IBD is characterized by inflammation involving a complex interplay between innate and adaptive immunity, non-immune cells, and intestinal microbes. To understand the contributions of innate immunity to inflammation during colitis we have developed a chronic model of innate immune-mediated colitis wherein RAG1-/- mice are crossed with mice expressing TNFAIP3 in intestinal epithelial cells (villin-TNFAIP3 mice). These villin-TNFAIP3 x RAG1-/- (TRAG) mice develop early onset, 100% penetrant, chronic colitis driven by gut microbes. To understand the inflammatory signals that lead to innate colitis in this model, we crossed TRAG mice with TNFα-/- mice (FRAG mice) and compared the severity of colitis in TRAG vs. FRAG mice. In addition, we characterized TNFα-producing myeloid populations from the lamina propria of TRAG mice. Finally, we assessed the expression and localization of proteins involved in cell death pathways, to unravel the mechanisms driving innate-immune mediated colitis. Our results show that, compared to RAG1-/- mice, TRAG mice have significantly increased numbers of lamina propria leukocytes, including increased numbers of neutrophils, inflammatory monocytes, macrophages, dendritic cells, and innate lymphoid cells. TRAG mice lacking TNFα (FRAG mice) had significantly lower numbers of all these leukocyte subsets. Consistent with this, FRAG mice had significantly lower histological colitis scores, compared to TRAG mice. Inflammatory monocytes, monocytes, macrophages, and neutrophils were found to be the main sources of TNFα production, while TNFα production was null or minimal in dendritic cells, natural killer cells, ILC1, ILC3 and IC2. Macrophages and inflammatory monocytes in TRAG mice were characterized by the expression of iNOS, whereas macrophages and monocytes of FRAG and RAG1-/- mice were notably marked by Arginase1 expression. Localization of active caspase3, phosphor-MLKL and TUNEL positive cells indicated extensive epithelial crypt cell death in TRAG mice that was markedly suppressed in FRAG mice. Together our results show that TNFα drives innate immune mediated colitis, potentially through induction of IEC necroptosis. TNFAIP3 is well known as a negative regulator of receptor-mediated NFκB and MAPK activation and therefore attempts to increase TNFAIP3 expression are considered promising avenues for prevention of inflammation. The present study, indicating that intestinal epithelial cell expression of TNFAIP3 drives TNFα-induced IEC death and innate colitis, highlights potential tissue-specific roles for TNFAIP3 that should be considered in the paradigm of TNFAIP3 as a target for prevention of inflammation.
We have developed an innate immune model of colitis that occurs in the absence of adaptive immunity, is early-onset, 100% penetrant, chronic, and dependent on microbes. villin-TNFAIP3 transgenic mice, crossed to RAG1-/- mice (aka TRAG mice) develop innate immune-mediated colitis that is not observed in villin-TNFAIP3 or RAG1-/- littermates. Antibiotic treatment prevents colitis in TRAG mice. Thus, we sought to investigate the microbes that drive chronic innate immune colitis in the absence of adaptive immunity. To examine the spatial organization of bacteria, mucus and intestinal epithelium, bacteria and mucus were localized in Methyl-Carnoys-fixed sections of WT, villin-TNFAIP3, RAG1-/- and TRAG colons and mucus layers were collected by laser capture microdissection (LCM) for evaluation of bacterial populations. To test antibiotic effectiveness in colitis prevention, TRAG mice were treated with single antibiotic solutions of ampicillin, neomycin, metronidazole, or vancomycin, or with all antibiotics combined in a cocktail, and colitis was scored along the proximal-distal axis of the colon. Microbial populations along the proximal-distal axis of the gut were compared between RAG1-/- and TRAG mice by 16S sequencing. Microbial invasion of the mucus layer was abundant in villin-TNFAIP3 and in TRAG mice, but not WT or RAG1-/- mice. LCM indicated that a subset of microbes, of the classes Gammaproteobacteria and Actinobacteria, were over-represented in the mucus layer of TRAG mice. These included increased abundance of genera Serratia, Acinetobacter, Pseudomonas and Corynebacterium and decreased Bifidobacterium. Differences in antibiotic effectiveness were observed along the proximal-distal axis of the colon, with neomycin being most effective in prevention of inflammation in the cecum, while ampicillin was most effective in the distal colon. The proximal-distal communities of microbes in RAG1-/- mice were distinct and tightly grouped within the cecum, proximal colon, and distal colon, whereas this structure was lost in TRAG mice. In the RAG1-/- gut, the relative abundance along the proximal-distal axis decreased for Firmicutes (mainly Lachnospiraceae and Ruminococcacaea) and Proteobacteria (mainly Helicobacteriaceae and Desulfovibrionbacteriaceae) but increased for Actinobacteria (mainly Bifidobacteriaceae) and Bacteroidetes (mainly Bacteroidales). Unlike RAG1-/- mice, in TRAG mice the abundance of Bacteroidetes decreased along the proximal-distal gut axis. TRAG mice had higher abundance of Bifidobacterium animalis, Bacteroidetes acidifaciens and Lactobacilli and lower abundance of Lachnospiraceae, along the proximal-distal axis of the gut, compared to RAG1-/-mice. The cross-sectional and proximal-distal biogeography of microbes in RAG1-/- mice is disrupted in TRAG mice, which may contribute to the innate colitis observed in this model.
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