Therapeutic implications of Inflammasome in Inflammatory bowel disease

Abstract

Inflammatory bowel disease (IBD) remains a persistent health problem with a global burden surging over 6.8 million cases currently. Clinical pathology of IBD is complicated; however, hyperactive inflammatory and immune responses in the gut is shown to be one of the persistent causes of the disease. Human gut inflammasome, the activator of innate immune system is believed to be a primary underlying cause for the pathology and is largely associated with the progression of IBD. To manage IBD, there is a need to fully understand the role of inflammasome activation in IBD. Since inflammasome potentially play a significant role in IBD, systemic modulation of inflammasome may provide an effective therapeutic and clinical approach to control IBD symptoms. In this review we have focused on this association between IBD and gut inflammasome, and recent advances in the research and therapeutic strategies for IBD. We have discussed inflammasomes and their components, outcomes from the experimental animals and human studies, inflammasome inhibitors, and developments in the inflammasome targeted therapies for IBD.

INTRODUCTION

The human gut microbiome is stringently regulated by the intestinal immune system. However, under certain conditions, disruption in gut homeostasis may cause an over-reacted inflammatory immune response. These unproportioned inflammatory immune responses may lead to Crohn’s disease (CD) or Ulcerative colitis (UC) depending on the area they are affecting (1). Both conditions are cumulatively referred to as inflammatory bowel disease (IBD). The pathophysiology and etiology of IBD are complicated. This involves genetic, environmental, and several other factors. Likewise, gut inflammasome, which has been associated with several other inflammatory and autoimmune disorders can also be one of the factors in the development of IBD. Abolition or remission of mucosal inflammation is an ultimate therapeutic aim for IBD treatment. Studies have shown that targeting inflammasome can reduce inflammation in IBD (2). In this review, we have focused on this association between IBD and gut inflammasome, and recent advances in the research and therapeutic strategies for IBD.

INFLAMMATORY BOWEL DISEASE

IBD is a group of Ulcerative colitis and Crohn’s disease, both affecting gastrointestinal system. In 1875, two English physicians Samuel Wilks and Walter Moxon first described possible condition of ulcerative colitis in a woman who died with recurrent diarrhea and fever (3, 4). Whereas, in 1932 American physicians Burrill Crohn, Leon Ginzberg, and Gordon Oppenheimer described the CD (5). Since then, physicians and researchers are working to improve their understanding on the pathophysiology and immunology of these debilitating disorders. Symptoms of both CD and UC include intermittent abdominal pain, weight loss, diarrhea, rectal bleeding, fever, and fatigue. Those who suffer, have increased mortality with a risk of cancer, blood clots, and primary sclerosing cholangitis (6). IBD may occur at any age through adolescents to adults irrespective of the sex. Genetic, environmental, and immunological factors contribute in unison towards compromising the intestinal epithelial barrier function increasing the risk of disease (7).

Ulcerative colitis involves rectum and extending proximally to the large intestine. UC patients develop intestinal lesions which are mostly superficial and extend proximally, but in severe disease condition deep intestinal ulcerations are observed. Complications with UC include anemia, toxic colitis due to the damage to entire thickness of colon wall that can lead to ileus, toxic megacolon and finally colon cancer (1 out of every 200 patients with UC develops colon cancer) (8). Crohn’s disease can involve the entire gastrointestinal tract, however mostly affecting the distal end of small intestine (terminal ileum) and colon (9). Most patients with CD presents chronic inflammation, strictures, intestinal stenosis and fistulas (3). CD patients also encounter malnutrition as damage to small intestine significantly affect the absorption of nutrients (10).

Epidemiology

There is a worldwide increase in the incidence of IBD. IBD, which is incurable reduces the quality of life, increasing disability and thus places a heavy burden on the population and health system. The incidence of IBD varies geographically and a considerable difference between east and west exists. However, this difference between eastern and western countries is shrinking rapidly (11). According to a recent study there were 6.8 million cases of IBD globally in 2017 (1). The age standardized prevalence of IBD between 1990 to 2017 was 464.5 in United States of America, 449.6 in United Kingdom, 520 in Canada, 136.2 in China, and 16.2 in India per 100,000 populations (1). The highest prevalence of UC was 505 per 100,000 population in Northern Europe followed by prevalence of 286 and 44 per 100,000 population in North America and South Asia respectively (12). The highest incidences of UC were 57 in Northern Europe, 23.14 in North America, and 6 in South Asia per 100,000 populations (12). For CD, the highest prevalence was observed in Western Europe (322 per 100,000) followed by North America (318 per 100,000), North Europe (262 per 100,000) and East Europe (200 per 100,000) (12). The highest incidence was in North America (23 per 100,000), Southern (15 per 100,000) and Eastern (14 per 100,000) Europe. Asian countries are also showing increase in both prevalence and incidence of CD. The average prevalence of CD in Asia was 18 and incidence was 4 per 100,000 population (12). The reported numbers can be far more than expected as only partial data is available from most of the underdeveloped countries. Overall, the available data suggest a continuous surge in IBD incidences.

Treatment and disease management

There is no specific treatment available to cure IBD. The ultimate goal of IBD treatment is to achieve clinical remission and then after maintaining this achieved remission of disease condition. The primary line of treatment includes 5-aminosalicylic acid (5-ASA; sulfasalazine, mesalamine), steroids (prednisone, budesonide), and immunomodulators (6-mercaptopurine, methotrexate, azathioprine) (13, 14). Depending on the severity of disease condition and using recent algorithms, treatment approach may vary (15). Additional drugs for the management and inducing the remission in IBD patients include use of calcineurin inhibitors or antibiotics (cyclosporine, tacrolimus, ciprofloxacin, metronidazole), and anti-tumor necrosis factor-α (TNF-α) antibodies or other biologicals (infliximab, adalimumab) (13, 14). However, at least 90% of the IBD patients experience relapse of disease condition once in a lifetime (16, 17). To avoid morbidity in such individuals, surgery is the only option. About 15-47% of patients on medical therapy require surgical intervention (16). Depending on the existing prevalence of IBD in USA, total annual direct cost for the treatment of IBD can be USD 35 billion.

Existing medications to treat IBD are associated with the increased risk of side-effects and complications. The side-effects existing drugs can cause are renal toxicity, hemolytic anemia, osteoporosis, insomnia, pancreatitis, bone marrow suppression, non-Hodgkin lymphoma, hepatosplenic T-cell lymphoma, fatigue, diarrhea, nausea and vomiting (18). Individuals on long-term IBD therapy are vulnerable to other opportunistic infections (8), cervical cancer (19), skin cancer (20), or poor compliance to vaccinations (21, 22). Considering these drawbacks of existing drugs, new therapeutic options are being developed for the treatment of IBD (15, 18).

