Effects Of Enterococcus Faecium EF1on Small Intestinal Mucosal Barrier Functions Of Piglets

Enterococcus faecium has been widely used as probiotics due to their functioning ability to improve intestinal health, exhibit a growth-enhancing effect, and enhance the immunity of the host. Aprobiotic strain Enterococcus faecium EF1isolated from a healthy piglet was evaluated for its ability to modulate intestinal mucosal barrier functions with both in vivo and in vitro studies.The main contents and results are as follows:Trial1:The effects of Enterococcus faecium EF1on small intestinal mucosal barrier functions of pre-weaning piglets were studied to improve our understanding of the underlying mechanisms of this probiotic strain.6litters newborn piglets ([Large White×Landrace]×Duroc)(no significant deviation in body weight) were randomly divided into two groups,3litters per group. Immediately after birth and before the first sucking, piglets of control group were administered10%sterilized skim milk2mL piglet-1day-1by oral gavage, and the probiotics group received10%sterilized skim milk2mL piglet-1day-1with addition of viable Enterococcus faecium EF1(5～6×108CFU/mL) for the first time, and other two of oral gavages were carried on the alternative odd days3rd and5th day post partum. Piglets were housed in standard farrowing crates with sows and subjected to routine management practices. The diet of sows contained no added antibiotics throughout the trial. From day12onward, piglets had free access to a supplemented pre-starter feed and ad libitum access to water. The feeding trial was conducted for25days. Results showed that oral administration of Enterococcus faecium EF1increased daily body weight gain by30.73%(P<0.01) while decreased diarrhea rate by43.21%(P<0.05). No significant modifications were found for the diversity of intestinal flora, but the composition of microflora was improved, which would exert beneficial effects by decreasing the growth of gram-negative bacteria (e.g., Bacteroidetes spp., Proteobacteria spp., and Fusobacteria spp.) while allowing an increase in the number of gram-positive bacteria (e.g., Firmicutes spp.) when Enterococcus faecium EF1were administered. As compared with control group, jejunal mucosal sIgA production was up-regulated significantly, while the lys expression was down-regulated in the probiotics group. In addition, Enterococcus faecium EF1enhanced DAO activities but decreased ITF content in both jejunal and ileal mucosa thereby improving small intestinal integrity in sucking piglets, ocln expression was down-regulated obviously in jejunal mucosa induced by Enterococcus faecium EF1, while zo-1expression was not significantly changed, and level of serum DAO activity increased, indicating the mucosal permeability increased. When Enterococcus faecium EF1were administered, the concentrations of many pro-inflammatory cytokines, including ileal mucosal TNF-a, jejunal mucosal IFN-y, as well as IL-1, IL-6, IL-12, and IL-8in both jejunal and ileal mucosa decreased dramatically, but the production of anti-inflammatory cytokine IL-10in both jejunal and ileal mucosa and jejunal mucosal TGF-β1, as well as tgf-β1mRNA expression in the jejunal mucosa significantly increased, thus enhancing the immune tolerance of the small intestine. Moreover, jejunal mucosal TNF-a secretion increased and tnf-α mRNA expression levels also improved significantly in probiotics group, indicating a local physiological inflammatory immune response occurred in jejunal mucosa. However, ileal mucosal TGF-(31and IFN-y production were similar in both probiotics and control group. A further important finding was that levels of tlr2, tlr9, and traf6mRNA epression were up-regulated in probiotics group, suggesting they would be involved in signaling pathway of immuno-modulation by Enterococcus faecium EF1. Taken together, oral administration of Enterococcus faecium EF1could promote growth performance and decreased diarrhea rate through regulating microbial ecological balance, functions of chemical and physical barrier, as well as innate immune responses in pre-weaning piglets.Trial2:The effects of Enterococcus faecium EF1on small intestinal mucosal barrier functions of weaning piglets were evaluated. In this trail, we used the same experimental protocol as in Trial1. Piglets were weaned