ABSTRACT Despite our vastly improved understanding of pulmonary alveolar proteinosis (PAP) ? a syndrome of surfactant accumulation and respiratory failure that occurs in multiple distinct diseases; clinically, PAP remains diagnosed by methods unable to identify the causative disease (e.g., lung biopsy) and no drug is FDA-approved to treat it. While numerous PAP-causing diseases exist, Primary PAP (caused by GM-CSF autoantibodies or CSF2RA/B mutations) accounts for more than 90% of cases. In the prior funding period, we developed `research tests' that comprise the only means now available to specifically diagnose Primary PAP. Disruption of GM-CSF signaling in alveolar macrophages (AMs) in Primary PAP impairs GM-CSF-dependent surfactant clearance by AMs. Pulmonary GM-CSF is also critical for other functions including AM maturation, self-renewal, and population size and, consequently, is vital to surfactant homeostasis, alveolar stability, lung function, and host defense. It is widely-believed that the loss of GM-CSF signaling causes PAP by reducing the intrinsic ability of AMs to catabolize phospholipids but no such mechanism has ever been identified. Based on our Preliminary Data, we identified a novel mechanism that challenges the current concept of PAP pathogenesis and has identified molecular targets that we are now exploiting to develop novel diagnostics and therapeutics. This proposal seeks to test the following central hypothesis: cholesterol toxicity, not reduced phospholipid catabolism, drives the pathogenesis of impaired surfactant clearance of AMs in Primary PAP. We also hypothesize that GM-CSF is required constitutively to enhance cholesterol clearance by AMs via PU.1/ CEBP?-mediated expression of PPAR? (and its downstream target ABCG1) and post-translational activation of PPAR? by unsaturated fatty acids arising from surfactant phospholipid metabolism. In Aim 1 we will determine the mechanism of AM dysfunction caused by the loss of GM-CSF signaling in man, monkeys, and mice with Primary PAP. In Aim 2, we will evaluate lung cholesterol:choline ratio for bronchoscopic diagnosis of PAP, serum cholestenoic acid for monitoring PAP disease severity, and GM-CSF signaling in whole blood as a multifunctional diagnostic test. In Aim 3, we will validate a novel molecular target for pharmacotherapy of PAP. We expect to 1) determine molecular and cellular pathogenesis of diseases causing PAP in >90% of patients and inform mechanisms by which AMs regulate surfactant homeostasis in health and disease; 2) develop novel biomarker-based `research tests' to facilitate diagnosis of PAP, monitor disease severity, accelerate clinical research, and provide new tools to practicing clinicians; and 3) validate new targets for pharmacotherapy of PAP. Results are expected to led to improved healthcare delivery by practicing physicians, improving quality of life for people living with PAP, and to refocus PAP research to molecular targets for which FDA-approved drugs could be repurposed as therapy of PAP. Results may have broader implications for role of cholesterol metabolism in lung diseases beyond PAP, and for the pathogenesis of atherosclerotic cardiovascular disease.