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

• 批准号: 8336081
• 负责人: THOMAS LETO
• 金额: $ 122.08万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 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/8336081
• 关键词: 4-ethoxymethylene-2-phenyl-2-oxazoline-5-one; Affect; Anabolism; Animal Model; Antioxidants; Apical; Apoptosis; Asthma; Autocrine Communication; Bacterial Infections; Biochemical; Biological Assay; Blood Vessels; Brain; Caspase; Cell Aging; Cell Culture Techniques; Cell Differentiation process; Cells; Chronic; Chronic Granulomatous Disease; Cretinism; Cystic Fibrosis; DTR gene; Defect; Disease; Double-Stranded RNA; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor Receptor; Epithelial; Epithelial Cells; Exhibits; Exocrine Glands; Extracellular Matrix; Family; Flagellin; Gastrointestinal tract structure; Gene Expression; Gene Expression Profiling; Genes; Genetic Polymorphism; Growth Factor; Hepatitis C; Hormones; Host Defense; Hydrogen Peroxide; Immune; Immune response; Immunocompromised Host; Individual; Infection; Inflammatory; Inflammatory Bowel Diseases; Inflammatory Response; Interleukin-6; Investigation; Isoenzymes; Kidney; Ligands; Link; Lipopolysaccharides; Liver; Lung; Mediating; Microbe; Microbial Biofilms; Modeling; Molecular; Mouse Strains; Mucins; Mucous Membrane; Mus; Mutation; NADPH Oxidase; Natural Immunity; Nox enzyme; Oxidases; Oxidation-Reduction; Oxidative Stress; Oxygen; Paracrine Communication; Pathogenesis; Pathway interactions; Patients; Pattern; Peroxidases; Phagocytes; Phenotype; Poly I-C; Predisposition; Process; Production; Pseudomonas; Pseudomonas aeruginosa; Pyocyanine; Reactive Oxygen Species; Role; Salivary Glands; Signal Pathway; Signal Transduction; Single Nucleotide Polymorphism; Small Interfering RNA; Stimulus; Superoxides; Surface; System; TLR3 gene; Technology; Thyroid Gland; Tissues; Toxin; Transforming Growth Factor alpha; Tumor Necrosis Factor Receptor; Tumor Necrosis Factor-alpha; Type III Secretion System Pathway; Viral; Virulence Factors; Virus Diseases; airway epithelium; antimicrobial; base; cellular targeting; cystic fibrosis patients; cytokine; defense response; human TNF protein; inhibitor/antagonist; insight; interest; killings; lactoperoxidase; microbial; microbicide; mouse model; neutralizing antibody; novel; pathogen; programs; receptor; reconstitution; response; tissue/cell culture

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.

这个项目探索了天然的抗微生物防御和炎症机制，涉及宿主故意产生的活性氧物种(ROS)。循环中的吞噬细胞产生高水平的ROS，作为重要的杀微生物剂响应感染或炎症刺激，这归因于NADPH氧化酶的激活。慢性肉芽肿性疾病(CGD)患者患有NADPH氧化酶(基于NOX2或PHOX)缺乏，导致对微生物感染和异常炎症反应的易感性增加。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、肾、甲状腺和唾液腺、脑和血管组织中。这些氧化酶产生的ROS提供氧化还原信号，在对感染、氧气感应、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(细胞凋亡)的反应中影响基因表达模式。一些非吞噬细胞的NOx酶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，并由细胞因子或病原体相关分子模式的识别来诱导或激活。最近，我们发现，成熟的纤毛呼吸道上皮细胞产生足够的DUOX衍生的过氧化氢来支持乳过氧化物酶介导的对几种呼吸道病原体的杀灭，而新鲜生长的铜绿假单胞菌在多种微生物因素(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激活。相比之下，过度生长的假单胞菌会分泌一种微生物毒素(绿青素)，这种毒素会竞争性地抑制DUOX活性，因为它会产生细胞内的超氧化物，并对宿主细胞施加氧化压力。在这场宿主和微生物之间的氧化还原战争中，后一过程代表了假单胞菌在生物被膜形成过程中的反攻适应。 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青素在慢性呼吸道感染发病机制中的重要性。当假单胞菌在免疫受损个体(即囊性纤维化患者)的慢性感染肺部的生物膜中过度生长时，该因子是对法定信号的反应。我们展示了(纯化的)绿色素介导的氧化应激对分离的呼吸道上皮细胞的影响，概括了晚期囊性纤维化疾病的许多表型特征，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以及炎症细胞刺激和趋化因子。这些反应最初是通过基于微阵列的基因表达谱来检测的，它鉴定了大约286个被绿青素上调的基因。分泌的细胞因子和可能的EGF受体配体的ELISA检测证实了许多诱导基因的重要性。然后，利用生化、药理学和抗体中和的方法建立了绿青素诱导的粘蛋白高分泌的机制模型，在该模型中，几种分泌的表皮生长因子受体(EGFR)配体(IL-β、IL-6、转化生长因子-α、肿瘤坏死因子-α、HB-EGF)通过自分泌或旁分泌信号通路促进粘蛋白的分泌。这些发现表明，针对这种毒素对治疗进展期囊性纤维性疾病的影响的治疗方法具有潜在的实用价值，并且它们为最近的发现提供了见解，这些发现表明，将绿青素长期注入小鼠呼吸道会产生囊性纤维性变的表型。 在其他合作研究中，我们检测了呼吸道上皮细胞对多肌苷-多胞苷酸(PolyI：C)的先天免疫反应，作为通过TLR3途径模拟病毒双链RNA信号。Poly I：C可诱导可溶性肿瘤坏死因子受体胞外结构域(STNFR1)脱落，从而下调细胞对肿瘤坏死因子-α的反应。我们证明，受体脱落需要两条途径：一条是DUOX2介导的过氧化氢释放，另一条是caspase介导的细胞凋亡。Poly I：C可引起呼吸道细胞过氧化氢的产生和sTNFR1的脱落，这种作用可被siRNA介导的DUOX2基因敲除、氧化酶抑制剂或抗氧化剂抑制。这些发现揭示了新的机制，通过这些机制可以调节呼吸道上皮对病毒感染的先天免疫和炎症反应。 我们在DUOX重构技术方面的进展已经确定了几个DUOX单核苷酸多态(SNPs)或突变，这些SNPs或突变改变了氧化酶功能或细胞靶向，可能与先天性甲状腺功能减退或改变对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)的易感性有关。在对炎症性肠病易感性改变的小鼠品系之间发现的其他DUOX多态正在研究氧化酶功能的变化。