Palmitoylation at a conserved cysteine residue facilitates gasdermin D-mediated pyroptosis and cytokine release.

Gasdermin D (GSDMD)-mediated pyroptotic cell death drives inflammatory cytokine release and downstream immune responses upon inflammasome activation, which play important roles in host defense and inflammatory disorders. Upon activation by proteases, the GSDMD N-terminal domain (NTD) undergoes oligomerization and membrane translocation in the presence of lipids to assemble pores. Despite intensive studies, the molecular events underlying the transition of GSDMD from an autoinhibited soluble form to an oligomeric pore form inserted into the membrane remain incompletely understood. Previous work characterized S-palmitoylation for gasdermins from bacteria, fungi, invertebrates, as well as mammalian gasdermin E (GSDME). Here, we report that a conserved residue Cys191 in human GSDMD was S-palmitoylated, which promoted GSDMD-mediated pyroptosis and cytokine release. Mutation of Cys191 or treatment with palmitoyltransferase inhibitors cyano-myracrylamide (CMA) or 2-bromopalmitate (2BP) suppressed GSDMD palmitoylation, its localization to the membrane and dampened pyroptosis or IL-1ß secretion. Furthermore, Gsdmd-dependent inflammatory responses were alleviated by inhibition of palmitoylation in vivo. By contrast, coexpression of GSDMD with palmitoyltransferases enhanced pyroptotic cell death, while introduction of exogenous palmitoylation sequences fully restored pyroptotic activities to the C191A mutant, suggesting that palmitoylation-mediated membrane localization may be distinct from other molecular events such as GSDMD conformational change during pore assembly. Collectively, our study suggests that S-palmitoylation may be a shared regulatory mechanism for GSDMD and other gasdermins, which points to potential avenues for therapeutically targeting S-palmitoylation of gasdermins in inflammatory disorders.

TL1A/DR3 signaling regulates the generation of pathogenic Th9 cells in experimental inflammatory bowel disease.

OBJECTIVE: Death receptor 3 (DR3) and its ligand tumor necrosis factor like ligand 1A (TL1A), are involved in the regulation of the balance between effector and regulatory T cells in IBD. New evidence suggests a role of IL-9-secreting Th9 cells in the pathogenesis of ulcerative colitis (UC), although the molecular pathways through which IL-9 and Th9 cells may mediate intestinal inflammation in Crohn's disease (CD) are still unclear. DESIGN: We investigated the role of DR3 signaling in the differentiation of Th9 cells in mouse models of CD-like ileitis and colitis, including SAMP1/YitFc (SAMP) mice. RESULTS: Polarized-Th9 cells with functional DR3 from SAMP WT (Th9WT) harbor a pro-inflammatory signature compared to DR3-deficient Th9 cells that were obtained from DR3-/-xSAMP mice (Th9KO). Conversely, ablation of DR3 signaling generated anti-inflammatory responses, as reflected by higher numbers of IL-10 producing cells in DR3-/-xSAMP mice. Additionally, RNA-seq and phosphoproteomic analyses showed that inflammatory pathways are significantly more activated in Th9WT than in Th9KO cells. Finally, in the T-cell adoptive transfer model, Th9KO cells were less colitogenic than Th9WT, while IL-9 blockade diminished the severity of intestinal inflammation, indicating a crucial role of functional DR3 receptor in Th9 cells pathogenicity. CONCLUSION: We describe herein that a functional DR3 receptor is required for the pathogenicity of Th9 cells, thus, constituting a novel mechanism by which TL1A/DR3 signaling mediates experimental CD-like ileitis. The TL1A/DR3/Th9 pro-inflammatory pathway may offer a novel therapeutic target for patients with CD.

Host-microbial crosstalk relies on "tuft" love.

How commensals influence intestinal immunity is incompletely understood. In this issue of Immunity, Eshleman et al. demonstrate that microbiota-derived butyrate restrains tuft cell development via HDAC3 modulation in intestinal epithelial cells, showing how microbial metabolites impact intestinal type 2 immunity.

Association between metabolic syndrome components and gingival bleeding is women-specific: a nested cross-sectional study.

