The complex role of inflammasomes in the pathogenesis of Inflammatory Bowel Diseases – Lessons learned from experimental models

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The complex role of inflammasomes in the pathogenesis of Inflammatory Bowel Diseases – Lessons learned from experimental models Mo`nica Aguilera a,b,1, Trevor Darby c,2, Silvia Melgar c,* a b c

Department of Cell Biology, Physiology and Immunology, Universitat Auto`noma de Barcelona, Bellaterra 08193, Barcelona, Spain Neuroscience Institute, Universitat Auto`noma de Barcelona, Bellaterra 08193, Barcelona, Spain Alimentary Pharmabiotic Centre, University College Cork, National University of Ireland, Cork, Ireland

A R T I C L E I N F O

A B S T R A C T

Article history: Available online xxx

Inflammasomes are a large family of multiprotein complexes recognizing pathogen-associated molecular pattern molecules (PAMPs) and damage-associated molecular patterns (DAMPs). This leads to caspase-1 activation, promoting the secretion of mature IL-1b, IL-18 and under certain conditions even induce pyroptosis. Inflammatory Bowel Diseases (IBD) is associated with alterations in microbiota composition, inappropriate immune responses and genetic predisposition associated to bacterial sensing and autophagy. Besides their acknowledged role in mounting microbial induced host responses, a crucial role in maintenance of intestinal homeostasis was revealed in inflammasome deficient mice. Further, abnormal activation of these functions appears to contribute to the pathology of intestinal inflammation including IBD and colitis-associated cancer. Herein, the current literature implicating the inflammasomes, microbiota and IBD is comprehensively reviewed. ß 2014 Published by Elsevier Ltd.

Keywords: Inflammasomes Microbiota IBD Colitis-associated cancer Caspase-1

Contents 1. 2. 3. 4.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The inflammasomes – definitions and components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Inflammatory Bowel Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Caspase-1, ASC and IL-1 family members in IBD-pathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Caspase-1 and Asc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. IL-1b and IL-18 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Animal models of IBD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IBD and genetic associations to the inflammasome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The inflammasomes and their association to IBD pathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . NLRP1 inflammasome. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1. NLRP3 Inflammasome – a dual role in gut homeostasis and IBD pathology . . . . . . . . . . . . . . . . . . . . . . . 7.2. NLRP6 inflammasome – a regulator of gut homeostasis and microbiota composition . . . . . . . . . . . . . . . 7.3. NLRP12 inflammasome – a regulatory inflammasome in IBD? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.4. NLRC4 inflammasome – a discriminator of commensal and pathogenic bacteria in intestinal infections 7.5. The PYHIN inflammasomes (IFI16, AIM2, MNDA and IFIX). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.6. The inflammasome, the intestinal microbiota and IBD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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* Corresponding author at: Alimentary Pharmabiotic Centre, Biosciences Institute, 4th Floor, University College Cork, Cork City, Ireland. Tel.: +353 21 4901384; fax: +353 21 4901436. E-mail addresses: [email protected] (M. Aguilera), [email protected] (T. Darby), [email protected] (S. Melgar). 1 Edifici V (Veterinary School), Despatx V0-131, Universitat Auto`noma de Barcelona, Spain. Tel.: +34 935814781. 2 Alimentary Pharmabiotic Centre, Biosciences Institute, 4th floor University College Cork, Cork, Ireland. Tel.: +353 21 4901794; fax: +353 21 4901794. http://dx.doi.org/10.1016/j.cytogfr.2014.04.003 1359-6101/ß 2014 Published by Elsevier Ltd.

Please cite this article in press as: Aguilera M, et al. The complex role of inflammasomes in the pathogenesis of Inflammatory Bowel Diseases – Lessons learned from experimental ?models. Cytokine Growth Factor Rev (2014), http://dx.doi.org/10.1016/ j.cytogfr.2014.04.003

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The inflammasomes and their contribution to colitis-associated colon cancer (CAC) Conclusions and future perspectives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction The collected data in the last decade, including the discovery of genes important for bacterial recognition and a dysregulation in microbial flora composition, points toward a crucial role for the innate immune system in Inflammatory Bowel Diseases (IBD) pathophysiology. The intestinal mucosa is in persistent contact with the commensal microbiota resulting in a state of physiological inflammation and when this equilibrium is altered it can lead to chronic inflammation such as IBD and colorectal associated cancer (CAC) [1,2]. Mammalian hosts have developed mechanisms that confer tolerance to the commensal microbiota or that initiate immune responses against invading pathogens. These are supported by an array of receptors, the so called pattern recognition receptors (PRRs). These include Toll-like receptors (TLRs) – detects microbes on the cell surface and in endosomes; and nucleotide oligomerization domain (NOD)-like receptors (NLRs) – detects microbes in intracellular compartments [3–5]. PRRs recognize different conserved pathogen associated patterns (PAMPs) and play important roles in host–bacteria interactions. Within the cytosolic compartment, PAMPs and DAMPs (damage-associated molecular patterns) can also be sensed by a subset of PRRs belonging to the NLR and HIN200 family called the inflammasomes. These are a group of proteins that scaffold and detect harmful stimuli playing an important role in innate immunity. Their function as mediators of inflammation in the pathophysiology of several chronic inflammatory conditions including IBD has recently emerged. The collected data so far indicate a far more complex impact of inflammasomes in regulating gastrointestinal inflammation and gut homeostasis than anticipated. However, a major understanding of inflammasome biology is gained from animal studies and therefore their contribution to human inflammatory disorders such as IBD is still yet to be fully elucidated. Herein, we sought to comprehensively review the current literature implicating the inflammasomes and their attributed contribution to IBD pathology. We will also discuss the complex crosstalk between inflammasomes, mucosal immune responses and the intestinal microbiota as learned from experimental models. 2. The inflammasomes – definitions and components Inflammasomes were first identified in 2002 by the group of Tschopp [6]. They were originally described as activators of inflammatory caspases and as regulators of IL-1b processing and therefore proposed to be vital players in innate immunity. It is now well acknowledged that inflammasomes contribute to host protection by inducing immune responses such as secretion of the cytokines IL-1b/IL-18 and pyroptosis, a programmed cell death dependent on caspase-1, which limits microbial invasion. However, over-activation of these responses appears to be linked to inflammatory conditions including obesity, IBD and diabetes [7–9]. Inflammasomes are high-molecular weight (700 kDa) cytosolic multi-protein complexes found in cells of the innate immune system including macrophages, monocytes, dendritic cells, neutrophils; cells of the adaptive immune system such as T cells and in nonimmune cells such as epithelial cells. Inflammasomes are formed by a sensor protein, an adaptor protein and an inflammatory caspase. The sensor proteins are PRRs that act as scaffolds, and could be a

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member of the NLR or a pyrin (PYD) and HIN domain-containing protein (PYHIN) family. The members of the NLR family include the NOD (NOD1, NOD2, NOD3/NLRC3, NOD4/NLRC5, NOD5/NLRX1, CIITA), NLRP or NALP (NLRP 1–14) and IPAF (NLRC4 and NAIP) subfamilies. Structurally, all of these NLR proteins have a nucleotidebinding domain (NBD), which is necessary for ATP-dependent oligomerization of inflammasome assembling and a carboxyterminal leucine-rich repeat (LRR), which is involved in autoregulation and in sensing activation signals. They can also contain a PYD or/and a N-terminal caspase recruitment CARD domain, which facilitates downstream signaling by homotypic protein–protein interactions [7,10]. Other inflammasomes that contain a PYHIN rather than NLR domain include absent in melanoma 2 (AIM2) and IFNg-inducible protein 16 (IFI16). AIM2 and IFI16 have DNA-binding HIN domains and can therefore recognize DNA from virus [11–14]. In Table 1 we list the structures, cell subsets and activators of inflammasome-associated components involved in gastrointestinal responses. The apoptosis-associated speck-like protein (ASC), is an adaptor protein needed for the assembly of the inflammasome complex, and whose ultimate goal is to activate caspase-1 and -5, which are important for the cleavage of pro-IL-1b/IL-18 into mature IL-1b and IL-18 [6]. A third caspase, caspase-8 has recently been shown to cleave pro-IL-1b under certain conditions e.g. Salmonella infection [15,16]. The inflammasomes can sense an array of stimuli via PRRs, including PAMPs from the bacterial cell wall (e.g. lipopolysaccharides (LPS), lipoproteins or flagellin), bacterial and viral nucleic acids and fungal cell wall components (e.g. zymosan and mannan) or from DAMPs including ATP, uric acid, amyloid b, asbestos, silica, hyaluronan and heparin sulfate [5,11,17]. These recognition patterns appear to be redundant since one receptor can recognize several stimuli and one stimulus can activate several receptors. Caspase-1 is a protease implicated in the proteolytic cleavage of pro-inflammatory cytokines, and can also induce pyroptosis (presenting features of both apoptosis and necrosis) as a response to PAMP and DAMP signals [9]. Caspase-1 processes the cytokines IL-1b, IL-18 and IL-33. IL-1b is implicated in systemic and local immune responses caused by infection, injury and immunological challenges resulting in fever and promoting leukocyte migration and Th17 responses [18,19]. In contrast, IL-18 does not have pyrogenic activity and its induction is a result of IFNg and IL-12 activation [5,8,19]. In contrast to IL-1b and IL-18, IL-33 is biologically active and caspase-1 processing appears to render its inactivity [19,20]. The inflammasomes can be regulated by cell-extrinsic or cellintrinsic mechanisms. For cell-extrinsic regulation there are different mediators that positively or negatively regulate and prime inflammasomes, including IFN-b and g for AIM2 [21,22], certain bacteria strains or components such as Salmonella typhimurium or flagellin for NLRC4 [23,24] or other mediators such as inducible nitric oxide synthase (iNOS) or nitric oxide (NO) for NLRP3 [5] (Table 1). Cell-intrinsic regulatory agents include ion fluxes (low intracellular K+ or Cl levels), oxidative states (e.g. reactive oxygen species inhibitors suppressing NLRP3) and/or autophagy (e.g. ATG16L1) (Table 1). Moreover, inflammasomes can also be regulated by interacting with host proteins e.g. CARD-containing proteins such as CARD8 sequesters caspase-1 resulting in inhibition of the functional inflammasome complex [5].

