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

氧气、肺先天免疫和肺泡上皮细胞 GM-CSF

• 批准号: 10266006
• 负责人: Robert Paine
• 金额: --
• 依托单位: VA SALT LAKE CITY HEALTHCARE SYSTEM
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-04-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10266006
• 关键词: Address; Adult Respiratory Distress Syndrome; Affect; Alveolar; Alveolar Macrophages; Animal Model; Area; Atelectasis; Atmosphere; Binding Sites; Biological; Biological Response Modifiers; CCL2 gene; Cell Line; Cells; Chronic Bronchitis; Chronic Obstructive Airway Disease; Colony-Stimulating Factor Gene; Complex; Data; Defect; Epithelial Cells; Exposure to; Floods; Functional disorder; Generations; Genetic Transcription; Granulocyte-Macrophage Colony-Stimulating Factor; Heart failure; Host Defense; Human; Hyperoxia; Hypoxia; Hypoxia Inducible Factor; Immune; Impairment; In Vitro; Individual; Infection; Inflammation Mediators; Inflammatory; Innate Immune Response; Interleukin-6; Investigation; Laboratories; Lead; Literature; Lung; Lung diseases; Mechanical ventilation; Mediating; Mental Depression; Messenger RNA; MicroRNAs; Molecular; Morbidity - disease rate; Mus; NF-kappa B; NOS2A gene; Natural Immunity; Newborn Infant; Oxidative Stress; Oxygen; Participant; Phagocytosis; Pneumonia; Population; Predisposition; Promoter Regions; Proteins; Pulmonary Emphysema; Pulmonary Fibrosis; Recombinant Granulocyte-Macrophage Colony-Stimulating Factors; Recording of previous events; Resolution; Risk; Role; Signal Transduction; Specificity; Surface; Therapeutic Intervention; Transgenic Organisms; Variant; Veterans; Work; alveolar epithelium; base; cell injury; cell type; chemokine; chromatin remodeling; experimental study; granulocyte; in vivo; innate immune function; insight; macrophage; military veteran; mortality; mouse model; novel; novel therapeutic intervention; prevent; promoter; prototype; response; transcription factor; ventilation

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.

肺先天免疫反应依赖于高度调节的多细胞网络来防御 与外界互动的巨大表面积。这些反应的中断使得 宿主对肺炎和随后全身性感染传播易感。这项提议是为了调查 肺内氧合的局部变化对宿主防御网络关键方面的影响。 不均匀通气是许多肺部疾病的共同特征，包括纤维化肺病， 慢性阻塞性肺病和肺炎，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种先天免疫网络的关键参与者。以前的工作从这个 实验室确定，高氧（一种与生物学相关的氧化应激形式）会导致特定的 抑制肺泡上皮细胞表达粒细胞-巨噬细胞集落刺激因子（GM-CSF）， CSF），导致肺泡巨噬细胞功能障碍和对致死性肺炎的易感性增加。的 这种抑制的机制涉及由于在细胞中诱导特异性microRNA而加速mRNA周转。 肺泡上皮细胞在对该应用的初步研究中，我们发现缺氧具有深刻的 但对肺泡上皮细胞（AEC）天然免疫功能的影响却截然不同。暴露在大气中 1%氧气导致主要先天免疫原性鼠AEC表达的显著抑制， 包括GM-CSF、CCL 2和IL-6的分子，而不诱导细胞损伤。同时，缺氧也会导致 炎症巨噬细胞的吞噬作用和关键介质的表达减少，表明 缺氧引起肺先天免疫应答的广泛抑制。GM-CSF在AEC中的应用 原型，我们发现mRNA周转在缺氧中没有改变，但转录大大增加。 降低，因为是近端启动子区的GM-CSF基因的可及性。我们还发现NF- 在缺氧条件下，AEC的kB活化降低，这可能解释了缺氧对AEC的一系列影响。 先天性免疫介质。这一新的发现与许多其他细胞类型的观察相反， 缺氧诱导NF-κ B活化，强调了细胞特异性对这些反应的重要性。最后， 小鼠缺氧48小时后，GM-CSF mRNA和蛋白的表达明显降低， 肺匀浆，与肺泡巨噬细胞功能受损有关。巨噬细胞缺陷是 通过用GM-CSF体内处理逆转。这一应用基于一个中心假设，即区域 肺中的缺氧导致局部宿主防御的抑制，从而导致对 肺炎的延迟清除。我们将在三个具体目标的实验中探索这一假设， 从初级AEC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GM-CSF表达、AM吞噬作用和 细菌清除。为此目的的实验将证实GM-CSF在缺氧诱导的细胞凋亡中的中心作用。 缺陷宿主防御和阐明缺氧诱导因子（HIF）的贡献，诱导一氧化氮 一氧化氮合酶（iNOS）抑制NF-κ B活化和GM-CSF表达。具体目标2将 确定缺氧抑制AEC先天免疫反应的分子机制。聚焦 对GM-CSF表达的影响，这一特定目标的工作将确定HIF-激活、NO- 介导的NF-κ B信号转导的变化和ATP诱导的染色质重塑的变化。第3章将 使用机械通气的羔羊，确定缺氧对体内局部先天性免疫防御的影响 表明肺部局部缺氧一旦成功完成，这项工作将解决重要的 与局部缺氧对肺炎易感性的贡献有关的问题，并将提出新的 治疗干预的机会。