Project Summary Our intestines comprise over 50% of our bodies immune cells and tightly maintains a constant state of homeostasis against foreign stimulus like food particles and bacteria. This immune “tolerance” is broken during chronic intestinal inflammation as seen during inflammatory bowel diseases (IBD). Adverse CD4+ THelper responses perpetuate the chronic pathology observed in IBD, thus understanding the molecular mechanisms that control their function is central to endeavors of finding novel therapeutic treatments. A physiologic adaptive response to inflammation is a marked shift in the supply and demand of metabolites that results in limited oxygen availability (now termed inflammatory hypoxia) and activation of the transcription factors hypoxia- inducible-factors (HIFs). While there are multiple affects of hypoxia on T cell gene function, the effects on post- transcriptional regulation are underexplored. To investigate hypoxia-elicited tissue protective signaling we performed a screen of CD4+ T cell-specific miRNAs and identified a selective induction of miR-29a. Studies with genetic models identified a role of Hif-2α in miR-29a induction and subsequent experiments demonstrated repression of the miR-29a target-genes Tbet and IFNγ (canonical TH1 markers) during hypoxia. Mice with a T- cell-intrinsic deficiency in Hif-2α displayed elevated Tbet levels in CD4+ T cells and exacerbated inflammation during experimental colitis. Based on these preliminary studies, we hypothesize that Hif-2α induction of miR- 29a suppresses TH1 CD4+ cell activation. In this proposal we will address this hypothesis with three integrated lines of investigation. Firstly, we will examine how Hif-2α transcriptionally induces miR-29a and the functional requirements for Hif-2α in CD4+ THelper differentiation. Second, we will assess the role of miR-29a in the regulation of T cell mediated colitis. Lastly, in proof of concept studies we will target miR-29a stabilization in vivo during experimental colitis. Collectively these studies aim to dissect the Hif-2α-miR-29a-Tbet axis in the regulation of CD4+ T cell-mediated intestinal inflammation.! !