and destabilization of meibomian lipid
EY021725], and grants from Foundation Fighting films caused by increasing amounts of ceramides
Blindness Inc., USA and Research to Prevent Blindness and cholesterol. Invest Ophthalmol Vis Sci 54:1352–
Inc., USA. The authors gratefully acknowledge the edito- 1360. https://doi.org/10.1167/iovs.12-10662
rial help received from Dr. Dianna A. Johnson, Emeritus 12. Baeyens A, Fang V, Chen C, Schwab SR (2015)
Professor, UTHSC and Richard C. Grambergs, UTHSC, Exit Strategies: S1P Signaling and T Cell
Memphis, TN. Migration. Trends Immunol 36:778–787. https://doi.
org/10.1016/j.it.2015.10.005
13. Barak A, Morse LS, Goldkorn T (2001) Ceramide:
a potential mediator of apoptosis in human retinal
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Roles of Ceramides and Other
Sphingolipids in Immune Cell 15
Function and Inflammation
Sabrin Albeituni and Johnny Stiban
Abstract lin resistance, graft-versus-host disease,

Ceramides are bioactive sphingolipids that immune suppression in cancer, multiple scle-
support the structure of the plasma membrane rosis, and inflammatory bowel disease.
and mediate numerous cell-signaling events in
eukaryotic cells. The finding that ceramides Keywords
act as second messengers transducing cellular Ceramides · Immune cells · Sphingolipids ·
signals has attracted substantial attention in Inflammation · Disease
several fields of Biology. Since all cells con-
tain lipid plasma membranes, the impact of
various ceramides, ceramide synthases,
ceramide metabolites, and other sphingolipids
has been implicated in a vast range of cellular
functions including, migration, proliferation, Abbreviations
response to external stimuli, and death. The
roles of lipids in these functions widely differ 3KS 3-keto-sphinganine
among the diverse cell types. Herein, we dis- acyl-CoA fatty acyl-coenzyme A
cuss the roles of ceramides and other sphingo- aSMase acid sphingomyelinase
lipids in mediating the function of various ATM adipose tissue macrophages
immune cells; particularly dendritic cells, C1P ceramide 1-phosphate
neutrophils, and macrophages. In addition, we CerS ceramide synthases
highlight the main studies describing effects CNS central nervous system
of ceramides in inflammation, specifically in COX-2 cyclooxygenase-2
various inflammatory settings including insu- CTL cytotoxic T lymphocytes
CXCR2 C-X-C motif chemokine receptor
type 2
DAG diacylglycerol
S. Albeituni
Department of Oncology, St. Jude Children’s DC dendritic cells
Research Hospital, Memphis, TN, USA DSS dextran sulfate sodium
J. Stiban (*) EAE autoimmune encephalomyelitis
Department of Biology and Biochemistry, Birzeit ERK extracellular signal-regulated
University, West Bank, Palestine kinases

170 S. Albeituni and J. Stiban
fMLP N-formylmethionine-leucyl- 15.1 Introduction

phenylalanine
GalCer galactosylceramides Ceramides are sphingolipids (SLs) that along
G-CSF granulocyte-colony stimulating with sterols and glycerolipids constitute the
factor “fluid” part of the plasma membrane of eukary-
GI gastrointestinal otic cells. Ceramides are biologically active
GVHD Graft-Versus-Host Disease metabolites of the SL family, composed of a
HIV human immunodeficiency virus sphingoid base that mainly consists of the
IBD Inflammatory Bowel Disease 18-carbon amino alcohols sphinganine (Sa) or
IFNγ interferon gamma sphingosine (So) covalently bound to a long
IL- interleukin fatty acyl side-chain [1, 2]. Conjugation of vari-
iNOS inducible nitric oxide synthase ous headgroups to ceramide leads to the produc-
IRS-1 insulin receptor substrate-1 tion of sphingomyelin (choline phosphate
JNK c-Jun N-terminal kinase group), ceramide 1-phosphate (C1P) (phosphate
LacCer lactosylceramide group), glucosylceramide (glucose), galactosyl-
LipC6 nanoliposome-loaded C6-ceramide ceramides (GalCer) (galactose), or diverse gly-
LPS lipopolysaccharide colipids of the ganglioside and globoside
MAPK mitogen-activated protein kinase families (addition of various saccharides) [1, 3,
M-CSF macrophage-colony stimulating 4]. Apart from the differences in the headgroups,
factor the variations in the number of carbons of the
MDSC myeloid-derived suppressor cells sphingoid base, the length of the fatty acyl side-
MS Multiple Sclerosis chain, and location of double bonds lead to the
NETs neutrophil extracellular traps diversification of ceramide structure and biologi-
Nlrp3 Nod-like receptor pyrin domain- cal function [5].
containing-3 In eukaryotic cells, ceramide generation
NO nitric oxide occurs via three main pathways: de novo synthe-
PI3K phosphatidylinositol 3 kinase sis, sphingomyelin hydrolysis, and the salvage
PKB protein kinase B pathway [6, 7]. In the de novo biosynthetic path-
PKCζ protein kinase C zeta way, ceramide synthesis is first mediated by ser-
PRR pattern recognition receptors ine palmitoyltransferase (SPT) that transfers
ROS reactive oxygen species serine to fatty acyl-coenzyme A (acyl-CoA),
S1P sphingosine 1-phosphate leading to the generation of 3-keto-sphinganine
Sa sphinganine (3KS), which is then reduced by 3KS reductase
SK sphingosine kinase producing the saturated amino alcohol Sa. Sa is
SLs sphingolipids later N-acylated through the action of any of the
SMase sphingomyelinase 6 identified ceramide synthases (CerS) forming
So sphingosine dihydroceramide which is finally converted to
SPT serine palmitoyltransferase ceramide via the action of dihydroceramide
TAM tumor-associated macrophages desaturases [8–10]. The second major reaction
TCR T cell receptor that results in ceramide production is sphingomy-
TEM tumor microenvironment elin hydrolysis. In this reaction ceramides are
TGFβ transforming growth factor beta generated via hydrolysis of the phosphocholine
TLRs toll-like receptor head group from sphingomyelin by sphingomy-
TNFα tumor necrosis factor alpha elinase (SMase) enzymes [11, 12]. The third
Treg regulatory CD4+ T cells major pathway by which ceramide is produced is
15 Roles of Ceramides and Other Sphingolipids in Immune Cell Function and Inflammation 171
the SL recycling or salvage pathway. In this path- has been recently reported by Tidhar et al., that
way, complex SLs are catabolized to So which is 11 key amino acid residues might be critical in
subsequently N-acylated to ceramide. A more determining the acyl chain length specificity of
complex network of enzymes involving SMases, CerS2, CerS4, and CerS5 [21].
ceramidases, and CerS are implicated in the path- Functional CerS are important players for
way [13]. the wellbeing of cells. Loss of CerS has led to
In mammals CerS family consists of 6 abnormalities in mouse models (Table 15.1):
enzyme isoforms, named CerS1-6 [9, 14, 15]. CerS1 knockout mice suffered from reduced
Different CerS are distributed differentially in ganglioside levels and Purkinje cell loss leading
cells and tissues in the body (Fig. 15.1) [14, 16]. to impaired behavioral and motor development
All CerS transfer acyl-CoA of variable lengths [17, 22]. CerS2 knockout mice developed hepa-
to the amine group of a sphingoid base [2]. Each tocarcinoma and cerebral degeneration [28–30].
CerS has a higher specificity towards the transfer In addition, CerS3 loss resulted in disruption of
of acyl-CoA of a certain length [9]. CerS1 shows the skin barrier and spermatogenic arrest [18,
specificity towards the transfer of C18-CoA [17]; 31]. While CerS4 deficient mice developed
CerS2 is specific to C20–26 [16]; CerS3 acylates alopecia [32] due to destabilization in epider-
sphingoid bases with very long-chain fatty acyl mal stem/progenitor cell homeostasis [35].
CoAs (>26) [18]. CerS4 is specific for the trans- Interestingly, CerS5 loss led to improved adi-
fer of C18–22 acyl CoAs [19], whereas the pre- pose tissue health and function after consump-
ferred substrates for CerS5 and CerS6 are C14–18 tion of high fat diet [20]. Deficiency of CerS6
CoAs [20], and C14–16 CoAs [16], respectively. led to behavioral abnormalities and abnormal
Even though the specificity of CerS towards dif- clasping of the hind limbs in mice [34].
ferent CoAs has been well established, little is Ceramides are the simplest SLs composed of
known about the CerS regions that determine two hydrophobic tails and a simple rather than
such specificity, because the crystal structure of complex hydrophilic head, consisting of a
different CerS has not been solved. However, it hydroxyl group [36]. Because ceramides are
Fig. 15.1 Tissue distribution of CerS isoforms in mice. Tissue distribution of all CerS isoforms based on mRNA levels
[14, 16]. The percentage next to organ pictures represents the relative amount of each CerS isoform in that organ
Table 15.1 Cellular abnormalities resulting from CerS [41]). In all, ceramides are not mere structural
deficiency
components in membranes. Along with So,
Deficient sphingosine 1-phosphate (S1P), C1P and lyso-
CerS
isoforom Abnormalitya Reference(s)
sphingomyelin, ceramides have been implicated
CerS1 Purkinje cell loss; motor Ginkel et al. as bioactive lipids [42] that act as second mes-
development impairment [17] and Zhao sengers and regulate cellular functions including
et al. [22] apoptosis and stress responses [43], tumor cell
CerS2 Delayed liver Jin et al. [23] death and metabolism [44], and cytokine signal-
regeneration after partial
ing and inflammation [45, 46]. Interestingly,
hepatectomy
Increased intestinal Chen et al. numerous evidences point to the ability of
permeability [24] ceramides to self-assemble into protein-
Pheochromocytoma Park et al. [25] permeable channels [47] in artificial membranes
Impaired neutrophil Barthelmes [48, 49], mitochondria [50–53] and lysosomes
migration et al. [26] [54] that are upstream to caspase-dependent
Enhanced liver Chen et al.
apoptotic cell death.
tumorigenesis [27]
Cerebral degeneration, Imgrund et al.
Despite the enormous progress in understand-
myelin sheath defects [28] and ing the effects of ceramides in regulating key
Pewzner-Jung events in cellular biology, their role in regulating
et al. [29, 30] immune cell function and inflammatory diseases
CerS3 Skin barrier deformation Jennemann
has only gained momentum in the last two
et al. [18]
Blocked spermatogenesis Rabionet et al.
decades. Herein, we discuss the main research
[31] findings describing the roles of ceramides in reg-
CerS4 Altered lipid Ebel et al. [32] ulating the function of various immune cells,
composition in skin; including dendritic cells (DC) and neutrophils,
alopecia and the modulation of T cell function and
CerS5 Improved glucose Gosejacob
homeostasis and adipose et al. [20]
macrophages in different disease settings. In

tissue health following addition, we will address the main findings high-
high fat dieta lighting ceramide function in various inflamma-
CerS6 Protection against the Scheffel et al. tory diseases.
development of colitisa [33]
Behavioral problems Ebel et al. [34]
a
Abnormality may not necessarily be detrimental to cells,
it may be beneficial, but still it is a change from normal
15.2 C
eramides and Other SLs
conditions in Immune Cell Function
15.2.1 Dendritic Cells
hydrophobic and usually composed of a C18 Unraveling the mechanisms of DC develop-

sphingoid base and a long C14–26 N-linked-fatty ment, differentiation, maturation, antigen
acyl group, these lipids are embedded in the uptake, processing, and presentation have lied in
membrane making it harder to study their physi- the core of immunology research since their dis-
ological functions. For this reason, more perme- covery by Steinman more than four decades ago
able ceramide analogs with short chains have [55, 56]. The main function associated with DC
been used in the laboratory (C2, C6, and C8) to is linking the innate and adaptive immune
better understand ceramide biological function responses through the uptake of foreign antigens
[37]. Nevertheless, numerous studies have been and subsequent presentation to T cells in order
performed on the biophysical effects of cerami- to mount effective inflammatory and immune
des in membranes ([30, 38–40]; Stiban et al. responses. In early studies, the role of ceramides
in regulating DC function was initially associ- The study of ceramides in DC gained further
ated with induction of apoptosis by C2-ceramide attention due to the structural resemblance
[57]. Similarly, induction of DC apoptosis was between the toxic component of LPS, lipid A,
linked to increased accumulation of ceramides and ceramide (Fig. 15.2) [62]. It was suggested
in DC cultured with tumor supernatants, subse- that LPS mediates its function by mimicking
quently leading to down regulation of the fol- ceramides since, similar to LPS inducing DC
lowing survival signaling pathways, maturation, C2-ceramide reduces micropinocyto-
phosphatidylinositol 3 kinase (PI3K), Akt sis and antigen presentation to T cells by DC
kinase, Bcl-xL, and NF-κB [58]. However, [60]. However, while both LPS and C2-ceramide
induction of ceramide accumulation in DC by induce c-Jun N-terminal kinase (JNK), only LPS
factors that cause DC maturation including, activates extracellular signal-regulated kinases
CD40L, interleukin 1β (IL-1β), tumor necrosis (ERKs) and NF-κB. In addition, LPS stimulates
factor alpha (TNFα), and the gram-negative bac- the production of ceramides regardless of whether
terial endotoxin lipopolysaccharide (LPS) did macrophages are genetically responsive or unre-
not induce DC apoptosis [59, 60]. These contro- sponsive to LPS [63]. Therefore, this further sup-
versial findings were later reconciled by Franchi ported that LPS exerts its function by inducing
and colleagues demonstrating that ceramide- ceramide accumulation and not by interacting
induced cell death in DC is only exacerbated in with ceramide-producing enzymes or as a struc-
the absence of serum, while in the presence of tural mimic of ceramide.
serum and LPS, DC survival was achieved by Moreover, ceramides play important roles in
the action of cellular ceramidases that deacylate DC during viral infections. GalCer on epithelial
ceramides to So, thus, preventing ceramide cells binds the human immunodeficiency virus
accumulation and DC apoptosis [61]. (HIV)-1 envelope glycoproteins gp120 [64, 65]
Fig. 15.2 Structural similarity between Lipid A of structure to ceramides. The 2D structures were
LPS and ceramides. Two representative ceramides are obtained from PubChem and were redrawn to increase
included for comparison, the most abundant C16- resolution. PubChem CID: 9877306 (Lipid A),
ceramide and the phosphorylated C12-ceramide 1-phos- 71314646 (C16-ceramide) and 5283580 (C12-ceramide
phate. The blue triangle identifies the comparable 1-phosphate).
and gp41 [66, 67]. In addition, GalCer is essen- neutrophil extracellular traps (NETs) in
tial in the formation of membrane lipid rafts that response to inflammatory stimuli are crucial for
allow for the internalization of HIV-1 through mounting an effective frontline immune
endocytosis and transcytosis [68]. Interestingly, response against invading microorganisms,
GalCer is present in monocyte-derived immature especially extracellular pathogens [72]. Since
DC and human primary DC isolated from the isolation of the free long-chain base So from
mucosa, suggesting that DC mediates HIV-1 human neutrophils [73], the role of ceramides in
transfer to its target cells through GalCer [69]. mediating and regulating neutrophil function
Ceramides also stabilized the membrane for has gained considerable traction. This was par-
measles virus entry in DC, as Dendritic Cell- ticularly due to early studies linking TNFα sig-
specific intercellular adhesion molecule-3-grab- naling, a potent inducer of superoxide and
bing non-integrin ligation on DC results in apoptosis in neutrophils [74], to ceramide func-
sphingomyelinase activation and subsequent tion [75]. In HL-60 human promyelocytic leu-
ceramide accumulation in DC exposed to either kemia cells, TNFα causes early sphingomyelin
mannan or measles virus [70]. hydrolysis and ceramide production [76]. In
The role of ceramides was further extended addition, in a cell-free system both sphingomy-
beyond viral entry in DC. It has been demon- elin content and ceramide concentration were
strated that local administration of a ceramide reported to be increased in response to TNFα
analog C8-ceramide causes the induction of DC [77]. It was therefore hypothesized that cerami-
maturation, secretion of the pro-inflammatory des might regulate neutrophil function in
cytokines IL-12p70 and TNFα, and enhanced response to TNFα. However, later studies have
virus-specific T cell responses in murine models proposed that ceramides activated a negative
of chronic lymphocytic choriomeningitis feedback loop to inhibit superoxide production
virus clone 13 and influenza virus [71]. in human neutrophils. It has been shown that
Collectively, these findings demonstrated ceramides not only did not mediate TNFα-
that DC apoptosis, maturation, and antigen pre- induced superoxide production in human neu-
senting capacity, are finely tuned by the action trophils [78] but also C2-ceramide inhibited the
of endogenous ceramides or treatment with 20:4 (n-6)-mediated superoxide formation in
exogenous ceramide analogs. In addition, human neutrophils [79]. C2-ceramide also inhib-
ceramides are key structural components of ited respiratory burst of N-formylmethionine-
lipid rafts in DC required for binding, uptake, leucyl-phenylalanine (fMLP)-stimulated
and internalization of viruses. These steps are adherent neutrophils [80–82]. Modulation of
key for initiating DC response allowing for viral neutrophil response to polarity in response to a
internalization, virus peptide processing, and chemotactic agent such as fMLP has been
presentation to T cells that subsequently kill shown to be dependent on neutral sphingomye-
virus-infected cells. Since these findings linase activity that converts sphingomyelin to
strongly suggest that ceramides are key effec- ceramide through the modulation of Rac1/2/
tors of DC function, it is important to note that RhoA GTPases [83]. These combined results
future studies elucidating the role of ceramides suggested that induction of ceramides might
on the various DC subsets (e.g. plasmacytoid delay the response of neutrophils to TNFα to
DC, migratory classical DC, tissue resident allow for neutrophil extravasation and migration
classical DC) could be of great therapeutic rele- prior to superoxide production.
vance in numerous immunologic diseases. Ceramides have also been implicated in the
modulation of phagocytosis and migration in
neutrophils. C2-ceramides reduced phagocytosis
15.2.2 Neutrophils of IgG-opsonized erythrocytes by fMLP-
activated neutrophils through the inhibition of
Neutrophil extravasation, migration, production MAP kinase activation and tyrosine phosphoryla-
of cytokines and superoxide, and formation of tion of ERK1 and ERK2 [84]; and by inhibiting
phospholipase D (PLD) function required for strated to be not only relevant in supporting the
phagocytosis [85, 86]. Similarly, in COS-1 mon- membrane structure but also as a transducer of
key kidney immortalized cells transfected with cellular signals. In vitro studies have revealed
FcγIIA receptor, inhibition of ceramide synthesis that LacCer enhances the upregulation of the
led to enhanced phagocytosis [87]. On the other integrin, CD11b, on neutrophil surface, inducing
hand, chemotaxis, phagocytosis and NETs for- neutrophil adherence to endothelium, and medi-
mation in fMLP-stimulated neutrophils were ating the production of ROS through activation
enhanced by the selective estrogen receptor of NADPH oxidase [96]. NADPH oxidase acti-
antagonist, tamoxifen, through induction of vation in neutrophils results from the association
ceramide accumulation, and subsequently proof LacCer in lipid rafts with the Src family
tein kinase C zeta (PKCζ) activation [88]. These kinase Lyn, and subsequent activation of PI3K,
conflicting findings might suggest the opposing mitogen-activated protein kinase (MAPK) and
roles of various ceramides on neutrophil PKC [97]. In addition, LacCer with long-chain
function. fatty acids (C24) are required for the coupling of
A more direct role of ceramides is also preva- Lyn to LacCer lipid rafts resulting in the produc-
lent in neutrophils. Ceramides are pro-apoptotic tion of superoxides and induction of migration
metabolites whose concentration in cells rises in neutrophils [98–100]. Association of Lyn with
prior to the execution of the apoptotic pathway. the LacCer containing C24-fatty acid chain is
Neutrophil apoptosis is mediated by C16- and necessary for the phagocytosis of mycobacteria
C24-ceramides via caspase activation. This is cor- by neutrophils. Interestingly, LacCer-enriched
related with the ability of granulocyte-macro- lipid rafts are modulated by pathogenic bacteria
phage colony-stimulating factor, a neutrophil to evade neutrophil-mediated killing. For
survival factor, to reduce the accumulation of instance, Mycobacterium tuberculosis prevented
ceramides in neutrophils [89]. During early neu- phagolysosome formation in neutrophils through
trophil apoptosis ceramide is generated by acid binding of bacterial mannose-capped lipoarabi-
SMase (aSMase). In aSMase −/− mice, neutro- nomannan to LacCer rafts in neutrophils [101].
phil apoptosis is delayed compared to WT mice In addition, inhibition of inflammation in neu-
[90]. In an anti-microbial setting, Pseudomonas trophils has also been associated with SL biology.
auroginosa release pyocyanin that induce reac- Ceramides inhibit immune cell responses by
tive oxygen species (ROS), which subsequently binding to CD300f inhibitory receptor in sepsis
activates mitochondrial SMase, therefore, [102], LPS-induced skin inflammation [103],
increasing mitochondrial ceramide levels and allergic responses [104], and experimental colitis
inducing cytochrome c release from mitochon- [105]. Interestingly, disruption of ceramide-
dria. This initiates cell death in neutrophils [91]. CD300f binding induces neutrophil infiltration in
In addition, the enzyme sphingomyelin synthase, sepsis and skin inflammation [102]. Ceramide
which mediates the transfer of choline phosphate metabolites have also been implicated in the
to ceramide from phosphatidylcholine, leads to modulation of neutrophil function. For instance,
the production of sphingomyelin and diacylglyc- chemotactic migration of neutrophils in response
erol (DAG) [92, 93] and mediates neutrophil to IL-8 and fMLP is inhibited by S1P, an
killing of fungus Cryptococcus neoformans [94]. immediate metabolite of ceramide [102].

Nevertheless, the aforementioned negative Moreover, IL-8 gene expression and secretion
regulation by ceramides was not reported upon was shown to be induced by S1P in lung H441
stimulation with the glycosphingolipid, lactosyl- epithelial cells [107]. Similarly, C1P dampens
ceramide (LacCer). In neutrophils, LacCer com- inflammation in a model of LPS-induced lung
pose more than two thirds of the glycolipid inflammation. Particularly, C16-C1P and synthetic
molecules in the plasma membrane and is mainly C8-C1P analog inhibit NF-κB activation in neu-
associated with a pro-inflammatory phenotype trophils and LPS-mediated IL-8 production
[95]. This large composition was later demon- [108].
These observations make it difficult, at a first 15.2.3 Macrophages

glance, to conclude whether ceramides induce
or inhibit neutrophil function. It is important to As the name implies, macrophages or the ‘big
note that neutrophils are short-lived innate eaters’, along with DC and monocytes, compose
immune cells that sense extracellular pathogens the mononuclear phagocytic system [109, 110].
early on during infection. Therefore, early on Due to their phagocytic nature, macrophages
during activation, it is possible that ceramide have the capacity to engulf debris, dead cells, and
induction in neutrophils leads to a delay in the foreign pathogens [110, 111]. The heterogeneity
production of TNFα allowing for neutrophil and plasticity of macrophages enable these cells
extravasation. However, it is not yet clear to mount a spectrum of pro-inflammatory or anti-
whether, as neutrophil activation progresses, a inflammatory responses to various stimuli [111,
certain threshold of ceramides is required to turn 112]. Due to their vast distribution throughout the
on the apoptotic program in neutrophils. Data body, understanding macrophage biology has
using ceramide analogs must be interpreted with been a high pursuit since their description by
caution, since the use of ceramide analogs might Metchnikoff in the late 1800s. It is therefore not
not correspond to the physiologic concentration surprising that such a cell type with high mem-
of ceramide during infection. Nevertheless, the brane activity has recently gained considerable
importance of understanding ceramide biology attention from lipid biochemists and immunolo-
in neutrophils has started to attract more atten- gists alike [113, 114].
tion, especially in studies describing ceramide Macrophages recognize pathogen-associated
inhibitory role in multiple models of inflamma- molecular patterns through pattern recognition
tion including models of sepsis, skin inflamma- receptors (PRR), including toll-like receptors
tion, allergic responses, and experimental (TLRs) [115, 116]. The role of ceramides in
colitis. Thus, these results may possibly be open- modulating the ability of macrophages to sense
ing new doors for the inclusion of novel and respond to microbes has been documented.
ceramide inhibitors that dampen neutrophil acti- Global lipidome analysis revealed that ceramides
vation in diseases in which neutrophils are the accumulated in all cellular membranes of acti-
main cause of pathophysiology. The effects of vated macrophages in response to TLR4 stimula-
different SLs on neutrophil function are summa- tion with Kdo2-lipid A [117–119]. Furthermore,
rized in Table 15.2. ceramide production was induced in macro-
Table 15.2 SLs in Neutrophil Function

