Ex vivo anti-inflammatory effects of probiotics for periodontal health

Abstract

Background: Probiotic bacteria with anti-inflammatory properties have the potential to be of therapeutic benefit in gingivitis. Objective: To evaluate the effects of potential probiotic strains on inflammatory mediators involved in early gingivitis using an ex vivo inflammation model. Methods: Strains were screened in viable and attenuated forms for effects on bacterial lipopolysaccharide (LPS)-stimulated release of interleukins (IL)-1β, -6 and -8, tumor necrosis factor-α, prostaglandin E₂ and 8-isoprostane from human primary monocytes, and then, if anti-inflammatory effects were shown, on IL-1β-stimulated release of inflammatory mediators from primary gingival fibroblasts. Lead strains were evaluated for optimal dosing, batch-to-batch variation and functional consistency in toothpaste. Results: Twenty-one of 73 strains showed anti-inflammatory effects in monocytes; of which, seven showed effects in both viable and attenuated forms. Seven of 14 strains showed effects in fibroblasts. Strains Lactobacillus paracasei LPc-G110(SYBIO-15) and Lactobacillus plantarum GOS42(SYBIO-41) induced statistically significant dose-dependent reductions in the release of multiple inflammatory mediators from monocytes, which were consistent across batches. Viable L. paracasei LPc-G110 tooth paste significantly reduced IL-6, IL-8 and prostaglandin E₂ release from monocytes versus placebo. Conclusion: Strains L. paracasei LPc-G110 and L. plantarum GOS42 have potential for use as probiotics in oral care products to reduce gingival inflammation.

Keywords: Gingivitis; bacteria; biofilms; dental plaque; inflammation; oral hygiene; probiotics.

Figure 1.

Screening of probiotic strains for effects on lipopolysaccharide (LPS)-stimulated inflammatory mediators in primary monocytes. Effects of different concentrations of Lactobacillus paracasei LPc-G110 in viable (a) and attenuated (b) forms. Effects of different concentrations of Lactobacillus plantarum GOS42 in viable (c) and attenuated (d) forms. Graphs show single experiments with biological replicates. Data are expressed as means ± standard deviation. The colony forming units per ml corresponding to each concentration of each strain are shown in Supplementary Table 1.

Figure 2.

Effects of Lactobacillus paracasei LPc-G110 on interleukin (IL) -1β-stimulated release of IL-6 and IL-8 from gingival fibroblasts. Effects of viable (a) and attenuated (b) L. paracasei LPc-G110 on IL-6. Effects of viable (c) and attenuated (d) L. paracasei LPc-G110 on IL-8. Graphs show single experiments with biological triplicates. P-values are from matched one-way analysis of variance followed by Dunnett’s multiple comparisons test – compared to IL-1β-stimulated control. The colony forming units per ml corresponding to each concentration of L. paracasei LPc-G110 are shown in Supplementary Table 2.

Figure 3.

Effects of Lactobacillus plantarum GOS42 on interleukin (IL) -1β-stimulated release of IL-6 and IL-8 from gingival fibroblasts. Effects of viable (a) and attenuated (b) L. plantarum GOS42 on IL-6. Effects of viable (c) and attenuated (d) L. plantarum GOS42 on IL-8. Graphs show single experiments with biological triplicates. P values are from matched one-way analysis of variance followed by Dunnett’s multiple comparisons test – compared to IL-1β-stimulated control. The colony forming units per ml corresponding to each concentration of L. plantarum GOS42 are shown in Supplementary Table 2.

Figure 4.

Dose-dependency of effects of Lactobacillus paracasei LPc-G110 on lipopolysaccharide (LPS)-stimulated release of inflammatory mediators from primary monocytes. Effects of viable (a) and attenuated (b) L. paracasei LPc-G110 at increasing concentrations (0.05%, 0.1%, 0.25%, 0.5%, 0.75%, 1.0%, 2.5%, 5.0% v/v); graphs show results from four independent experiments, each performed with biological triplicates and technical duplicates (n = 6 per experiment). All data are expressed as means ± standard deviation. P-values are from matched one-way analysis of variance followed by Dunnett’s multiple comparisons test – compared to LPS-stimulated control. The colony forming units per ml corresponding to each concentration of L. paracasei LPc-G110 are shown in Supplementary Table 3.

Figure 5.

Dose-dependency of effects of Lactobacillus plantarum GOS42 on lipopolysaccharide (LPS)-stimulated release of inflammatory mediators from primary monocytes. Effects of viable (a) and attenuated (b) L. plantarum GOS42 at increasing concentrations (0.05%, 0.1%, 0.25%, 0.5%, 0.75%, 1.0%, 2.5%, 5.0% v/v); graphs show results from three independent experiments, each performed with biological triplicates and technical duplicates (n = 6 per experiment). All data are expressed as means ± standard deviation. P values are from matched one-way analysis of variance followed by Dunnett’s multiple comparisons test – compared to LPS-stimulated control. The colony forming units per ml corresponding to each concentration of L. plantarum GOS42 are shown in Supplementary Table 4.

Figure 6.

Batch-to-batch variation of effects of viable Lactobacillus paracasei LPc-G110 on lipopolysaccharide (LPS)-stimulated inflammatory mediators in primary monocytes. Graphs show the effects of four different batches of viable L. paracasei LPc-G110 at different concentrations on interleukins (IL) -1β (a) and -6 (b), and prostaglandin E₂ (PGE₂) (c); results are from four independent experiments, each performed with biological triplicates and technical duplicates (n = 6 per experiment). All data are expressed as means ± standard deviation. The colony forming units per ml corresponding to each concentration for each batch are shown in Supplementary Table 3.

Figure 7.

Batch-to-batch variation of effects of viable Lactobacillus plantarum GOS42 on lipopolysaccharide (LPS)-stimulated inflammatory mediators in primary monocytes. Graphs show the effects of three different batches of viable L. plantarum GOS42 at different concentrations on interleukins (IL) -1β (a) and -6 (b), and prostaglandin E₂ (PGE₂) (c); results are from three independent experiments, each performed with biological triplicates and technical duplicates (n = 6 per experiment). All data are expressed as means ± standard deviation. The colony forming units per ml corresponding to each concentration for each batch are shown in Supplementary Table 4.

Figure 8.

Anti-inflammatory effects of Lactobacillus paracasei LPc-G110 formulated in toothpaste. Viability of primary monocytes when exposed to different concentrations of viable (a) or attenuated (b) L. paracasei LPc-G110 toothpaste, or placebo toothpaste. Effects of viable L. paracasei LPc-G110 toothpaste on interleukins (IL) -6 (c) and -8 (d), PGE₂ (e) and 8-isoprostane (f). Effects of attenuated L. paracasei LPc-G110 toothpaste on IL-6 (g), PGE₂ (h) and 8-isoprostane (i). Results are from a single experiment with two biological and two technical replicates. P values are from matched one-way analysis of variance followed by Dunnett’s multiple comparisons test – compared to placebo control. The colony forming units per g corresponding to each concentration of each probiotic toothpaste are shown in Supplementary Table 5.