Antimicrobial Peptides Role in Human Health and Disease 2016 Harder

Birkhäuser Advances in Infectious Diseases
Jürgen Harder
Jens-M. Schröder
Editors
Antimicrobial
Peptides
Role in Human Health and Disease
Jürgen Harder • Jens-M. Schröder
Editors
Antimicrobial Peptides
Role in Human Health and Disease
Series editors
Stefan H. Kaufmann
Max Planck Institute for Infection Biology, Berlin, Germany
Andrew A. Mercer
Department of Microbiology and Immunology, University of Otago
Dunedin, New Zealand
Olaf Weber
Bonn, Germany
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Editors
Jürgen Harder Jens-M. Schröder
Department of Dermatology Department of Dermatology
University-Hospital Schleswig-Holstein University-Hospital Schleswig-Holstein
Campus Kiel Campus Kiel
Kiel Kiel
Germany Germany

ISBN 978-3-319-24197-5 ISBN 978-3-319-24199-9 (eBook)
DOI 10.1007/978-3-319-24199-9
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Contents
1 Antimicrobial Peptides in Cutaneous Wound Healing . . . . . . . . . . . . . . 1

Ole E. Sørensen
2 Antimicrobial Peptides as Endogenous Antibacterials
and Antivirals at the Ocular Surface . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Fabian Garreis, Martin Schicht, and Friedrich Paulsen
3 Function of Antimicrobial Peptides in Lung Innate Immunity . . . . . . 33
Frederik Seiler, Robert Bals, and Christoph Beisswenger
4 Antimicrobial Peptides: Maintaining Sterility
of the Urinary Tract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
Brian Becknell and John David Spencer
5 Antimicrobial Peptides in the Gut . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
Maureen J. Ostaff, Eduard F. Stange, and Jan Wehkamp
6 Metal Sequestration: An Important Contribution of Antimicrobial
Peptides to Nutritional Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
Steven Damo and Thomas E. Kehl-Fie
7 Regulation of Antimicrobial Peptide Gene Expression by
Vitamin D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
Adrian F. Gombart
8 Dichotomous Roles of Cationic Polypeptides Targeting HIV . . . . . . . 115
Alexander M. Cole and Amy Liese Cole
v
vi Contents
9 Antimicrobial Peptides in Host Defense: Functions

Beyond Antimicrobial Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
Kim Alan Brogden, Amber M. Bates, and Carol L. Fischer
10 Antimicrobial Peptides: Do They Have a Future as
Therapeutics? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
Michael Zasloff
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
Chapter 1
Antimicrobial Peptides in Cutaneous
Wound Healing
Ole E. Sørensen
Abstract Injury that breached the physical skin barrier increases the likelihood of
infection. The wound healing process is divided into hemostasis, inflammation, pro-
liferation, and tissue remodeling. Antimicrobial peptides play a major role for the
antimicrobial defense at all these stages in wound healing, but the main sources of
antimicrobial peptides vary with the different stages of wound healing coming from
plasma proteins, neutrophils, and keratinocytes. Apart from being part of the antimi-
crobial defense, antimicrobial peptides play other important roles in wound healing
as in angiogenesis, attraction of leukocytes, resolution of inflammation, and prolif-
eration. Future studies will demonstrate whether antimicrobial peptides can be used
therapeutically to improve the wound healing processes and reduce scar formation
in chronic wounds.
1.1 Introduction
The intact skin constitutes a very efficient physical barrier toward surrounding
microbes. Indeed, skin infections are rarely found in intact healthy skin. However,
injury or wounding causes breach in the physical barrier of the skin increasing the
likelihood of infections. Keeping the wound free of overt infection is a prerogative
for successful wound healing (Edwards and Harding 2004). Indeed, chronic non-
healing cutaneous wounds are most often infected with Staphylococcus aureus or
Pseudomonas aeruginosa (Edwards and Harding 2004). Antimicrobial peptides
(AMPs) play a major role for the antimicrobial defense during wound healing.
Indeed, studies of the remarkable ability of the African frog Xenopus laevis to keep
its wounds free of infections under non-sterile conditions led to the first identifica-
tion of antimicrobial peptides (AMPs) from the skin (Zasloff 1987).
O.E. Sørensen
Nuclear Biology Laboratory, Division of Infection Medicine, BMC, B14, Department of
Clinical Sciences, Lund University, Tornavägen 10, Lund SE-221 84, Sweden
e-mail: [email protected]
© Springer International Publishing Switzerland 2016 1

J. Harder, J.-M. Schröder (eds.), Antimicrobial Peptides: Role in Human Health
and Disease, Birkhäuser Advances in Infectious Diseases,
DOI 10.1007/978-3-319-24199-9_1
2 O.E. Sørensen
Wound healing following injury is traditionally divided into four stages: (1) hemostasis,
(2) inflammation, (3) proliferation, and (4) tissue remodeling (Singer and Clark 1999).
AMPs are found at all stages of wound healing; however, the sources of AMPs are differ-
ent at each of these stages of wound healing.
1.2 Generation of AMPs at the Different Stages of Wound

Healing
1.2.1 Injury
Injury by itself leads to generation of AMPs. In frog skin, the injury-induced ner-
vous stimulation leads to release of antimicrobial peptides from skin glands
(Simmaco et al. 1998). Though similar mechanisms have not been described in
mammals, injury does lead to activation of proteases that release membrane-bound
growth factor like HB-EGF and amphiregulin that possess antimicrobial activity
(Malmsten et al. 2007). Furthermore, these growth factors play a major role as
inducers of the epidermal AMP expression at later stages in the wound healing pro-
cess (Sørensen et al. 2006, 2008; Roupé et al. 2010). Surely, tissue injury may gen-
erate additional AMPs but a systematic study of antimicrobial peptides released by
tissue injury is still to come.
1.2.2 Hemostasis
After injury, there is extravasation of plasma proteins into the wound with activation
of complement and coagulation cascades. Activation of the complement system
leads to generation of fragments of C3a with antimicrobial activity (Nordahl et al.
2004; Sonesson et al. 2007; Pasupuleti et al. 2007). The coagulation cascade is one
of the principal host defenses in insects and activation of the human coagulation
cascade leads to cleavage of several proteins like kininogen, fibrinogen, and throm-
bin involved in the coagulation cascade, which leads to generation of several anti-
microbial peptides (Frick et al. 2006; Påhlman et al. 2013; Papareddy et al. 2010).
Thrombin knockout mice have increased susceptibility to infection with
Staphylococcus aureus infection in their limited life span (Mullins et al. 2009),
which may indicate an important role of the thrombin-derived AMPs.
1.2.3 Inflammation
After hemostasis follows influx of neutrophils followed by monocytes and lympho-

cytes to the wounds in the inflammatory stage of wound healing (Singer and Clark
1999). Neutrophils contain large amounts of antimicrobial peptides (Levy 1996;
1 Antimicrobial Peptides in Cutaneous Wound Healing 3
Borregaard and Cowland 1997; Borregaard et al. 2007) important for the microbici-
dal activity of these cells (Flannagan et al. 2009). During the inflammatory phase,
the major AMPs present in the wound will be derived from the neutrophils. The
AMPs in neutrophils are found both in neutrophil granules and cytosol (Levy 1996;
Borregaard and Cowland 1997). Neutrophils even produce an AMP like elafin after
migration to the skin (Theilgaard-Mönch et al. 2004); however, the main antimicro-
bial peptides found in neutrophils are the α-defensins (HNPs) in azurophil granules
(Ganz et al. 1985), cathelicidins in specific granules (Sørensen et al. 1997), and
calgranulins (S100A8/S100A9) in the cytosol (Hessian et al. 1993).
The main antimicrobial activity of neutrophil defensins is exerted in the neutro-
phil phagolysosome (Joiner et al. 1989), but neutrophil defensins are also secreted
to the exterior (Ganz 1987; Faurschou et al. 2002). Apart from their direct antibacte-
rial and antiviral activity (Ganz et al. 1985; Daher et al. 1986), defensins also boost
bacterial phagocytosis by macrophages (Soehnlein et al. 2008a). Neutrophil defen-
sins have other functions of importance in wound healing. Human neutrophil defen-
sins have chemotactic activity toward monocytes (Territo et al. 1989), T cells
(Chertov et al. 1996), and immature dendritic cells (Yang et al. 2000a). Furthermore,
these peptides are mitogenic for epithelial cells and fibroblasts (Murphy et al. 1993).
Neutrophil defensins do not seem to have a nonredundant function in wound heal-
ing since mice neutrophils lack defensins (Eisenhauer and Lehrer 1992).
The cathelicidins present in specific granules (or large granules in rudiments) are
stored as inactive proteins in the granules and the biological function is unleashed
following extracellular cleavage with serine proteases from azurophil granules
(Zanetti 2004). In porcine and bovine neutrophils, the cathelicidins are processed
by elastase (Panyutich et al. 1997; Scocchi et al. 1992) while proteinase 3 is respon-
sible for the processing of the human cathelicidin hCAP-18 to the antimicrobial
peptide LL-37 (Sørensen et al. 2001), a peptide with broad-spectrum antimicrobial
activity (Turner et al. 1998). The elastase-mediated processing porcine cathelicidins
has been shown to be important for clearance of bacteria from wounds (Cole et al.
2001). Cathelicidins have other important functions in wound healing, both in the
recruitment of mononuclear cells and in the tissue regeneration. It has long be rec-
ognized that neutrophils play a major role for the subsequent recruitment of mono-
cytes (Ward 1968), and it has now been recognized that LL-37 plays a role for the
recruitment of monocytes (Soehnlein et al. 2008b). Indeed, LL-37 has been shown
to be a chemotactic factor toward monocytes as well as T cells and neutrophils
(Yang et al. 2000b). Porcine and murine cathelicidins have importance for angio-
genesis (Li et al. 2000; Koczulla et al. 2003) and both the porcine PR-39 and the
human LL-37 induce expression of VEGF (Rodriguez-Martinez et al. 2008).
Furthermore, treatment of wounds with the gene delivery of the human cathelicidin
LL-37 promotes wound healing (Jacobsen et al. 2005; Steinstraesser et al. 2014;
Carretero et al. 2008). Transactivation of the epidermal growth factor (EGFR)
occurs in cutaneous wound healing (Tokumaru et al. 2000) and LL-37 has been
shown to cause EGFR transactivation (Tjabringa et al. 2003) and thereby induce
keratinocyte migration (Tokumaru et al. 2005). Furthermore, LL-37 suppresses
both keratinocyte and neutrophil apoptosis (Chamorro et al. 2009; Nagaoka et al.
2006).
4 O.E. Sørensen
S100A8/S100A9 constitutes 40 % neutrophil cytosolic protein (Edgeworth et al.

1991) and is a potent antifungal agent (Steinbakk et al. 1990). In wounds, neutrophil-
derived S100A8/S100A9 is probably released from dying neutrophils or from neu-
trophil extracellular traps (NETs) and contributes to killing of Candida albicans
(Urban et al. 2009). Apart from the direct antifungal effect, S100A9 enhances
microbicidal activity of neutrophils by enhancing phagocytosis (Simard et al. 2011)
and S100A8/S100A9 is important for neutrophil accumulation in response to LPS
(Vandal et al. 2003) and induces neutrophil chemotaxis and adhesion (Ryckman
et al. 2003). S100A8/S100A9 mediates proinflammatory activities mediated by
binding to TLR-4 (Vogl et al. 2007) or the receptor for advanced glycation end
products (RAGE) (Hofmann et al. 1999). Additionally, calgranulins have functions
that may limit tissue damage or are anti-inflammatory. S100A8/S100A9 is very
sensitive to oxidation (Raftery et al. 2001; Lim et al. 2008) and may, thus, act as
oxidant scavenger. S100A8 induces expression of the anti-inflammatory cytokine
IL-10 and protects against tissue injury (Hiroshima et al. 2014) and may, thus, play
a role in resolution of inflammation.
1.2.4 Proliferative Phase
As the inflammatory cells recede from the wound, the epidermal keratinocytes
become a major source for AMPs during the proliferative phase of wound healing.
While non-injured epidermis contains constitutively expressed AMPs like hBD-1
and RNase 7, the expression of many AMPs is induced during wound healing and
inflammation. Indeed, many AMPs like hBD-2, hBD-3, RNase 7, and psoriasin
were originally isolated from inflamed epidermis (Gläser et al. 2005; Harder et al.
1997, 2001; Harder and Schröder 2002). Though some epidermal expression of the
human cathelicidin hCAP-18/LL-37 may also be found in the inflammatory stage of
wound healing (Dorschner et al. 2001), the peak expression of epidermal AMPs is
found during the proliferative phase of wound healing (Roupé et al. 2010). The
AMPs with increased epidermal expression during the proliferative phase of wound
healing include hBD-2 (Schmid et al. 2001), hBD-3 (Sørensen et al. 2006), psoria-
sin (S100A7) (Lee and Eckert 2007), S100A8/S100A9 (Thorey et al. 2001),
S100A15 (Roupé et al. 2010), elafin (vanBergen et al. 1996), SLPI (Schmid et al.
2001; Wingens et al. 1998), lactoferrin (Roupé et al. 2010), midkine (Frick et al.
2011), and NGAL (Sørensen et al. 2006). Since the various defensins arise from a
common ancestral gene (Bevins et al. 1996), it is remarkable that neutrophils and
keratinocytes share many of the same AMPs and antimicrobial proteins (see
Table 1.1); however, the AMPs in keratinocytes are induced only during wound
healing and inflammation while the AMPs in neutrophils are synthesized during
normal neutrophil differentiation in the bone barrow (Borregaard et al. 2005).
However, this means that the antimicrobial proteins and peptides are present in the
wound over and extended time, though the cellular source varies.
Table 1.1 Antimicrobial peptides and proteins present in both neutrophils and keratinocytes
AMPs in neutrophils and keratinocytes
Neutrophils Keratinocytes
Azurophil granules
α-defensins (HNP-1-4) β-defensins (hBD-1-3)
Lysozyme Lysozyme
Specific and gelatinase granules
Lactoferrin, NGAL, hCAP-18/LL-37 Lactoferrin, NGAL, hCAP-18/LL-37
Transcobalamin, SLPI, lysozyme Transcobalamin, SLPI, lysozyme
Cytosol
Calgranulins (S100A8/S100A9) Calgranulins (S100A8/S100A9)
Produced following extravasation
Elafin Elafin
Keratinocyte AMPs not present in neutrophils
Psoriasin, RNase7, S100A15
Since α- and β-defensins arise from an ancestral gene, it is remarkable how many other antimi-
crobial peptides and proteins found in keratinocytes are also found in neutrophils. See text for
references
The expression of some AMPs during wound healing is dependent on inflamma-

tion, i.e., cytokines from infiltrating inflammatory cells as in the case of the IL-1-
dependent hBD-2 expression (Liu et al. 2003; Sørensen et al. 2005). However, the
expression of other AMPs, like hBD-3, is induced by the injury-induced EGFR
activation in epidermal keratinocytes, even in the absence of inflammatory cells
(Sørensen et al. 2006; Roupé et al. 2010), thus directly linking growth and tissue
regeneration with AMP expression. Deficiency in the injury-induced hBD-3 expres-
sion has been linked to the severity of skin infections and nasal carriage of S. aureus
(Zanger et al. 2010, 2011; Nurjadi et al. 2013). Additionally, many AMPs like
S100A8/S100A9, SLPI, and NGAL are induced both by injury-induced GFR acti-
vation and proinflammatory cytokines (Roupé et al. 2010; Mork et al. 2003;
Sørensen et al. 2003; Liang et al. 2006).
Though AMP expression mainly is induced during wound healing and inflamma-
tion, inflammatory stimuli seem to downregulate the expression of certain AMPs
during wound healing. The antimicrobial chemokine CXCL14 is normally expressed
in the epidermis (Maerki et al. 2009) but downregulated during wound healing and
inflammation (Frick et al. 2011; Maerki et al. 2009). Likewise, RNase 7 protects
healthy skin from Staphylococcus aureus infection (Simanski et al. 2010). The
expression of RNase 7 is induced both by IFN-γ/IL-17 (Simanski et al. 2013) and
significantly by EGFR activation in injured skin ex vivo (Wanke et al. 2011). Though
RNase 7 is significantly induced through EGFR activation in injured skin ex vivo,
the expression level of RNase 7 is the same in skin wounds in vivo as in non-injured
skin (Roupé et al. 2010) indicating that some inflammatory mediators may down-
regulate the injury-induced RNase 7 expression.
6 O.E. Sørensen
The epidermal AMPs induced by wound healing generate a broad spectrum on

antibacterial activity, e.g., hBD-3 against S. aureus and S. pyogenes (Harder et al.
2001), psoriasin against E. coli (Gläser et al. 2005), and calgranulins against C.
albicans (Steinbakk et al. 1990). Though, for example, psoriasin has been impli-
cated in defense against E. coli (Gläser et al. 2005) and hBD-3 and RNase 7 against
S. aureus (Zanger et al. 2010; Simanski et al. 2010), the appearance of AMPs during
the proliferative phase of wound healing raises the question of whether these pep-
tides may play additional roles beyond antimicrobial defense of the wound. Notably,
some AMPs seem to remain intracellular or cell associated while other AMPs are
secreted. hBD-2 is readily secreted into the medium from multilayer epidermal
keratinocyte cell cultures (Sørensen et al. 2005), while hBD-3 remains cell associ-
ated, both in multilayer epidermal keratinocytes cell cultures (Sørensen et al. 2005)
and in whole epidermis (Sørensen et al. 2006). Though, the antimicrobial S100A
proteins psoriasin (S100A7), calgranulins (S100A8/S100A9), and S100A15 are
cytosolic, at least psoriasin is also found extracellularly (Gläser et al. 2005). For
AMPs to interact with other cells, the AMPs must – at least partially – be found
extracellularly.
Numerous cytokine-line functions have been attributed to hBD-2. hBD-2 acti-
vates dendritic cells through TLR-4 (Biragyn et al. 2002) and is a chemoattractant
toward immature dendritic cells and memory T cells through CCR6 (Yang et al.
1999) and toward neutrophils, monocytes, and macrophages through CCR2 (Röhrl
et al. 2010). hBD-2 has been found to promote intestinal wound healing in vitro
(Otte et al. 2008) and stimulate proliferation, migration, and cytokine production of
epidermal keratinocytes (Niyonsaba et al. 2007).
Likewise, non-antimicrobial functions have been attributed to hBD-3 including
chemoattractant properties (Röhrl et al. 2010), antagonism of CXCR4 (Feng et al.
2006), and activation of mast cells with increase of vascular permeability (Chen
et al. 2007). However, the fact that hBD-3 appears to remain cell associated in the
skin (Sørensen et al. 2005 2006) questions whether these functions play a major role
during wound healing.
The non-antimicrobial functions of psoriasin include chemotactic activity toward
T lymphocytes and neutrophils (Jinquan et al. 1996) mediated by binding to RAGE
(Wolf et al. 2008). This also promotes proliferation of endothelial cells (Shubbar
et al. 2012). Psoriasin induces VEGF (Shubbar et al. 2012) and the expression of
keratinocyte differentiation markers (Hattori et al. 2014) as wells as strengthens the
tight junction barrier in the skin (Hattori et al. 2014).
Though SLPI (secretory leukocyte protease inhibitor) is also found in neutro-
phils, keratinocytes are the major source of SLPI in wound healing (Jacobsen et al.
2008). SLPI is found to be secreted both in multilayer epidermal keratinocytes cell
culture (Sørensen et al. 2003) and whole epidermis (Sørensen et al. 2006). SLPI has
antimicrobial activity against bacteria (Hiemstra et al. 1996), fungi (Tomee et al.
1997), and HIV-1 (McNeely et al. 1995). During wound healing, the secreted SLPI
has nonredundant functions (Ashcroft et al. 2000) by inhibiting the elastase-
mediated cleavage the epithelial growth factor proepithelin to the growth-inhibitory
epithelin (Zhu et al. 2002).
1.2.5 Tissue Remodeling Phase
Tissue remodeling in cutaneous wound healing involves increased expression of

collagen VI (Betz et al. 1993) that has antimicrobial activity (Abdillahi et al. 2012).
Interestingly, collagen VI expression in fibroblasts is induced by neutrophil defen-
sins (Li et al. 2006). The regulation of expression of antimicrobial proteins and
peptides in the underlying connective tissue of skin will likely receive more interest
after the finding that the normal skin microbiome extends to the connective tissue
(Nakatsuji et al. 2013).
1.2.6 Chronic Wounds
Though many studies demonstrate how microbial products induce AMP expression
in keratinocytes mainly through TLR activation (Abtin et al. 2008; Büchau et al.
2007, 2008; Li et al. 2013; Nagy et al. 2005; Gariboldi et al. 2008; Gerstel et al.
2009; Liu et al. 2002), the possible significance of this in acute wound healing in
noninfected wounds is not clear. The bacteria-induced AMP expression may be
more important in chronic wounds with infections (Edwards and Harding 2004).
Furthermore, in this instance, the bacterial proteases could play contradictory roles,
either by induction of AMP expression through protease-activated receptors (Chung
et al. 2004) or by AMP degradation (Schmidtchen et al. 2002). Chronic wounds like
chronic venous ulcers are characterized by chronic inflammation with continuous
recruitment of inflammatory cells, and indeed neutrophil AMPs like neutrophils
defensins are found in high amounts (Lundqvist et al. 2008). The epidermal expres-
sion of hBD-2 and psoriasin is induced in chronic venous ulcers (Butmarc et al.
2004; Dressel et al. 2010), while the expression of LL-37 is decreased (Heilborn
et al. 2003). In diabetic wounds, the high glucose levels may suppress the expres-
sion of both hBD-2 (Lan et al. 2012) and hBD-3 (Lan et al. 2011) in keratinocytes.
To understand the role of AMPs in chronic wound pathology or chronic wound
infections, more detailed studies are needed to delineate the AMP expression in dif-
ferent types of chronic wounds and how this is related to wound infection or under-
lying disease such as diabetes.
1.3 Concluding Remarks and Future Perspective
AMPs provide part of the antimicrobial defense during wound healing. Topical
application of antimicrobials do not improve normal wound healing (Lipsky and
Hoey 2009), demonstrating adequate antimicrobial defenses. While the AMPs orig-
inating from the coagulation and complement cascades, the neutrophils, and epi-
dermal keratinocytes undoubtedly contribute to the antimicrobial defense in the
8 O.E. Sørensen
wound healing process, it is difficult to decipher the role of individual AMPs due to
overlapping antimicrobial activities. Apart from SLPI (Ashcroft et al. 2000), no
AMP has been demonstrated to possess nonredundant functions during wound
healing.
Though mice models provide useful insight, it is important to note that differ-
ences exist between humans and mice – also when it comes to both wound healing
and AMPs. Wound contractions are important for wound healing in rodents such as
mice, but not in humans (Davidson 1998). Neutrophils and keratinocytes are major
sources of AMPs during wound healing, but mice have far fewer neutrophils than
humans (Mestas and Hughes 2004) and the mouse epidermis contains far fewer
keratinocytes than human epidermis. Accordingly, though no directly comparative
studies exist, it seems reasonable that the mouse wounds will contain fewer AMPs
than human wounds. There will also be qualitative difference in the AMPs. The
mouse contains many more different β-defensins than humans (Schutte et al. 2002),
while there will be no neutrophil α-defensins in mouse wounds (Eisenhauer and
Lehrer 1992). The neutrophil α-defensins play a role for the antimicrobial function
of the neutrophils (Sørensen et al. 2014), and this will undoubtedly be important for
the antimicrobial defense also in wounds.
While even infected wounds only in some instances benefit from topical treat-
ment with antibiotics (Lipsky and Hoey 2009), gene delivery of AMPs has been
found to have beneficial effects for wound healing (Jacobsen et al. 2005;
Steinstraesser et al. 2014; Carretero et al. 2008). This clearly indicates that AMPs
have beneficial effects in wound healing beyond its antimicrobial properties. AMPs
are generated at all stages of wound healing and it does seem like AMPs participate
in the regulation of some aspects of the wound healing processes, for instance,
LL-37 plays a role for recruitment of monocytes (Soehnlein et al. 2008b).
Resolution of inflammation is now recognized as a regulated process (Serhan and
Savill 2005; Ortega-Gomez et al. 2013), and in wounds, AMPs may play an impor-
tant role here. Dying and necrotic neutrophils are anti-inflammatory due to the
release of neutrophil defensins (Miles et al. 2009) and calgranulin S100A8, which
induce the anti-inflammatory cytokine IL-10. Later, IL-10 may play a role for
downregulation of AMP expression in keratinocytes (Howell et al. 2005). Both
defensins and LL-37 promote proliferation of keratinocytes (Niyonsaba et al. 2007;
Heilborn et al. 2003). Accordingly, AMPs may play a role for inflammation, resolu-
tion of inflammation, and proliferation during wound healing. One of the para-
doxes of psoriasis, a disease with very prominent AMP expression (Harder and
Schröder 2005), is the lack of scarring following the chronic inflammation
(Nickoloff et al. 2006). Future studies will further address the role of AMPs for
regulation of the wound healing process and scar formation. This will hopefully
pave the way for new treatment modalities for chronic wounds and wound
infection.
Acknowledgments This work was supported by grants from the Alfred Österlunds Stiftelse, the
Royal Physiografic Society Lund, Greta och Johan Kocks Stiftelse, Petrus och Augusta Hedlunds
Stiftelse, and the Swedish Research Council.
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Chapter 2
Antimicrobial Peptides as Endogenous
Antibacterials and Antivirals at the Ocular
Surface
Fabian Garreis, Martin Schicht, and Friedrich Paulsen
Abstract Ocular surface infections are a significant cause of blindness worldwide

particularly in developing countries and are mainly caused by bacteria, fungal spe-
cies, and parasites as well as by viruses. Staphylococcus epidermidis, Staphylococcus
aureus, Streptococcus pneumonia, and Pseudomonas aeruginosa are reported to be
the most common bacteria associated with keratitis where the most frequent viruses
leading to ocular surface infection are herpes simplex, adenovirus, and vaccinia
virus. The eye and its adnexa have evolved a bulk of defense strategies to prevent
microbial invasion. These include important components of the innate defense sys-
tem in form of classical antimicrobial compounds as well as members of the cat-
ionic antimicrobial peptide family, distinct surfactant proteins, and potential new
candidate molecules contributing to antimicrobial protection. Several of them are
studied at the ocular surface not only for their antimicrobial properties, but based on
easy topical application possibilities for their potential therapeutic effects. As sev-
eral reviews already summarized the current knowledge with regard to the eye, this
chapter will only briefly recapitulate the current knowledge of antimicrobial com-
pound expression in the eye and then will focus on infectious keratitis resulting from
bacterial and viral infection. In addition, the potential of using such peptides as
therapeutics for treating bacterial and viral ocular surface infections will be
elucidated.
2.1 Introduction
The ocular surface and its adnexal structures comprise the cornea, conjunctiva
with the bulbar, the fornical, and palpebral parts, the main lacrimal gland, the
F. Garreis • M. Schicht • F. Paulsen (*)

Department of Anatomy II, Friedrich Alexander University Erlangen Nürnberg,
Universitätsstraße 19, 91054 Erlangen, Germany

DOI 10.1007/978-3-319-24199-9_2
18 F. Garreis et al.
glands of the eyelids (including the meibomian glands, the glands of Moll, and
accessory lacrimal glands), as well as the nasolacrimal system (or efferent tear
ducts with the upper and lower puncta, the paired lacrimal canaliculi, the lacri-
mal sac, and the nasolacrimal duct). The nasolacrimal system collects the tear
fluid from the ocular surface and conveys it into the nasal cavity. All the other
structures contribute to formation of the preocular tear film. The tear film
together with the glands and cells that produce it (lacrimal glands, accessory
lacrimal glands, ocular surface epithelia, meibomian glands, glands of Moll, and
the sensory and motor nerves connecting these) has been assigned the general
term lacrimal functional unit (LFU) (Stern et al. 1998). Disease in or damage to
any component of the LFU can destabilize the tear film and lead to dry eye dis-
ease (DED), a condition expressed by signs and/or symptoms of ocular irrita-
tion, redness, and foreign body sensation. DED afflicts millions of people around
the world and makes the ocular surface more vulnerable to infection (Gayton
2009).
2.2 Tear Film Composition
The tear film coats the cornea and conjunctiva and is a complex structure com-
posed of an inner mucous layer that is part of the glycocalyx and is attached to the
epithelial cells (composed mainly of membrane-bound mucins), an aqueous com-
ponent consisting, beside water (99 %) of ions, soluble mucins and a wide range
of different proteins and peptides (such as trefoil factor family peptides that func-
tion as linker molecules for mucins to form gels), as well as an outer, anterior-most
lipid component that prevents evaporation. Ocular mucins and trefoil factor family
(TFF) peptides take a variety of different functions which provide protection at the
ocular surface. Mucins are a family of high molecular weight, heavily glycosyl-
ated hydrophilic proteins. Membrane-anchored mucins, MUC1, MUC4, und
MUC16, are part of the epithelial glycocalyx and provide a continuous anatomic
barrier across the ocular surface which prevents pathogen penetration. The mem-
brane-spanning molecules also have signaling capabilities that influence epithelial
activity. The negative charge of the mucins (glycocalyx) occurs in an accumulation
of positive-charged tear components, like lysozyme, SLPI, as well as AMPs (see
below), which enhance the local concentration of antimicrobial agents at the ocu-
lar surface. The high molecular secreted mucins are distinguished in gel-forming
mucins (mainly MUC5AC from conjunctival goblet cells and MUC5B from the
lacrimal gland) and small soluble mucins (MUC7 from the lacrimal gland). The
secreted mucins as well as TFFs are responsible for the rheological properties of
the tear film, enabling movement and spreading of the tear film. TFF peptides have
many other physiological functions in addition to their rheological properties,
such as promotion of epithelial cell migration, antiapoptotic properties, induction
of cell scattering, epithelial restitution, and neuropeptide functions (Paulsen and
Berry 2006).
2 Antimicrobial Peptides as Endogenous Antibacterials and Antivirals 19
2.2.1 Antimicrobial Compounds at the Ocular Surface,

in the Lacrimal Apparatus, and in the Tear Film
Like other epithelia, also the ocular surface is constantly in contact with various
microorganism and pathogen-associated molecular patterns (PAMPs). The ocular
surface epithelia and associated structures of the lacrimal apparatus are effective in
counteracting invasion and colonization of microorganisms. They produce the tear
film that contains up to 1500 different proteins (Zhou et al. 2012). More than 90 %
of the total amount of tear proteins are four antimicrobial compounds: lysozyme,
lactoferrin, tear lipocalin and secreted immunoglobulin A (sIgA). The concentration
of these four proteins in the tear fluid is in the mg/ml range. These tear compounds
are well known and show broad antimicrobial activity. Lysozyme cleaves the pepti-
doglycan backbone of bacterial cell walls and is also able to digest fungal cell walls.
Lactoferrin binds free iron at the ocular surface and inhibits bacterial and fungal
growth. It has also been shown to have iron-independent anti-HIV activity. Tear
lipocalin binds microbial siderophores (iron chelating compounds) and thus has a
bacteriostatic effect. Lysozyme, lactoferrin, and tear lipocalin are built mainly by
the acinar cells of the main lacrimal gland as well as by accessory lacrimal glands
in the eyelids (glandulae conjunctivales [glands of Krause and Wolfring]). In con-
trast, sIgA is produced as a dimer by subepithelial plasma cells, transported via
transcytosis, bound to a protein termed “secretory component,” through the epithe-
lium and then is secreted into the tear film (Mcdermott 2013; Tiffany 2008).
In addition to these principal antimicrobial factors, tear fluid contains a great
bulk of other tear compounds, several of them with antimicrobial activity. The con-
centration of these compounds is in the range of μg/ml to pg/ml. Beside their anti-
microbial activity, several of these compounds have no antimicrobial activity as
main biological function and several are with yet unknown function (Mcdermott
2013). Among those constituents with antimicrobial activity is secretory phospholi-
pase A2 (sPLA2). It binds to anionic bacterial membranes of Gram-positive bacte-
ria due to its cationic domains and kills bacteria by its enzymatic activity. sPLA2
shows activity against Gram-positive bacteria in the physiological tear environment
(Qu and Lehrer 1998). Antimicrobial activity and anti-inflammatory properties
were shown for members of the whey acidic protein (WAP) family like secretory
leukocyte protease inhibitor (SLPI) as well as elafin, which are characterized by a
cysteine-rich region with four intramolecular disulfide bonds. SLPI was identified
by its antiprotease activity and shows also activity against Gram-positive and Gram-
negative bacteria, fungi, and HIV (Sallenave 2010). High amounts could be detected
in closed-eye tears. SLPI may inhibit neutrophil elastase derived from immigrating
neutrophilic granulocytes and protects the ocular surface from enzymatic degrada-
tion effects during the sleep (Sathe et al. 1998). Furthermore, bactericidal/
permeability-increasing protein (BPI) was initially identified in neutrophils, but it is
also expressed by ocular surface epithelial cells and could be detected in tears. BPI
binds lipopolysaccharides (LPS) liberated from Gram-negative bacteria (Peuravuori
et al. 2006). Clinical trials with recombinant modified BPI molecule (rBPI21)
showed reduced mortality of Gram-negative bacteria-induced sepsis (Domingues