SL Function Modulation Reference(s)
Ceramide Superoxide Inhibition Robinson et al. [79], Nakamura et al. [82], Ahmed and Berridge
production [80], and Fuortes et al. [81]
Extravasation and Activation
migration
Phagocytosis Inhibition Suchard et al. [84], Hinkovska-Galcheva et al. [85], Suchard
et al. [86]
Chemotaxis Activation Corriden et al. [88]
Apoptosis Activation Manago et al. [91]
Inflammation Inhibition Izawa et al. [102], Shiba et al. [103], Izawa et al. [104], and
Matsukawa et al. [105]
LacCer Adherence to Activation Arai et al. [96]
endothelium
ROS production Activation Iwabuchi and Nagaoka [97], Chiricozzi et al. [98], Iwabuchi
Migration Activation et al. [99], and Sonnino et al. [100]
S1P Chemotaxis Inhibition Kawa et al. [106]
C1P Inflammation Inhibition Baudiss et al. [108]
phages stimulated with TLR4 in combination 15.3 C

eramides and Other SLs
with palmitate, suggesting that lipotoxic condi- in Inflammatory Diseases
tions induce de novo ceramide synthesis in mac-
rophages. Increased ceramide production was Most of what is known today about the roles of
also associated with induced TNFα and IL1β pro- ceramides and other SLs in modulating immune
duction by macrophages [150]. In addition, pal- cell responses arises from a plethora of studies
mitate and LPS synergistic effect was also shown performed by lipid biochemists around the world.
to induce NLRP3 inflammasome activation, In the current era of lipidomics and targeted lipid
resulting in induced IL-1β and IL-18 production therapeutics, lipid biology and metabolism have
[121] in a manner independent of the SPT sub- also started to attract many immunologists
unit Sptlc2 [122]. Interestingly, ceramide accu- worldwide. In this section we present the main
mulation has also been implicated in macrophage studies highlighting the roles of SLs in some
response to oxidative stress in a TLR2-dependent major inflammatory diseases.
manner [123]. In the aforementioned study,
ceramide accumulation led to suppression of
mitochondrial respiration shifting the use of the 15.3.1 Insulin Resistance
citric acid cycle metabolites to the production of
the antioxidant glutathione. Insulin Resistance refers to the impaired
Ceramide accumulation has also been associ- response of cells to insulin, resulting in a reduced
ated with induced cell death in macrophages uptake of glucose and glucose accumulation in
[124]. It has been demonstrated that apoptosis in the bloodstream. Insulin resistance is the main
adipose triglyceride lipase-deficient macrophages hallmark of type 2 diabetes mellitus and is highly
is mediated by increased C16-ceramide concentra- associated with induction of cardiovascular dis-
tions [125]. Interestingly, withdrawal of macro- ease, obesity, and various types of cancer [128].
phage-colony stimulating factor (M-CSF) from Early studies established a strong link between
bone marrow-derived macrophages resulted in ceramide accumulation and insulin resistance by
enhanced SPT activation and ceramide produc- demonstrating that SLs inhibited glucose trans-
tion prior to apoptosis [126]. This finding was port into 3T3-L1 mouse adipose fibroblasts
further supported by the partial rescue of aSMase- [129] and that bacterial SMase and the short-
deficient macrophages following M-CSF with- chain ceramides, C2 and C6, reduced insulin-
drawal, suggesting that apoptosis was at least in induced tyrosine phosphorylation of insulin
part mediated by ceramides. Additionally, receptor substrate- 1 (IRS-1) downstream the
ceramide accumulation was shown to mediate glucose transporter, GLUT4, in insulin-sensitive
apoptosis in macrophages exposed to a lipotoxic rat hepatoma Fao cells [130]. Interestingly, inhi-
diet consisting of saturated fatty acids [127]. bition of IRS-1 was later demonstrated to be
Altogether, these studies suggest a crucial role mediated by TNFα via induction of tumor necro-
for ceramides in modulating macrophage func- sis factor receptor and SMase activity [131].
tion and metabolism early and late during infec- However, inhibition of IRS-1 activity by cerami-
tion. The ability of ceramides to induce des seems to be a result of protein kinase B
downstream signaling for the production of pro- (PKB) activity rather than direct inhibition of
inflammatory cytokines following an infection, IRS-1 function [132–136]. Further investigation
allows macrophages to persist and orchestrate of the mechanisms of PKB inhibition by cerami-
activation of immune cells that subsequently pro- des demonstrated that C2-ceramide inhibited
duce survival factors such as M-CSF. Once the nuclear translocation of Akt1 [137], reduced
infection is cleared and macrophage survival fac- phosphorylation of serine 473 in PKB [138],
tors are no longer high, ceramides mediate mac- inhibited translocation of Akt/PKB to the plasma
rophage apoptosis winding down the cycle of membrane and promoted dephosphorylation of
immune cell activation. Akt/PKB by protein phosphatase 2A [139–141].
Recent studies have also demonstrated induction deficient mice fed with high-fat diet are protected
of PKR/JNK activation [142, 143]. from obesity, glucose intolerance, and insulin
Further investigations aimed to determine resistance suggesting that targeting CerS6 could
whether ceramides induce insulin resistance. be beneficial for the treatment of obesity and type
Analysis of muscle biopsies from insulin intoler- 2 diabetes [20, 156]. It is worth noting that the
ant obese individuals revealed an increased products of CerS5 (C16-ceramide) and CerS2
accumulation of ceramides [144–147]. In addi- (C24-ceramide) antagonized each other’s ability
tion, exogenous C2-ceramide induced apoptosis to form channels in mitochondrial outer mem-
in skeletal muscle myotubes, reducing the cell branes to induce apoptosis [157]. This may be
capacity to uptake glucose, a process that can be another mechanism by which different CerS
reversed by the CerS inhibitor, fumonisin B1 affect insulin resistance and tolerance
[148, 149]. In muscle cells, C2-ceramide also differentially.
mediated insulin resistance by inhibiting Rac Ceramide accumulation has been linked to
activation, thus reducing GLUT4 translocation inflammatory pathways mainly involving signal-
to the plasma membrane in response to insulin ing events downstream of TLR4. More specifi-
[150]. These studies suggested that targeting cally, Holland et al., was the first to provide a link
ceramide accumulation could protect muscles between lipotoxicity and inflammation in the
against insulin resistance. In line with this induction of insulin intolerance [158].
hypothesis, Bruce and coworkers demonstrated Particularly, saturated fatty acids induced de
that overexpression of sphingosine kinase 1 novo ceramide synthesis via TLR4 activation,
(SK1) led to reduced ceramide accumulation which also altered the metabolic program of
and insulin resistance in mice given high-fat diet skeletal muscle, inducing insulin resistance.
[151]. Inhibition of CerS and ceramide synthesis However, Galbo et al. showed that lipid-induced
with the S1P analog, FTY720 [152], also insulin resistance is a result of increased accumu-
reduced insulin tolerance in mice on high-fat lation of DAG and induced DAG-PKCε signal-
diet [153]. Furthermore, in obese rodents, blocking rather than induced TLR4-ceramide pathway
ade of de novo ceramide synthesis with the SPT) [159]. These conclusions were based on the
inhibitor, myriocin, improved glucose tolerance observation that knockdown of TLR4 or the
[128, 154]. adaptor protein MyD88 prevented hepatic steato-
Since CerS are required for de novo ceramide sis in mice fed with a saturated fat diet through
generation, several studies have focused on the reduction of appetite but not hepatic insulin sig-
role of CerS in inducing insulin tolerance. naling. When mice were given saturated fat by
Overexpression of CerS1, CerS2, CerS4, CerS5, oral gavage, loss of TLR4 or MyD88 did not pro-
and CerS6 in L6 myotubes induced ceramide tect mice from hepatic insulin resistance.
production; however, none of the CerS was able Interestingly, another link between ceramides
to inhibit insulin signaling [139]. Given the and inflammatory pathways in insulin tolerance
opposing role of CerS2 and CerS6 in inflamma- involves stimulation of the Nod-like receptor
tion, current studies have been focusing on the pyrin domain-containing-3 (Nlrp3) inflamma-
role of these CerS in insulin resistance in vivo. some [160]. In obese mice, ceramides activated
CerS2 haploinsufficient mice have altered pat- Nlrp3 inflammasome and IL-1β secretion
terns of ceramide acylation, leading to reduced through caspase-1 activation in adipose tissue
levels of very-long-chain ceramides and increased macrophages in a Nlrp3-dependent manner. This
levels of long-chain C16-ceramide as a compensa- induction subsequently led to T cell activation.
tory mechanism leading to insulin resistance However, this study does not exclude the possi-
when fed with high-fat diet [155]. In agreement bility that inducers other than ceramides present
with these findings, CerS6 expression and levels in the diet induced caspase-1 activation. Further
of C16-ceramide are induced in the adipose tissue studies are required to establish whether cerami-
of obese humans. Moreover, CerS6- and CerS5- des are truly sensed by PRR in immune cells.
15.3.2 Graft-Versus-Host Disease 15.3.3 Immune Suppression

in the Tumor
Graft-versus-host disease (GVHD) is a major Microenvironment
complication of allogeneic hematopoietic stem
cell transplantation. It is an inflammatory Immunotherapy has recently been employed as
response mediated primarily by donor T cells, a novel strategy to eradicate tumors. During
resulting in destruction of host tissues including tumor development, immune cell responses
skin, liver, and gastrointestinal (GI) tract [161]. include massive tumor infiltration and produc-
The role of ceramides in mediating T cell cyto- tion of cytokines in an attempt to contain tumor
toxic function has only been recently explored growth and kill tumor cells. If not contained,
in two studies of mouse models of tumor growth leads to the establishment of a
GVHD. Rotolo et al. reported that aSMase in tumor microenvironment (TEM) characterized
the host mediated GVHD [162]. Adoptive trans- by induced hypoxia [167] and lactic acid accu-
fer of allogeneic T cells to aSMase-deficient mulation leading to suppression of effector
hosts reduced morbidity and mortality. This CD8+ CTL [168]. CD8+ T cell responses are
induction in survival resulted from reduced inhibited by suppressive immune cells of vari-
inflammatory responses, cytokine storm, CD8+ ous types that initially infiltrated the tumor to
T cell proliferation and activation, and apopto- contain growth but reversed its effector pheno-
sis of host hepatocytes, skin and intestinal cells. type in the TEM. Among these suppressive
Interestingly, aSMase is required for the forma- immune cells are tumor-associated macrophages
tion of ceramide-rich platforms on target cells (TAM), myeloid-derived suppressor cells
for cytotoxic T lymphocytes (CTL) efficient (MDSC), and regulatory CD4+ T (Treg) cells
killing. Further investigation of requirement of [111]. Recently, it has been appreciated that
ceramide bioactive function in donor T cells by tumor cells gain advantage over immune cells
Sofi and colleagues demonstrated that CerS6- by altering their glucose and lipid metabolism.
deficient donor T cells reduced GVHD [163]. In Despite many studies describing the effects of
addition, T cells lacking CerS6 had an aberrant alterations in glucose metabolism in immune
T cell receptor (TCR) signal transduction due to cell function, only recently has similar altera-
the reduction of tyrosine phosphorylation and tions in immune cell lipid metabolism been con-
CD3-PKCθ colocalization required for T cell sidered [169].
proliferation and response to pro-inflammatory S1P has been implicated in the polarization of
cytokines, particularly interferon gamma macrophages to an M2 anti-inflammatory pheno-
(IFNγ). Furthermore, inhibition of Cer6S with type in the TEM. During tumor progression, apop-
its specific inhibitor, ST1072 [164], reduced T totic cells are released in the TEM serving as a
cell proliferation and IFNγ production. This source for S1P [170]. Interestingly, S1P released
could also be a result of reduced IL-2 secretion from engulfed apoptotic cells by macrophages
by aSMase-deficient T cells upon TCR stimula- induces macrophage survival through the activa-
tion as previously reported [165]. Moreover, tion of PIRK, ERK/2, and Ca+2 [171] and polariza-
aSMase has also been described to be required tion to an M2 phenotype through the inhibition of
for proper TCR signaling downstream CD3/ inducible nitric oxide synthase (iNOS) and reduc-
CD28 activation in CD4+ T cells, since block- tion of TNFα and IL-8 production [172–175].
ade of aSMase bioactivity with imipramine Knock down of the S1P-producing enzyme, sphin-
impaired PLCγ1, JNK, ERK, Akt, and mTOR gosine kinase 2 (SK2) in MCF-7 breast carcinoma
phosphorylation downstream TCR [166]. More cells delayed tumor growth in a xenograft model
studies are required to further elucidate the in nude mice by promoting macrophage polariza-
roles of the aSMase and lipid metabolism in tion to an M1 pro-inflammatory phenotype char-
driving T cell activation and its role in GVHD acterized by increased expression of nitric oxide
pathogenesis. (NO), TNFα, IL-12, and major histocompatibility
complex class II and reduced expression of IL-10 mune responses against myelin in the central ner-
and CD206,which are markers associated with vous system (CNS) [181]. In experimental
macrophage suppression [176]. autoimmune encephalomyelitis (EAE), de novo
Modulation of TAM function by ceramides synthesis of C16-ceramide by CerS6 is required for
has been also achieved in a mouse model of the production of pro-inflammatory TNFα and
hepatocellular cancer through the administration iNOS in macrophages in response to IFNγ [182].
of nanoliposome-loaded C6-ceramide (LipC6) Upregulation of CerS6 in EAE led to further specu-
[177]. LipC6 exerts its modulatory function by lations on its role in driving MS. Interestingly,
acting as a ROS scavenger, therefore inducing upregulation of CerS6 and TNFα mRNA expres-
macrophage polarization from an M2 to an M1 sion was found to be higher in females compared to
phenotype and resulting in reversal of CD8+ T male littermates in spontaneous relapsing remitting
cell exhaustion and induction of CD8+ cytotoxic EAE [183]. This increase was correlated with the
function against tumor cells. The effect of ability of females to initiate anti-inflammatory
ceramides has also been explored in suppressive responses during the course of the disease, suggest-
MDSC in mice bearing CMS4-metastic tumors. ing that CerS6 could promote anti-inflammatory
Treatment of tumor-bearing mice with acid responses. This observation was later confirmed
ceramidase inhibitor, LCL521, reduced MDSC with experiments showing that EAE progression is
accumulation in tumors without reducing tumor worsened in CerS6 knockout mice [184]. In addi-
growth. In addition, LCL521 treatment led to an tion, EAE was enhanced in chimeric mice in which
increased accumulation of C16-ceramide that only leukocytes were CerS6-deficient, providing
resulted in cathepsin-mediated cell death of further evidence that leukocytes lacking CerS6
tumor and MDSC-like J774 cells [178]. drive the exacerbated phenotype. Moreover,
Suppression of CD8+ T cell function in tumors expression of genes driving leukocyte migration
also results from suppression mediated by Treg and CNS infiltration (e.g. CCL2, CCL5, CXCL2)
cells. To our knowledge, there have been no stud- was increased, especially in CerS6-deficient neu-
ies reporting the effect of ceramides on Treg cell trophils due to increased C-X-C motif chemokine
function in tumor-bearing mice. However, recent receptor type 2 (CXCR2) in response to
studies have described the effect of ceramide granulocyte-colony stimulating factor. On the con-
accumulation on Treg cells in healthy WT and trary, CerS2-deficient mice had delayed develop-
aSMase-deficient mice. Treg cell frequency and ment of EAE. This delay in disease onset was
suppressive function are induced in aSMase-defi- correlated with the reduced expression of
cient mice [179]. In addition, this was associated CXCR2 in CerS2-deficient neutrophils [26]. In
with increased percentage of induced Treg (iTreg) addition, the induction of ceramides was also asso-
cells from aSMase−/− CD4+ T cells treated with ciated with disease development in EAE, since
transforming growth factor beta (TGFβ) and aSMase deficiency protected mice from disease
IL-2. Moreover, aSMase product C6-ceramide [185]. Collectively, these findings suggest that
induced a Th17-associtated cytokine in CD4+ T CerS6 and CerS2 have opposing roles in driving
cell hampering Treg cell induction, suggesting disease progression in EAE.
that aSMase is a negative regulator of Treg cell Excitingly, the S1P receptor agonist, fingoli-
development [180]. The relevance of these find- mod FTY720, was FDA approved in 2010 as a
ings remains to be further explored in models of first-line therapy for the treatment of MS [186,
inflammation for further development of lipid- 187]. FTY720 is phosphorylated in vivo by SK to
targeted therapies. form FTY720-P, which binds with a high affinity
to S1P receptors and competes with its natural
ligand, S1P. Therefore, FTY720 functionally
15.3.4 Multiple Sclerosis antagonizes S1P by strongly binding to S1PR
leading to internalization and inhibition of the
Multiple sclerosis (MS) is a chronic inflammatory receptor [188–190]. By modulating the S1P
disease that results in demyelination due to autoim- receptor, fingolimod prevented autoreactive S1P
expressing inflammatory T cells from exiting the COX-2 expression, and neutrophil infiltration
lymph nodes, therefore, inhibiting CNS infiltra- [203–205]. In support of these findings, high
tion by autoreactive T cells. Subsequently, this SK1 expression has also been reported in intesti-
resulted in reduced destruction of myelin sheath nal mucosa of patients with ulcerative colitis
surround the axons of nerve cells [188–191]. This [205]. Currently, modulators of S1P receptor,
is a perfect example of the potential outcome of such as ozanimod (RPC1063) and etrasimod
modulating lipid metabolism in immune cells (APD334) are being tested in clinical trials for
driving forward the era of lipid therapeutics. the treatment of IBD [206, 207], further empha-
sizing the importance of investigating the role of
SL metabolism in this disease setting.
15.3.5 Inflammatory Bowel Disease Along with inhibition of S1P receptor func-
tion, modulation of CerS has also been explored
Inflammatory Bowel Disease (IBD) is a group of in IBD, particularly CerS2 and CerS6. In a mouse
diseases, including ulcerative colitis and Crohn’s model of colitis, loss of CerS2 destabilized the
disease, characterized by chronic inflammation epithelial barrier and tight junction in the intesti-
of gastrointestinal tract. Pathology is mediated nal membrane leading to increased intestinal per-
by leukocytes infiltration and massive production meability. CerS2-deficient mice have reduced
of pro-inflammatory cytokines leading to intesti- expression of junctional adhesion molecule A
nal damage [192, 193]. The first link between SL [208] and tight junction protein ZO-1 [209]. This
metabolism and IBD originated from studies outcome is associated with reduced levels of
showing that TNFα, a cytokine that induces the very-long acyl chain ceramides (C24) and
generation of S1P and synthesis of cyclooxygen- increased levels of long-chain sphingoid bases
ase-2 (COX-2) [194, 195], was induced in and C16-ceramides [208]. These findings further
patients with IBD [192, 196]. In addition, TNFα suggest that CerS2 is protective against colitis as
blockade alleviated clinical symptoms in mouse opposed to EAE in which CerS2-deficient mice
models of disease [197, 198]. Since ceramides are protected [26]. Interestingly, further explora-
also induced the activation of MAPK, a key tion of the role of CerS6 in colitis has led to
inducer of inflammatory responses [199], follow opposing conclusions. Scheffel et al. reported
up studies demonstrated that inhibition of that the transfer of CerS6-deficient CD4+ T cells
ceramide accumulation by targeting SMase or is protective in colitis [33]. On the contrary,
S1P receptor diminished clinical manifestations Helke et al. showed that in a model of DSS-
of disease in mouse models of colitis. For exam- induced colitis, loss of CerS6 exacerbates inflam-
ple, in a dextran sulfate sodium (DSS) model of mation [210]. Interestingly, pathology in
colitis, inhibition of SMase with a sphingomy- CerS6-deficient mice is not a result of reduced
elin analogue-7 reduced the formation of intestinal permeability but is associated with
ceramide, levels of cytokines, and intestinal enhanced neutrophil infiltration. However, more
injury [200]. In addition, administration of studies are needed to further elucidate the role of
FTY720 reduced disease symptoms in a mouse CerS in various models of colitis and its imple-
model of colitis, associated with a reduced pro- mentation in the development of CerS targeted
duction of the pro-inflammatory cytokine therapies in humans with IBD.
IL-12p70 and Th1 cytokines, and upregulation of
CD4+ Foxp3+ suppressive Treg cells [201].
Furthermore, administration of a new S1P recep- 15.4 Conclusions and Future
tor agonist, KRP-203, resulted in reduced lym- Directions
phocyte infiltration and production of
pro-inflammatory Th1 cytokines in the colonic While it has been thoroughly reviewed in other
mucosa [202]. Similarly, chemical and genetic cells, the roles of ceramides in immune cells have
inhibition of SK1 that is responsible for the pro- not been proportionally presented. In this chapter
duction of S1P reduced manifestations of colitis, we attempted to summarize the significant effects
of ceramides in immune cells and immune dis- nisms of ceramides in immune cell function and
eases. Numerous studies have highlighted the other cell types. Since diversity of ceramides,
importance of ceramides in a variety of pathways similar to other membrane and bioactive lipids,
necessary for the development, function, and arises from the different possible combinations
metabolism of immune cells. Ceramide accumu- of head groups and/or degree of saturation, the
lation has been described as a key step mediating effect of each type of SL and ceramide on every
and regulating various immune cell functions, immune cell opens endless possibilities in target-
including regulation of immune cell responses to ing various ceramides to retune immune cell
viruses, bacteria, and other foreign pathogens, function in cancer, transplantation, diabetes, MS,
migration, phagocytosis, and cytokine produc- IBD and many other diseases.
tion. For instance, ceramides have been impli-
cated in the modulation of responses to LPS, Acknowledgements The authors are indepted to Ms.
cytokine production, antigen presentation, and Yara Khodour for her diligent redrawing of Fig. 15.2 and
her full chapter proof. Dr. Johnny Stiban acknowledges
viral entry in DC; control of migration, cytokine the receipt of two grants from Birzeit University that
and superoxide production, and formation of allowed him to pursue this endeavor (Grant #240193 and
NETs in neutrophils; and response to TLR stimu- #241109).
lation, inducing apoptosis, and regulating
oxidative stress in macrophages. Moreover,

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Bioactive Lipids in Cancer,
Inflammation and Related 16
Diseases
Acute and Chronic Mild Traumatic Brain
Injury Differentially Changes Levels
of Bioactive Lipids in the CNS Associated
with Headache
Emma Leishman, Phillip E. Kunkler,
Joyce H. Hurley, Sally Miller,
and Heather B. Bradshaw
Abstract mTBI increased arachidonic acid and

Headache is a common complaint after mild repeated mTBI increased prostaglandins in
traumatic brain injury (mTBI). Changes in all 3 tissue types. mTBI affected multiple
the CNS lipidome were previously associ- TRPV agonists, including N-arachidonoyl
ated with acrolein-induced headache in ethanolamine (AEA), which increased in the
rodents. mTBI caused similar headache-like TNC and CER after single mTBI. After
symptoms in rats; therefore, we tested the repeated mTBI, AEA increased in the TG,
hypothesis that mTBI might likewise alter but decreased in the TNC. Common to all tis-
the lipidome. Using a stereotaxic impactor, sue types in single and repeated mTBI was an
rats were given either a single mTBI or a increase the AEA metabolite, N-arachidonoyl
series of 4 mTBIs 48 h apart. 72 h later for glycine, a potent activator of microglial
single mTBI and 7 days later for repeated migration. Changes in the CNS lipidome
mTBI, the trigeminal ganglia (TG), trigemi- associated with mTBI likely play a role in
nal nucleus (TNC), and cerebellum (CER) headache and in long-term neurodegenera-
were isolated. Using HPLC/MS/MS, ~80 lip- tive effects of repeated mTBI.
ids were measured in each tissue and com-
pared to sham controls. mTBI drove Keywords
widespread alterations in lipid levels. Single mTBI · Endocannabinoids · TRP · Lipidomics
· Lipoamine · Acyl amino acid · Acyl amide ·
E. Leishman · S. Miller · H. B. Bradshaw (*) Docosahexaenoyl ethanolamine ·
Department of Psychological and Brain Sciences, N-arachidonoyl glycine · BV-2
Indiana University, Bloomington, IN, USA
P. E. Kunkler · J. H. Hurley
Stark Neurosciences Institute, Indiana University
School of Medicine, Indianapolis, IN, USA

