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: 8946288
• 负责人: THOMAS LETO
• 金额: $ 96.03万
• 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/8946288
• 关键词: 4-ethoxymethylene-2-phenyl-2-oxazoline-5-one; Address; Affect; Agonist; Allergic; Anabolism; Animal Model; Apical; Apoptosis; Bacteria; Bacterial Infections; Biochemical; Blood Vessels; Breast Epithelial Cells; Calcium; Caspase-1; Cell Aging; Cell Culture Techniques; Cell membrane; Cells; Characteristics; Chronic; Chronic Granulomatous Disease; Chronic Hepatitis C; Cirrhosis; Colitis; Colon; Complex; Cretinism; Cystic Fibrosis; Cytoplasmic Granules; Cytoskeleton; DNA; Data; Defect; Disease; Disease Progression; Dominant-Negative Mutation; Duct (organ) structure; Enzymes; Epithelial; Epithelial Cells; Excision; Exocrine Glands; Extracellular Matrix; Family; Fibronectins; Fibrosis; Gastrointestinal tract structure; Generations; Genetic Polymorphism; Glutathione; Goals; Growth Factor; H1299; Hepatitis C; Hepatitis C virus; Hepatocyte; Histone H4; Hormones; Host Defense; Human; Hydrogen Peroxide; Immune; Immune response; In Vitro; Infection; Inflammation; Inflammation Mediators; Inflammatory; Inflammatory Response; Influenza; Intervention; Ionomycin; Isoenzymes; Kidney; Knockout Mice; Laboratories; Lead; Leukocyte Elastase; Link; Liver; Liver diseases; Lung; MADH3 gene; MCF10A cells; MDA MB 231; MEKs; Mammary gland; Mediating; Molecular; Mucous body substance; Mus; Mutant Strains Mice; Mutation; NADPH Oxidase; Natural Immunity; Neoplasm Metastasis; Neutrophil Infiltration; Oxidases; Oxidation-Reduction; Oxidative Stress; Oxygen; PTK2 gene; Pathway interactions; Pattern; Pattern Recognition; Peroxidases; Phagocytes; Phosphorylation; Predisposition; Primary carcinoma of the liver cells; Process; Production; Protein p53; Proteins; Pseudomonas; Pseudomonas aeruginosa; Reactive Oxygen Species; Repression; Respiratory Burst; Respiratory System; Respiratory physiology; Respiratory tract structure; Role; Salivary; Salivary Glands; Signal Transduction; Silicon Dioxide; Source; Structure; Surface; System; Thapsigargin; Thyroid Gland; Tissues; Transforming Growth Factor beta; Viral; Work; aerosolized; airway obstruction; antimicrobial; base; cancer cell; cell motility; cystic fibrosis airway; cystic fibrosis patients; cytokine; epithelial to mesenchymal transition; extracellular; inflammatory marker; insight; interest; killings; macrophage; microbial; mouse model; mutant; neoplastic cell; neutrophil; novel; overexpression; pathogen; prevent; programs; response; signal processing; tumor; tumor progression; tumorigenesis

