MOE Key Laboratory of Tumor Molecular Biology and Key Laboratory of Functional Protein Research of Guangdong Higher Education Institutes, Institute of Life and Health Engineering, College of Life Science and Technology, Jinan University, Guangzhou, 510632, China.
Department of Nephrology, Affiliated Huadu Hospital, Southern Medical University (People's Hospital of Huadu District), Guangzhou, China.
Inflamm Res. 2023 Aug;72(8):1567-1581. doi: 10.1007/s00011-023-01765-5. Epub 2023 Jul 13.
Intercellular communication between macrophages and peritoneal mesothelial cells (PMCs) has been suggested as a key factor regulating peritonitis development. Here, we explored whether PPARγ (peroxisome proliferator-activated receptor gamma) can be packaged into macrophage exosomes to mediate intercellular communication and regulate peritonitis.
Macrophage exosomes were isolated by ultracentrifugation and identified by nanoparticle tracking analysis and transmission electron microscopy. Proteomic analysis of macrophage-derived exosomes was performed using mass spectrometry. Co-culture models of supernatants or exosomes with PMCs, as well as a mouse peritonitis model induced by lipopolysaccharide (LPS), were employed.
In this study, using stable Raw264.7 cells overexpressing GFP-FLAG-PPARγ (OE-PPARγ), we found that PPARγ inhibited LPS-induced inflammatory responses in Raw264.7 cells and that PPARγ was incorporated into macrophage exosomes during this process. Overexpression of PPARγ mainly regulated the secretion of differentially expressed exosomal proteins involved in the biological processes of protein transport, lipid metabolic process, cell cycle, apoptotic process, DNA damage stimulus, as well as the KEGG pathway of salmonella infection. Using co-culture models and mouse peritonitis model, we showed that exosomes from Raw264.7 cells overexpressing PPARγ inhibited LPS-induced inflammation in co-cultured human PMCs and in mice through downregulating CD14 and TLR4, two key regulators of the salmonella infection pathway. Pretreatment of the PPARγ inhibitor GW9662 abolished the anti-inflammatory effect of exosomes from Raw264.7 OE-PPARγ cells on human PMCs.
These results suggested that overexpression of PPARγ largely altered the proteomic profile of macrophage exosomes and that exosomal PPARγ from macrophages acted as a regulator of intercellular communication to suppress LPS-induced inflammatory responses in vitro and in vivo via negatively regulating the CD14/TLR4 axis.
巨噬细胞与腹膜间皮细胞（PMCs）之间的细胞间通讯被认为是调节腹膜炎发展的关键因素。在这里，我们探讨了过氧化物酶体增殖物激活受体γ（PPARγ）是否可以被包装到巨噬细胞外泌体中，以介导细胞间通讯并调节腹膜炎。
通过超速离心分离巨噬细胞外泌体，并通过纳米颗粒跟踪分析和透射电子显微镜进行鉴定。使用质谱法对巨噬细胞衍生的外泌体进行蛋白质组学分析。使用上清液或外泌体与 PMCs 的共培养模型以及脂多糖（LPS）诱导的小鼠腹膜炎模型进行实验。
在这项研究中，我们使用稳定过表达 GFP-FLAG-PPARγ（OE-PPARγ）的 Raw264.7 细胞，发现 PPARγ 抑制了 LPS 诱导的 Raw264.7 细胞中的炎症反应，并且在此过程中 PPARγ 被包裹到巨噬细胞外泌体中。PPARγ 的过表达主要调节了参与蛋白质运输、脂质代谢过程、细胞周期、凋亡过程、DNA 损伤刺激以及沙门氏菌感染的 KEGG 途径的差异表达外泌体蛋白的分泌。通过共培养模型和小鼠腹膜炎模型，我们表明过表达 PPARγ 的 Raw264.7 细胞的外泌体通过下调 CD14 和 TLR4（沙门氏菌感染途径的两个关键调节剂）抑制了共培养的人 PMCs 和小鼠中的 LPS 诱导的炎症。用 PPARγ 抑制剂 GW9662 预处理可消除 Raw264.7 OE-PPARγ 细胞外泌体对人 PMCs 的抗炎作用。
这些结果表明，PPARγ 的过表达极大地改变了巨噬细胞外泌体的蛋白质组图谱，并且巨噬细胞来源的外泌体中的 PPARγ 作为细胞间通讯的调节剂，通过负调控 CD14/TLR4 轴，在体外和体内抑制 LPS 诱导的炎症反应。
