PROJECT SUMMARY This project focuses on the mechanisms by which IL-10 differentially regulates and promotes mast cell (MC) hyperplasia, heterogeneity, activation and cellular functions during the development of IgE and non-IgE-mediated allergic responses to food antigens. We have demonstrated a key role for the immunomodulatory cytokine IL-10 in driving MC expansion, activation, and cytokine production during food allergy. IL-10-primed MCs exhibited enhanced proliferation, IgE-mediated passive anaphylaxis, and the production of TH2-type cytokines. This suggests a novel role for IL-10 in the homeostasis and function of MCs. However, the mechanisms by which IL-10 promotes MC activation are not very clear and remain to be determined. Furthermore, whether these proinflammatory effects of IL-10 on MCs extend to specific MC lineages, are global in nature, or context-dependent also remain to be examined. This project combines hypothesis testing and targeted mechanistic approaches to further probe the mechanisms by which IL-10 regulates MC functions. These include understanding whether IL-10 differentially regulates MC responses to distinct antigenic stimuli, assessment of its effects on heterogeneous MC subsets, and determination of whether its effects are controlled by the source cell type. The long-term overall goals are to: 1) identify cell-intrinsic and extrinsic factors by which IL-10 promotes or suppresses distinct MC populations and regulates their effector function during type 2 inflammatory responses; and 2) examine the systemic and functional local tissue consequences of regulation of MC expansion and activation by IL-10. Aim 1 will elucidate whether IL-10 promotes IgE-mediated mucosal MC responses to physiological food antigens such as peanut and egg allergen and examine its effects on non-IgE-mediated pathways of allergic sensitization including IL-33-induced anaphylaxis. Whether the effects of IL-10 also extend to connective tissue MCs will be assessed and the phenotypic and transcriptomic profiles of both MC subsets will be mapped. Aim 2 will assess the contributions of cell-intrinsic and paracrine IL- 10 to MC hyperplasia and effector function. Aim 3 will identify the effects of source-dependent IL- 10 and determine whether TH2 or non-TH2-derived IL-10 can differentially regulate MC function. Given the known pleiotropic pathologic and regulatory effects of IL-10 during various T and MC- mediated immune responses, it is important to further identify the mechanisms underlying the proinflammatory effects of IL-10 and its ability to promote rather than suppress MC-dependent responses. As such, these studies are not only relevant to the mission of NIAID but may also serve to identify precision-based approaches for therapeutic interventions.