NQO1: Linking Oxidant Stress to Inflammation in Airway Epithelial Cells

描述（由申请人提供）：个体对臭氧的反应差异很大，有些个体对臭氧肺毒性的敏感性阈值要低得多。因此，一个复杂的个人遗传因素可能控制臭氧暴露后氧化剂信号的传输和传播。流行病学研究支持野生型NADPH醌氧化还原酶1（NQO 1）基因型在臭氧存在下作为哮喘易感因素的作用。NQO 1催化醌类的专性2电子还原，被认为是细胞内抗氧化剂。我们的初步数据表明，NQO 1需要臭氧诱导的IL-8/ KC的表达，增加嗜酸性气道炎症，气道高反应性小鼠。这提出了一个难题，以解释如何氧化还原酶，消除抗氧化剂传播ROS信号连接氧化应激炎症反应。我们将使用原代人气道上皮细胞和小鼠（野生型和NQO 1-null）模型系统来测试以下前所未有的假设机制，以解释NQO 1如何将氧化应激与上皮炎症联系起来：NQO 1和臭氧产生的ROS是臭氧暴露后气道炎症的中心调节因子。我们建议，NQO 1调节细胞内的氧化还原环境，导致异前列腺素（isoP）的平衡的转变。在NQO 1存在下，ROS和F2-isoP激活NF-β B，导致IL-8/ KC表达增加和嗜酸性炎症增加。在缺乏NQO 1的情况下，细胞有利于A2-isoP的产生，其抑制NF-β B活化，导致阻断IL-8/ KC表达和嗜酸性炎症的矛盾效应。具体目标是：目标1a：为了确定臭氧暴露后，NQO 1是否通过上调中性粒细胞趋化因子KC/ IL-8介导嗜中性粒细胞炎症和气道高反应性。目标1b：为了确定是否NQO 1的表达在结构气道上皮细胞和/或造血细胞是肺对臭氧的反应所必需的。目标2a：为了确定NQO 1是否改变细胞氧化还原状态，诱导相对还原环境，如通过还原：氧化β-生育酚、还原：氧化泛醌和还原：氧化谷胱甘肽的水平所确定的。目标2b：为了确定在臭氧暴露后，NQO 1是否导致异前列烷产生的变化，F2- isoP形成的相对增加，相反，体内NQO 1的损失是否导致A2-isoP形成的增加。目的3a：确定在臭氧暴露后，F2-isoP和/或臭氧产生的ROS是否通过NF-β B活化上调IL-8/KC表达。目的3b：确定在NQO 1不存在的情况下，通过A2-isoP抑制NF-B从I β释放是否废除了这种调节。

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.