Project Summary/Abstract Regulatory T cell (Treg) immunosuppression is critical for maintaining immune tolerance to a diverse array of potential antigens in the intestinal mucosa. In patients with inflammatory bowel disease (IBD), chronic intestinal inflammation overwhelms local Treg function, allowing inflammation to persist. Our previous work and that of others have identified important roles for 17β-estradiol (estrogen, E2) signaling in promoting Treg differentiation and function. E2 signals through two nuclear receptors, alpha and beta (ERα, ERβ), to modulate gene transcription in target cells. Although they share high sequence homology, ERα and ERβ mediate distinct and often opposing functions on gene regulation. In previously published work, we showed that shifting the balance of E2 signaling towards ERα is generally pro-inflammatory, whereas shifting towards ERβ is generally protective. In recent preliminary studies using IBD patient samples, we observed significantly diminished ERβ expression in intestinal biopsy tissues and peripheral T cells from females with active Crohn’s disease (CD). We also found that ERβ-deficient T cells are resistant to ex vivo, TGFβ-dependent Treg differentiation, and that deletion of ERβ in a spontaneous ileitis model (SAMP/YitFC, “SAMP” mice) results in significant impairment of Treg transcriptional and functional responses, contributing to exacerbated inflammation. Therefore, the goal of this project is to determine the molecular and cellular mechanism(s) by which altered E2 signaling impacts Treg differentiation and function, contributing to intestinal inflammation. The mechanisms by which E2 signaling cross-talks with inflammatory signals to influence immune cell function are poorly understood. This proposal seeks to address this knowledge gap through our central hypothesis that dysregulated E2 signaling contributes to Treg transcriptional remodeling and loss-of-function, facilitating sustained intestinal inflammation in IBD. In Aim 1, we propose to delineate the molecular mechanisms by which ERα- and ERβ cross-talk with signaling downstream of TGFβ in primary T cells, influencing TGFβ-dependent Foxp3 expression and function. Aim 2 will determine the functional impact of rebalancing Treg-specific E2 signaling on intestinal inflammation in vivo, testing our hypothesis that augmenting Treg-specific ERβ signaling may prevent and/or rescue intestinal inflammation. Experiments will include adoptive transfer of ERβ-expressing Tregs to SAMP mice, as well as in vivo assays using a T cell-dependent colitis model. In Aim 3, we plan to determine the transcriptional and functional effects of rebalancing E2 signaling in CD patient Tregs using novel MaxCyte transfection technology, assessing ERα- and ERβ-specific effects on (i) TGFβ-dependent Foxp3 induction in naïve T cells and (ii) ex vivo suppressive function of Tregs. Successful completion of our proposed Aims will provide key mechanistic insight into the functional...