et al. 2009). The amino acid derivate β-lysin (3,6-diaminohexanoic acid) produced
by platelets was also found in tears and showed antimicrobial peptide-like charac-
teristics. β-Lysin interacts with bacterial cell membranes of Gram-positive bacteria,
inhibits bacterial enzymes, and enhances phagocytosis (Yeaman 2010).
2.2.2 Surfactant Proteins
During recent years, the well-known lung surfactant proteins (SPs) have also been
described in other mucosae such as in the ocular system (Schicht et al. 2010). Here,
they have been associated with surface tension regulating activities of the tear film
as well as with immunological functions. Tear SP-D has been shown to protect the
ocular surface from infection with Gram-negative bacteria, in particular
Pseudomonas aeruginosa (Mcdermott 2013). With regard to virus infection, SP-A
and SP-D are able to bind and agglutinate viruses (such as influenza A virus, adeno-
virus, hepatitis B virus [HBV], and herpes simplex virus [HSV]) and they have been
shown to initiate phagocytosis by macrophages (Harrod et al. 1999; Kishore et al.
2006; Levine et al. 2001). In chronic herpes virus infection, a protein misfolding
shall lead to functional loss of SP-C with losing of the surface regulatory properties
(Lawson et al. 2008). Also, the recently discovered surfactant protein H (SFTA3)
has been demonstrated to occur at the ocular surface (Schicht 2012). Also, this sur-
factant protein has immunological functions by mediating activation of macro-
phages (Diler et al. 2014). However, so far, there exist no data demonstrating any
function of surfactant proteins with regard to viral eye infection.
2.2.3 Antimicrobial Peptides (AMPs)
AMPs are defined as small endogenous proteins displaying direct antimicrobial activ-
ity. In general, AMPs have no specific consensus amino acid sequence but most of
them have an amphipathic character and are positively charged under physiological pH
conditions. AMPs have been detected across a wide spectrum of different species. In
February 2015, the Antimicrobial Peptide Database (APD; http://aps.unmc.edu/AP/
main.php) lists 2493 AMPs: 244 from bacteria (bacteriocins), 2 from archaea, 7 from
protists, 13 from fungi, 312 from plants, and 1874 from animals. In human, 108 differ-
ent AMPs have been described to date (Wang 2014). According to their secondary
structure, AMPs can be classified into four groups: (i) AMPs with linear α-helical
domains (e.g., human cathelicidin LL-37), (ii) AMPs with β-sheets and disulfide bonds
(e.g., α- and β-defensins), (iii) AMPs with extended structures (e.g., indolicidin), and
(iv) AMPs with loop structures (cyclic defensins, bactenecin) (Lai and Gallo 2009).
Various different human AMPs with activity against bacteria, fungi, and viruses
have been described and characterized (Wang 2014). Figure 2.1 provides an over-
view about known AMPs at the ocular surface and lacrimal structures.
Glands of Moll
hBD-1,-2 Lacrimal gland
LL-37 hBD-1,-2,-3
Psoriasin
Cornea
epithelium
hBD-1,-2,-3,-9
LL-37
RNase-7
Psoriasin
LEAP1/Hepcidin
Tears
HNP 1-3
LL-37,
hBD-2,-3
Histatin 5
Dermcidin
Psoriasin
Conjunctiva
epithelium
hBD-1,-2,-3,-9
LL-37
RNase-7
Psoriasin
Meibomian
glands
Psoriasin
Fig. 2.1 Expression of antimicrobial peptides with antibacterial and antiviral activity at the ocular
surface structures, in the lacrimal gland, and in tears (Partly modified from Kolar and Mcdermott
(2011))
2.2.3.1 Human Defensins
Human defensins are small in size (29–45 amino acids), are cationic, and are char-
acterized by the presence of six conserved cysteine residues. Based on the distribu-
tion of the cysteines and the linkage of disulfide bonds, defensins are classified into
three subfamilies, referred to α-, β-, and θ-defensins (theta or minidefensins).
Humans express six α-defensins and up to 31 β-defensins. α-Defensins and
β-defensins differ by their organization of the three disulfide bonds (Hazlett and Wu
2011). θ-Defensins are not translated in humans due to premature stop codons pre-
venting their expression (Lehrer et al. 2012). Interestingly, pseudogenes of
θ-defensins, named retrocyclins, show antimicrobial activity against the gut bacte-
rium E. coli as well as against HIV-1 (Tran et al. 2008; Wang et al. 2004). Human
α-defensins have been divided into human neutrophil peptides (HNP) 1–4 and
human defensin (HD)-5 and human defensin-6. HNP1–HNP4 were identified in
particularly high concentrations in azurophil granules of neutrophils but also in
other immune cells. In contrast, HD-5 and HD-6 are expressed by Paneth cells in the
small intestine as well as different epithelial cells (Lehrer et al. 2012). Human
β-defensins (hBD) 1–3 are expressed by epithelial cells and also in different immune
cells (Semple and Dorin 2012).
At the ocular surface, α-defensins and hBD-1 are constitutively expressed
whereas hBD-2 and hBD-3 show a low basal expression at the ocular surface but
enhanced expression after stimulation with different pro-inflammatory stimuli,
including cytokines and bacterial compounds (Garreis et al. 2010). In addition,
hBD-9 (DEFB109) has been identified at the ocular surface. Interestingly, hBD-9
shows a reduced ex vivo gene expression in patients with keratitis and also a down-
regulation in human corneal limbal epithelial cells after cocultivation with
Acanthamoeba spp. and bacteria from keratitis patients (Abedin et al. 2008; Otri
et al. 2012).
Both α- and β-defensins have broad activity against different microbes, including
various bacteria and viruses in vitro (Gwyer Findlay et al. 2013; Klotman and Chang
2006; Wilson et al. 2013). In vivo, only α-defensin concentrations are generally
within the range of direct antimicrobial activity whereas the concentrations of con-
stitutively expressed β-defensins often do not reach levels sufficient for a direct
antimicrobial activity (Wilson et al. 2013). Interestingly, various studies indicate
that the local β-defensin concentration can be increased markedly by various inflam-
matory and (patho)physiological processes. HBD-1 becomes a potent antimicrobial
peptide after reduction of disulfide bridges against gut pathogens (Schroeder et al.
2011). In addition, all human defensins have also several immunomodulatory prop-
erties (see below).
2.2.3.2 Human Cathelicidin LL-37
In humans, only one member of the cathelicidin family is functionally expressed:

human cationic antimicrobial peptide-18 (hCAP-18). This 18 kDa peptide is
encoded by the CAMP gene and cleaved by proteinase 3 into the 37 amino acid
long-active peptide LL-37 (starts with two leucines) and the N-terminal cathelin
domain. Human LL-37 is characterized as cationic, amphipathic peptide with an
α-helical structure and a broad-spectrum antimicrobial activity against various
pathogens (Vandamme et al. 2012). It is stored in neutrophils, inducible in most
epithelial cells and leukocytes, and secreted into various body fluids, including
tears. Human LL-37 and its derivatives are also able to inhibit and destroy biofilms
and have antifungal and antiviral activity (Vandamme et al. 2012). Moreover, LL-37
modulates the adaptive immune system, stimulates angiogenesis, and supports reep-
ithelialization (Vandamme et al. 2012).
2.2.3.3 Other AMPs
Beside defensins and cathelicidins, also other AMPs and other antimicrobial com-
pounds have been demonstrated to show antibacterial and antiviral activity at the ocu-
lar surface. Thus, a group of human inhibitors of neutrophil serine proteases known as
SLPI (secretory leukocyte protease inhibitor) and elafin (also known as peptidase
inhibitor 3 or skin-derived antileucoprotease [SKALP]) have been associated with
anti-HIV activity of vaginal fluid (Ma et al. 2004; Pillay et al. 2001). The antiviral
activity does not depend on inhibition of serine proteases. It is associated with binding
of host cell membrane-associated proteins such as scramblase and/or annexin II (Ma
et al. 2004; Tseng and Tseng 2000). The antifungal peptide histatins (histidine-rich
cationic peptides) are present in high amounts in both human saliva and tears and are
also discussed for their antiviral activity. A histatin-5 derivate affects HIV-1 replica-
tion by promoting host cell entry (Groot et al. 2006). Psoriasin (S100A7), a highly
potent AMP against Escherichia coli, was detected in elevated concentrations in
human tears and in tissues of the ocular surface and lacrimal apparatus (Garreis et al.
2011). Two RNases from eosinophils, eosinophil-derived neurotoxin (RNase 2/EDN),
and eosinophil-cationic protein (RNase 3/ECP) have been shown to inhibit respira-
tory syncytial viruses (RSV) and HIV infection in vitro (Domachowske et al. 1998).
RNase 2 antiviral activity depends on ribonucleolytic activity. In addition, RNase 2
functions as chemoattractant and can activate immune cells via Toll-like receptor
(TLR)-2 and myeloid differentiation factor 88 (Myd88) signaling. RNase 3 is able to
permeabilize microbial membranes and stimulate histamine release from mast cells
(Wiesner and Vilcinskas 2010). In addition, also RNase 5 (angiogenin) acts antivirally
against HIV with a yet unsolved mechanism (Cocchi et al. 2012). In contrast to
RNases 2 and 3, which have not yet been analyzed at the ocular surface and in tears,
tear fluid contains very high amounts of RNase 5 (Sack et al. 2005). However, neither
the cellular source and whether and how it is functionally active at the ocular surface
is unclear yet. A further RNase has been described at the ocular surface: RNase 7.
However, RNase 7 has not yet been associated with antiviral activity (Boix and
Nogues 2007). It plays an important role as antibacterial factor in cutaneous defense
(Simanski et al. 2012) and its expression in corneal epithelial cells, which is regulated
by tears and specific microRNAs, suggests that RNase 7 may also participate to pro-
tect the ocular surface against bacterial infection (Mun et al. 2013). The liver-expressed
antimicrobial peptide (LEAP), 1/hepcidin, is expressed at the ocular surface and
shows increased expression in viral keratitis (Mohammed et al. 2011).
2.3 Antimicrobial Activity of AMPs
AMPs are an essential part of the innate immune defense at the ocular surface, with
both direct antimicrobial activity and immunomodulatory functions. AMPs demon-
strate direct antimicrobial activity against a broad spectrum of ocular pathogens,
including Gram-positive and Gram-negative bacteria, fungi, and various viruses.
2.3.1 Antibacterial Mechanism
Direct antimicrobial activity is based on the charge-dependent interaction of posi-

tively charged AMPs with the negatively charged surface of microorganisms. The
microbial cell membrane contains a huge proportion of acidic phospholipids con-
tributing to a negative charge of the surface. In contrast, eukaryotic cell membranes
have much fewer negatively charged phospholipids and contain high amounts of
cholesterol. This shall be responsible for the selective antimicrobial activity as well
as a low cytotoxic effect of AMPs on eukaryotic cell membranes. Aggregation and
integration of cationic AMPs into the lipid bilayer lead to expansion of the outer
leaflet and to a local membrane thinning. The detailed mechanism of the electro-
static interaction, however, is still being under discussion yet. All models so far
available show a permeabilization of the microbial cell membrane, which leads to
loss of essential intracellular components and finally to cell death (Wiesner and
Vilcinskas 2010). Interestingly, transfer and/or interaction with peptidoglycan in the
bacterial cell wall are largely unknown. Furthermore, charge-independent mecha-
nisms of AMP activity have also been described, which contain partly specific inter-
actions with membrane receptors or intracellular molecules (Wiesner and Vilcinskas
2010). For example, buforin, a histone H2A-derived antimicrobial peptide, binds
nucleic acids of bacteria (Jang et al. 2012). Further studies showed the direct inter-
action of AMPs with the cell cycle proteins as well as heat shock proteins resulting
in bacterial and fungal growth (Kragol et al. 2001; Lobo et al. 2007). Recent studies
have shown the ability of various AMPs and optimized derivatives to act against
various stages of microbial biofilm formation including multidrug-resistant strains
in biofilms (Di Luca et al. 2014).
2.3.2 Antiviral Mechanisms
Several AMPs have an antiviral activity against different viruses including human
immunodeficiency virus (HIV), herpes simplex virus (HSV), influenza A virus
(IAV), and non-enveloped viruses, like adenovirus (AV) and human papillomavirus
(HPV). Figure 2.2 shows various antiviral mechanisms by AMPs. These include:
• Direct interaction (“virolysis”)
• Blockage of host cell surface receptors
• Inhibition of viral fusion to host cells
• Aggregation of viruses
• Inhibition of viral replication
• Activation of adaptive immune response
Most of the antiviral properties of AMPs are based on direct interaction between
cationic AMPs and different components of the virus. The major compound of
enveloped viruses is the negatively charged lipid bilayer. Cationic AMPs interact
Fig. 2.2 Overview of the main antiviral mechanisms of antimicrobial peptides (AMPs). AMPs
show activity against various cell surface targets (virus and/or host cell) as well as intracellular
targets. In vitro, cationic AMPs have been shown to decrease viral infection by following mecha-
nism: 1 direct interaction with envelope virus (“virolysis”), 2 blocking of viral entry into host cell
by binding of viral and/or host cell receptors, 3 suppressing viral fusion with host cell, 4 extracel-
lular aggregation of viruses, 5 activation of adaptive immune response, and 6 inhibition of viral
replication. Arrow indicates blockage of viral transcription
with negatively charged phospholipids of the viral envelope. This leads to a desta-
bilization and neutralization of the virus (“virolysis”) and additionally to an inhibi-
tion of viral fusion with host cells (Wilson et al. 2013). Furthermore, AMPs interact
with viral attachment proteins (glycoproteins as well as capsid proteins) and endog-
enous host cell receptors. These blocking mechanisms inhibit the viral binding to
host cells and inhibit virus infection (Wilson et al. 2013). A critical step in virus
infection is the penetration of the virus (genome) into the host cell. Enveloped
viruses have to fuse their lipid bilayer with the cell membrane of the host cell. Non-
enveloped viruses penetrate cell membranes by specific viral capsid proteins.
Several studies demonstrate inhibition of the viral fusion process by AMPs through
preventing penetration of host cell membranes (Wilson et al. 2013). In addition,
AMPs initiate accumulation and aggregation of viruses in vitro. It has not been
clarified yet whether AMP-mediated aggregation inhibits virus infection in vivo.

Aggregation could affect binding to host cells and enhance opsonization by macro-
phages (Wilson et al. 2013). Furthermore, AMPs block viral infection by inhibiting
intracellular viral reproduction through blockage of viral transcription, protein pro-
duction, assembly, and release of new virus particles. This antiviral activity is based
on the opportunity of AMPs to interact with viral and/or host cell compounds. At
last, AMPs, especially at physiological concentrations, activate the adaptive immune
system and demonstrate immunomodulatory properties. AMPs induce expression
of pro-inflammatory molecules as well as chemokines, enhance phagocytosis, and
act as chemotaxins for various immune cells (Choi et al. 2012). This immunomodu-
latory properties link the innate to the adaptive (cellular) immune defense.
2.4 Keratitis
Microbial keratitis is an infectious disease of the cornea that is characterized by

inflammation and infiltration by leukocytes. A range of microorganisms, including
fungi, bacteria, protozoa, and viruses, have been identified to induce microbial kera-
titis. Moreover, the use of contact lens and corneal trauma are common risk factors.
Keratitis can progress rapidly with corneal destruction as well as pathological
wound healing and requires immediate medical treatment.
2.4.1 Bacterial Keratitis
Various studies have investigated AMP activity at the ocular surface related to bac-
terial infection. Staphylococcus epidermidis, Staphylococcus aureus, Streptococcus
pneumonia, and Pseudomonas aeruginosa are reported to be the most common
pathogens associated with bacterial keratitis (Karsten et al. 2012). P. aeruginosa
infection is the most common cause for bacterial keratitis in contact lens wearers.
As described before, human defensins and LL-37 are expressed at the ocular surface
and show varying levels of antibacterial activity against common ocular bacteria in
vitro. Antimicrobial activity occurs in a low micromolar range, and AMPs show
large variability in their efficiency in killing specific pathogens (McDermott 2013).
Generally, the concentrations of AMPs in tears are too low for direct antibacterial
activity. Furthermore, some AMPs show a reduced antibacterial activity in the pres-
ence of tear-specific salt concentration as well as in contact with ocular mucins.
However, studies have also shown an elevated secretion of AMPs under inflamma-
tory conditions or after bacterial infection. In addition, synergistic and/or additive
interactions between different AMPs and other antimicrobial compounds of the tear
film have been described. These effects may compensate the low AMP concentra-
tion in tears (McDermott 2013). Animal models of bacterial keratitis have demon-
strated that CRAMP (mouse homolog of LL-37) and mouse beta-defensins 2 and
3 (putative orthologs of hBD-2) are important to prevent P. aeruginosa infection in

vivo (Huang et al. 2007; Wu et al. 2009). Moreover, various studies reveal that
human defensins and LL-37 also stimulate migration and proliferation of corneal
epithelial cells and therefore also contributing to wound healing processes. In addi-
tion, defensins and LL-37 recruit and activate various immune cells by chemotaxis
and cytokine production. This contributes to an activation of the adaptive immune
defense at the ocular surface. For further information, the reader is referred to the
review of A. McDermott (McDermott 2013).
2.4.2 Herpes Simplex Virus Keratitis
Herpes simplex virus type 1 (HSV-1) is the most common cause of viral keratitis
(Fig. 2.3) and also the most common cause of irreversible cornea-derived blindness
in developed nations (Karsten et al. 2012). Worldwide, an estimated ten million
persons suffer from HSV keratitis, with about two million individuals left with
impaired vision (Rowe et al. 2013). The incidence lies between 5.9 and 20.7/105 of
the population per year and with a prevalence of 149/105 in the developed countries
(for review, see Kaye and Choudhary 2006). The initial (not necessarily primary)
sites of herpetic eye involvement usually manifest as a blepharitis (infection of the
lid rim), conjunctivitis, or corneal epithelial keratitis. More often, a younger age
group is infected and tends to be more severely infected, especially in the develop-
ing world, where malnutrition and several diseases as well as the lack of access to
treatment may be present.
As already mentioned, defensins are beside LL-37 the most abundant group of
AMPs in human. Their antiviral activity already was defined in the mid-1980s.
Studies of AMPs interacting with HSV-1 revealed direct inactivation of the virus by
HNP1 produced by neutrophils (Daher et al. 1986). Since that time, the antiviral
activity of various AMPs against HSV infection has been followed up by an
Fig. 2.3 Herpes simplex

virus (HSV) keratitis.
Fluorescein staining of the
cornea shows typical HSV
lesions consisting of a
linear branching corneal
ulcer (dendritic ulcer).
With kind permission of
Prof. Gerd Geerling
(Department of
Ophthalmology, University
Hospital Düsseldorf,
Heinrich Heine University
of Düsseldorf, Düsseldorf,
Germany)
increasing level of research interest. Thus, α-defensins have been demonstrated to

prevent HSV penetration through blocking viral glycoproteins as well as by binding
of host cell receptors. HNP1–HNP3 prevent HSV infection through blockage of
HSV surface glycoprotein B (gB) and in addition show postinfection effects, sug-
gesting unknown effects with regard to viral replication (Hazrati et al. 2006). In
contrast, HNP4 reduces HSV infection but do not bind to viral gB. This α-defensin
shows high affinity against heparan sulfate, which is a preferred host cell receptor
for attachment. Only hBD-3 inhibits HSV-1 infection by similar mechanisms.
HBD-1 and HBD-2 show low affinity to gB and cellular glycosaminoglycans and
are not able to reduce HSV infection (Hazrati et al. 2006). In cervical epithelium of
the human uterus, θ-defensins from rhesus monkeys and modified derivates (retro-
cyclins) protect the epithelial cells from HSV infection by inhibiting viral adhesion
and entry. Retrocyclin (RC)-2 has no direct HSV activity but decreases viral infec-
tion by cross-linking viral glycoproteins (Yasin et al. 2004). A prophylactic applica-
tion of RC2 that was applied in a murine HSV-mediated keratitis model demonstrated
reduced viral titers, reduced symptoms of blepharitis, corneal vascularization, and
stromal disease. However, RC2 had no effect if it was applied after HSV infection
(Brandt et al. 2007).
2.4.3 AMPs and Other Viral Keratitis Forms
With regard to other frequent viral ocular surface infections such as adenovirus or
vaccinia virus infection and in addition to other viruses, no data exist in a context
with AMPs.
2.4.4 AMPs for Treating Ocular Surface Infections
Here, the reader is referred to a very recent review by Curtis R. Brandt (Brandt
2014). This review presents an overview about antimicrobial drugs available for the
treatment of ocular surface infections. In that review, it is also discussed that given
the nature of peptides, topical applications are the most likely use to be successful
for treating keratitis. Such peptides would be effective against drug-resistant patho-
gens and might act synergistically if used in combination therapy. Although hun-
dreds of peptides with antimicrobial properties have been isolated or synthesized,
only a handful have been tested so far against ocular pathogens and even fewer have
been tested in animal models. The review summarizes the currently available infor-
mation on the use of these peptides to treat keratitis, outlines some of the problems
that have been identified, and discusses future studies that will be needed. As out-
lined in the review, most of the peptides that have been tested so far have shown
activity at concentrations that do not warrant further development, but nevertheless,
there are promising candidates such as, for example, RC2, as mentioned above, or
the coupling of acyclovir (ACV) to the 3-OS heparan sulfate (a co-receptor for
HSV-1 glycoprotein) binding G2 (a M13 phage peptide) raising the possibility that
peptides and peptide-drug conjugates can be developed to treat viral keratitis.
A further strategy to prevent microbial keratitis is the attachment of modified AMPs
to contact lenses or contact lens cases. Contact lens-mediated microbial keratitis, in
particular by P. aeruginosa, Fusarium spp., and Acanthamoeba (an ocular parasite), is
associated with microbial adherence and biofilm formation. In the last decade, various
antimicrobial strategies, such as cationic metals and peptides, selenium, quorum-sens-
ing inhibitors, and various biocidal and non-biocidal agents, have been developed and
tested with promising results in various animal models (Dutta and Willcox 2014).
Covalent binding of cationic selenium to contact lenses revealed inhibition of bacterial
colonization as well as reduction of acute red eye formation and bacterial ulceration in
a rabbit model (Mathews et al. 2006). Fimbrolide-coated lenses (a quorum-sensing
inhibitor) reduced bacterial and Acanthamoeba adhesion without negative ocular
responses in a 1-month animal model and in an overnight human trial (Zhu et al. 2008).
Moreover, contact lenses coated with melamine, a synthetic cationic hybrid AMP pre-
pared by combining active regions of the AMPs protamine (from salmon sperm) and
melittin (from bee venom), reduced adhesion to contact lenses and prevented coloniza-
tion against a broad spectrum of ocular pathogens (Dutta et al. 2013). In addition,
melamine-coated contact lenses showed reduced ocular inflammation and corneal
infiltrates and less epithelial defects in some animal keratitis models (Cole et al. 2010).
Acknowledgment We would like to thank Jörg Pekarsky, Department of Anatomy II, FAU
Erlangen, Germany, for drawing Figures 2.1 + 2.2 and Gerd Geerling, Director and Head,
Department of Ophthalmology, Heinrich Heine University of Düsseldorf, Düsseldorf, Germany,
for providing us with Figure 2.3. Supported by Deutsche Forschungsgemeinschaft (DFG) grant
PA738/9-2.
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Chapter 3
Function of Antimicrobial Peptides in Lung
Innate Immunity
Frederik Seiler, Robert Bals, and Christoph Beisswenger
Abstract The innate immune system of the lung is a complex network of different
cellular and noncellular components protecting the lung from inhaled pathogens.
Antimicrobial peptides (AMP) are produced by epithelial and myeloid cells as part
of this system. AMPs, such as defensins and cathelicidin, are small cationic peptides
with a broad microbicidal activity against respiratory bacteria, viruses, and fungi.
However, their functions go beyond antimicrobial activity and include modulation
of the innate and adaptive immune response to infection as well as lung repair after
injury. Thus, AMPs are involved in pathophysiological processes of many lung dis-
eases, such as acute and chronic lung infection, chronic obstructive pulmonary dis-
ease, cystic fibrosis, and lung cancer.
3.1 The Innate Immune Network of the Lung
The human lung is continuously exposed to a broad array of airborne pathogens

such as bacteria, viruses, and fungi. Given its specific requirement to ensure an
effective gas exchange, the lung has a unique structure that is characterized by a
large contact surface combined with an extremely thin epithelial layer. Hence, the
lung is a potential portal of entry for inhaled microbes. In order to avoid coloniza-
tion of the lower airways with inhaled pathogens and prevent local and systemic
infection, the lung is protected by a complex innate immune network consisting of
various cellular and noncellular components.
The first line of defense is represented by the airway epithelium, a pseudostrati-
fied epithelium of basal cells, ciliated cells, secretory Clara cells, and goblet cells.
F. Seiler • R. Bals • C. Beisswenger (*)

Department of Internal Medicine V – Pulmonology, Allergology, Respiratory and
Environmental Medicine, Saarland University, Kirrberger Str,
Building 61.4, Homburg/Saar 66421, Germany

DOI 10.1007/978-3-319-24199-9_3
34 F. Seiler et al.
Its function within the innate immune system exceeds well-known mechanisms
like barrier formation and mucociliary clearance. The epithelium of the respiratory
tract is an immunologically active tissue, which exerts functions such as pathogen
recognition, pathogen neutralization, and activation of further immune mecha-
nisms. These include resident alveolar macrophages as well as inflowing neutro-
phils. Those professional immune cells have a high capability to neutralize
microbial pathogens. In addition, the release of immunomodulatory mediators
from epithelial and myeloid cells induces activation of the adaptive immune
response.
Small cationic antimicrobial peptides (AMPs) from the families of defensins and
cathelicidins play a key role within the innate immune network of the lung.
3.2 Expression of Antimicrobial Peptides in the Lung
AMPs are expressed by various cell types present in the lung during health and
disease. The main AMPs expressed in the lung are α-defensins, β-defensins, and
cathelicidins. Table 3.1 gives an overview on AMPs of the human lung.
3.2.1 Defensins
3.2.1.1 α-Defensins
α-Defensins comprise the human neutrophil peptides (HNP) 1–4 and the epithelial
α-defensins HD-5 and HD-6 (Doss et al. 2010). Different to other epithelial tissues
of the human body, the airway epithelium hardly produces α-defensins; only HD-5
has been detected in airway epithelial cells in small amounts (Frye et al. 2000). The
main sources of α-defensins in the human lung are neutrophilic granulocytes. They
Class Peptide name Gene name

α-Defensins HNP-1 DEFA1
HNP-2 DEFA1
HNP-3 DEFA3
HNP-4 DEFA4
HD-5 DEFA5
β-Defensins hBD-1 DEFB1
hBD-2 DEFB4A
hBD-3 DEFB103A
hBD-4 DEFB104A
Table 3.1 Antimicrobial
peptides of the human lung Cathelicidins LL-37/hCAP18 CAMP
3 Function of Antimicrobial Peptides in Lung Innate Immunity 35
constitutively express α-defensins, which are stored in azurophilic granula and rep-
resent their main protein content. Upon infection, activation of innate immunity
triggers the influx of neutrophils into the lung, where they neutralize pathogens
through phagocytosis and degranulation of their azurophilic granula (Ganz et al.
1985; Selsted and Ouellette 2005).
In human alveolar macrophages, α-defensins are virtually absent; only rabbits
have been found to express α-defensins in their alveolar macrophages (Ganz et al.
1985; Selsted and Ouellette 2005).
3.2.1.2 β-Defensins
The principal β-defensins found in the human lung are human β-defensin (hBD) 1–4
(Doss et al. 2010). Their main sources are epithelial cells and submucous glands
(Kao et al. 2003; Bals et al. 1998a). However, β-defensins (hBD-1 and hBD-2) are
also released from myeloid cells such as alveolar macrophages and dendritic cells
(Duits et al. 2002). The expression of β-defensins shows a gene-specific behavior:
hBD-1 is constitutively expressed by the epithelium, ensuring a constant basic anti-
microbial activity of the airway surface liquid (McCray and Bentley 1997; Singh
et al. 1998). In contrast, hBD-2, hBD-3, and hBD-4 are not released by default, but
only if required; high levels can only be detected in the case of infection or inflam-
mation (Hess et al. 2010; Harder et al. 2001; Yanagi et al. 2005; Scharf et al. 2010a,
2012; Benincasa et al. 2009). Compared to hBD-3 and hBD-4, which are predomi-
nantly expressed in other epithelial tissues, hBD-2 is mainly expressed in the respi-
ratory tract (Bals et al. 1998a).
3.2.2 Cathelicidin
In the lung, the only human cathelicidin LL-37/hCAP-18 is expressed by different

cell types, namely, myeloid cells such as alveolar macrophages (Rivas-Santiago
et al. 2008), neutrophils (Rivas-Santiago et al. 2008; Cowland et al. 1995), and mast
cells (Di Nardo et al. 2003), as well as airway epithelial cells (Bals et al. 1998b). It
can be detected in bronchoalveolar lavage fluid (Agerberth et al. 1999). Similar to
α-defensins, cathelicidin is constitutively expressed by neutrophils and stored in
granula until release (Larrick et al. 1995). Degranulation is triggered by external
stimulation such as TLR or cytokine receptor activation. Since degranulation can
also be induced by cathelicidin itself, a feedforward loop is initiated and results in
a cathelicidin burst with very high local concentrations (Vandamme et al. 2012). In
granula, cathelicidin is stored as an inactive precursor protein that is processed to
its active form by extracellular cleavage upon degranulation (Vandamme et al.
2012). The mechanism that activates the cathelicidin precursor protein in airway
epithelial cells, which do not store it in granula, has not been identified yet
(Vandamme et al. 2012).
36 F. Seiler et al.
3.3 Regulation of Antimicrobial Peptide Expression

in the Lung
3.3.1 Defensins
In the lung, β-defensin expression is triggered by common respiratory pathogens

including the most common causes of pneumonia. Airway epithelial cells express
β-defensins upon stimulation with Streptococcus pneumoniae (Scharf et al. 2012;
García et al. 2001), Haemophilus influenzae (Seiler et al. 2013), Legionella pneu-
mophila (Scharf et al. 2010a), and Pseudomonas aeruginosa (García et al. 2001;
Harder et al. 2000). β-Defensins are also induced by the most common causes of
pulmonary tuberculosis, Mycobacterium tuberculosis and Mycobacterium bovis
(Méndez-Samperio et al. 2007; Rivas-Santiago et al. 2005). Viral pathogens result-
ing in increased expression of β-defensins include respiratory syncytial virus (Kota
et al. 2008), rhinovirus (Proud et al. 2004; Duits et al. 2003), and, at least in mice,
influenza virus (Chong et al. 2008). Finally, β-defensins are induced by fungal
pathogens such as Aspergillus fumigatus (Alekseeva et al. 2009).
The cellular components of lung innate immunity are able to recognize and spe-
cifically react to pathogens through detection of conserved microbial structures
called pathogen-associated molecular patterns (PAMPs) by pattern recognition
receptors. These include transmembrane Toll-like receptors (TLRs) as well as cyto-
solic NOD-like and RIG-I-like receptors and others. Activation of pattern recogni-
tion receptors leads to induction of intracellular signaling pathways like the MAP
kinase cascade and activation of transcription factors such as NF-κB, eventually
resulting in gene expression. Apart from the constitutively expressed hBD-1, the
expression of β-defensins is largely regulated by TLR signaling. The role of cytosolic
pattern recognition receptors in the regulation of β-defensins remains to be
discovered.
There are ten human members of the TLR family. In the lung, the whole array of
TLRs is expressed by airway epithelial cells and resident innate immune cells like
alveolar macrophages and neutrophils (Kovach and Standiford 2011; Kawasaki and
Kawai 2014; Futosi et al. 2013; Parker and Prince 2011). Many of them have been
shown to upregulate β-defensin expression upon stimulation with their specific ago-
nists. In particular, the expression of β-defensins is induced by TLR2 (Hertz et al.
2003; Wang et al. 2003), TLR3 (Seiler et al. 2013; Proud et al. 2004), TLR4
(MacRedmond et al. 2005), TLR5 (Froy 2005), TLR6 (Froy 2005), and TLR9
(Platz et al. 2004). Since the members of this list can be activated by bacterial,
mycobacterial, viral, and fungal PAMPs (Akira et al. 2006), the innate immune net-
work of the lung can potently respond to different respiratory infections.
Apart from TLR-related β-defensin upregulation, lung infection also induces the
expression of various proinflammatory cytokines derived from epithelial, innate,
and adaptive immune cells. TNF-α and IL-1β are released by alveolar macrophages
during bacterial infection (Hess et al. 2010) and upregulate hBD-2 in airway epithe-
lial cells in vitro and in vivo (Hess et al. 2010; Harder et al. 2000; Albanesi et al.
2007). hBD-2 expression is also stimulated by IL-17A (Kao et al. 2004), which is
derived from Th17 cells during lung infection (Ye et al. 2001), and airway epithelial
cell-derived IL-17C (Kusagaya et al. 2014). Upon viral infection, interferon-gamma
release is triggered, for example, by TLR3 (Akira 2009) and leads to increased
hBD-3 expression (Albanesi et al. 2007).
Stimulation of TLRs and cytokine receptors leads to activation of intracellular
signaling processes like mentioned above. Therefore, TLR and cytokine receptors
induce β-defensin expression in an NF-κB- or MAP kinase-dependent manner
(McCray and Bentley 1997; Scharf et al. 2010b). In fact, hBD-2 has been shown to
be a direct target gene of the transcription factors NF-κB and the MAP kinase target
AP-1 (Wehkamp et al. 2004; O’Neil et al. 1999). With a vitamin D response element
within its promoter sequence, hBD-2 further is a target gene of vitamin D; however,
vitamin D reactivity of the hBD-2 gene is not as strong as it is described for cathe-
licidin (Wang et al. 2004).
3.3.2 Cathelicidin
In contrast to β-defensins, the expression of cathelicidin is not primarily induced

upon infectious or inflammatory stimulation but is regulated by vitamin D. Airway
epithelial as well as myeloid cells express the vitamin D receptor (Hansdottir et al.
2008; Liu et al. 2006). Stimulation of these cells with the active form of vitamin D,
1,25-dihydroxyvitamin D3, induces the expression of cathelicidin (Wang et al.
2004; Schauber et al. 2007a; Gombart et al. 2005). The promoter of the correspond-
ing gene contains a vitamin D response element, which makes it a direct vitamin D
target gene (Wang et al. 2004). Infectious stimuli can also indirectly trigger catheli-
cidin expression: it has been shown that TLR activation upregulates the expression
of the vitamin D receptor in human macrophages, which leads to an increased
expression of cathelicidin (Liu et al. 2006). Because the effects of vitamin D rely on
its conversion from its inactive proform to 1,25-dihydroxyvitamin D3 by the
hydroxylase CYP27B1, cathelicidin expression is largely influenced by modulation
of this reaction. TLR stimulation leads to activation of CYP27B1, resulting in
increased levels of active vitamin D (Liu et al. 2006). Conversion of vitamin D to its
active form is also induced by IFN-γ in alveolar macrophages (Koeffler et al. 1985).
1,25-dihydroxyvitamin D3 further amplifies the innate immune response by upreg-
ulation of TLR2 and CD14 (Schauber et al. 2007a, b).
3.4 Microbicidal Functions of Antimicrobial Peptides

in the Lung
AMPs exert antimicrobial activity against many common respiratory pathogens,

including bacterial, viral, and fungal species. The human β-defensins hBD-2 and
38 F. Seiler et al.
hBD-3 are bactericidal against the typic pathogen of community-acquired pneu-

monia, S. pneumoniae (Scharf et al. 2012). In vitro microbicidal activity against
P. aeruginosa and Escherichia coli, frequent causes of nosocomial and ventilator-
associated pneumonia, has been shown for hBD-2 (Harder et al. 2000). hBD-3 is
further bactericidal against Staphylococcus aureus and Enterococcus faecium
(Harder et al. 2001; Chen et al. 2005). hBD-4 has antimicrobial activity against
P. aeruginosa (Yanagi et al. 2005).
The antimicrobial spectrum of cathelicidin includes a broad array of bacterial
pathogens, including gram-positive species like S. pneumoniae and gram-negative
species such as P. aeruginosa (Benincasa et al. 2009; Bals et al. 1998b; Saiman
et al. 2001; Felgentreff et al. 2006).
Apart from bacteria, a couple of respiratory viruses are targeted by AMPs: influ-
enza virus, parainfluenza virus, and respiratory syncytial virus (Klotman and Chang
2006; Tripathi et al. 2013). AMPs have also been shown to act against Candida spp.
and Aspergillus spp., the most frequent species causing respiratory mycosis
(Alekseeva et al. 2009; Aerts et al. 2008).
While there is much evidence for the microbicidal effect of AMPs in vitro, it is
hard to assess their impact in the human lung. Due to their chemical structure, salt
concentrations and pH in healthy or inflamed lung tissue may alter AMP function
in vivo. In addition, physiological concentrations of AMPs may be significantly
lower than the minimal inhibitory concentrations assessed by in vitro approaches
(Bals et al. 1998b; Bowdish et al. 2005); thus, particular AMPs may not reach
microbicidal concentrations in vivo. However, it has to be considered that in the
human lung, AMPs are part of a well-orchestrated immune network with many
synergistic capacities. AMPs can act in mutual synergism (García et al. 2001; Chen
et al. 2005; Tripathi et al. 2013) as well as cooperative with other antimicrobial
substances such as lactoferrin and lysozyme (Bals et al. 1998a). The effect of
AMPs can also be enhanced by surfactant proteins (Tripathi et al. 2013) and even
bacterial components (Iwase et al. 2010). Finally, AMPs have been shown to act
synergistic with antibiotic agents (Xiong et al. 1999; Scott et al. 1999a, b; Hancock
and Scott 2000).
3.5 Beyond Antimicrobial Activity: Modulatory Functions

