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

本节目探讨先天抗微生物防御和炎症机制，涉及宿主蓄意活性氧（ROS）的产生。循环吞噬细胞产生高水平的活性氧，在感染或炎症刺激下作为重要的杀微生物剂，这归因于NADPH氧化酶的激活。慢性肉芽肿病（CGD）患者患有NADPH氧化酶（Nox2-或phox-基）缺乏，导致对微生物感染的易感性增强和异常炎症反应。我们目前的重点是研究在非吞噬细胞（Nox1, Nox4, Duox1, Duox2）中表达的相关Nox家族NADPH氧化酶的细胞机制，特别是在粘膜表面（肺和胃肠道），肝脏，肾脏，甲状腺和唾液腺，大脑和血管组织。这些氧化酶产生的活性氧提供氧化还原信号，在感染、氧感应、生长因子、激素、细胞因子、细胞分化、细胞衰老、细胞程序性死亡（凋亡）等反应过程中影响基因表达模式。几种非吞噬性Nox酶也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，并由细胞因子或病原体相关分子模式的识别诱导或激活。最近，我们发现成熟的纤毛气道上皮细胞产生足够的过氧化氢来支持乳酸过氧化物酶介导的几种气道病原体的杀伤，并且新鲜生长的铜绿假单胞菌在多种微生物因子（脂多糖、鞭毛蛋白和III型分泌系统）的作用下诱导气道上皮Duox活化。相比之下，过度生长的假单胞菌分泌一种微生物毒素（pyocyanin），当它产生细胞内超氧化物并对宿主细胞施加氧化应激时，它会竞争性地抑制Duox活性。后一个过程在宿主和微生物之间的“氧化还原战”中代表了假单胞菌在建立生物膜期间的反攻适应。

This program explores innate anti-microbial defense and inflammatory mechanisms involving deliberate reactive oxygen species (ROS) production by the host. 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 (Nox2- or phox-based) deficiencies, resulting in enhanced susceptibility to microbial infections and aberrant inflammatory responses. Our current focus investigates 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. ROS produced by these oxidases 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). Several non-phagocytic Nox enzymes also 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. Recently, we found that mature ciliated airway epithelial cells produce sufficient Duox-derived hydrogen peroxide to support lactoperoxidase-mediated killing of several airway pathogens, and that freshly grown Pseudomonas aeruginosa elicits airway epithelial Duox activation in response to multiple microbial factors (lipopolysaccharide, flagellin and the type III secretion system). In contrast, overgrown Pseudomonas secretes a microbial toxin (pyocyanin) that competitively inhibits Duox activity as it produces intracellular superoxide and imposes oxidative stress on host cells. The latter process in this 'redox warfare' between host and microbe represents a counter-offensive adaptation of Pseudomonas during the establishment of biofilms. In 2011, we have studied in detail the importance of the redox-active Pseudomonas aeruginosa virulence factor, pyocyanin, in the pathogenesis of chronic Pseudomonas airway infections. This factor is produced in response to quorum signals when Pseudomonas is overgrown in biofilms of chronically infected lungs of immunocompromised individuals (i.e., cystic fibrosis patients). We showed the effects of (purified) pyocyanin-mediated oxidative stress imposed on isolated airway epithelial cells recapitulate many of the phenotypic features of advanced cystic fibrosis disease, including mucin hypersecretion (mucin5a and mucin2) and release of pro-inflammatory cytokines and inflammatory cell-stimulating and chemotactic agents. These responses were detected initially by microarray-based gene expression profiling, which identified some 286 genes upregulated by pyocyanin. The importance of many of the induced genes was confirmed by ELISA assays of secreted cytokines and putative EGF receptor ligands. Biochemical, pharmacological, and antibody-neutralizing approaches were then used to establish a mechanistic model for pyocyanin-induced mucin hypersecretion in which several secreted epidermal growth factor receptor (EGFR) ligands (IL-beta, IL-6, TGF-alpha, TNF-alpha, HB-EGF) were shown to act through autocrine or paracrine signaling pathways to promote mucin secretion. These findings suggest potential utility of therapies aimed at the effects of this toxin for treating advance cystic fibrosis disease, and they provide insights into recent findings showing that chronic pyocyanin administration into mouse airways produces a cystic fibrosis-like phenotype. In other collaborative studies, we examined innate immune responses of airway epithelial cells to polyinosinic-polycytidylic acid (poly I:C), as a mimic of viral double-stranded RNA signaling through TLR3 pathways. Poly I:C was shown to induce shedding of a soluble TNF receptor ectodomain (sTNFR1), which could down-regulating cellular responses to TNF-alpha. We demonstrated that receptor shedding requires two pathways: one involving Duox2-mediated hydrogen peroxide release and the other involving caspase-mediated apoptosis. Poly I:C triggered hydrogen peroxide production and sTNFR1 shedding from airway cells, which was suppressed by siRNA-mediated Duox2 knockdown, oxidase inhibitors, or antioxidants. These findings reveal novel mechanisms by which innate immune and inflammatory responses of airway epithelium to viral infection can be modulated. Our advances in Duox reconstitution technology have identified several Duox single nucleotide polymorphisms (SNPs) or mutations that alter oxidase function or cellular targeting, which may relate to congenital hypothyroidism or altered susceptibilities to airway infectious or inflammatory disease (cystic fibrosis, asthma, bacterial or viral infection). Other Duox polymorphisms identified between mouse strains that exhibit altered susceptibilities to inflammatory bowel disease are being investigated for alterations in oxidase function.