RevIews

Inflammasomes in the gastrointestinal

tract: infection, cancer and gut
microbiota homeostasis
Si Ming Man
Abstract | Inflammasome signalling is an emerging pillar of innate immunity and has a central
role in the regulation of gastrointestinal health and disease. Activation of the inflammasome
complex mediates both the release of the pro-inflammatory cytokines IL-1β and IL-18 and the
execution of a form of inflammatory cell death known as pyroptosis. In most cases, these
mediators of inflammation provide protection against bacterial, viral and protozoal infections.
However, unchecked inflammasome activities perpetuate chronic inflammation, which
underpins the molecular and pathophysiological basis of gastritis, IBD, upper and lower
gastrointestinal cancer, nonalcoholic fatty liver disease and obesity. Studies have also highlighted
an inflammasome signature in the maintenance of gut microbiota and gut–brain homeostasis.
Harnessing the immunomodulatory properties of the inflammasome could transform clinical
practice in the treatment of acute and chronic gastrointestinal and extragastrointestinal
diseases. This Review presents an overview of inflammasome biology in gastrointestinal health
and disease and describes the value of experimental and pharmacological intervention in the
treatment of inflammasome-associated clinical manifestations.

The immune system has an extraordinary capacity to the AIM2-like receptor (ALR) family; and pyrin from the
recognize and respond to a range of microbial patterns tripartite motif-containing protein (TRIM) family5
and danger signals. In the gastrointestinal tract, tril- (Fig. 1). Several other sensors have been suggested to
lions of microorganisms colonize the mucosal surface activate the inflammasome, including the NLR family
and lumen and constantly release immunomodulatory members NLRP6, NLRP7, NLRP9 (NLRP9b in mice)
molecules that interact with and shape the immune and NLRP12, the DNA sensor IFNγ-inducible protein
system1. Most of these microorganisms are symbionts 16 (IFI16) and the RNA sensor retinoic acid-inducible
or so-called commensals and do not generally evoke a gene I protein (RIG-I; also known as DDX58); however,
detrimental inflammatory response. However, patho- the ability of these sensors to form inflammasome com-
gens have the ability to invade the mucosal barrier and plexes is still uncertain6. On activation, inflammasome
underlying tissue, inducing the production of cytokines, sensors recruit the adaptor protein apoptosis-associated
chemokines and antimicrobial molecules. speck-like protein containing a caspase activation and
A central and rapid mechanism triggering an inflam- recruitment domain (ASC; also known as PYCARD)
matory response occurs through activation of the innate and the cysteine protease caspase 1, and form a single
immune signalling complex called the inflammasome2,3. cytoplasmic aggregate known as the inflammasome
The inflammasome is formed in a variety of immune speck6. Caspase 1 is activated in this complex, inducing
and non-immune cells when a subset of cytosolic proteolytic cleavage of the pro-inflammatory cytokines
Department of Immunology pattern-recognition receptors known as inflamma­some pro-IL-1β and pro-IL-18 and the pore-forming protein
and Infectious Disease, The sensors recognizes pathogen-associated molecular gasdermin D, which results in the release of bioactive
John Curtin School of Medical patterns (PAMPs) or danger-associated molecular pat- IL-1β and IL-18 into the extracellular milieu and pyrop-
Research, The Australian terns (DAMPs)4. To date, members from three families tosis, respectively7. Bioactive IL-1β and IL-18 and cer-
National University,
Canberra, Australia.
of pattern-recognition receptors are known to have an tain DAMPs can be secreted through the gasdermin D
established role in the assembly of an inflammasome: pores before rupture of the host cell membrane, cell lysis
e-mail: siming.man@
anu.edu.au NLRP1, NLRP3, NLRC4 and neuronal apoptosis inhibi­ and cell death8–10.
https://doi.org/10.1038/ tory proteins (NAIPs) from the NOD-like receptor Inflammation and cell death responses triggered by
s41575-018-0054-1 (NLR) family; absent in melanoma 2 (AIM2) from the inflammasome contribute to the pathogenesis of a

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Reviews

inflammasome requires initial recognition of the ligand

Key points
by NAIP23–27. The single NAIP encoded in the human
• Inflammasomes are expressed in both immune and non-immune cells, contributing genome is capable of recognizing the needle and inner
to their functional ties to infection, IBD, cancer, autoinflammation and autoimmune rod components of the type 3 secretion system (T3SS)
conditions. and flagellin subunits of bacteria28–30. Of the mouse
• Inflammasome sensors function by recognizing and responding to a pathogen NAIPs, NAIP5 and NAIP6 directly bind flagellin sub-
(lipopolysaccharide, microbial DNA or bacterial flagellin) or to a danger signal units, and NAIP1 and NAIP2 bind the needle and
(ion flux, self-DNA or ATP).
inner rod components of the T3SS, respectively28,31–34.
• Inflammasomes control the magnitude of inflammation and cell death in response Ligand-bound NAIPs then recruit and induce oligo­
to pathogen-associated molecular patterns or danger-associated molecular patterns,
merization of NLRC4 in order to catalyse the assem-
which, in part, determines a protective or detrimental outcome in the host.
bly of a functional NAIP–NLRC4 inflammasome
• Experimental and pharmacological interventions have yielded success in the
complex35,36 (Fig. 1).
treatment of inflammasome-mediated disorders, such as autoinflammatory
The inflammasome sensors NLRP1, NLRP3 and
enterocolitis.
pyrin do not bind directly to a ligand but respond to
• The inflammasome–gut microbiota axis and its relevance to health and disease are
a subset of cellular events triggered by PAMPs and
influenced by genetic, environmental and experimental factors.
DAMPs. Human NLRP1, rat NLRP1 and mouse NLRP1b
• The deep and complex relationship between inflammasomes, pathogens and the
are activated by any protease capable of mediating site-
microbiota provides an exciting platform for basic and clinical research with which
to understand health and disease.
specific proteolytic cleavage at the amino terminus or
function to find domain (FIIND) of NLRP1 (refs37–40),
such as the lethal factor component of the anthrax
spectrum of acute and chronic diseases in the gastro­ lethal toxin41. NLRP3 indirectly responds to a mul-
intestinal tract, including infection, gastritis, IBD, cancer, titude of PAMPs and DAMPs, including pathogens,
nonalcoholic fatty liver disease (NAFLD) and obesity11,12. toxins, crystalline substances and host and microbial
Inflammasomes might also support a healthy gut micro- metabolites14. These NLRP3 activators trigger physio­
biota and regulate metabolic and gut–brain homeostasis, logical aberrations in the cell, signified by potassium
with a majority of studies focusing on bacteria of the gut efflux42–44, lysosomal rupture45, mitochondria disrup-
microbiota13,14. This Review describes the central roles tion and release of either mitochondrial reactive oxygen
of inflammasome signalling in the gastrointestinal tract, species (ROS) or oxidized mitochondrial DNA46–49 or
highlighting its major functionalities in shaping the out- calcium influx and reduction in cAMP levels50. These
come of acute and chronic gastrointestinal diseases in signals converge on a pathway that drives activation
the context of infectious agents, genetic susceptibili- of the NLRP3 inflammasome, which is mediated by
ties, physiological aberrations and the changing land- serine/threonine-protein kinase NEK7 (Fig. 1).
scape of the bacterial gut microbiota. I also discuss the The most recently described inflammasome sensor,
potential translation and efficacies of pharmacological pyrin, responds to inactivation of host small GTPases
interventions targeting the inflammasome pathways in of the RHO family51 (Fig. 1). RHO-inactivating events
gastrointestinal diseases. occur as a result of infection by bacteria capable of
secreting RHO-inactivating toxins, such as infection
Inflammasome biology and activation by the diarrhoeal pathogen Clostridium difficile51,52, or
An inflammasome complex is generally categorized as a result of host genetic mutations, such as mutations
into a canonical or non-canonical inflammasome. S208A and S242R in MEFV (which encodes pyrin),
The inflammasome sensors NLRP1, NLRP3, NLRC4, which impair either actin dynamics or phosphorylation
NAIPs, AIM2 and pyrin are capable of assembling a of pyrin53,54.
canonical inflammasome complex15 (Fig. 1). The canoni- On activation, inflammasome components assem-
cal inflammasome is a protein complex comprising one ble a single speck of 1 μm in diameter55–59. Cryoelectron
or more inflammasome sensors, ASC and caspase 1. microscopy analysis revealed that ASC and caspase 1 act
The non-canonical inflammasome refers to an activa- in a prionoid-like fashion to form filamentous structures
tion pathway of the NLRP3 inflammasome that spe- and catalyse formation of the inflammasome speck59–62.
cifically requires mouse caspase 11, human caspase 4 Although the use of cryoelectron microscopy analysis
or human caspase 5 (ref.16). The current conceptual of overexpressed inflammasome proteins can provide
view is that inflammasome sensors either directly bind potentially ground-breaking insights into inflammas-
and interact with a ligand, such as flagellin, DNA or ome structures and assembly, these techniques can lead
lipopolysaccharide (LPS), or indirectly sense a physio- to artefactual observations and would require further
logical aberration, such as ion fluxes and mitochondrial experimental support.
dysfunction17. The former case is exemplified by AIM2, Caspase 1 contains a CARD. Inflammasome sen-
caspase 4, caspase 5, caspase 11 and NAIPs. Direct sors that do not carry their own CARD must first
binding between double-stranded DNA (dsDNA) recruit ASC — a bipartite protein composed of a pyrin
and AIM2, independently of the DNA sequence, via domain and a CARD, the former can interact with the
electro­static interaction initiates activation of the AIM2 pyrin domain of CARD-less inflammasome sensors,
inflammasome18–21. Similarly, direct binding between and the latter can subsequently bind the CARD of
cytosolic LPS and caspase 4, caspase 5 or caspase 11 caspase 1 (ref.63). Indeed, inflammasome sensors that
initiates activation of the non-canonical NLRP3 have a CARD, such as NLRC4 and NLRP1, can activate
inflammasome pathway22. Activation of the NLRC4 inflammasome responses or induce pyroptosis without