BACKGROUND: Metabolic syndrome (MetS) is a cluster of atherosclerotic risk factors that increases cardiovascular risk. MetS has been associated with periodontitis, but the contribution of single MetS components and any possible sexual dimorphism in this relation remain undetermined. METHODS: Using the third National Health and Nutrition Examination Survey (NHANES III), we performed a nested cross-sectional study to test whether individuals aged > 30 years undergoing periodontal evaluation (population) exposed to ≥ 1 MetS component (exposure) were at increased risk of bleeding/non-bleeding periodontal diseases (outcome) compared to nonexposed individuals, propensity score matched for sex, age, race/ethnicity, and income (controls). The association between MetS components combinations and periodontal diseases was explored overall and across subgroups by sex and smoking. Periodontal health status prediction based on MetS components was assessed. RESULTS: In total, 2258 individuals (n. 1129/group) with nested clinical-demographic features were analyzed. Exposure was associated with gingival bleeding (+ 18% risk for every unitary increase in MetS components, and triple risk when all five were combined), but not with stable periodontitis; the association was specific for women, but not for men, irrespective of smoking. The only MetS feature with significant association in men was high BP with periodontitis. CRP levels significantly increased from health to disease only among exposed women. MetS components did not substantially improve the prediction of bleeding/non-bleeding periodontal disease. CONCLUSION: The observed women-specific association of gingival bleeding with single and combined MetS components advances gender and precision periodontology. Further research is needed to validate and expand these findings.

The gasdermin protein family: emerging roles in gastrointestinal health and disease.

Since the identification and characterization of gasdermin (GSDM) D as the main effector of inflammatory regulated cell death (or pyroptosis), literature on the GSDM family of pore-forming proteins is rapidly expanding, revealing novel mechanisms regulating their expression and functions that go beyond pyroptosis. Indeed, a growing body of evidence corroborates the importance of GSDMs within the gastrointestinal system, underscoring their critical contributions to the pathophysiology of gastrointestinal cancers, enteric infections and gut mucosal inflammation, such as inflammatory bowel disease. However, with this increase in knowledge, several important and controversial issues have arisen regarding basic GSDM biology and its role(s) during health and disease states. These include critical questions centred around GSDM-dependent lytic versus non-lytic functions, the biological activities of cleaved versus full-length proteins, the differential roles of GSDM-expressing mucosal immune versus epithelial cells, and whether GSDMs promote pathogenic or protective effects during specific disease settings. This Review provides a comprehensive summary and interpretation of the current literature on GSDM biology, specifically focusing on the gastrointestinal tract, highlighting the main controversial issues and their clinical implications, and addressing future areas of research to unravel the specific role(s) of this intriguing, yet enigmatic, family of proteins.

Single-cell Transcriptomics Reveal the Importance of Distinct Epithelial Cell Populations in Ileal-specific, Treatment-naïve, and Treated Crohn's Disease Patients.

The advent of single-cell technologies has revolutionized analyses of IBD-specific processes by identifying important, often novel, mucosal cells subpopulations and their associated functions. We discuss recent findings reporting transcriptomic and cellular diversity of treatment-naïve and treated patients with ileal-specific CD.

The enigmatic roles of epithelial gasdermin B: Recent discoveries and controversies.

Gasdermin B (GSDMB) belongs to a family of structurally related proteins [(i.e., gasdermins (GSDMs)]. It distinguishes itself from other members by the lack of autoinhibition but clear bioactivity of its full-length form, its preference to bind to phosphatidylinositol phosphates and sulfatides, and the ability to promote both lytic and nonlytic cellular functions. It is the only gasdermin that lacks a mouse ortholog, making in vivo mechanistic studies challenging to perform. GSDMB is abundantly expressed in epithelial cells lining organs that directly interface with the external environment, such as the gastrointestinal tract, with emerging evidence supporting its role in enteric infections, inflammatory bowel disease (IBD), and colorectal cancer. This review discusses the unique features of GSDMB among other gasdermin family members and controversies surrounding GSDMB-dependent mammalian inflammatory cell death (i.e., pyroptosis), including recent discoveries revealing both lytic and nonlytic functions of epithelial-derived GSDMB, particularly during gut health and disease.

