ALTERED MICROBIAL BIOGEOGRAPHY IN INNATE IMMUNE-MEDIATED COLITIS

Abstract

Abstract 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.