This program explores innate immune, pro-inflammatory, and other signaling processes dependent on deliberate reactive oxygen species (ROS) production by Nox family NADPH oxidases. The prototypical NADPH oxidase complex of circulating phagocytes (Nox2-based) is well known for its functions in microbial killing. Non-phagocytic enzymes (Nox1, Nox4, Duox1, Duox2) are highly expressed in epithelial cells, notably along mucosal surfaces (lung and gastrointestinal tract), liver, kidney, thyroid and exocrine glands (salivary, mammary), and in vascular tissues. ROS produced by these oxidases affect cell migration, differentiation, cellular senescence, programmed cell death (apoptosis), oxygen sensing, and responses to growth factors, hormones, cytokines or recognition of danger- and pathogen-associated molecular patterns (DAMPs and PAMPs). In 2014, we explored the molecular basis for Nox4 induction by transforming growth factor-beta (TGF-beta) and its role in cell migration in normal and metastatic tumor cells. We initially identified Nox4 as a novel source of TGF-beta-inducible ROS in hepatitis C virus (HCV)-infected hepatocytes and suggested links between excess Nox4-derived ROS with chronic HCV infection and hallmarks of liver disease progression (fibrosis, cirrhosis, and hepatocellular carcinoma). TGF-beta induces the epithelial-to-mesenchymal transition (EMT) leading to increased cell plasticity at the onset of cancer cell invasion and metastasis. Recent studies showed that p53 affects TGF-beta;/SMAD3-mediated signaling, cell migration, and tumorigenesis. We previously demonstrated that the Nox4 is a TGF-beta/SMAD3-inducible source of reactive oxygen species (ROS) affecting cell migration and fibronectin expression, an EMT marker, in normal and metastatic breast epithelial cells. Our current studies investigated effects of wild type p53 (WT-p53) and mutant p53 proteins on TGF-beta;-regulated Nox4 expression and cell migration. We found that WT-p53 is a potent suppressor of TGF-beta-induced Nox4, ROS production, and cell migration in p53-null lung epithelial (H1299) cells overexpressing WT p53. In contrast, metastatic tumor-associated mutant p53 proteins (R175H or R280K) caused enhanced Nox4 expression and cell migration in both TGF-beta-dependent and TGF-beta-independent pathways. Knockdown of endogenous mutant p53 (R280K) in TGF-beta-treated MDA-MB-231 metastatic breast epithelial cells resulted in decreased Nox4 protein and reduced phosphorylation of FAK, a key regulator of cell motility. Expression of WT-p53 or dominant-negative Nox4 decreased TGF-beta-mediated FAK phosphorylation, whereas mutant p53 (R280K) increased phospho-FAK. Furthermore, knockdown of WT-p53 in MCF-10A normal human breast epithelial cells increased basal Nox4 expression, whereas p53-R280K could override endogenous WT-p53 repression of Nox4. These findings define novel opposing functions for WT-p53 and mutant p53 proteins in regulating Nox4-dependent signaling in TGF-beta;-mediated cell motility. The studies highlight the importance of a TGF-beta-SMAD3-mutantp53-Nox4 axis in cell migration and tumor metastasis and reveal novel targets for intervention against cancer progression and metastatic disease. In work related to our long-term interests in redox-based host-pathogen interactions, we explored release of inflammatory mediators by neutrophils exposed to Pseudomonas aeruginosa. Cystic fibrosis (CF) airways are characterized by chronic bacterial infections (typically Pseudomonas