Akkermansia muciniphila and Its Pili-Like Protein Amuc_1100 Modulate Macrophage Polarization in Experimental Periodontitis

Abstract

Periodontitis is a chronic inflammatory disease triggered by dysbiosis of the oral microbiome. Porphyromonas gingivalis is strongly implicated in periodontal inflammation, gingival tissue destruction, and alveolar bone loss through sustained exacerbation of the host response. Recently, the use of other bacterial species, such as Akkermansia muciniphila, has been suggested to counteract inflammation elicited by P. gingivalis In this study, the effects of A. muciniphila and its pili-like protein Amuc_1100 on macrophage polarization during P. gingivalis infection were evaluated in a murine model of experimental periodontitis. Mice were gavaged with P. gingivalis alone or in combination with A. muciniphila or Amuc_1100 for 6 weeks. Morphometric analysis demonstrated that the addition of A. muciniphila or Amuc_1100 significantly reduced P. gingivalis-induced alveolar bone loss. This decreased bone loss was associated with a proresolutive phenotype (M2) of macrophages isolated from submandibular lymph nodes as observed by flow cytometry. Furthermore, the expression of interleukin 10 (IL-10) at the RNA and protein levels was significantly increased in the gingival tissues of the mice and in macrophages exposed to A. muciniphila or Amuc_1100, confirming their anti-inflammatory properties. This study demonstrates the putative therapeutic interest of the administration of A. muciniphila or Amuc_1100 in the management of periodontitis through their anti-inflammatory properties.

Keywords: Akkermansia muciniphila; Porphyromonas gingivalis; host-microbe interactions; periodontitis; probiotics.

FIG 1

A. muciniphila and Amuc_1100 reduce P. gingivalis-induced alveolar bone loss. Mice were divided at random into the following groups for gavage: PBS (control), P. gingivalis (Pg), A. muciniphila (Akk), P. gingivalis and A. muciniphila (Akk/Pg), Amuc_1100 (1100), and Amuc_1100 and P. gingivalis (1100/Pg). Mice were then monitored for 6 weeks. (A) Alveolar bone loss was observed after staining. (B) Alveolar bone loss was determined by the distance between the CEJ and ABC (in micrometers). All values are represented as means ± SEM (n, 6/group). Results were analyzed by one-way ANOVA, with differences considered significant at a P value of ≤0.05. ***, P ≤ 0.001. (Asterisks indicate comparisons with P. gingivalis; tildes indicate comparisons with the control.)

FIG 2

A. muciniphila and Amuc_1100 increase the presence of M2 macrophages during P. gingivalis-induced experimental periodontitis. (A to C) SMLNs were isolated at the endpoint of the gavage period and analyzed for the presence of M1 macrophages (CD11b⁺ CD80⁺) (C) and M2 macrophages (CD11b⁺ CD206⁺) (B). Treatment groups were as follows: PBS (control), P. gingivalis (Pg), A. muciniphila (Akk), P. gingivalis and A. muciniphila (Akk/Pg), Amuc_1100 (1100), and Amuc_1100 and P. gingivalis (1100/Pg). All values are represented as means ± SEM (n, 6/group). Results were analyzed by one-way ANOVA, with differences considered significant at a P value of ≤0.05. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. (Asterisks indicate comparisons with P. gingivalis; tildes indicate comparisons with the control.)

FIG 3

A. muciniphila and Amuc_1100 increase the secretion of the anti-inflammatory mediator IL-10 by BMM infected with P. gingivalis. BMM were exposed to the control (Us), P. gingivalis alone (Pg) (MOI, 20:1), A. muciniphila alone (Akk) (MOI, 40:1), A. muciniphila and P. gingivalis (Akk/Pg), Amuc_1100 alone (1 μg/ml or 10 μg/ml), or Amuc_1100 and P. gingivalis (Amuc_1100/Pg) (1 μg/ml or 10 μg/ml). TNF-α (A) and IL-10 (B) concentrations were determined after 8 h of stimulation. All values are represented as means ± SEM (n, 5/group). Results were analyzed by one-way ANOVA, with differences considered significant at a P value of ≤0.05. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. (Asterisks indicate comparisons with P. gingivalis.)

FIG 4

A. muciniphila and Amuc_1100 increase the levels of anti-inflammatory mediators in the gingival tissue during P. gingivalis infection. (A to C) Gingival tissues isolated at the endpoint of the gavage period were analyzed for mRNA expression. Mice were divided into the following groups: PBS (control), P. gingivalis (Pg), A. muciniphila (Akk), P. gingivalis and A. muciniphila (Akk/Pg), Amuc_1100 (1100), and Amuc_1100 and P. gingivalis (1100/Pg). The relative expression of TNF-α (A), IL-10 (B), and CXCL10 (C) mRNAs was determined by quantitative real-time PCR using TaqMan assays. The fold change was calculated using control tissue as a baseline and β-actin as a normalizing control. (D to F) Gingival tissue lysates were also analyzed for the protein concentrations of TNF-α (D), IL-10 (E), and CXCL10 (F) via ELISA. All values are represented as means ± SEM (n, 6/group). Results were analyzed by one-way ANOVA, with differences considered significant at a P value of ≤0.05. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. (Asterisks indicate comparisons with P. gingivalis; tildes indicate comparisons with the control.)

FIG 5

Increased expression of IL-10 and CXCL10 in the gingival tissues of mice exposed to A. muciniphila or Amuc_1100 correlated with reduced alveolar bone loss. Gingival tissues isolated at the endpoint of the gavage period were analyzed for mRNA expression. Mice were divided into the following groups: PBS (control), P. gingivalis (Pg), A. muciniphila (Akk), P. gingivalis and A. muciniphila (Akk/Pg), Amuc_1100 (1100), and Amuc_1100 and P. gingivalis (1100/Pg). The relative expression of mRNA was determined by quantitative real-time PCR using TaqMan assays. The fold change was calculated using control tissue as a baseline and β-actin as a normalizing control. All values are represented as means ± SEM (n, 6/group). Results were analyzed by Pearson’s correlation analysis, with differences considered significant at a P value of ≤0.05.