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: 10692034
• 负责人: THOMAS LETO
• 金额: $ 76.15万
• 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/10692034
• 关键词: Affect; Agonist; Anabolism; Animal Model; Anti-Inflammatory Agents; Antimetastatic Agent; Apoptosis; Area; Biochemical; Breast; CYBA gene; Cell Aging; Cell Culture Techniques; Cell Mobility; Cell Proliferation; Cell secretion; Cells; Chronic Granulomatous Disease; Clinical; Colon; Communication; DNA Sequence Alteration; Data; Data Set; Defect; Detection; Disease; Disease Progression; Dominant-Negative Mutation; Ductal Epithelium; Enzymes; Epithelial Cells; Exhibits; Extracellular Matrix; Family; Gastrointestinal tract structure; Genes; Genetic Polymorphism; Growth Factor; H1299; Hematopoietic; Hepatitis C; Homologous Gene; Hormones; Host Defense; Hot Spot; Human; Hydrogen Peroxide; Hypoxia; Immune; Immunophenotyping; In Vitro; Infection; Inflammation; Inflammatory; Inflammatory Bowel Diseases; Inflammatory Response; Informatics; Investigation; Isoenzymes; KRAS2 gene; Kidney; Link; Liver; Liver neoplasms; Longitudinal Studies; Lung; Lung Neoplasms; MADH3 gene; Malignant Neoplasms; Mammary Neoplasms; Mediating; Modeling; Molecular; Mucous Membrane; Mutation; NADPH Oxidase; National Human Genome Research Institute; Natural Immunity; Neoplasm Metastasis; Nox enzyme; Outcome; Oxidases; Oxidation-Reduction; Pancreas; Pancreatic Ductal Adenocarcinoma; Patients; Pattern; Peroxidases; Phagocytes; Predisposition; Primary Neoplasm; Process; Production; Prognosis; Proteins; RANTES; Reactive Oxygen Species; Research; Research Personnel; Respiratory System; Respiratory Tract Infections; Role; Signal Pathway; Signal Transduction; Site; Surface; Syndrome; System; TP53 gene; The Cancer Genome Atlas; Tissues; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cell Line; Tumor Tissue; Tumor-associated macrophages; Variant; Viral; Work; angiogenesis; antifibrotic treatment; antimicrobial; autocrine; base; cancer cell; cancer type; cell motility; chemokine; cytokine; epithelial wound; exome sequencing; experimental study; follow-up; genetic variant; inhibitor; insight; interest; loss of function; macrophage; microbial; microbicide; migration; monocyte; mouse model; mutant; neoplastic cell; pancreatic cancer cells; pancreatic ductal adenocarcinoma model; paracrine; pathogen; pathogenic fungus; patient population; programs; receptor; reconstitution; recruit; response; senescence; therapeutic target; translational goal; tumor; tumor microenvironment; tumor progression; wound closure; wound healing

This program explores innate immune, pro-inflammatory, and signaling functions of NOX family NADPH oxidases. Current research focuses on non-phagocytic NADPH oxidases (NOX1, NOX4, DUOX1, DUOX2) expressed primarily in epithelial cells, as well as NOX2 in hematopoietic cells. Reactive oxygen species (ROS) production by NOX enzymes relays redox signals in responses to cytokines, chemokines, growth factors, hormones, and danger- and pathogen-associated molecular patterns (DAMPs and PAMPs). In addition to serving direct microbicidal roles, NOX-derived ROS regulate cell migration, proliferation, differentiation, senescence, apoptosis, tumor invasiveness and metastasis. In 2022, we have explored functions of several NOX family NADPH oxidase components in two areas of investigation: 1) studies on genetic variants of NOX and DUOX components associated with innate antimicrobial defense and barrier function defects or inflammatory disease, 2) studies on roles of NADPH oxidases in cancer progression. Our interests in rare NOX and DUOX genetic variants in patients with innate immune defects and inflammatory disease syndromes originated with identification of NOX2 component defects in patients with chronic granulomatous disease (CGD). As whole exome sequencing (WES) data has become widely available from patients of our clinical collaborators, we have explored functional consequences of oxidase defects using