Please cite this article in press as: Aguilera M, et al. The complex role of inflammasomes in the pathogenesis of Inflammatory Bowel Diseases – Lessons learned from experimental ?models. Cytokine Growth Factor Rev (2014), http://dx.doi.org/10.1016/ j.cytogfr.2014.04.003

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Table 1 Description of Inflammasome associated components. Family subtype

Domain structure in humansa

GI expression

Activatorsb

References

Human

Mice

NLR family PYD domain NLRP1/NALP1/CARD7

Nalp1a,b,c

CARD-PYD-6xLRR-NACHT

Intestinal epithelial cells Immune cells Paneth cells

Lethal toxin (fr. Bacillus antrachis) MDP

[5,76,80, 81,84,87]

NLRP3/NALP3/Cryopyrin

Nalp3

PYD-9xLRR-NACHT

Intestinal epithelial cells Immune cells (DC, MØ)

[5,32,58,75,78, 89,92,95,98,149]

NLRP6/NALP6

Nalp6

PYD-5xLRR-NACHT

NLRP7/NALP7/NOD12

–

PYD-9xLRR-NACHT

– Nalp12

PYD-6xLRR-NACHT PYD-8xLRR-NACHT

NLRP14/NALP14/NOD5

Nalp14

PYD-11xLRR-NACHT

Microbial diacylated lipopeptides NDc Yersinia pestis; S. typhimurium; Listeria spp. NDc

[84,150]

NRLP11/NALP11/NOD17 NRLP12/NALP12

Small and large intestine Colonic myofibro-blasts Intestinal epithelial cells Germ cells; BMDM/MØ Paneth cells Paneth cells Colon Myeloid cells Myeloid cells

ATP; amyloid b; alum; asbestos; glucose hyaluronan; MSU; cholesterol; silica; ROS; pore forming toxins; ss-RNA and dsRNA; M-proteins; Hemozoin; b-glucans; hyphae; LPS; CpG DNA ATP Microbiota

IPAF/NAIP domain NLRC4/Ipaf/CARD12

Ipaf

CARD-14xLRR-NACHT

Intestinal epithelial cells MØ, T cells, monocytes Small and large intestine

Naip (a, b, c, d, e, f, g) (1, 2, 3, 4, 5, 6, 7) Naip2

3xBIR-NACHT

Intestinal epithelial cells, MØ Small and large intestine

PYHIN-AIM2-like family AIM2

Aim2

PYD-HIN200

IFI16

Ifi204

PYD-2HIN200

PYHIN1/IFIX

Pyhin1/Ifix

PYD-HIN200

Immune cells (MØ) and intestinal epithelial cells Small and large intestine Epithelial, immune and fibroblast cell lines Immune cells (MØ)

NAIP/BIRC1

[43,81, 113,115]

[64,84] [81,120–122, 126,127,151] [64]

Flagellin; S. typhimurium; L. pneumophila; Pseudomonas spp.; Yersinia spp.; Listeria spp. T3SS and T4SS ROD proteins; Flagellin; S. typhimurium; L. monocytogenes; T3SS; TEA domain protein 1 (TEAD1)

[3,17,24,41,78, 81,94,128, 130–133,141]

dsDNA; L. monocytogenes; Francisella tularensis

[7,8,14,149, 152]

dsDNA

[13,14]

DNA

[13,154]

[81,128,129,131, 149,152,153]

Inflammasome adaptor proteins ASC Asc

PYD-CARD

Immune cells (Monocytes, neutrophils and MØ). Epithelium of small intestine and colon

Fas; Inflammasomes

[33,155,156]

Inflammasome related caspases Caspase-1 Caspase-1

CARD

ASC Inflammasomes Dectin 1; cycloheximide; ripoptosome; inflammasome independent: TNFR and FAS ER stress

[32–34]

Caspase-8

Caspase-8

2xDED

Small intestine and colon Macrophages DC

Caspase-4 (ICH2)

Caspase-4 or -11 –

CARD

NDc

CARD

NDc

LPS NALP1

[6]

–

CARD

Intestinal epithelial cells MØ Small intestine

NDc

[1,157]

Caspase-5 (casp1-related or ICH3) 6 isoforms Caspase-12

Family subtype Human

Mice

Inflammasome related cytokines IL-1b

Il-1b

[5]

[32]

GI expression

Activatorsb

References

Widely expressed Hematopoietic cells and tissue MØ

NF-kB Casp-1 MALT1-ASC-casp8 ATP (by P2X7) Proteinase 3 LPS (by TLR4)

[5,19,51,52]

Please cite this article in press as: Aguilera M, et al. The complex role of inflammasomes in the pathogenesis of Inflammatory Bowel Diseases – Lessons learned from experimental ?models. Cytokine Growth Factor Rev (2014), http://dx.doi.org/10.1016/ j.cytogfr.2014.04.003

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4 Table 1 (Continued ) Family subtype Human

Mice

IL-18 (IGIF, IL1F4) 2 isoforms

Il-18

GI expression

Activatorsb

References

MØ DC Epithelial cells

Constitutively expressed Casp-1; Casp-4; IFNU; LPS; Flagellin; Nigericin; ATP

[5,19,52]

a PYD – pyrin domain, also known as PAAD/DAPIN; LRR – leucine-rich repeat domain; NACHT – nucleoside triphosphatase (NTPase) domain; CARD – caspase activation and recruitment domain; BIR – baculovirus inhibitor of apoptosis protein repeat, also known as IAP (inhibitor of apoptosis domain); HIN200 – interferon-inducible p200, also known as IFI200; DED – death domain. b MDP – muramyl dipeptide; BCL2 – B-cell lymphoma 2; BCLXL – B-cell lymphoma-extra large; PKR – protein kinase receptor; PKC – protein kinase C; MSU – monosodium urate crystals; ROS – reactive oxygen species; M proteins – virulence factor that can be produced by certain Streptococcus; GBPs – guanylate binding proteins; BRCC3 deubiquitinase of NLRP3; IAPs – inhibitor of apoptosis proteins; HSP90 – heat-shock protein 90; SGT1 – ubiquitin ligase-associated protein; MØ – macrophage; NOD – nucleotide-binding oligomerization domain; FAF1 – FAS-associated factor, apoptotic inducer; UBA – ubiquitin-associated domain; T3SS – bacterial type III secretion system; T4SS – bacterial type IV secretion systems; TEAD1 – transcriptional enhancer factor TEF-1; YAP – yes-associated protein; CAD – caspase-activated DNase or carbamoyl phosphate synthetase aspartate transcarbamylase dihydroorotase; poly(IC) – polyinosine-polycytidylic acid; ds – double stranded; ss – single stranded; DC – dendritic cell; MHC-II – major histocompatibility complex class II; Ripoptome – multiprotein complex that induced cell death due to genotoxic stress or depletion of apoptosis protein (IAP); ER – endoplasmic reticulum; LPS – lipopolysaccharide; TNF – tumor necrosis factor; IL – interleukin; Casp – caspase; IFN – interferon; PRR – pattern recognition receptor; MALT – mucosa-associated lymphoid tissue. c ND – not determined.