194 E. Leishman et al.
16.1 T
raumatic Brain Injury, were also neuroprotective in rodent TBI models
Headache, and Endogenous by reducing blood-brain barrier disruption, as
Cannabinoids measured by sodium fluorescein uptake, which
prevented the entry of pro-inflammatory immune
~40 million people worldwide endure a mild cells, and by attenuating microglial activation, as
traumatic brain injury (mTBI; also known as con- measured by immunohistochemistry [21].
cussion) each year [1], accounting for over 75% Cannabinoid receptor activation is hypothesized
of brain injuries [2]. Headache is a common com- to reduce headache pain [4, 18], suggesting con-
plaint after mTBI [2–6]. In studies of TBI patient vergent biochemistry between headache and TBI.
populations in the United States, around 70% of
participants reported having headaches during
the first-year post-injury [3, 5]. Demonstrating 16.2 Lipoamines as Endogenous
that post-mTBI headache is a global phenome- Activators of Transient
non, a study of TBI patients in China found that, Receptor Potential (TRP)
regardless of TBI severity, 58% of patients had Channels
headaches at 3 months post-injury, 54% at
6 months, and 49% at 1 year [6]. In contrast, only TRP channels are ubiquitous ligand-gated ion
12% of the patients reported pre-injury head- channels that serve a variety of functions through-
aches [6]. Often, post-mTBI headaches are out the body [22–31]. There are 28 known TRP
migraine-like [4, 6–9]. For example, in the study channels in mammalian systems; however, they
by Xu and colleagues, around 70% of the head- are arguably understudied to date as their endog-
aches described by TBI patients were classified enous ligands, distributions, and signaling prop-
as migraine-type [6]. Activation of the trigemino- erties are still in question [26, 29, 31]. In the
vascular system (TVS), the trigeminal innerva- context of disease, and more specifically the
tion of cranial vasculature [10], is hypothesized symptom of pain, TRP vanilloid type 1 (TRPV1)
to underlie migraine pain [11]. Sensitization can has been the focus of intense study and may rep-
occur after repeated TVS activation [11]. After resent the TRP channel being the most studied
the initial mTBI, there is a secondary inflamma- overall [23, 27, 28, 31–34]. More than a dozen
tory biochemical insult that occurs days later [12, bioactive lipids have been shown to activate
13]. These biochemical changes may promote TRPV1 [31, 35–40]. Our group recently identi-
headaches through the activity of inflammatory fied 8 of these [41]. Given the ubiquity of TRPs,
mediators, many of which are bioactive lipids, it is not surprising that they appear to have inter-
that are produced during chronic immune connected responses with other TRPs and
responses [4, 8]. GPCRs. A combination of TRPV1-TRP ankyrin
Bioactive lipids such as endogenous cannabi- 1 (TRPA1) activity has been observed in an ani-
noids (eCBs), the endogenous activators of CB1 mal model for headache wherein ablation of
and CB2 cannabinoid receptors [14–17], help TRPV1 in the trigeminal ganglia (TG) inhibited
regulate inflammation [17] and have emerging TVS activation driven by TRPA1 agonists [42].
roles in the TVS [18]. Brain levels of the eCB We recently showed that TRPA1 activation by
2-arachidonoyl glycerol (2-AG) [15, 16] signifi- acrolein drives increases in TRPV1 endogenous
cantly increased in a mouse model of TBI, start- bioactive lipids in the CNS [43].
ing at 4 h post-injury [19]. As 2-AG was Many endogenous TRPV ligands are
neuroprotective after TBI [12, 19, 20], it was lipoamines, which are conjugations of fatty acids
hypothesized that the increase in 2-AG was a and amines [22, 44–46]. The eCB N-arachidonoyl
compensatory mechanism to mitigate the damage ethanolamine (AEA) [14] is a lipoamine that acti-
of pro-inflammatory mediators released after vates TRPV1, as well as cannabinoid receptors
injury [19]. The effects of 2-AG were mostly [35]. Additional lipoamines activate TRPV1 such
mediated by CB1 [19]. However, CB2 agonists as the N-acyl ethanolamines (NAEs) N-oleoyl
16 Bioactive Lipids in Cancer, Inflammation and Related Diseases 195
ethanolamine (OEA) [37, 38], N-linoleoyl etha- measured in the acrolein model [54], showing
nolamine (LEA), and N-docosahexaenoyl etha- these very different models have similar effects
nolamine (DEA) [41], and certain lipoamines on measures of headache-like pain in rodents.
derived from arachidonic acid (AA) such as Given that headache driven by acrolein exposure
N-arachidonoyl GABA (A-GABA) [41] and was linked to alterations in bioactive lipids [43],
N-arachidonoyl taurine (A-Taur) [39]. we wanted to know whether headache caused by
N-arachidonoyl serine (A-Ser) was neuroprotec- a very different model, mTBI, would drive simi-
tive in a mouse model of TBI, and these effects lar alterations in lipid levels and whether the
were attenuated with a TRPV1 antagonist, as effects of repeated mTBIs would differ from the
well as a CB2 antagonist [47, 48]. effects of a single mTBI. To test the hypothesis
that acute and repeated mTBI alters the CNS lipi-
dome, we performed targeted lipidomics screens
16.3 B
ioactive Lipid Levels of the TG, TNC and the CER, from rats exposed
Change in a Model to single or repeated mTBI. Analyzing 74
of Headache lipoamine structural analogs, 2-acyl glycerol
structural analogs, prostaglandins (PGs), and free
Showing cross-talk with TRPV1, TRPA1 is fatty acids through targeted high-pressure liquid
expressed in sensory neurons [49]. The TRPA1 chromatography-coupled tandem mass spec-
agonist acrolein is an irritant and a major compo- trometry (HPLC/MS/MS) we were able to deter-
nent of air pollution [50, 51]. Molecular mecha- mine how this lipidome is modified. Both single
nisms of inflammatory pain and hypersensitization and repeated mTBI modified many bioactive lip-
of the TVS caused by irritants like acrolein par- ids including increased levels of the AEA metab-
tially depend on TRPA1 activity [52, 53]. In a rat olite [58] and GPR18 ligand [59], N-arachidonoyl
model of headache driven by exposure to acro- glycine (NAGly), and alterations of TRPV1 and
lein, TVS sensitization [53] and headache-like CB ligands. Increased levels of PGs were associ-
behaviors [54] were correlated with altered levels ated with repeated mTBI. These findings may
of bioactive lipids in the TVS and the cerebellum shed new light on how bioactive lipids in the
(CER), such as eCBs and endogenous lipoamine CNS play a role in headache associated with TBI.
TPRV1 ligands [43]. TVS tissues analyzed were
the TG, where cell bodies of trigeminal nerve
fibers are located [10], and the trigeminal nucleus 16.4 mTBI Protocols, Tissue
caudalis (TNC), part of the brainstem which Collection, and HPLC/MS/MS
receives sensory signals from the TG and proj- Analysis
ects them onto thalamo-cortical pathways [55].
These experiments provided evidence that acro- All animal procedures were approved by the
lein exposure drives changes in CNS lipids, sup- Institutional Animal Care and Use Committee of
porting a working hypothesis that both TRPA1 Indiana University School of Medicine and were
and TRPV1 activity play a role in headache. in accordance with the ethics guidelines set by
Exposure to a single mTBI caused headache- the International Association for the Study of
like symptoms in rats [56, 57]. Specifically, Pain [60]. In total, 24 adult male (170–250 g)
mTBI caused periorbital allodynia, meaning that Sprague-Dawley rats (Harlan, Indianapolis, IN,
stimuli that were non-nociceptive before the USA) were used: 12 were assigned to the single
injury became painful after the injury [56, 57]. mTBI paradigm and 12 were assigned to the
Providing more evidence of alterations in the repeated mTBI paradigm. Rats were housed in
TVS that could support headache post-TBI, lev- pairs with food and water available ad libitum,
els of pro-nociceptive peptides were upregulated and were kept on a standard light/dark cycle.
in the TNC after TBI [56, 57]. Periorbital allo- Using an Impact One stereotaxic impactor (Leica
dynia was a marker of headache-like symptoms Microsystems, Wetzlar, Germany) with a 3 mm
diameter tip, mTBI was induced by 4.0 m/s (MRM) tailored for each group of structurally
impact to 2.0 mm depth on the right hemisphere similar compounds (Fig. 16.1). HPLC/MS/MS
adjacent to bregma and sagittal sutures. This was data was analyzed using Analyst software
performed once on single mTBI rats and per- (Applied Biosystems) as previously described
formed 4 times, with 48 h between each impact, [41, 43, 46, 65–72]. Chromatograms were gener-
on repeated mTBI rats. 6 of the 12 rats in each ated by determining the retention time of ana-
paradigm (single or repeated) received mTBI and lytes from the C18 analytical column with a
the other 6 received a sham treatment. [M−1] or [M + 1] parent peak and a fragmenta-
To better model concussion, the injury was a tion peak corresponding to the programmed val-
closed-head injury and the rats did not undergo ues. Extraction efficiency was calculated with the
craniotomy [61]. Confirming a mild injury, the d8NAGly or d8AEA-spiked recovery vial as a
impact did not damage the skull. As reviewed by standard as done previously [41, 43, 46, 65–72].
Fehily and Fitzgerald, other rodent models of For each individual lipid in the TG, TNC and
repeated mTBI use similar parameters [62]. For CER, concentrations in moles per gram of wet
example, a study by David Brody’s group tissue weight adjusted for percent recovery from
described modeling repeated mTBI in mice by mTBI animals were compared to sham using a
striking the exposed, intact skull with a rubber tip one-way ANOVA. Statistical tests were carried
at 5.0 m/s to a depth of 3.3 mm [63]. Although out using SPSS Statistics (IBM, Armonk, NY,
this injury caused cognitive deficits, lasting USA) and significance was defined as p < 0.05
microglial activation and disruption to white- and trending significance was defined as p < 0.10.
matter axonal cytoskeletal integrity, there was no Using ANOVAs instead of more flexible t-tests
damage to the skull or any readily observable provides more constraints on this type of larger
damage to the underlying brain tissue [63]. data sets.
Bolton and Saatman tested various impact depths As a first level of analysis presented here, the
in a closed-head mouse model of repeated mTBI percentage of the detected lipids affected by sin-
and found that 2.0 mm impact depth did not dam- gle and repeated mTBI in each tissue type was
age the skull but caused gliosis which was wors- calculated. To calculate the percent of the lipids
ened when mTBI was repeated every 24 h [64]. detected that were unaffected by mTBI, the num-
Thus, our parameters are consistent with models ber of changes (p < 0.10) in a tissue type for a
employed by other groups studying mTBI in specific condition (single or repeated) was first
rodents. Rats were sacrificed via decapitation, subtracted from the number of lipids detected in
either 72 h after the single mTBI or 1 week after that tissue (which represents the number of pos-
the last session in repeated mTBI. Brains were sible changes). This number was then divided by
immediately removed. Following removal of the the number of lipids detected and multiplied by
brain, the left TG was removed from the skull and 100. For example, in the TNC in the repeated
frozen on dry ice. The brains were dissected into mTBI condition, there were 73 lipids detected
targeted regions: TNC and CER, and frozen on and 29 of them changed with repeated mTBI,
dry ice. TG, TNC and CER samples were stored meaning that 44 did not change. 44 divided by 73
at −80 °C before being processed for lipid times 100 is 60.27%, so 60.27% of the detected
extraction. lipidome did not change with repeated mTBI in
Tissue extracts were performed as previously the TNC. This percentage can be viewed in the
described [41, 43, 46, 65–72]. Lipids were ana- left, blue columns of Fig. 16.2. The next portion
lyzed using an Applied Biosystems API 3000 of Fig. 16.2 (middle, green) gives the percentage
triple quadrupole mass spectrometer with elec- of the lipids detected in each tissue that increased
trospray ionization (Foster City, CA, USA) as in mTBI. Sticking with the repeated mTBI TNC,
previously described [43]. Levels of each com- of the 29 changes that were detected, 14 of them
pound were determined by running each sample were increases. 14 is then divided by the number
using a multiple-reactions monitoring method of lipids detected (73 in this case) and multiplied
N-acyl alanine [M - H]- Fragment N-acyl proline [M - H]- Fragment
N-palmitoyl alanine 326.5 88.09 N-palmitoyl proline 352.53 114.12
N-stearoyl alanine 354.55 88.09 N-stearoyl proline 380.59 114.12
N-oleoyl alanine 352.53 88.09 N-oleoyl proline 378.31 114.12
N-linoleoyl alanine 350.52 88.09 N-linoleoyl proline 376.56 114.12
N-arachidonoyl alanine 374.5 88.09 N-arachidonoyl proline 400.58 114.12
N-docosahexaenoyl alanine 398.56 88.09 N-docosahexaenoyl proline 424.6 114.12
N-acyl dopamine [M - H]- Fragment N-acyl serine [M - H]- Fragment
N-oleoyl dopamine 416.3 123.2 N-palmitoyl serine 342.3 74
N-arachidonoyl dopamine 438.4 123.2 N-stearoyl serine 370.3 74
N-acyl ethanolamine [M + H]+ Fragment N-oleoyl serine 368.3 74
N-palmitoyl ethanolamine 300.29 62.1 N-linoleoyl serine 366.27 74
N-stearoyl ethanolamine 328.3 62.1 N-arachidonoyl serine 390.3 74
N-oleoyl ethanolamine 326.3 62.1 N-docosahexaenoyl serine 414.3 74
N-linoleoyl ethanolamine 324.3 62.1 N-acyl taurine [M - H]- Fragment
N-arachidonoyl ethanolamine 348.3 62.1 N-palmitoyl taurine 362.6 124
N-docosahexaenoyl ethanolamine 372.6 62.1 N-stearoyl taurine 390.6 124
N-acyl GABA [M - H]- Fragment N-oleoyl taurine 388.6 124
N-palmitoyl GABA 340.54 102.1 N-arachidonoyl taurine 410.6 124
N-stearoyl GABA 368.58 102.1 N-acyl tryptophan [M - H]- Fragment
N-oleoyl GABA 366.57 102.1 N-palmitoyl tryptophan 441.63 203.1
N-linoleoyl GABA 364.54 102.1 N-stearoyl tryptophan 469.68 203.1
N-arachidonoyl GABA 388.57 102.1 N-oleoyl tryptophan 467.67 203.1
N-docosahexaenoyl GABA 412.59 102.1 N-linoleoyl tryptophan 465.65 203.1
N-acyl glycine [M - H]- Fragment N-arachidonoyl tryptophan 489.67 203.1
N-palmitoyl glycine 312.26 74.2 N-docosahexaenoyl tryptophan 513.69 203.1
N-stearoyl glycine 340.3 74.2 N-acyl tyrosine [M - H]- Fragment
N-oleoyl glycine 338.3 74.2 N-palmitoyl tyrosine 418.59 180.18
N-linoleoyl glycine 336.3 74.2 N-stearoyl tyrosine 446.65 180.18
N-arachidonoyl glycine 360.3 74.2 N-oleoyl tyrosine 444.63 180.18
N-docosahexaenoyl glycine 384.3 74.2 N-linoleoyl tyrosine 442.61 180.18
N-acyl leucine [M - H]- Fragment N-arachidonoyl tyrosine 466 180.18
N-palmitoyl leucine 368.58 130.1 N-docosahexaenoyl tyrosine 490.66 180.18
N-stearoyl leucine 396.63 130.1 N-acyl valine [M - H]- Fragment
N-oleoyl leucine 394.61 130.1 N-palmitoyl valine 354.31 116.31
N-linoleoyl leucine 392.6 130.1 N- stearoyl valine 382.6 116.14
N-docosahexaenoyl leucine 440.64 130.1 N-oleoyl valine 380.59 116.14
N-acyl methionine [M - H]- Fragment N-linoleoyl valine 378.58 116.14
N-palmitoyl methionine 386.62 148.2 N-docosahexaenoyl valine 426.62 116.14
N-stearoyl methionine 414.64 148.2 2-acyl glycerol [M + H]+ Fragment
N-oleoyl methionine 412.65 148.2 2-palmitoyl glycerol 331.5 239.5
N-linoleoyl methionine 410.64 148.2 2-oleoyl glycerol 357.5 265.2
N-arachidonoyl methionine 434.66 148.2 2-linoleoyl glycerol 355.5 245
N-docosahexaenoyl methionine 458.68 148.2 2-arachidonoyl glycerol 379.3 287.5
N-acyl phenylalanine [M - H]- Fragment Free Fatty Acids [M - H]- Fragment
N-palmitoyl phenylalanine 402.59 164.1 Oleic acid 281.5 263
N-stearoyl phenylalanine 430.65 164.1 Linoleic acid 279.5 261
N-oleoyl phenylalanine 428.63 164.1 Arachidonic acid 303.5 285
N-linoleoyl phenylalanine 426.61 164.1 PhosphoNAEs [M - H]- Fragment
N-arachidonoyl phenylalanine 450.64 164.1 PhosphoLEA 403.5 58.5
N-docosahexaenoyl phenylalanine 474.66 164.1 Prostaglandins [M - H]- Fragment
PGE2 351.2 315
PGF2α 353.3 309.2
6-keto-PGF1α 369.3 206.9
Fig. 16.1 Lipids in HPLC/MS/MS screening library parent ion is the only ion allowed to pass through the first
with parent ion and fragment ion masses. Lipids are quadrupole of the API3000. The parent ion is then frag-
grouped by amide family and all members of that lipid mented into the collision chamber and an abundant frag-
family are screened in a multiple reactions monitoring ment can be selected as the fragment ion. Only the
(MRM) method. The parent ion mass is also listed: nega- selected fragment ion can pass from the collision chamber
tive ionization mode, resulting in a [M – H]− parent ion, is into the third quadrupole. Therefore, unknown lipids are
used for all methods except the N-acyl ethanolamine and matched to known standards according to retention time
2-acyl glycerol methods, which uses positive ionization from the analytical column and according to their mass
and generates a parent ion with a mass of [M + H]+. The fingerprint
% Lipids % Lipids % Lipids

detected detected detected
unchanged increased decreased
by mTBI by mTBI by mTBI
Cerebellum Single 53.85% 46.15% 0.00%
Repeated 75.64% 21.80% 2.56%
Trigeminal Ganglia Single 77.50% 18.75% 3.75%
Repeated 63.64% 35.06% 1.30%
Trigeminal Nucleus Single 63.16% 36.84% 0.00%
Repeated 60.27% 19.18% 20.55%
Fig. 16.2 Summary of effects of single and repeated or repeated mTBI. The middle, green column represents
mild traumatic brain injury (mTBI) on lipid levels in the percentage of lipids detected that increased with single
the rat cerebellum, trigeminal ganglia, and trigeminal and repeated mTBI and the right, orange column repre-
nucleus caudalis. The left, blue column represents the sents the percentage of lipids detected that decreased with
percentage of the lipids detected within each of tissue type single and repeated mTBI relative to sham. See Methods
(row) with concentrations that were unaffected by single for more details on how percentages were calculated
by 100 to yield the percentage increased Immortalized BV2 microglia cells were a gift
(19.18%). The right portion of Fig. 16.2 shows from Dr. N. Stella at University of Washington,
the percentage of the lipids detected that Seattle. Boyden migration chamber assays and
decreased with single or repeated mTBI. The per- analyses were performed identically to those pre-
centage was calculated by taking the number of viously described in our laboratory [59, 73].
lipids with decreased concentrations in each tis-
sue and dividing it by the number of lipids
detected in that tissue. For the TNC, 15 lipids 16.4.1 mTBI Has Broad Effects
decreased with repeated mTBI, giving a percent- on the CER, TG, and TNC
age of 20.55% for the proportion of the lipidome Lipidome
that decreased. When the 3 different percentages
for a tissue are added, they should equal 100. For Of the 74 lipoamines in our screening library
the repeated mTBI TNC, 60.27 plus 19.18 plus (Fig. 16.1), over 50 were detected in the TG,
20.55 equals 100. TNC, and CER. If a lipid was detected in the
Analyzed lipidomics data from the TG, TNC, samples from injured rats it was also found in the
and CER are represented in tabular format illus- corresponding sham samples. In both single and
trating both the direction and magnitude of change. repeated mTBI, 78 lipids were detected in the
For example, the mean level of AEA was CER (92% of the 85 lipids in the screening
9.20 × 10−11 moles per gram in the CER of the rats library). For the TG and TNC, different numbers
subjected to a single mTBI and 7.86 × 10−11 moles of lipids were detected after single and repeated
per gram in the corresponding sham CER. mTBI. In the TG, 80 lipids were detected in the
9.20 × 10−11 divided by 7.86 × 10−11 is 1.17, assign- single condition (94%) and 77 in the repeated
ing it 1 up arrow in the figures because the magni- condition (91%). Fewer lipids were detected in
tude of change was between 1 and 1.5 times higher the TNC (single: 76 lipids – 89%; repeated: 73
than sham. Providing an example of a decrease, lipids – 86%). Tables listing the lipids detected
the average level of AEA in the TNC of rats and their mean levels in each tissue type in sham
exposed to repeated mTBI was 5.22 × 10−12 moles and mTBI tissues are available upon request from
per gram and the average level of AEA in the TNC the authors.
of the sham rats was 7.32 × 10−12 moles per gram; The first level of analysis examined the pro-
5.22 × 10−12 divided by 7.32 × 10−12 equals 0.71, portion of the lipids detected that were
and the reciprocal of 0.71 is 1.41, meaning that the unchanged, upregulated, or downregulated with
decrease is between 1 and 1.5 times sham levels mTBI relative to sham. Effects of single mTBI
and giving it 1 down arrow on our scale. on the lipidome were mostly tissue-dependent
(Figs. 16.2, 16.3, 16.4, and 16.5). The highest increases in a lipid’s concentration. In contrast,
percentage of changes in lipid levels occurred in changes measured in the TG included decreases
the CER (36 lipids – 46% of the lipids detected), (15 increases, 3 decreases).
followed by the TNC (28 lipids – 37%). The area Repeated mTBI affected a similar number of
with the lowest percentages of changes in lipid the detected lipids (Figs. 16.2, 16.3, 16.4, and
levels was the TG (18 lipids – 22.5%). All of the 16.5). However, the specific tissue most affected
changes driven in the TNC and CER were shifted in repeated mTBI. The highest percent-
Cerebellum Trigeminal Ganglia Trigeminal Nucleus

Lipid Species mTBI X 1 mTBI X 4 mTBI X 1 mTBI X 4 mTBI X 1 mTBI X 4
N-acyl ethanolamine
N-palmitoyl ethanolamine ↓
N-stearoyl ethanolamine
N-oleoyl ethanolamine ↓
N-linoleoyl ethanolamine ↑ ↑ ↑ ↓
N-arachidonoyl ethanolamine ↑ ↑ ↑ ↓
N-docosahexaenoyl ethanolamine ↑ ↓ ↑ ↑ ↑ ↓
N-acyl glycine
N-palmitoyl glycine ↑
N-stearoyl glycine ↑ ↑ ↑ ↓
N-oleoyl glycine ↑ ↑ ↑
N-linoleoyl glycine ↑
N-arachidonoyl glycine ↑ ↑ ↑ ↑ ↑ ↑
N-docosahexaenoyl glycine ↑ ↑ ↑
N-acyl taurine
N-palmitoyl taurine ↑ ↑
N-stearoyl taurine ↑ ↓ ↑
N-oleoyl taurine ↑ ↑ ↑
N-arachidonoyl taurine ↑ ↑ ↑ ↑
2-acyl glycerol
2-palmitoyl glycerol ↑↑ ↑ ↑↑
2-oleoyl glycerol ↑ ↓
2-linoleoyl glycerol ↑ ↑ ↑
2-arachidonoyl glycerol ↑ ↑
Free Fatty Acids
Oleic acid ↑ ↑
Linoleic acid ↑ ↑ ↑ ↑ ↑ ↓
Arachidonic acid ↑ ↑ ↑ ↑ ↑
Prostaglandins
PGE2 ↑ ↑ ↑↑
PGF2α ↑ ↑↑ ↑ ↑↑ ↑
6-ketoPGF1α ↑ ↑ ↓ ↑↑ ↑ ↑
Fig. 16.3 Comparison of effects of single or repeated colors represent increases, whereas orange colors repre-
mild traumatic brain injury (mTBI) on levels of N- sent decreases. Darker colors represent significant
acyl ethanolamines, N-acyl glycines, N-acyl taurines, changes of p < 0.05 and lighter colors represent trending
2-acyl glycerols, free fatty acids, and prostaglandins. changes of p < 0.10, as determined by one-way
Levels of targeted bioactive lipids in the cerebellum, tri- ANOVA. The number of arrows indicates the magnitude
geminal ganglia, and trigeminal nucleus caudalis of rats of the difference between mTBI and sham. One arrow
given a single mTBI (mTBI × 1) or 4 repeated mTBIs indicates a magnitude difference of less than 1.5 fold
(mTBI × 4) were compared to respective sham rats. and 2 arrows indicate a 1.5–1.99 fold change. A blank
Cells with shaded arrows indicate a significant or trend- cell indicates that there was no change in the lipid’s level
ing change in that lipid with mTBI. The arrow color due to mTBI. See Methods for more detailed description
indicates the direction of change relative to sham. Green of analysis
Fig. 16.4 Comparison of effects of single or repeated geminal nucleus caudalis (TNC) of rats exposed to a sin-
mild traumatic brain injury (mTBI) on levels of N- gle mild traumatic brain injury (single mTBI – black bars)
docosahexaenoyl ethanolamine (DEA) and N- or sham (open bars) and of rats exposed to a series of 4
arachidonoyl glycine (NAGly). Panel A shows bar mTBIs (repeated mTBI – black bars) or sham (open bars).
graphs for mean levels in moles per gram tissue of DEA in Error bars are ± standard error of the mean. Asterisk (∗)
the cerebellum (CER), trigeminal ganglia (TG), and tri- indicates a difference between mTBI and sham of p < 0.05
age of changes occurred in the TNC (29 lipids – mental toxin, acrolein [43]. This means that
40% of the lipids detected), followed by the TG increases in NAGly may serve as a marker of
(28 lipids – 36%). Unlike in single mTBI, headache caused by very different stimuli, mTBI
where the most changes were detected in the and TRPA1 activation. There are multiple path-
CER, in repeated mTBI the CER had the fewest ways that regulate NAGly levels. NAGly is a
changes detected (19 lipids – 24%). The ratio of metabolite of AEA and can be formed from AEA
increases and decreases also shifted, with via 2 enzymatic pathways, one mediated by
decreases occurring in every tissue type. In the alcohol dehydrogenase and the other mediated
TG and CER, most of the changes were by fatty acid amide hydrolase (FAAH) [58].
increases (27 increases of 28 changes for TG Indeed, in FAAH knockout (KO) mice, brain
and 17/19 for CER). The TNC stands out, levels of NAGly are significantly reduced,
because it was the only tissue type where most whereas AEA levels are significantly higher
of the changes were decreases, suggesting func- compared to wild-type (WT) [69]. The concur-
tional specialization in this area that can poten- rent reduction in AEA and other NAEs and
tially underlie a more dynamic response to increase in NAGly in the TNC after repeated
injuries (15/29; Figs. 16.2, 16.3, 16.4, and mTBI could potentially be driven by an increase
16.5). Given that the CER was the most affected in AEA metabolism via FAAH. NAGly produc-
area after a single mTBI, a single mTBI may tion can also be mediated by glycine N-acyl
therefore have a stronger effect on bioactive transferase enzymes [75] and by the newly-char-
lipid production in sites closer to the injury, acterized enzyme peptidase M20 domain con-
such as the CER, whereas the effects of repeated taining 1 (PM20D1) [76, 77]. PM20D1 regulates
mTBI on bioactive lipid levels may spread to mitochondrial uncoupling via regulation of
more distant areas. endogenous lipoamines. As mitochondrial dys-
function is a hallmark of neuroinflammation
after TBI [13], the relationship between
16.4.2 Effects of mTBI on Bioactive PM20D1, TBI, and lipoamine regulation should
Lipids with Relatively More be further investigated.
Well-Studied Biosynthetic All other N-acyl glycine lipids that changed
Pathways and Protein Targets with a single mTBI showed an increase
(Fig. 16.3). After a single mTBI, N-oleoyl gly-
16.4.2.1 N -Acyl Glycine Lipid Species cine (O-Gly) increased in all 3 tissue types, along
Are Upregulated After mTBI with the increase in NAGly, which was still pres-
Levels of NAGly increased in all 6 groups in the ent after repeated mTBI. Alterations in O-Gly
mTBI experiments (Figs. 16.3 and 16.4). have previously been associated with TBI in
Increases in NAGly were also found after rodent models, as O-Gly was upregulated in the
repeated exposure to low levels of the environ- mouse insular cortex 24 h after TBI [78]. Levels
Fig. 16.4 (continued) whereas the hash sign (#) indicates nucleus caudalis (TNC) of rats exposed to a single mild
a difference between mTBI and sham of p < 0.10, as traumatic brain injury (single mTBI – black bars) or sham
determined by one-way ANOVA. After single mTBI, (open bars) and of rats exposed to a series of 4 mTBIs
DEA increased in all 3 tissue types, corresponding to (repeated mTBI – black bars) or sham (open bars). Error
green cells with 1 up arrow in Fig. 16.4. After repeated bars are ± standard error of the mean. Asterisk (∗) indi-
mTBI, DEA decreased in the CER and TNC, correspond- cates a difference between mTBI and sham of p < 0.05
ing to orange cells with 1 down arrow in Fig. 16.4, but whereas the hash sign (#) indicates a difference between
increased in the TG, corresponding to a green cell with 1 mTBI and sham of p < 0.10, as determined by one-way
up arrow in Fig. 16.4. Panel B shows bar graphs for mean ANOVA. In all 3 tissue types, levels of NAGly increased
levels in moles per gram tissue of NAGly in the cerebel- after single and repeated mTBI, corresponding to green
lum (CER), trigeminal ganglia (TG), and trigeminal cells with 1 up arrow in Fig. 16.4
Cerebellum Trigeminal Ganglia Trigeminal Nucleus

