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：
8336273
负责人：
THOMAS LETO
金额：
$ 31.29万
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/8336273
关键词：
Affect Animal Model Animals Anti-Bacterial Agents Antibodies Antigen Receptors Antioxidants Area Arthritis Autoimmune Diseases Autoimmunity Biological Biological Models CD28 gene CD3 Antigens CD4 Positive T Lymphocytes CD95 Antigens Cell Differentiation process Cell Lineage Cells Chemistry Clinical Protocols Communicable Diseases Cytokine Signaling Cytoplasm Data Development Disease Enzymes Family Family member Flow Cytometry Generations Goals Health Homologous Gene Host Defense Human Hydrogen Peroxide Immune Immune response Immune system Immunologic Deficiency Syndromes In Vitro Inflammatory Inflammatory Response Inositol Interleukin-17 Investigation Knockout Mice Knowledge Location Lymphocyte Lymphocyte Subset Lymphoid Cell Mediating Membrane Molecular Multiple Sclerosis Mus NADP NADPH Oxidase Outcome Oxidants Oxidation-Reduction Oxygen Pathogenesis Pathologic Processes Phagocytes Phase Play Process Production Protein Isoforms Proteins Reactive Oxygen Species Receptor Signaling Regulation Regulatory T-Lymphocyte Research Role Signal Transduction Signal Transduction Pathway Signaling Molecule Sorting - Cell Movement Specificity Stimulus Superoxides System T cell differentiation T-Lymphocyte Testing Transactivation Wild Type Mouse Work adaptive immunity base computerized data processing cytokine design effective intervention genetic manipulation magnetic beads mouse model novel novel therapeutics pathogen programs receptor response translational study tripolyphosphate

项目摘要

This program explores the role of reactive oxygen species (ROS) as specific signaling molecules in the adaptive immune system through genetic manipulation of the Nox/Duox family of NADPH oxidases. These enzymes are membrane flavocytochromes that catalyze NADPH-dependent reduction of molecular oxygen to generate superoxide and/or hydrogen peroxide. Phagocytes produce large amounts of ROS in response to infectious or inflammatory stimuli through the prototypic NADPH oxidase (Nox) containing gp91phox (Nox2). Recent discovery of multiple homologues of gp91phox (Nox1, Nox3-5, Duox1, Duox2) has opened studies on the roles of Nox-derived ROS in non-phagocytic cells. In non-phagocytic cells, Nox enzymes produce lower levels of ROS that can act as signaling molecules. We have studied T lymphocytes as a model system because of their well-established signaling function and their critical roles in human health and disease. Our studies of the functions of Nox family members in lymphocytes provide opportunities to establish distinct roles of deliberate ROS generation in adaptive immune responses to diverse pathogens and their roles in autoimmunity or immunodeficiency. Although originally understood as an anti-bacterial mechanism employed by phagocytes, our research revealed that ROS intentionally generated by several NADPH oxidase family members play specific signaling roles in TCR-stimulated T cells. We showed that TCR stimulation induces three kinetically distinct ROS generation phases in vitro. Early H2O2 generation comes from Duox1, activated downstream of inositol 1,4,5 triphosphate receptor 1; one of the later responses comes from Nox2, activated downstream of the Fas receptor. Our data suggest that the different ROS generation phases require receptor-receptor transactivation processes involving different activation mechanisms and locations. In terms of cytokine production, Nox2-derived late-phase ROS inhibit Th1 and augment Th2 cytokine production, whereas early-phase ROS from Duox1 augment both Th1 and Th2 cytokine production. In 2011, we have developed complete and lymphoid cell-targeted (conditional) Duox1-deficient mouse models in order to examine our hypothesis on the role of Duox1 as a positive regulator of TCR function within whole animals. These studies will allow investigations of the impact of Duox1-based signaling on immunodeficiency and autoimmune disease. In other studies, we found that TCR-stimulated CD4+ T cells from Nox4-deficient mice induced much greater amounts of IL-17 secretion compared with cells from wild-type mice. CD4+ T cells were isolated from wild type and Nox4-deficient mice by cell marker-based negative selection using magnetic beads, and CD4+ T cell subpopulations were sorted by flow cytometry. Sorted T cells were stimulated on anti-CD3/anti-CD28 antibodies-coated plates in various conditions and their cytokine production was examined. These studies identified a novel role for Nox4-derived ROS in differentiation of T helper subsets. The data suggest that ROS generation from a NADPH oxidase homologue Nox4 play regulatory roles in T cell differentiation and that Nox4-derived ROS inhibit differentiation of the inflammatory TH-17 cell lineage. Our studies suggest there are three separate signaling processes based on ROS generated from different Nox isoforms, producing different biological outcomes in TCR-stimulated T cells. The response of the immune system is multifaceted and studies in animal models are critical in understanding roles of these distinct oxidant-generating systems in host defense, adaptive immunity and related inflammatory immune processes.

