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

这个项目探索了天然的抗微生物防御和炎症机制，涉及宿主故意产生活性氧物种(ROS)的能力。循环中的吞噬细胞在感染或炎症刺激下产生高水平的ROS。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ROS的前体。慢性肉芽肿性疾病(CGD)患者患有NADPH氧化酶缺陷，导致对微生物感染和异常炎症反应的易感性增加。该项目探索了吞噬NADPH氧化酶(基于PHOX或NOX2的系统)的细胞调控机制，并表征了在非吞噬细胞(NOX1、NOX3、NOX4、NOX5、DOX1、DUOX2)中表达的相关的NOX家族NADPH氧化酶。我们正在研究几种非髓系组织中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。这些非吞噬的NOx酶中有几种也参与宿主防御和炎症过程，因为它们主要表达在上皮细胞的顶端表面，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还可以提供氧化还原信号，影响分化、细胞衰老、细胞程序性死亡(细胞凋亡)、氧气感应或对感染、生长因子、激素或细胞因子的反应过程中的基因表达模式。 2009年，随着我们开发了通过共表达新发现的DUOX成熟因子(又名DUOX成熟因子)来高效重组重组酶的系统，我们加深了对双氧合酶(Duox1和DUOX2)的生物合成、亚细胞靶向和激活过程的理解。DUOX激活剂(多沙))。这些研究为这些酶作为专用过氧化氢生成器的功能提供了洞察力，而所有其他NOx酶则产生超氧化物。我们鉴定了几个Duoxa1剪接变异体，表明α亚型有效地将DUOX靶向质膜，而伽马变异体靶向DUOX到细胞内的小泡，在那里它可能服务于细胞内氧化还原相关的功能(信号传递，增殖，迁移)。DUOX和DuoxA蛋白中基于高尔基体的碳水化合物修饰表明DUOX在高尔基体后的隔室中变得活跃，而稳定的DUOX-Duoxa二聚体复合体的检测表明DUOX成熟因子是过氧化氢产生复合体的一部分。DuoxA1-α亚型在呼吸道上皮细胞中的主要表达与其在顶端质膜上的检测一致，DUOX提供细胞外过氧化氢以支持呼吸道表层基于乳过氧化物酶的抗菌活性。我们在DUOX重组技术方面的进展正被用于筛选各种DUOX或DuoxA单核苷酸多态(SNPs)的影响，以及可能与呼吸道感染或炎症性疾病(囊性纤维化、哮喘、细菌或病毒感染)的遗传易感性改变有关的氧化酶功能或细胞靶向改变的突变。 在相关研究中，我们开发了极化的人类呼吸道上皮细胞模型(气液界面培养)，以检测依赖DUOX的抗菌药物对病毒和细菌病原体的反应。我们已经证明，成熟的原代人类支气管上皮细胞产生足够的DUOX衍生的过氧化氢来杀死几种呼吸道病原体(铜绿假单胞菌、洋葱伯克霍尔德氏菌和金黄色葡萄球菌)。原始和重建的呼吸道模型正被用于研究DUOX对病原体暴露的相关反应，包括DUOX亚细胞靶向、ROS产生和下游氧化还原相关的细胞变化。这些关于呼吸道上皮细胞-病原体相互作用的实验将得到DUOX缺乏动物模型感染研究的补充。 在旨在探索NOX4(或Renox)功能作用的努力中，我们正在研究NOX4基因缺失的小鼠。NOX4基因缺陷小鼠在非应激状态下表现出正常的寿命和表型。基因芯片研究的重点是识别其他氧化剂生成或清除系统的变化，以探索在NOX4缺陷小鼠中维持正常氧化还原稳态的机制。NOX4是一种结构性的活性酶，与其作为氧气感应酶的作用是一致的。我们正在研究NOX4在肾脏氧气感知和造血中的作用，因为ROS被认为提供了调节肾脏促红细胞生成素合成的反馈信号。我们已经证明，NOX4水平直接对转化生长因子-β(TGF-β)或肾细胞中的缺氧和肝细胞中的丙型肝炎病毒(HCV)做出反应，我们正在探索这些影响的机制基础。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(肝硬变)和与低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，我们对基于NOX1的多组分氧化酶的兴趣主要集中在酵母双杂交筛选实验中确定的NOX激活剂1(NOXA1)的功能配对。定点突变实验的目的是在全细胞模型中确定Noxa1伙伴参与Nox1的激活。

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.