Role of Reactive Oxygen Species in Lymphocyte Development and Function

这个项目通过对NOX/DUOX家族NADPH氧化酶的遗传操作，探索了活性氧簇(ROS)作为B和T淋巴细胞中特定信号分子的作用。这些酶催化NADPH依赖的分子氧还原生成超氧化物或过氧化氢。吞噬细胞通过含有gp91Phox(又名NOX2)的原型NADPH氧化酶产生大量ROS，以响应感染或炎症刺激。虽然最初被认为是吞噬细胞的抗菌机制，但我们的研究发现，由几个NOX家族成员故意产生的ROS在B细胞受体(BCR)刺激的B细胞和T细胞受体(TCR)刺激的T细胞中发挥着特定的信号作用。我们对淋巴细胞的研究正在探索NOX家族成员在对不同病原体的适应性免疫反应以及自身免疫或免疫缺陷中的作用。 我们最近发现，基于DUOX1和NOX2的NADPH氧化酶在体外与白介素4(IL-4)和抗IgM共刺激的脾B淋巴细胞中具有不同的信号功能。与野生型或DUOX1-/-细胞相比，NOX2缺陷的B细胞表现出更强的细胞凋亡率和更低的IgM、IgG1和IgG2a生成。DUOX1基因缺陷的细胞增殖增强，这与几种BCR信号中间产物(Akt、BCAP和Rgs16)的表达增强有关。 2017年，我们扩大了上述发现，研究了T细胞依赖和独立抗原(分别为硝基苯基-锁眼血蓝蛋白(NP-KLH)和硝基苯基-脂多糖(NP-LPS))免疫整个动物的反应。DUOX1基因缺陷小鼠、NOX2基因缺陷小鼠和野生型小鼠血清中抗NP-内毒素(IgM，免疫后7天)和抗NP-KL H(免疫后14天，免疫后14天)的反应无显著差异。然而，免疫小鼠脾的免疫组织化学染色显示DUOX1基因敲除小鼠的脾细胞明显不同，与野生型和NOX2基因敲除小鼠相比，具有强烈反应性生发中心和增殖标记染色的卵泡数量显著增加。这些发现与DUOX1基因敲除小鼠在体外观察到的刺激的脾B细胞增殖增强是一致的。综上所述，我们的研究结果表明，DUOX1可以抑制B细胞的增殖，但对全身免疫球蛋白的产生或类别转换的影响可能很小。鉴于IL-4在B淋巴细胞中诱导DUOX1的独特作用，未来的工作将研究DUOX1对感染的组织特异性适应性免疫功能。

This program explores roles of reactive oxygen species (ROS) as specific signaling molecules in B and T lymphocytes through 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., NOX2). Although originally understood as an anti-bacterial mechanism deployed by phagocytes, our research revealed that ROS intentionally generated by several NOX family members play specific signaling roles in B cell receptor (BCR)-stimulated B cells and T cell receptor (TCR)-stimulated T cells. Our studies in lymphocytes are exploring roles of NOX family members in adaptive immune responses to diverse pathogens as well as in autoimmunity or immunodeficiencies. We recently showed that DUOX1- and NOX2-based NADPH oxidases serve distinct signaling functions in splenic B lymphocytes co-stimulated ex vivo with interleukin-4 (IL-4) and anti-IgM. NOX2-deficient B cells showed enhanced apoptosis and diminished IgM, IgG1 and IgG2a production relative to wild type or DUOX1-/- cells. DUOX1-deficient cells exhibited enhanced proliferation, which was correlated with enhanced expression of several BCR signaling intermediates (Akt, BCAP and RGS16). In 2017, we extended the above findings by examining whole animal responses to immunization with T cell dependent and independent antigens (nitrophenyl-keyhole limpet hemocyanin (NP-KLH) and nitrophenyl-lipopolysaccharide (NP-LPS), respectively). No significant differences in serum anti-NP-LPS (IgM, 7 days post-immunization) or anti-NP-KLH (IgG, 14 days post-immunization) were detected when comparing these responses in DUOX1-deficient, NOX2-deficient and wild type mice. However, immuno-histochemical staining of spleens from immunized mice revealed distinct differences in DUOX1 knockout mice, which showed markedly enhanced numbers of follicles with intense reactive germinal center and proliferation marker staining relative to that observed in wild type and NOX2 knockout mouse spleens. These findings are consistent with the enhanced stimulated splenic B cell proliferation observed in vitro with DUOX1 knockout mice. Together, our findings suggest that DUOX1 can suppress B cell proliferation, but may have little influence on systemic immunoglobulin production or class switching. Given the unique induction of DUOX1 by IL-4 in B lymphocytes, future work will examine tissue-specific adaptive immune functions of DUOX1 in response to infection.