Nutri Co., Ltd.

Porphyromonas gingivalis, a gram-negative obligate anaerobic bacterium, is a major pathogen involved in the onset and progression of periodontal disease, a chronic inflammatory disorder observed in approximately two-thirds of the Japanese population older than age 30 years. P gingivalis cells produce and secrete gingipain, a powerful proteolytic enzyme, on their surfaces and in external environments.

The effects of heat-killed Enterococcus faecalis (HkEf), a lactic acid bacterium, on the growth of P gingivalis were evaluated in vitro by measuring the viable cell count of P gingivalis and gingipain activity.

HkEf solution (1.63 or 163 mg/mL) was added to 1 mL P gingivalis culture to generate a final HkEf concentration of 0.64 or 64 mg/mL. The cultures were incubated anaerobically. The number of viable P gingivalis cells and gingipain activity were measured after incubation for 0, 12, 24, 48, and 72 hours. The number of viable P gingivalis cells was calculated by counting the number of colonies after culture. Gingipain activity was quantified by adding a chromogenic substrate to P gingivalis culture medium and measuring the absorbance of the reaction solution with a plate reader. Mean (SE) was calculated for viable cell counts and gingipain activity, and Wilcoxon rank-sum test was used to test for significant differences.

The counts of viable P gingivalis cells in the control group increased as incubation time progressed for 12, 24, 48, and 72 hours; similar results were observed in the low-concentration HkEf group. In the high-concentration HkEf group, the increase in the viable cell count was significantly inhibited compared with that of the control group. Furthermore, gingipain activity in the low- and high-concentration HkEf groups was significantly inhibited over time compared with that of the control group. Although the pH of the culture solution tended to decrease in the high-concentration HkEf group, it was not considered to have affected the growth of P gingivalis.

HkEf exhibits inhibitory effects on the growth of P gingivalis and gingipain activity.

We aimed to examine the mechanism of heat-killed Enterococcus faecalis (HkEf) immunostimulatory effect when orally administered. Immunocompetent splenocytes from mice orally administered HkEf were assessed using flow cytometry. Immunocompetent cells were determined by culturing splenocytes with serum or blood derived-exosomes. In vitro studies evaluated the reaction between mouse splenocytes and exosomes purified and isolated from mice bone marrow-derived macrophages (BMDMs) treated with HkEf. Levels of dendritic cells, red pulp macrophages, and inactive NK cells were significantly higher in the HkEf-treated group. Red pulp macrophages and inactive NK cells were increased in splenocytes cultured with blood-derived exosomes from mice administered HkEf. Further, mouse splenocytes cultured with the HkEf-stimulated BMDMs-derived exosomes group had significantly higher levels of red pulp macrophages. Thus, HkEf was involved in host immunostimulation and exosomes were identified as mediators in immune response signaling. Further verification of the mechanism would be needed to fill in the gap between the present results and conclusions.

Previously, the mechanism underlying the immunostimulatory effect of orally administered heat-killed Enterococcus faecalis (HkEf) mediated by exosomes secreted after intestinal macrophage phagocytosis was clarified. In the present study, mouse bone marrow-derived dendritic cells (DCs) were used as intestinal DCs. In vitro studies evaluating the response of mouse spleen cells to exosomes purified and isolated from intestinal DCs stimulated with HkEf showed a significant increase in standard DCs and plasmacytoid DCs (pDCs), indicating a positive effect on DCs, mainly pDCs, and possibly contributing to their immunostimulatory effects. The microRNAs (miRNAs) of exosome fractions derived from intestinal DCs and macrophages were also examined using bone marrow progenitor cells. HkEf stimulation released miRNA-rich exosomes, which acted as an immune response signal mediator, and increased various miRNAs other than miR-146 that are essential for refilling innate immune cells. In the present study, the effect mediated by miRNA-rich exosomes on the distant immune system from intestinal immunity (macrophages, DCs) was observed via orally administered HkEf, a Lactobacillus preparation. The findings of the present study revealed that this mechanism can potentially prevent systemic infection.