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A3011 - Nocardia Survival and Virulence in Pulmonary Alveolar Proteinosis
Author Block: C. McCarthy1, M. Crist1, J. C. Gardner2, D. Black1, P. I. Arumugam1, M. Wessendarp1, T. Suzuki1, C. Chalk1, B. C. Carey1, B. C. Trapnell1; 1Translational Pulmonary Science Center, Cincinnati Children's Hospital, Cincinnati, OH, United States, 2Pulmonary, Critical Care, and Sleep Medicine, University of Cincinnati School of Medicine, Cincinnati, OH, United States.
Rationale: Nocardia species account for a disproportionally high percentage of infections in pulmonary alveolar proteinosis (PAP), a rare syndrome of progressive alveolar surfactant accumulation resulting in hypoxemic respiratory failure. Primary PAP occurs as a result of disrupted granulocyte/macrophage-colony stimulating-factor (GM-CSF) signalling, and recent evidence shows that the primary pathogenic defect is a reduction in macrophage cholesterol clearance. Because Nocardia can utilize cholesterol as a carbon energy source we hypothesize that the increased cholesterol content of alveolar macrophages contributes to the increased infection risk of Nocardia in PAP. Methods: Nocardia asteroides GUH-2 was used to infect cells and mice. Bone marrow derived macrophages (BMDMs) were generated from C57BL/6 (Wild-Type/WT) and Csf2rb-/- mice. Liposomes were generated with pharmaceutical-grade surfactant (Survanta) with or without cholesterol. BMDMs with and without liposomes were exposed to N.asteroides (4 colony forming units (CFU)/macrophage); intracellular CFUs and virulence (measured by superoxide-dismutase (SOD)) were assessed at various timepoints. In vivo experiments involved 8wk old mice; 1x107 CFU of N.asteroides was delivered by intranasal inhalation. Mice were sacrificed at specific timepoints and lung homogenates (LH) assessed for bacterial load and virulence. Results: N.asteroides survival was significantly increased in Csf2rb-/- BMDMS (8.6 CFU/macrophage) compared to WT BMDMs (2.2 CFU/macrophage) 24h after exposure (p=0.0108). Withdrawal of GM-CSF from WT BMDMs, 72 hours prior to N.asteroides exposure, resulted in increased survival of N.asteroides (4.73 CFU/macrophage) compared to WT BMDMs with GM-CSF present (0.93 CFU/macrophage) (p=0.0357). N.asteroides demonstrated increased virulence (measured by SOD) in Csf2rb-/- BMDMs compared to WT at 0h, 4h and 24h (p=0.0066). Exposure of BMDMs to liposomes containing cholesterol increased N.asteroides virulence in both WT and Csf2rb-/- macrophages. Survanta plus cholesterol in WT BMDMs increased the mean level of SOD (151+/-18.6 AU/107 CFU) compared to no lipid (Mean SOD=69.9+/-11.57 AU/107 CFU) (p=0.0206). In vivo exposure of WT and Csf2rb-/- mice to N.asteroides resulted in significantly different clearance rates and SOD levels. In WT mice there was significantly lower numbers of N.asteroides 72h post infection (4.67x103 CFU/LH) compared to Csf2rb-/- mice (2.625x104 CFU/LH) (p=0.0015). N.asteroides was completely cleared from the lungs of WT mice 5 days post-infection, while Csf2rb-/- mice continued to demonstrate N.asteroides up to 15days post-infection (1.25x103 CFU/LH) and SOD levels were significantly higher (270+/-36.68 AU/LH) compared to WT mice (0.8323+/-0.701 AU/LH) (p=0.0018). Conclusion: Macrophages require GM-CSF to control Nocardia infection in vitro and in vivo. Nocardia appears to be more virulent in cholesterol-laden macrophages irrespective of GM-CSF signalling.