of Antimicrobial Peptides in the Lung
In primitive invertebrate species like the common model organism Caenorhabditis

elegans, there is no cellular immune system. Apart from rather unspecific mecha-
nisms such as pathogen avoidance and physical isolation, their resistance against
infectious agents largely depends on the microbicidal activity of their AMPs. In the
course of evolution, AMPs became part of increasingly complex immune systems
in which their role changed from solely antimicrobial agents to versatile modulators
of different physiologic systems.
3.5.1 Modulation of Lung Immunity
Within the innate immune network of the lung, AMPs are one of the first defense
mechanisms to face invading pathogens. Apart from their antimicrobial activity,
AMPs have many immunomodulatory features that are similar to those of classic
cytokines. For example, AMPs act as chemoattractants for different immune cells,
recruiting them to sites of inflammation. α-Defensins have been shown as chemo-
tactic factors for monocytes, dendritic cells, and T cells (Yang et al. 2000a, 2001;
Chertov et al. 1996). β-Defensins chemoattract monocytes, macrophages, and neu-
trophils via the chemokine receptor CCR2, immature dendritic cells and T cells via
CCR6, and mast cells in a phospholipase C-dependent manner (Soruri et al. 2007;
Yang et al. 1999; Röhrl et al. 2010; Niyonsaba 2002). Finally, cathelicidin is capable
of recruiting neutrophils, monocytes, and T cells via FPRL1 (Yang et al. 2000b;
Kurosaka et al. 2005), and mast cells via phospholipase C (Niyonsaba et al. 2002),
and takes part in the regulation of dendritic cell maturation and differentiation
(Davidson et al. 2004; Kandler et al. 2006).
In addition to their chemokine nature, AMPs are able to act as endogenous TLR
agonists. For example, mouse β-defensin 2 activates TLR4 (Biragyn et al. 2002),
and β-defensin 3 is capable of activating TLR1 and TLR2 (Funderburg et al. 2007).
By that, AMPs can induce cytokine expression and activation of immune cells:
β-defensins induce IL-6, IL-8, IL-10, and MCP-1 in monocytes (Boniotto et al.
2006) and IL-1β and IL-6 in dendritic cells (Biragyn et al. 2002). In myeloid and
epithelial cells, IL-8 expression is upregulated by α-defensins (Khine et al. 2006).
β-Defensin 2 leads to dendritic cell maturation in mice (Biragyn et al. 2002), and
β-defensin 3 activates monocytes and neutrophils (Funderburg et al. 2007).
Cathelicidin has been shown to indirectly enhance TLR signaling (Lai et al. 2011).
Cathelicidin further induces IL-8 and MCP-1 in a MAPK-dependent manner (Scott
et al. 2002; Tjabringa et al. 2003; Bowdish et al. 2004; Yu et al. 2007). Mast cell
degranulation and histamine release are mediated by β-defensins and cathelicidin
(Schiemann et al. 2009; Niyonsaba et al. 2001).
Excessive activity of the immune system can cause self-damage of the host by
immunopathological processes. Therefore, balancing of pro- and anti-inflammatory
signals is crucial. Above immunostimulation, AMPs also take part in the fine-tuning
of the immune response by additionally regulating anti-inflammatory mechanisms.
Activation of the complement system, which can be induced in the lung (Varsano
et al. 2000), is inhibited by β-defensin 2 (Bhat et al. 2007). β-Defensin 3 has been
shown to inhibit ERK and, via downmodulation of TRIF and MyD88, TLR signal-
ing, leading to a decreased production of proinflammatory cytokines in macrophages
and dendritic cells (Semple et al. 2010, 2011; Pingel et al. 2008). α-Defensins have
an anti-inflammatory effect on macrophages, reducing production of reactive oxy-
gen species and inhibiting the expression of proinflammatory cytokines (Miles et al.
2009). However, compared to PAM3CSK4, another TLR1/2 agonist, β-defensin
3-derived TLR1/2 activation induces a pro- rather than an anti-inflammatory cyto-
kine pattern in monocytes (Funderburg et al. 2011).
40 F. Seiler et al.
Excessive immunostimulation by bacterial PAMPs is considered to be a key event

in the pathogenesis of septic shock. Cathelicidin inhibits proinflammatory cytokine
release from neutrophils and macrophages upon stimulation with the gram-negative
endotoxin LPS (Mookherjee et al. 2006; Alalwani et al. 2010; Scott et al. 2011). In
particular, cathelicidin antagonizes LPS by preventing the formation of the LPS/LBP
complex, which is required for TLR4-dependent LPS recognition (Scott et al. 2000;
Kirikae et al. 1998). Synthetic mimics of AMPs further intercept the classical gram-
positive PAMP and TLR2 agonist LTA (Scott et al. 1999a). The role of cathelicidin at
the crossroads of inflammation is further underlined by its influence on bacterially
stimulated neutrophils: cathelicidin increases their antimicrobial activity and phagocy-
totic capacity. In cathelicidin-deficient neutrophils, antimicrobial activity is impaired;
instead, they react with more production of proinflammatory cytokines (Alalwani et al.
2010). Thus, AMPs may limit the adverse effects of extensive immune cell activation.
On the other hand, AMPs seem to especially promote airway epithelial innate
immune mechanisms: cathelicidin leads to a decrease in epithelial permeability,
thereby preventing transepithelial invasion of bacterial pathogens (Byfield et al.
2011). It further induces internalization of LPS into airway epithelial cells, which
leads to activation of endosomal TLR4 and consequently LPS-induced epithelial
cytokine production (Shaykhiev et al. 2010). Moreover, cathelicidin facilitates sig-
naling via TLR3, a receptor for viral dsRNA (Lai et al. 2011). In synergy with other
inflammatory mediators and TLR agonists, cathelicidin activates cytokine produc-
tion in epithelial cells even in low concentrations (Filewod et al. 2009; Nijnik et al.
2012). Cathelicidin is also able to activate NF-κB and induce cytokine production
in airway epithelial cells on its own (Pistolic et al. 2009).
The pro- and anti-inflammatory capacities of AMPs in different settings are still
incompletely understood and even seem to be self-contradictory to some degree.
Anyway, their position within the hierarchically structured immune network of the
lung is strategic: at the first line of defense, they ensure an early answer to pathogen
exposition, namely, through direct microbicidal activity, PAMP interception, and
immunological activation of resident cells. In the next step, they trigger more potent
mechanisms of the innate and adaptive immunity while simultaneously contributing
to attenuate those mechanisms in order to avoid immunopathology.
In summary, AMPs are key players in the initiation and amplification of the
innate immune response and contribute to activation of adaptive immunity. At the
same time, they seem to protect the host from overshooting inflammation by coor-
dination of pro- and anti-inflammatory signaling.
3.5.2 Modulation of Lung Injury and Repair
Infection and inflammation leading to epithelial injury are characteristic hallmarks

of acute and chronic lung diseases. Termination and resolution of inflammation and
maintenance of tissue homeostasis are active and coordinated processes which
include the expression of immune cell-derived and epithelial factors such as
pro- and anti-inflammatory cytokines, the keratinocyte growth factor (KGF), and
the epidermal growth factor (EGF) (Tjabringa et al. 2003; Campbell et al. 2011;
Puddicombe et al. 2000; Michelson et al. 1999). AMPs qualify as mediators of
wound healing and tissue repair as the expression of AMPs by inflammatory and
epithelial cells is increased under infectious and inflammatory conditions and epi-
thelial injury induced by infectious agents leads to an increased risk that pathogens
invade the host (Steinstraesser et al. 2008; Lai and Gallo 2009). Indeed, there is
strong evidence that AMPs are involved in epithelial repair processes, such as cell
proliferation, epithelial regeneration, and reepithelialization (Lai and Gallo 2009;
Heilborn et al. 2003; Tokumaru et al. 2005; Carretero et al. 2008; Beisswenger and
Bals 2005; Murphy et al. 1993; Aarbiou et al. 2004).
The function of AMPs in the repair of epithelial injury has been particularly stud-
ied in wound healing of the skin. Cathelicidin, for instance, induces reepithelializa-
tion after skin injury via transactivation of the EGF receptor (Heilborn et al. 2003;
Tokumaru et al. 2005; Carretero et al. 2008). Cathelicidin also plays an important role
in angiogenesis and thereby contributes to wound repair (Koczulla et al. 2003). As
cathelicidins and defensins induce proliferation of diverse airway epithelial cell lines
and primary airway epithelial cells in vitro, it is suggested that AMPs also mediate
wound healing in the lung via the activation of pro-proliferative signaling cascades
(Beisswenger and Bals 2005; Murphy et al. 1993). Cathelicidin activates airway epi-
thelial cell via metalloproteinase-mediated cleavage of membrane-anchored EGF
receptor ligands as well as MAP kinase-dependent signaling pathways (Tjabringa
et al. 2003) and stimulates airway epithelial wound closure in an EGF receptor-, G
protein-coupled receptor-, and MAP kinase-dependent manner (Shaykhiev et al.
2005). A function of cathelicidins in tissue repair is further supported by a mouse
study which associates deficiency of the mouse cathelicidin CRAMP with increased
lung injury during infection with gram-negative bacteria (Kovach et al. 2012).
Like cathelicidin, α-defensins from human neutrophils induce proliferation of
airway epithelial cell lines via activation of MAP kinase signaling cascades (Aarbiou
et al. 2002) and enhance wound closure in an EGF-dependent manner (Aarbiou
et al. 2004). α-Defensins also increase proliferation of lung fibroblasts and collagen
synthesis (Han et al. 2009).
However, in vitro studies also suggest that cathelicidins and defensins induce the
release of inflammatory mediators by airway epithelial cells and that AMPs are
cytotoxic at higher concentrations (Tjabringa et al. 2003; Shaykhiev et al. 2005;
Sakamoto et al. 2005). Therefore, the proinflammatory, cytotoxic, and pro-
proliferative functions of AMPs may also have adverse consequences by perpetuating
inflammation and potentially contribute to epithelial injury in lung diseases
(Shaykhiev et al. 2005; Sakamoto et al. 2005). Moreover, concentrations of AMPs
have been found to be increased in severe lung diseases. Plasma concentrations of
α-defensins, for instance, are increased in pulmonary fibrosis (Mukae et al. 2002)
and β-defensins are increased in bronchoalveolar lavage fluids of patients with
diffuse panbronchiolitis and in bronchiolitis obliterans syndrome after lung
transplantation (Ross et al. 2004; Hiratsuka et al. 2003). Cathelicidin associates
with bronchial inflammation in cystic fibrosis lung disease (Chen et al. 2004).
42 F. Seiler et al.
In summary, AMPs may contribute to tissue regeneration and epithelial regen-

eration in the lung during infection and inflammation by inducing proliferation of
structural cells, but they also potentially contribute to epithelial injury and fibrotic
remodeling during airway inflammation when present at high concentrations.
3.6 Clinical Perspectives
3.6.1 Acute Respiratory Tract Infections
Respiratory tract infections are very common. Especially pneumonia is a major cause
of global morbidity and mortality in children as well as in adults (Klugman and Garau
2009). Due to their microbicidal properties (“endogenous antibiotics”), the role of
AMPs in acute respiratory tract infection has drawn much attention. AMP levels are
increased in patients with pneumonia. High levels of β-defensins and cathelicidin can
be detected in plasma or sputum of patient with acute pneumonia (Hiratsuka et al.
1998; Herr et al. 2009; Schaller-Bals et al. 2002; Ishimoto et al. 2006).
The important role of AMPs in respiratory tract infection is especially underlined
by studies in animal models. Cathelicidin treatment has been shown to reduce pro-
inflammatory cytokine release during MRSA pneumonia and improves the histo-
pathological outcome (Hou et al. 2013). In cathelicidin-deficient mice, the immune
response to gram-negative pneumonia with K. pneumoniae is delayed, eventually
resulting in more inflammation and increased lung injury (Kovach et al. 2012).
During gram-negative pneumonia with P. aeruginosa, overexpression of β-defensin
2 in rats reduces bacterial load in the lung as well as inflammation and lung injury
and increases survival rate (Shu et al. 2006; Hu et al. 2010). In β-defensin 1-defi-
cient mice, clearance of H. influenzae in the lung is impaired (Moser et al. 2002).
The effect of AMPs is also modulated by host-microbe interactions. The S. aureus
virulence factor staphylokinase acts synergistically with cathelicidin to promote
fibrinolysis during mouse pneumonia, which leads to a more invasive infection (Braff
et al. 2007). Staphylokinase is also able to inactivate α-defensins (Jin et al. 2004).
While there are natural limitations in evaluation of the in vivo impact of AMPs in
human pneumonia, the high AMP levels found in patients with pneumonia together
with the studies mentioned above suggest a crucial role of AMPs in the response to
lung infection.
3.6.2 Chronic Lung Diseases
Cigarette smoke is the major risk factor for the development of the chronic obstruc-
tive pulmonary disease (COPD). COPD is characterized by chronic inflammation of
the lung leading to tissue destruction, emphysema, and loss of pulmonary function
(Sethi and Murphy 2008; Sethi 2010). Stable COPD patients are frequently colo-
nized with bacterial pathogens (e.g., H. influenzae); recurrent infections of the
respiratory tract and chronic bronchitis are common in COPD (Sethi and Murphy
2008; Sethi 2010; Moghaddam et al. 2011; Garmendia et al. 2012). Microbial infec-
tions of the lung evoke harmful inflammatory responses that contribute to clinical
deterioration of COPD patients (Sethi 2010). Even in healthy individuals, smoking
suppresses pulmonary host defense making the lung more susceptible for microbial
colonization and infection (Herr et al. 2009; Sethi 2010; Mehta et al. 2008). Ex vivo
and in vitro studies showed that the expression of AMPs is inhibited by cigarette
smoke and that COPD is associated with skewed AMP expression in the respiratory
tract. Analyses of human samples showed that current or former smoking is associ-
ated with significantly reduced levels of the β-defensin hBD-2 in pharyngeal wash-
ing fluids and sputum from patients with acute pneumonia (Herr et al. 2009). In
vitro studies demonstrated that exposure to cigarette smoke and reactive oxygen
species inhibit the expression of hBD-2 in respiratory epithelial cells in response to
inflammatory mediators or bacteria (Herr et al. 2009). The effects of cigarette smoke
on β-defensin expression are likely due to the suppression of cellular signaling cas-
cades by cigarette smoke, such as NF-κB and AP-1 signaling (Pace et al. 2012;
Kulkarni et al. 2010; Laan et al. 2004). In currently smoking COPD patients, hBD-2
expression is decreased in central but not in distal airways, inversely correlating
with cigarette smoke exposure (Pace et al. 2012). This has been underlined by the
finding that hBD-2 mRNA expression in peripheral lung samples has been found to
be increased in COPD patients and was associated with higher IL-8 expression and
poor lung function (Liao et al. 2012). Cathelicidin has also been shown to be
increased in small airways of COPD patients, where it may contribute to airway
remodeling (Sun et al. 2014). In addition, a proteomic study showed that the
α-defensins are increased in bronchoalveolar lavage fluids of COPD patients
(Merkel et al. 2005). In summary, the expression pattern of AMP seems to be
deranged in COPD with low antimicrobial activity in the central airways. Altered
AMP expression may lead to airway colonization with pathogens in smokers and
patient with COPD, providing reservoirs for recurrent infection. Enhanced suscep-
tibility to recurrent respiratory tract infections subsequently leads to chronic inflam-
mation, aberrant expression of AMPs, and disease progression (Herr et al. 2009;
Sethi 2010).
Studies also suggest that skewed expression of AMPs and inhibition of the bacte-
ricidal activity of AMPs play a role in cystic fibrosis (CF). AMPs are detectable in
sputum or bronchoalveolar lavage fluid of CF patients with α-defensins and catheli-
cidin being present at high levels (Felgentreff et al. 2006; Chen et al. 2004; Soong
et al. 1997). Moreover, enhanced levels of cathelicidin correlate with severity of CF
lung disease (Chen et al. 2004). β-Defensin levels do not seem to correlate with the
severity of inflammation, but decreased levels of hBD-2 are associated with severity
of CF (Chen et al. 2004; Dauletbaev et al. 2002; Bals et al. 2001). In vitro, the bac-
tericidal activity of cationic AMPs is salt sensitive. Thus, it is suggested that increased
salt concentrations in airway fluids of CF patients contribute to chronic lung infec-
tion by inhibiting bactericidal functions of AMPs (Bals et al. 1998a). In line with this
hypothesis, bactericidal activity of hBD-1 has been shown to be decreased in air-
ways of CF patients and CF airway fluids fail to kill S. aureus and P. aeruginosa
(Goldman et al. 1997; Bals et al. 1999). However, in a human bronchial xenograft
44 F. Seiler et al.
model, salt-independent dysfunction of antimicrobial activity was corrected by ade-

novirus-mediated gene transfer of CFTR (Bals et al. 2001). Thus, the role of altered
salt concentrations in airway fluids of CF patients is controversial and yet unknown
mechanisms may contribute to the reduced bactericidal activity in the fluids lining
the mucosal surfaces of CF patients. Excessive mucus production, for instance, also
potentially contributes to impaired bactericidal activities of AMPs in CF patients as
mucus interferes with AMPs (Felgentreff et al. 2006).
3.6.3 Lung Cancer
For several tumor types such as ovarian cancer and malignant melanoma, adverse
effects of AMPs on tumor development and progression have been shown. In lung
cancer, overexpression of different AMPs was found. Cathelicidin is overexpressed
in up to 25 % of NSCLC samples (Von Haussen et al. 2008). It is also released by
myeloid cells in the tumor microenvironment (Li et al. 2014). Cathelicidin has been
shown to transactivate EGFR in airway epithelial cells (Tjabringa et al. 2003).
EGFR signaling is one of the critical pathways in lung carcinogenesis (Mitsudomi
and Yatabe 2010). Through interaction with the EGFR pathway, cathelicidin pro-
motes tumor growth in vitro and in vivo (Von Haussen et al. 2008), which is further
induced by cigarette smoke (Li et al. 2014).
As for β-defensins, hBD-1 and hBD-2 are overexpressed in about 50–60 % of
NSCLC tissue samples (Shestakova et al. 2008). In serum of patients with lung
cancer, concentrations of hBD-1 and hBD-2 are significantly higher as compared to
healthy controls and even patients with pneumonia. Therefore, hBD-1 and hBD-2
have even been proposed as lung tumor markers (Arimura et al. 2004).
AMPs take part in the orchestration of important cellular functions that contrib-
ute to oncogenesis and tumor promotion, as they modulate proliferation, cell
migration, and angiogenesis. Thus, given their central role in chronic lung disease,
AMPs might link chronic airway inflammation to the development of lung cancer.
In contrast to cathelicidin and β-defensins, the α-defensin HNP-1 has been shown
to act as an inhibitor of tumor growth. HNP-1 is cytotoxic to tumor cells (Van
Wetering et al. 1997; Okrent et al. 1990) and induces apoptosis in lung cancer and
other cells (Xu et al. 2008; Lichtenstein 1991). This even led to successful gene
therapy approaches with direct intratumoral administration of plasmid DNA encod-
ing HNP-1 in a murine xenograft tumor model (Xu et al. 2008).
3.7 Conclusions
In the human lung, AMPs are abundantly expressed by different cell types, includ-
ing epithelial and immune cells. The expression of defensins is largely triggered
upon stimulation with PAMPs and inflammatory mediators, whereas cathelicidin
expression mainly underlies a vitamin D-dependent mechanism. At least in vitro,

AMPs are capable of neutralizing a broad array of bacterial, viral, and fungal spe-
cies, including the most common respiratory pathogens. While their microbicidal
impact in vivo is still not completely clear, it is now recognized that the abilities of
AMPs within a complex immune network by far exceed antimicrobial activity.
AMPs take part in the initiation, amplification, and modulation of lung innate
immunity. Remarkably, AMPs seem to be involved in balancing of pro- and anti-
inflammatory signaling, thus protecting the host from excessive immunostimula-
tion. Thus, AMPs seem to have an important role in acute lung infection. However,
in the context of chronic inflammation and infection-related lung diseases, AMPs
can be dysregulated and even act adversely: AMP dysfunction may contribute to the
pathogenesis of COPD, asthma, and cystic fibrosis. Furthermore, while AMPs con-
tribute to lung repair after injury, they may also have harmful effects regarding
fibrogenesis and lung tumor growth.
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Chapter 4
Antimicrobial Peptides: Maintaining Sterility
of the Urinary Tract
Brian Becknell and John David Spencer
Abstract Due to its close proximity to the gastrointestinal tract, the normally ster-
ile urinary tract is constantly challenged by microbial invasion. To counter this
microbial assault, the urinary tract has developed a highly effective antimicrobial
“shield” that can rapidly eliminate invading pathogens or prevent their growth.
During recent years, considerable advances have been made in our understanding of
the immune mechanisms that contribute to urinary tract sterility. Recent evidence
indicates that cationic antimicrobial peptides contribute to the innate host defense of
the urinary tract. This chapter reviews the published literature on the role(s) of anti-
microbial peptides (AMPs) in maintaining urinary tract sterility.
4.1 Introduction
Urinary tract infections (UTI) are one of the most common and serious bacterial
infections encountered by physicians (Bachur and Harper 2001; Foxman et al. 2000).
Nearly half of all women develop a UTI requiring antimicrobial therapy during their
lifetime (Foxman et al. 2000). In 2013, aggregate hospital charges for inpatient UTI
management exceeded $640 million US dollars (Spencer et al. 2011a). Specific sub-
populations have increased UTI risk, including pregnant women, the elderly, patients
with diabetes or multiple sclerosis, patients with acquired immunodeficiency dis-
eases, patients with urologic anomalies, and those having urinary tract intervention
such as catheter insertion. Although UTI is not typically associated with significant
morbidities, UTI does increase the risk of premature delivery and fetal mortality
among pregnant women (Foxman et al. 2000). Long-term complications of UTI
include renal insufficiency, renal scarring, hypertension, and chronic kidney disease.
B. Becknell • J.D. Spencer (*)

Department of Pediatrics, Division of Nephrology, Center for Clinical and Translational
Research, The Research Institute at Nationwide Children’s, 700 Children’s Drive,
Columbus, OH 43205, USA

DOI 10.1007/978-3-319-24199-9_4
54 B. Becknell and J.D. Spencer
To date, no treatment strategy has been proven to be effective in the prevention of

UTI sequelae. Moreover, antibiotic resistance in uropathogenic bacteria has been
increasing in large part due to antibiotic overuse (Spencer et al. 2014a).
4.2 UTI Pathogenesis and Roles for Antimicrobial Peptides

in the Host Response
UTI refers to the presence of microbial pathogens within the urinary tract. The site
of infection classifies UTI—infection localized to the bladder is referred to as “cys-
titis” and infection in the kidney is “pyelonephritis.” Cystitis typically presents with
lower urinary tract symptoms—including dysuria, urgency, and urinary frequency.
Pyelonephritis is often associated with more severe or systemic symptoms includ-
ing fever, back/flank pain, and vomiting. Ascending infection may result in bactere-
mia and present as the systemic inflammatory response syndrome or overt septic
shock (i.e., urosepsis).
Escherichia coli (E. coli) is the most frequent bacterial pathogen responsible for
UTI—accounting for 85–90 % of cases. Uropathogenic E. coli (UPEC) are thought to
originate from the fecal flora, spread across the perineum, and enter the bladder through
the urethra. Before invading the urothelium, UPEC must overcome several intrinsic
characteristics of the urinary tract. Proposed functional mechanisms contributing to
urinary tract sterility include barrier formation by uroepithelial cells, unidirectional
flow of urine, regular bladder emptying, mucous production, the urinary microbiome,
and alterations in the urinary ionic composition (Spencer et al. 2014a; Sobel 1997).
The microbial virulence of UPEC has been linked to many factors (Ragnarsdottir
and Svanborg 2012; Mulvey et al. 1998, 2000). The most prominent is type I fim-
briae, which are filamentous bacterial appendages that are capped by FimH, a
mannose-binding adhesion protein. Type I fimbriae promote tight bacterial binding
to a matrix of uroplakin complexes on the surface of superficial bladder epithelial
cells (Mulvey et al. 2000). After binding, UPEC invades the bladder uroepithelium
where it may establish a state of commensalism or cause a severe, symptomatic
infection characterized by a rapid innate host response with cytokine secretion and
recruitment of leukocytes to the infection source (Weichhart et al. 2008).
In the kidney, UPEC binds to the apical surfaces of the kidney’s collect duct. Within
the collecting duct, UPEC specifically targets the intercalated cells (Chassin et al.
2011; Paragas et al. 2014). The signaling pathways activated by UPEC have been
investigated in primary cultures of medullary collecting duct cells dissected from the
kidneys of LPS-sensitive C3H/HeOuJ mice (Chassin et al. 2011). Signaling pathway
analysis demonstrates that UPEC stimulates the expression of pro-inflammatory
mediators in the medullary collecting ducts via TLR4-mediated, MyD88-dependent,
TRIF-independent NF-kB and MAPK-activated pathways and also via a TLR4-
independent, MyD88-independent pathway. The TLR4-independent pathway results
from activation of the TNF receptor-associated factor-2 (TRAF2) and apoptosis sig-
nal-regulatory kinase 1 (ASK1)-JNK pathway (Chassin et al. 2011; Vandewalle 2008).
4 Antimicrobial Peptides: Maintaining Sterility of the Urinary Tract 55
Kidney Urinary tract Nephron and Collecting Tubule

Distal Convoluted Tubule
Renal pelvis HBD1
HD5
RNase7 Glomerulus
Proximal Tubule
Ureter LL-37
HD5
LL37
RNase7
Urethra
Bladder Loop of Henle
HD5
HD5 HBD1
RNase7 HBD2 Collecting Tubule
HD5 HBD1
HBD2
HD5
RNase7
Fig. 4.1 Defined AMP in the kidney and urinary tract. Left panel: AMP identified in the human
lower and upper urinary tract. Right panel: AMPs identified in the nephron and collecting tubule
of human kidney (Image reused with permission from Spencer et al. (2014a))
Antimicrobial peptides (AMP) may be induced through TLR-4-mediated and

TLR-4-independent pathways. Bacterial attachment may immediately induce uro-
epithelial AMP production (Spencer et al. 2014a; Zasloff 2007). Alternatively,
AMPs may be constitutively produced by the uroepithelium and limit bacterial
attachment by direct antimicrobial activity. AMPs may also contribute to urinary
tract sterility by depleting vital nutrients required for bacterial growth or serving as
chemoattractants for local leukocytes. The following sections outline the published
literature on the function(s) of AMPs in the urinary tract. Figure 4.1 shows an over-
view about identified AMP in the kidney and urinary tract.
4.3 Cathelicidin
The human cathelicidin LL-37 is an amphipathic, α-helical AMP expressed on all epi-
thelial surfaces, myeloid bone marrow cells, and circulating neutrophils. Cathelicidin
possesses antimicrobial activity against viruses and Gram-positive and Gram-negative
bacteria. Moreover, it acts as a chemoattractant for neutrophils and tissue-derived
monocytes by interacting with their fMLP receptors. The optimal chemoattractant
concentrations of cathelicidin are in the micromolar range, considerably higher than

observed for traditional chemokines (Zasloff 2007; Yang et al. 2007).
There is limited but convincing evidence that LL-37 plays a role in maintaining
urinary tract sterility (Ali et al. 2009). Chromek et al. demonstrate that LL-37 is
constitutively expressed in the human upper and lower urinary tract. They detected
low levels of cathelicidin in the urine of healthy children (0.2–5.9 ng/mL). Urinary
LL-37 levels increased during cystitis and pyelonephritis (0–312.5 ng/mL)
(Chromek et al. 2006). The authors concluded that urinary cathelicidin originates
from the uroepithelium as there was only a small correlation with urinary leuko-
cytes and myeloperoxidase. They supported this finding through immunostaining
localizing cathelicidin production to the renal tubular epithelium in noninfected
human renal biopsy specimens. When they exposed these biopsy specimens to
UPEC, LL-37 production increased. Additionally, when renal epithelial (A498 and
hPTC cells) and uroepithelial cells (J82 and UROtsa cells) were challenged with
UPEC, mRNA expression of the LL-37 gene CAMP increased within 5 min. This
mRNA induction was followed by prolonged LL-37 peptide secretion into the sur-
rounding medium. These data suggest that cathelicidin production originates from
the uroepithelium and is designed to facilitate an immediate and sustained response
to microbial insult (Chromek et al. 2006; Chromek 2015).
In the same study, Chromek et al. evaluated the biological relevance of cathelici-
din in vivo using a mouse model of pyelonephritis. Immunofluorescent staining
suggests that Cramp, the murine homologue of human cathelicidin, is upregulated
during the acute stages of pyelonephritis in the renal tubular epithelium. During
more advanced stages of infection, cathelicidin is also released from leukocytes,
indicating that urinary cathelicidin originates from both the urothelium and leuko-
cytes. Deletion of the Cramp gene leads to increased bacterial attachment to bladder
uroepithelium compared to wild-type mice. Cathelicidin-deficient mice also experi-
enced a higher rate of ascending infection compared to wild-type animals with
increased renal bacterial burden. Thus, cathelicidin appears to participate in epithe-
lial antimicrobial defense, recruitment of immune cells, and neutrophil killing of
pathogens. During UTI, epithelial cells rapidly increase production of cathelicidin
to protect the urinary tract from bacterial invasion, and the “second wave” of cathe-
licidin comes from leukocytes (Chromek et al. 2006).
4.4 Defensins
Defensins, characterized by a 15–20 amino acid sequence including six cysteine resi-
dues, are one of the most studied families of AMPs. Defensins typically have broad-
spectrum antimicrobial activity against Gram-positive and Gram-negative bacteria,
viruses, fungi, and protozoa (Lehrer et al. 1993). Along with their direct antimicrobial
properties, defensins play a role in cell-mediated immunity as chemoattractants for
immature dendritic cells (Zasloff 2007). Defensins are initially synthesized as
pre-pro-proteins and undergo processing to become mature, biologically active peptides.

