Bacillus licheniformis PF9 improves barrier function and alleviates inflammatory responses against enterotoxigenic Escherichia coli F4 infection in the porcine intestinal epithelial cells

Background: Enterotoxigenic Escherichia coli(ETEC) F4 commonly colonizes the small intestine and releases enterotoxins that impair the intestinal barrier function and trigger inflammatory responses. Although Bacillus licheniformis(B. licheniformis) has been reported to enhance intestinal health, it remains to be seen whether there is a functional role of B. licheniformis in intestinal inflammatory response in intestinal porcine epithelial cell line(IPEC-J2) when stimulated with ETEC F4.Methods: In the present study, the e ects of B. licheniformis PF9 on the release of pro-inflammation cytokines, cell integrity and nuclear factor-κB(NF-κB) activation were evaluated in ETEC F4-induced IPEC-J2 cells.Results: B. licheniformis PF9 treatment was capable of remarkably attenuating the expression levels of inflammation cytokines tumor necrosis factor-α(TNF-α), interleukin(IL)-8, and IL-6 during ETEC F4 infection. Furthermore, the gene expression of Toll-like receptor 4(TLR4)-mediated upstream related genes of NF-κB signaling pathway has been significantly inhibited. These changes were accompanied by significantly decreased phosphorylation of p65 NF-κB during ETEC F4 infection with B. licheniformis PF9 treatment. The immunofluorescence and western blotting analysis revealed that B. licheniformis PF9 increased the expression levels of zona occludens 1(ZO-1) and occludin(OCLN) in ETEC F4-infected IPEC-J2 cells. Meanwhile, the B. licheniformis PF9 could alleviate the injury of epithelial barrier function assessed by the trans-epithelial electrical resistance(TEER) and cell permeability assay. Interestingly, B. licheniformis PF9 protect IPEC-J2 cells against ETEC F4 infection by decreasing the gene expressions of virulence-related factors(including lux S, est A, est B, and elt) in ETEC F4.Conclusions: Collectively, our results suggest that B. licheniformis PF9 might reduce inflammation-related cytokines through blocking the NF-κB signaling pathways. Besides, B. licheniformis PF9 displayed a significant role in the enhancement of IPEC-J2 cell integrity.