HIF-1α介导NF-κB通路在甲状腺相关眼病B10细胞功能异常中的机制研究

Immune imbalance plays an important role in the pathogenesis of thyroid-associated ophthalmopathy (TAO), and the B10 cells play an inhibitory role in immune regulation by secreting IL-10. Our study found that there was functional abnormality in the peripheral human B10 cells from TAO patients, while activation of NF-κB could up-regulate of IL-10 producing in B10 cells. However, the mechanism of B10 cells dysfunction in TAO is still unknown. Higher hypoxia-inducible factor-1α (HIF-1α) level in orbital tissues and peripheral blood of TAO patients has been reported previously. Recent studies demonstrated that HIF-1α had a key function in increasing number of B10 cells and driving IL-10 expression, and there is a crosstalk between HIF-1α and NF-κB in the control of the immune response in different immune cell types. Therefore, it is hypothesized that elevated HIF-1α in TAO can regulate the proliferation of B10 cells and its secretion of IL-10 through the NF-κB signaling pathway, which is involved in the pathogenesis of TAO. For this end, this study intends to analysis the molecular mechanism of NF-κB pathway mediated by HIF-1α in the regulation of proliferation and secretion of B10 cells with the technologies including siRNA, and use in-vitro co-cultures to investigate the effect of HIF-1α level on the regulation of CD4 + T cells by B10 cells. Ultimately, this research will clarify the pathogenesis role of HIF-1α in the function of B10 cells in TAO, and provide a new immune perspective for the prevention and treatment of TAO.

免疫失衡在甲状腺相关眼病（TAO）发病中起重要作用，B10细胞亚群通过分泌IL-10发挥免疫调节抑制功能。本课题研究发现，TAO患者外周血B10细胞发生功能失调，而活化NF-κB上调B10细胞表达IL-10，但B10细胞及其功能失调的机制不明。在TAO患者的眼眶组织和外周血中HIF-1α水平上调，最新研究发现HIF-1α可调节B10细胞增殖及IL-10分泌，且HIF-1α与NF-κB通路存在负反馈相互作用。故提出假设：TAO患者中增高的HIF-1α通过NF-κB信号通路，调控了B10细胞的活化增殖、IL-10分泌，参与TAO发病。为此，本研究拟利用siRNA等技术深入研究NF-κB通路在HIF-1α调控B10细胞增殖分泌中的分子机制，采用体外刺激共培养探讨HIF-1α水平对B10细胞调控CD4+T细胞的影响，阐明HIF-1α介导B10细胞在TAO发病中的作用，为防治提供新免疫视角。

B10细胞在自身免疫性疾病中发挥了正向免疫调控功能。活动期TAO患者B10细胞的频率高于正常人，静止期TAO患者B10细胞频率与正常人相比无统计学差异。活动期TAO患者B10细胞的诱导效率低于正常人，静止期TAO患者的B10细胞的诱导效率与正常人相比无统计学差异。B10细胞频率与TAO疾病活动性呈正相关，与TAO病程无关。吸烟是TAO进展加重的危险因素，低氧分压是眼眶成纤维细胞微环境的重要构成，我们研究发现，吸烟是活动性TAO患者IL-10表达上调的危险因素；同时，吸烟也是外周血HIF-1α表达水平上调的影响因素，其对TAO患者的影响最为显著。为此，我们构建了HIF-1α慢病毒体系，在体外实现了TAO眼眶成纤维细胞HIF-1α高表达。采用了RNA二代测序技术，发现眼眶成纤维细胞可能通过细胞焦亡途径导致其炎症化水平改变，后续通过分子生物学检测，表明HIF-1α通过细胞焦亡途径上调了眼眶成纤维细胞炎症化水平，且HIF-1α造成了成纤维细胞纤维化能力增强。在对铁死亡通路的基因表达进行富集时，还揭示了HIF-1α导致眼眶成纤维细胞炎症化水平上调，可能与铁死亡通路有关。