Immunomodulation Of Lactobacilli On Intestinal Epithelial Cells Induced By Lipopolysaccharides And Its Signaling Pathway

Lactobacilli are one of probiotics that are widely used for maintaining animal intestinal health. Lactobacilli can modulate animal gastrointestinal immune responses via different components and have beneficial effects on host. However, the immunomodulatory mechanisms of Lactobacilli on host are still unclear. Therefore, it is important to clarify how Lactobacilli function during immune responses in intestines and to analyse key target protein in cell signalling during Lactobacilli-intestine interaction, which is necessary to the selection and breeding of highly efficient Lactobacillus probiotics in practices.The aim of this study is to evaluate the immunomodulatory effects and signalling mechanisms of Lactobacillus rhamnosus GG(LGG) and its components(surface-layer protein [SLP], DNA, CpG oligodeoxynucleotides [CpG ODN] and exopolysaccharides [EPS]) on inflammatory porcine intestinal epithelial cells(IECs) stimulated by lipopolysaccharide(LPS) and to find key functional components of LGG. Meanwhile, the immunomodulatory effects and signalling mechanisms of two Lactobacillus reuteri strains(Lactobacillus reuteri ZJ617 with high adhesive ablitiy and Lactobacillus reuteri ZJ615 with low adhesive abilty) were inverstigated on C57BL/6 mice model in vivo. The mRNA expressions of inflammatory cytokines and Toll-like receptors(TLRs) in IEC and mice intesintal tissues were measured by qRT-PCR. The activations of MAPK and NF-κB signalings were detected by western blotting, immunohistochemistry and immunofluorescence.Compared with LPS stimulation alone group in IPEC-J2 cells, pretreatment of IPEC-J2 cells with LGG, SLP or EPS significantly alleviated LPS-induced inflammatory cytokines(IL-6, IL-12 and TNF-α) and TLRs(TLR2, TLR4 and TLR9) activations at mRNA level. LGG, SLP and EPS also attenuated LPS-induced MAPK and NF-κB signalings activations in IPEC-J2 cells. Compared with LPS stimulation alone group in IPEC-J2 cells, CpG ODN significantly increased the IL-12, TNF-α and TLR9 mRNA levels and also enhanced NF-κB signalling activation in LPS-stimulated cells(P<0.05). Compared with LPS stimulation alone group in C57BL/6 mice without Lactobacilli administration, pretreatment of mice with 108 CFU/mL LGG or ZJ615 significantly decreased LPS-induced inflammatory cytokines(IL-6, IL-12 and TNF-α) and TLRs(TLR2, TLR4 and TLR9) mRNA levels in the intestinal tissues of mice. LGG and ZJ615 suppressed the activations of MAPK and NF-κB signalings in LPS-induced intestinal tissues. Compared with LPS stimulation alone group in C57BL/6 mice without Lactobacilli administration, pretreatment of mice with 108 CFU/mL ZJ617 significantly increased anti-inflammatory cytokine IL-10 mRNA levels in mice intestinal tissues, and activated NF-κB signalings in LPS-induced intestinal tissues(P<0.05).The results indicated that LGG alleviates inflammatory signalling in LPS-induced porcine IECs and LPS-induced mice models by modulating TLR2, TLR4 and TLR9 expression, inhibiting MAPK and NF-κB signalling and leading to the decreased expression of pro-inflammatory cytokines. The components(SLP, EPS and CpG ODN) extracted from LGG exerted immunomodulatory effects on the porcine IECs, especially CpG ODN, which exhibited a strong immunostimulatory effect. Lactobacilli with different adhesive ability showed different immunoregulatorty effects on the intestines. In this study, we find that immunomodulatory effects of ZJ615 with low adhesive ability are similar to LGG.