Nox family NADPH oxidases: roles in innate immunity and inflammatory disease

该项目探索先天抗微生物防御和炎症机制，涉及宿主故意产生活性氧 (ROS) 的能力。循环吞噬细胞产生高水平的 ROS，作为响应感染或炎症刺激的重要杀菌剂，这归因于 NADPH 氧化酶的激活。慢性肉芽肿病 (CGD) 患者缺乏 NADPH 氧化酶，导致对微生物感染和异常炎症反应的易感性增强。该项目目前的重点是探索调节非吞噬细胞（Nox1、Nox4、Duox1、Duox2）中表达的相关Nox家族NADPH氧化酶的细胞机制，特别是粘膜表面（肺和胃肠道）、肝脏、肾脏、甲状腺和唾液腺、大脑和血管组织。其中几种非吞噬性 Nox 酶在宿主防御和炎症过程中发挥作用，因为它们主要在上皮细胞的顶端表面表达，并由细胞因子或通过识别病原体相关分子模式诱导或激活。这些氧化酶产生的 ROS 还提供氧化还原信号，影响感染、氧传感、生长因子、激素、细胞因子、细胞分化、细胞衰老、程序性细胞死亡（细胞凋亡）反应期间的基因表达模式。 2010 年，我们探索了宿主对两种模型病原体的先天免疫反应，涉及非吞噬细胞产生过量的 ROS：1) 铜绿假单胞菌，作为一种细菌病原体，在气道上皮细胞中引发 Duox 衍生的 ROS；2) 丙型肝炎病毒 (HCV)，作为受感染肝细胞中过量 Nox4 衍生的 ROS 的引发剂，可导致促纤维化肝损伤（肝硬化）在慢性感染的患者中。使用人类气道上皮细胞模型，我们比较了暴露于几种气道病原体（铜绿假单胞菌、洋葱伯克霍尔德菌和金黄色葡萄球菌）的影响，结果表明，只有铜绿假单胞菌通过需要细胞外钙和暴露于新鲜生长的细菌的机制触发 Duox1 衍生的过氧化氢释放。通过比较宿主上皮细胞对各种假单胞菌突变体的反应，我们得出结论，几种微生物因子协同作用以引发 Duox 活性：微生物表面因子（鞭毛蛋白和脂多糖）能够粘附到宿主细胞，从而促进更有效的 3 型分泌系统依赖性 Duox1 激活。这些发现表明了一种从健康气道中消除这种病原体的机制。 相比之下，我们发现过度生长的假单胞菌培养物作为慢性感染肺部生物膜中建立的假单胞菌转化状态的模型，产生氧化还原活性毒力因子（绿脓菌素），可以竞争性抑制 Duox 活性并产生细胞内 ROS。我们探讨了气道上皮细胞慢性绿脓素暴露引起的氧化应激的影响。两天的绿脓素暴露（8微摩尔）会引发多种促炎细胞因子以及表皮生长因子受体（EGFR）配体的分泌，从而触发主要气道粘蛋白的转录和释放。这些反应再现了慢性假单胞菌感染引起的晚期囊性纤维化疾病表型的许多特征，表明气道上皮中绿脓素介导的氧化应激是气道炎症、粘液过度分泌和循环炎症细胞募集的主要决定因素。 我们对 HCV 感染的先天氧化反应的研究正在探索与转化生长因子-β (TGF-B) 水平升高和肝纤维化相关的肝损伤原因。感染或转染 HCV 或单独 HCV 核心蛋白的肝细胞显示 ROS 产量增加以及 Nox4 mRNA 和蛋白质水平增加。相比之下，表达Nox4短发夹RNA（RNA干扰）或Nox4的截短、显性阴性形式的肝细胞在转染HCV时表现出ROS产生减少。人类和小鼠 Nox4 的启动子都证明了 HCV 对 Nox4 mRNA 的转录调节。对与一系列 Nox4 启动子片段 (0.7-2.4 kb) 相关的荧光素酶报告基因进行分析，确定了调节 Nox4 表达的 HCV 响应调控区域；这些人类 Nox4 启动子片段也对 TGF-B1 有反应。此外，HCV 核心依赖性 Nox4 诱导被 TGF-B 中和抗体或显性负性 TGF-B 受体 II 型的表达所阻断。 总的来说，这些发现确定 HCV 通过自分泌 TGF-B 依赖性信号传导机制作为 Nox4 表达的调节剂。这些数据提供证据表明，HCV 诱导的 Nox4 有助于 ROS 的产生，这可能与慢性 HCV 诱导的肝病有关。 在探索 Nox4（或 Renox）的其他功能作用的过程中，我们正在对 Nox4 基因被删除的小鼠进行表征。 Nox4 缺陷小鼠在无应激状态下表现出正常的寿命和表型。基因微阵列研究的重点是识别其他氧化剂生成或清除系统的补偿性改变，以探索 Nox4 缺陷小鼠维持正常氧化还原稳态的机制。 Nox4 具有组成型活性，与其作为氧敏感酶的作用一致。我们正在研究 Nox4 在氧传感和造血中的拟议作用，因为 ROS 被认为提供调节肾促红细胞生成素合成的反馈信号。未来的工作将检查 Nox4 缺陷小鼠对各种应激源的反应，以评估 Nox4 在缺氧、感染或炎症期间氧化还原稳态和基于氧化还原的信号传导中的潜在作用。 我们在 Duox 重建技术方面取得的进展正用于筛选假定的 Duox（或 DuoxA）单核苷酸多态性 (SNP) 或改变氧化酶功能或细胞靶向的突变的影响，这可能与气道感染或炎症性疾病（囊性纤维化、哮喘、细菌或病毒感染）的易感性改变有关。

This program explores innate anti-microbial defense and inflammatory mechanisms involving the host's ability to deliberately produce reactive oxygen species (ROS). Circulating phagocytes generate high levels of ROS that serve as important microbicidal agents in response to infectious or inflammatory stimuli, which is attributed to NADPH oxidase activation. Patients with chronic granulomatous disease (CGD) suffer from NADPH oxidase deficiencies, resulting in enhanced susceptibility to microbial infections and aberrant inflammatory responses. The current focus of this project explores cellular mechanisms regulating related Nox Family NADPH oxidases expressed in non-phagocytic cells (Nox1, Nox4, Duox1, Duox2), notably on mucosal surfaces (lung and gastrointestinal tract), the liver, kidney, thyroid and salivary glands, brain, and vascular tissues. Several of these non-phagocytic Nox enzymes serve in host defense and inflammatory processes, as they are expressed predominately on apical surfaces of epithelial cells and are induced or activated by cytokines or by recognition of pathogen-associated molecular patterns. ROS produced by these oxidases also provide redox signals that affect gene expression patterns during responses to infection, oxygen sensing, growth factors, hormones, cytokines, cell differentiation, cellular senescence, programmed cell death (apoptosis). In 2010, we explored host innate immune responses to two model pathogens involving excess ROS production by non-phagocytic cells: 1) Pseudomonas aeruginosa, as a bacterial pathogen that elicits Duox-derived ROS in airway epithelial cells and 2) Hepatitis C virus (HCV), as an elicitor of excess Nox4-derived ROS in infected hepatocytes, which can lead to pro-fibrotic liver injury (cirrhosis) in chronically infected patients. Using human airway epithelial cell models we compared the effects of exposure to several airway pathogens (Pseudomonas aeruginosa, Burkholderia cepacia, and Staphylococcus aureus) and showed only P. aeruginosa