Monocyte culture as a model for in vitro testing of pharmaceutical compositions

This study aimed to investigate the immunotropic effects of a pharmaceutical composition based on a probiotic strain metabolites, and plant components of various origin using an in vitro model of human monocyte culture. The authors propose a mixture of exometabolites from Saccharomyces boulardii (S. boulardii) as the basis of all suggested formulations aimed at 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 donors. Mononuclear cells (MNCs) were isolated by gradient centrifugation. The monocytes were seeded into 24-well plates, co-cultured with the studied compositions (experimental samples) and without compositions (controls). Monoclonal antibodies were used to identify cell markers of 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, whereas the M1 phenotype was assessed using CD80 and CD86 antibodies. It was proven that co-cultivation with St. John’s wort extract induced cultured monocytes to acquire the M1 phenotype, while a significantly increased expression of all surface markers for TLR4, CD80, and CD86 was observed. In contrast, the coriander extract (mixture #1) significantly inhibited the expression of M2 phenotype markers compared with unstimulated cells. The percentage of M2 monocytes (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, i.e. TLR4, CD80, CD86. The reducing effect of M1 marker expression persisted after addition of S. boulardii metabolites only for TLR4 and CD86 compared with the control. A subpopulation of monocytes with TLR4+, CD204+, CD206+, CD163+ phenotype, coexpressing the M1 and M2 polarization markers was detected during co-cultivation with both mixtures #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). Hence, the mixtures under study cause M1/M2 monocyte polarization depending on the composition, thus recommending its potential usage in cosmetology and medical practice, depending on etiology of the disease.