Immunoregulatory effect of bifidobacteria strains in porcine intestinal epithelial cells through modulation of ubiquitin-editing enzyme A20 expression

Abstract

Background: We previously showed that evaluation of anti-inflammatory activities of lactic acid bacteria in porcine intestinal epithelial (PIE) cells is useful for selecting potentially immunobiotic strains.

Objective: The aims of the present study were: i) to select potentially immunomodulatory bifidobacteria that beneficially modulate the Toll-like receptor (TLR)-4-triggered inflammatory response in PIE cells and; ii) to gain insight into the molecular mechanisms involved in the anti-inflammatory effect of immunobiotics by evaluating the role of TLR2 and TLR negative regulators in the modulation of proinflammatory cytokine production and activation of mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) pathways in PIE cells.

Results: Bifidobacteria longum BB536 and B. breve M-16V strains significantly downregulated levels of interleukin (IL)-8, monocyte chemotactic protein (MCP)-1 and IL-6 in PIE cells challenged with heat-killed enterotoxigenic Escherichia coli. Moreover, BB536 and M-16V strains attenuated the proinflammatory response by modulating the NF-κB and MAPK pathways. In addition, our findings provide evidence for a key role for the ubiquitin-editing enzyme A20 in the anti-inflammatory effect of immunobiotic bifidobacteria in PIE cells.

Conclusions: We show new data regarding the mechanism involved in the anti-inflammatory effect of immunobiotics. Several strains with immunoregulatory capabilities used a common mechanism to induce tolerance in PIE cells. Immunoregulatory strains interacted with TLR2, upregulated the expression of A20 in PIE cells, and beneficially modulated the subsequent TLR4 activation by reducing the activation of MAPK and NF-κB pathways and the production of proinflammatory cytokines. We also show that the combination of TLR2 activation and A20 induction can be used as biomarkers to screen and select potential immunoregulatory bifidobacteria strains.

Figure 1. Selection of bacterial strains with immunomodulatory capacities.

Bacteria were evaluated by studying (A) the mitogenic assay and expression of cytokines in porcine Peyer_$B!G_(Bs patches (B), mesenteric lymphoid nodes (C), and porcine intestinal epithelial cells (D). Cells were pre-treated with different bacterial strains for 48 h, and then the mitogenic activity or the expression of MCP-1, IL-6, and IL-8 were measured. The results represent four independent experiments. Different letters indicate significant differences (P<0.05).

Figure 2. 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 pre-treated with different bacterial strains for 48 h and stimulated with heat-stable ETEC PAMPs. The expression of MCP-1, IL-6, and IL-8 mRNAs (A) and proteins levels (B) were studied at 12 h post-stimulation. The results represent four independent experiments. Different letters indicate significant differences (P<0.05).

Figure 3. Western blot analysis of NF-κB pathway activation on porcine intestinal epithelial (PIE) cells after challenge with heat-stable Enterotoxigenic Escherichia coli (ETEC) pathogen-associated molecular patterns (PAMPs).

PIE cells were pre-treated with different bacterial strains for 48 h and stimulated with heat-stable ETEC PAMPs. Levels of the counter-regulatory factor IκBα were studied at the indicated times post-stimulation. Different letters indicate significant differences (P<0.05).

Figure 4. Western blot analysis of activation of p38 and JNK and ERK mitogen-activated protein kinases in porcine intestinal epithelial (PIE) cells after challenge with heat-stable Enterotoxigenic Escherichia coli (ETEC) pathogen-associated molecular patterns (PAMPs).

PIE cells were pre-treated with different bacterial strains for 48 h and then stimulated with heat-stable ETEC PAMPs. Phosphorylation of p38, JNK, and ERK was studied at the indicated times post-stimulation. Different letters indicate significant differences (P<0.05).

Figure 5. Expression of toll-like receptor negative regulators in porcine intestinal epithelial (PIE) cells.

PIE cells were pre-treated with different bacterial strains for 48 h, stimulated with heat-stable ETEC PAMPs, and the expression of IRAK-M, SIGIRR, Bcl-3, MKP-1, Tollip, and A20 negative regulators was studied at the indicated time points. The results represent four independent experiments. Different letters indicate significant differences (P<0.05).

Figure 6. Role of TLR2 in the immunomodulatory effect of Bifidobacterium longum BB536 and Bifidobacterium breve M-16V in porcine intestinal epithelial (PIE) cells.

PIE cells were pre-treated with BB536 or M-16V strains in the presence or absence of anti-TLR2 antibodies. Untreated PIE 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 A20 mRNA levels and MCP-1, IL-6, and IL-8 protein levels were studied at 12 h post-stimulation. The results represent four independent experiments. Different letters indicate significant differences (P<0.05).

Figure 7. Proposed mechanism for the immunomodulatory effect of Bifidobacterium longum BB536 and Bifidobacterium breve M-16V in porcine intestinal epithelial cells.

The effect of bifidobacteria strains is compared with the previously studied immunobiotic strain, Lactobacillus jensenii TL2937 .