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.

该项目探讨了依赖于 Nox 家族 NADPH 氧化酶有意产生活性氧 (ROS) 的先天免疫、促炎症和其他信号传导过程。循环吞噬细胞的原型 NADPH 氧化酶复合物（基于 Nox2）以其杀灭微生物的功能而闻名。非吞噬酶（Nox1、Nox4、Duox1、Duox2）在上皮细胞中高度表达，特别是在粘膜表面（肺和胃肠道）、肝、肾、甲状腺和外分泌腺（唾液、乳腺）以及血管组织中。这些氧化酶产生的 ROS 影响细胞迁移、分化、细胞衰老、程序性细胞死亡（细胞凋亡）、氧感应以及对生长因子、激素、细胞因子的反应或对危险和病原体相关分子模式（DAMP 和 PAMP）的识别。 2014年，我们探索了通过转化生长因子-β（TGF-β）诱导Nox4的分子基础及其在正常和转移性肿瘤细胞中细胞迁移中的作用。我们最初确定 Nox4 是丙型肝炎病毒 (HCV) 感染的肝细胞中 TGF-β 诱导型 ROS 的新来源，并提出过量的 Nox4 衍生的 ROS 与慢性 HCV 感染和肝病进展标志（纤维化、肝硬化和肝硬化）之间的联系。肝细胞癌）。 TGF-β 诱导上皮间质转化 (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-β/SMAD3 介导的信号传导、细胞迁移和肿瘤发生。我们之前证明，Nox4 是 TGF-β/SMAD3 诱导的活性氧 (ROS) 来源，影响正常和转移性乳腺上皮细胞中的细胞迁移和纤连蛋白表达（EMT 标志物）。我们目前的研究调查了野生型 p53 (WT-p53) 和突变型 p53 蛋白对 TGF-β 调节的 Nox4 表达和细胞迁移的影响。我们发现，在过表达 WT p53 的 p53 缺失肺上皮 (H1299) 细胞中，WT-p53 是 TGF-β 诱导的 Nox4、ROS 产生和细胞迁移的有效抑制剂。相比之下，转移性肿瘤相关突变p53蛋白（R175H或R280K）导致TGF-β依赖性和TGF-β非依赖性途径中Nox4表达和细胞迁移增强。在 TGF-β 处理的 MDA-MB-231 转移性乳腺上皮细胞中敲低内源突变体 p53 (R280K) 导致 Nox4 蛋白减少，并减少 FAK（细胞运动的关键调节因子）的磷酸化。 WT-p53 或显性失活 Nox4 的表达降低了 TGF-β 介导的 FAK 磷酸化，而突变体 p53 (R280K) 则增加了磷酸化 FAK。此外，在MCF-10A正常人乳腺上皮细胞中敲低WT-p53会增加Nox4的基础表达，而p53-R280K可以克服内源性WT-p53对Nox4的抑制。这些发现定义了 WT-p53 和突变型 p53 蛋白在调节 TGF-β 介导的细胞运动中 Nox4 依赖性信号传导方面的新的相反功能。这些研究强调了 TGF-β-SMAD3-mutantp53-Nox4 轴在细胞迁移和肿瘤转移中的重要性，并揭示了干预癌症进展和转移性疾病的新靶标。 在与我们对基于氧化还原的宿主-病原体相互作用的长期兴趣相关的工作中，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。囊性纤维化 (CF) 气道的特点是慢性细菌感染（通常是铜绿假单胞菌）、中性粒细胞大量募集、粘液产生过多、炎症和氧化应激增强。中性粒细胞主要颗粒成分髓过氧化物酶 (MMPO) 和人中性粒细胞弹性蛋白酶 (HNE) 是 CF 气道中的炎症标志物，其水平升高与肺功能不良相关。在这里，我们发现，在铜绿假单胞菌存在的情况下，人类中性粒细胞会释放大量的中性粒细胞胞外陷阱（NET）。细菌缠绕在 NET 中并与细胞外 DNA 共存。 MPO、HNE 和瓜氨酸组蛋白 H4 都与假单胞菌触发的 NET 中的 DNA 相关。铜绿假单胞菌的实验室菌株和 CF 分离株均可诱导人中性粒细胞释放 DNA、MPO 和 HNE。铜绿假单胞菌会诱导中性粒细胞发生强烈的呼吸爆发，这是细胞外 DNA 释放所必需的； NADPH 氧化酶抑制可抑制假单胞菌诱导的活性 MPO 和 HNE 的释放。阻断 MEK/ERK 信号传导仅对假单胞菌诱导的 DNA 释放产生最小程度的抑制，而抑制细胞骨架则有效地阻止 DNA 释放。这些研究共同表明，假单胞菌诱导的 NET 形成是导致晚期 CF 气道疾病的炎症状况和气道阻塞特征增强的重要机制。此外，他们还可以解释为什么一些 CF 患者受益于雾化 DNAse 疗法，这种疗法可以溶解 NET 结构。 我们还研究了钙和钙激活的 NADPH 氧化酶在 NLRP3 炎性体激活和 IL-1B 中的参与；释放以更好地了解巨噬细胞中的炎症小体信号传导。在含钙或无钙培养基中，用 NLRP3 炎性体激动剂（ATP、二氧化硅晶体）或钙动员激动剂（毒胡萝卜素、离子霉素）在体外处理小鼠或人巨噬细胞。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。单独增加细胞质钙会导致 IL-1B；分泌。钙去除会阻止 caspase-1 激活。人类巨噬细胞表达 Duox1，这是一种钙调节的 NADPH 氧化酶，可产生活性氧。然而，Duox1 缺陷的小鼠巨噬细胞显示正常的 IL-1B；发布。人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先天免​​疫和炎症反应的研究中，我们正在开发细胞培养和基因敲除小鼠模型，以探索 Nox1 和 Duox 同工酶的关键作用。我们对 Duox1 和 Duox2 缺陷小鼠模型进行了表征，目的是检查气道上皮对甲型流感感染或过敏性哮喘挑战的反应。我们发现，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。唾液腺管中 Duox2 的免疫染色表明，这些突变小鼠中的缺陷 Duox2 失去了野生型 Duox2 的浓缩顶端质膜定位模式特征。与 F. Chu 的合作研究表明，在谷胱甘肽过氧化物酶 1/2 双敲除小鼠中观察到的自发性结肠炎中存在品系特异性 Duox2 多态性。其他研究表明，胞质过氧化物酶 peroxiredoxin6 支持结肠上皮细胞中基于 Nox1 的 ROS 生成和细胞迁移，再次证明粘膜 ROS 生成器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。