宿主自噬介导的弓形虫清除作用新机制研究

Toxoplasma gondii is one of the world-wide distributed opportunistic pathogenic protozoa, that parasitize in all the warm-blooded animals, including humans. The infection by T. gondii has already become a major threat to the population with compromised immunity, leading to the toxoplasmic encephalitis, toxoplasmosis ophthalmopathy of the patients. In the previous studies, we demonstrated that the LC3 production was first induced in the mouse macrophages RAW264.7 infected by the tachyzoites of the RH strain of T. gondii, then attenuated gradually to the uninfected level. Meanwhile, SIRT1 production was found to be upregulated by T. gondii infection along time. When challenged by SIRT1 activator or inhibitor, the infected macrophages showed regulation of LC3 production, accompanied with the alteration of intracellular tachyzoites population. These results gave us a hint that SIRT1 was likely to play an essential role in the interaction between host and the parasites. Based on the previous studies, we assume that host may employ autophagy to eliminate T. gondii through SIRT1 by regulating autophagy and autophagy-related proteins. To suppress this scavenging effect, T. gondii may also exploit SIRT1 to attenuate autophagy of host in the mid to late stages of infection. We aim to achieve our goal by inspecting the changes at cellular level, the infected animal model and also the molecular mechanism underlying, thereby depicting the host autophagic killing towards T. gondii centering on SIRT1 and establishing the SIRT1 signal network utilized by the parasites to resist the host. The fulfillment of this project helps to explain the immune evasion of T. gondii, to explicit the potential of SIRT1 for its application as an anti-toxoplasma therapeutic drug, and to lay the foundation for the further exploration.

刚地弓形虫是一种体内机会致病性原虫，其感染可导致免疫力低下人群出现严重临床症状。在前期研究中我们发现弓形虫感染鼠巨噬细胞后可导致胞内重要自噬蛋白LC3及组蛋白去乙酰化酶SIRT1水平改变，且此现象随感染时间推移呈现动态变化。同时，对SIRT1的干扰可改变胞内自噬蛋白水平，并直接影响胞内感染虫数。本项目在前期基础上提出，宿主可通过自噬清除弓形虫，而SIRT1可能在此清除效应中具有重要作用，表现为宿主通过SIRT1调控自噬及自噬蛋白的表达而清除虫体，另一方面，弓形虫也可利用SIRT1抑制宿主的自噬性清除作用。项目拟从分子、细胞及动物感染模型层面阐明SIRT1在宿主自噬介导的弓形虫清除效应中的作用及机制，并构建以SIRT1为核心的弓形虫抑制宿主自噬性清除信号网络。本项目的实施有助于揭示SIRT1在宿主杀伤与弓形虫免疫逃避过程中的重要地位，阐明SIRT1作为抗弓形虫药物靶标的潜在应用价值。

弓形虫是一种机会性致病性原生动物，可以感染几乎所有种类的温血动物，包括人类。弓形虫在胞内可以逃避宿主的免疫杀伤作用，这一过程被称为免疫逃避。本项目聚焦于评估Sirtuin1 (SIRT1) （SIRT1为烟酰胺腺嘌呤二核苷酸(NAD)依赖的组蛋白去乙酰酶(HDACs)sirtuins (SIRTs)家族成员之一]在小鼠RAW264.7巨噬细胞系的弓形虫RH强毒株感染模型中所起的作用。在本研究中，我们评估并观察了受感染细胞内SIRT1活性、表达和定位的变化，并评估了它在弓形虫感染期间参与CD154/IFN-γ杀伤途径和自噬通路的情况和程度。通过总结实验数据，我们发现，弓形虫感染宿主巨噬细胞系后，可上调胞内SIRT1的活性，表达，并能一定程度促使SIRT1从细胞核内向胞质迁移。同时，我们还发现，在弓形虫侵入宿主细胞过程中，SIRT1可能通过以下途径影响弓形虫的清除: SIRT1通过抑制IRGM1从而抑制细胞内弓形虫的清除，和/或SIRT1通过下调自噬激活抑制细胞内弓形虫的杀伤。本项目研究成果揭示弓形虫感染对宿主细胞内SIRT1具有上调作用，抑制或激动SIRT1可分别减少或增加细胞速殖子的数量，而对IRGM1表达和对自噬通路的影响可能是SIRT1调控弓形虫胞内增殖的原因。