Inflamm-Aging-Related Cytokines of IL-17 and IFN- γ Accelerate Osteoclastogenesis and Periodontal Destruction

Abstract

Periodontal disease (PD), as an age-related disease, prevalent in middle-aged and elderly population, is characterized as inflammatory periodontal tissue loss, including gingival inflammation and alveolar bone resorption. However, the definite mechanism of aging-related inflammation in PD pathology needs further investigation. Our study is aimed at exploring the effect of inflamm-aging-related cytokines of interleukin-17 (IL-17) and interferon-γ (IFN-γ) on osteoclastogenesis in vitro and periodontal destruction in vivo. For receptor activator of nuclear factor-κB ligand- (RANKL-) primed bone marrow macrophages (BMMs), IL-17 and IFN-γ enhanced osteoclastogenesis, with the expression of osteoclastogenic mRNA (TRAP, c-Fos, MMP-9, Ctsk, and NFATc1) and protein (c-Fos and MMP-9) upregulated. Ligament-induced rat models were established to investigate the role of IL-17 and IFN-γ on experimental periodontitis. Both IL-17 and IFN-γ could enhance the local inflammation in gingival tissues. Although there might be an antagonistic interaction between IL-17 and IFN-γ, IL-17 and IFN-γ could facilitate alveolar bone loss and osteoclast differentiation.

Figure 1

Effect of IL-17 or/and IFN-γ on differentiation of preosteoclasts. (a–k) In condition 1, BMMs were cultured with M-CSF (30 ng/ml) for 3 days and then with 20 ng/ml RANKL for another 3 days. On day 6, IL-17 or/and IFN-γ of different concentrations was added. (l, m) The results of TRAP staining of each group are presented (scale bar = 500 μm). Osteoclasts were recognized as TRAP-positive multinucleated cells (≥3 nuclei). (l) The number of TRAP-positive osteoclasts and (m) the area of TRAP-positive osteoclasts were counted and presented (mean ± standard deviation, n = 3). ^#p < 0.05 compared with the control group; ^&p < 0.05 compared with the IL-17+IFN-γ group.

Figure 2

Effects of IL-17 (1 ng/ml) or/and IFN-γ (0.2 ng/ml) on the expression of osteoclastogenic genes in vitro. The mRNA expression of osteoclast-related genes (a) c-Fos, (b) NFATc1, (c) Ctsk, (d) MMP-9, and (e) TRAP was detected with different concentrations of IL-17 or/and IFN-γ using RT-qPCR. Data were standardized to GAPDH expression and shown as a fold change relative to the control group (mean ± standard deviation, n = 3). ^#p < 0.05 compared with the control group; ^&p < 0.05 compared with the IL-17+IFN-γ group.

Figure 3

Role of IL-17 (5 μg/ml) or/and IFN-γ (1 μg/ml) on local gingival inflammation in an experimental periodontitis rat model. (a) HE staining showed the periodontal morphology in the interradicular regions of the first maxillary molars of the NC, NS, IL-17, IFN-γ, and IL-17+IFN-γ groups (scale bar = 100 μm). (b) Quantitative analysis was performed on the number of inflammatory cells of each group (mean ± standard deviation, n = 5). ^∗p < 0.05 compared with the NC group, ^#p < 0.05 compared with the NS group, and ^&p < 0.05 compared with the IL-17+IFN-γ group. T: tooth; AB: alveolar bone.

Figure 4

Effect of IL-17 (5 μg/ml) or/and IFN-γ (1 μg/ml) on proinflammatory cytokine expression in vivo. Immunohistochemical staining was used to examine the level of (a) IL-1β, (b) TNF-α, and (c) IL-6 expression in gingival tissues (scale bar = 20 μm). Quantitative analysis results of (d) IL-1β-, (e) TNF-α-, and (f) IL-6-positive cells are shown. The mRNA level of (g) IL-1β, (h) TNF-α, and (i) IL-6 in gingival tissues after 1 week and 2 weeks was detected by RT-qPCR. Data were presented by mean ± standard deviation, n = 5. ^∗p < 0.05 compared with the NC group, ^#p < 0.05 compared with the NS group, and ^&p < 0.05 compared with the IL-17+IFN-γ group.

Figure 5

Effect of IL-17 (5 μg/ml) or/and IFN-γ (1 μg/ml) on alveolar bone loss and osteoclast differentiation. (a) Reconstructed micro-CT images showed the buccal view of the first maxillary molars. The distance from CEJ to ABC indicated bone loss, shown by the red lines. Quantitative analysis results of the (b) CEJ-ABC distance and (c) BV/TV are shown as mean ± standard deviation. (d) TRAP staining was used to evaluate the formation of osteoclasts (red arrows) for the NC, NS, IL-17, IFN-γ, and IL-17+IFN-γ groups (scale bar = 50 μm). (e) Quantitative analysis was performed on the number of TRAP-positive multinucleated cells in the interradicular regions of the first maxillary molars (mean ± standard deviation, n = 5). ^∗p < 0.05 compared with the NC group, ^#p < 0.05 compared with the NS group, and ^&p < 0.05 compared with the IL-17+IFN-γ group. ABC: alveolar bone crest; CEJ: cemento-enamel junction; BV: bone volume; TV: tissue volume; T: tooth; AB: alveolar bone.