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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

基本信息

批准号：
7732482
负责人：
Thomas Leto
金额：
$ 156.89万
依托单位：
NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
依托单位国家：
美国
项目类别：
财政年份：
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/7732482
关键词：
4-ethoxymethylene-2-phenyl-2-oxazoline-5-one Affect Anabolism Anemia Angiotensin II Animal Model Animals Antimicrobial Resistance Apical Apoptosis Binding Biochemical Blood Vessels Brain Burkholderia cepacia C2 Domain Cell Aging Cell membrane Cell model Cells Chemistry Chronic Chronic Granulomatous Disease Chronic Obstructive Airway Disease Chronic Obstructive Asthma Cirrhosis Colon Complementary DNA Cystic Fibrosis Cytosolic Phospholipase A2 Defect Disease Enzymes Epithelial Cells Erythropoiesis Erythropoietin Exhibits Exocrine Glands Extracellular Matrix Family Feedback Future Gastrointestinal tract structure Gene Expression Genes Genetic Polymorphism Genus Cola Goals Granulomatous Growth Factor Hematology Hepatitis C virus Hepatocyte Homeostasis Hormones Host Defense Human Hydrogen Peroxide Hypoxia Immune Immunocompromised Host Infection Inflammation Mediators Inflammatory Inflammatory Response Interferon Type II Interleukin-13 Interleukin-4 Isoenzymes Kidney Light Link Liver Lung Mediating Membrane Mouse Strains Mucous Membrane Mus Mutation Myelogenous NADP NADPH Oxidase Natural Immunity Organism Oxidants Oxidases Oxidation-Reduction Oxidative Stress Oxygen Pathology Patients Pattern Pattern Recognition Peptides Peroxidase Peroxidases Phagocytes Phagocytosis Phagosomes Phenotype Predisposition Process Production Protein Kinase C Pseudomonas aeruginosa RNA Splicing Range Reactive Oxygen Species Recombinants Recruitment Activity Reporter Respiratory Burst Role SH3 Domains Salivary Glands Serum Signal Pathway Signal Transduction Site Source Staphylococcus aureus Stimulus Superoxides Surface System Thinking Thyroid Gland Tissues Transforming Growth Factor beta Urine Variant Viral Virulence Factors Work angiogenesis antimicrobial arachidonate base cytokine design extracellular inhibitor/antagonist insight interest kidney cell killings lactoperoxidase microbial microbicide mouse model neutrophil neutrophil cytosol factor 40K neutrophil cytosol factor 67K novel novel therapeutics pathogen pathogen exposure phospholipase C beta programs promoter reconstitution response superoxide-generating NADPH oxidase tissue/cell culture

