There are limited effective treatments for autoimmune and inflammatory diseases in the GI tract like Inflammatory Bowel Disease. The treatments that are available can have toxic side-effects and part of the barrier to developing better alternatives is that mucosal immunity remains incompletely understood. During disease, there is a breakdown in the body's natural tolerance mechanisms and that allows inflammation to progress unopposed. There is a growing body of evidence that supports a role for regulatory T cells (Tregs) in suppressing these deleterious immune responses but these cells are not fully understood and the cellular dynamics have not been explored. In this application, we propose to use functional immunoimaging to study Tregs as they modulate inflammation in the intestine and explore the role of biotin and calcium in this process. There are several additional approaches to define phenotype and functional status of the tissue and cells being studied which will supplement the imaging findings. Notably, there are no published reports on studying Tregs in this manner during colitis. The experiments have been designed to use two mouse models for colitis, an adoptive-transfer model and a spontaneous model that develops with biotin deficiency. The experiments have also been developed in such a way as to facilitate translation to the clinic. We will use two-photon microscopy which allows for real-time imaging of lymphocytes interacting in live tissue to define the cellular immunoregulatory events that explain how Tregs function to suppress colitis. In the first aim of the proposal, our studies are focused on the mesenteric lymph node to look at induction and progression of immune responses hypothesizing that Tregs physically displace pathogenic T cells from Dendritic Cells. We will also perform experiments using an S1P1 agonist which is a pharmacological agent believed to prevents effector T cells from leaving the lymph node and which may work synergistically with Tregs to prevent pathogenic T cells from reaching the colon. A similar agent has already shown promising results in clinical trials for Ulcerative Colitis. In the second aim of the proposal, our studies focus on the intestinal wall where inflammation and tissue destruction create the disease symptoms. Again, we will be focused on the role of Tregs in this process. We have seen that biotin deficient mice develop spontaneous disease that completely resolves with biotin replacement which directly links biotin to intestinal inflammation. If biotin supplementation can be shown to be effective, it would represent a low cost treatment without side-effects. Finally, the third aim focuses on how Tregs modulate calcium signaling in effector T cells in both the mesenteric lymph node and the colon. Calcium signaling is necessary for T cells to develop an immunological synapse with Dendritic Cells for activation and pathogenesis. We believe that Tregs hijack this process to induce the release of effector T cells from Dendritic Cells preventing disease. Additionally, we will study the effects of a novel therapeutic agent called an Orai1 channel blocker. Orai1 has been identified as the calcium channel in this system and we believe its blockade will also be effective in modulating immune responses in the GI tract. Furthermore, Orai1 blockade seems to have no effect of Treg function and we will examine if it can be used to augment Treg-mediated suppression of inflammation in the mesenteric lymph node and the colon. We anticipate that these three independent, but interrelated aims will enhance our understanding of the mechanism of Tregs and how they affect inflammation in the GI tract. Furthermore, we hope that the studies on the pharmacologic agents mentioned will advance their implementation for clinical use and benefit the Veterans we serve.
Inflammatory conditions in the GI tract like Inflammatory Bowel Disease (Crohn's Disease and Ulcerative Colitis) represent a burden to our Veterans and our healthcare system because these are chronic conditions which dramatically affect quality of life and are expensive to treat. In general, these diseases arise when the immune system malfunctions and cannot distinguish between infectious agents and normal tissue. Approximately 1 in 2,000 people in the US will develop disease in any given year and studies show more than a 2-fold increase in the number of Veterans with these problems since 1998. The direct medical cost is estimated to be over $18,000 per person per year and is likely to keep increasing. In this proposal, we will be studying three potential treatments as well as the underlying immune process that leads to disease. Our hope is that with a better understanding of the disease, we will be able to develop more effective and safer treatments.
