巨噬细胞亚群在肝脏损伤修复中的功能研究

Distinct macrophage populations or macrophages with different phenotypes are specifically involved in various discrete functions during all stages of tissue injury repair and regeneration. Liver is a newly identified peripheral lymphoid organ as well as highly regenerative organ. However, the precise mechanism by which and how specific subsets of hepatic macrophages are involved in the process of injury or regeneration remain obscure. We studied the roles of specific macrophage subsets in a mouse model of drug-induced self-limiting liver injury model. We show that there are different macrophage subsets, including resident Kupffer cells, Ly6Chi monocyte-derived macrophages and Ly6Clo monocyte-derived macrophages, and they have undergone dynamic changes during different phases of inflammation and resolution. We then performed proteomic profiling of these macrophage subsets, which suggested Ly6Chi macrophages are pro-inflammation while Ly6Clo macrophages are pro-resolution. Furthermore, Ly6Clo macrophages derived from an in situ phenotypic switch of recruited Ly6Chi macrophages and KC may played a role in this phenotype switch. A comprehensive investigation of (1) the precise mechanism by how the Ly6Clo macrophages stimulate liver regeneration; (2) more solid evidence of an in situ phenotypic switch from Ly6Chi macrophages to Ly6Clo macrophages; (3) the precise function and mechanism of KC in the macrophages phenotype switch, are required.

从损伤到再生、修复的不同阶段，组织内会出现不同亚群或不同表型的巨噬细胞，各司其职，参与组织损伤与修复。肝脏是一个新的免疫器官和高度再生器官，各巨噬细胞亚群如何分工合作，调控肝组织损伤、再生与修复鲜有研究。我们前期研究利用药物诱导的急性自限性肝损伤模型，观察到肝脏中存在三种巨噬细胞亚群：组织固有的枯否细胞（KC）、单核来源的巨噬细胞（Ly6Chi Mφ和Ly6Clo Mφ），并发现这三群细胞伴随着组织损伤与修复，出现各自截然不同的动力学变化。利用质谱技术手段分析这三群细胞的蛋白表达特性，提示Ly6Chi Mφ可能促进炎症发生，而Ly6Clo Mφ可能促进损伤修复。我们还发现KC可能调控Ly6Chi Mφ向Ly6Clo Mφ的转换。本项目将继续研究：Ly6Clo Mφ促进肝损伤修复的功能；肝内Ly6Chi Mφ向Ly6Clo Mφ转换；KC调控Ly6Chi Mφ向Ly6Clo Mφ的转换。

炎症是组织损伤或病原微生物入侵机体引发的复杂反应，以帮助机体抵御感染，清除坏死的组织细胞，恢复组织稳态。研究发现炎症反应深度参与组织的再生和修复过程。巨噬细胞是重要的先天免疫细胞，具有极强的异质性和可塑性。研究表明巨噬细胞能维持组织稳态和促进组织损伤修复。然而巨噬细胞亚群在肝脏损伤修复中发挥何种作用，以及亚群之间的转换机制如何，均不十分清楚。. 本研究利用APAP诱导的急性肝损伤模型，鉴定了肝损伤修复过程中巨噬细胞的亚群变化及其功能，并探索了巨噬细胞亚群转换的细胞和分子调节机制。（1）在肝脏损伤修复过程中，出现两群单核来源巨噬细胞亚群，分别是损伤时期的Ly6Chi Mφ和修复时期的Ly6Clo Mφ。利用CD11b-DTR小鼠选择性地清除两个亚群，我们发现清除Ly6Clo Mφ后肝脏修复严重受损。体外共培养实验也表明Ly6Clo Mφ能显著促进肝细胞增殖，而Ly6Chi Mφ则不能。质谱鉴定表明Ly6Clo Mφ显著上调促修复相关蛋白。（2）CD45.1+ Ly6Chi移植实验证明炎性Ly6Chi Mφ可转换为促修复表型的Ly6Clo Mφ。（3）PMN产生的 ROS通过活化AMPK诱导巨噬细胞的表型转换。αLy6G清除PMN和Nox2-/-小鼠巨噬细胞转换减弱，肝脏修复受损。体外实验证明，H2O2促进Ca2+内流进入巨噬细胞并活化CaMKKβ及下游的AMPK。AMPK缺失后，巨噬细胞表型转换和肝脏修复受损。. 综上，本研究表明，在肝脏损伤修复的不同时期有不同亚群的巨噬细胞在发挥作用。炎性Ly6Chi Mφ在PMN来源的ROS的诱导下逐渐转换为促修复表型的Ly6Clo Mφ，进而促进肝脏损伤修复。机制上，ROS通过活化Ca2+-CaMKKβ-AMPK通路诱导巨噬细胞的表型转换。本研究表明参与炎症起始的巨噬细胞和中性粒细胞也参与促进了炎症的恢复和组织的修复，并阐明了其中的分子机制。