GSK-3β通过调控Breg细胞分化和功能减轻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 remains a major cause of early graft loss and a risk factor that impairing long-term recipient post-transplant survival. Recent studies have shown the critical role of regulatory B cells(Breg) in regulating immunological response of transplantation. We conducted a preliminary study and found that GSK-3β could regulate Treg differentiation and function leading to reduced acute rejection. We also found that the proportion of Breg in the peripheral blood was lower in patients with acute rejection as compared with that of non-acute-rejection patients. Meanwhile, higher levels of GSK-3β activation in Breg cells were found in patients with acute rejection. Moreover, GSK-3β inhibition by SB216763 significantly enhanced Breg differentiation, resulting in increased Treg differentiation from CD4+ naive T cells by Breg co-culture in vitro. We hypothesize that inhibition of GSK-3β promotes Breg differentiation and enhances their regulation of naive CD4 + T cell and Kupffer cell differentiation and immune response in the liver graft, leading to alleviated graft rejection and better transplant tolerance after liver transplantation. To verify this hypothesis, we plan to take advantages of lentiviral vector transduction, RNA interference and molecular inhibitors for interference of GSK-3β activation to investigate the roles of GSK-3β in the liver transplant tolerance both in vivo and vitro. This project could provide new ideas and strategies for improving the patient long-term outcome after liver transplantation.

肝移植术后排斥严重影响患者生存，目前调控Breg分化已成为减轻肝移植排斥反应的热点。在前期研究中我们发现GSK-3β可调节Treg分化和功能减轻排斥反应，还发现急性排斥患者外周血Breg与Treg同步减少，呈正相关，且Breg的GSK-3β活性升高，体外抑制GSK-3β使B细胞向Breg分化增多，并增强Breg抑制CD4+CD25-T细胞的增殖和促进naïve CD4+T细胞向Treg分化。为此提出假说：抑制GSK-3β活性促进Breg分化，并增强Breg调节naiveCD4+T细胞和肝内Kupffer等免疫细胞的分化和功能促进移植肝免疫耐受。本项目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调控Breg分化进而影响移植肝内外多种免疫细胞分化和功能，最终诱导移植肝免疫耐受的机制。项目成功实施将为提高肝移植长期疗效提供新策略。

肝移植术后排斥严重影响患者生存，目前调控Breg分化已成为减轻肝移植排斥反应的热点。回顾性分析19例肝移植患者，分为急性排斥组和肝功能稳定组，动态检测移植患者外周血中CD19+CD24hiCD27+ （mBreg）和CD19+CD24hiCD38hi（tBreg）数目，发现mbreg频率的降低与急性同种异体排斥反应发生率的增加有关，mbreg也可以作为抗排斥治疗的疗效评价指标。我们发现mBreg细胞在肝移植后发生急性排斥的患者中显著降低，且其分泌IL-10和TGF-β能力也显著降低。GSK-3β/NFATc1通路在体内外调控mBreg细胞的分化和免疫抑制功能中发挥了关键作用。因此，关注GSK-3β可为Breg临床试验和治疗提供新的思路。肝脏含有不同的效应性淋巴细胞亚群，它们由一种称为Tregs的T细胞亚群来控制。由于Treg/Th17比率的增加与急性/慢性肝衰竭患者的更好的生存相关。急性肝损伤的治疗选择是在受损的肝脏中富集Treg，改变效应器和调节性淋巴细胞之间的平衡，使之趋向于免疫抑制的Treg。吲哚胺2，3-双加氧酶(IDO)是一种含血红素的酶，在诱导肝脏免疫耐受中起关键作用。它将色氨酸转化为犬尿氨酸，抑制效应T细胞的增殖，促进Tregs的生成。我们通过使用α-半乳糖基神经酰胺(a-GalCer)诱导的肝炎，建立了依赖自然杀伤T细胞(NKT)的急性肝损伤小鼠模型, 我们强调了间充质干细胞(MSC)衍生的IDO对减轻急性肝功能衰竭的重要性。MSCs通过以IDO依赖的方式降低肝脏NKT细胞的肝毒性来保护急性肝损伤。