Pg, Fn and Pi periodontal pathogens promote periodontal, cervical and systemic immune disorders together with periodontal microbiota dysbiosis during an HFD

Pg, Fn and Pi,31 periodontal pathogens, are drivers of the development of periodontitis in mice. Here, to demonstrate that periodontitis is an aggravating risk factor for diet-induced metabolic disease, we generated a unique mouse model. First, periodontitis was induced by colonising 5-week-old WT C57Bl6/J female mice with all three pathogens; then, mice were fed with an NC or a diabetogenic/not obesogenic HFD (figure 1A).

We validated our model by showing periodontal pathogens-induced mandibular ABL, a feature of periodontitis, on NC. Moreover, this parameter was worsened on HFD (figure 1B, C). Then, we studied the periodontal tissue looking for a putative inflammatory status. As shown in figure 1D, NC-fed colonised mice displayed a significant increased gene expression for all the analysed cytokines. Moreover, the pro-inflammatory effect due to TNF-α and PAI-1 was increased by HFD (figure 1D). Subsequently, given this evidence, we analysed histological sections of hemi-mandibles. We showed by H&E staining that cells infiltrated the periodontal tissue (figure1E) after the colonisation with the periodontal pathogens on NC. In addition, we characterised the cell types by immunostaining and showed an increased macrophage (cells F4/80+), lymphocyte T (cells CD3+) and lymphocyte B (cells CD20+) number in the same experimental conditions. In response to the HFD treatment, the number of cells increased when compared to NC-fed mice. Additionally, in colonised HFD-fed mice the number of immune cells even further increased over that of NC, NC-Co and HFD mice, showing the impact of the dietary treatment and of the colonisation on the inflammatory process in periodontal tissue (figure 1E).

Next, to identify whether periodontitis and local inflammation may be associated with an impaired immune system, we quantified local (cervical lymph node) and systemic (spleen) adaptive and innate immune system cells. HFD feeding increased the number of cells in both cervical lymph node and spleen when compared to NC-fed mice. Interestingly, periodontitis blunted this increase in HFD-fed mice only (figure 1F, H). In the latter, this variation was due to a strong reduction in the frequency of innate CD11b+ cells (gating on CD3-CD19-CD11c-CD11b+) in cervical lymph nodes and spleen, whereas periodontitis increased the frequency of T-lymphocyte and B-lymphocyte (CD4+, CD8+ and CD19+) during HFD only (figure 1G, I).

To further explore the systemic effect of periodontitis, we analysed immune cells in blood, where the above reported modifications were confirmed for all cell types and especially for dendritic cells (CD11b+CD11c+) and innate CD11b+CD11c− cells (figure 1J). By contrast, periodontitis had no significant impact on any cell type whatever the tissue under NC (figure 1F–J). Indeed, the periodontal colonisation increased the number of antibodies anti-Pg on NC, whereas the HFD treatment reduced IgG serum levels and antibodies anti-Pg in colonised mice only (table 1). Conversely, HFD increased IgG serum levels independently of colonisation at both 2 and 3 months of treatment (table 1). Moreover, the periodontitis increased blood IL-6 on HFD and decreased blood interferon-γ concentrations on HFD and NC at 3 months (table 1).

Then, to evaluate whether the change in immune features was linked to a periodontal microbiota dysbiosis, we studied the composition of periodontal microbiota on both NC-fed and HFD-fed colonised mice. On NC, colonisation significantly increased the genus Lactococcus, as shown by the cladogram based on the LDA effect size (figure 1K). On HFD, colonisation significantly increased the Porphyromonadaceae family, in accordance with Pg colonisation (figure 1K). We also showed that HFD induces a periodontal microbiota dysbiosis based on the significant increase in genera Bacteroides, Clostridium and Ruminococcus. The LDA scores and the details (Phyla to Species) for all the periodontal microbiota changes are reported in online supplementary figure S1. Interestingly, periodontitis affected the overall periodontal microbiota profile of HFD-fed mice to a greater extent than NC-fed mice, as reported by cluster analysis at the family level (figure 1L).

We also investigated whether colonisation with periodontal pathogens may induce changes in the gut microbiota. In NC-fed mice, the above periodontal microbiota changes were associated with tiny modifications of gut microbiota due to colonisation, which increased members of Actinobacteria and Deltaproteobacteria groups (see online supplementary figure S2A). In HFD-fed mice, we also observed tiny modifications of gut microbiota due to colonisation (only unclassified bacteria), whereas HFD increased the intestinal proportion of Proteobacteria (see online supplementary figure S2B). All the gut microbial profiles in each group from Phyla to Species are provided in online supplementary figure S2C–H.

Altogether, this data set provides evidence that periodontal pathogens induce periodontitis in mice, alter periodontal microbiota and that the subsequent periodontitis aggravates HFD-induced periodontal and systemic inflammation.