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.

Immunological Regulation of Intestinal Fibrosis in Inflammatory Bowel Disease.

Intestinal fibrosis is a late-stage phenotype of inflammatory bowel disease (IBD), which underlies most of the long-term complications and surgical interventions in patients, particularly those with Crohn's disease. Despite these issues, antifibrotic therapies are still scarce, mainly due to the current lack of understanding concerning the pathogenetic mechanisms that mediate fibrogenesis in patients with chronic intestinal inflammation. In the current review, we summarize recent evidence regarding the cellular and molecular factors of innate and adaptive immunity that are considered critical for the initiation and amplification of extracellular matrix deposition and stricture formation. We focus on the role of cytokines by dissecting the pro- vs antifibrotic components of the immune response, while taking into consideration their temporal association to the progressive stages of the natural history of IBD. We critically present evidence from animal models of intestinal fibrosis and analyze inflammation-fibrosis interactions that occur under such experimental scenarios. In addition, we comment on recent findings from large-scale, single-cell profiling of fibrosis-relevant populations in IBD patients. Based on such evidence, we propose future potential targets for antifibrotic therapies to treat patients with IBD.

In this review, authors describe the cellular and molecular immunological mechanism(s) of intestinal fibrosis in IBD, with a particular focus on animal models of intestinal fibrosis.

Parabacteroides distasonis induces depressive-like behavior in a mouse model of Crohn's disease.

Patients with inflammatory bowel disease (IBD) are particularly susceptible to behavioral diagnoses, and the microbiome has been repeatedly implicated in the pathogenesis of IBD. The intestinal microbiome's ability to affect behavior has become increasingly recognized and studied. The so-called 'psychobiome' has been linked to a plethora of neurological and psychological diagnoses, including autism and Parkinson's disease. Despite the ability of many bacterial species within the human intestinal microbiome to synthesize neurotransmitters, it has never been previously reported that a single bacterial species is sufficient to induce depression. Here, we demonstrate that our mouse model of Crohn's disease (CD)-like ileitis, the SAMP1/YitFc (SAMP1), does not exhibit baseline behavioral abnormalities. By comparison, SAMP6 mice develop depressive-like behavior that is associated with a rise in the GABA-producing bacterial genus Parabacteroides. We finally demonstrate that administration of Parabacteroides distasonis into our SAMP1 mice induces depressive-like behavior. Colonization with P. distasonis was not associated with increased intestinal inflammation or alterations in other measures of behavior. The intestinal environment of CD may be particularly conducive to colonization with P. distasonis and subsequent induction of depressive-like behavior. To our knowledge, this is the first report of a bacterial species specifically inducing depressive-like behavior.

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.

Novel Insights Into the Interactions Between the Gut Microbiome, Inflammasomes, and Gasdermins During Colorectal Cancer.

Colorectal cancer (CRC) is one of the most prevalent and deadly forms of cancer in Western countries. Inflammation is a well-known driver of colonic carcinogenesis; however, its role in CRC extends beyond colitis-associated cancer. Over the last decades, numerous associations between intestinal dysbiosis and CRC have been identified, with more recent studies providing mechanistic evidence of a causative relationship. Nonetheless, much remains to be discovered regarding the precise implications of microbiome alterations in the pathogenesis of CRC. Research confirms the importance of a bidirectional crosstalk between the gut microbiome and the mucosal immune system in which inflammasomes, multiprotein complexes that can sense "danger signals," serve as conduits by detecting microbial signals and activating innate immune responses, including the induction of microbicidal activities that can alter microbiome composition. Current evidence strongly supports an active role for this "inflammasome-microbiome axis" in the initiation and development of CRC. Furthermore, the gasdermin (GSDM) family of proteins, which are downstream effectors of the inflammasome that are primarily known for their role in pyroptosis, have been recently linked to CRC pathogenesis. These findings, however, do not come without controversy, as pyroptosis is reported to exert both anti- and protumorigenic functions. Furthermore, the multi-faceted interactions between GSDMs and the gut microbiome, as well as their importance in CRC, have only been superficially investigated. In this review, we summarize the existing literature supporting the importance of the inflammasome-microbiota axis, as well as the activation and function of GSDMs, to gain a better mechanistic understanding of CRC pathogenesis.

