Probiotic metabolites from Bacillus coagulans GanedenBC30TM support maturation of antigen-presenting cells in vitro

Figure 1 CD14 expression on mononuclear phagocytes. Mononuclear phagocytes present in 3-d peripheral blood mononuclear cell cultures exposed to either the Ganeden Bacillus coagulans 30 (GBC30) metabolites (MET), cell wall-enriched (CW), or MET fractions, were identified using electronic gating of the flow cytometry data by gating on forward scatter/side scatter followed by gating for CD14 positivity. A comparison was made between cells that were untreated (UT), exposed to lipopolysaccharide (LPS) or to the different GBC30 fractions. A: Comparison of CD14 mean fluorescence intensity showed a dose-dependent increase in CD14 expression in cells treated with crude MET. A milder increase was seen for cells treated with crude CW. The baseline indicates CD14 expression on untreated cells; B: The increase in CD14 expression was primarily caused by high molecular weight compounds present in MET; C: The percent of CD14^bright cells in the mononuclear phagocyte population was decreased by all fractions of MET; D: The percent of CD14^dim cells in the mononuclear phagocyte population was increased by treatment of cells with all MET fractions. Bar graphs show data from 1:200 dilutions of each MET fraction and lipopolysaccharide (1 μg/mL). ^aP < 0.05, ^bP < 0.01 and ^cP < 0.001. For each data point, the mean + SD are shown for each duplicate data set. Graphs show data representative of 1 out of 3 experiments. MFI: Mean fluorescence intensity.

Figure 2 Expression of the co-stimulatory molecules CD80 and CD86 on CD14^bright mononuclear phagocytes from 3-d peripheral blood mononuclear cell cultures. A: Comparison between the effects of serial dilutions of Ganeden Bacillus coagulans 30 (GBC30) crude metabolites (MET) or cell wall enriched (CW) fractions on CD80 expression on CD14^bright cells showed dose-dependent decreases in CD80 expression. Both MET and CW reduced CD80 expression to levels similar to those seen with Lipopolysaccharide (LPS) treatment; B: A comparison of the effects of size-fractionated MET on CD80 expression on CD14^bright cells shows that all MET fractions reduce expression; C: Comparison between the effects of serial dilutions of crude MET or CW on CD86 expression on CD14^bright cells showed dose-dependent increases in CD86 expression when cells were exposed to the three most concentrated dilutions of MET. Treatment of cells with CW resulted in a uniform modest decrease in CD86 expression; D: The effect on increased CD86 expression is present only in the crude preparation of MET. Bar graphs show data from 1:200 dilutions of each MET fraction and lipopolysaccharide (1 μg/mL). ^aP < 0.05, ^bP < 0.01 and ^cP < 0.001. For each data point, the mean + SD are shown for each duplicate data set. Graphs show data representative of 1 out of 3 experiments. MFI: Mean fluorescence intensity; UT: Untreated.

Figure 3 Expression of the co-stimulatory molecules CD80 and CD86 on CD14^dim mononuclear phagocytes from 3-d peripheral blood mononuclear cell cultures. A: Comparison between the effects of serial dilutions of Ganeden Bacillus coagulans 30 (GBC30) crude metabolites (MET) or cell wall enriched (CW) fractions on CD80 expression on CD14^dim cells showed that both MET and CW led to increased expression; B: The increase in CD80 expression following treatment of cells with MET was due to high molecular weight compounds; C: Comparison of CD86 expression on CD14^dim cells exposed to crude fractions of MET or CW resulted in increased CD86 expression; D: Size-selected fractions of MET did not have uniform effects on CD86 expression on CD14^dim cells. Crude MET increased expression while 3-30 kDa and < 3 kDa fractions decreased expression. Bar graphs show data from 1:200 dilutions of each MET fraction and lipopolysaccharide (1 μg/mL). ^aP < 0.05, ^bP < 0.01 and ^cP < 0.001. For each data point, the mean + SD are shown for each duplicate data set. Graphs show data representative of 1 out of 3 experiments. MFI: Mean fluorescence intensity; UT: Untreated; LPS: Lipopolysaccharide.

Figure 4 Changes in the percent of CD14+ CD16+ and CD14+ CD16- cell populations following exposure of 3-d peripheral blood mononuclear cell cultures to Ganeden Bacillus coagulans 30. A: Exposure of cells to serial dilutions of Ganeden Bacillus coagulans 30 (GBC30) crude metabolites (MET) led to a strong dose-dependent decrease in CD14 CD16 double positive cells while exposure to cell wall enriched (CW) did not reduce this cell population; B: Treatment of cells with size-selected MET fractions show that all fractions of MET reduce the number of CD14+ CD16+ cells; C: The percent of CD14+ CD14- cells in peripheral blood mononuclear cell cultures increased in cultures treated with crude MET and CW. Treatment with MET resulted in a very strong dose-dependent increase while CW treatment produced a milder increase at the two highest concentrations; D: Treatment of cells with size-selected MET fractions show that only fractions containing high molecular weight compounds increase the number of CD14+ CD16- cells. Bar graphs show data from 1:200 dilutions of each MET fraction and lipopolysaccharide (1 μg/mL). ^aP < 0.05, ^bP < 0.01 and ^cP < 0.001. For each data point, the mean + SD are shown for each duplicate data set. Graphs show data representative of 1 out of 3 experiments. UT: Untreated; LPS: Lipopolysaccharide.