Guangdong Provincial Key Laboratory of Pharmaceutical Bioactive Substances, Guangdong Pharmaceutical University, Guangzhou, PR China.
Guangzhou Chest hospital, Guangzhou, PR China.
Acta Trop. 2023 Mar;239:106815. doi: 10.1016/j.actatropica.2023.106815. Epub 2023 Jan 3.
Liver injury is a common clinical feature of Plasmodium spp. infection and contributes to multi-organ failure of severe malaria. Malaria-derived exosomes (MD-Exos) have recently engaged as key mediators in parasite-host interactions, modulating the subsequent pathogenic process. However, the role of MD-Exos in malaria-related liver injury and the underlying mechanisms remain unclear. Herein, exosomes from C57BL/6 mice infected with or without P. berghei ANKA serum (namely inf-Exos or un-Exos) were isolated and characterized by transmission electron microscopy, western blotting, and nanoparticle tracking analysis. The miRNAs profiling between inf-Exos and un-Exos were generated using RNA-seq and qPCR. The functions of inf-Exos on liver injury were investigated after two types of exosomes injected into mice intravenously (i.v.), by examining histopathological and apoptotic changes, macrophage polarization, and pro-inflammatory response. The infected red blood cells-stimulated mouse Raw264.7 macrophage cells targeted by inf-Exos or un-Exos were cultured for further study and verification the potential mechanisms. We found that both inf-Exos and un-Exos displayed a typical cup-shaped structure with a diameter of 60-200 nm, and had a positive expression of exosomal markers (e.g., CD9, CD63, and CD81). Compared with infected control mice, the treatment of inf-Exos but not un-Exos dramatically enhanced peripheral blood parasitemia and ECM incidence, exacerbated liver histopathological damage, elevated numbers of liver apoptotic cells, CD68and CD86 macrophages. The CD68-TREM-1 macrophages in liver tissues and the mRNA levels of pro-inflammatory cytokines (e.g., iNOS, TNF-α, IL-1β, and IL-6) were increased by inf-Exos treatment in vivo. Meanwhile, the treatment of inf-Exos resulted in a substantial increase of the mRNA levels of CD86, iNOS, TNF-α, IL-1β, and IL-6, but led to a remarkable decrease of Bcl-6 and SOCS-1 in Raw264.7 cells stimulated with iRBC in vitro. Notably, compared to un-Exos, five types of miRNAs (including miR-10a-5p, miR-10b-5p, miR-155-5p, miR-205-5p, and miR-21a-5p), that were previously reported to target Bcl-6 or SOCS-1, present higher abundance on inf-Exos, as demonstrated by RNA-seq and qPCR. Collectively, our data suggest that inf-Exos exacerbate malaria-induced liver pathology via triggering excessive pro-inflammatory response and promoting macrophage M1 polarization. Our findings will provide new insights into the roles of inf-Exos in malaria parasite-host interaction and pathogenesis of liver injury.
肝损伤是疟原虫感染的常见临床特征，并导致严重疟疾的多器官衰竭。疟原虫来源的外泌体（MD-Exos）最近被认为是寄生虫-宿主相互作用中的关键介质，调节随后的致病过程。然而，MD-Exos 在疟疾相关肝损伤中的作用及其潜在机制尚不清楚。在此，通过透射电子显微镜、western blot 和纳米颗粒跟踪分析，从感染或未感染 P. berghei ANKA 血清的 C57BL/6 小鼠中分离并表征外泌体（分别命名为 inf-Exos 或 un-Exos）。使用 RNA-seq 和 qPCR 生成 inf-Exos 和 un-Exos 之间的 miRNA 图谱。通过检查组织病理学和细胞凋亡变化、巨噬细胞极化和促炎反应，研究两种外泌体静脉注射（i.v.）进入小鼠后 inf-Exos 对肝损伤的作用。培养受感染的红细胞刺激的 inf-Exos 或 un-Exos 靶向的 Raw264.7 巨噬细胞细胞以进一步研究和验证潜在机制。我们发现，与感染对照小鼠相比，inf-Exos 而非 un-Exos 处理显著增强外周血疟原虫血症和 ECM 发生率，加重肝组织病理学损伤，增加肝细胞凋亡细胞数量，CD68 和 CD86 巨噬细胞。体内 inf-Exos 处理增加了肝脏组织中 CD68-TREM-1 巨噬细胞和促炎细胞因子（如 iNOS、TNF-α、IL-1β 和 IL-6）的 mRNA 水平。同时，体外用 iRBC 刺激 Raw264.7 细胞后，inf-Exos 处理导致 CD86、iNOS、TNF-α、IL-1β 和 IL-6 的 mRNA 水平显著增加，但导致 Bcl-6 和 SOCS-1 的 mRNA 水平显著降低。值得注意的是，与 un-Exos 相比，五种 miRNA（包括 miR-10a-5p、miR-10b-5p、miR-155-5p、miR-205-5p 和 miR-21a-5p）的丰度更高，这些 miRNA 先前被报道可靶向 Bcl-6 或 SOCS-1，这通过 RNA-seq 和 qPCR 得到证实。总之，我们的数据表明 inf-Exos 通过触发过度的促炎反应和促进巨噬细胞 M1 极化来加剧疟疾引起的肝病理。我们的发现将为 inf-Exos 在寄生虫-宿主相互作用和肝损伤发病机制中的作用提供新的见解。