The activation of the inflammasome requires at least 2 signals; one, also known as priming step, induced by TLR activation of NFkB, which promotes ASC and pro-caspase-1 signaling which, in turn, cause the transcription of pro-IL-1b, e.g. LPS priming of NLRP3 [7]; and a second signal originating from the cytoplasmatic NLRs that activates the inflammasome resulting in the cleavage of cytokines (summarized in Fig. 1). The 2nd signal which activates the NLRs can be mediated by several activators as discussed above. The best studied activation cascade for NLRP3 inflammasome consists of: 1) The purinergic receptors e.g. P2X7 are activated by the endogenous ligand ATP, resulting in pore formation in the cell membrane whereby K+ effluxes from the cell leading to the formation of pannexin-1 channels allowing traffic of PAMPs and DAMPs leading to NLRP3 activation. 2) Phagocytosis of crystalline or particulate ligands activates the inflammasome after rupture of the phagolysosomes. 3) DAMPs and PAMPs can also induce ROS which in turn can activate the inflammasomes (Fig. 1). The collected data thus far suggests that microorganism ligands and cellular damage are necessary for inflammasome signaling [3,5].

IL-10 and IL-13 whereas CD presents a Th1/Th17 profile associated with high IFNg, IL-12 and IL-17 levels [25–27]. 4. Caspase-1, ASC and IL-1 family members in IBD-pathology Recognition of PAMPs/DAMPs by NLRs leads to caspase-1 activation, which leads to the processing of the cytokines IL-1b/IL18. Both UC and CD present elevations in several innate cytokines including IL-8, IL-6, IL-1b and IL-18 [25–27]. An enhanced production of IL-1b and IL-18 in the inflamed mucosa of IBD patients was described in the late 1980s and late 1990s, respectively [28,29]. In addition, evidence of increased caspase-1 activity was identified in intestinal tissue and in macrophages from patients with both UC and CD [30,31] (Tables 1 and 3). These reports represent the early links on inflammasome alterations and IBD pathophysiology. In this section we will summarize the contribution of caspase-1 and ASC and IL-1 family members to the pathology of IBD. Table 1 summarizes different activators of caspase-1, -3, -4 and -8, ASC and inflammasome related cytokines IL-1 and IL-18.

3. Inflammatory Bowel Disease 4.1. Caspase-1 and Asc Inflammatory Bowel Diseases is a group of chronic inflammatory conditions affecting the gastrointestinal tract, mainly represented by ulcerative colitis (UC) and Crohn’s disease (CD). These are debilitating disorders affecting the patient’s quality of life and with unknown etiology. However, the collected evidence indicates that genetically susceptible individuals with a dysregulated immune response can, under certain environmental factors, develop bowel inflammation. Alterations in epithelial barrier function, the commensal microbiota (dysbiosis), diet and the intestinal innate and adaptive immune system are the main factors implicated in these conditions. Recently a new group of Escherichia coli, adherent-invasive E. coli (AIEC), was recovered from biopsies of CD patients but not UC, suggesting AIEC may be involved in the pathology of CD [25,26]. Although UC and CD share some clinical features including abdominal pain, fever, bowel diarrhea with blood and/or mucus excretion their location and inflammatory profile differs. UC usually starts in the rectum and extends throughout the colon, with the inflammation characteristically restricted to the mucosal surface. One characteristic feature of UC is the formation of crypt abscesses, formed by extravasation of neutrophils through the intestinal epithelium [27]. Contrary to UC, CD can affect the entire gastrointestinal tract although the terminal ileum and proximal colon are mainly affected. The inflammation in CD is generally transmural and characterized by the formation of granulomas, fissures and fistulas [25]. The cytokine profile in UC is an atypically T helper (Th)-2, associated with high levels of IL-5,

Caspase-1 is an intracellular cysteine protease whose major function is in the processing and mature release of IL-1b and IL-18. ASC and inflammasomes activate caspase-1 whereas caspase-12, proteinase inhibitor-9, CARD8 and intact pyrins are described to be inhibitors of caspase-1 [32–34]. Initial studies in caspase-1 / mice and in mice treated with Pralnacasan, a caspase-1 inhibitor or in mice treated with IL-1R antagonist (IL-1Ra) and exposed to Dextran Sodium Sulfate (DSS) revealed amelioration of colitis. The improvement in colitis was due to a reduction in disease symptoms, intestinal pathology and colonic IL-1b and IL-18 expression [35–37]. Furthermore, caspase-1 activation of intestinal epithelial cell lines and in IL-10 / mice led to an altered intestinal permeability which was reversed upon addition of caspase-1 inhibitor [38]. In contrast, recent studies have reported worsening of DSS-induced colitis in caspase-1 / mice. This was attributed to a deficiency in epithelial cell restitution and associated with the critical role of IL-18 for the early induction of tissue repair [39,40] (Table 2). In contrast to the results obtained in caspase-1 / mice, Asc / mice showed amelioration of colitis and no defect in epithelial regeneration [1], suggesting that caspase-1 is required for tissue repair and epithelial cell regeneration. The results from Asc / mice are most likely linked to the dual role of this adaptor protein functioning both as an activator of the inflammasomes and as an inhibitor of the NF-kB pathway [41,42]. In contrast, a second study reported that Asc / mice grow normally but spontaneously develop colonic crypt

Please cite this article in press as: Aguilera M, et al. The complex role of inflammasomes in the pathogenesis of Inflammatory Bowel Diseases – Lessons learned from experimental ?models. Cytokine Growth Factor Rev (2014), http://dx.doi.org/10.1016/ j.cytogfr.2014.04.003

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Fig. 1. Components of the inflammasomes, their assembly and activation. Representative image showing the general mechanisms of activation of the inflammasomes largely based on NLRP3 inflammasome signaling. A large variety of endogenous and exogenous (microbial) molecules (DAMPs and PAMPs) can induce the inflammasome cascade. Two signals are needed to secrete the inflammasome related cytokines IL-1b and IL-18 and other cytokines (such us IL-6 and TNFa), one comes from TLRs and the other one from the cytoplasmatic NLRs to, at the end, induce inflammation or pyroptosis.

hyperplasia, an alteration in the crypt-to-villus ratio in the terminal ileum, enlargement of Peyer’s patches and were more susceptible to DSS-induced colitis and CAC. A similar profile was observed in Nlrp6 / mice [40,43] (Table 2). Future studies aiming to dissect caspase-1 and Asc function should investigate the expression and regulation of these genes in the intestinal epithelium e.g. in conventional caspase-1/Asc mice or in intestinal epithelial cells lacking or overexpressing these genes and exposed to IBDassociated bacteria. 4.2. IL-1b and IL-18 IL-1b is a pleiotropic cytokine regulating a wide range of functions and this cytokine is critical for the pathology of several chronic conditions including IBD, diabetes and CAC [9,44]. IL-1b is

produced by cells of the innate immune system (macrophages, dendritic cells, monocytes) and from non-immune cells such as epithelial cells. Caspase-1 regulates the cleavage of inactive pro-IL1b to active mature IL-1b upon NLR-stimulation. Secreted IL-1b binds to IL-1R1 forming an IL-1R complex; which recruits MyD88 and phosphorylates several kinases (IRAK), accompanied by the translocation of NF-kB to the nucleus, thereby activating proinflammatory genes. Increased IL-1b secretion was identified in macrophages and dendritic cells in the lamina propria and in epithelial cells of IBD patients [28,30]. High levels of IL-1b correlated well with disease activity and with the presence of active lesions in IBD tissue [45,46] (Tables 1 and 3). In addition, high levels of IL-1b were also associated with disease progression in several animal models of colitis [47,48] and treatment with IL-1 blocking agents reduced disease severity [18,35] (Table 2). In

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Table 2 Summary of intestinal inflammation and cancer outcome in mice deficient of inflammasome components. Murine Modela

Insultb

Inflammation/CACc

Disease pathology

Role in homeostasis

Microbiota involvement

References

Nlrp3

/

C. rodentium 2% DSS 3% DSS AOM/2% DSS 2.5% DSS 2% DSS 5% DSS 3  2.5%DSS AOM/2.5% DSS

Acute Acute Acute CAC Acute Acute Acute Chronic CAC

More susceptible Less inflammation More susceptible Similar to WT More susceptible Less susceptible More susceptible More susceptible More susceptible

NDd ND Yes ND Yes Yes Yes Yes Yes

ND Yes ND ND Yes ND ND ND ND

[135] [43] [108] [141] [110] [106] [58]

Nlrp6

/

3.5% DSS AOM/2% DSS 2% DSS

Acute CAC Acute

More susceptible More susceptible More susceptible

Yes Yes Yes

ND ND Yes

[40,112,113]

3% DSS AOM/3% DSS

Acute CAC

More susceptible More susceptible

Yes Yes

ND ND

[126]

C. rodentium 2% DSS AOM/2% DSS AOM/2% DSS

Acute Acute CAC CAC

More susceptible More susceptible More susceptible Similar to WT

ND Yes Yes Yes

ND ND ND ND

[135] [24,40,140]

More susceptible Alteration in IL-18 secretion

Yes Yes

ND ND

[134,136]

[135] [43] [40] [58]

Nlrp12

Nlrc4

/

Aim2

/

/

F. novicida mCMV

Caspase-1

Asc

/

Pycard

Il-1b

Il-18

/

/

/

/

[43]

[58]

