透明质酸在人早孕期母-胎免疫耐受中的调节作用及其机制研究

The Th2 immune bias and promotion of regulatory T cells (Treg) at the maternal-fetal interface build an immune environment favoring successful pregnancy. Our data indicated that human maternal-fetal interface expressed high level of Hyaluronan (HA), and HA content and molecular weight were significantly decreased from unexplained miscarriage compared with normal pregnancy. High molecular size of HA-CD44 interaction enhanced proliferation of decidual stromal cells and invasion of trophoblasts while inhibited their apoptosis, implying that HA played an important role in decidualization, implantation and placentation. HMW-HA instructed CD14+ peripheral monocytes/macrophages to up-regulate the negative co-stimulatory molecules, to down-regulate the positive co-stimulatory molecules, and to secret high level of IL-4, IL-10 and TGF-β1, thus presenting the immunotolerant phenotype. The aim of the project is to ellucidate the regulatory mechanism of HA on maternal-fetal immunotolerance in human early pregnancy. By simulating microenvironment in the body, we established co-culture system of decidual immune cells, decidual stromal cells and trophoblasts to investigate how HA induced Naïve CD4+ T cells to differentiate into Th2 and Treg subtypes. Our project showed the innovation of immune modulation of HA at the maternal-fetal interface, and shed new light on the pathogenesis and prevention of spontaneous abortion.

母-胎界面Th2型免疫偏倚和调节性T细胞优势是成功妊娠的标志。我们已发现：人母-胎界面表达高水平的透明质酸，正常早孕透明质酸分泌水平和平均分子量显著高于不明原因自然流产患者；高分子量透明质酸通过CD44受体相互作用能改善蜕膜基质细胞和滋养细胞的生物学行为，提示透明质酸有助于蜕膜化、胚胎植入和胎盘形成；高分子量透明质酸能诱导CD14+的外周单核巨噬细胞上调负性协同刺激分子，下调正性协同刺激分子，分泌高水平的IL-4，IL-10 和TGF-β1，呈现免疫耐受表型。本项目进一步解析透明质酸在早孕期母-胎免疫耐受的调节功能及其机制。通过模拟体内微环境，我们建立蜕膜免疫细胞、蜕膜基质细胞与滋养细胞的共培养系统，探究母-胎界面透明质酸调控Naïve CD4+ T细胞向Th2和Treg方向的功能分化及其参与对话的分子机制。本项目具有源头创新性，为人类自然流产的病因及防治提供了新的视角。

人早孕期母-胎界面大量表达透明质酸（HA）及其受体CD44，绒毛与蜕膜组织均表达HA和CD44。HA主要表达于胞浆和胞外基质，CD44表达于胞膜，透明质酸主要由早孕蜕膜胞基质细胞（DSCs）和滋养细胞（Tros）分泌，正常早孕HA分泌水平和平均分子量显著高于不明原因自然流产患者，妊娠激素显著促进HA的分泌。高分子量HA能促进DSCs的增殖，抑制其凋亡；而高、中分子量HA能促进Tros的增殖，抑制其凋亡。中分子量HA显示出比高分子量HA更强的促进Tros的侵袭效应。HA抑制肽和CD44中和性抗体能完全或部分阻断HA对DSCs和Tros的生物学功能的升调节作用。高分子量HA能活化PI3K/AKT和MAPK/ERK1/2信号通路，PI3K/AKT抑制剂LY294002和MEK1/2抑制剂U0126阻断相应的信号通路可逆转HA对DSCs和Tros的生物学行为的升调节作用。在蜕膜免疫细胞中CD14+巨噬细胞和调节性T细胞高表达CD44。经HMW-HA刺激后，蜕膜CD14+巨噬细胞CD80、CD86表达下降，PD-L1和PD-L2表达上调；但对HLA-DR和ICOS-L无显著影响；HA抑制肽和CD44中和性抗体可抵消HA对CD14+单核巨噬细胞的调节作用。HMW-HA促进CD14+巨噬细胞分泌IL-10和TGF-β1，而LMW-HA抑制对CD14+巨噬细胞分泌IL-10和TGF-β1。正常蜕膜CD14+巨噬细胞较流产蜕膜CD14+巨噬细胞CD80、CD86表达水平下降，IL-12p40分泌减少, PD-L1和PD-L2上升，IL-10和TGF-β1水平增加。HMW-HA的单核巨噬细胞诱导naïve CD4+T细胞上调CTLA-4和PD-1的表达，但对CD28和ICOS无显著影响；上调Th2 型细胞因子IL-4、IL-10和转录因子GATA-3的表达，下调Th1型细胞因子IFN-γ、TNF-α和转录因子T-bet的表达，母-胎局部呈Th2免疫偏移；且上调CD4+T细胞TGF-β1和Foxp3的表达，有利于Treg 扩增和功能增强；但对CD4+T细胞IL-17A无明显影响，对RORγt有下调趋势。高水平和成熟状态的HA有利于维持正常妊娠，而低水平和HA的降解产物可能导致不明原因自然流产。