Lipid Species mTBI X 1 mTBI X 4 mTBI X 1 mTBI X 4 mTBI X 1 mTBI X 4
N-acyl alanine
N-palmitoyl alanine ↑ ↑ ↑
N-stearoyl alanine ↑ ↑↑ ↓
N-oleoyl alanine ↑ ↑ ↑ ↑
N-arachidonoyl alanine ↑↑↑ ↑↑↑ ↑
N-docosahexaenoyl alanine ↑↑ ↑↑
N-acyl GABA
N-palmitoyl GABA ↓ ↑↑ ↑
N-stearoyl GABA ↑ ↑ ↓
N-oleoyl GABA ↑ ↓ ↓
N-linoleoyl GABA ↑ BAL BAL
N-arachidonoyl GABA ↑ ↑ ↑ ↑
N-docosahexaenoyl GABA ↓
N-acyl leucine
N-palmitoyl leucine ↑
N-stearoyl leucine ↑ ↓
N-oleoyl leucine ↓
N-linoleoyl leucine ↑
N-docosahexaenoyl leucine ↑
N-acyl methionine
N-palmitoyl methionine ↑
N-stearoyl methionine ↑ ↑
N-oleoyl methionine ↑
N-arachidonoyl methionine ↑
N-docosahexaenoyl methionine BAL ↑↑↑ BAL BAL
N-acyl phenylalanine
N-stearoyl phenylalanine ↓
N-oleoyl phenylalanine ↑
N-arachidonoyl phenylalanine ↑ ↑ ↑ ↑ ↑
N-docosahexaenoyl phenylalanine ↓↓
N-acyl proline
N-oleoyl proline ↑ ↑
N-arachidonoyl proline BAL BAL BAL ↑↑ BAL BAL
N-acyl serine
N-palmitoyl serine ↑ ↑
N-stearoyl serine ↑
N-oleoyl serine ↑
N-linoleoyl serine ↑
N-arachidonoyl serine ↑ ↑ ↑
N-acyl tryptophan
N-stearoyl tryptophan ↑
N-acyl tyrosine
N-palmitoyl tyrosine ↑
N-oleoyl tyrosine ↑
N-linoleoyl tyrosine ↑↑↑ BAL
N-arachidonoyl tyrosine ↑ ↑ ↑
N-acyl valine
N-palmitoyl valine ↑
N- stearoyl valine ↑ ↑ ↑
N-docosahexaenoyl valine ↑↑ BAL BAL
phosphoLEA
phosphoLEA BAL ↑↑ BAL ↑↑
Fig. 16.5 Effects of single and repeated mild trau- tophan, N-acyl tyrosine, and N-acyl valine families of
matic brain injury (mTBI) on additional bioactive bioactive lipids, and effects on phosphoLEA. Levels of
lipids in the cerebellum, trigeminal ganglia, and tri- targeted bioactive lipids in the cerebellum, trigeminal
geminal nucleus. Specifically, this heatmap shows ganglia, and trigeminal nucleus caudalis of rats given a
effects of mTBI on members of the N-acyl alanine, single mTBI (mTBI × 1) or 4 repeated mTBIs (mTBI × 4)
N-acyl GABA, N-acyl leucine, N-acyl methionine, N-acyl were compared to respective sham rats. Only lipids that
phenylalanine, N-acyl proline, N-acyl serine, N-acyl tryp- had significant or trending changes with either single or
of the TRPV1 agonist N-docosahexaenoyl gly- tained up to 1 week after repeated mTBI suggests
cine (D-Gly) [41] also increased in the CER and broad increases in neuroinflammation [79].
N-stearoyl glycine (S-Gly) also increased in the Microglia can release pro-inflammatory lipid sig-
TG. The TNC had the most changes in N-acyl naling molecules such as PGs [4]. It is possible
glycine levels after a single mTBI, with levels of that the increases in PGs observed after repeated
all 6 N-acyl glycines increasing. After repeated mTBI are a consequence of increased microglial
mTBI, the increase in NAGly was the only activation. Experiments are planned to see if
increase in an N-acyl glycine in the TNC. In the microglia release PGs when incubated with
TNC, S-Gly decreased after repeated mTBI, NAGly.
which was the only mTBI-driven decrease mea- Novel data shown here demonstrates that
sured in N-acyl glycines. S-Gly changed in the NAGly is not the only N-acyl glycine that can
opposite direction in the CER, increasing after induce microglial migration. At 10 μM,
repeated mTBI. D-Gly increased in the TG only N-palmitoyl glycine, S-Gly, O-Gly, N-linoleoyl
(Figs. 16.3 and 16.4). glycine, NAGly, and D-Gly induced migration
in BV-2 microglia (Fig. 16.6a, b). This demon-
16.4.2.2 N AGly and Additional N-Acyl strates how BV2 microglia might not be
Glycine Lipids Drive responding to the identity of the acyl chain but
Microglial Migration are instead sensitive to glycine-containing
NAGly drives dose-dependent migration in BV2 lipoamines. Follow-up studies should examine
microglia via activation of GPR18, which can be how combining these compounds affects
inhibited by A-Ser [59]. A-Ser’s effects on migration, as levels of multiple N-acyl glycines
microglia could underlie some of its reported change with mTBI. Unpublished data from the
neuroprotective actions after mTBI [47, 48] Hurley lab confirmed that there were increased
because microglial migration is understood to be numbers of microglia in the corpus callosum of
a marker of neuroinflammation [47]. Other stud- mTBI rats, suggesting some effects on migra-
ies have shown an increase in microglial activation (personal communication). Highlighting
tion after mTBI, with microglia rapidly migrating potential cross-talk between TRPA1 and
towards damaged tissue [79]. However, the utility microglia, endogenous TRPA1 ligands released
of the microglial response is complex [13]. by microglia in the spinal cord are hypothe-
Initially, microglia may be useful at clearing sized to contribute to central sensitization after
away damaged axonal tissue, which can promote a peripheral nerve injury. Both TRPA1 antago-
regeneration of damaged axons [13]. However, nists and blockers of microglia can reduce cen-
sustained microglial activation does not seem to tral pain hypersensitivity in animal models
be as beneficial and appears to promote inflam- [80]. Therefore, it is worth investigating
mation rather than regeneration [13, 79]. The fact whether TRPA1 antagonists or microglial
that the increase in NAGly was widespread, blockers can similarly reduce headache pain
occurring in sites distant from the injury, and sus- secondary to TBI.
Fig. 16.5 (continued) repeated mTBI in at least 1 tissue magnitude of the difference between mTBI and sham.
type are shown here. Cells with shaded arrows indicate a One arrow indicates a magnitude difference of less than
significant or trending change in that lipid with 1.5 fold, 2 arrows indicate a 1.5–1.99 fold change, and 3
mTBI. The arrow color indicates the direction of change arrows indicate a 2–2.99 fold change. A blank cell indi-
relative to sham. Green colors represent increases, cates that there was no change in the lipid’s level due to
whereas orange colors represent decreases. Darker colors mTBI. BAL stands for below analytical limits and means
represent significant changes of p < 0.05 and lighter col- that lipid was not detected in all samples in that tissue
ors represent trending changes of p < 0.10, as determined type. See Methods for more detailed description of
by one-way ANOVA. The number of arrows indicates the analysis
Fig. 16.6 Cellular migration activity of N-acyl gly- level when compared to vehicle. Panel B: At 10 μM,
cines in BV2 microglial cells. Panel A: At 10 μM, N-arachidonoyl glycine (NAGly), N-linoleoyl glycine
N-docosahexaenoyl glycine (DocGly), N-arachidonoyl (LinGly), N-stearoyl glycine (StrGly), and N-palmitoyl
glycine (NAGly), N-linoleoyl glycine (LinGly), and glycine (PalGly) caused cellular migration that was sig-
N-oleoyl glycine (OlGly) caused cellular migration that nificantly greater than the vehicle control. 4 plates were
was significantly greater than the vehicle control. 7 plates analyzed with 20 repeats for each condition per plate. ∗
were analyzed with 20 repeats for each condition per indicates a mean difference significant at the 0.05 level
plate. ∗ indicates a mean difference significant at the 0.05 when compared to vehicle
16.4.3 N-Docosahexaenoyl TNC after repeated mTBI, along with N-palmitoyl

Ethanolamine (DEA) May Play ethanolamine (Figs. 16.3 and 16.4). Reductions
a Key Role in the Effects in NAEs in the TNC after repeated mTBI could
of mTBI with a Potential be driven by a decrease in biosynthesis via N-acyl
Implication in Resolving phosphatidyl ethanolamine-specific phospholi-
Pathways pase D (NAPE-PLD) [70]. In NAPE-PLD KO
mice, brain levels of NAGly either stayed the
As the most affected NAE, the TRPV1 agonist same or increased while AEA decreased [70],
DEA [41] increased in all single mTBI groups consistent with what was seen in the TNC after
but decreased in the CER and the TNC with repeated mTBI. Increased levels of PGs in the
repeated mTBI. The eCB AEA increased in the TNC after repeated mTBI (Fig. 16.3) are also
CER and TNC after a single mTBI, but not in the consistent with NAPE-PLD blockade, as NAPE-
TG. LEA also increased in the CER and TNC, PLD KO mice have higher levels of PGs in the
meaning that multiple TRPV1 agonist NAEs [41] CNS compared to WT [70].
were elevated in these tissues after a single The omega-3 fatty acid, docosahexaenoic
mTBI. Repeated mTBI drove a tissue-specific acid (DHA) is a precursor for DEA and can be
mixture of increases and decreases in NAEs, with neuroprotective after TBI [81–85]. For example,
increases restricted to the TG. The only change in rats were subjected to an impact acceleration
NAEs in the CER was a decrease in DEA. In con- injury, which causes diffuse axonal injury [81].
trast, DEA increased in the TG, along with LEA For the next 30 days, injured rats were given
and AEA. OEA decreased slightly in the TG. In vehicle or DHA [81]. In rats supplemented with
contrast to single mTBI where LEA, AEA, and DHA, plasma levels of DHA increased and the
DEA increased, these NAEs decreased in the ratio of AA to eicosapentaenoic acid, a general
marker of inflammation, decreased [81]. In rats effects are due to conversion into pro-resolving
given vehicle, plasma levels of DHA were sig- mediators such as D-series resolvins and neuro-
nificantly reduced [81]. DHA had a protective protectin [81].
effect and decreased the number of amyloid pre- The primary biosynthetic pathways for these
cursor protein-positive and caspase-3 positive DHA-derived lipids, 15-lipoxygenase
(apoptotic) neurons in the brainstem [81]. The (15-LOX) can convert DHA into
same group performed a similar follow-up 17S-hydroperoxydocosahexaenoic acid
study, but here they gave rats DHA for 30 days (17S-H(p)-DHA), whereas cyclooxygenase 2
before the injury instead of after the injury [83]. can convert DHA into the enantiomer,
Similar results were uncovered, with DHA hav- 17R-hydroperoxydocosahexaenoic acid
ing a neuroprotective effect against TBI, sug- (17R-H(p)-DHA) [86]. 5-lipoxygenase
gesting that DHA can also be used as a (5-LOX) converts 17S-H(p)-DHA to resolvin
prophylactic to protect the brain against the con- D1, whereas the same enzyme converts
sequences of injury [83]. 17R-H(p)-DHA into aspirin-triggered resolvin
In another study, rats were given a DHA- D1 [86]. Alternatively, 17S-H(p)-DHA and
supplemented diet or a control diet for 30 days 17R-H(p)-DHA can first be converted into
before receiving a mild fluid percussion injury epoxide intermediates which are then hydro-
[82]. Rats on the control diet experienced learn- lyzed to produce neuroprotection D1 [86].
ing difficulties after the injury, as measured by Resolvin D1 can reduce immune cell infiltra-
the Morris water maze, whereas rats that tion and reduces inflammatory pain [87].
received DHA did not show these deficits [82]. Likewise, neuroprotectin D1 reduces inflam-
Furthermore, injury-related decreases in BDNF, matory pain and can also inhibit pain signals
CREB, and synapsin I, were not measured in via TRPV1 at intrathecal doses of just 0.1–
the DHA-supplemented rats and lower levels of 10 ng [88]. Given that TRPV1 ligands are ele-
oxidized proteins were measured in the hippo- vated after a single mTBI, it is possible that
campus of the DHA-rats, suggesting a reduc- neuroprotectin D1 could reduce TVS sensitiza-
tion in oxidative stress [82]. DHA also appears tion and therefore decreases headache pain.
to protect against inflammation after TBI, as Pro-resolving mediators are currently being
rats that were given daily injections of DHA for investigated as neuroprotective therapies for
21 days showed a reduction in markers of acti- brain injuries [89], as they are highly potent anti-
vated microglia, which were elevated after TBI inflammatory bioactive lipids [90]. Treating mice
[85]. Of the activated microglia that were still with diffuse axonal injury with aspirin-triggered
present in the CNS of the DHA-treated rats, resolvin D1 for 3 days pre-injury and 7 days post-
more of these expressed the anti-inflammatory injury partially rescued the cognitive deficits and
marker CD206 and fewer expressed the pro- motor deficits, as measured by the novel object
inflammatory marker CD16/32 [85]. On the recognition task and the rotorod task [90].
other hand, when rats are raised on a diet low in Neuroprotectin D1 formation increased in the
DHA and other omega-3 fatty acids, they show mouse hippocampus after cerebral artery occlu-
worsened recovery from TBI-related motor def- sion, where it inhibited immune cell infiltration
icits and increased neuronal death compared to and pro-inflammatory gene induction [91].
rats on a control diet, as well as a 70% loss of Neuroprotectin D1 also inhibited cytokine pro-
DHA in the brain [84]. This data is an impor- duction in neuronal cell cultures [91].
tant reminder that diet can influence levels of Highlighting the neuroprotective effect of this
bioactive lipids and that a diet rich in omega-3 bioactive lipid, the infarct volume in the hippo-
polyunsaturated fatty acids can have measur- campus was significantly reduced 48 h after the
able health benefits. Although metabolites of cerebral artery occlusion injury in mice given
DHA were not quantified, the authors hypothe- intracerebroventricular infusions of neuroprotec-
sized that some of DHA’s neuroprotective tin D1 [91].
Decreases in DEA after repeated mTBI could [41] and in a rodent model of headache driven by
potentially be driven by conversion to oxygenated acrolein exposure [43], suggesting that these
metabolites. DEA has been identified as a precursor molecules are sensitive to inflammatory insults.
for several bioactive lipids, which are formed via The actions of multiple NAEs at TRP channels
similar pathways as the biosynthesis of resolvins and may override the analgesic effects of AEA via
neuroprotectin from DHA [92]. For example, incu- CB1 [18], and prolonged exposure to elevated lev-
bating mouse brain homogenates with DEA gener- els of NAEs has been associated with a nocicep-
ated 17- hydroxydocosahexaenoyl ethanolamide tive phenotype in response to capsaicin [66].
(17-HDHEA), 4,17-dihydroxydocosahexaenoyl
ethanolamide, as well as smaller amounts of
7,17-dihydroxydocosahexaenoyl ethanolamide, 16.4.4 Additional Bioactive Lipids
10,17-dihydroxydocosahexaenoyl ethanolamide Effected by mTBI
(10,17-diHDHEA), and 15-hydroxy-16(17)-epoxy-
docosapentaenoyl ethanolamide (15-HEDPEA) 16.4.4.1 N-Acyl Taurines
[92]. 10,17-diHDHEA and 15-HEDPEA were ago- All 4 N-acyl taurines, including the TRPV1 and
nists at CB2 with EC50’s in the low nanomolar range TRPV4 agonist [39] A-Taur, increased in the
and blocked platelet-leukocyte aggregation forma- CER and TNC after a single mTBI. However,
tion in human whole blood at concentrations of just none of the targeted N-acyl taurines changed in
10pM [92]. As CB2 ligands were neuroprotective the TG. Fewer changes in N-acyl taurines were
after TBI [21], it is possible that these DEA-derived measured after repeated mTBI and there were no
lipids have neuroprotective effects via that receptor. longer increases in all 4 N-acyl taurines in the
Highlighting the potency of these resolving lipids, CER and TNC. Instead, N-stearoyl taurine
injections of 15-HEDPEA reduced lung injury after decreased in the CER and A-Taur increased in the
reperfusion at doses as low as 1 μg/mouse [92]. TG, which was the only change in N-acyl tau-
Given that these lipids are so potent, they might be rines associated with mTBI in the TG. A-Taur
bioactive at concentrations below the detection lim- also increased in the TNC, as did N-oleoyl tau-
its of our HPLC/MS/MS system. It might instead be rine (Fig. 16.3) [43].
a more viable strategy to measure the activity of
5-LOX after mTBI and acrolein exposure, as this is 16.4.4.2 2-Acyl Glycerols
a rate-limiting enzyme for the formation of resolvins After a single mTBI, 2-acyl glycerol levels were
from DHA and might also be important for the for- only affected in the TNC. Here, there were
mation of oxygenated metabolites of DEA [93]. increases in the eCB 2-AG, as well as 2-palmitoyl
Indeed, blocking 5-LOX with siRNA in head and glycerol (2-PG) and 2-linoleoyl glycerol (2-LG).
neck squamous cell carcinoma cell lines attenuated This makes the TNC the only tissue where levels
the formation of reactive oxygen species and blocked of both of the eCBs, AEA and 2-AG, were
the ability of DEA to inhibit cell proliferation [93]. affected by a single mTBI. Whether levels of
15-LOX is also an important enzyme to monitor, 2-acyl glycerols increased or decreased after
because it mediates the conversion of DEA to repeated mTBI was dependent on tissue type.
17-hydroperoxydocosahexaenoyl ethanolamide, a Changes in 2-acyl glycerols were increases in the
precursor for the CB2 agonists 10,17-diHDHEA and CER and the TG, but decreases in the TNC. In the
15-HEDPEA, as well as 17-HDHEA [92]. CER, 2-PG and 2-LG increased. In contrast, all 4
The fact that multiple NAEs were altered with 2-acyl glycerols in the screening library signifi-
mTBI is important because there are emergent cantly increased in the TG. Only 1 change in
signaling properties when multiple TRPV1- 2-acyl glycerol levels was measured in the TNC –
ligand NAEs are combined in that the potency is a reduction in 2-oleoyl glycerol (Fig. 16.3).
increased when multiple agonists are present As 2-AG has been shown to be neuroprotec-
[43]. Multiple NAEs also changed in brain tis- tive in multiple TBI studies [12, 19, 20], increases
sues of rats in a model of peripheral inflammation in 2-AG in the TG after repeated mTBI and in the
TNC after single mTBI may be part of a neuro- or deleted. If effects on the lipidome require
protective and regenerative response that medi- DAGL, then more direct measurements of DAGL
ates some of the moderate spontaneous recovery activity can be performed after mTBI or acrolein
that occurs after TBI [74]. mTBI also impacted exposure. Recently, a fluorescent probe was
structurally analogous lipids to the eCB 2-AG, developed to measure DAGL activity [101]. This
especially in the in the TG, where repeated mTBI probe can be used in activity based protein profil-
increased 4 different 2-acyl glycerols. This impli- ing (ABPP) assays in mouse brain proteasomes
cates eCB system enzymes as potential mediators [102]. By performing ABPP after acute and
of mTBI’s effects on lipid levels because these repeated mTBI and after acute and chronic acro-
enzymes regulate lipid levels beyond eCB sub- lein exposure, the impact on DAGL activity can
strates [69, 70, 94]. A decrease in 2-acyl glycerol be quantified. ABPP can also be employed to
hydrolysis via monoacylglycerol lipase (MAGL) detect off-target serine hydrolase activity [103],
could increase 2-acyl glycerol levels, and we which may reveal a more widespread impact of
have shown that MAGL deletion causes wide- mTBI on enzymes important for maintaining lev-
spread increases in 2-acyl glycerols in the mouse els of eCBs and other bioactive lipids.
brain [69]. However, these increases in 2-acyl
glycerols likely came at the expense of levels of 16.4.4.3 T he Potential Link
AA and PGs [69]. Increases in AA and PGs were Between TRPV1 and 2-Acyl
measured in the TG after repeated mTBI, making Glycerol Lipids
MAGL inhibition an unlikely mechanism. What upstream changes can affect DAG produc-
Diacylglycerol lipases (DAGL) synthesize tion? Phospholipase C (PLC) intracellular path-
2-acyl glycerols from their diacylglycerol (DAG) ways stimulate the release of DAGs from
precursors, so an increase in DAGL activity could membrane phospholipids [104]. An earlier study
upregulate 2-acyl glycerols [95]. The DAGLα demonstrated that TBI increased PLC activity in
isoform appears more important for biosynthesis the cat brain [105]. Activation of TRP receptors
of 2-AG in the brain, whereas the DAGLβ iso- can drive PLC pathways [36]. Indeed, we have
form appears more important in the periphery shown that capsaicin activation of TRPV1
[96]. There is evidence that levels of AA and increases 2-acyl glycerol production [43].
AEA can change in the same direction as 2-AG Increases in levels of multiple TRPV1 ligands
upon a change in DAGL activity. Along with like LEA, AEA, and DEA in the TG after repeated
2-AG levels, AEA levels were downregulated in mTBI could potentially increase PLC signaling,
the cerebellum, striatum, and hippocampus of a leading to an increase in DAG substrates for
line of DAGLα KO mice [97]. Another line of 2-acyl glycerol production. AA itself can also
DAGLα KO had an 80% reduction in whole brain activate PLC pathways [106] and levels of AA
2-AG and AA levels and a 40% reduction in AEA were upregulated after repeated mTBI in the TG.
[98]. A third line of DAGLα KO had reduced lev-
els of 2-AG and AA in the forebrain, but did not 16.4.4.4 Free Fatty Acids and PGs
detect a significant reduction in AEA in this area, After a single mTBI, increases in AA and linoleic
hinting at some regional specificity for the acid (LA) were common to all 3 tissues. Oleic
contribution of DAGLα activity to AEA levels acid (OA) also increased in the CER, meaning
[99]. Reductions in whole-brain 2-AG, AA, and that all 3 free fatty acids screened for increased
AEA also occur when DAGLα is pharmacologi- after single mTBI in this tissue type. After
cally inhibited [100]. It isn’t yet known whether repeated mTBI, it was the TG that had increases
increasing DAGL activity causes concurrent in all 3 free fatty acids. AA and LA increased in
increases in AA and AEA, which occurred in the the TG, but OA did not significantly change in
TG after repeated mTBI. this tissue. The only decrease in free fatty acid
Follow-up studies can determine if the effects levels was restricted to the TNC, where LA
of mTBI are abolished when DAGL is inhibited decreased after repeated mTBI (Fig. 16.3).
PGs were also affected by mTBI. A single have some activity at TRP channels. Many of
mTBI increased levels of 2 different PGs, PGF2α these lipids have little data about them beyond
and 6-ketoPGF1α in the CER. In the TG, PGF2α those generated by our laboratory from which to
increased, whereas 6-ketoPGF1α decreased. This compare; therefore, we will have to rely on those
was the only measured decrease in PG levels. In few reports. The lipids are subdivided into
contrast, 2 PGs increased in the TNC, PGE2 and lipoamine “families” as characterized by the
6-ketoPGF1α. After repeated mTBI, PGF2α and amine moiety and each section is labeled with
6-ketoPGF1α increased in all 3 tissue types. In the this family for ease of referencing those that
CER and TG, PGE2 also increased, such that all 3 might be of particular interest to a reader.
PGs in the screening library increased in the CER
and TG (Fig. 16.3). N-Acyl GABAs N-linoleoyl GABA (L-GABA),
Increases in AA in CNS tissues were mea- A-GABA, and N-docosahexaenoyl GABA
sured after mTBI. AA can be directly released (D-GABA) are ligands at TRPV1 [41]. However,
from phospholipids via phospholipase A2, or D-GABA did not change after single mTBI. After
indirectly via PLC [107]. Levels of AA are ele- single mTBI, change in L-GABA was restricted
vated in the CSF of TBI patients and higher levels to the CER, where it increased along with
of AA 1 week post-injury were correlated with A-GABA and 2 additional N-acyl GABAs.
worse outcomes [108]. Increased levels of AA Changes in N-acyl GABAs were decreases in the
are hypothesized to underlie increases in PGs in TG after single TBI and were restricted to N-acyl
the brain after TBI [105] and here there were GABAs that lack TRPV1 activity. N-palmitoyl
increases in levels of all 3 PGs in the CER and GABA changed in the opposite direction in the
TG after repeated mTBI, where levels of AA also TNC, as it increased after a single mTBI, along
increased. with A-GABA. Fewer N-acyl GABAs changed in
Levels of bioactive lipids are differentially the CER after repeated mTBI, as an increase in
affected by mTBI, mostly increasing after single A-GABA was the only detected change.
and repeated mTBI. However, decreases in bio- Decreases in N-acyl GABAs were no longer
active lipid levels were more common after found in the TG after repeated mTBI and instead
repeated mTBI compared to single mTBI, espe- 2 N-acyl GABAs with saturated acyl moieties
cially in the TNC. After a single mTBI, certain increased. On the other hand, 3 N-acyl GABAs
changes were measured in all 3 tissue types and decreased in the TNC, including D-GABA. This
all of these were increases relative to sham. was the only change in D-GABA associated with
Specifically, DEA, O-Gly, NAGly, LA, and AA mTBI. However, A-GABA increased in the
increased. After repeated mTBI, fewer changes TNC. This means that increases in A-GABA
were common to all 3 tissue types, although were present in the CER and TNC after both sin-
levels of PGs increased in all 3 tissues, along gle and repeated mTBI, but other changes in
with NAGly. Thus, DEA levels changed in every N-acyl GABAs were sensitive to the number of
tissue type and NAGly was upregulated in the mTBIs (Fig. 16.5).
CER, TG, and TNC, after a single or repeated
mTBI (Figs. 16.3 and 16.4).
N-Acyl Prolines Although N-docosahexaenoyl
proline is a TRPV1 antagonist [41], this lipid was
16.4.5 Effects of mTBI on Lipoamine not detected in any tissue type. This is likely to do
Families That Include a TRPV with a poor signal to noise characteristic of our
Ligand mass spectrometric analysis than due to a lack of
endogenous production given that all other com-
The following section summarizes some of the binations are measurable. Even with the poor sig-
effects on the lipids that were measured in these nal to noise of this lipoamine family in general,
models but are lipids that have members that we are still able to measure many of the members
and showed that those N-acyl prolines only single mTBI increased levels of the TRPV2/4
changed after repeated mTBI. N-oleoyl proline agonist N-palmitoyl tyrosine [41] and A-Tyr. An
increased in the CER and TNC, whereas increase in A-Tyr in the TNC was the only change
N-arachidonoyl proline (A-Pro) increased in the in N-acyl tyrosines measured after repeated
TG, which was the only tissue where it was mTBI (Fig. 16.5).
detected in all samples (Fig. 16.5).
N-Acyl Valines In all 3 tissue types, exposure to

N-Acyl Serines A single mTBI increased 4 dif- a single mTBI increased levels of the TRPV3
ferent N-acyl serines in the CER, including A-Ser. antagonist [41] N-stearoyl valine (S-Val). In the
The only other change in an N-acyl serine after a CER, levels of N-palmitoyl valine and
single mTBI was an increase in N-palmitoyl ser- N-docosahexaenoyl valine, which is also a
ine in the TNC. Repeated mTBI increased A-Ser TRPV3 antagonist [41], additionally increased.
in the CER and TG. N-linoleoyl serine also Repeated mTBI did not affect N-acyl valine lev-
increased in the TG but no changes in N-acyl ser- els (Fig. 16.5).
ines were measured in the TNC in the repeated
mTBI paradigm. Levels of the TRPV1 agonist To summarize the effects mTBI on endoge-
N-docosahexaenoyl serine [41] did not change nous TRPV ligands, more endogenous TRPV
with mTBI (Fig. 16.5). ligands were affected after a single mTBI com-
pared to after repeated mTBI,. A single mTBI
elevated levels of TRPV ligands, whereas
N-Acyl Tryptophans N-acyl tryptophans con- repeated mTBI produced a mixture of increases
taining polyunsaturated acyl chains are agonists and decreases in levels of TRPV ligands. In the
at TRPV4 [41]. However, these particular lipids mTBI model, changes in TRPV ligands com-
were either not detected or unchanged by mon to all 3 tissue types were only uncovered
mTBI. The only mTBI-driven change in an after a single mTBI, specifically, the TRPV1
N-acyl tryptophan was an increase in N-stearoyl agonist DEA [41] and the TRPV3 antagonist
tryptophan measured in the TG after repeated S-Val [41] increased.
mTBI (Fig. 16.5).
16.4.6 Effects of mTBI on Orphan

