Cui Bin, Chen Xiao-Jie, Sun Jie, Li Shi-Peng, Zhou Guang-Peng, Sun Li-Ying, Wei Lin, Zhu Zhi-Jun
Liver Transplantation Center, Beijing Friendship Hospital, Capital Medical University, Beijing 101100, China; Department of Neurosurgery, Aviation General Hospital, Beijing 100012, China.
Liver Transplantation Center, Beijing Friendship Hospital, Capital Medical University, Beijing 101100, China; Clinical Center for Pediatric Liver Transplantation, Capital Medical University, Beijing 101100, China; National Clinical Research Center for Digestive Diseases, Beijing 101100, China.
Int Immunopharmacol. 2023 Jan;114:109541. doi: 10.1016/j.intimp.2022.109541. Epub 2022 Dec 21.
Exosomes exert considerable influence in mediating regulatory T (Treg) cells differentiation, which attach great importance to attenuating acute cellular rejection after liver transplantation (LT). And, miRNAs are known to play essential roles in cell-cell communication delivered by exosomes. However, the function of exosomal miRNAs in regulating Treg cells after LT remains unknown. Here, we performed an expression profiling analysis of exosome-miRNAs from human plasma after LT and investigated their immunoregulatory effects on Treg cells.
Fifty-eight LT patients and nine donors were included in this report. miRNA profiles in plasma exosomes were analyzed using next-generation sequencing. Flow cytometry, HE and multiplex immunofluorescent staining were used to identify Treg cells in the liver and peripheral blood. A lentiviral vector system was used to overexpress miR-193b-3p in dendritic cells (DCs), and exosomes isolated from these transfected cells were co-cultured with spleen lymphocytesin vitro. A quantitative Real-time PCR and enzyme-linked immunosorbent assay were used to detect the expression of cytokines.
Treg cell infiltration was increased in the liver along with Th17 and CD8+ T cell, and it was down-regulated in peripheral blood in the acute rejection group. High-throughput sequencing revealed that miR-193b-3p was markedly up-regulated in plasma exosomes of non-rejection LT patients. The NLRP3 inflammasome was screened as a target for miR-193b-3p based on target prediction and functional enrichment analyses. Exosomal miR-193b-3p derived from DCs increased Treg cells as demonstrated in vitro. miR-193b-3p overexpression down-regulated NLRP3 as well as the inflammatory cytokines IL-1β and IL-17A while increasing levels of the cytokines IL-10 and TGF-β.
DC derived exosomal miR-193b-3p promoted Treg cells by inhibiting NLRP3 expression. These findings not only provide a new perspective on the mechanisms, but also hold great promise for the treatment or prevention of liver allograft rejection.
外泌体在介导调节性T（Treg）细胞分化方面发挥着重要作用，这对于减轻肝移植（LT）后的急性细胞排斥反应至关重要。而且，已知微小RNA（miRNA）在由外泌体传递的细胞间通讯中起关键作用。然而，LT后外泌体miRNA在调节Treg细胞中的功能仍不清楚。在此，我们对LT后人血浆中外泌体-miRNA进行了表达谱分析，并研究了它们对Treg细胞的免疫调节作用。
本报告纳入了58例LT患者和9名供体。使用下一代测序分析血浆外泌体中的miRNA谱。采用流式细胞术、苏木精-伊红（HE）染色和多重免疫荧光染色来鉴定肝脏和外周血中的Treg细胞。使用慢病毒载体系统在树突状细胞（DC）中过表达miR-193b-3p，并将从这些转染细胞中分离的外泌体与脾淋巴细胞在体外共培养。采用定量实时聚合酶链反应（qRT-PCR）和酶联免疫吸附测定（ELISA）检测细胞因子的表达。
在急性排斥反应组中，肝脏中Treg细胞浸润与辅助性T细胞17（Th17）和细胞毒性T细胞（CD8+T）一起增加，而外周血中Treg细胞浸润下调。高通量测序显示，在未发生排斥反应的LT患者的血浆外泌体中，miR-193b-3p显著上调。基于靶标预测和功能富集分析，NOD样受体蛋白3（NLRP3）炎性小体被筛选为miR-193b-3p的靶标。如体外实验所示，源自DC的外泌体miR-193b-3p增加了Treg细胞。miR-193b-3p过表达下调了NLRP3以及炎性细胞因子白细胞介素-1β（IL-1β）和白细胞介素-17A（IL-17A），同时增加了细胞因子白细胞介素-10（IL-10）和转化生长因子-β（TGF-β）的水平。
DC来源的外泌体miR-193b-3p通过抑制NLRP3表达促进Treg细胞。这些发现不仅为相关机制提供了新的视角，也为肝移植排斥反应的治疗或预防带来了巨大希望。
