Project Summary Obesity cultivates complex immune responses in white adipose tissue (WAT), which may contribute to the development of insulin resistance and type 2 diabetes mellitus (T2DM). However, there still exists a gap in knowledge on cellular mechanisms regulating “obesity-specific” immune responses that precludes the development of novel immune therapies to treat obesity and related insulin resistance and T2DM. We were one of the first to report T cells in WAT in obesity. Further studies from us and others showed that WAT conventional T cells promote, and regulatory T cells (Tregs) protect against, insulin resistance in obesity. Nonetheless, the mechanisms underlying WAT Treg homeostasis and mediating their metabolic benefits remain incompletely understood. This application aims to investigate microRNA-30a (miR-30a) regulation of WAT Tregs and its impacts on WAT immune response and metabolism in obesity. This proposal builds on our novel pilot data: 1) miR-30a expression in WAT correlated positively with insulin sensitivity in obese mice and people; 2) enforced miR-30a expression in WAT promoted whole-body insulin sensitivity in obese mice; 3) miR-30a levels were high in WAT Tregs in lean but reduced in obese mice; 4) enforced miR-30a expression in WAT of obese mice increased WAT Treg numbers; 5) specific overexpression of miR-30a in Tregs increased WAT Treg numbers and improved insulin sensitivity in mice fed high-fat diet. Therefore, we hypothesize that miR-30a in WAT stimulates expansion of WAT Tregs and improves Treg functions to increase insulin sensitivity. We will test this hypothesis by pursuing three aims: Aim 1 will define effects of cell (Treg vs adipocyte)-restricted miR- 30a on WAT immune response and metabolism in obesity and establish cell-specific roles of miR-30a; Aim 2 will build on our additional pilot data that miR-30a delivery in WAT suppresses STAT1 (a key inflammatory transcription factor) and establish how miR-30a targeting of Stat1 governs WAT Treg homeostasis in obesity; Aim 3 will build on our novel data that compared to diet high in saturated fat, diet high in unsaturated fat induces higher levels of WAT miR-30a and Tregs in mice and examine dietary regulation of miR-30a and its role in WAT immune response and metabolism. We will accomplish these aims using gain-of-function and loss-of-function approaches in genetically modified animal models, cutting-edge immunophenotyping and transcriptomics, gold standard clamp for insulin sensitivity, and other state-of-the-art tools. Upon completing these aims, we expect to have uncovered a novel mechanism that may help develop new therapies that leverage the immune system to treat obesity and T2DM.