Production of biologically active human interleukin-10 by Bifidobacterium bifidum BGN4

Abstract

Background: Bifidobacterium spp. are representative probiotics that play an important role in the health of their hosts. Among various Bifidobacterium spp., B. bifidum BGN4 exhibits relatively high cell adhesion to colonic cells and has been reported to have various in vivo and in vitro bio functionalities (e.g., anti-allergic effect, anti-cancer effect, and modulatory effects on immune cells). Interleukin-10 (IL-10) has emerged as a major suppressor of immune response in macrophages and other antigen presenting cells and plays an essential role in the regulation and resolution of inflammation. In this study, recombinant B. bifidum BGN4 [pBESIL10] was developed to deliver human IL-10 effectively to the intestines.

Results: The vector pBESIL10 was constructed by cloning the human IL-10 gene under a gap promoter and signal peptide from Bifidobacterium spp. into the E. coli-Bifidobacterium shuttle vector pBES2. The secreted human IL-10 from B. bifidum BGN4 [pBESIL10] was analyzed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), Western Blotting, and enzyme-linked immunosorbent assay (ELISA). More than 1,473 ± 300 ng/mL (n = 4) of human IL-10 was obtained in the cell free culture supernatant of B. bifidum BGN4 [pBESIL10]. This productivity is significantly higher than other previously reported human IL-10 level from food grade bacteria. In vitro functional evaluation of the cell free culture supernatant of B. bifidum BGN4 [pBESIL10] revealed significantly inhibited interleukin-6 (IL-6) production in lipopolysaccharide (LPS)-induced Raw 264.7 cells (n = 6, p < 0.0001) and interleukin-8 (IL-8) production in LPS-induced HT-29 cells (n = 6, p < 0.01) or TNFα-induced HT-29 cells (n = 6, p < 0.001).

Conclusion: B. bifidum BGN4 [pBESIL10] efficiently produces and secretes significant amounts of biologically active human IL-10. The human IL-10 production level in this study is the highest of all human IL-10 production reported to date. Further research should be pursued to evaluate B. bifidum BGN4 [pBESIL10] producing IL-10 as a treatment for various inflammation-related diseases, including inflammatory bowel disease, rheumatoid arthritis, allergic asthma, and cancer immunotherapy.

Keywords: Bifidobacterium bifidum; Bioactive; Expression vector; Human interleukin-10; Recombinant; Secretion.

Fig. 1

Alignment of nucleotide sequences of the original and codon-optimized hIL-10 gene (* is the modified sequence from the original hIL-10 gene during codon-optimization)

Fig. 2

a The structure of pBESIL10. b Restriction assay of pBES2 and pBESIL10. pBESIL10 is constructed with Pgap (gap promoter), the signal peptide of gene bbif_1761, hIL-10 (human Interleukin-10 ORF gene) optimized for Bifidobacterium bifidum, and terminator into pBES2. CmR and AmpR are chloramphenicol and ampicillin resistance genes; Ori is the origin of replication from pUC19; Mob and Rep are plasmid–encoded protein of MG1 from Bifidobacterium longum MG1 [38]. b Lane 1: 1 kb DNA ladder; Lane 2, 3, 4, 5: Xba1–Acc65I restriction of pBES2 shows one fragment (7.5 kb), as the other fragment is too small; Lane 6: Xba1–Acc65I restriction of pBESIL10 shows a 0.9 kb fragment (Insert fragment with Pgap, signal peptide, hIL-10 (optimized) and terminator connected, white arrow) with cut pBES2 (7.5 kb)

Fig. 3

Bacterial growth and hIL-10 production over time. a Culturing recombinant BGN4 graph at OD_600nm. b Quantification of hIL-10 produced by BGN4 [pBESIL10] Cell Free Culture Supernatant (CFCS) during culture in pH-controlled MRS medium by sandwich ELISA. (c) Western blot detection of human IL-10 protein. a The 120 mL of cultured recombinant bacteria were centrifuged and inoculated into 1.2 L of a fresh MRS broth (pH 6.5) at 0 h each. BGN4 cells were cultured until reaching a pH of 6.0, and a diluted ammonia solution was added to maintain a pH of 6.8 during the bacteria culture. b The pH was maintained at 6.8 during cultivation and the negative control BGN4 [pBES2] CFCS did not show detectable hIL-10 at the same condition. All data are shown as the mean ± standard deviation (n = 4). The amount of hIL-10 were: (2 h) 128 ± 46 ng/mL, (3 h) 189 ± 54 ng/mL, (4 h) 327 ± 20 ng/mL, (5 h) 554 ± 42 ng/mL, (6 h) 956 ± 129 ng/mL, (7 h) 1,124 ± 170 ng/mL, (8 h) 1,171 ± 114 ng/mL, (9 h) 1,416 ± 300 ng/mL and (10 h) 1,473 ± 300 ng/mL, respectively. (c) All CFCS were concentrated 40-fold by different precipitation methods. Lanes 1, 3, 5: BGN4 [pBES2] control strain grown in buffered MRS medium (pH 6.8); Lanes 2, 4, 6, 7: BGN4 [pBESIL10] strain grown in buffered MRS medium (pH 6.8); Lane C: positive control (recombinant hIL-10 protein (1 μg)); Lanes 1, 2: ethanol precipitation; Lanes 3, 4: acetone precipitation; Lanes 5, 7: chlorophenol methanol precipitation; Lane 6: TCA precipitation (other supernatant precipitation data and cell pellet data are not shown)

Fig. 4

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