MONOCYTE CULTURE AS A MODEL FOR IN VITRO TESTING OF PHARMACEUTICAL COMPOSITIONS IN VITRO

Abstract

This study aimed to investigate the immunotropic effects of a pharmaceutical composition based on metabolites of a probiotic strain and plant components of various origins on human monocytes in vitro. The authors proposed using exometabolites of S. boulardii as the basis of all proposed formulations for the regeneration of human epithelial cells. The compositions included St. John's wort extract and coriander oil. The effectiveness of these pharmaceutical compositions was evaluated using monocytes isolated from adult humans. Mononuclear cells (MNCs) were isolated by gradient centrifugation. Monocytes were seeded into 24-well plates, co-cultured with the studied compositions (experiment) and without compositions (control). Monoclonal antibodies were used to identify the surface markers of the monocyte/macrophage lineage. Phenotypic analysis of monocytes was performed using a CytoFLEX flow cytometer. The M2 phenotype was characterized using CD204, CD163, and CD206 antibodies, while the M1 phenotype was assessed using CD80 and CD86 antibodies. It was experimentally proved that co-cultivation with St. John's wort extract induced cultured monocytes to acquire the M1 phenotype, while a significant increase in the expression of all surface markers for TLR4, CD80, and CD86 was observed. In contrast, coriander extract significantly suppressed the expression of M2 phenotype markers compared with unstimulated cells. The percentage of M2 monocytes of the CD204+, CD206+, and CD163+ phenotypes was increased after co-cultivation with both oil and after co-cultivation with S. boulardii metabolites for all the studied markers. Exposure to coriander oil and mixture No. 1 significantly reduced the gene expression of all M1 markers: TLR4, CD80, CD86. The lowering effect of M1 marker expression persisted after the addition of S. boulardii metabolites only for TLR4 and CD86 compared with the control. A subpopulation of monocytes of the TLR4+, CD204+, CD206+, CD163+ phenotype, coexpressing the M1 and M2 polarization markers was detected during co-cultivation with mixtures of both 2 and 3. The percentage of hybrid TLR4 +M2 monocytes was significantly higher in mixture 2 compared with mixture 3 (p<0.001 and p<0.05, respectively). Thus, the various mixtures studied cause polarization of monocytes in M1/M2, depending on the composition of the composition, which will allow it to be recommended for use in cosmetology and potentially medical practice, depending on the etiology of the disease.