PROJECT SUMMARY - ESI PROJECT Lung transplant outcomes are the worst among solid organ transplants in large part due to the nearly universal development of chronic lung allograft dysfunction (CLAD) in lung transplant recipients. CLAD is characterized by progressive fibrosis of the small airways and less commonly of the alveolar spaces. Our group has contributed to an evolving model of lung fibrosis that is initiated by injury to the small airways and alveolar epithelium and can be interrupted by successful epithelial repair. We focus on an intermediate epithelial cell population expressing genes characteristic of both AT2 and AT1 cells. These “transitional cells” are expanded during postnatal development and in several models of lung injury and fibrosis, but are rare in the normal adult lung. In a recently published paper (Nature 2023), we reported that lung epithelial-specific knockout of a gene necessary to complex I of the mitochondrial electron transport chain during development (Ndufs2) results in failed postnatal alveologenesis. Single-cell RNA sequencing revealed an accumulation of transitional cells in mice lacking epithelial Ndufs2 with upregulation of genes induced by the integrated stress response (ISR). In our preliminary studies, knockout of Ndufs2 in adult AT2 cells results in spontaneous lung fibrosis that was fatal after several months. A small molecule inhibitor of the ISR (ISRIB) rescued postnatal alveologenesis and attenuated bleomycin- and asbestos-induced fibrosis in adult mice. More importantly, ISRIB improved CLAD in a mouse model. Activation of the ISR results in a global inhibition of translation accompanied by activation of a transcriptional program mediated by ATF4, both of which are inhibited by ISRIB. We hypothesize that impaired mitochondrial function in the airway and alveolar epithelium, induced by ischemia-reperfusion injury during transplantation, activates the ISR to enhance ATF4-mediated transcription, precluding normal epithelial differentiation and allograft repair after PGD- (Project 1) or acid- (Project 2) induced lung injury. The resulting failed repair leads to the recruitment of profibrotic MoAM and progressive fibrosis (CLAD, Project 3). We will test this hypothesis in the following interrelated specific aims: Aim 1: To determine whether mitochondrial dysfunction causes pathological ISR activation to preclude normal lung allograft repair following lung transplant. Aim 2: To test whether epithelial ATF4 is necessary and sufficient to preclude alveolar epithelial differentiation and promote fibrosis during CLAD. We propose a series of causal experiments in a murine model of lung transplantation to link mitochondrial dysfunction, activation of the ISR, and failed epithelial differentiation to the development of CLAD. With Core B and C, we pair our experiments with samples collected and analyzed from lung transplant patients with a goal of credentialling mitochondrial function and the ISR as targets for the...