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

该计划探讨先天性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，以响应感染或炎症刺激。这归因于NADPH氧化酶介导的超氧化物的产生，超氧化物是重要的杀微生物剂的ROS的前体。慢性肉芽肿病（CGD）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易感性增强。该项目探索调节吞噬NADPH氧化酶（phox或Nox 2为基础的系统）的细胞机制，并表征在非吞噬细胞中表达的相关Nox家族NADPH氧化酶（Nox 1，Nox 3，Nox 4，Nox 5，Duox 1，Duox 2）。我们正在研究几种非骨髓组织中ROS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）、脑和血管组织。这些非吞噬性Nox酶中的几种也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上表达，并且由促炎细胞因子或微生物因子的识别诱导或激活。由这些酶产生的ROS还可以提供氧化还原信号，其在分化、细胞衰老、程序性细胞死亡（凋亡）、氧感测或对感染、生长因子、激素或细胞因子的响应期间影响基因表达模式。 在2009年，我们已经推进了我们对双氧化酶（Duox 1和Duox 2）的生物合成、亚细胞靶向和活化过程的理解，因为我们已经开发了通过共表达新鉴定的Duox成熟因子（a.k.a. Duox活化剂（Duoxa））。这些研究提供了关于这些氧化酶作为专用过氧化氢发生器的功能的见解，而所有其他Nox酶都产生超氧化物。我们鉴定了几种Duoxa 1剪接变体，表明α亚型有效地将Duox靶向质膜，而γ变体将Duox靶向细胞内囊泡，在那里它可能发挥细胞内氧化还原相关功能（信号传导，增殖，迁移）。基于高尔基体的碳水化合物的Duox和DuoxA蛋白质的修饰表明，Duox成为活跃的后高尔基隔室和稳定的Duox-Duoxa二聚体复合物的检测表明，Duox成熟因子的功能作为过氧化氢生成复合物的一部分。气道上皮细胞中DuoxA 1-α亚型的主要表达与其在顶端质膜上的检测一致，Duox在顶端质膜上提供细胞外过氧化氢以支持气道表面层中基于乳过氧化物酶的抗微生物活性。我们在Duox重建技术方面的进展正在用于筛选各种Duox或DuoxA单核苷酸多态性（SNP）的影响，以及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）遗传易感性改变相关的氧化酶功能或细胞靶向改变的推定突变。 在相关研究中，我们开发了极化的人气道上皮模型（气液界面培养物），以检查Duox对病毒和细菌病原体的依赖性抗菌反应。我们已经证明，成熟的原代人支气管上皮细胞产生足够的Duox衍生的过氧化氢杀死几个气道病原体（铜绿假单胞菌，洋葱伯克霍尔德菌，金黄色葡萄球菌）。主要和重建的气道模型被用来检查Duox相关的反应，病原体暴露，包括Duox亚细胞靶向，活性氧产生，和下游氧化还原相关的细胞变化。这些关于气道上皮细胞-病原体相互作用的实验将通过Duox缺陷动物模型中的感染研究来补充。 为了探索Nox 4（或Renox）的功能作用，我们正在描述Nox 4基因缺失的小鼠。Nox 4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确定其他氧化剂产生或清除系统的改变，以探索维持Nox 4缺陷小鼠正常氧化还原稳态的机制。Nox 4是一种组成型活性酶，与其作为氧敏感酶的预期作用一致。 我们正在研究Nox 4在肾氧传感和造血中的作用，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。我们已经表明，Nox 4水平直接响应于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），我们正在探索这些效应的机制基础。未来的工作将研究Nox 4缺陷小鼠对这些因素的反应，以评估Nox 4在纤维化疾病（肝硬化）和与缺氧相关的氧化还原稳态（血管生成，贫血，血管调节）中的潜在作用。 最后，我们的兴趣，在多组分的Nox 1为基础的氧化酶的目的是在酵母双杂交筛选实验中确定的Nox激活剂1（Noxa 1）的功能合作伙伴的特征。 定点诱变实验的目的是在全细胞模型中建立参与Noxa 1激活的Noxa 1伴侣。

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 in response to infectious or inflammatory stimuli. This is attributed to NADPH oxidase-mediated production of superoxide, a precursor of ROS that are important microbicidal agents. 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 phagocytic NADPH oxidase (phox or Nox2-based system) and is characterizing related Nox Family NADPH oxidases expressed in non-phagocytic cells (Nox1, Nox3, Nox4, Nox5, Duox1, Duox2). 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. 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 can also provide redox signals that affect gene expression patterns during differentiation, cellular senescence, programmed cell death (apoptosis), oxygen sensing, or responses to infection, growth factors, hormones or cytokines. In 2009, we have advanced our understanding of processes involved in the biosynthesis, subcellular targeting and activation of the dual oxidases (Duox1 and Duox2) as we have developed systems for efficient reconstitution of the recombinant enzymes by co-expressing newly-identified Duox maturation factors (a.k.a. Duox activators (Duoxa)). These studies have provided insight on the function of these oxidases as dedicated hydrogen peroxide generators, whereas all other Nox enzymes generate superoxide. We identified several Duoxa1 splice variants, showed that the alpha isoform efficiently targets Duox to the plasma membrane, while the gamma variant targets Duox to intracellular vesicles, where it may serve intracellular redox-related functions (signaling, proliferation, migration). Golgi-based carbohydrate modifications in Duox and DuoxA proteins suggest that Duox becomes active within post-Golgi compartments and the detection of stable Duox-Duoxa dimeric complexes suggests that the Duox maturation factors function as part of the hydrogen peroxide-generating complex. Predominant expression of DuoxA1-alpha isoform in airway epithelial cells is consistent with its detection on the apical plasma membrane, where Duox provides extracellular hydrogen peroxide to support lactoperoxidase-based antimicrobial activity in the airway surface layer. Our advances in Duox reconstitution technology are being used to screen effects of various Duox or DuoxA single nucleotide polymorphisms (SNPs) and putative mutations for alterations in oxidase function or cellular targeting that may relate to altered genetic susceptibility to airway infectious or inflammatory disease (cystic fibrosis, asthma, bacterial or viral infection). In related studies, we developed polarized human airway epithelial models (air-liquid interface cultures) to examine Duox-dependent antimicrobial responses to viral and bacterial pathogens. We have shown that mature primary human bronchial epithelial cells produce sufficient Duox-derived hydrogen peroxide to kill several airway pathogens (Pseudomonas aeruginosa, Burkholderia Cepacia, and Staphylococcus aureus). The primary and reconstituted airway models are being used to examine Duox-related responses to pathogen exposure, including Duox subcellular targeting, ROS production, and downstream redox-related cellular changes. These experiments on airway epithelial cell-pathogen interactions will be complemented by infection studies in Duox-deficient animal models. In efforts aimed at exploring functional roles of Nox4 (or Renox), we are characterizing mice in which the Nox4 gene is deleted. Nox4-deficient mice exhibit a normal life-span and phenotype in the unstressed state. 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 is a constitutively active enzyme, consistent with its proposed role as an oxygen-sensing enzyme. We are investigating the proposed role of Nox4 in renal oxygen sensing and hematopoiesis, since ROS are thought to provide feedback signals regulating renal erythropoietin synthesis. We have shown that 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, and we are exploring the mechanistic basis for these effects. Future work will examine 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, vascular regulation). Finally, our interests in the multi-component Nox1-based oxidase are aimed at characterizing functional partners of Nox activator 1 (Noxa1) identified in yeast two-hybrid screening experiments. Site-directed mutagenesis experiments are aimed at establishing involvement of Noxa1 partners in Nox1 activation in whole cell models.