C. rodentium 2% DSS AOM/2% DSS 5% DSS 3  2.5% DSS AOM/2.5%DSS 3.5% DSS 3  2% DSS and IL-1Ra treatment 3.5% DSS and pralnacasan

Acute Acute CAC Acute Chronic CAC Acute Chronic Acute

More susceptible More susceptible More susceptible More susceptible More susceptible More susceptible Less susceptible Modest effect Reduced inflammation

ND ND ND Yes Yes Yes Yes No Yes

ND Yes ND ND ND ND ND ND ND

2% DSS AOM/2% DSS

Acute CAC

More susceptible More susceptible

ND ND

Yes Yes

[43] [40]

5% DSS 3  2.5% DSS AOM/2.5% DSS

Acute Chronic CAC

More susceptible More susceptible More susceptible

Yes Yes Yes

ND ND ND

[58]

C. rodentium 2.5% DSS 3% DSS and anti-IL-1b treatment

Acute Acute Acute

ND Yes ND

ND ND ND

[135] [36] [158]

H. hepaticus and CD4+CD45RBHi T cells transfer model and anti-IL-1b treatment

Chronic

More susceptible More susceptible Reduce inflammation IL-1b increase inflammation Reduce inflammation IL-1b increase inflammation

ND

ND

[18]

C. rodentium 3% DSS AOM/2% DSS

Acute Acute CAC

More susceptible More susceptible More susceptible

ND Yes Yes

ND Yes Yes

[135] [40,108]

[35,53] [37]

a

Mouse deficiency and/or treatment. C. Rodentium – Citrobacter rodentium; DSS – dextran sodium sulfate; AOM – azoxymethane; TNBS – trinitrobenzene sulfonic acid; WT – wild type; mCMV – mouse cytomegalovirus; H. hepaticus – Helicobacter hepaticus. c Acute – acute inflammation; Chronic – chronic inflammation; CAC – colitis-associated colorectal cancer. d ND – not determined. b

contrast, treatment with IL-1 blocking agents such as Anakinra (used in rheumatoid arthiris patients) proved to worsen disease in CD patients [49]. a-Defensins and IL-1Ra are described to act as inhibitors of IL-1b [5,19,50–52]. Table 1 summarizes activators of IL-1b. IL-18 is a cytokine that regulates both Th1 and Th2 induced responses. IL-18 is expressed in an array of hematopoietic and nonhematopoietic cell lineages including intestinal macrophages, intestinal epithelial cells and Kuppfer cells (Tables 1 and 3) [19,29,30]. IL-18 signals via an IL-18R complex through MyD88/ IRAK-pathways in a similar pattern as IL-1b [19]. Contrary to Il1b / mice, Il-18 / and Il-18r1 / mice are more susceptible to DSS-induced colitis, most likely due to the critical role played by IL18 in the early phases of wound healing [43,53,54]. In support of this regulatory role, IL-18 derived from the colonic epithelial cell is

highly elevated in the early phases of DSS-induced colitis [1,54], caspase-1 / mice treated with exogenous IL-18 showed accelerated recovery from colitis and epithelial regeneration [53,55] and adoptive transfer of Il-18+ myeloid cells to caspase-1 / mice failed to rescue the DSS-induced phenotype [1,8,9] (Table 2). The mechanism(s) by which IL-18 promotes early tissue repair remains unclear [55], although the adaptor protein MyD88 was shown to regulate intestinal epithelial repair via prostaglandins/COX-2 signaling pathways [1,55]. Nevertheless, the protective role of IL-18 appears to be related to the early phase of colitis, since excessive IL-18 production is associated with chronic inflammation and IL-18 blocking agents ameliorate it [5,39,53,56]. In agreement with these, IL-18 is highly expressed in the epithelium of patients with inactive CD, and as disease severity progresses IL18 epithelial expression decreases while IL-18+ macrophages

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Table 3 Expression and genetic susceptibility of inflammasome components in human IBD. Inflammasome component

Sourcea

Outcomeb

Reference

NLRP1

mRNA of peripheral blood cells and monocytes (CD patients) mRNA of Paneth cells

2 SNPs associated to colonic and inflammatory CD

[64,84]

No major differences

(healthy, CD and UC patients) NLRP3

DNA (CD patients) PBC and Monocytes (CD patients) Colon biopsies (healthy and CD patients) DNA (CD patients) DNA (CD patients)

6 SNPs in a regulatory region downstream of NLRP3 associated to CD Decreased NLRP3 expression and rs4353135 genotype Decreased LPS-induced IL-1b secretion associated to rs6672995 High NLRP3 expression NLRP3 loci described as a risk factor 4 SNPs associated to CD-susceptibility No significant associations to British CD cohort

[38,39,42,43,62,64, 67,68]

NLRP3 and CARD8

DNA (CD patients)

Combined polymorphisms in C10X and Q705K alleles in CARD8 and NALP3

[66]

NLRP3 and NOD2

DNA (CD patients)

The polymorphism in CARD8 with NLRP3 conferred a protective effect against Crohn’s disease (and vice versa)

[159]

NLRP7

mRNA of Paneth cells (healthy, CD and UC patients)

No differences

[84]

NLRP11

DNA (CD patients) mRNA of Paneth cells (healthy, CD and UC patients)

2 SNPs weakly associated to CD No differences

[64,84]

NLRP14

DNA (CD patients)

Weak association to CD

[64]

Caspase-1

Tissue (UC and CD patients) IBD-macrophages mRNA of Paneth cells (healthy, CD and UC patients)

Active caspase-1 increased in UC and CD tissue Increased active caspase-1 from IBD-macrophages Expression decreased in the metaplastic Paneth cells of the CD patients

[30,31,84,112,113]

IL-1b

DNA (IBD patients) IBD-macrophages

No association of IL-1b to UC or CD No association of IL-1b or IL1R to CD Increased IL-1b expression from IBD-Macrophages and epithelial cells

[28,30,160,161]

IL-18

DNA (IBD patients) DNA (IBD patients) Colon biopsies (healthy and IBD patients)

An SNP related to IL-18 gene was more frequent in CD females Two alleles associated to extent of disease in UC No association to CD Increased IL-18 expression in macrophages, dendritic cells and epithelial cells in active CD

[29,162,163]

IL18R

DNA (CD and UC patients)

IL1RL1, IL18R1, IL18RAP and SLC9A4 genes identified as IBDsusceptibility genes CARD9 variant, associated predominantly with UC patients

[164]

a b

CD – Crohn’s disease; UC – ulcerative colitis; PBC – peripheral blood cells. SNPs – single-nucleotide polymorphisms.

increases in patients with active CD. None of these changes in IL-18 are associated to patients with UC [29,57] (see Tables 1 and 3). Overall, the experimental and human data indicate that IL-18 plays a dual role in intestinal homeostasis and inflammation.

5. Animal models of IBD In the last decade, over 40 animal models have been described in studies of IBD. Many of these models have provided significant insights into IBD pathogenesis and facilitated the evaluation of novel therapeutic avenues. These models are generally divided into 3 categories – chemically induced models, spontaneous models (mainly due to genetic manipulation) and models dependent on transfer of cells to immunodeficient recipients [56]. Among these, DSS-induced colitis, 2,4,6-trinitrobenzene sulfonate (TNBS)-induced colitis, the IL-10 / mouse and the SCID-transfer model are the most well accepted animals models in IBD research. Although these models do not develop IBD per se, they do present different profiles of the disease (e.g. innate and adaptive immune responses), reflect different inflammatory phases of both diseases (e.g. acute and chronic inflammation) or display clinical aspects of

IBD (e.g. diarrhea). Increments in IL-1b and IL-18 have been associated with intestinal inflammation in several of these models and this is why they have been utilized as tools to examine the contribution of the inflammasomes to IBD pathology. The colitisassociated cancer (CAC) model, where mice are injected with the carcinogen azoxymethane (AOM) in conjunction with 2–3 cycles of DSS treatment thereby resulting in an acceleration in tumor progression, has been an important tool when examining the involvement of the inflammasome in epithelial homeostasis [2,9,58].

