Recent studies have shown that alterations of the immune response in the gastrointestinal mucosa are key components of the mechanism of the probiotic action of beneficial bacteria. Most of the beneficial effects of probiotics are due to the action of their structural components and metabolites. Macrophages are first-line defense cells of the immune system, which not only participate in the detection, phagocytosis and destruction of harmful microorganisms, but also determine the nature of the subsequent immune response by presenting antigens to T-cells and initiating inflammation by releasing cytokines. We researched the effect of two types of cell-free extracts (CFEs) containing probiotic derivatives (structural components and metabolites of bacteria) Bifidobacterium bifidum 1 (BbCFE) and Lactobacillus reuteri DSM 17938 (LrCFE) on the activity of mouse peritoneal macrophages and on the ability of peripheral human blood mononuclear cells to produce cytokines. CFEs were obtained by culturing probiotics in their own disintegrates and then removing cells and cell debris by centrifugation and filtration. Peritoneal macrophages were isolated from mice. Some of them were infected in vitro by Salmonella thyphimurium. Uninfected and infected macrophages were incubated in culture medium containing (30% vol) or not containing CFEs at 37 °С in a microaerobic atmosphere (5% СО2) for 18 hours. After incubation, peritoneal macrophages were lysed. The obtained suspensions were centrifuged and supernatants were carefully collected. Macrophages activity was assessed by the nitrites level, superoxide dismutase (SOD), lactate dehydrogenase (LDH) activity and antiinflammatory cytokines levels in supernatants using spectrophotometric method. Peripheral mononuclear cells were isolated from the blood of healthy volunteers. The ability of peripheral mononuclear blood cells to produce antiinflammatory cytokines was evaluated after cell stimulation with lipopolysaccharide (LPS) and incubation with or without CFEs. Cytokine levels in supernatants were determined using enzyme-linked immunosorbent assay (ELISA). After infection with S. thyphimurium in macrophages, nitrite levels increased 5.5-fold, SOD activity 4.8-fold, and LDH 2-fold. Both studied CFEs exerted a similar effect on the macrophages’ activity. Addition of BbCFE to the incubation medium of infected macrophages resulted in a 4-fold decrease in nitrite levels, and the addition of LrCFE was accompanied by a decrease in nitrite levels to levels in intact cells. Under the influence of both CFEs, the activity of SOD and LDH was significantly reduced and did not differ significantly from the activity of these enzymes in intact cells. BbCFE and LrCFE did not have a significant effect on nitrite levels, SOD and LDH activity in intact macrophages. Under the influence of BbCFE, there was a 2-fold decrease in the production of TNF, a 2-fold increase in IL10 production, and a 30% increase in IL6 production by mononuclear cells. LrCFE caused a decrease in TNF production by 26.7% and IL6 by 36%, and IL10 by 1.9 times. Thus, the studied CFEs normalized the nitrite levels in peritoneal macrophages infected with S. thyphymurium and infection-induced activation of SOD and LDH enzymes. This demonstrates their ability to modulate oxidative processes in macrophages. In addition, under the influence of the investigated CFEs, there was a decrease in the production of pro-inflammatory cytokines (TNFα and IL-6) and increased production of anti-inflammatory cytokine (IL-10) by human peripheral mononuclear cells. The results of the study indicate the ability of CFEs by influencing the functions of innate immunity cells to restrict the inflammatory response and oxidative stress. Based on this, CFEs can be considered as promising agents for the treatment of inflammatory diseases.
Structural components of cells and metabolites of probiotics with biologically active potential, along with the study of effectiveness, require a series of tests to ensure their safety. The study aims to test the cytotoxicity and potential of structural and metabolic derivatives of Bifidobacterium bifidum and Lactobacillus reuteri to affect the immunocompetent cells using in vitro tests that characterize the metabolic activity of test-cells. Structural components of probiotic bacteria were obtained by the physical method of disintegration – cyclic freezing-thawing. Metabolic derivatives were obtained by cultivation of producers – bifidobacteria and lactobacilli in their own disintegrates. Cultures of mouse embryonic fibroblasts and splenocytes were used as the test cells. MTT and Alamar Blue® were used as redox indicators. According to the MTT test, filtrates that contain structural and metabolic derivates at a concentration of 5% and 10% in the incubation medium did not cause significant changes in the metabolic activity of the embryonic mouse fibroblasts. An increase of up to 20% of content in the incubation medium of filtrates of lactobacilli disintegrates led to a reduction of metabolic activity of test cells by 52.7 ± 6.2%, of filtrates of bifidobacteria disintegrates – by 26.5 ± 6.5%, of filtrates of lactobacterium culture – by 15.7 ± 6.9%, of filtrates of bifidobacterium cultures - by 40.4 ± 6.8%. According to the Alamar Blue® test, filtrates that contained only structural derivatives of lactobacilli and bifidobacteria at concentrations of 5% and 10%, as well as filtrates that contained a complex of structural and metabolic derivatives at a concentration of 5%, did not cause significant changes in the reducing ability of mouse splenocytes. At concentrations of 10%, filtrate containing a complex of structural and metabolic derivatives of lactobacilli, caused the inhibition of metabolic activity of splenocytes by 14.6 ± 3.5%, and bifidobacteria – by 10.0 ± 2.8%. With the contents of the incubation medium at 20% concentration, the filtrate of the disintegrates of lactobacilli decreased the metabolic activity of splenocytes by 12.2 ± 3.0%, and the filtrate of lactobacillus cultures that were grown on their own disintegrates – by 43.2 ± 3.3%. Increasing the content of the disintegrate filtrate and the bifidobacteria culture that were grown on their own disintegrates in the culture medium by up to 20% led to a decrease the metabolic activity of splenocytes by 38.0 ± 2.0%. Thus, the research has shown: the orientation of changes in cellular metabolism under the influence of the studied biologically active derivatives is similar in all model systems, and their intensity depends on the type of test cells, regenerative substrates and concentration of the agent of influence in model systems. The obtained results stimulate further exploration of the immunotropicity of the investigated derivatives of probiotic bacteria and can be used for development of new immunobiological preparations.
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