TLR9介导的STAT3通路缺陷导致HIES患儿记忆性B细胞发育障碍的分子机制的研究

The hyper IgE syndrome (HIES), or job syndrome, is a rare primary immune deficiency disorder which characterized by eczema, recurrent infections of the skin and lung and excessively elevated serum IgE levels. Additional clinical features include but not limited to a characteristic facial appearance, scoliosis, retained primary teeth, joint hyper-extensibility and recurrent bone fractures following minor trauma. Autosomal dominant HIES, the most common disease in this group, results from dominant negative STAT3 mutations. HIES has been studied for almost 50 years since HIES patient was first described by Davis et al (1966) in two sisters with eczema, cold boils, and pneumonias. However, the precise pathophysiology of HIES is still unclear. ..In our previous study, we analyzed B-cell phenotype in 8 HIES patients with STAT3 mutations and 10 age-matched healthy controls. Four-color flow cytometry of PBMC was used to analyze different B cell subpopulations. The result shows that the percentage of memory B cells is reduced in HIES patients compared with healthy controls, which suggests that the STAT3 signaling pathway might contribute to the development of memory B cells. The reduced number of memory B cells could explain not all, but at least the susceptibility to recurrent infections in HIES patients. Our further study shows that the memory B cells production decreases when CpG is used to culture human centroblast which treated with STAT3 inhibitor. This result indicates that TLR9 mediated activation of STAT3 signal pathway might be important in the differentiation of human GC B cells into memory B cells. Thus, we hypothesize that TLR9 mediated STAT3 signal pathway deficiency might contribute to the memory B cell reduction in HIES patients...In this study, we aim to find out the molecular mechanism by which memory B cell is reduced in HIES patients with STAT3 mutation. Firstly, we will purify B cell and naïve B cell from HIES patients’ peripheral blood. Through a series of bio-experiments, including flow cytometry, ELISPOT, ELISA, etc., we will compare the difference between HIES patients and healthy controls on the B cell proliferation, differentiation, antibodies production and antigen presenting function. Secondly, we will purify centroblasts from human tonsils, cultured with CD40L and IL-2 to mimic GC environment. Using this in vitro culture system, we will study the impact of TLR9 mediated STAT3 signal pathway deficiency on the B cell proliferation and differentiation. Finally, we will identify gene and cytokine regulatory networks underlying B cell differentiation by high-throughput approaches (Flowcytomix, Microarray and CHIP-sequencing). As a whole, our study will aim at revealing the comprehensive mechanism of memory B cell reduction in HIES patients, which will provide new insights into pathogenesis and treatment of this disease.

高IgE综合征（HIES）是一种罕见的原发性免疫缺陷病，临床表现复杂，其发病机制至今不甚明了。我们发现，STAT3缺陷的HIES患儿记忆性B细胞减少；当用TLR9的配体CpG处理STAT3表达抑制的生发中心母细胞，其记忆性B细胞的生成明显减少。因此我们推测，TLR9介导的STAT3通路缺陷可能导致HIES患儿记忆性B细胞发育障碍。本课题拟分离HIES患儿外周血B细胞和初始B细胞，研究TLR9介导的STAT3通路缺陷对HIES患儿B细胞增殖、分化、抗体分泌和抗原提成能力的影响；同时，我们拟在体外模拟GC微环境，研究STAT3通路缺陷对TLR9诱导的B细胞增殖和分化的影响及其分子机制；最后，我们还将构建TLR9介导的STAT3信号通路对B细胞分化的细胞因子和基因调控网络，以期全面阐明STAT3缺陷的HIES患儿记忆性B细胞发育障碍的分子机制，为HIES提供新的治疗思路和新的治疗靶点。

STAT3基因突变导致的常染色体显性遗传高IgE综合征（Autosomal dominant form of Hyper IgE syndrom, AD-HIES）是一种罕见的原发性免疫缺陷病，其临床主要表现为顽固性湿疹样皮炎、反复皮肤和肺部感染，并伴有血嗜酸性粒细胞、血清IgE水平的显著增高。此外，许多患者还表现出与免疫无关的症状，包括颅面、牙齿、骨骼肌、神经及心血管异常等。本课题报道了17例AD-HIES综合征患者的临床表现、实验室检查结果和基因突变频谱，并与世界其他国家的大规模队列进行对比分析，阐明了中国AD-HIES患者的疾病频谱，对中国AD-HIES患儿的诊断和治疗具有一定的指导意义。在此基础上，我们还受邀对不同类型的HIES综合征进行了综述分析，这部分内容已经发表在相关SCI杂志上。通过本项目研究，我们发现：（1）AD-HIES患儿B细胞发育障碍。与同龄健康对照组相比，AD-HIES患儿外周血Naïve B细胞增多，记忆性B细胞，B1细胞以及记忆性B1均降低。（2）AD-HIES患儿B细胞应对Toll样受体（Toll like receptors, TLRs）激动剂刺激时的免疫应答反应缺陷，表现为细胞增殖能力降低，细胞共活化分子表达降低，IgG的生成水平降低，IL-10表达降低。（3）通过结合RNA-seq和CHIP-seq高通量测序的结果，我们构建了TLRs介导的B细胞活化的基因调控网络。通过后续的实验验证，我们发现，STAT3可能通过直接调控BCL6，ICAM1和IL-7等B细胞发育和B细胞功能密切相关的基因的表达，来调控B细胞应对TLRs激动剂的免疫应答反应。本项目初步阐明了STAT3缺陷的HIES患者B细胞TLRs通路缺陷的分子机制，为HIES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SCI论文17篇，其中包括14篇SCI论著，2篇中文论著和1篇SCI综述。在本项目基础上获得浦东新区科技进步奖2等奖1项，培养博士研究生2名，硕士研究生1名。

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