6. IBD and genetic associations to the inflammasome Genome-Wide Association Studies have so far identified 163 susceptibility loci for IBD, with at least 30 loci classified specifically to CD and 23 loci to UC. The bacterial recognition receptor NOD2 was the first gene identified to confer a higher CD risk and mutations in NOD2 are found in up to 25% of CD patients [59–61]. Other SNPs associated with the recognition of bacteria and CD susceptibility are genes involved in autophagy, i.e. ATG16L1 and immunity-related GTPases family M (IRGM), while genes involved

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in the adaptive immune responses e.g. IL-18R and IL-23R are associated with both UC and CD [3,60,62]. The NLRP3 related NLRs NOD1 and NOD2 are found in the cell cytosol where they recognize the bacterial peptidoglycans muramyldipeptide (MDP and g-DGlu-mDAP (iE-DAP), NOD2 and NOD1 ligands, respectively) resulting in the activation of the transcription factors NF-kB and AP-1 in Paneth cells, epithelial cells and antigen-presenting cells, leading to the release of pro-inflammatory mediators such as IL-1b [63]. Recent reports have linked members of the NLR family with CD susceptibility, these include NLRP1 (located on chromosome 17), NLRP3 and NLRP11 (located on chromosome 19) and NLRP14 (located on chromosome 11) [64], where the mutations are mostly found in the C-terminal leucine-rich repeats [59]. Furthermore, two studies described that the polymorphisms in the NLRP3 gene, either alone or in combination with CARD8 gene, resulted in decreased NLRP3 expression and altered IL-1b production which were linked to chronic auto inflammatory syndromes [65] and Crohn’s disease [9,66,67]. In addition, homozygosity for the NLRP3 risk allele was associated with the lowest level of IL-1b secretion from LPS-stimulated monocytes. However, a third study failed to confirm an association between NLRP3 and CD patients in a large British cohort [68]. The difference in the results from these studies may be attributed to many factors, including the heterogeneity of the population, differences in technical and analytical approaches, design of study. Overall, further work is required for confirmation of the association between any of the identified NLRP SNPs and CD and their subsequent functional effects on inflammasome induced responses to IBD pathology. IBD is associated with alterations in the ability of patients to sense the gut microbiota and many IBD patients present with a dysbiosis in their intestinal microbiota. The interaction between NOD2/CARD15 and ATG16L1 (allele T300A) and also IRGM [59,69], has linked the alteration in bacterial recognition in IBD to autophagy pathways thus underlying their importance in IBD, and in particular CD, pathogenesis. Furthermore, NOD1 and NOD2 also connect bacterial sensing to autophagy through ATG16L1 via a RIP2/NF-kB independent pathways [70], and TRIF-dependent activation of caspase-1 leading to IL-1b and IL-18 secretion [59,71,72]. It is suggested that in the absence of autophagy (or mitochondrial autophagy), damaged mitochondria accumulate and generate excessive ROS and release mitochondrial DNA into the cytoplasm of cells, events that trigger activation of the NLRP3 inflammasome [73,74]. The specific role of NOD-ATG16L1 autophagosome regulation and how mitochondrial ROS and/or mitochondrial DNA enhance the inflammasome-mediated release of IL1b and IL-18 is still unclear [11]. The collected data so far indicate that NODs, inflammasomes and autophagy may have a protective role within the gut. Table 3 summarizes the genetic association and expression of inflammasome components in patients with CD and UC. 7. The inflammasomes and their association to IBD pathology The persistent chronic inflammation in IBD patients has been widely associated to alterations in the adaptive immune response represented by a Th1/Th17 profile in CD patients and atypical Th2 profile in UC patients [25–27]. The collected evidence to date indicates that a dysfunction in the innate immune system greatly contributes to IBD-pathology. The inflammasomes were originally described as major players of the innate immune system and therefore they were not considered as major contributors to chronic inflammatory disorders. However, collected data generated in experimental models of autoimmunity and chronic inflammatory disorders have revealed a more complex crosstalk between the inflammasomes and the innate and adaptive immune responses. The inflammasomes are formed as a result of

simultaneous expression of all components within a specific cell type. Several of its components such as caspase-1 and Asc are widely expressed and an array of different ligands can be recognized by one NLR e.g. NLPR3, suggesting a cell-specific regulation of the inflammasome-induced response. This has become evident especially in the gut, where multiple cell types interact with commensal microbiota and potential pathogens, and therefore different cell types (epithelial vs. hematopoetic cells) can mount different and often complementary functions during intestinal immune responses. Below we will summarize the collected data on the NLRP1, NLRP3, NLRP6, NLRP12, NLRC4 and AIM2 inflammasomes and their relationship with intestinal homeostasis and IBD pathology. Fig. 2 summarizes the inflammasome related-responses associated to healthy and diseased intestine. Table 1 describes different gastrointestinal inflammasome components and its activators. Table 2 summarizes the reports on inflammasome components in models of experimental IBD and CAC. 7.1. NLRP1 inflammasome NLRP1 (NALP1) was the first inflammasome identified in 2002 [6]. The NLRP1 inflammasome complex encompasses caspase-1, caspase-5 and the adaptor proteins, ASC and CARDINAL. Human NLRP1 gene contains two signal transduction domains (PYD and CARD), unlike mice which present several paralogs (Nlrp1a, Nlrp1b, Nlrp1c), with Nlrp1b lacking the N-terminal PYD domain [75,76]. Human NLRP1 is genetically linked to several autoimmune and auto-inflammatory diseases including vitiligo, autoimmune thyroid diseases, and type I diabetes [77]. A recent study found a weak association between NLRP1 and CD susceptibility, although a higher association to disease behavior such as colonic disease was apparent [64]. Known activators of NLRP1 include MDP (human and mice) and the lethal factor (LeTx) toxin secreted by Bacillus anthracis (mice) [6,76,78]. NLRP1 is mainly localized to the nucleus [4] and can be expressed by a wide range of cells including granulocytes, monocytes, dendritic cells, B and T cells, Langerhans cells and in the epithelium of the stomach, intestines and lung [79– 81]. The adaptor ASC does not seem essential for the activation of caspase-1 in mouse Nlrp1b macrophages [82,83]. Furthermore, recent studies suggest that murine Nlrp1 plays a major role in cell death independent of IL-1b and IL-1R [77]. There are currently no human data supporting a similar function for human NLRP1. In the context of IBD, no major differences in NLRP1 or NLRP7 mRNA expression were reported in Paneth cells of IBD patients compared to controls (Tables 1 and 3) [84]. Chronic stimulation of primary human monocyte-derived macrophages (MDM) with the NOD2 ligand MDP was partly dependent on NRLP1-inflammasome. The caspase-1 activated IL-1 secretion led to a reduction in proinflammatory cytokines and up-regulation of bacteria killing [85]. In line with these results, NOD2 stimulation in vivo led to improvement of experimental colitis, although it is currently unknown if this response is dependent on NLRP-1 [86]. The contribution of NLRP1 in experimental models of IBD or in intestinal homeostasis has yet to be investigated. 7.2. NLRP3 Inflammasome – a dual role in gut homeostasis and IBD pathology NLRP3 (NALP3/cryopyrin) is one of the best characterized inflammasomes to date. The NLRP3 inflammasome uses the adaptor molecule ASC and caspase-1 to convert pro-IL-1b and pro-IL-18 into their active forms. NLPR3 can be activated by an array of stimuli including microorganisms such as viruses (Sendai and influenza viruses and adenovirus [87]), fungi (Candida albicans and Saccharomyces cerevisiae [88]) and bacteria (Staphylococcus

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Fig. 2. Model depicting the contribution of the inflammasomes to intestinal homeostasis and inflammation. Representative scheme showing the normal (homeostasis) and altered intestinal regulatory functions (disease) regulated by inflammasome responses. As represented, microbial compartment have an active role in the maintenance of intestinal homeostasis (e.g. tissue repair and proliferation). Different inflammasomes (NLRP3, NLRP6, NLRP12 and NLRC4) are expressed in both hematopoietic and nonhematopoietic compartment affecting host-defenses and homeostasis. Altered immune responses and microbial composition lead to aberrant responses and inflammation. Regulation of the inflammasome involves epithelial compartment (NLRC4, NLRP6 and NLRP12), the hematopoietic cells (NLRP3 and 12) and stromal cells (NLRP6).

aureus and Listeria monocytogenes [89,90]); individual bacterial components such as LPS, single stranded RNA (ssRNA), doublestranded RNA (dsRNA), peptidoglycans (PGN), CpG DNA, MDP and bacterial pore-forming toxins [74,78,91]. Many DAMPs can also activate NLRP3, including extracellular ATP signaling via purinergic P2X7 receptors, monosodium urate crystals (MSU), amyloid-b, environmental insults (e.g. silica, asbestos), potassium efflux, reactive oxygen species and cathepsins [3,78,92–95] (all summarized in Table 1). Overall, the exact activation mechanism for NLRP3 is still unknown, but it is believed to be mediated by 2 mechanisms, including priming and activating signals as described under the inflammasome section and in Fig. 1. Certain microbial stimuli can indirectly activate NLRP3 via NF-kB signaling with support of a secondary stimuli e.g. a virulence factor and independent of TLR or ATP signaling [96,97]. In contrast, extracellular ATP can directly activate the inflammasome by the induction of K+ efflux. Furthermore, certain microbial toxins require pre-stimulation of TLR or pannexin-1 receptors to induce NLRP3 expression [82] (Fig. 1). Due to the varied range of activators it is rather unlikely that all of these ligands bind directly to NLRP3 [78]. On the contrary, antioxidants like N-acetyl-cysteine, glyburide and antagonists of P2X7R are substances described to act as inhibitors to NLRP3 inflammasome [5,32,58,98].