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 Reviews

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Fig. 1 | Inflammasome complexes. The inflammasome sensors NLRP1b, NLRP3, NLRC4, AIM2 and pyrin are all capable
of forming a canonical inflammasome complex containing the adaptor protein ASC and the cysteine protease caspase 1.
NLRP1b and NLRC4 also recruit caspase 1 without ASC owing to the presence of a CARD domain in their structure64–66.
Activation of NLRP3 and NLRC4 requires the kinase NEK7 and the NLR family members neuronal apoptosis inhibitory
pro­teins (NAIPs), respectively. Caspase 1 cleaves the precursor cytokines pro-IL-1β and pro-IL-18 and the pore-forming
protein gasdermin D. The active fragment of gasdermin D oligomerizes and forms pores on the cell membrane, resulting
in pyroptosis73–80. These pores also allow passive release of biologically active IL-1β and IL-18 from the cell. The non-canonical
inflammasome is defined by a requirement for human caspase 4, human caspase 5 or mouse caspase 11 for the activation
of the NLRP3 inflammasome complex16. Activation of these caspases leads to cleavage of gasdermin D and pyroptosis73–75.
The pore-forming fragment of gasdermin D activates the NLRP3 inflammasome and caspase 1-dependent maturation
of IL-1β and IL-18 (refs73,74).

ASC, whereas inflammasome sensors that have a pyrin Bacteria

domain but not a CARD, such as NLRP3 and AIM2, Bacteria carry an array of PAMPs, some of which are
require ASC for inflammasome activation23,51,56,64–66. delivered into the host cytoplasm, where they activate
Although it has been known for decades that the inflammasome (Fig. 3). A thematic overview of the
caspase 1 induces proteolytic cleavage of the substrates mechanisms of inflammasome activities in the recog-
pro-IL-1β and pro-IL-18 (refs 67–70) , how caspase 1 nition of bacteria and host defence, with a focus on
mediates pyroptosis was unknown71,72. Studies have gastrointestinal pathogens, is provided herein.
now identified the pore-forming protein gasder-
min D as a substrate of inflammatory caspases73–75. NAIP–NLRC4 and caspase 11 — direct sensors of
Caspase 1 and caspase 11 cleave gasdermin D between pathogen-associated molecular patterns. Flagellin,
its Asp276 and Gly277 residues73,74, yielding an active T3SSs and LPS of bacteria are some of the most potent
30–31 kDa amino-terminal fragment that oligomerizes activators of the innate immune system5. Flagellated
and forms pores on the host cell membrane76–80 (Fig. 1). bacteria that are of clinical importance in the gut and
The pores are 10–21 nm in diameter, and biologically are recognized by the NAIP–NLRC4 inflammasome
active and pleotropic forms of the cytokines IL-1β and include Salmonella enterica subsp. enterica serovar
IL-18 and certain DAMPs are released through them, Typhimurium, Escherichia coli, Shigella flexneri and
even before lytic demise of the cell8–10,76–80 (Fig. 1). These Listeria monocytogenes (Fig. 4a). Flagellin subunits of
cytokines and DAMPs collectively regulate intestinal bacteria delivered into the cytoplasm by T3SSs are rec-
inflammation, host defence, gut barrier functions and ognized directly by NAIP sensors, triggering activation
host–microbiota homeostasis81,82. Thus, secretion of of the NLRC4 inflammasome24–27. Indeed, studies have
cytokines and execution of cell death via the inflam- shown that mice lacking NAIPs or NLRC4 are highly
masome are critical to the functional activity of the susceptible to infection by S. Typhimurium 55,84–87,
gastrointestinal tract. S. flexneri26,88,89, L. monocytogenes90, the mouse-specific
enteropathogen Citrobacter rodentium91,92 and other
Infectious diseases extragastrointestinal pathogens that carry flagellin
Microbial pathogens from all domains of life are cap­ and/or the T3SS 63, highlighting the physiological
able of colonizing and infecting the mammalian gut. importance of the NAIP–NLRC4 inflammasome in
These pathogens continue to pose serious concerns the host defence against bacterial infection. The release
to public health and cause substantial morbidity and of IL-1β as a result of NAIP–NLRC4 inflammasome
mortality worldwide83. The inflammasome operates activation drives IL-1 receptor (IL-1R) signalling to
in intestinal cells and immune cells as a cytosolic promote neutrophil recruitment to the intestine and
sentinel that serves to protect the host from invading prevent colonization of gastrointestinal bacteria such
microorganisms (Fig. 2). as S. Typhimurium85,93,94.

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NAIPs and NLRC4 are expressed in both immune bacteria that naturally escape the pathogen-containing
cells and enterocytes (Fig. 2). Infection of intestinal vacuole, such as S. flexneri and Burkholderia thailan-
macro­phages with S. Typhimurium, but not with the densis, transport their LPS into the cytoplasm of macro­
commensal bacteria Bacteroides fragilis, Enterococcus fae- phages, where the hexa-acylated lipid A portion of the
calis or Lactobacillus plantarum, triggers activation of the LPS binds and activates caspase 4, caspase 5 and caspase 11
NAIP–NLRC4 inflammasome85, suggesting that innate (refs 22,97,98) . Indeed, the naturally cytosolic bacteria
immune detection of flagellin and the T3SS enables Burkholderia pseudomallei, B. thailandensis and a
the host immune cell to, in part, discriminate between genetically engineered strain of S. Typhimurium that
pathogenic and commensal bacteria (Fig. 4b). In addition, are incapable of remaining in the pathogen-containing
intestinal macrophages, unlike bone marrow-derived vacuole of macrophages and thus enter the host cyto-
macrophages, are relatively unresponsive to activators plasm aberrantly, are rapidly cleared from the host in
of the NLRP3 inflammasome owing to rapid protea­ a caspase 11-dependent manner 99,100. Furthermore,
somal degradation of NLRP3 and pro-IL-1β95, poten- outer membrane vesicles carrying LPS are shed by
tially representing a mechanism to restrain activation E. coli and most Gram-negative bacteria101. These outer
of an inflammasome sensor in a cell type that normally membrane vesicles are phagocytosed by macrophages,
exposes and responds to a plethora of signals. leading to LPS-induced activation of caspase 11 and
In addition to the NAIP–NLRC4 inflammasome, LPS the non-canonical NLRP3 inflammasome even in the
from Gram-negative bacteria including S. Typhimurium, absence of bacterial invasion of the host cell102.
E. coli and C. rodentium have been found to be recog- Emerging evidence indicates that pyroptosis exe-
nized by caspase 4, caspase 5 and caspase 11 on the basis cuted by NAIP–NLRC4 or caspase 11 is an effective
of cell culture and mouse studies16. LPS from these bacte- host-protective mechanism that either drives bacteria
ria is liberated in the cytoplasm through a host-mediated out of infected host cells or removes an infected cell in
process requiring interferon-inducible GTPases96. These its entirety from the host63. In the former scenario, the
interferon-inducible effector proteins rupture the mem- expelled bacteria released from an infected macrophage
brane of the pathogen-containing vacuole and/or the could be free or trapped within a cellular corpse com-
bacterial cell membrane96. Cytosolic Gram-negative posed of pyroptotic debris, both of which are cleared
by neutrophils84,103. In the latter scenario, enterocytes
in the mouse intestine infected with bacteria, such as
S. Typhimurium, can be physically extruded in their
Caspa e 1
IL- as 4
IL- β e 5
sp se

rin 2
s

entirety from the gut epithelium into the lumen for

NLRP1
NLRP3
NL P6
NLRP7
NL P9
Py P1
Caspa

NL IPs
NLRC
AS 2

NA18
CaC

R
R

a
M

1
AI

Enterocytes removal93,104 (Fig. 4c). Rearrangement of the cytoskeleton

Gastric cells in enterocytes is required for epithelial cell extrusion,
Keratinocytes and caspase 8 has been implicated in this extrusion
Kupffer cells process during S. Typhimurium infection owing to
Monocytes (blood-derived) the ability of NLRC4 to engage caspase 8-dependent
Macrophages (monocyte-derived) cell death58,93,94,104.
Neutrophils (blood-derived) Activation of the NAIP–NLRC4 inflammasome
Peripheral blood mononuclear cells
can also prevent further bacterial uptake and enhance
intracellular bactericidal activity in macrophages105
c
NLRP6 RP1