several heterologous NOX and DUOX expression systems. Collaborative studies with investigators in the IPS/LCIM (A. Hsu, S. Holland) characterized several DUOX1 and DUOXA1 loss-of-function variants linked to pulmonary and disseminated Coccidiodomycosis. We reconstituted signaling pathways linking DUOX1/DUOXA1 activation with Dectin1 fungal pathogen receptor detection and signaling. We also showed rare NOX1 variants detected in patients with inflammatory bowel disease exhibit variable protein stabilities, diminished NADPH oxidase activity, and lower NOX1-dependent cell migration in our reconstituted model of colon epithelial wound closure and barrier function (NHGRI collab.). We also characterized several partially functional CYBA, CYBB, and NCF1 variants linked to enhanced susceptibility to microbial infection and inflammation in several atypical CGD cases. Our interests in NOX4 function in cancer originated with work in lung, breast, liver, and pancreatic tumor cell lines showing that NOX4 is induced in a TGF-beta- and SMAD3-dependent manner in tumors bearing TP53 hot spot mutations (PMID: 22728268; PMID: 28574838). We recently conducted an in-depth pan-cancer informatic analysis of primary human tumors of 23 cancer types in The Cancer Genome Atlas (TCGA) and validated our in vitro observations demonstrating roles of NOX4 in promoting programs of cancer progression in tumors with TP53 hotspot mutations, regardless of tumor tissue origin (PMID: 33557266). NOX4 expression correlates positively with gene programs associated with tumor cell proliferation, invasiveness, and angiogenesis and negatively with programs of cell apoptosis in tumors with mutant TP53; the opposite was observed in tumors with wild-type TP53. Thus, TP53 mutations switch NOX4 from acting as a protective oxidase predicting a favorable prognosis to one with deleterious effects on cancer progression and clinical outcome in late-stage cancers. In this last year, related follow-up cell culture experiments have explored NOX4-dependent chemotactic and inflammatory signaling between macrophages and tumor cells as a model of the tumor microenvironment (TME). We investigated the roles of mutant p53-induced NOX4 on the cancer cell secretome and the effects of NOX4-based signaling in the TME. We found the secretome from p53-null H1299 lung epithelial cells stably expressing mutant p53 proteins (R248Q or R273H) promotes the migration and invasion of naive H1299, as well as chemotactic recruitment of THP-1 monocytes. These effects were diminished when the secreting tumor cells were transfected with dominant negative NOX4 (P437H). Immunoblot-based cytokine array analysis revealed tumor cell secretion of CCL5 was mutant p53 and NOX4-dependent, promoting autocrine and paracrine-mediated cell migration and invasion, whereas neutralization of CCL5 reduced autocrine-mediated H1299 cell mobility. Furthermore, we showed neutralization of CCL5 and TGF-beta released by M2-polarized macrophages have a significant role in this TME crosstalk by promoting H1299 cell migration and invasion. Our findings provide further insight into NOX4-based communication in the tumor microenvironment and its potential as a therapeutic target affecting metastatic disease progression. Finally, in related long-term studies in a mouse model of pancreatic ductal adenocarcinoma (PDAC), with targeted mutations in TP53 and K-Ras, we observed 10-15-fold enhanced NOX4 and NOX2 levels relative to normal pancreatic tissue, consistent with our TCGA informatics analysis of human PDAC datasets. Highest NOX4 expression was detected in ductal epithelial tumor cells, as well as in adjacent fibrotic stomal tissue, whereas highest NOX2 (Cybb) expression was detected in tumor associated macrophages. Current work is exploring whether the absence of functional NOX4 or NOX2 genes affects tumor cell escape from primary tumor sites or modifies the access or immunophenotype of inflammatory cells within the tumor microenvironment.