In humans, defensins are classified into one of two families depending on their disulfide-
bridging pattern—the α-defensins or the β-defensins. The clusters of genes encoding the
α-defensin subfamily and the majority of members of the β-defensin subfamily are
located on chromosome 8p22 and 8p23 (Liu et al. 1997).
4.5 α-Defensins
The α-defensins HNP1–HNP4 are primarily found in neutrophils where they pro-
vide non-oxidative microbicidal activity. HNPs encounter pathogens after they are
secreted onto the cell’s surface via degranulation or after a pathogen undergoes
phagocytosis and the phagocytic vacuole fuses with the neutrophilic granule (Ganz
2003). Regarding the urinary tract, Ihi et al. demonstrated that urinary levels of
HNP1–HNP3 significantly increased in the setting of UPEC and Enterococcus fae-
calis UTI (6.5 ± 1.1 pg/μL to 29 ± 5.7 pg/μL) (Ihi et al. 1997). Similarly, Tikhonov
et al. demonstrated that urinary HNP1 increased eightfold in patients with chronic
pyelonephritis compared to control patients and patients with glomerulonephritis.
Urinary HNP1 levels correlated with urinary IL-8 levels as well as leukocyte count,
suggesting that urinary HNP1 may reflect neutrophil recruitment to the infection
site (Tikhonov et al. 1997). No microbiological information has been published in
regard to the HNPs and UTI.
The expression and function of epithelial human defensin HD5 have been
reported mostly in the small intestine where it is secreted by Paneth cells into the
intestinal crypts. HD5 has also been described in the male and female reproductive
tracts, with evidence suggesting that it is inducible and important in eradicating
infection (Porter et al. 2005; Quayle et al. 1998). Recombinant mature HD5 has
been shown to have bactericidal activity against uropathogenic bacteria, including
Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus epidermidis,
Enterococcus faecium, and UPEC (Wang et al. 2010).
In the urinary tract, HD5 has been localized to the uroepithelium of the kidney,
ureter, and bladder (Spencer et al. 2012). Schwaderer et al. demonstrate that HD5
gene and protein expression were significantly greater in human kidney biopsy
specimens from patients with pyelonephritis compared to noninfected controls.
Their data also show that secreted HD5 was not routinely detected in culture-
negative human urine samples; however, HD5 levels significantly increased in urine
samples infected with UPEC (300–670 ng HD5/mg urine creatinine). Although uri-
nary HD5 did not reach concentrations likely to be directly antimicrobial against
common uropathogens, the mucosal surface concentrations may be higher (Spencer
et al. 2012). Urinary HD5 levels are higher in patients who have undergone ileal
neobladder reconstruction and ileal conduit urinary diversion (Porter et al. 1998;
Townes et al. 2011).
4.6 β-Defensins
The human β-defensins, encoded by over 28 genes, are widely expressed in human
epithelia and certain family members exhibit antimicrobial activity toward Gram-
positive and Gram-negative bacteria (Ali et al. 2009; Schutte et al. 2002). Of the
β-defensins, human β-defensin-1 (HBD1, encoded by the DEFB1 gene) and human
β-defensin-2 (HBD2, encoded by DEFB4) have been described in the human urinary
tract. DEFB1 mRNA is constitutively expressed by the epithelial lining of the kid-
ney’s loop of Henle, the distal tubule, and the collecting duct (Valore et al. 1998).
HBD1 is translated as a 68 amino acid pro-peptide and undergoes variable amino-
terminal processing to a 36–47 amino acid (Valore et al. 1998; Zucht et al. 1998).
Mature HBD1 is constitutively detected in sterile urine (10–100 μg/L) and levels
increase up to threefold in patients with UTI (Valore et al. 1998; Hiratsuka et al.
2000). Although these urinary levels of HBD1 are insufficient to kill invading bacte-
ria, HBD1 may provide a fast-acting antimicrobial coating of urothelium and prevent
infection by inhibiting bacterial attachment to the urothelium (Valore et al. 1998).
Defb1 encoding mouse BD1 (mBD1) has been considered orthologous to HBD1
based on conserved gene structure, expression pattern, and antimicrobial activity,
though the amino acid sequences of mBD1 and HBD1 are only 51 % identical
(Huttner et al. 1997; Bals et al. 1998; Morrison et al. 1998). Defb1 is transcribed
within lower urinary tract and the kidney’s distal tubules and collecting ducts (Bals
et al. 1998; Becknell et al. 2013). The mature mBD1 peptide exhibits salt-sensitive
antimicrobial activity toward Gram-positive and Gram-negative bacteria (Bals et al.
1998; Morrison et al. 1998). Mice lacking both copies of Defb1 (Defb1−/−) exhibit
increased incidence of spontaneous bacteriuria compared to Defb1+/− and wild-type
controls, with Staphylococcus species predominating among urine isolates
(Morrison et al. 2002). Wild-type mice undergoing transurethral inoculation of
UPEC exhibit significant reduction in bladder Defb1 transcript levels within 2 h of
infection that persists up to 2 days. These results suggest that successful UPEC
colonization of the urinary tract is achieved through local inhibition of Defb1
expression. However, when Defb1−/− animals were challenged with UPEC, no dif-
ference in upper or lower tract bacterial burden was observed relative to wild-type
controls (Becknell et al. 2013).
Additional Defb family members are detectable at the mRNA level in the murine
urinary tract. Defb3 (encoding mBD3) and Defb14 (encoding mBD14) mRNA are
enriched in mouse bladder, compared to ureter and kidney (Becknell et al. 2013).
Both mBD3 and mBD14 exhibit bactericidal activity toward UPEC in the low-
micromolar range. Other Defb transcripts, such as Defb2, Defb28, Defb29, and
Defb42, are enriched or exclusively expressed in murine ureter and kidney, compared
to bladder (Becknell et al. 2013). The lack of available antibodies has limited the
study of the levels and distribution of these mBD peptides in the urinary tract. The
establishment of mouse strains with deletions in one or more of Defb family mem-
bers will facilitate studies that determine their biological relevance within the uri-
nary tract (Zhou et al. 2013; Navid et al. 2012).
4.7 Hepcidin
Hepcidin, also known as liver-expressed antimicrobial peptide-1 (LEAP-1), is

produced in the liver and excreted in the urine (urinary concentrations range from
10 to 30 μg/L). It is translated in the liver as an 84 amino acid pre-pro-peptide.
After processing and excretion through the kidneys, a 25 amino acid peptide
(hepc-25) is the predominant form in the urine (but shorter urinary peptides are
also detected). Hepcidin does not show sequence similarity to any of the other
described AMPs but structurally resembles the defensin family given the four
disulfide bridges in its tertiary structure (Park et al. 2001; Krause et al. 2000).
Hepcidin has broad-spectrum antimicrobial activity against E. coli (ML-35), S.
epidermidis, S. aureus, C. albicans, and group B Streptococcus (Park et al. 2001).
Hepcidin also plays an important role in iron homeostasis. Genetically modified
mice, engineered to overexpress hepcidin, die shortly after birth secondary to severe
iron deficiency. These two findings indicate that hepcidin may participate in the
innate defense of the urinary tract by direct antimicrobial activity and/or reduction
of available iron—which is an essential nutrient for uropathogens (Park et al. 2001;
Weinstein et al. 2002).
4.8 Lactoferrin and Lipocalin
Lactoferrin inhibits bacterial growth through free iron chelation or the effects of
lactoferricin, a bactericidal protein generated by the proteolytic cleavage of lacto-
ferrin (Bellamy et al. 1992). Lactoferrin is detectable in human urine; however,
these levels are relatively low (14–145 ng/mL). In the human kidney, lactoferrin is
expressed in the cells lining the distal collecting ducts of the medulla (Abrink et al.
2000). Lipocalin 2 (LCN2) is a member of the large lipocalin protein family, which
has a wide range of biological functions. Several reports indicate that lipocalin 2
(LCN2) mediates a bacteriostatic effect by sequestering iron (Berger et al. 2006; Flo
et al. 2004; Holmes et al. 2005). Specifically, LCN2 binds the secreted siderophore
enterochelin (Ent), which UPEC and other pathogens release into the extracellular
milieu to acquire essential iron. Upon complexing with Ent:Fe3+, LCN2 reroutes
Ent:Fe3+ for degradation and prevents iron transfer to bacteria. Without iron, uro-
pathogens are unable to grow, facilitating immune clearance (Paragas et al. 2014).
Thus, Lcn2-deficient mice demonstrate increased susceptibility to bacterial infec-
tions when challenged with intraperitoneal E. coli (Berger et al. 2006).
Recently, Paragas et al. observed a significant induction of Lcn2 in the urine of
mice with UTI compared with uninfected animals. The degree of Lcn2 upregulation
correlated with the number of infecting bacteria in the urine and reduction of bacte-
rial load with antibiotics resulted in decreased Lcn2 production. A similar correla-
tion between bacterial load and LCN2 was also seen in the clinical setting—with
urinary levels increasing tenfold in patients with UTI (30–300 ng/mL) compared to
healthy controls (7.4–36.3 ng/mL). These results provide clinical support for LCN2
production in immune defense of the urinary tract (Paragas et al. 2014). Additionally,
using an Lcn2 global knockout mouse, Paragas et al. demonstrate that after transure-
thral UPEC inoculation, Lcn2−/− mice have higher bladder bacterial burden than
their littermates and take longer to clear the infection. Both wild-type and Lcn2−/−
mice were comparably infected with an Ent mutant UPEC strain, indicating that the
antimicrobial actions of Lcn2 on uropathogens require iron acquisition (Paragas
et al. 2014).
Using an Lcn2-Luc2-mCherry reporter mouse, this same research group demon-
strates that Lcn2 is constitutively produced by the thick ascending limbs of the loop
of Henle and the α-intercalated cells of the renal collecting tubule (Paragas et al.
2011, 2014). During experimental UTI, GFP-labeled UPEC bound the kidney’s
α-intercalated cells and induced Lcn2 expression in a TLR-4- and NF-κB-sensitive
fashion (Paragas et al. 2014; van Adelsberg et al. 1994; Xie et al. 2008).
4.9 Ribonuclease A Superfamily
Four lineages of the ribonuclease (RNase) A superfamily encode proteins associ-

ated with host defense: (1) angiogenins, (2) eosinophil RNases, (3) RNase 6, and (4)
RNase 7 and RNase 8 (Rosenberg 2008; Simanski et al. 2010; Becknell et al. 2015).
To date, RNase 6 and RNase 7 have been evaluated in the urinary tract and during
UTI. Both RNases 6 and 7 have potent antimicrobial activity against common
Gram-positive and Gram-negative uropathogens (Becknell et al. 2015; Spencer
et al. 2011b, 2013). The mechanisms accounting for the antimicrobial properties of
these RNase A proteins are not completely understood (Boix and Nogues 2007).
The bactericidal activity of RNase 7, for example, has been linked to its capacity to
permeate and disrupt the bacterial cell membrane, which is an action independent of
its ribonuclease activity (Huang et al. 2007; Harder and Schroder 2002). Moreover,
RNase 7 is highly cationic (isoelectric point 10.7), and the cationic charge is neces-
sary for antibacterial activity. Finally, distinct regions of the RNase 7 peptide appear
to be responsible for its antimicrobial activity against various pathogens (Huang
et al. 2007; Wang et al. 2013).
In the urinary tract, our research group has demonstrated that RNase 6 and RNase
7 expression differ. Using human biopsy samples and a murine model of experimen-
tal UTI, Becknell et al. demonstrate that RNase 6 expression localized to resident
urinary tract leukocytes or infiltrating granulocytes and macrophages in mouse cys-
titis bladders, mouse pyelonephritis kidneys, and human pyelonephritis kidneys.
RNase 6 was not routinely detected in noninfected human or mouse kidney and
bladder tissues. With UPEC infection, RNase 6 peptide production markedly
increased, likely representing the recruitment of RNase 6 expression leukocytes to
the infected kidney (Becknell et al. 2015).
In contrast, RNase 7 is an epithelial-derived AMP that is constitutively produced
by the uroepithelium of the lower urinary tract and the α- and β-intercalated cells of
the renal collecting tubule. When urinary RNase 7 is neutralized in human urine
specimens in vitro, urinary bacterial growth increases (Spencer et al. 2011b, 2014b).
During UTI, urine from children with UPEC infection contained about twice the
concentration of RNase 7 as the urine of noninfected controls. Similarly, human
kidney biopsies from patients with pyelonephritis demonstrate that tissue concen-
trations of RNase 7 protein are about twice what was measured in normal controls.
What distinguishes the expression of RNase 7 within the urinary tract from the other
previously described AMPs is the considerably greater amount secreted. Whereas
median urinary concentrations of cathelicidin and HBD1 are 1.6 × 10−5 μM and
2.5 × 10−5 μM, respectively, RNase 7 is present at around a 1000-fold greater con-
centrations (Spencer et al. 2011b, 2013; Zasloff 2013). These results suggest that
RNase 7 provides a front-line antimicrobial shield that permits uropathogenic
organisms from invading the urothelium (Zasloff 2013). If this barrier is breached
during microbial assault, other defenses like RNase 6 or other AMPs are activated
to combat infection.
4.10 Tamm-Horsfall Protein (THP)
THP or uromodulin is the most abundant protein in human urine, and mounting
evidence points to its role in maintaining urinary tract sterility. Mice deficient in
Thp (Thp−/−) exhibit delayed clearance of UPEC and higher UPEC recovery from
urine and bladders following experimental UTI (Bates et al. 2004). This was con-
firmed independently by Mo and colleagues, who further demonstrated that this
protective effect of Thp existed specifically toward type 1-fimbriated UPEC (Mo
et al. 2004). This may be explained by the observation that type 1-fimbriated UPEC
interacts with THP’s mannose residues and relies on these structures for attachment
to uroplakin plaques on bladder urothelium (Pak et al. 2001). While THP does not
possess bactericidal activity in vitro, it is capable of inhibiting bacterial attachment
to cultured kidney epithelial cells (Hawthorn et al. 1991).
4.11 AMPs as Therapeutics
The therapeutic potential of AMPs to prevent or treat UTI has been given added
incentive as antibiotic resistance toward uropathogenic bacteria has been increasing.
The use of AMPs does require some degree of caution as AMPs are powerful bio-
molecules that can elicit native cell death if applied in high concentration (Ali et al.
2009). The most straightforward method of AMP UTI therapy is direct application to
the source of infection (i.e., through bladder washes) or oral therapy. The use of oral
lactoferrin B effectively decreased infection and inflammation in the mouse urinary
tract after UPEC infection (Haversen et al. 2000). An alternative possibility for using
AMPs therapeutically would be to induce or augment their natural production using
homeopathic therapies like specific nutrients and vitamins. Treatment of human

bladder cell lines with 25-hydroxyvitamin D3 and 1,25-hydroxyvitamin D3 boosted
cathelicidin production and antibacterial activity against UPEC. Moreover, bladder
biopsy specimens from postmenopausal women treated with 25-hydroxyvitamin D3
rapidly show increased CAMP expression in response to UPEC infection compared
to biopsy specimens from women who did not receive vitamin D supplementation
(Hertting et al. 2010). Since urothelium and intercalated cells have emerged as the
chief parenchymal sources of AMPs, future efforts to induce AMP production should
focus on these cell populations. As some of the basic issues that control urinary tract
AMP expression and function are clarified, strategies and possible agents for pre-
venting and treating UTI can be developed to limit antibiotic overuse.
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Chapter 5
Antimicrobial Peptides in the Gut
Maureen J. Ostaff, Eduard F. Stange, and Jan Wehkamp
Abstract The gut represents a unique interface toward our environment. It not only
facilitates digestion and resorption but also battles ingested pathogens, while also
controlling an immense community of commensal microorganisms. To aid with the
latter, it produces a wide range of innate immune mediators, such as antimicrobial
peptides (AMPs), which can combat viruses, bacteria, and fungi. Gut AMPs have
differing activity ranges and modes of action, so their expression varies depending
on the present conditions and threats. The most famous examples for site-specific
AMPs are probably the two α-defensins HD5 and HD6. In a homeostatic state, they
are exclusive to the Paneth cells of the small intestine. Since the importance of gut
microbiota has become more and more evident, research on AMPs has also
increased. This is particularly obvious in the case of inflammatory bowel diseases,
but also noticeable in other disorders. Defects in the AMP machinery have been
linked to increased susceptibility to infections, chronic inflammation, and distur-
bances in commensal composition. Recently, even a role in colon cancer has been
proposed. The gut provides a complex and challenging environment for the study of
interactions between AMPs and microbes; and while we are now widely aware of
their crucial role in keeping us healthy, more research is needed to fully uncover the
involved multi-level crosstalk of their actions. Such investigations might one day
help us in fully understanding the mechanisms of various diseases. Even more, they
might aid in developing new anti-infectious, antiinflammatory, and maybe even
antitumorigenic drugs.
M.J. Ostaff
Dr. Margarete Fischer Bosch Institute of clinical pharmacology and University of Tübingen
E.F. Stange
Department of Gastroenterology, Robert Bosch Hospital, Stuttgart, Germany
J. Wehkamp (*)
Department of Internal Medicine I, University Hospital Tuebingen,
Otfried-Müller-Straße 10, Tuebingen 72076, Germany

DOI 10.1007/978-3-319-24199-9_5
68 M.J. Ostaff et al.
5.1 Introduction
The human gut has always been a fascinating topic for scientists and laypersons
alike. We tend to trust our proverbial gut instinct and sometimes wallow in hard to
digest life lessons. We all have experienced moments when our gut gravely interfered
with daily routines, whether it was an exam-related nervous stomach, a bad onset of
abdominal influenza, food poisoning, travelers’ diarrhea, or indigestion after enjoy-
ing food that was simply not good for us. A particularly recent advance in the field
involves an increasing awareness of the importance of commensal microbiota. While
early medical research focused primarily on pathogens, contemporary studies often
favor analyses on the role of regular microbes as drivers of gut health or disease.
Since AMPs are tightly involved in regulating the composition of these gut inhabit-
ants, studies on their expression and functional role have also experienced somewhat
of a renaissance. The following paragraphs will now aim at discussing this excep-
tional host microbe situation in the gut and the role of AMP in mediating health and
disease in this context.
5.2 The Exceptional Situation of the Gastrointestinal Tract

as a Host–Microbe Interface
Our gastrointestinal (GI) tract includes the mouth, esophagus, stomach, as well as
the small and large intestine ending with the rectum and anus. All these parts per-
form specific tasks aimed at an optimized uptake of nutrients, while the prevention
of inflammation and infection by food-borne and resident microbes has to be
ensured. The GI system consequently represents a particularly tightly controlled
host–microbe interface and is characterized by distinct expression patterns of vari-
ous antimicrobial peptides. In the mouth, bacteria can form a structurally and func-
tionally organized biofilm designated as dental plaque. While distinct interindividual
differences in the microbial composition among healthy individuals exist, within
one person, the oral microbiota can remain relatively stable over time. Thereby,
Firmicutes, Proteobacteria, Bacteroidetes, and Actinobacteria seem to be the most
abundant phyla (Bik et al. 2010). When however a shift in one of the critical param-
eters controlling this ecological homeostasis occurs, disease can follow. One of the
most prevalent oral diseases is caries, and clinical studies have associated it with an
increase in acid-forming or acid-tolerating bacteria, especially streptococci and
lactobacilli, which can demineralize enamel (Marsh 2006). The adjacent GI com-
ponent, the esophagus, forms an exception in the gut, as it is not meant to retain
food. However, it is nonetheless characterized by a distinct microbial community.
While it’s most abundant phyla, Firmicutes, Proteobacteria, Bacteroidetes, and
Actinobacteria, are shared with the oral cavity, and it also displays a similar level
of complexity, the esophagus also shows specific features, e.g., significantly higher
5 Antimicrobial Peptides in the Gut 69
amounts of Prevotella, whereas Neisseria appear less frequent (Fillon et al. 2012).
The stomach represents the first GI component which holds ingested food for some-
what longer periods of time. It should consequently provide sound conditions for
microbial growth. However, the high acidity in gastric juices generates a unique
ecological environment which limits bacterial survival. While early on it was
thought that the gastric acid kills indeed all entering bacteria, leaving it unsuitable
for colonization, later studies unveiled large numbers of acid-resistant bacteria,
stemming from the transient oral and food-borne microbiota, including Streptococci,
Neisseria, and Lactobacilli (Wang and Yang 2013). A 2006 study by Bik et al. had
already detected 128 different phylotypes among 8 bacterial phyla, dominated by
Firmicutes, Bacteroidetes, Actinobacteria, and Fusobacteria, suggesting a much
greater diversity within this ecosystem than previously described (Bik et al. 2006).
Different from the stomach, the presence and importance of colonizing microbiota
in the small and large intestines has been acknowledged early on. A role in, for
instance, the development of a proper intestinal morphology and function was
already accepted in the 1970s (Thompson and Trexler 1971) based on evidence
from different germ-free or gnotobiotic animal models. An influence of bacteria has
also been implicated in epithelial cell renewal rates (Heitman et al. 1980; Sun et al.
2004) and the development of Peyer’s patches (ileum-specific immune tissue) and
other gut-associated lymphoid tissue (Hooper and Gordon 2001). Intestinal bacteria
are involved in optimal food utilization, by degrading nutrients, aiding in digestion,
absorption, and vitamin synthesis while also inhibiting pathogen growth (Saulnier
et al. 2009; Hooper and Gordon 2001). In return, the intestine offers its commensals
an ideal habitat with constant temperatures and a never ending nutritional supply.
The generation of a beneficial microbial symbiosis is essential for the health in the
intestinal tract, but it can also influence peripheral bodily functions and even impact
on the brain (Sommer and Backhed 2013; Stilling et al. 2014). Most intestinal bac-
teria belong to the phyla Bacteroidetes or Firmicutes, but their numbers and com-
position vary locally and are in large parts dependent on the present local immune
defense strategies. While the ileum, for example, contains a somewhat fairly large
amount of aerobic and anaerobic bacteria such as Enterobacter and Lactobacilli,
the most densely populated intestinal region is found in the colon with numbers of
up to 1012 bacteria per gram of luminal content and predominately anaerobic repre-
sentatives such as Bacteroides, Bifidobacteria, Fusobacteria, Clostridium, and
Peptostreptococci (Sears 2005).
Many recent studies try to map and understand the relationship between the host
and its intestinal microbiota. Similar to what has been found in studies on oral plaque,
their composition is varying between individuals, especially when comparing groups
from different geographical locations, but it is also shifting among one individual,
depending on age (Yatsunenko et al. 2012). Additional environmental factors, such as
a specific nutritional lifestyle, or certain medications, can further impact on microbial
composition (David et al. 2014; Ji et al. 2014; Voreades et al. 2014). Due to its high
variability and dynamic, defining an “ideal gut microbiota” is challenging.
Nonetheless, many diseases, including cancer, obesity, autism, and, above all,
inflammatory bowel diseases (IBD), have been associated with more or less distinct
alterations (Uronis et al. 2009; Sartor 2014; Turnbaugh et al. 2009; Sekirov et al.
2010). The reliability of some of these associations seems however greatly dependent
on the effect scope, study design, sampling, and analysis (Hanage 2014). Changes in
the microbiota in IBD patients however show a high effect size with robust observa-
tions reported by several independent groups (Frank et al. 2011; Gophna et al. 2006;
Walker et al. 2011; Sartor 2014). While mostly a reduced overall diversity and an
increase in Proteobacteria with a parallel decrease in Firmicutes are seen, more stud-
ies are indispensable until microbiota might be considered a possible diagnostic tool
or a promising therapeutic target in the treatment of this disorder (Matsuoka and
Kanai 2015).
In general, even in health, the GI tract is constantly challenged by a remarkable
and diverse community of commensals. It is therefore surprising that infections or
bacterial translocations are rather rare and limited to highly pathogenic strains or
predisposing disease states. This is in big parts to the effective generation of a highly
competent arsenal of antimicrobial peptides (AMP) with activity against bacteria,
viruses, protozoa, and fungi, reflecting the different threats and challenges met at GI
mucosae. Most of these AMP, such as β-defensins, are thereby generated by gut
epithelial cells themselves, but some are also contributed by specialized resident
immune cells.
Many members of the gastrointestinal AMP arsenal are at least in part dependent
on so-called pattern recognition receptors (PRR) for either their transcriptional
induction or their secretion. PRRs include, among others, the outer membrane Toll-
like receptors (TLRs) and the intracellular NOD-like receptors (NLR) and detect
highly conserved microbial structures, called pathogen- (or microbial)-associated
molecular pattern (PAMP or MAMP). Upon binding their ligand, PRRs can activate
innate immune reactions, which often occur via MyD88 and NF-κB or MAP kinases
(Abreu et al. 2005). These events then lead to a proinflammatory response but also
the release of stored AMP and/or their de novo synthesis (Takeda and Akira 2004;
Bevins 2003). A relatively low expression of some TLRs in the gut, as well as a
basolateral location upon epithelial cells of others, allows an effective recognition
of invading pathogens, but also tolerance toward commensals without generating
constant inflammatory conditions (Abreu 2010; Cario 2010). The following chap-
ters will take a closer look on the different AMPs found in the gut and how their
expression and function is related to different gastrointestinal disorders.
5.3 AMP in the Upper Gastrointestinal Tract
The upper gastrointestinal tract, for all intents and purposes, includes the esopha-
gus, the stomach, and the duodenum. However, in the framework of this chapter,
when speaking of the upper GI tract, it is referred to the mouth, esophagus, and
stomach. The duodenum is included below when discussing the situation present in
the small intestine.
Starting with the oral cavity, major AMPs, which control the microbial environ-
ment in this compartment, include human β-defensins (hBDs), histatins, the cathe-
licidin LL-37, the antifungal and antibacterial lactoferrin, as well as lysozyme. As
a whole, they not only limit an overgrowth of commensal microorganisms but also
prevent colonization by pathogens (Melino et al. 2014; van’t Hof et al. 2014; Hans
and Madaan 2014). While the β-defensin hBD-4 has not been reported to be
expressed in oral epithelial cells, the constitutively expressed hBD-1, as well as
the inducible hBD-2 and hBD-3, can be found in oral mucosa, gingiva, and tongue
epithelia as well as in salivary glands (Hans and Madaan 2014). The latter two
hBDs are thereby upregulated during infection, as it can occur in gingivitis and
periodontitis, and are also involved in response to regular plaque-forming bacteria.
A recent study by Wang et al. could, for instance, show that the bacteria found in
oral biofilms from healthy individuals have the potential to induce antibacterial or
immunomodulatory reactions in oral epithelial cells during early stages of bacte-
ria–host interactions in a distinct species-dependent way. This includes the asso-
ciation of a transcriptional upregulation of hBD-2 and hBD-3 with an abundance
of Prevotella and the absence of species of the Streptococcus cluster (Wang et al.
2015). One mechanism by which oral bacteria have been reported to mediate an
upregulation of inducible β-defensins is based on cytokine-mediated pathway acti-
vation, e.g., via IL-17 signaling, which has an important role in defending the oral
cavity against Candida albicans infections (Trautwein-Weidner et al. 2015). C.
albicans can cause oral candidiasis in immune-deficient patients, as it is, for
instance, the case in human immunodeficiency virus (HIV) carriers (Samaranayake
et al. 2009). Aside from a functional involvement of β-defensins, which has been
demonstrated in mouse models (Tomalka et al. 2015), lysozyme and lactoferrin
can also play an important role in limiting oral C. albicans growth (Samaranayake
et al. 2001). Similar reports also exist for histatins, which are secreted by the
parotid and submandibular salivary glands and are found in saliva in high concen-
trations. Their antimicrobial function is based on multiple modes of actions includ-
ing the induction of a non-lytic ATP loss from actively respiring cells, the
disruption of the cell, as well as the generation of reactive oxygen species, which
can all lead to cell death (Kavanagh and Dowd 2004). Interestingly, C. albicans
however does not necessarily submit to oral AMPs without a fight. Several resis-
tance mechanisms have been reported which can deter antifungal activities. One is
the secretion of a Msb2 glycoprotein, that can function as a broad-range anti-AMP
protectant; another is the use of efflux transporters to shuttle AMP out of the cell,
while an adaptive modification of the fungal membrane can also convey resistance
against membrane-interacting AMPs such as hBD-2 (Swidergall and Ernst 2014).
In addition to oral manifestations, Candida infections can also target the
esophagus.
Here again, a role for β-defensins, in particular the inducible hBD-2 and hBD-3,
in response to an autocrine IL-1β loop and an activation of the epidermal growth
factor receptor (EGFR) by endogenous transforming growth factor-α (TGF-α) have
been reported (Pahl et al. 2011). Their upregulation might thereby help in protecting
the host against invasive candidiasis by limiting the infection to the mucosal surface
(Kiehne et al. 2005). An interesting observation regarding esophageal antimicrobial

defense was made by our group in 2008: Whereas expression levels of inducible
hBDs as well as the antiproteases elafin and psoriasin were much higher in the
esophagus than in the stomach or duodenum, cationic tissue extracts from this loca-
tion exhibited a comparably weaker killing activity against C. albicans (Hosaka
et al. 2008). This suggests that certain AMPs in the stomach mediate potent antifun-
gal properties and that these might be lacking in the esophagus.
As mentioned earlier, due to its low pH, the stomach represents an unusual
niche. To protect itself from the high acidity in the lumen, the stomach epithelium
generates a two-layered mucus system. This acts as a diffusion barrier for hydro-
chloric acid with surface epithelial cells secreting bicarbonate, creating a pH gradi-
ent toward the lumen (Johansson et al. 2013). However, one particularly well-studied
gastric pathogen, the gastritis-causing Helicobacter pylori, uses this to its advan-
tage. It has the ability to achieve motility by reducing viscoelasticity of mucins (the
glycoproteins making up the mucus barrier), allowing it to reach the higher pH
region at the epithelial surface (Celli et al. 2009). This interesting bacterium can
additionally also sidestep the host’s epithelial antimicrobial line of defense. Host
AMPs exert a major influence on shaping microbial communities. It is therefore
not surprising that the ability to evade them provided certain pathogenic strains
with a selective evolutionary advantage (Gruenheid and Le 2012; Koprivnjak and
Peschel 2011). H. pylori in particular utilizes host cholesterol to change its mem-
brane makeup gaining resistance against LL-37 (McGee et al. 2011), and, while
inducing hBD-2, it seemingly avoids the upregulation of hBD-3 (Bauer et al.
2013). The latter, while having the potential to kill H. pylori effectively, can conse-
quently not help in eradicating it from gastric epithelia, while hBD-2, which is
readily induced, only shows minor activity against the pathogen (Nuding et al.
2013).
5.4 Small Intestinal AMP
Regarding its antimicrobial peptide defense, the small intestine holds an exceptional
position. This metabolically most active GI compartment is characterized by a
largely puckered surface which contains invaginations, called crypts of Lieberkühn,
and protrusions, known as villi; both are thereby crucial in enlarging the mucosal
surface to optimize nutrient resorption but also offer up a particularly large host–
microbe interface. While the villi are more leaflike in the duodenum, they appear in
fingerlike structures in the ileum (Helander and Fandriks 2014). It is important to
acknowledge that the bacterial density rises steadily from the proximal duodenum,
over the jejunum in the middle, to the distal ileum. This is accompanied by an
increasing number of Paneth cells toward the ileum. Paneth cells are important
secretory specialists and specific to the small intestine, where they are found at the
bottom of crypts. They generate a wide arsenal of antimicrobial factors making
them crucial players in the regulation of gut microbiota while also mediating defense
against pathogens (Clevers and Bevins 2013). Expressing the human α-defensins
HD5 and HD6, as well as lysozyme, the lectin Reg3γ and phospholipase A2 group
IIA (sPLA2, also known as PLA2G2A), among others, the Paneth cell represents
the main supplier of small intestinal AMP. To a smaller extent, other small intestinal
epithelial cells deliver additional mediators like hBD-1, hBD-2, hBD-3, elafin, and
LL-37.
Paneth cells store AMPs in vesicles and secrete them mainly upon bacterial
stimulation, allowing enteric defensin concentrations of up to 15–100 mg/ml in the
crypt of mice models (Ayabe et al. 2000; Selsted and Ouellette 2005) or 50–250 μg/
ml of HD5 alone, in human studies (Ghosh et al. 2002). This outreaches an effec-
tive antimicrobial concentration by far and ensures a sterile crypt microenviron-
ment (Ericksen et al. 2005; Ouellette and Bevins 2001). The highly conserved
Paneth cell α-defensin genes include two exons and are produced as prepro-defen-
sins, meaning they initially include a signaling sequence and a pro-segment, which
is promptly separated. The still inactive propeptide is stored in granules and acti-
vated upon release by targeted cleavage (Bevins and Salzman 2011). With respect
to their genetic organization, HD5 and HD6 exhibit a relatively similar 5′- putative
promoter region, sharing several similar transcription factor binding sites, includ-
ing multiple Wnt response elements (Beisner et al. 2014). This might explain the
almost parallel regulation of HD5 and HD6 in many setups. It was indeed shown
by several studies that the Wnt pathway can directly impact on HD5 and HD6
expression, while it is interestingly also directly involved in Paneth cell maturation
(Ostaff et al. 2013; Andreu et al. 2008). Even though they are transcriptionally
regulated in a similar way, the mature Paneth cell α-defensin peptides differ greatly
in their antimicrobial function. HD5 is a very potent defense mediator with high
activity at low concentrations against several bacteria, fungi, and viruses (Bevins
and Salzman 2011). It furthermore possesses chemotactic properties (Grigat et al.
2007). Using transgenic HD5 mice, a direct influence on the composition of intes-
tinal microbiota and a direct effect against invading Salmonella could be demon-
strated (Salzman et al. 2003, 2010). Less is however known about its mode of
action. It was shown to form dimers, and, depending on its amphipathic structure,
it likely kills bacteria by disrupting their cell wall (Clevers and Bevins 2013).
Moreover, HD5 is able to inactivate bacterial toxins, probably by their unfolding,
making them susceptible to proteolysis (Kudryashova et al. 2014). The second
enteric α-defensin, HD6, has not yet been as rigorously studied. Although it was
shown to exhibit antiviral properties (Doss et al. 2009), its antimicrobial activity
has long been evasive. Quite recently, Bevins and his colleagues discovered that
HD6 can form nanonets in vivo, which actively trap bacteria (Chu et al. 2012), a
strategy similar to what has been reported on neutrophil defensins (Brinkmann and
Zychlinsky 2007). Furthermore, it was shown that under reducing conditions – as
present in the gut – HD6 also possesses antimicrobial properties aimed at commen-
sals (Schroeder et al. 2015).
The study of mice models allowed the elucidation of essential functions of
Paneth cells in fending of commensal but also pathogenic threats. A lack of
Paneth cell α-defensin (termed cryptdins in mice)-activating enzyme promotes a
high susceptibility to orally administered pathogens (Wilson et al. 1999), while

an induction of an extensive release of AMPs by Paneth cell degranulation via
TLR9 stimulation has, for instance, been shown to conversely protect mice
against Salmonella (Rumio et al. 2004). In addition to TLR9, another important
link between a pattern recognition receptor and Paneth cells lies in the function
of the intracellular NOD2 receptor, which recognizes bacterial muramyl dipep-
tide. NOD2 is specifically expressed in crypts, mainly in Paneth cells, but not in
small intestinal villus structures (Lala et al. 2003). NOD2 knockout mice have
been reported to display an impaired cryptdin expression (Kobayashi et al. 2005),
but these effects have recently been under discussion (Shanahan et al. 2014).
Another group suggested a secondary mechanism via a negative crosstalk
between NOD2 and other PRRs, resulting in an overreaction of TLRs when
NOD2 is not functional (Amendola et al. 2014). Even newer work by Tan et al.
however suggests that NOD2 has a dual role in Paneth cells, depending on their
differentiation state. Their work showed that while NOD2 itself can slightly
upregulate expression of enteric α-defensins mainly via NF-κB activation, it can
also strongly downregulate their levels during Paneth cell lineage differentiation
in large parts by inhibiting the MAPK pathway (Tan et al. 2015a, b).
Interestingly, a person’s nutritional status and lifestyle might play a crucial role
in intestinal antimicrobial defense. Malnutrition, for example, which is linked to
diarrhea and inflammation, has the potential to increase intestinal permeability and
translocation of bacteria. It has furthermore been associated with differences in
AMP expression and a dysbiosis of resident microbiota (Hashimoto et al. 2012).
Paneth cells seem to be particularly affected by a lack of nutrition, as they show
reduced AMP levels and granule aberrations in a starvation mouse model (Hodin
et al. 2011). A link between Paneth cell AMPs and intestinal barrier effectiveness
became evident in a rat model of liver cirrhosis. Translocation of common intestinal
microbiota has in this case been related to lower epithelial α-defensin levels in the
respective animals (Teltschik et al. 2012). Another study utilizing a mouse model of
graft-versus-host disease (GVHD) also linked diminished Paneth cell antimicrobi-
als to an increase of normally rare septicemia causing E. coli in expense of symbi-
otic diversity (Eriguchi et al. 2012).
Even though in health, Paneth cells are restricted to the small bowel, in disease,
they can arise at other gastrointestinal locations, an observation which is hypothe-
sized to be an “on-demand” defense mechanism, changing the pattern of local
antimicrobial immunity. Their occurrence is often linked to the presence of intesti-
nal metaplasia, which can be found in the esophagus (Barrett’s esophagus) and in
the distal stomach during gastritis. While Barrett’s esophagus develops most likely
in response to longstanding exposure to gastric acid and bile salts, gastric intestinal
metaplasia has been associated with H. pylori colonization (Shen et al. 2005).
Paneth cells can furthermore arise in colonic tissue during intense mucosal
challenges as it can, for instance, be seen in chronic inflammation such as Crohn’s
disease or ulcerative colitis (Wehkamp et al. 2002), which will later be discussed in
detail.
5.5 Colonic AMP
Related to microbes, the large intestine represents the most densely populated
region in the human body. It is therefore not surprising that it possesses a thick,
complex mucus barrier formed by a high amount of resident goblet cells that helps
to shield its epithelium. Different from a somewhat thin and much looser layer
found in the small intestine, it is made up by a dual system. The two colonic mucus
layers encompass an adherent film as well as a mobile coating, which is continu-
ously shuffled distally, ensuring optimized passage of luminal content, potential
harmful substances, but also microorganisms which might colonize it. Of note, the
inner colon mucus layer is thereby rapidly renewed and converted into the outer one
by a host controlled endogenous proteolytic process (Johansson et al. 2011). The
glycoprotein Muc2 is the most important protein component of these layers, and a
genetic ablation of this mucin leads to a spontaneous and chronic inflammation in
mice which resembles ulcerative colitis in humans (Wenzel et al. 2014). An impor-
tant function of colonic mucus is the retention of AMPs, which ensures comparably
high concentrations of these important innate immune mediators in close proximity
to the epithelial surface (Antoni et al. 2013).
Colonic epithelia lack the villus structures found in the small intestine, but crypts
are present, which normally do not sport Paneth cells. Hence, a multitude of non-
Paneth cell epithelial antimicrobials play a front role in ensuring a tight control of
colonic commensals. Among these are LL-37, elafin, as well as another antiprote-
ase, the secretory leukocyte protease inhibitor SLPI, and ß-defensins. β-defensins,
while sharing common properties, such as most AMPs (positive charge, disulfide
bonds) (Boman 2003), exhibit vast differences regarding their antimicrobial effec-
tiveness and in their modes of action. They share this apparent paradox with HD5
and HD6, which, as discussed above, also display quite differing functions. A strik-
ing example within the family of ß-defensins is provided by hBD-1. hBD-1 has
previously shown to exhibit only weak antimicrobial activity as compared to hBD-2
or others. The reason for this perceived lack of function became evident in the work
of Schroeder et al. Thereby, a quite strong activity against various commensal gut
bacteria and the facultative pathogenic fungus C. albicans only emerged after a
biochemical activation in a reducing environment, as it is, for instance, present in
the colon (Schroeder et al. 2011). In addition, a physiological activation via the
thioredoxin system has also been demonstrated using the colonic epithelial cell line
Caco-2 (Jaeger et al. 2013).
The important role of colonic AMPs has been demonstrated in mouse models.
An example for a direct link between antimicrobial function and colonic health
could be demonstrated in mice lacking cathelicidin (mCRAMP in mice, LL-37 in
humans), which is normally upregulated during colitis, but also in ileitis, depending
on the level of inflammation (Kubler et al. 2009; Schauber et al. 2006; Koon et al.
2011). The cathelicidin knockout model strikingly showed more severe symptoms
and mucosal disruption in a dextran sodium sulfate (DSS) chemically induced coli-
tis approach (Koon et al. 2011). Interestingly, the different abnormalities in this
DSS-challenged knockout model, including an increase in cytokine production and

