LILRB4在孕期弓形虫感染致不良妊娠结局中的作用机制研究

Infection of Toxoplasma gondii (T. gondii) during human pregnancy can cause unfavorable pregnancy outcomes such as miscarriage, stillbirth, or fetal abnormalities. It has been demonstrated that T. gondii infection would destroy the immuno-microenvironment at the maternal-fetal interface, lead to the abnormal pregnancy outcomes. DC and macrophages are proved to play vital roles in the maintenance of normal pregnancy, and LILRB4 as an important immune inhibitory molecular is expressed on both decidual DC and decidual macrophages during the whole gestation. Our previous studies have found that T. gondii infection can cause dysfunction of decidual DC and decidual macrophages, which is closely related to the occurrence of adverse pregnancy outcomes. Furthermore, we found that the level of LILRB4 expression on decidual DC and decidual macrophages were decreased after T. gondii infection. However, whether LILRB4 play an important part in the abnormal pregnancy caused by T. gondii infection and what are the detailed mechanisms, still need to be clarified. . In this study, we intend to detect the expression level of LILRB4, functional molecules and cytokines on decidual DC and decidual macrophages with T. gondii infection in vivo and in vitro, and analyze the relationship between the expression level of LILRB4 and functional molecules as well as cytokines, and then illuminate whether LILRB4 of decidual DC and decidual macrophages participated in the occurrence of adverse pregnancy outcomes caused by T. gondii infection. . We aim to demonstrate the effects and mechanisms of LILRB4 on the occurrence of adverse pregnancy outcomes during T. gondii infection and search the molecular target to prevent and treat the congenital toxoplasmosis. Until now, there has been no relative report.

孕期弓形虫感染最大的危害是可导致流产、死胎、先天性弓形虫病等不良妊娠结局的发生，严重影响着新生儿的出生质量，但其发生的分子机制仍待进一步阐明。近期研究已证实，LILRB4是表达于蜕膜DC和蜕膜巨噬细胞上的主要免疫抑制性分子，有利于维持母胎耐受。本室研究发现，弓形虫感染所致蜕膜DC和巨噬细胞的功能异常与不良妊娠结局的发生密切相关；且弓形虫感染后两类细胞表面LILRB4表达水平均明显改变，那么，弓形虫感染后LILRB4表达水平的改变是否是通过影响以上细胞的免疫耐受功能进而导致不良妊娠结局的发生呢？亟待进一步探索。本研究拟在本室大量前期研究的基础上，通过体外建立人蜕膜DC和蜕膜巨噬细胞弓形虫感染体系、体内采用LILRB4基因敲除孕鼠建立弓形虫感染的不良妊娠结局模型，探索LILRB4在孕期弓形虫感染致不良妊娠结局中的作用机制，为最终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。

维持正常妊娠有赖于母胎界面免疫微环境的稳定，打破这种微环境就可能导致不良妊娠结局的发生。LILRB4为免疫抑制性受体，在母胎界面主要表达在蜕膜DC和蜕膜巨噬细胞上，是维持这两类细胞参与母胎免疫耐受的关键分子。本室研究发现，弓形虫感染能导致蜕膜DC和蜕膜巨噬细胞LILRB4表达水平降低，且弓形虫感染的LILRB4基因敲除（LILRB4-/-）孕鼠较感染的野生孕鼠发生了更为严重的不良妊娠结局。然而，LILRB4在孕期弓形虫感染致不良妊娠结局发生中的详细机制尚未阐明，有待于深入研究。.本项目通过体内采用LILRB4-/-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DC和蜕膜巨噬细胞弓形虫感染体系，深入探索了LILRB4在孕期弓形虫感染致不良妊娠结局发生中的作用机制；本项目还研究了TGF-β在改善弓形虫感染致不良妊娠结局中的分子机制。此外，采用蛋白质组学研究方法对弓形虫感染所致人蜕膜免疫细胞差异表达蛋白进行了筛选和分析。.主要结果如下：.⑴弓形虫感染导致蜕膜DC表面LILRB4表达水平降低，使得蜕膜DC各亚群比例失调，表面CD80、CD86和HLA-DR（MHC-II）的表达水平升高，以及母胎界面主要细胞因子表达失衡，导致蜕膜DC免疫活性升高而耐受功能降低，是不良妊娠结局发生的重要免疫机制。.⑵弓形虫感染所致蜕膜巨噬细胞LILRB4表达水平降低，进一步导致蜕膜巨噬细胞M2型功能分子CD163、CD209、CD206和Arg-1等的表达水平下降，而M1型功能分子CD80、CD86、iNOS和TNF-α等的表达水平升高，导致蜕膜巨噬细胞功能向M1极化，免疫耐受功能减弱，是不良妊娠结局发生的又一重要机制。.⑶给予弓形虫感染的孕鼠TGF-β治疗可改善弓形虫感染所致不良妊娠结局。其机制为：TGF-β治疗可促进蜕膜Treg细胞的分化、蜕膜Treg细胞表面抑制性分子CTLA-4和PD-1的表达水平上调，还可纠正母胎界面主要细胞因子失衡。.⑷采用串联质谱标签标记定量技术，从5510种检测到的人蜕膜免疫细胞蛋白样本中筛选出181种与弓形虫感染相关的差异表达蛋白，这些蛋白大多参与了滋养层细胞侵袭、胎盘发育和宿主免疫应答等生命活动。.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，为最终预防和治疗先天性弓形虫病的发生、降低出生缺陷奠定了理论基础。