Pulmonary innate immune responses depend upon a highly regulated multicellular network to defend an enormous surface area of interaction with the external world. Disruption of these responses renders the host susceptible to pneumonia and subsequent systemic spread of infection. This proposal is to investigate the impact of local variation in oxygenation within the lung on key aspects of this host defense network. Heterogeneous ventilation is a common feature of many pulmonary conditions, including fibrotic lung diseases, COPD, and pneumonia and especially the acute respiratory distress syndrome (ARDS), resulting in regional hypoxia. Alveolar epithelial cells are key participants in this innate immune network. Prior work from this laboratory determined that hyperoxia, a biologically relevant form of oxidative stress, results in specific suppression of alveolar epithelial cell expression of granulocyte-macrophage colony stimulating factor (GM- CSF), leading to alveolar macrophage dysfunction and increased susceptibility to lethal pneumonia. The mechanism of this suppression involves accelerated mRNA turnover due to induction of specific microRNA in alveolar epithelial cells. In preliminary studies for this application we have found that hypoxia has a profound but very different effect on alveolar epithelial cell (AEC) innate immune function. Exposure to an atmosphere of 1% oxygen results in significant suppression of primary murine AEC expression of key innate immune molecules including GM-CSF, CCL2 and IL-6, without inducing cellular injury. In parallel, hypoxia also leads to diminished phagocytosis and expression of key mediators by inflammatory macrophages, suggesting that hypoxia causes a broad depression of pulmonary innate immune responses. Using GM-CSF in AEC as a prototype, we found that mRNA turnover was not altered in hypoxia, but that transcription was greatly decreased, as was accessibility of the proximal promoter region of the GM-CSF gene. We also found that NF- kB activation was decreased in AEC exposed to hypoxia, perhaps explaining the effects of hypoxia on a range of innate immune mediators. This novel finding is in contrast to observations in many other cell types in which hypoxia induces NF-kB activation, emphasizing the importance of cell specificity for these responses. Finally, mice exposed to hypoxia for 48h demonstrate greatly reduced expression of GM-CSF mRNA and protein in lung homogenates, in association with impaired alveolar macrophage function. The macrophage defect was reversed by in vivo treatment with GM-CSF. This application is based on the central hypothesis that regional hypoxia in the lung leads to suppression of local host defense, resulting in increased susceptibility to, and delayed clearance of, pneumonia. We will explore this hypothesis in experiments with three Specific Aims that progress from primary AEC, to multicellular networks, to animal models. Specific Aim 1 will determine the mechanisms by which h...