CYTOKINOGENESIS BY TLR-INDUCED ACTIVATION OF PERIPHERAL BLOOD MONOCYTES IN PATIENTS WITH MULTIPLE SCLEROSIS

Tupotilov O. V., Kolyada T. I.

CLINICAL AND EXPERIMENTAL MEDICINE

Scentific article

Peripheral monocytes play an important role in all stages of the development of multiple sclerosis (MS), in particular they are involved in the formation of a pro-inflammatory context on both sides of the bloodbrain barrier, and also have a wide range of immunoregulatory functions. Analysis of the ability of peripheral blood monocytes to activate when stimulated by TLR4 and TLR7/8, including the features of cytokinogenesis, is important for characterizing changes in the functional state of these cells, understanding their pathogenetic role in RRMS and progressive types (PMS) of disease. 48 patients with MS and 27 practically healthy persons (control group) were examined in this study. The in vitro experiment included the isolation of peripheral blood monocytes and their cultivation in three parallel series: intact cells; with the addition of the TLR4 stimulator; with the addition of the TLR7/8 stimulator. Peripheral blood monocytes were isolated on a double percoll gradient and resuspended with a complete RPMI medium supplemented with 1 μg/ml LPS E. Coli or ssRNA40/LyoVec (Invivogen, USA) until the final cell concentration in the wells of the plate reached 1×105 cell/ml and the final volume reached 0.2 ml, and then were incubated in 96-well plates for 24 hours at t=37 °C under 5% CO2 atmosphere. The assay of TNF-α, IL-1β, IL-1RA, IL-6, IL-10, IL-12 in culture supernatants was performed by ELISA. For each experimental series, the IL-1RA/IL-1β ratio was calculated. For the LPS and ssRNA40/LyoVec series, the reserve capacity index (RCi) of monocytes was calculated as the ratio of the production of each of the cytokines in the series with the addition of the appropriate stimulator and the series with intact cells. Results. It was found a decrease in the ratio of spontaneous production of IL-1RA and IL-1β and an increased RCi for LPS- and ssRNA-induced production of IL-6 against a background of decreased RCi for production of TNF-α, IL-1β and IL-10 in comparison with healthy persons (p<0.05). The RCi for ssRNA40/LyoVec-induced production of IL-6 and IL-10 was lower, and IL-1RA was higher compared to stimulation with LPS. Peripheral monocytes of patients with relapsing-remitting MS were also characterized by a decreased spontaneous production of IL-12p70 and an increased RCi for LPS- and ssRNA40/LyoVec-induced production of this cytokine. In patients with progressive type of disease, a high level of spontaneous production of IL-12p70 and IL-1RA was found against a background of reduced RCi for stimulated production of IL-12p70. By TLR4-stimulation it was found an increase in level of IL-6, a decrease in RCi for production of IL-1RA and a decrease in a ratio of induced production of IL-1RA and IL-1β, and by TLR7/8 stimulation – an increase in the level of IL-1β (p <0.05). Conclusions. The obtained results demonstrate differences in the state of cytokinogenesis when stimulated by TLR4 and TLR7/8 and may indicate the presence of functional and phenotypic alterations of peripheral blood monocytes, depending on the type of course of the disease.

multiple sclerosis, monocytes, cytokines, Toll-like receptors

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«Bulletin of problems biology and medicine» Issue 3 (145), 2018 year, 181-187 pages, index UDK 612.017.11:616.832-004.2

10.29254/2077-4214-2018-3-1-145-181-187