Periodontitis increases HFD-induced metabolic diseases

To demonstrate that periodontal pathogen-induced periodontitis may represent an aggravating risk factor for diet-induced metabolic diseases, we characterised glucose metabolism in response to the nutritional stress. Our data show that periodontitis aggravated the HFD-induced glucose intolerance by the first and up to the third month of treatment (figure 2A, C, E). These data were associated with a progressive and significant increase in the fat/lean mass ratio (figure 2B, D, F). Furthermore, according to the time course, periodontitis increased plasma leptin and insulin concentrations on HFD at 3 months (table 1). Insulin resistance (indexed by the glucose-infusion rate (GIR)), as assessed by the euglycemic/hyperinsulinemic clamp technique, was induced by the periodontitis in HFD-fed mice only (figure 2G). Importantly, ABL was strongly and significantly correlated (R²=0.52; p<0.0001) with insulin resistance (figure 2H).

Then, we analysed by PCoA whether correlations may exist between families abundance in periodontal microbiota and metabolic parameters such as GIR, glycaemic index (GI3), fasted glucose (FG3), ABL, BW3, cytokines for gingival inflammation, blood immunoglobulins levels and gingival (IgG) cytokines at 3 months. We found that Lactobacillaceae abundance in periodontal microbiota positively and significantly correlated with GIR (figure 2I, up-left inset). By contrast, Porphyromonadaceae abundance was negatively and significantly correlated with GIR (figure 2I, up-right inset), but positively with ABL (figure 2I, down-right inset). Overall, the PCoA allowed identifying a specific cluster for each group, despite a tiny superposition between the NC-Co and the HFD group. Furthermore, Porphyromonadaceae abundance was associated with gingival TNF-α expression (figure 2I, down-left inset), whereas we also found a positive and significant correlation between ABL and fasted glycaemia at 3 months (figure 2I, middle-right inset).

Altogether, these data show that periodontitis aggravates HFD-induced glucose intolerance and insulin resistance together with changes in the periodontal microbiota, among which some of them are associated with the metabolic phenotypes.

Pg colonisation recruits cells from the adaptive immune system to control glucose tolerance

To demonstrate that the adaptive immune system was triggered by the change in periodontal microbial ecology and was a causal mechanism responsible for the deleterious impact of the periodontal pathogens on metabolic disease, we first transferred the cervical lymph-node cells from mice with or without periodontitis to healthy recipient mice (figure 3A). In such conditions, the glucose tolerance was similar in both groups of recipient mice (not shown), suggesting that other factors were required to trigger the metabolic disease. Hence, we challenged the recipient mice with the periodontal pathogens during 4 weeks after the cell transfer (figure 3A) and demonstrated that the glucose tolerance was improved in mice that received the immune cells from the infected mouse when compared to those that received immune cells from a non-infected mouse (figure 3B, C). BW gain was not significantly affected (not shown). Then, to study the impact of the colonisation and of the transfer of cervical immune cells from mice with periodontal disease on HFD-induced glucose intolerance, we then challenged the colonised and transferred mice for 4 weeks with HFD. After 4 weeks of HFD, the IPGTT is showing that the protection, although still significantly present in glycaemic index, is starting to vanish (figure 3D, E). The data show that when the HFD-fed mice were transferred with immune cells from mice with periodontitis, the glucose tolerance is improved although modestly (figure 3D, E). By contrast, the transfer of immune cells by itself, without colonising the recipient mice, was not sufficient to impact glucose tolerance (figure 3B–E). However, glucose metabolism could be impacted when the immune cells were specifically adapted to the periodontal pathogens. This suggests that both nutritional stress and periodontitis are required factors to trigger metabolic phenotypes.

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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).

In a second set of experiments, to further validate the role of the adaptive immune system on the control of glucose tolerance, we immunised the lymphocytes to the periodontal pathogens by treating mice with different sets of inactivated periodontal pathogens (figure 4A). The intramuscular treatment with the three inactivated periodontal pathogens prevented the above reported aggravating effects of periodontitis on HFD-induced glucose intolerance at 1 month (see online supplementary figure S3A–F), 2 months (not shown) and 3 months (figure 4B–F). Importantly, this preventive effect was due to Pg since the treatment of the mice with this unique bacterium was sufficient to protect against periodontitis-induced metabolic diseases. Moreover, the specific treatment by Pg prevented the decrease of antibodies anti-Pg observed on HFD after periodontal colonisation at 1 and 3 months (see online supplementary figures S3G and S4G). At 3 months of HFD, the treatment with Pg protected against the periodontal colonisation-induced ABL in periodontal tissue (figure 4H).

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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).

The LPS from Pg enhance HFD-induced metabolic diseases

In the quest for molecular determinants responsible for Pg-aggravated diet-induced metabolic diseases, we evaluated the impact of LPS from Pg (figure 5A). A 1-month continuous infusion of a low rate of Pg-LPS aggravated HFD-induced glucose intolerance and insulin resistance (figure 5B–D). Furthermore, the fat to lean ratio was significantly increased in mice pre-treated with Pg-LPS when compared to saline (vehicle)-pre-treated mice (figure 5E), suggesting that the LPS from Pg could be a major molecular mechanism explaining the metabolic impact of periodontitis on metabolic disease.

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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).