A recent report described that live Gram negative bacteria activate the NLRP3 inflammasome in a non-canonical way, supported by a complex of NLRP3, caspase-1, caspase-11 (human caspase-4 and -5) and ASC [99]. The pathway is dependent on caspase-11 and requires 3 signals for its activation [99,100]. These consist of TLR-priming, followed by a 2nd signal, which can be triggered by either bacterial mRNA activating NLRP3 or by a TLR4-TRIF dependent type I IFN-response, leading to procaspase-11 and IFNRa-expression, with caspase-11 promoting caspase-1 activation. However, the specific mechanisms regulating caspase-11 and -1 activation in response to Gram negative bacteria are yet to be elucidated. It was recently reported that bacterial LPS can also activate caspase-11 in a TLR4-independet manner triggering the non-canonical pathway thereby revealing a new innate immune recognition of LPS [101]. Caspase-11 can also independently induce pyroptosis as a response to Gram negative bacteria [100]. NLRP3 is also regulated by autophagy in a negative way. It has been shown that autophagy can regulate IL-1b secretion both by targeting pro-IL-1b turnover and by regulation the activation of the NLRP3 inflammasome [102]. Cells carrying the NOD2- or ATG16L1mutations associated to CD patients that are infected with bacteria display increased pyroptosis accompanied with an elevated proinflammatory state. Similarly, ATG16L1 hypermorphic mice present

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hyper-activation of inflammasomes resulting in higher secretion of IL-1b and IL-18 and a worsened phenotype in experimental colitis [3,72]. Similarly to NRLP1, NLRP3 has been implicated in homeostasis and inflammation through chronic NOD2 stimulation in human MDM [85]. A recent study also highlighted that E. coli strains isolated from patients with IBD could induce IL-1b secretion from murine macrophages via NLRP3-inflammasome [103]. NLRP3 expression is widely found in mucosal sites [64,81], especially, in epithelial cells (lung and gastrointestinal) and in immune cells including granulocytes, monocytes, dendritic cells, macrophages, T- and B cells and in the CNS [67,79,80,95]. High NLRP3 expression was associated to the ulcerated colonic tissue of CD patients and in the colon of mice with acute and chronic colitis [67]. Thus, the collected data suggest a promoting inflammatory role of NLRP3 in the pathology of IBD. So far, studies in mice deficient in Nlrp3 and challenged for colitis development have revealed contradictory results. Nlrp3 / mice have a healthy growth but challenge with DSS results in reduced colitis severity as reported in several studies [58,104] (Table 2). In addition, transmission of endogenous bacterial flora from wild type mice to Nlrp3 / mice increased colitis susceptibility and antibiotic treatment prevented colitis progression. Moreover, mice treated with Fc11a-2, an NLPR3-dependent inhibitor of pro-caspase-1, IL-1b and IL-18, attenuated acute colitis [105]. The alleviation in disease severity in these studies were associated to improved clinical signs of disease, reductions in immune cell infiltration and pro-inflammatory cytokine expression and improved histological parameters [58,104–106]. In line with these, a recent study in Il-10 / mice, presenting chronic colitis identified activation of the NLRP3 inflammasome and potentiation of a Th17 phenotype regulated by high IL-1b production from macrophages [107]. Overall, these reports support a pro-inflammatory contribution of NLRP3 to colitis pathology. However, other groups have also reported higher susceptibility of Nlrp3 / mice to DSS-induced colitis. These were associated with worsening colonic inflammation, alterations in epithelial barrier due to deficiency in epithelial cell proliferation [108] and increased permeability and translocation of bacteria through the intestinal wall [9,82,106,109]. Overall, these results suggest a protective role of NLRP3 inflammasome in acute DSS colitis [106,108,110] (Table 2). In agreement with these findings, mice deficient in Asc, Il-18, Il-18r, Myd88 and caspase-1 show worsening of TNBS-induced colitis [8,104,109]. Recent evidence has indicated that NLRP3 from non-hematopoietic cells are vital for the protective role on the epithelium, whereas NLRP3 from hematopoietic cells confers protection against the intestinal inflammation [2,8,10,58]. In line with the protective effect on the epithelium, the NLRP3 inflammasome seems to regulate epithelial derived IL-18, important for maintaining intestinal homeostasis [38]. Furthermore, lack of NLRP3 expression resulted in impaired bdefensin production due to an altered microbiota composition in Nlrp3 / compared to wild type mice [82,110] indicating a secondary mechanism by which NLRP3 confers protection. The contradictory results in the phenotype of Nlrp3 / mice can be attributed to several factors including differences in environmental conditions (microbiota composition, protocol to induce colitis), the genetic background and the gender of the animals used [111]. Future studies in these animals should contain controlled parameters such as microbiota composition, animal house health status and general protocol in order to allow comparisons between different groups. However, it is still difficult to attribute the diverse findings entirely to differences in protocol. Colonization studies using IBD-associated bacteria and examination of human immune responses to relevant NLRP3 activators are necessary to elucidate the role of the NLRP3 inflammasome in IBD-pathology.

7.3. NLRP6 inflammasome – a regulator of gut homeostasis and microbiota composition NLRP6 (NALP6/PYPAF5) contains one DAPIN domain that binds to ASC. NLRP6 may mediate activation of caspase-1 via ASC, with subsequent activation of NF-kB and also stimulate cAMP accumulation. NLRP6 is predominantly expressed in epithelial cells in the small and large intestine and to lesser level in hematopoietic cells, with higher expression in granulocytes and lymphocytes compared to macrophages and dendritic cells [43,112,113] and in myofibroblasts [83] (Table 1). NLRP6 is confined to the cytoplasm and co-localizes with ASC [82]. There are no specific ligands for NLRP6 although ATP, nucleotides and the microbiota have been proposed as NLRP6 ligands (http://www.uniprot.org/uniprot/P59044). Specific PAMPs and DAMPs can also induce NLRP6 expression in isolated epithelial and hematopoietic cells [43]. NLRP6 inflammasome function remains undetermined and it is not known whether IL-1b and IL-18 production via NLRP6 is ASC and/or caspase-1 dependent or if other caspases or adaptor molecules are involved [112]. To date, the collected data does not indicate an inflammatory role of NLRP6 in intestinal inflammation. On the contrary, it points toward a regulatory role of NLRP6 in intestinal homeostasis. Nlrp6 / mice grow normally but spontaneously develop colonic crypt hyperplasia, present alterations in crypt-to-villus ratio in the terminal ileum, enlargement of Peyer’s patches and are more susceptible to DSS-induced colitis and CAC [43]. This impairment in colonic inflammation is accompanied by worsening of mucosal permeability and elevated pro-inflammatory cytokine release [43,112–114] (Table 2). In line with its suggested regulatory role, Nlrp6 deficiency in hematopoietic cells led to higher susceptibility to colitis-associated tumorogenesis and Nlrp6 deficiency in colonic epithelial cells led to reduced IL-18 levels accompanied by an alteration in the microbiota composition [43] (Table 2). However, it is still plausible that NLRP6 can be up-regulated in specific cell subsets under certain conditions. Indeed, a reduction in small intestinal Nlrp6 expression was identified in mice exposed to chronic stress which subsequently led to gastrointestinal inflammation. Administration of the PPARg agonist rosiglitazone induced Nlrp6 expression in epithelial cells and reversed the stress-induced pathology in the mice [115]. It is currently unknown whether the level of NLRP6 is reduced in tissue/cells from other intestinal inflammation models or in patients with IBD or if PPARg agonists can be utilized as novel strategies to target the recovery of NLRP6 inflammasome function in intestinal epithelial cells. Interestingly, a reduced expression of PPARg was identified in colonic epithelial cells of UC patients [116] and treatment with PPARg agonists such as rosiglitazone have provided encouraging outcomes in experimental colitis and in human UC trials [117–119]. Thus, the collected data support a regulatory role of NLRP6 in intestinal homeostasis by promoting epithelial derived mediators such as IL18 and by monitoring the intestinal microbiota composition dampening the blooming of harmful bacteria (discussed in the next section). Future investigations should be aiming to identify targets such as PPARg agonists to correct NLRP6 expression in the inflamed intestine. 7.4. NLRP12 inflammasome – a regulatory inflammasome in IBD? NLRP12 (Monarch-1) is predominantly expressed in myeloid cells [120]. It is associated with ASC to produce active IL-1b [11,121] and is a negative regulator of TLR – driven NF-kB activation. NLRP12 inhibitory function requires ATP binding to its NACHT domain [122]. However, the assembly of the NLRP12 inflammasome is as yet unknown. Contrary to other NLRs, NLRP12 expression is downregulated upon TLR-activation with M. tuberculosis, TNFa or IFNg