(Fig. 4d). Unlike macrophages and enterocytes, neutro-

D

NL P3 –NL
IL- de e 11
in
Gaspa e 1

phils are resistant to pyroptosis despite expressing all

1β rm

RP b
rin 2
R a
s
s

3
NLRC4
NLRP1

NL P9
Py 1
Caspa

NL s
NLRC
AS 2

P
NA18

b essential components of the inflammasome, including

CaC

R
M

I
IL-
AI

Dendritic cells (bone marrow-derived) gasdermin D10,106 (Fig. 2). This cell-intrinsic resistance to
Enterocytes pyroptosis enables neutrophils to phagocytose extracel-
Hepatocytes lular bacteria or bacteria entrapped in pyroptotic debris,
Gastric epithelium secrete an abundant amount of IL-1β to amplify local
Goblet cells
Intestinal endothelial cells
inflammation and generate ROS to facilitate intracellular
Intestinal myofibroblasts pathogen killing84,106 (Fig. 4d).
Intestinal phagocytes
Lamina propria inflammatory monocytes NLRP3 and pyrin — sensors of physiological aberra-
Liver immune cells tions. Bacteria that either evade or fail to activate the
Macrophages (bone marrow-derived) NAIP–NLRC4 inflammasome or caspase 11 can be rec-
Neutrophils ognized by other inflammasome complexes22,98 (Fig. 4).
Expression reported Of clinical importance in this bacterial category are
Expression not reported or unknown the Gram-negative bacterium Helicobacter pylori and
several toxin-producing foodborne bacteria, such as
Fig. 2 | Expression of inflammasome sensors and related molecules by cell type.
Staphylococcus aureus. H. pylori, the most common
a | Expression in humans. b | Expression in mice. References for expression data are given
in Supplementary table 1. AIM2, absent in melanoma 2; ASC, apoptosis-associated bacterial cause of gastric cancer107, encodes flagella
speck-like protein containing a caspase activation and recruitment domain (CARD); and flagellin A (FlaA) subunits whose structures evade
NAIP, neuronal apoptosis inhibitory protein; NLRC, nucleotide-binding domain, detection by the NAIP–NLRC4 inflammasome 108.
leucine-rich repeat-containing protein (NLR) family CARD domain-containing protein; Instead, H. pylori infection induces ROS production,
NLRP, NACHT, LRR and PYD domains-containing protein. potassium efflux and lysosomal destabilization in the

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 Reviews

Bacteria Campylobacter jejuni Citrobacter rodentium Citrobacter rodentium Listeria Clostridium

Clostridium difficile Escherichia coli Escherichia coli monocytogenes difficile
Helicobacter pylori Salmonella enterica Listeria monocytogenes
Listeria monocytogenes subsp. enterica serovar Salmonella enterica
Proteus mirabilis Typhimurium subsp. enterica serovar
Staphylococcus aureus Shigella flexneri Typhimurium
Yersinia enterocolitica Vibrio cholerae Shigella flexneri

Other Virus Not known Not known Virus Not known

pathogens • Adenovirus • Adenovirus
• Enterovirus
Protozoan
• Entamoeba histolytica
Helminth
• Schistosoma mansoni

Activators PAMPs and DAMPs, LPS, lipid A Bacterial flagellin Microbial Toxin-induced
including microbial and host and the T3SS and RHO GTPase
toxins, RNA, oxidized (needle and inner mammalian modification
DNA–RNA hybrids, phospholipids rod proteins) DNA
mitochondrial DNA,
ATP, K+ efflux and ROS

NLRP3 Non-canonical NAIP–NLRC4 AIM2 Pyrin

inflammasome inflammasome inflammasome inflammasome inflammasome
NLRP3 NAIP NLRC4 AIM2 Pyrin

Caspase 4
ASC Caspase 5
Caspase 1 Caspase 11

Fig. 3 | Inflammasomes recognize gastrointestinal bacteria, viruses, protozoa and helminths. Pathogens carry a
plethora of pathogen-associated molecular patterns (PAMPs). These PAMPs either directly bind and activate an
inflammasome sensor or induce a physiological change in the cell that is sensed by an inflammasome sensor5. Pathogens
also cause substantial damage to the host cell, a process that induces the liberation of danger-associated molecular
patterns (DAMPs) that activate the inflammasome15. AIM2, absent in melanoma 2; ASC, apoptosis-associated speck-like
protein containing a caspase activation and recruitment domain (CARD); LPS, lipopolysaccharide; NAIP, neuronal
apoptosis inhibitory protein; NLRC4, nucleotide-binding domain, leucine-rich repeat-containing protein (NLR) family
CARD domain-containing protein 4; NLRP3, NACHT, LRR and PYD domains-containing protein 3; ROS, reactive oxygen
species; T3SS, type 3 secretion system.

host cell, ultimately leading to activation of the canon- inflammasome sensors. The Gram-positive bacterium
ical NLRP3 inflammasome109–113. Further studies have C. difficile is a causative agent of diarrhoea associated
shown that the H. pylori virulence factors cag patho- with the use of antibiotics, toxic megacolon and colonic
genicity island and urease B subunit potentiate acti- perforation117. The toxins toxin A (TcdA) and toxin B
vation of the NLRP3 inflammasome and secretion of (TcdB) secreted by C. difficile can inactivate host trans-
IL-1β109,112,113. Excessive production of IL-1β could be a forming protein RHOA at the GTPase switch I region;
vital link between H. pylori infection and tumorigenesis this RHO-inactivating activity is sensed by pyrin, result-
in the stomach. Indeed, a study found that a transgenic ing in the assembly of the pyrin inflammasome51,52.
mouse strain engineered to overexpress human IL-1β The physiological function of pyrin in the gastro­
in the stomach is prone to the development of gastric intestinal tract remains unexplored, and further studies
cancer, either spontaneously or after colonization with are required to clarify its role. However, wild-type mice
Helicobacter felis, a model organism for H. pylori114. treated with the IL-1R antagonist anakinra are protected
The specific inflammasome involved in the develop- from C. difficile-toxin-induced intestinal injury118. This
ment of gastric cancer is not known; however, an asso- finding indicates that activation of the pyrin inflamma­
ciation between polymorphisms in the genes encoding some in response to C. difficile infection might lead to
NLRP3 and caspase 1 and patients with gastric cancer excessive IL-1-mediated inflammation and pathology
has been reported115. Furthermore, H. pylori infection that is detrimental to the host. Pharmacological inhibi-
suppresses the expression of the microRNA molecule tors of microtubules are efficacious in blocking the pyrin
miR-22 in gastric epithelial cells, which leads to elevated inflammasome in cell culture systems54,119,120, but their
expression of NLRP3 and cellular proliferation116. These clinical applications against pyrin-mediated pathology
studies suggest that activation of the inflammasome by have not been tested in animal models.
Helicobacter spp. infection contributes to the molecular The haemolysin of the Enterobacteriaceae mem-
basis of gastritis and gastric cancer. ber Proteus mirabilis can activate NLRP3 and drive
Toxins secreted by bacteria can induce a state of inflammation in the mouse intestine 121. Similarly,
intracellular physiological aberration that is sensed by α-haemolysins, β-haemolysins and γ-haemolysins of