INFLAMMASOME PATHWAY

Inflammasome is a cytoplasmic multiprotein constituent of the innate immune system. They exist across all immune (macrophages, dendritic cells, etc.) and epithelial cells. Inflammasome is a fundamental and often tightly regulated host defense system that specifically involves sensing microbial, fungal, viral, nematode, and other harmful agents (23). Recognition of pathogen-associated molecular patterns (PAMPs) or danger-associated molecular patterns (DAMPs) by pattern recognition receptors (PRR) triggers inflammasome assembly and subsequent induction of adaptive immune responses (24). Depending on the stimulus, inflammasome can be activated via canonical caspase-1, or noncanonical caspase-4/5 pathways (caspase-11 in mouse) (25). To date, several kinds of inflammasomes including nucleotide-binding domain and leucine-rich repeat receptors (NLR) pyrin domain-containing family (NLRP), NLR family caspase recruitment domain-containing protein-4 (NLRC4), and double-stranded DNA sensors absent in melanoma-2 (AIM2) have been described.

NLRP3 inflammasome

NLRP3 is a tripartite assembly of NLRP3 protein, caspase-1, and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) (Figure 1). NLRP3 protein complex contains an amino-terminal pyrin domain (PYD), N-terminal domains, a central nucleotide-binding and oligomerization (NACHT) domain, and a carboxy-terminal leucine-rich repeat domain (LRR). A cryo-electron microscopy structure of NLRP3 has been recently determined (26). The atomic model building on a 3.8-A resolution map showed earring-shaped structure of NLRs (PYD-deleted) containing a curved LRR domain and NACHT domain. The earring-shaped structural characteristics were similar to NLRC4 (27), NOD2 (28) and NLR apoptosis inhibitory protein 5 (NAIP5) (29). The caspase-1 has amino-terminal caspase activation and recruitment domain (CARD), a central large catalytic domain (p20), and a carboxy-terminal small catalytic subunit domain (p10) (30). Formation of an active NLRP3 assembly is triggered on sensing stimulations like extracellular ATP, potassium (K+) efflux, calcium (Ca2+) signaling, reactive oxygen species (ROS), toxins, and mitochondrial dysfunction. The NLRP3 then polymerizes through interactions with the NACHT domains, initiating a homotypic pyrin-pyrin domain interaction through ASC (31). Multiple ASC filaments further segregate to form an ASC speck (32). Finally, ASC speck interacts with caspase-1 through CARD-CARD interaction, enabling enzymatic cleavage and activation of caspase-1 (30). The activated caspase-1 enzymatically cleaves pro-interleukin (IL)-1β and pro-IL-18 to their mature forms. The release of IL-1β and IL-18 cytokines triggers inflammatory signals through pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and IL-6, nuclear factor-κB (NF-κB) pathway, and gene transcription.

Figure 1: NLRP3 inflammasome structure.

NLRP3 inflammasome active complex is a multimeric assembly consisting of NLRP3 protein, ASC, and caspase-1. Once triggered, inflammasome assembly is formed by homotypic interactions between PYD-PYD domain interactions between NLRP3 protein and ASC. Multiple ASC filaments then segregate to form an ASC speck and interact with caspase-1 through CARD-CARD interactions. Created with Biorender.com. (PDB database protein accession # NLRP3; 6NPY, ASC; 3J63, Caspase-1; 5FNA)

The canonical activation of NLRP3 is by a two-step process (Figure 2). In the first priming step, PAMPs or DAMPs recognition by toll-like receptors (TLR) initiates the NF-κB-mediated signaling and activation pathway. This downstream events to NF-κB pathway upregulates the transcriptional and translational induction of inflammasome sensors, NLRP3, proIL-1β, and proIL-18. In the second activation step, NLRP3 protein, ASC, and caspase-1 are assembled into an active NLRP3 inflammasome assembly, which converts caspase-1, proIL-18, and proIL-1β to their active forms. The formation of active NLRP3 assembly also induces programmed necrosis or pyroptosis in cells by the formation of gasdermin-D (GSDMD; a pore-forming protein) (25).

Figure 2: NLRP3 activation pathway.

On detection of stimuli, the first step of NLRP3 inflammasome activation initiates starting from the activation of the NF-κB pathway and subsequent transcription of components from the NLRP3 inflammasome complex. In the second canonical activation step of the NLRP3 inflammasome, an active assembly of NLRP3 inflammasome complex is formed which is triggered with the efflux or influx of ions, reactive oxygen species among other signals. Activated and fully formed NLRP3 inflammasome complex then converts IL-1β, IL-18 cytokines, and gasdermin D into their activated forms. In the non-canonical activation of the NLRP3 inflammasome, sensing of cytosolic LPS by caspases triggers the formation of an active NLRP3 inflammasome assembly by the efflux of K+ ions subsequently leading to the release of cytokines and other mediators. Created with Biorender.com.

GSDMD-mediated pyroptosis is a crucial component in executing inflammasome triggered innate immune responses. Recent developments redefine pyroptosis as GSDMD-mediated programmed necrosis (33). Caspase-1 once activated, cleaves at aspartate-275 site within the GSDMD-C terminal linker loop (34). Recent study uncovers more specific structural details of this engagement (35). The GSDMD-C terminal linker loop binds to the exosite and catalytic groove of caspase-1 via hydrogen bonds forming a dual-site engagement for more specific recognition and efficient cleavage of GSDMD. The linker loop between C-terminal (repressor domain) and N-terminal (pore-forming domain) of GSDMD is responsible for the autoinhibition of GSDMD (36). Whereas the N-terminal domain of GSDMD is in-charge for forming the membrane pores and inducing pyroptosis (34). The GSDMD-N terminal domain is itself sufficient to drive pyroptosis. GSDMD-N domain can form pores on phosphoinositides on the plasma membrane, disrupting the osmotic potential and K+ efflux, causing cell swelling and eventually cell lysis (36). GSDMD-pore probably even allows the release of IL-1β and IL-18 cytokines (37). However, the possibility of releasing these cytokines through pyroptotic membrane rupture or through normal cytokine release mechanism in conjunction with the timing of cell death is still unclear.

The canonical activation and oligomerization of inflammasome was triggered by the efflux of K+ or Cl- ions, flux of Ca2+ ions, lysosomal disruption, mitochondrial dysfunction, and metabolic changes (38). Additional stimuli for canonical NLRP3 activation are: (1) the disassembly of trans-Golgi network can cause aggregation and activation of NLRP3, (2) bacterial lipopolysaccharide (LPS) can initiate NLRP3 activation through TLR4 ligand, (3) priming step that can induce post-translational modifications of NLRP3, and recently discovered (4) the post-transcriptional deubiquitination of NLRP3 (39).

