Bifidobacterium breve MCC-117 Induces Tolerance in Porcine Intestinal Epithelial Cells: Study of the Mechanisms Involved in the Immunoregulatory Effect

Abstract

Bifidobacterium breve MCC-117 is able to significantly reduce the expression of inflammatory cytokines in porcine intestinal epithelial (PIE) cells and to improve IL-10 levels in CD4(+)CD25(high) Foxp3(+) lymphocytes in response to heat-stable enterotoxigenic Escherichia coli (ETEC) pathogen-associated molecular patterns (PAMPs), while the immunoregulatory effect of B. adolescentis ATCC15705 was significantly lower than that observed for the MCC-117 strain. Considering the different capacities of the two bifidobacterium strains to activate toll-like receptor (TLR)-2 and their differential immunoregulatory activities in PIE and immune cells, we hypothesized that comparative studies with both strains could provide important information regarding the molecular mechanism(s) involved in the anti-inflammatory activity of bifidobacteria. In this work, we demonstrated that the anti-inflammatory effect of B. breve MCC-117 was achieved by a complex interaction of multiple negative regulators of TLRs as well as inhibition of multiple signaling pathways. We showed that B. breve MCC-117 reduced heat-stable ETEC PAMP-induced NF-κB, p38 MAPK and PI3 K activation and expression of pro-inflammatory cytokines in PIE cells. In addition, we demonstrated that B. breve MCC-117 may activate TLR2 synergistically and cooperatively with one or more other pattern recognition receptors (PRRs), and that interactions may result in a coordinated sum of signals that induce the upregulation of A20, Bcl-3, Tollip and SIGIRR. Upregulation of these negative regulators could have an important physiological impact on maintaining or reestablishing homeostatic TLR signals in PIE cells. Therefore, in the present study, we gained insight into the molecular mechanisms involved in the immunoregulatory effect of B. breve MCC-117.

Keywords: Toll-like receptor 2; Toll-like receptors negative regulators; anti-inflammatory activity; bifidobacteria; porcine intestinal epithelial cells.

Fig. 1.

Expression of cytokines in porcine intestinal epithelial (PIE) cells after stimulation with heat-stable Enterotoxigenic Escherichia coli (ETEC) pathogen-associated molecular patterns (PAMPs). PIE cells were pretreated with Bifidobacterium breve MCC-117 or Bifidobacterium adolescentis ATCC15705 for 48 hours and, stimulated with heat-stable ETEC PAMP and then the expression of MCP-1, IL-6 and IL-8 was studied at hour 12 post stimulation. The results represent four independent experiments. Significantly different from control: *p<0.05; **p<0.01.

Fig. 2.

Western blot analysis of activation of NF-kB and IP3 K pathways in porcine intestinal epithelial (PIE) cells after challenge with heat-stable Enterotoxigenic Escherichia coli (ETEC) pathogen-associated molecular patterns (PAMPs). PIE cells were pretreated with Bifidobacterium breve MCC-117 or Bifidobacterium adolescentis ATCC15705 for 48 hours and, stimulated with heat-stable ETEC PAMPs. Levels of the counter-regulatory factor IκBα and phosphorylation of IP3 K were studied at the indicated times post stimulation. For the evaluation of IkBa degradation, the expressions were compared with the values obtained at time 0 that were set as 1. For the phosphorylation of PI3 K, the phosphorylated protein/total protein ratio was calculated, and the differences were compared with the values obtained at time 0 that were set as 1. Significantly different from the control at the same time point: *p<0.05; **p<0.01.

Fig. 3.

Western blot analysis of the activation of p38, JNK and ERK mitogen-activated protein kinases in porcine intestinal epithelial (PIE) cells after challenge heat-stable Enterotoxigenic Escherichia coli (ETEC) pathogen-associated molecular patterns (PAMPs). PIE cells were pretreated with Bifidobacterium breve MCC-117 or Bifidobacterium adolescentis ATCC15705 for 48 hours and, stimulated with heat-stable ETEC PAMPs. Phosphorylation of p38, JNK and ERK was studied at the indicated times post stimulation. For the phosphorylation of p38, JNK and ERK, each phosphorylated protein/total protein ratio was calculated, and the differences were compared with the values obtained at time 0 that were set as 1. Significantly different from the control at the same time point: *p<0.05; **p<0.01.

Fig. 4.

Expression of toll-like receptor negative regulators in porcine intestinal epithelial (PIE) cells. PIE cells were pretreated with Bifidobacterium breve MCC-117 or Bifidobacterium adolescentis ATCC15705 for 48 hours and, stimulated with heat-stable ETEC PAMPs. Then, the expression of IRAK-M, SIGIRR, Bcl-3, Tollip and A20 negative regulators was studied at the indicated time points. The results represent four independent experiments. Significantly different from control at the same time point: *p<0.05; **p<0.01.

Fig. 5.

Role of TLR2 in the immunomodulatory effect of Bifidobacterium breve MCC-117 in porcine intestinal epithelial (PIE) cells. PIE cells were pretreated with Bifidobacterium breve MCC-117 or Bifidobacterium adolescentis ATCC15705 plus anti-TLR2 antibodies. Untreated adherent cells were used as controls. PIE cells were stimulated with heat-stable Enterotoxigenic Escherichia coli (ETEC) pathogen-associated molecular patterns (PAMPs), and then the expression of MCP-1, IL-6 and IL-8 was studied at hour 12 post stimulation. The results represent four independent experiments. Significantly different from the anti-TLR2 untreated group: *p<0.05.