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the Gram-positive foodborne bacterium S. aureus medi- the human, but not the mouse, genome) is a sensor of
ate activation of the NLRP3 inflammasome in mouse bacterial lipopeptides; its activation in macrophages
macro­phages and mice122,123, and the pore-forming impairs intracellular replication of L. monocytogenes
cytolysin listeriolysin O of the Gram-positive food- or S. aureus129. Further studies have shown that, in
borne bacterium L. monocytogenes potentiates activation macro­phages or embryonic fibroblasts, inflammasome
of the NLRP3, NAIP–NLRC4 and AIM2 inflamma­ components accumulate on the phagosome and pro-
somes90,124–128. Infection with either L. monocytogenes mote phagosome–lysosome fusion and phagosomal
or S. aureus activates caspase 1 in human macrophages acidification27,105,130–132. The functional diversity of the
via NLRP3 and NLRP7 (ref.129). NLRP7 (encoded in inflammasome in both immune and non-immune cells

a IL-1β and IL-18 TNF, IL-6 and KC Type I interferons

Citrobacter rodentium Type III interferons
Citrobacter rodentium
Salmonella Bacterium
enterica subsp. enterica Rotavirus EMCV
serovar Typhimurium TLR Mucus secretion

Autophagy

NLRP6
NLRP6 dsRNA ssRNA
NF-κB Pyroptosis
NLRP12
Pyroptosis DHX9 NLRP6
NAIP–NLRC4 and NLRP9b DHX15
caspase 11–NLRP3 ASC MAVS
inflammasome Caspase 1
Goblet cell
Enterocyte Mitochondrion

b Intestinal macrophages Bone marrow-derived c Pyroptosis

macrophages
Pathogenic Commensal Infection Extrusion
bacterium bacterium
Intestinal
IL-18 epithelium

IL-1β Unresponsive IL-1β, TNF IL-1β, TNF Lumen

Lamina propria
and IL-6 and IL-6
d
Bacterial uptake IL-1β

ROS

Pyroptotic
corpse
Pyroptosis

Expelled
Pyroptotic bacterium
IL-1β and IL-18 macrophage Neutrophil

Fig. 4 | Inflammasomes and related molecules contribute to the killing and clearance of gastrointestinal pathogens
in intestinal cells and immune cells. a | Inflammasomes mediate host protection against Gram-negative bacteria by
inducing the secretion of IL-1β and IL-18 and pyroptosis55,59,84,85,91,92,254,255. NLRP6 and NLRP12 negatively regulate
inflammation133,134,256,257. NLRP6 responds to Toll-like receptor (TLR)-induced autophagy in goblet cells and mediates
secretion of mucus137. RNA-bound DEAH box protein 9 (DHX9) interacts with NLRP9b, inducing the assembly of an
inflammasome complex161. The NLRP6–DHX15 complex binds to viral RNA and induces the production of type I and type III
interferons165. b | Intestinal macrophages can discriminate pathogens from commensals85. c | Activation of caspase 1,
caspase 8 or caspase 11 leads to cell death, which removes and extrudes the infected enterocyte from the epithelium93,94,104.
d | The inflammasome can reduce bacterial load by inhibiting bacterial uptake, which limits macrophage movement and
stiffness, and can promote the production of reactive oxygen species (ROS)105. Pyroptotic macrophages liberate either
whole bacteria or bacteria entrapped within pore-induced intracellular traps103. These entities are phagocytosed by
neutrophils84,106. ASC, apoptosis-associated speck-like protein containing a caspase activation and recruitment domain
(CARD); dsRNA, double-stranded RNA; EMCV, encephalomyocarditis virus; KC, keratinocyte chemoattractant (also known
as CXCL1); MAVS, mitochondrial antiviral-signalling protein; NAIP, neuronal apoptosis inhibitory protein; NF-κB, nuclear
factor-κB; NLRC4, nucleotide-binding domain, leucine-rich repeat-containing protein (NLR) family CARD domain-containing
protein 4; ssRNA, single-stranded RNA.

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ensures host protection against a range of bacteria with and increase susceptibility to sepsis150, whereas certain
preferential tropisms and virulence strategies. strains of E. coli, such as strain O21:H+, engage activation
of the NAIP–NLRC4 inflammasome to inhibit the body
NLRP6 and NLRP12 — enigmatic sensors. In line wasting induced by S. Typhimurium infection151. Thus, the
with the functional versatility of other inflammasome inflammasome influences the magnitude of inflammation
sensors, NLRP6 and NLRP12 have multiple functions towards bacterial infection and, in part, determines either
during bacterial infection. The functions of these sen- a protective or detrimental outcome in the host.
sors are enigmatic owing to the lack of expert con-
sensus on their biological functions. Both NLRP6 Viruses
and NLRP12 can negatively regulate activation of the Norwalk and Norwalk-like viruses, rotavirus, astro­
nuclear factor-κB (NF-κB) and mitogen-activated pro- viruses, adenoviruses, caliciviruses and coronaviruses
tein kinase (MAPK) pathways in macrophages infected are among the leading causes of acute gastroenteritis
with L. monocytogenes, S. Typhimurium, E. coli or other worldwide152. Evidence has emerged that inflammas-
non-gastrointestinal bacteria (Fig. 4a), resulting in a state omes respond to some of these gastrointestinal viruses.
of reduced inflammation that can be readily exploited Earlier studies had demonstrated that the DNA virus
by bacterial pathogens during infection of a cell or a adenovirus induces the production of IL-1β in circu-
mouse133–136. Others have proposed that NLRP6 and lation and in hepatocytes and Kupffer cells of mice153.
NLRP12 mediate activation of caspase 1 in response to Further functional studies have shown that adenovirus,
infection by C. rodentium and Yersinia pestis, respec- specifically its virion component, triggers activation
tively137,138, and that NLRP12 functions in dampening of the NLRP3 inflammasome and secretion of IL-1β
the cell-intrinsic migratory capacity of neutrophils139. in human THP-1 macrophages and primary mouse
A further study suggests that the C57Bl/6J mice that are macrophages 154. Adenovirus-induced secretion of
used widely in research harbour a missense mutation IL-1β in human monocyte-derived macrophages and
in the Nlrp12 gene that impairs the ability of NLRP12- peripheral blood mononuclear cells partially requires
expressing macrophages to draw neutrophils to the site Toll-like receptor 9 (TLR9)155,156, presumably owing
of inflammation in response to bacterial infection140. to TLR9-mediated sensing of viral DNA as the virus
These conflicting studies highlight that the biological pene­trates the cell membrane and enters the endo-
functions of NLRP6 and NLRP12 should be revisited some. Injection of an adenovirus vector into mice trig-
and should take into account the genetic background gers NLRP3-dependent production of IL-1β and other
of the mouse strains, cell types, bacterial agents and the pro-inflammatory cytokines, highlighting a physio-
experimental approaches used. logical role for the NLRP3 inflammasome in licensing
an innate immune response to adenovirus in vivo154.
Detrimental roles of the inflammasome. Overt activation This finding is also consistent with the observation that
of the inflammasome is detrimental, especially during IL-1R, which is activated downstream of inflamma­
systemic bacterial dissemination, which might arise from somes, incites an acute and deleterious inflammatory
intestinal perforation. Mice lacking caspase 11 or the signalling cascade in mice infected with adenovirus153.
pyroptosis-inducing effector gasdermin D are remark- The magnitude of the inflammatory response is reduced
ably resistant to LPS-induced or E. coli-induced endo- in mice lacking IL-1R or in wild-type mice treated with
toxaemia compared with wild-type mice16,73,97,98,141–144, an anti-IL-1 antibody153, suggesting that blockade of
suggesting that caspase 11-mediated pyroptosis drives IL-1 signalling prevents overt inflammation that might
lethality in endotoxaemia. Deletion of the Nlrp3 gene in otherwise be damaging to the host.
mice offers protection against polymicrobial endotoxae- Similar to adenovirus, enterovirus EV71, a spor­
mia following caecal ligation and puncture145,146. In this adic cause of diarrhoea and a common cause of hand,
model of systemic inflammation, the lack of inflammas- foot and mouth disease in humans157, is sensed by the
ome activity impairs the production of pro-inflammatory NLRP3 inflammasome158,159. Inflammasome-mediated
and endogenous lipid mediators but increases the production of IL-18 is crucial for the protection against
levels of pro-resolving lipid mediators145. Moreover, entero­virus EV71 in mice158,159. Administration of IL-18
LPS-induced endotoxaemia promotes secretion of bile to infected mice reduces viral loads and the production
acids from hepatocytes in mice; bile acids are a form of of pro-inflammatory cytokines159. The precise viral
DAMP that either synergize with ATP to activate the components of enterovirus EV71 that activate NLRP3
NLRP3 inflammasome147 or signal through the mem- are unknown; it is possible that the single-stranded
brane receptor G protein-coupled bile acid receptor 1 RNA (ssRNA) viral genome and/or the structural
(GBAR1) to inhibit the NLRP3 inflammasome148. proteins of the capsid might trigger activation of the
In other models of systemic inflammation, activa- NLRP3 inflammasome.
tion of the NAIP–NLRC4 inflammasome by systemic The double-stranded RNA (dsRNA) virus rotavirus
delivery of cytosolic flagellin to mice results in the pro- is one of the most common causes of gastroenteritis
duction of inflammatory lipid mediators called eicosa- in children160. The role of inflammasomes in the host
noids149. Synthesis of eicosanoids initiates fluid loss into defence against this virus was revealed when the poorly
the mouse intestine and peritoneal cavity and ultimately characterized NLR member NLRP9b was found to
causes rapid host death within 30 min149. The presence of mediate sensing of rotavirus and drive the production
a multidrug-resistant E. coli pathobiont in the gut can also of IL-18 and pyroptosis in organoids generated from
trigger activation of the NAIP–NLRC4 inflammasome the small intestine of mice161. Furthermore, mice lacking