N-Acyl Tyrosines Multiple N-acyl tyrosines Bioactive Lipids
increased in the CER and TNC after single
mTBI. The CER had the most changes, with Those lipids for which we have no known molec-
increases in N-oleoyl tyrosine (O-Tyr), and the ular targets and are collectively described as
TRPV4 agonists [41] N-linoleoyl tyrosine “orphan lipids” are discussed here. Some of these
(L-Tyr) and N-arachidonoyl tyrosine (A-Tyr). lipoamine families have multiple members that
Although O-Tyr does not directly activate or demonstrate consistent changes across models
inhibit a TRPV channel, a mixture of N-acyl tyro- and represent potentially important targets to
sines including O-Tyr activated TRPV2, TRPV3, focus future research efforts.
and TRPV4 in transfected HEK cells [41]. Thus,
the elevated levels in multiple N-acyl tyrosines N-Acyl Alanines N-acyl alanine species were
might affect signaling via TRPV2-4. The increase responsive to mTBI. N-acyl alanines did not
in L-Tyr was a larger magnitude change than the change in the TNC after a single TBI, whereas 4
other increases, with levels of L-Tyr being twice different N-acyl alanines increased in the CER
as high in the mTBI CER relative to the sham and TG. The increase in N-arachidonoyl alanine
CER. On the other hand, no changes in N-acyl (A-Ala) was of a larger magnitude than the other
tyrosines were measured in the TG. In the TNC, increases, with levels of A-Ala being over twice
as high in the mTBI CER and TG compared to mTBI. Specifically, repeated mTBI increased
sham. In contrast, none of the N-acyl alanines phosphoLEA in the CER and TG (Fig. 16.5) [43].
changed in the TG after repeated mTBI and only Interestingly, these increases did not always
2 changed in the CER. In the repeated condition, accompany an increase in LEA.
the TNC had the most changes, with increases in
4 N-acyl alanines, including A-Ala, and with a Multiple orphan lipids, which lack molecular
decrease in N-stearoyl alanine (Fig. 16.5). targets, change with mTBI suggesting that a
wider variety of signaling systems may be
affected. Those orphan lipids that stand out
N-Acyl Leucines N-acyl leucines only changed include A-Phe, which increased in all 3 tissue
after exposure to 4 mTBIs and did not change after types after a single mTBI. mTBI affected certain
a single mTBI. N-linoleoyl leucine and lipoamines derived from AA, both orphan and
N-docosahexaenoyl leucine increased in the CER, with known molecular targets. For example,
whereas N-palmitoyl leucine and N-stearoyl leu- A-Ala robustly increased in the CER and TG fol-
cine (S-Leu) increased in the TG. In contrast, lowing a single mTBI, A-Met increased in the
S-Leu decreased in the TNC, as did N-oleoyl leu- TNC after repeated mTBI, and A-Pro increased
cine (Fig. 16.5). in the TG after repeated mTBI. In all 3 tissue
types, a single exposure to mTBI increased levels
of A-Phe. Increases in A-Phe were also found in
N-Acyl Methionines Alterations in N-acyl the TG and TNC after repeated mTBI. mTBI
methionines were restricted to the CER and TG might therefore have more of an effect on
after a single mTBI. 2 N-acyl methionines enzymes that preferentially act on substrates con-
increased in each of these tissue types and taining AA.
N-stearoyl methionine increased in both tissue
types. These changes were no longer present
after repeated mTBI. Only the TG and TNC had 16.5 E
ffects on Bioactive Lipids
changes in N-acyl methionines in this condition. Change Over Time But Are
There was a large increase in N-docosahexaenoyl More Similar When
methionine in the TG and a more modest increase Headache-Like Symptoms
in N-arachidonoyl methionine (A-Met) in the Are Present
TNC (Fig. 16.5).
Effects of mTBI and acrolein exposure were
quite different when comparing acute acrolein
N-Acyl Phenylalanines In all 3 tissue types, a exposure effects to mTBI effects. Specifically,
single exposure to mTBI increased levels of bioactive lipids were more likely to
N-arachidonoyl phenylalanine (A-Phe). Single decrease after the acute acrolein exposure [43].
mTBI also increased N-oleoyl phenylalanine in These lipids tended to increase after mTBI. One
the TNC only. Changes in N-acyl phenylalanines explanation for these differences is that the tissue
were not measured in the CER after 4 mTBIs and was collected immediately after the single 4 h
increases in A-Phe were restricted to the TG and acrolein exposure, whereas tissue was collected
TNC. 2 additional N-acyl phenylalanines at least 72 h later in the case of mTBI. It wasn’t
decreased in the TNC (Fig. 16.5). until after repeated acrolein exposure that
headache-like behaviors appeared [54], whereas
after the single mTBI exposure, there were mea-
PhosphoLEA PhosphoNAEs are intermediates surable headache-like behaviors. Similar altera-
for NAE production [109]. PhosphoLEA is the tions in lipid levels were observed between the
only phosphoNAE that is available in the screen- models at time points when headache behavior
ing library. This lipid only changed after repeated was measurable (4 acrolein exposures or 1 mTBI
exposure). One key finding is that chronic acro- Systemic pretreatment with the TRPA1 antago-
lein exposure increased levels of multiple TRPV nist AP-18 prevented the sensitization of the TVS
ligands and increased levels of NAGly [43], as due to chronic acrolein [11]. In acrolein-exposed
did mTBI. Thus, bioactive lipids may serve as a rats treated with AP-18, changes in dural blood
marker for the physiological changes in head- flow in response to either TRPA1 or TRPV1 ago-
ache, regardless of the original stimulus that dis- nist challenges did not sensitize the responses,
rupted the TVS. Follow-up studies should implicating TRPA1 in the process of sensitization
examine whether there are similar changes in regardless of the exact receptor action of the
lipid levels immediately after the mTBI, as these agent [11]. The biochemical similarities between
could give insight into the lipid milieu that even- the 2 models (acrolein and mTBI), such as
tually gives rise to inflammation and pain. increases in NAGly [43], as well as the similari-
Providing further evidence that the effects of ties in central and peripheral sensitization [11,
painful insults on bioactive lipids are dynamic 54], suggest that the same therapy may work in
and dependent on time post-insult, in another both models. A follow up study will test the
rodent model of inflammatory pain, we also hypothesis that pretreatment with AP-18 prevents
observed decreases in bioactive lipids in CNS tis- headache driven by mTBI.
sues 1 h after intraplantar carrageenan applica- Novel headache therapeutics that block
tion [41]. For example, A-GABA decreased in microglial activation are currently being devel-
the cerebellum and brainstem and OEA decreased oped [13, 112]. It is possible that attenuating
in the thalamus. At 3 h post-carrageenan, microglial activation could be a therapy for head-
increases were observed in multiple NAEs in all ache due to mTBI, as both share changes in neu-
6 brain areas tested (striatum, hippocampus, cer- roinflammatory mediators that drive microglial
ebellum, thalamus, midbrain, and brainstem) activation [4, 13]. Strengthening the hypothesis
[41]. These effects at 3 h included increases in that blocking microglial migration represents a
NAGly in the striatum, hippocampus, and cere- strategy for treating mTBI, A-Ser, which attenu-
bellum [41]. Therefore, the effects of peripheral ated NAGly-driven microglial migration [59],
insults on bioactive lipids take some time to cen- was also neuroprotective after TBI [47, 48].
tralize, whereas the effects of central insults like A-Ser levels were elevated in the CER and TG
mTBI may take some time to fully permeate the post-repeated mTBI, potentially representing a
periphery. Overall, the effects of peripheral compensatory strategy to reduce the microglial
inflammation, acrolein exposure, and mTBI on activation and migration that occurs after
bioactive lipids look more similar when pain- TBI. Because GPR18 activation drives microglial
related behaviors emerge. migration [59], it should be tested whether
GPR18 antagonists are effective at preventing
microglial migration after mTBI, as this might
16.5.1 Implications for Therapeutics represent a novel strategy to reduce inflamma-
tion. FAAH inhibition also represents a strategy
Current therapeutics for headache are not partic- to reduce NAGly levels [69, 113]. Lipidomics
ularly effective, underscoring the need to better studies have revealed that genetic [69] and phar-
understand the mechanisms that cause headache macological [113] inhibition of FAAH increases
and develop new drugs that target these mecha- levels of AEA and other NAEs but at the expense
nisms [110]. TRPA1 may represent a novel target of other AA-derived lipoamines such as NAGly.
for treating headache [111]. Studies in the TRPA1 Treatment with the FAAH inhibitor URB597
KO mouse [52] suggest that TRPA1 is required reduced microglial and astrocytic activation after
for pain and hypersensitivity associated with TBI in rats and was associated with improvement
chronic inflammation, as TRPA1 functions as a in behavioral measures [20]. The contribution of
gatekeeper of the release of endogenous inflam- NAGly to these findings has not yet been
matory mediators from sensory neurons [24]. investigated.
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Novel Anti-inflammatory
and Vasodilatory ω-3 17
Endocannabinoid Epoxide
Regioisomers
Lauren N. Carnevale and Aditi Das
Abstract apentaenoyl ethanolamide (EPEA) by

Accumulating evidence suggests that diets cytochrome P450 (CYP) epoxygenases to
rich in ω-3 polyunsaturated fatty acids form epoxydocosapentaenoic acid-
(PUFAs) offer protection against vascular ethanolamides (EDP-EAs) and epoxyeicosa-
inflammation, neuroinflammation, hyperten- tetraenoic acid-ethanolamides (EEQ-EAs),
sion, and thrombosis. Recently, biochemical respectively. The EDP-EAs and EEQ-EAs are
studies have demonstrated that these benefits endogenously produced in rat brain and
are partially mediated by their conversion to peripheral organs. Additionally, EDP-EAs and
ω-3 endocannabinoid epoxide metabolites. EEQ-EAs dose-dependently decrease pro-
These lipid metabolites originate from the inflammatory IL-6 cytokine and increased
epoxidation of ω-3 endocannabinoids, doco- anti-inflammatory IL-10 cytokine.
sahexanoyl ethanolamide (DHEA) and eicos- Furthermore, the EEQ-EAs and EDP-EAs
attenuate angiogenesis and cell migration in
cancer cells, induce vasodilation in bovine
L. N. Carnevale coronary arteries, and reciprocally regulate
Department of Biochemistry, University of Illinois platelet aggregation in washed human plate-
Urbana-Champaign, Urbana, IL, USA
lets. Taken together, the ω-3 endocannabinoid
A. Das (*) epoxides represent a new class of dual acting
Department of Biochemistry, University of Illinois
Urbana-Champaign, Urbana, IL, USA molecules that display unique pharmacologi-
cal properties.
Department of Comparative Biosciences, University
of Illinois Urbana-Champaign, Urbana, IL, USA
Keywords
Center for Biophysics and Quantitative Biology,
University of Illinois Urbana-Champaign, Cytochrome p450 · Epoxygenase ·
Urbana, IL, USA Neuroinflammation · Endocannabinoid ·
Beckman Institute for Advanced Science and Epoxyeicosatrienoic acids · Cannabinoid
Technology, University of Illinois Urbana- receptors 1 and 2 · Omega-3 fatty acids
Champaign, Urbana, IL, USA
Department of Bioengineering, University of Illinois
Urbana-Champaign, Urbana, IL, USA
Division of Nutritional Sciences, University of
Illinois Urbana-Champaign, Urbana, IL, USA

220 L. N. Carnevale and A. Das
Abbreviations 5] pulmonary, intestinal, and reproductive tissues

[6]. Three years after CB1 was cloned, a second
2-AG 2-arachidonoyl-glycerol cannabinoid receptor, termed CB2, was identi-
2-DHG 2-docosahexaenoyl-glycerol fied. While both CB1 and CB2 are GPCRs (Gi/o)
2-EPG 2-eicosapentaenoyl-glycerol activated by phytocannabinoids, their tissue dis-
AA arachidonic acid tribution and resulting physiological effects dif-
AEA anandamide fer. For example, CB2 is primarily expressed in
cAMP cyclic adenosine monophosphate the spleen and immune cells with the highest lev-
CB1 cannabinoid receptor 1 els occurring in B lymphocytes, macrophages,
CB2 cannabinoid receptor 2 and natural killer cells [6–8]. Furthermore, dur-
CYP cytochrome P450 ing times of injury or stress, the resident immune
DHA docosahexaenoic acid cells, microglial cells, express CB2 thereby sug-
DHEA docosahexaenoyl ethanolamide gesting a role in abating neuroinflammation.
eCB endocannabinoid These results prompted the design of CB2 ago-
EDP-EA docosapentaenoic acid ethanolamide nists to modulate neuroinflammation [9].
EEQ-EA epoxyeicosatetraenoic acid The discovery of CB1 and CB2 prompted the
ethanolamide researchers to identify the endogenous ligands of
EPA eicosapentaenoic acid these receptors. The first endocannabinoid identi-
EPEA eicosapentaenoyl ethanolamide fied was arachidonoyl ethanolamide (AEA), which
FAAH fatty amide hydrolase was extracted from pig brain and could readily dis-
GPCR G-Protein coupled receptor place the potent cannabinoid receptor ligand, [3H]-
MAGL monoacylglycerol lipase HU243 (Ki = 52 nM). AEA was identified to be an
sEH soluble epoxide hydrolase agonist at the CB1 receptor and was later named
Δ9-THC Δ9-tetrahydrocannabinol anandamide. The name anandamide was derived
from ‘ananda,’ the Sanskrit word for ‘bliss’ due to
the compound’s ability of modulate neurobehav-
ioral activity [10]. Accordingly, AEA and all future
17.1 Introduction ligands for CB1 and CB2 were collectively termed
the endocannabinoids (eCBs) since they are
17.1.1 The Endocannabinoid System endogenously produced molecules that bind and
activate cannabinoid receptors. The second eCB,
The main psychoactive component of Cannabis 2-arachidonoyl glycerol (2-AG), was discovered
sativa, Δ9-tetrahydrocannabinol (Δ9-THC), was just shortly after AEA. It was shown to activate the
identified in 1987. Howlett’s group showed that CB2 with greater potency than at the CB1 [11].
the neurobehavioral effects of Δ9-THC were Since the discovery of AEA and 2-AG, several
mediated through a G-protein (Gi/o) coupled other eCBs have been discovered, including, but
receptor (GPCR). Shortly thereafter, Pfizer devel- are not limited to, 2- arachidonoylglycerylether
oped the synthetic cannabinoid, CP 55,940. (noladin ether), N-arachidonoyl dopamine
Later, CP 55,940 and Δ9-THC helped identify a (NADA), N-oleoylethanolamide (OEA),
cannabinoid (CB) receptor [1]. The first identi- 2-docosahexaenoyl glycerol, (2-DHG), virod-
fied cannabinoid receptor, termed cannabinoid hamine (O-AEA), eicosapentaenoic acid ethanol-
receptor 1 (CB1), is the most abundant GPCR amide (EPEA) and docosahexaenoyl ethanolamide
located within the central nervous system (CNS) (DHEA) [10, 12–15]. Several other eCB conge-
[1–3]. More specifically, it is found at high levels ners have been identified that may not directly
in the hippocampus, pre-frontal cortex, dorsal bind cannabinoid receptors but can bind to other
root ganglion, cerebellum, and substantia nigra. receptors such as TRPPV1, GRP119, GPR55, and
Although CB1 is most abundant in the CNS, it is GPR18. In addition, many of these receptors inter-
found to a lesser extent in the cardiovascular, [4, act with phytocannabinoids [5, 16, 17].
17 Novel Anti-inflammatory and Vasodilatory ω-3 Endocannabinoid Epoxide Regioisomers 221
Cellular eCB levels are carefully regulated Following their uptake, PUFAs are mobilized
through multiple biosynthetic and degradative via transport proteins to tissues where they esteri-
enzymes. For instance, the eCBs originate from fied and are temporarily stored in the cell mem-
dietary ω-3 and ω-6 polyunsaturated fatty acids brane (Fig. 17.1). For example, AEA is stored as
(PUFAs). PUFAs are class of lipids that contain its membrane precursor N-arachidonoyl phos-
two or more cis carbon-carbon double bonds phatidylethanolamine (NAPE) [18] and is
(points of unsaturation) and are identified as ω-3 released from the membrane by phospholipase
or ω-6 based on the location of the first carbon- D. This catabolic process yields a free eCB [19].
carbon double bond from the terminal methyl After biosynthesis, the eCBs are tightly regulated
group. ω-6 eCBs derived from arachidonic acid by degradative enzymes through hydrolytic inac-
(AA), such as AEA and 2-AG, are the most well tivation [20]. For example, fatty acid amide
studied eCBs. With the recent interest in the hydrolase (FAAH) catalyzes the hydrolytic cleav-
dietary effects of ω-3 PUFAs, there has been an age of AEA to form AA and ethanolamine [21].
increased interest in ω-3 eCBs. The ω-3 eCBs are FAAH is the principal AEA and DHEA-
derived from the ω-3 PUFA docosahexaenoic hydrolyzing enzyme distributed throughout the
acid (DHA), eicosapentanoic acid (EPA, timn- nervous system [22] On the other hand, monoac-
odonic acid), or α-linoleic acid (ALA). ylglycerol lipase (MAGL) is the degradative
Fig. 17.1 Overview of the ω-3 Endocannabinoid to the eCBs DHEA and EPEA, respectively. The metabo-
Epoxide Biosynthetic Pathway. Dietary consumption of lism of DHEA and EPEA by cytochrome P450 epoxygen-
ω-3 polyunsaturated fatty acids results in the storage of ases produces six (DHEA) or five (EPEA) constitutional
DHA and EPA esters within the plasma membrane. Upon isomers at each carbon-carbon double bond. DHEA epox-
cleavage by a phospholipase, DHA and EPA are cleaved ide metabolites are termed EDP-EAs while the EPEA
from their membrane precursor and undergo biosynthesis epoxide metabolites are termed EEQ-EAs
enzymes for 2-AG and 2-DHG [20, 23]. Overall, metabolites with novel anti-inflammatory and
these enzymes convert the eCBs back into their potential anti-pain properties as well as vasoac-
free fatty acid form, which can be esterified and tive activities.
stored in the phospholipid membrane bilayer or
used for cellular functions [24].
Based on the widespread tissue distribution of 17.1.2 Endocannabinoid Metabolism
the cannabinoid receptors there is a plethora of by Cytochrome P450 (CYP)
biochemical processes that utilize cannabinoid Epoxygenases
signaling [25–27]. The main role of CB1 is to
mediate neurobehavioral activities, such as appe- In humans, there are 57 CYPs which are
tite regulation and executive functions, while the involved in xenobiotic metabolism, steroid syn-
prominent function of CB2 is to mediate an anti- thesis, and fatty acid metabolism [43]. These
inflammatory immune response. More specifi- enzymes are distributed throughout the human
cally, CB2 is involved in neuroinflammation body, with the highest levels reported in the
through its immunoregulatory activity in murine liver. In 2008, the Hollenberg group was the
brain microglia [5, 28]. Overall, these activities first to demonstrate the metabolism of AEA by
suggest that CB1 and CB2 are neuroprotective CYPs [44]. This represented a novel metabolic
[29–32]. Given the prevalence of pathway for AEA that led to the formation of
neuroinflammatory- based diseases such as the emerging class of ω-6 derived eCB epox-
Alzheimer’s disease (AD), multiple sclerosis ides. Importantly, it was demonstrated that the
(MS), and Parkinson’s disease (PD), selective AEA epoxide, 5,6-EET-EA, had 300-fold selec-
agonists for CB2 activation or modulation of tivity for the CB2 compared to CB1 and 1000-
eCB levels have promising therapeutic potential fold greater affinity for CB2 compared to AEA,
for the control of neuroinflammation [29, 33–37]. indicating unique biological activity of the
Additionally, accumulating evidence suggests epoxides compared to their parent molecule
that eCBs target alternative receptors such as the [44]. To identify CYPs involved in the conver-
nuclear peroxisome proliferator-activated recep- sion of both AEA and 2-AG, the Hollenberg lab
tors (PPAR) and the heat sensing receptor tran- utilized LC-MS/MS to determine that CYP iso-
sient vanilloid type-1 (TRPV1), [38–40] forms, CYP3A4, CYP4F2, and CYP2D6,
prompting studies regarding their role in noci- metabolize AEA in human liver and kidney
ceptive pain. microsomes as well as in brain microsomal and
While the eCBs directly bind to CB1 and mitochondrial preparations [45]. Furthermore,
CB2 receptors, they also serve as substrates for the Das group discovered the role of cardiac
the eicosanoid synthesizing enzymes. CYP, CYP2J2, in the metabolism of 2-AG and
Cyclooxygenases, lipoxygenases, and epoxy- AEA. This finding inspired future studies on
genases oxidize fatty acids to bioactive metabo- CYP2J2-mediated ω-3 and ω-6 eCB metabo-
lites that have novel physiological and lism [12, 13, 46]. CYP2J2 converts PUFAs to
cannabinoid receptor activities that are unique bioactive fatty acid epoxides which display
compared to their parent molecule [5, 41]. anti-inflammatory activities and enhance
Previously, the Serhan group reported the metab- wound repair [47–49]. CYP epoxygenases
olism of DHEA by the lipoxygenase pathway. mediate the metabolism of eCBs to eCB epox-
DHEA is metabolized to 10,12-diHDHA-EA ides [13, 50, 51]. More recent studies have
and 15-HEDPEA, which reduce leukocyte infil- shown that CYP2J2 and other CYPs metabolize
tration (chemotaxis) during inflammation. They ω-3 eCBs such as DHEA and EPEA to more
can activate the CB2 receptor with EC50 values at potent anti-inflammatory epoxide metabolites,
3.9 nM and 1 nM, respectively [42]. In this chap- as illustrated in Figs. 17.1 and 17.2. This meta-
ter, we will discuss the monooxygenation of bolic pathway consists of CYP epoxygenases,
eCBs by epoxygenases to produce eCB epoxide which belong to the cysteinato-hemoprotein
Fig. 17.2 Endocannabinoid Signaling Regulates tively). These metabolites are more potent at CB2 than
Inflammation and Arterial Tone. Inflammatory stimuli their eCB parent molecule and display CB2 selectivity.
such as LPS up-regulates cytochrome P450 expression 19,20-EDP-EA and 17,18-EEQ-EA diminish pro-
which metabolize eCBs DHEA and EPEA to epoxide inflammatory NO and IL-6 while they elevate anti-
metabolites (19,20-EDP-EA and 17,18-EEQ-EA, respec- inflammatory marker IL-10. In addition, DHEA, EPEA as
well as their epoxide metabolites are vasoactive
superfamily and diflavin enzyme, CYP reduc- MS/MS) detected DHEA in rat brain
tase (CPR), which shuttles electrons from (128.9 ± 27.9 pmol/g), heart (57.7 ± 24.4 pmol/g),
NADPH to the CYP [52]. kidney (49.6 ± 22.2 pmol/g), spleen
(22.9 ± 1.3 pmol/g), liver (195.1 ± 48.7 pmol/g),
pig brain (265.5 ± 100 pmol/g), and human
17.2 Endocannabinoid Epoxides plasma (13.3 ± 0.4 pmol/mL). Interestingly,
dietary supplementation with DHA increased
17.2.1 ω-3 Endocannabinoid DHA the levels of DHEA at the expense of AEA,
Ethanolamide (DHEA) thereby providing a method for elevating the
levels of ω-3 eCBs in vivo [13, 15]. Although an
Encouraged by studies that identified AEA-like exact biosynthetic pathway for DHEA has yet to
fatty acid ethanolamides across several tissues, be determined, it is believed that DHEA is pro-
the Di Marzo group sought to identify their ω-3 duced in a similar manner to AEA. It has been
eCB counterparts. This group demonstrated that suggested that a membrane precursor,
ω-3 eCB, DHEA is located in bovine retina. N-docosahexaenoyl phosphatidylethanolamine
Furthermore, DHEA was found at twofold (NDHPE) is hydrolyzed by phospholipase D2 to
higher levels compared to AEA in the brain produce free DHEA, however, more studies are
(3.77 ± 0.66 ng/g and 1.88 ± 0.46 ng/g, respec- required to confirm this hypothesis [55]. Since
tively) [15, 53]. Additionally, DHEA has been DHEA is structurally similar to AEA and
detected under peripheral inflammation in rat showed a high potential for physiological rele-
contralateral spinal tissue [54]. More recently, a vance, there was significant interest in investi-
targeted lipidomics method using liquid chro- gating its physiological activities in vitro and in
matography tandem mass spectrometry (LC- vivo. Additionally, DHEA displays immune-
modulatory activity in RAW264.7 cells. DHEA showed that epoxygenation of DHEA by CYP
dose-dependently reduced monocyte chemoat- epoxygenases led to an altered CB1 and CB2
tractant protein 1 (MCP-1) mRNA, prostaglan- activity profile compared to the DHEA parent
din and thromboxane B2 levels produced by molecule. For example, 19,20-EDP-EA was
COX-2 [56]. observed to be more potent at CB2 compared to
DHEA and had a ten-fold lower activity at CB1
(EC50 at CB1: 108 nM and CB2: 280 nM)
17.2.2 ω-3 Endocannabinoid DHEA (Fig. 17.2) [13]. As 19,20-EDP-EA, was observed
Epoxides to display an altered cannabinoid receptor profile
(Epoxydocosapentaenoic compared to DHEA, it would be interesting to
Acid-Ethanolamide, EDP-EA) characterize the other five DHEA epoxide regioi-
somers produced by the cross-talk of CYP
Recently, the Das group showed that DHEA is a metabolism pathways. From similar studies on
substrate for enzymes in the CYP epoxygenase other regioisomers, it is expected that DHEA
pathway. They designed a targeted lipidomics epoxide regioisomers and enantiomers will dis-
LC-MS/MS method in the multiple reaction play different activities at the CB1 and CB2
monitoring mode (MRM) to simultaneously receptors.
detect and quantify five DHEA epoxide
(EDP-EA) regioisomers in rat brain, heart, kid-
ney, spleen, liver tissues as well as in mouse 17.2.3 Anti-Inflammatory Activity
microglial cells. These isomers share the same of DHA Ethanolamide
molecular weight but differ in the order in which Epoxides (EDP-EAs)
their atoms are connected. Due to the reported
upregulation of CYP epoxygenases in LPS- Multiple studies indicate that eCBs combat
stimulated microglia, the Das lab sought to microglial-promoted neuroinflammation via CB2
investigate these molecules under inflammatory activation [58]. In fact, there is a current drive
conditions [44, 57]. By monitoring the produc- towards the development of CB2-selective ago-
tion of EDP-EAs in LPS-stimulated microglia nists to combat the inflammatory pathology asso-
treated with DHEA, they confirmed that EDP- ciated with progression of neurodegenerative
EAs are produced by CYPs as inhibitors of disease. As the DHEA epoxides (EDP-EAs) are
CYPs reduce their levels. It was further demon- naturally produced by microglia and the microglia
strated that the CYP isozyme, CYP2J2, is express CYPs, the Das group examined the anti-
responsible for this action. Current research is inflammatory effects of these molecules in an in
going on to identify other brain CYPs that are vitro model of neuroinflammation. As mentioned
involved in eCB metabolism. Interestingly, the briefly above, microglial cells are the innate
production of the terminal epoxide, immune cells of the brain, which continuously
19,20-EDP-EA is favored over the other regioi- survey the brain parenchyma for harmful stimuli.
somers in a reconstituted CYP2J2-CYP When harmful stimuli are encountered, microglia
Reductase (CPR) Nanodisc system and by brain exhibit a pro-inflammatory phenotype to promote
microsomes [13]. More research is needed to defense. As microglia clear the harmful stimuli, a
further understand why certain regioisomers are phenotype change occurs that promotes an inflam-
preferred by CYPs. mation-resolution phase. This phenotype switch-
As previous studies consistently showed ing mechanism is classically known as
DHEA as a weak ligand at both CB1 and CB2, macrophage polarization. Out of the EDP-EAs
the Das lab tested to see if DHEA and its epoxide tested, 19,20-EDP-EA showed the most potent
metabolites activated or bound to CB receptors dose-dependent reduction of pro- inflammatory
[56]. Using the PRESTO-Tango assay, they markers. (Fig. 17.2). Correspondingly, it decreased
pro-inflammatory cytokine IL-6 and increased the 17.2.5 ω-3 Endocannabinoid EPA
production of IL-10 [13]. Additionally, the anti- Ethanolamide (EPEA)
inflammatory effects of 19,20-EDP-EA were con-
firmed using freshly isolated primary piglet The report that several fatty acid ethanolamides
microglial cells stimulated with LPS, which sup- other than AEA have unique activities at the can-
pressed IL-6 production, and thus corroborated nabinoid receptors sparked a study in 1996 by the
the findings in immortalized BV-2 cells. Based on Martin and Razdan groups, which sought to
these findings, it is possible that the DHA-derived examine the structure-activity relationships of
eCB epoxides mediate anti-inflammatory activi- AEA analogs at CB1 [60, 61]. Previous to this
ties in addition to other pathways. Taken together, study, AEA was the only endogenously produced
this indicates that the EDP-EAs and their stable fatty acid ethanolamide to bind and activate the
derivatives may be utilized for treating neuroin- cannabinoid receptors. By understanding the
flammatory conditions. structural requirements necessary for ligands to
bind CB1 it would be possible to develop more
potent ligands for both biological and therapeutic
17.2.4 Vasoactivity of DHA applications. Cannabinoid structure-activity rela-
Ethanolamide Epoxide tionships (SAR) indicated that a phenolic
(EDP-EA) hydroxyl, a lipophilic side chain, and an appro-
priately oriented carbocyclic ring system are
It is well known that CYP epoxygenases convert required for cannabinoid receptor activity. One of
ω-3 PUFAs to PUFA epoxides. For example, the AEA analogs tested in their study was EPA
DHA is converted to DHA epoxides (EDPs). ethanolamide (EPEA). Based on molecular
These molecules are vasoactive in nature, and are dynamic simulations, EPEA was suggested to
more effective at relaxing pre-constricted vessels possess some affinity for CB1 [61].
compared to CYP-derived AA epoxides (EETs) Alongside these studies, the Di Marzo group
[59]. The Das lab demonstrated that CYP determined the in vivo localization of EPEA in
epoxygenase- mediated metabolism of DHEA mouse brains. EPEA was detected at
produced a vasodilatory eCB epoxides, 0.18 ± 0.09 pmol/mg of brain lipid extract, which
19,20-EDP-EA. Compared to 19,20-EDP, as was the lowest level of all of the fatty acid etha-
shown in Fig. 17.1, EDP-EA had a two-fold nolamides detected in their study. EPEA was
higher EC50 at relaxing bovine coronary arteries found at five-fold lower levels than DHEA [53].
(Fig. 17.2) [13]. Although CYP-derived DHEA Compared to EPEA, its parent molecule EPA
epoxides are vasoactive molecules, they do so was found at 443 ± 167 ng/g mice brain.
with reduced potency relative to their parent mol- However, dietary supplementation with EPA
ecule. Furthermore, 19,20-EDP-EA exerts anti- increased the endogenous levels of EPEA,
angiogenic effects in HMVEC cells. Angiogenesis thereby providing a dietary delivery method for
plays several roles in pathological and non- elevating ω -3 eCBs [15]. Furthermore, when
pathological states. As eCBs are regarded as anti- mice were fed a high ω -3 diet consisting of DHA
cancer molecules through their anti-angiogenic and EPA phospholipids, the levels of EPEA
effects, the Das lab tested the ability of increased in adipose tissue, corroborating the
19,20-EDP-EA to inhibit angiogenesis in link between diet and the eCB system. More
HMVEC cells. 1 μM and 3 μM 19,20-EDP-EA recently, a targeted lipidomics method using
decreased tubulogensis by 31 and 75%, respec- LC-MS/MS detected EPEA in rat brain
tively. Previous to this study, the anti-angiogenic (6.2 ± 1.6 pmol/g), heart (9.4 ± 6 pmol/g), kidney
actions of the EDP metabolites were attributed to (37.1 ± 21 pmol/g), spleen (9.2 ± 4.1 pmol/g),
their inhibition of VEGF-stimulated cell migra- liver (80.8 ± 20.1 pmol/g), and pig brain
tion via an unknown epoxyeicosanoic metabolic (2.2 ± 0.4 pmol/g) [13]. Interestingly, EPEA is
pathway [13]. found at a significantly lower level than DHEA
in rat brain (DHEA: 128.9 ± 27.9 pmol/g). molecule EPEA. For example, 17,18-EEQ-EA
Together, these findings support that supplemen- had a lower EC50 at CB2 than EPEA (EC50
tation with ω -3 fatty acids can modulate eCB 17,18-EEQ-EA: 1.4 nM and EC50 EPEA:
levels and may increase the bioavailability of 2.1 nM). However, compared to EPEA (EC50
DHA and EPA for physiological function. CB1: 0.1 nM), 17,18-EEQ-EA had a lower
Although the biosynthesis of EPEA has not potency at CB1 (EC50 CB1: 18.5 nM), thus
been proved, the well-studied transacylation- showing a CB2-selectivity for 17,18-EEQ-EA
PDE pathway suggests that n-acyl ethanol- [13] (Fig. 17.2). As mentioned above, discover-
amides are biosynthesized from ing novel CB2-selective molecules may aid in
glycerophospholipids via N-acylphosphatidyl the development of anti-inflammatory therapeu-
ethanolamine (NAPE). Through this pathway, a tics that overcome any potential psychoactive
Ca2+-dependent N-acyltransferase and NAPE- activities that may be mediated through CB1
hydrolyzing PLD render the fatty acid ethanol- activation.
amide free from its membrane precursor [56].
This pathway is similar to the biosynthesis of
AEA and may support the idea of a conserved 17.2.7 Anti-inflammatory Activity
biosynthesis for fatty acid ethanolamides. of EPA Ethanolamide Epoxides
(EEQ-EAs)
17.2.6 ω-3 Endocannabinoid Epoxide Since EPA and EPEA have been shown to pos-
Epoxyeicosatetraenoic Acid- sess anti-inflammatory activities, the anti-
Ethanolamide (EEQ-EA) inflammatory activities of EEQ-EAs were studied
in brain microglia. When BV-2 microglia were
In 2017, the Das lab showed that EPEA, like stimulated with LPS, the levels of EPEA and
DHEA, is a substrate for the CYP epoxygenase total EEQ-EA were elevated. Interestingly, EPEA
pathway. Using a targeted lipidomics LC-MS/MS levels significantly surpassed DHEA levels after
approach they detected and quantified all five 12 h of LPS stimulation (DHEA:
EPEA epoxide (EEQ-EA) regioisomers in rat 6.7 ± 4.6 pmol/106 cells and EPEA:
brain, heart, kidney, spleen, liver tissues as well 2042 ± 939.2 pmol/106 cells). Furthermore, in
as in mouse microglial cells. They found that LPS-stimulated BV-2 cells, 17,18-EEQ-EA
LPS-stimulated and EPEA-treated mouse (10 μM) dose-dependently reduced NO in a CB2-
microglia could convert EPEA to the EEQ-EAs dependent manner, which was attenuated with
via CYP epoxygenases. They further determined CB2 antagonist, AM-630, treatment. In addition,
that one of the CYP isozymes responsible for this 17,18-EEQ-EA dose-dependently decreased IL-6
action is CYP2J2. Using recombinantly expressed and increased IL-10 (Fig. 17.2). Interestingly,
proteins, CYP2J2-CPR Nanodiscs showed a 17,18-EEQ-EA was a more potent inhibitor of
favored production of 17,18-EEQ-EA, as shown NO production than EPA epoxide (17,18-EEQ).
in Fig. 17.1, compared to other regioisomers [13]. This evidence shows that the CYP epoxygenase
To further characterize EPEA and its epoxide pathway may play be responsible for the anti-
metabolites, we were interested in testing inflammatory nature of EPA through conversion
whether the EEQ-EAs were eCBs. Previous of EPEA to its epoxide metabolite. Lastly, similar
studies consistently showed EPEA as a weak to 19,20-EDP-EA, the effects of 17,18-EEQ-EA
agonist for CB1 and CB2 [56]. Through the were confirmed in freshly isolated piglet microg-
PRESTO-Tango assay, the Das lab showed that lia cells, thereby corroborating the anti-
epoxidation of EPEA by CYPs led to an altered inflammatory effects of 17,28-EEQ-EA in
CB1 and CB2 activity compared to their parent primary cells.
17.2.8 Vasoactivity of EPA been shown to be anti-proliferative, anti-

