蜕膜巨噬细胞在孕期弓形虫感染致不良妊娠结局发生中的作用机制研究

Primary infection by Toxoplasma gondii (T. gondii) during human pregnancy can cause unfavorable pregnancy outcomes. Macrophages represent one of the major leukocyte subsets in the uterine decidua and comprise about 20%~25% of the total decidual leucocytes. Decidual macrophages are characterized by an immuno-suppressive phenotype and M2 polarization, supporting feto-maternal immune tolerance. Decidual macrophages have a role not only in presenting antigens as professional APCs, but also as important players in embryo implantation, vascular remodeling of uterine spiral artery and development of placentas. Dysfunction of macrophages or disequilibrium of M1/M2 is closely related to pathological pregnancy. Recent studies showed that macrophages can be classical activated (or M1) by infection of T. gondii, and then M2 phenomenon at the maternal-fetal interface maybe disrupted which would lead to abnormal pregnancy. Nevertheless, such reports are not found presently.. In this study, the role of decidual macrophages in T. gondii infection-induced pregnancy failures will be studied. The pregnant C57BL/6 mice will be infected by YFP-T. gondii tachyzoite of RH strain on gestational day (gd) 8. The infected and control pregnant mice will be sacrificed on gd 14 and the pregnancy outcomes will be recorded. The levels of cytokines TNF-α and IL-10 in tissue homogenate, peripheral blood and decidual macrophages will be measured. The expression of iNOS and arginase 1 on decidual macrophage will also be detected. The relationship between pregnancy outcomes and levels of cytokines TNF-α and IL-10, expression of iNOS and arginase 1 will be analyzed. In the in-vitro assays, the purified first trimester human decidual macrophages will be infected with YFP-T. gondii. After 24-hour culture, levels of cytokines TNF-α and IL-10 in culture supernatants, expression and activity of iNOS and arginase 1, and effect of decidual macrophages on trophoblast cells will be assayed. . The results of this study might reveal the role of decidual macrophages in the immune mechanism of abnormal pregnancy outcomes caused by T. gondii infection, and provide the basis of measures to reduce the neonatal birth defects.

孕期弓形虫感染最大的危害是导致不良妊娠结局的发生。巨噬细胞可占蜕膜白细胞总数的20%～25%，在功能上类似于替代激活的M2型巨噬细胞，参与母胎免疫耐受、维持正常妊娠；其除作为专职APC递呈抗原外，还参与胚胎植入、子宫血管重塑、胎盘生长发育等过程；蜕膜巨噬细胞功能异常或M1/M2平衡失调常与病理性妊娠密切相关。资料表明，弓形虫感染可经典激活某些组织来源的巨噬细胞，增加其细胞毒性；然而弓形虫感染对蜕膜巨噬细胞功能有何影响，此影响与其感染所致不良妊娠结局的发生有何关系，国内外未见报道。本研究拟结合本课题组前期对“孕期弓形虫感染致不良妊娠结局发生的机制”研究基础上，从蜕膜巨噬细胞分泌的细胞因子、胞内iNOS与arginase 1的表达与活性以及其对滋养层细胞的作用三方面，揭示蜕膜巨噬细胞在孕期弓形虫感染致不良妊娠结局发生中的作用机制，为寻找减少弓形虫感染后不良妊娠结局发生的有效方法奠定理论基础。

刚地弓形虫是一类机会致病性原虫，孕妇初次感染可引发流产、早产、死胎等不良妊娠结局，以及新生儿先天性弓形虫病的发生。正常妊娠的维持有赖于母胎界面免疫微环境的稳定，其主要由蜕膜免疫细胞及其产生的细胞因子等成分组成。蜕膜巨噬细胞是除NK细胞外数量最多的一群免疫细胞，可占蜕膜免疫细胞总数的20%-25%左右。正常妊娠时，蜕膜巨噬细胞的表型表现为M2型，即高表达CD163、CD206、CD209、Arg-I及IL-10等M2型分子，而低表达CD80、CD86、iNOS和TNF-α等M1型分子，故正常妊娠是一种特殊的M2现象，这对于维持正常妊娠母胎免疫耐受至关重要。实验室数据和临床资料都显示，蜕膜巨噬细胞表型和功能异常与不良妊娠结局的发生密切相关。那么，孕期弓形虫感染是否通过影响蜕膜巨噬细胞的表型和功能，而进一步导致不良妊娠结局发生呢？. 本项目采用体内和体外实验，分别检测了弓形虫感染对蜕膜巨噬细胞表面M1型和M2型膜功能分子、胞内精氨酸代谢酶以及主要细胞因子表达水平的影响，以期阐明蜕膜巨噬细胞在孕期弓形虫感染致不良妊娠结局发生中的作用机制。主要研究结果如下：. (1) 弓形虫感染导致蜕膜巨噬细胞表面M1型膜相关功能分子（CD80、CD86）表达上调，而M2型膜相关功能分子（CD163、CD206和CD209）的表达水平下调。. (2) 弓形虫感染导致蜕膜巨噬细胞胞内参与精氨酸代谢的关键酶 iNOS（M1型分子）表达水平上调，而Arg-I（M2型分子）表达水平下调。. (3) 弓形虫感染导致蜕膜巨噬细胞TNF-α、IL-12和IL-10细胞因子表达升高，同时TNF-α/IL-10和IL-12/IL-10比值也显著升高。. (4) 弓形虫感染导致蜕膜巨噬细胞表型由M2型向M1型极化，这使得蜕膜巨噬细胞维持母胎免疫耐受的功能发生紊乱，最终可能参与了弓形虫感染所致不良妊娠结局的发生。. 本研究结果初步阐明了蜕膜巨噬细胞在孕期弓形虫感染致不良妊娠结局中的作用机制，为寻找减少弓形虫感染后不良妊娠结局发生的有效方法奠定了理论基础。