apoptotic cells, as well as an impaired mucus production, were reversed by intrarec-
tal administration or gene therapy with mCRAMP (Tai et al. 2013). Evidence for a
likely protective role of AMPs in humans comes from studies using probiotic bac-
teria, which have been demonstrated to lead to a potent induction of hBD-2 in vitro
using Caco-2 cells, as well as in vivo, utilizing stool samples from healthy volun-
teers before and after probiotic therapy (Mondel et al. 2008; Schlee et al. 2007,
2008). This boosting effect on epithelial antimicrobial defense might be one reason
why these health-promoting bacteria have been successfully used in remission
maintenance in the chronic inflammatory disorder ulcerative colitis.
A quite new view on the role of AMPs and gut health has emerged with some
recent studies, suggesting that hBDs are expressed by and have certain functions in
cancer. Recent work by Uraki et al. on hBD-3 could show that, while colonic cancer
cells do not express this AMP, the production of hBD-3 by tumor-infiltrating mono-
cytes can have potent antitumor effects. hBD-3 can significantly inhibit the migra-
tion of colon cancer cells, with one mechanism including a downregulation of the
metastasis-associated 1 family, member 2 (MTA2) (Uraki et al. 2014). Interestingly,
colorectal cancer (CRC) has also been linked to a dysbiosis of gut microbiota in
fecal samples (Sobhani et al. 2011; Ohigashi et al. 2013). An additional specific
approach in six CRC patients could furthermore elucidate how tumor tissue itself
harbors a distinct microbiome with strikingly different features from adjacent non-
malignant tissue (Marchesi et al. 2011). Colorectal cancer progression is generally
coupled to a deregulation of different signals and pathways, among others, an aug-
mentation of canonical Wnt (Goel and Boland 2010). Proinflammatory cytokines
have been shown to enhance Wnt in this setting, and patients with chronic colonic
inflammation have been shown to bear an increased risk for CRC development
(Saleh and Trinchieri 2011). An impact of microbiota in this context has also been
investigated. In azoxymethane (AOM), chemically induced colonic inflammation of
conventional, Bacteroides vulgatus mono-associated, and germ-free IL-10(−/−) and
MyD88(−/−) mice, Uronis et al. could show that the risk for colitis-associated can-
cer seems to be TLR/MyD88 dependent. It was further shown that the cancer risk
can be altered by manipulation of intestinal microbes (Uronis et al. 2009). In a simi-
lar approach, Arthur et al. could demonstrate that specific bacterial abilities hold
cancer-promoting effects and argued that colitis may foster the expansion of
microbes with such characteristics (Arthur et al. 2012). Since AMPs are critical in
controlling the enteric microbiota, they might well have a role in bacteria-driven
cancer development. Unfortunately this hypothesis has yet to be tested. Functions of
AMPs related to orchestrating innate and adaptive immune responses have however
already been studied in this context. In mice it could be shown that mBD-14 (the
mouse ortholog of hBD-3) recruits macrophages which can indirectly promote
tumorigenesis by inducing proinflammatory cytokines (Rohrl et al. 2012a, b).
However, together with the abovementioned antimetastatic role of hBD-3 and addi-
tional in vitro and mice model results showing antiinflammatory influences of
hBD-3 on monocytes and macrophages (Semple et al. 2010, 2011), its role in
inflammation-promoted cancer seems to be complex. If and how the manipulation
of AMP expression or a direct alteration of gut microbiota might be a therapeutic

strategy in colon cancer, it provides nonetheless interesting questions for future
research.
5.6 Functional Role of AMP in Inflammatory Bowel Diseases
The most well-known associations between antimicrobial peptides and gastrointes-

tinal disease can be found in multiple subgroups of inflammatory bowel diseases
(IBD), recurring and severe inflammations in the intestinal tract. Indicators like a
distorted epithelial architecture, lymphocyte and plasma cell infiltrate, polymorpho-
nuclear cryptitis, crypt abscesses, and basal lymphoid aggregates are used to distin-
guish IBD from regular intestinal inflammations (Le et al. 1995). The most common
forms of IBD are ulcerative colitis (UC) and Crohn’s disease (CD). Whereas the
former is typically restricted to the rectum and colon, spreading out continuously
from the rectum in a proximal direction, CD can occur at many sites, predominantly
in the ileocecal zone, the terminal ileum, and colon. The cardinal symptom of UC,
bloody, slimy diarrhea, may be associated with abdominal pain and tenesmus.
Clinical symptoms of CD are characterized as crampy pain and diarrhea, mostly
without blood. As both diseases progress, intestinal complications may arise. In
CD, fistulas, abscesses, stenosis, and strictures are the most common complications,
whereas in UC, bleeding, toxic megacolon, perforations, and the inflammation-
related rise of colon carcinoma are the most prevalent problems. Both diseases may
also be accompanied by high temperature and weight loss as well as extraintestinal
complications, potentially affecting the skin, eyes, joints, liver, and bile ducts. Even
though many pathologic features might differ in UC and CD, about 5 % of cases
cannot be finally diagnosed and are classified as “indeterminate” colitis (IC) (Odze
2003).
A variety of factors of environmental, genetic, immunological, and microbial
nature contributes to the development of inflammatory bowel diseases.
Epidemiological investigations showed an increased incidence in families and an
especially high concordance rate among monozygotic twins, uncovering a stronger
genetic influence in CD and somewhat less of a role, but a notable leverage in UC
(Ellinghaus et al. 2015). The importance of environmental factors becomes evident in
a continuously rising prevalence level, especially since the beginning of the last cen-
tury. The rise in IBD specifically affects developed nations and has been attributed to
a high standard of hygiene, new dietary habits, and the industrialization of food pro-
duction and preservation (Dixon et al. 2015; Ananthakrishnan 2015). Due to this
complex and multifactorial character, current pathogenesis concepts try to incorpo-
rate a balanced role of both, environment and genetics. One well-established concept
is based on the presence of diminished epithelial defenses in genetically predisposed
individuals and a microbial dysbiosis. The latter can thereby in part be a consequence
of the former, but is also largely influenced by the environment. This dual pathogen-
esis aspect highlights a loss of the balanced and beneficial host–microbe relationship
as it is normally found in a healthy gut (Fig. 5.1), as the main disease driver (Ostaff
et al. 2013). Intestinal epithelia of IBD patients consequently display mucosal adher-
ent bacteria, activated T cells, and antibodies aimed not toward pathogens but regular
gut inhabitants (MacPherson et al. 1996; Sartor 2001; Duchmann et al. 1995).
Regarding to genetics, the intracellular PRR NOD2, also known as CARD15, a
receptor for muramyl dipeptide, a minimal bioactive peptidoglycan motif common to
all bacteria, represents the foremost CD susceptibility factor (Hugot et al. 2001;
Ogura et al. 2001; Girardin et al. 2003). Its association with the disease was the first
major genetic mechanism underlining the importance of innate defenses and a crucial
role of bacteria–host interactions. NOD2 mutations were later specifically associated
with the clinical phenotype of small intestinal (or ileal) Crohn’s disease. Since NOD2
is mainly expressed in Paneth cells (Lala et al. 2003) and, as discussed above,
involved in Paneth cell-mediated small intestinal antimicrobial defense, this location-
specific association appears plausible.
The pathogenesis of small intestinal (or more specific ileal) CD is, to a substan-
tial extent, based on defects in the Paneth cell. When comparing ileal CD patients to
healthy individuals, but also patients with colonic CD or UC, both Paneth cell
α-defensins show reduced expression levels. In contrast, other Paneth cell products,
such as lysozyme and sPLA2, seem to be unchanged (Wehkamp et al. 2005). The
specific reduction of Paneth cell HD5 and HD6 appears to be particularly marked in
CD patients with a NOD2 frameshift mutation, while other NOD2 SNPs seem to be
inconsequential in this regard (Bevins et al. 2009; Wehkamp et al. 2004, 2005).
Fig. 5.1 Balanced

host–microbe interactions
at the epithelial gut barrier
are characterized by an
effective generation of
site-specific AMPs and a
respective adequate mucus
layer
Another functional single nucleotide polymorphism (SNP) has also been associated
with particularly low Paneth cell defensin levels. It is present in the Wnt coreceptor
LRP6, which showed a genetic association with early-onset ileal CD (Koslowski
et al. 2012). The intensified decrease in both cases, NOD2 and LRP6, has been
found to be independent from current inflammation. Additional mechanisms under-
lying the reduced α-defensin expression also affect the Wnt pathway. Patients with
ileal CD show diminished mRNA expression of the transcription factor TCF-4, the
Wnt coreceptor LRP6, and also the Wnt pathway transcription factor TCF1
(Koslowski et al. 2012; Beisner et al. 2014; Wehkamp et al. 2007). While also an
SNP in the TCF4 promoter showed an association with ileal CD (Koslowski et al.
2009), a causal relationship between Wnt/TCF-4 and α-defensin levels could be
underlined in a knockout mouse model. In these animals, a 50 % reduction of TCF-4
mRNA promoted a significant drop of Paneth cell cryptdins and subsequent dimin-
ished antibacterial activity (Wehkamp et al. 2007). Aside from controlling the
expression of various Paneth cell genes, among them HD5 and HD6, Wnt also
plays, as mentioned, an essential role in the maturation of these specialized secre-
tory cells, while also governing the proliferation of intestinal epithelia. An involve-
ment of important Wnt pathway components in ileal CD supports the idea that a
disrupted Paneth cell differentiation and a disturbed epithelial regenerative homeo-
stasis might potentially be relevant in disease pathogenesis. Aside from NOD2,
TCF-4, and LRP6, the autophagy factor ATG16L1 (autophagy-related protein 16-1)
is another genetically associated factor with an important role for Paneth cell func-
tion. ATG16L1 is involved in cellular recycling processes which are important for
cell and tissue homeostasis. In a genome-wide association study, an SNP in
ATG16L1 was associated with CD and preceded changes in Paneth cell granules in
homozygous carriers. Similar aberrations were thereby also present in ATG16L1
hypomorphic mice (Cadwell et al. 2008). An additional study in pediatric patients
furthermore found activation of autophagy in Paneth cells, irrespective of inflamma-
tion and independent of the disease-associated variants (Thachil et al. 2012). A
deregulated autophagy might therefore be an additional and general characteristic in
ileal CD with a particular impact on Paneth cell biology.
In small intestinal CD, the involvement of Paneth cell defects clearly highlights
a role for antimicrobial defense defects in patients. The colonic manifestation of
the disease also shows distinct AMP-related aberrations, such as an attenuated
induction of hBD-2, as well as an inflammation-related decrease in hBD-1,
although the respective mechanisms are generally less well understood. While in
the subgroup of UC, colonic expression levels of hBD-2 are readily induced on a
high extent during inflammation, CD patients show a much lower upregulation of
this important defense mediator in active disease (Wehkamp et al. 2003). This has
originally been linked to lower copy numbers of the hBD-2 gene (Fellermann et al.
2006), but multiple reports could not confirm or rather contradicted this association
(Bentley et al. 2009; Aldhous et al. 2009). However, based on functional studies,
investigating the antimicrobial killing activity of mucosal protein extracts against
E. coli, Nuding et al. could clearly show that, while antibacterial activity was
majorly enhanced in UC, colonic CD patients have a severely inhibited killing
capacity against this gut bacterium (Nuding et al. 2007). Additional inducible
AMPs associated with a lower inducibility in CD are the cathelicidin LL-37 and
the antimicrobial serine protease inhibitors elafin and SLPI (secretory leukocyte
protease inhibitor). The constitutively expressed hBD-1 on the other hand shows
reduced levels in the colon of IBD in general, which is however especially pro-
nounced in colonic CD (Peyrin-Biroulet et al. 2010). Interestingly, this might partly
be explained by a functional promoter SNP that has previously been associated
with protection from colonic CD in a European cohort (Peyrin-Biroulet et al. 2010).
Furthermore, thioredoxin, an enzyme involved in the redox activation of hBD-1,
also exhibits a reduction in active colonic CD, but this can however also be seen in
UC patients (Jaeger et al. 2013).
While as mentioned, general mucosal production of antimicrobial factors seems
to be enhanced in UC (Nuding et al. 2007), patients are nonetheless characterized
by a diminished epithelial barrier protection. This is related to distinct aberrations
in the mucus layer. As previously discussed, one important function of colonic
mucus is the retention of AMPs, keeping them close to the mucosa to ensure that
commensal bacteria cannot attach to and/or translocate over the intestinal barrier.
Specific detrimental changes of the mucus structure, regarding a reduction in thick-
ness and continuity, have been repeatedly described in UC patients (Johansson et al.
2013). It is easy to imagine how such aberrations can considerably weaken the
mucosal barrier. Acute inflamed areas have even been described to be sometimes
completely depleted of mucus. In contrast, patients with CD show a comparable or
even thicker mucus layer than what is seen in healthy controls. Additional changes
in UC patients related to mucus promote a simplification of mucin glycosylation
patterns, potentially allowing it to be easily degradable by bacterial enzymes and
also include alterations in the content of phosphatidylcholine and lysophosphatidyl-
choline (Antoni et al. 2014). Whether such changes are primary or secondary, with
regard to the presence of inflammation, has not yet been definitively determined.
While some studies have shown an inflammation dependency of mucus-related
alterations in active versus non-active UC, genetic links also highlight that the
observed effects are likely not an exclusive result of continuous inflammatory pro-
cesses. A higher susceptibility for UC was, for instance, found in carriers of rare
alleles in the MUC3 gene (Kyo et al. 1999), and an association of MUC4 and
MUC13 variants with ulcerative colitis has also been described (Moehle et al.
2006). It was furthermore demonstrated that UC patients show an impairment in the
inducibility of certain secretory and especially goblet cell differentiation factors
when compared with active CD. A significant inflammation-related upregulation of
the secretory differentiation factors Hath1 and the goblet cell differentiation factor
KLF4, as it can be seen in active CD, is absent in ulcerative colitis (Gersemann et al.
2009).
Taken together, in UC a diminished mucus layer provides a less effective reser-
voir for AMPs and a reduced protection against bacterial invasion, while in CD,
distinct impairments related to the production of AMPs disturb the host–microbe
balance at the epithelial barrier (Fig. 5.2). The underlying mechanisms of IBD-
related barrier defects are various and very complex. We are certainly only begin-
Fig. 5.2 An impaired barrier, as it, for instance, occurs when AMP expression is diminished, and
a dysbiosis of commensals can lead to an imbalanced host–microbe interaction. This can promote
the activation of ongoing and over-shooting inflammatory immune responses, which characterize
inflammatory bowel diseases
ning to understand all the detailed aspects, so the study of interactions between the
different factors and their control, as well as environmental influences, is sure to
provide long-ranging potential for scientific research. A better understanding of the
mucosal barrier, and in particular the role of AMPs, will however be the key to open
up new therapeutic avenues.
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Chapter 6
Metal Sequestration: An Important
Contribution of Antimicrobial Peptides
to Nutritional Immunity
Steven Damo and Thomas E. Kehl-Fie
Abstract First-row transition elements are essential for all forms of life. During
infection invading microbes must obtain these nutrients from their host. Vertebrates
take advantage of this fact to combat invaders by sequestering essential nutrients, a
defense known as nutritional immunity. The most well-characterized aspect of this
defense is the iron-withholding response. Advances in elemental imaging have
revealed that zinc and manganese are also sequestered during infection. The impor-
tance of nutritional immunity to host defense is emphasized by the increased sus-
ceptibility to infection when levels of these metals are elevated. This chapter will
discuss iron, zinc, and manganese availability during infection, the impact of with-
holding these metals from invading pathogens, and the antimicrobial peptides uti-
lized by the host to restrict the availability of these essential nutrients.
6.1 Introduction
Transition elements such as iron (Fe), zinc (Zn), and manganese (Mn) are essential
for all forms of life. This essentiality stems from their numerous structural and cata-
lytic roles. The importance of these metals to life is further emphasized by analyses
of protein databases, which suggest that 30 % of all proteins utilize a metal cofactor
(Andreini et al. 2008). Fe is highly versatile and is utilized by bacteria in numerous
ways including in mononuclear enzymes, such as ribonucleotide reductase and
superoxide dismutase; in Fe-S-containing enzymes, such as succinate dehydroge-
nase; and in heme-containing enzymes, such as cytochrome oxidase (Andreini et al.
2008; Py and Barras 2010; Mayfield et al. 2011). In bacteria, 4–6 % of all proteins
S. Damo
Department of Life and Physical Sciences, Fisk University, Nashville, TN, USA
T.E. Kehl-Fie, PhD ()
Department of Microbiology, University of Illinois Urbana-Champaign,
601 South Goodwin Avenue, MC-110, Urbana, IL 61801, USA

DOI 10.1007/978-3-319-24199-9_6
90 S. Damo and T.E. Kehl-Fie
are predicted to utilize Zn either catalytically, as in alcohol dehydrogenases, lyases,

hydrolases, and Cu/Zn superoxide dismutases, or structurally, as in the Fur family
of transcriptional regulators (Andreini et al. 2006; Vallee and Auld 1990; Waldron
and Robinson 2009). Mn contributes to numerous processes and can serve as a
cofactor for glycolytic enzymes, such as phosphoglycerate mutase and pyruvate
kinase; signaling proteins, such as ppGpp synthetases; and superoxide dismutases
(Kehres and Maguire 2003; Papp-Wallace and Maguire 2006).
During infection bacteria must acquire all of their nutrients from the host.
Vertebrates take advantage of this fact by restricting the availability of essential transi-
tion elements. This host defense, known as nutritional immunity, is critical for defend-
ing against numerous pathogens (Hood and Skaar 2012; Weinberg 1974, 2009). The
most well-characterized aspect of nutritional immunity is Fe restriction, but it has
become apparent that the host also limits access to Zn and Mn to combat invaders
(Kehl-Fie and Skaar 2010; Hood and Skaar 2012). This chapter will discuss the avail-
ability of essential transition metals during infection, the impact of withholding these
metals on invading pathogens, and the host proteins produced by immune cells, which
are utilized to restrict access to these essential nutrients during infection.
6.2 Nutrient Metal Availability During Infection
6.2.1 Iron Availability
While Fe is the most abundant transition element on earth, in vertebrates, free Fe is

effectively nonexistent. The scarcity of Fe is driven by several factors (Cassat and
Skaar 2013). First, Fe is highly compartmentalized, with the vast majority of this
metal located in red blood cells in the form of heme bound to hemoglobin. Second,
any released Fe is rapidly bound by transferrin. In response to infection, Fe avail-
ability is further restricted by the host. This further reduction in availability is driven
by reduced intestinal Fe absorption, increased production of transferrin, and release
of lactoferrin from neutrophil granules at sites of infection (Fig. 6.1). Additionally,
phagocytes express natural resistance-associated macrophage protein 1 (NRAMP1),
which removes Fe and Mn from the phagolysosome (Papp-Wallace and Maguire
2006). The importance of restricting Fe is seen in the increased ability of pathogens
to replicate in Fe overloaded tissues and the increased susceptibility to infection of
people suffering from Fe overload, whether due to environmental or genetic factors
(Weinberg 1974, 2009).
In an attempt to obtain Fe during infection, pathogens express a myriad of differ-
ent importers (Wandersman and Delepelaire 2004; Cassat and Skaar 2013). While
the specific repertoire varies between pathogens, almost all bacteria express some
form of Fe uptake system, which contributes to virulence. These systems include Fe
transporters as well as heme and hemoglobin uptake systems. These latter systems
allow bacteria to acquire Fe following the lysis of red blood cells. To counter these
systems, extracellular hemoglobin and heme are rapidly scavenged by haptoglobin
6 Antimicrobial Peptides and Nutritional Immunity 91
Zn Zn
Epithelial Cells
Fe
Granules Fe
Fe
Fe
Fe Virulence Factors
Cytoplasm Zn
Zn Essential Processes
Mn
Zn
Neutrophils Pathogens
Inhibited
S100A7 S100A15
Processes
Calprotectin Transferrin
S100A12 Lactoferrin
Fig. 6.1 Antimicrobial peptides sequester iron, zinc, and manganese during infection
and hemopexin, respectively. Bacteria also express receptors that allow them to
utilize host Fe scavenging proteins including transferrin, haptoglobin, and hemo-
pexin directly as nutrient sources. To acquire Fe, bacteria also produce siderophores,
which have a higher affinity for Fe than transferrin. More recently it has become
apparent that the host produces proteins that bind bacterial siderophores, such as
lipocalin-2, preventing them from facilitating bacterial Fe acquisition. Lipocalin-2
is capable of binding Enterobactin and other similar siderophores (Goetz et al.
2002). An eight-stranded beta barrel protein, lipocalin-2, binds siderophores with
subnanomolar affinity using a highly specific binding site comprised of lysine and
arginine residues that interact tightly with the negatively charged siderophores
(Goetz et al. 2002). To counter this defense, pathogens express “stealth sidero-
phores,” which cannot be bound by lipocalin-2 (Abergel et al. 2006; Raffatellu et al.
2009). The numerous strategies employed by host and pathogen reinforce the impact
that wining the struggle for nutrient metals can have on the outcome of infection.
6.2.2 Zinc Availability
While Fe sequestration is the most well-characterized nutrient-withholding

response, it has become apparent that Zn and Mn (discussed below) are also with-
held from invading pathogens (Corbin et al. 2008; Kehl-Fie et al. 2013). However,
unlike Fe whose availability is always restricted, Zn and Mn appear to be specifi-

cally limited in response to infection. This realization has been largely driven by the
application of advanced elemental imaging technologies, such as laser ablation
inductively coupled plasma mass spectrometry (LA-ICP-MS), to the study of infec-
tion (Corbin et al. 2008; Kehl-Fie et al. 2013). LA-ICP-MS produces a two-
dimensional image of metal distribution within a tissue. The prototypic example of
Zn restriction is the staphylococcal abscess (Corbin et al. 2008; Kehl-Fie et al.
2013). Utilization of LA-ICP-MS to investigate metal distribution during S. aureus
infection revealed that the staphylococcal abscess is rendered virtually devoid of
Zn, while the surrounding healthy tissue is metal replete. While the abscess is Zn
deplete, total Zn levels in the organ remain unchanged (Kehl-Fie et al. 2013).
Currently, the host factors responsible for sequestering Zn during infection are
unknown. However, potential candidates include a subset of the S100 family of
proteins (discussed below), such as calprotectin (CP), a heterodimer of S100A8 and
S100A9 (also known as calgranulin A and B, Mrp8 and Mrp14, L1, and the
CF-antigen), S100A12 (calgranulin C), and S100A7 (psoriasin), which are capable
of binding Zn with high affinity (Kehl-Fie et al. 2011; Brophy et al. 2012; Moroz
et al. 2011) (Fig. 6.1). While the specific expression profile of these proteins varies,
they can be produced by epithelial cells and neutrophils. Furthermore, they are fre-
quently found at high concentrations at sites of infection and can inhibit bacterial
growth by sequestering Zn (Kehl-Fie and Skaar 2010; Lee and Eckert 2007; Gläser
et al. 2005). Supporting the idea that the host is a Zn-limited environment, loss of
Zn uptake systems in Campylobacter jejuni, Salmonella enterica, Haemophilus
ducreyi, uropathogenic E. coli, Brucella abortus, Streptococcus pyogenes,
Acinetobacter baumannii, and Yersinia pestis results in reduced bacterial virulence
(Davis et al. 2009; Ammendola et al. 2007; Lewis et al. 1999; Sabri et al. 2009; Kim
et al. 2004; Weston et al. 2009; Campoy et al. 2002; Hood et al. 2012; Bobrov et al.
2014). Notably, in an oral gastric model of Salmonella typhimurium infection, Zn
sequestration appears to benefit the pathogen due to the expression of Zn importers,
which allow Salmonella to outcompete the microbiota for Zn (Liu et al. 2012).
6.2.3 Manganese Availability
The observation that NRAMP1 removes Mn from the phagolysosome and contrib-
utes to controlling infection provided some of the first evidence that restricting Mn
availability contributes to host defense (Papp-Wallace and Maguire 2006). The use
of LA-ICP-MS has significantly expanded our knowledge regarding the scope of
Mn sequestration during infection. Similar to Zn, LA-ICP-MS revealed that the
staphylococcal abscess, but not the surrounding tissue, is rendered devoid of Mn
(Corbin et al. 2008; Kehl-Fie et al. 2013). The localized Mn sequestration does not
appear to impact total Mn levels in the organ (Kehl-Fie et al. 2013). Further investi-
gation revealed that calprotectin (CP) contributes to this restriction, as CP-deficient
mice do not remove Mn from staphylococcal liver abscesses (Corbin et al. 2008)
(Fig. 6.1). In addition to binding Zn, CP also binds Mn tightly, and the ability to
sequester this metal contributes to the antimicrobial activity of the protein (Damo
et al. 2013; Kehl-Fie et al. 2011). Mice that lack CP are more susceptible to bacterial
and fungal pathogens, including S. aureus, A. baumannii, Klebsiella pneumoniae,
and Candida albicans (Corbin et al. 2008; Kehl-Fie et al. 2013; Hood et al. 2012;
Achouiti et al. 2012). CP is not the only host protein that contributes to Mn seques-
tration, as CP-deficient mice still remove Mn from staphylococcal kidney abscesses
(Kehl-Fie et al. 2013). Similar to Fe and Zn, loss of Mn uptake systems in S.
typhimurium, B. abortus, Y. pestis, S. aureus, Streptococcus pneumoniae, and S.
pyogenes results in reduced virulence (Dintilhac et al. 1997; Berry and Paton 1996;
Anderson et al. 2009; Bearden and Perry 1999; Horsburgh et al. 2002; Janulczyk
et al. 2003; Sun et al. 2009; Kehl-Fie et al. 2013; Janakiraman and Slauch 2000).
The importance of sequestering Mn is highlighted by the observation that a staphy-
lococcal strain lacking Mn importers has a virulence defect in wild-type mice but is
able to grow as well as the parental strain in the livers of CP-deficient mice (Kehl-
Fie et al. 2013).
6.3 The Impact of Metal Limitation
Host-imposed metal limitation can impact the host-pathogen interaction in two

ways. First, metal limitation causes directed changes in gene expression by invad-
ers, which are intended to minimize the impact of nutrient limitation. Second, metal
starvation can inactivate bacterial metalloenzymes disrupting essential metabolic
pathways and processes. It should be noted that while the impact of metal starvation
is likely to have some common aspects, the specific response and processes dis-
rupted are likely to be as diverse as the pathogens that infect the human host. The
directed bacterial response is frequently controlled by metal-responsive transcrip-
tion factors, such as Fur (Fe), Zur (Zn), and MntR (Mn), which directly sense intra-
cellular metal availability (Lee and Helmann 2007). Generally, these regulators
repress expression when their cognate metal is available but not when it is limiting.
These transcription factors frequently regulate the expression of metal uptake sys-
tems and metal-independent isozymes. They can also activate metal-sparing
responses, as observed by Fur regulation of the small RNA RhyB in E. coli.
Expression of RhyB leads to reduced expression of nonessential Fe-dependent pro-
cesses increasing Fe availability for essential Fe-dependent processes (Lee and
Helmann 2007). Metal limitation often indicates interaction with a host, and in
some bacteria, metal-responsive regulators also regulate the expression of virulence
factors (Troxell and Hassan 2013).
In addition to the directed changes, host-imposed metal limitation can also inac-
tivate metal-dependent processes. While the specific processes that are inhibited by
nutritional immunity are largely unknown, the increased susceptibility to infection
when metals are not sequestered indicates that pathogens experience metal starva-
tion during infection. This idea is supported by studies using wild-type and
CP-deficient mice, which found that host-imposed Mn starvation inhibits

Mn-dependent staphylococcal superoxide dismutase activity during infection
(Damo et al. 2013; Kehl-Fie et al. 2011). The inhibition of SOD activity in turn
renders S. aureus more sensitive to neutrophil-mediated killing (Kehl-Fie et al.
2011). These results indicate that host-imposed metal starvation not only reduces
bacterial growth but can also enhance the efficacy of the immune response by inhib-
iting virulence factors. While the impact of nutritional immunity on invading patho-
gens is still being elucidated, it is clear that withholding Fe, Zn, and Mn is a critical
weapon utilized by the host.
6.4 Antimicrobial Peptides Involved in Metal Sequestration
6.4.1 Lactoferrin
Lactoferrin is a member of the transferrin family of Fe-binding proteins. Transferrin

transports Fe within blood and other bodily secretions and contributes to the consti-
tutive restriction of this metal. Lactoferrin is present within the granules of poly-
morphonuclear leukocytes where it acts as a crucial component of the innate
immune response to infection (Orsi 2004). Additionally, lactoferrin can be found in
breast milk, tears, and saliva. Similar to other members of the transferrin family,
lactoferrin is an ~80 kDa protein comprised of two domains, an N-terminal domain
(N lobe) and a C-terminal domain (C lobe). In lactoferrin, the two lobes are con-
nected via a short helical stretch. This differs from transferrin where this linker is
unstructured. The N lobe and C lobe share ~40 % sequence identity with each other
resulting in two homologous halves (Wally and Buchanan 2007). Each lobe is com-
prised of two alpha/beta fold subdomains with the metal-binding site located at a
deep pocket between the subdomains. Hence, each lactoferrin molecule can bind
two Fe atoms. The ligands responsible for metal chelation are conserved throughout
the transferrin family and in lactoferrin are Y92, Y192, D60, and H253 (Mizutani
et al. 2012). Additionally, two oxygen ligands are provided by CO32−, which is syn-
ergistically bound with Fe. Fe binding is tightly regulated and coordinated through
conformational changes in lactoferrin (Baker and Baker 2004). In the apo state,
lactoferrin forms an open structure, with the alpha/beta subdomains rotated as rigid
bodies between 20° and 50° away from each other (Anderson et al. 1990). Upon Fe
binding, the alpha/beta subdomains of each lobe close forming a much more com-
pact structure. These conformational dynamics are critical for the controlled bind-
ing and release of Fe. It is suggested that CO32− ion binds first to the open
conformation (Baker and Baker 2004). This creates a four coordinate Fe-binding
site together with the two tyrosines (Khan et al. 2001). Once Fe is bound, conforma-
tional sampling will select for the closed state and allow D60 and H253 to act as
ligands and complete the full coordination site. This mechanism allows for the high-
affinity (Kd ~ 10−20 M) hexadentate coordination of Fe, while also permitting revers-
ibility (Baker and Baker 2004).
6.4.2 S100 Proteins
The S100 subclass of EF hand family of calcium (Ca)-binding proteins are small,
acidic, alpha helical proteins that are found in vertebrates. Their expression is tissue
specific, and their regulation has been correlated with a wide range of diseases
including autoimmune disorders, cancer, and neurological disorders (Heizmann
et al. 2002). S100 proteins are approximately 100 amino acids long and typically
form homodimers arranged in an antiparallel fashion. Each subunit contains two EF
hand Ca-binding motifs connected by a hinge region.
The first crystal structure of a Zn-bound S100 protein was determined for
S100A7 (Brodersen et al. 1999). This structure revealed the presence of two identi-
cal Zn-binding sites located at opposite ends of the dimer interface containing three
histidines and one aspartic acid. In the S100A7 dimer, each binding site is com-
prised of H86 and H90, arranged in an HXXXH motif from one subunit and H17
and D24 from the other. These binding sites are distinct from the Ca-binding sites,
which are characteristic of S100 proteins. While the binding sites are distinct, the
presence of either Ca or a transition metal increases the affinity for the other (Brophy
et al. 2012; Hayden et al. 2013; Moroz et al. 2011). This three His Asp site is the
canonical transition metal-binding site in S100 proteins. Zn-bound structures have
also been determined for S100B, S100A12, and S100A15 and suggest that the
canonical binding site is largely conserved; however, there is some variation (Murray
et al. 2012; Ostendorp et al. 2011; Moroz et al. 2009, 2011). For example, while
S100A15 shares 93 % sequence identity with S100A7, originally it was thought to
not bind Zn, as the conserved aspartate in the canonical binding site has been
replaced by glycine. However, the crystal structure of Zn-bound S100A15 reveals a
unique binding motif where a chloride ion acts as the fourth ligand, replacing the
conserved aspartate (Murray et al. 2012).
Of the S100 proteins, the metal-binding properties of CP have received consider-
able attention. This interest is due to the demonstrated contribution of CP to host
defense and the sequestration of Mn during infection (Corbin et al. 2008; Kehl-Fie
et al. 2013; Hood et al. 2012; Achouiti et al. 2012). CP comprises ~50 % of the total
protein in the neutrophil cytoplasm and can be expressed by epithelial cells when
induced by proinflammatory cytokines such as IL-22 (Blaschitz and Raffatellu 2010;
Gebhardt et al. 2006). At sites of infection, CP concentration can be in excess of 1 mg/
ml (Clohessy and Golden 1995). CP is capable of binding two Zn ions with picomolar
affinity (Kd) but only one Mn ion with nanomolar or subnanomolar affinity (Kehl-Fie
et al. 2011; Brophy et al. 2012). Consistent with CP imposing metal starvation, the
addition of excess Mn and Zn or mutation of both transition metal-binding sites elimi-
nates the antimicrobial activity of CP (Corbin et al. 2008; Kehl-Fie et al. 2011).
Due to the heterodimeric nature of CP, this protein has two nonidentical transi-
tion metal-binding sites. The first site (S1) was originally predicted to be comprised
of residues H17 and H27 from S100A8 and H91 and H95 from S100A9, with a
fourth histidine replacing the aspartate found in the canonical S100 transition metal-
binding site (Kehl-Fie et al. 2011; Korndorfer et al. 2007). However, subsequent
investigations revealed that two additional histidines, H103 and H105 from S100A9,
also contribute to metal binding (Damo et al. 2013). The second site (S2) is com-
prised of H83 and H87 of S100A8 and H20 and D30 from S100A9 and is effectively
identical to the canonical site found in other S100 proteins (Kehl-Fie et al. 2011;
Korndorfer et al. 2007). Site 1 binds both Mn and Zn tightly, while S2 only binds Zn
tightly (Damo et al. 2013; Brophy et al. 2013). A high-resolution crystal structure
of Mn-bound CP revealed that H103 and H105 from a C-terminal extension of
S100A9 also contribute to Mn binding (Damo et al. 2013). The six histidines in S1
form an almost perfect octahedral coordination site, with the C-terminal extension
wrapping around the Mn ion. Subsequent solution studies confirmed the near-
perfect octahedral geometry and the importance of H103 and H105 to Mn binding
(Brophy et al. 2013). While loss of the two histidines disrupts the ability of S1 to
bind Mn tightly, it appears to have negligible impact on Zn binding (Damo et al.
2013). The C-terminal extension is unique to S100A9 among the S100 proteins and
provides an explanation for why none of the other S100 proteins assayed to date
bind Mn (Damo et al. 2013; Brophy et al. 2013). Similar to other S100 proteins,
there appears to be communication between the EF hands and the transition metal-
binding sites, as the presence of Ca increases the affinity of CP for Zn and Mn. In
the absence of Ca, CP has negligible affinity for Mn, and Zn binding is substantially
weaker (Brophy et al. 2012; Hayden et al. 2013). This altered affinity has been pro-
posed to serve as a switch, allowing CP to accumulate to high levels in the Ca-poor
cytosol of neutrophils without negatively impacting the cell but bind Mn and Zn
tightly when released into the Ca-rich extracellular space.
6.5 Conclusions
As invading pathogens must obtain essential nutrients from their host, nutritional
immunity is a powerful host defense mechanism. While canonically associated with
the Fe-withholding response, the application of advanced elemental analysis has
revealed that other essential metals, such as Mn and Zn, are also withheld from
invaders. The expansion of nutritional immunity to include Mn and Zn leads to
more questions. What are the host factors that restrict the availability of these metals
during infection? What are the bacterial processes that are disrupted by nutritional
immunity? How do successful pathogens overcome this host defense, allowing
them to cause disease? Answering these questions will aid in the development of
therapeutics that are intended to augment the efficacy of nutritional immunity.
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Chapter 7
Regulation of Antimicrobial Peptide Gene
Expression by Vitamin D
Adrian F. Gombart
Abstract In the mid-2000s, several investigators discovered that antimicrobial

peptide (AMP) gene expression was regulated by the vitamin D pathway. This rev-
elation provided a potential explanation for the ability of vitamin D to enhance the
antimicrobial activity of immune cells like macrophages that had been observed in
the 1980s. Further, this finding provided a mechanism that could explain the
observed importance of vitamin D in maintaining the epithelial barrier defenses of
the skin and gut. As reviewed in this chapter, an abundance of in vitro evidence
demonstrates the regulation of AMPs by vitamin D. Nevertheless, there is a lack of
in vivo data that demonstrates just how this regulation plays a role in the immune
response against infection. This is due, in part, to the current lack of a viable animal
model as the regulation of antimicrobial peptide gene expression occurs only in
humans and nonhuman primates. Generation of an appropriate animal model and/or
carefully designed human and primate studies should provide a clearer picture of
the role that this pathway plays in barrier function and the immune response.
7.1 Introduction
A decade ago three groups simultaneously discovered the regulation of human anti-
microbial peptide gene expression by vitamin D. In screening the human genome
for vitamin D response elements (VDREs), White and colleagues identified poten-
tial VDREs in the cathelicidin antimicrobial peptide (CAMP) and β-defensin 4
(DEFB4) genes. They demonstrated induction of these two genes by treatment of
isolated human keratinocytes, monocytes and neutrophils, and human cell lines
with 1α,25-dihydroxyvitaminD3 [1,25(OH)2D3] concomitant with increased secre-
tion of bactericidal activity from treated cells (Wang et al. 2004). Ståhle and
A.F. Gombart, PhD

