Role of Reactive Oxygen Species in Lymphocyte Development and Function

本课程通过对NOX/DUOX家族NADPH氧化酶的遗传操作，探索活性氧（ROS）作为B和T淋巴细胞中特异性信号分子的作用。这些酶催化NADPH依赖性的分子氧还原以产生超氧化物或过氧化氢。吞噬细胞通过含有gp 91 phox的原型NADPH氧化酶（也称为，CYBB或NOX 2）。虽然最初被理解为吞噬细胞部署的氧化剂依赖性微生物杀伤机制，但我们的研究表明，由其他NOX同系物有意产生的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细胞中发挥特定的信号传导作用。我们在淋巴细胞中的研究正在探索NOX家族成员在对不同病原体的适应性免疫应答中的作用，以及在免疫缺陷和自身免疫中的作用。几种NOX家族氧化酶与自身免疫炎性疾病样过程相关，如关节炎、狼疮和炎性肠病。约40-50%的具有N 0X 2缺陷的慢性肉芽肿性疾病患者患有自身免疫并发症和极早发型炎性肠病（VE 0 IBD）。其他几种NOX同工酶的缺陷与炎症性肠病有关，尽管这些氧化酶缺陷是否表现在淋巴细胞或其他造血细胞谱系中仍然很清楚。 2019年，我们完成了Duox 1参与B淋巴细胞对IL-4和B细胞受体刺激反应的研究。我们发现Duox 1在体外用IL-4处理的小鼠B淋巴细胞中被诱导，并且通过B细胞受体（BCR）的共刺激产生归因于Duox 1的ROS信号。有趣的是，受刺激的Duox 1缺陷型B细胞比野生型或NOX 2缺陷型B细胞在体外增殖更快，这与几种BCR信号传导中间体（Akt、BCAP和RGS 16）的表达增强相关。此外，我们证明了过氧化氢清除剂过氧化氢酶通过增强野生型CD 19 + B细胞的体外增殖来模拟Duox 1缺乏的效果。相比之下，NOX 2缺陷型B细胞显示出相对于野生型或DUOX 1-/-细胞增强的凋亡和减少的IgM、IgG 1和IgG 2a产生。这些观察结果与全动物免疫结果相关：用T细胞依赖性（TD）或非依赖性（TI）抗原免疫的Duox 1敲除小鼠产生正常的血清IG水平，但用TI抗原，硝基苯基-脂多糖（NP-LPS），导致更多的和扩大的脾脏反应性（Ki 67和花生抗原阳性）与野生型或NOX 2缺陷型小鼠相比，Duox 1敲除小鼠的生殖中心。我们的研究结果表明Duox 1可能在限制对BCR刺激的早期增殖反应中发挥作用，特别是对于避免T细胞识别的病原体。因此，由不同的NOX同工酶产生的ROS对受刺激的淋巴样细胞群的命运和功能具有不同的影响。未来的工作应该探索Duox 1在长寿命浆细胞的产生或Th 2免疫占主导地位的感染模型中的作用。

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., CYBB or NOX2). Although originally understood as an oxidant-dependent microbial killing mechanism deployed by phagocytes, our research revealed that ROS intentionally generated by other NOX homologues 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 immunodeficiencies and autoimmunity. Several NOX family oxidases have been associated with auto-immune inflammatory disease-like processes, such as arthritis, lupus and inflammatory bowel disease. About 40-50% of chronic granulomatous disease patients with NOX2 deficiencies suffer from autoimmune complications and very early onset inflammatory bowel disease (VEOIBD). Deficiencies in several other NOX isozymes have been linked to inflammatory bowel disease, although it is remains clear whether these oxidase defects are manifested in lymphoid or other hematopoietic cell lineages. In 2019, we completed studies on the involvement of Duox1 in B lymphocyte responses to IL-4 and B cell receptor stimulation. We showed that Duox1 is induced in murine B lymphocytes treated with IL-4 in vitro and that co-stimulation through B cell receptors (BCR) generates a ROS signal attributable to Duox1. Interestingly, stimulated Duox1-deficient B cells proliferate in vitro more rapidly than wild type or NOX2-deficient B cells, which was correlated with enhanced expression of several BCR signaling intermediates (Akt, BCAP and RGS16). Furthermore, we demonstrated that the hydrogen peroxide scavenger catalase mimics the effect of Duox1 deficiency by enhancing proliferation of wild type CD19+ B cells in vitro. In contrast, NOX2-deficient B cells showed enhanced apoptosis and diminished IgM, IgG1 and IgG2a production relative to wild type or DUOX1-/- cells. These observations were correlated with whole animal immunization findings: the Duox1 knockout mice immunized with T cell-dependent (TD) or -independent (TI) antigens produce normal serum Ig levels, but immunization with the TI antigen, nitrophenyl-lipopolysaccharide (NP-LPS), resulted in more numerous and enlarged splenic reactive (Ki67 and peanut antigen positive) germinal centers in Duox1 knockout mice when compared with wild type or NOX2-deficient mice. Our results suggest Duox1 may have a role in limiting early proliferative responses to BCR stimulation, particularly with pathogens that avoid T cell recognition. Thus, the ROS generated by distinct NOX isozymes have different effects on the fate and function of stimulated lymphoid cell populations. Future work should explore roles for Duox1 in generation of long-lived plasma cells or in infection models where Th2-based immunity prevails.