GSDMB is increased in IBD and regulates epithelial restitution/repair independent of pyroptosis.

Gasdermins are a family of structurally related proteins originally described for their role in pyroptosis. Gasdermin B (GSDMB) is currently the least studied, and while its association with genetic susceptibility to chronic mucosal inflammatory disorders is well established, little is known about its functional relevance during active disease states. Herein, we report increased GSDMB in inflammatory bowel disease, with single-cell analysis identifying epithelial specificity to inflamed colonocytes/crypt top colonocytes. Surprisingly, mechanistic experiments and transcriptome profiling reveal lack of inherent GSDMB-dependent pyroptosis in activated epithelial cells and organoids but instead point to increased proliferation and migration during in vitro wound closure, which arrests in GSDMB-deficient cells that display hyper-adhesiveness and enhanced formation of vinculin-based focal adhesions dependent on PDGF-A-mediated FAK phosphorylation. Importantly, carriage of disease-associated GSDMB SNPs confers functional defects, disrupting epithelial restitution/repair, which, altogether, establishes GSDMB as a critical factor for restoration of epithelial barrier function and the resolution of inflammation.

Interleukin 33 Triggers Early Eosinophil-Dependent Events Leading to Metaplasia in a Chronic Model of Gastritis-Prone Mice.

BACKGROUND & AIMS: Interleukin (IL)33/IL1F11 is an important mediator for the development of type 2 T-helper cell (Th2)-driven inflammatory disorders and has also been implicated in the pathogenesis of gastrointestinal (GI)-related cancers, including gastric carcinoma. We therefore sought to mechanistically determine IL33's potential role as a critical factor linking chronic inflammation and gastric carcinogenesis using gastritis-prone SAMP1/YitFc (SAMP) mice. METHODS: SAMP and (parental control) AKR mice were assessed for baseline gastritis and progression to metaplasia. Expression/localization of IL33 and its receptor, ST2/IL1R4, were characterized in corpus tissues, and activation and neutralization studies were both performed targeting the IL33/ST2 axis. Dissection of immune pathways leading to metaplasia was evaluated, including eosinophil depletion studies using anti-IL5/anti-CCR3 treatment. RESULTS: Progressive gastritis and, ultimately, intestinalized spasmolytic polypeptide-expressing metaplasia (SPEM) was detected in SAMP stomachs, which was absent in AKR but could be moderately induced with exogenous, recombinant IL33. Robust peripheral (bone marrow) expansion of eosinophils and local recruitment of both eosinophils and IL33-expressing M2 macrophages into corpus tissues were evident in SAMP. Interestingly, IL33 blockade did not affect bone marrow-derived expansion and local infiltration of eosinophils, but markedly decreased M2 macrophages and SPEM features, while eosinophil depletion caused a significant reduction in both local IL33-producing M2 macrophages and SPEM in SAMP. CONCLUSIONS: IL33 promotes metaplasia and the sequelae of eosinophil-dependent downstream infiltration of IL33-producing M2 macrophages leading to intestinalized SPEM in SAMP, suggesting that IL33 represents a critical link between chronic gastritis and intestinalizing metaplasia that may serve as a potential therapeutic target for preneoplastic conditions of the GI tract.

IL-33 promotes recovery from acute colitis by inducing miR-320 to stimulate epithelial restitution and repair.

