Changes in the spectrum of extracellular vesicles produced by THP-1 cells during polarization toward M1 or M2 macrophages

BACKGROUND: Macrophages are capable of secreting extracellular vesicles that exert a wide range of biological effects, including modulation of the immune response under pathological conditions. AIM: The work aimed to compare the qualitative and quantitative composition of extracellular vesicles produced by THP-1 cells depending on the concentration and duration of activation with phorbol 12-myristate 13-acetate and the direction of polarization toward M1 or M2 macrophages. METHODS: THP-1 cells were activated with different concentrations of phorbol 12-myristate 13-acetate (100 and 10 ng/mL). Polarization toward M1 macrophages was induced using IFN-γ and LPS, and toward M2 using IL-4 and IL-13. Cells and their extracellular vesicles were immunophenotyped for CD80, CD64, HLA-DR, CD206, CD209, and CD163. Relative gene expression levels of IL-1β, IL-6, IL-8, IL-12p40, TNFα, CXCL10, CD163, CD206, CCL22, IL-10, FN, and GAPDH were assessed. The size and concentration of extracellular vesicles were measured by nanoparticle tracking analysis. The protein composition of extracellular vesicles was additionally assessed for the presence of tetraspanin receptors (CD9, CD63, CD82, and CD81) and flotillin-1. RESULTS: Activation of cells with high doses of phorbol 12-myristate 13-acetate followed by polarization toward M1, compared to M2, led to increased expression of CD80, CD209, and CD163. Regardless of the applied activation–polarization protocol, THP-1 cells were distributed into distinct, compact clusters according to the results of discriminant analysis of gene expression levels. Activation was accompanied by a more than 10-fold increase in extracellular vesicle production. High-dose phorbol 12-myristate 13-acetate activation followed by M1 polarization resulted in secretion of the highest number of extracellular vesicles (188×108 [185×108; 202.5×108] particles/mL), of larger size (134 ± 6.1 nm), and expressing CD63 and CD82. However, their flotillin-1 content was reduced. CONCLUSION: Thus, high-dose phorbol 12-myristate 13-acetate activation of THP-1 cells is more effective for subsequent polarization. Depending on the applied polarization protocol, cells produce extracellular vesicles differing in both quantity and composition.