弓形虫感染滋养层及羊膜细胞中典型炎症小体激活机制及其与不良妊娠关系

Toxoplasma gondii (T. gondii) can transmit to placenta through blood circulation during pregnancy, resulting in placenta inflammation, fetal infection, and adverse pregnancy outcomes. It has been shown that four typical inflammasomes (NLRP1, NLRP3, NLRC4 and AIM2) are activated by T. gondii infection in immune cells. However, whether T. gondii would activate inflammasomes in placental cells and thus impact adverse pregnancy outcomes remains unknown. We previously found that T. gondii and it’s excretory secretory antigen (ESA) up-regulates the expression levels of NLRP1, NLRP3, NLRC4, AIM2, ASC and IL-1β in trophoblasts. These results indicate that T. gondii infection indeed activates inflammasomes in trophoblasts. Yet, the underlying mechanisms and it’s relationship with adverse pregnancy are not clear. In the present study, by using both in vitro (i.e., trophoblasts and amnion cells) and in vivo (i.e., human placenta and amnion tissue, gene-knockout mice) models of T. gondii infection, we will employ diverse cellular and molecular biology approaches such as gene knockdown, qRT-PCR, Western blot and transmission electron microscopy to further investigate the difference in inflammasome activation in placental and amniotic tissues derived from various pregnancy stages, the detailed mechanism of inflammasome activation, alterations in cell behavior as well as their association with adverse pregnancy outcomes. The results will reveal the mechanism of T. gondii-induced inflammasome activation in placental cells and it’s relationship with adverse pregnancy, providing a sound theoretical basis for the prevention and treatment of T. gondii infection during pregnancy.

孕期感染弓形虫通过血液循环到达胎盘，引起胎盘炎症反应和胎儿感染，导致不良妊娠结局。弓形虫感染可激活免疫细胞4种典型炎症小体（NLRP1、NLRP3、NLRC4、AIM2），但是否激活胎盘细胞中炎症小体未见报道，炎症小体激活与不良妊娠结局关系不明。我们发现弓形虫及其排泄分泌抗原（ESA）可诱导滋养层细胞NLRP1、NLRP3、NLRC4、AIM2、ASC、IL-1β表达，提示弓形虫感染可激活滋养层细胞中炎症小体，但激活机制及其与不良妊娠关系有待阐明。我们拟通过弓形虫及ESA处理滋养层和羊膜细胞、胎盘和羊膜组织、基因敲除小鼠，采用shRNA干扰、qRT-PCR、免疫印迹、透射电镜等方法，观察不同孕期胎盘及羊膜组织中炎症小体激活差异，了解炎症小体激活机制、细胞生物学行为变化及不良妊娠结局。研究结果将阐明弓形虫感染胎盘细胞中典型炎症小体激活机制及其与不良妊娠关系，为防治孕期弓形虫感染提供理论依据。

刚地弓形虫是一种专性细胞内寄生原虫，入侵宿主细胞时释放排泄分泌抗原（ESA）。孕期感染弓形虫可经胎盘传播导致不良妊娠结局。弓形虫感染可激活免疫细胞炎症小体，但是否激活胎盘来源细胞炎症小体及其与不良妊娠结局关系未见报道。本研究中，我们成功建立弓形虫感染或ESA处理胎盘滋养层、羊膜细胞模型，并优化典型炎症小体激活最佳条件，我们发现弓形虫感染对三种胎盘细胞（BeWo、HTR8 SV/neo和WISH）微管网络产生不同程度的破坏作用，通过降低线粒体膜电位诱导胎盘细胞毒性；此外，我们还采用弓形虫感染胎盘滋养层及羊膜新鲜组织，检测炎症小体相关基因表达变化，结果显示弓形虫感染可以激活胎盘滋养层及羊膜新鲜组织中炎症小体并诱导细胞焦亡。同时我们明确了NLRP1、NLRP3、NLRC4和AIM2参与弓形虫感染诱导的胎盘细胞焦亡。弓形虫感染胎盘细胞产生胞浆活性氧和线粒体活性氧，且活性氧参与了弓形虫诱导的炎症小体激活和焦亡，此外，我们还发现组织蛋白酶B（CatB）-ASC信号通路也参与弓形虫感染诱导的胎盘细胞焦亡。最后，我们成功建立弓形虫感染炎症小体基因敲除小鼠模型（NLRP3-/- ，Aim2-/-），感染弓形虫可导致胎儿发育明显迟缓，甚至有流产发生，表明炎症小体（NLRP3和Aim2）基因敲除可明显加重弓形虫感染导致的不良妊娠结局。综上，我们通过本研究清晰阐明了典型炎症小体在弓形虫感染滋养层及羊膜细胞中激活机制及其与不良妊娠关系，为防治妊娠期弓形虫感染提供理论依据，也为研究其它疾病炎症小体激活及作用机制提供借鉴。