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: 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
• 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

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.

该项目通过对 NADPH 氧化酶的 Nox/Duox 家族进行基因操作，探讨活性氧 (ROS) 作为特异性信号分子在适应性免疫系统中的作用。这些酶是膜黄细胞色素，催化 NADPH 依赖性分子氧还原，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，以响应感染或炎症刺激。最近发现的 gp91phox 的多个同源物（Nox1、Nox3-5、Duox1、Duox2）开启了对 Nox 衍生 ROS 在非吞噬细胞中作用的研究。在非吞噬细胞中，Nox 酶产生较低水平的 ROS，可充当信号分子。我们将 T 淋巴细胞作为模型系统进行研究，因为它们具有完善的信号传导功能及其在人类健康和疾病中的关键作用。我们对 Nox 家族成员在淋巴细胞中的功能的研究为确定 ROS 生成在针对不同病原体的适应性免疫反应中的独特作用及其在自身免疫或免疫缺陷中的作用提供了机会。尽管最初被理解为吞噬细胞采用的抗菌机制，但我们的研究表明，由几种 NADPH 氧化酶家族成员有意产生的 ROS 在 TCR 刺激的 T 细胞中发挥特定的信号传导作用。 我们发现 TCR 刺激在体外诱导三个动力学上不同的 ROS 生成阶段。 早期 H2O2 的产生来自 Duox1，激活肌醇 1,4,5 三磷酸受体 1 的下游；随后的反应之一来自 Nox2，它在 Fas 受体下游被激活。我们的数据表明，不同的 ROS 生成阶段需要涉及不同激活机制和位置的受体-受体反式激活过程。在细胞因子的产生方面，Nox2 衍生的晚期 ROS 抑制 Th1 并增加 Th2 细胞因子的产生，而来自 Duox1 的早期 ROS 则增加 Th1 和 Th2 细胞因子的产生。 2011 年，我们开发了完整的淋巴细胞靶向（条件性）Duox1 缺陷小鼠模型，以检验我们关于 Duox1 在整个动物中作为 TCR 功能正调节因子的作用的假设。 这些研究将有助于研究基于 Duox1 的信号传导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，来自 Nox4 缺陷小鼠的 TCR 刺激的 CD4+ T 细胞诱导了更多的 IL-17 分泌。 使用磁珠通过基于细胞标记的阴性选择从野生型和 Nox4 缺陷小鼠中分离 CD4+ T 细胞，并通过流式细胞术对 CD4+ T 细胞亚群进行分选。 在不同条件下，在抗 CD3/抗 CD28 抗体包被的板上刺激分选的 T 细胞，并检查它们的细胞因子产生。这些研究确定了 Nox4 衍生的 ROS 在 T 辅助细胞亚群分化中的新作用。数据表明，NADPH 氧化酶同源物 Nox4 产生的 ROS 在 T 细胞分化中发挥调节作用，并且 Nox4 衍生的 ROS 抑制炎症 TH-17 细胞谱系的分化。 我们的研究表明，存在三个基于不同 Nox 异构体产生的 ROS 的独立信号传导过程，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，动物模型研究对于理解这些不同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。