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NLRP9b are substantially more susceptible to rotavi- E. histolytica170. Further studies indicated that the para­
rus infection, harbour a higher viral load and develop site integrin-binding cysteine protease E. histolytica
more severe intestinal pathology than wild-type mice161. cysteine protease gene 5 (CP5) mediates activation of
Mouse NLRP9b and its human counterpart, NLRP9, the host α5β1 integrin during parasite–macrophage
are strongly and specifically expressed in ileal epithelial contact, leading to the release of ATP and subsequent
cells and primary intestinal epithelial cells, respectively161 activation of the canonical NLRP3 inflammasome171.
(Fig. 2). Both mouse NLRP9b and human NLRP9 coop- The importance of the inflammasome in E. histolytica
erate with the RNA sensor DEAH box protein 9 (DHX9) infection is partially inferred by the observation
to sense dsRNA161 (Fig. 4a), implicating the existence of that recombinant IL-1β and IL-18 are cleaved and
an RNA-sensing NLRP9 inflammasome complex in the inactivated by E. histolytica CP5 (refs172,173).
host defence against rotavirus infection. A protective Similar to pathogenic protozoa, the commensal gut
role of inflammasomes was also observed in a previous protozoan Tritrichomonas musculis in mice also triggers
study showing that administration of bacterial flagel- activation of the inflammasome in intestinal epithelial
lin to mice — causing activation of NLRC4 and the cells in mice174. In this context, T. musculis–induced
membrane-bound flagellin sensor TLR5, which induced activation of the inflammasome promotes IL-18 secre-
the production of IL-18 and IL-22, respectively — tion, T helper 1 (TH1) cell and TH17 cell immunity and
mediates clearance of rotavirus within 24 h (ref.162). protection against S. Typhimurium infection174, high-
Recapitulating the beneficial effects of flagellin, coadmin­ lighting a favourable outcome of this host–protozoan
istration of IL-18 and IL-22 simi­larly drives anti- engagement. However, intestinal colonization by
rotavirus immunity in infected mice, supporting the view T. ­musculis also drives sustained inflammation that ele-
that inflammasome signalling can mediate protection vates the likelihood of developing T cell-driven colitis
against rotavirus infection. and sporadic colorectal tumours in mice174. The pre-
Similar to NLRP9, NLRP6 is strongly expressed in cise inflammasome complex triggered by T. musculis
the intestinal tract of humans and mice163,164 (Fig. 2). In is unknown; however, it is likely that protozoa-induced
concert with the RNA sensor DHX15, NLRP6 interacts activation of the inflammasome in the gastrointestinal
with viral RNA and mediates the recognition of the tract is a double-edged sword that contributes to both
ssRNA viruses encephalomyocarditis virus and murine health and disease.
norovirus165 (Fig. 4a). In this case, NLRP6 does not
assemble an inflammasome. Instead, the RNA-bound Intestinal inflammation, IBD and cancer
NLRP6–DHX15 complex migrates to the mitochondria, The ability of inflammasomes to respond to danger
where it interacts with the signalling adaptor mitochon- signals broadens their clinical relevance to diseases
drial antiviral-signalling protein (MAVS) to induce the other than infectious diseases175,176. Genetic analyses in
production of type I and type III interferons and viral humans have pinpointed an association between muta-
clearance165 (Fig. 4a). These studies underscore emerg- tions in genes encoding NLRs and intestinal inflam-
ing roles of the inflammasome and associated signalling mation, autoinflammatory conditions and cancer14,177.
molecules in antiviral defence of the gastrointestinal The contribution of individual inflammasome sensors
tract. The relative contribution of IL-1β and IL-18 secre- and related molecules in the pathogenesis of intestinal
tion versus pyroptosis in driving immunity to viruses inflammation, IBD and cancer is discussed herein.
remains largely unknown. The current availability of
mice lacking gasdermin D will enable exploration of this NLRP3
emerging research area. NLRP3 is a global sensor of PAMPs and DAMPs and
is expressed in multiple cell types (Fig. 2), contribut-
Protozoa ing to its links to a plethora of clinical manifestations.
Protozoal pathogens cause several neglected tropical Genetic analysis identified an association between six
diseases and are public health risks largely affecting polymorphisms located in a region 4.7 kb downstream
the developing world166. Inflammasomes are known of the NLRP3 gene and the development of Crohn’s dis-
to recognize major protozoal pathogens, including ease in some, but not all, European populations178,179.
Leishmania spp., Plasmodium spp. and Toxoplasma Peripheral blood mononuclear cells derived from
spp. 167. However, information concerning the bio­ patients with Crohn’s disease who were homozygous for
logical importance of inflammasomes in the context any one of these six polymorphisms have an impaired
of gastrointestinal protozoa is limited. The foodborne ability to express NLRP3 and secrete IL-1β in response
protozoan Entamoeba histolytica causes amoebiasis, to LPS stimulation in vitro178. A further study reported
which is characterized by dysentery, amoebic colitis, that a heterozygous NLRP3 polymorphism, resulting in
amoeboma (the formation of fibrotic tissue masses in a mutation at residue Q705K, is associated with poor
the intestinal wall) and invasive disease of the brain, survival in patients with advanced colorectal cancer
lung and liver168. There is evidence to support the idea (CRC)180. Global expression analysis of genes encod-
that E. histolytica infection activates caspase 1 and ing cytosolic innate immune sensors revealed that the
induces secretion of IL-1β and IL-18 in both human expression of genes encoding NLRP3 and other inflam-
and mouse macrophages169. Pharmacological blockade masome sensors is substantially reduced in tumour
of potassium efflux inhibits activation of caspase 1, tissues compared with non-tumour-associated tissues
IL-1β-release and pyroptosis in THP-1 macrophages or in patients with CRC181. Similar observations have
intestinal epithelial cells that have been infected with been made in mice during colitis-associated CRC182.