Department of Biochemistry and Biophysics, Linus Pauling Institute, Oregon State
University, 307 Linus Pauling Science Center, Corvallis, OR 97331, USA

DOI 10.1007/978-3-319-24199-9_7
102 A.F. Gombart
colleagues demonstrated induction of CAMP in human keratinocytes in vitro by

1,25(OH)2D3 and in vivo after topical application of the 1,25(OH)2D3 analog calci-
potriol to the skin of human volunteers (Weber et al. 2005). Our group discovered
induction of CAMP by 1,25(OH)2D3 in acute myeloid leukemia (AML), immortal-
ized keratinocytes, and colon cancer cell lines as well as normal human bone mar-
row (BM)-derived macrophages and fresh BM cells from two normal individuals
and one acute myelogenous leukemia patient (Gombart et al. 2005). Each group
demonstrated the requirement of a VDRE at about 700 base pairs upstream of the
transcription start site in the CAMP gene by site-directed mutagenesis or deletion
(Wang et al. 2004; Weber et al. 2005; Gombart et al. 2005). In the DEFB4 gene
promoter, the VDRE is located about 1200 base pairs upstream of the transcription
start site (Wang et al. 2004).
Interestingly, we observed a lack of this regulation in the mouse and discovered
from a comparison of various mammalian genomes an evolutionarily conserved
VDRE in a short interspersed nuclear element (SINE) in the CAMP promoter of
primates that was absent in other mammalian genomes (Gombart et al. 2005).
Further, we demonstrated that the VDRE in the CAMP gene originated from the
exaptation of an AluSx SINE in the lineage leading to humans, apes, Old World
monkeys, and New World monkeys and remained under purifying selection for the
last 55–60 million years (Gombart et al. 2009a). Taken together, these findings
revealed a novel activity of 1,25(OH)2D3 and the vitamin D receptor (VDR) in regu-
lation of primate innate immunity due to an evolutionarily fixed, Alu-mediated
divergence in steroid hormone nuclear receptor gene regulation between humans/
primates and other mammals (Gombart et al. 2009a). Regulation of a murine homo-
log for DEFB4 by vitamin D has not been described.
In a series of subsequent in vitro experiments, Modlin and colleagues demon-
strated that vitamin D was required for the induction of CAMP by Toll-like receptor
(TLR) signaling, and findings indicated that insufficient serum 25-hydroxyvitamin
D [25(OH)D] levels could lead to a lack of CAMP gene expression by macrophages
in response to infection (Liu et al. 2006). In the current model, TLR signaling
induces CYP27B1 and VDR expression. CYP27B1 activity hydroxylates 25(OH)D
resulting in the production of the active 1,25(OH)2D that binds to the VDR and thus
induces target genes including CAMP (Liu et al. 2006).
Activation of the vitamin D pathway by TLR signaling was identified in lung and
skin epithelial cells, but was not observed in cell lines derived from the colon
(Hansdottir et al. 2008; Schauber et al. 2007; Lagishetty et al. 2010). Wounding of
the skin enhanced TLR2 function which enabled keratinocytes to respond to
pathogen-associated molecular patterns, activate the vitamin D pathway, and
increase CAMP levels in the skin to protect against infection (Schauber et al. 2007).
Interestingly, the induction of CAMP gene expression by 1,25(OH)2D3 was
observed in keratinocytes and monocytes, but not in epithelial cells of the colon
(Schauber et al. 2006). In contrast butyrate induced CAMP in colonic cells, but not
significantly in keratinocytes and monocytes suggesting that responses to these
stimuli were cell type and microenvironment specific (Schauber et al. 2006). Several
studies have demonstrated that butyrate and other histone deacetylase inhibitors
7 Regulation of Antimicrobial Peptide Gene Expression by Vitamin D 103
together with 1,25(OH)2D3 cooperatively or synergistically induce CAMP gene

expression in monocytes/macrophages, keratinocytes, and lung and colon cells
(Gombart et al. 2007; Schauber et al. 2008; Mily et al. 2013; van der Does et al.
2014; Kulkarni et al. 2014).
7.2 The CAMP Gene
A majority of the interest in vitamin D-mediated regulation of antimicrobial peptide

(AMP) gene expression has focused on induction of the CAMP gene because it is
more robustly upregulated as compared to DEFB4 (Wang et al. 2004). The CAMP
gene encodes an 18-kDa proprotein called hCAP18. It is processed to release a
peptide called LL-37 that is expressed by neutrophils and macrophages for killing
bacteria and by epithelial cells in barrier defense (Lehrer and Ganz 2002; Gombart
2009; White 2010). In addition, the LL-37 peptide can chemoattract T cells, den-
dritic cells, neutrophils, and monocytes (Chertov et al. 1996; Yang et al. 2001),
which could allow LL-37 to influence cellular traffic at sites of infection or inflam-
mation. Also, LL-37 affects dendritic cell activation and subsequent priming of T
cells when added exogenously (Davidson et al. 2004), demonstrating that adaptive
immune responses may be regulated by LL-37. The hCAP18 protein is present in γδ
T cells, B cells, monocytes, and NK cells of the peripheral blood (Agerberth et al.
2000) following a general hierarchy of protein expression with neutrophils showing
the highest levels, monocytes intermediate levels, and lymphocytes the lowest levels
(Lowry et al. 2014). In the lymphocyte population, B cells, NK cells, CD4+ T cells,
and CD8+ T cells all have similar levels of hCAP18 expression (Lowry et al. 2014).
CAMP is secreted by tissues exposed to the environment and in saliva and seminal
fluid (Frohm Nilsson et al. 1999; Murakami et al. 2002; Malm et al. 2000). It is criti-
cal for host barrier defense as mice lacking it are susceptible to infection (Gombart
2009; Nizet et al. 2001; Chromek et al. 2006).
7.3 The Vitamin D Pathway
The most effective way to acquire vitamin D is through synthesis in the skin or
consumption of a purified supplement as diet, which is a poor source (Holick 2011).
Ultraviolet B rays provided by natural or artificial sunlight cleave the B-ring of
7-dehydrocholesterol in the skin to produce cholecalciferol or vitamin D3. This is
absorbed into the blood and hydroxylated in the liver by the cytochrome p450
enzyme CYP27A1 to calcidiol or 25(OH)D3. This form (together with 25(OH)D2;
see below) is measured in the serum as an indicator of vitamin D status (Holick
2011). 25(OH)D3 is converted to its bioactive form, calcitriol or 1,25(OH)2D3, by
the mitochondrial 1α-hydroxylase enzyme CYP27B1 in the kidney. A fungal-
derived form of vitamin D is created by the UVB exposure of ergosterol to generate
104 A.F. Gombart
ergocalciferol. This form of vitamin D is hydroxylated in the liver to 25(OH)D2 and

in the kidney to 1,25(OH)2D2 (Holick 2011). Both 1,25(OH)2D3 and 1,25(OH)2D2
bind to the VDR a steroid hormone nuclear receptor/transcription factor that binds
to VDREs and recruits cofactors to activate and/or repress the expression of target
genes (Mangelsdorf et al. 1995; Christakos et al. 1996).
Synthesis of 1,25(OH)2D in the kidney is essential for efficient uptake of dietary
calcium in the gut and to maintain bone health. A drop in circulating Ca2+ levels
stimulates the production of parathyroid hormone (PTH) which induces CYP27B1
expression by primary renal tubules. Increased 1,25(OH)2D production activates
Ca2+ transporter expression via the VDR in the small intestine, thereby increasing
circulating Ca2+ and suppressing PTH production (Holick 2011). In a negative feed-
back loop, activated VDR binds to the CYP27B1 promoter and represses its expres-
sion. Also, VDR induces fibroblast growth factor-23 in osteocytes which inhibits
secretion of PTH and represses CYP27B1 expression and induces expression of
CYP24A1, a mitochondrial enzyme that catabolizes both 1,25(OH)2D and 25(OH)
D to limit 1,25(OH)2D levels and prevent hypercalcemia (Paz et al. 2007; Saito et al.
2003; Zierold et al. 1995).
Abundant epidemiological, clinical, and basic research has implicated vitamin D
in preventing cancer, autoimmune disorders, cardiovascular disease, and infections
(Grober et al. 2013). The synthesis of 1,25(OH)2D in nonrenal tissues and cells
likely mediates these additional health benefits (Hewison et al. 2004). The extrare-
nal synthesis of 1,25(OH)2D occurs in lung, colon, parathyroid glands, bone, skin,
and macrophages and is considered important for optimal immune response at sites
of infection (Hewison et al. 2004).
7.4 Vitamin D and Immunity
The role of vitamin D in regulating the adaptive immune response is highlighted by

numerous lines of evidence. The VDR is expressed in T and B cells, monocytes,
macrophages, dendritic cells (DCs), and neutrophils (Provvedini et al. 1983; Bhalla
et al. 1983; Deluca and Cantorna 2001; Adorini et al. 2004; Kreutz et al. 1993;
Brennan et al. 1987; Takahashi et al. 2002; Mangelsdorf et al. 1984). 1,25(OH)2D3
inhibits Th17 development, increases the frequency of Th2 and regulatory T cells,
decreases Th1 development, and modulates T-cell proliferation and cytokine expres-
sion (Lemire et al. 1995; Boonstra et al. 2001; Penna and Adorini 2000; Daniel et al.
2008). 1,25(OH)2D3 also promotes tolerance in dendritic cells and T cells and inhib-
its B-cell differentiation into plasma cells (Adorini et al. 2004; Mathieu and Adorini
2002; Chen et al. 2007). Overall vitamin D mediates an antiinflammatory response
and promotes tolerance in the adaptive response.
In addition to responding to circulating 1,25(OH)2D3, dendritic cells, macro-
phages, and T cells can actively produce it (Hewison 2012). Initially, extrarenal
production of 1,25(OH)2D3 by macrophages from some granulomatous disease
patients was reported (Barbour et al. 1981; Adams et al. 1983). In vitro studies with
normal macrophages indicated that CYP27B1 activity was induced as part of the
immune response (Koeffler et al. 1985; Reichel et al. 1986). DCs confer specific
homing properties upon T cells during the adaptive immune response, and DCs
derived from the skin are able to synthesize 1,25(OH)2D3 from vitamin D3. This, in
turn, induces expression of CC chemokine receptor 10 in T cells and suppresses
expression of gut-homing receptors which enable T cells to migrate toward the che-
mokine CCL27 that is secreted by epidermal keratinocytes. These findings demon-
strate that DCs produce locally high levels of 1,25(OH)2D3 to regulate T-cell
epidermal tropism (Sigmundsdottir et al. 2007).
The production of potentially high local levels of 1,25(OH)2D3 is most likely
important for intracrine and paracrine influences on the interactions between vita-
min D, the immune system, and pathogens (Hewison 2012). During the mid-1980s,
it was demonstrated that both 25(OH)D3 and 1,25(OH)2D3 increased the capacity of
human monocytes to control Mycobacterium tuberculosis (Mtb) growth (Davies
1985; Rook et al. 1986). Nearly 20 years later, as described above, we and others
discovered that vitamin D increased expression of the CAMP gene (Wang et al.
2004; Weber et al. 2005; Gombart et al. 2005). In addition, the human β-defensin 2
or DEFB4 gene was identified as a vitamin D inducible antimicrobial peptide gene,
but its induction by vitamin D or TLR activation is much less robust than CAMP
(Wang et al. 2004; Liu et al. 2006). These observations offered a mechanism by
which vitamin D could directly enhance killing of Mtb.
7.5 Cooperative Induction of Antimicrobial Peptide Gene

Expression by Multiple Signaling Pathways
Robust induction of DEFB4 by vitamin D requires activation of additional signaling

pathways. Co-treatment of monocytes with IL-1 and 1,25(OH)2D3 induced binding
of both NF-κB and VDR to the DEFB4 promoter and was much more effective in
inducing gene expression (Wang et al. 2004; Liu et al. 2009). Also, in the presence
of muramyl dipeptide (MDP), the intracellular pattern recognition receptor
nucleotide-binding oligomerization domain protein 2 (NOD2) activates NF-κB, and
there is a modest induction of the DEFB4 gene (Voss et al. 2006; Wang et al. 2010);
however, treatment with 1,25(OH)2D3 prior to addition of MDP strongly induces the
DEFB4 gene (Wang et al. 2010). It was shown that 1,25(OH)2D3 strongly induced
expression of NOD2 in primary human monocytic and epithelial cells which ampli-
fied the MDP signal (Wang et al. 2010). In total, studies have shown that the vitamin
D pathway alone is insufficient to induce robust expression of DEFB4, and activa-
tion of additional signaling pathways is required (Liu et al. 2009; Wang et al. 2010).
Several published studies have demonstrated that cytokine expression also mod-
ulates vitamin D-mediated CAMP and DEFB4 expression. In human macrophages,
TLR2/TLR1 signaling induces IL-15 expression which increases IL-32 which is
essential for induction of CYP27B1 and the VDR (Krutzik et al. 2008; Montoya
et al. 2014). The subsequent increased conversion of 25(OH)D3 to 1,25(OH)2D3 by
106 A.F. Gombart
CYP27B1 activates the VDR and induces CAMP expression and antimicrobial
activity against Mtb (Krutzik et al. 2008; Montoya et al. 2014). In human mono-
cytes, Th1 cytokine IFN-γ upregulates TLR2/TLR1 induction of CYP27B1 and the
bioconversion of 25(OH)D3 to 1,25(OH)2D3 which enhances induction of CAMP
(Edfeldt et al. 2010). Further, vitamin D is required for IFN-γ-mediated activity of
human macrophages (Fabri et al. 2011). On the other hand, the Th2 cytokine IL-4
induces CYP24A1 expression which leads to the catabolism of 25(OH)D3 and
downregulation of CAMP expression (Edfeldt et al. 2010). In contrast, the Th2 cyto-
kine IL-13 enhances CAMP expression by 25(OH)D3 due to increased CYP27B1
expression and synthesis of 1,25(OH)2D3 (Schrumpf et al. 2012). No effect is
observed with IL-17 in monocytes, but in the presence of 1,25(OH)2D3, IL-17
enhances CAMP expression in human keratinocytes via activation of the Act1 and
MEK/ERK pathway (Peric et al. 2008). In addition to IL-4, other cytokines can
inhibit antimicrobial peptide (AMP) expression. In macrophages, IFN-γ-induced
vitamin D-dependent AMP expression was suppressed by IFN-β and IL-10 (Teles
et al. 2013). Similarly, in placental cells, IL-10 inhibited β-defensin and CAMP
expression, while 1,25(OH)2D3 treatment could override the suppression (Olmos-
Ortiz et al. 2015). Further, TNF-α and 1,25(OH)2D3 enhanced β-defensin, and
TNF-α reduced both basal and 1,25(OH)2D3-induced CAMP expression (Olmos-
Ortiz et al. 2015). Taken together, the differential effect of T-cell cytokines on
CAMP and DEFB4 expression represents mechanisms by which adaptive immune
responses can regulate innate immune antimicrobial peptide defenses against patho-
gens. It remains to be determined how these various signaling pathways work
together in vivo during infection.
7.6 Fighting Infection Through Increased AMP Expression
Historically, sources of vitamin D were used as treatments for tuberculosis

(Martineau et al. 2007). In the 1940s, physicians effectively treated cutaneous Mtb
infection with high-dose vitamin D2, but this fell out of favor with the advent of
effective antibiotics (Martineau et al. 2007; Dowling 1946; Gaumond 1948). In the
1980s, epidemiological studies pointed to a correlation between higher rates of
tuberculosis and vitamin D deficiency (Davies 1985). Further, 1,25(OH)2D3 was
shown to enhance intracellular killing by human monocytic cells (Rook 1986).
Knockdown of either DEFB4 or CAMP expression in monocytes/macrophages
decreased killing of Mtb indicating their importance for fighting infection (Liu et al.
2009). The induction of CAMP by vitamin D is required for promoting autophagy
to kill Mtb (Hoyer-Hansen et al. 2005; Wang et al. 2008; Yuk et al. 2009). Additional
findings support a paracrine macrophage-lung epithelial cell signaling pathway that
is driven by IL-1β and 1,25(OH)2D3 (Verway et al. 2013). In this model, 1,25(OH)2D3
increased IL-1β secretion in Mtb-infected macrophages. The secreted IL-1β induced
DEFB4 expression from airway epithelial cells which enhanced control of Mtb
growth in co-cultured macrophages in vitro (Verway et al. 2013). Taken together
these studies support an important role for vitamin D in modulating the immune
response to Mtb infection.
These findings have renewed interest in potentially using vitamin D to treat
tuberculosis. A review of clinical trials and case series indicates that numerous stud-
ies are methodologically flawed (Martineau et al. 2007) or an insufficient vitamin D
dose was used (Wejse et al. 2009). Two small randomized studies indicate some
benefit from vitamin supplementation of TB patients (Nursyam et al. 2006; Morcos
et al. 1998). More recently, pulmonary tuberculosis patients receiving standard ther-
apy and a 100,000 IU dose of vitamin D3 every 2 weeks showed accelerated sputum
conversion if they possessed the tt genotype of the vitamin D receptor as compared
with placebo (Martineau et al. 2011). Further, vitamin D supplementation acceler-
ated resolution of inflammation during tuberculosis treatment (Coussens et al.
2012). A recent randomized, double-blinded, multicenter, placebo-controlled clini-
cal study involving 258 patients showed that 600,000 IU vitamin D3 once per month
for 2 months led to a significant increase in average weight gain and lower residual
disease by chest x-ray as compared to placebo (Salahuddin et al. 2013).
Deficiencies in vitamin D are associated with poor outcomes in HIV-infected
individual, bacterial vaginosis in the first trimester of pregnancy, increased influ-
enza A infections, and increased respiratory tract infections (Bodnar et al. 2009;
Villamor 2006; Aloia and Li-Ng 2007; Sabetta et al. 2010). Supplementation with
vitamin D lowered the incidence of seasonal flu in school children, the elderly, and
African-American women and lowered the severity of respiratory tract infections
(Urashima et al. 2010; Avenell et al. 2007; Aloia et al. 2005; Kenny et al. 2012). In
contrast, vitamin D supplementation did not reduce the incidence and duration of
severity of upper respiratory tract infection (Li-Ng et al. 2009). In a meta-analysis
of 11 placebo-controlled studies involving 5660 patients, vitamin D showed a pro-
tective effect against respiratory tract infections with once-daily dosing being better
than bolus doses (Bergman et al. 2013). The authors noted that there was significant
heterogeneity and evidence of publication bias in the field and warned that results
should be carefully interpreted (Bergman et al. 2013). It should be noted that in all
of these studies including those with tuberculosis, the role of CAMP induction in
these outcomes is unknown. Future studies must optimize dose, dosing frequency,
and target populations that are deficient in vitamin D to detect modest effects.
7.7 The Impact of Vitamin D on CAMP Levels
To date, in vivo studies demonstrating that vitamin D status or supplementation

affects the levels of CAMP/hCAP18 are inconclusive. High levels of hCAP18 are
found in the blood; therefore, we hypothesized that vitamin D levels may correlate
with hCAP18 levels (Sorensen et al. 1997; Gombart et al. 2009b). In an early study
on dialysis patients, we found only a modest positive correlation between hCAP18
and 1,25(OH)2D, but not 25(OH)D levels, but high hCAP18 levels were associated
with a significant decrease in 1-year mortality (Gombart et al. 2009b). For sepsis
108 A.F. Gombart
patients, a positive association between 25(OH)D and hCAP18 levels was observed
in all patients (Jeng et al. 2009). In healthy individuals, a positive association
between hCAP18 and 25(OH)D levels was observed at levels of 25(OH)D below
32 ng/ml, but not above (Bhan et al. 2011; Dixon et al. 2012). In the elderly and in
atopic dermatitis patients and normal controls, a positive correlation was observed
without applying a cutoff (Alvarez-Rodriguez et al. 2012; Kanda et al. 2012). On
the other hand, in cord blood samples, patients with active TB and patients with
pneumonia, a correlation between serum 25(OH)D and hCAP18 was not observed
(Yamshchikov et al. 2010; Mandic Havelka et al. 2010; Leow et al. 2011).
Supplementation of atopic dermatitis patients with 4000 IU/day vitamin D for 3
weeks increased CAMP in skin lesions and unaffected skin, but a second study with
more patients was negative (Hata et al. 2008, 2014). Several studies using high-dose
supplementation (50,000–60,000 IU/week) did not observe increased hCAP18 in
the blood (Adams et al. 2009; Alvarez et al. 2013; Das et al. 2014). In a randomized
controlled trial in patients with severe sepsis, 1,25(OH)2D3 did not increase plasma
hCAP18 levels (Leaf et al. 2014). In a study of 15 hereditary vitamin D-resistant
rickets patients (possess a nonfunctional VDR) and 17 normal controls, it was
shown that VDR is required for induction of CAMP by vitamin D in adherent
mononuclear cells cultured for 24 h, but basal expression of CAMP in various cell
types, fluids, or tissue samples was not determined (Tiosano et al. 2013). Additional
studies are required to determine the effect of vitamin D status or treatment on in
vivo CAMP expression, particularly on the in vivo induction of CAMP in immune
cells like macrophages during infection.
7.8 Vitamin D-Mediated Regulation of AMPs:

An Animal Model
The difficulty in determining the role CAMP in mediating the effects of vitamin D
on the immune response is the lack of a good animal model that replicates the path-
way as it is found in humans. As described earlier, vitamin D does not regulate
CAMP expression in mice or other mammals (Gombart et al. 2009a). In addition,
work from our own group and others revealed a striking difference in the use of
vitamin D by human versus murine macrophages. As described above, activation of
human macrophages by TLR ligands induces expression of CYP27B1 and the bio-
conversion of 25(OH)D3 to 1,25(OH)2D3. This, in turn, leads to the induction of
various VDR target genes including CAMP. In contrast, TLR activation of murine
macrophages does not induce CYP27B1 expression; thus, bioactive 1,25(OH)2D3 is
not synthesized by murine macrophages (Kapetanovic et al. 2012; Ooi et al. 2014),
and vitamin D target genes are not induced (our unpublished findings). This major
difference in the utilization of vitamin D by macrophages highlights the importance
of caution when using the mouse model to elucidate the role of vitamin D on
immune function in humans. Macrophages are very likely important for producing
locally high levels of 1,25(OH)2D3 at sites of infection in humans, but not in mice.
7.9 Conclusion
An abundance of in vitro evidence exists to demonstrate the regulation of AMPs,

particularly CAMP, by vitamin D. Also, historical, epidemiological, and clinical
data is consistent with the vitamin D-CAMP pathway providing protection against
infection. Nevertheless, there is a paucity of in vivo data that demonstrates that
induction of CAMP mediates important antibacterial or viral activities that are
attributed to vitamin D. Due to the current lack of a viable animal model, this evi-
dence will need to come from carefully designed human and/or primate studies.
Outstanding questions remain on how vitamin D status or supplementation affects
CAMP and DEFB4 expression and can active forms of vitamin D increase levels of
AMPs to improve immunity.
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Chapter 8
Dichotomous Roles of Cationic Polypeptides
Targeting HIV
Alexander M. Cole and Amy Liese Cole
Abstract Cationic antimicrobial polypeptides have classically been considered

the principal effector molecules of innate host defense against microbes and viruses.
This chapter describes the role of human cationic antimicrobial polypeptides in
modulating HIV-1 infection and is presented in the biologically relevant context of
sexual HIV-1 transmission. The cationic polypeptides described herein have been
identified in either the genital or anorectal mucosa, which are the main target tis-
sues for HIV-1 transmission, or are contained in human semen that is the source of
most transmitted HIV-1. Emphasis is placed not only on the ability of cationic
polypeptides to inhibit HIV-1 infection but also on the growing body of evidence
suggesting an increasing number of cationic polypeptides enhance HIV-1, often
robustly.
8.1 Redefining the Role of Cationic “Antimicrobial”

Peptides and Proteins in HIV-1 Infection
and Transmission
Worldwide, by the end of 2013, the HIV/AIDS pandemic had killed nearly 40 mil-
lion people, and an estimated 35 million people at the time were infected with
HIV [www.who.int]. The use of antiretroviral therapy (ART) to combat the virus
has been steadily increasing, and the number of people globally receiving ART
approaches 13 million. With such a large number of people receiving ART, viral
resistance to most of the available drugs is inevitable, and thus, to control the
A.M. Cole, PhD (*) • A.L. Cole

Division of Molecular Microbiology, Burnett School of Biomedical Sciences,
University of Central Florida College of Medicine, 4110 Libra Drive, Building 20,
Room 236, Orlando, FL 32816, USA

DOI 10.1007/978-3-319-24199-9_8
116 A.M. Cole and A.L. Cole
pandemic, additional strategies must be employed in concert with ART. While the
field still remains hopeful, essentially all vaccines targeting HIV-1 have shown
little promise or outright failure in the clinic (Haynes et al. 2014). Newer strate-
gies to prevent transmission of HIV-1 have demonstrated potential, including pre-
exposure prophylaxis (PrEP) targeting the infected, and to a lesser extent
uninfected, partner in a serodiscordant couple to reduce viral load and subsequent
viral transmission (McMahon et al. 2014). Conversely, topical antiretroviral
microbicides that are applied by the receptive partner to their own vagina or rec-
tum have not been successful in the clinic due to myriad factors, including product
adherence and psychosocial issues (Van Damme and Szpir 2012). Ensuring bio-
compatibility of microbicides with the milieu of the vagina or rectum has been
more recently realized (Trifonova et al. 2006), as several older-generation micro-
bicides likely failed due to incompatibility with the mucosal environment (Morris
and Lacey 2010).
Developing a mucosal vaccine or a topical microbicide that would be applied
to the anogenital mucosa to prevent viral transmission requires a comprehensive
understanding of the local mucosal environment to increase the likelihood of suc-
cess. While there are many topics that could be covered in this realm, including
the architecture and cellular composition of the respective mucosal tissues, this
chapter is focused particularly on cationic effector peptides and proteins that are
produced by certain immune cells and epithelia and often liberated into secre-
tions that overlie the mucosa. Historically, these cationic peptides and proteins
have been referred to as “antimicrobial polypeptides” or “AMPs,” due to their
broad-spectrum activity against bacteria, fungi, and viruses such as HIV-1
(Zasloff 1992), and more recently termed “host defense polypeptides” or “HDPs,”
because of expanded activities that are related to innate immunity but not neces-
sarily due to direct antimicrobial action (Mansour et al. 2014). Either definition
implies that these polypeptides are involved in protection of the host, and indeed
this is the ascribed function for many peptides and proteins detailed in other
chapters of this book.
Our chapter instead examines the dichotomous roles of cationic polypeptides in
their abilities to modulate HIV-1 infection. The first half of the chapter begins in a
similar vein as most reviews on the topic, by outlining the capacity of human cat-
ionic polypeptides to inhibit HIV-1 infection and transmission. The second part
presents a growing body of evidence that suggests many human cationic polypep-
tides are in fact enhancers of HIV-1 infection. As presented below, the distinction
between inhibiting and promoting HIV-1 infection is not mutually exclusive for
certain classes of polypeptides. Note also that all cationic polypeptides are bio-
logically relevant in the context of HIV-1 infection and transmission – they have
either been identified in the genital or anorectal mucosal areas that are the main
target tissues for HIV-1 transmission or are contained in human semen that is the
source of most transmitted HIV-1. Due to space constraints, background on the life
cycle of HIV-1 and events in sexual transmission of HIV-1 are not covered in this
chapter, but the reader is instead referred to an excellent review on the subject
(Haase 2011).
8 Dichotomous Roles of Cationic Polypeptides Targeting HIV 117
8.2 Cationic Polypeptides That Primarily Inhibit