Defective and/or delayed wound healing has been implicated in the pathogenesis of several chronic inflammatory disorders, including inflammatory bowel disease (IBD). The resolution of inflammation is particularly important in mucosal organs, such as the gut, where restoration of epithelial barrier function is critical to reestablish homeostasis with the interfacing microenvironment. Although IL-33 and its receptor ST2/ILRL1 are known to be increased and associated with IBD, studies using animal models of colitis to address the mechanism have yielded ambiguous results, suggesting both pathogenic and protective functions. Unlike those previously published studies, we focused on the functional role of IL-33/ST2 during an extended (2-wk) recovery period after initial challenge in dextran sodium sulfate (DSS)-induced colitic mice. Our results show that during acute, resolving colitis the normal function of endogenous IL-33 is protection, and the lack of either IL-33 or ST2 impedes the overall recovery process, while exogenous IL-33 administration during recovery dramatically accelerates epithelial restitution and repair, with concomitant improvement of colonic inflammation. Mechanistically, we show that IL-33 stimulates the expression of a network of microRNAs (miRs) in the Caco2 colonic intestinal epithelial cell (IEC) line, especially miR-320, which is increased by >16-fold in IECs isolated from IL-33-treated vs. vehicle-treated DSS colitic mice. Finally, IL-33-dependent in vitro proliferation and wound closure of Caco-2 IECs is significantly abrogated after specific inhibition of miR-320A. Together, our data indicate that during acute, resolving colitis, IL-33/ST2 plays a crucial role in gut mucosal healing by inducing epithelial-derived miR-320 that promotes epithelial repair/restitution and the resolution of inflammation.

A signalling cascade of IL-33 to IL-13 regulates metaplasia in the mouse stomach.

OBJECTIVE: Alternatively activated macrophages (M2) are associated with the progression of spasmolytic polypeptide-expressing metaplasia (SPEM) in the stomach. However, the precise mechanism(s) and critical mediators that induce SPEM are unknown. DESIGN: To determine candidate genes important in these processes, macrophages from the stomach corpus of mice with SPEM (DMP-777-treated) or advanced SPEM (L635-treated) were isolated and RNA sequenced. Effects on metaplasia development after acute parietal cell loss induced by L635 were evaluated in interleukin (IL)-33, IL-33 receptor (ST2) and IL-13 knockout (KO) mice. RESULTS: Profiling of metaplasia-associated macrophages in the stomach identified an M2a-polarised macrophage population. Expression of IL-33 was significantly upregulated in macrophages associated with advanced SPEM. L635 induced metaplasia in the stomachs of wild-type mice, but not in the stomachs of IL-33 and ST2 KO mice. While IL-5 and IL-9 were not required for metaplasia induction, IL-13 KO mice did not develop metaplasia in response to L635. Administration of IL-13 to ST2 KO mice re-established the induction of metaplasia following acute parietal cell loss. CONCLUSIONS: Metaplasia induction and macrophage polarisation after parietal cell loss is coordinated through a cytokine signalling network of IL-33 and IL-13, linking a combined response to injury by both intrinsic mucosal mechanisms and infiltrating M2 macrophages.

IL-33 activates tumor stroma to promote intestinal polyposis.

Tumor epithelial cells develop within a microenvironment consisting of extracellular matrix, growth factors, and cytokines produced by nonepithelial stromal cells. In response to paracrine signals from tumor epithelia, stromal cells modify the microenvironment to promote tumor growth and metastasis. Here, we identify interleukin 33 (IL-33) as a regulator of tumor stromal cell activation and mediator of intestinal polyposis. In human colorectal cancer, IL-33 expression was induced in the tumor epithelium of adenomas and carcinomas, and expression of the IL-33 receptor, IL1RL1 (also referred to as IL1-R4 or ST2), localized predominantly to the stroma of adenoma and both the stroma and epithelium of carcinoma. Genetic and antibody abrogation of responsiveness to IL-33 in the Apc(Min/+) mouse model of intestinal tumorigenesis inhibited proliferation, induced apoptosis, and suppressed angiogenesis in adenomatous polyps, which reduced both tumor number and size. Similar to human adenomas, IL-33 expression localized to tumor epithelial cells and expression of IL1RL1 associated with two stromal cell types, subepithelial myofibroblasts and mast cells, in Apc(Min/+) polyps. In vitro, IL-33 stimulation of human subepithelial myofibroblasts induced the expression of extracellular matrix components and growth factors associated with intestinal tumor progression. IL-33 deficiency reduced mast cell accumulation in Apc(Min/+) polyps and suppressed the expression of mast cell-derived proteases and cytokines known to promote polyposis. Based on these findings, we propose that IL-33 derived from the tumor epithelium promotes polyposis through the coordinated activation of stromal cells and the formation of a protumorigenic microenvironment.

