内皮细胞源外泌体miRNAs调控抗汉坦病毒感染免疫的作用及机制研究

Hemorrhagic fever with renal syndrome (HFRS) caused by hantavirus is a serious public health problem in China. Endothelial cells are the main targets for hantavirus, but infection per se is not lytic. This suggests that hantavirus-induced immune response itself plays a pivotal role in the cascade of events leading to endothelial dysfunction. The mechanism of hantavirus-induced modulation of host cellular immunity and antiviral response remain poorly understood, especially the long-distance regulation and paracrine effect.. Exosomes are nano-sized extracellular vesicles released upon fusion of multivesicular bodies with the cellular plasma membrane. Exosomes were shown to contain selectively sorted functional proteins, lipids, and RNAs, mediating cell-to-cell communications and hence playing a role in the physiology of the healthy and diseased pathogens. An extensive body of evidence has shown that exosomes play a crucial role for antiviral innate immune response.. Little is known about the functional relevance of exosomes in hantavirus infection and disease pathogenesis. We have found that exosomes derived from hantavirus-infected HUVECs (Exo-HV) contain viral S gene, CD81(exosomal marker) without infectivity. Exo-HV reduced replication of hantavirus in the exosomes recipient cells. We examined the miRNA profile in Exo-HV and clustering analysis revealed that Exo-HV expressed distinct pattern of miRNAs compared to the control with 304 significant expressed miRNAs. We also identified six miRNAs(including let-7a-3p, let-7b-3p, miR-15b-5p, miR-96-5p, miR-4429, miR-326) in exosomes, which are involved in ERK/MAPK pathway. Thus, we speculate that during a course of hantavirus infection, exosomes are released from host cells, deliver miRNAs in the absence of virus contact and modulate the antiviral immune response in the recipient cells by targeting ERK/MAPK pathway.. We will examine morphological characterization, transcriptomics profiling analysis of Exo-HV, which will provide the overall information of Exo-HV. We will test exosomes from multiple endothelial cells (HUVEC, HMEC-1, HRGEC) to confirm its anti-hantavirus effect. We will also determine the binding, uptake and localization of Exo-HV to the recipient HUVECs, macrophage, NK cells and dendritic cells. Furthermore, we will explore the antiviral effect of Exo-HV miRNA in the recipient cells and the involved signaling pathway(mainly on ERK/MAPK pathway), and define the key miRNAs transferred intercellularly via exosomes that benefit the host immune response and confer hantavirus resistance to the bystander cells. In addition, we will verify these miRNAs and pathway obtained from in vitro experiment in the exosomes from HFRS patients.. This work will provide insight into the relationship of exosomes, virus and host cells, which will be beneficial in expanded understanding the mechanism of anti-hantavirus immune response as well as the development of novel treatment of HFRS.

汉坦病毒（HV）感染抗病毒免疫机制尚未完全阐明。外泌体（Exo）介导的miRNA转移在病原体感染和免疫应答中有重要作用。本课题组前期研究显示HV感染HUVEC释放的Exo（Exo-HV）具有远程抗病毒作用，结合miRNA芯片及预实验结果我们提出内皮细胞源Exo-HV 转运miRNAs至不同受体细胞靶向调控ERK/MAPK为核心的信号通路，发挥抗病毒免疫作用。. 本项申请拟进一步全面分析Exo-HV生物学特性及miRNAs表达谱，明确多种内皮细胞源Exo-HV阻断HV感染的作用，重点研究Exo-HV miRNAs对受体内皮细胞、免疫细胞中ERK/MAPK通路表达的调控及其在抗病毒免疫中的关键作用，最终阐明内皮细胞源Exo-HV在“病毒感染HUVEC→循环外泌体→未感染靶细胞”的旁分泌过程中横向转运miRNAs发挥抗病毒作用的分子免疫机制，为新型抗病毒药物以及疫苗的研发提供新的思路。

汉坦病毒感染会导致潜在的致命疾病，例如肾综合征出血热（HFRS）。但是目前尚无有效的治疗方法或FDA批准的针对汉坦病毒的市售疫苗。外泌体是细胞之间信息交换的新载体。许多研究表明，病毒感染细胞释放的外泌体可用于阻断或促进受体细胞中的病毒感染，但人们对外泌体在抗汉坦病毒免疫中的作用了解甚少。在本研究中，我们的结果表明汉坦病毒感染导致外泌体分泌上调以及miR-145-5p进入外泌体的包装具有差异。进一步提供的证据表明，miR-145-5p在外泌体中优先富集。外泌体miR-145-5p可以转移到人脐静脉内皮细胞（HUVEC），通过直接靶向编码HTNV 76-118糖蛋白的M RNA，同时miR-145-5p可以促进I型干扰素反应以及干扰素刺激因子的表达，从而阻止了病毒复制。总之，我们的研究表明由HTNV感染的细胞分泌的外泌体选择性地包装了高表达的miR-145-5p，可以通过靶向HTNV 76-118的M RNA并诱导I型干扰素来抑制受体细胞中的病毒复制，发挥抗病毒免疫作用，最终阐明内皮细胞源Exo-HV在“病毒感染HUVEC→循环外泌体→未感染靶细胞”的旁分泌过程中横向转运miRNAs发挥抗病毒作用的分子免疫机制，为新型抗病毒药物以及疫苗的研发提供新的思路。