Enterococcus faecalis from healthy infants modulates inflammation through MAPK signaling pathways

Abstract

Colonizing commensal bacteria after birth are required for the proper development of the gastrointestinal tract. It is believed that bacterial colonization pattern in neonatal gut affects gut barrier function and immune system maturation. Studies on the development of faecal microbiota in infants showed that the neonatal gut was first colonized with enterococci followed by other microbiota such as Bifidobacterium. Other studies showed that babies who developed allergy were less often colonized with Enterococcus during the first month of life as compared to healthy infants. Many studies have been conducted to elucidate how bifidobacteria or lactobacilli, some of which are considered probiotic, regulate infant gut immunity. However, fewer studies have been focused on enterococi. In our study, we demonstrate that E. faecalis, isolated from healthy newborns, suppress inflammatory responses activated in vivo and in vitro. We found E. faecalis attenuates proinflammatory cytokine secretions, especially IL-8, through JNK and p38 signaling pathways. This finding shed light on how the first colonizer, E.faecalis, regulates inflammatory responses in the host.

Figure 1. Phylogeny tree of 25 bacterial strains isolated from new born infants according to their 16sRNA sequences.

Figure 2. IL-8 secretion in IECs.

IL-8 secretions in Caco-2 (A), HCT116 (B) and HT-29 (C) with the treatment of Enterococcus. A total number of 10⁷ cfu/ml bacteria were added into the cells for 6 h. Supernatants were harvested for cytokine assay as described in Materials and Methods. (D) HCT116 cells were co-cultured with E. faecalis (EC1, EC3, EC15, EC16) and L. rhamnosus GG (L.GG) and S. typhimurium (Salm) with a multiplicity of infection (MOI) of 100 for 2 h, 4 h, 6 h and 24 hours. Three independent experiments were compiled to produce the data shown. Data were expressed as mean value ±SD. Student's T-test was used for statistical analysis as described in Materials and Methods. * p<0.05.

Figure 3. IL-8 secretions in HCT116 cells.

IL-8 production in HCT116 with the treatment of different conditional medium (CM) (from coculture supernatant, from bacteria and from cell supernatant), cell inserts, UV killed and sonicated bacteria. Whole live bacteria were used as control as described in Materials and Methods. Three independent experiments were done and data were expressed as mean value ±SD. Student's T-test was used for statistical analysis as described in Materials and Methods. * p<0.05.

Figure 4. Cytokine secretions in HCT116.

IL-8 (A) and TNF-α (C) production in HCT116 with the treatment of 2 ng/ml of IL-1β and 10⁷ cfu/ml of S. typhimurium and E. faecalis (EC16 and EC2) as described in Materials and Methods. (B) IL-8 production in HCT116 with the treatment of TNF-α and E. faecalis EC16 as described in Materials and Methods. Data was expressed as mean value ±SD. Student's T-test was used for statistical analysis as described in Materials and Methods. *p<0.05, **P<0.01.

Figure 5. IPA and Realtime PCR analysis.

Ingenuity pathway analysis (A) and Real time PCR (B) on E.faecalis treated HCT116 cells. All pathways and genes showed in the figure were significantly (p<0.05) regulated in HCT116 cells with the treatment of E. faecalis EC16 for 6 h at a MOI of 100. Experiments were done on 3 biological replicates.

Figure 6. Protein expression in HCT116JNK expression (A) at 30 mins, c-JUN (B) and P38 (C) expression at 1 h in HCT116 with the treatment of 2 ng/ml of IL-1β and 10⁷ cfu/ml of S. typhimurium, E. faecalis EC16 as described in Materials and Methods.

Cells were then lysed and proteins were tested using western blot. Three independent experiments were done.

Figure 7. E.faecalis treated DSS mild colitis model.

Colon lengths (cm) (A) and Mice weight change (B) with 1.5% DSS with or without EC16 and L.GG treatment as described in Material and Method. Real time PCR on IL-1β (C) and TNF-α (D) expression in colon. Each group contains 6–8 mice. Data was expressed as mean value ±SD. Student's T-test was used for statistical analysis as described in Materials and Methods. * p<0.05, ** P<0.01.