HIV-1 Infection
8.2.1 Lysozyme and Lactoferrin
Human lysozyme is a 14.6 kDa polypeptide identified by Sir Alexander Fleming in

the early 1920s as the first known cationic polypeptide with bactericidal activity
against a broad spectrum of microbes (Fleming 1922; Gallo 2013). Its presence is
ubiquitous in mucosal fluids, and although the concentration of lysozyme is relatively
low (approximately 13 μg/ml) in vaginal fluid from healthy donors (Valore et al.
2002), concentrations as high as 1 mg/g have been observed in cervical mucus plugs
(Hein et al. 2002). Lysozyme has been reported to lower the ability of virally infected
primary T cells and monocytes to produce HIV-1 (Lee-Huang et al. 1999). Another
group later reported that the anti-HIV activity exhibited by lysozyme was likely due
to its ability to bind viral RNA (Steinrauf et al. 1999), although how lysozyme enters
HIV-1 virions or infected T cells and monocytes remains unclear. Subsequent studies
revealed a core nonapeptide region that exhibited potent activity against HIV-1 at low
to mid-nanomolar concentrations, by preventing viral entry (Lee-Huang et al. 2005).
Although this particular nine residue lysozyme-derived peptide has not been identi-
fied in vivo, flanking trypsin cleavage sites suggest that cleavage might occur in bio-
logically favorable conditions to liberate this highly active antiretroviral peptide.
Lactoferrin is a larger (78 kDa) protein that is an abundant constituent of specific
granules of the neutrophil as well as certain epithelia. This protein has been ascribed
many functions, including binding and sequestering essential iron to inhibit microbial
growth. Similar to lysozyme, concentrations are low in healthy vaginal fluids (Valore
et al. 2002), but rise to nearly 100 μg/g in cervical mucus plugs (Hein et al. 2002).
Lactoferrin has been shown to prevent HIV-1 infection by binding to the V3 loop of the
HIV-1 envelope protein gp120, thus inhibiting viral binding to the surface or entry into
target CD4+ T cells. Although the individual concentrations of lysozyme and lactoferrin
are modest, their efficacy in preventing HIV-1 infection might be better realized in con-
cert with other cationic polypeptides. Indeed, well over a dozen peptides and proteins
related to host defense have been identified in the cervicovaginal fluids of healthy women
(Venkataraman et al. 2005; Dasari et al. 2007). While the anti-HIV-1 activity of each
polypeptide by itself at its biological concentration measured in cervicovaginal fluid was
not sufficient to prevent HIV-1 infection ex vivo, the activity of the fluid was realized
only with the sum total of the cationic polypeptide components, suggesting additive or
synergistic potential of many cationic polypeptides (Venkataraman et al. 2005).
8.2.2 Whey Acidic Protein (WAP) Motif-Based Polypeptides
Trappin-2/elafin and secretory leukocyte protease inhibitor (SLPI) are 10–12 kDa
members of the whey acidic protein (WAP) motif family (Bingle and Vyakarnam
2008; Moreau et al. 2008). Reducing inflammation in the female reproductive tract
has been attributed to their antiprotease activity targeting neutrophil elastase and pro-
teinase-3 (Horne et al. 2008). The ability of trappin-2/elafin to prevent HIV-1 infec-
tion is supported by several lines of evidence (Ghosh et al. 2010). Only uterine cells
of the upper female reproductive tract (FRT) can be induced to express trappin-2/
elafin, while epithelia of both the upper and lower FRT can express trappin-2/elafin
constitutively (Ghosh et al. 2010). Importantly, the antiretroviral activity of trappin-2/
elafin was measured against HIV-1 that utilizes either coreceptor required for entry
(CXCR4 or CCR5), revealing that trappin-2/elafin targeted HIV-1 virions directly in
dose-dependent manner. Trappin-2/elafin concentrations are highest during the secre-
tory phase of the menstrual cycle, and HIV-infected patients contain less of this pro-
tein in their cervicovaginal lavage fluids than uninfected patients (Ghosh et al. 2010),
suggesting hormonal and other regulatory factors likely modulate its expression.
SLPI’s capacity to prevent HIV infection is equivocal. One report suggested that
very high (milligram per mL) concentrations of SLPI could not prevent HIV-1 infec-
tion (Turpin et al. 1996). Other studies revealed that very low concentrations (high
nanomolar) could block HIV-1 or uncoat the viral capsid independent of SLPI’s
function as a protease (McNeely et al. 1995, 1997). Moreover, reduced SLPI levels
in the cervicovaginal fluids of women with bacterial vaginosis or sexually transmit-
ted infections (STIs) is a potential cofactor for increased incidence of HIV-1 acqui-
sition (Draper et al. 2000). Indirect evidence in another study suggested that elevated
SLPI levels were associated with decreased perinatal acquisition of HIV, which was
specific to SLPI and not other cationic polypeptides measured (Pillay et al. 2001).
Further study is warranted to unmask the true antiretroviral role for SLPI, especially
in combination with other antiviral factors that may potentiate its action.
8.2.3 Defensins
Human defensins are a thoroughly researched family of antimicrobial peptides

comprised of three classes: α-defensins, β-defensins, and θ-defensins, each of which
is categorized largely on the canonical disulfide bonding pattern of their six cysteine
residues (Ganz 2003). Although α-defensins and β-defensins are produced as
mature peptides by human cells and tissues, under normal conditions, θ-defensin
genes are transcribed but not translated due to a premature termination codon pres-
ent in the signal sequence that precludes the production of θ-defensin peptides (Cole
et al. 2002; Nguyen et al. 2003). It is remarkable that these expressed pseudogenes
have remained otherwise intact for tens of millions of years, retaining nearly 90 %
nucleotide identity with θ-defensins from our evolutionarily distant relative, the rhe-
sus macaque (Nguyen et al. 2003). Based on human θ-defensin genetic information,
θ-defensin peptides called “retrocyclins” were recreated using solid-phase synthetic
approaches. Retrocyclins were shown to be remarkably active against most lab-
adapted strains and worldwide clinical isolates of HIV-1 tested (Cole et al. 2002;
Owen et al. 2004; Gupta et al. 2013) by interfering with the fusogenic complex
needed for HIV-1 entry into target cells (Munk et al. 2003; Gallo et al. 2006). In a
study that utilized aminoglycosides to suppress the premature termination codon in
retrocyclins, intact retrocyclin peptides could be produced by promyelocytes and
vaginal epithelia and were active against HIV-1 (Venkataraman et al. 2009). Given
that θ-defensins are thought to be produced from the end-to-end ligation and mac-
rocyclization of two nonapeptides (Tang et al. 1999), this study also suggested that
the complex molecular machinery required to produce retrocyclin peptides remains
comparatively intact in human cells. It is therefore tempting to speculate whether
these antiretroviral retrocyclin peptides can still be produced by modern-day
humans under natural conditions that at least partially suppress the premature termi-
nation codon.
Human β-defensins (HBDs) are produced primarily by epithelial cells (Lehrer
and Ganz 2002), and although there are at least six β-defensin peptides produced
throughout the body, only HBD2 and HBD3 have been reported to exert activity
against HIV-1 (Klotman and Chang 2006). HBD2 and HBD3 downmodulate
CXCR4, one of two chemokine coreceptors required for HIV-1 entry into target
CD4+ cells (Quinones-Mateu et al. 2003), while only HBD3 antagonizes the CXCR4
receptor (Feng et al. 2006). In vaginal fluid (Valore et al. 2002) and cervical mucus
plugs (Hein et al. 2002), the concentration of HBD2 is present at nanograms per mL
concentrations, which are far below the low microgram per mL levels required for
anti-HIV-1 activity in vitro (Quinones-Mateu et al. 2003). Nevertheless, the physi-
ological amounts of HBD2 are within the range required to chemoattract potential
targets of HIV-1. HBD2 is a ligand for CCR6-expressing cells, including CD45RO+/
CD4+ T cells and immature dendritic cells (Hoover et al. 2002), while HBD2 and
HBD3 are ligands for CCR2-expressing cells, including macrophages/monocytes
and neutrophils (Rohrl et al. 2010). While HBD2 and HBD3 may unlikely directly
affect HIV-1 infection in vivo, their upregulation by inflammatory processes and the
ability of HBDs to recruit additional target cells to the genital and other mucosae
together would likely increase the incidence of viral transmission.
Human α-defensins are further divided into two subcategories, human neutrophil
peptides 1–4 (HNP1-4) and human defensins 5 and 6 (HD5, HD6). With regard to
HIV-1 infection, perhaps this class of defensins is the most intriguing: several HNPs
are active against HIV-1, while HD5 and HD6, which will be discussed in the next
section, are robust enhancers of HIV-1 infection. HNPs are present at very high con-
centrations in neutrophil azurophil granules where they can reach concentrations in
the millimolar range, comprising approximately one-third of the granules’ total pro-
tein content (Harwig et al. 1992; Valore and Ganz 1992; Ganz and Lehrer 1997). The
first mention of an anti-HIV-1 peptide began with a short communication describing
how α-defensins isolated from rabbits, rats, and guinea pigs could inhibit HIV-1
replication and reduce HIV-mediated cytopathology in a T-cell line (Nakashima
et al. 1993). Similarities between α-defensins and a looped region of the fusogenic
HIV-1 gp41 glycoprotein suggested an antiviral mechanism that prevented viral
entry or fusion (Monell and Strand 1994). More recently, an elegant study revealed
that HNP1 had a dual mode of action against HIV-1 by interfering with protein
kinase C activity as well as acting directly on the virus (Chang et al. 2005). Although
HNPs are intrinsically antiretroviral, these peptides are only liberated from neutro-
phils, and thus, their presence also signifies subclinical or clinical inflammation.
Similar to the upregulation of HBD2 and HBD3 that typifies epithelial inflamma-
tion, neutrophil-mediated inflammation can in turn render the anogenital mucosa
more susceptible to HIV-1 infection through the recruitment of CD4+ target cells.
8.3 Cationic Polypeptides That Enhance HIV-1 Infection

and Transmission
8.3.1 Human Defensin-5 and Human Defensin-6
The previous section of this chapter described various human cationic antimicrobial
polypeptides that reportedly inhibit HIV-1 infection. Yet as has been observed for
defensins, the presence of HNPs and HBDs in the anogenital mucosa could poten-
tially be a double-edged sword, more carefully honed on the side of inflammation-
mediated enhancement of HIV-1 infection. Two other α-defensins reportedly
augment HIV-1 infection. Originally isolated from Paneth cells located at the base of
the crypts of Lieberkühn within the small intestine (Jones and Bevins 1992, 1993;
Ouellette 2005), HD5 and HD6 were found to be stored as inactive propeptides and
proteolytically activated by trypsin upon release of secretory granules (Ghosh et al.
2002). Similar to other defensins, these α-defensins have classically been considered
broad-spectrum antimicrobial peptides. In the female reproduction tract (FRT), HD5
has been localized to vaginal and ectocervical epithelia, granules within endocervi-
cal epithelium, and the surface of the endocervix (Quayle et al. 1998). Expression of
HD5 is highest during the secretory phase of the menstrual cycle (Quayle et al.
1998), suggesting hormonal influence on expression and periods in which the FRT,
particularly the cervix, is potentially more susceptible to HIV-1 infection. While all
other defensins have been shown to inhibit HIV-1 infection in vitro, HD5 and HD6
were instead found to enhance HIV-1 infection by promoting viral attachment to
target cells (Rapista et al. 2011) and could also antagonize the anti-HIV-1 activity of
polyanion topical microbicides (Ding et al. 2011). Given that the cervix is likely the
primary site for initial HIV-1 infection in women (Miller et al. 2005; Pudney et al.
2005; Salazar-Gonzalez et al. 2009), HD5 and HD6 expressed by cervical epithelia
may be vital determinants for establishing HIV-1 infection in the FRT.
8.3.2 Semen-Derived Enhancers of Viral Infection (SEVI)
Human semen and seminal plasma contain numerous cationic polypeptides, many
of which are proteolytic fragments of larger proteins. Proteomic analyses of seminal
plasma identified at least 52 separate cationic polypeptides, twenty of which were
derived from the semenogelin I and II parent proteins during the process of semen
liquefaction that follows ejaculation (Martellini et al. 2009). From this fluid, an
individual semenogelin-derived peptide “SG-1” was purified, which exhibited
activity against HIV-1 in vitro at sub-physiological concentrations (Martellini et al.
2009). While seminal plasma clearly contains this and potentially other cationic
antiviral components, greater emphasis has been placed on studying cationic poly-
peptides within this fluid that enhance HIV-1 infection. An important study reported
that semen-mediated enhancement of HIV transmission is due to a cationic peptide
“PAP286” derived from prostatic acid phosphatase, which forms amyloid fibrils
called semen-derived enhancers of viral infection or “SEVI” (Munch et al. 2007).
Further in vitro studies confirmed the role of SEVI in enhancing HIV-1 infection
(Roan et al. 2009; Olsen et al. 2010; Easterhoff et al. 2011; Martellini et al. 2011),
implicating that the cationic properties of PAP286 and SEVI underlie this ability
(Roan et al. 2009). The HIV-enhancing effects of SEVI may be diminished in vivo
due to resident proteases, such as prostate-specific antigen (PSA), matriptase and
prostasin, that were shown to cleave PAP286 and prevent SEVI fibril formation
(Martellini et al. 2011). Other studies have suggested that endogenous Zn2+ within
seminal plasma might protect the fibrils from proteolysis (Olsen et al. 2012), which
may explain why SEVI fibrils were detected in fresh human semen, at least for cer-
tain donors (Usmani et al. 2014a, b). Seminal plasma has also been shown to
enhance SEVI fibril formation, in part due to anionic components such as inorganic
phosphate and sodium bicarbonate (Olsen et al. 2012). Cationic polypeptide frag-
ments from semenogelins have also been shown to form SEVI-like amyloid fibrils
that can enhance HIV-1 infection (Roan et al. 2014). Given that many peptides and
proteins can form amyloid fibrils, it is possible that other similar enhancers of HIV-1
infection will be uncovered in semen or perhaps also in cervicovaginal and anorec-
tal fluids. Collectively, while both antiviral and viral-enhancing cationic polypep-
tides have been described in semen and seminal plasma, factors such as SEVI that
promote viral infection might outpace those that act to inhibit infection.
8.3.3 Other Endogenous Polypeptide Enhancers

of HIV Infection
Several other human cationic peptides and proteins have also been reported to
enhance HIV-1 infection. The 18 kDa cathelicidin protein hCAP18 can be proteo-
lytically processed to at least three mature forms, LL-37, ALL-38, and FALL-39,
each of which differs only by one or two amino-terminal residues (Agerberth et al.
1995; Cowland et al. 1995; Larrick et al. 1995; Sorensen et al. 2003). LL-37 is
found in neutrophils and is expressed by a variety of mucosal epithelia, including
the FRT where it has been immunolocalized to inflamed ectocervical epithelia.
While the direct antibacterial actions of LL-37 are well known, this peptide can also
bind the G protein-coupled receptor, N-formyl peptide receptor 2 (FPR2), to exert
chemotactic and immunomodulatory functions. Using this mechanism, LL-37 can
antagonistically ligate FPR2 to downregulate chemokine coreceptors necessary for
HIV-1 to bind and enter peripheral blood mononuclear cells, including primary
CD4+ cells (Bergman et al. 2007). The level of LL-37 in healthy vaginal fluid has
been reported in the mid-to-high nanogram per mL range (Valore et al. 2002), which
is within the concentration necessary to exert FPR2-mediated anti-HIV-1 activity in
vitro. Conversely, LL-37 produced by herpes simplex virus-2 (HSV-2)-infected
keratinocytes has also been reported to increase the susceptibility of Langerhans
cells to HIV-1, which was neutralized by blocking LL-37 production (Ogawa et al.
2013).
Two additional polypeptides from neutrophils can also serve to promote HIV-1
infection. Cathepsin G is a serine protease that has been identified in human cervi-
covaginal fluids (Venkataraman et al. 2005). This protease can enhance HIV-1
infection of macrophages (Moriuchi et al. 2000) likely through its binding of the
gp120 envelope glycoprotein of HIV-1 (Avril et al. 1994, 1995). The mechanism
likely involves Gi protein-mediated signal transduction as cells treated with pertus-
sis toxin did not increase viral infection (Moriuchi et al. 2000). Additionally, cathep-
sin G can cleave RANTES, a natural ligand of the HIV-1 coreceptor CCR5, thus
reducing RANTES’ natural antiviral capacity (Lim et al. 2006). Calprotectin is a
heterodimeric protein composed of an 8 kDa polypeptide (termed MRP-8, S100A8,
or calgranulin A) and a 14 kDa polypeptide (termed MRP-14, S100A9, or calgranu-
lin B) and is produced by neutrophils, monocytes, and various epithelia. Within the
neutrophil, calprotectin accounts for over one-third of the cytoplasmic protein con-
tent (Brandtzaeg et al. 1995). In normal vaginal fluid, its levels are in the low to
mid-microgram per mL range, but due to its presence in neutrophils, much higher
concentrations can be found in inflammatory conditions. A study that isolated the
8 kDa subunit of calprotectin from cervicovaginal secretions revealed that it could
increase expression of HIV-1 in latently infected monocytes (Hashemi et al. 2001).
Interestingly, another study showed that the 14 kDa subunit could stimulate the anti-
HIV-1 activity of NK cells by ligating CD85j (Arnold et al. 2013). Assessing the
activity of both subunits together would be important in determining if one activity
will supersede the other or if their combined effects on HIV-1 infection will be
neutralized. Nevertheless, local mucosal inflammation that induces epithelia-
derived LL-37 and calprotectin and promotes neutrophil influx and granular release
of LL-37, calprotectin, and cathepsin G could serve to increase susceptibility to
HIV-1 infection and transmission.
8.4 Looking Forward
Since the discovery of cationic antimicrobial polypeptides, the field has expended
much time and energy in promoting the ability of these molecules to serve as broad-
spectrum antibiotics, antifungals, and antivirals – with good reason. Many of these
polypeptides are highly effective against microbes and viruses, and a good number
could serve as scaffolds for the design and development of next-generation therapeu-
tics and preventatives to combat pathogens that are resistant to current antibiotic
treatments. In the body, a number of cationic polypeptides appear to have a primary

or supportive role to directly control infection, influence immune responses to
microbes, or amplify other host defense processes. Indeed, the field would have had a
difficult time establishing itself without promoting these important, beneficial roles of
cationic polypeptides. Over the last decade, it has been clear that an increasing num-
ber of cationic polypeptides act in roles inimical to defending the host, and as profiled
in this chapter, many can promote rather than suppress HIV-1 infection. We are only
beginning to understand the complex regulation of cationic polypeptides, how they
work in concert to modulate HIV-1 infection and transmission, and how they might
adversely affect antiretroviral therapies such as topical microbicides currently being
developed. While stopping short of suggesting that the field rename these molecules
once again, the terms “antimicrobial peptides” and “host defense peptides” are either
becoming outdated or should only be applied to a particular subset of molecules.
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Chapter 9
Antimicrobial Peptides in Host Defense:
Functions Beyond Antimicrobial Activity
Kim Alan Brogden, Amber M. Bates, and Carol L. Fischer
Abstract Antimicrobial peptides are well known for their important roles in
host defense by enhancing the barrier function and limiting microbial popula-
tions of the skin and mucosa. However, many of these peptides are now known to
have additional roles assisting innate and adaptive immune functions. To facili-
tate innate immunity, antimicrobial peptides activate complement, chemoattract
cells (e.g., monocytes, macrophages, T cells, neutrophils, immature dendritic
cells, and mast cells), enhance phagocytosis, and modulate the production of
chemokines and proinflammatory cytokines in other cells. At local sites of initia-
tion, antimicrobial peptides can act as opsonins to enhance phagocytosis by
monocytes and phagocytes and can activate cells. In the latter, for example, treat-
ment of osteoblasts and osteoblast-like MG63 cells with human beta-defensin
(HBD)2 increases their proliferation rates. Treatment of osteoblast-like MG63
cells with HBD2 and HBD3 increases transcript levels of osteogenic markers for
differentiation, increases antileukoprotease (ALP) levels, and enhances mineral-
ized nodule formation. To facilitate adaptive immunity, antimicrobial peptides
assist the uptake of antigens by monocytes or other antigen-presenting cells and
later direct the process toward a Th1 or Th2 adaptive immune response. More
commonly though, antimicrobial peptides induce a mixed response characterized
by Th1-/Th2-specific antibodies and Th1/Th2 cytokines from antigen-exposed
splenocytes of immunized animals. Finally, antimicrobial peptides can be
detected in the margins around both oral and cutaneous wounds, and there is
K.A. Brogden, PhD (*)

Dows Institute for Dental Research, College of Dentistry, The University of Iowa,
Iowa City, IA 52242, USA
Department of Periodontics, College of Dentistry, The University of Iowa,
A.M. Bates • C.L. Fischer
Dows Institute for Dental Research, College of Dentistry, The University of Iowa,

DOI 10.1007/978-3-319-24199-9_9
130 K.A. Brogden et al.
growing evidence to suggest they also play a dynamic role in wound healing by
improving wound angiogenesis, vascularization, and reepithelialization.
9.1 Introduction
Antimicrobial factors in normal tissues and fluids were described as early as 1888
(Skarnes and Watson 1957). These factors, isolated from extracts of tissues, serum,
serous fluids, and leukocytes, later became the well-known members of innate
immunity: antibodies, complement, lysozyme, histones, and protamines (Skarnes
and Watson 1957). Small, linear, basic peptides (called tissue basic polypeptides)
with antimicrobial activity in normal tissues and fluids were also described as early
as 1947 (Bloom et al. 1947; Bloom and Prigmore 1952; Bloom and Blake 1948).
These peptides contained lysine (29–30 %) and arginine (3.5 %) amino acid resi-
dues with isoelectric points between pI 10 and 11.2. They were thought to be
attracted to the negatively charged surfaces of microbial cells via electrostatic bond-
ing and to alter microbial membrane integrity. These peptides are likely the group
we now know as the antimicrobial peptides (Skarnes and Watson 1957). The early
history of antimicrobial peptide discovery and research can be found in two excel-
lent reviews by Skarnes and Watson (1957) and Nakatsuji and Gallo (2012).
Almost immediately after the discovery of cationic peptides with antimicrobial
activity, investigators began to assess their secondary functions, and many of these
peptides did indeed have additional roles in innate and adaptive immunology
(Nakatsuji and Gallo 2012). This was not an unusual finding as the inverse was also
found to be true, and some other physiologically important peptides had antimicro-
bial activity. For example, some neuropeptides, peptide hormones, and chemokines
were found to have antimicrobial activities (Brogden et al. 2005; Cole et al. 2001;
Yang et al. 2003). These results clearly suggest that peptides with antimicrobial
activity are multifunctional in a variety of situations.
In this chapter, we present the alternate functions of peptides with antimicrobial
activity, a topic of a number of excellent comprehensive reviews (Yang et al. 2001,
2002, 2004; Yang and Oppenheim 2004; Bowdish et al. 2005; Pingel et al. 2007;
Rehaume and Hancock 2008; Semple et al. 2010; Semple and Dorin 2012; Greer
et al. 2013). We start by presenting the roles of antimicrobial peptides in innate
immunity and their ability to chemoattract and activate cells (Table 9.1). This
includes recent discoveries that antimicrobial peptides influence the properties of
human mesenchymal stem cells (hMSCs) and osteoblasts in addition to epithelial
cells, keratinocytes, and immune cells of myeloid or lymphoid origin. We then pres-
ent the roles of antimicrobial peptides in adaptive immunity and their ability to
influence Th1, Th2, and mixed Th1 and Th2 responses (Table 9.2). Third, we pres-
ent the ability and conditions of antimicrobial peptides to modulate chemokine and
proinflammatory cytokine responses, an exciting area of current research by a vari-
ety of investigators (Table 9.3). Finally, we present the roles of antimicrobial pep-
tides in wound healing, angiogenesis, and autoimmune diseases.
9 Antimicrobial Peptides in Host Defense: Functions Beyond Antimicrobial Activity 131
Table 9.1 Antimicrobial peptides regulate innate immunity by chemoattracting inflammatory

cells, enhancing phagocytosis, enhancing the production of proinflammatory mediators, and
regulating complement activation
Chemoattract cells
Cathelicidins LL-37 and CRAMP chemoattract monocytes, neutrophils, macrophages, and
peripheral blood leukocytes (Kurosaka et al. 2005; An et al. 2005)
α-, β-defensins chemoattract monocytes, immature dendritic cells, neutrophils, macrophages,
CD4+ T cells (CD45 RA+), and CD8+ T cells (Chertov et al. 1996; Fleischmann et al. 1985;
Ichinose et al. 1996; Territo et al. 1989; Yang et al. 1999, 2000; Li et al. 2014)
Enhance phagocytosis
α-defensins enhance macrophage phagocytosis in a variety of species (Ichinose et al. 1996;
Fleischmann et al. 1985)
Enhance the production of proinflammatory mediators
α-, β-defensin-treated epithelial cells and monocytes produce IL-1, IL-8, IL-10, and TNF-α
(Chertov et al. 1996; Van Wetering et al. 1997; Chaly et al. 2000)
HBD3 induces production of Gro-α, MDC, MCP-1, MIP-1α, MIP-1β, and VEGF in
monocytes and macrophages (Petrov et al. 2013)
LL-37 induces production of Gro-α, MDC, MCP-1, MIP-1α, MIP-1β, and VEGF in
monocytes and macrophages (Petrov et al. 2013)
Degranulate mast cells
α-, β-defensins degranulate mast cells and release histamine and prostaglandin D2 (Yamashita
and Saito 1989; Befus et al. 1999; Chertov et al. 2000; Niyonsaba et al. 2001)
Regulate complement
Defensins regulate complement activation (Prohaszka et al. 1997; van den Berg et al. 1998)
9.2 Antimicrobial Peptides in Innate Immunity
By definition, innate immunity is a nonspecific defense against mechanical injury

and damage, chemical exposure, or microbial infection in barrier surfaces like the
skin or mucosa. It also protects from internal exposure to abnormal cells (Martin
2014). Cellular components such as macrophages, dendritic cells, neutrophils, and
granulocytes and humoral components such as lactic acid, fatty acids, lysozyme,
and complement are often involved. Cells produce inducible humoral components
after stimulation of surface receptors with microbe-associated molecular pattern
(MAMP) or pathogen-associated molecular pattern (PAMP) molecules that include
lipopolysaccharides, peptidoglycans, or nucleic acids. Exposed cells release che-
mokines and proinflammatory cytokines via a variety of receptor signaled path-
ways. Cells also release antimicrobial peptides after exposure to PAMPs directly or
can release antimicrobial peptides after exposure to chemokines and proinflamma-
tory cytokines (Liu et al. 2013b; Jan et al. 2006). Antimicrobial peptides activate
complement, attract neutrophils, enhance phagocytosis, and complete the cycle by
enhancing the production of chemokines and proinflammatory cytokines in other
cells (Yang et al. 2002).
Antimicrobial peptides can enhance the barrier function of the skin or mucosa.
For example, in the skin, HBD3 regulates cell permeability and membrane tight
junctions in keratinocytes. HBD3 enhances the expression of claudins (e.g., 1–5, 9,
Table 9.2 Antimicrobial peptides influence Th1, Th2, and mixed Th1/Th2 adaptive immune
responses, often with adjuvant-like activities
Enhance Th1 responses
Murine β-defensin 2 (mDF2β) induces potent cell-mediated responses and antitumor
immunity when genetically fused with nonimmunogenic tumor antigens (Biragyn et al.
2001, 2002; Biragyn 2005)
Mice receiving L1210 cells expressing mDF2β have responses strong NK and CTL responses
with enhanced IL-12 and IFN-γ production, protecting them from lethal challenge with
L1210 cells (Ma et al. 2006)
Zebra fish immunized with zebra fish β-defensin 2 (zfBD2) develops a Th1 immune response
with an upregulated IFN-γ response (Garcia-Valtanen et al. 2014)
Enhance Th2 responses
Cationic peptide KLKL5KLK with ovalbumin induces a Th2 response. Immunized mice
produce ovalbumin-specific IgG1, but not IgG2, and splenocytes from immunized mice,
stimulated with ovalbumin, produce IL-4 and IL-5, but not IFN-γ (Fritz et al. 2004)
Enhance mixed Th1/Th2 responses
Melittin enhances a mixed Th1/Th2 response to tetanus toxoid in mice. Total IgG and IgG2a
responses are increased (Bramwell et al. 2003)
CRAMP enhances ovalbumin-specific IgG1, IgG2a, IgG2b, and IgG3 and Th1/Th2 cellular
ovalbumin-specific responses in mice (Kurosaka et al. 2005)
LL-37 enhances Th1 and Th2 humoral, cytotoxic, and protective responses in mice when
fused with M-CSF receptor cloned from J6-1 leukemia cells (M-CSFRJ6-1). Splenocytes
from immunized mice, stimulated with M-CSFRJ6-1, produce IFN-γ (An et al. 2005)
HNP-1, HNP-2, HNP-3 enhance keyhole limpet hemocyanin-specific and ovalbumin-
specific IgG1, IgG2a, and IgG2b responses, and splenocytes from immunized mice
stimulated with keyhole limpet hemocyanin produce KLH-specific IFN-γ (Th1 cytokine)
and IL-4 (Th2 cytokine), and splenocytes from immunized mice stimulated with
ovalbumin produce greater amounts of IL-5, IL-6, IL-10, and IFN-γ (Th1 and Th2
cytokines) (Lillard et al. 1999; Tani et al. 2000)
11, 14–17, 20, 23, and 25) and the location of claudins in cell membranes and ele-
vates transepithelial electrical resistance (Kiatsurayanon et al. 2014). This occurs
via HBD3-mediated pathways involving Rac1, atypical protein kinase C, glycogen
synthase kinase, and phosphatidylinositol 3-kinase (Kiatsurayanon et al. 2014).
Once produced, cathelicidins and defensins then attract a variety of cells to their
sites of induction (Table 9.1). LL-37, CRAMP, α-defensins, and β-defensins all are
reported to chemoattract monocytes, macrophages, T cells, neutrophils, immature
dendritic cells, and mast cells.
At the site of initiation, antimicrobial peptides then can act as opsonins
(Fleischmann et al. 1985), enhance phagocytosis by monocytes and phagocytes
(Ichinose et al. 1996), and activate cells. They can also degranulate mast cells. For
example, human, rabbit, and guinea pig α-defensins activate and degranulate mast
cells releasing histamine and prostaglandin D2 (Yamashita and Saito 1989; Befus
et al. 1999; Niyonsaba et al. 2001).
An additional and well-known function is the ability of antimicrobial peptides,
particularly defensins, to regulate complement activation (Prohaszka et al. 1997;
van den Berg et al. 1998). Here, C1q of C1 binds to HNP-1, HNP-2, and HNP-3,
Table 9.3 Peptides and proteins in oral secretions with antiinflammatory and proinflammatory
properties
Antiinflammatory activities
LL-37
Attenuates agonist-induced, chemokine, and proinflammatory cytokine responses in
macrophages (Scott et al. 2000), lung epithelial cells (Scott et al. 2000), peripheral blood
mononuclear cells (Molhoek et al. 2009), and whole blood leukocytes (Walters et al. 2010)
Attenuates MAPK pathway activation of p38 and ERK responses in gingival fibroblasts
(Inomata et al. 2010)
α-defensins
Inhibit the production of proinflammatory cytokines from macrophages (Miles et al. 2009)
Attenuate a chemokine and proinflammatory cytokine response in mice (Kohlgraf et al. 2010)
β-defensins
HBD3 attenuates agonist-induced, chemokine, and proinflammatory cytokine responses in
dendritic cells (Pingel et al. 2008; Harvey et al. 2013), in THP-1 human myelomonocytic
cells (Semple et al. 2010), peripheral blood monocyte-derived macrophages (Semple et al.
2010), and in RAW264.7 murine macrophages (Semple et al. 2010)
DEFB114 attenuates MAPK pathway p42/44 response and attenuates an agonist-induced
TNF-α response in RAW264.7 murine macrophages (Yu et al. 2013)
DEFB123 attenuates an agonist-induced MAPK pathway activation of p42/44 and p38 and
attenuates an agonist-induced TNF-α response in RAW264.7 murine macrophages
(Motzkus et al. 2006)
DEFB126 attenuates an agonist-induced proinflammatory response in RAW264.7 murine
macrophages (Liu et al. 2013a)
Attenuate a chemokine and proinflammatory cytokine response in mice (Kohlgraf et al. 2010)
θ-defensins
Retrocyclin RTD-1 attenuates agonist-induced, chemokine, and proinflammatory cytokine
responses in human peripheral blood leukocytes (Schaal et al. 2012)
Histatins
Histatin 5 attenuates agonist-induced, chemokine, and proinflammatory cytokine responses in
gingival fibroblasts (Imatani et al. 2000) and dendritic cells (Borgwardt et al. 2014)
CEMA (cecropin-melittin hybrid)
CEMA blocks the binding of LPS to LPS-binding protein, attenuates agonist-induced,
chemokine, and proinflammatory cytokine responses in murine macrophages (Scott et al.
2000)
Proinflammatory activities
LL-37
50–100 μg/ml enhances an agonist-induced IL-8 response in epithelial cells (Scott et al. 2002)
β-defensins
Pre-stimulation or post-stimulation of dendritic cells and mice with HBD3 enhances an
agonist-induced chemokine and proinflammatory cytokine response (Harvey et al. 2013)
Pre-stimulation of macrophages with MBD14 enhances an agonist-induced chemokine and
proinflammatory cytokine response (Barabas et al. 2013)
HD5 upregulates expression of genes involved in cell survival and inflammation in an
NF-kB-dependent fashion in epithelial cells (Lu and de Leeuw 2013)
and complement in normal human serum is activated by HNP-released C4b

(Prohaszka et al. 1997).
Antimicrobial peptides clearly influence the properties of a variety of cell types
including epithelial cells, keratinocytes, and immune cells of myeloid or lymphoid
origin, and these properties are listed in Table 9.1. They also influence the properties
of hMSCs and osteoblasts. Treatment of hMSCs, osteoblasts, and osteoblast-like
MG63 cells with HBD2 increases their proliferation rates (Warnke et al. 2013; Kraus
et al. 2012), and treatment of osteoblast-like MG63 cells with HBD2 and HBD3
increases their transcript levels of osteogenic markers for differentiation, increases
ALP levels, and enhances mineralized nodule formation (Kraus et al. 2012).
9.3 Antimicrobial Peptides in Adaptive Immunity
Adaptive immunity is an acquired resistance produced after antigenic exposure in

the form of antibody production together with the development of cell-mediated
immunity. The adaptive immune system is organized around highly specialized
cells including antigen-presenting cells and two classes of specialized lymphocytes,
T and B cells, with a variety of functions (Dunkelberger and Song 2010). These
cells display a diverse repertoire of antigen-specific recognition receptors. This
enables specific identification and elimination of pathogens, tailoring of immune
responses, and long-lived immunological memory.
Antimicrobial peptides are known to play roles in adaptive immune responses and
exert their influence at numerous steps in the process. Early in the process, antimi-
crobial peptides can facilitate the uptake of antigen by monocytes or other antigen-
presenting cells (Fritz et al. 2004) and later direct the process toward a Th1, Th2, or
mixed Th1/Th2 adaptive immune response (Table 9.2).
9.3.1 Induced Th1 Responses
Cells expressing defensins or vaccines containing defensins induce strong Th1

responses resulting in protection from lethal cell challenges and appear to have
clinical promise in combating cancer (Biragyn 2005). There are three nice examples
of antimicrobial peptide-induced Th1 responses. In one example, mice receiving
L1210 cells expressing murine β-defensin 2 (mDF2β) developed strong CTL and
NK cell responses with enhanced IL-12 and IFN-γ production, which protected
them from lethal challenge with L1210 cells (Ma et al. 2006). In another example,
mDF2β-based vaccines elicited potent cell-mediated responses and antitumor
immunity when genetically fused with another nonimmunogenic tumor antigen
(Biragyn et al. 2001). The fusion proteins, consisting of mDF2β linked to a tumor
antigen, acted directly on immature dendritic cells as an endogenous ligand for
TLR-4 and upregulated co-stimulatory molecules, induced dendritic cell matura-
tion, and induced the production of lymphokines (Biragyn et al. 2002).
Zebra fish immunized with a plasmid encoding zebra fish β-defensin 2 (zfBD2),
and the glycoprotein G of the spring viremia of carp virus (gpGsvcv) developed a Th1
immune response with an upregulated IFN-γ response (Garcia-Valtanen et al. 2014).
Expression of zfBD2 upregulated IS mx genes related to the activation of the type I
IFN system. It also induced the transcription of proinflammatory cytokine genes
tnfα and il1β, increased the presence of mhc2 transcripts related to MHC class II
presentation of antigens, enhanced granzyme and NK lysine transcripts related to
immune cytotoxic responses cytotoxic responses, and mediated recruitment of Th
cells at the injection site.
9.3.2 Induced Th2 Responses
In the presence of an antigen, some cationic peptides induce immunized animals to

produce primary Th2-specific antibodies and Th2 cytokines from antigen-exposed
splenocytes of immunized animals. Cationic peptide KLKL5KLK is one of these
peptides. Mice immunized with KLKL5KLK with ovalbumin produce ovalbumin-
specific IgG1, but not IgG2, and splenocytes from immunized mice, stimulated with
ovalbumin, produce IL-4 and IL-5, but not IFN-γ (Fritz et al. 2004).
9.3.3 Induced Th1/Th2 Responses
More commonly than the above two examples, antimicrobial peptides induce
immunized animals to produce a mixed response characterized by producing Th1-/
Th2-specific antibodies and Th1/Th2 cytokines from antigen-exposed splenocytes
of immunized animals. Melittin from bees enhances a mixed Th1/Th2 response to
tetanus toxoid in mice. Melittin increases tetanus toxoid total IgG and IgG2a anti-
body responses (Bramwell et al. 2003). Similarly CRAMP enhances mixed Th1/
Th2 antigen-specific immune responses to ovalbumin in mice. CRAMP increases
ovalbumin IgG1, IgG2a, IgG2b, and IgG3 antibody responses (Kurosaka et al.
2005). LL-37 enhances a mixed Th1/Th2 humoral, cytotoxic, and protective
response in mice when LL-37 was fused with M-CSFRJ6-1, an M-CSF receptor
cloned from J6-1 leukemia cells (M-CSFRJ6-1). Splenocytes from immunized mice,
stimulated with M-CSFRJ6-1, produced IFN-γ (An et al. 2005).
A mixed Th1/Th2 response is also induced by defensins. HNP-1, HNP-2, and
HNP-3 enhance keyhole limpet hemocyanin (KLH) IgG1, IgG2a, and IgG2b anti-
body responses (Tani et al. 2000), and HNP-1, HNP-2, and HNP-3 and human
β-defensins enhance ovalbumin-specific IgG1, IgG2a, and IgG2b antibody
responses (Lillard et al. 1999; Brogden et al. 2003). T cells from KLH-immunized
mice, stimulated with KLH, produce KLH-specific IFN-γ (Th1 cytokine) and IL-4
(Th2 cytokine) (Tani et al. 2000). T cells from ovalbumin immunized mice, stimu-
lated with ovalbumin, produce greater amounts of CD4+ Th1 and Th2 cytokines
(IFN-γ, IL-5, IL-6, and IL-10) (Lillard et al. 1999; Brogden et al. 2003).
More recently, a human adenovirus vector expressing mDF2β (e.g., HAd-

mDF2β) was found to chemoattract murine bone marrow-derived immature den-
dritic cells and increase their surface expression levels of CD40, CD80, and CD86
activation markers (Vemula et al. 2013). Immunization with the HAd-mDF2β vec-
tor prior to immunization with a human adenovirus-hemagglutinin-nucleoprotein
vaccine significantly increases hemagglutinin inhibition antibody titers and
increases nucleoprotein-147 epitope-specific CD8+ T cells. Immunization also
decreases virus titers of VNH5N1-PR8/CDC-RG in the lungs of challenged mice.
9.4 Antimicrobial Peptides Modulate Chemokine

and Cytokine Responses
Cells treated with antimicrobial peptides alone or with a microbial antigen have
both proinflammatory and antiinflammatory activities: a dichotomy that is not
entirely well understood (Harvey et al. 2013). These proinflammatory and antiin-
flammatory activities appear to be dependent upon a number of conditions that
include antimicrobial peptide concentration, antimicrobial peptide association with
proinflammatory agonists, or the temporal order of peptide exposure to cells, with
respect to agonist exposure to cells.
9.4.1 Exposure of Cells to Low Concentrations

of Antimicrobial Peptides Is an Antiinflammatory Event
Generally, cells exposed to <1.0–10.0 μg/ml antimicrobial peptide do not produce

much of a chemokine and proinflammatory cytokine response. 0.003–0.03 μg/ml
HNP alone does not induce TNF-α or IL-1β expression in resting monocytes (Chaly
et al. 2000), and 5.0 μg/ml mBD14, HBD2, or HBD3 does not produce TNF-α in
murine bone marrow-derived macrophages (Barabas et al. 2013).
9.4.2 Exposure of Cells to High Concentrations

of Antimicrobial Peptides Is a Proinflammatory Event
Generally, cells exposed to 10.0 to >100.0 μg/ml antimicrobial peptide produce

higher amounts of chemokines and proinflammatory cytokines in a dose-related
fashion. Monocytes and macrophages treated with 20.0 μg/ml HBD3 or 20.0 μg/ml
of LL-37 produce Gro-α, MDC, MCP-1, MIP-1α, MIP-1β, and VEGF (Petrov et al.
2013). Keratinocytes treated with 30 μg/ml HBD2, HBD3, or HBD4 produce ele-
vated IL-6, IL-10, IP-10 (CXCL10), MCP-1 (CCL2), MIP-3α (CCL20), and
RANTES (CCL5) (Niyonsaba et al. 2007). RAW264.7 murine macrophages treated
with 50.0–100.0 μg/ml LL-37 produce 200, 400, and >1,000 pg/ml MCP-1 (CCL2)
(Scott et al. 2002); and A549 human epithelial cells treated with 10–100 μg/ml
LL-37 produce 300–1,200 pg/ml IL-8 (Scott et al. 2002). Epithelial cells treated
with 100.0 μg/ml HNP-1, HNP-2, and HNP-3 induce ~17,000 pg/ml IL-8 (Van
Wetering et al. 1997).
9.4.3 Antimicrobial Peptide Binding to Proinflammatory

