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Extracellular vesicles (EVs) are increasingly recognized as important mediators of intercellular communication. In this study, we aimed to further characterize the role of macrophage-derived EVs in immune responses against hepatitis C virus (HCV) and the potential of polyunsaturated fatty acids (PUFAs) to modulate this modality of innate immunity. To this end, EVs were isolated from interferon-stimulated macrophage cultures or from serum of patients with acute or chronic hepatitis C. EVs were characterized by electron microscopy, flow cytometry, RNA-sequencing, and Western blot analysis. The effect of EVs on replication of HCV was assessed in coculture models. Functional analyses were performed to assess the impact of PUFAs on EV-mediated antiviral immunity. We found that macrophages secreted various cytokines shortly after stimulation with type I and II IFN, which orchestrated a fast but short-lasting antiviral state. This rapid innate immune answer was followed by the production of macrophage-derived EVs, which induced a late, but long-lasting inhibitory effect on HCV replication. Of note, exposure of macrophages to PUFAs, which are important regulators of immune responses, dampened EV-mediated antiviral immune responses. Finally, EVs from patients with hepatitis C exhibited long-lasting antiviral activities during IFN therapy as well. The antiviral effect of EVs from Caucasian and Japanese patients differed, which may be explained by different nutritional uptake of PUFAs. In conclusion, our data indicate that macrophage-derived EVs mediate long-lasting inhibitory effects on HCV replication, which may bridge the time until efficient adaptive immune responses are established, and which can be blunted by PUFAs.

细胞外囊泡（Extracellular vesicles, EVs）如今愈发被认为是细胞间通讯的重要介导因子。本研究旨在进一步阐明巨噬细胞来源的细胞外囊泡在抗丙型肝炎病毒（hepatitis C virus, HCV）固有免疫应答中的作用，以及多不饱和脂肪酸（polyunsaturated fatty acids, PUFAs）调控这类固有免疫模式的潜力。为此，我们从干扰素刺激的巨噬细胞培养物，或急性、慢性丙型肝炎患者的血清中分离得到细胞外囊泡。通过电子显微镜、流式细胞术（flow cytometry）、RNA测序（RNA-sequencing）及蛋白质印迹法（Western blot）对所获细胞外囊泡进行表征。在共培养模型中评估细胞外囊泡对丙型肝炎病毒复制的影响，并开展功能分析以探究多不饱和脂肪酸对细胞外囊泡介导的抗病毒免疫的调控作用。研究发现，巨噬细胞在经I型和II型干扰素刺激后会迅速分泌多种细胞因子，从而构建起快速但持续时间较短的抗病毒状态。这一快速固有免疫应答之后，巨噬细胞会分泌细胞外囊泡，这类囊泡可对丙型肝炎病毒复制产生延迟但持久的抑制效应。值得注意的是，作为重要免疫调控因子的多不饱和脂肪酸，可削弱细胞外囊泡介导的抗病毒免疫应答。此外，丙型肝炎患者来源的细胞外囊泡在干扰素治疗期间同样展现出持久的抗病毒活性。高加索人群与日本患者来源的细胞外囊泡所发挥的抗病毒效应存在差异，这或许可通过不同人群对多不饱和脂肪酸的营养摄入差异加以解释。综上，本研究数据表明，巨噬细胞来源的细胞外囊泡可对丙型肝炎病毒复制产生持久的抑制效应，这类囊泡或可填补高效适应性免疫应答建立前的免疫空白，且其功能可被多不饱和脂肪酸所抑制。