Role of Reactive Oxygen Species in Lymphocyte Development and Function

本项目通过研究NOX/DUOX家族NADPH氧化酶的遗传缺陷或基因操作，探索活性氧（ROS）作为淋巴样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CYBB或NOX2）的原型NADPH氧化酶对感染或炎症刺激产生大量ROS。我们在淋巴细胞中的研究正在探索NOX家族成员在各种病原体的适应性免疫反应以及免疫缺陷和自身免疫中的氧化还原信号作用。在适应性免疫系统细胞（B细胞、T细胞和树突状细胞）中检测到几种NOX家族氧化酶（即NOX2、DUOX1、NOX5），并与自身免疫性炎症样疾病过程相关，如关节炎、狼疮和炎症性肠病。约40%的慢性肉芽肿性疾病NOX2缺乏患者患有自身免疫性并发症和极早发性炎症性肠病（VEOIBD）。其他几种氮氧化物同工酶的缺乏与炎症性肠病有关，尽管尚不清楚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。

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.