CD4+T细胞过度O-GlcNAc修饰在中枢神经系统自身免疫性疾病发病机制中作用的研究

O-GlcNAc modification, namely O-GlcNAcylation, is an essential post-translational modification with a single β-N-acetylglucosamine linked to Ser or Thr residues of various nucleocytoplasmic proteins. It contributes to various cellular cascades, including gene expression and protein trafficking, and also is related with immunoregulation. Dysregulation in O-GlcNAcylation is assumed to be tightly linked to chronic diseases, such as cancers, diabetes and neurodegenerative diseases. However, the effect of O-GlcNAcylation on the pathogenesis of autoimmune disease is still unknown. Previous results showed that the O-GlcNAcylation level in CD4+ T cells from MS patients was upregulated, and the expression of OGT & OGA were different, which was supposed to be related with the activation of immune system. This phenomena will be further investigated to identify the signaling pathway, through which O-GlcNAcylation modulates T cell immunity to affect the progress of EAE. The two fundamental scientific issues will be resolved in this project, including how the O-GlcNAcylation in CD4+ T cells affects the clinical scores, pathological features and immune responses of EAE, and what molecular mechanism is involved in the regulation of O-GlcNAcylation on T cell immunity. The outcome of this project can provide new target for the therapy of AID, and lay the foundation for the application of modulation in O-GlcNAcylation level to improve the clinical symptoms of AID.

O-GlcNAc修饰是一种动态的蛋白质翻译后修饰，与免疫系统功能调控相关，但O-GlcNAc修饰是否参与自身免疫性疾病（AID）的发生与发展尚不清楚。前期实验表明多发性硬化（MS）患者CD4+ T细胞O-GlcNAc修饰水平增高及相关酶表达水平变化与其免疫活化程度相关。本课题将利用小鼠模型，通过分子生物学、细胞生物学和蛋白质组学等方法对此进行进一步研究，明确O-GlcNAc修饰调控T细胞免疫进而影响实验性自身免疫性脑脊髓炎（EAE）疾病表现与病理特征的信号转导途径。本课题拟解决以下两个基本科学问题：1）分析EAE中CD4+ T细胞O-GlcNAc修饰水平变化对其临床评分、病理特征及免疫应答的影响；2）揭示O-GlcNAc修饰调控T细胞免疫的分子生物学机制。此项研究将为AID诊疗策略的开发提供新的思路与靶点，为通过调节O-GlcNAc修饰水平改善自身免疫性疾病临床症状奠定实验基础。

O-GlcNAc糖基化修饰是一种动态可逆的蛋白质翻译后修饰，能够参与免疫细胞的功能调控，但其在自身免疫性疾病（AID）发病机制中的作用尚不清楚。前期实验中，我们发现多发性硬化（MS）患者CD4+ T细胞O-GlcNAc糖基化修饰水平增高；同时，在MS患者外周血单核细胞中microRNA 15b（miR-15b）下调表达，且其表达水平与O-GlcNAc糖基转移酶（OGT）表达水平负相关。因此，我们推测miR-15b可能通过调节OGT表达水平，影响关键蛋白的O-GlcNAc糖基化修饰水平，进而调控CD4+ T细胞功能。我们构建MS经典动物模型——实验性变态性脑脊髓炎（EAE）小鼠模型，在其CD4+ T细胞中特异性过表达或下调miR-15b的表达水平，探索调控O-GlcNAc糖基化修饰对MS的治疗作用。通过对不同干预小鼠的临床评分以及病理学检测，结果显示，在CD4+ T细胞中特异性过表达miR-15b能够抑制炎性细胞的浸润，减小脱髓鞘病灶，改善EAE小鼠的临床症状。对EAE小鼠免疫细胞亚群的分析发现，过表达miR-15b能够抑制CD4+ T细胞向Th17细胞分化。体外实验也表明，在CD4+ T细胞中过表达miR-15b能够抑制其激活和增殖，对CD4+ T细胞向Th17细胞分化也具有抑制作用。进一步的研究表明，OGT是miR-15b的下游调控靶蛋白，而OGT能够通过NF-κB通路调控Th17细胞分化关键细胞因子RORγt的表达，从而调节CD4+ T细胞向Th17细胞分化；在EAE小鼠体内过表达OGT能够抵消miR-15b过表达的作用。这说明miR-15b过表达通过与OGT的mRNA结合，下调OGT的表达水平，从而减少NF-κB信号通路中c-Rel和p65的糖基化水平，降低其转录活性，减少二者下游细胞因子RORγt的表达，进而抑制CD4+ T细胞向Th17细胞分化，减少炎性细胞因子的分泌，改善EAE的临床表现。此项研究将为AID诊疗策略的开发提供新的思路与靶点，为通过调节O-GlcNAc修饰水平改善自身免疫性疾病临床症状奠定实验基础。