弓形虫TgMIF调控宿主ERK/MAPK/NF-κB信号轴驱动巨噬细胞的极化及其致病机制研究

Toxoplasma gondii is a foodborne parasitic protozoon that can infect virtually all warm-blooded animals, including humans. Different strains of Toxoplasma have evolved specific effector molecules to modulate the immune responses of different hosts. The Toxoplasma rhoptry kinase ROP16 carried by type I and III strains, which activates STAT6, drives macrophages to alternative activatied cells (M2); this leads to less inflammation and enhanced parasite survival, but also to a higher mortality rate of hosts. The Toxoplasma dense granule protein GRA15 carried by II strain, which activates NF-κB, drives macrophages toward classical activatied cells (M1); this leads to effective control of acute infection and results in the chronic stage of infection. Our previous study found that these above two effectors in strains with genotype Chinese 1 (the virulent Wh3 strain and the less virulent Wh6 strain) prevalent in China have the same genotype and phenotypic characteristics, suggesting that there are other unknown effector molecules required for macrophage polarization. Our preliminary data indicated that TgMIF, a Toxoplasma ortholog of Macrophage migration Inhibitory Factor, was highly expressed in type II and Chinese 1 Wh6 strains, and activated ERK/MAPK and NF- κ B pathways, inducing macrophages to polarize toward M1. Our results strongly suggested that TgMIF might be another important effector molecule inducing macrophage polarization, but the mechanism of regulating immune response is unclear. The present study will analyze the polarization of macrophages infected with Tgmif knockout transgenic strains, such as ME49mif-/- and Wh6mif-/- strains. In addition, we will uncover the signal pathway and illuminate the mechanism by using recombinant TgMIF viruses and these Tgmif knockout transgenic strains. Using shRNA, over-expression and inhibitors of signal pathway, we will define the related key molecules in ERK/MAPK/NF-κB axis targeted by TgMIF. So far, no relevant approach has been seen. The results will provide a deep insight into the pathogenesis of Toxoplasma genotype Chinese 1(Wh6 strain) prevalent in China and provide important clues to their widespread success in establishing chronic infection.

弓形虫毒力分子与其地域分布、基因型、宿主免疫应答以及感染的结局密切相关：宿主感染早期ROP16I/III诱导巨噬细胞(Mφ)向M2极化，虫体快速增殖和播散；而GRA15II使Mφ向M1极化，虫体被杀灭或抑制，宿主进入慢性隐性感染状态。课题组前期发现我国流行的弓形虫Chinese1强毒株（Wh3）和弱毒株（Wh6）中上述两个效应分子具有相同的基因型和表型特征，提示存在着尚未认知的Mφ极化诱导因子。我们发现TgMIF在II型和Wh6弱毒株高表达，并可活化ERK/MAPK和NF-κB通路，诱导Mφ向M1极化，提示TgMIF是又一个重要的效应分子，但机制不明。本研究构建Tgmif敲除虫株和TgMIF慢病毒，体内外研究TgMIF对Mφ极化的影响；采用抑制剂、过表达或shRNA在ERK/MAPK/NF-κB信号途径免疫应答轴中发现关键作用因子，为阐明我国流行的Wh6弱毒株成囊及隐性感染提供一种新机制。

弓形虫弱毒株是感染人类的主要虫株，在人群中广泛传播。本项目研究揭示了弓形虫源性的巨噬细胞迁移抑制因子（Toxoplasma Macrophage Migration Inhibitory Factor, TgMIF）是弓形虫弱毒株引起的急性弓形虫病致肝损伤的重要致病因子。TgMIF可引起肝实质细胞NLRP3炎症小体活化和随后的细胞焦亡。同时，TgMIF可募集Ly6Chi巨噬细胞，使其释放促炎性细胞因子，进而导致肝实质细胞凋亡。以上结果说明TgMIF通过诱导组织剧烈的炎症反应去抑制虫体增殖，但是同时会导致致命性的肝损伤。与之相一致的是，我们在II型弱毒株上敲除TgMIF可显著减轻炎症性肝损伤并延长小鼠的存活时间。此外，本项目进一步证实TgMIF调控宿主ERK/MAPK/NF-κB信号轴驱动巨噬细胞向M1方向极化。本项目的研究不仅能阐明TgMIF调控宿主免疫应答的致病分子机制，也为弓形虫病预防和针对性治疗提供新的思路。