NQO1: Linking Oxidant Stress to Inflammation in Airway Epithelial Cells

描述(申请人提供)：个人对臭氧的反应差异很大，有些人对肺部毒性的臭氧敏感性阈值要低得多。因此，在臭氧暴露后，个体遗传因素的复合体可能控制着氧化剂信号的传递和繁殖。流行病学研究支持野生型NADPH苯醌氧化还原酶1(NQO1)基因在臭氧存在时作为哮喘易感因素的作用。NQO1催化对苯二酚的专有2电子还原反应，被认为是细胞内的抗氧化剂。我们的初步数据表明，NQO1是臭氧诱导的IL-8/KC表达、中性粒细胞气道炎症增加和小鼠气道高反应性所必需的。这就提出了一个难题来解释循环使用抗氧化剂的氧化还原酶如何传播ROS信号，将氧化剂应激与炎症反应联系起来。我们将使用原代人类呼吸道上皮细胞和小鼠(野生型和NQO1缺失)模型系统来测试以下前所未有的假设机制，以解释NQO1如何将氧化应激与上皮炎症联系起来：NQO1和臭氧产生的ROS是臭氧暴露后呼吸道炎症的中央调节因子。我们认为，NQO1调节细胞内的氧化还原环境，导致异前列腺素(Isoprostanes，ISOP)平衡的改变。在NQO1存在下，ROS和F2-IsoP激活了NF-B，导致IL-8/KC表达增加，中性粒细胞炎症加重。在没有NQO1的情况下，细胞有利于A2-IsoP的产生，从而抑制NF-B的激活，导致阻断IL-8/KC表达和中性粒细胞炎症的矛盾效应。具体目标是：目的1a：确定臭氧暴露后，NQO1是否通过上调中性粒细胞趋化因子KC/IL-8来介导中性粒细胞炎症和气道高反应性。目的1b：确定NQO1在结构上皮细胞和/或造血细胞中的表达是否是肺对臭氧反应所必需的。目的2a：确定NQO1是否改变细胞的氧化还原状态，诱导由还原型：氧化(-生育酚)、还原：氧化泛醌和还原：氧化谷胱甘肽水平决定的相对还原环境。目的2b：确定在臭氧暴露后，NQO1是否引起异前列腺素生成的改变，同时F2-IsoP的形成相对增加，反之，NQO1在体内的丢失是否会导致A2-IsoP的形成增加。目的3a：确定臭氧暴露后，F2-isP和/或臭氧产生的ROS是否通过激活核因子-B上调IL-8/KC的表达。目的3b：确定在没有NQO1的情况下，这一调节是否被A2-isP抑制核因子-(B)从I(B)释放而取消。

DESCRIPTION (provided by applicant): Individual responses to ozone vary widely with some individuals having a much lower threshold of ozone sensitivity for pulmonary toxicity. Therefore, a complex of individual genetic factors likely controls the transmission and propagation of oxidant signaling following ozone exposure. Epidemiologic studies support the role of the wild-type NADPH quinone oxidoreductase1 (NQO1) genotype as an asthma susceptibility factor in the presence of ozone. NQO1 catalyzes the obligate 2-electron reduction of quinones and is considered an intracellular antioxidant. Our preliminary data demonstrate that NQO1 is required for ozone-induced IL-8/ KC expression, increased neutrophilic airway inflammation, and airway hyperresponsiveness in mice. This poses a conundrum to explain how an oxidoreductase that recycles antioxidants propagates ROS signaling linking oxidant stress to an inflammatory response. We will use primary human airway epithelial cells and mouse (wild-type and NQO1-null) model systems to test the following unprecedented hypothetical mechanism to explain how NQO1 links oxidant stress to epithelial inflammation: NQO1 and ozone-generated ROS are central regulators of airway inflammation following ozone exposure. We propose that NQO1 regulates the intracellular redox environment resulting in a shift in the balance of isoprostanes (isoP). In the presence of NQO1, ROS and F2-isoP activate NF-(B, resulting in increased IL-8/ KC expression and increased neutrophilic inflammation. In the absence of NQO1, the cell favors A2-isoP production, which inhibits NF-(B activation, causing the paradoxical effect of blocking IL-8/ KC expression and neutrophilic inflammation. The specific aims are: Aim 1a: To determine whether following ozone exposure, NQO1 mediates neutrophilic inflammation and airway hyperresponsiveness via upregulation of the neutrophil chemokines KC/ IL-8. Aim 1b: To determine whether NQO1 expression in structural airway epithelial cells and/or in hematopoetic cells is required for pulmonary responses to ozone. Aim 2a: To determine whether NQO1 alters the cellular redox state, inducing a relative reducing environment as determined by levels of reduced: oxidized (-tocopherol, reduced: oxidized ubiquinone and reduced: oxidized glutathione. Aim 2b: To determine whether following ozone exposure, NQO1 causes a shift in isoprostane production with a relative increase in F2- isoP formation and conversely, a loss of NQO1 in vivo, causes increased formation of A2-isoP. Aim 3a: To determine whether following ozone exposure, F2-isoP and/or ozone-generated ROS upregulate IL-8/KC expression by NF-(B activation. Aim 3b: To determine whether this regulation is abrogated in the absence of NQO1 by A2-isoP inhibition of NF-(B release from I(B.