at25days of age and fed the pre-starter diet during the first week following weaning. Results showed that as compared to control group, oral administration of Enterococcus faecium EF1could increase daily body weight gain of weaning piglets by320.84%(P<0.01) while decrease diarrhea incidence by71.42%(P<0.05). As compared with control, Enterococcus faecium EF1declined the diversity of intestinal flora, but the composition of micro flora improved, the change in amount of Firmicutes bacteria was not significant, while the number of Proteobacteria spp. reduced markedly. However, the amount of Bacteroidetes spp was increased obviously, indicating that the balance of microflora improved and carbohydrate digestion and absorption enhanced. No changes were observed in sIgA concentration in the jejunal mucosa, while pH value in the stomach reduced markedly. There was an increase in the jejunal mucosal DAO activity and a decrease in serum DAO activity and in jejunal mucosal ITF level. Moreover, the serum endotoxin concentrations maintained at a very low level, suggesting that epithelial integrity was sustained. Furthermore, Enterococcus faecium EF1down-regulated the production of TGF-β1significantly while secretion of TNF-a, IL-1β and IL-12was enhanced in the jejunal mucosa. Whereas production of IL-10, IL-6, IFN-γ, and IL-8unchanged, indicating that pro-inflammatory response was activated to increase the capability to inhibit pathogenic bacteria invation. Taken together, Enterococcus faecium EF1given after birth promoted growth performance and improved the intestine health, prevented detrimental effects of weaning stress on jejunal mucosal integrity, the capacity of digestion and absorption, as well as microbial ecological balance, and enhanced functions of chemical barrier and mechanical barrier, as well as innate immune responses in the piglets during the first week of weaning.Trial3:The study aimed at determining the effects of Enterococcus faecium EF1on Caco-2cells.1) Adhesion. Caco-2cells were co-cultured with Enterococcus faecium EF1(EF group) and E. coli K88(EC group), respectively. In addition, Coco-2cells were co-incubated with both Enterococcus faecium EF1and E. coli K88(EF+EC group), or Caco-2cells were pre-cultured with Enterococcus faecium EF1and then following co-incubating with E. coli K88(EF-EC group), or Caco-2cells were co-cultured with E. coli K88firstly, and then co-incubated with Enterococcus faecium EF1(EC-EF group), the adhesion rate of E. coli K88was examined, respectively. Results showed that a higher adhesion in EF group than in EC group. As compared with EC group, the adhesion rate of E. coli K88declined significantly in EF+EC group, EF-EC group and EC-EF group. Taken together, the capacity of Enterococcus faecium EF1to adhere to Caco-2cells was greater than that of E. coli K88. Moreover, Enterococcus faecium EF1had activity against E. coli K88adhesion by exclusion, displacement and competition.2) The effects of Enterococcus faecium EF1on metabolic activity and membrane integrity of Caco-2cells. Caco-2cells were co-cultured with PBS (CT group), Enterococcus faecium EF1(EF group), and E. coli K88(EC group), respectively. In addition, Caco-2cells were pre-incubated with Enterococcus faecium EF1and then followed by challenging with E. coli K88(EF-EC group). Both the intracellular and extracellular AKP and LDH activities were measured. Results showed that as compared with CT group, intracellular AKP activity decreased significantly in EF group, while it was much higher than that in EC group. However, intracellular LDH content increased clearly in EF group. Furthermore, both extracellular AKP and LDH activities declined obviously in EF group. In addition, when compared with EC group, no significant change in intracellular AKP activity was observed, whereas a remarkable up-regulation of LDH concentration was found. Moreover, both extracellular AKP and LDH activities declined markedly in EF group. These results indicated that Enterococcus faecium EF1was capable of protecting Caco-2cells monolayer integrity from the damage induced by E. coli K88and maintaining the normal physiological level of AKP and LDH.3) The immune modulations of Enterococcus faecium EF1on Caco-2cells and the underlying mechanisms. Caco-2cells were co-cultured with PBS (CT group), Enterococcus