Pulmonary alveolar proteinosis (PAP) is a syndrome characterized by accumulation of surfactant in alveolar macrophages (AMs) and alveoli resulting in respiratory failure and increased mortality from infection. For nearly 4 decades, the only available therapy was whole lung lavage, a highly invasive procedure performed at few centers in which one lung is mechanically ventilated while the other is repeatedly filled with saline and the chest is percussed vigorously to physically remove surfactant. No advances in pharmacologic therapy occurred due to a lack of pathogenic insight until PAP was discovered in GM-CSF-/- mice, a finding that transformed our concepts of the biological role of GM-CSF and led to novel diagnostics and therapy for PAP. My laboratory has contributed significantly to our understanding that GM-CSF is critical for surfactant homeostasis, AM ontogeny, neutrophil and AM functions, and innate immunity, and that in ~90% of patients, PAP is caused by a high level of GM-CSF autoantibodies (GMAbs). Current evidence suggests GM-CSF regulates surfactant homeostasis via the transcription factors PU.1 and PPAR? by stimulating expression of the lipid transporter, ABCG1: all three are deficient in AMs in GM-CSF-deficient mice and PAP patients. Notwithstanding, questions remain regarding the 1) natural history of PAP, 2) mechanism by which loss of GM-CSF signaling causes PAP, and 3) roles and relationship of PU.1 and PPAR3 in mechanisms by which GM-CSF regulates surfactant clearance and immune functions in AMs. We will use our novel primate model of autoimmune PAP, AM cell lines and PAP biomarkers;an existing murine model of hereditary PAP;and autoimmune and hereditary PAP patients to test our central hypothesis: PAP is caused by reduced GM-CSF?PU.1?PPAR3?ABCG1-dependent excretion of neutral lipids from AMs, which impairs their ability to clear surfactant. This hypothesis will be addressed in 3 specific aims focusing to GM-CSF regulation of myeloid cells.
In Aim 1, we will determine the natural history of autoimmune PAP, critical threshold of GMAbs and their effects on myeloid immune functions in our primate model and PAP patients.
In Aim 2, the roles of PU.1, PPAR?, and ABCG1 in hereditary PAP caused by CSF2RA or B mutations will be evaluated in vitro using lentiviral-mediated expression in macrophages from mice or humans with hereditary PAP, and in vivo by transplanting ABCG1-transduced bone marrow into CSF2RB-/- mice.
In Aim 3, we will determine if GM-CSF regulates surfactant clearance and immune functions in AMs via the PU.1-dependent regulation of PPAR? using novel AM cell lines that do not spontaneously express PU.1, or that also respond to GM-CSF. The transcriptional program that GM-CSF regulates in AMs will be examined in vivo free of secondary effects of surfactant by using our primate model. We will determine if the PPAR? agonist pioglitazone restores AM surfactant clearance in vitro in cells from mice and humans with PAP and in vivo using CSF2RB-/- mice. Anticipated results have implications for PAP pathogenesis and therapy, surfactant homeostasis, and GMAb therapy of common inflammatory diseases.

Public Health Relevance

GM-CSF is a cytokine regulator of alveolar macrophage function, surfactant homeostasis, and host defense. Disruption of GM-CSF signaling, due to GM-CSF autoantibodies or GM-CSF receptor mutations, causes pulmonary alveolar proteinosis (PAP), a syndrome of respiratory insufficiency due to surfactant accumulation in alveoli caused by reduced clearance by alveolar macrophages. In Aim 1, we will use a novel primate model of PAP and PAP patients to determine the natural history and level of GM-CSF autoantibodies causing PAP. In Aim 2, we will use gene transfer to determine the role PU.1, PPAR?, and ABCG1, in the pathogenesis of hereditary PAP and surfactant clearance by alveolar macrophages in vitro and in vivo (in mice). In Aim 3, we will determine if GM-CSF regulates PPAR3 in a PU.1-dependent manner and test whether pioglitazone, an FDA-approved PPAR? activator, is effective as therapy of hereditary PAP in vitro and in vivo (in mice). Anticipated results are relevant to surfactant homeostasis, pathogenesis and therapy of PAP, and the use of GM-CSF autoantibodies to treat common diseases like asthma and rheumatoid arthritis.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Lung Cellular, Molecular, and Immunobiology Study Section (LCMI)
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Eu, Jerry Pc
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