Project Summary Airway hyperresponsiveness (AHR) is a fundamental feature of asthma and is the hallmark of the disease in the majority of patients with asthma. Our lab has been working to understand the underlying immunopathogenesis of AHR and recently discovered using three-dimensional quantitative imaging of the airway wall that in asthma there is a shift in mast cells (MC)s from the submucosa to the airway epithelium that is closely related to the severity of AHR as well as features of type-2 (T2) inflammation. This shift is critical as we identified a IL-33 dependent feed-forward loop in which MCs regulate the expression of epithelial IL-33 (IL33), indicating that MCs serve not only as effector cells but also regulate T2 inflammation through IL-33. This feed-forward loop is accentuated in airway epithelial cells (AEC)s from individuals with asthma indicating that the unique microenvironment that is established by the epithelium in asthma is critical for the propagation of AHR and T2 inflammation through this feed-forward interaction between MCs and the airway epithelium. The major goals of this research are to identify the underlying basis for MC infiltration of the airway epithelium, the molecular details of this interaction between MCs and primary AECs from individuals with asthma and the in vivo relevance of the IL-33-MC-axis using selected murine models of asthma. Given that mature MCs arise from circulating progenitors, we hypothesize that the number of MC progenitors (MCP)s in the airways is increased in asthma, and these cells function in concert with the susceptible airway epithelium to orchestrate T2 inflammation. We will directly address our hypothesis by using airway samples and primary AECs from individuals with and without asthma and/or allergic sensitization, combined with AEC- MC coculture models, and murine models of asthma in which we examine the function of the IL-33-MC-axis. In specific aim 1, we investigate the differences in the number and proliferation potential of MCPs in the airways and peripheral blood of subjects with asthma and AHR, relative to non-asthmatic control subjects with and without allergic sensitization. We further examine the expansion of MCPs in the airways following allergen challenge in individuals with asthma. In specific aim 2, we determine how the asthmatic airway epithelium interacts with MCs to regulate T2 inflammation, using an ex vivo model of primary AECs from subjects with and without asthma to examine this feed-forward mechanism. In specific aim 3, we use selective IL-33 receptor deletion in MCs in an in vivo model to understand how IL-33 acts through MCs to regulate the development of T2 inflammation and AHR. The completion of these studies using airway samples, phenotyped AECs, and sophisticated murine models will move the field forward through a better understanding of the interplay between AECs and intraepithelial MCs that serve to regulate AHR and the T2 immune response.