Project Summary/Abstract: Mast cells (MCs) expand within the epithelium and sub-epithelium during prevalent and burdensome human mucosal disease, including asthma, nasal polyposis, food allergy and eosinophilic esophagitis, where they are thought to play a central role in disease pathobiology. MCs take on discrete protease expression profiles in each location, with sup-epithelial MCs co-expressing tryptase and chymase (MCTC) while epithelial MCs express tryptase alone (MCT). Although MCT preferentially expand during human type 2 mucosal inflammatory disease, very little is known about how these cells influence tissue inflammation in part because all existing systems for in vitro study of MCs give rise to MCTC. We have recently developed a novel cell culture system for directing the selective in vitro differentiation of MCT or MCTC from human peripheral blood CD34+ cells, allowing in-depth study of MCT effector function of phenotypes for the first time. This proposal seeks to use our culture system to test the central hypothesis that MCTs within the epithelium have a discrete effector phenotype and that a more comprehensive understanding of the effector function linked to this phenotype will enhance our understanding of disease endotypes in nasal polyposis. A related hypothesis is that in vitro activation profiles of MCTC poorly predict. In support of these hypotheses, preliminary findings indicate that in MCT and MCTC have differential capacity to generate the pro-inflammatory eicosanoids and type 2 inflammation-linked cytokines IL-5 and IL-13 following activation, mimicking transcriptional differences between the two phenotypes in vivo, and that IL-4 differentially regulates upregulation of the high affinity IgE receptor FceR1a in each population. Aim 1 of this proposal seeks to comprehensively define production of cytokines, chemokines and growth factors in vitro-derived MCT vs MCTC in response to a range of activating stimuli, as well as determine the transcriptional changes associated with each. Aim 2 characterizes the differential effects of several inflammation-associated mediators on MCT vs MCTC activation, based on differential expression of cell surface receptors and signal transduction components between the two subsets. Aim 3 datasets generated through aim 1 and 2 to probe existing scRNA-seq data and datasets generated by a collaborator assessing eicosanoids in the nasal lavage of nasal polyposis patients at baseline and following a series of therapeutic interventions, in an attempt to use our in vitro findings to better understand MC effector function in vivo. Completion of these aims will greatly expand our understanding MC effector capacity differential effector capacity of human MC subsets in human disease and is a critical first step towards a more comprehensive understanding of the role of MCs in human disease pathobiology.