OVERALL SUMMARY This proposal seeks continued support for the UCSF Asthma and Allergic Diseases Cooperative Research Center’s longstanding and productive efforts to understand how type 2 immune responses in the airway act on epithelial cells to produce muco-obstructive pathology, a central feature of severe asthma and a major contributor to fatality from this disease. Our Center has made major contributions to identifying type 2 high asthma as the major asthma endotype, demonstrating that the type 2 cytokine IL-13 acts directly on airway epithelial cells to induce pathological changes in mucus, and showing that mucus plugging is a persistent feature of asthma that is associated with type 2 responses and with increased asthma severity. The overall objective of this proposal is to understand molecular mechanisms that account for alterations in secretory cell and mucus function that are important in severe asthma. The overarching hypothesis that serves as the focus of this renewal is that local type 2 immune responses induce IL-13-mediated changes in epithelial gene expression and that these changes, which involve several novel molecular mechanisms not previously explored, alter differentiation of secretory cells and production and secretion of mucins, leading to mucus plugging and airway obstruction. The proposal includes two highly related projects, each of which focuses on molecules and pathways that have previously unknown roles in secretory cell biology and mucus dysfunction. Project 1 will determine the roles of the tetraspanin TSPAN8, which we recently found to be required for stimulated release of MUC5AC, the major component of mucus plugs, and investigate the contributions of cysteine proteases and cystatin inhibitors implicated in our preliminary studies of mucociliary clearance in IL-13-stimulated primary human bronchial epithelial cells (HBECs). In addition, project 1 will directly assess the relationships between type 2 immune responses, epithelial alterations, and mucus plugging by analyzing lung tissue and HBECs from individuals with asthma and controls. Project 2 focuses on understanding new findings showing key roles for airway epithelial miRNAs in the miR-141/200 family and particular portions of the ER stress response in secretory cell differentiation, pathologic mucus production, and mucus plugging in asthma. Every aim of both projects depends upon a Clinical Subject and Biospecimen Processing and Analysis Core that will study banked lung specimens (for detailed molecular studies of mucus plugs, including from fatal asthma), conduct a new bronchoscopy-based study to obtain freshly isolated cells, provide banked human bronchial epithelial cells, and store, analyze, and disseminate data. The Administrative Core will coordinate our Center and our interactions with other AADCRCs and NIAID. The proposed studies will provide new mechanistic insights that are highly relevant to the pathogenesis of severe asthma and may lead to novel the...