Oxygen, Pulmonary Innate Immunity and Alveolar Epithelial Cell GM-CSF

肺天然免疫反应依赖于高度调节的多细胞网络来防御 与外部世界相互作用的巨大表面积。这些响应的中断会导致 容易感染肺炎和随后的全身性感染的宿主。这项建议是为了调查 肺内氧合的局部变化对这一宿主防御网络的关键方面的影响。 异质通风是许多肺部疾病的共同特征，包括纤维化肺部疾病， 慢性阻塞性肺病、肺炎，特别是急性呼吸窘迫综合征(ARDS)，导致区域性 缺氧。肺泡上皮细胞是这一先天免疫网络的关键参与者。之前的工作来自于此 实验室确定，高氧是一种与生物相关的氧化应激形式，它会导致特定的 粒细胞-巨噬细胞集落刺激因子抑制肺泡上皮细胞表达 脑脊液)，导致肺泡巨噬细胞功能障碍，增加对致死性肺炎的易感性。这个 这种抑制的机制涉及到由于诱导特定的microRNA而加速mRNA的周转。 肺泡上皮细胞。在对这一应用的初步研究中，我们发现缺氧具有深远的 但对肺泡上皮细胞(AEC)天然免疫功能的影响却大相径庭。暴露在大气中 1%氧气显著抑制原代小鼠AEC的关键先天免疫表达 包括GM-CSF、CCL2和IL-6在内的分子，不会引起细胞损伤。同时，低氧也会导致 炎性巨噬细胞对关键介质的吞噬和表达减少，提示 低氧会导致肺部天然免疫反应的广泛抑制。GM-CSF在AEC中的应用 我们发现，在低氧条件下，mRNA的周转没有改变，但转录却发生了很大的变化 GM-CSF基因近端启动子区域的可及性降低。我们还发现，核因子- 缺氧使血管内皮细胞KB活性降低，这可能解释了缺氧对血管内皮细胞的影响。 与生俱来的免疫介质。这一新发现与在许多其他类型的细胞中观察到的结果形成了对比 低氧诱导核因子-kB的激活，强调了细胞特异性对这些反应的重要性。最后， 缺氧48h小鼠脑内GM-CSF mRNA和蛋白表达显著降低 肺组织匀浆，与肺泡巨噬细胞功能受损有关。巨噬细胞缺陷是 经GM-CSF体内治疗后逆转。此应用程序基于以下中心假设：地区性 肺内的低氧导致局部宿主防御的抑制，从而增加对 肺炎的延迟清除。我们将在有三个具体目标的实验中探索这一假设 从原发血管内皮细胞，到多细胞网络，再到动物模型。具体目标1将决定 体内低氧条件抑制肺组织GM-CSF、AM吞噬功能表达及机制的研究 细菌清除。为此目的的实验将证实GM-CSF在低氧诱导的 宿主防御缺陷及低氧诱导因子(HIF)和诱导型一氧化氮的作用 氧化氮合酶(INOS)抑制核因子-kB活化和GM-CSF表达。特定目标2将 确定低氧抑制AEC先天免疫反应的分子机制。调焦 在GM-CSF的表达上，这一特定目的的工作将确定HIF-激活、NO-1和HIF-1的作用。 介导的核因子-kB信号的改变和三磷酸腺苷诱导的染色质重塑的改变。具体目标3将 利用机械通气法测定体内低氧对局部天然免疫防御的影响 显示肺部局部缺氧。在成功完成后，这项工作将解决重要的 有关局部缺氧对肺炎易感性的影响的问题，将提出新的 治疗干预的机会。

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 hypoxic conditions in vivo suppress lung expression of GM-CSF, AM phagocytosis and bacterial clearance. Experiments for this aim will confirm the central role of GM-CSF in the hypoxia-induced defect in host defense and elucidate the contributions of hypoxia inducible factors (HIF), and inducible nitric oxide synthase (iNOS) to suppression of NF-kB activation and GM-CSF expression. Specific Aim 2 will determine the molecular mechanisms by which hypoxia suppresses AEC innate immune responses. Focusing on GM-CSF expression, work in this specific aim will determine the contributions of HIF-activation, NO- mediated changes in NF-kB signaling and ATP-induced changes in chromatin remodeling. Specific Aim 3 will determine the effects of hypoxia on local innate immune defenses in vivo, using mechanically ventilated lambs demonstrating regional hypoxia in the lung. When successfully completed, this work will address important questions related to the contribution of local hypoxia to vulnerability to pneumonia and will suggest novel opportunities for therapeutic intervention.