In response to different stimuli (e.g., infections), naive macrophages polarize into M1 macrophages, which have the potential to secrete numerous pro-inflammatory cytokines and extracellular vesicles (EVs). EVs are important mediators of intercellular communication. Via horizontal transfer, EVs transport various molecules (e.g., proteins, DNA, and RNA) to target cells. This in vitro study elucidated that M1-EVs from macrophages induced by interferon-γ (IFN-γ) and lipopolysaccharide (LPS) 24 h (M1), but not M0-EVs from untreated macrophages (M0), shifted M0 into M1 phenotype via activating the nuclear factor-κB pathway. The characteristics of these EVs were assessed by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and a western blot assay. RAW 264.7 cells were incubated with M1-EVs (experimental group) or PBS (sham group) or M0-EVs (control group) for 24 h. The viability, change of shape, and phenotype differentiation of the macrophages were identified by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometry, and immunofluorescence staining. The TLR4-NFκB pathway of RAW264.7 macrophages was assessed by a western blot assay. M1-EVs but not M0-EVs were incorporated by the RAW264.7 cells and directly induced polarization of RAW264.7 macrophages to M1 macrophages. This polarization was demonstrated by significant upregulation of the M1 macrophage marker CD86 in the experimental group (49.93 ± 5.0%) as compared with that in the control and sham groups (1.22% and 1.46%, respectively) and significant upregulation of iNOS in the experimental group (75 ± 5.0%) as compared with that in the control and sham groups (0%). Furthermore, cell viability was higher (1.3 times) in the experimental group as compared with that in both the sham and control groups. The regulatory mechanism of M1-EVs on RAW 264.7 macrophages polarization and activation was triggered by the activation of the TLR4-NFκB signaling pathway. Based on our observations, we conclude that M1-EVs play an important role in the M1 macrophage auto-polarizing loop. These data clearly demonstrate an important role for macrophage-derived EVs in cellular differentiation. Further studies are needed to elucidate the potential of these EVs in the modulation of inflammatory stimuli.

中文翻译：

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M1但不是M0的细胞外小泡通过TLR4-NFκB途径体外诱导RAW264.7巨噬细胞极化。

响应不同的刺激（例如感染），幼稚的巨噬细胞极化为M1巨噬细胞，这可能分泌多种促炎性细胞因子和细胞外囊泡（EVs）。电动汽车是细胞间通讯的重要媒介。电动汽车通过水平转移将各种分子（例如蛋白质，DNA和RNA）运输到靶细胞。这项体外研究阐明了干扰素-γ（IFN-γ）和脂多糖（LPS）诱导的巨噬细胞在24小时（M1）产生的M1-EVs，而非未经处理的巨噬细胞（M0）产生的M0-EVs通过以下方式将M0转化为M1型：激活核因子-κB途径。这些电动汽车的特性通过透射电子显微镜（TEM），纳米颗粒跟踪分析（NTA）和Western blot分析进行评估。RAW 264。将7个细胞与M1-EVs（实验组）或PBS（假手术组）或M0-EVs（对照组）孵育24小时。通过3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四唑溴化物（MTT）测定，流式细胞术和免疫荧光染色鉴定巨噬细胞的生存力，形状变化和表型分化。RAW264.7巨噬细胞的TLR4-NFκB通路通过蛋白质印迹分析进行评估。RAW264.7细胞掺入了M1-EV，但未掺入M0-EV，并直接将RAW264.7巨噬细胞极化为M1巨噬细胞。与对照组和假手术组（分别为1.22％和1.46％）相比，实验组中的M1巨噬细胞标记CD86显着上调（分别为49.93±5.0％）和iNOS显着上调证明了这种极化组（75±5。与对照组和假手术组（0％）相比）。此外，与假手术组和对照组相比，实验组的细胞活力更高（1.3倍）。M1-EVs对RAW 264.7巨噬细胞极化和激活的调控机制是由TLR4-NFκB信号通路的激活触发的。根据我们的观察，我们得出结论，M1-EV在M1巨噬细胞自极化环路中起重要作用。这些数据清楚地证明了巨噬细胞源电动汽车在细胞分化中的重要作用。需要进一步的研究来阐明这些电动汽车在调节炎症刺激方面的潜力。实验组与假手术组和对照组相比，是实验组的3倍）。M1-EVs对RAW 264.7巨噬细胞极化和激活的调控机制是由TLR4-NFκB信号通路的激活触发的。根据我们的观察，我们得出结论，M1-EV在M1巨噬细胞自极化环路中起重要作用。这些数据清楚地证明了巨噬细胞源电动汽车在细胞分化中的重要作用。需要进一步的研究来阐明这些电动汽车在调节炎症刺激方面的潜力。实验组与假手术组和对照组相比，是实验组的3倍）。M1-EVs对RAW 264.7巨噬细胞极化和激活的调控机制是由TLR4-NFκB信号通路的激活触发的。根据我们的观察，我们得出结论，M1-EV在M1巨噬细胞自极化环路中起重要作用。这些数据清楚地证明了巨噬细胞源电动汽车在细胞分化中的重要作用。需要进一步的研究来阐明这些电动汽车在调节炎症刺激方面的潜力。我们得出的结论是，M1-EV在M1巨噬细胞自极化环路中起重要作用。这些数据清楚地证明了巨噬细胞源电动汽车在细胞分化中的重要作用。需要进一步的研究来阐明这些电动汽车在调节炎症刺激方面的潜力。我们得出的结论是，M1-EV在M1巨噬细胞自极化环路中起重要作用。这些数据清楚地证明了巨噬细胞源电动汽车在细胞分化中的重要作用。需要进一步的研究来阐明这些电动汽车在调节炎症刺激方面的潜力。