IL-33 Drives Eosinophil Infiltration and Pathogenic Type 2 Helper T-Cell Immune Responses Leading to Chronic Experimental Ileitis.

Although a clear association has been established between IL-33 and inflammatory bowel disease, mechanistic studies to date, primarily using acute murine models of colitis, have yielded contradicting results, demonstrating both pathogenic and protective roles. We used a well-characterized, spontaneous model of inflammatory bowel disease [ie, SAMP1/YitFc (SAMP) mice] to investigate the role of IL-33 during chronic intestinal inflammation. Our results showed marked eosinophil infiltration into the gut mucosa with increased levels of eotaxins and type 2 helper T-cell (Th2) cytokines as disease progressed and became more severe, which could be reversed upon either eosinophil depletion or blockade of IL-33 signaling. Exogenous IL-33 administration recapitulated these effects in ilea of uninflamed (parental) control AKR/J mice. Human data supported these findings, showing colocalization and up-regulation of IL-33 and eosinophils in the colonic mucosa of inflammatory bowel disease patients versus noninflamed controls. Finally, colonization of commensal flora by fecal material transplantation into germ-free SAMP and the presence of the gut microbiome induced IL-33, subsequent eosinophil infiltration, and mounting of Th2 immune responses, leading to exacerbation of chronic intestinal inflammation characteristic of SAMP mice. These data demonstrate a pathogenic role for IL-33-mediated eosinophilia and activation of Th2 immunity in chronic intestinal inflammation that is dependent on the gut microbiome. Targeting IL-33 may represent a novel therapeutic approach to treat patients with inflammatory bowel disease.

Central role of IL-17/Th17 immune responses and the gut microbiota in the pathogenesis of intestinal fibrosis.

PURPOSE OF REVIEW: Intestinal fibrosis is a serious, yet common, outcome in patients with inflammatory bowel disease (IBD). Despite advances in developing novel treatment modalities to control chronic gut inflammation characteristic of IBD, no effective antifibrotic therapies exist to date. As such, a deeper understanding of the molecular mechanisms underlying intestinal fibrosis and the availability of relevant animal models are critical to move this area of investigation forward. RECENT FINDINGS: Emerging concepts in the pathogenesis of intestinal fibrosis include the central role of interleukin (IL)-17 and Th17 immune responses, although their precise contribution to chronic inflammation and IBD remains controversial. Other novel mediators of intestinal fibrosis, such as tumor necrosis factor-like ligand 1A and components of the renin-angiotensin system, support the importance of IL-17. Additionally, recent studies utilizing novel mouse models highlight the significance of the gut microbiota and link components of bacterial sensing, including nucleotide-binding oligomerization domain-containing protein 2, to IL-17/Th17 immune responses in the development of inflammation-associated intestinal fibrosis. SUMMARY: Recent progress in identifying key mediators, novel animal models, and important mechanistic pathways in the pathogenesis of intestinal fibrosis holds promise for the development of effective antifibrotics in an area of significant, unmet clinical need.

Mechanisms and models for intestinal fibrosis in IBD.

Intestinal fibrosis is a common outcome in IBD leading to significant morbidity that, to date, has no effective medical treatment. Current knowledge regarding potential mechanism(s) of intestinal fibrogenesis and stricture formation is limited, due in large part to the lack of relevant animal models. Although conventional models possess aspects that are advantageous to study specific mechanisms involved in gut fibrosis, most lack the features of a spontaneously occurring process leading to the formation of intestinal fibrotic lesions following mucosal inflammatory events and the ability to investigate the natural course of disease over time. This review aims to discuss established and novel animal models of gut fibrosis, particularly focusing on the advantages and disadvantages of each model system and the insights they bring to our understanding of the mechanisms of fibrogenesis. In fact, recent enhancements to existing models and the expansion of novel animal models of gut fibrosis is opening up multiple avenues for investigation which should stimulate progress in our mechanistic understanding of intestinal fibrogenesis and facilitate the development of effective pharmacotherapy in an area of significant unmet need.

