我国弓形虫优势基因型效应分子ROP16/GRA15多态性及其致不良妊娠结局的机制研究

Toxoplasma gondii is an intracellular protozoon. The vertical transmission of T.gondii causes abortion, constituting a serious threat to humans and leading to great losses of livestock production. It has been reported that no parasites，and their DNAs as well, are detectable in some diseased samples with abnormal pregnancy outcomes, suggesting that an immunity-related pathogenesis induced by T. gondii might be involved. Toxoplasma virulence effectors possess the genotype-associated polymorphism. ROP16 and GRA15 carried by type I,III and II strains drive macrophages to M2 or M1, respectively,inducing a remarkably different immune consequences. It is hypothesized that strains with genotype Chinese 1(Wh3) prevalent in China may have the pathogenesis differing from the strains with archetypical types due to the fact that Wh3 is characterized by sharing ROP16I/III and GRA15II polymorphisms. Both effectors polymorphisms noted in type Chinese 1 strains may induce imbalance at maternal-fetal interface and account for the failure of pregnancy caused by Toxoplasma infection in some cases. The present study will uncover the pathway of trophoblast apoptosis induced by transfected macrophages and illuminate the mechanism by which Wh3 strain induces mouse abortion,by using mouse bone marrow macrophages(mBMMφ)transfection with recombinant ROP16I/III and GRA15II viruses, transwell culture of polarized macrophages with trophoblasts and Th17 cells, in vivo cells inoculation, and rop16I/III-knock out parasite infection of pregnant ICR mice. So far, no relevant approach has been available. The results will provide a deep insight into the pathogenesis of abnormal pregnancy caused by Toxoplasma genotype predominantly circulating in China.

弓形虫寄生在有核细胞内，其垂直传播严重危害人类健康和畜牧业生产。近年发现，部分抗体阳性孕妇甚至实验感染动物流产的病理组织中难以查见虫体和DNA，推测免疫机制是妊娠失败的主要原因。弓形虫毒力分子具有基因型相关的多态性：I/III和II型虫株ROP16和GRA15的多态性分别驱动巨噬细胞向M2和M1极化，诱导宿主迥异的免疫应答。我国的弓形虫优势基因型虫株Wh3兼具I/III型ROP16和II型GRA15的多态性，其诱导母-胎界面的免疫失衡致流产可能具有独特的致病机制。本课题采用ROP16I/III和GRA15II重组病毒转染巨噬细胞，诱导的M2和M1分别与滋养细胞和Th17共培养、体内转输、ROP16基因敲除虫株感染等，揭示上述两个效应分子诱导的滋养细胞凋亡和Wh3虫株致流产的机制，为基因型相关的致病机制和我国流行的优势基因型弓形虫病的防治研究具有重要的理论与实际意义。此项研究国内外未见报告。

我国人兽间流行的弓形虫的基因型为Chinese 1（本课题组首先命名），虫株具有独特的基因标志和效应分子（ROP16， GRA15）以及致病性（本课题组首先发现）。鉴于弓形虫抗体阳性的孕妇常难以从胎盘和死胎组织中分理处虫体，本课题假设部分的不良妊娠结局是由虫株效应分子 诱导的母胎界面免疫耐受失衡所致。通过构建ROPI-和GRA15II-慢病毒体外感染巨噬细胞，采用FCM以及标志物（Arg-1，iNOS等）检测，以及全基因组表达谱芯片转录水平分析活化的通路，分别验证了M2和M1的极化及机制；继而采用本课题组分离和保种的Chinese1 的Wh3株感染巨噬细胞，观察到了类似的M1 偏移；将表达GRA15II的巨噬细胞被动转输导致孕鼠，见到了死胎，胎盘发育迟缓、出血，吸收等。孕鼠胎盘、脾细胞中Th17数升高，Treg阳性细胞数减少，血清IL-17A， IL-23和IFN-γ水平显著上调。以上结果显示，弓形虫GRA15II效应分子（而非活虫感染）可以诱导巨噬细胞向M1偏移，后者参与了母胎界面生理性免疫耐受失衡，与不良妊娠结局有关。我们又采用CRISPR/Cas9 敲除了Wh3株的ROP16I/III的基因，构建了以GRA15II为背景的虫株(Wh3ΔROP16I/III),以此感染BALB/c孕鼠，论证我国Chinese 1基因型虫株携带的GRA15II是否通过驱动M1的偏移，导致了母胎免疫耐受的失衡。结果发现，ROP16I/III 敲除虫株体外感染的巨噬细胞与滋养细胞共培养可诱导后者的凋亡。虽然ROP16I/III敲除虫株与野生型相比毒力未见明显变化，都导致了不良妊娠结局，但是前者感染的孕鼠IL-17、IL-12和IFN-γ显著高表达，而脾脏和胎盘中的Tregs 显著减少；相反，Th2细胞因子IL-4, IL-10 和 TGF-β1水平明显下调。 我们的结果表明，我国Chinese 1型弓形虫感染导致不良妊娠结局不仅是虫体的直接入侵，而且与gra15II诱导的宿主M1-Th1免疫应答有关，后者颠覆了母胎界面的免疫耐受，是妊娠失败的成因之一。.此外，基于GRA15II对巨噬细胞的靶向极化作用，我们还发现，GRA15II-M输注肝癌荷瘤小鼠，可显著抑制癌肿大小和增长速度，还可减轻感染所致的肝脏纤维化，提示GRA15II具有潜在的免疫治疗价值。