Agonists Is an Antiinflammatory Event
Antimicrobial peptides readily bind to microbial lipopolysaccharides, adhesins, and

toxins with rapid association rate constants, slower dissociation rate constants, and
high affinity (Caccavo et al. 2002; Wang et al. 2003, 2006; Owen et al. 2004a, b; Liu
et al. 2013a; Yu et al. 2013; Dietrich et al. 2008; Pingel et al. 2008). This binding
generally alters the physiological properties of the agonist (Gough et al. 1996;
Bowdish and Hancock 2005; Motzkus et al. 2006; Scott et al. 2011; Kim et al. 2005,
2006; Giesemann et al. 2008; Yeom et al. 2011).
The binding of antimicrobial peptides to microbial lipopolysaccharides, adhes-
ins, and toxins also alters binding of these agonists to cell surface receptors (Gallo
and Hooper 2012). Lactoferrin is a good example. It inhibits the interaction of LPS
with CD14 on cell surfaces by competing with the LPS-binding protein (Molhoek
et al. 2009), and it blocks DC-SIGN-gp120 interaction and prevents dendritic cell-
mediated HIV type 1 transmission (Groot et al. 2005). HBD3 alters the binding of
Porphyromonas gingivalis hemagglutinin B (HagB) to the surface of dendritic cells
(Van Hemert et al. 2012) and HNP-1, HBD1, HBD2, HBD3, DEFB104, and LL-37,
all inhibit binding of Alexa Fluor 546-labeled P. intermedia and T. forsythia LPS to
THP-1 human monocytes (Lee et al. 2010).
It also appears that this changes agonist-induced signal transduction.
Antimicrobial peptides can selectively attenuate agonist-induced signal transduc-
tion including MAPK pathways involving p38, c-Jun NH2-terminal protein kinases
(JNK), or extracellular signal-regulated kinase (ERK). DEFB114 attenuates an
LPS-induced activation of p42/44 responses in RAW264.7 murine macrophages
(Yu et al. 2013), and DEFB123 (Motzkus et al. 2006) and DEFB126 (Liu et al.
2013a) attenuate an LPS-induced activation of p42/44 and p38 responses in
RAW264.7 murine macrophages. LL-37 attenuates a P. gingivalis extract-induced
activation of p38 and ERK responses in human gingival fibroblasts (Inomata et al.
2010). Modulation of the TLR response by LL-37 occurs at least partly through
inhibition of p38 phosphorylation (Walters et al. 2010).
The resulting chemokine and proinflammatory cytokine output is noticeably
reduced, and this is a very popular area of research. Lactoferrin attenuates an LPS-
induced IL-1β, IL-6, and ICAM-1 mRNA response in bovine aortic endothelial
cells (Yeom et al. 2011). Cathelicidins also attenuate chemokine and proinflamma-
tory cytokine output. LL-37 attenuates periodontopathogenic LPS-induced IL-8
responses in human periodontal ligament fibroblasts and gingival fibroblasts
(Suphasiriroj et al. 2013); P. aeruginosa LPS-induced IL-8 response in THP-1

human monocyte cells (Scott et al. 2011); LPS-induced TNF-α response in mouse
bone marrow-derived macrophages and tissue macrophages (Brown et al. 2011);
LPS-induced, S. aureus lipoteichoic acid-induced, and Mycobacterium
lipoarabinomannan-induced TNF-α response in RAW264.7 murine macrophages
and IL-8 and MCP-1 (CCL2) response in A549 human lung epithelial cells (Scott
et al. 2000); and P. gingivalis extract-induced IL-6, IL-8, and IP-10 (CXCL10)
responses in human gingival fibroblasts (Inomata et al. 2010).
Similarly, defensins attenuate agonist-induced chemokine and cytokine
responses. HNP-1 attenuates an LPS-induced IL-1β, but not TNF-α, response in
human monocytes (Shi et al. 2007); DEFA1-3 attenuates P. aeruginosa-induced
TNF-α, IL-8, IL-6, and IL-1β responses in human monocyte-derived macrophages
(Miles et al. 2009); and HNP-1 and HNP-3 attenuate P. intermedia LPS-induced
IL-1β, IL-8, and ICAM-1 responses in THP-1 human monocytes and HGF cells
(Lee et al. 2010). HBD1, HBD2, HBD3, and DEFB104A attenuate P. intermedia
LPS-induced IL-1β, IL-8, and ICAM-1 responses in THP-1 human monocytes and
HGF cells (Lee et al. 2010); HBD3 attenuates the IL-6, IL-10, GM-CSF, and TNF-α
responses of HagB-induced human myeloid dendritic cells (Pingel et al. 2008);
DEFB114 attenuates an LPS-induced TNF-α response in RAW264.7 murine mac-
rophages (Yu et al. 2013); and DEFB123 (Motzkus et al. 2006) and DEFB126 (Liu
et al. 2013a) attenuate an LPS-induced IL-6 (e.g., DEFB126) and TNF-α (e.g.,
DEFB123, DEFB126) response in RAW264.7 murine macrophages. Finally,
θ-defensin retrocyclin RTD-1 inhibits a TLR2, 4, and 5 agonist-induced TNF-α,
IL-1α, IL-1β, IL-6, IL-8, MCP-1 (CCL2), MIP-1α (CCL3), and MIP-1β (CCL4)
responses in human peripheral blood leukocytes (Schaal et al. 2012).
Histatin 5 suppresses the induction of IL-6 and IL-8 in P. gingivalis outer mem-
brane protein-induced human gingival fibroblasts, and this activity is more effective
when outer membrane protein is incubated with histatin 5 before addition to the cell
culture (Imatani et al. 2000). Histatin 5 also attenuates a P. gingivalis hemagglutinin
B (HagB)-induced chemokine and proinflammatory cytokine response in dendritic
cells (Borgwardt et al. 2014). 20.0 mM histatin 5, mixed with 0.02 mM HagB, atten-
uates an HagB-induced CCL3/MIP-1α, CCL4/MIP-1β, and TNF-α responses.
9.4.4 The Order of Peptide Exposure to Cells, with Respect

to Agonist Exposure to Cells, Is a Proinflammatory Event
This, too, is a recent and very exciting area of research. Antimicrobial peptides
given before or after a proinflammatory agonist induce cells to produce higher con-
centrations of proinflammatory mediators. When given together, LL-37 attenuates
LPS-induced TNF-α responses (Scott et al. 2011; Brown et al. 2011). However,
when THP-1 monocytes are treated with LL-37 for 1 h before the addition of LPS
(Scott et al. 2011) or 3 h after incubation with LPS (Brown et al. 2011), attenuation
is abolished. In yet another example, pre-mixing of leukocytes and E. coli for up to
2 h, followed by addition of retrocyclin RTD-1, led to a reduction of TNF-α levels

(46–93 %) by RTD-1 at each time point (Schaal et al. 2012).
In our work, HBD3 given before or after a proinflammatory agonist induces
human dendritic cells, murine JAWSII dendritic cells and mice to produce higher
concentrations of proinflammatory mediators (Harvey et al. 2013). HBD3 (0.2, 2.0,
or 20.0 μM) given to human myeloid dendritic cells pre- (1 h before), co-, or post-
(1 h after) HagB treatment (0.02 or 0.2 μM) displayed a concentration-dependent
ability to both attenuate and enhance the chemokine and proinflammatory cytokine
response. Timing is important, and MIP-1α (CCL3), MIP-1β (CCL4), and TNF-α
responses to 0.02 μM HagB are both enhanced and attenuated when 0.2 and 2.0 μM
HBD3 is given pre-/post- or co-HagB exposure, respectively.
9.5 Wound Healing and Angiogenesis
Wound healing occurs in three phases: an inflammatory phase, a proliferative

phase, and a maturational phase (Sinno and Prakash 2013), all involving multiple
steps in hemostasis, inflammation, remodeling, formation of granulation tissue,
and reepithelialization (Ramos et al. 2011). The process involves various cells like
fibroblasts, keratinocytes, endothelial cells, growth factors, extracellular matrix
components, and chemokines and cytokines. Antimicrobial peptides can be
detected in the margins around both oral and cutaneous wounds, and there is a
growing body of evidence to suggest that they also play a dynamic role in the
wound healing process at multiple steps. Furthermore, a large number of antimi-
crobial peptides do have properties that have the ability to speed wound healing
and angiogenesis.
Early steps involve cell migration and proliferation. Histatins and LL-37 induce
fibroblast migration, HBD2 promotes keratinocyte migration (Niyonsaba et al.
2007), and LL-37 induces human microvascular endothelial cell and human umbili-
cal vein endothelial cell migration (Ramos et al. 2011). Histatins and LL-37 also
induce fibroblast proliferation, HBD2 increases keratinocyte proliferation
(Niyonsaba et al. 2007; Warnke et al. 2013), and LL-37 induces human microvascu-
lar endothelial cell and human umbilical vein endothelial cell proliferation (Ramos
et al. 2011).
Later steps involve wound angiogenesis, vascularization, and reepithelialization,
and LL-37 again is particularly active. LL-37 stimulates angiogenesis (Nakatsuji
and Gallo 2012) and induces the formation of tubule-like structures (Ramos et al.
2011). Another cathelicidin CRAMP has angiogenic properties, too (Kurosaka
et al. 2005). LL-37 also stimulates reepithelialization (Heilborn et al. 2003; Ramos
et al. 2011; Nakatsuji and Gallo 2012).
One unique mechanism, discussed in detail below, involves LL-37 produced by
keratinocytes in injured skin (Lande et al. 2007). Here LL-37 combines with self-
DNA released from injured or dead tissues and initiates immune responses in dam-
aged skin enhancing resistance to infection and initiating wound healing.
9.6 Autoimmune Functions
Defensins and LL-37 bind to CpG, self-DNA, and RNA (Tewary et al. 2013; Frasca
and Lande 2012; Lande et al. 2007; Gilliet and Lande 2008). This interaction forms
complexes. HBD3 + human genomic DNA forms large complex DNA nets (Tewary
et al. 2013), and LL-37 + DNA forms aggregated and condensed structures (Lande
et al. 2007). These complexes are readily taken up by plasmacytoid dendritic cells in
a TLR9 (e.g., HBD3/DNA; LL-37/DNA)-dependent manner in the endocytotic path-
way and induce the production of IFN-α (Tewary et al. 2013; Lande et al. 2007). In
mice, CpG + HBD3 complexes administered intravenously alone induce proinflam-
matory cytokines in serum, administered subcutaneously alone induce the formation
of local inflammatory cell infiltrates, and administered intraperitoneally with an
immunogen like ovalbumin enhance both cellular and humoral responses to ovalbu-
min. Nakatsuji and Gallo suggest that this is an important normal physiological and
immunological function leading to the attraction of various immune cells (Nakatsuji
and Gallo 2012). Tewary and colleagues suggest that these complexes could improve
vaccine formulations and enhance immune responses (Tewary et al. 2013). However,
they also point out that these complexes are found to be a constituent of circulating
immune complexes isolated from sera in patients with autoimmune diseases (Tewary
et al. 2013). Frasca and Lande (2012) and Gilliet and Lande (2008) also suggest that
this mechanism may lead to autoimmune and autoinflammatory diseases.
There is a growing body of evidence that suggests defensins and LL-37 have
roles in autoimmune and autoinflammatory diseases like psoriasis, rosacea, ulcer-
ative colitis, rheumatic joint disease, and systemic lupus erythematosus (Frasca and
Lande 2012; Vordenbaumen et al. 2010). Subjects with autoimmune disease have
increased circulating concentrations of α- and β-defensins (Vordenbaumen et al.
2010). In the sera of subjects with systemic lupus erythematosus, concentrations of
HBD2 correlate with red blood cell count, dsDNA antibody titers, systemic lupus
erythematosus disease activity index, and clinical transverse myelitis and myositis
(Vordenbaumen et al. 2010). Similarly, serum HNP concentration correlates with
subject white blood cell counts and clinical transverse myelitis and rash
(Vordenbaumen et al. 2010). The relative amounts of HNP mRNA from neutrophils
correlate with C3c concentrations, systemic lupus erythematosus disease activity
index, and clinical renal involvement and rash (Vordenbaumen et al. 2010).
Clearly these are interesting findings, and further work is needed to clarify the
roles of defensins and LL-37 (and other antimicrobial peptides) in the pathophysiol-
ogy of autoimmune and autoinflammatory diseases (Frasca and Lande 2012; Gilliet
and Lande 2008).
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Chapter 10
Antimicrobial Peptides: Do They Have
a Future as Therapeutics?
Michael Zasloff
Abstract Antimicrobial peptides of higher organisms have been studied for the
past 25 years and their importance as components of innate immunity is now well
established. The basic simplicity of the chemical structure of antimicrobial peptides
along with the lower likelihood of the emergence of resistance compared with con-
ventional antibiotics have made them attractive candidates for development as ther-
apeutics. In this chapter, I describe the stories behind three drug candidates currently
in clinical trials: Pexiganan, Plectasin, and Brilacidin. Each of these compounds has
faced specific challenges in development and has a high likelihood of reaching com-
mercialization. Antimicrobial peptides appear to be coming of age as therapeutics.
10.1 Introduction
I have been asked to address the question as to whether antimicrobial peptides have
a future as therapeutics. Antimicrobial peptides of multicellular organisms were
characterized in the 1980s from insects, mammals, and frogs (Zasloff 2002). As yet,
antimicrobial agents of this class have not yet been approved as drugs. It is reason-
able to ask what the problems have been that have interfered with their commercial-
ization. In this piece I will focus on three examples of antimicrobial peptides that
have advanced deeply into development but faced specific challenges that inter-
rupted their progress: Pexiganan developed by Magainin Pharmaceuticals; plecta-
sin, developed by Novozymes; and brilacidin, developed by Polymedix. Although
there are other compounds that could be included, I have selected these because I
am personally most familiar with these three, and I believe the lessons learned apply
broadly to all compounds of this class destined for therapeutic development.
M. Zasloff, MD, PhD

Department of Surgery, MedStar Georgetown Transplant Institute,
Georgetown University Hospital,
3800 Reservoir Road NW, Washington, DC 20007, USA

DOI 10.1007/978-3-319-24199-9_10
148 M. Zasloff
Fig. 10.1 The amino acid sequences of pexiganan, magainin 1 and magainin 2, are compared.
Each peptide has an amide at the carboxyl terminus. Lysine residues are highlighted in gray
10.2 Pexiganan
Pexiganan was developed as a topical anti-infective. It is a 22 amino acid analogue

of magainin 2, the natural peptide produced by Xenopus laevis (Zasloff 1987; Ge
et al. 1999a, b). Pexiganan differs from magainin 2 by the substitution of 5 lysines
and the deletion of a glutamic acid (Fig. 10.1), which dramatically extends the anti-
microbial spectrum of the peptide while only minimally increasing its lytic activity
against mammalian cells. These substitutions were conceived using the principle
that linear antimicrobial peptides capable of forming alpha helical structures in
membranes would likely exhibit greater antimicrobial potency by increasing the
density of positively charged amino acids on the hydrophilic face of the alpha helix.
The decision to develop this peptide as a locally applied therapeutic was based on
the results of extensive in vivo assessments of the anti-infective properties of magainin
2 in systemic infections. It was evident that the therapeutic index of this peptide was
too narrow to be advanced as a systemically administered drug. Pexiganan, although
more active in vitro than magainin 2, did not exhibit a larger therapeutic window.
Initially we chose to develop pexiganan against the superficial infection, impetigo,
but subsequently focused on its use for the treatment of infected diabetic foot ulcers.
Infected diabetic ulcers remain a major medical problem. About 50,000 amputa-
tions are performed annually on the lower limbs of diabetics due to the progressive and
irreversible destruction of soft tissue and bone that often follows the appearance of a
minor infection on the foot. Although the etiology of these infections is not fully
understood, patients are advised to attend to even the most minor signs of injury on the
foot, including debridement of open wounds and the administration of broad-spectrum
antibiotics if any indication of infection is evident (swelling, redness, purulent dis-
charge). The infections are complex polymicrobial, and no specific microorganism is
implicated in the destructive outcome (Ge et al. 2002). The antibiotics must be admin-
istered until control of the infection is clinically apparent, generally about 1 month of
treatment. Unfortunately, many individuals find these broad-spectrum antibiotics
10 Antimicrobial Peptides: Do They Have a Future as Therapeutics? 149
intolerable, due to side effects such a diarrhea, and frequently stop taking them despite
being fully aware of the dire consequences of progressive infection.
Pexiganan was developed as a locally applied alternative to a systemically
administered broad-spectrum antibiotic. The antibiotic spectrum was broad enough
to cover the range of microbes known to be present on the diabetic ulcer. Furthermore,
as a topical agent, high local concentrations in the superficial soft tissues could be
achieved easily, surpassing the concentrations that could be reached systemically.
We chose as our intended diabetic patients those with the most superficial of infected
ulcers, excluding those cases where deep penetration of a topically applied ointment
could not occur, including those with soft tissue infections in close proximity to
bone. We argued that high concentrations of pexiganan would also reduce the likeli-
hood of selection of resistant organisms and thus could be used repeatedly over the
course of a patient’s lifetime.
The choice of developing pexiganan as a topical agent was also influenced by sev-
eral practical and strategic considerations. The primary structure of the peptide makes
it susceptible to proteolytic cleavage, especially by trypsin-like enzymes. As a conse-
quence, the intact peptide does not pass from the soft tissue into the systemic circula-
tion. This property permitted us to drastically reduce very costly preclinical toxicology
prior to clinical evaluation, since chronic systemic exposure to an intact peptide would
not occur following long-term local application to an open wound. In addition, a
therapeutic course of local administration would require relatively small amounts of
peptide compared with systemic administration. Based on the minimal inhibitory
concentrations of pexiganan, one could estimate that intravenous dosing would
require about 1 g daily, not very different from the dosing of most antibiotics currently
in use. At the time we began the development process, the chemical synthesis of pexi-
ganan cost about $1000 per gram, using our proprietary solution-phase process. Only
if this peptide were dramatically more effective than the less expensive agents in use
would it make sense to develop it for systemic use, and we did not believe that this
was the case. As a topical agent, however, much lesser amounts of peptide could be
applied directly to a site of infection, and the cost of goods relative to the anticipated
price of the commercial product made sense. Eventually, we were able to bring the
cost of production of pharmaceutical grade pexiganan down to about $100 per gram.
We conducted two extensive pivotal phase III clinical trials in the late 1990s
involving about 1000 subjects (Lipsky et al. 2008). Topically applied pexiganan was
evaluated against orally administered ofloxacin in what is a called an “inferiority”
study. The hypothesis being tested was whether topically applied pexiganan exhib-
ited was less effective than orally administered ofloxacin, an antibiotic approved for
deep soft tissue infections (no specific agent had been approved for diabetic foot
infections, so the FDA permitted us to use this antibiotic as the comparator). In both
treatment arms, the wounds were debrided of dead tissue as part of the study. Ideally,
it would have been scientifically more exciting to have conducted a “superiority”
trial, where pexiganan treatment was compared to simple wound debridement (a
placebo-controlled trial), but all parties, including the FDA and participating physi-
cians and patients, felt this to be unethical.
Pexiganan proved itself to be as effective as oral ofloxacin in the treatment of
infected diabetic foot ulcers over about a month of treatment. Of particular interest
150 M. Zasloff
was that pexiganan-resistant bacteria did not appear on the treated wounds. In con-
trast, the microbes cultured from the wounds of those subjects treated with ofloxacin
shifted to a more resistant concentration range following the month of therapy. The
rates and extent of wound healing were comparable as were the impact on the clini-
cal appearance of the treated ulcers. As anticipated, those receiving topical treat-
ment reported fewer side effects than those taking the oral therapeutics.
Despite the positive outcome, the FDA Advisory Panel voted 7–5 against
approval of pexiganan. Their primary concern was the uncertainty over the “placebo”
outcome. Suppose these individuals had not been treated with any antibiotic, just
surgical curettage, what would the outcome have been? Despite being presented
with the historical data then available, the panel was not convinced. Indeed, Dr.
Julie Parsonnet of Stanford University suggested to the panel that all a doctor had to
do to treat an infected ulcer was “to cut out the infected tissue and drop it into a trash
can and forget the antibiotics!” (She herself had never treated such a patient, nor had
most members of that FDA panel). Several panelists could not believe that it was
possible for a topical agent to be as effective as we had claimed. In response to the
Advisory Panel, the FDA requested that we repeat a trial using a placebo arm. We
did not believe this to be practical, and the pexiganan program was halted.
Over the past few years, pexiganan’s development as a topical agent for the treat-
ment of infected diabetic foot ulcers has been taken up by Dipexium Pharmaceuticals,
Inc. Working closely with the FDA and many of the American experts in the treat-
ment of infected diabetic foot ulcers, the company has designed a pivotal phase III
placebo-controlled trial which should both fulfill the requirements of a superiority
study and also satisfy any concerns regarding the placing of untreated patients at
risk. Within the next few years, we will learn of this study’s outcome.
10.3 Plectasin
The discovery and characterization of plectasin was first reported in 2005 (Mygind
et al. 2005) (Fig. 10.2). A team of scientists at Novozymes, headed by Dr. Hans
Henrik Kristensen, had exploited a novel method for the discovery of antimicrobial
peptides from natural sources. The team had created a cDNA library from a wild
mushroom like fungus (Pseudoplectania nigrella). By reengineering the library, the
team searched for the expressed gene products that would be secreted (i.e., that con-
tained “signal” sequences). Of the cDNAs identified, one exhibited the primary
sequence of a defensin antimicrobial peptide. Using several fungal- and yeast-based
expression systems, the Novozymes team biosynthetically produced the peptide in
sufficient amounts to begin characterization. The defensin, named “plectasin,” was
found to have a primary, secondary, and tertiary structure very similar (surprisingly!)
to defensins from insects and mussels. Of particular interest was its antibacterial
spectrum. Of the bacteria studied, plectasin exhibited its greatest potency against
Streptococcus pneumonia (Mygind et al. 2005). In vivo, plectasin demonstrated
potency when administered systemically that equaled or exceeded conventional anti-
biotics such as penicillin. In addition, the antibiotic, following parenteral
Fig. 10.2 A representative

tertiary structure of
plectasin as determined by
solution NMR presented
beneath the primary
structure (Mygind et al.
2005)
administration to the mouse, could be recovered intact from its urine. Plectasin was
essentially nontoxic in early preclinical experiments. Its mechanism of action was
established and shown to involve interaction with lipid II (Schneider et al. 2010).
Almost all of the clinical isolates of Pneumococcus studied exhibited a minimal
inhibitory concentration at sub-micromolar values, suggesting that most infections
would be effectively treated initially with this antibiotic. Furthermore, because plec-
tasin was of fungal origin, it could be expressed robustly in several recombinant
vectors used for the industrial production of commodity substances, such as enzymes
used in the food industry. Hence, it could potentially be produced inexpensively and
in large amounts.
From one perspective plectasin was an ideal antibiotic. Here was a narrow-
spectrum antibiotic that a physician could call upon to treat an infection caused by
Pneumococcus. In a setting where a specific bacterial organism is involved, a spe-
cific antibiotic should be used. Since many strains of Pneumococcus have devel-
oped resistance to beta-lactams, and plectasin retained activity against
penicillin-resistant Pneumococcus, plectasin could become the antibiotic of choice
for the treatment of pneumococcal infections.
Unfortunately, the economics of drug development intervened. The market for a
pneumococcal-specific antibiotic was too small to justify the expensive investment
required to bring the antibiotic through development into clinical use. The
Novozymes team began a long and highly creative series of structure-activity stud-
ies to extend the spectrum of plectasin so as to include human pathogens such as
Staphylococcus aureus. The Kristensen team succeeded creating the plectasin
derivative NZ2114 which was effective against systemic S. aureus infections in sev-
eral animal models (Andes et al. 2009; Xiong et al. 2011). Similar to plectasin,
NZ2114 could be produced at commercial viable expression levels through recom-
binant expression in a yeast system (Zhang et al. 2014).
In late 2009 Sanofi-Aventis entered a partnership with Novozymes to lead the
clinical development of NZ2114 and in early 2010 announced that planning for a
phase 1 clinical program had begun. Very little public information has been dis-
closed since then.
152 M. Zasloff
10.4 Brilacidin
Linear amphipathic alpha helical cationic antimicrobial peptides target the external
membranes of microbes. In some cases they damage the permeability of the mem-
brane; in others they translocate into to the cytoplasm and cause functional distur-
bance. Molecules that adopt comparable secondary structures can exhibit antimicrobial
activity, and many synthetic mimetics of the naturally occurring linear peptides have
been synthesized. In 2002 DeGrado reported the synthesis and characterization of a
novel family of arylamide amphipathic anionic polymers that exhibited many of the
physical and biological properties of antimicrobial peptides, such as magainin (Tew
et al. 2002). These mimetics could be synthesized from inexpensive monomers via
classical polymerization methods and were resistant to proteolytic hydrolysis. Through
structure-activity refinements, potency against Gram-positive bacteria was optimized
while maintaining minimal lytic activity against human erythrocytes, resulting in the
creation of brilacidin (Fig. 10.3) (Choi et al. 2009). This molecule adopts a planar
secondary structure, with guanidinyl and pyridinyl groups positioned on one edge of
the scaffold and trifluoromethane groups on another. As reported recently, brilacidin
acts by selectively damaging the microbial membrane, similar to linear antimicrobial
peptides and the lipopeptide in commercial use, daptomycin (Mensa et al. 2014).
Brilacidin was initially licensed to Polymedix, Inc., and subsequently to
Cellceutix, Inc. It has been advanced in development as a parenteral drug for the
treatment of acute skin and skin structure infections caused by Gram-positive bac-
teria, positioned to compete with drugs such as daptomycin.
Brilacidin advanced successfully through phase 1 and phase 2 clinical trials. On
October 23, 2014, Cellceutix reported unpublished results of a phase 2b randomized
double-blind study comparing brilacidin to daptomycin for the treatment of Gram-
positive acute skin and skin structure infections (http://cellceutix.com/cellceutix-
releases-confidence-interval-statistics-showing-clinical-success-rates-for-brilacidin-
in-treatment-of-absssi/#sthash.UpDWgko4.dpbs). Two-hundred and fifteen subjects
were enrolled. The primary end point was clinical success defined as reduction of at
least 20 % in area of the infected site when evaluated 48–72 h after the first dose of the
NH HN
NH H O H N N H O H NH
( ) N N N N ( )
H2N N 4 4 N NH2
H O O O O H
F F F F
F F
Brilacidin
Fig. 10.3 The chemical structure of brilacidin is shown

study drug. As reported by Cellceutix: “In treated patients assessed at 48–72 h, 47/51
(92.2 %), 46/48 (95.8 %), 51/52 (98.1 %), and 45/48 (93.8 %) achieved clinical suc-
cess in the brilacidin 0.6 mg/kg single-dose group, brilacidin 0.8 mg/kg single-dose
group, brilacidin 1.2 mg/kg 3-day group, and daptomycin 7-day group, respectively.
All three brilacidin treatment arms (two single-dose regimens and one 3-day dose
regimen) reached the primary endpoint, with the clinical success rate for each dosing
regimen statistically comparable to the clinical success rate of the FDA-approved
7-day dosing regimen of daptomycin. All brilacidin treatment regimens were well
tolerated. There were six severe adverse events (SAE) reported across the study, none
of which were considered related to brilacidin by the principal investigator.” Brilacidin
will be advanced into a phase 3 clinical trial.
10.5 Conclusions
The three compounds that I have discussed are advancing through the therapeutic
development process. Unless they face clinical failure, due either to a lack of effi-
cacy or an unacceptable toxicity, they will represent the first “graduating class” of
antimicrobial peptides.
Each of the three highlights several attractive characteristics of antimicrobial
peptides:
1. Antimicrobial resistance does not readily occur.
2. The mechanism of action can be reproduced with molecules as structurally dis-
tant from a linear peptide as brilacidin.
3. Certain naturally occurring peptides from fungal sources with very narrow anti-
biotic spectra, such as plectasin, could be developed for the treatment of specific
bacterial infections. Many uncharacterized fungal defensins have already been
identified (Zhu 2008).
4. Naturally occurring antimicrobial peptides, such as plectasin, exhibit acceptable
therapeutic indices and can serve as the models upon which the structural prin-
ciples that govern bio-distribution of therapeutically effective antimicrobial pep-
tides can be deciphered.
5. Antimicrobial peptides and mimetics can be commercially synthesized
cost-effectively.
6. As yet, no class specific toxicity has become apparent.
Although it seems like an eternity that we have been awaiting the introduction of
antimicrobial peptides into the clinic, we are now on the threshold of seeing this
happen. We are now facing a medical crisis some have called the “End of the
Antibiotic Era.” Resistance of both Gram-positive and Gram-negative human patho-
gens to our conventional antibiotics has spread throughout the world. Infections that
could be treated routinely several years ago now present as life-threatening medical
challenges. The introduction of antimicrobial peptides as therapeutic drugs could
not be happening at a more critical moment in time than now.
154 M. Zasloff
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Birkhäuser Advances in Infectious Diseases

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Birkhäuser Advances in Infectious Diseases

Library of Congress Control Number: 2015960250

Springer Cham Heidelberg New York Dordrecht London

Printed on acid-free paper

Springer International Publishing AG Switzerland is part of Springer Science+Business Media

1 Antimicrobial Peptides in Cutaneous Wound Healing . . . . . . . . . . . . . . 1

9 Antimicrobial Peptides in Host Defense: Functions

© Springer International Publishing Switzerland 2016 1

1.2 Generation of AMPs at the Different Stages of Wound

After hemostasis follows influx of neutrophils followed by monocytes and lympho-

S100A8/S100A9 constitutes 40 % neutrophil cytosolic protein (Edgeworth et al.

1.2.4 Proliferative Phase

The expression of some AMPs during wound healing is dependent on inflamma-

The epidermal AMPs induced by wound healing generate a broad spectrum on

1.2.5 Tissue Remodeling Phase

Tissue remodeling in cutaneous wound healing involves increased expression of

1.2.6 Chronic Wounds

1.3 Concluding Remarks and Future Perspective

Abdillahi SM, Balvanovic S, Baumgarten M, Mörgelin M (2012) Collagen VI encodes antimicro-

Fabian Garreis, Martin Schicht, and Friedrich Paulsen

Abstract Ocular surface infections are a significant cause of blindness worldwide

F. Garreis • M. Schicht • F. Paulsen (*)

© Springer International Publishing Switzerland 2016 17

2.2 Tear Film Composition

2.2.1 Antimicrobial Compounds at the Ocular Surface,

showed reduced mortality of Gram-negative bacteria-induced sepsis (Domingues

2.2.2 Surfactant Proteins

2.2.3 Antimicrobial Peptides (AMPs)

2.2.3.1 Human Defensins

2.2.3.2 Human Cathelicidin LL-37

In humans, only one member of the cathelicidin family is functionally expressed:

2.2.3.3 Other AMPs

2.3 Antimicrobial Activity of AMPs

2.3.1 Antibacterial Mechanism

Direct antimicrobial activity is based on the charge-dependent interaction of posi-

2.3.2 Antiviral Mechanisms

clarified yet whether AMP-mediated aggregation inhibits virus infection in vivo.

Microbial keratitis is an infectious disease of the cornea that is characterized by

2.4.1 Bacterial Keratitis

3 (putative orthologs of hBD-2) are important to prevent P. aeruginosa infection in

2.4.2 Herpes Simplex Virus Keratitis

Fig. 2.3 Herpes simplex

increasing level of research interest. Thus, α-defensins have been demonstrated to

2.4.3 AMPs and Other Viral Keratitis Forms

2.4.4 AMPs for Treating Ocular Surface Infections

Abedin A, Mohammed I, Hopkinson A et al (2008) A novel antimicrobial peptide on the ocular

Vandamme D, Landuyt B, Luyten W et al (2012) A comprehensive summary of LL-37, the facto-

Frederik Seiler, Robert Bals, and Christoph Beisswenger

3.1 The Innate Immune Network of the Lung

The human lung is continuously exposed to a broad array of airborne pathogens

F. Seiler • R. Bals • C. Beisswenger (*)

© Springer International Publishing Switzerland 2016 33

3.2 Expression of Antimicrobial Peptides in the Lung

Class Peptide name Gene name

In the lung, the only human cathelicidin LL-37/hCAP-18 is expressed by different

3.3 Regulation of Antimicrobial Peptide Expression

In the lung, β-defensin expression is triggered by common respiratory pathogens

In contrast to β-defensins, the expression of cathelicidin is not primarily induced