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Inflammatory bowel disease (IBD) is associated with risk variants in the human genome and dysbiosis of the gut microbiome, though unifying principles for these findings remain largely undescribed. The human commensal Bacteroides fragilis delivers immunomodulatory molecules to immune cells via secretion of outer membrane vesicles (OMVs). We reveal that OMVs require IBD-associated genes, ATG16L1 and NOD2, to activate a noncanonical autophagy pathway during protection from colitis. ATG16L1-deficient dendritic cells do not induce regulatory T cells (Tregs) to suppress mucosal inflammation. Immune cells from human subjects with a major risk variant in ATG16L1 are defective in Treg responses to OMVs. We propose that polymorphisms in susceptibility genes promote disease through defects in \"sensing\" protective signals from the microbiome, defining a potentially critical gene-environment etiology for IBD.

Abstract Background Intestinal fibrosis is a serious complication of Crohn’s disease. Numerous cell types including intestinal epithelial and mesenchymal cells are implicated in this process, yet studies are hampered by the lack of personalized in vitro models. Human intestinal organoids (HIOs) derived from induced pluripotent stem cells (iPSCs) contain these cell types, and our goal was to determine the feasibility of utilizing these to develop a personalized intestinal fibrosis model. Methods iPSCs from 2 control individuals and 2 very early onset inflammatory bowel disease patients with stricturing complications were obtained and directed to form HIOs. Purified populations of epithelial and mesenchymal cells were derived from HIOs, and both types were treated with the profibrogenic cytokine transforming growth factor β (TGFβ). Quantitative polymerase chain reaction and RNA sequencing analysis were used to assay their responses. Results In iPSC-derived mesenchymal cells, there was a significant increase in the expression of profibrotic genes (Col1a1, Col5a1, and TIMP1) in response to TGFβ. RNA sequencing analysis identified further profibrotic genes and demonstrated differential responses to this cytokine in each of the 4 lines. Increases in profibrotic gene expression (Col1a1, FN, TIMP1) along with genes associated with epithelial-mesenchymal transition (vimentin and N-cadherin) were observed in TGFβ -treated epithelial cells. Conclusions We demonstrate the feasibility of utilizing iPSC-HIO technology to model intestinal fibrotic responses in vitro. This now permits the generation of near unlimited quantities of patient-specific cells that could be used to reveal cell- and environmental-specific mechanisms underpinning intestinal fibrosis. Lay Summary Intestinal fibrosis is a serious complication of Crohn’s disease and novel in vitro models are urgently needed to study this. We describe an induced pluripotent stem cell–derived modeling system whereby a near unlimited number of both epithelial and mesenchymal cells could be used in a personalized intestinal fibrosis model. Graphical Abstract

TL1A is a member of the TNF superfamily and its expression is increased in the mucosa of inflammatory bowel disease patients. Moreover, a subset of Crohn's disease (CD) patients with the risk TL1A haplotype is associated with elevated TL1A expression and a more severe disease course. To investigate the in vivo role of elevated TL1A expression, we generated two transgenic (Tg) murine models with constitutive Tl1a expression in either lymphoid or myeloid cells. Compared to wildtype (WT) mice, constitutive expression of Tl1a in either lymphoid or myeloid cells showed mild patchy inflammation in the small intestine, which was more prominent in the ileum. In addition, mice with constitutive Tl1a expression exhibited enhanced intestinal and colonic fibrosis compared to WT littermates. The percentage of T cells expressing the gut homing chemokine receptors CCR9 and CCR10 was higher in the Tl1a Tg mice compared to WT littermates. Sustained expression of Tl1A in T cells also lead to increased Foxp3+ Treg cells. T cells or antigen presenting cells (APC) with constitutive expression of Tl1a were found to have a more activated phenotype and mucosal mononuclear cells exhibit enhanced Th1 cytokine activity. These results indicated an important role of TL1A in mucosal T cells and APC function and showed that up-regulation of TL1A expression can promote mucosal inflammation and gut fibrosis.

