P-165 Colonization of Lymphopenic Mice with Dysbiotic Microflora Markedly Increases the Severity and Incidence of Chronic Colitis Following T Cell Transfer

Abstract

It is well-known that intestinal microbiota are required for the induction of chronic gut inflammation in the CD4+CD45RBhigh→ RAG-1-/- mouse model of chronic colitis. Following our relocation from LSU Health Sciences Center (LSUSHC) to Texas Tech University Health Sciences Center (TTUHSC), we observed a significant reduction in the incidence and severity of disease (30-40%) compared to our previous 15 year history of ∼85% at LSUHSC. The objectives of this study were to: a) ascertain whether differences in the gut microbiota may account for the differences in disease incidence and b) determine whether colonization of RAG-1-/- (RAG ko) mice with feces obtained from colitic mice increases the incidence and severity of colitis in the T cell transfer model. DNA from freshly frozen feces was isolated using standard protocols and 16S rRNA sequencing was performed using the Roche 454 platform. For some studies, mice were colonized (via gastric gavage) with 40 mg donor feces 1 week prior to adoptive transfer of naïve (CD4+CD45RBhigh) T-cells. Microbiome analysis revealed marked shifts in the relative abundance of several major bacterial communities present in feces obtained from LSUHSC versus TTUHSC mice. For example, the relative abundance of Firmicutes and Bacteroidetes was ∼70 and 20%, respectively in healthy TTUHSC-RAG ko mice whereas these same 2 phyla represented 48 and 50%, respectively in healthy LSUHSC-RAG ko animals. Interestingly, the fecal microbiota from TTUHSC-RAG ko mice revealed significantly higher abundances of Proteobacteria, Verrucomicrobia and Tenericutes when compared to the microbiota obtained from healthy LSUHSC-RAG ko mice. Not surprisingly, induction of chronic colitis in LSUHSC-RAG ko mice via adoptive transfer of naïve T cells, produced a marked dysbiosis most notably characterized by an increased abundance of Verrucomicrobia together with corresponding and marked decrease in Bacteroidetes. Adoptive transfer of naive T cells into healthy TTUHSC-RAG ko mice that were first colonized with healthy LSUHSC-RAG ko feces induced mild-to-moderate colitis in ∼80% of the mice at 8 weeks post T cell transfer. In addition, we found that T cell transfer into TTUHSC-RAG ko mice that were first colonized with feces from colitic LSUHSC-RAG ko mice induced robust colitis in >90% of these recipients (called LSUHSCc→TTUHSC mice). Furthermore, T cell transfer into TTUHSC-RAG ko mice that had been colonized with feces from colitic LSUHCc→TTUHSC mice induced moderate-to-severe colitis in virtually all (>98%) of these recipients (called TTUHSCc→TTUHSC mice). Importantly, colonization of healthy wild type mice or TTUHSC-RAG ko mice (in the absence of T cell transfer) with colitic feces from TTUHSCc→TTUHSC mice did not induce disease over the 8 week observation period. We conclude that colonization of healthy RAG ko mice with dysbiotic microbiota obtained from colitic mice markedly increases the severity and incidence of chronic colitis in the T cell transfer model. Our data also suggest that the increase in severity and incidence of disease is not due to the presence of intestinal pathogens and can only be produced in mice with a dysregulated immune system (supported by grants from the DOD-W81XWH-11-1-0666 and NIH- RO1 DK 091269).