DIFFERENTIAL EXTRACELLULAR VESICLE PRODUCTION BY THP-1 CELLS ACTIVATED AND POLARIZED INTO M1 OR M2 MACROPHAGES

Macrophages are capable of secreting extracellular vesicles (EVs) that exhibit a wide range of biological effects, including modulation of the immune response during pathological conditions. The aim of this study was to compare the qualitative and quantitative composition of EVs produced by the THP-1 cell line, depending on the concentration and duration of activation with phorbol-12-myristate-13-acetate (PMA) and the direction of polarization into M1 or M2 macrophages. Cells were activated with different concentrations of PMA (100 ng/ml and 10 ng/ml). Polarization of cells into M1 macrophages was carried out by treatment with IFN-γ and LPS; into M2 - IL-4 and IL-13. Cells and their EVs were immunophenotyped for CD80, CD64, HLA-DR, CD206, CD209, and CD163. The relative expression of the genes IL-1β, IL-6, IL-8, IL-12p40, TNFα, CXCL10, CD163, CD206, CCL22, IL-10, FN, and GAPDH was evaluated in the cells. The size and concentration of EVs were assessed using nanoparticle tracking analysis (NTA). Additionally, the protein composition of EVs was evaluated for the presence of tetraspanin receptors (CD9, CD63, CD82, and CD81) and flotillin-1 protein. Activation of cells with high doses of PMA followed by polarization towards M1 compared to M2 resulted in increased expression of CD80, CD209, and CD163. Regardless of the activation polarization protocol used, THP-1 cells were distributed into distinct compact clusters based on discriminant analysis of gene expression levels. Cell activation was accompanied by an increase in EV production by more than 10 times. After activation with high doses of PMA, subsequent polarization towards M1 led to the secretion of the largest amount of EVs (188108 (185108; 202.5*108) particles/ml) of a larger size (134 ±6.1 nm) and expressing CD63 and CD82. However, the content of flotillin-1 in them was reduced. Thus, activation of THP-1 cells with high doses of PMA is more effective for subsequent polarization. Depending on the polarization protocol used, cells produce EVs that differ in quantitative and qualitative composition.