Macrophage-derived extracellular vesicles regulate concanavalin A-induced hepatitis by suppressing macrophage cytokine production.

Acute liver failure is a clinical syndrome of severe hepatic dysfunction. Immune cells play an important role in the failure. In recent years, the immunoregulatory function of extracellular vesicles (EVs) has been reported; therefore, it is inferred that EVs have some role in an immune mediated liver injury. In this study, we investigated the immunoregulatory function of EVs in a concanavalin A (Con A)-induced liver injury. The mouse model was prepared by a single intravenous administration of 15 mg/kg Con A, in which there was a significant increase in serum EVs number. In in vitro study, the secreted EVs number was also significantly increased in Con A-treated RAW264.7, a mouse macrophage cell line, but not Hepa1-6, a mouse hepatoma cell line. In in vitro EVs treatment study, the EVs from Con A-treated mice serum and Con A-treated RAW264.7 suppressed the inflammatory cytokines production in Con A-stimulated RAW264.7. miRNA sequencing analysis showed that mmu-miR-122-5p and mmu-miR-148a-3p were commonly increased in these EVs and the EVs-treated cells. The enriched pathways in the miRNAs predicted target genes included the inflammatory response pathways. mRNA levels of the target genes in the pathways (mitogen-activated protein kinase, Phosphoinositide 3-kinase/Akt and Rho/Rho associated coiled-coil containing protein kinase pathways) were decreased in the EVs-treated cells. In in vivo RNA interference study, knock down of liver RAB27A, an EVs secretion regulator, significantly exacerbated the Con A-induced liver injury. These data suggest that macrophage-derived EVs play an important role in a Con A-induced liver injury through the immunoregulation. Overall design: miRNA profiles in RAW264.7 cells and extracellular vesicles from mouse serum or RAW264.7 culture supernatant were analyzed with MiSeq system (Illumina).

急性肝衰竭（acute liver failure）是一类以严重肝功能异常为特征的临床综合征。免疫细胞在肝衰竭的发生发展中发挥关键作用。近年来，细胞外囊泡（extracellular vesicles, EVs）的免疫调节功能已有诸多报道，因此推测细胞外囊泡在免疫介导的肝损伤中具有潜在调控作用。本研究针对刀豆蛋白A（concanavalin A, Con A）诱导的肝损伤模型，探究了细胞外囊泡的免疫调节功能。实验通过单次静脉注射15 mg/kg的Con A构建小鼠肝损伤模型，该模型的血清中细胞外囊泡数量显著升高。体外实验中，经Con A处理的小鼠巨噬细胞系RAW264.7所分泌的细胞外囊泡数量同样显著升高，而小鼠肝癌细胞系Hepa1-6则未出现该变化。体外细胞外囊泡处理实验显示，来自Con A处理小鼠血清以及Con A处理RAW264.7的细胞外囊泡，可抑制Con A刺激的RAW264.7细胞炎性细胞因子的产生。miRNA测序分析表明，mmu-miR-122-5p与mmu-miR-148a-3p在上述细胞外囊泡及经细胞外囊泡处理的细胞中均呈共同高表达。上述miRNA的预测靶基因富集通路涵盖炎性反应相关通路。经细胞外囊泡处理的细胞中，这些通路内靶基因的mRNA水平（包括丝裂原活化蛋白激酶通路、磷脂酰肌醇3-激酶/Akt通路及Rho/Rho相关卷曲螺旋包含蛋白激酶通路）均显著下调。体内RNA干扰实验证实，敲低细胞外囊泡分泌调控因子肝脏RAB27A的表达，可显著加重Con A诱导的肝损伤。综上，本研究数据表明巨噬细胞来源的细胞外囊泡可通过免疫调节在Con A诱导的肝损伤中发挥重要作用。实验整体设计：采用MiSeq系统（Illumina）对RAW264.7细胞、小鼠血清及RAW264.7细胞培养上清中的细胞外囊泡的miRNA表达谱进行分析。