Paneth cells regulate host-microbial homeostasis and defects in autophagy and host defense pathways have been associated with inflammatory bowel diseases (IBD). Genetic variants in TL1A (TNFSF15) and its receptor DR3 (TNFRSF25) have been associated with IBD. TL1A expression is increased in IBD patients, particularly in TL1A risk allele carriers. However, effects of TL1A on Paneth cells, resident microbiota, and development of ileitis remain unknown. TL1A overexpression in mice induces Paneth cell hyperplasia and morphological abnormalities preceding the development of ileitis. In Crohn's disease (CD) patients, ileal TL1A expression was associated with abnormal Paneth cell phenotypes. We confirmed direct effects of TL1A on Paneth cells in human iPSC-derived human intestinal organoids and mouse Paneth cell-enriched organoids. Resident microbiota was required for TL1A-mediated Paneth cell dysfunction, and ileitis. Tl1a-tg mice were enriched in short chain fatty acid-producing bacteria and the metabolite acetate. Acetate supplementation in WT or Tl1a-tg mice caused ileal inflammation, suggesting that acetate is sufficient to cause ileitis. DR3-deficiency in Paneth cells resulted in Paneth cell abnormalities and microbiome composition changes. Our findings provide a mechanistic link between overexpression of TL1A in CD patients, Paneth cell dysfunction, and enrichment of acetate-producing bacteria and acetate that promotes ileal inflammation. ### Competing Interest Statement Cedars-Sinai has financial interests in Prometheus Biosciences, Inc., a company which has access to the data and specimens in Cedars-Sinai's MIRIAD Biobank (including data and specimens used in this study) and seeks to develop commercial products. DPBM and SRT own stock in Prometheus Biosciences Inc. SRT and DPBM are consultants for Prometheus Biosciences. DPBM has consulted for Takeda, Sandoz Immunology, Gilead, Pfizer, Boehringer Ingelheim, Qu Biologics, and Bridge Biotherapeutics. EEA, NJ, SRT, KSM are co-inventors on US patent no. PCT/US22/51440 "Microbial metabolites on intestinal inflammation" filed on November 30, 2023. The other authors declare that they have no competing interests.

IntroductionThe maintenance of intestinal homeostasis depends on a complex interaction between the immune system, intestinal epithelial barrier, and microbiota. Alteration in one of these components could lead to the development of inflammatory bowel diseases (IBD). Variants within the autophagy gene ATG16L1 have been implicated in susceptibility and severity of Crohn’s disease (CD). Individuals carrying the risk ATG16L1 T300A variant have higher caspase 3-dependent degradation of ATG16L1 resulting in impaired autophagy and increased cellular stress. ATG16L1-deficiency induces enhanced IL-1β secretion in dendritic cells in response to bacterial infection. Infection of ATG16L1-deficient mice with a persistent strain of murine norovirus renders these mice highly susceptible to dextran sulfate sodium colitis. Moreover, persistent norovirus infection leads to intestinal virus specific CD8+ T cells responses. Both Toll-like receptor 7 (TLR7), which recognizes single-stranded RNA viruses, and ATG16L1, which facilitates the delivery of viral nucleic acids to the autolysosome endosome, are required for anti-viral immune responses.Results and discussionHowever, the role of the enteric virome in IBD is still poorly understood. Here, we investigate the role of TLR7 and ATG16L1 in intestinal homeostasis and inflammation. At steady state, Tlr7-/- mice have a significant increase in large intestinal lamina propria (LP) granzyme B+ tissue-resident memory CD8+ T (TRM) cells compared to WT mice, reminiscent of persistent norovirus infection. Deletion of Atg16l1 in myeloid (Atg16l1ΔLyz2) or dendritic cells (Atg16l1ΔCd11c) leads to a similar increase of LP TRM. Furthermore, Tlr7-/- and Atg16l1ΔCd11c mice were more susceptible to dextran sulfate sodium colitis with an increase in disease activity index, histoscore, and increased secretion of IFN-γ and TNF-α. Treatment of Atg16l1ΔCd11c mice with the TLR7 agonist Imiquimod attenuated colonic inflammation in these mice. Our data demonstrate that ATG16L1-deficiency in myeloid and dendritic cells leads to an increase in LP TRM and consequently to increased susceptibility to colitis by impairing the recognition of enteric viruses by TLR7.ConclusionIn conclusion, the convergence of ATG16L1 and TLR7 signaling pathways plays an important role in the immune response to intestinal viruses. Our data suggest that activation of the TLR7 signaling pathway could be an attractive therapeutic target for CD patients with ATG16L1 risk variants.