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[123]. In contrast, pathogenic bacteria such as S. typhimurium and Listeria species can activate the NLRP12 inflammasome through NFkB activation [11] (Table 1). NLRP12 can control the migration of dendritic cells and myeloid cells to the site of infection, and once the infection is controlled, NLRP12 expression is decreased to prevent excessive inflammation [124]. To date, no endogenous or microbial ligands have been identified for NLRP12 [125]. Nlrp12 / mice grow normally but are more susceptible to DSS-induced colitis [11,126,127]. The severity of colon inflammation was associated with an increased secretion of pro-inflammatory mediators due to a failure in reducing NF-kB and ERK activation in Nlrp12 / macrophages [126,127]. Studies indicated that both hematopoietic and non-hematopoietic cells expressing NLRP12 are implicated in the acute and chronic inflammation [127] (Table 2). With regards to NLRP12 and intestine regulation, its exact molecular mechanisms, its cellular contribution, its expression in IBD tissue or genetic susceptibility or its effect on microbial composition have yet to be elucidated. 7.5. NLRC4 inflammasome – a discriminator of commensal and pathogenic bacteria in intestinal infections The NLRC4 (IPAF/CARD12) belongs to the NLR family and forms a complex with ASC, caspase-1 and NLR apoptosis-inhibitory protein (NAIP) 5 (BIRC1/NLRB1) leading to the activation of caspase-1, IL-1b/IL-18 and pyroptosis in response to Gram negative bacteria. NLRC4 is activated by flagellin or by the rod complex of bacterial type III secretion system (T3SS, for S. enterica, P. aeruginosa and the aflagellated Shigella flexneri) or T4SS (for Legionella pneumophila). Flagellin deficient S. enterica, P. aeruginosa, L. pneumophila and Yersinia pestis can also activate NLRC4 via PrgJ, a protein belonging to the basal body rod component of T3SS [3,78,94]. These unexpected findings were attributed to the ability of the cytosolic NAIP5 to link different flagellin structures to NLRC4 [24]. NAIP5 does not have a caspase-1 domain, instead it requires phosphorylation of NLRC4 to activate caspase-1 and inflammasome [17,128]. In contrast, NLRC4 can activate caspase-1 independently of NAIP5 and ASC [41,129]. One NAIP gene has been described in humans whereas seven paralogs have been associated to mice [130]. It is currently unknown which mechanisms regulate NAIP proteins association to NLRC4 e.g. why Naip5 is crucial for the activation of caspase-1 in response to L. pneumophila while it is dispensable for S. enterica infections, even though flagellin from both bacteria binds to Naip5 [24,131] (Table 1). It is also believed that NLRC4 may recognize additional ligands besides flagellin, although the identity of these molecules remains to be uncovered [78]. Furthermore, flagellin induced caspase-1 activation is independent of the flagellin receptor TLR5 [82]. NLRC4 has also been suggested to participate in the apoptotic pathways downstream of p53 [132]. NLRC4 is expressed in hematopoietic cells (such us monocytes, macrophages and T cells), in the small intestine and in the colon [81] (Table 1). To date, the main function of NLRC4 is promoting host defense. This was shown in an elegant study by Franchi and colleagues, where intestinal mononuclear phagocytes were shown to be able to discriminate between pathogenic and commensal bacteria via NLRC4-induced IL-1b production [133]. In addition, Nlrc4 / mice do not develop colitis upon anti-IL-10R injection but do develop severe colitis upon DSS challenge and upon infection with pathogenic bacteria such as S. typhimurium infection and Citrobacter rodentium [24,111,134,135] (Table 2). Interestingly, the protective function of NLRC4 was dependent on macrophage responses including IL-1b/IL-18 production [24,135]. The data also indicate that NLRC4 is not required for intestinal homeostasis or for intestinal epithelial cell recovery [10,24,133]. It is currently unknown if the expression of NLRC4 is altered in IBD tissue or if

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NLRC4 inflammasome can contribute to discriminate between the bacteria associated with IBD e.g. high ratio of Proteobacteria spp. including AIEC, from commensal bacteria populations. Similarly to NLRP6, future studies should be aimed to identify targets that increase or normalize NLRC4 expression to promote host defense against altered bacteria populations. 7.6. The PYHIN inflammasomes (IFI16, AIM2, MNDA and IFIX) AIM2 is the best known inflammasome in this family. AIM2 was originally identified in a screen for suppressors of melanoma tumorigenicity and it contains the HIN200 and PYD domains. The PYHIN inflammasome consist of a family of IFN-inducible proteins encoded by structurally related murine (Ifi202a, Ifi202b, Ifi203, Ifi204 and D3) and human (IFI16, MNDA and AIM2) genes. AIM2 is mainly confined to the cytoplasm, whereas IFI16 is primarily found in the nucleus. PYHIN inflammasomes are expressed in hematopoietic cells and in the small intestine [21,101,134]. The HIN200 domain of AIM2 binds to dsDNA [75], DNA of certain viruses and bacteria including mouse cytomegalovirus (mCMV) [13,14,136], Francisella tularensis [134,137] and L. monocytogenes [101,136], and endogenous DNA ligands (derived from self cells) (Table 1). Both AIM2 and IFI16 lack the CARD domain, explaining why the HIN200 domain connects with the adaptor ASC through a PYD domain thereby regulating caspase-1 activation and IL-1b production [12]. Activation of these inflammasomes encodes the transcription of type I IFNs and IFN-regulated genes [11]. Studies in mice deficient in Aim2 indicated the requirement for this inflammasome in order to mount a response against certain bacterial pathogens e.g. L. monocytogenes and C. rodentium [134– 137] (Table 2), suggesting AIM2 may contribute to the response against certain intestinal infections. The role of PYHIN inflammasomes in gut homeostasis and intestinal inflammation is yet to be discovered. 8. The inflammasome, the intestinal microbiota and IBD Gut commensal microbiota protect the host from pathogens by means of competing for nutrients or the biologic niche. Moreover, commensal microbiota communicates to the host mainly via the innate immune system. The influence of the microbiota on the host immune system is however bi-directional as the immune system also influences the composition and the potential responses of the microbiota in order to maintain intestinal homeostasis. Toll-like receptors are in the frontline in both the extracellular and intracellular compartments but cytosolic receptors (NLRs) have a higher importance in balancing the feedback mechanisms. The cytosolic location of inflammasomes is of high significance aiming to recognize invading pathogens and degrading molecules from phagocytosed bacteria or virus which results in the activation of inflammasomes. Therefore, deficiencies in components of the innate immune response can alter the commensal community as described in Nod2 / mice. These mice harbor an altered microbiota composition compared to their wild type counterparts, and show a reduced ability to prevent colonization with pathogens such as C. rodentium [3,110,138]. However, if the physiological equilibrium between the microbiota and the host immune response is modified due to alterations in gut commensal microbiota composition, the intestinal innate immune responses will also be altered and promote a chronic inflammatory state. In the following section we summarize the findings on the crosstalk between intestinal microbiota and the host by means of inflammasome platforms. Several groups have reported alterations in the microbiota composition of Nlrp6 / when compared to wild type mice [40,43,115]. This alteration was not revealed in mice deficient for other inflammasome components, such as Nlrc4 and Aim2 [43].

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Interestingly, the colitogenic profile was associated with an increase in Bacteroidetes (mainly, the genus Prevotellaceace) and TM7 and a concomitant decrease in Lactobacillus spp. in the Nlrp6 / mice. In addition, the altered microbiota in Asc / and Nlrp6 / mice appears to be a crucial driver of the colitogenic phenotype since transfer of their microbiota to wild type co-housed mice resulted in a worsening colitis phenotype [43] (Table 2). A higher production of the chemokine CCL5/RANTES induced by the ‘‘inflammasomedeficient’’ microflora on the colonic epithelial cells led to an enhanced pro-inflammatory cell influx (including macrophages/T cells) into the lamina propria of Nlrp6 / mice [111]. CCL5 was recently shown to enhance epithelial cell proliferation via the local activation of the IL-6 pathway and reduction in IL-18 leading to enhanced inflammation-induced colorectal cancer in Nlrp6 / and Asc / mice [11,40]. Treatment with antibiotics of Nlrp6 / mice, reduced among others the Prevotollaceace genus, and improved the colitis phenotype and transfer of their microbiota into co-housed wild type mice did not promote colitis [112]. Overall, these findings indicate a crucial function of NLRP6 in regulating the function of the commensal microbiota. In addition, the high expression of NLRP6 in intestinal epithelium suggests an important role for this NLR in the recovery from epithelial injury and in maintaining epithelial homeostasis [112]. IL-1b and IL-18 are also directly implicated in regulating the composition of the gut commensal microbiota as they can regulate the secretion of antimicrobial peptides, like defensins. Similarly, mice deficient in Nlrp3 showed altered b-defensins levels most likely due to an altered microbiota [110]. In addition to the mentioned functions, NLRP6 appears to act as a negative regulator of innate immunity, since mice deficient for NLRP6 were resistant to infection with certain bacterial strains namely L. monocytogenes, Salmonella, and E. coli [139]. Inflammasome-induced pyroptosis in injured or infected epithelial cells may also affect the recovery/regeneration of the epithelium exemplified by increased proliferation and reduction in mucus-producing goblet cells. These features affect the permeability of the epithelium allowing bacteria to penetrate it and be recognized by lamina propria immune cells, thereby affecting the severity of inflammation [111]. The collected data so far, pinpoints NLRP6 as the main regular of colonic microbial ecology. Changes in the microbial diversity across the inflammasome deficient mouse life span and in pediatric vs adult IBD patient are needed to reveal the long term interaction between the microbiome, inflammasomes and IBD pathophysiology. Also, molecular mechanisms leading to inflammasome signaling due to altered human IBD microbiota composition and/or in IBD specific bacteria deserve a deeper characterization. 9. The inflammasomes and their contribution to colitisassociated colon cancer (CAC) Patients suffering from chronic inflammation and long term IBD have a higher risk of developing CAC [44]. This tumorigenesis progression has been related to detrimental epithelial responses such as epithelial proliferation and tissue recovery, persistent immune inflammatory responses such as elevated cytokines, chemokines and ROS production and alterations in the gut microbiota composition. The collected data so far links a protective function of inflammasomes in colitis-associated cancer. The AOM/DSS-model has been extensively employed to examine the role of inflammasomes in the development of intestinal tumors. Using this model, it was shown that IL-18 is a key protective cytokine, largely based on its critical role in epithelial recovery as previously discussed under the IL-18 section [19,39,53,55]. In addition, mice lacking components of the functional NLRP3 inflammasome i.e. Asc, Myd88 and caspase-1 / and mice deficient

