PROJECT SUMMARY/ABSTRACT Asthma is an airway obstructive disease that is a major cause of morbidity in the US and worldwide. Approximately half of people with asthma present with type 2 high (T2-high) asthma, which is an endotype of asthma commonly characterized by high levels of type 2 cytokines, such as IL-4 and IL-13, within the airway. These cytokines induce airway epithelial remodeling, causing goblet cell hyperplasia. Goblet cells are responsible for the production and secretion of mucus, and one hallmark of Th2-high asthma is mucus hypersecretion and the development of mucus plugs within airways. The Erle lab has recently identified that IL- 13 stimulation in human bronchial epithelial cell (HBEC) cultures causes the overproduction of MUC5AC, and also leads to MUC5AC “tethering”, where MUC5AC protein adheres to the apical surface of epithelial cells. This tethering causes a significant reduction in mucociliary clearance (MCC), and is hypothesized to contribute to the formation of mucus plugs in asthma patients. However, it is not yet known what changes induced by IL-13 lead to mucus tethering. Recent RNA sequencing analysis of both IL-13 stimulated HBEC cultures and airway cells from T2-high asthma patients revealed significantly elevated levels of CST1 and CST4, genes encoding for Cystatin 1 (CST1) and Cystatin 4 (CST4), respectively. Cystatin proteins function as cysteine protease inhibitors, but their role in airway epithelia function has not been characterized. Downstream functional analyses have shown that deletion of either CST1 or CST4 rescues the IL-13-dependent loss in MCC, suggesting an important role for both cystatins in T2-high asthma pathogenesis. The studies in this proposal seek to characterize the role of CST1 and CST4 in T2-high asthma. CST1 and CST4 will be knocked out of HBEC cultures prior to IL-13 stimulation in order to determine how they influence mucin secretion, turnover, and/or tethering. In addition, we will identify the endogenous cysteine proteases that are inhibited by CST1 and CST4, and use knockout and overexpressing HBEC cultures to test if these cysteine proteases regulate MCC and mucus tethering. Finally, in vitro enzyme assays and short-circuit current analysis of HBEC cultures will be used to determine if endogenous cysteine proteases target MUC5AC and/or ENaC in order to regulate MCC. Understanding the mechanisms that control mucus tethering and impaired MCC in asthma will provide fundamental insights into the progression of the disease and could contribute to the development of future therapeutics for asthma patients. This proposed project will be performed as part of a postdoctoral fellowship at UCSF and will utilize comprehensive institutional support and resources in order to prepare the applicant for a research career.