Department of Orthopedics, Trauma and Reconstructive Surgery, RWTH Aachen University Hospital, Pauwelsstraße 30, 52074, Aachen, Germany.
Experimental Orthopaedics and Trauma Surgery, RWTH Aachen University Hospital, Pauwelsstraße 30, 52074, Aachen, Germany.
Eur J Med Res. 2023 Nov 9;28(1):506. doi: 10.1186/s40001-023-01427-6.
Extracellular vesicles (EVs) mediate cell-to-cell communication by horizontally transferring biological materials from host cells to target cells. During exposure to pathogens, pathogen-associated molecular patterns (e.g., lipopolysaccharide, LPS) get in contact with endothelial cells and stimulate the secretion of endothelial cell-derived EVs (E-EVs). The triggered EVs secretion is known to have a modulating influence on the EVs-receiving cells. Macrophages, a major component of innate immunity, are polarized upon receiving external inflammatory stimuli, in which toll-like receptor4 (TLR4)-nuclear factor kappa B (NFκB) pathway plays a key role. However, the functions of LPS-induced E-EVs (E-EVs) in modulating macrophage phenotype and activation remain elusive. We collected the EVs from quiescent endothelial cells (E-EVs) and E-EVs to detect their stimulatory role on NR8383 macrophages. Isolated EVs were characterized by transmission electron microscopy (TEM), western blot assay, and nanoparticle tracking analysis (NTA). NR8383 macrophages were stimulated with ELPS-EVs, ENor-EVs, or PBS for 24 h. Hereafter, the uptake of EVs by the macrophages was investigated. Upon EVs stimulation, cellular viability was determined by MTT assay, while macrophage phenotype was analyzed by flow cytometry and immunofluorescence analysis. Furthermore, a western blot assay was conducted to evaluate the potentially involved TLR4-NFκB pathway. Interestingly, upon exposure to LPS, endothelial cells secreted significantly higher amounts of EVs (i.e., E-EVs) when compared to quiescent cells or cells in PBS. The E-EVs were also better internalized by NR8383 macrophages than E-EVs. The cellular viability of E-EVs-treated macrophages was 1.2 times higher than those in the E-EVs and PBS groups. In addition, E-EVs modulated NR8383 macrophages towards a proinflammatory macrophage M1-like phenotype. This was indicated by the significantly upregulated expressions of proinflammatory macrophage biomarkers CD86 and inducible nitric oxide synthase (iNOS) observed in E-EVs-treated macrophages. The TLR4-NFκB signaling pathway was substantially activated in E-EVs-treated macrophages, indicated by the elevated expressions of makers TLR4 and phosphorylated form of nuclear factor kappa B p65 subunit (p-NFκBp65). Overall, our results indicate that E-EVs play a crucial role in macrophage phenotype modulation under inflammatory conditions.
细胞外囊泡 (EVs) 通过将生物物质从宿主细胞横向转移到靶细胞来介导细胞间通讯。在暴露于病原体时，病原体相关分子模式（例如脂多糖，LPS）与内皮细胞接触并刺激内皮细胞衍生的 EVs（E-EVs）的分泌。已知触发的 EVs 分泌对接收 EVs 的细胞具有调节作用。巨噬细胞是先天免疫的主要组成部分，在接收到外部炎症刺激时会发生极化，其中 Toll 样受体 4 (TLR4)-核因子 kappa B (NFκB) 途径发挥关键作用。然而，LPS 诱导的 E-EVs（E-EVs）在调节巨噬细胞表型和激活中的功能仍不清楚。我们从静止的内皮细胞 (E-EVs) 和 E-EVs 中收集 EVs，以检测它们对 NR8383 巨噬细胞的刺激作用。通过透射电子显微镜 (TEM)、western blot 分析和纳米颗粒跟踪分析 (NTA) 对分离的 EVs 进行了表征。NR8383 巨噬细胞用 LPS-EVs、ENor-EVs 或 PBS 刺激 24 小时。此后，研究了巨噬细胞对 EVs 的摄取。在 EVs 刺激后，通过 MTT 测定法测定细胞活力，通过流式细胞术和免疫荧光分析分析巨噬细胞表型。此外，进行了 western blot 分析以评估潜在涉及的 TLR4-NFκB 途径。有趣的是，与静止细胞或 PBS 中的细胞相比，LPS 暴露后内皮细胞分泌的 EVs（即 E-EVs）明显增多。NR8383 巨噬细胞对 E-EVs 的内化也优于 E-EVs。E-EVs 处理的巨噬细胞的细胞活力比 E-EVs 和 PBS 组高 1.2 倍。此外，E-EVs 将 NR8383 巨噬细胞向促炎巨噬细胞 M1 样表型调节。这是通过 E-EVs 处理的巨噬细胞中观察到的促炎巨噬细胞生物标志物 CD86 和诱导型一氧化氮合酶 (iNOS) 的表达显著上调来表明的。TLR4-NFκB 信号通路在 E-EVs 处理的巨噬细胞中被显著激活，这表明标记物 TLR4 和核因子 kappa B p65 亚单位的磷酸化形式 (p-NFκBp65) 的表达升高。总体而言，我们的结果表明，E-EVs 在炎症条件下的巨噬细胞表型调节中起着至关重要的作用。
