肝细胞特异缺失ATG5对弓形虫急性感染小鼠肝脏损伤的影响与机制研究

It is known that ATGs of immune-derived host cells mediate the killing of Toxoplasma gondii by IFN-γ, but the effect of ATGs of on the infection of T. gondii in non-immune-derived host cells is not clear. We have found previously that T. gondii infected hepatocyte-specific Atg5 knockout mice caused increased parasite burden along with the aggravated pathological damage of liver, leading to the shorten survival of mice. Further studies showed that deletion of the autophagy protein Atg5 specifically in hepatocyte result in significantly increased of IFN-γ and TNF-α.Neutralization of IFN-γ markedly abolished pathological changes and parasite burden, meanwhile the expression of TNF-α expression was down-regulated. This study suggests that loss of Atg5 in hepatocytes may affect the killing effect of IFN-γ on T. gondii, leading to the immunopathology of liver and decreased survival of mice. In view of this, this project firstly intends to further demonstrate whether loss of Atg5 in hepatocytes affects the killing effect of IFN-γ on T. gondii. Then, Next, we will screen regulatory molecules of IFN-γ that may be involved in regulating the killing effect of IFN-γ on T. gondii in hepatocytes through RNAseq. We intend to explore a new mechanism of IFN-γ killing mediated by ATGs in non-immune host cells. Finally, we validated whether ATG5 knockout mice could affect the expression of TNF-α through the increase of parasite burden, leading to severe immunopathology. This is an supplementary research to the ATGs-mediated IFN-γ killing of T. gondii, and also provides an important theoretical foundation for the immunological control of the parasite.

已知免疫源性宿主细胞ATGs介导了IFN-γ对弓形虫的杀伤，但非免疫源性宿主细胞ATGs对弓形虫感染的影响并不清楚。课题组前期发现弓形虫感染肝细胞ATG5条件敲除小鼠后，肝脏虫荷增加、病变加重，小鼠死亡提前；进一步发现，IFN-γ和TNF-α表达显著上调，而体内耗竭IFN-γ后，肝脏虫荷差异消失、病变减轻，TNF-α下调。提示肝细胞Atg5可能影响IFN-γ对弓形虫的杀伤，促发肝脏炎症病理反应，导致小鼠死亡。鉴此，本项目拟进一步证实肝脏是否通过Atg5影响IFN-γ对弓形虫的杀伤；并通过RNAseq，筛选出可能参与调节IFN-γ杀伤肝细胞内弓形虫的效应分子，探讨非免疫源性宿主细胞内ATGs介导IFN-γ杀伤的新机制；最后验证ATG5是否通过虫荷增加影响TNF-α的表达，导致肝脏严重损伤。这是对现有ATGs介导IFN-γ杀伤弓形虫研究的重要补充，也为弓形虫免疫防治提供重要的理论依据。

机体免疫状态影响急性弓形虫感染的发展和转归，而免疫细胞因子IFN-γ 是宿主杀灭和控制弓形虫扩散的关键效应分子。IFN-γ杀灭胞内弓形虫受到自噬和自噬相关蛋白的调控。本研究证实了弓形虫感染肝脏Atg5条件缺失小鼠导致小鼠肝脏严重的病理损伤，弓形虫感染诱导的炎症细胞因子IFN-γ和TNF-α是导致肝脏病理损伤的主要介导者，CD4+T是肝脏淋巴细胞中上调的IFN-γ的主要来源。我们将进一步检测肝脏淋巴细胞中TNF-α的分泌，并通过体外实验检测ATG5介导IFN-γ杀伤弓形虫的机制。我们的实验结果是现有ATGs介导IFN-γ杀伤弓形虫研究的重要补充，也有助于我们理解弓形虫肝病的发生机制。