Gut immunopathological signatures in Rag1 hypomorphic mice

Abstract

Inflammatory bowel disease (IBD) has been reported in ~15% of patients with partial RAG deficiency. To investigate the pathophysiology of intestinal disease, we studied the interplay of T-cell immune dysregulation and microbiome abnormalities in a mouse model (Rag1w/w) homozygous fora hypomorphic Rag1 mutation (R972W) whose orthologue variant (R975W) has been reported in patients. Analysis of gut tissue in Rag1w/w mice revealed severe spontaneous colitis with lymphocytic infiltrates, which become histologically evident after weaning. To assess the relative contribution of genetic and environmental factors on the pathogenesis of gut inflammation, we performed flow cytometry and single-cell RNA-seq of lamina propria (LP) T cells and microbiome studies of fecal specimens in Rag1w/w and Rag1+/+ mice. Rag1w/w mice showed skewing towards a Th1/Th17 phenotype in CD4+ cells infiltrating the LP. Analysis of the fecal microbiota composition revealed a severe restriction of microbial diversity in Rag1w/w mice, with progressive depletion of three genera of firmicutes (Lachnospiraceae, Oscillibacter, Intestinimonas) producing butyrate. Principal component analysis of metabolomics data of fecal specimens clustered Rag1w/w separately from Rag1+/+ mice, with a severe defect in the amount of butyrate in the former. Consistent with an important role of butyrate in facilitating priming of IL-10 producing Tr1 cells, Il10 expression was markedly reduced in CD4+ LP T-cells from Rag1w/w compared to Rag1+/+ mice. Furthermore, scRNAseq of LP CD4+ T-cells revealed reduced presence of Tr1 cells in Rag1w/w mice, indicating that microbiome abnormalities contribute to the intestinal immune dysregulation. Treatment of Rag1w/w mice with vedolizumab (aiming at blocking gut homing of pathogenic T cells) had no effect on intestinal inflammation and on restriction of microbiome diversity. By contrast, both use of broad-spectrum antibiotics and bone marrow transplantation (BMT) significantly improved the inflammation and normalized the number of LP CD4+ cells. However, the LP CD4+ T cells of antibiotic-treated mice retained a strong Th1/Th17 signature, whereas only BMT rescued the phenotype, and induced normalization of microbiome diversity and restoration in the relative abundance of firmicutes. These data provide mechanistic insights into the pathophysiology of IBD associated with partial RAG deficiency, and confirm a curative role of bone marrow transplantation.