Andrographis paniculata has in vitro inhibitory activity against TNF-α, IL-1β and NF-κB. A pilot study of A. paniculata extract (HMPL-004) suggested similar efficacy to mesalamine for ulcerative colitis. A randomized, double-blind, placebo-controlled trial evaluated the efficacy of A. paniculata extract (HMPL-004) in 224 adults with mild-to-moderate ulcerative colitis. Patients were randomized to A. paniculata extract (HMPL-004) 1,200 mg or 1,800 mg daily or placebo for 8 weeks. In total, 45 and 60% of patients receiving A. paniculata 1,200 mg and 1,800 mg daily, respectively, were in clinical response at week 8, compared with 40% of those who received placebo (P=0.5924 for 1,200 mg vs. placebo and P=0.0183 for 1,800 mg vs. placebo). In all, 34 and 38% of patients receiving A. paniculata 1,200 mg and 1,800 mg daily, respectively, were in clinical remission at week 8, compared with 25% of those who received placebo (P=0.2582 for 1,200 mg vs. placebo and P=0.1011 for 1,800 mg vs. placebo). Adverse events developed in 60 and 53% of patients in the A. paniculata 1,200 mg and 1,800 mg daily groups, respectively, and 60% in the placebo group. Patients with mildly to moderately active ulcerative colitis treated with A. paniculata extract (HMPL-004) at a dose of 1,800 mg daily were more likely to achieve clinical response than those receiving placebo.

Background and Aims. Recent evidences reveal that epithelial to mesenchymal transition (EMT) exacerbates the process of intestinal fibrosis. Tumor necrosis factor-like ligand 1A (TL1A) is a member of the tumor necrosis family (TNF), which can take part in the development of colonic inflammation and fibrosis by regulating immune response or inflammatory factors. The purpose of this study was to elucidate the possible contribution of TL1A in onset and progression of intestinal inflammation and fibrosis through EMT. Methods. Colonic specimens were obtained from patients with inflammatory bowel disease (IBD) and control individuals. The expression levels of TL1A and EMT-related markers in intestinal tissues were evaluated. Furthermore, the human colorectal adenocarcinoma cell line, HT-29, was stimulated with TL1A, anti-TL1A antibody, or BMP-7 to assess EMT process. In addition, transgenic mice expressing high levels of TL1A in lymphoid cells were used to further investigate the mechanism of TL1A in intestinal fibrosis. Results. High levels of TL1A expression were detected in the intestinal specimens of patients with ulcerative colitis and Crohn’s disease and were negatively associated with the expression of an epithelial marker (E-cadherin), while it was positively associated with the expression of interstitial markers (FSP1 and α-SMA). Transgenic mice with high expression of TL1A were more sensitive to dextran sodium sulfate and exhibited severe intestinal inflammation and fibrosis. Additionally, the TGF-β1/Smad3 pathway may be involved in TL1A-induced EMT, and the expression of IL-13 and EMT-related transcriptional molecules (e.g., ZEB1 and Snail1) was increased in the intestinal specimens of the transgenic mice. Furthermore, TL1A-induced EMT can be influenced by anti-TL1A antibody or BMP-7 in vitro. Conclusions. TL1A participates in the formation and process of EMT in intestinal fibrosis. This new knowledge enables us to better understand the pathogenesis of intestinal fibrosis and identify new therapeutic targets for its treatment.

We present a genome-wide association study of ileal Crohn disease and two independent replication studies that identify several new regions of association to Crohn disease. Specifically, in addition to the previously established CARD15 and IL23R associations, we identified strong and significantly replicated associations (combined P < 10 −10 ) with an intergenic region on 10q21.1 and a coding variant in ATG16L1 , the latter of which was also recently reported by another group. We also report strong associations with independent replication to variation in the genomic regions encoding PHOX2B , NCF4 and a predicted gene on 16q24.1 ( FAM92B ). Finally, we demonstrate that ATG16L1 is expressed in intestinal epithelial cell lines and that functional knockdown of this gene abrogates autophagy of Salmonella typhimurium . Together, these findings suggest that autophagy and host cell responses to intracellular microbes are involved in the pathogenesis of Crohn disease.

