Role of NOS2 in Sepsis-Induced ARDS

描述（由申请人提供）：败血症代表了对感染的全身反应，随后的炎症级联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）是一个破坏性肺损伤的过程，它使败血症复杂化，并在初始感染治疗后很长时间内促进宿主炎症反应的传播。ARDS导致表面活性剂丧失、肺泡塌陷和难治性低氧血症。虽然机械通气对于支持ARDS患者是必要的，但由于气道高压对受损肺实质的输送，对肺的持续损伤是不可避免的。因此，通过维持表面活性剂表达来改善肺损伤的新分子策略有望改善脓毒症的总体结果。长期以来，人们一直认为，表面活性剂的显著损失是通过一氧化氮（NO）及其代谢物破坏蛋白质而发生的。脓毒症和ARDS期间一氧化氮的过量产生是由于实质细胞和炎症细胞中NOS2表达上调。我们和其他研究人员报道了NOS2衍生的NO干扰表面活性剂表达并传播肺功能障碍的新机制。在小鼠内毒素肺损伤模型中，我们证明nos2来源的NO下调肺上皮细胞中表面活性剂蛋白b的表达。尽管全身NOS2抑制剂并没有提高人类败血症患者的生存率，外源性表面活性剂替代也没有显示出对ARDS的益处，但我们假设，调节肺中局部NOS2表达的能力代表了一种令人兴奋的方法，可以恢复ARDS期间“可再生”表面活性剂的表达。我们的总体假设是，肺上皮细胞中nos2来源的NO通过调节表面活性剂蛋白的表达，在脓毒症期间肺损伤的传播中起关键作用。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NOS2在脓毒症诱导的肺损伤小鼠模型中的作用，该模型与人类ARDS模型相似。野生型和nos2缺陷小鼠将进行盲肠结扎穿刺（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、表面活性剂功能和表面活性剂蛋白的表达。在AIM 2中，我们将研究急性肺损伤时肺上皮细胞中NOS2表达对表面活性物质表达和功能的直接影响。我们将使用两只转基因动物和适当的对照，选择性地修饰肺上皮细胞内SPB和/或NOS2的表达。在AIM 3中，我们将探讨nos2衍生的NO在调节人表面活性剂蛋白b表达中的作用。我们将进行转染分析、体内结合研究（染色质免疫沉淀测定）和s -亚硝基化测定，实验对象为人和小鼠转化细胞系，以及从野生型和nos2缺陷小鼠分离的小鼠原代远端肺上皮细胞。

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.