aeruginosa), robust neutrophil recruitment, excess mucus production, enhanced inflammation and oxidative stress. Neutrophil primary granule components, myeloperoxidase (MMPO) and human neutrophil elastase (HNE), are inflammatory markers in CF airways, and their increased levels are associated with poor lung function. Here, we showed that human neutrophils release large amounts of neutrophil extracellular traps (NETs) in the presence of P. aeruginosa. Bacteria are entangled in NETs and colocalize with extracellular DNA. MPO, HNE, and citrullinated histone H4 are all associated with DNA in Pseudomonas-triggered NETs. Both laboratory strains and CF isolates of P. aeruginosa induce DNA, MPO, and HNE release from human neutrophils. P. aeruginosa induces a robust respiratory burst in neutrophils that is required for extracellular DNA release; NADPH oxidase inhibition suppresses Pseudomonas-induced release of active MPO and HNE. Blocking MEK/ERK signaling results in only minimal inhibition of DNA release induced by Pseudomonas, whereas inhibition of the cytoskeleton effectively prevents DNA release. Together these studies suggest Pseudomonas-induced NET formation is an important mechanism contributing to the enhanced inflammatory conditions and airway obstruction characteristic of advanced CF airway disease. Furthermore, they may explain why some CF patients benefit from aerosolized DNAse-based therapies, which can dissolve NET structures. We also studied involvement of calcium and calcium-activated NADPH oxidases in NLRP3 inflammasome activation and IL-1B; release to better understand inflammasome signaling in macrophages. Murine or human macrophages were treated in vitro with NLRP3 inflammasome agonists (ATP, silica crystals) or calcium mobilizing agonists (thapsigargin, ionomycin) in calcium-containing or calcium-free medium. Our data show that calcium is essential for IL-1B; release in human macrophages. Increases in cytosolic calcium alone lead to IL-1B; secretion. Calcium removal blocks caspase-1 activation. Human macrophages express Duox1, a calcium-regulated NADPH oxidase that produces reactive oxygen species. However, Duox1-deficient murine macrophages show normal IL-1B; release. Human macrophage inflammasome activation and IL-1B; secretion requires calcium but does not involve NADPH oxidases. In other studies addressing oxidative innate immune and inflammatory responses of mucosal epithelial tissues of the respiratory and gastrointestinal tracts, we are developing cell culture and knockout mouse models to explore critical roles of Nox1 and Duox isozymes. We have characterized Duox1- and Duox2-deficient mouse models with goals of examining airway epithelial responses to Influenza A infection or allergic asthmatic challenges. We showed that a Duox2 V674G mutation associated with congenital hypothyroidism leads to impaired subcellular targeting and ROS release. Immunostaining of Duox2 in salivary gland ducts showed that the defective Duox2 in these mutant mice loses its condensed apical plasma membrane