The non-canonical activation of NLRP3 occurs through the activation pathways of human caspases-4/5 and mouse caspase-11 (Figure 2). Activation of GSDMD by the enzymatic cleavage from caspases-4/5 or 11 leads to the K+ efflux and autocrine activation of NLRP3, which subsequently leads to the maturation of IL-1β and IL-18 cytokines. The non-canonical pathway of inflammasome activation is independent of LPS detection by TLR. Until now it was believed that the CARD domain of caspase-4/5/11 recognizes cytosolic LPS which is internalized through phagocytosis, inducing the oligomerization and cleavage of GSDMD (40-43). However, recent studies reported the necessity of interferon-induced guanylate binding proteins (GBP) for detection of cytosolic LPS by caspase-4/5/11 and non-canonical inflammasome activation (44-47). GBP acts as a sensor for cytosolic LPS and their interactions are through electrostatic interactions between Lipid A and inner core of LPS and hydrophobic LPS binding sites CD14 and MD-2 of GBP (44). GBP-LPS complex then recruits and activates caspase-4 for subsequent release of cytokines.

Recently an alternative pathway for NLRP3 activation in human and porcine monocytes has also been discovered (48). This involves TLR4 activation by LPS. The binding of extracellular ATP to ATP-gated ion channel-P2X7 triggers the downstream TLR4 signaling cascade (TLR-4-TRIF-RIPK1-FADD) activating caspase-8 and ultimately formation of NLRP3 inflammasome assembly. The mechanism was independent of pyroptosome formation, pyroptosis and K+ efflux for inflammasome activation. The LPS and ATP co-stimulation of THP-1 macrophages increased the expression of RIPK1 and activation of caspase-8 (49). The alternative pathway of NLRP3 activation has been involved in the development of inflammation and autoimmunity (50-52). However, additional components of this pathway are yet to be explored.

NLRP1 inflammasome

NLRP1 is a first inflammasome discovered and has PYD, NACHT and LRR domains. The interactions between human NLRP1 (hNLRP1) and caspase-1 CARD is through electrostatic interactions (53). NLRP1 oligomerization can directly recruit caspase-1 via the C-terminal CARD domain independent of ASC (54). The hNLRP1 and mouse NLRP1b homolog have a C-terminal extension containing a function-to-find domain (FIIND) which is not commonly observed in other NLRP’s (55). The autolytic proteolysis within the FIIND domain between the “found in ZO-1 and UNC5” and “conserved in UNC5, PIDD, and Ankyrin; (UPA)” sub-domains generates two non-covalent polypeptide chains (N-terminal and C-terminal fragments) (56). FIIND domain is required for the activation of NLRP1 assembly (57), as inhibition of the autoproteolytic activity of FIIND domain blocks the activation of NLRP1 inflammasome (57, 58). Studies have shown that direct proteolytic cleavage of NLRP1 by anthrax lethal toxin (LT) or IpaH7.8, a Shigella flexneri ubiquitin ligase secreted effector is required for the NLRP1 activation, formation of IL-1β and pyroptosis (58, 59). The activation of NLRP1 by IpaH7.8 needed E3 ligase and host proteosome but independent of N-end rule ubiquitin ligase Ubr2 (59, 60). Studies have also shown that selective inhibition of dipeptidyl peptidases 8 and 9 (DPP8/9) activate NLRP1 in human THP-1 and mouse RAW 264.7 macrophage cell lines to induce pyroptosis without efficiently activating IL-1β (60-62). DPP8/9 can directly bind to the FIIND domain mediating the NLRP1 inflammasome activation (55, 63). However, underlying molecular mechanisms of this pathway are still subject to further investigation.

NLRP6 inflammasome

NLRP6 (PYPAF5) is a newly discovered inflammasome involved in the regulation of NF-κB and mitogen-activated protein kinase (MAPK) pathways (64). NLRP6 can be regulated at both transcriptional and post-translational levels by transcription factor peroxisome proliferator-activated receptor-γ (PPAR-γ), TNF-α and miRNA-331-3p (65-67). Activation of NLRP6 mediates the formation of an active inflammasome assembly with ASC and caspase-1 or 11 leading to the production of IL-18 and IL-1β. Some of the activators of NLRP6 includes deubiquitinase Cyld (68), carcinogenic bacterial lipoteichoic acid (LTA) (69), and LPS (70). Interestingly, LTA-induced formation of NLRP6 assembly has been shown to follow the non-canonical pathway of inflammasome activation (71).

The structure of NLRP6 resembles NLRP3 with few differences in the PYD component (72, 73). Recent cryo-EM and crystal structure of NLRP6 showed that the N-terminal NLRP6^PYD filament is a core element to recruit inflammasome assembly (72). The NLRP6 constructs (full-length NLRP6, NLRP6^PYD, and NLRP6^PYD+nucleotide-binding domain, NBD) were able to induce ASC-PYD polymerization. Unlike NLPR3, PYD of NLRP6 at higher concentrations is independently capable of nucleating ASC-PYD polymerization. The NLRP6^PYD is a hollow cylindrical structure assembly of right-handed helix containing six antiparallel α-helices. The α2 and α3 loop of NLRP6^PYD are involved and facilitates confirmational changes and molecular interactions in the NLRP6 inflammasome assembly (72).

NAIP/NLRC4 inflammasome

NAIP/NLRC4 inflammasome is important during lung, spleen and liver infections, sepsis, and intestinal inflammation. The NAIP/NLRC4 complex is formed upon detection of bacterial flagellin and type-3 secretion system components as activating ligands and induce activation of caspase-1 and pyroptosis (74-76). NLRC4 cannot directly recognize activation signals and thus activation of NLRC4 is through NAIP. NAIP as a sensor and NLRC4 as adaptor protein forms a single inflammasome complex (77). A three domain structure of NLRC4 complex comprises of N-terminal homotypic interaction domain, a central NBD domain, and a series of C-terminal LRR (77). Upon activation, a single NAIP protein forms assembly with 10 to 11 NLRC4 molecules leading to a multi-subunit disk-like structure of NAIP/NLRC4 inflammasome (27). NLRC4 can directly bind to caspase-1 through CARD-CARD domain independent of ASC. However, binding to ASC can efficiently activate caspase-1 (78). Other reviews have detailed discussion on NAIP/NLRC4 inflammasome (77, 79).

