子孢子感染诱导肝细胞自噬并促进其发育的分子机制研究

Autophagy is not only one of the recently discovered intracellular innate immune responses to restrict the development of a variety of intracellular microorganisms, but also the primary target for microorganisms to evade or suppress the host innate immune responses. Our preliminary data showed that infection of sporozoites could induce autophagy of exo-erythrocytic forms by hepatocytes. To our surprise, induction of autophagy couldn't inhibit, but significantly promote the development of exo-erythrocytic stage in vitro. However, the underlined mechanism of sporozoite-induced hepatocyte autophagy and its promotive role on liver-stage development is still unknown. Here, we firstly would like to investigate whether the sporozoite-induced hepatocyte autophagy is dependent on the activation of mTOR/Beclin-1/ATG5. Then, we'll observe whether the parasite could still normally replicate in the autolysosome. Finally, we would also like to investigate whether the hepatocyte autophagy could reduce the production of IFN-α/β in the infected hepatocytes through removing the malaria parasite RNA, and/or inhibite the apoptosis of the infected hepatocytes, in order to support the growth of liver-stage. This study could not only shed new light on the escape strategy of exo-erythrocytic stage, but also provide novel clues to design prophylactic stragies against the liver-stage.

自噬是近年来受到高度关注的一种非常重要的固有免疫应答机制，是胞内病原体与宿主相互作用的重要焦点。课题组前期研究发现，子孢子感染后能诱导肝细胞发生自噬，并自噬其中的疟原虫。有意思的是，肝细胞自噬后不但不能清除被自噬的疟原虫，而且还能显著促进肝期疟原虫的发育。然而，目前尚不清楚子孢子感染诱导肝细胞自噬并促进其自身发育的具体分子机制。鉴此，本项目拟首先观察子孢子感染是否通过mTOR/Beclin-1/ATG5的活化诱导肝细胞的自噬；然后，观察肝期疟原虫能否在自噬溶酶体内存活和发育；最后，观察肝细胞自噬能否通过清除疟原虫RNA抑制其刺激感染肝细胞产生IFN-α/β，以及抑制感染肝细胞发生凋亡，从而抑制宿主的抗疟原虫固有免疫应答；探讨子孢子感染诱导肝细胞自噬及其促进肝期疟原虫发育的分子机制。这不但有助于我们揭示新的肝期疟原虫免疫逃避机制，还可为设计新的红外期干预措施提供重要的靶点和思路。

研究报道非选择性自噬能促进肝期疟原虫的发育，但是关于选择性自噬在肝期疟原虫发育中的作用和机制并不清楚。我们发现疟原虫子孢子的感染能诱导肝细胞对肝期疟原虫的选择性样自噬。自噬诱导剂雷帕霉素处理能显著地促进肝细胞对肝期疟原虫的选择性自噬以及肝细胞的经典自噬，并促进疟原虫在小鼠肝脏的发育。虽然雷帕霉素处理选择性自噬体与溶酶体的融合，但是一些肝期疟原虫仍然能在肝细胞内存活并增殖。进一步的研究发现，肝期疟原虫能显著地抑制自噬溶酶体的成熟和酸化。因此，本研究证实，虽然经典自噬能促进肝期疟原虫的发育，但是肝期疟原虫可能通过抑制自噬溶酶体的酸化而存活，从而揭示了肝期疟原虫的一种新的免疫逃避机制。