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，复杂的个体遗传因素可能控制臭氧暴露后氧化剂信号的传播和传播。流行病学研究支持野生型 NADPH 醌氧化还原酶 1 (NQO1) 基因型在臭氧存在下作为哮喘易感因素的作用。 NQO1 催化醌的专性 2 电子还原，被认为是一种细胞内抗氧化剂。我们的初步数据表明，NQO1 是臭氧诱导的 IL-8/KC 表达、小鼠中性粒细胞气道炎症增加和气道高反应性所必需的。这就提出了一个难题，无法解释回收抗氧化剂的氧化还原酶如何传播将氧化应激与炎症反应联系起来的 ROS 信号。我们将使用原代人类气道上皮细胞和小鼠（野生型和 NQO1-null）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 是臭氧暴露后气道炎症的中央调节剂。我们认为 NQO1 调节细胞内氧化还原环境，导致异前列烷 (isoP) 平衡发生变化。在 NQO1 存在的情况下，ROS 和 F2-isoP 激活 NF-(B，导致 IL-8/KC 表达增加，中性粒细胞炎症增加。在 NQO1 不存在的情况下，细胞有利于 A2-isoP 的产生，A2-isoP 抑制 NF-(B 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： 目标 1a：确定臭氧暴露后是否NQO1 通过上调中性粒细胞趋化因子 KC/IL-8 介导中性粒细胞炎症和气道高反应性 目标 1b：确定气道结构上皮细胞和/或造血细胞中的 NQO1 表达是否是肺部对臭氧反应所必需的 目标 2a：确定 NQO1 是否会改变细胞氧化还原状态，从而诱导相对还原环境（根据还原：氧化水平确定）。 （-生育酚，还原型：氧化泛醌，还原型：氧化型谷胱甘肽。目标 2b：确定臭氧暴露后，NQO1 是否会导致异前列腺素生成发生变化，同时 F2-isoP 形成相对增加，相反，体内 NQO1 的损失会导致 A2-isoP 形成增加。目标 3a：确定臭氧暴露后，F2-isoP 和/或臭氧生成的 ROS 是否会增加通过 NF-(B 激活上调 IL-8/KC 表达。目标 3b：确定在 NQO1 不存在的情况下，A2-isoP 抑制 I(B.

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.