AIM2 inflammasome

AIM2 is a ALR family of a non-NLR cytosolic protein that can activate caspase-1 and subsequently cytokines (80-82). AIM2 is different from other inflammasomes due to its ability of detecting DNA from its characteristic HIN200 domain (25, 80). Normally, DNA is not present in the cytosol of eukaryotic cells, hence detection of pathogenic DNA from multiple origins or even self-DNA can trigger AIM2 inflammasome responses (25). Recent studies have also showed detection of RNA by AIM2 (83). AIM2 consists of a N-terminal PYD domain and C-terminal HIN200 domain. The N-terminal PYD domain interacts with ASC, while the HIN200 domain recognizes DNA sequences. The optimal length of DNA sequences to be recognized by AIM2 is approximately 80 base pairs (84). In normal inactive state, AIM2^HIN confines AIM2^PYD domain, restricting its ability to form PYD-PYD interactions (84). The binding of dsDNA to AIM2^HIN by oligonucleotide/oligosaccharide binding folds of HIN or independent interactions, release PYD domain further enabling it to form PYD-PYD interactions (85). Binding of AIM2^PYD to ASC completes the formation of an active inflammasome assembly to perform downstream actions. However, few studies contradicted the autoinhibitory model of AIM2^PYD activation, the studies suggested the oligomerization-driven activation of AIM2^PYD assembly (86, 87). The regulation of AIM2 activation and assembly has been through PYD-only proteins (POP) (88). POP1 and POP2 binds to ASC^PYD, whereas POP3 binds directly to AIM2^PYD. Binding of POP3 to AIM2^PYD blocks PYD-PYD interactions with ASC and subsequent activation of caspase-1 and cytokine release. Similarly, CARD-only proteins (COPs) and decoy proteins belonging to the ALR family can regulate activation of AIM2 inflammasome assembly (89).

Pyrin inflammasome

Pyrin is primarily expressed in neutrophils, monocytes, eosinophils and dendritic cells. Human pyrin contains four functional domains (90): 1) PYD domain (1-92) at N-terminal end which is responsible for interaction with ASC and subsequently activation of caspase-1 and cytokine release. 2) A zinc finger B-box domain (370-412) and 3) a coiled-coil (CC) domain (420-440). Both these domains are involved in the oligomerization of pyrin and organization of cytoskeleton. 4) The C-terminal B30.2/SPRY domain is responsible for direct interactions with caspase-1 and with the other inflammasome components (91).

Activation of pyrin has been associated with the disruption in actin filaments and cytoskeleton assembly (92). Pyrin activation is triggered on pathogen-induced modifications in Rho guanosine triphosphatases (Rho GTPases; RhoA) of host cells resulting in formation of pyrin-ASC inflammasome complex (93). However, pyrin does not directly sense the modifications in RhoA but the resulting activation of downstream RhoA signals (94). RhoA modifications can disrupt actin cytoskeleton assembly triggering activation of pyrin inflammasome assembly. Studies using the regulators of actin cytoskeleton assembly like WDR1, vinblastine or colchicine showed changes in the formation of pyrin inflammasome assembly (95, 96). Individuals deficient in wdr1 gene showed increase in the actin polymerization and suffer with IL-18 dependent autoinflammatory phenotype and thrombocytopenia (96–98). These studies highlight the engagement of inflammasome components with structural organization of cells. Moreover, bacterial toxins can activate pyrin inflammasome (99), however very few have studied the association between pyrin inflammasome and microbiome-mediated intestinal inflammation (100–102).

Other NLR inflammasomes

Besides the above discussed inflammasomes, over the year’s scientists have also identified additional inflammasomes. NLRP7 has been shown to activate in response to microbial acylated lipopeptides and induce ASC-dependent formation of inflammasome assembly (103). NLRP9 is a other inflammasome originally linked to the reproductive system, however recent studies have highlighted NLRP9’s role in the intestine on rotavirus infection (104). NLRP2 and NLRP5 are also other inflammasomes associated with the early embryonic arrest and infertility (105).

ROLE OF INFLAMMASOME IN IBD

Essential microbiota residing in the human intestinal tract is not part of the immune system. Hence, even a minor invasion of this microbiota through a tightly regulated intestinal barrier can trigger cellular inflammatory responses (Figure 3). The gut inflammasome primary regulates the host defense responses to microbial pathogens limiting their ingress through the intestinal tract. This does mean the possibilities of their involvement in the progression of IBD.

Figure 3: Inflammasome activation in inflammatory bowel disease (IBD).

In response to a pathogenic stimulus, NLRP3 inflammasome is activated releasing IL-1β and IL-18 cytokines. The overall response during normal gut homeostasis is regulated and restricted to the elimination of infiltrating pathogens only. However, during IBD due to the genetic mutations, loss of mucosal layer, defects in barrier functionality, and several other contributing factors uncontrolled inflammasome activation and responses are triggered. This leads to the infiltration of innate immune cells and cumulatively there is a hyper-aggressive pro-inflammatory immune response in the gut mucosa. Created with Biorender.com.

Experimental animal studies

Several studies demonstrate the protective effects of gut inflammasome in the development of IBD. Mice lacking components of inflammasome assembly NLRP3, NLRC4, IL-1β, Caspase-1/11, and ASC showed more susceptibility to colitis with exacerbation in clinical disease symptoms, and increased mortality when challenged with chemically induced colitis compared to their wild-type (WT) mates (106–112). Caspase-1^−/− and pycard^−/− (ASC containing CARD) mice demonstrated severe acute and chronic dextran sulfate sodium (DSS)-induced colitis with significantly increased morbidity, mortality and clinical disease score compared to the WT colitis mice (106). Further studies from other groups reiterated these observations and showed that in the absence of NLRP3 or even the downstream components of NLRP3 pathway like ASC and caspase-1 are sufficient to exacerbate colitis condition (107, 113). These studies also concluded that both the activation of inflammasome components and subsequent release of cytokines are equally important in attenuating colitis. Subsequently, another study showed a significantly lower intestinal levels of IL-10 and TGF-β in nlrp3^−/− colitis mice (108). Both IL-10 and TGF-β play prominent roles in downregulating intestinal inflammation (114). The neutrophils isolated from nlrp3^−/− mice displayed increased apoptosis and impaired chemotaxis to neutrophil chemotaxis factors (keratinocyte-derived chemokine (KC), leukotriene B4, WKTMVm and C5). Concurrent observation of impaired neutrophil migration in Nlrp3^−/− mice was observed in another study that showed decreased KC-induced release of intracellular calcium elevation, Rac activation, and actin assembly formation in nlrp3^−/− neutrophils (115). Defective neutrophil chemotaxis has been observed in individuals with CD and can be responsible for impaired innate immune response towards microbial invasion (116). Nlrp3^−/− and caspase-1^−/− colitis mice showed loss of intestinal barrier integrity (106, 107). Caspase-1^−/− mice were extremely susceptible to develop colitis and continued to lose their body weight with severe hematochezia (110). Caspase-1^−/− mice showed reduced claudin-3 expression and intestinal epithelial cell (IEC) proliferation.