项目摘要

This program explores innate anti-microbial defense and inflammatory mechanisms involving the host's ability to deliberately produce reactive oxygen species (ROS). Neutrophils and other circulating phagocytes generate high levels of ROS in response to infectious or inflammatory stimuli in a process known as the respiratory burst. The response is attributed to the activity of NADPH oxidase, which produces superoxide, a precursor of ROS that are important microbicidal agents and mediators of inflammation. Patients with chronic granulomatous disease (CGD) suffer from NADPH oxidase deficiencies, resulting in enhanced susceptibility to microbial infections and aberrant inflammatory responses. This project explores the cellular mechanisms regulating the respiratory burst oxidase in phagocytes (phox or Nox2-based system) and is characterizing related oxidant-generating NADPH oxidases expressed in non-immune cells (Nox1, Nox3, Nox4, Nox5, Duox1, Duox2), now known as "Nox family" NADPH oxidases. We are studying sources of ROS in several non-myeloid tissues, notably colon, kidney, liver thyroid and salivary glands, mucosal surfaces (lung and gastrointestinal tract), brain, and vascular tissues. Our recent evidence indicates several of these non-phagocytic Nox enzymes also serve in host defense and inflammatory processes, since they are expressed predominately on apical surfaces of epithelial cells and are induced or activated by pro-inflammatory cytokines or recognition of microbial factors. ROS produced by these enzymes also provide redox signals that can affect gene expression patterns during differentiation, cellular senescence, programmed cell death (apoptosis), oxygen sensing, or responses to infection, growth factors, cytokines, or hormones. In the last year, we have explored the functional importance of dual oxidases (Duox1 and Duox2) in human airway epithelial cells and have developed the means for their reconstitution in transfected cell models. Duox targeted to the apical surfaces of bronchial epithelial cells provides extracellular hydrogen peroxide needed to support the well-documented anti-microbial activities of lactoperoxidase. We examined airway epithelial cell Duox expression and activity in response to differentiation, pathogen exposure, and pro-inflammatory cytokines. We demonstrated Duox- and lactoperoxidase-dependent microbial killing of Pseudomonas aeruginosa, Burkholderia Cepacia, and Staphylococcus aureus, organisms that commonly infect airways of immunocompromised patients, including those with Cystic Fibrosis. We explored host-pathogen interactions involved in oxidative antimicrobial resistance as well as the adaptive microbial counter-defenses to these oxidants. We identified specific effects of the Pseudomonas aeruginosa virulence factor, pyocynin, showing that it can act as a cell permeable, redox-active inhibitor of Duox activity and expression, as it consumes intracellular NADPH and imposes oxidative stress on airway cells. The findings provide novel insight on the adaptive capabilities of this opportunistic pathogen and shed light on pathology of chronic infections encountered in Cystic Fibrosis. We confirmed that airway Duox isozymes are induced by interferon-gamma, IL-4, and IL-13, suggesting roles in airway viral and microbial infection and in inflammatory airway disease (i.e., asthma, chronic obstructive pulmonary disease). We are also exploring determinants enabling delivery of active Duox to the plasma membrane, where it supports extracellular antimicrobial peroxidases. Active recombinant forms of Duox co-expressed along with essential maturation factors have been produced in whole transfected cells. We identified multiple splice variants of the Duox1 maturation factor (DuoxA1) capable of targeting Duox1 to distinct subcellular sites. These recombinant systems will be used to screen for Duox and maturation factor genetic polymorphisms associated with altered oxidase targeting and function. In efforts aimed at exploring functional roles of the renal oxidase (Nox4 or Renox), we are characterizing mouse strains in which the Nox4 gene is deleted. We are investigating the proposed role of Nox4 in renal oxygen sensing and erythropoiesis, since ROS are thought to provide feedback signals regulating renal erythropoietin synthesis. The renal oxidase is a constitutively active enzyme, consistent with its proposed role as an oxygen-sensing enzyme. Surprisingly, Nox4-deficient mice exhibit a normal phenotype in the unstressed state. Hematology as well as serum and urine chemistries (i.e., urine hydrogen peroxide levels) are normal in these animals. Related gene microarray studies are focused on identifying alterations in other oxidant generating or scavenging systems to explore mechanisms maintaining normal redox homeostasis in Nox4-deficient mice. Nox4 levels respond directly to Transforming Growth Factor-beta (TGF-beta) or hypoxia in renal cells and to hepatitis C virus (HCV) in hepatic cells. Furthermore, the Nox4 promoter, fused to Nox4 cDNA or to other reporters, responds to hypoxia, TGF-beta and HCV. Based on these findings, future work will examine the responses of Nox4-deficient mice to these factors to assess potential roles of Nox4 in fibrotic disease (cirrhosis) and redox homeostasis related to hypoxia (angiogenesis, anemia). In studies aimed at defining the sources of ROS responsive to angiotensin II-mediated cell stimulation, we used reconstituted Nox1 and Nox2 transfected cell models to show involvement of a cell signaling pathway comprising AT(1)R, Galpha(q/11), phospholipase C-beta, and protein kinase C. Finally, our long-standing interests in the phagocytic oxidase are focused on the activating roles of cytosolic regulators, p40phox and cytosolic phospholipase A2 (cPLA2). We have shown that cPLA2 is recruited to the membrane during cellular activation through direct interactions with the p47phox oxidase component, involving the cPLA2 C2 domain and the p47phox PX domain. Peptides designed from these interacting domains were shown to be effective inhibitors of the oxidase in intact neutrophils. We also explored the role of p40phox as a positive oxidase regulator in cells undergoing phagocytosis, as it functions as an adaptor promoting retention of other regulators (p47phox and p67phox) on phagosomes. We also showed that p40phox recruitment to phagosomal membranes involves arachidonate-dependent exposure of its membrane-binding PX domain. Finally, we also obtained evidence for direct interactions between SH3 domains of p47phox and the catalytic gp91phox (Nox2) component. These findings broaden our understanding of the mechanisms of phagocytic oxidase activation and may suggest novel therapeutic strategies for modifying oxidase activity.

