Bifidobacterium Strains Present Distinct Effects on the Control of Alveolar Bone Loss in a Periodontitis Experimental Model

Abstract

Periodontitis is an inflammatory disease induced by a dysbiotic oral microbiome. Probiotics of the genus Bifidobacterium may restore the symbiotic microbiome and modulate the immune response, leading to periodontitis control. We evaluated the effect of two strains of Bifidobacterium able to inhibit Porphyromonas gingivalis interaction with host cells and biofilm formation, but with distinct immunomodulatory properties, in a mice periodontitis model. Experimental periodontitis (P+) was induced in C57Bl/6 mice by a microbial consortium of human oral organisms. B. bifidum 162^2A [B+ (1622)] and B. breve 110^1A [B+ (1101)] were orally inoculated for 45 days. Alveolar bone loss and inflammatory response in gingival tissues were determined. The microbial consortium induced alveolar bone loss in positive control (P + B-), as demonstrated by microtomography analysis, although P. gingivalis was undetected in oral biofilms at the end of the experimental period. TNF-α and IL-10 serum levels, and Treg and Th17 populations in gingiva of SHAM and P + B- groups did not differ. B. bifidum 162^2A, but not B. breve 110^1A, controlled bone destruction in P+ mice. B. breve 110^1A upregulated transcription of Il-1β, Tnf-α, Tlr2, Tlr4, and Nlrp3 in P-B+(1101), which was attenuated by the microbial consortium [P + B+(1101)]. All treatments downregulated transcription of Il-17, although treatment with B. breve 110^1A did not yield such low levels of transcripts as seen for the other groups. B. breve 110^1A increased Th17 population in gingival tissues [P-B+ (1101) and P + B+ (1101)] compared to SHAM and P + B-. Administration of both bifidobacteria resulted in serum IL-10 decreased levels. Our data indicated that the beneficial effect of Bifidobacterium is not a common trait of this genus, since B. breve 110^1A induced an inflammatory profile in gingival tissues and did not prevent alveolar bone loss. However, the properties of B. bifidum 162^2A suggest its potential to control periodontitis.

Keywords: Bifidobacterium; P. gingivalis; immune modulation; periodontitis; probiotics.

FIGURE 1

Mean and SD of weight gain in grams after 45 days of experimental period of C57Bl/6 mice submitted to different treatments: SHAM (negative control), P + B- (positive control), P-B+ (1101) (B. breve 110^1A), P + B+ (1101) (microbial consortium + B. breve 110^1A), P-B+ (1622) (B. bifidum 162^2A) and P + B+ (1622) (Microbial consortium + B. bifidum 162^2A). *Statistically significant difference in relation to negative control (SHAM), # Statistically significant difference in relation to positive control (P + B-). ANOVA, Tukey’s multiple comparison, p <0.05%. Data representative of two independent experiments (n = 8 mice/per group).

FIGURE 2

Alveolar bone analysis determined by Microtomography in the interproximal region of first and second M at the right maxilla of C57Bl/6 mice submitted to different treatments for 45 days: SHAM (negative control), P + B- (positive control), P-B+ (1101) (B. breve 110^1A), P + B+ (1101) (microbial consortium + B. breve 110^1A), P-B+ (1622) (B. bifidum 162^2A) and P + B+ (1622) (microbial consortium + B. bifidum 162^2A). (A) Representative images of alveolar bone. All data were obtained in the region between the red points. Data on Alveolar bone volume (ABV) (Average and sd) determined in pixels³ (B), Percentage of alveolar bone volume (Average and sd) and (C) Percentage of total porosity (Average and sd) (D) of the different groups. * Statistically significant difference in relation to negative control (SHAM), # Statistically significant difference in relation to positive control (P + B-). ANOVA, Tukey’s multiple comparison, p <0.05%. Data representative of two independent experiments (n = 8 mice/per group).

FIGURE 3

Bifidobacteria alter transcription of genes encoding cytokines and receptors for PAMPS in gingival tissues. Relative transcription of Il-1β (A) and Tnf-α (B), Il-17 (C), Tlr2 (D), Tlr4 (E) and Nrlp3 (F), determined by RT-qPCR in gingival tissues of C57Bl/6 mice submitted to different treatments for 45 days of experimental period: SHAM (negative control), P + B- (positive control), P-B + (1101) (B. breve 110^1A), P + B+ (1101) (microbial consortium + B. breve 110^1A), P-B+ (1622) (B. bifidum 162^2A) and P + B+ (1622) (microbial consortium + B. bifidum 162^2A). * Statistically significant difference in relation to negative control (SHAM), # Statistically significant difference in relation to positive control (P + B-). & Statistically significant difference in relation to P-B+ (1101). ANOVA, Tukey’s multiple comparison, p <0.05%. Data representative of two independent experiments (n = 8 mice/per group).

FIGURE 4

Treg populations remained unchanged in gingival tissues of C57Bl/6 mice submitted to different treatments: SHAM (negative control), P + B- (microbial consortium), P-B+ (1101) (B. breve 110^1A), P + B+ (1101) (microbial consortium + B. breve 110^1A), P-B+ (1622) (B. bifidum 162^2A) and P + B+ (1622) (microbial consortium + B. bifidum 162^2A). In (A) representative flow cytometry diagram showing the gating of CD4⁺, FoxP3+ Treg cells. In (B) Average percentages of CD4⁺, FoxP3+ Treg cells. No differences among the groups. ANOVA, Tukey’s multiple comparison, p>0.05%. Facs plots represent the results of one of two independent experiments with similar results (n = 2 pooled samples from 4 mice/per group).

FIGURE 5

Oral administration of B. breve 110^1A increases Th17 population in the gingival tissue. Th17 population in the gingival tissue of C57Bl/6 mice submitted to different treatments: SHAM (negative control), P + B- (microbial consortium), P-B+ (1101) (B. breve 110^1A), P + B+ (1101) (microbial consortium + B. breve 110^1A), P-B+ (1622) (B. bifidum 162^2A) and P + B+ (1622) (microbial consortium + B. bifidum 162^2A). In (A) representative flow cytometry diagram showing the gating of CD4⁺, RORγt⁺ Th17 cells. In (B) Average percentages of CD4⁺, RORγt⁺ Th17 cells. *Statistically significant difference in relation to negative control (SHAM), # Statistically significant difference in relation to positive control (P + B-). ANOVA, Tukey’s multiple comparison, p<0.05%. Facs plots represent the results of one of two independent experiments with similar results (n = 2 pooled samples from 4 mice/per group).

FIGURE 6

Serum levels (pg/ml) of IL-10 in C57Bl/6 mice submitted to different treatments for 45 days: SHAM (negative control), P + B- (positive control), P-B+ (1101) (B. breve 110^1A), P + B+ (1101) (microbial consortium + B. breve 110^1A), P-B+ (1622) (B. bifidum 162^2A) and P + B+ (1622) (Microbial consortium + B. bifidum 162^2A). * Statistically significant difference in relation to negative control (SHAM), # Statistically significant difference in relation to positive control (P + B-). ANOVA, Tukey’s multiple comparison, p <0.05%. Data representative of two independent experiments (n = 8 mice/per group).