Caspases dysfunction has been associated with intestinal epithelial inflammation and disruption of epithelial barrier function and carry a direct ability to regulate IL-18 and IL-1β cytokines. Caspase-11^−/− mice showed increased susceptibility to acute DSS-colitis, increased mortality and morbidity and impaired epithelial barrier permeability compared to their WT mates (117–119). Moreover, the caspase-1 expression was increased in caspase-11^−/− mice and can confer to the levels of IL-1β in these animals. The role of TRIF was not observed in caspase-11 mediated effects on intestinal inflammation, however IFN-γ induced STAT1 expression and phosphorylation was corelated with the severity of DSS-colitis mice and partially dependent on caspase-11 (119, 120). STAT1 signaling in IECs was observed to play a role in protective effects of caspase-11 in colitis (119). Further study established that the caspase-1 induced STAT1 activation in IEC was independent of IFN-γ but dependent on LPS/IL-1β-mediated activation (120). This presents a new paradigm in caspase-11 mediated STAT1 activation in IECs via IL-1β released from non-canonical inflammasome.

IL-18 contributes towards gut microbiota development, intestinal barrier function, and mucosal renewal (121). Recent studies implicate that IL-18 expression is expressed only in intestinal epithelium and this expression is independent of NLRP3 activation but dependent on Caspase-1 (122, 123). In concordance with this observation, previous studies have attributed colonic epithelial assault to significantly ablated levels of mature IL-18 in caspase-1/11^−/− colitis mice (107, 117). Susceptibility to develop colitis and colorectal cancer was reported in IL-18^−/− mice (2, 124–126). On the contrary, on treating caspase-11^−/− colitis mice with recombinant IL-18, animals showed significant attenuation in colitis disease pathology. These studies highlight the importance of IL-18 in imparting the protection from intestinal inflammation.

However, recent studies consider NLRP6 as a critical player in the maintenance of gut homeostasis, mucosal renewal, and proliferation, but not IL-18 (127–129). Gene knockdown studies presents the protective role of NLRP6 against intestinal inflammation. NLRP6 is essentially expressed in IECs and impaired expression of nlrp6 develops severe colitis in mice challenged with DSS compared to their WT mates (129, 130). Nlrp6^−/− mice exhibited changes in terminal ileum, colonic crypt hyperplasia, and increased size of Peyer’s patches with formation of germinal centers. Colonic explants from nlrp6^−/− mice also produced significantly less IL-18. However, recent study showed no change in the expression of IL-18 despite the differential NLRP6 expression in human colonic biopsies from CD and UC patients (131). In agreement to this study, Normand et al. (132) showed no NLRP6-dependent changes in the IL-18 expression of colonic biopsies from nlrp6^−/− and WT mice. This source of IL-18 can be from other inflammasomes or cells adjacent to intestinal microenvironment. Seregin et al. (133) showed that NLRP6 from lamina propria cells and not IECs was upregulated in response to DSS-treatment in IL-10^−/− mice. Moreover, adoptive transfer of Ly6C^hi monocytes from WT mice to nlrp6^−/− mice reduced DSS-induced mortality, intestinal injury, intestinal permeability and inflammation. The adoptively transferred Ly6C^hi monocytes migrated to the colon. The study concluded NLRP6-mediated IL-18 secretion by inflammatory monocytes recruited to colon in response to DSS-induced intestinal assault. These monocytes, in turn, upregulate TNFα and promote epithelial repair and recovery. The study presents a whole new paradigm of NLRP6-mediated protective effects in intestinal inflammation and homeostasis. The correlation between TNF-α and NLRP6 remains to be fully elucidated. Furthermore, the underlying mechanisms of association between IL-18 and NLRP6 is more complex. Elucidating their specific roles is important although challenging. NLRP6 deficient mice also displayed reduced intestinal mucus layer (IML) (134), a common feature in the IBD patients (135). While this notion was contradicted by Volk et al. (136), they showed that the IML formation and function are independent of NLRP6 activation (136).

The importance of GSDMD is continuously emerging as a regulator of downstream processes in inflammasome pathway. However, little is known on the roles of GSDMD in the context of intestinal inflammation. The distribution of GSDMD protein was observed in the lamina propria myeloid cells and E-cadherin+ IECs (137). DSS-induced assault to intestinal epithelium in mice significantly increased the expression and activation of GSDMD in both myeloid cells and IECs promoting pyroptosis in colitis (137). Role of GSDMD was protective in DSS-colitis mice, as the condition got exacerbated in gsdmd^−/− mice independent of the microbiota influence. The study outlined GSDMD as a negative regulator of cyclic GMP–AMP synthase (cGAS)-dependent inflammation and cGAS-inhibitor RU.521 treatment attenuated colitis severity in gsdmd^−/− mice.

Immunosuppressive activity of IL-10 is critical in restoring anti-inflammatory pathways in IBD. Individuals with IL-10R mutations spontaneously develop CD (138). IL-10 deficient mice spontaneously develop colitis and are even an experimental animal model to study colitis. IL-10 has been correlated with NLRP3 upregulation in the intestinal mucosa. Liu et al. (139) showed elevated NLRP3 activity in intestinal epithelial cells and colonic macrophages of IL-10^−/− mice. NLRP3 expression was increased in IL-10^−/− mice even before colitis onset suggesting a colitogenic role of NLRP3. Likewise, IL-10^k/o chronic colitis mice showed significantly increased colonic IL-1β protein levels that got reversed following inhibition of inflammasome activation with IL-1R antagonist or caspase-1 inhibitors (140).

Recently, the CD risk factor, immunity related GTPase M (IRGM) has been shown as a negative regulator of NLRP3 activation. Irgm1^−/− mice showed aggravated DSS-colitis development compared to their Irgm1^+/+ littermate controls. Expression of NLRP3, IL-1β, TNF-α, and IL-18 genes was also significantly increased in Irgm^−/− animals compared to their littermate controls (141). Recent studies showed novel role of IRGM2 as an inhibitor of caspase-11 when challenged with bacterial LPS (142, 143). The Irgm2^−/− mice when challenged with endotoxemia exhibited increased serum levels of TNF-α, IL-1β and IL-18 cytokines, increased weight loss and mortality (142). However, LPS challenged Irgm2^−/− capsase-11^−/− mice did not show increased serum levels of TNF-α, IL-1β and IL-18 cytokines and less mortality. The LPS primed Irgm^−/− bone marrow derived macrophages (BMDM) showed pyroptosis, reduced cell viability, IL-1β and IL-18 secretion and ASC speck formation. All these effects were abolished on ectopic expression of Irgm2 in Irgm2^−/− BDBM or on deletion of caspase-11. Moreover, the observed effects of IRGM2 were independent of NLRP3 activation (142, 143). Further studies are warranted to explore the role of IRGM in IBD pathogenesis and utilize IRGM as a therapeutic target for treating intestinal inflammation. NEK7 a component of NLRP3 is also shown to modulate NLRP3 activation and subsequently induce pyroptosis in DSS-induced chronic colitis in mice (144).

