Role of NOS2 in Sepsis-Induced ARDS

描述(申请人提供)：败血症代表全身对感染的反应，随之而来的炎症级联导致多器官衰竭和高死亡率。急性呼吸窘迫综合征(ARDS)是一种破坏性的肺损伤过程，使脓毒症复杂化，并在初始感染得到治疗后很长一段时间内促进宿主炎症反应的传播。ARDS导致肺表面活性物质丢失、肺泡塌陷和顽固性低氧血症。虽然机械通气对ARDS患者的支持是必要的，但随着高呼吸道压力传递到受损的肺实质，对肺的持续损害是不可避免的。因此，通过保留表面活性物质的表达来改善肺损伤的新的分子策略有望改善脓毒症的整体预后。长期以来，人们一直认为，一氧化氮(NO)及其代谢物会破坏蛋白质，从而导致表面活性物质的大量损失。在脓毒症和ARDS期间，NO的过量产生是由于实质细胞和炎性细胞中NOS2表达上调所致。我们和其他研究人员报道了一种新的机制，即NOS2产生的NO干扰表面活性物质的表达并传播肺功能障碍。在内毒素性肺损伤的小鼠模型中，我们证明了NOS2来源的NO下调肺上皮细胞表面活性蛋白B的表达。尽管全身性NOS2抑制剂没有改善人类脓毒症的存活率，外源性表面活性物质替代也没有显示出在ARDS中的益处，但我们推测，调节肺中NOS2局部表达的能力代表着一种令人兴奋的方法，可以在ARDS期间恢复可再生的表面活性物质的表达。我们的总体假设是，肺上皮细胞中NOS2来源的NO通过调节表面活性物质蛋白的表达，在脓毒症时肺损伤的传播中发挥关键作用。为了检验我们的假设，我们提出了三个具体目标。在目标1中，我们将确定NOS2在脓毒症诱导的肺损伤小鼠模型中的作用，该模型反映了人类ARDS。野生型和NOS2缺陷小鼠将接受盲肠结扎和穿孔(CLP)和抗生素治疗，然后进行低潮气量机械通气。我们将评估NOS2的表达，肺生理，细胞炎症，血管渗漏，细胞因子水平，表面活性物质功能和表面活性蛋白的表达。在AIM 2中，我们将研究在急性肺损伤过程中肺上皮细胞NOS2表达对肺表面活性物质表达和功能的直接影响。我们将使用两只转基因动物和适当的对照组，选择性地改变肺上皮细胞中SPB和/或NOS2的表达。在AIM 3中，我们将探讨NOS2来源的NO在调节人表面活性蛋白B表达中的作用。我们将在人和小鼠转化细胞系以及从野生型和NOS2缺陷小鼠分离的原代小鼠肺远端上皮细胞中进行转基因分析、体内结合试验(染色质免疫沉淀试验)和S亚硝化试验。公共卫生相关性：脓毒症代表全身对感染的反应，随之而来的炎症级联导致多器官衰竭和高死亡率。急性呼吸窘迫综合征(ARDS)是一种破坏性的肺损伤过程，使脓毒症复杂化，并在初始感染得到治疗后很长一段时间内促进宿主炎症反应的传播。这项提案的目标的实现为获得对脓毒症引起的ARDS的病理生物学的新见解带来了重大希望，这些新的见解可以为这些毁灭性综合征的新治疗策略的开发提供信息。

DESCRIPTION (provided by applicant): Sepsis represents the systemic response to infection, and the ensuing inflammatory cascade leads to the development of multi-organ failure and high mortality. The acute respiratory distress syndrome (ARDS) represents a process of devastating lung injury that complicates sepsis and contributes to propagation of the host inflammatory response long after the initial infection has been treated. ARDS results in loss of surfactant, alveolar collapse, and refractory hypoxemia. While mechanical ventilation is necessary to support patients with ARDS, ongoing damage to the lung is inevitable with delivery of high airway pressures to injured lung parenchyma. Thus, novel molecular strategies that target amelioration of lung injury through preservation of surfactant expression hold promise for improving overall outcomes from sepsis. It has long been proposed that significant surfactant loss occurs through protein destruction by nitric oxide (NO) and its metabolites. Excessive production of NO during sepsis and ARDS results from upregulation of NOS2 expression in parenchymal and inflammatory cells. We and other investigators reported a novel mechanism by which NOS2- derived NO interferes with surfactant expression and propagates lung dysfunction. In a murine model of endotoxemic lung injury, we demonstrated that NOS2-derived NO downregulates expression of surfactant protein-B in lung epithelial cells. Although systemic NOS2 inhibitors have not improved survival in human sepsis, and exogenous surfactant replacement has not shown benefit in ARDS, we postulate that the ability to modulate localized NOS2 expression in the lung represents an exciting approach toward restoring "renewable" surfactant expression during ARDS. Our overall hypothesis is that NOS2-derived NO in lung epithelial cells plays a critical role in propagation of lung injury during sepsis through modulating expression of surfactant proteins. To test our hypothesis, we propose three Specific Aims. In AIM 1, we will determine the role of NOS2 in a murine model of sepsis-induced lung injury that mirrors human ARDS. Wild type and NOS2-deficient mice will be subjected to cecal-ligation and puncture (CLP) and antibiotic treatment, followed by low tidal volume mechanical ventilation. We will assess NOS2 expression, lung physiology, cellular inflammation, vascular leak, cytokine levels, surfactant function and expression of surfactant proteins. In AIM 2, we will examine the direct effect of NOS2 expression in the lung epithelial cell on surfactant expression and function during acute lung injury. We will selectively modify SPB and/or NOS2 expression within the lung epithelial cell using two transgenic animals and appropriate controls. In AIM 3, we will explore the role of NOS2-derived NO on regulation of human surfactant protein-B expression. We will perform transfection analyses, in vivo binding studies (chromatin immunoprecipitation assays), and S-nitrosylation assays in human and murine transformed cell lines and in primary murine distal lung epithelial cells isolated from wild-type and NOS2-deficient mice. PUBLIC HEALTH RELEVANCE: Sepsis represents the systemic response to infection, and the ensuing inflammatory cascade leads to the development of multi-organ failure and high mortality. The acute respiratory distress syndrome (ARDS) represents a process of devastating lung injury that complicates sepsis and contributes to propagation of the host inflammatory response long after the initial infection has been treated. Achievement of the AIMS of this proposal hold significant promise for gaining novel insights into the pathobiology of sepsis-induced ARDS that can inform the development of new treatment strategies for these devastating syndromes.