Ethanolamide Epoxide tumorigenic, and can inhibit metastasis [66, 67].
(EEQ-EA) The Das lab recently revealed that DHEA and
EPEA epoxides display anti-cancer activities.
Since it is known that CYP epoxygenases EDP-EAs are not only abundant in osteosarcoma-
metabolizes EPA to vasoactive epoxide metabo- derived lung tumors (20–80% increased levels
lites, the Das lab tested the vasodilatory activity compared to healthy lungs) but they also possess
of eCB EPEA and eCB epoxide 17,18-EEQ-EA anti-inflammatory and anti-tumorigenic proper-
in isolated bovine coronary arteries [59, 62]. ties in an in vitro model of osteosarcoma [68].
Utilizing isometric tension measurements to More specifically, Roy et al. examined the ability
monitor the ability of these molecules to relax of EDP-EA regioisomers 7,8-EDP-EA,
preconstricted arteries, they determined that 10,11-EDP-EA, and 13,14-EDP-EA to inhibit
17,18-EEQ-EA, like 19,20-EDP-EA, dose- migration of cancer cells and induce apoptosis.
dependently relaxed constricted bovine coro- Additionally, in 143B human osteosarcoma cells,
nary arteries (Fig. 17.2). On the other hand, the 10,11-EDP-EA showed the highest ability to
ED50 values determined from 17,18-EEQ-EA induce apoptosis in a CB1-dependent manner
was two-fold greater than the ED50 value of (EC50 at CB1: 0.43 nM). A time-dependent
17,18-EEQ (ED50 of 17,18-EEQ: 0.47 ± 0.01 μM scratch assay revealed that 10,11-EDP-EA
and ED50 of 17,18-EEQ-EA: 1.1 ± 0.6 μM), thus has anti-migratory potential, an indicator of
showing that eCB epoxides are vasoactive but metastasis. In order to increase the stability of
not as potent as their PUFA epoxides. It has 10,11-EDP-EA, derivatives were synthesized to
been previously been shown that EPA epoxides prevent degradation by FAAH. Based on a previ-
act as endothelial-derived hyperpolarizing fac- ous characterization of CB1-binding and meta-
tors that inhibit platelet aggregation. To follow- bolic stability of AEA’s functional groups, a
up with these observations, the Das lab revealed R-1′-methyl isomer (10,11-EDP-IA), a cyclopro-
that 17,18-EEQ-EA dose-dependently inhibits pyl derivative (10,11-EDP-CA), and a n-propyl
platelet aggregation and angiogenesis [13]. derivative (10,11-EDP-NA) was prepared.
Overall these endocannabinoid epoxides are Through LC-MS/MS quantification of metabo-
vasoactive. lites, the rate of hydrolysis of 10,11-EDP-EA to
10,11-EDP was decreased by nearly 100-fold
with the derivatives 10,11-EDP-NA,
17.3 Anti-tumorigenic Properties 10,11-EDP-IA, and 10,11-EDP-CA. Furthermore,
of the ω-3 Endocannabinoid these stable derivatives were successful at inhib-
Epoxides iting migration in osteosarcoma cells and
reduce angiogenesis. Taken together, this novel
A particular consequence of unresolved inflam- class of eCB epoxides have shown potential to
mation is the progression towards tumor forma- reduce tumor progression through activation of
tion. The microenvironment of a tumor is highly the CB1 receptor. In addition, this was one of the
inflammatory and contains a carcinogenic milieu first studies to demonstrate the potential utility of
that promotes tumor progression via pro- cannabinoid-like molecules in the treatment of
inflammatory cytokines, growth factors, and sev- bone cancer. Furthermore, these studies provide
eral adhesion molecules [63]. Several studies the grounds for future work to determine the
have reported the anti-inflammatory, pro- CB1-dependent anti-pain behavior of these mol-
resolving, and anti-tumorigenic activity of ω-3 ecules, which would be highly beneficial for the
fatty acids, DHA and EPA [64, 65]. Upon conver- development of multi-modal bone cancer
sion to their epoxides, the EEQs and EDPs have therapies.
17.4 Conclusions and Future disease, hypertension, and nociceptive pain [21,

Perspectives 69, 70]. It needs to be evaluated if FAAH and
sEH inhibitors increase the levels of these dual
The inflammatory response leads to release of functional endocannabinoid epoxides.
lipid mediators that help in regulating the inflam- Although it was shown that the ω-3 eCBs,
mation. There is a strong interest in identifying DHEA and EPEA are metabolized by CYP
anti-inflammatory lipid mediators that can epoxygenases to form the epoxides, the identity
resolve the ongoing inflammation. In this chap- of the specific CYP enzyme is unclear. It has
ter, we review a novel class of anti-inflammatory been shown that the ω-6 eCB 2-AG, AEA and
lipid mediators that emanate from the cross-talk ω-3 eCBs DHEA and EPEA are metabolized by
of the two important biochemical pathways: the CYP2J2 to produce epoxide metabolites [50,
CYP-epoxygenase pathway and the endocan- 71]. Several papers from Hollenberg laboratory
nabinoid biosynthetic pathway. The ω-3 eCBs, have identified other CYPs such as CYP3A4
DHEA and EPEA are metabolized by CYP epox- and CYP2D6 are involved in metabolizing
ygenases to form the epoxides, EDP-EA and eCBs [51]. However, there needs to be a sys-
EEQ-EAs. These eCB epoxides are multi- tematic approach to delineate which CYPs are
functional molecules as they can target multiple involved in the endocannabinoid metabolism in
receptors including the cannabinoid receptors the body.
and unknown epoxide receptor. Their effects on The EDP-EA and EEQ-EA metabolites were
inflammation resolution, cancer and vasodilation successfully detected in several tissues. Detection
have briefly been explored through multiple stud- of lipid metabolites is often challenging as these
ies and show promise for the development of lipid metabolites are unstable and the levels at
therapeutics for cerebrovascular and neuroin- which they are detected in blood and tissues are
flammatory diseases. lower than their endogenous levels at the site of
Although evidence points to CB1 and CB2 formation and site of action. The levels of
receptors as the primary modulator of the anti- EDP-EA and EEQ-EA in the tissues are within
inflammatory and vasodilatory effects of the the pmol/g tissue range, however, their local con-
omega-3 eCB epoxides, the studies also indicate centration is likely to be greater than plasma lev-
that the eCB epoxides are anti-inflammatory and els. This is because they are locally produced and
vasoactive through an unknown receptor. In order act in a localized manner on the receptors unlike
to target hypertension and other vascular condi- most hormones and neuropeptides. For instance,
tions through the eCB system, identification of it is hypothesized that eCBs are synthesized on-
this receptor and competing metabolic enzymes demand from their esterified membrane precur-
would be highly beneficial. Hence, it is important sors in response to a variety of chemical and
to establish the receptor systems through which neuronal stimuli and are rapidly degraded by
these molecules elicit their physiological FAAH and sEH. Local measurement of lipid
response. Additionally, it is critical to map all the metabolite is technically challenging. As a result,
primary synthetic and degradative pathways of the levels of these molecules extracted from tis-
these eCB epoxides. While it was shown that sues does not mirror the local concentrations in
these eCB epoxides are substrates for two degra- the intracellular and synaptic environment. In
dative pathways mediated by sEH and FAAH, it addition, it is important to note that many of these
is possible that they are synthesized and stored in lipids are esterified in the membrane yet the lev-
membrane and released by phospholipases. els that are reported within the literature reflect
Current therapeutics, such as FAAH and sEH the abundance of free lipids often in healthy tis-
inhibitors, increase the endogenous levels of sues. Future studies are needed to discern eCB
PUFA epoxides and eCBs, and have since shown and eCB epoxide levels in disease states as the
promise for the treatment of inflammatory-based expression of the enzymes such as CYP epoxy-
genases change during inflammation. Another References

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phrs.2015.04.001
Overview of Lipid Biomarkers
in Amyotrophic Lateral Sclerosis 18
(ALS)
Andres Trostchansky
Abstract 18.1 Introduction

Amyotrophic lateral sclerosis (ALS) is a multi-
factorial neurodegenerative disease involving Amyotrophic lateral sclerosis (ALS) is a neuro-
motor neuron (MN) degeneration in the spinal degenerative disease without treatment and a
cord, brain stem and primary motor cortex. The short life expectancy upon clinical symptoms
existence of inflammatory processes around appear [1]. This underscores the necessity for an
MN and axonal degeneration in ALS has been expansion of the current knowledge and underly-
shown. Unfortunately, none of the successful ing biological mechanisms driving disease.
therapies in ALS animal models has improved Recent research shows the presence of inflamma-
clinical outcomes in patients with tory processes around motor neurons (MN) and
ALS. Therefore, the detection of blood bio- axonal degeneration, being evident through the
markers to be used as screening tools for disease accumulation of reactive astrocytes and activated
onset and progression has been an expanding microglia [2]. Many treatments have been tested
research area with few advances in the develop- on different ALS animal models. Unfortunately,
ment of drugs for the treatment of ALS. In this no successful therapy with promising results in
review, we will address the available data ana- animal models has improved clinical outcomes in
lyzing regarding the relationship of lipid metab- patients with ALS [3]. This is partly due to the
olism and lipid derived- products with ALS. We lack of specific blood biomarkers, which can act
will address the advances on the studies about as screening tools to identify individuals at risk
the role that lipids plays at the onset, progres- of developing the disease, as well as the lack of
sion and lifespan extension of ALS patients. clinical studies. The development of novel medi-
cal treatments has lead to an increase in life
Keywords expectancy, especially in the western world.
ALS · Lipid biomarkers · Mass spectrometry However, the former is accompanied by an incre-
· Cell signaling ment on the appearance of cognitive dysfunctions
related to aging in addition to the development of
neurodegenerative diseases. Overall, these
A. Trostchansky (*) changes represent a significant challenge from
Departamento de Bioquimica and Center for Free
Radical and Biomedical Research (CEINBIO), the scientific and medical point of view. The cur-
Facultad de Medicina, Universidad de la Republica, rent review aims to analyze the recent data
Montevideo, Uruguay

234 A. Trostchansky
obtained about lipid help in the development of processing and compromised mitochondria
drugs for the treatment of ALS. homeostasis, increased oxidative stress, excito-
toxicity, reduced neurotrophic support, and glial
inflammatory responses [7]. Familiar ALS has
18.2 Amyotrophic Lateral been studied using active (SOD1G93A, SOD1G37R)
Sclerosis mice mutants which have a phenotype character-
ized by progressive paralysis and death caused by
Amyotrophic lateral sclerosis is a multifactorial the degeneration of MN, in addition to gliosis
neurodegenerative disease caused by genetic and within the spinal cord, brain stem and cortex [6].
non-inheritable components leading to MN These features suggest a gain of toxic function of
degeneration in the spinal cord, brain stem and the mutant SOD might play a role in the neurode-
primary motor cortex [1]. It is well established generative process. In addition, oxidative stress is
that ALS affects both cortical as well as cranial an important contributor to neuronal death in
and spinal MN with variable evolution. The ALS involving astrocytes [8]. Current research
majority of ALS cases are sporadic, however has shown that ALS is not a single cell type dis-
there exists a 10% of familial cases, some of ease, since it involves microglia and astrocytes
which arise from mutations of the protein surrounding the MN. In fact, our laboratory
Superoxide Dismutase 1(SOD1) [1]. The average reported astrocytes promote neuronal loss by
age of onset of the disease is 55 years, beginning mechanisms involving alterations in mitochon-
later in women than men, and with 90% of the drial functionality, increased production of nitric
patients dying within 5 years after the appearance oxide and nerve growth factor [2, 8–13]. Glial
of the clinical symptoms. For a definitive diagno- cells are the principle innate immune cell of the
sis of ALS the progressive spread of symptoms CNS and pathology associated with these cells is
involving upper and lower motor neurons is referred to as neuroinflammation, a hallmark of
required, according to the revised El Escorial and ALS [14, 15].
the Awaji criteria [4, 5]. The most used clinical Importantly, there exists an increase of neuro-
scores for disease progression is the ALS func- toxic molecules such as pro-inflammatory cyto-
tional rating scale-revised (ALSFRS-R) [5]. This kines, reactive oxygen species (ROS) and
scale is employed to evaluate the pace of disease pro-inflammatory lipid-derived compounds.
progression, predict survival and assess the These molecules may cause further neuronal
effects of disease-modifying drugs in therapeutic damage leading to further glial cell activation
trials. The scale is based on clinical symptoms so resulting in a positive feedback loop of neuroin-
it is imperative to discover biomarkers that allow flammation. Indeed, neuroinflammatory pro-
early diagnosis of the disease. In addition, bio- cesses have been detected in ALS patients as well
markers will aid for therapeutic drug design to as in transgenic models of the disease [16]. Based
improve the quality of life of those affected due on this background, many treatments aim to
to the lack of effective pharmacological treat- inhibit or reduce the pro-inflammatory action of
ments for ALS [5]. The pathogenesis of ALS con- microglia and astrocytes and counteract the pro-
sists of two stages: an early neuroprotective stage gression of the disease. Unfortunately, none of
and a later neurotoxic stage. Multiple mecha- the successful therapies in ALS animal models
nisms have been described for MN death includ- has improved clinical outcomes in patients with
ing glutamate toxicity, mitochondrial dysfunction, ALS [3]. This can be ascribed to different factors.
protein misfolding and apoptosis [6]. However, For example, decreasing or deleting single pro-
ALS appears as a composite syndrome where the inflammatory factors such as TNF-α, IL1-β, and
aberrant cellular pathways may not be ascribed inducible nitric oxide synthase has had little-to-no
solely from a protein misfolding issue, but cellu- effect on overall survival of SOD1G93A mice [8,
lar physiology aspects like deficiencies in RNA 12]. These suggest that a multiplicity of pro-
18 Lipids in ALS 235
inflammatory cytokines can compensate the arachidonic acid (AA) and docosahexaenoic acid
absence of any single factor, and it is unlikely (DHA). Since these PUFA are the precursors of
that continuing efforts to target a single factor in important cell signaling molecules. As examples,
humans will provide significant therapeutic ben- DHA and AAs can be oxidized to give rise to
efit in patients with ALS. Considering the impor- prostaglandins or leukotrienes. The oxidation of
tance of the disease and the fact that inflammatory DHA produces neuroprotectinD1, a signaling
processes are involved, the identification of blood molecule that promotes cell survival under stress.
biomarkers to identify individuals at risk of The latter is part of the denominated specialized
developing the disease or for their recruitment in pro-resolving lipid mediators (SPM) giving to
clinical studies in order to analyze benefits in the these PUFA precursors an important role in neu-
development of drugs for the treatment of ALS roinflammation [20, 21]. The presence of these
would be of biological relevance. Below we will compounds has been implicated in neuronal sig-
discuss the identification and use of lipid-derived naling processes controlling neurogenesis, brain
products as biomarkers since research in the field vesicular activity, central glucose homeostasis,
and the optimization of lipidomic techniques are mood and cognition [22]. Other example is
giving novel and important new data for using Prostaglandin E2 (PGE2), synthetized by
them as disease biomarkers. Prostaglandin Endoperoxide H Synthase-2
(PGHS-2) from AA, which promotes inflamma-
tion after binding to its receptor [23]. Their role
18.3 R
ole of Lipids in Central as biomarkers or footprints of ALS will be dis-
Nervous System and ALS cussed later.
In many neurodegenerative diseases, an accu-
Lipids are implicated in a wide variety of biologi- mulation of ceramides has been observed. The
cal processes and can be classified into five cate- latter is considered toxic, since it has been shown
gories: fatty acids, triacylglycerols (TAGs), to promote neuronal death by oxidative stress and
phospholipids, sterol lipids and sphingolipids. apoptosis in both animal models and patients
The brain requires a near constant source of [24–28]. Moreover it has been proposed in
metabolites to maintain function, and contains SOD1G93A mice that sphingomyelin-associated
the second highest concentration of lipids in the ROS production leads to MN death through the
human body [17]. Although the consensus is that p75 factor [12]; in addition, it is reported that an
glucose metabolism almost completely satisfies increase of sphingomyelin in the spinal cord of
the brain energy requirements, it has recently ALS patients mediates MN death via oxidative
been shown that approximately 20% of the total stress [24]. From a structural-functional point of
energy requirement of the brain is met through view, it has been shown that membrane fluidity in
the oxidation of fatty acids, and that this fatty the brain and spinal cord decreases significantly
acid oxidation may take place entirely in astro- over the course of disease in ALS mice [29]. In
cytes [18]. Besides this fact, perhaps the most fact, neuronal membranes rich in phosphatidyl-
essential role for lipids in the brain is as compo- ethanolamine and phosphatidylserine are signifi-
nents of the membrane cellular machinery. This cantly less fluid. Thus, the increase in DHA
is important in the brain since changes in mem- results in more rigid membranes with an impor-
brane fluidity and many signaling processes tant impact of signaling activities in ALS brains
occur in specific intracellular compartments. [30].
Importantly, fatty acids and their derivatives have Along with neuronal degeneration, alterations
been well characterized as drivers of intracellular of lipid metabolism have been reported in
signaling processes [19]. ALS. Different studies have shown that muscle
Two major polyunsaturated fatty acids denervation has a role in the promotion of
(PUFAs) are present at high levels at the brain: abnormal lipid metabolism. In fact, Body Mass
236 A. Trostchansky
Index (BMI) at diagnosis remains the only meta- 18.4 L

