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

Nox 家族 NADPH 氧化酶：在先天免疫和炎症性疾病中的作用

• 批准号: 8156865
• 负责人: THOMAS LETO
• 金额: $ 145.86万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8156865
• 关键词: Disease; Family; Inflammatory; NADPH Oxidase; Natural Immunity; Role

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).

该计划探讨了先天的抗微生物防御和炎症机制，涉及宿主故意生产活性氧（ROS）的能力。循环吞噬细胞会产生高水平的ROS，这些ROS响应于传染性或炎症刺激，这是NADPH氧化酶的激活。患有慢性肉芽肿性疾病（CGD）的患者患有NADPH氧化酶缺乏症，导致对微生物感染和异常炎症反应的敏感性增强。该项目的当前重点探讨了调节在非噬细胞细胞（NOX1，NOX4，DUOX1，DUOX2）中表达的相关NOX家族NADPH氧化酶的细胞机制，尤其是在粘膜表面（肺和胃肠道），肝脏，肾脏，肾脏，甲状腺和乳腺癌和脑，脑和脑，脑，脑，脑，脑，脑，脑，脑，脑，脑，脑，脑，脑，脑，脑，脑，脑，脑，脑，脑，脑，脑，脑，脑，脑，脑，脑，脑，脑，脑，脑，脑，脑，脑，脑，脑，脑，脑，脑，脑，脑，脑，脑，脑，脑，脑，脑，脑，脑，脑，脑，脑，脑，脑，脑，脑，脑和乳液。这些非斑点NOX酶中的几种用于宿主防御和炎症过程，因为它们主要在上皮细胞的顶端表面表达，并被细胞因子或通过病原体相关的分子模式诱导或激活。这些氧化酶产生的ROS还提供了氧化还原信号，该信号会影响感染，氧气感应，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）的反应期间基因表达模式。 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长期感染患者的促纤维化肝损伤（肝硬化）。使用人类气道上皮细胞模型，我们比较了接触几种气道病原体的影响（铜绿假单胞菌，伯克霍尔德氏菌和金黄色葡萄球菌），仅显示铜绿假单胞菌触发了DUOX1衍生的氢根氢，需要通过一种机制释放氧化氢和膨胀的机制，需要新鲜的甲基钙化。通过比较宿主对各种假单胞菌突变体的上皮细胞反应，我们得出的结论是，几种微生物因子起着引起DUOX活性的作用：微生物表面因子（鞭毛蛋白和脂多糖）能够对宿主细胞进行粘附，从而促进一种更有效的类型-3分泌系统依赖性Duox1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，我们表明，过度生长的假单胞菌培养物是在慢性感染肺生物膜中建立的假单胞菌状态转化状态的模型，产生了氧化还原活性毒力因子（pyocyanin），可以竞争地抑制Duox活性并产生内细胞内的ROS。我们探索了由气道上皮细胞暴露于慢性增生酸引起的氧化应激的影响。为期两天的增自氨酸暴露（8微摩尔）引起了几种促炎性细胞因子以及表皮生长因子受体（EGFR）配体的分泌，这些配体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的晚期囊性纤维化疾病表型的许多特征，这表明气道上皮中的增生素蛋白介导的氧化应激是气道炎症，粘液过度分泌和循环炎性细胞的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肝损伤的原因，该原因与增加的生长因子-BETA（TGF-B）水平和肝纤维化有关。被HCV感染或转染的肝细胞单独感染或转染HCV核蛋白，显示ROS产生增加，NOX4 mRNA和蛋白质水平升高。相比之下，表达NOX4短发夹RNA（RNA干扰）或截短的，Dominannt阴性的NOX4的肝细胞在用HCV转染时显示出ROS的产生降低。人类和鼠NOX4的启动子均表现出HCV对NOX4 mRNA的转录调控。分析与一系列NOX4启动子片段（0.7-2.4 kb）相关的荧光素酶报道者确定了HCV反应性调节区调节NOX4表达；这些人NOX4启动子片段也对TGF-B1有反应。此外，HCV核心依赖性诱导NOX4被TGF-B中和抗体阻止或显性负TGF-B受体II型的表达。 总的来说，这些发现通过自分泌TGF-B依赖性信号传导机制将HCV确定为NOX4表达的调节剂。这些数据提供了HCV诱导的NOX4有助于ROS产生的证据，这可能与慢性HCV诱导的肝病有关。 在探索NOX4（或Renox）其他功能作用的努力中，我们正在表征删除NOX4基因的小鼠。缺乏NOX4的小鼠在无重理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产生或清除系统中的补偿改变，以探索在NOX4缺陷小鼠中维持正常氧化还原稳态的机制。 NOX4具有组成性活跃，与其作为氧气酶的作用一致。我们正在研究NOX4在氧气传感和造血的拟议作用，因为ROS被认为提供了调节肾红细胞生成素合成的反馈信号。未来的工作将检查NOX4缺陷小鼠对各种压力源的反应，以评估NOX4在氧化还原稳态中的潜在作用和在暴露于缺氧，感染或炎症的过程中基于氧化还原的信号传导。 我们在DUOX重建技术方面的进步被用来筛选假定的DUOX（或DUOXA）单核苷酸多态性（SNP）的效果，或在改变氧化酶功能或细胞靶向方面的突变，这可能与对空气性感染性或炎症性疾病（ASTHMA，ASTHMA，ASTHMA，ASTHMA，细菌性，细菌感染）的易感性改变有关。