Role of Reactive Oxygen Species in Lymphocyte Development and Function

该计划通过对NOX/DUOX家族NADPH氧化酶的遗传缺陷或遗传操作的研究，探索活性氧（ROS）作为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）。我们在淋巴细胞中的研究正在探索NOX家族成员在对各种病原体的适应性免疫应答以及免疫缺陷和自身免疫中的氧化还原信号传导作用。几种NOX家族氧化酶（即，在适应性免疫系统的细胞（B细胞、T细胞和树突状细胞）中检测到（例如，NOX 2、DUOX 1、NOX 5），并且已经与自身免疫炎性疾病样过程（例如，关节炎、狼疮和炎性肠病）相关。约40%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，尽管目前尚不清楚这些氧化酶缺陷是否表现在淋巴或其他造血细胞谱系中。 2020年，我们继续与Sullivan博士和Kelsen博士（费城儿童医院）合作研究在其原发性免疫缺陷患者队列中检测到的NOX 5遗传缺陷的特征。到目前为止，我们已经确定了三个不同的NOX 5功能丧失蛋白变体的患者非常早发性炎症性肠病（VEOIBD）的基础上分析NOX 5 NADPH氧化酶活性观察到三个重建的细胞模型。所有这三种突变都是罕见的，并且预测具有有害的功能效应，尽管检测到的蛋白质水平与野生型NOX 5相当。基于最近的NOX 5晶体学数据，所有突变的位置都发生在参与NOX 5电子载体功能的高度保守区域内：一个是在跨膜螺旋4（TM 4）的开始处靠近TM 3和TM 5的外膜小叶血红素结合位点的框内3密码子缺失，第二个是在显示结合内小叶血红素丙酸酯加合物的保守位点处的错义突变，第三个是在脱氢酶（flavo）结构域的高度保守区段中的错义突变，其与含TM血红素的结构域B环接触。这些突变的蛋白质不仅丧失了N 0X 5活性，而且还表现出共表达的野生型N 0X 5的氧化酶活性的剂量依赖性抑制。因此，所有三种突变似乎都对野生型等位基因产生显性负效应，这与在具有单等位基因NOX 5基因缺陷的患者中观察到的疾病表现一致;此外，这些发现支持了NOX 5作为自相关四聚体复合物发挥作用的观点。正在进行的研究试图解释淋巴结中的NOX 5氧化酶缺陷如何在这些患者的炎症性疾病过程中表现出来。

This program explores roles of reactive oxygen species (ROS) as specific signaling molecules in lymphoid cells through studies on inherited deficiencies or 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). Our studies in lymphocytes are exploring redox signaling roles of NOX family members in adaptive immune responses to diverse pathogens, as well as in immunodeficiencies and autoimmunity. Several NOX family oxidases (i.e., NOX2, DUOX1, NOX5) are detected in cells of the adaptive immune system (B cells, T cells and dendritic cells) and have been associated with auto-immune inflammatory disease-like processes, such as arthritis, lupus and inflammatory bowel disease. About 40% 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 remains unclear whether these oxidase defects are manifested in lymphoid or other hematopoietic cell lineages. In 2020, we have continued collaborative studies with Drs. Sullivan and Kelsen (Childrens Hospital of Philadelphia) on the characterization of NOX5 genetic defects detected in their cohort of patients with primary immune deficiencies. Thus far, we have identified three distinct NOX5 loss-of-function protein variants in patients with very early onset inflammatory bowel disease (VEOIBD) based on analysis of NOX5 NADPH oxidase activities observed in three reconstituted cell models. All three mutations are rare and predicted to have deleterious functional effects, although the proteins are detected at levels comparable to wild-type NOX5. Based on recent NOX5 crystallographic data, the locations of all mutations occur within highly conserved regions involved in electron carrier functions of NOX5: One is an in-frame 3-codon deletion at the beginning of transmembrane helix 4 (TM4) close to the outer membrane leaflet heme binding sites of TM3 and TM5, the second is a missense mutation at a conserved site shown to bind the inner leaflet heme propionate adduct, and the third is a missense mutation in a highly conserved segment of the dehydrogenase (flavo)domain that makes contact with the TM heme-containing domain B-loop. These mutated proteins have not only lost NOX5 activity, but also exhibit dose-dependent suppression of oxidase activity of co-expressed wild-type NOX5. Thus, all three mutations appear to exert dominant-negative affects on the wild-type allele, consistent with the disease manifestations observed in patients with monoallelic NOX5 gene defects; furthermore, these finding support the notion that NOX5 functions as a self-associated, tetrameric complex. Ongoing studies are attempting to decipher how NOX5 oxidase deficiencies in lymphoid nodules are manifested in inflammatory disease processes in these patients.