triggers Duox1-derived hydrogen peroxide release through a mechanism requiring extracellular calcium and exposure to freshly grown bacteria. By comparing host epithelial cell responses to various Pseudomonas mutants, we concluded that several microbial factors act cooperatively to elicit Duox activity: microbial surface factors (flagellin and lipopolysaccharide) enable adhesion to host cells, thereby promoting a more efficient Type-3 Secretion System-dependent Duox1 activation. These findings suggest a mechanism by which this pathogen is eliminated from healthy airways. In contrast, we showed that over-grown Pseudomonas cultures, as a model of the transformed state of Pseudomonas established in biofilms of chronically infected lungs, produce a redox-active virulence factor (pyocyanin) that can competitively inhibit Duox activity and produce intracellular ROS. We explored the effects of oxidative stress caused by chronic pyocyanin exposure of airway epithelial cells. Two-day pyocyanin exposure (8 micromolar) elicits secretion of several pro-inflammatory cytokines as well as epidermal growth factor receptor (EGFR) ligands that trigger transcription and release of the major airway mucins. These responses reproduce many features of the advanced cystic fibrosis disease phenotype with chronic Pseudomonas infection, suggesting that pyocyanin-mediated oxidative stress in the airway epithelium is a major determinant in airway inflammation, mucus hyper-secretion, and recruitment of circulating inflammatory cells. Our studies on innate oxidative responses to HCV infection are exploring causes of hepatic injury linked to increased transforming growth factor-beta (TGF-B) levels and hepatic fibrosis. Hepatocytes infected or transfected with HCV, or HCV core protein alone, showed increased ROS production along with increased Nox4 mRNA and protein levels. In contrast, hepatocytes expressing Nox4 short hairpin RNA (RNA interference) or a truncated, dominannt-negative form of Nox4 showed decreased ROS production when transfected with HCV. The promoters of both human and murine Nox4 demonstrated transcriptional regulation of Nox4 mRNA by HCV. Analysis of luciferase reporters tied to a series of Nox4 promoter fragments (0.7-2.4 kb) identified HCV-responsive regulatory regions modulating Nox4 expression; these human Nox4 promoter fragments were also responsive to TGF-B1. Furthermore, HCV core-dependent induction of Nox4 was blocked by TGF-B-neutralizing antibodies or the expression of dominant negative TGF-B receptor type II. Collectively, these findings identified HCV as a regulator of Nox4 expression through an autocrine TGF-B-dependent signaling mechanism. These data provide evidence that HCV-induced Nox4 contributes to ROS production that may be related to chronic HCV-induced liver disease. In efforts exploring other functional roles of Nox4 (or Renox), we are characterizing mice in which the Nox4 gene is deleted. Nox4-deficient mice exhibit a normal lifespan and phenotype in the unstressed state. Gene microarray studies are focused on identifying compensating alterations in other oxidant generating or scavenging systems to explore mechanisms maintaining normal redox homeostasis in Nox4-deficient mice. Nox4 is constitutively active, consistent with its proposed role as an oxygen-sensing enzyme. We are investigating the proposed role of Nox4 in oxygen sensing and hematopoiesis, as ROS are thought to provide feedback signals regulating renal erythropoietin synthesis. Future work will examine responses of Nox4-deficient mice to various stressors to assess potential roles of Nox4 in redox homeostasis and redox-based signaling during exposure to hypoxia, infection, or inflammation. Our advances in Duox reconstitution technology are being used to screen effects of putative Duox (or DuoxA) single nucleotide polymorphisms (SNPs) or mutations in altering oxidase function or cellular targeting, which may relate to altered susceptibilities to airway infectious or inflammatory disease (cystic fibrosis, asthma, bacterial or viral infection).