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However, how reduced expression of these genes is initially tumour burden in inflammasome-deficient mice that had
triggered or executed mechanistically is unknown. been administered AOM and DSS; the same treatment
Several groups have found that mice lacking NLRP3, also ameliorated colitis in inflammasome-deficient mice
ASC or caspase 1 and administered a combination of exposed to the chemical colitogen oxazolone183,187,204,211,214.
the DNA-damaging agent azoxymethane (AOM) and the IL-18 functionally exerts healing properties184,185,187,214 and
chemical colitogen dextran sodium sulfate (DSS) are triggers natural killer cell-mediated killing of colonic
highly sensitive to the development of colitis and tumour cells that have metastasized to the liver215.
colitis-associated CRC183–188. DSS-induced activation of Another study challenged the view that IL-18 is beneficial
the inflammasome in mice also causes a loss of enteric in intestinal inflammation and reported that the IL-18
neurons and onset of abnormal gut motility189. The activ- and IL-18 receptor signalling pathway drives depletion
ity of the NLRP3 inflammasome during DSS-induced of mucus-secreting goblet cells, such that a loss of the
colitis is controlled by the microRNA molecule miR- mucous layer augments sensitivity to DSS-induced coli-
223, which binds the miR-223-binding site within the tis in mice216. Similarly, a transgenic mouse strain over­
3′ untrans­lated region of NLRP3 (ref.190). However, the state expressing IL-18 develops aggressive colitis in response to
of hyporesponsiveness of colonic macrophages to NLRP3 DSS owing to increased levels of infiltrating macrophages
activators is lost during DSS-induced colitis95, which in the colon217. One possibility that might reconcile the
might fuel inflammation. Indeed, studies in mice have also diametrically opposing roles of IL-18 is the ability of
reported a deleterious or non-existent role for NLRP3 or this cytokine to downregulate the soluble IL-22 recep-
caspase 1 and caspase 11 in colitis and colitis-associated tor called IL-22-binding protein (IL-22BP)218. IL-22BP
CRC191–194. The biological basis of these discrepancies modulates the bioavailability of IL-22, a cytokine
is unclear; however, differences in gut microbiota, diet capable of suppressing early intestinal damage but also
and/or housing conditions might affect the activity of promoting tumour development over time218 (Fig. 5a).
the inflammasome (the differences in the microbiota Inappropriate activation of the NLRP3 inflam-
are specifically discussed later). Indeed, a ketogenic masome can result in damage to the small intestine214.
diet or caloric restriction that increases the prod­uction In mice, enteropathy induced by NSAIDs, such as indo-
of the ketone body β-hydroxybutyrate from the liver methacin, can be attenuated through the use of neutral-
contributes to the inhibition of the NLRP3 inflammas- izing antibodies to IL-1β or inhibitors of the NLRP3
ome195. Similarly, omega-3 fatty acids signal through the inflammasome, including the ATP scavenger apyrase,
metabolite-sensing receptors G protein-coupled receptor the P2X purinoceptor 7 (P2X7) antagonist brilliant blue
40 (GPR40; also known as FFAR1) and GPR120 (also G and the cytoskeletal inhibitor colchicine214. These
known as FFAR4) to inhibit activation of the NLRP3 studies support the view that unwarranted activation of
inflammasome and prevent diet-induced insulin resist- the NLRP3 inflammasome is a catalyst for small intesti-
ance196. By contrast, short-chain fatty acids from dietary nal damage. The availability of more specific inhibitors
fibres bind to the metabolite-sensing receptor GPR43 of the NLRP3 inflammasome, such as MCC950 and
(also known as FFAR2) on enterocytes, stimulating CY-09 (refs219–221), enables clinical trials to explore new
potassium efflux that drives activation of the NLRP3 options for the treatment of human diseases182.
inflammasome and protection against colitis197,198. A diet
rich in cholesterol or saturated fatty acids also promotes NLRC4
activation of the NLRP3 inflammasome in mice199–203, Studies in humans have revealed that gain-of-function
whereby a cholesterol-rich diet increases inflammation mutations in the gene encoding NLRC4 are associated
and tumour burden199, but a high-fat diet reduces glu- with enterocolitis and autoinflammation222–224. For exam-
cose tolerance and insulin sensitivity200. These studies ple, a substitution mutation within the HD1 domain of
provide evidence to indicate that dietary components, the NLRC4 protein is linked to recurrent enterocolitis
in association with the gut microbiota, are critical mod- and autoinflammation in several members of the same
ulators of inflammasome activity and susceptibility to family222. A newborn baby from this family and carrying
the development of intestinal inflammation, cancer and this mutation died 23 days after birth owing to severe
metabolic syndromes. gastro­intestinal complications, fever and systemic inflam-
Activation of the NLRP3 inflammasome leads to mation222. A further study identified that a heterozygous
secretion of both IL-1β and IL-18. Although injection of de novo substitution mutation in the nucleotide-binding
recombinant IL-1β into mice has been shown to mediate domain of NLRC4, potentially causing destabiliza-
protection against DSS-induced colitis204, studies have tion and autoactivation of the protein, was linked to
found variable levels of IL-1β in the colon tissue of macrophage activation syndrome, gastrointestinal
inflammasome-deficient mice in response to pathology, splenomegaly and systemic inflammation223.
DSS163,184,185,204–211. These findings would suggest that the Monocytes or macrophages carrying the aforemen-
role of IL-1β is not always coupled with inflammasome- tioned disease-associated mutations undergo spontane-
mediated protection of intestinal ­inflammation and ous activation of the inflammasome and pyroptosis and
colitis-associated CRC. secrete increased levels of IL-1β and IL-18 (refs222–224),
The protective role of the NLRP3 inflammasome in suggesting that unchecked inflammasome activation
colitis and CRC is attributed in part to the secretion of contributes to the molecular basis of these autoinflam-
IL-18 (Fig. 5a). Mice lacking IL-18 are highly susceptible matory conditions. Of clinical importance is that the
to the development of colitis and CRC183,212,213. Injection of aforementioned patient with the substitution mutation
recombinant IL-18 reduces intestinal inflammation and in NLRC4 responded to treatment with the recombinant

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a Anti-tumour Inflammation
b
Oncogenic assault immunity
(AOM and/or
DSS)
Gut microbiota IL-18 IL-22BP IL-22

PAMPs and Flagellin, T3SS Flagellin, T3SS

DAMPs and DAMPs? and DAMPs?
DAMPs DAMPs
Proliferation NLRC4 NAIPs
P
STAT3 STAT3
Host DNA
Tumorigenesis Tumorigenesis Tumorigenesis
Enterocyte Inflammasome

c Growth factors d Inflammatory Antitumour

mediators immunity

TLR Taurine Histamine IL-18 AMPs

AMPs GFR Spermine Mucus
Microbial DNA
secretion

? ? PI3Ks NLRP6 NLRP6

NLRC3 ROS
NLRP6
AIM2 DNA-PK p85 p110α NLRP12

AKT and/or MYC AKT and mTOR

Host DNA
Tumorigenesis Tumorigenesis
Sentinel goblet cell

Fig. 5 | Development of intestinal inflammation and cancer is regulated by the inflammasome–microbiota axis.
a | Oncogenic assaults such as azoxymethane (AOM) and dextran sodium sulfate (DSS) cause damage, leading to the
release of danger-associated molecular patterns (DAMPs). Bacteria can invade enterocytes and introduce pathogen-
associated molecular patterns (PAMPs) into the host cell. DAMPs and PAMPs are sensed by inflammasomes183–185,187,188,204.
IL-18 promotes downregulation of soluble IL-22-binding protein (IL-22BP), which controls the ability of IL-22 to suppress
inflammation or induce tumorigenesis in the gut218. b | Nucleotide-binding domain, leucine-rich repeat-containing
protein (NLR) family CARD domain-containing protein 4 (NLRC4) and neuronal apoptosis inhibitory proteins (NAIPs)
can block cellular proliferation and tumorigenesis192,226. c | DNA-dependent protein kinase (DNA-PK) induces colorectal
tumorigenesis via activation of AKT and the transcription factor MYC205,206. This response is inhibited by absent in
melanoma 2 (AIM2)205,206. AIM2 also triggers the production of antimicrobial peptides (AMPs) in intestinal epithelial
cells to modulate the gut microbiota207,208. A similar negative regulatory role for NLRC3 has been described229.
d | NLRP6 and NLRP12 contribute to the pathogenesis of gastrointestinal infection, acute colitis and colorectal
cancer133,134,163,234–236,244,248,256,257. Question marks denote unknown mediators. GFR, growth factor receptor; mTOR,
mechanistic target of rapamycin; NLRP, NACHT, LRR and PYD domains-containing protein; PI3Ks, phosphoinositide
3-kinases; ROS, reactive oxygen species; STAT3, signal transducer and activator of transcription 3; T3SS, type 3
secretion system; TLR, Toll-like receptor.

IL-1R antagonist anakinra223. A newly identified patient response to AOM and DSS192,226. Mechanistically, NLRC4
with the heterozygous de novo mutation in NLRC4, who inhibits cellular proliferation and promotes apoptosis
also experienced enterocolitis and skin rash, responded via an undefined mechanism192, whereas NAIPs inhibit
to a combined blockade therapy targeting IL-1β and proliferation in enterocytes by blocking phosphoryl-
IL-18 (ref. 225) . These studies exemplify the success ation and activation of the transcription factor signal
of experimental targeted therapies in the treatment of transducer and activator of transcription 3 (STAT3)
inflammasome-mediated disorders. independently of NLRC4 (ref.226) (Fig. 5b). A possible
In animal studies, deletion of the genes encoding scenario is that either spontaneous activation or loss of
NLRC4 or NAIPs heightens the susceptibility of mice to function associated with the NAIP or NLRC4 signalling
the development of colitis and colitis-associated CRC in axis could upset the equilibrium of the gut ecosystem

