细胞外囊泡(EV)通过将生物材料从宿主细胞水平转移到靶细胞来介导细胞与细胞的通讯。在接触病原体期间,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EV(E-EV)的分泌。已知触发的EV分泌对EV接收细胞具有调节作用。巨噬细胞,先天免疫的主要组成部分,在接受外部炎症刺激时极化,其中Toll样受体4(TLR4)-核因子κB(NFκB)通路起关键作用。然而,LPS诱导的E-EV(ELPS-EV)在调节巨噬细胞表型和激活方面的功能仍然难以捉摸。我们从静止内皮细胞(ENor-EV)和ELPS-EV中收集EV,以检测它们对NR8383巨噬细胞的刺激作用。分离的电动汽车通过透射电子显微镜(TEM)进行表征,westernblot检测,和纳米粒子跟踪分析(NTA)。用ELPS-EV刺激NR8383巨噬细胞,ENOR-EV,或PBS24小时。此后,研究了巨噬细胞对EV的摄取。在电动汽车刺激时,细胞活力通过MTT测定,而巨噬细胞表型通过流式细胞术和免疫荧光分析进行分析。此外,进行了蛋白质印迹分析以评估可能涉及的TLR4-NFκB通路.有趣的是,暴露于LPS后,内皮细胞分泌的电动汽车量明显更高(即,ELPS-EV)与静止细胞或PBS中的细胞相比。ELPS-EV也比ENor-EV更好地被NR8383巨噬细胞内化。ELPS-EV处理的巨噬细胞的细胞活力比ENor-EV和PBS组高1.2倍。此外,ELPS-EV调节NR8383巨噬细胞向促炎巨噬细胞M1样表型。这通过在ELPS-EV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)的表达显着上调来表明。TLR4-NFκB信号通路在ELPS-EVs处理的巨噬细胞中基本上被激活,标记TLR4和磷酸化形式的核因子κBp65亚基(p-NFκBp65)的表达升高。总的来说,我们的结果表明,在炎症条件下,E-EV在巨噬细胞表型调节中起着至关重要的作用.
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 (ELPS-EVs) in modulating macrophage phenotype and activation remain elusive. We collected the EVs from quiescent endothelial cells (ENor-EVs) and ELPS-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., ELPS-EVs) when compared to quiescent cells or cells in PBS. The ELPS-EVs were also better internalized by NR8383 macrophages than ENor-EVs. The cellular viability of ELPS-EVs-treated macrophages was 1.2 times higher than those in the ENor-EVs and PBS groups. In addition, ELPS-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 ELPS-EVs-treated macrophages. The TLR4-NFκB signaling pathway was substantially activated in ELPS-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.