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

Nishibayashi R, Inoue R, Harada Y, Watanabe T, Makioka Y, Ushida K - PLoS ONE (2015)

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Animal Science, Kyoto Prefectural University, Kyoto, Kyoto, Japan.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

Effect of stimulation by rRNA and mRNA from E. faecalis EC-12 on IL-12 production.Fragmented 23S rRNA and 16S rRNA, and mRNA from E. faecalis EC-12 was transfected using pLa to PMA-differentiated THP-1 cells. After 24 h of incubation, the concentration of IL-12 protein in the culture supernatant was measured using enzyme-linked immunosorbent assay. Bars sharing the same letter are not significantly different at P < 0.01, mean ± SD, n = 4.
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pone.0129806.g004: Effect of stimulation by rRNA and mRNA from E. faecalis EC-12 on IL-12 production.Fragmented 23S rRNA and 16S rRNA, and mRNA from E. faecalis EC-12 was transfected using pLa to PMA-differentiated THP-1 cells. After 24 h of incubation, the concentration of IL-12 protein in the culture supernatant was measured using enzyme-linked immunosorbent assay. Bars sharing the same letter are not significantly different at P < 0.01, mean ± SD, n = 4.

Mentions: When cells were stimulated using 23S rRNA, significantly higher IL-12 production was observed than in the case of cells stimulated using equimolar 16S rRNA and equimass mRNA (P < 0.01). In addition, between equimass 23S rRNA and 16S rRNA, IL-12 concentration in the culture supernatant was still higher in 23S rRNA-stimulated cells than in 16S rRNA-stimulated cells (23S: 15.6 ± 3.6 pg/mL vs 16S: 11.9 ± 4.5 pg/mL; Fig 4).


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

Nishibayashi R, Inoue R, Harada Y, Watanabe T, Makioka Y, Ushida K - PLoS ONE (2015)

Effect of stimulation by rRNA and mRNA from E. faecalis EC-12 on IL-12 production.Fragmented 23S rRNA and 16S rRNA, and mRNA from E. faecalis EC-12 was transfected using pLa to PMA-differentiated THP-1 cells. After 24 h of incubation, the concentration of IL-12 protein in the culture supernatant was measured using enzyme-linked immunosorbent assay. Bars sharing the same letter are not significantly different at P < 0.01, mean ± SD, n = 4.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4470910&req=5

pone.0129806.g004: Effect of stimulation by rRNA and mRNA from E. faecalis EC-12 on IL-12 production.Fragmented 23S rRNA and 16S rRNA, and mRNA from E. faecalis EC-12 was transfected using pLa to PMA-differentiated THP-1 cells. After 24 h of incubation, the concentration of IL-12 protein in the culture supernatant was measured using enzyme-linked immunosorbent assay. Bars sharing the same letter are not significantly different at P < 0.01, mean ± SD, n = 4.
Mentions: When cells were stimulated using 23S rRNA, significantly higher IL-12 production was observed than in the case of cells stimulated using equimolar 16S rRNA and equimass mRNA (P < 0.01). In addition, between equimass 23S rRNA and 16S rRNA, IL-12 concentration in the culture supernatant was still higher in 23S rRNA-stimulated cells than in 16S rRNA-stimulated cells (23S: 15.6 ± 3.6 pg/mL vs 16S: 11.9 ± 4.5 pg/mL; Fig 4).

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Animal Science, Kyoto Prefectural University, Kyoto, Kyoto, Japan.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus