Takumi Watanabe

Diverse Immune Responses to Orally Administered Heat-Killed Cell Preparation of Enterococcus faecalis Strain EC-12 in Murine Immune Tissues

Diverse immune responses to an orally administered heat-killed cell preparation of Enterococcus faecalis strain EC-12 (EC-12) among jejunal-Peyer’s patches (PPs), ileal-PPs, mesenteric lymph nodes (MLNs), and spleen were compared by real-time PCR in mice. Intriguingly, distinct responses to EC-12 were observed in the various tissues. This study indicates a site-specific response to orally administered bacteria, particularly in jejunal- and ileal-PPs.

Evaluation of the heat-killed and dried cell preparation of Enterococcus faecalis against villous atrophy in early-weaned mice and pigs

Early weaning induces villous atrophy in the small intestine (SI) of piglets. Oral administration of live lactic acid bacteria (LAB) can improve villous shortening. In this study, we evaluated the oral administration of a heat-killed and dried cell preparation of Enterococcus faecalis (a LAB) strain EC-12 against villous atrophy in early-weaned mice (Experiment 1) and pigs (Experiments 2 and 3). Twelve 16-days-old mice were divided into two groups in Experiment 1: gavage of EC-12 (10 mg/kg body weight (BW)/day), or control. On day 21, SI was collected. Eighteen 21-day-old pigs were divided into two groups in Experiment 2: gavage of EC-12 (10 mg/kg BW/day), or control. After 10 days, the villous height of jejunum was measured. Six 21-day-old pigs were divided into two groups in Experiment 3: the basal diet supplemented with EC-12 at 0.05%-fed group, or the basal diet-fed group. After 10 days, the villous height of jejunum was measured. The villous heights in SI were significantly higher by EC-12 administration in all experiments. EC-12 successfully improved the villous atrophy in the early-weaned mice and pigs when EC-12 was administered orally.

RNA of Enterococcus faecalis Strain EC-12 Is a Major Component Inducing Interleukin-12 Production from Human Monocytic Cells

Interleukin-12 (IL-12) is an important cytokine for the immunomodulatory effects of lactic acid bacteria (LAB). Using murine immune cells, we previously reported that the RNA of Enterococcus faecalis EC-12, a LAB strain exerting probiotic-like beneficial effects, is the major IL-12-inducing immunogenic component. However, it was recently revealed that bacterial RNA can be a ligand for Toll-like receptor (TLR) 13, which is only expressed in mice. Because TLR13 is not expressed in humans, the immuno-stimulatory and -modulatory effects of LAB RNA in human cells should be augmented excluding TLR13 contribution. In experiment 1 of this study, the role of LAB RNA in IL-12 induction in human immune cells was studied using three LAB strains, E.faecalis EC-12, Lactobacillus gasseri JCM5344, and Bifidobacterium breve JCM1192. RNase A treatment of heat-killed LAB significantly decreased the IL-12 production of human peripheral blood mononuclear cells on stimulation, while RNase III treatment revealed virtually no effects. Further, IL-12 production against heat-killed E. faecalis EC-12 was abolished by depleting monocytes. These results demonstrated that single stranded RNA (ssRNA) of LAB is a strong inducer of IL-12 production from human monocytes. In experiment 2, major receptor for ssRNA of E. faecalis EC-12 was identified using THP-1 cells, a human monocytic cell line. The type of RNA molecules of E. faecalis EC-12 responsible for IL-12 induction was also identified. IL-12 production induced by the total RNA of E. faecalis EC-12 was significantly reduced by the treatment of siRNA for TLR8 but not for TLR7. Furthermore, both 23S and 16S rRNA, but not mRNA, of E. faecalis EC-12 markedly induced IL-12 production from THP-1 cells. These results suggested that the recognition of ssRNA of E. faecalis EC-12 was mediated by TLR8 and that rRNA was the RNA molecule that exhibited IL-12-inducing ability in human cells.

Rapid Induction of an Immune Response in Rat Peyer's Patch after Oral Administration of Enterococcus faecalis Strain EC-12

A rapid and sharp immune response induced in Peyers patches (PPs) by a single gavage of a heat-killed Enterococcus faecalis strain EC-12 (EC-12) was demonstrated. EC-12 was observed inside the PPs 2.5 h post administration and induction of TNF-α and CD69 gene expression was observed at the same time. The immune response in PPs disappeared 24 h post administration.

Oral Administration of EC-12 Increases the Baseline Gene Expression of Antiviral Cytokine Genes, IFN-γ and TNF-α, in Splenocytes and Mesenteric Lymph Node Cells of Weaning Piglets.

Weaning piglets are continuously exposed to various viruses. The antiviral effects of lactic acid bacteria (LAB) have been confirmed mainly in humans and mice, while few studies have been conducted in livestock. In this study, we evaluated the effect of oral administration of Enterococcus faecalis strain EC-12 (EC-12) on the gene expressions of antiviral cytokines in weaning piglets. Piglets were allocated to the EC-12-administered group (E group) and the no-treatment control group (C group). The small intestinal tissue, the mesenteric lymph node (MLN) cells and the splenocytes were collected from the piglets. The tissue and cells were co-cultured with a live vaccine of porcine reproductive respiratory syndrome virus, porcine epidemic diarrhea virus or EC-12. After the incubation, the gene expressions of IFN-γ and TNF-α in the tissue and cells were evaluated. The gene expressions of IFN-γ in the MLN cells and TNF-α in the splenocytes were significantly higher in the E group than in the C group. However, the increase in the gene expression of antiviral cytokines was observed independently of the antigen treatments. The results of the present study suggest that oral administration of EC-12 did not increase the response of immune cells to specific viral antigens but increased the baseline gene expression of antiviral cytokines.