Porphyromonas gingivalis induces an inflammatory response via the cGAS-STING signaling pathway in a periodontitis mouse model

Abstract

Periodontitis is an inflammatory disease initiated by periodontopathogenic bacteria in the dental plaque biofilms. Understanding the role of Porphyromonas gingivalis (P. gingivalis), a keystone pathogen associated with chronic periodontitis, in the inflammatory response is crucial. Herein, we investigated whether P. gingivalis infection triggers the expression of the type I IFN gene and various cytokines and leads to activation of the cGAMP synthase-stimulator of IFN genes (cGAS-STING) pathway both in vitro and in a mouse model. Additionally, in an experimental model of periodontitis using P. gingivalis, Sting^Gt mice showed lower levels of inflammatory cytokines and bone resorption than wild-type mice. Furthermore, we report that a STING inhibitor (SN-011) significantly decreased inflammatory cytokine production and osteoclast formation in a periodontitis mouse model with P. gingivalis. In addition, STING agonist (SR-717) -treated periodontitis mice displayed enhanced macrophage infiltration and M1 macrophage polarization in periodontal lesions compared with that in vehicle-treated periodontitis mice. In conclusion, our results demonstrate that the cGAS-STING signaling pathway may be one of the key mechanisms crucial for the P. gingivalis-induced inflammatory response that leads to chronic periodontitis.

Keywords: Porphyromonas gingivalis; RANKL; cGAS-STING signaling pathway; macrophage polarization; osteoclast; periodontal disease; proinflammatory cytokines.

Figure 1

Successful construction of a Porphyromonas gingivalis-infected animal model of periodontitis. C57BL/6 mice were used to establish the periodontitis model, and uninfected mice served as the control group (n = 4 mice per group). (A) Schematic of the mouse periodontitis model. (B) Three-dimensional reconstruction images and analysis of the cementoenamel junction to alveolar bone crest (CEJ-ABC) distances of maxilla with periodontitis mice (scale bar: 500 μm). Representative X-ray images (scale bar: 1.0 mm) (C) Gingipain (RgpA, KGP) expression analysis of gingival tissues from periodontitis mice by RT–PCR. GAPDH was used as the endogenous control in RT–PCR analysis of mouse specimens. Values are expressed as the mean ± SEM. The p value is indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 2

Porphyromonas gingivalis affects the viability of HGFs and induces the generation of ROS in HGFs. The cells were incubated with P. gingivalis. Cells without any treatment (MOI 0 for 0 h) were used as controls. (A) Diagram of the cell experimental procedure. (B) Cell viability after incubation with P. gingivalis at an MOI of 100, 50, or 0 for 0 h, 4 h, or 6 h as determined by MTT assay. (C) HGFs were preincubated with P. gingivalis (MOI 100 for 6 h) to detect ROS-induced fluorescence (scale bar, 1,000 μm). Statistical analysis of ROS-positive cells per field in the three groups. The data are expressed as the mean ± the SEM of four independent experiments (n = 4). The p value is indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns, no significance; MOI, multiplicity of infection; ROS, reactive oxygen species.

Figure 3

Porphyromonas gingivalis activates the cGAS-STING signaling pathway in vitro. HGFs were infected with P. gingivalis at an MOI of 100 for 6 h. Untreated cells (MOI 0 for 0 h) were used as controls. (A) Total lysates were harvested, and cGAS and STING protein levels in HGFs were confirmed by Western blotting. β-Actin served as a control. (B) Transcript levels of cGAS and STING were measured by qRT–PCR and normalized to uninfected cells under each treatment using the threshold cycle (2^−ΔΔCt) method with GAPDH as the reference gene. (C) Supernatants were collected, and the levels of IL-1β, IL-6, TNF-a, and IFN-β were analyzed by ELISA. (D) Cells were also collected to measure the gene expression of RANKL by qRT–PCR. (E) Western blotting analysis of RANKL expression in HGFs. Values are expressed as the mean ± SEM (n = 4). The p value is indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns, no significance.

