Effect of Salivary Exosomal miR-25-3p on Periodontitis With Insulin Resistance

Abstract

Periodontitis is caused by an oral microbial dysbiosis-mediated imbalance of the local immune microenvironment, which is promoted by insulin resistance and obesity. The prevalence and severity of periodontitis is higher in patients with type 2 diabetes than in healthy individuals, possibly because of differences in immune responses. The level of glycemic control also affects the saliva profile, which may further promote periodontal disease in diabetes patients. Therefore, we compared the salivary exosomal miRNA profiles of patients with type 2 diabetes with those of healthy individuals, and we found that exosomal miR-25-3p in saliva is significantly enriched (by approximately 2-fold, p < 0.01) in obese patients with type 2 diabetes. We also identified CD69 mRNA as a miR-25-3p target that regulates both activation of γδ T cells and the inflammatory response. Knockdown of CD69 increased (by approximately 2-fold) interleukin-17A production of γδ T cells in vitro. To evaluate the role of exosomal miRNA on progression of periodontitis, we analyzed regional immune cells in both periodontal tissues and lymph nodes from mice with periodontitis. We found that diet-induced obesity increased the population of infiltrating pro-inflammatory immune cells in the gingiva and regional lymph nodes of these mice. Treatment with miR-25-3p inhibitors prevented the local in vivo inflammatory response in mice with periodontitis and diet-induced obesity. Finally, we showed that suppression of interleukin 17-mediated local inflammation by a miR-25-3p inhibitor alleviated (by approximately 34%) ligature-induced periodontal alveolar bone loss in mice. Taken together, these data suggest that exosomal miR-25-3p in saliva contributes to development and progression of diabetes-associated periodontitis. Discovery of additional miR-25-3p targets may provide critical insights into developing drugs to treat periodontitis by regulating γδ T cell-mediated local inflammation.

Keywords: diabetes; exosome; inflammation; insulin resistance; miRNA; periodontitis; saliva.

Figure 1

Diet-induced obesity promotes the local inflammatory response in male mice with periodontitis. (A–C) Representative flow cytometry plots and percentages of CD44⁺CD62L⁻ and CD44⁻CD62L⁻ cells in the CD4⁺ and CD8⁺ T cell populations from submandibular lymph nodes of either NCD-fed (n = 5) or HFD-fed (n = 5) male mice with periodontitis 9 days after ligature placement. (D–G) Frequency of TNF-α- or IL-17A-producing cells in the populations of CD4⁺ or CD8⁺ T cells in periodontitis-induced mice fed with either a NCD or a HFD. (H) Frequency of γδ T cells in submandibular lymph nodes of periodontitis-induced mice fed with either a NCD or a HFD. (I–K) Representative flow cytometry plots and percentages of either TNF-α- or IL-17A-producing cells in the population of γδ T cells from periodontitis-induced mice fed with either a NCD or a HFD. Data represent mean values of more than three independent experiments. Data are expressed as the mean ± SEM. *p < 0.05, **p < 0.01 [(C, G, H, K): two‐tailed t tests].

Figure 2

Salivary exosomal miRNA profiles in healthy individuals and in patients with type 2 diabetes. (A) Heatmap showing differentially enriched miRNAs in salivary exosomes from patients with type 2 diabetes as compared with those from normoglycemic healthy individuals. (B) Expression of differentially enriched miRNAs in salivary exosomes was measured by real-time PCR. (C) Co-culturing γδ T cells with salivary exosomes (from either healthy individuals or patients with type 2 diabetes) with anti-CD3 (2 μg/mL), anti-IL-2 (10 ng/mL), and isopentenyl pyrophosphate (5 μg/mL). (D) The population of IL‐17A-producing cells under culture conditions of γδ T cell activation at the indicated concentrations of miR-25-3p inhibitors. (E) Transcription of Il17a and Rorc in differentiated CD4⁺ T cells under T_H17 differentiation-inducing culture conditions at the indicated concentrations of miR-25-3p inhibitors. (F, G) γδ T cells were transfected with either control siRNA or anti-CD69 siRNA for 48 hr. Expression levels of IL-17A were measured by using qPCR and FACS analysis. Data represent mean values of more than three independent experiments. Data are expressed as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 (B, C, F, G), two‐tailed t tests; (E), one‐way ANOVA.

