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: 10014043
• 负责人: THOMAS LETO
• 金额: $ 112.38万
• 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/10014043
• 关键词: Adenocarcinoma; Affect; Agonist; Alleles; Anabolism; Animal Model; Animals; Anti-Inflammatory Agents; Antimetastatic Agent; Antiviral Agents; Apoptosis; Area; B-Lymphocytes; Binding; Biochemical; Breast; CCL2 gene; COS-7 Cell; Cell Aging; Cell Culture Techniques; Cell Differentiation process; Cell Line; Cell model; Cells; Characteristics; Chemotaxis; Chronic Granulomatous Disease; Collaborations; Colon; DNA Sequence Alteration; Defect; Disease; Disease Progression; Dominant-Negative Mutation; Ductal Epithelial Cell; Encapsulated; Engineering; Enzymes; Epithelial; Epithelial Cells; Exhibits; Extracellular Matrix; Family; Fibroblasts; Functional disorder; Gastrointestinal tract structure; Generations; Genes; Genetic Polymorphism; Goals; Growth Factor; Guanosine Triphosphate Phosphohydrolases; Hematopoietic; Hepatitis C; Homologous Gene; Hormones; Host Defense; Hot Spot; Hydrogen Peroxide; Hypoxia; Immune; Immunologic Deficiency Syndromes; Impairment; Infection; Inflammatory; Inflammatory Bowel Diseases; Inflammatory Infiltrate; Inflammatory Response; Investigation; Isoenzymes; KRAS2 gene; Kidney; Knock-in; Link; Liver; Lung; Lymphoid; Lymphopenia; Lymphopoiesis; MADH3 gene; Mediating; Membrane; Modeling; Molecular; Mucous Membrane; Mus; Mutation; Myelogenous; NADPH Oxidase; National Human Genome Research Institute; Natural Immunity; Neoplasm Metastasis; Nox enzyme; Oxidases; Oxidation-Reduction; Pancreas; Pancreatic Ductal Adenocarcinoma; Pancreatic duct; Pathway interactions; Patients; Pattern; Peroxidases; Phagocytes; Phenotype; Population; Predisposition; Process; Production; Protein Isoforms; Proteins; Proto-Oncogene Proteins c-akt; RANTES; Reactive Oxygen Species; Recurrence; Research; Research Personnel; Respiratory Tract Infections; Rest; Role; Signal Transduction; Superoxides; Surface; T-Lymphocyte; TP53 gene; Therapeutic Intervention; Transforming Growth Factor beta; Transgenic Mice; Tumor Cell Migration; Tumor Tissue; Variant; Work; Wound Healing; angiogenesis; antimicrobial; base; cell motility; cell transformation; chemokine; cofactor; cytokine; disease phenotype; early onset; exome sequencing; formyl peptide; genetic variant; inhibitor/antagonist; insight; interest; macrophage; microbial; mutant; neoplastic cell; neutralizing antibody; neutrophil; novel; overexpression; pathogen; programs; protein expression; reconstitution; recruit; response; senescence; tumor; tumor microenvironment; tumor progression

This program explores innate immune, pro-inflammatory, and signaling functions of NOX family NADPH oxidases. The current research focuses on non-phagocytic NADPH oxidases (NOX1, NOX4, NOX5, DUOX1, DUOX2) expressed primarily in epithelial cells, as well as NOX2 and NOX5-based oxidases in hematopoietic cells. Deliberate reactive oxygen species (ROS) production by the epithelial enzymes relays redox signals in responses to cytokines, growth factors, hormones, and danger- and pathogen-associated molecular patterns (DAMPs and PAMPs). NOX enzymes also participate in cell migration, proliferation, tumor invasiveness and metastasis, cell differentiation, senescence, apoptosis, and microbial killing. In 2019, we explored functions of several NOX family NADPH oxidase components in three areas of investigation: 1) studies on genetic variants of NOX components linked to immunodeficiencies, 2) studies on NOX defects associated with inflammatory bowel disease, and 3) studies on the interplay of NOX4 and NOX2 in the tumor microenvironment using mice with conditional expression of mutant TP53 and KRAS and established tumor and macrophage-like cell lines. In collaboration with other LCIM investigators (Amy Hsu and Steven Holland), we characterized two novel mono-allelic RAC2 mutations associated with combined immune deficiency in several patients, resulting in severe T- and B-cell lymphopenia, myeloid dysfunction, and recurrent respiratory infections. Whole exome sequencing identified RAC2 E62K and N92T mutations in several patients whose neutrophils exhibited functional defects, including enhanced markers of spontaneous activation, excess