Role of NOS2 in Sepsis-Induced ARDS

描述（由申请方提供）：脓毒症代表对感染的全身反应，随后的炎症级联反应导致多器官衰竭和高死亡率的发展。急性呼吸窘迫综合征（ARDS）是一种破坏性肺损伤的过程，它使脓毒症复杂化，并在初始感染得到治疗后很长时间内促进宿主炎症反应的传播。ARDS导致表面活性物质丧失、肺泡萎陷和顽固性低氧血症。虽然机械通气对于支持患有ARDS的患者是必要的，但是随着高气道压力输送到受伤的肺实质，对肺的持续损伤是不可避免的。因此，通过保护表面活性剂表达来靶向改善肺损伤的新分子策略有望改善脓毒症的总体结局。长期以来，人们一直认为，通过一氧化氮（NO）及其代谢产物对蛋白质的破坏，会发生显著的表面活性剂损失。脓毒症和ARDS期间NO的过量产生是由于实质和炎症细胞中NOS 2表达上调所致。我们和其他研究人员报道了一种新的机制，通过这种机制，NOS 2衍生的NO干扰表面活性物质的表达，并传播肺功能障碍。在小鼠内毒素性肺损伤模型中，我们证明了NOS 2衍生的NO下调肺上皮细胞表面活性蛋白B的表达。虽然全身性NOS 2抑制剂没有改善人败血症的存活率，外源性表面活性剂替代在ARDS中也没有显示出益处，但我们假设调节肺中局部NOS 2表达的能力代表了一种令人兴奋的方法，可以在ARDS期间恢复“可再生”表面活性剂表达。我们的总体假设是，在肺上皮细胞中的NOS 2衍生的NO通过调节表面活性蛋白的表达在脓毒症期间肺损伤的传播中起着关键作用。为了验证我们的假设，我们提出了三个具体目标。在AIM 1中，我们将确定NOS 2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N 0 S2缺陷型小鼠将经受盲肠结扎和穿孔（CLP）和抗生素治疗，然后进行低潮气量机械通气。我们将评估NOS 2表达、肺生理学、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物质蛋白的表达。在AIM 2中，我们将研究急性肺损伤时肺上皮细胞中NOS 2表达对表面活性物质表达和功能的直接影响。我们将使用两个转基因动物和适当的对照选择性地改变肺上皮细胞内SPB和/或NOS 2的表达。在AIM 3中，我们将探讨NOS 2衍生的NO对人表面活性蛋白-B表达的调节作用。我们将进行转染分析，在体内结合研究（染色质免疫沉淀试验），和S-亚硝基化试验在人类和小鼠转化细胞系和原代小鼠远端肺上皮细胞分离的野生型和NOS 2缺陷小鼠。公共卫生相关性：脓毒症代表对感染的全身反应，并且随后的炎症级联反应导致多器官衰竭和高死亡率的发展。急性呼吸窘迫综合征（ARDS）是一种破坏性肺损伤的过程，它使脓毒症复杂化，并在初始感染得到治疗后很长时间内促进宿主炎症反应的传播。该提案的目标的实现对于获得脓毒症诱导的ARDS的病理生物学的新见解具有重要意义，这些新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.