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TL1A overexpression in Crohn’s Disease patients and mice alters Paneth cell biology and intestinal microbiota in promoting ileal inflammation

ABSTRACT: Small intestinal 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) have been associated with susceptibility to and severity of IBD. TL1A expression is increased in IBD patients, particularly in TL1A risk allele carriers. However, the effects of TL1A on the function of ileal Paneth cells and downstream effect on commensal microbiota and development of ileitis has not been investigated. Here, we demonstrate that TL1A overexpression in mice (Tl1a-tg) induces Paneth cell hyperplasia and morphological abnormalities that precedes the development of spontaneous ileitis. In Crohn’s disease (CD) patients, ileal TL1A expression was associated with abnormal Paneth cell phenotypes. Deficiency of the TL1A receptor DR3 in Paneth cells resulted in Paneth cell abnormalities suggesting a direct effect of TL1A on Paneth cells which we confirmed in human CD patient-derived Paneth cell enriched organoid cultures. Commensal microbiota were required for TL1A-mediated Paneth cell dysfunction, and development of spontaneous ileitis. Overexpression of TL1A results in dysbiosis with an enrichment in short chain fatty acid-producing bacteria Bifidobacterium and Allobaculum and metabolomic analysis revealed an increase of the metabolite acetate. Acetate supplementation in germ-free or SPF housed mice caused ileal inflammation and elevated IFN-g secretion, suggesting that acetate is sufficient to cause ileitis. Our findings provide a mechanistic link between overexpression of TL1A in CD patients with TNFSF15 risk variants, Paneth cell dysfunction, intestinal dysbiosis and enrichment of acetate-producing bacteria that promote ileal inflammation.

ORGANISM(S): Mus musculus

PROVIDER: GSE228942 | GEO | 2026/04/01

REPOSITORIES: GEO

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Project description:This UH2/UH3 demonstration project entitled "Effects of Crohn's disease risk alleles on enteric microbiota" is focused on characterizing intestinal associated microbiota in patients with ileal Crohn's disease (ileal CD), ulcerative colitis (UC) and control patients without inflammatory bowel diseases (non-IBD). We hypothesize that genetic factors that affect Paneth cell function, contribute to compositional changes in intestinal microbiota. These changes in microbiota may lead to reduction of protective commensal organisms and increased numbers of aggressive organisms that incite intestinal inflammation. This hypothesis is being tested by high throughput 16S rRNA sequence analysis of de-identified ileal and colonic tissues that have been archived at Washington University St. Louis, University of North Carolina, Mount Sinai Hospital and the Cleveland Clinic. Multivariate analysis of the metagenomic data will be conducted with genotyping metadata (highly reproducible CD risk alleles, including NOD2 and ATG16L1) and phenotyping metadata (e.g. age, gender, race, body mass index, medications and smoking.) Shotgun sequencing will be performed on selected fecal specimens linked to ileal tissues to identify additional, or auxiliary, or synergistic pathogenic factors or other functional changes in the microbiome. Because members of this research team have observed that a chronic viral infection is required for the Paneth cell defect in Atg16l1 hypomorphic mice, a major focus of these studies will be towards identifying potential viral triggers for the defective Paneth cell phenotype in individuals harboring the ATG16L1 risk allele. Novel genetic probes for protective and aggressive organisms will be developed by mining bacterial genome and shotgun sequencing data. Genomic sequences will be produced for candidate protective and aggressive strains (e.g. adherent-invasive strains of E. coli) isolated from human intestinal tissues where there is limited existing genome information. Quantitative qPCR assays using the novel as well as established genetic probes will be conducted to test the hypothesis that an imbalance between protective and aggressive organisms is associated with genetic factors that affect Paneth cell function. Our combined expertise in multiple disciplines across multiple institutions, our demonstrated ability to collect a large number of well-phenotyped samples with longitudinal clinical information that will be linked to host response and morphologic studies, and our consortium's capacity for high-throughput sequencing will be used to investigate how alterations in human microbiome relate to CD risk alleles and CD pathogenesis.

Project description:Background & Aims Variants in the tumor necrosis factor superfamily member 15 gene (TNFSF15, also called TL1A) have been associated with risk for inflammatory bowel diseases (IBD). TL1A affects expression of multiple cytokines to promote mucosal inflammation. Little is known about the TL1A-response pathways that regulate cytokine expression. We investigated T-cell gene expression patterns to determine the mechanisms by which TL1A regulates cytokine production, and whether these associate with outcomes of patients with Crohn’s disease (CD).Methods Peripheral T cells isolated from normal donors were cultured with TL1A. We performed gene expression profile analysis, by RNA sequencing, of subsets of interferon gamma (IFNG)-producing and non-producing cells, purified by flow cytometry. Unsupervised hierarchical clustering analysis was used to identify gene expression differences between these subsets. Ribonuclease T2 gene (RNASET2) expression and methylation were assessed by quantitative trait loci analyses. Clinical characteristics of patients (complications, resistance to therapy, recurrence time) were associated with single nucleotide polymorphisms in RNASET2. We performed motif screening to identify polymorphisms that disrupt transcription factor binding sites. Levels of RNASET2 were knocked down with small interfering RNA in CD4+ T cells and the effect on protein expression was determined by proteomic analysis and cytokine production. Cell aggregation was measured by flow cytometry. ResultsWe identified 764 genes with at least a 2-fold difference in TL1A-mediated expression between IFNG-secreting and non-secreting T cells (P<1 × 10−5). Many of these genes were located near IBD susceptibility variants. RNASET2 was the only IBD risk-associated gene with greater than 5-fold downregulation in the IFNG-secreting subset. RNASET2 disease risk variants were associated with decreased expression in peripheral and mucosal tissues and DNA hypermethylation in CD patients requiring surgical intervention. RNASET2 disease risk variants were associated in CD patients with more complicated disease or resistance to therapy, defined in part by failed response to treatment, increased length of intestinal resection, shorter time to repeat surgery, and high Rutgeerts score (>2) in post-operative endoscopy. The RNASET2 variant rs2149092 was predicted to disrupt a consensus binding site for the transcription factor ETS within an enhancer region. Expression of RNASET2 correlated with expression of ETS. RNASET2 knockdown in T cells increased expression of IFNG and ICAM1 and induced T cells aggregation. A blocking antibody against LFA1, disrupting the LFA1-ICAM1 interaction, reduced T-cell production of IFNG. Conclusions We identified decreased expression of RNASET2 as a component of TL1A-mediated increase in production of IFNG and as a potential biomarker for patients with severe CD. Further study of the role of RNASET2 in regulating mucosal inflammation may lead to development of novel therapeutic targets.

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TL1A overexpression in Crohn’s Disease patients and mice alters Paneth cell biology and intestinal microbiota in promoting ileal inflammation

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