ipidomic Analysis in ALS
bolic independent prognostic factor for survival Animal Models and Patients
in ALS [31]. Studies to determine the role of lipid
supplementation and serum lipid profile on ALS Metabolomics studies search for small molecules
onset, progression or fate [4, 32–34] have been present in cells, tissues or biological samples,
carried out, but with unfavorable outcomes. For whereas the observation of modifications in the
example, supplementation with omega-3 fatty levels of these molecules, in addition to physio-
acids (e.g. eicosapentaenoic acid, EPA, before logical modifications of signaling pathways may
clinical symptoms appear, in SOD1G93A mice help to elucidate where these changes are occur-
increased the progression of the disease and ring, (e.g. intracellularly). When these molecules
shortened life span of the transgenic animals are lipids, the studies are called Lipidomics [43].
[35]. In addition, markers of oxidative stress such The chance that a small molecule (e.g. fatty acid)
as 4-hydroxynonenal were also observed [36]. can be used as a biomarker relies on the existing
High fat diets exert a modest decrease in disease communication between the brain and the periph-
progression in a mouse model of ALS [37, 38]. In ery. The former must be accompanied to the
contrast to other pathologies, such as cardiovas- capacity of different metabolites to cross the
cular diseases, dyslipidemia is a good prognostic blood-brain barrier (BBB) and therefore be
factor for ALS. When looking to ALS transgenic detected in plasma. In addition to the non-
mice, they are leaner, hypolipidemic and present invasive capacity to obtain blood samples from
a higher metabolic intake of fatty acids in muscle patients, promotes that the metabolites present in
than control animals [34]. plasma are an ideal source of biomarkers as
Dyslipidemia, reduced body mass and molecular traces of the disease. Besides, in neu-
increased resting energy expenditure are often rodegenerative diseases the hemato-encephalic
present in ALS patients [39, 40]. Importantly, barrier can be compromised, increasing the pos-
ALS is characterized by important alterations in sibilities that these metabolites can be detected in
energy homeostasis [37]. Leptin levels in ALS plasma [43].
patients appeared significantly decreased, sug- Lipidomic studies have shown that different
gesting lower fat reserves [41]. Reports from the classes of lipids can be used as biomarkers of
literature show a high LDL/HDL cholesterol ALS onset and progression when compared to
ratio or elevated content of total cholesterol and healthy volunteers or other neurodegenerative
triglycerides in ALS patients that were associated patients, as well as to follow preliminary studies
with better prognosis or slower disease progres- with pharmacological compounds that have
sion [33, 42]. impact on signaling pathways involved in neuro-
In summary, neuronal lipid metabolism in inflammation. Below we will discuss these bio-
ALS is dysregulated affecting energy use, struc- markers in terms of the complexity of the lipids
tural integrity, and signaling processes. Oxidative analyzed and the oxidative status of these
stress is increased since neurons metabolize a products.
greater proportion of lipid substrates. When look-
ing at the structural level, altered lipid metabo-
lism leads to cytoskeletal defects and 18.4.1 Saturated and Unsaturated
neuromuscular junction denervation. Finally, Fatty Acids
altered lipid metabolism disrupts important bio-
logical signaling processes, altering neurotrans- Nutritional status in conditions such as obesity
mitter synthesis and release and impairing and cancer can be followed by looking the blood
intracellular transport. palmitoleate to palmitate (16:1/16:0) and stearate
to oleate (18:0/18:1) ratios [4, 44, 45]. Recent
studies have shown that levels of 16:1 and 18:1 18.4.2 Complex Lipids
fatty acids, as well as of the enzyme that
introduces the instaurations at the carbon chain, Complex lipids as triglycerides (TG), total cho-
stearoyl-CoA desaturase, are significantly lesterol (TC), phospholipids (PL) were all ana-
increased in blood cells from ALS patients com- lyzed as biomarkers of ALS onset, progression or
pared to healthy controls [4, 44, 45]. It is impor- drug effect on clinical symptoms [43]. The levels
tant to note that the ratio of different of TG and TC in ALS patients were comparable
monounsaturated fatty acids was strongly to those found in control subjects. A recent study
increased in ALS patients. In particular, 16:1 and analyzed the composition of lipids in cerebrospi-
18:1 levels increased significantly in blood cells, nal fluid (CSF) from ALS patients compared to
and higher oleate levels were also observed in controls [43]. In this study, the authors demon-
serum [4]. Fatty acids levels in the blood corre- strated that phosphatidylcholine PC(36:4),
lated with ALSFRS-R, the 16:1/16:0 ratio in ceramide and glucosylceramide levels were
blood cells negatively correlated with ALSFRS-R higher in ALS patients than in control subjects.
decline over a six-month period [4]. Circulating Bioinformatics and statistical analysis showed
free fatty acids were more abundant in ALS six different metabolites that can be followed as
patients, suggesting increased lipid breakdown distinctive between ALS and control groups.
by adipocytes [46]. In fact, a decline of These compound corresponded to the PC deriva-
ALSFRS-R was also significantly associated tives PC (36:2p), PC(36:4p), PC(40:6p), the
with the levels of 16:1 itself. Survival rates were sphingomyelin (SM) derivatives SM(d34:0), and
greater when associated to higher 16:1/16:0 SM(d39:1), and the TG (Triglyceride)
ratios, with a prolonged life expectancy of almost (16:1/18:1/18:2) [43]. The PC and SM com-
11 months in the population of patients with a pounds levels were higher in ALS patients, in
high 16:1/16:0 ratio, as compared to patients with contrast to TG which was lower in ALS patients
low 16:1/16:0 ratio. Higher levels of palmitoleate than in controls. It is worth noting the higher
itself were also significantly associated with level of PC (36:4p and 36:4e) was the strongest
extended life span. In contrast to what was discriminant factor identified by all the statistical
observed for the 16:1/16:0 ratio, no changes in approaches used [43]. However, and importantly,
survival were observed associated to the higher levels of SM(d43:2) were associated to a
18:1/18:0 ratio [4]. Moreover, BMI or circulating lesser decline of the ALSFRS-r score [43].
leptin content did not correlate with ALSFRS-R Similar studies were performed using trans-
decline, nor prognosticated survival based on genic ALS mice. In these experiments, the results
multivariate analysis. In fact, statistical analysis were quite different from those of humans, high-
showed that blood cell 16:1/16:0 ratio was an lighting the difficulties in transferring the data
independent prognostic factor for survival with obtained in animals to humans. In this case the
age, BMI, site of onset and ALSFRS-R as vari- complex lipids, which were found to be discrimi-
ables [4]. The 16:1/16:0 index is an easy-to- nant between control and ALS mice, were PC
handle parameter that predicts survival of ALS (36:2), PC(36:4), PC(40:6). These three lipids
patients. The enzymatically and non-were found in higher levels in the ALS groups
enzymatically oxidation of unsaturated fatty than in controls [43].
acids increase during oxidative stress and may We have previously discussed that accumula-
reflect the presence of an inflammatory process. tion of ceramides (Cer) are toxic for MN [16]. By
Lipid peroxidation susceptibility, assessed by the contrast, increasing the formation and accumula-
peroxidability index (PI) [47], was 16% lower in tion of Cer-derived agents may be protective by
the blood cell fraction of ALS patients as com- reducing ceramide synthesis thus limiting the
pared to controls, independent of the omega-6/ direct toxic effect on MN [16]. The membrane
omega-3 (AA/EPA) index which did not show lipids Glycosphingolipids (GSLs) are a heteroge-
significant modification [4]. neous lipid groups formed through the covalent
238 A. Trostchansky
linkage of a glycan moiety to Cer, especially The involvement of AA metabolites in ALS

abundant in the CNS with important bioactive was also supported by the increased mRNA and
metabolic roles, growth factor signaling and par- protein levels of 5-lipoxygenase (5-LOX)
ticipation on neuroinflammation [48, 49]. All of observed in SOD1G93A mice at 120 days of age.
these activities are thought to participate in ALS Of therapeutic interest, oral administration of the
disease pathogenesis. When cervical spinal cord 5-LOX and tyrosine kinase inhibitors nordihy-
gray and white matter samples from ALS patients droguaiaretic acid, significantly extended lifes-
were analyzed, the major isoforms of Cer, e.g. pan and slowed motor dysfunction in this animal
GalCer, GlcCer, LacCer, GM3, GM1, GL3, and model [15]. We have recently reported changes
SPM were significantly elevated [16]. This on the levels of some of AA-derived compounds
increase was not due to changes on the enzymes in SOD1G93A mice [50]. In addition, we have
activities that catalyze the formation of these recently published changes in levels of LOX-
products. These results were confirmed by using derived products in SOD1G93A mice at different
an inhibitor of the enzyme that produces GlcCer, stages of the disease. We observed, when the
which increased Cer levels and accelerated dis- clinical symptoms appear, a significant increase
ease progression in SOD1G93A mice [16]. of 12-hydroxyeicosatetraenoic acid (12-HETE)
in both plasma and brain whereas no changes
were observed in age-matched non-transgenic
18.4.3 Oxidized Lipids mice. Similarly, 15-hydroxyeicosatetraenoic acid
(15-HETE) levels were also higher in SOD1G93A
Oxidized lipids can be used as blood biomarkers brains. Prostaglandin levels were also increased
for diseases progression or drug treatments. at day 90 in plasma from SOD1G93A compared
Importantly, oxidized lipids involves enzymatic to non-Tg being similar in both types of animals
derived compounds with relevant biological and at later stages of the disease [50].
signaling actions. Arachidonic acid is the precur- Unexpected results also were reported when
sor of a wide variety of anti- or pro-inflammatory analyzing oxidized lipids in ALS mice models. In
compounds when metabolized by the PGHS or a recent study, the neuroprotective effect of EPA
Lipoxygenase (LOX) pathways. These com- was analyzed [35]. The treatment with EPA
pounds can be followed in small samples of resulted in an enhancement of neuroinflamma-
blood and used as disease biomarkers [43, 50]. In tion, faster disease progression and hastened
fact, ALS mice as well as patients with sporadic death for SOD1G93A mice [35]. The authors pro-
ALS have increased levels of prostaglandin E2 pose that the observed results are due to the
(PGE2) [23]. This compound is formed by the greater susceptibility to be peroxidized and there-
action of PGE synthase-1 on PGH2 synthesized fore toxic end products of EPA.
by oxidation of AA by PGHS. Furthermore, the
protein levels of microsomal PGE synthase-1 and
PGHS-2, which catalyze PGE2 biosynthesis, are 18.5 Conclusions
significantly increased in the spinal cord of ALS
mice [23]. In ALS patients, PGE2 levels are ALS is a neurodegenerative disease, with a short
increased in the serum and the CSF [51]. The life expectancy, for which efficient treatments are
pharmacological inhibition of PGE2 receptor or still missing. Since many factors affect the onset
the silencing of the gene coding for PGHS-2 can and progression of the disease, it is important to
lower neuroinflammation in SOD1G93A mice, pre- find biomarkers that may aid the design of drugs
serve motor functions and extend survival [52, that specifically influence and mitigate the dam-
53]. Since PGE2 can exert different biological age, prevent MN loss and neuromuscular dener-
actions depending of the tissue, its use as a blood vation. The high presence at CNS, the capacity to
or tissue disease biomarker needs further cross the BBB and be found at plasma suggest
studies. that lipid products can be used as prognostics
indicators of ALS. The analysis we did in this 11. Pehar M, Cassina P, Vargas MR, Castellanos R, Viera
L, Beckman JS, Estevez AG, Barbeito L (2004)
review suggests that the benefits of detecting Astrocytic production of nerve growth factor in motor
these biomarkers may help in designing and per- neuron apoptosis: implications for amyotrophic lat-
forming clinical trials whose aim should be not eral sclerosis. J Neurochem 89(2):464–473
only increase life expectancy but also quality of 12. Pehar M, Vargas MR, Robinson KM, Cassina P, Diaz-
Amarilla PJ, Hagen TM, Radi R, Barbeito L, Beckman
life. JS (2007) Mitochondrial superoxide production and
nuclear factor erythroid 2-related factor 2 activation
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Flavonoids Ability to Disrupt
Inflammation Mediated by Lipid 19
and Cholesterol Oxidation
Carlo Barnaba and Ilce G. Medina-Meza
Keywords items are: flavonols, flavones, flavanones, flavan-

Flavonoids · Cardiovascular disease · 3-ols, proanthocyanidins, and anthocyanins [44,
Inflammation · Lipid oxidation · Cholesterol 67]. Figure 19.1 depicts the major classification
oxidation · LDL · Macrophages · Oxysterols · of flavonoids according to their chemical struc-
Resveratrol · Dietary antioxidants · Oxidative ture. Their occurrence in food matrices has been
stress extensively reviewed [39, 44], and has been sub-
ject of extensive research in the last decades.
Table 19.1 contains a few examples of com-
pounds from each of the subcategory, with the
19.1 Introduction fruit (berry) in which they are commonly found.
The monomeric unit of flavonoids can dimerize
Flavonoids are plant secondary metabolites that and polymerize to form other important high
act as protectants against harmful effects of UV-B molecular weight molecules; this is the case of
radiation inasmuch as biotic stress, conferring at proanthocyanidins, that are polymers of flavan-3-
the same time pigmentation of fruits and leaves ols or flavanols. Not only do these compounds act
[67]. The term “flavonoid” refers to phenolics as plant protectants, but they can also be very
having a basic skeleton of diphenylpropane beneficial to human health. Cohorts studies per-
(C6-C3-C6), which consists of two aromatic formed in the early ‘90 have shown that dietary
rings linked through three carbons that usually consumption of flavonoids was inversely associ-
form an oxygenated heterocycle [25, 52]. ated with morbidity and mortality from coronary
Flavonoids are broken down into several different heart disease [31, 32]. These findings have
sub-categories based on their chemical structure. opened an intensive field of research on the
The main subclasses commonly found in food effects of flavonoids and flavonoids-rich food
extracts in cardiovascular diseases (CVD) pro-
gression, particularly in the modulating CVD-
C. Barnaba associated oxidative stress and inflammation. In
Department of Pharmacology and Toxicology,
Michigan State University, East Lansing, MI, USA this short review, we will summarize the current
findings in flavonoids beneficial effects in pre-
I. G. Medina-Meza (*)
Department of Biosystems and Agricultural venting CVD through inhibition of initial stages
Engineering, Michigan State University, of CVD progression. Given the magnitude of sci-
East Lansing, MI, USA entific literature in the field, we will focus on two

244 C. Barnaba and I. G. Medina-Meza
Fig. 19.1 Main classification of phenolic compounds
strictly mechanistic aspects: inhibition of ize its bioavailability [46]. In terms of flavonoids,
chemical-induced LDL oxidation, and the effect the primary route of administration is ingestion.
of flavonoids in the monocyte/macrophages acti- Once ingested, flavonoids undergo sulfation,
vation pathways. methylation and glucuronidation in the small
intestine and liver by respective enzymes [26,
44]. These metabolic forms are the ones com-
19.2 Flavonoid Absorption monly found in plasma, rather than the aglycone
and Bioavailability [10, 38, 44, 46, 64]. Several studies performed in
humans have shown that after ingestion of flavo-
The average US daily flavonoid intake is noid-rich food, maximum plasma levels are
189.7 mg/d, subdivided in flavan-3-ols (87%), observed after 1.5–2 h; however, only sub- or
flavanones (7.6%), flavonols (6.8%), and others low-micromolar concentration of the correspond-
[12]. Quercetin is the most important contributor ing aglycone are retrieved. This is the case of
to the estimated intake of flavonoids, mainly green tea catechins (0.6–1.8 μM, depending on
from the consumption of apples and onions [52]. the considered compound) [74], quercetin from a
Data regarding flavonoids bioavailability – i.e. vegetable and fruit-enriched diet (0.14 μM) [17],
the portion of the compound ingested that is as well as from ingestion of apples and onions
digested, absorbed and metabolized – are still (1.5–3.4 μM) [33], among others. From
incomplete. Plasma or urine concentration of a these studies, it is clear that only a small percent-
certain compound is typically used to character- age of flavonoids (<2%) of the initial intake is
19 Flavonoids Ability to Disrupt Inflammation Mediated by Lipid and Cholesterol Oxidation 245
Table 19.1 Individual flavonoids according to their subclass, as found in common berries
Flavonoid subclass Compound Berry species
Flavonols Kaempferol Blueberry, sour cherry, cranberry, raspberry, strawberry
Quercetin Blueberry, sour cherry, cranberry, raspberry, strawberry
Myricetin Blueberry, cranberry, strawberry
Isoharmnetin Sour cherry
Flavones Apigenin –
Luteolin –
Flavanones Hesperitin –
Narigenin Strawberry
Flavan-3-ols (-)-Epicatechin Blueberry, sour cherry, cranberry, raspberry, strawberry
(-)-Epicatechin 3-gallate Strawberry
(-)-epigallocatechin Cranberry, raspberry, strawberry
(-)-epigallocatechin 3-gallate Cranberry, raspberry, strawberry
(+)-catechin Blueberry, sour cherry, cranberry, raspberry, strawberry
(+)-gallocatechin Strawberry
Proanthocyanidins Monomer Blueberry, cranberry, raspberry
Dimer Blueberry, cranberry, raspberry
Trimer Blueberry, cranberry, raspberry
4-6mers Blueberry, cranberry, raspberry
7-10mers Blueberry, cranberry, raspberry
Polymers Blueberry, cranberry,
Anthocyanidins Cyanidin Blueberry, sour cherry, cranberry, raspberry, strawberry
Delphinidin Blueberry, cranberry, raspberry, strawberry
Malvidin Blueberry, cranberry, raspberry, strawberry
Pelargonidin Blueberry, cranberry, raspberry, strawberry
Peonidin Blueberry, sour cherry, cranberry, raspberry, strawberry
Petunidin Raspberry, strawberry
absorbed. Compounds that are not absorbed from actually decrease as the dose increases, suggest-
the intestine migrate to the colon wherein they ing that there is an absorption saturation point
interact with the microflora. The flavonoids that [10]. Because of the overall poor absorption and
re-enter circulation can now be broken into low low bioavailability of flavonoids, a variety of
aglycones by the microbiota and further into low strategies have been explored in order to improve
molecular weight compounds that can be these inefficiencies. Increasing metabolic stabil-
absorbed [64]. ity, intestinal absorption and changing the absorp-
Different polyphenols have different absorption site have been considered to improve
tion rates and metabolic conversions due to their flavonoid bioavailability [52, 64].
variable structures. Regarding flavonols, studies
performed in the Caco-2 model cell line have
shown that the several glycosylated forms are 19.3 Lipid and Cholesterol
poorly absorbed [15, 26]. Aglycones can pass Oxidation in LDL
through the gut wall because microflora hydro- and Atherosclerosis
lyzes the sugar moiety [25]. However, there is
also a compound specificity in the absorption 19.3.1 Chemical-Induced Oxidation
mechanism, i.e. quercetin and daidzein gluco- of LDL
sides seemed to be directly absorbed by active
transport [26]. Not only does polyphenol struc- There is a generous literature regarding ex vivo or
ture affect the absorption, but so can the dose and in vitro oxidation of LDL, and it is out of the
food matrix in which they are contained. It has scope of this chapter a detailed revision of the
been found that the absorption efficiency can field, which can be found elsewhere [42]. Here,
we will summarize the main strategies used to ity [42]. LDL can be mildly or highly oxidized
assess LDL oxidation and how these experimen- (MM-LDL and the proper ox-LDL, respec-
tal setups are relevant for flavonoids- centered tively), although there is no general consensus
antioxidant studies. LDL are complex macromol- about the active species present in each stage
ecules, made of a lipid core surrounded by a sin- [11, 42, 50].
gle non-exchangeable lipoprotein, known as
apolipoprotein (ApoB). The average lipid core
has been calculated to contain 600 molecules of 19.3.2 Cholesterol Oxidation in LDL
free cholesterol, 1600 molecules of cholesteryl
esters (mainly arachidonate and linoleate esters), Cholesterol is a natural occurring compound that
700 molecules of phospholipids (64% PC, 1.5% has several crucial functions within the body. It
PE, 26% SM, and 11% LPC), 180 molecules of is a key component of all cellular membranes, a
triacylglycerides (TAGs), and approximately 10 precursor to steroid hormones and is involved in
molecules of the lipophilic antioxidant many signal transduction pathways [27].
α-tocopherol [37, 42]. Several exchangeable Cholesterol transport in humans is a complex
lipoproteins are also associated to LDL particle, process. LDL are the lipoproteins that carry
and their function is to modulate interactions esterified cholesterol from the liver to the periph-
with specific cell receptors [7, 16]. In particular, eral tissue [71]. In humans, a balance of choles-
high concentrations of ApoC-III in ApoB lipo- terol is critical for many physiological
proteins have been reported among patients with processing, since accumulation of this molecules
coronary disease and coronary atherosclerotic in cell membrane can affect the function of a
lesions, as well as metabolic syndrome and type variety of enzymes, transporters and receptors.
2 diabetes [16, 61]. Cholesterol is susceptible of being oxidized by
Ex vivo and in vitro oxidation of LDL is an naturally occurring oxidative agents, like reac-
experimental strategy largely adopted to either tive oxygen and nitrogen species (ROS and RNS,
characterize products of lipid oxidation, or respectively), that can alter its chemical back-
assess the ability of antioxidants to dampen bone by oxygenating carbons at several posi-
oxidation itself. Oxidation can be triggered via tions. Commonly, auto-oxidation of cholesterol
several mechanisms, including but not limited (i.e. by chemical and physical means) occurs at
to free radicals generation, Fenton reaction, and position C-7 proximal to the C5-C6 double bond,
peroxynitrite oxidation agents [19]. α,α′- or to the C-25 tertiary carbon [45, 47]. However,
azodiisobutyramidine dihydrochloride (AAPH) first oxidative derivatives can be further oxidized
has been widely used as ROS inducer, since it and go subsequent chemical rearrangements. To
degrades to highly reactive radical species that date, more than 80 compounds, broadly known
oxidized molecular oxygen to peroxy radicals as cholesterol oxidation products (COPs) have
[19]. Fenton reaction is triggered by micromo- been identified. COPs are more polar than the
lar solution of Cu2+ (or less commonly Fe2+) parent cholesterol, thus present different chemi-
salts, which are able to abstract a proton from cal and biophysical properties in cell environ-
preformed lipid peroxides to generate peroxyl ment. Their different chemistry is the major
radicals [28]. Finally, peroxynitrite is obtained source of health- associated issues, since they
from a variety of precursor, including linsido- can interfere with cholesterol homeostasis at
mine (SIN-1) [62, 65]. Oxidation of LDL can several level. It is not the scope of this chapter to
potentially affect each of its constituents, detail the putative biological effects due to the
including the apoB, and cross-reactions exposure of COPs, as from cell, in vivo and
between different fractions (i.e. protein/lipid (marginally) clinical studies. The reader can
chemical interaction) can exponentially multi- refer to our recent review [45] or other compel-
ply the chemistry of the oxidative products, to a ling works in the area. Briefly, in vitro works
variety of lipids with reported biological activ- using several cell lines have reported that admin-
istration of high concentration of COPs – either the biomarkers of oxidative stress: PL-esterified
individually or as a mixture of compounds – can isoprostanes are known to have pro-inflamma-
trigger several physiological responses able to tory action, and stimulate monocyte adhesion to
induce pro-inflammatory, pro- fibrogenic, and the endothelial cell [1, 2]. Oxysterols, in partic-
pro-apoptotic effects. A consistent body of liter- ular 7-ketocholesterol, 7α-OH and 7β-OH cho-
ature has also assessed the cytotoxic, mutagenic lesterol, and cholesterol epoxides are abundant
and carcinogenic properties of COPs. As afore- in LDL, and are believed to exert cytotoxic
mentioned, cholesterol homeostasis is strictly effects on monocyte-macrophages ([5, 13]).
linked to COPs accumulation in fluids and cell Although toxic, ROS-induced COPs (particu-
compartments [45]. larly B-ring derivatives) seem unable to activate
liver-X receptors (LXR-α and β) and
peroxisome-proliferator-activated (PPARs)
19.3.3 OxLDL Activate Monocytes/ receptors in macrophages, and so trigger the
Macrophages Pathway anti-atherogenic response [6, 37, 69]. On the
other side, enzymatic side-chain COPs like
The mechanism by which LDL activates mono- 22-OH, 25-OH and 27-OH cholesterols can trig-
cytes in the intima towards their conversion to ger both LXRα and PPARs [69], which is
macrophages is still controversial, although a explained by specific structural requirements
rough phenomenological description has been needed for these ligand-receptor interactions to
provided [59]. There is scientific consensus that occur [36]. Other inflammatory mechanisms
oxLDLs, rather than the parent LDL, are the pri- have been proposed for B-ring oxysterols, as
mary stimulus leading endothelial activation 7-keto and 7-OH cholesterol isomers. A increas-
and subsequent inflammatory response. Under ing literature body has demonstrated that B-ring
particular conditions, LDL can cross the endo- oxysterols can stimulate inflammatory signaling
thelial tissue of the blood vessels and being in monocytes by interacting with Toll-like
incorporated into the intima. According to the receptors [18], particularly TLR-4 [34]. 7β-OH
current response-to-injury hypothesis, oxidation cholesterol stimulate the increase of IL-8 secre-
of LDLs favors the expression of cell adhesion tion via the MEK/ERK1/2 pathway [69].
molecules by endothelial cells, such as the cell Oxidation of the fatty acid moiety of
adhesion molecule-1 (ICAM-1) and the vascu- cholesteryl-esters also forms compounds – like
lar adhesion molecule-1 (VCAM-1) [59]. These 9-oxononanoyl cholesterol – able to activate
cell adhesion molecules favor LDL migration adhesion of monocytes [5, 11]. These oxidative
towards the intima. ROS and RNS-induced CE (OxCE) are also able to trigger inflammation
action on LDLs can also occur at the intima through several mechanisms, including TLR-
level [23, 40]. oxLDL-induced activation of mediated response, as recently discussed in detail
macrophages is the second stage of atheroscle- by Miller and Shyy [50].
rosis development. Both chemical and enzy- The discussed inflammatory responses trigger
matic oxidative processes act on the whole LDL the differentiation of monocytes, as well as their
chemical structure, including phospholipids, recruitment from the vascular vessels to the arte-
cholesterol (free and esterified), apolipoprotein rial wall, via trans-endothelial migration [43, 59].
and TAGs [11]. Lipids species derived from oxi- The monocyte recruitment molecular process is
dation are responsible to “activate” monocytes, still under debate, and involve several signaling
via specific interaction with membrane recep- mechanisms, as discussed elsewhere [43]. In the
tors. Extensive oxidation is necessary for LDL intima, monocytes then differentiate into macro-
to bind macrophages receptors SR-A or CD36, phages, that uptake oxLDL via scavenging recep-
including up to 50% cholesterol oxidation, apoB tors, resulting in the transformation of
fragmentation, and diffused oxidation of unsat- macrophages to foam cells. Accumulation of
urated fatty acids [6, 37]. Isoprostane are one of foam cells and platelets is the boundary stone for
the formation of the atherosclerotic lesions showed that the contribution of the different
known as fatty streaks. functional groups to the inhibition of LDL oxi-
dation is well correlated to the energy needed to
form the flavonoid radical by abstraction of a
19.4 Flavonoids Against LDL hydrogen atom. In other words, it is possible to
Oxidation classify flavonols according to their inhibition
potential towards radical and copper induced
19.4.1 Mechanisms of Flavonoids oxidation, being more effective those com-
Action Against Lipid pounds having two adjacent hydroxyls at the
Oxidation B-ring, like quercetin and catechin. Solubility
and partitioning behavior of flavonoids could
Several studies in recent years have examined the also play a role in defining their scavenging
relation between flavonoids and oxidation or activity, considering that their effectiveness as
inflammation. The powerful antioxidant activity LDL oxidative protectors relies in their ability to
exhibited by flavonoids is due to their action as physically interact with the lipid core [48].
free radical scavengers, singlet oxygen quench- However, Paganga et al. [54] demonstrated that
ers, and metal ion chelators [26]. Several in vitro superiority of quercetin vs. catechin in inhibiting
systems have been used to evaluate the antioxi- LDL peroxidation cannot be accounted to parti-
dant potential of flavonoids. In this chapter, we tioning considerations, but to the exceptional
will focus in LDL oxidation and macrophages ability of the former to chelate copper. Another
activation. interesting SAR study was performed by Yi and
LDL oxidation, as aforementioned, is the first coworkers [75], where twenty-three 4-oxo-flavo-
critical step in triggering the inflammatory cas- noids (i.e. C-4 is oxidized to ketone function)
cade that finally brings to atheroma formation were assessed against oxLDL-induced endothe-
and consequent CVD manifestation. Early works lial dysfunction. Similarly to the previous stud-
by Frankel’s group [20, 48] have extensively ies [48, 49, 68], flavonols – morin, myricetin,
explored the ability of fruits flavonoids extracts fisetin and quercetin – showed higher activity in
to dampen oxidation in LDL. Flavonoids possess micromolar range. The promising “hits” shared
lower redox potential than lipids, and are thus higher oxy-functionalization at the flavan ring:
oxidized by ROS and RNS, resulting in more 3′,4′-o-dihydroxyl on B-ring, a 3-hydroxyl on
stable and less-reactive ortho-semiquinone radi- C-ring, a 2,3-double bond and a 5,7-m-dihy-
cal compared to lipid radicals and peroxy radi- droxyl on the A-ring were all required for the
cals [3, 4, 29]. When evaluated individually, observed inhibitory effect.
several pure flavonoids (catechin, quercetin,
cyanidin, caffeic acid, and ellagic acid) showed
up to 95% inhibition of Cu2+-induced hexanal 19.4.2 Dietary Flavonoids and LDL
production in micromolar ranges (2.5–7.5 μM). Oxidation
However, specific binary and ternary mixtures of
flavonoids showed antagonistic effects, particu- A substantial amount of literature has used
larly if caffeic and ellagic acids coexisted in grape and wine as flavonoids-enriched matrix to
equal amounts [48]. Dissimilarities among flavo- leverage their LDL-protectant function, mainly
noids AO activity can be explained taking into supported by early hypothesis regarding the so-
account differences in their chemical structures; called “French paradox” formulated by epide-
particularly, structural variation in the C-ring miologists in the early ‘80. The “French
and the oxidative state of C3 seemed to confer paradox” is the observation of low coronary
different reactivity [29, 48]. A structure-activity heart disease (CHD) death rates despite high
relationship (SAR) study by Vaya et al. [68] intake of dietary cholesterol and saturated fat in
southern French diet [58]. Red wine contains up intrinsic antioxidants are still present. Similar
to 250 mg/mL phenols, depending on type and results have been obtained with flavonoids
variety [44]. Experiments performed using Petit extract from different species of berries [30,
Syrah wine showed that 5 μM concentration of 51], as well as other fruits [44].
individual catechin, myricetin, epicatechin, An obvious following step in CVD-related fla-
rutin and quercetin were able to inhibit LDL vonoids research are in vivo and clinical studies.
oxidation by Cu2+ in the range 60–75% [63], Various studies have exposed either animal mod-
higher than α-tocopherol. More interesting, pro- els or humans subjects to flavonoids-enriched
cyanidins (i.e. oligomeric forms of flavonols) diets, recording controversial results that par-
were higher inhibitors of hexanal formation (up tially debunked the “anti-oxidative claim” [44].
to 80% at 5 μM), which can be explain by their Part of this controversy is due to the lack of reli-
better ability to interact with the LDL constitu- able biomarkers for measuring plasma antioxi-
ents. A more extensive work performed on 14 dant activity, lack of long-term studies and
grapes varieties demonstrated similar effects on underestimation of gut and liver bio-
the inhibition of hexanal generation, with the transformation [44, 73]. A few examples of in
additional information that the inhibitory poten- vivo works follow, and a good critical review can
tial was positively correlated with the content of be found elsewhere [44].
phenols, and in less extent anthocyanins and fla- O’Reilly and co-workers coupled in vitro
vonols [49]. An interesting paper by Frémont assessment of a flavonoid mixture against Cu2+
and co-workers more deeply investigated the oxidation of LDL with dietary intervention
effects of wine extract and individual flavonoids study. All the tested flavonoids – including quer-
in in vitro LDL oxidation by copper and AAPH cetin, luteolin, catechin, and kaempferol, among
[21]. Their results discriminated between che- others – showed sub-micromolar ability to halve
lating agents (resveratrol), which are active TBARS value and decrease LDL peroxides.
against Cu2+ oxidation, and radical scavengers Surprisingly, when 32 subjects were exposed to
(catechin) that inhibit AAPH lipid peroxide for- a high-flavonoids diet, no significant effects
mation. Furthermore, in their studies highly were found in ex vivo LDL oxidation [53]. From
unsaturated cholesteryl-esters, rather than phos- the in vitro effects on LDL, Pignatelli and others
pholipids, were mostly affected by both oxida- [56] administered red and white wine to 20
tion mechanisms, and this was demonstrated by healthy volunteers, finding a significant reduc-
altered electrophoresis mobility of apoB after tion in plasma conjugate dienes, as well as uri-
oxidation. These effects were negligible in nary PGF-2α-III, a marker of oxidative stress.
white wines, which contain lower amounts of Flavonols from concentrated cranberry juice
flavonoids, although imposing a longer grape- decreased plasma oxLDL and cell adhesion mol-
skin contact during winemaking can led to “red- ecules ICAM-1 and VCAM-1 in 30 men, over
like” white wines with analogue antioxidant three successive periods of 4 weeks [60]. In their
capacity [22]. Beside grape, other flavonol-rich review, Lotito and collaborators [44] list a few
dietary extracts have been tested as antioxidants works that failed to demonstrate in vivo efficacy
against LDL chemically-induced oxidation. of flavonoids in inhibit plasma oxidation. The
Viana et al. [70] used an extract of Vaccinium conclusion is that, although epidemiology stud-
myrtillus containing anthocyan, catechin, chal- ies are fairly convincing about their antioxidant
cone and other flavonoids to dampen Cu2+- effects, is unlikely that flavonoids per se acts
induced LDL oxidation. They showed a against LDL oxidation, given their poor bio-
significant increase of the lag phase of lipid per- availability. Likely, observed effects are due to
oxidation associated with the presence of vita- upregulation of endogenous antioxidants
min E, indicating that the effects of flavonoids enzymes, like those of the uric acid biosynthetic
are limited to the early stages of oxidation, when pathway.
19.4.3 Flavonoids and Inflammatory several flavonoids – including kaempferol,

