Periodontitis induced by Porphyromonas gingivalis drives periodontal microbiota dysbiosis and insulin resistance via an impaired adaptive immune response

Abstract

Objective: To identify a causal mechanism responsible for the enhancement of insulin resistance and hyperglycaemia following periodontitis in mice fed a fat-enriched diet.

Design: We set-up a unique animal model of periodontitis in C57Bl/6 female mice by infecting the periodontal tissue with specific and alive pathogens like Porphyromonas gingivalis (Pg), Fusobacterium nucleatum and Prevotella intermedia. The mice were then fed with a diabetogenic/non-obesogenic fat-enriched diet for up to 3 months. Alveolar bone loss, periodontal microbiota dysbiosis and features of glucose metabolism were quantified. Eventually, adoptive transfer of cervical (regional) and systemic immune cells was performed to demonstrate the causal role of the cervical immune system.

Results: Periodontitis induced a periodontal microbiota dysbiosis without mainly affecting gut microbiota. The disease concomitantly impacted on the regional and systemic immune response impairing glucose metabolism. The transfer of cervical lymph-node cells from infected mice to naive recipients guarded against periodontitis-aggravated metabolic disease. A treatment with inactivated Pg prior to the periodontal infection induced specific antibodies against Pg and protected the mouse from periodontitis-induced dysmetabolism. Finally, a 1-month subcutaneous chronic infusion of low rates of lipopolysaccharides from Pg mimicked the impact of periodontitis on immune and metabolic parameters.

Conclusions: We identified that insulin resistance in the high-fat fed mouse is enhanced by pathogen-induced periodontitis. This is caused by an adaptive immune response specifically directed against pathogens and associated with a periodontal dysbiosis.

Keywords: BACTERIAL INFECTION; BACTERIAL PATHOGENESIS; DIABETES MELLITUS; DIET; IMMUNE RESPONSE.

Figure 1

Oral colonisation with Porphyromonas gingivalis (Pg), Fusobacterium nucleatum (Fn) and Prevotella intermedia (Pi) induces periodontitis associated with local and systemic immune disorders. (A) Mice were colonised with Pg, Fn and Pi or by vehicle solution for 1 month and then randomised into four groups: normal chow (NC, blue bar, n=6), normal chow colonised (NC-Co, purple bar, n=6), high-fat diet (HFD red bar, n=7) and high-fat diet colonised (HFD-Co, green bar, n=10). (B) Hemi-mandible for each group, as reconstructed by micro-CT (Viva CT40; Scanco Medical, Bassersdorf, Switzerland). (C) Alveolar bone loss (yellow line) for each group. (D) Tumour necrosis factor (TNF)-α, plasminogen activator inhibitor (PAI)-1, interleukin (IL)1-β and IL-6 expression in periodontal tissue. (E) Histological examination for hemi-mandibles (PT, periodontal tissue; T, tooth) stained with H&E, F4/80, CD3 and CD20 antibodies: cells count is shown on the right side of each panel series. Arrows show infiltrated cells within the anatomical area and insets show a magnification of the slide. Number and relative abundance of immune cell types explored at 3 months in cervical lymph nodes (F and G) and in spleen (H and I) and the blood (J). (K) Linear discriminant analysis effect size (LEfSe) analysis-based cladogram for periodontal microbiota of each group. (L) Complete linkage clustering using Euclidean distance. Data (mean±SEM) and one-way analysis of variance (ANOVA) followed by Tukey's test used for *p<0.05 and ****p<0.0001 when compared to HFD, ^§p<0.05; ^§§p<0.001 ^§§§§p<0.0001 when compared to NC and ^$p<0.05 when compared to NC-Co.

Figure 1

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Figure 2

Periodontitis enhances high-fat diet (HFD)-induced metabolic disorders in mice. Glycaemic profiles (mg/dL) during an intraperitoneal glucose-tolerance test (IpGTT; normal chow (NC, blue bar, n=6), normal chow colonised (NC-Co, purple bar, n=6), high-fat diet (HFD, red bar n=7) and high-fat diet colonised (HFD-Co, green bar, n=10)) and glycaemic indexes as inset; ratio fat/lean for each group during 1 month (A and B), 2 months (C and D) and 3 months (E and F). (G) Insulin sensitivity evaluated by the euglycemic-hyperinsulinemic clamp technique. (H) Correlation between glucose infusion rate (GIR) and alveolar bone loss (ABL). (I) Principal coordinate analysis (PCoA) between dominant bacterial families from periodontal microbiota (abundance >1%, detected at least in one mouse) and metabolic parameters such as ABL, GIR, gingival inflammation (TNFaG, IL1bG, PAI1G, IL6G, where ‘G’ stands for gingival), immunoglobulin G score, glycaemic index, fasted glycaemia and body weight at 3 months (IgG3, GI3, FG3, BW3, respectively): the three insets represent the correlation between GIR and Lactobacillaceae family, Porphyromonadaceae family and Porphyromonadaceae family with ABL. Data are mean±SEM. Significant results when *p<0.05; **p<0.01 and ***p<0.001 when compared to HFD, ^§p<0.05 and ^§§§p<0.001 when compared to NC and ^$p<0.05 when compared to NC-Co as determined by two-way analysis of variance (ANOVA) with Bonferroni's post-test for (A), (C) and (E) and one-way ANOVA followed by Tukey's post-test for (B), (D), (F) and (G).