Chronic intestinal inflammation, as seen in inflammatory bowel disease (IBD), results from an aberrant and poorly understood mucosal immune response to the microbiota of the gastrointestinal tract in genetically susceptible individuals. Here we used serological expression cloning to identify commensal bacterial proteins that could contribute to the pathogenesis of IBD. The dominant antigens identified were flagellins, molecules known to activate innate immunity via Toll-like receptor 5 (TLR5), and critical targets of the acquired immune system in host defense. Multiple strains of colitic mice had elevated serum anti-flagellin IgG2a responses and Th1 T cell responses to flagellin. In addition, flagellin-specific CD4(+) T cells induced severe colitis when adoptively transferred into naive SCID mice. Serum IgG to these flagellins, but not to the dissimilar Salmonella muenchen flagellin, was elevated in patients with Crohn disease, but not in patients with ulcerative colitis or in controls. These results identify flagellins as a class of immunodominant antigens that stimulate pathogenic intestinal immune reactions in genetically diverse hosts and suggest new avenues for the diagnosis and antigen-directed therapy of patients with IBD.

Ribonuclease T2 gene (RNASET2) variants are associated in genome wide association studies (GWAS) with risk for several autoimmune diseases, including Crohn’s disease (CD). In T cells, a functional and biological relationship exists between TNFSF15-mediated enhancement of IFN−γ production, mucosal inflammation and RNASET2. Disease risk variants are associated with decreased mRNA expression and clinical characteristics of severe CD; however, functional classifications of variants and underlying molecular mechanisms contributing to pathogenesis remain largely unknown. In this study we demonstrate that allelic imbalance of RNASET2 disease risk variant rs2149092 is associated with transcriptional and post-transcriptional mechanisms regulating transcription factor binding, promoter-transactivation and allele-specific expression. RNASET2 mRNA expression decreases in response to multiple modes of T cell activation and recovers following elimination of activator. In CD patients with severe disease necessitating surgical intervention, preoperative circulating RNASET2 protein levels were decreased compared to non-IBD subjects and rebounded post-operatively following removal of the inflamed region, with levels associated with allelic carriage. Furthermore, overexpression or treatment with recombinant RNASET2 significantly reduced IFN-γ secretion. These findings reveal that RNASET2 cis- and trans-acting variation contributed regulatory complexity and determined expression and provide a basis for linking genetic variation with CD pathobiology. These data may ultimately identify RNASET2 as an effective therapeutic target in a subset of CD patients with severe disease.

The 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 have been implicated in susceptibility and severity of Crohn's disease (CD). Individuals carrying the risk 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. However, 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, mice have a significant increase in large intestinal lamina propria (LP) granzyme B tissue-resident memory CD8 T (T ) cells compared to WT mice, reminiscent of persistent norovirus infection. Deletion of in myeloid ( ) or dendritic cells ( ) leads to a similar increase of LP T . Furthermore, and 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 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 T and consequently to increased susceptibility to colitis by impairing the recognition of enteric viruses by TLR7. In 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 risk variants.

Acute severe ulcerative colitis (UC) remains a significant clinical challenge and the ability to predict, at an early stage, those individuals at risk of colectomy for medically refractory UC (MR-UC) would be a major clinical advance. The aim of this study was to use a genome-wide association study (GWAS) in a well-characterized cohort of UC patients to identify genetic variation that contributes to MR-UC.MethodsA GWAS comparing 324 MR-UC patients with 537 non-MR-UC patients was analyzed using logistic regression and Cox proportional hazards methods. In addition, the MR-UC patients were compared with 2601 healthy controls.ResultsMR-UC was associated with more extensive disease (P = 2.7 × 10−6) and a positive family history of UC (P = 0.004). A risk score based on the combination of 46 single nucleotide polymorphisms (SNPs) associated with MR-UC explained 48% of the variance for colectomy risk in our cohort. Risk scores divided into quarters showed the risk of colectomy to be 0%, 17%, 74%, and 100% in the four groups. Comparison of the MR-UC subjects with healthy controls confirmed the contribution of the major histocompatibility complex to severe UC (peak association: rs17207986, P = 1.4 × 10−16) and provided genome-wide suggestive association at the TNFSF15 (TL1A) locus (peak association: rs11554257, P = 1.4 × 10−6).ConclusionsA SNP-based risk scoring system, identified here by GWAS analyses, may provide a useful adjunct to clinical parameters for predicting the natural history of UC. Furthermore, discovery of genetic processes underlying disease severity may help to identify pathways for novel therapeutic intervention in severe UC. (Inflamm Bowel Dis 2010)