Activation Pathways genistein, quercetin and dadzein – were able to
reduce STAT-1 and NF-kB activation (both
The immune system is integrated by a highly iNOS transcription factors), and suppress iNOS
complex regulated group of cells that may inter- expression, whereas others (including narin-
act in a cell-cell manner and may also respond genin and pelargonidin) had no effect on STAT-
to intercellular messages including hormones 1. A similar mechanism has been proposed to
and cytokines. Diet, pharmacological agents, explain the strong inhibition of NO generation
pollutants, and food chemicals display remark- and iNOS expression in activated macrophages
able actions, at both pharmacological and bio- by resveratrol [66], although a similar study
chemical level, that affect the function of contradicts these findings [72]. Resveratrol can
inflammatory and immune cells, including mac- dampen the pro- inflammatory phenotype by
rophages [24]. We already discussed the func- activating sirtuin-1, a type III histone deacety-
tion of monocytes and derived-macrophages in lase that suppresses NF-kB factor [55].
triggering the inflammatory effect during the Resveratrol downstream regulation mode of the
formation of the atheroma and CVD progres- mitochondrial biogenesis via sirtuin-1 activa-
sion. Macrophage can proliferate in the pres- tion seems to be strongly dose-dependent [9,
ence of a specific growth factor, M-CSF, and in 57]. It is important to notice that these in vitro
the presence of an inducer such as lipopolysac- studies, as well others [41] were performed at
charide (LPS), they stop proliferating adopt a relatively high concentration of resveratrol, far
phenotype that is characterized by the expres- from the ones normally observed in plasma after
sion of early cytokines and NO [14]. Several ingestion (see Sect. 19.2).
flavonoids – as genistein – can interact with
enzymes and receptors involved in the genera-
tion of the inflammatory processes, especially 19.5 Conclusions
tyrosine and serine-threonine protein kinases. In
monocytes, those are responsible for the pro- A few considerations follow from the above
duction of cytokines, such as TNF-α, IL-6 and review. First, several epidemiology studies con-
IL-1β. Kaempferol and quercetin inhibitory ducted in the last decades have demonstrated
effects against tyrosine kinases was reflected in health benefits due to regular intake of flavonoid-
a significant antiproliferative effects on M-CSF- rich foods. Second, the chemistry of flavonoids
activated macrophages [14]. Aglycones and makes them prone to show radical-scavenging
conjugated-derivative showed anti-inflamma- and metal-chelating properties, that can be suc-
tory activity: an interesting study by Kawai and cessfully used to dampen in vitro lipid oxidation,
co-workers [38] found that quercetin-3-glucoro- in particular Cu2+ and free radical-induced oxida-
nide and quercetin disrupt expression of tion of plasma LDL. This has been achieved both
CD36 in murine macrophages cell lines. In vivo using fruit and vegetable extracts, as well as indi-
studies on mice showed that epigallocatechin-3- vidual pure flavonoids standards, with inhibitory
gallate can inhibit LPS-induced and interferon- concentration in the order of 5–10 μM. However,
γ-activated nitric oxide synthase (iNOS) gene clinical studies performed in healthy subjects
expression, inasmuch as iNOS activity in cul- were only partially able to replicate these in vitro
tured macrophages, and so reducing nitric oxide and ex vivo findings by administration of a vari-
production and subsequently oxidative stress ety of dietary regimes with high content in flavo-
[8]. Direct inactivation of iNOS was explained noids. The lack of in vitro-in vivo correlation can
also by the ability of certain flavonoids to bind be attributed to several factors, that can be sum-
arginine and the iNOS cofactor tetrahydrobiop- marized in: i) reduced adsorption (0 to 1–2 μM in
terin [8]. A more extended study performed by plasma) and short lifetime, that make unlikely an
Hämäläinen and co-workers [35] showed that actual antioxidant effect, ii) biotransformation to
flavonoids-conjugates, with unknown or reduced 8. Chan MM-Y, Fong D, Ho C-T, Huang H-I (1997)
Inhibition of inducible nitric oxide synthase gene
biological activity. In order to positively merge expression and enzyme activity by epigallocatechin
the in vitro observations with the diffused epide- gallate, a natural product from green tea. Biochem
miological studies, research should be focused in Pharmacol 54(12):1281–1286
validate those speculated mechanisms in more 9. Chang YP, Ka SM, Hsu WH, Chen A, Chao LK, Lin
CC, Hsieh CC, Chen MC, Chiu HW, Ho CL (2015)
physiological settings. Among various aspects Resveratrol inhibits NLRP3 inflammasome activation
worth of research interests, the authors believe by preserving mitochondrial integrity and augmenting
that priority should be given to a deep under- autophagy. J Cell Physiol 230(7):1567–1579
standing of the “fate” of flavonoids in our organ- 10. Charron CS, Kurilich AC, Clevidence BA, Simon PW,
Harrison DJ, Britz SJ, Baer DJ, Novotny JA (2009)
ism, a critical effort that should consider Bioavailability of anthocyanins from purple carrot
adsorption, bioavailability, and the actions of juice: effects of acylation and plant matrix. J Agric
intrinsic metabolism, inasmuch as gut microbi- Food Chem 57(4):1226–1230
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Acknowledgements This work has been partially funded
of pro-inflammatory markers in primary bone marrow-
by Michigan State University start-up to I.G.M.M. Mr.
derived mouse macrophages by naturally occurring
Matthew Schweiss is acknowledged for his help in prepar-
flavonoids: analysis of the structure–activity relation-
ing Table 19.1.
ship. Biochem Pharmacol 72(8):1010–1021
15. Dai J-y, Yang J-l, Li C (2008) Transport and metab-
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62. Shafiee M, Carbonneau M-A, Urban N, Descomps B, inhibition of oxidized low-density lipoprotein (LDL)-
Leger CL (2003) Grape and grape seed extract capaci- induced vascular endothelial dysfunction. Int J Mol
ties at protecting LDL against oxidation generated by Sci 12(9):5471–5489
Index
A Colonic inflammation, 2, 115–118, 120

Age related macular degeneration (AMD), Colorectal cancer (CRC), 2, 115–120
155, 157, 158, 160 Conjunctiva, 2, 14–23, 160
Allergic eye disease, 5 Cornea, 3–9, 13–17, 19, 83, 155, 156, 160
Amyotrophic lateral sclerosis (ALS), 233–239 COX-inhibition, 94, 96
Anandamide, 77, 108, 220 Cyclooxygenase (COX), 2, 18, 38, 52, 77, 90, 91,
2-Arachidonoyl glycerol (2-AG), 77–79, 81, 83, 84, 86, 107, 126, 136, 137, 222
194, 206, 207, 220–222, 228 Cyclooxygenase-2 (COX-2), 5, 6, 71, 72, 77–80, 83, 84,
Astrocytes, 30, 135, 142, 155, 159, 233–235 86, 93, 94, 96, 108, 109, 115, 116, 119, 136,
Autoimmunity, 19, 134, 135, 141, 153, 156, 157, 150, 152, 153, 181, 224
160, 180 Cytochrome P450 (CYP), 2, 107, 108, 115–121,
221–226, 228
Cytokines, 4, 6, 7, 9, 28–30, 38, 39, 49, 50, 52, 55, 66,
B 71, 90, 91, 117, 120, 138, 139, 150, 151,
Bacteria, 3, 5, 6, 8, 14, 15, 23, 66, 175, 182 153–156, 158–160, 172, 174, 177, 179–182,
Biomarkers, 2, 32, 47, 49, 74, 101–109, 117, 233–239, 205, 225, 227, 250
247, 249
Blood-brain barrier (BBB), 28–31, 134, 142, 150, 194,
236, 238 D
Bone, 31, 94, 139, 159, 177, 246 Delirium, 27
Brain, 1, 14, 28, 66, 78, 104, 125, 134, 150, 194, 234 Demyelination, 134, 135, 180
BV-2, 29, 203, 225, 226 Diabetic retinopathy (DR), 2, 119, 155, 158–160
Dietary antioxidants, 246
Disease, 1, 3, 14, 29, 37, 46, 66, 81, 102, 134, 151,
C 172, 194, 222, 233, 243
Cancer, 1, 2, 80, 90, 91, 93, 95–97, 105, 106, 115–120, n-3 Docosapentaenoic acid (DPA), 57, 66
153, 180, 182, 194–204, 227, 228, 236 Docosahexaenoyl ethanolamine (DEA), 195, 200–201,
Cancer-associated fibroblasts (CAFs), 91–93, 95, 97 204–209, 212
Cannabinoid (CB), 2, 142, 194, 212, 220, 222, 224, Dry eye disease, 19, 160
225, 227, 228
Cannabinoid receptors (CB1 and CB2), 194, 220, 222
Cardiac disorders, 37–42 E
Cardiac repair, 53 Eicosanoids, 1, 2, 19, 38, 52, 55, 71, 73, 82, 86, 89,
Cardiovascular disease (CVD), 1, 2, 37–41, 46, 57, 58, 103–105, 107–109, 115–121, 126, 135–138,
68, 69, 105, 106, 118, 177, 150, 153, 222
236, 243, 248–250 Endocannabinoid (eCB), 2, 78, 80, 194, 195,
Cell signaling, 235 204, 206, 207, 220–229
Ceramide-1-phosphate (C1P), 151, 152, 160, Endopthalmitis, 8
170, 175, 176 Epoxyeicosatrienoic acids (EETs), 107, 116, 118, 119,
Ceramides, 2, 151, 170, 235 135, 142, 143, 225
Cholesterol oxidation, 243–251 Epoxygenases, 1, 107, 221–226, 228, 229

https://doi.org/10.1007/978-3-030-21735-8
256 Index
F M
Flavonoids, 2, 243–251 Macrophages, 4, 16, 28, 38, 47, 67, 79, 129, 137, 150,
220, 244
Maresins (MaR), 1, 5, 17–19, 29, 31, 38,
G 51, 53–55, 66, 67
Glaucoma, 2, 5, 159 Mass spectrometry (MS), 19, 71, 82, 83, 103–105, 108,
Goblet cells (GC), 2, 14–23 120, 126, 134–139, 142, 143, 160, 195–198,
206, 208, 223
Memory, 27, 28
H Metabolomics, 101–109, 120, 236
Healing, 5–7, 9, 19, 28, 31, 48 Microglia, 29, 30, 137, 155, 156, 159, 194, 196,
Heart failure, 2, 37, 41, 46, 105 198, 203–205, 211, 212, 220, 222, 224,
Hemorrhage, 117, 125, 127–129, 157 226, 233, 234
Hemorrhagic transformation, 125, 127 Microsomal prostaglandin E synthase-1 (mPGES-1),
12-Hydroxyeicosatetraenoic acid (12-HETE), 18, 55, 56, 89–97, 137
126, 129, 238 Mild traumatic brain injury (mTBI), 194–196,
15-Hydroxyeicosatetraenoic acid (15-HETE), 56, 83, 198–212
126, 238 mPGES-1 inhibition, 93, 95, 96
Mucins, 15, 16, 20
Myocardial infarction (MI), 38–40, 46, 48, 106
I
Immune cells, 2, 14, 29, 49, 51, 52, 91, 93,
120, 135, 136, 139, 150, 151, 154, 156, N
157, 160, 170–182, 194, 205, 220, N-arachidonoyl glycine (NAGly), 195, 200–201,
224, 234, 250 203, 204, 208, 211, 212
Infection, 1, 3, 5–9, 28, 47, 49, 54, 71, 72, 150, 151, Neuroblastoma, 2, 89–97
173, 176, 177 Neurodegeneration, 29, 30, 135, 155
Inflammation, 1, 3, 17, 28, 37, 47, 66, 81, Neuroimmunology, 134–143
104, 134, 149, 172, 203, 222, Neuroinflammation, 2, 27–32, 154, 155, 157, 201, 203,
235, 243 212, 220, 222, 234–236, 238
Ischemia, 40, 52, 126, 127, 158 Neuroprotection, 29, 129, 142, 205, 212
Ischemic stroke, 2, 125–129
O
K Ocular surface, 2, 3, 7–9, 13–23, 160
Keratitis, 1, 3–9 Omega-3 fatty acids, 54, 204, 205
Oxidative stress, 39, 40, 54, 157, 158, 177, 182,
205, 234–237, 243, 247, 249, 250
L Oxysterols, 247
Lactosylceramide (LacCer), 151, 154, 160,
175, 176, 238
Leukocytes, 3, 5, 7, 9, 19, 37, 40, 41, 47–49, P
51, 52, 54, 57, 58, 66–69, 71, 73, Phospholipases A2 (PLA2), 2, 21–23, 136, 139, 142
150, 154, 157, 159, 180, 181, Platelet-activating factor (PAF), 139
206, 222 Polyunsaturated fatty acids (PUFA), 4, 29, 38, 41, 51, 55,
Leukotriene B4 (LTB4), 6, 38, 53, 54, 73, 119, 138 105–107, 115, 116, 118, 126, 135,
Lipid biomarkers, 233–239 205, 221, 222, 225, 235
Lipid mediators, 1, 3–9, 28, 29, 38, 39, 50, 52, 54, 57, Pro-resolving mediators, 2, 7–9, 49, 51, 54–56, 58,
67, 69, 73, 86, 89, 105, 108, 139, 65–74, 205
142, 143, 228, 235 Prostaglandin E2 (PGE2), 2, 4, 20, 38, 79, 80, 89–91,
Lipid oxidation, 2, 246, 248, 250 93–95, 97, 107, 108, 119, 154, 235, 238
Lipidomics, 55, 103–105, 107, 137, 177, 195, 198, Prostaglandin glycerol ester, 77–86
211, 223–226, 235, 236 Prostaglandins (PGs), 2, 4, 19, 38, 56, 66, 77–86,
Lipoamines, 194, 195, 198, 201, 203, 208–211 107, 126, 150, 195, 224, 235
Lipoxins (LX), 1, 4, 5, 17–19, 29, 30, 38, 40, Psychiatric disorders, 101–109
51, 53, 54, 56, 57, 66, 107, 138
Lipoxygenase (LOX), 1, 2, 4, 6, 18, 38, 49,
52, 53, 55, 57, 119, 125–129, 138, 150, R
205, 222, 238 Resolution, 4, 28, 38, 47, 67, 83, 105, 224
Low-density lipoprotein (LDL), 236, 244–248 Resolution of inflammation, 1, 4, 6, 28, 31, 39,
Lysophosphatidic acid (LPA), 139–142 47–57, 67, 107
Index 257
Resolvin D1 (RvD1), 2, 5–8, 13–23, 29, 40, 41, 51–57, T

67, 68, 205 Targeted therapy, 90
Resolvins (Rv), 1, 2, 5, 13, 17–19, 29, 38, 40, 51–53, Tear film, 5, 14–17, 19, 23, 160
56–58, 66, 67, 71, 138, 205, 206, 212 Tissue plasminogen activator (tPA), 125,
Resveratrol, 249, 250 127–129
Transgenic mouse, 83, 84, 118
Transient receptor potential (TRP), 194, 195,
S 206–208
Secretion, 2, 9, 14–17, 19–23, 50, 54, 81, 92, 129, Tumor microenvironment (TEM), 2, 91–93, 95, 97, 117,
151, 157, 158, 174, 175, 178, 179, 247 179, 180
Signaling pathways, 2, 8, 17, 20–23, 28, 29, 40, 47, 55, Tumor-promoting inflammation, 92–94, 97
91, 115–121, 129, 142, 149, 173, 229, 236
Soluble epoxide hydrolase (sEH), 2, 107–109,
116–120, 228 U
Specialized pro-resolving mediators (SPMs), 1, 4, 7–9, Uveitis, 2, 5, 155–157
14, 17–20, 27–32, 37–42, 46–56, 66, 67, 69, 72,
73, 138, 142, 143, 235
Sphingolipids, 2, 105, 139, 149–160, 170–182, 235 V
Sphingosine-1-phosphate (S1P), 30, 139–142, 151, Vascular inflammation, 56, 69
153–158, 160, 172, 175, 176, 178, 179, 181
STAT6, 128
Statins, 2, 65–74 W
Surgery, 3, 8, 27–32, 46 Warfarin, 127, 128

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Advances in Experimental Medicine and Biology 1161

The Role of Bioactive

The Role of Bioactive

ISSN 0065-2598 ISSN 2214-8019 (electronic)

© Springer Nature Switzerland AG 2019

10 Metabolomics Biomarkers for Precision Psychiatry................. 101

Kenneth V. Honn, Ph.D., Distinguished

clinical trials, and he holds 17 US patents, 7 of

Darryl C. Zeldin, M.D., Scientific Director,

and clinical research fellows. As the NIEHS sci-

© Springer Nature Switzerland AG 2019 1

2.1 Infectious Keratitis ally characterized by rapid progression, focal

© Springer Nature Switzerland AG 2019 3

Fig. 2.1 Schematic of major SPM pathways involved in the cornea

2.3 Current Treatments

results in a larger epithelial defect. Unfortunately, of infection. The effect on infiltrating

oxygenase in counter-regulating inflammation during D1 analogue controls maturation of dendritic cells

Abstract in human tears and induces goblet cell mucin

© Springer Nature Switzerland AG 2019 13

Fig. 3.1 Scehmatic diagram of the eye and ocular surface

Fig. 3.2 An electron

also works by activating a matrix metalloprotein- 3.2 Specialized Pro-resolving

b Human Emotional Tears 2D Loading Plot

c Total SPMs and PG Gender

Fig. 3.6 Schematic RvD1

increases IP3 and

3.4.3 Summary of Pathways Acknowledgements Dr. Dartt is supported by the

Abstract postoperative neuroinflammation and neuro-

© Springer Nature Switzerland AG 2019 27

­ olecules is required to balance and orchestrate

References USA 113:E6686–E6695. https://doi.org/10.1073/

30. Block ML, Zecca L, Hong JS (2007) Microglia-­

tial. Immunity 46:957–967. https://doi.org/10.1016/j. pro-­

Keywords We can consider acute inflammation as an

© Springer Nature Switzerland AG 2019 37

5.2 Cells and Mediators in CVD Thromboxane B2 (TXB2) and leukotriene B4

Prostaglandins Resolvins E Resolvins D

remodelling. Br J Pharmacol 172(23):5647–5660. 37. Martinet W, Meyer I, Verheye S, Schrijvers DM,

G. V. Halade (*) · B. Tourki

© Springer Nature Switzerland AG 2019 45

6.1 Introduction Currently, 5.7 million people are diagnosed

(DHA), eicosapentaenoic acid (EPA), and acti- 6.3.1 Neutrophils – Digest

Table 6.1 Role of SPMs in cardiac repair in different experimental models

6.4.3 Maresins dual roles as counter-regulators of inflammation,

Abstract biological actions, actively reprogramming

© Springer Nature Switzerland AG 2019 65

damaged tissues as well as to defend the organ- (5S-hydroxy-6R-(S-cysteinylglycinyl)-7E,9E,

Fig. 7.2 PDn-3 DPA

P. J. Kingsley · L. J. Marnett (*)

© Springer Nature Switzerland AG 2019 77

compounds – referred to as endocannabinoids lipids is mainly based on three observations:

co-­ordination with the ammonium ion PGF2α-ethanolamide (PGF2α-EA) in the spinal

Despite the relative abundance of reports of 8.5.1 P

Fig. 8.2 Western blot of Tg WT

Abstract geted therapies. Cancer-associated fibroblasts

K. Larsson (*) · P.-J. Jakobsson

© Springer Nature Switzerland AG 2019 89

PGH 2 PGF synthase PGF2α

tions remains to be proven [32, 33]. PGE 9.3 PGE2 in the Tumor

Proliferation, survival, migration, invasion,

10 Metabolomics Biomarkers for Precision Psychiatry................. 101

2.1 Infectious Keratitis ally characterized by rapid progression, focal

2.3 Current Treatments

also works by activating a matrix metalloprotein- 3.2 Specialized Pro-resolving

3.4.3 Summary of Pathways Acknowledgements Dr. Dartt is supported by the

olecules is required to balance and orchestrate

30. Block ML, Zecca L, Hong JS (2007) Microglia-

tial. Immunity 46:957–967. https://doi.org/10.1016/j. pro-

5.2 Cells and Mediators in CVD Thromboxane B2 (TXB2) and leukotriene B4

6.1 Introduction Currently, 5.7 million people are diagnosed

(DHA), eicosapentaenoic acid (EPA), and acti- 6.3.1 Neutrophils – Digest

6.4.3 Maresins dual roles as counter-regulators of inflammation,

co-ordination with the ammonium ion PGF2α-ethanolamide (PGF2α-EA) in the spinal

tions remains to be proven [32, 33]. PGE 9.3 PGE2 in the Tumor

Carlson and coworkers, a shift was found during 9.5 Prostaglandin E2

The levels of PGE2 not only depend on COX 9.5.2 COX-Inhibitors

9.8 Conclusions (www.nnbcr.se), Märta and Gunnar V Philipson

post-traumatic stress disorder; mood disorders, 10.2 Omics-Based Strategies

filing approach characterizing 181 proteins and 10.4 Overview of Metabolomics

of analytes in targeted metabolomics, offering brain-specific alterations in glucoregulatory

Depression is heterogeneous in its presenta- 10.6 Polyunsaturated Fatty Acids

percentages of CD3+ and CD+ lymphocytes Schizophrenia is a serious psychiatric disorder

10.8 Conclusions psychopathology of psychiatric disorders, metab-

13.1 Neuro-immune Interactions ing difficulties, numbness/tingling, vision prob-

13.1.2 Experimental Autoimmune since NOD mice develop spontaneous diabetes

from motor dysfunction associated with EAE 13.2.4 Lipoxygenases (LOs)

13.3 Platelet-Activating Factor 13.4 Lysophospholipid (LP)

PAF (1-O-alkyl-2-acetyl-sn-glycero-3-phospho LP is a derivative of a phospholipid in which one

expansion of Lin−Sac-1+cKit+ hematopoietic pro- Knockout and antagonist studies targeting

inflammatory responses [111]. The involvement 14.2.4 Lactosylceramide

and induces angiogenesis. LacCer mediated 14.3.2 Sphingolipidoses and Ocular

tent low-grade inflammation. It has been reported 14.3.6 Sphingolipids and Glaucoma

fMLP N-formylmethionine-leucyl- 15.1 Introduction

15.2.1 Dendritic Cells

These observations make it difficult, at a first 15.2.3 Macrophages

15.3.2 Graft-Versus-Host Disease 15.3.3 Immune Suppression

16.4.3 N-Docosahexaenoyl TNC after repeated mTBI, along with N-palmitoyl

16.4.6 Effects of mTBI on Orphan

17.2.8 Vasoactivity of EPA been shown to be anti-proliferative, anti-

17.4 Conclusions and Future disease, hypertension, and nociceptive pain [21,

Abstract 18.1 Introduction

Keywords items are: flavonols, flavones, flavanones, flavan-

19.4.3 Flavonoids and Inflammatory several flavonoids – including kaempferol,