In vitro immunomodulatory effect of Bifidobacterium bifidum and Lactobacillus reuteri cell free extracts
Authors: Knysh O. V. , Pogorila M. S. , Voyda Y. V. 
Published: May 2020

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.