PD-1/Tim-3信号分子调控蜕膜巨噬细胞极化参与子痫前期发病的机制研究

Pre-eclampsia (PE) is a placenta-originated disease characterized by over-activation of maternal adaptive immunity and innate immunity. Monocyte/macrophage are the bridges of the two kinds of immunity, which play a critical role in the development of placenta. Our team has undertaken a series of studies on the pathogenesis of PE in view of the adaptive immunity and innate immunity. Our recent findings show that the pro-inflammative subsets of peripheral monocytes increased in PE patients, and were closely correlated with its severity. The published studies indicated that the peripheral monocytes in PE were easily differentiated into the pro-inflammative subsets of macrophages in deciduas (namely, M1 subset), but not into the immune-regulative subsets (M2 subset). And the new researches have shown that the biased polarization of macrophages is related with some diseases in humans. However, it remains unclear about the factors and mechanism on regulating the polarization of DM to M1 or M2 subsets. PD-1 and Tim-3 are important negative regulative signals, which can regulate the polarization of DM. As a result, we hypothized that the dysregulation of DM polarization by PD-1/Tim-3 at the first trimester of gestation would participate in the pathogenesis of PE. In this study, by clinical observation, in-vitro study and animal model experiments, we continue to determine the factors that regulate the DM polarization and its mechanism. Moreover, by setting up PE-like animal model, we further investigate the therapeutic effects by targeting PD-1/Tim-3 signals. We aim to elucidate the important molecular immunological mechanism of PE, to discover the potential therapeutic target, and to provide supporting evidence for the clinical prevention and treatment of PE.

子痫前期(PE)是一种母体适应性免疫和固有免疫均过度激活的胎盘源性疾病。单核/巨噬细胞是连接这两种免疫反应的桥梁，且在胎盘形成中发挥着重要作用。课题组前期从适应性免疫和固有免疫角度对PE的发病机制进行了系列研究，近期发现PE外周血中“促炎型”单核细胞明显增加，且与疾病严重程度密切相关。另有研究表明，PE患者外周单核细胞易在子宫局部分化为促炎型(M1型)蜕膜巨噬细胞(DM)，而非免疫调节型(M2型)DM。然而，调节DM向M1或M2型极化的原因和机制尚不是十分清楚。PD-1和Tim-3是重要的负性调节信号分子，它们参与巨噬细胞极化的调节。我们推测：PD-1/Tim-3调控早期妊娠中DM极化参与PE的发生。本项目通过临床现象观察、体外实验和动物模型三个方面，延续探讨PE中调节DM极化的原因、分子机制，以及以PD-1/Tim-3为靶点的治疗效果。为深入研究PE发病机制和其防治提供新思路和新靶点。

本项目探讨了PD-1/PD-L1和Tim-3/Gal-9信号通路调节蜕膜巨噬细胞(DM)极化平衡在PE中的作用、机制及干预效果。通过采集临床样本分析了正常妊娠(NP)和子痫前期(PE)妇女M1和M2型DM比例及PD-1和Tim-3在这两种DM亚型上的表达。结果表明PE患者DM存在向M1型极化偏移，且PD-1和Tim-3在M1型DM上低表达。对NP和PE妇女胎盘组织中PD-L1和Gal-9蛋白及mRNA的表达进行检测，发现PE胎盘PD-L1和Gal-9低表达。表明PD-1和Tim-3分子分别通过PD-1/PD-L1和Tim-3/Gal-9信号通路影响巨噬细胞M1/M2型极化平衡参与PE发生。通过尾静脉注射脂多糖(LPS)成功建立了PE样大鼠模型，外源性给予PD-L1 Fc和Gal-9 Fc来激活PD-1/PD-L1和Tim-3/Gal-9信号通路，可逆转PD-1和Tim-3异常导致的DM极化异常。从母鼠血压、尿蛋白、胎盘及子代发育、肝肾病理改变、滋养细胞浸润和螺旋动脉重塑、胎儿血管生成等方面评价干预PE样大鼠模型的效应和作用机制。发现用PD-L1 Fc活化PD-1/PD-L1信号通路或（和）用Gal-9 Fc活化Tim-3/Gal-9信号通路，在逆转DM向M1极化的同时，对PE样大鼠及其子代均起到保护性作用；且PD-L1 Fc和Gal-9 Fc联合干预降低PE样大鼠血压和蛋白尿的效果更明显，起效时间也更早，在E9时即有明显的效果。体外机制研究发现：①LPS可诱导M0型巨噬细胞向M1型极化，M1型巨噬细胞表达PD-1和Tim-3减少；②给予PD-L1 Fc和Gal-9 Fc单独或二者联合干预LPS诱导的M1型巨噬细胞，可抑制M1型极化，促进M2型极化效应；分泌TNF-α减少，而IL-10增加；③PD-L1 Fc的干预效应主要通过结合PD-1进而抑制胞内PI3K-AKT-mTOR信号通路实现，Gal-9 Fc的干预效应主要通过结合Tim-3以及胞内的ERK(AKT)/GSK3β/β-catenin信号通路实现，二者之间通过ERK、GSK3β等信号分子发挥协同作用。本项目进一步解析了PE的分子免疫学发生机制、发现了潜在的治疗靶点，为开创联合以PD-1和Tim-3信号分子为靶点探索PE的治疗策略提供了新思路和理论依据。