Tim-3在孕期弓形虫感染致不良妊娠结局中的作用机制研究

Infection with Toxoplasma gondii(T. gondii) during human pregnancy can cause unfavorable pregnancy outcomes such as miscarriage, stillbirth, or fetal abnormalities. It has been demonstrated that Tim-3 is expressed in the decidual immune cells as an inhibitory molecule, promoting the maintaince of normal pregnancy. Our previous study has found that the abnormal pregancy in Tim-3-KO pregnanct mice caused by T. gondii-infection was even worse than the infected wild type mice, suggesting that Tim-3 might play an important part in the abnormal pregancy caused by T. gondii-infection. We also found that the expression level of Tim-3 on the decidual NK cells, macrophages and Treg cells surface were significantly decrease after T. gondii-infection. But, whether Tim-3 plays an important part in the abnormal pregancy caused by T. gondii-infection and what are the detailed machenism, still need to be clarified. In this study, we intend to establish T. gondii-infection system by human decidual NK cells, macrophages and Treg cells in vitro, and the abnormal pregancy animal model after T. gondii-infection by Tim-3-KO pregnanct mice in vivo, in order to explore the effects and mechanisms of Tim-3 on the incidence of adverse pregnancy outcome during T. gondii infection and search the molecular target to prevent and treat the congenital toxoplasmosis.

弓形虫感染最大的危害是导致流产、死胎、先天性弓形虫病等不良妊娠结局的发生，但作用机制仍不十分清楚。已证实，Tim-3是表达于蜕膜免疫细胞上的抑制性分子，有利于正常妊娠的维持；本室研究发现，Tim-3基因敲除小鼠孕期弓形虫感染后较野生感染鼠发生更严重的不良妊娠结局，提示Tim-3在孕期弓形虫感染致不良妊娠结局中发挥重要作用。本室还发现，弓形虫感染后蜕膜NK细胞、巨噬细胞、Treg表面Tim-3表达水平显著降低，那么，弓形虫感染后是否是通过影响Tim-3的表达水平，进而导致三类蜕膜免疫细胞功能异常在不良妊娠结局发生中发挥着重要作用呢？值得深入探索。本研究拟体外建立人蜕膜NK、巨噬细胞、Treg细胞弓形虫感染体系，体内采用Tim-3基因敲除孕鼠建立弓形虫感染致不良妊娠结局的动物模型，探索Tim-3在弓形虫感染致不良妊娠结局中的作用机制，为建立有效预防和治疗先天性弓形虫病的手段和方法奠定理论基础。

孕期弓形虫感染最大危害是导致流产、死胎等不良妊娠结局的发生，目前认为其发生机制与弓形虫感染导致母胎界面免疫微环境紊乱密切相关。Tim-3为免疫抑制性受体，表达于多种蜕膜免疫细胞，参与维持母胎免疫耐受。本室发现，弓形虫感染可导致蜕膜NK细胞、巨噬细胞和DC Tim-3表达水平降低，且感染的Tim-3-/-孕鼠较感染野生鼠发生更严重的不良妊娠结局，提示Tim-3在弓形虫感染致不良妊娠结局发生中扮演重要角色。本项目通过体内采用野生孕鼠和Tim-3-/-孕鼠建立弓形虫感染致不良妊娠结局模型和体外建立人蜕膜NK细胞、巨噬细胞及DC弓形虫感染体系，深入探索了Tim-3在孕期弓形虫感染致不良妊娠结局中的作用机制。此外，在本项目经费支持下，还研究了B7-H4在弓形虫感染致不良妊娠结局中的作用机制。.重要结果如下：①弓形虫感染可下调蜕膜NK细胞Tim-3的表达，进而导致其活化性受体与抑制性受体平衡失衡，并通过PI3K-AKT通路促进Granzyme B和IFN-γ表达，以及通过JAK-STAT1/3通路促进其Perforin和IL-10产生，这使得其杀伤活性增强，耐受功能减弱，是导致不良妊娠结局发生的重要分子机制。②弓形虫感染可致蜕膜巨噬细胞Tim-3表达显著下调，进而引起M2型蜕膜巨噬细胞向M1型极化，导致其母胎耐受功能减弱，是弓形虫感染致不良妊娠结局发生的重要分子机制。③弓形虫感染可造成蜕膜DC Tim-3表达显著下调，进而导致其耐受功能紊乱；弓形虫感染致Tim-3下调可通过SRC-STAT3通路降低蜕膜DC IL-10表达，损害其母胎耐受功能，促进不良妊娠结局的发生。④弓形虫感染导致蜕膜DC B7-H4表达下调，进而诱导其CD80、CD86和MHC-II/HLA-DR表达上调，并通过JAK-STAT通路抑制其IDO和IL-10生成，促进IL-12产生，使得蜕膜DC功能紊乱，最终导致不良妊娠结局的发生。⑤弓形虫感染可下调蜕膜巨噬细胞B7-H4表达，进而通过PETN/PI3K/AKT和JAK2/STAT1通路影响其功能分子表达，导致其母胎耐受功能紊乱；过继转输B7-H4+蜕膜巨噬细胞可有效改善弓形虫感染所致不良妊娠结局的严重程度。.以上研究进一步揭示了孕期弓形虫感染致不良妊娠结局发生的作用机制，为最终建立有效干预不良妊娠结局发生的手段和方法奠定理论基础。