ObjectivePerianal Crohn's disease (pCD) occurs in up to 40% of patients with CD and is associated with poor quality of life, limited treatment responses and poorly understood aetiology. We performed a genetic association study comparing CD subjects with and without perianal disease and subsequently performed functional follow-up studies for a pCD associated SNP in Complement Factor B (CFB). DesignImmunochip-based meta-analysis on 4056 pCD and 11 088 patients with CD from three independent cohorts was performed. Serological and clinical variables were analysed by regression analyses. Risk allele of rs4151651 was introduced into human CFB plasmid by site-directed mutagenesis. Binding of recombinant G252 or S252 CFB to C3b and its cleavage was determined in cell-free assays. Macrophage phagocytosis in presence of recombinant CFB or serum from CFB risk, or protective CD or healthy subjects was assessed by flow cytometry. ResultsPerianal complications were associated with colonic involvement, OmpC and ASCA serology, and serology quartile sum score. We identified a genetic association for pCD (rs4151651), a non-synonymous SNP (G252S) in CFB, in all three cohorts. Recombinant S252 CFB had reduced binding to C3b, its cleavage was impaired, and complement-driven phagocytosis and cytokine secretion were reduced compared with G252 CFB. Serine 252 generates a de novo glycosylation site in CFB. Serum from homozygous risk patients displayed significantly decreased macrophage phagocytosis compared with non-risk serum. ConclusionpCD-associated rs4151651 in CFB is a loss-of-function mutation that impairs its cleavage, activation of alternative complement pathway, and pathogen phagocytosis thus implicating the alternative complement pathway and CFB in pCD aetiology.

Expression of death receptor 3 in intestinal epithelial cells regulates intestinal permeability and cellular localization of tight junction proteins during homeostasis. As a result, Dr3DIEC mice are more susceptible to acute colitis and show severely impaired epithelial barrier regeneration. BACKGROUND & AIMS: Tumor necrosis factor (TNF) superfamily member tumor necrosis factor-like protein 1A (TL1A) has been associated with the susceptibility and severity of inflammatory bowel diseases. However, the function of the tumor necrosis factor-like protein 1A and its receptor death receptor 3 (DR3) in the development of intestinal inflammation is incompletely understood. We investigated the role of DR3 expressed by intestinal epithelial cells (IECs) during intestinal homeostasis, tissue injury, and regeneration. METHODS: Clinical phenotype and histologic inflammation were assessed in C57BL/6 (wild-type), Tl1a(-/-) and Dr3(-/-) mice in dextran sulfate sodium (DSS)-induced colitis. We generated mice with an IEC-specific deletion of DR3 (Dr3(Delta IEC)) and assessed intestinal inflammation and epithelial barrier repair. In vivo intestinal permeability was assessed by fluorescein isothiocyanate dextran uptake. Proliferation of IECs was analyzed by bromodeoxyuridine incorporation. Expression of DR3 messenger RNA was assessed by fluorescent in situ hybridization. Small intestinal organoids were used to determine ex vivo regenerative potential. RESULTS: Dr3(-/-) mice developed more severe colonic inflammation than wild-type mice in DSS-induced colitis with significantly impaired IEC regeneration. Homeostatic proliferation of IECs was increased in Dr3(-/-) mice, but blunted during regeneration. Cellular localization and expression of the tight junction proteins Claudin-1 and zonula occludens-1 were altered, leading to increased homeostatic intestinal permeability. Dr3DIEC mice recapitulated the phenotype observed in Dr3(-/-) mice with increased intestinal permeability and IEC proliferation under homeostatic conditions and impaired tissue repair and increased bacterial translocation during DSSinduced colitis. Impaired regenerative potential and altered zonula occludens-1 localization also were observed in Dr3(Delta IEC) enteroids. CONCLUSIONS: Our findings establish a novel function of DR3 in IEC homeostasis and postinjury regeneration independent of its established role in innate lymphoid cells and T-helper cells.