该程序通过基因操纵NADPH氧化酶的NOX/DUOX家族来探讨活性氧（ROS）作为自适应免疫系统中特定信号分子的作用。这些酶是膜黄素色素，可催化NADPH依赖性分子氧的减少，从而产生超氧化物和/或过氧化氢。吞噬细胞通过含有GP91Phox（NOX2）的原型NADPH氧化酶（NOX）产生大量ROS，以响应传染性或炎症刺激。最近发现GP91Phox的多个同源物（NOX1，NOX3-5，DUOX1，DUOX2）已经开放了NOX衍生的ROS在非噬细胞中的作用的研究。在非吞噬细胞中，NOX酶产生的ROS水平较低，可以充当信号分子。由于其公认的信号传导功能及其在人类健康和疾病中的关键作用，因此我们研究了T淋巴细胞作为模型系统。我们对NOX家族成员在淋巴细胞中功能的研究提供了机会，可以在适应性免疫反应中对各种病原体的自适应免疫反应及其在自身免疫或免疫缺陷中的作用中建立不同的作用。尽管最初被理解为吞噬细胞采用的抗细菌机制，但我们的研究表明，多个NADPH氧化酶家族成员故意产生ROS在TCR刺激的T细胞中起特定的信号作用。我们表明，TCR刺激在体外诱导了三个动力学上不同的ROS产生阶段。 H2O2早期的生成来自Duox1，肌醇1,4,5三磷酸受体1的下游激活。后来的一个响应来自NOX2，即FAS受体的下游激活。我们的数据表明，不同的ROS生成阶段需要受体受体反式激活过程，涉及不同的激活机制和位置。在细胞因子的产生方面，NOX2衍生的后期ROS抑制了Th1并增强Th2细胞因子的产生，而DUOX1的早期相结合ROS均增强了Th1和Th2细胞因子的产生。在2011年，我们开发了完全且淋巴细胞靶向（条件）DUOX1缺陷小鼠模型，以研究我们对Duox1作为TCR功能在整个动物中的阳性调节剂的作用的假设。这些研究将允许研究基于DUOX1的信号对免疫缺陷和自身免疫性疾病的影响。在其他研究中，我们发现与野生型小鼠的细胞相比，NOX4缺陷小鼠的TCR刺激的CD4+ T细胞引起了更多的IL-17分泌。通过使用磁珠基于细胞标记的负选择，从野生型和NOX4缺陷小鼠中分离CD4+ T细胞，通过流式细胞仪对CD4+ T细胞亚群进行分类。在抗CD3/抗CD28抗体涂层的板上刺激分类的T细胞在各种条件下，并检查了其细胞因子的产生。这些研究确定了NOX4衍生的ROS在T辅助子集的分化中的新作用。数据表明，从NADPH氧化酶同源物NOX4产生ROS在T细胞分化中起调节作用，并且NOX4衍生的ROS抑制了炎症性TH-17细胞谱系的分化。我们的研究表明，基于不同NOX同工型产生的ROS，有三个独立的信号传导过程，在TCR刺激的T细胞中产生不同的生物学结果。免疫系统的反应是多方面的，在动物模型中的研究对于理解这些独特的氧化剂生成系统在宿主防御，适应性免疫和相关炎症免疫过程中的作用至关重要。