superoxide generation, abnormal macropinocytosis and impaired chemotaxis in response to formyl peptide. We engineered these RAC2 mutant proteins for expression in transfected CHO-K1 and COS-7 cell lines to investigate the cellular and molecular mechanistic pathways responsible for the patients' aberrant myeloid and lymphoid phenotypes. Transfected models reconstituted with NOX2 components and either RAC2 mutant protein demonstrated exuberant ROS generation in both resting and stimulated cells. The over-expressed mutant proteins also exhibited enhanced RAC2-PAK1 binding and increased activated phospho-AKT, responses characteristic of dominant active RAC2 molecules that account for the enhanced membrane ruffling and macropinosome formation observed in the transfected models and patients neutrophils. Transgenic mice expressing Rac2 E62K exhibited the same myeloid and lymphoid disease phenotypes, including B and T cell lymphopenia, excess neutrophil superoxide generation and cytoskeletal and migratory defects. Together, these findings provide novel insights on roles for RAC2 in neutrophil function and lymphopoiesis distinct from those of the more widely expressed RAC1 GTPase homologue. Other work is exploring defects in several NOX isoforms associated with inflammatory bowel disease. Several NOX1 variants show partial or complete loss of superoxide generation when co-expressed with the other NOX1-supportive cofactors in transfected cell models (collaboration with D. Kastner's group, NHGRI). The defects in oxidase activity were correlated with diminished NOX1-dependent cell migration in transfected colon epithelial cells. Thus, the lower NOX1 oxidase activities associated with inflammatory bowel disease may reflect compromised NOX1-dependent epithelial barrier functions and enhanced inflammatory responses to microbial exposure. We also explored functional effects of a deletion in NOX5 detected by whole exome sequencing in a patient with early onset inflammatory bowel disease (collaboration with K. Sullivan and J Kelsen, CHOP). We showed the mutation not only impairs NOX5 superoxide generation but has dominant-negative effects on wild type NOX5 activity. Our studies on NOX involvement in cancer progression originated from observations showing that NOX4 induction by TGF-beta and SMAD3 is enhanced in several epithelial tumors bearing TP53 'hot spot' mutations that support tumor cell migration, invasiveness, and angiogenesis, processes that all appear to be NOX4-dependent. We are currently exploring roles for NOX4 and NOX2 in inflammatory signaling within the tumor microenvironment in cell culture models and in mice that develop pancreatic ductal adenocarcinoma (PDAC) as a consequence of conditional knock-in of TP53 and KRAS mutations. The cell culture studies demonstrated that mutant p53 mediates NOX4-dependent secretion of chemokines (CCL2 and CCL5) that not only stimulate tumor cell migration, but also recruit macrophages. Macrophages, in turn, cross-talk by secreting CCL5 and TGF-beta to promote tumor cell mobilization, which can be inhibited by neutralizing antibodies against these factors. These observations have revealed novel NOX-related inflammatory pathways with potential for therapeutic intervention. In related whole animal studies, we detected high NOX4 expression in pancreatic ductal epithelial cells transformed by mutant p53 expression, as well as in the fibroblast layer encapsulating the adenocarcinoma. In contrast, highest NOX2 expression is observed within infiltrating inflammatory cells surrounding the adenocarcinoma. Thus, the two oxidases over-expressed in PDAC are detected in different cell populations in these tumor tissues and appear to serve distinct roles that can either suppress or support metastatic disease progression. Ongoing work will investigate whether Nox4 or Nox2 gene deficiencies affect PDAC progression or overall survival in mice to determine whether these oxidases represent worthwhile targets for suppressing pancreatic metastatic disease progression.

本项目探讨NOX家族NADPH氧化酶的先天免疫、促炎和信号功能。目前的研究重点是主要在上皮细胞中表达的非吞噬性NADPH氧化酶（NOX1、NOX4、NOX5、DUOX1、DUOX2），以及造血细胞中基于NOX2和NOX5的氧化酶。在细胞因子、生长因子、激素以及危险和病原体相关分子模式（DAMPs和PAMPs）的响应中，上皮酶有意产生活性氧（ROS）传递氧化还原信号。氮氧化物酶还参与细胞迁移、增殖、肿瘤侵袭和转移、细胞分化、衰老、凋亡和微生物杀伤等过程。