The Importance of Th2 Immune Responses in Mediating the Progression of Gastritis-Associated Metaplasia to Gastric Cancer.

Gastric cancer is one of the leading causes of cancer deaths worldwide, with chronic gastritis representing the main predisposing factor initiating the cascade of events leading to metaplasia and eventually progressing to cancer. A widely accepted classification distinguishes between autoimmune and environmental atrophic gastritis, mediated, respectively, by T cells promoting the destruction of the oxyntic mucosa, and chronic H. pylori infection, which has also been identified as the major risk factor for gastric cancer. The original dogma posits Th1 immunity as a main causal factor for developing gastritis and metaplasia. Recently, however, it has become evident that Th2 immune responses play a major role in the events causing chronic inflammation leading to tumorigenesis, and in this context, many different cell types and cytokines are involved. In particular, the activity of cytokines, such as IL-33 and IL-13, and cell types, such as mast cells, M2 macrophages and eosinophils, are intertwined in the process, promoting chronic gastritis-dependent and more diffuse metaplasia. Herein, we provide an overview of the critical events driving the pathology of this disease, focusing on the most recent findings regarding the importance of Th2 immunity in gastritis and gastric metaplasia.

Candida tropicalis Affects Candida albicans Virulence by Limiting Its Capacity to Adhere to the Host Intestinal Surface, Leading to Decreased Susceptibility to Colitis in Mice.

Candida (C.) infections represent a serious health risk for people affected by inflammatory bowel disease. An important fungal virulence factor is the capacity of the fungus to form biofilms on the colonized surface of the host. This research study aimed to determine the effect of a C. tropicalis and C. albicans co-infection on dextran sodium sulfate (DSS)-induced colitis in mice. The colitis severity was evaluated using histology and a colonoscopy. The mice were mono-inoculated with C. albicans or C. tropicalis or co-challenged with both species. The mice were administered 3% DSS to induce acute colitis. The biofilm activity was assessed using (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl] 2H-tetrazoliumhydroxide (XTT) and dry-weight assays. The abundance of C. albicans in the colon tissues was assessed by immunohistochemistry. The co-challenged mice showed a decreased colitis severity compared to the mono-inoculated mice. The dry-weight assay demonstrated a marked decrease in C. albicans biofilm production in a C. albicans culture incubated with C. tropicalis supernatant. Immunohistochemical staining showed that C. albicans was more abundant in the mucosa of C. albicans mono-inoculated mice compared to the co-inoculated group. These data indicate an antagonistic microbial interaction between the two Candida species, where C. tropicalis may produce molecules capable of limiting the ability of C. albicans to adhere to the host intestinal surface, leading to a reduction in biofilm formation.

The role of IL-33 in gut mucosal inflammation.

Interleukin (IL)-33 is a recently identified cytokine belonging to the IL-1 family that is widely expressed throughout the body and has the ability to induce Th2 immune responses. In addition, IL-33 plays a key role in promoting host defenses against parasites through the expansion of a novel population of innate lymphoid cells. In recent years, a growing body of evidence has shown that the proinflammatory properties displayed by IL-33 are detrimental in several experimental models of inflammation; in others, however, IL-33 appears to have protective functions. In 2010, four different research groups consistently described the upregulation of IL-33 in patients with inflammatory bowel disease (IBD). Animal models of IBD were subsequently utilized in order to mechanistically determine the precise role of IL-33 in chronic intestinal inflammation, without, however, reaching conclusive evidence demonstrating whether IL-33 is pathogenic or protective. Indeed, data generated from these studies suggest that IL-33 may possess dichotomous functions, enhancing inflammatory responses on one hand and promoting epithelial integrity on the other. This review focuses on the available data regarding IL-33/ST2 in the physiological and inflammatory states of the gut in order to speculate on the possible roles of this novel IL-1 family member in intestinal inflammation.

