ERRα在调控Kupffer细胞表型重塑及诱导肝移植免疫耐受中的作用及机制研究

Macrophage polarization refers to development of a specific phenotype important for tissue homeostasis or host defense in response to environmental cues. Kupffer cells (KCs), the most important resident macrophages of the liver, played a dual role in the pathological changes after liver transplantation, and the mechanism is closely associated with its activation and polarization.Activated KCs polarize towards various functional phenotypes depending on the pathogen and cytokines expressed in the microenvironment. The best characterized activation phenotypes are classical activation(also termed M1) and alternative activation (M2). M1 macrophages are effective at host defense and leading to actue rejection,and M2 macrophages are important for resolution of inflammation and induction of immune tolerance.Our previous studies found the method to induce the functional phenotype of KCs towards M2 via regulating the expression of microRNA-155, XBP1, HDAC11 and NF-κB, respectively.But the effect was not satisfactory.A recent study reported that estrogen-related receptor α (ERRα)can effectively inhibit macrophage inflammatory response via controlling the activity of A20, Sirt1, NF-κB and HIF1α and these molecules were closely associated with M2-polarized macrophage phenotype. But so far there is no related research in solid organ transplantation, especially in liver. We hereby put forward a hypothesis that up-regulation of ERRα in KCs may induce liver transplant tolerance via shifting the phenotype of KCs to M2. To clarify the role of the molecule in liver tolerance induction, we will separate KCs and establish liver transplantation model in rat, using ERRα gene knock-in or knock-out mice to enhance or inhibit ERRα expression both in vitro and in vivo. Once the hypothesis of up-regulation of ERRα in KCs mediating the self-tolerance induction in liver transplantation is corroborated, this molecule can become a novel target for treatment of acute rejection.

Kupffer细胞（KCs）在肝移植免疫中起着即促进排斥反应又诱导免疫耐受的双重角色，其原因与其自身表型相关。项目组前期发现：通过单一调控KCs内microRNA-155、XBP1、HDAC11、NF-κB等调节因子，可诱导KCs表型重塑，但其持续性及稳定性欠佳。新近发现雌激素相关受体α(ERRα) 能从多个层面有效抑制巨噬细胞炎症反应，其涉及多个分子与调控巨噬细胞表型相关分子密切相关，是潜在的调控KCs表型重塑的“开关”分子。但相关研究尚未涉及移植领域,更未涉及KCs。我们认为该分子在调控KCs表型重塑及诱导肝移植免疫耐受中也起重要作用。因此，本项目选择肝KCs为靶细胞，以ERRα为切入点，分离KCs并建立肝移植模型，采用基因敲除、敲入、慢病毒及纳米技术，沉默或增强ERRα表达，探讨ERRα在调控KCs表型重塑中的作用及相关分子机制，并阐明该分子在诱导移植肝脏免疫耐受形成中的作用机制。

研究背景：临床器官移植后长期服用免疫抑制剂所引起的感染、肿瘤发生/复发等并发症严重影响患者生存质量和移植疗效。因此，诱导移植术后免疫耐受一直是移植界的研究热点。不同于其他实质器官，一系列动物模型及临床研究显示，移植肝脏具有免疫特惠现象，即“自发性免疫耐受”，其形成与肝脏内存在Kupffer细胞（KCs）等特异性免疫细胞密切相关。新近发现雌激素相关受体α (Estrogen-related receptor α, ERRα)从多条途径参与了负性调控巨噬细胞炎症反应，其机制涉及通过增强A20及Sirt1蛋白负性调控NF-κB活性、影响HIF1α稳定性及代谢重塑等。综上，我们不难发现ERRα分子能从多个层面有效抑制巨噬细胞炎症反应，其涉及多个分子与调控巨噬细胞表型相关分子密切相关。主要研究内容：（1）明确了ERRα在急性排斥反应中具有保护作用；（2）阐明了ERRα的保护作用与促进其凋亡清除、M2型极化有密切关系；（3）探索了ERRα相关的A20/NF-κB和Sirt1/STAT3信号通路在该过程中的调控效应。重要结果与关键数据：（1）经WB、免疫荧光检测，肝移植后急性排斥组中KCs内的ERRα降低，提示ERRα与急性排斥反应呈负相关；（2）过表达KCs内ERRα后，肝移植后急性排斥组病理损伤程度、肝功能标志物水平均显著改善，提示其有利于减轻急性排斥程度；（3）过表达KCs内ERRα后，肝移植后急性排斥组中凋亡细胞显著下降，提示ERRα促进了凋亡细胞清除；（4）而阻断KCs M2型极化后，凋亡细胞下降被逆转，提示ERRα促进了凋亡细胞清除是依赖于KCs M2型极化的；（5）过表达KCs内ERRα后，肝移植后急性排斥组中M2型KCs数量、抗炎因子表达量均显著增高，提示其有利于KCs M2型极化；（6）经WB、免疫荧光染色检测发现，ERRα抑制了A20/NF-κB信号通路，激活了Sirt1/STAT3信号通路；（7）促进A20/NF-κB通路活化或抑制Sirt1/STAT3信号活化，逆转了ERRα引起的KCs M2极化，证明ERRα相关的A20/NF-κB和Sirt1/STAT3信号通路是其促进KCs M2型极化的具体机制。科学意义：证明了ERRα是潜在的调控巨噬细胞表型重塑的关键分子，探索了其在肝脏移植免疫调控中的具体机制，可能为肝脏移植后诱导免疫耐受提供新思路。