Rubicon调控非经典自噬(LAP)在Kupffer细胞降解凋亡细胞及诱导肝移植免疫耐受中的作用

Previous studies demonstrated that the induction and effective clearance of apoptotic cell by Kupffer cell (KC) are closely related to the formation of immune tolerance. When phagocytizing apoptotic cells are more than KC degradation ability, it will induce inflammatory response. How to enhance the ability of KC to degrade apoptotic cells and induce immune tolerance in liver transplantation is an important problem to be solved. Recent study described that the pathway of LC3 (microtubule-associated protein 1 light chain3)-associated phagocytosis (LAP) has shed some light on this issue, and the Rubicon (Run domain protein as Beclin-1-interactingand cysteine-rich-containing) is the key component for activating the LAP pathway. Our preliminary shows Rubicon involved in the immunoregulation after liver transplantation and regulation of Rubicon activity in KC has a significant effect on the degradation of the apoptotic cells and the balance of pro-inflammatory/anti-inflammatory. We hereby put forward a hypothesis that enhancement of the LAP by Rubicon may induce liver transplant tolerance. To clarify the role of the molecule in liver tolerance induction, we will separate KC and establish liver transplantation model in rat, using knock-out or knock-in of Rubicon to inhibit or enhance Rubicon expression both in vitro and in vivo. Once the hypothesis of up-regulation of LAP by Rubicon in KC mediating tolerance induction in liver transplantation is corroborated, this molecule can become a novel target for treatment of acute rejection.

Kupffer细胞（KC）介导的细胞凋亡及有效清除与免疫耐受微环境形成密切相关；当KC吞噬大量凋亡细胞超出其自身降解能力，则会诱导炎性反应。如何增强KC降解凋亡细胞能力进而诱导免疫耐受是亟待解决的重要问题。新近研究发现非经典自噬途径—LAP是吞噬细胞有效降解凋亡细胞的关键途径，Rubicon是启动LAP的核心蛋白，调控Rubicon蛋白活性可直接影响LAP功能。课题组预实验证实Rubicon参与肝移植术后免疫调控，改变其活性可以影响凋亡细胞降解及促炎/抗炎免疫平衡。因此，增强Rubicon的表达可能有利于形成免疫耐受微环境。本项目选择肝脏KC为靶细胞，以Rubicon为切入点，采用基因敲除、敲入及纳米技术，沉默或增强Rubicon表达，通过KC体外培养和大鼠肝移植体内实验，探讨Rubicon调控LAP在KC降解凋亡细胞中的作用及机制，阐明该途径在调节免疫微环境及诱导免疫耐受中的作用。

肝移植术后急性排斥反应与患者术后生存密切相关。我们既往的研究结果表明，增强Kupffer细胞（KC）清除凋亡细胞的能力可以有效缓解急性排斥反应。非经典自噬（LAP）途径是吞噬细胞降解凋亡细胞的关键途径，而Rubicon作为组成PI3KC3复合物的重要蛋白之一，在LC3相关吞噬作用（LAP）过程中起关键作用。本项目选择KC为靶细胞，利用RNA干扰及病毒转染技术，判断Rubicon沉默及过表达对KC抗原呈递作用及吞噬功能的影响；通过诱导产生凋亡T细胞与KC共培养，判断Rubicon对KC通过LAP介导的凋亡细胞降解功能的影响，并探讨其免疫抑制机制；建立肝移植动物模型，观察Rubicon活性对供肝免疫反应的影响。研究发现，在体外，随着Rubicon的过表达，LC3相关吞噬作用（LAP）必需的蛋白有明显的增加，LC3 II的募集以及吞噬体、溶酶体的融合有明显的增加，这证实了Rubicon介导的LAP促进了KC降解和清除凋亡T细胞的能力。此外，Rubicon的过表达促进了KC的M2极化，与对照组相比，实验组CD163、IL-10、TGF-β等抑炎因子随着Rubicon的过表达有明显的增加。凋亡T细胞降解产物多不饱和脂肪酸，可以激活PPARγ信号，进而诱导KC向M2极化。在体内，过表达Rubicon有效地减轻了急性排斥模型的肝功能损伤。由此表明Rubicon通过LAP途径调控KC降解凋亡细胞在诱导和维持移植肝脏免疫耐受中起重要作用。