Candida tropicalis Infection Modulates the Gut Microbiome and Confers Enhanced Susceptibility to Colitis in Mice.

BACKGROUND & AIMS: We previously showed that abundance of Candida tropicalis is significantly greater in Crohn's disease patients compared with first-degree relatives without Crohn's disease. The aim of this study was to determine the effects and mechanisms of action of C tropicalis infection on intestinal inflammation and injury in mice. METHODS: C57BL/6 mice were inoculated with C tropicalis, and colitis was induced by administration of dextran sodium sulfate in drinking water. Disease severity and intestinal permeability subsequently were evaluated by endoscopy, histology, quantitative reverse-transcription polymerase chain reaction, as well as 16S ribosomal RNA and NanoString analyses (NanoString Technologies, Seattle, WA). RESULTS: Infected mice showed more severe colitis, with alterations in gut mucosal helper T cells (Th)1 and Th17 cytokine expression, and an increased frequency of mesenteric lymph node-derived group 2 innate lymphoid cells compared with uninfected controls. Gut microbiome composition, including changes in the mucin-degrading bacteria, Akkermansia muciniphila and Ruminococcus gnavus, was altered significantly, as was expression of several genes affecting intestinal epithelial homeostasis in isolated colonoids, after C tropicalis infection compared with uninfected controls. In line with these findings, fecal microbiome transplantation of germ-free recipient mice using infected vs uninfected donors showed altered expression of several tight-junction proteins and increased susceptibility to dextran sodium sulfate-induced colitis. CONCLUSIONS: C tropicalis induces dysbiosis that involves changes in the presence of mucin-degrading bacteria, leading to altered tight junction protein expression with increased intestinal permeability and followed by induction of robust Th1/Th17 responses, which ultimately lead to an accelerated proinflammatory phenotype in experimental colitic mice.

NOD2 drives early IL-33-dependent expansion of group 2 innate lymphoid cells during Crohn's disease-like ileitis.

Innate lymphoid cells (ILCs) are enriched at barrier surfaces, including the gastrointestinal tract. While most studies have focused on the balance between pathogenic group 1 ILCs (ILC1s) and protective ILC3s in maintaining gut homeostasis and during chronic intestinal inflammation, such as Crohn's disease (CD), less is known regarding ILC2s. Using an established murine model of CD-like ileitis, i.e., the SAMP1/YitFc (SAMP) mouse strain, we showed that ILC2s, compared with ILC1s and ILC3s, were increased within draining mesenteric lymph nodes and ilea of SAMP versus AKR (parental control) mice early, during the onset of disease. Gut-derived ILC2s from CD patients versus healthy controls were also increased and expanded, similarly to ILC1s, in greater proportion compared with ILC3s. Importantly, we report that the intracellular bacteria-sensing protein, nucleotide-binding oligomerization domaining-containing protein 2, encoded by Nod2, the first and strongest susceptibility gene identified for CD, promoted ILC2 expansion, which was dramatically reduced in SAMP mice lacking NOD2 and in SAMP mice raised under germ-free conditions. Furthermore, these effects occurred through a mechanism involving the IL-33/ST2 ligand-receptor pair. Collectively, our results indicate a functional link between NOD2 and ILC2s, regulated by the IL-33/ST2 axis, that mechanistically may contribute to early events leading to CD pathogenesis.

Cytokine-Mediated Regulation of Innate Lymphoid Cell Plasticity in Gut Mucosal Immunity.

