Role of Reactive Oxygen Species in Lymphocyte Development and Function

该计划通过对NADPH氧化酶的Nox/Duox家族的遗传操作，探索活性氧（ROS）作为适应性免疫系统中特异性信号分子的作用。这些酶是膜黄细胞色素，其催化分子氧的NADPH依赖性还原以产生超氧化物和/或过氧化氢。吞噬细胞通过含有gp 91 phox（Nox 2）的原型NADPH氧化酶（Nox）响应感染或炎症刺激而产生大量ROS。最近发现的gp 91 phox的多个同源物（Nox 1，Nox 3 -5，Duox 1，Duox 2）开启了对Nox衍生的ROS在非吞噬细胞中的作用的研究。在非吞噬细胞中，Nox酶产生较低水平的ROS，其可以充当信号分子。我们已经研究了T淋巴细胞作为一个模型系统，因为它们的完善的信号功能和它们在人类健康和疾病中的关键作用。我们对淋巴细胞中Nox家族成员的功能的研究提供了机会，以建立在对不同病原体的适应性免疫应答中故意产生ROS的不同作用及其在自身免疫或免疫缺陷中的作用。虽然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作用。 我们发现，TCR刺激诱导三个动力学不同的ROS生成阶段在体外。 早期H2 O2产生来自Duox 1，在肌醇1，4，5三磷酸受体1的下游被激活;后来的反应之一来自Nox 2，在Fas受体的下游被激活。我们的数据表明，不同的ROS生成阶段需要受体-受体的反式激活过程涉及不同的激活机制和位置。在细胞因子产生方面，Nox 2衍生的晚期ROS抑制Th 1细胞因子产生并增加Th 2细胞因子产生，而Duox 1衍生的早期ROS增加Th 1和Th 2细胞因子产生。 2011年，我们开发了完整的淋巴细胞靶向（条件性）Duox 1缺陷小鼠模型，以检验我们关于Duox 1作为整个动物体内TCR功能正调节因子的假设。 这些研究将允许调查基于Duox 1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，来自Nox 4缺陷小鼠的TCR刺激的CD 4 + T细胞诱导更大量的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Nox 4缺陷型小鼠中分离CD 4 + T细胞，并通过流式细胞术分选CD 4 + T细胞亚群。 在各种条件下在抗-CD 3/抗-CD 28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Nox 4衍生的ROS在T辅助细胞亚群分化中的新作用。这些数据表明，从NADPH氧化酶同源物Nox 4产生的ROS在T细胞分化中起调节作用，并且Nox 4衍生的ROS抑制炎性TH-17细胞谱系的分化。 我们的研究表明，有三个独立的信号传导过程的基础上产生的ROS从不同的Nox亚型，产生不同的生物学结果在TCR刺激的T细胞。免疫系统的反应是多方面的，动物模型的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。

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.