Effect on intestinal microbiota

Deficiencies in the inflammasome pathway may have stunted innate immune response to microbiota and promote intestinal assault. Inflammasome pathway may drive specific alterations in the intestinal microbiota, which in turn promote disease in individuals by either exposing them to these pro-colitogenic microbiota or disruption in their normal gut epithelial barrier function due to a variety of inflammatory insults. NLRC4 and interleukin-1 receptor (IL-1R) deficient mice exhibited a reduced immune response to salmonella and Citrobacter rodentium infections respectively (145, 146). Similarly, mice lacking NLRP3, NLRC4, caspase-1, and ASC showed higher sensitivity to C. rodentium, Clostridium difficile infections and establishment of Akkermansia muciniphila in nlrp6^−/− mice (130, 147, 148). Moreover, in-spite of housing in the same environment, nlrp3^−/− mice displayed differences in intestinal microbiota than WT mice with detection of potentially pathogenic bacterium from species Enterobacteriaceae and Mycobacterium (108). Elinav et al. (129) showed that colonic microbiota of NLRP6, IL-18, ASC, and caspase-1 deficient mice dominated colitiogenic TM7 and Prevotellaceae (Bacteroidetes phyla) bacterial strains. However, another study contradicted these observations using littermate-controlled nlrp6^−/− mice and ex-germ-free littermate-controlled asc^−/− mice (149). Importantly, recent studies have also contradicted the assumption that inflammasomes can influence shaping of intestinal microbiota. Lemire et al. (150) showed that NLRP6 had no dominant influence in shaping the structure of intestinal bacterial community and both WT and nlrp6^−/− littermates exhibited similar microbial community. Different from this point of view of whether inflammasomes disrupts normal gut microbiota structure, Meng et al. (123) observed improvement in colitis and colorectal cancer in NLRP3^R258W mutation experimental mice. NLRP3^R258W mutation has been associated with the hyperactivation of inflammasome, autoinflammatory disorders and excessive production of IL-1β. If NLRP3 increases susceptibility to intestinal inflammation as discussed in another section of this review, NLRP3^R258W mutation should exacerbate the colitis condition. However, intrinsically pro-inflammatory condition and intestinal assault due to the hyperactivation of NLRP3^R258W mutation was compensated by the reshaping of local microbiota and induction of T-regulatory milieu. The findings open an interesting new avenue of inflammasome research in intestinal disorders.

Studies contradicting protective role of inflammasome in intestinal inflammation

Although most studies support the notion that inflammasomes activation reduced the pathology in IBD, several studies contradict this view. Proponents of this theory demonstrated the detrimental effects of inflammasome activation in IBD. Animal studies showed no influence of NLRP6-ASC in curating the gut microbiota and susceptibility to DSS-induced colitis in NLRP6 and ASC deficient mice compared to the WT mice (149, 150). Thus, these studies undermine the protective effects of the inflammasome activation in IBD as otherwise suggested elsewhere (129). In another study, NLRP6 aggravated the allogeneic immune-mediated gastrointestinal graft-versus-host disease independent of changes in the composition of gut microbiome (151). Continued observations showed that mice lacking caspases-1 or NLRP3 exhibited significantly less severe clinical and pathological symptoms of colitis than their WT counterparts (152–154). These mice also showed reduced levels of IL-1β and IL-18 cytokines in the gut suggesting a prominent role of these cytokines in the progression of inflammasome-mediated intestinal inflammation. Recently, Fan et al. (155) showed no effect of increased caspase-11 activity on the severity of spontaneous chronic DSS-colitis in IL-10^−/− mice. The effect of caspase-11 on the severity of chronic colitis was also not pronounced in double knockout caspase-11^−/− IL-10^−/− mice. However, the different effects of caspase-11 were observed between acute and chronic DSS models of colitis over a repeated and longer period of experimental follow-up. The investigators could not specify exact reasons for this discrepancy in their research outcomes. Another study observed improvement in the clinical DSS-colitis symptoms, reduced mortality and reduced goblet cell loss in caspase-1^−/− mice compared to their WT mates normalized for intestinal microbiota (156). Contradicting observations on the protective role of GSDMD in colitis is published recently (157). The severity of acute DSS-induced colitis got attenuated in the gsdmd^−/− mice. Other than the widely recognized pyroptosis inducing role of GSDMD, this study reported a novel non-pyroptotic role of GSDMD. GSDMD was shown to form IL-1β–containing small extracellular vesicles in IECs. Following a stimulated activation of caspase-8 and subsequent cleavage of GSDMD, a GSDMD-NEDD4 complex is assembled on the NLRP3/caspase-8 complex to produce mature IL-1β. This complete GSDMD-complex is loaded on LC3-II+ vesicles to release out of the cell and release mature IL-1β.

Human studies

In concordance with the above findings, human research also highlights the role of the inflammasome in IBD. Peripheral blood mononuclear cells from 60% of CD patients showed higher NLRP3 activation compared to the cells from 28.6% of control individuals (158). Similarly, colonic biopsies from quiescent or active UC and CD patients showed upregulated expression of NLRP3, IL-1β, caspase-1, and ASC (159). The colon biopsies from UC and CD patients reported 131-fold and 3.9 fold increase of NLRP6 expression in ileal CD and colonic CD patients respectively (131). The NLRP6 was localized to the IECs, myofibroblasts, neutrophils, and monocytic lineage cells of the lamina propria in CD patients evaluated. The colon biopsies from similar patients also showed increase in the mRNA levels of IL-1β, nlrp1, nlrp3, nlrc4, nlrp12 and aim2. NLRP3 was expressed by the intestinal neutrophils and lamina propria cells but not in the epithelial cell layer of active UC patients (139, 159). A genome-wide study suggested an association between NLRP7 signaling and inflammasome formation during IBD pathogenesis (160). Human studies showed upregulated inflammasome activity in CD patients with higher levels of IL-1β and IL-18 in intestinal cells (112). A study published in 1999, showed increased levels of IL-18 mRNA in the colon biopsies from CD patients (161). The localization of mature IL-18 was more in the IECs and LPMCs of CD patients. Individuals with human immunodeficiency virus (HIV) infection showed a concomitant increase in the circulating levels of IL-18 and LPS and reduced levels of IL-18 binding protein (IL-18BP) compared to the control subjects (162, 163). These individuals often demonstrate intestinal inflammation and compromised intestinal epithelial integrity. The disbalance between the circulating levels of IL-18 and IL-18BP in CD patients can be one reason behind intestinal inflammation and compromised barrier integrity (164). This discrepancy among the serum levels of IL-18 and IL-18BP was also reported in children with IBD (165). Similarly, excessive level of IL-1β is considered to increase susceptibility for intestinal inflammation. In patients with CARD8 mutation and IL-10 receptor abnormalities, IL-1β was shown to trigger intestinal inflammation independent of NLRP3 (112, 166, 167). Blocking IL-1β successfully suppressed inflammation (166). In a recent study, the nucleotide polymorphism in IL-18 and IL-1β genes among CD and UC patients has been corelated with a poor response to anti-TNF-α therapy (168).

