Fig. 4
From: Production of biologically active human interleukin-10 by Bifidobacterium bifidum BGN4
Bioassay with Raw 264.7 cells and HT-29 cells treated with 10% (v/v) cell free culture supernatant (CFCS) of BGN4 [pBES2] and BGN4 [pBESIL10] or rhIL-10 protein. rhIL-10 protein is an active recombinant human interleukin-10 protein, 150 ng/mL of which was similar to the 10% (v/v) of hIL-10 secreted by the BGN4[pBESIL10] 10 h cultured group. Viability of Raw 264.7 cells cultured in DMEM media for 24 h without LPS (a) and with 100 ng/mL of LPS (b). The inhibition of nitric oxide (NO) (c). The effects of CFCS of BGN4 [pBESIL10] on IL-6 production in (d) LPS-stimulated RAW 264.7 macrophage cells, IL-8 production in LPS- (e) or TNFα- (f) stimulated on HT-29 cells. Each data point represents the mean + standard deviation (n = 6). a, b Raw 264.7 cells (105/well) were treated with 10% (v/v) CFCS of BGN4 [pBES2], BGN4 [pBESIL10] or rhIL-10 protein. Control: 10% (v/v) MRS; P6, P10: CFCS of BGN4 [pBES2] prepared at 6 h or 10 h of cultivation in buffered MRS; 10P6, 10P10: CFCS of BGN4 [pBESIL10] prepared at 6 h or 10 h of cultivation in buffered MRS; rhIL-10: 1 μg/mL of recombinant hIL-10 protein. There was no statistically significant difference among the treatment groups in both graphs with p < 0.05. c, d Raw 264.7 cells (5 × 105 /well) were cultured in DMEM media for 6 h with 100 ng/mL of LPS and 10% (v/v) CFCS of bacteria. c LPS: LPS 100 ng/mL treated only; P6, P10: CFCS of BGN4 [pBES2] prepared at 6 h or 10 h of cultivation in buffered MRS with LPS 100 ng/mL; 10P6, 10P10: CFCS of BGN4 [pBESIL10] prepared at 6 h or 10 h of cultivation in buffered MRS with LPS 100 ng/mL; rhIL-10: 1 μg/mL of recombinant hIL-10 protein with LPS 100 ng/mL; CON: DMEM media only. Untreated control group exposure data was below the detection limit. For all samples: p < 0.0001 (against LPS-stimulated group). d Control: 10% (v/v) MRS; RAW: Raw 264.7 cells without any treatment; P6: CFCS of BGN4 [pBES2] 6 h cultivation in buffered MRS; 10P6: CFCS of BGN4 [pBESIL10] 6 h cultivation in buffered MRS; rhIL-10: 0.1 μg/mL of recombinant hIL-10 protein. e, f HT-29 cells (5 × 105/well) were incubated in DEMEM culture media for 4 h with 5% (v/v) CFCS of bacteria. Control: 5% (v/v) MRS; HT-29: HT-29 cells without any treatment; P10: CFCS of BGN4 [pBES2] 10 h cultivation in buffered MRS; 10P10: CFCS of BGN4 [pBESIL10] 10 h cultivation in buffered MRS; rhIL-10: recombinant hIL-10 protein 0.1 μg/mL; LPS: LPS 1 μg/mL; TNFα: TNFα 0.5 ng/mL. ####p < 0.0001, the control group versus the untreated group; *p < 0.1, **p < 0.01, ***p < 0.001, ****p < 0.0001, the treated group was significantly different from the control group