in Nlrp6 exhibit more extensive tumorigenesis. The mechanisms for this is still unclear, but the increased tumor burden appear to be associated with a general reduction in IL-18 production accompanied by an elevated inflammatory response and increased destruction of the epithelial cells [2,8,44,104,110,114,140]. Furthermore, the hematopoietic compartment was linked to the tumorigenesis protection in mice lacking Nlrp3 while is unknown for Nlrp6 [40,112,113,126]. In contrast, and as stated previously for NLRP12 and colitis, both the non- and hematopoietic compartment have been linked to the tumor suppression activity most likely due to NLRP12 ability to reduce NF-kB and ERK pathways, although the non-hematopoietic compartment appear to primarily contribute to this response [114,126,127]. Similarly to the above mentioned studies, Nlrc4 / mice develop more tumors in the AOM/DSS model [24,114,141], which was not dependent on colonic inflammation [140]. Using bone marrow chimera animals it was shown that the NLRC4 tumor suppressive functions were associated with the non-hematopoietic compartment, suggesting that NLRC4 regulates epithelial proliferation and apoptosis. In contrast, no difference were reported by Allen and colleagues in Nlcr4 / mice exposed to AOM/DSS [58] suggesting that differences in animal facility status may affect the responses in these mice indirectly implicating microbiota inflammation-induced tumorigenesis [140]. Infection with pathogens such as Helicobacter and Streptococcus species and mono-colonization with commensal bacteria such as E. coli promoted tumor progression in colitis susceptible mice such as Il-10 / mice [42,142]. Alterations of the microbiota are linked to both experimental and human IBD and in recent years also to colorectal cancer and CAC [40,114,143–145]. The molecular mechanisms leading to inflammasome signaling associated to altered human IBD microbiota composition/bacteria and its contribution to tumor progression remains to be elucidated. 10. Conclusions and future perspectives A decade has passed since the first description of the inflammasomes. This area has evolved enormously and great progress has been made in elucidating the mechanisms and functions of inflammasomes in response to microbes and other stimuli. New inflammasome components have been identified and their contribution to host defense against pathogenic microbes is becoming clearer. The use of mice deficient in inflammasome components has hugely contributed to the elucidation of their contribution to the pathology of several chronic inflammatory conditions including intestinal inflammation. The initial reports on elevated levels of IL-1b and caspase-1 over a decade ago represent the early links on inflammasome and their potential contribution to IBD pathophysiology [28,30,31]. Twenty years later, progress has been made to identify and characterized the activators (microbes, particles, etc.), the sensors (NLRP3, NLRC4, etc.) and the adaptors (ASC) responsible for caspase-1 activation and IL-1b processing. In the last 5 years, reports have also highlighted an additional role for the inflammasomes, not only as being crucial mediators of host defense but also being crucial regulators of intestinal homeostasis. In addition, a more evident role of the intestinal microbiota in shaping the inflammasome response has also been uncovered [5,43,111,146,147]. The collected data so far points toward a complexity of inflammasomes in intestinal health and disease due to their dual role in host defense and intestinal homeostasis. This complexity appears to be related to cell-specific responses, especially in the intestine with the close interaction of different cell types to a high bacterial load and to the type of inflammatory cells (e.g. acute vs. chronic). A plausible explanation for the discrepancies in several

Please cite this article in press as: Aguilera M, et al. The complex role of inflammasomes in the pathogenesis of Inflammatory Bowel Diseases – Lessons learned from experimental ?models. Cytokine Growth Factor Rev (2014), http://dx.doi.org/10.1016/ j.cytogfr.2014.04.003

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studies is most likely due to the variation in microbiota composition present in the individual animal facilities [114]. The use of animal models, especially mice, in elucidating novel mechanisms associated to IBD pathology has proven crucial in the past. Considering the reports on alterations in microbiota composition in IBD patients and in mice deficient of inflammasome components, the design of future experimental studies should be performed under a standardization of environmental conditions including microbial status and diet, both of which able to shown to influence each other and the subsequent disease phenotype [148]. In spite of the great progress on elucidating the role of the inflammasomes in experimental models of IBD, a few reports have been published using tissue/cells from IBD patients. To date, only a few inflammasomes SNPs have been associated with CD and none have been associated with UC, which suggests that the genetics of these components may not be that crucial for IBD pathology. Instead, the CD-susceptibility mutations associated with bacteria recognition and autophagy e.g. NOD2, ATG16L1, IRGM may directly affect the inflammasome response, as exemplified in the ATG16L1 hypermorphic mice [3,72]. This is a highly exciting area and future studies may clarify the intricate interactions between the inflammasomes and autophagy and their contribution to orchestrate the innate and adaptive immune responses in intestinal health and disease. In conclusion, the last decade has uncovered a complex role of innate immune responses e.g. inflammasomes, PRRs and microbial composition to intestinal inflammation and tumorigenesis. Our present understanding of inflammasomes to human IBD is still rather limited. Therefore, future investigations should aim to translate the experimental findings into human models to reveal novel understandings on these complex conditions, which will potentially lead to the discovery of new treatments for these debilitating diseases.

Conflict of interest The authors declare there are no conflicts of interest. Acknowledgments The authors would like to thank Dr. David Clarke for his critical reading of the manuscript. This publication has emanated from research conducted with the financial support of Science Foundation Ireland (SFI) under Grant Numbers SFI/12/RC/2273 and 12/RC/ 2273. M. Aguilera was awarded an internship grant to spend at the Alimentary Pharmabiotic Centre (EEBB-I-13-07758) from the Spanish Government (Ministerio de economı´a y competitividad).

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Mo`nica Aguilera received her DVM degree from Universitat Auto`noma de Barcelona. She has a Master in Neurosciences and is a Ph.D. student at Universitat Auto`noma de Barcelona – Institut de Neurocie`ncies. She is in the last year of her thesis. Her current research is focused on how gut commensal microbiota influences visceral pain (intestinal neuro-immune interactions). She did an internship at Alimentary Pharmabiotic Center (University College Cork) focusing on inflammasomes responses in intestinal epithelial cells and its relevance to Inflammatory Bowel Disease pathology.

Trevor Darby obtained his B.Sc. degree in Applied Biosciences from Cork Institute of Technology as well as an M.Sc. in Biotechnology from University College Cork. He is a final year Ph.D. student at the Alimentary Pharmabiotic Center and the Department of Medicine, University College Cork. His current research involves the molecular analysis of inflammatory mechanisms associated with Escherichia coli and Inflammatory Bowel Diseases.

Silvia Melgar received her B.Sc. in Molecular Biology and a Ph.D. in Immunology from Umea˚ University, Sweden. In 2002, she joined AstraZeneca R&D, Sweden, as a post-doctoral fellow followed by a position as Senior research scientist within the disease area of Inflammatory Bowel Diseases (IBD). In 2008, she joined the Alimentary Pharmabiotic Center (APC) at University College Cork under the GlaxoSmithKline (GSK)-APC collaboration as an investigator (APC) and principal scientist (GSK). In 2012, she became Senior Research Fellow in the APC. Her research interests include host–bacterial interactions in in vivo models of IBD and colitis-associated cancer and the contribution of environmental triggers such as diet in the pathophysiology of these diseases.

Please cite this article in press as: Aguilera M, et al. The complex role of inflammasomes in the pathogenesis of Inflammatory Bowel Diseases – Lessons learned from experimental ?models. Cytokine Growth Factor Rev (2014), http://dx.doi.org/10.1016/ j.cytogfr.2014.04.003