localization pattern characteristic of wild type Duox2. Collaborative studies with F. Chu implicate strain-specific Duox2 polymorphisms in spontaneous colitis observed in glutathione peroxidase1/2 double knockout mice. Other work shows the cytosolic peroxidase, peroxiredoxin6, supports Nox1-based ROS generation and cell migration in colon epithelial cells, again demonstrating how mucosal ROS generators and scavengers can collaborate in processes affecting epithelial barrier formation, innate immunity, and inflammation.

该计划探讨了先天免疫，促炎和其他信号传导过程，取决于NOX家族NADPH氧化酶的故意活性氧（ROS）。循环吞噬细胞（基于NOX2）的原型NADPH氧化酶复合物以其在微生物杀伤中的功能而闻名。在上皮细胞中高度表达了非斑点细胞酶（NOX1，NOX4，DUOX1，DUOX2），特别是沿粘膜表面（肺和胃肠道），肝，肾脏，甲状腺和外在腺体（乳液，乳液，乳腺，乳腺，乳腺）以及在血管组织中。这些氧化酶产生的ROS会影响细胞迁移，分化，细胞衰老，程序性细胞死亡（凋亡），氧气感应以及对生长因子，激素，细胞因子或对危险和病原体相关的分子模式（DAMP和PAMPS）的识别。 在2014年，我们通过转化生长因子β（TGF-β）及其在正常和转移性肿瘤细胞中细胞迁移中的作用来探索NOX4诱导的分子基础。我们最初将NOX4确定为肝炎病毒（HCV）感染的TGF-BetA诱导ROS的一种新来源，并提出了与慢性HCV感染过多的NOX4衍生的ROS之间的联系，并具有肝脏疾病的慢性HCV感染和肝病进展的标志（纤维化进展，纤维化，肝硬化和Hepatocatomalulor Carcinomal carcinoma carcinoma carcinoma）。 TGF-β诱导上皮到间质转变（EMT），导致癌细胞侵袭和转移开始时细胞可塑性增加。最近的研究表明，p53影响TGF-beta；/smad3介导的信号传导，细胞迁移和肿瘤发生。我们先前证明了NOX4是影响细胞迁移和纤连蛋白表达（在正常和转移性乳房上皮细胞中）的TGF-beta/Smad3诱导源的反应性氧（ROS）。我们目前的研究调查了野生型p53（WT-P53）和突变p53蛋白对TGF-β的影响；调节的NOX4表达和细胞迁移。我们发现WT-P53是TGF-BETA诱导的NOX4，ROS产生和细胞迁移的有效抑制剂，p53-null肺上皮（H1299）细胞过表达WT p53。相反，转移性肿瘤相关的突变体p53蛋白（R175H或R280K）在TGF-beta依赖性和与TGF-beta的途径中均导致NOX4表达和细胞迁移增强。 TGF-BETA处理的MDA-MB-231转移性乳腺上皮细胞中内源性突变体P53（R280K）的敲低导致NOX4蛋白减少，并降低了FAK的磷酸化，FAK是细胞运动的关键调节剂。 WT-P53或显性阴性NOX4的表达降低了TGF-BETA介导的FAK磷酸化，而突变体P53（R280K）的表达增加了磷酸化-FAK。此外，在MCF-10A中敲低WT-P53正常人乳腺上皮细胞增加了基础NOX4的表达，而P53-R280K可能会覆盖NOX4的内源性WT-P53抑制。这些发现定义了WT-P53和突变体p53蛋白在调节TGF-BetA中NOX4依赖性信号传导方面的新型相对功能；介导的细胞运动。该研究强调了TGF-beta-Smad3-Mutantp53-Nox4轴对细胞迁移和肿瘤转移的重要性，并揭示了针对癌症进展和转移性疾病的干预的新靶标。 在与我们在基于氧化还原的宿主 - 疾病相互作用中的长期利益有关的工作中，我们探索了暴露于铜绿假单胞菌的中性粒细胞释放炎症介质的释放。囊性纤维化（CF）气道的特征是慢性细菌感染（通常是铜绿假单胞菌），强大的中性粒细胞募集，粘液过量产生，增强的炎症和氧化应激。中性粒细胞原代颗粒成分，髓过氧化物酶（MMPO）和人类嗜中性粒细胞弹性酶（HNE）是CF气道中的炎症标志物，其水平增加与肺功能差有关。在这里，我们表明，在存在铜绿假单胞菌的情况下，人类中性粒细胞释放了大量的中性粒细胞外陷阱（NET）。细菌纠缠在网中，并与细胞外DNA共定位。 MPO，HNE和Citrullated组蛋白H4都与假单胞菌触发的网中的DNA有关。铜绿假单胞菌的实验室菌株和CF分离株诱导了人类嗜中性粒细胞的DNA，MPO和HNE释放。铜绿假单胞菌诱导细胞外DNA释放所需的嗜中性粒细胞中的强大呼吸爆发； NADPH氧化酶抑制抑制了假单胞菌诱导的活性MPO和HNE的释放。阻断MEK/ERK信号传导仅导致假单胞菌诱导的DNA释放极少抑制，而对细胞骨架的抑制有效地阻止了DNA释放。这些研究共同表明，假单胞菌诱导的净形成是一种重要的机制，该机制有助于增强的炎症状况和气道障碍物的高级CF气道疾病特征。此外，他们可以解释为什么一些CF患者受益于基于雾化的DNase疗法，从而可以溶解净结构。 我们还研究了钙和钙激活的NADPH氧化酶参与NLRP3炎性体激活和IL-1B。释放以更好地了解巨噬细胞中的炎性体信号传导。在含钙或无钙的培养基中，用NLRP3炎性体激动剂（ATP，硅晶体）或钙动员激动剂（Thapsigargin，Ionymycin）在体外用鼠或人类巨噬细胞处理。我们的数据表明，钙对于IL-1B至关重要。在人类巨噬细胞中释放。单独的胞质钙增加导致IL-1B；分泌。钙去除阻断caspase-1激活。人类巨噬细胞表达DUOX1，这是一种产生活性氧的钙调节的NADPH氧化酶。但是，DUOX1缺陷的鼠巨噬细胞显示出正常的IL-1B。发布。人类巨噬细胞炎性体激活和IL-1B；分泌需要钙，但不涉及NADPH氧化酶。 在其他针对呼吸道和胃肠道粘膜上皮组织氧化性的免疫和炎症反应的研究中，我们正在发展细胞培养和敲除小鼠模型，以探索NOX1和DUOX同工母的关键作用。我们已经表征了DUOX1和DUOX2缺陷的小鼠模型，该模型的目标是检查对流感感染或过敏性哮喘挑战的气道上皮反应。我们表明，与先天性甲状腺功能减退症相关的DUOX2 V674G突变会导致亚细胞靶向和ROS释放受损。 DUOX2在唾液腺管道中的免疫染色表明，这些突变小鼠中的有缺陷的DUOX2失去了其凝结的顶端质膜定位模式的野生型DUOX2的特征。 F. chu的协作研究暗示了在谷胱甘肽过氧化物酶1/2双基因敲除小鼠中观察到的自发性结肠炎中菌株特异性的DUOX2多态性。其他工作表明，胞质过氧化物酶（过氧化物蛋白氧化物6）支持基于NOX1的ROS的产生和细胞迁移，再次证明了粘膜ROS发电机和清除剂如何在影响上皮屏障形成，先天免疫和炎症的过程中进行协作。