GSK-3β通过影响IL-17+Treg的分化参与肝移植排斥反应的机制研究

Liver transplantation has become a lifesaving procedure for patients with chronic end-stage liver disease, however, rejection is still a major cause of early graft loss and a risk factor for long-term recipient post-transplant survival. Recently, Th17 and regulatory T (Treg) cells are thought to be critically involved in mediating and regulating immunological response of transplantation. Our fingdings in previos National Nature Science Foundation showed that GSK3β inhibition promoted human iTreg differentiation and suppressive function, while inhibiting Th17 differentiation. We also found that the proportion of IL17+Treg, the intermediate between Treg and Th17, in peripheral blood was higher in patients with acute rejection when compared with that of non-acute-rejection patients. Meanwhile, the propotion of IL17+Treg decreased after succesive anti-rejection therapy. The protein level of GSK-3β gradually increased in Treg, IL17+Treg and Th17. Moreover, we also found GSK-3β inhibitor significantly enhanced Treg differentiation and inhibited Th17 cell differentiation from IL17+Treg. We hypothesize that inhibition of GSK-3β alleviates the rejection of liver transplantation via regulating Treg/Th17 cell differentiation from IL17+Treg and enhancing IL17+Treg suppressive function. To verify this hypothesis, flow cytometry, Western Blot, lentiviral vector transduction and RNA interference were applied to investigate the role of GSK-3β in liver transplantation in vivo and vitro. This research could provide new ideas and methods to improve the long-term outcome of liver transplantation.

肝移植术后排斥严重影响患者生存，目前调控Treg/Th17分化已成为减轻排斥的热点。在前一项国自然项目中发现GSK-3β可调节Treg/Th17平衡和Treg功能，还发现排斥患者外周血中Treg与Th17的中间体IL17+Treg比例明显升高，抗排斥治疗后下降，GSK-3β表达在Treg、IL17+Treg和Th17之间逐渐升高，体外抑制GSK-3β促进IL17+Treg向Treg分化抑制其向Th17分化，并增强其免疫功能。为此我们提出假说：抑制GSK-3β活性促进IL17+Treg向Treg分化及增强其免疫调节功能，减轻肝移植排斥反应。为此我们将建立大鼠肝移植模型，采用流式细胞仪、病毒载体转染等手段，从分子、细胞、动物模型等方面研究，明确GSK-3β通过调控IL17+Treg分化进而影响Treg/Th17平衡及功能最终减轻排斥的分子机制。本项目的成功实施将为提高肝移植长期疗效提供新策略。

IL17+Treg也可以被看作是Treg的一种不稳定的状态，向Th17转化的一种亚群细胞，因此在本课题的实施过程中，我们还重点关注了Treg在不同环境中的稳定性问题，并且有重要发现即人牙龈组织来源的间充质干细胞（GMSCs）具有抑制Treg细胞向Th1和/或类Th17细胞转化以及稳定Foxp3的表达的作用，进一步研究还发现GMSCs通过影响Treg的分化和功能明显抑制小鼠急性移植物抗宿主病（GVHD）。我们还研究了microRNA对Treg分化和功能的影响，发现miR-142-3p调控ATG16L1表达影响tTreg的自噬，提高了tTreg的生存率和功能。我们在实验过程中还发现肝移植发生急性排斥的患者Treg的数量、Treg/Th17的比例发生明显变化，同时还发现Breg（CD19+CD24++CD27+，mBreg）的数量及比例也发生明显变化，并且Breg的数量与Treg的数量呈正相关，考虑到Breg可以调节Treg分化，因此我们我们推测肝移植急性排斥的发生可能也与Breg密切相关，回顾性分析19例肝移植患者，发现mBreg频率的降低与AR发生率的增加有关，mBreg也可以作为抗排斥治疗的疗效评价指标。本课题还资助了临床研究：肝移植术后肺部空洞的诊断和治疗。发现肝移植术后肝移植患者的曲霉菌感染和结核分枝杆菌感染引起的空洞很难用CT扫描来区分，但是可以通过结合临床特征、痰培养、GM测定、PPD和痰涂片来确诊，早期诊断和治疗可以导致更好的预后。最后还总结不同固有淋巴细胞（ILC）亚群在移植免疫中的具体作用及其相关机制，为进一步研究ILC在肝移植免疫中的作用提供理论基础。