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。中性粒细胞和其他循环吞噬细胞在称为呼吸爆发的过程中响应感染或炎症刺激而产生高水平的 ROS。该反应归因于 NADPH 氧化酶的活性，它产生超氧化物，超氧化物是 ROS 的前体，是重要的杀菌剂和炎症介质。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。该项目探索调节吞噬细胞（基于 phox 或 Nox2 的系统）中呼吸爆发氧化酶的细胞机制，并表征在非免疫细胞中表达的相关氧化剂生成 NADPH 氧化酶（Nox1、Nox3、Nox4、Nox5、Duox1、Duox2），现在称为“Nox 家族”NADPH 氧化酶。我们正在研究几种非骨髓组织中活性氧的来源，特别是结肠、肾脏、肝脏、甲状腺和唾液腺、粘膜表面（肺和胃肠道）、大脑和血管组织。我们最近的证据表明，其中一些非吞噬性 Nox 酶也参与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，并由促炎细胞因子或微生物因子的识别诱导或激活。这些酶产生的 ROS 还提供氧化还原信号，可影响分化、细胞衰老、程序性细胞死亡（细胞凋亡）、氧感应或对感染、生长因子、细胞因子或激素的反应期间的基因表达模式。去年，我们探索了双氧化酶（Duox1 和 Duox2）在人气道上皮细胞中的功能重要性，并开发了在转染细胞模型中重建它们的方法。 Duox 靶向支气管上皮细胞的顶端表面，提供细胞外过氧化氢，以支持乳过氧化物酶的抗菌活性。我们检测了气道上皮细胞 Duox 的表达和活性，以响应分化、病原体暴露和促炎细胞因子。我们证明了 Duox 和乳过氧化物酶依赖性微生物对铜绿假单胞菌、洋葱伯克霍尔德菌和金黄色葡萄球菌的杀灭作用，这些微生物通常感染免疫功能低下患者（包括囊性纤维化患者）的气道。我们探索了参与氧化抗菌药物耐药性的宿主-病原体相互作用以及对这些氧化剂的适应性微生物反防御。我们确定了铜绿假单胞菌毒力因子绿脓菌素的具体作用，表明它可以作为 Duox 活性和表达的细胞渗透性氧化还原活性抑制剂，因为它消耗细胞内 NADPH 并对气道细胞施加氧化应激。这些发现为这种机会性病原体的适应能力提供了新的见解，并揭示了囊性纤维化中遇到的慢性感染的病理学。我们证实气道 Duox 同工酶是由干扰素 γ、IL-4 和 IL-13 诱导的，表明其在气道病毒和微生物感染以及炎症性气道疾病（即哮喘、慢性阻塞性肺疾病）中发挥作用。我们还在探索能够将活性 Duox 传递到质膜的决定因素，在质膜上它支持细胞外抗菌过氧化物酶。 Duox 的活性重组形式与必需的成熟因子共表达，已在全转染细胞中产生。我们鉴定了 Duox1 成熟因子 (DuoxA1) 的多个剪接变体，能够将 Duox1 靶向不同的亚细胞位点。这些重组系统将用于筛选与氧化酶靶向和功能改变相关的 Duox 和成熟因子遗传多态性。为了探索肾氧化酶（Nox4 或 Renox）的功能作用，我们正在鉴定 Nox4 基因缺失的小鼠品系。我们正在研究 Nox4 在肾氧传感和红细胞生成中的拟议作用，因为 ROS 被认为提供调节肾红细胞生成素合成的反馈信号。肾氧化酶是一种组成型活性酶，与其作为氧敏感酶的作用一致。令人惊讶的是，Nox4 缺陷小鼠在无应激状态下表现出正常的表型。这些动物的血液学以及血清和尿液化学（即尿液过氧化氢水平）均正常。