PROJECT SUMMARY/ABSTRACT The mucosal immune system fosters a delicate regulatory environment which provides protection from gastric pathogens while maintaining oral tolerance to food. Tolerance to food antigens occurs through the induction of peripheral regulatory T cells (pTreg) and their regulatory suppressive functions. Moreover, dendritic cells (DCs) play a central role in maintaining tolerance to dietary antigens. However, enteric virus infections, like reovirus, can elicit pathological processes leading to the initiation of T helper 1 (Th1) immunity against dietary gluten resulting in loss of oral tolerance (LOT) and celiac disease (CeD). CeD is a digestive disorder characterized by an inflammatory CD4 T helper 1 (Th1) immune response to dietary gluten found in wheat, barley, and rye. Even though 30% of people carry the genetic alleles predisposing to CeD, only 1% develop disease, suggesting that additional genetic and environmental factors such as enteric viral infections contribute to pathogenesis. The high prevalence of CeD and the challenges associated with maintaining a gluten-free diet make CeD a significant burden in modern health care. New strategies to promote oral tolerance and to prevent or revert LOT in CeD patients upon exposure to gluten antigens are still needed. We hypothesized that certain gut microbes have the capacity to protect against virus-mediated LOT and development of CeD. By using our previously defined virus mediated LOT CeD model, we discovered that the gut colonizing protist Tritrichomonas arnold (T. arnold) promotes oral tolerance and protects against virus-mediated LOT. Moreover, our studies using human stool samples support a role for Tritrichomonas spp. colonization in protecting against development of CeD. Mechanistically, our findings show that T. arnold promotes oral tolerance by engaging dietary antigen presenting DCs and restraining the reovirus induced proinflammatory program of dietary antigen presenting DCs and thus limit their ability to promote Th1 immune responses. In this proposal we will use conceptually and technically innovative approaches to further gain insights in how T. arnold-derived compounds modulate dietary antigen presenting DC responses under homeostatic conditions and inflammatory microbial triggers. Furthermore, using our LOT CeD mouse model we will interrogate a druggable candidate pathway identified in our T. arnold DC-RNA-sequencing screen that has been associated with tolerogenic immune responses. The findings of this proposal will provide insights into novel approaches for preventing LOT in at-risk individuals and re-establishing oral tolerance to gluten in CeD patients.