faecium EF1(EF group), and E. coli K88(EC group), respectively. In addition, Caco-2cells were pre-incubated with Enterococcus faecium EF1and then followed by infecting with E. coli K88(EF-EC group). Cytokines of IL-10and APRIL secretion, as well as the differential expression profile of innate and adaptive immune response related genes in Caco-2cells were measured. Results showed that as compared with CT group, IL-10level increased significantly in group EF, while no IL-10secretion was detectable in group EC. The production of APRIL improved markedly, while the level was much lower than that in EC group, indicating that Enterococcus faecium EF1induced both immune tolerance and activation. In addition, the APRIL content was much less in group EF-EC than that in group EC, while IL-10concentration was much higher in group EF-EC than that in CT group, suggesting that Enterococcus faecium EF1may attenuate the pro-inflammatory response in Caco-2cells induced by E. coli K88challenge to promote homeostasis. Furthermore, we demonstrated that Enterococcus faecium EF1served a dual role in modulating genes involved in the innate and adaptive immune responses by PCR array technology. The mRNA levels of24genes were significantly increased, including ccl2, cdld, cxcr4, defb4, dmbtl, ifnal, ifnbl, ill0, illf5, illf6, illrapl2, illrl2, illrn, ly96, ncf4, pglyrp3, ptafr, sftpd, tlr3, tlr4, tlr6, tlr8, tlr10and tnf while the levels of adora2a, casp1, casp4, cdl4, colecl2, ifngrl, ill2rb2, illf7, illf8, illf9. illrl, irakl, mif nlrc4, pglyrp1, pglyrp2, ppbp, tlr2, tollip and traf6were noticeably suppressed. These genes are involved in innate immune response, inflammatory response and host defense to bacteria. Among those, five genes (dmtbl, tlr10, tlr6, illf6and tnf) may contribute to immuno-modulating properties of Enterococcus faecium EF1.Trial4:Effects of Enterococcus faecium EF1on production of cytokines, prostaglandin E2and superoxide anion in a murine macrophage cell line, RAW264.7was investigated. RAW264.7cells were co-cultured with PBS (CT group), Enterococcus faecium EF1(EF group), and E. coli K88(EC group), respectively. In addition, RAW264.7cells were pre-incubated with Enterococcus faecium EF1and then followed by challenging with E. coli K88(EF-EC group). The results showed that as compared with CT group, an increased release of IL-10, TNF-α, IFN-γ, IL-6, and O2·-in EF group were observed, while these cytokines concentrations were far lower than that in group EC. However, no significant affects on IL-1β, IL-12, and PGE2secretion were examined, indicating that Enterococcus faecium EF1was capable of triggering a moderate innate inflammatory response on direct contact with RAW264.7. In addition, when RAW264.7cells were firstly treated with Enterococcus faecium EF1and then infected with E. coli K88, the production of IL-10, TNF-α, IFN-γ, IL-1β, and PGE2in response to E. coli K88were apparently increased, while O2-significantly suppressed, suggesting that Enterococcus faecium EF1may enhance the ability of macrophages to prevent E. coli K88invation and keep the balance of immune response by improving both pro-inflammatory and anti-inflammatory cytokines production, as well as regulating seretion of PGE2and O2.To sum up, these findings revealed that orally administered Enterococcus faecium EF1after birth was effective to improve growth and decrease diarrhea incidence by reversing microbiota disruption, improving functions of intestinal mucosal chemical and physical barrier, as well as innate immune response in small intestine in both of pre-weaning and weaning piglets. Enterococcus faecium EF1resisted against infection with E. coli K88by preventing adhesion, protecting metabolic activity and integrity of membrane of intestinal epitheliums, attenuating pro-inflammatory response in intestinal epitheliums, as well as promoting inflammatory response in macrophages. Furthermore, Enterococcus faecium EF1served a dual role in triggering innate immune response in mucosa, Caco-2cells, as well as in RAW264.7macrophage cells, and exhibiting both pro-inflammatory and anti-inflammatory activities, which would contribute to the improvement of intestinal immune homeostasis. Toll-like receptor signaling pathway might be involved in the observed immuno-modulating property of Enterococcus faecium EF1.