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.

Impaired estrogen signaling underlies regulatory T cell loss-of-function in the chronically inflamed intestine.

Signaling of 17ß-estradiol (estrogen) through its two nuclear receptors, α and ß (ERα, ERß), is an important mechanism of transcriptional regulation. Although ERs are broadly expressed by cells of the immune system, the mechanisms by which they modulate immune responses remain poorly understood. ERß-specific signaling is reduced in patients with chronic inflammatory diseases, including systemic lupus erythematosus and inflammatory bowel disease, and our previous work suggests that dysregulation of ERß-specific signaling contributes to enhanced intestinal inflammation in female SAMP/YitFC mice, a spontaneous model of Crohn's disease-like ileitis. The present study builds on these prior observations to identify a nonredundant, immunoprotective role for ERß-specific signaling in TGF-ß-dependent regulatory T cell (Treg) differentiation. Using a strain of congenic SAMP mice engineered to lack global expression of ERß, we observed dramatic, female-specific exacerbation of intestinal inflammation accompanied by significant reductions in intestinal Treg frequency and function. Impaired Treg suppression in the absence of ERß was associated with aberrant overexpression of Tsc22d3 (GILZ), a glucocorticoid-responsive transcription factor not normally expressed in mature Tregs, and ex vivo data reveal that forced overexpression of GILZ in mature Tregs inhibits their suppressive function. Collectively, our findings identify a pathway of estrogen-mediated immune regulation in the intestine, whereby homeostatic expression of ERß normally functions to limit Treg-specific expression of GILZ, thereby maintaining effective immune suppression. Our data suggest that transcriptional cross-talk between glucocorticoid and steroid sex hormone signaling represents an important and understudied regulatory node in chronic inflammatory disease.

Epithelial-derived gasdermin D mediates nonlytic IL-1ß release during experimental colitis.

Gasdermin D (GSDMD) induces pyroptosis via the pore-forming activity of its N-terminal domain, cleaved by activated caspases associated with the release of IL-1ß. Here, we report a nonpyroptotic role of full-length GSDMD in guiding the release of IL-1ß-containing small extracellular vesicles (sEVs) from intestinal epithelial cells (IECs). In response to caspase-8 inflammasome activation, GSDMD, chaperoned by Cdc37/Hsp90, recruits the E3 ligase, NEDD4, to catalyze polyubiquitination of pro-IL-1ß, serving as a signal for cargo loading into secretory vesicles. GSDMD and IL-1ß colocalize with the exosome markers CD63 and ALIX intracellularly, and GSDMD and NEDD4 are required for release of CD63+ sEVs containing IL-1ß, GSDMD, NEDD4, and caspase-8. Importantly, increased expression of epithelial-derived GSDMD is observed both in patients with inflammatory bowel disease (IBD) and those with experimental colitis. While GSDMD-dependent release of IL-1ß-containing sEVs is detected in cultured colonic explants from colitic mice, GSDMD deficiency substantially attenuates disease severity, implicating GSDMD-mediated release of IL-1ß sEVs in the pathogenesis of intestinal inflammation, such as that observed in IBD.

Winnie-APCMin/+ Mice: A Spontaneous Model of Colitis-Associated Colorectal Cancer Combining Genetics and Inflammation.

(1) Background: Colorectal cancer (CRC) is among the best examples of the relationship between inflammation and increased cancer risk. (2) Methods: To examine the effects of spontaneous low-grade chronic inflammation on the pathogenesis of CRC, we developed a new murine model of colitis-associated cancer (CAC) by crossing Mucin 2 mutated mice (Winnie) with ApcMin/+ mice. (3) Results: The resulting Winnie-ApcMin/+ model combines an inflammatory background with a genetic predisposition to small intestinal polyposis. Winnie-ApcMin/+ mice show an early occurrence of inflammatory signs and dysplastic lesions in the distal colon with a specific molecular signature. (4) Conclusion: The Winnie-ApcMin/+ model is a perfect model to demonstrate that chronic inflammation represents a crucial risk factor for the onset and progression of tumoral lesions in individuals genetically predisposed to CRC.