Mucosal barriers are active sites that encounter a bombardment of antigenic stimuli derived from both the commensal flora and a variety of pathogens, as well as from environmental insults. As such, the ability to mount appropriate innate immune responses is an important first line of defense that confers protection to the host. Central to innate immunity are innate lymphoid cells (ILCs), which were first described a decade ago, and represent a family of heterogeneous cells driven by specific transcription factors and exhibit distinct cytokine profiles that are shared with their CD4+ T-helper cell counterparts. ILCs are particularly enriched at mucosal surfaces, and the tissue microenvironment and cytokine milieu in which ILCs reside are critical factors that drive the behavior and overall function of these cells. In fact, ILCs situated at mucosal barriers must be able to temper their response to a constant exposure of environmental antigens, but also promptly react to pathogens or signals that are potentially harmful to the host. In this context, the ability of ILCs to readily transdifferentiate in response to their dynamic surroundings has become a vigorous area of research, and defining specific mechanism(s) of ILC plasticity is at the advent of discovery. This review will summarize what is currently known regarding the network of cytokines and regulatory elements that enable ILCs to readily transform, based on the range of diverse signals and signal gradients they encounter that lead to either protective or pathogenic function(s), with focus on the gut mucosal immune system.

Death-Domain-Receptor 3 Deletion Normalizes Inflammatory Gene Expression and Prevents Ileitis in Experimental Crohn's Disease.

Background: TNF-like cytokine 1A (TL1A) and its functional receptor, death-domain-receptor-3 (DR3), are multifunctional mediators of effector and regulatory immunity. We aimed to evaluate the functional role and therapeutic potential of TL1A/DR3 signaling in Crohn's disease-like ileitis. Methods: Ileitis-prone SAMP1/YitFc (SAMP) and TNFΔARE/+ mice were rendered deficient for DR3 or TL1A by microsatellite marker-assisted backcrossing. Pathological and immunological characteristics were compared between control and knockout mice, and mucosal immunophenotype was analyzed by Nanostring microarray assay. The therapeutic effect of pharmacological TL1A neutralization was also investigated. Results: DR3 deficiency was associated with restoration of a homeostatic mucosal immunostat in SAMP mice through the regulation of several pro- and anti-inflammatory genes. This led to suppression of effector immunity, amelioration of ileitis severity, and compromised ability of either unfractionated CD4+ or CD4+CD45RBhi mucosal lymphocytes to transfer ileitis to severe combined immunodeficient mice recipients. TNF-driven ileitis was also prevented in TNFΔARE/+xDR3-/- mice, in association with decreased expression of the pro-inflammatory cytokines TNF and IFN-γ. In contrast to DR3, TL1A was dispensable for the development of ileitis although it affected the kinetics of inflammation, as TNFΔARE/+xTL1A-/- demonstrated delayed onset of inflammation, whereas administration of a neutralizing, anti-TL1A antibody ameliorated early but not late TNFΔARE/+ ileitis. Conclusion: We found a prominent pro-inflammatory role of DR3 in chronic ileitis, which is only partially mediated via interaction with TL1A, raising the possibility for additional DR3 ligands. Death-domain-receptor-3 appears to be a master regulator of mucosal homeostasis and inflammation and may represent a candidate therapeutic target for chronic inflammatory conditions of the bowel.

Central role of the gut epithelial barrier in the pathogenesis of chronic intestinal inflammation: lessons learned from animal models and human genetics.

The gut mucosa is constantly challenged by a bombardment of foreign antigens and environmental microorganisms. As such, the precise regulation of the intestinal barrier allows the maintenance of mucosal immune homeostasis and prevents the onset of uncontrolled inflammation. In support of this concept, emerging evidence points to defects in components of the epithelial barrier as etiologic factors in the pathogenesis of inflammatory bowel diseases (IBDs). In fact, the integrity of the intestinal barrier relies on different elements, including robust innate immune responses, epithelial paracellular permeability, epithelial cell integrity, as well as the production of mucus. The purpose of this review is to systematically evaluate how alterations in the aforementioned epithelial components can lead to the disruption of intestinal immune homeostasis, and subsequent inflammation. In this regard, the wealth of data from mouse models of intestinal inflammation and human genetics are pivotal in understanding pathogenic pathways, for example, that are initiated from the specific loss of function of a single protein leading to the onset of intestinal disease. On the other hand, several recently proposed therapeutic approaches to treat human IBD are targeted at enhancing different elements of gut barrier function, further supporting a primary role of the epithelium in the pathogenesis of chronic intestinal inflammation and emphasizing the importance of maintaining a healthy and effective intestinal barrier.