Gut Microbiota Modulate CD8 T Cell Responses to Influence Colitis-Associated Tumorigenesis

Abstract

The gut microbiome plays important physiological roles, including aiding in digestion of non-digestible starches and fibers, production of nutrients, protection against pathogens, as well as inducing mucosal immune system development. Disturbances to the composition of the gut microbiome are associated with intestinal disease including inflammatory bowel disease (IBD) and colorectal cancer (CRC), where CRC is the third most common cancer in both men and women in the United States. There is increasing evidence that CRC patients have altered microbiomes compared to healthy controls; specific bacteria known to be increased in CRC patients include E. coli with the pks pathogenicity island, enterotoxigenic B. fragilis and F. nucleatum. Additionally, specific microbiota have been shown to modulate host immunity, but the mechanisms by which the gut microbiota can contribute to colon carcinogenesis remain to be fully elucidated. Our lab is interested in studying the interplay between the gut microbiome and CRC development. Using the azoxymethane (AOM)/dextran sulfate sodium (DSS) mouse model of colitis-associated colorectal cancer (CAC), our lab discovered two colonies of specific pathogen free (SPF) C57BL/6J wild type (WT) mice housed in the same mouse room that develop differential tumor burdens. Mice from the “WT1” colony developed five tumors on average while mice from the “WT2” colony developed 15 tumors on average. The increased tumor susceptibility in WT2 mice can be directly attributed to the gut microbiome as germ-free (GF) WT mice, where GF mice are sterile and have no microbiota, colonized with WT2 bacteria developed more tumors compared to that of GF WT mice colonized with WT1 bacteria. Additionally, 16S rRNA gene sequencing of fecal bacteria from WT1 and WT2 mice revealed distinct microbiomes with certain bacteria consistently associated with high or low tumor numbers. As the microbiome can promote altered immunity, we further examined SPF WT1 and WT2 immune compositions. Immunologically, naïve and acutely-inflamed (day 12 of AOM/DSS) WT2 mice have increased colon lamina propria (LP) CD8+ IFNγ+ T cells compared to WT1 mice as measured by flow cytometry. Bone marrow-derived dendritic cells (BMDCs) stimulated with WT2 bacteria produced increased IL-12, a cytokine that induces increased IFNγ activity by CD8 T cells, compared to BMDCs stimulated with WT1 bacteria. Additionally, introduction of WT2 microbiota promoted an increase in colon LP CD8+ IFNγ+ T cells in GF WT and microbiome-depleted SPF WT1 mice. However, in tumor-bearing WT2 mice, there was decreased tumor-infiltrating CD8+ T cells with reduced IFNγ production, where these cells are increasingly exhausted. GF Rag1-/- mice as well as SPF CD8-/- mice inoculated with WT2 gut microbiota developed fewer tumors than SPF WT2 mice, suggesting that the gut microbiome of WT2 mice increases tumor susceptibility, in part, through an effect on CD8 T cells. Altogether, our data reveals a potential novel role of microbiota in altering colon CD8 T cell function that ultimately impacts colon cancer risk.