Figure 4

The Porphyromonas gingivalis infection-driven cGAS-STING signaling pathway induces type I IFN gene expression in mouse models of periodontitis. The periodontitis model was established using C57BL/6 mice, and untreated animals served as the control group (n = 4 mice per group). (A) Mouse gingival cells were preincubated with DCFH-DA (10 mM) to detect ROS-induced fluorescence. Statistical analysis of ROS-positive cells per field in the two groups. Uninfected mice were used as controls (scale bar, 1,000 μm). (B) Mouse gingival tissue lysates were collected to determine the levels of cGAS and STING by Western blotting. (C) Mouse gingival tissue was collected to measure the gene expression of cGAS and STING by qRT–PCR. (D) Serum samples were collected, and the levels of IL-6, IL-1β, TNF-a and IFN-β were analyzed by ELISA. (E,F) Mouse gingival tissues were also collected to measure the gene expression of IFN-β (E) and RANKL (F) by qRT–PCR. Values are expressed as the mean ± SEM. The p value is indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns, no significance.

Figure 5

Porphyromonas gingivalis decreases proinflammatory cytokine synthesis in periodontitis Sting^Gt mice via the cGAS-STING signaling pathway. Sting^Gt and C57BL/6 mice were used to construct periodontitis models, with C57BL/6 periodontitis mice serving as controls (n = 4 mice per group). (A) Diagram of the STING transgenic (Sting^Gt) mouse periodontitis model. (B) Images showing H&E staining following infection with P. gingivalis. The images shown are 14 dpi for all groups (scale bar, 200 μm). Each image is representative of a group of 4 mice at 14 dpi. Semiquantitative analysis of H&E pictures was also performed. (C) Serum samples were collected, and the levels of IL-6, IL-1β, TNF-a and IFN-β were analyzed by ELISA. (D) Mouse gingival tissues were also collected to measure the gene expression of IFN-β by qRT–PCR. (E–G) The gingival cells were harvested and stained with mAbs specific for F4/80, CD11c, CD206, CCR2, and CCL2 for flow cytometric analysis. The results are expressed as M1 macrophages: F4/80 + CD11c+; M2 macrophages: F4/80 + CD206+. The relative proportion of M1 macrophages and M2 macrophages is shown in (E). The relative proportion of CCR2 (F) and CCL2 (G) in gingival cells induced by P. gingivalis. The results shown are 14 dpi for all groups. Data are expressed as the mean ± SEM. The p value is indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns, no significance.

Figure 6

Porphyromonas gingivalis decreases alveolar bone loss in periodontitis Sting^Gt mice via the cGAS-STING signaling pathway. (A) Three-dimensional reconstruction images and analysis of the cementoenamel junction to alveolar bone crest (CEJ-ABC) distances of maxilla with periodontitis mice (scale bar: 500 μm). Representative X-ray images (scale bar: 1.0 mm) (B) Mouse periodontal tissues were also collected to measure the gene expression of RANKL by qRT–PCR.

Figure 7

Suppression of cGAS/STING ameliorates Porphyromonas gingivalis-induced periodontal disease. P. gingivalis-induced periodontitis mouse groups were injected intraperitoneally with SR-717 (30 mg/kg), SN-011 (10 mg/kg) and vehicle (5% DMSO in PBS) for 7 days of daily dosing and euthanized after the last treatment. Mice with PBS-treated periodontitis served as controls (n = 4 mice per group). (A) Mouse gingival tissue lysates were collected to determine the protein levels of cGAS and STING by Western blotting. (B) Serum samples were collected, and the levels of IL-6, IL-1β, TNF-a and IFN-β were analyzed by ELISA. (C) H&E staining showed hemi-maxillae histopathological changes after treatment with small molecule modulators (scale bar, 200 μm). Semiquantitative analysis of H&E pictures was also performed. (D–F) Gingival cells were harvested and stained with mAbs specific for F4/80, CD11c, CD206, CCR2, and CCL2 for flow cytometric analysis. The results are expressed as M1 macrophages: F4/80 + CD11c+; M2 macrophages: F4/80 + CD206+. The relative proportion of M1 macrophages and M2 macrophages is shown in (D). The relative proportion of CCR2 (E) and CCL2 (F) in periodontal ligament cells induced by P. gingivalis. (G) Mouse gingival tissues were also collected to measure the gene expression of RANKL by qRT–PCR. Data are expressed as the mean ± SEM. The p value is indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns, no significance.