Extracellular vesicles derived from endothelial cells modulate macrophage phenotype in vitro

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 (NF kappa B) pathway plays a key role. However, the functions of LPS-induced E-EVs (E-LPS-EVs) in modulating macrophage phenotype and activation remain elusive. We collected the EVs from quiescent endothelial cells (E-Nor-EVs) and E-LPS-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-NF kappa B pathway. Interestingly, upon exposure to LPS, endothelial cells secreted significantly higher amounts of EVs (i.e., E-LPS-EVs) when compared to quiescent cells or cells in PBS. The E-LPS-EVs were also better internalized by NR8383 macrophages than E-Nor-EVs. The cellular viability of E-LPS-EVs-treated macrophages was 1.2 times higher than those in the E-Nor-EVs and PBS groups. In addition, E-LPS-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-LPS-EVs-treated macrophages. The TLR4-NF kappa B signaling pathway was substantially activated in E-LPS-EVs-treated macrophages, indicated by the elevated expressions of makers TLR4 and phosphorylated form of nuclear factor kappa B p65 subunit (p-NF kappa Bp65). Overall, our results indicate that E-EVs play a crucial role in macrophage phenotype modulation under inflammatory conditions.

Automated summary

Extracellular vesicles (EVs) are involved in cell-to-cell communication by transferring biological materials. A study found that pathogen-associated molecular patterns, such as lipopolysaccharide (LPS), can stimulate the secretion of specific types of EVs (E-LPS-EVs) from endothelial cells. These E-LPS-EVs can modulate macrophage phenotype and activation, promoting inflammatory response. This discovery enhances our understanding of the importance of EVs in regulating macrophages under inflammatory conditions.