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and drive intestinal inflammation and tumorigenesis. (CPT-11), a topoisomerase I inhibitor used in the
Immunization of mice with tumour cell lines express- treatment of a variety of cancers, elicits the release of
ing flagellin — to trigger activation of the NAIP–NLRC4 self-DNA, subsequent activation of AIM2 and secre-
and TLR5 pathways – induces clearance of tumour cells tion of IL-1β and IL-18 in enterocytes, leading to intes-
by innate immune cells and tumour-specific CD4+ and tinal mucositis, late-onset diarrhoea and microbiota
CD8+ T cells227. Thus, modulation of the NAIP–NLRC4 disturbance in mice232,233. Deletion of Aim2 or phar-
and/or the IL-1–IL-18 pathways might provide a macological inhibition of the AIM2 inflammasome
promising avenue for cancer immunotherapy. in mice reduces the incidence of irinotecan-induced
diarrhoea without dampening the anticancer efficacy
AIM2 and other signalling molecules of irinotecan232.
The inflammasome-associated sensor AIM2 also con- Fur t her studies have shown t hat NLRP6
tributes to the pathogenesis of colitis and CRC205–208. (refs 137,163,234–237 ) , NLRP1b (ref. 204 ) and caspase 11
The levels of AIM2 expression in tumour tissues often (refs209–211) are important innate immune rheostats of
predict survival in patients with CRC228. Patients whose the gut, providing additional targets for experimental
tumour cells lack AIM2 expression are threefold more therapies. NLRP6 controls intestinal inflammation and
likely to die within 5 years of diagnosis than patients tumorigenesis via multiple mechanisms (discussed further
whose tumour cells retain some level of expression228. later), including modulation of IL-18 secretion163,234,235,237,
Reflecting the scenario in humans, the expression of curtailing the colonization of pro-colitogenic gut bac-
the Aim2 gene is markedly reduced in inflamed and teria163 and promoting secretion of mucus in goblet
tumour-associated tissues of mice, and mice lacking cells137,236 (Fig. 5d). NLRP6 expression in enterocytes and
AIM2 are hypersusceptible to both colitis-associated inflammatory monocytes has been reported to mediate
and spontaneous colorectal tumorigenesis205,206. AIM2 protection against DSS-induced colitis163,235,237. NLRP1b
has functions independent of the inflammasome that regulates activation of the inflammasome and secretion
drive protection against both colitis-associated and of IL-1β and IL-18 specifically in non-haematopoietic
spontaneous CRC, the latter of which arises from a cells 204, whereas caspase 11 triggers IL-1β–IL-18-
mutation in the mouse homologue of the human dependent and IL-1β–IL-18-independent func-
APC gene or from aberrant β-catenin activation205,206. tions209–211, with both sensors mediating protection
AIM2 suppresses the ability of the DNA-binding against DSS-induced colitis. A side-by-side comparison
kinase DNA-dependent protein kinase (DNA-PK) to revealed that, under isobiotic conditions with normal-
activate RAC serine/threonine-protein kinase (AKT) ized gut microbiota across genotypes, mice lacking
and transcription factor MYC proto-oncogene pro- caspase 1, but not mice lacking caspase 11, were hyper-
tein, ultimately preventing excessive proliferation of sensitive to DSS-induced colitis188. Evidence from this
stem cells in the colon205,206 (Fig. 5c). A similar negative study would seem to favour a role for canonical inflam-
regulatory effect of the orphan receptor NLRC3 on masome complexes over the non-canonical inflammas-
the AKT–mechanistic target of rapamycin (mTOR) ome in this mouse model of intestinal inflammation.
pathways activated during colitis-associated CRC Although these studies point towards a protective role
or spontaneous CRC has been described 229 (Fig. 5c). of inflammasomes in the context of intestinal inflam-
Pharmacological inhibitors of the AKT–mTOR mation and tumorigenesis, a study found that deletion
pathway are efficacious against the development of of the genes encoding the mucous layer components
colitis-associated or spontaneous CRC in mice lack- core 1-derived and core 3-derived intestinal O-glycans
ing either AIM2 or NLRC3 (refs205,206,229). The precise in mice causes colonic mucous barrier breach, spon-
identity or source of the ligand(s) activating these taneous colitis and the development of colorectal
sensors in the colon is not known. It is possible that tumours, and further deletion of the genes encoding
self-DNA liberated from the damaged intestinal bar- caspase 1 and caspase 11 reduces the propensity of
rier or DNA derived from the gut microbiota might colitis and colorectal tumorigenesis239. These findings
activate AIM2 (ref.230) (Fig. 5c). Consistent with this illustrate that in the presence of pre-existing immuno-
hypothesis, AIM2 can localize to DNA in the nucleus logical dysregulation triggering overt inflammation,
of mouse intestinal epithelial cells and bone marrow blockade of inflammasome signalling might inhibit
cells following dsDNA breaks caused by ionizing radi- an inflammatory circuitry that normally fuels dis-
ation or chemotherapeutic agents231. The generation ease progression. Development of safe and efficacious
of conditional mouse models that enable deletion modulators of AIM2, NLRP1 and NLRP6 and investi-
or re-expression of AIM2 and other related sensors gations into the bilateral interaction between inflam-
in all cells or in certain cell types once tumours are masomes and the gut ecosystem would create new
established would provide additional insights into the avenues for the treatment of intestinal inflammation
molecular and cellular mechanisms of the operation and cancer.
of these sensors. These studies would also aid further
assessment of the translational potential of targeting Inflammasome–gut microbiota axis
these sensors in cancer immunotherapy. The gut microbiota has an important role in the
Indeed, therapeutic inhibition of the AIM2 inflam- development and progression of a range of clini-
masome might, in some cases, reduce the adverse cal manifestations, especially in association with the
gastro­intestinal effects associated with chemotherapy232. gastrointestinal tract. Evidence supporting this view
The chemotherapeutic and cytotoxic agent irinotecan includes the ability of postoperative diversion of the