Dectin-1 recognizes β-glucan in fungal cell walls, and activation of Dectin-1 in dendritic cells (DCs) influences immune responses against fungi. Although many studies have shown that DCs activated via Dectin-1 induce different subsets of T helper cells according to different cytokine milieus, the mechanisms underlying such differences remain unknown. By harnessing polymorphic Candida albicans and polystyrene beads of different sizes, we find that target size influences production of cytokines that control differentiation of T helper cell subsets. Hyphal C. albicans and large beads activate DCs but cannot be phagocytosed due to their sizes, which prolongs the duration of Dectin-1 signaling. Transcriptomic analysis reveals that expression of Il33 is significantly increased by larger targets, and increased IL-33 expression promotes TH9 responses. Expression of IL-33 is regulated by the Dectin-1-SYK-PLCγ-CARD9-ERK pathway. Altogether, our study demonstrates that size of fungi can be a determining factor in how DCs induce context-appropriate adaptive immune responses.

The intestinal immune system and microbiota are emerging as important contributors to the development of metabolic syndrome, but the role of intestinal dendritic cells (DCs) in this context is incompletely understood. BATF3 is a transcription factor essential in the development of mucosal conventional DCs type 1 (cDC1). We show that Batf3-/- mice developed metabolic syndrome and have altered localization of tight junction proteins in intestinal epithelial cells leading to increased intestinal permeability. Treatment with the glycolysis inhibitor 2-deoxy-D-glucose reduced intestinal inflammation and restored barrier function in obese Batf3-/- mice. High-fat diet further enhanced the metabolic phenotype and susceptibility to dextran sulfate sodium colitis in Batf3-/- mice. Antibiotic treatment of Batf3-/- mice prevented metabolic syndrome and impaired intestinal barrier function. Batf3-/- mice have altered IgA-coating of fecal bacteria and displayed microbial dysbiosis marked by decreased obesity protective Akkermansia muciniphila, and Bifidobacterium. Thus, BATF3 protects against metabolic syndrome and preserves intestinal epithelial barrier by maintaining beneficial microbiota.

The maintenance of intestinal homeostasis depends on a complex interaction between microbiota, intestinal epithelial barrier and immune system. Alteration in of one of these components could lead to the development of chronic inflammatory diseases such as inflammatory bowel diseases (IBD). Recent studies have associated obesity with more severe IBD. In obesity, pro-inflammatory macrophages accumulate in adipose tissue and their numbers correlate with inflammation and insulin resistance. BATF3 is a transcription factor implicated in the development of conventional dendritic cells type 1 (cDC1). Here, we show that aged Batf3−/− mice developed obesity as characterized by increased body weight, fasting glucose levels (Batf3−/−: 213, WT: 115 mg/dl, p < 0.005), size of abdominal white adipocyte, and development of hepatosteatosis. We also observed increased intestinal permeability in Batf3−/− mice (FITC-dextran gavage; p < 0.01) suggesting impaired epithelial barrier function. Feeding high-fat diet for 2 months further enhanced the metabolic phenotype in Batf3−/− mice, leading to increased fasting glucose levels (300 vs. 246 mg/dl, p < 0.05), and increased adipocyte size. Moreover, during high-fat diet Batf3−/− mice were more susceptible to dextran sulfate sodium (DSS) colitis with more severe cecal inflammation, increased gut and mesenteric fat M1 macrophages (p < 0.05). Our data suggest that deficiency of the transcription factor BATF3 and lack of cDC1 alters the intestinal epithelial barrier, promotes the development of obesity, and increases susceptibility to DSS. In conclusion, BATF3-dependent cDC1 play an important role in maintaining the intestinal epithelial barrier and are protective for the development of obesity.

