NFIL3导致的高IgE综合症IgE类别转换重组增强的机制研究

Hyper-IgE syndrome (HIES) is a primary immunodeficiency with extremely high serum IgE levels which accompanied with atopic dermatitis, susceptibility to recurrent staphylococcal skin abscesses and pneumonia. To date, three genes such as STAT3, TYK2 and DOCK8 have been identified with 90% HIES patients resulted from STAT3 mutation (STAT3-HIES). In addition, the IgE level is associated with pathogenesis of allergy, parasitic infection and tumor formation. Despite the mechanism of recurrent bateria infections of HIES has been elucidated, the exact clinical importance and pathogenesis of pathologically increased IgE levels remains unclarified. Thus, deepening mechanic study on the IgE regulation is of great academic significance in clinical intervention and pathogenesis of HIES..It is reported that levels of extracellular signal cytokine and the prolonged half-life of IgE are not the causes of dramatically increased IgE levels in STAT3-HIES patients. According to our preliminary work, we found that the slight increase of IgE-secreting plasma cells couldn't explain the tremendously increased IgE level and that the key class switch recombination enzyme (AID) was up-regulated in STAT3-HIES patients. Intriguingly, we found that deregulation of immunoglobulin class switch recombination (CSR) in IgE secreting plasma cells in STAT3-HIES patients might play a key role in dramatically increased IgE levels. Nuclear factor IL-3 regulated (NFIL3) is a newly discovered transcriptional factor. During STAT3-HIES IgE-secreting plasma cells differentiating, NFIL3 was significantly upregulated. The CSR of IgE was down-regulated in STAT3-defeciency mice as well as NFIL3-defeciency mice, however IL-4, a STAT3-independent cytokine, promotes NFIL3 expression by STAT6 dependent manner. Thus, we hypothesize that NFIL3 may play a key role in dramatically increased IgE levels in STAT3-HIES patients..By using human STAT3-HIES as study model, our study set out to apply Epstein-Barr virus transformed B lymphocytes technique to construct immortalized STAT3-HIES B cell line and integrate ChIP, Co-IP, Luciferase, EMSA and RNA interference assays and bioinfomatics analysis to decipher the role of NFIL3 in pathological IgE class swith recombination of HIES from transcriptional regulation and epigenetics modification views of point. We mostly focus on NFIL3 expression levels and its location in cells, as well as its role in CSR enhancement, IgE-secreting plasma cells differentiation, we also put an emphasis on NFIL3 target genes and signaling pathways. In-depth insight of the pathogenic role of NFIL3 within human STAT3-HIES has great significance in clarifying the pathogenesis of HIES and exploiting effective targeting interventions to improve clinical outcomes. Also, it can provide valuable clues for the clinical treatment of IgE-related diseases, such as parasite infection and malignant diseases.

STAT3突变的高IgE综合症(HIES)IgE病理性增高，但其增高机制和临床意义尚不清楚。HIES IgE增高与信号刺激和半衰期无关，我们发现其IgE类别转换重组障碍。敲除STAT3，依赖STAT3的IgE类别转换重组缺失。NFIL3缺失IgE类别转换重组障碍，而IL-4可通过STAT6促进NFIL3高表达，因此我们推测NFIL3是导致HIES IgE增高的另外一条通路。我们以人HIES为研究模型，通过EBV转染建立HIES永生B细胞，利用细胞和分子生物学方法、整合生物信息学分析，拟从转录调控和表观修饰层面探讨NFIL3对HIES IgE类别转换重组影响，研究包括NFIL3表达和核浆分布，其对CSR增强作用、诱导IgE浆细胞分化、作用靶基因和信号传导通路等。此为阐明HIES发病机制和探索靶向干预提供理论依据；也为筛选有效抗IgE药物治疗过敏、寄生虫感染和肿瘤等IgE相关疾病开辟新途径。

高IgE综合征（Hyper IgE syndrome, HIES）是一种极为罕见的原发性免疫缺陷病，临床主要表现为湿疹、反复皮肤和肺部感染，并伴有血清IgE水平显著升高。STAT3基因突变导致的常染色显性遗传（AD）HIES占HIES患者的大多数。目前，AD-HIES患者血清IgE水平极度增高的分子机制仍然不甚明了。在本项研究中，我们共收集了17例AD-HIES患者，对其临床表现，实验室检查结果进行了详尽的分析。我们在17例STAT3突变的患者中发现了11种不同的突变，其中包括1种新发突变。这是目前为止中国最大规模样本的HIES报道，极大地促进了中国临床医师对于HIES患者的认知能力。我们对部分HIES患者的淋巴细胞表型进行分析。结果表明， STAT3 突变并未影响到B 淋巴细胞早期分化发育，也不影响浆细胞的发育；但可影响各类记忆性B细胞以及B1细胞的分化，导致其比例降低；然而IgE+B细胞以及IgE+记忆性B细胞升高均升高，这可能是导致HIES患者血清IgE升高的原因之一。HIES患儿类别转换型记忆性B细胞降低，说明STAT3突变可导致抗体的类别转换障碍。当利用IL-4与CD40L协同刺激B细胞，NFIL3基因的表达量在STAT3突变的HIES患者中的表达量明显高于正常对照。IL-4与CD40L协同刺激EBV转化的永生化B细胞株，IgE的水平在HIES患者中高于正常人，而IgE的水平在NFIL3沉默型的正常人以及HIES患者细胞株中均降低；同时NFIL3 KD后可降低CSR关键基因的表达水平，说明NFIL3参与了IL4诱导的HIES患儿血清IgE的生成过程。染色质免疫共沉淀联合荧光报告基因实验证明，NFIL3可结合Iε启动子，并调控Iε的转录。最后，我们还发现，STAT3表达抑制后，TLR9信号通路介导的B细胞活化障碍，导致B细胞的增殖、凋亡及表面共刺激分子表达障碍。. 综上所述，本研究报道了17例HIES患者，是中国最大规模样本的HIES报道。我们发现，STAT3突变可导致HIES患者各类记忆性B细胞分化障碍，IL4介导的NFIL3表达水平的升高可能是导致HIES患者血清IgE水平升高的原因；NFIL3可直接调控Iε基因的表达，进一步促进抗体的类别转换。抑制STAT3表达导致TLR9信号通路介导的B细胞活化障碍。

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