该计划探讨了先天免疫，促炎和NOX家族NADPH氧化酶的信号功能。目前的研究主要集中在主要在上皮细胞中表达的非吞噬性NADPH氧化酶（NOX 1，NOX 4，DUOX 1，DUOX 2）以及造血细胞中的NOX 2。由NOX酶产生的活性氧（ROS）响应于细胞因子、趋化因子、生长因子、激素以及危险和病原体相关的分子模式（DAMP和PAMP）而传递氧化还原信号。除了发挥直接的杀微生物作用外，NOX衍生的ROS还调节细胞迁移、增殖、分化、衰老、凋亡、肿瘤侵袭和转移。 于2022年，我们在两个研究领域探索了几种NOX家族NADPH氧化酶组分的功能：1）与先天性抗微生物防御和屏障功能缺陷或炎症性疾病相关的NOX和DUOX组分的遗传变异研究，2）NADPH氧化酶在癌症进展中的作用研究。 我们对先天性免疫缺陷和炎性疾病综合征患者中罕见的NOX和DUOX遗传变异的兴趣起源于慢性肉芽肿病（CGD）患者中NOX 2组分缺陷的鉴定。由于全外显子组测序（WES）数据已从我们的临床合作者的患者中广泛获得，我们已经使用几种异源NOX和DUOX表达系统探索了氧化酶缺陷的功能后果。与IPS/LCIM研究者的合作研究（A.徐氏S. Holland）表征了与肺和播散性球孢子菌病相关的几种DUOX 1和DUOXA 1功能丧失变体。我们重建了连接DUOX 1/DUOXA 1激活与Dectin 1真菌病原体受体检测和信号传导的信号通路。我们还发现，在炎症性肠病患者中检测到的罕见NOX 1变体表现出可变的蛋白质稳定性，降低的NADPH氧化酶活性，以及在我们的结肠上皮伤口闭合和屏障功能重建模型中较低的NOX 1依赖性细胞迁移（NHGRI collab.）。我们还描述了几种部分功能性CYBA，CYBB和NCF 1变体，这些变体与几种非典型CGD病例中对微生物感染和炎症的易感性增强有关。 我们对NOX 4在癌症中的功能的兴趣源于对肺、乳腺、肝脏和胰腺肿瘤细胞系的研究，表明NOX 4在携带TP 53热点突变的肿瘤中以TGF-β和SMAD 3依赖性方式诱导（PMID：22728268; PMID：28574838）。我们最近在癌症基因组图谱（TCGA）中对23种癌症类型的原发性人类肿瘤进行了深入的泛癌症信息学分析，并验证了我们的体外观察结果，证明了NOX 4在促进具有TP 53热点突变的肿瘤的癌症进展程序中的作用，无论肿瘤组织来源如何（PMID：33557266）。NOX 4表达与肿瘤细胞增殖、侵袭性和血管生成相关的基因程序呈正相关，与突变型TP 53肿瘤中的细胞凋亡程序呈负相关;在野生型TP 53肿瘤中观察到相反的情况。因此，TP 53突变将NOX 4从预测有利预后的保护性氧化酶转变为对晚期癌症的癌症进展和临床结果具有有害影响的保护性氧化酶。 在过去的一年中，相关的后续细胞培养实验探索了巨噬细胞和肿瘤细胞之间的NOX 4依赖性趋化和炎症信号传导，作为肿瘤微环境（TME）的模型。我们研究了突变型p53诱导的NOX 4对癌细胞分泌组的作用以及基于NOX 4的信号传导在TME中的作用。我们发现来自p53缺失的H1299肺上皮细胞的分泌组稳定表达突变型p53蛋白（R248 Q或R273 H），促进幼稚H1299的迁移和侵袭，以及THP-1单核细胞的趋化性募集。当用显性负性NOX 4（P437 H）转染分泌肿瘤细胞时，这些作用减弱。基于免疫印迹的细胞因子阵列分析显示，肿瘤细胞分泌CCL 5是突变型p53和NOX 4依赖性的，促进自分泌和旁分泌介导的细胞迁移和侵袭，而CCL 5的中和降低了自分泌介导的H1299细胞迁移率。此外，我们发现M2极化巨噬细胞释放的CCL 5和TGF-β的中和通过促进H1299细胞迁移和侵袭在TME串扰中具有显著作用。我们的研究结果为肿瘤微环境中基于NOX 4的通信及其作为影响转移性疾病进展的治疗靶点的潜力提供了进一步的见解。 最后，在胰腺导管腺癌（PDAC）小鼠模型的相关长期研究中，在TP 53和K-Ras中具有靶向突变，我们观察到相对于正常胰腺组织，NOX 4和NOX 2水平提高了10-15倍，这与我们对人类PDAC数据集的TCGA信息学分析一致。在导管上皮肿瘤细胞以及邻近的纤维化造口组织中检测到最高的N 0X 4表达，而在肿瘤相关巨噬细胞中检测到最高的N 0X 2（Cybb）表达。目前的工作是探索功能性NOX 4或NOX 2基因的缺乏是否会影响肿瘤细胞从原发性肿瘤部位逃逸，或改变肿瘤微环境中炎症细胞的进入或免疫表型。