Project Summary: For many patients with advanced lung disease, lung transplantation remains the only viable therapeutic option to extend life. However, the 5-year survival for patients following lung transplantation is 54%, the lowest among solid organ transplant recipients. Chronic lung allograft dysfunction (CLAD) is the leading cause of morbidity and late mortality after lung transplantation. Up to a third of lung transplant recipients will develop CLAD within 3 years post-transplant. Currently, there are no effective therapies to prevent or treat CLAD. Hence, the identification of disease biomarkers and novel therapeutic targets are urgently needed to prevent or treat CLAD and extend survival after lung transplantation. Distinct clinical phenotypes of CLAD have been identified, including bronchiolitis obliterans syndrome (BOS) and restrictive allograft syndrome (RAS), however clinical and pathologic overlap between these endotypes suggests shared pathogenic mechanisms. We present preliminary data demonstrating that pathogenic profibrotic monocyte-derived alveolar macrophages (MoAM), maintained via CSF1 signaling, represent a shared pathogenic mechanism between different forms of CLAD. Based on our published and preliminary data, we propose to test hypothesis that autocrine, T cell- or neutrophil-derived CSF1 signaling drives development of CLAD via maintenance of pathogenic MoAM. We will test this hypothesis in two Specific Aims that include rigorous mechanistic experiments in mouse models of CLAD (both BOS and RAS). We will credential our findings in humans by leveraging our expertise in electronic health record analysis, machine learning approaches, and cellular and molecular analysis of small samples that can be safely obtained from patients during standard-of-care surveillance procedures. Aim 1: To determine whether cell type-specific CSF1/CSF1R signaling maintains MoAM to drive CLAD pathogenesis. In Aim 1.1. will determine whether pathogenic macrophages are ontogenetically, transcriptomically, and spatially distinct between BOS and RAS endotypes of CLAD. In Aim 1.2 we will use pharmacologic CSF1R blockade and cell type-specific genetic ablation of CSF1 to determine the role of autocrine, T cell- or neutrophil-derived CSF1 in the maintenance of pathogenic MoAM and development and progression of BOS and RAS endotypes of CLAD. Aim 2: To test whether emergence of pathogenic MoAM and loss of Tregs serve as cellular and molecular biomarkers predictive of CLAD (BOS and RAS) development in lung transplant patients. With Project 1, we will examine the impact of the presence and titers of LRA and PGD severity on these outcomes. With Project 2, we will examine the association between esophageal dysfunction and gastroesophageal reflux on the recruitment and retention of MoAM after transplantation. With Project 4, we will determine whether PGD-induced mitochondrial dysfunction in the epithelium results in enhanced recruitment of MoAM and CLAD progres...