Inflammation mobilizes copper metabolism to promote colon tumorigenesis via an IL-17-STEAP4-XIAP axis.

Copper levels are known to be elevated in inflamed and malignant tissues. But the mechanism underlying this selective enrichment has been elusive. In this study, we report a axis by which inflammatory cytokines, such as IL-17, drive cellular copper uptake via the induction of a metalloreductase, STEAP4. IL-17-induced elevated intracellular copper level leads to the activation of an E3-ligase, XIAP, which potentiates IL-17-induced NFκB activation and suppresses the caspase 3 activity. Importantly, this IL-17-induced STEAP4-dependent cellular copper uptake is critical for colon tumor formation in a murine model of colitis-associated tumorigenesis and STEAP4 expression correlates with IL-17 level and XIAP activation in human colon cancer. In summary, this study reveals a IL-17-STEAP4-XIAP axis through which the inflammatory response induces copper uptake, promoting colon tumorigenesis.

Novel Pharmacological Therapy in Inflammatory Bowel Diseases: Beyond Anti-Tumor Necrosis Factor.

Inflammatory bowel diseases (IBDs) are chronic conditions of the gastrointestinal tract in which dysregulated immune responses cause persistent inflammation of the gut mucosa. Biologic therapy with anti-TNF blockers has revolutionized the therapeutic management of IBD for their remarkable efficacy and potential impact on disease course and for many years has represented the sole treatment option for patients refractory or intolerant to conventional therapy. In recent years, more molecules, both biologically and chemically synthetized, have been developed as potential therapeutic options for IBD that target different molecular pathways aside from TNF blockade, and which have been proposed as targets for novel drugs. This is particularly relevant for the present, as well as future, management of IBD, considering that some patients are refractory to anti-TNF. This review will summarize the pharmacological options, either currently available or in the pipeline, for market approval to treat IBD, besides anti-TNF strategies, based on their mechanism(s) of action. We will also analyze the current evidence for effectiveness and safety, as well as offer perspective, regarding the potential implementation for such therapies in the future.

TWEAK/Fn14 Is Overexpressed in Crohn's Disease and Mediates Experimental Ileitis by Regulating Critical Innate and Adaptive Immune Pathways.

BACKGROUND & AIMS: Crohn's disease (CD) is a debilitating inflammatory disorder that affects more than 1.6 million people in North America alone. Members of the tumor necrosis factor superfamily are key regulators of intestinal inflammation; specifically, tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and its receptor, fibroblast growth factor-inducible 14 (Fn14), are involved in normal and pathologic tissue remodeling. Our aim was to determine the role of TWEAK/Fn14 in CD and a murine model of CD-like ileitis (ie, SAMP1/YitFc [SAMP] strain). METHODS: SAMP mice deficient in Fn14 (SAMP × Fn14-/-) were developed and a detailed time-course study was performed evaluating ileal tissues by histology and stereomicroscopy, as well as quantitative polymerase chain reaction and NanoString technology (Seattle, WA). Reciprocal bone marrow chimeras were generated to assess the relevance of Fn14 in hematopoietic vs nonhematopoietic compartments. Surgically resected intestinal tissues and mucosal biopsy specimens from patients with CD, ulcerative colitis, and healthy controls were analyzed for the expression of TWEAK/Fn14 by quantitative polymerase chain reaction, Western blot, immunohistochemistry, and immunofluorescence. RESULTS: SAMP × Fn14-/- showed a marked decrease in ileitis severity at 20 weeks of age compared with SAMP WT controls. Bone marrow chimeras showed that Fn14 was required in both hematopoietic and nonhematopoietic compartments for ileitis to develop. Transcriptome data showed multiple cellular pathways regulated by Fn14 signaling. Finally, increased expression of TWEAK and Fn14 was observed in tissue lesions from CD patients compared with ulcerative colitis and healthy controls. CONCLUSIONS: TWEAK/Fn14 are up-regulated in CD, and also mediate experimental CD-like ileitis, by regulation of multiple innate and adaptive cellular pathways. Therefore, TWEAK/Fn14 may represent a novel therapeutic target for the treatment of small intestinal inflammation in CD.