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Figure 3

Immune cells transfer from cervical lymph nodes from periodontitis mice reduce colonisation-induced glucose intolerance. (A) Immune cells from cervical lymph nodes from donor mice with or without periodontitis were transferred to recipient mice. Then, each group was colonised by Porphyromonas gingivalis (Pg), Fusobacterium nucleatum (Fn) and Prevotella intermedia (Pi) in periodontal tissue for 4 weeks. Intraperitoneal glucose-tolerance tests were performed in recipient mice after transfer (not shown), after colonisation (B) and after 4 weeks of high-fat diet (HFD) (D); (C) and (E) glycaemic index. After colonisation (±): HTC+NC-Co (black bar n=4): healthy transfer+colonisation, PTC+NC-Co (green bar n=4) periodontitis transfer+colonisation, HTC+NC (blue bar n=4) and PTC+NC (purple bar n=4) periodontitis transfer+no colonisation and after 4 weeks of HFD: HTC+HFD-Co (black bar n=4): healthy transfer+colonisation, PTC+HFD-Co (green bar n=4) periodontitis transfer+colonisation, HTC+HFD (blue bar n=4) and PTC+HFD (purple bar n=4) periodontitis transfer+no colonisation. Data are mean±SEM. Significant results when ***p<0.001 when compared to HTC+NC as determined by one-way analysis of variance (ANOVA) followed by Tukey's test (C) and two-way ANOVA with Bonferroni's post-test (B).

Figure 4

Pre-treatment with inactivated Porphyromonas gingivalis (Pg) prevents periodontitis-aggravated glucose intolerance in high-fat diet (HFD)-fed mice. (A) Mice were injected by 10⁶ colony-forming unit (CFU) of inactivated Pg or inactivated Fusobacterium nucleatum (Fn) or inactivated Prevotella intermedia (Pi) or a mix of all inactivated bacteria or vehicle solution. One month later, mice were colonised by Pg, Fn, Pi and/or by vehicle solution for 1 month and then randomised into seven groups: NC-Vehicles (vehicle+normal chow, black bar, n=4), HFD (vehicle+HFD, red bar, n=4), high-fat diet colonised (HFD-Co) (vehicle+HFD+colonisation, green bar, n=4), HFD-Co+I B mix (inactivated mix bacteria+colonisation+HFD, black blue bar, n=4), HFD-Co+I Pg (inactivated Pg+colonisation+HFD, purple bar, n=4), HFD-Co+I Fn (inactivated Fn+colonisation+HFD, light blue bar, n=4) and HFD-Co+I Pi (inactivated Pi+colonisation+HFD, orange bar, n=4). Intraperitoneal glucose-tolerance test (IpGTT) and glycaemic index were assessed for each group after 3 months of HFD (B–F); (G) measurement of immunoglobulin G antibodies specific to lipopolysaccharide (LPS) of Pg in blood. (H) Alveolar bone loss was explored after experimental procedures for each group. Data are mean±SEM. Significant results when: **p<0.01, ***p<0.001 and ****p<0.0001 when compared to HFD vehicles, ^§p<0.05 and ^§§§§p<0.0001 when compared to NC vehicles and ^#p<0.05 and ^####p<0.0001 when compared to HFD-Co as determined by two-way analysis of variance (ANOVA) with Bonferroni's post-test for (B), (C), (D) and (E) and one-way ANOVA followed by Tukey's post-test for (F) and (G).

Figure 5

Lipopolysaccharide (LPS) from Porphyromonas gingivalis (Pg) aggravates high-fat diet (HFD)-induced metabolic diseases. (A) Mice were implanted with Pg-LPS or vehicle releasing minipumps for 1 month; then, mice were fed an HFD for 1 month. After 1 month of diet, glucose-tolerance (B), glycaemic index (C), insulin sensitivity (E) and ratio fat/lean mass (D) were assessed in each group: (Pg-LPS, red bar, n=7) or (NaCl, black bar, n=7). Significant results when: *p<0.05, **p<0.01 and ***p<0.001 when compared to HFD+vehicle as determined by one-way analysis of variance (ANOVA) followed by Tukey's test, except for ‘B’ (two-way ANOVA and Bonferroni's post-test).