T helper 9 (TH9) cells are important for the development of inflammatory and allergic diseases. The TH9 transcriptional network converges signals from cytokines and antigen presentation but is incompletely understood. Here, we identified TL1A, a member of the TNF superfamily, as a strong inducer of mouse and human TH9 differentiation. Mechanistically, TL1A induced the expression of the transcription factors BATF and BATF3 and facilitated their binding to the Il9 promoter leading to enhanced secretion of IL-9. BATF- and BATF3-deficiencies impaired IL-9 secretion under TH9 and TH9-TL1A-polarizing conditions. In vivo, using a T-cell transfer model, we demonstrated that TL1A promoted IL-9-dependent, TH9 cell-induced intestinal and lung inflammation. Neutralizing IL-9 antibodies attenuated TL1A-driven mucosal inflammation. Batf3−/− TH9-TL1A cells induced reduced inflammation and cytokine expression in vivo compared to WT cells. Our results demonstrate that TL1A promotes TH9 cell differentiation and function and define a role for BATF3 in T-cell-driven mucosal inflammation.

Hemidesmosome integrity protects the colon against colitis and colorectal cancer Adèle De Arcangelis, Hussein Hamade, Fabien Alpy, Sylvain Normand, Emilie Bruyère, Olivier Lefebvre, Agnès Méchine-Neuville, Stéphanie Siebert, Véronique Pfister, Patricia Lepage, Patrice Laquerriere, Doulaye Dembele, Anne Delanoye-Crespin, Sophie Rodius, Sylvie Robine, Michèle Kedinger, Isabelle Van Seuningen, Patricia Simon-Assmann, Mathias Chamaillard, Michel Labouesse, Elisabeth Georges-Labouesse

Objective Epidemiological and clinical data indicate that patients suffering from IBD with long-standing colitis display a higher risk to develop colorectal high-grade dysplasia. Whereas carcinoma invasion and metastasis rely on basement membrane (BM) disruption, experimental evidence is lacking regarding the potential contribution of epithelial cell/BM anchorage on inflammation onset and subsequent neoplastic transformation of inflammatory lesions. Herein, we analyse the role of the α6β4 integrin receptor found in hemidesmosomes that attach intestinal epithelial cells (IECs) to the laminin-containing BM. Design We developed new mouse models inducing IEC-specific ablation of α6 integrin either during development (α6ΔIEC) or in adults (α6ΔIEC-TAM). Results Strikingly, all α6ΔIEC mutant mice spontaneously developed long-standing colitis, which degenerated overtime into infiltrating adenocarcinoma. The sequence of events leading to disease onset entails hemidesmosome disruption, BM detachment, IL-18 overproduction by IECs, hyperplasia and enhanced intestinal permeability. Likewise, IEC-specific ablation of α6 integrin induced in adult mice (α6ΔIEC-TAM) resulted in fully penetrant colitis and tumour progression. Whereas broad-spectrum antibiotic treatment lowered tissue pathology and IL-1β secretion from infiltrating myeloid cells, it failed to reduce Th1 and Th17 response. Interestingly, while the initial intestinal inflammation occurred independently of the adaptive immune system, tumourigenesis required B and T lymphocyte activation. Conclusions We provide for the first time evidence that loss of IECs/BM interactions triggered by hemidesmosome disruption initiates the development of inflammatory lesions that progress into high-grade dysplasia and carcinoma. Colorectal neoplasia in our mouse models resemble that seen in patients with IBD, making them highly attractive for discovering more efficient therapies.