相关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的改变，以探索维持 Nox4 缺陷小鼠正常氧化还原稳态的机制。 Nox4 水平直接对肾细胞中的转化生长因子-β (TGF-β) 或缺氧以及肝细胞中的丙型肝炎病毒 (HCV) 做出反应。此外，与 Nox4 cDNA 或其他报告基因融合的 Nox4 启动子对缺氧、TGF-β 和 HCV 做出反应。基于这些发现，未来的工作将检查Nox4缺陷小鼠对这些因素的反应，以评估Nox4在纤维化疾病（肝硬化）和与缺氧（血管生成、贫血）相关的氧化还原稳态中的潜在作用。在旨在确定对血管紧张素 II 介导的细胞刺激有反应的 ROS 来源的研究中，我们使用重建的 Nox1 和 Nox2 转染细胞模型来显示包含 AT(1)R、Galpha(q/11)、磷脂酶 C-β 和蛋白激酶 C 的细胞信号传导通路的参与。最后，我们对吞噬氧化酶的长期兴趣集中在胞质调节因子 p40phox 和胞质磷脂酶 A2 (cPLA2) 的激活作用上。我们已经证明，在细胞激活过程中，cPLA2 通过与 p47phox 氧化酶成分直接相互作用（涉及 cPLA2 C2 结构域和 p47phox PX 结构域）而被招募到膜上。由这些相互作用的结构域设计的肽被证明是完整中性粒细胞中氧化酶的有效抑制剂。我们还探讨了 p40phox 作为正氧化酶调节剂在细胞吞噬作用中的作用，因为它作为接头促进其他调节剂（p47phox 和 p67phox）在吞噬体上的保留。我们还表明，p40phox 招募至吞噬体膜涉及其膜结合 PX 结构域的花生四烯酸依赖性暴露。最后，我们还获得了 p47phox 的 SH3 结构域与催化 gp91phox (Nox2) 成分之间直接相互作用的证据。这些发现拓宽了我们对吞噬氧化酶激活机制的理解，并可能提出改变氧化酶活性的新治疗策略。

项目成果

期刊论文数量（32）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Cystosolic phospholipase A2alpha is targeted to P47phox-PX domain of the assembled NADPH oxidase via a novel binding site in its C2 domain.

胞浆磷脂酶 A2alpha 通过其 C2 结构域中的新结合位点靶向组装的 NADPH 氧化酶的 P47phox-PX 结构域。

DOI：
10.1074/jbc.m804674200
发表时间：
2008
期刊：
The Journal of biological chemistry
影响因子：
0
作者：
Shmelzer,Zeev;Karter,Maria;Eisenstein,Miriam;Leto,ThomasL;Hadad,Nurit;Ben-Menahem,David;Gitler,Daniel;Banani,Shirly;Wolach,Baruch;Rotem,Meir;Levy,Rachel
通讯作者：
Levy,Rachel

Genetic requirement of p47phox for superoxide production by murine microglia.

p47phox 小鼠小胶质细胞产生超氧化物的遗传要求。