Role of Reactive Oxygen Species in Lymphocyte Development and Function

活性氧在淋巴细胞发育和功能中的作用

• 批准号: 10272142
• 负责人: THOMAS LETO
• 金额: $ 23.45万
• 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/10272142
• 关键词: Adaptive Immune System; Affect; Alleles; Antigens; Area; Arthritis; Autoimmune Process; Autoimmunity; B-Lymphocytes; Binding; Binding Sites; Cell Lineage; Cell model; Cells; Chronic; Chronic Granulomatous Disease; Clinical Protocols; Codon Nucleotides; Communicable Diseases; Complex; Data; Defect; Dendritic Cells; Development; Disease; Dominant-Negative Mutation; Dose; Electrons; Enzymes; Exhibits; Family; Family member; Generations; Genes; Goals; Hematopoietic; Heme; Human; Hydrogen Peroxide; Immune; Immune system; Immunologic Deficiency Syndromes; Inflammatory; Inflammatory Bowel Diseases; Inherited; Investigation; Isoenzymes; Link; Location; Lupus; Lymphocyte; Lymphoid; Lymphoid Cell; Mediating; Membrane; Missense Mutation; Molecular; Mutate; Mutation; NADP; NADPH Oxidase; Nodule; Nox enzyme; Oxidases; Oxidation-Reduction; Oxidative Stress; Oxidoreductase; Oxygen; Pathogenesis; Pathologic Processes; Patients; Pediatric Hospitals; Phagocytes; Philadelphia; Play; Process; Production; Propionates; Protein Isoforms; Proteins; Reactive Oxygen Species; Regulation; Role; Signal Transduction; Signaling Molecule; Site; Stimulus; Superoxides; T-Lymphocyte; Variant; adaptive immune response; adduct; base; cohort; early onset; genetic manipulation; loss of function; mouse model; negative affect; pathogen; programs; protein function; reconstitution; response; translational study

This program explores roles of reactive oxygen species (ROS) as specific signaling molecules in lymphoid cells through studies on inherited deficiencies or genetic manipulation of NOX/DUOX family NADPH oxidases. These enzymes catalyze NADPH-dependent reduction of molecular oxygen to generate superoxide or hydrogen peroxide. Phagocytes produce large amounts of ROS in response to infectious or inflammatory stimuli through the prototypic NADPH oxidase containing gp91phox (a.k.a., CYBB or NOX2). Our studies in lymphocytes are exploring redox signaling roles of NOX family members in adaptive immune responses to diverse pathogens, as well as in immunodeficiencies and autoimmunity. Several NOX family oxidases (i.e., NOX2, DUOX1, NOX5) are detected in cells of the adaptive immune system (B cells, T cells and dendritic cells) and have been associated with auto-immune inflammatory disease-like processes, such as arthritis, lupus and inflammatory bowel disease. About 40% of chronic granulomatous disease patients with NOX2 deficiencies suffer from autoimmune complications and very early onset inflammatory bowel disease (VEOIBD). Deficiencies in several other NOX isozymes have been linked to inflammatory bowel disease, although it remains unclear whether these oxidase defects are manifested in lymphoid or other hematopoietic cell lineages. In 2020, we have continued collaborative studies with Drs. Sullivan and Kelsen (Childrens Hospital of Philadelphia) on the characterization of NOX5 genetic defects detected in their cohort of patients with primary immune deficiencies. Thus far, we have identified three distinct NOX5 loss-of-function protein variants in patients with very early onset inflammatory bowel disease (VEOIBD) based on analysis of NOX5 NADPH oxidase activities observed in three reconstituted cell models. All three mutations are rare and predicted to have deleterious functional effects, although the proteins are detected at levels comparable to wild-type NOX5. Based on recent NOX5 crystallographic data, the locations of all mutations occur within highly conserved regions involved in electron carrier functions of NOX5: One is an in-frame 3-codon deletion at the beginning of transmembrane helix 4 (TM4) close to the outer membrane leaflet heme binding sites of TM3 and TM5, the second is a missense mutation at a conserved site shown to bind the inner leaflet heme propionate adduct, and the third is a missense mutation in a highly conserved segment of the dehydrogenase (flavo)domain that makes contact with the TM heme-containing domain B-loop. These mutated proteins have not only lost NOX5 activity, but also exhibit dose-dependent suppression of oxidase activity of co-expressed wild-type NOX5. Thus, all three mutations appear to exert dominant-negative affects on the wild-type allele, consistent with the disease manifestations observed in patients with monoallelic NOX5 gene defects; furthermore, these finding support the notion that NOX5 functions as a self-associated, tetrameric complex. Ongoing studies are attempting to decipher how NOX5 oxidase deficiencies in lymphoid nodules are manifested in inflammatory disease processes in these patients.

该项目通过对NOX/DUOX家族NADPH氧化酶遗传缺陷或遗传操作的研究，探索作为特定信号分子的ROS在淋巴细胞中的作用。这些酶催化NADPH依赖的分子氧还原生成超氧化物或过氧化氢。吞噬细胞通过含有gp91Phox(又名CyBB或NOX2)的原型NADPH氧化酶产生大量ROS，以响应感染或炎症刺激。我们在淋巴细胞中的研究正在探索NOX家族成员在对不同病原体的适应性免疫反应中的氧化还原信号作用，以及在免疫缺陷和自身免疫中的作用。在获得性免疫系统的细胞(B细胞、T细胞和树突状细胞)中检测到几种NOX家族氧化酶(即NOX2、DUOX1、NOX5)，它们与自身免疫性炎症性疾病样过程有关，如关节炎、狼疮和炎症性肠病。约40%的NOX2缺乏的慢性肉芽肿性疾病患者患有自身免疫性并发症和极早发病的炎症性肠病(VEOIBD)。其他几种NOX同工酶的缺失与炎症性肠病有关，尽管目前尚不清楚这些氧化酶缺陷是否表现在淋巴系或其他造血细胞系中。 2020年，我们继续与Sullivan博士和Kelsen博士(费城儿童医院)合作研究在他们的原发免疫缺陷患者队列中发现的NOX5基因缺陷的特征。到目前为止，我们已经根据在三个重组细胞模型中观察到的NOX5 NADPH氧化酶活性的分析，在极早发型炎症性肠病(VEOIBD)患者中识别出三个不同的NOX5功能丧失蛋白变体。这三种突变都很罕见，预计会产生有害的功能影响，尽管检测到的蛋白质水平与野生型NOX5相当。根据最近的结晶学数据，所有突变的位置都发生在与NOX5的电子载体功能有关的高度保守的区域：一个是跨膜螺旋4(TM4)开始处靠近TM3和TM5外膜血红素结合位点的框内3密码子缺失，第二个是与内小叶血红素丙酸加合物结合的保守位点的错义突变，第三个是脱氢酶(FEHO)结构域高度保守的片段上的错义突变，该片段与TM含血红素的结构域B-环接触。这些突变蛋白不仅失去了NOX5的活性，而且对共表达的野生型NOX5的氧化酶活性也表现出剂量依赖性的抑制作用。因此，所有三个突变似乎都对野生型等位基因产生显性-负面影响，这与单等位基因NOX5基因缺陷患者的疾病表现一致；此外，这些发现支持NOX5作为自相关的四聚体复合体发挥作用的观点。正在进行的研究正在试图破译淋巴结节中的NOX5氧化酶缺陷是如何在这些患者的炎症疾病过程中表现出来的。