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NOX家族NADPH氧化酶的先天免疫，促炎和信号传导功能。当前的研究侧重于非斑点NADPH氧化酶（NOX1，NOX4，DUOX1，DUOX2），主要在上皮细胞中表达，以及造血细胞中的NOX2。 NOX酶产生的活性氧（ROS）在对细胞因子，趋化因子，生长因子，激素以及与病原体相关的分子模式（潮湿和PAMPS）的反应中中继氧化还原信号。除了发挥直接杀生作用外，NOX衍生的ROS还调节细胞迁移，增殖，分化，衰老，凋亡，肿瘤侵入性和转移。 在2022年，我们探索了两个研究领域的几个NOX家族NADPH氧化酶成分的功能：1）研究NOX和DUOX成分与与先天性抗菌功能缺陷或炎症性疾病相关的NOX和DUOX成分的研究，2）研究NADPH氧化酶在癌症进展中的作用。 我们对罕见的NOX和DUOX遗传变异的兴趣对先天免疫缺陷和炎性疾病综合征的患者的兴趣起源于慢性肉芽肿性疾病（CGD）患者的NOX2成分缺陷。随着整个外显子组测序（WES）的数据已广泛可从我们的临床合作者的患者那里获得，我们使用多种异源NOX和DUOX表达系统探索了氧化酶缺损的功能后果。与IPS/LCIM（A. HSU，S。Holland）中研究人员的合作研究表征了几种与肺和传播球霉菌病有关的DUOX1和DUOXA1功能丧失变体。我们重组了将DUOX1/DUOXA1激活与Dectin1真菌病原体受体检测和信号传导联系起来的信号通路。我们还显示，在炎症性肠病患者中检测到的罕见NOX1变异表现出可变的蛋白质稳定性，NADPH氧化酶活性的降低以及NOX1依赖性细胞迁移在我们的结肠上皮伤口关闭和屏障功能（NHGRI COMPOR）中。我们还表征了几种与微生物感染的易感性增强的易感性和在几个非典型CGD病例中的易感性相关的几种部分功能性CYBA，CYBB和NCF1变体。 我们对NOX4在癌症中的功能的兴趣源于肺，乳腺癌，肝脏和胰腺肿瘤细胞系的作品，表明NOX4在带有TP53热点突变的肿瘤中诱导的TGF-beta-和Smad3依赖性方式是诱导的（PMID：22728268; PMID：PMID：285744838）。我们最近对癌症基因组中23种癌症类型的原发性人肿瘤（TCGA）进行了深入的泛伴奏信息分析，并验证了我们的体外观察结果，证明了NOX4在促进具有TP53热点突变的肿瘤中癌症进展方案中的作用，无论肿瘤组织起源于肿瘤组织，PMID：33355726666666666666666666666666666666666666）。 NOX4表达与与肿瘤细胞增殖，侵袭性和血管生成相关的基因程序正相关，并且与突变体TP53的肿瘤中的细胞凋亡程序呈负相关。在野生型TP53的肿瘤中观察到了相反的情况。因此，TP53突变将NOX4转化为一种保护性氧化酶，预测了对癌症进展和晚期癌症临床结果有害影响的预后。 去年，相关的随访细胞培养实验探索了巨噬细胞和肿瘤细胞之间NOX4依赖性的趋化性和炎症信号传导，作为肿瘤微环境（TME）的模型。我们研究了突变体p53诱导的NOX4在癌细胞分泌组中的作用以及基于NOX4的信号在TME中的作用。我们发现p53-Null H1299肺上皮细胞的分泌组稳定地表达突变体p53蛋白（R248Q或R273H）可促进NAIVE H1299的迁移和侵袭，以及THP-1单细胞细胞的化学术募集。当分泌肿瘤细胞用显性负NOX4（P437H）转染时，这些作用会减少。基于免疫印迹的细胞因子阵列分析表明，CCL5的肿瘤细胞分泌是突变体p53和NOX4依赖性，促进了自分泌和旁分泌介导的细胞迁移和侵袭，而CCL5的中和CCL5降低了自传介导的H1299细胞迁移率。此外，通过促进H1299细胞迁移和侵袭，我们显示了M2偏振巨噬细胞释放的CCL5和TGF-β的中和TGF-β在此TME串扰中具有重要作用。我们的发现提供了对肿瘤微环境中基于NOX4的通信的进一步见解，及其作为影响转移性疾病进展的治疗靶标的潜力。 最后，在胰腺导管腺癌（PDAC）的小鼠模型中相关的长期研究中，在TP53和K-RAS中具有靶向突变，我们观察到相对于正常胰腺组织，与正常的NOX2水平增强了10-15倍，与我们的TCGA信息分析人类PDAC数据集一致。在导管上皮肿瘤细胞以及相邻的纤维化静态组织中检测到最高的NOX4表达，而在肿瘤相关的巨噬细胞中检测到最高的NOX2（CYBB）表达。当前的工作是探索缺乏功能性NOX4或NOX2基因会影响肿瘤细胞从原发性肿瘤部位逃脱，还是修饰肿瘤微环境中炎性细胞的访问或免疫表型。