For this purpose, BIE cells were stimulated with the different LAB strains for 48 h, challenged with heat-stable ETEC PAMPs and the levels of the three pro-inflammatory cytokines were studied at hour 12 post-stimulation. MCP-1, IL-6 and IL-8 levels in BIE cells stimulated with OLL2768, MEP221101, MEP221105
and MEP221111 strains were significantly lower than those observed in the control. On the contrary, the other strains tested reduced one of the cytokines studied or had no effect (Additional file 1: Figure S1B). Considering that L. casei OLL2768 and L. casei MEP221111 showed the highest capacity to down-regulate IL-8 and also were able to reduce IL-6 and MCP-1 after heat-stable ETEC PAMPs challenge, one of these strains (L. casei OLL2768) was selected for the following experiments. To further confirm selleck chemical the immunoregulatory effect of L. casei OLL2768 and to obtain transcriptional data supported by protein detection of selected cytokines, we conducted KPT-330 solubility dmso ELISAs to evaluate the levels of IL-6 and MCP-1 proteins (Figure 3B). BIE cells were stimulated QNZ with L. casei OLL2768 or L. casei MEP221108 (negative control) and 48 h after
were challenged with heat-stable ETEC PAMPs. Challenge significantly increased levels of both IL-6 and MCP-1 proteins. Pretreatment of BIE cells with L. casei OLL2768 significantly reduced levels of MCP-1, however L. enough casei MEP221108 was not able to modify MCP-1 values (Figure 3B). Both L. casei OLL2768 and MEP221108 were able to reduce levels of IL-6 after the challenge with heat-stable ETEC PAMPs, however the effect of L. casei OLL2768 was significantly higher than those observed for MEP221108. In addition, we evaluated if the TLR2 agonist Pam3CSK4 was able to modulate IL-6 and MCP-1 synthesis. BIE cells pretreated Pam3CSK4 showed reduced levels of both cytokines
after heat-stable ETEC PAMPs challenge (Figure 3B). Effect of L. casei OLL2768 on MAPK and NF-κB pathways in BIE cells We next evaluated whether L. casei OLL2768 was able to attenuate heat-stable ETEC PAMPs-mediated pro-inflammatory responses by modulating the NF-κB pathway. Challenge of BIE cells with heat-stable ETEC PAMPs significantly reduced the levels of the counter-regulatory factor IκBα (Figure 4). BIE cells previously stimulated with L. casei OLL2768 or Pam3CSK4 did not show a significant degradation of IκBα indicating an inhibitory effect in NF-κB pathway (Figure 4). We also examined the relationship between MAPK activation and regulation of pro-inflammatory cytokines in BIE cells by L. casei OLL2768 (Figure 5). BIE cells were stimulated with OLL2768 strain, Pam3CSK4 or control medium and the activation profiles of p38, ERK and JNK were compared. As shown in Figure 5A and B, heat-stable ETEC PAMPs induced the phosphorylation of p38 and ERK, which reached a maximum between 5 and 10 minutes.