Role of Reactive Oxygen Species in Lymphocyte Development and Function

这个项目探索了活性氧簇(ROS)，如过氧化氢和超氧阴离子，在适应性免疫系统中作为信号的特定调节器的作用。受体刺激诱导产生ROS作为信号分子，在几种细胞类型中控制受体功能。过氧化氢等氧化剂与淋巴细胞激活有关，但这一观察背后的分子机制尚不清楚。尽管ROS的来源(S)及其作用机制尚不清楚，但T细胞受体(TCR)诱导的信号转导受到有意的受体介导的ROS产生的调节。吞噬细胞通过含有gp91Phox(NOX2)的原型NADPH氧化酶(NOX)产生大量的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衍生的ROS在非吞噬细胞中作用的研究。在非吞噬细胞中，NOx家族成员产生较低水平的ROS，这些ROS可以作为信号分子。我们对NOx家族成员在淋巴细胞中的功能的研究提供了机会，以确定故意产生的ROS在对不同病原体的适应性免疫反应中的不同作用，并调查与自身免疫或免疫缺陷的可能联系。 我们早期的研究表明，T细胞在TCR刺激下产生两个阶段的H_2O_2，而后一个阶段通过TCR-Fas受体的反式激活，涉及到吞噬基于NOX2/p47Phox的NADPH氧化酶。然而，目前尚不清楚TCR刺激如何触发早期过氧化氢的产生，以及这如何作为TCR信号的第二信使发挥作用。我们鉴定了钙依赖的、非吞噬的NADPH氧化酶Duox1和DuOX2在培养和原代培养的人类CD4+T细胞中的功能表达。负责TCR刺激的早期ROS产生的是Duox1，而不是DUOX2。Duox1的激活依赖于TCR诱导的酪氨酸激酶信号通路的激活。细胞内钙释放和可能的磷酸化都是产生依赖于Duox1的过氧化氢所必需的。Duox1来源的过氧化氢正向调节TCR信号和细胞因子的产生。我们之前的研究表明，由于ERK激活增强，NOX2缺陷的小鼠CD4+T细胞具有TH1偏斜的细胞因子谱。Duox1在TCR信号转导中显示出非常不同的作用。敲除人CD4+T细胞中的Duox1选择性地抑制过氧化氢的产生、近端信号分子的酪氨酸磷酸化和ERK的激活，提示Duox1在TCR信号转导中起积极作用。Duox1基因敲除导致人类CD4+T细胞产生的细胞因子减少，而没有任何明显的TH1或TH2型偏斜。 近端的TCR信号分子被认为处于磷酸化和去磷酸化状态之间的动态平衡。我们的研究支持通过一个正反馈回路放大TCR信号的方案，该回路涉及Duox1介导的H_2O_2产生。在TCR刺激下，蛋白酪氨酸磷酸酶SHP-2与近端的TCR信号分子结合，通过pY319的去磷酸化负调控ZAP-70。同时，依赖于TCR的信号通过Duox1导致局部过氧化氢的产生和SHP-2活性部位半胱氨酸的氧化。Duox1介导的SHP-2失活从而降低了其负调控活性，并通过Lck-TCR-ZAP-70途径进一步增强了TCR信号。在人类CD4+T细胞中，Duox1基因敲除也削弱了TCR刺激的持续钙内流、NFAT的激活和细胞因子的产生。

This program explores the roles of reactive oxygen species (ROS), such as H2O2 and superoxide anion, as specific regulators of signaling in the adaptive immune system. Receptor stimulation induces generation of ROS as signaling molecules, which control receptor function in several cell types. Oxidants such as H2O2 have been linked to lymphocyte activation, but the molecular mechanisms behind this observation are unclear. T cell receptor (TCR)-induced signaling is regulated by deliberate receptor-mediated ROS production, although the source(s) of ROS and their mechanism of action remain unknown. 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 family members produce lower levels of ROS that can act as signaling molecules. Our studies of the functions of Nox family members in the lymphocyte provide opportunities to establish distinct roles of deliberate ROS generation in adaptive immune responses to diverse pathogens and investigate possible links to autoimmunity or immunodeficiency. Our early studies showed that T cells exhibit two phases of H2O2 production upon TCR stimulation, and that the later phase involves a phagocytic Nox2/p47phox-based NADPH oxidase through TCR-Fas receptor transactivation. However, it remained unclear how TCR stimulation triggers early H2O2 generation and how this works as a second messenger in TCR signaling. We identified functional expression of calcium dependent, non-phagocytic NADPH oxidases, Duox1 and Duox2, in cultured and primary human CD4+ T cells. Duox1, not Duox2, is responsible for early TCR-stimulated ROS generation. Duox1 activation depends on TCR-induced activation of tyrosine kinase signaling pathways. Both calcium release from intracellular stores and possibly phosphorylation are necessary for Duox1-dependent H2O2 generation. Duox1-derived H2O2 positively regulates TCR signaling and cytokine production. Our previous studies showed that Nox2-deficient murine CD4+ T cells have a TH1-skewed cytokine profile due to enhanced ERK activation. Duox1 showed a very different role in TCR signaling. Knockdown of Duox1 in human CD4+ T cells selectively inhibits H2O2 generation, tyrosine phosphorylation of proximal signaling molecules and activation of ERK, suggesting a positive role in TCR signal transduction. Knockdown of Duox1 led to a decrease in cytokine production of human CD4+ T blasts without any obvious skewing to a TH1- or TH2-type profile. Proximal TCR signaling molecules are considered to be in a dynamic equilibrium between phosphorylated and dephosphorylated states. Our study supports a scheme of TCR signal amplification through a positive feedback loop involving Duox1-mediated H2O2 production. Upon TCR stimulation, the protein tyrosine phosphatase SHP-2 associates with proximal TCR signaling molecules and negatively regulates ZAP-70 through dephosphorylation of pY319. Concomitantly, TCR-dependent signaling leads to local production of H2O2 through Duox1 and oxidation of the active site cysteine of SHP-2. Duox1-mediated inactivation of SHP-2 thereby diminishes its negative regulatory activity and further enhances TCR signaling through the Lck-TCR-ZAP-70 pathway. In human CD4+ T cells, Duox1 knockdown also impaired TCR-stimulated sustained calcium influx, activation of NFAT, and cytokine production.