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faecal stream to prevent recurrent IBD in humans and of NLRP6 have been further characterized against the
the use of broad spectrum antibiotics in the amelior­ backdrop of intestinal inflammation, cancer and meta-
ation of chemical-induced colitis in mice177. The lat- bolic syndromes. NLRP6 senses the microbial metabolite
est developments in the field have suggested that taurine and drives the secretion of IL-18 and antimicro-
the inflammasome regulates gut microbiota compo- bial peptides to maintain a healthy gut microbiota and
sition, which would imply that the inflammasome– to promote antitumour immunity in mice163,248 (Fig. 5d).
gut microbiota axis might be a centrepiece in the By contrast, the microbial metabolites histamine and
development of intestinal inflammation, cancer and spermine suppress NLRP6-dependent IL-18 secretion
metabolic syndromes177,238,240,241. in mice, indicating that components of the gut micro­biota
can reciprocate and fine-tune the activities of NLRP6
Colitis and cancer (ref.248) (Fig. 5d). The NLRP6–gut microbiota axis mod-
The concept of inflammasomes controlling the bio- ulates the expression of the chemokine CC-chemokine
geography of intestinal bacteria was spearheaded by ligand 5 (CCL5) and the pro-inflammatory cytokines
studies showing that mice lacking NLRP6 harbour a IL-6 and TNF and, therefore, the overall inflamma-
pro-colitogenic gut microbiota that renders these mice tory state of the intestinal tract163,237,242. In the absence
more susceptible to DSS-induced colitis than wild-type of NLRP6, the dysbiotic gut microbiota developed as a
mice163. This pro-colitogenic signature is defined by an result induces the expression of CCL5 in enterocytes;
increased relative abundance of Prevotellaceae and the CCL5, in turn, mediates the secretion of IL-6 and drives
phylum Candidatus Saccharibacteria, reduced relative proliferation of epithelial cells and tumour formation
abundance of Lactobacillus spp. and the transmissi- in mice163,242. Deletion of the gene encoding CCL5 in
ble nature of the pro-colitogenic microbiota through mice prevents colitis and colitis-associated tumorigen­
means of cross-fostering and cohousing techniques163,242. esis despite these mice carrying a dysbiotic gut micro­
Furthermore, NLRP6 dampens spontaneous colitis biota163,242, placing dysbiosis upstream of CCL5-mediated
in mice lacking the anti-inflammatory cytokine IL-10 immune dysregulation. Furthermore, inflammatory
by reducing levels of the mucus-degrading bacterium monocytes residing in the lamina propria promote
Akkermansia muciniphila243. Carriage of a transmissible NLRP6-dependent and IL-18-dependent secretion of
pro-colitogenic gut microbiota has also been described TNF and feature a protective role in DSS-induced colitis
in mice lacking the inflammasome components AIM2, and colitis-associated tumorigenesis237.
NLRP3, ASC, caspase 1 or caspase 11 or mice lacking the The relationship between NLRP6 and intestinal
related NLR member NLRP12 (refs163,186,205,207–209,244,245). microorganisms is further exemplified by the ability of
Similar to the findings with NLRP6, reciprocal exchange NLRP6 to respond to Toll-like receptor (TLR)-induced
of the gut microbiota between wild-type mice and autophagy in goblet cells137. This NLRP6 response medi-
mutant mice lacking AIM2 or NLRP12 elevates the ates secretion of mucus via granule exocytosis and pre-
susceptibility of wild-type mice, but reduces the sus- vents colonization of the mouse gut by C. rodentium137.
ceptibility of mutant mice, to colitis-associated tumor- Further studies have shown that sentinel goblet cells
igenesis205,244, suggesting that the colitogenic potential located at the colonic crypt entrance execute NLRP6-
and transmissibility of the gut microbiota influence dependent secretion of mucin 2 (MUC2) to expel
disease outcome. intruding bacteria found in the inner mucous layer236
Other studies, however, challenged the idea that (Fig. 5d). In the small intestine of mice, a loss of endo­
NLRP6 controls the biogeography of the gut micro­ genous NLRP6 expression owing to stress is linked to
biota246,247. Phylogenetic analyses of wild-type mice and the development of intestinal pathology and changes
littermate-controlled mice lacking either NLRP6 or ASC to the relative abundance of Bacteroidetes, Clostridi­ales,
failed to reveal any difference in the composition of the Firmicutes, Lachnospiraceae, Lactobacillaceae, Rumino­
gut microbiota246. Lifetime separation of wild-type mice coccaceae and Streptococcaceae249. The peroxisome
and mice lacking NLRP6 also does not seem to promote proliferator-activated receptor-γ (PPARγ) agonist
alterations of their gut microbiota profile or differential rosiglitazone, among other functions, elevates the
susceptibility to DSS-induced colitis246, casting doubt expression of NLRP6 in the small intestine and pre-
over the previously suggested role of NLRP6 and ASC vents stress-related intestinal pathology in mice164,249.
in driving dysbiosis163. This study also argued that any Whether this therapeutic effect is directly or specifically
difference associated with the gut microbiota might dependent on the ability of rosiglitazone to modulate
be entirely dependent on the facilities used to house the expression of NLRP6 remains unclear.
the animals246. Thus, food sources, gut microbiota and An additional function of NLRP6 is its ability to
sterilization techniques used by different mouse vivar- suppress activation of the canonical NF-κB and MAPK
ium across the globe can influence the activation status pathways in response to infection by the gastrointesti-
of inflammasomes. Future experimental approaches nal pathogens L. monocytogenes, S. Typhimurium and
should be more tightly controlled to examine the role E. coli133 (Fig. 5d). This immunomodulatory pathway
of the gut microbiota in any disease context, including operates in the presence or absence of an altered gut
the use of littermate controls and careful evaluation of microbiota composition133, highlighting that certain
cage-to-cage and room-to-room effects and the overall biological functions of NLRP6 can clearly be uncoupled
sterility of the facility. from the gut microbiota and that previous studies iden-
Despite the uncertainty surrounding the nexus between tifying a connection with the gut microbiota should be
NLRP6 and the gut microbiota, the mechanistic functions interpreted with care and revisited.

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 Reviews

Nonalcoholic steatohepatitis, obesity and diet Conclusions

The influence of the inflammasome–gut microbiota Inflammasome signalling is a central pillar of innate
axis extends to the development of nonalcoholic steato­ immunity that triggers inflammation and cell death. The
hepatitis (NASH) and obesity. Mice lacking NLRP3, antimicrobial function of the inflammasome is largely
NLRP6, ASC, caspase 1 or IL-18 have a gut microbiota attributed to IL-1β and IL-18 and pyroptosis: IL-1β
profile that is associated with exacerbated hepatic stea- and IL-18 activate an inflammatory circuitry, whereas
tosis and inflammation and NASH progression, pheno- pyroptosis expels the entire infected cell from the body
types of which are transmissible to wild-type mice via gut or liberates concealed pathogen from the infected host
microbiota transfer245. A further study found that mice cell to facilitate pathogen elimination by other anti­
lacking ASC are prone to hepatosteatosis and obesity microbial mechanisms. These diverse and multifaceted
induction when fed a high-fat diet245. These abnormal- immune mechanisms are operated in both intestinal
ities are abrogated following treatment with the anti- epithelial cells and immune cells. Although micro­
biotics ciprofloxacin and metronidazole245, suggesting organisms across all kingdoms of life are detected by the
a connection with the gut microbiota in the context of inflammasome pathways, the mechanisms regulating
inflammasome signalling and obesity. activation of the inflammasome in response to many
A role for inflammasome sensors and diet in regu­ pathogens remain unclear, especially in the context of
lating microbial diversity and disease is further gastrointestinal viruses and protozoa.
demonstrated in mice lacking NLRP3, which when Uncontrolled inflammation and cell death underpin
fed a high-fat and high-carbohydrate diet develop the molecular basis of immunopathology and fuel the
microbial dysbiosis compared with mice lacking progression of chronic inflammatory conditions and can-
NLRP3 fed with standard chow; the degree of dysbio- cer. Inflammasome activities largely prevent the develop-
sis between the two groups seems to be more substan- ment of colitis and gastrointestinal cancer owing to their
tial than the changes observed between wild-type mice functions in tumour immunosurveillance, mucus produc-
that had been fed a high-fat, high-carbohydrate diet tion, cell-renewal, suppression of proliferation of intestinal
and wild-type mice that had been fed with chow250. epithelial stem cells and antitumorigenic responses. The
Compared with wild-type mice on a high-fat and specific PAMPs and DAMPs that trigger activation of
high-carbohydrate diet, the dysbiotic change in mice the inflammasome in the gut in non-infectious diseases
lacking NLRP3 on the same diet was associated with remain largely unknown. What is clear is that the develop-
elevated levels of triglyceride in the liver and faeces ment of intestinal inflammation and cancer is intimately
and increased intestinal permeability, liver injury and linked to dysregulated inflammasome signalling and/or
inflammation of the adipose tissue250. Moreover, a diet dysbiosis. This connection is in part owing to the ability
composed of high fat and high cholesterol induces a of the inflammasome and the gut microbiota to regulate
reduction in the relative abundance of Prevotella one another. Although advanced sequencing technol-
spp., which is associated with reduced expression of ogies have been informative in providing a snapshot of
pro-IL-1β in the intestine of mice carrying a mutation the global changes of the gut microbiota in the presence
that predisposes them to spontaneous inflammatory of inflammasome dysregulation, the causative microbial
bone disease251. species or population responsible for a specific clinical
manifestation has not been identified. Furthermore,
Gut–brain axis spatial and temporal changes to the gut microbiota over
The gut microbiota also operates at the interface the length of the intestine and over time with respect to
between inflammasome signalling and the gut–brain inflammasome dysregulation have not been investigated.
axis. An example of this relationship is the observation Nevertheless, further studies are needed in light of new
that inhibition of the inflammasome-mediated IL-1β evidence suggesting that the role of the inflammasome
signalling pathway in the small intestine in mice by S. in regulating the composition of the gut microbiota is not
Typhimurium-encoded E3 ubiquitin-protein ligase SlrP always apparent and is, in fact, heavily influenced by a
apparently prevents infection-induced anorexia owing multitude of genetic, environmental and experimental
to a blockade of signal transduction to the hypothala- factors associated with animal models246,247.
mus via the vagus nerve252. Furthermore, it was reported Given that experimental therapies have yielded
that mice lacking caspase 1 or treatment of wild-type success in the treatment of inflammasome-mediated
mice with a semisynthetic antibiotic minocycline, which disorders53,182,219,221,223,225, strategies targeting distinct
potentially functions to inhibit caspase 1 and modu- aspects of the inflammasome pathways will become an
lates the gut microbiota, are protected from developing emerging area of research and development. Discovery of
depressive-like and anxiety-like behaviours, even when novel small-molecule compounds for use in pharmaco­
experiencing chronic stress253. The increased resistance logical activation or inhibition of the inflammasome and
to stress in these mice is associated with an increase in the prevention and treatment of disease in humans
in the relative abundance of species of Akkermansia, will probably take priority in the next decade of inflam-
Blautia and Lachnospiraceae and a reduced abun- masome research. The deep and complex relationship
dance of Allobaculum spp., Bifidobacterium spp., between inflammasomes, pathogens and the micro­
Turicibacter spp. and Clostridium spp.253. These stud- biota provides an exciting platform for basic and clinical
ies highlight a complex and potentially emerging link research with which to understand health and disease.
between inflammasomes, microorganisms and the
gut–brain axis. Published online 5 September 2018

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