In summary, research evidence suggests differential roles of inflammasome regulation in IBD (Table 1). Recurrent intestinal tissue injury and prolonged inflammation can switch the protective effects of inflammasomes to detrimental effects. Moreover, the shaping of intestinal microbiota structure can have a fundamental role in differential research outcomes from these studies.

Table 1:

Role of Inflammasomes in inflammatory bowel disease

	Experimental animal studies	Human studies
NLR family proteins	• Attenuation or exacerbation of colitis • Maintenance of epithelial barrier integrity, repair and recovery • Gut homeostasis and mucosal renewal • Maintenance of intestinal mucus layer • Alteration or no effect on microbiota • Induction of T-cell activation	• Increased severity to intestinal inflammation • Increased activation in CD and UC patients • Increased expression in intestinal epithelial cells, neutrophils and lamina propria cells
Caspases	• Attenuation of colitis • Maintenance of intestinal epithelial barrier integrity • Release of IL-18 and IL-1β cytokines • Regulation of STAT1 signaling in IECs	• Increased activation in CD and UC patients
IL-18	• Attenuation or exacerbation of colitis • Controlling the outgrowth of colitogenic bacteria and maintenance of intestinal microbiota • Maintenance of intestinal barrier function and mucosal renewal • Induces release of other cytokines	• Increased severity to intestinal inflammation • Increased activation in CD patients • Maintenance of intestinal barrier integrity • Increased expression in intestinal epithelial cells and lamina propria cells
IL-1β	• No dominant role in intestinal inflammation • Activation of T-cell immune response	• Increased activation in CD and UC patients • Increased severity to intestinal inflammation

UC: Ulcerative colitis, CD: Crohn’s disease, IECs: Intestinal epithelial cells

INFLAMMASOME INHIBITORY MOLECULES AS A THERAPEUTIC APPROACH IN IBD

To date, several physiological, pharmacological, and inhibitors of inflammasome have been discovered. These inhibitors target either upstream or downstream effects of the inflammasome pathways and block NLRP3, TLR receptors, DAMPs, PAMPS, P2X7 receptor, ASC oligomerization, NACTH as well as inflammasome associated cytokines (Table 2).

Table 2:

Inflammasome inhibitors as therapeutics for IBD

Category of inhibitors	Drug/molecule	Experimental outcome/mechanism of action/clinical trial status	Reference
NLRP3 inhibitors	MCC950	• Significantly ameliorated colitis in mice • Reduced ASC oligomerization and colon levels of IL-1β and IL-18 cytokines • Binding to NACTH domain Walker B motif • Inhibits ATP hydrolysis	(169, 170, 174, 176)
	Oridonin	• Attenuated TNBS-induced CD and colitis in mice • Inhibits NLRP3 activation via covalently binding to NACTH domain and blocks interaction with NEK7	(178–180)
	Tranilast	• Directly inhibit NLRP3 by binding NACHT domain and block oligomerization • Attenuated gouty arthritis, cryopyrin-associated autoinflammatory syndromes, and type 2 diabetes	(208)
	OLT1177	• Reduced ATPase activity of recombinant NLRP3 protein • Prevented NLRP3-ASC and Nlrp3-caspase-1 interactions • Reduced caspase-1 activity, IL-1β and IL-18	(209)
	β-hydroxybutyrate	• Attenuates Muckle-Wells Syndrome, Familial Cold Autoinflammatory syndrome and Gout flares • Inhibits NLRP3 by blocking K+ efflux, reducing ASC oligomerization and speck formation • Reduces IL-1β and IL-18 cytokine maturation	(210, 211)
	NSAIDs	• Fenamate inhibited IL-1β release and ASC speck formation	(212)
	Dopamine	• Regulate NLRP3 inflammasome activation • Ameliorated neurotoxin-induced neuroinflammation, LPS-induced systemic inflammation, and monosodium urate crystal-induced peritoneal inflammation in mice	(213)
Micro RNA	miR-223	• Post-transcriptional regulator of NLRP3 • Nanoparticle-mediated overexpression of miR-223 ameliorated colitis	(186)
Antioxidant	Curcumin	• Reduces K+ efflux, ROS, and cathepsin B • Alleviated DSS-induced colitis in mice • Combined treatment of curcumin with mesalamine increased clinical remission in patients with colitis	(190, 191)
Antagonist	Anakinra	• IL-1β receptor antagonist • Initiated Phase-2 clinical trial for colitis	(203)
Pyroptosis inhibitor	Disulfiram	• Blocks GSDMD-mediated pore formation and IL-1β • Reduced mortality in LPS-induced sepsis in mice	(205)
	Dimethyl fumarate	• Inhibited GSDMD interaction with caspases • Ameliorated multiple sclerosis and familial Mediterranean fever in mice	(214)
	VX-765	• Inhibited atheroma and progression of atherosclerosis in mice and ameliorated multiple sclerosis • Reduced pyroptosis and IL-1β processing by caspase-1	(206, 207)

DSS: dextran sulfate sodium, TNBS: trinitrobenzene sulfonic acid, ROS: reactive oxygen species, NSAIDs: Non-steroidal anti-inflammatory drugs, VRAC: volume-regulated anion channel, GSDMD: gasdermin-D, LPS: lipopolysaccharide, CD: Crohn’s disease

Inhibitors that directly block NLRP3 or reduce the levels of NLRP3 are the ones that are primarily evaluated for inhibiting inflammasome. A pharmacological inhibitor of both canonical and non-canonical activation of NLRP3 can benefit by binding to the NACTH domain of the Walker B motif and by inhibiting ATP hydrolysis (169–172). Such an inhibitor, MCC950 has completed Phase-1 clinical trials for rheumatoid arthritis and can be developed as a potential treatment for IBD (173). MCC950 treatment alleviated the experimental chronic or acute colitis in mice by inhibiting ASC oligomerization, caspase-1 dependent activation of IL-1β, IL-18, and reducing the levels of pro-inflammatory cytokines (174, 175). In NOD2^k/o mice, MCC950 treatment significantly decreased the clinical and pathological symptoms of DSS-induced colitis (176). A recent study also showed that combinational therapy of metformin and MCC950 significantly ameliorated the DSS-induced colitis in mice (177). Treated animals showed reduced clinical and histopathological symptoms of colitis along with reduced TLR4/NF-kB signaling (effect of metformin) and inhibition of NLRP3 activation and caspase-1 activity (effect of MCC950) (177). The positive outcomes from these studies project MCC950 as a potential treatment for IBD. However, more studies are required in this direction. Oridonin, another plant-derived product and covalent inhibitor of NLRP3 was shown to suppress experimental colitis in mice (178–180).

