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

基本信息

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
项目类别：
财政年份：
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

项目摘要

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.

该程序通过研究NOX/DUOX家族NADPH氧化酶的遗传缺陷或遗传操纵，探讨了活性氧（ROS）作为淋巴样细胞中特定信号分子的作用。这些酶催化了NADPH依赖性分子氧的还原，从而产生超氧化物或过氧化氢。吞噬细胞通过含有GP91Phox的原型NADPH氧化酶（又称CYBB或NOX2）的原型NADPH氧化酶产生大量ROS。我们在淋巴细胞中的研究正在探索NOX家族成员在适应性免疫反应以及免疫缺陷和自身免疫性中的氧化还原信号传导作用。在自适应免疫系统（B细胞，T细胞和树突状细胞）的细胞中检测到几种NOX家族氧化酶（即NOX2，DUOX1，NOX5），并且与自身免疫性炎症性疾病样过程，例如关节炎，狼疮性和炎症性肠道疾病。 NOX2缺乏症患者中约有40％的慢性肉芽肿性疾病患者患有自身免疫性并发症和非常早期发作炎症性肠病（VEOIBD）。其他几种NOX同工酶中的缺陷已与炎症性肠病有关，尽管尚不清楚这些氧化酶缺陷是否表现在淋巴样或其他造血细胞谱系中。在2020年，我们继续与Drs进行合作研究。 Sullivan和Kelsen（费城儿童医院）关于原发性免疫缺乏症患者的同类中NOX5遗传缺陷的表征。到目前为止，我们已经根据在三个重构细胞模型中观察到的NOX5 NADPH氧化酶活性的分析来确定患有非常早期炎症性肠病（VEOIBD）患者的三种不同的NOX5功能丧失蛋白变异。尽管在与野生型NOX5相当的水平上检测到蛋白质，但所有三个突变都是罕见的，并且预计具有有害的功能作用。 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加合物，第三个是在高度保守的脱氢酶（Flavo）结构域中的错义突变，该域与含TM血红素的域B环接触。这些突变的蛋白质不仅失去了NOX5活性，而且还表现出对共表达野生型NOX5的氧化酶活性的剂量依赖性抑制。因此，这三个突变似乎对野生型等位基因产生了显着的阴性影响，这与单相关NOX5基因缺陷患者观察到的疾病表现一致。此外，这些发现支持了NOX5充当自我相关的四聚体配合物的观念。正在进行的研究试图破译淋巴结结节中NOX5氧化酶缺陷如何在这些患者的炎症性疾病过程中表现出来。