PROJECT SUMMARY The overall goal of Project 3 is to understand how increased misexpression of MUC5B in distal airways leads to honeycomb cyst formation, fibroblast accumulation, pro-fibrotic programming and ultimately fibrosis in response to distal airway and alveolar epithelial injury. Through a combination of in vivo studies in mouse models and in vitro studies with human lung epithelial cells and fibroblasts, Project 3 seeks to fill this knowledge gap by testing the hypothesis that MUC5B misexpression and persistent ER stress in distal airway epithelial cells creates susceptibility to apoptosis in both alveolar epithelial cells and distal airway epithelial cells leading to fibrosis and honeycomb cyst formation, respectively. We will address mechanistic questions about: (i) how MUC5B misexpression by distal airway epithelial cells creates vulnerability to apoptosis induction leading to fibroblast accumulation, pro-fibrotic programming, fibrosis and honeycomb cyst formation, (ii) the relative role(s) of alveolar and distal airway epithelial cell apoptosis in the development of fibrosis and honeycomb cysts, and (iii) from a remedial perspective, if prevention of epithelial cell apoptosis mitigates the development of pulmonary fibrosis and honeycomb cyst formation. While much has been learned about the role of alveolar epithelial cell apoptosis in the development of fibrosis, most of these studies were conducted prior to the discovery of increased IPF susceptibility conferred by MUC5B misexpression. Project 3 seeks to address this knowledge gap. Aim 1 will test the hypothesis that alveolar epithelial cell apoptosis, in the context of increased distal airway Muc5b misexpression, leads to fibrosis, but not honeycomb cyst formation in mice. This hypothesis will be tested using an alveolar epithelial cell ablation strategy in the context of Muc5b misexpression in distal airway epithelial cells to define the importance of alveolar epithelial cell apoptosis in the development of fibrosis. Aim 2 will test the hypothesis that induction of distal airway epithelial cell apoptosis in the context of increased distal airway Muc5b misexpression promotes honeycomb cyst formation in mice. This hypothesis with be tested using a distal airway epithelial cell ablation strategy. Aim 2 will also provide insight into the question of whether distal airway epithelial cell apoptosis and honeycomb cyst formation in the context of Muc5b misexpression indirectly leads to alveolar replacement and fibrosis. Lastly, Aim 3 will test the hypothesis that prevention of alveolar and distal airway epithelial cell apoptosis even in the context of increased distal airway Muc5b misexpression will prevent persistent fibrosis and honeycomb cyst formation. While increased MUC5B expression in distal airways is a risk factor for the development of fibrosis and honeycomb cysts in IPF patients, little is known about the fundamental mechanisms that connect these events. Though extensive integra...