NOX2突变导致CGD患者B细胞TLRs通路缺陷的分子机制研究

Chronic granulomatous disease (CGD) is a primary immunodeficiency disease caused by a defect in phagocyte NADPH oxidase (NOX2). In addition to infection, majority of CGD patients also suffered from autoimmune diseases, which indicated that NOX2 and its product reactive oxygen species (ROS) played a role not only in innate immunity, but also in adaptive immunity. ..In our previous studies, we found decreased memory B cells (both switched and unswitched memory B cells) and increased naïve B cells in CGD patients. Besides, impaired B cell proliferation, activation and immunoglobulin production in response to TLRs agonist was observed in NOX2-deficient CGD patients, which indicated that CGD patients had a defective B cell response to TLRs activation. Moreover, we found that clearance of intracellular ROS inhibited TLRs downstream signal pathway of STAT3 activation in B cells, which is one of the important signal pathway mediated B cell response to TLRs activation. Therefore, we speculated that lack of ROS in CGD patients led to abnormal TLRs-mediated signal signaling pathways, e.g., abnormal STAT3 signaling pathway, further resulted in defective B cell response to TLRs activation. ..In this study, we aim to investigate the effect of NOX2 mutation on TLRs-induced B cell immune response in CGD patients, and determine whether this deficiency is due to lack of ROS. Besides, we try to investigate the effect of ROS on TLRs-induced B cell immune response-related signaling pathway, further clarify the underlying molecular mechanism. Finally, we will identify gene regulatory networks underlying the defective B cell response to TLRs activation in CGD patients by high-throughput RNA sequencing. As a whole, our study aims at revealing the mechanism of defective B cell response to TLRs activation in CGD patients, which will provide new insights into pathogenesis and treatment of this disease.

慢性肉芽肿病（CGD）是由吞噬细胞NADPH氧化酶（NOX2）缺陷导致的原发性免疫缺陷病。除感染外，患者还常伴有自身免疫性疾病，提示NOX2及其产物活性氧（ROS）除在固有免疫，还在适应性免疫中发挥重要作用。我们前期研究发现，CGD患者记忆性B细胞减少；Toll样受体（TLRs）介导的B细胞免疫应答缺陷；清除ROS可抑制B细胞TLRs下游STAT3通路。因此推测，CGD患者ROS生成障碍可抑制STAT3等信号转导通路，导致TLRs介导的B细胞免疫应答缺陷。本项目拟研究NOX2突变对CGD患者TLRs介导的B细胞免疫应答的影响，明确其是否因ROS缺陷导致；研究ROS对B细胞TLRs下游相关信号通路的影响，明确其分子机制；最后构建CGD患者B细胞TLRs通路缺陷的基因调控网络，以全面阐明NOX2突变引起的ROS生成障碍导致CGD患者B细胞TLRs通路缺陷的分子机制，为其提供新的治疗思路和靶点。

慢性肉芽肿病（CGD）是一种罕见的原发性免疫缺陷病，以肺部、胃肠道、淋巴结、 皮肤、 肝脏等部位的反复感染为主要特征，严重者可危及生命。除感染外，高发的炎症性疾病和自身免疫性疾病也是影响患者生存质量的重要原因。在本研究中，我们研究了吞噬细胞NADPH氧化酶（NOX2）缺陷对于CGD患者B细胞Toll样受体（TLRs）通路介导的免疫应答的影响及其分子机制。主要包括以下研究内容：（1）从不同层次探讨了 CGD 患者适应性免疫特征的改变，包括 CGD患者淋巴细胞活性氧生成、淋巴细胞亚群分布和血清细胞因子分布；（2）研究了NOX2缺陷对于CGD患者B细胞TLRs通路免疫应答反应的影响；（3）探讨了NOX2缺陷导致CGD患者B细胞TLRs通路免疫应答异常的分子机制。通过本项目研究，我们发现：（1）CGD患者外周血T/B淋巴细胞的活性氧（ROS）生成均存在缺陷；（2）CGD患者淋巴细胞亚群分布异常，主要表现为中央记忆型T细胞的比例显著降低，以及记忆性B细胞、调节性B (Breg) 细胞的比例显著降低；（3）与对照组相比，CGD患者部分血清因子的水平发生改变；（5）CGD患者B细胞TLRs通路介导的免疫应答异常。最后，通过高通量的RNA-seq技术比较CGD患者和健康同龄对照外周血B细胞在TLRs激动剂刺激前后的差异表达基因，我们构建了CGD患者TLRs介导的B细胞活化异常的基因调控网络。本项目初步阐明了NOX2缺陷的CGD患者B细胞TLRs通路缺陷的分子机制，为CGD患者提供了新的治疗思路和新的治疗靶点。项目共发表受本项目资助的论著9篇。在本项目基础上培养博士研究生1名，硕士研究生1名，在读硕士研究生1名。