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）。循环吞噬细胞产生高水平的ROS，其作为重要的杀微生物剂响应于感染或炎症刺激，这归因于NADPH氧化酶活化。慢性肉芽肿病（CGD）患者患有NADPH氧化酶（Nox 2或phox）缺陷，导致对微生物感染和异常炎症反应的易感性增强。我们目前的重点是研究调节非吞噬细胞（Nox 1，Nox 4，Duox 1，Duox 2）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。由这些氧化酶产生的ROS提供氧化还原信号，其在对感染、氧感测、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（凋亡）的响应期间影响基因表达模式。 几种非吞噬性Nox酶也在宿主防御和炎症过程中起作用，因为它们主要在上皮细胞的顶端表面上表达，并且由细胞因子或通过识别病原体相关的分子模式诱导或激活。最近，我们发现成熟的纤毛气道上皮细胞产生足够的Duox衍生的过氧化氢，以支持乳过氧化物酶介导的几种气道病原体的杀伤，新鲜生长的铜绿假单胞菌eliminate气道上皮Duox激活响应多种微生物因素（脂多糖，鞭毛蛋白和III型分泌系统）。相比之下，过度生长的假单胞菌分泌的微生物毒素（绿脓菌素），竞争性抑制Duox活性，因为它产生细胞内的超氧化物，并对宿主细胞施加氧化应激。 在宿主和微生物之间的“氧化还原战争”中，后一个过程代表了假单胞菌在生物膜建立期间的反攻适应。 2011年，我们详细研究了氧化还原活性铜绿假单胞菌毒力因子绿脓菌素在慢性假单胞菌气道感染发病机制中的重要性。当假单胞菌在免疫功能低下个体的慢性感染肺的生物膜中过度生长时（即，囊性纤维化患者）。我们发现，（纯化）绿脓菌素介导的氧化应激对分离的气道上皮细胞的影响概括了许多晚期囊性纤维化疾病的表型特征，包括粘蛋白高分泌（粘蛋白5a和粘蛋白2）和释放促炎细胞因子和炎性细胞刺激剂和趋化剂。这些反应最初是通过基于微阵列的基因表达谱检测的，该基因表达谱鉴定了绿脓菌素上调的约286个基因。许多诱导的基因的重要性，证实了分泌的细胞因子和推定的EGF受体配体的ELISA测定。然后使用生物化学、药理学和抗体中和方法来建立绿脓菌素诱导的粘蛋白高分泌的机制模型，其中显示几种分泌的表皮生长因子受体（EGFR）配体（IL-β、IL-6、TGF-α、TNF-α、HB-EGF）通过自分泌或旁分泌信号传导途径起作用以促进粘蛋白分泌。这些研究结果表明，针对这种毒素治疗晚期囊性纤维化疾病的效果的疗法具有潜在的实用性，并且它们提供了对最近发现的见解，这些发现表明，慢性绿脓菌素给药至小鼠气道产生囊性纤维化样表型。 在其他合作研究中，我们检测了气道上皮细胞对聚肌胞苷酸（poly I：C）的先天免疫应答，聚肌胞苷酸是通过TLR 3途径模拟病毒双链RNA信号传导的。Poly I：C可诱导可溶性TNF受体胞外域（sTNFR 1）脱落，从而下调细胞对TNF-α的反应。我们证明，受体脱落需要两个途径：一个涉及Duox 2介导的过氧化氢释放，另一个涉及半胱天冬酶介导的细胞凋亡。Poly I：C触发过氧化氢产生和sTNFR 1从气道细胞脱落，这被siRNA介导的Duox 2敲低、氧化酶抑制剂或抗氧化剂抑制。这些发现揭示了新的机制，先天免疫和气道上皮细胞的炎症反应，病毒感染可以调制。 我们在Duox重建技术方面的进展已经确定了几种Duox单核苷酸多态性（SNP）或改变氧化酶功能或细胞靶向的突变，这可能与先天性甲状腺功能减退症或改变对气道感染或炎症性疾病（囊性纤维化、哮喘、细菌或病毒感染）的易感性有关。 在对炎症性肠病表现出改变的易感性的小鼠品系之间鉴定的其他Duox多态性正在研究氧化酶功能的改变。

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.