INNATE AN ADAPTIVE IMMUNITY TO MICROBIAL FLAGELLINS IN IBD

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Innate and Adaptive Immunity to Microbial Flagellins in IBD. Abnormalities of the host immune response to the intestinal microbiota is the basis of experimental IBD, and this is likely true in human IBD as well. We have cloned and sequenced a set of microbial proteins that serve as immunodominant antigens in experimental colitis. The largest group of these were previously unknown flagellins of the anaerobic commensal flora, which were recognized immunologically in multiple mouse models of IBD and in half of patients with Crohn's disease. Use of flagellins as probes has revealed an apparent deficiency in innate immunity in colitis-prone C3H/HeJBir mice (C3H), a response regulated by a C3H colitis-susceptibility gene locus on Chr 3 termed Cdcsl. Paradoxically this deficient innate response in C3H mice results in a increased T cell response to flagellins in vivo. Aim 1 will ask whether and how the dendritic cell and macrophage innate immune response to the microbiota is deficient in colitis-prone C3H compared to colitis-resistant C57BI/6 (B6) mice, and in C3H and B6 mice that are congenic for the Cdcs1 locus. Bacterial flagellins and other TLR ligands will be used as probes first, followed by challenge with gram-positive and gram-negative intestinal bacteria isolated from mice. The ability of C3H vs B6 dendritic cells to present microbial antigens to CD4 T cells will be assessed to test the hypothesis that the deficient acute C3H dendritic cell response to bacteria prolongs antigen presentation to T cells, thus resulting in a greater T cell responses in C3H mice. Further, we will ask whether flagellins can serve as adjuvants via Toll like receptor 5 (TLR5) for themselves or for other microbial antigens. Aim 2 will address for the first time whether there is a defined, repetitive, non-random pattern of spreading of the adaptive immune response to immunodominant enteric microbial antigens in IBD. Based on our preliminary data, the hypothesis to be tested is that a repetitive, hierarchical pattern of epitope spreading does occur in experimental IBD, similar to what has been observed in autoimmune diseases. We will determine if such epitope spreading is associated with disease progression, whether it requires TLR5, whether it is dependent on lnterleukin-23, and whether immunization with antigens recognized early in the spreading cascade can prevent reactivity to antigens recognized later and thus ameliorate intestinal inflammation. Aim 3 will define where, when, and how T cells become sensitized to microbial antigens in the colitis-prone host using two novel mouse lines, an interferon gamma-Thy1.1 reporter mouse and a TCR transgenic mouse reactive to CBiM bacterial flagellin. These studies will provide important new understanding of the mechanisms by which the host immune system responds to the microbiota and how an abnormal immune response to the microbiota results in chronic intestinal inflammation. Innate and adaptive response of Crohn's patients to these antigens will be assessed.