MicroRNAs (miR’s) can be used as post-transcriptional regulators of NLRP3 inflammasomes by negatively regulating NLRP3 protein production and activation (181). However, the role of miR-223 has been ambiguous in IBD (182–185). Intestinal biopsies from active IBD patients showed increased expression of miR-233 (186, 187). On the contrary, in mir-223^-/y mice, colitis was exacerbated with increased clinical disease pathology (184, 186). The condition got reversed following treatment with nanoparticle loaded hyperexpression of miR-223 resulting in reduced expression of NLRP3 and IL-1β (186). This suggested that, further studies are warranted before exploring miR-223 as a treatment for IBD.

Antioxidants are shown to reverse NLRP3 activation (188). Curcumin a hydrophobic polyphenol derived from turmeric has been investigated as a treatment for IBD (189). Curcumin treatment reduced clinical symptoms of murine DSS-colitis (190). DSS-induced ROS release, K+ efflux, and activation of cathepsin B were inhibited in curcumin-treated colitis mice (190). Clinical studies have also reported the therapeutic effects of curcumin in colitis patients (191, 192). However, one study showed no added advantage of curcumin treatment over placebo in randomized control trials (193). Similarly, flavonoid VI-16, plumercin, procyanidin, dimethyl fumarate, and caffeic acid phenethyl ester are found to be therapeutically effective in ameliorating experimental mouse models of colitis. The effects appear to be by inhibition of ROS release and NLRP3 (194–198).

Several other molecules inhibiting caspase-1, IL-1β, GSDMD, PAMP, and DAMP have also been proposed for regulating inflammasome activation in immune-mediated disorders. L-sulforaphane, a phytochemical derived from broccoli and a potential caspase-1 inhibitor is currently in clinical trials for several immune-mediated disorders like asthma, allergy, and rhinitis (199). Sulforaphane can also be explored for the treatment of IBD (200). Canakinumab (IL-1β antibody) and Anakinra (IL-1β receptor antagonist) are FDA approved therapies for cryopyrin-associated periodic syndromes and can be explored for the potential treatment for IBD (201, 202). Recently a Phase-2 clinical trial (Trial registration # ISRCTN43717130) investigates the use of anakinra in combination with corticosteroid treatment in patients with acute UC (203). Another study showed that IL-1Ra loaded alginate/chitosan microcapsule administration alleviated the clinical and pathological symptoms of DSS-colitis in mice (204). An FDA-approved drug disulfiram has been shown to inhibit pyroptosis in LPS-primed THP-1 macrophages by blocking both caspases and GSDMD-mediated pore formation (205). Disulfiram treatment improved survival rates, reduced TNF-α and IL-6 secretion and reduced GSDMD activation against LPS-induced experimental sepsis in mice (205). VX-765 is a specific inhibitor of caspase-1 used against myocardial infraction. VX-765 treatment reduced the caspase-1 activity, levels of p20 and IL-1β and inhibited pyroptosis in vascular smooth muscle cells treated with OxLDL (206). VX-765 also inhibited proinflammatory caspases and subsequent pyroptosis to reduce inflammation in experimental autoimmune encephalomyelitis (207). These studies provide evidence that inhibitors of pyroptosis (GSDMD and caspases inhibitors) can be used to reduce inflammation, however further studies are required to evaluate these drugs against intestinal inflammation.

FUTURE PERSPECTIVES

The function of the inflammasome in IBD is complex with no conclusive outcomes from clinical and experimental studies. This complexity of the inflammasome pathway and underexplored IBD pathology makes it difficult to completely understand the association between inflammasome activation and intestinal inflammation. Experimental approaches should be revisited using suitable and advance animal models with appropriate littermate controls, genetic background, considering the role of dietary components, housing conditions, and microbiota composition to establish the critical relationship between inflammasome and IBD. The development of advanced 3D organoid models may provide a niche in studying the role of the inflammasome in IBD. For now, targeting inflammasome appears to be a promising and lucrative treatment approach for IBD; clinical and long-term safety trials with a larger sample size should be conducted to determine the therapeutic efficacy of inflammasome inhibitory compounds in IBD. Advancing our understandings on the inflammasomes will aid in discovering novel pharmacological inhibitors. However, the variability in patient responses to therapeutics in IBD and combinational therapeutic efficacy of inflammasome targeting drugs along with the on-going remission following treatments is an important question to be answered. Furthermore, we do not know if abrupt and complete shutting of the inflammasome pathway in the gut may lead to severe side effects or even serious exacerbation of the inflammatory condition. The development of pharmacological drugs targeting upstream and downstream inflammasome pathways in the gut and clinical studies rationalizing the optimal required dosage is required. Overall, it is important to develop inflammasome-targeted IBD therapeutics and bring them to the clinic, which will benefit millions of patients who are suffering from IBD and colitis and will be a rewarding effort.

NONSTANDARD ABBREVIATIONS:

IBD

Inflammatory bowel disease

CD

Crohn’s disease

UC

Ulcerative colitis

5-ASA

5-aminosalicylic acid

PAMPs

pathogen-associated molecular patterns

DAMPs

danger-associated molecular patterns

PRR

pattern recognition receptors

NLR

nucleotide-binding domain and leucine-rich repeat receptors

NLRP

NLR pyrin domain-containing family

NLRC4

NLR family caspase recruitment domain-containing protein-4

AIM2

double-stranded DNA sensors absent in melanoma-2

NAIP

NLR apoptosis inhibitory protein

CARD

caspase activation and recruitment domain

ASC

apoptosis-associated speck-like protein containing CARD

PYD

amino-terminal pyrin domain

NACTH

N-terminal central nucleotide-binding and oligomerization domain

LRR

carboxy-terminal leucine-rich repeat domain

MAPK

mitogen-activated protein kinase

ATP

adenosine triphosphate

GBP

guanylate binding proteins

TRIF

TIR domain-containing adapter-inducing interferon-β

RIPK

receptor interacting protein kinases

FADD

Fas-associated protein with death domain

DSS

dextran sulfate sodium

IRGM

immunity-related GTPase M