Abstract/Summary This Project focuses on the mechanisms responsible for mast cell (MC) hyperplasia, heterogeneity, and activation that are characteristic of chronic rhinosinusitis with nasal polyposis (CRSwNP), especially in aspirin exacerbated respiratory disease (AERD). Studies in the current iteration of this AADCRC suggest that canonical mast cell subtypes expressing tryptase and chymase (MCTC) or tryptase alone (MCT) emerge from a novel CD117hiCD38hi transitional MC population that is enriched for markers of proliferation and activation. New data suggest that fibroblast-derived factors drive the emergence of this proliferating MC population from circulating MC progenitors (MCp), and that stromal cells conspire with cytokines (particularly IL-4Rα-active cytokines and IL-33) to drive different MC effector functions in correspondingly different anatomic compartments of the sinonasal mucosa. An unsupervised network analysis combined with functional studies in a novel 3-dimensional culture system reveals a likely feed-forward system between MC-derived LIF and fibroblast-derived IL-6 that may promote MC expansion and activation, and is greatly facilitated by IL-4Rα signaling at both MCs and fibroblasts. This Project combines hypothesis-testing and unbiased approaches to understand the developmental origins of MCs, the functional control of MC expansion and activation in T2I, and the mechanisms by which MCs in turn influence stromal cell function in this context. The long-term overall goals are to precisely identify the cell-intrinsic and -extrinsic factors most responsible for driving MC hyperplasia and influencing their effector function in respiratory T2I, to understand the local consequences of MC hyperplasia in tissue function, and to identify potential therapeutic targets. Aim 1 will map the molecular transitions between circulating MCp and their putative tissue descendants and determine the developmental origins of proliferation-enriched transitional MCs. Aim 2 will identify the mechanisms by which fibroblasts and EpCs facilitate the development of subepithelial MCTC and transitional MCs and potentiate their activation in the setting of T2I. Aim 3 will determine the role of stromal cell-amplified MC-derived mediators, including LIF, in driving MC-stromal cell interactions in T2I. The project is integral to the overall goals of the AADCRC, and involves substantial interactions with the other two Projects.