Probiotics Affect The Intestinal Epithelial Barrier And Gut Microbiota Of Pathogens-Infected Piglets Through Regulating EGFR/Akt Signalling

Background and Aims:Enterotoxigenic Escherichia coli expressing the F4 fimbriae(F4+ETEC)and Salmonella are the most common causes of bacteria-related post-weaning diarrhea in piglets.Probiotics represent the novel alternative to antibiotics for controlling pathogen infection,however,the exact model of action of probiotics remains largely unknown.The intestinal epithelial barrier is the first line of defense barrier against pathogen infection.The homeostasis of gut microbiota aids in managing enteric pathogen infection.The aim of the present study was to determine the role of intestinal epithelial barrier and gut microbiota in probiotics controlling enteric pathogen infection and explore the underlying mechanism.Methods:In this study,porcine intestinal epithelial J2 cells(IPEC-J2)were pre-incubated with or without Lactobacillus rhamnosus GG(LGG,108 CFU/mL)and then exposed to F4+ ETEC(107 CFU/mL)or Salmonella infantis(108 CFU/mL).Exact 21-day-old weaned piglets were orally administrated with 10 mL of LGG(109 CFU/mL)and then were infected with S.infantis constitutively expressing green fluorescent protein(5×1010 CFU/mL,10 mL).There were seven piglets each group.Low(3.9 × 107 CFU/mL,10 mL),moderate(7.8 × 107 CFU/mL,10 mL)or high(3.9 × 108 CFU/mL,10 mL)dose of a mixture of Bacillus subtilis and Bacillus licheniformis(BLS-mix)were orally administrated to 21-day-old weaned piglets before F4+ETEC challenge(109 CFU/mL,10 mL).There were six piglets each group.The number of adhered F4+ETEC and internalizated S.infantis in IPEC-J2 cells were determined using plate count method.The production of mucins in IPEC-J2 cells were determined by AB/PAS staining.The mRNA and protein expression of inflammatory cytokines and receptors were analyzed using Real-time PCR and ELISA.The expression of tight junction and autophagy proteins in IPEC-J2 cells and intestinal samples were determined using Western blotting.We also performed the high-throughput sequencing,quantitative PCR combined with culture-dependent analysis of the gut microbiota of feces and colon.Results:L.rhamnosus reduced the adhesion of F4+ ETEC and internalization of S.infantis in IPEC-J2 cells and attenuated F4+ETEC-induced mucin layer destruction and suppressed apoptosis.The relative expression of Toll-like receptor 4(TLR4)and NOD2 mRNA in IPEC-J2 cells increased at 3 h after F4+ETEC infection,but L.rhamnosus treatment attenuated these increases.Pre-incubation with L.rhamnosus suppressed the F4+ ETEC-induced increased concentration of tumor necrosis factor a.F4+ETEC-infected cells or L.rhamnosus-incubated cells had higer concentration of prostaglandin E2.Pre-incubation with L.rhamnosus decreased the expression of phosphorylated epidermal growth factor receptor(EGFR)protein and increased the expression of phosphorylated Akt,ZO-1 and occluding proteins.Pre-incubation with L.rhamnosus attenuated the S.infantis-induced increase expression of LC3 protein in IPEC-J2 cells and ileum and enhanced phosphorylation of EGFR and Akt.L.rhamnosus treatment inhibited the S.infantis-induced IPEC-J2 cell death.Probiotic BLS-mix attenuated F4+ETEC-induced increase of Bacteroides uniformis,Eubacterium eligens,Acetanaerobacterium and Sporobacter abundance,as well as expanded the populations of Lactobacillus gasseri and Lactobacillus salivarius.Low dose of BLS-mix treatment increased the populations of Clostridium and Turicibacter following F4+ ETEC challenge.However,administration of high-dose BLS-mix expanded the Proteobacteria population.Conclusions:L.rhamnosus protects intestinal epithelial cells from F4+ETEC infection,partly via the anti-inflammatory response involving synergism between TLR2 and NOD1.L.rhamnosus promotes the EGFR-independent Akt activation,increasing the tight junction integrity of intestinal epithelial cells and suppressing F4+ETEC invasion.L.rhamnosus inhibits the S.inantis-induced autophagic death of intestinal epithelial cells through EGFR-dependent Akt activation.Probiotic Bacillus reprograms the gut microbiota,with a corresponding increase in the populations of Clostridium,Lactobacillus and Turicibacter.