Figure 3

Inhibition of miR-25-3p attenuates periodontal inflammation in male mice with diet-induced obesity and periodontitis. (A–C) Representative flow cytometry plots and percentages of CD44⁺CD62L⁻ and CD44⁻CD62L⁻ cells in the CD4⁺ and CD8⁺ T cell populations from submandibular lymph nodes of either vehicle-treated (n = 5) or miR-25-3p inhibitor-treated (n = 5) male mice with periodontitis 9 days after ligature placement. (D, E) Representative flow cytometry plots and frequencies of CD4⁺CD25⁺Foxp3⁺ regulatory T cells in HFD-fed mice treated with either miR-25-3p inhibitor or vehicle. (F–I) Percentages of TNF-α- or IL-17A-producing cells in the population of CD4⁺ or CD8⁺ T cells in periodontitis-induced mice treated with either miR-25-3p inhibitor or vehicle. (J, K) Percentages of γδ T cells in submandibular lymph nodes of periodontitis-induced mice treated with either miR-25-3p inhibitor or vehicle. (L–N) Representative flow cytometry plots and percentages of TNF-α- or IL-17A-producing cells in the population of γδ T cells from periodontitis-induced mice treated with either miR-25-3p inhibitor or vehicle. Data represent mean values of more than three independent experiments. Data are expressed as the mean ± SEM. *p < 0.05, **p < 0.01 (C, E, I, K, N), two‐tailed t tests.

Figure 4

Treatment with miR-25-3p inhibitor decreases the populations of effector T cells and IL-17-producing cells in inflamed gingiva of obese male mice with periodontitis. (A, B) Size of the CD44⁺ population in inflamed gingiva-infiltrating CD4⁺ and CD8⁺ T cells from periodontitis-induced obese male mice treated with either miR-25-3p inhibitor (n = 5) or vehicle (n = 5). (C, D) Median fluorescence intensity of CD279 was measured in inflamed gingiva-infiltrating CD4⁺ and CD8⁺ T cells of periodontitis-induced obese mice treated with either miR-25-3p inhibitor or vehicle. (E) Representative flow cytometry plots and percentages in inflamed gingiva-infiltrating IL-17A-producing cells of the population of CD4⁺ T cells in periodontitis-induced obese mice treated with either miR-25-3p inhibitor or vehicle. (F) Representative flow cytometry plots and percentages in inflamed gingiva-infiltrating IL-17A-producing cells of the population of γδ T cells from periodontitis-induced obese mice treated with either miR-25-3p inhibitor or vehicle. (G, H) Synchrotron radiation micro-computed tomography analysis of periodontitis-induced bone loss in mice fed either a NCD or HFD and treated with either miR-25-3p inhibitor or vehicle. The upper red-dotted line indicates the cemento-enamel junction; the lower red-dotted line indicates the alveolar bone crest (left panel). (I) The distances from cemento-enamel junctions to alveolar bone crests were measured around the second molars on transaxial and sagittal sections along the buccal, palatal, and interdental axes. Distances were statistically analyzed in mice fed either a NCD or a HFD and treated with either miR-25-3p inhibitor or vehicle. Data represent mean value of more than three independent experiments. Data are expressed as the mean ± SEM. *p < 0.05, **p < 0.01 (A–F), two‐tailed t tests; (G–I), one‐way ANOVA.

Figure 5

Graphical abstract. Exosomal miR-25-3p in saliva is highly enriched in patients with type 2 diabetes. Salivary exosomal miR-25-3p promotes local inflammation and periodontal bone loss in mice by activating T_H17 cells and γδ T cells.