TREM-2阳性肝脏巨噬细胞群在肝衰竭中的来源形成、功能特性及作用机制研究

Intra-hepatic macrophages are a group of immune-competent cells with highly functional plasticity, which play an important role in the regulation of liver failure at stages of onset, propagation and recovery. Among them, hepatic macrophages with anti-inflammatory activity are the functional subgroup that promote inflammation appeasing and tissue repair. Therefore, to identify such macrophage subgroup is important to develop novel immunotherapy for liver failure. Recent studies have shown that TREM-2 is a cell-surface regulator that inhibits the monocyte / macrophage pro-inflammatory response. And our preliminary study found that TREM-2-positive monocytes/macrophages were expanded in peripheral blood of patients with liver failure and hepatic compartment of mouse models of liver failure. Therefore, it is suggested that TREM-2 positive macrophages may be a subset conferring anti-inflammatory activity in liver failure. In this project, we intend to measure the expansion of TREM-2-positive macrophages by collecting peripheral blood and liver tissue samples from patients with liver failure, as well as establishing mouse model of liver failure. We then investigate the ontogeny and shaping of this macrophage subset, and its transcriptomic, phenotypic and functional properties. We also explore the potential therapeutic effect on liver failure using TREM-2 knockout and TREM-2-positive macrophage adoptive transfer model. The study would shed light on novel immunotherapy for patients with liver failure.

肝内巨噬细胞是一群具有高度功能可塑性的免疫细胞群，其在调控肝衰竭发生、发展及恢复的各个阶段中均发挥重要作用。其中具有抑炎活性的肝脏巨噬细胞是促使肝脏炎症平息及组织修复的功能亚群，寻找和鉴定该肝脏巨噬细胞亚群是肝衰竭免疫细胞治疗的重要方向。最近研究表明TREM-2是抑制单核/巨噬细胞炎症应答的细胞表面调控分子，而本项目组前期研究发现TREM-2+单核/巨噬细胞在肝衰竭患者的外周血及小鼠模型肝组织内均显著增加，提示在肝衰竭中TREM-2+巨噬细胞可能是一群具有抑炎活性的巨噬细胞亚群。在本项目中，我们拟收集肝衰竭患者外周血及肝组织标本，同时利用肝衰竭小鼠模型，分析肝衰竭状态下的TREM-2+巨噬细胞的数量变化，探讨肝内TREM-2+巨噬细胞的来源及形成机制，研究其基因转录、表型及功能特性，并利用TREM-2基因敲除小鼠及过继转移模型研究对肝衰竭的治疗作用，为探索肝衰竭细胞治疗奠定基础。

肝内巨噬细胞是一群具有高度功能可塑性的免疫细胞群，其在调控肝衰竭发生、发展及恢复的各个阶段中均发挥重要作用。TREM-2+巨噬细胞可能是一群具有抑炎活性的巨噬细胞亚群。本课题组通过对肝衰竭患者肝组织标本进行单细胞测序，发现TREM-2表达的巨噬细胞显著增多，并在分别在对乙酰氨基酚（APAP）和刀豆蛋白（ConA）诱导的小鼠急性肝衰竭模型中验证发现肝脏TREM2和衔接蛋白DAP12 mRNA表达表现为早期短暂下降，后出现显著升高。进一步分析发现TREM-2表达巨噬细胞包含CD9+TREM2+巨噬细胞和TRFC+TREM2两个亚群，流式细胞术及免疫荧光验证高表达TREM2基因的CD9巨噬细胞和TFRC巨噬细胞在肝衰竭患者肝脏内明显富集。分化轨迹分析表明CD9巨噬细胞和TFRC巨噬细胞与单核细胞的分化轨迹有明显的重叠，证实了两群巨噬细胞的单核细胞起源，且CD9巨噬细胞是单核细胞的早期分化形式。进一步通过将单核细胞与肝衰竭患者的肝组织匀浆或细胞因子IL-6、TNF-α或IL-10进行培养，发现肝衰竭患者的肝组织匀浆或IL-10能够诱导单核细胞CD9、TFRC和CD163表达，提示肝衰竭中CD9+和TFRC+巨噬细胞的富集源于单核细胞对炎症肝脏中存在的炎症信号反应。内毒素+D-氨基半乳糖诱导的TREM2敲除小鼠急性肝衰竭模型的生存时间及生存率较野生型小鼠明显升高，而血清ALT及AST水平明显降低。与野生型小鼠组相比，TREM2敲除小鼠的骨髓来源巨噬细胞在内毒素刺激后炎症因子mRNA表达显著增加，其ERK 和p38 MAPK 通路更易被激活。该研究结果提供了一种潜在的肝衰竭的巨噬细胞治疗技术前景。