Hyperglycemia Aggravates Periodontitis via Autophagy Impairment and ROS-Inflammasome-Mediated Macrophage Pyroptosis

Abstract

Macrophage pyroptosis drives the secretion of IL-1β, which has been recently reported to be a featured salivary biomarker for discriminating periodontitis in the presence of diabetes. This study aimed to explore whether macrophage pyroptosis plays a role in the development of diabetes mellitus-periodontitis, as well as potential therapeutic strategies. By establishing a model of experimental diabetes mellitus-periodontitis in rats, we found that IL-1β and gasdermin D were highly expressed, leading to aggravated destruction of periodontal tissue. MCC950, a potent and selective molecule inhibitor of the NLRP3 inflammasome, effectively inhibited macrophage pyroptosis and attenuated alveolar bone losses in diabetes mellitus-periodontitis. Consistently, in vitro, high glucose could induce macrophage pyroptosis and thus promoted IL-1β production in macrophages stimulated by lipopolysaccharide. In addition, autophagy blockade by high glucose via the mTOR-ULK1 pathway led to severe oxidative stress response in macrophages stimulated by lipopolysaccharide. Activation of autophagy by rapamycin, clearance of mitochondrial ROS by mitoTEMPO, and inhibition of inflammasome by MCC950 could significantly reduce macrophage pyroptosis and IL-1β secretion. Our study demonstrates that hyperglycemia promotes IL-1β production and pyroptosis in macrophages suffered by periodontal microbial stimuli. Modulation of autophagy activity and specific targeting of the ROS-inflammasome pathway may offer promising therapeutic strategies to alleviate diabetes mellitus-periodontitis.

Keywords: MCC950; autophagy; hyperglycemia; oxidative stress; periodontal disease; pyroptosis.

Figure 1

Diabetes mellitus–periodontitis presented exacerbated bone resorption and aberrant activation of macrophage pyroptosis. (A) Representative 3D reconstructions and transverse section images of control (CON), diabetes mellitus (DM), ligature-induced periodontitis (Lig), and DM + Lig groups. The green area displays the exposure of the root (scale bar: 1 mm). (B) Bone volume/total volume (BV/TV). (C) Representative images of HE staining (upper panel) in coronal sections of periodontal tissue (scale bar: 1 mm). Representative images of TRAP staining (lower panel) in the furcation region of the second molar (scale bar: 200 μm). (D) Distances between the CEJ and the ABC. (E) Quantitative analysis of osteoclast number at the alveolar bone. (F) Immunohistochemistry staining against IL-1β and GSDMD of periodontal tissues (scale bar: 200 μm). Red arrows demonstrate GSDMD positive cells. (G) Quantitative analysis of IL-1β and GSDMD (total GSDMD and cleaved GSDMD) in the gingiva and the furcation of the second molar. (H) Representative images of immunofluorescence staining of macrophage marker (CD68) and pyroptosis marker (GSDMD) in the periodontium of the Lig group and the DM + Lig group. Nuclei were stained with DAPI (blue). Scale bar, 100 μm. (I) Pearson’s R value shows the degree of co-localization of GSDMD and CD68 determined using ImageJ software. The number of GSDMD/CD68 double-stained cells was analyzed. Data in (B,D,E,G,I) are shown as mean ± SD (n = 4). * p < 0.05. ** p < 0.01. *** p < 0.001. Ab, alveolar bone; G, gingiva; D, dentin; DM, diabetes mellitus; CEJ-ABC, cementoenamel junction and alveolar bone crest; AOD, average optical density.

Figure 1

Diabetes mellitus–periodontitis presented exacerbated bone resorption and aberrant activation of macrophage pyroptosis. (A) Representative 3D reconstructions and transverse section images of control (CON), diabetes mellitus (DM), ligature-induced periodontitis (Lig), and DM + Lig groups. The green area displays the exposure of the root (scale bar: 1 mm). (B) Bone volume/total volume (BV/TV). (C) Representative images of HE staining (upper panel) in coronal sections of periodontal tissue (scale bar: 1 mm). Representative images of TRAP staining (lower panel) in the furcation region of the second molar (scale bar: 200 μm). (D) Distances between the CEJ and the ABC. (E) Quantitative analysis of osteoclast number at the alveolar bone. (F) Immunohistochemistry staining against IL-1β and GSDMD of periodontal tissues (scale bar: 200 μm). Red arrows demonstrate GSDMD positive cells. (G) Quantitative analysis of IL-1β and GSDMD (total GSDMD and cleaved GSDMD) in the gingiva and the furcation of the second molar. (H) Representative images of immunofluorescence staining of macrophage marker (CD68) and pyroptosis marker (GSDMD) in the periodontium of the Lig group and the DM + Lig group. Nuclei were stained with DAPI (blue). Scale bar, 100 μm. (I) Pearson’s R value shows the degree of co-localization of GSDMD and CD68 determined using ImageJ software. The number of GSDMD/CD68 double-stained cells was analyzed. Data in (B,D,E,G,I) are shown as mean ± SD (n = 4). * p < 0.05. ** p < 0.01. *** p < 0.001. Ab, alveolar bone; G, gingiva; D, dentin; DM, diabetes mellitus; CEJ-ABC, cementoenamel junction and alveolar bone crest; AOD, average optical density.

Figure 2

MCC950 rescued the bone loss of diabetes mellitus–periodontitis in rats. (A) Representative 3D reconstructions and X-ray photos of the Lig + Vehicle, Lig + MCC950, DM + Lig + Vehicle, and DM + Lig + MCC950 groups. The green area displays the exposure of the root (scale bar: 1 mm). (B) BV/TV of the above 4 groups was measured. (C) Representative images of HE staining (scale bar: 1 mm). (D) Distances between the CEJ and the ABC. (E) Immunohistochemistry staining against IL-1β of periodontal tissues (Scale bar: 200 μm). (F) Quantitative analysis of IL-1β in the gingiva and the furcation of the second molar. (G) Representative images of immunofluorescence staining of CD68 and GSDMD in the periodontium of the DM + Lig + Vehicle and DM + Lig + MCC950 groups. Nuclei were stained with DAPI (blue). Scale bar, 100 μm. (H) The number of GSDMD/CD68 double-stained cells was analyzed. Data in (B,D,F,H) are shown as mean ± SD (n = 4). ** p < 0.01. *** p < 0.001. G, gingiva; D, dentin; Ab, alveolar bone; CEJ-ABC, cementoenamel junction and alveolar bone crest; AOD, average optical density.

Figure 3

High glucose aggravated LPS-induced macrophage pyroptosis and IL-1β production. (A,B) THP-1 macrophages were treated by different concentrations of glucose for 48 h. The expression of the GSDMD and N-GSDMD proteins was assessed. (C–G) Macrophages were cultivated in normal glucose (5.5 mM, NG) or high glucose (30 mM, HG) medium for 24 h, and were then stimulated with or without LPS (1 μg/mL) for 24 h. (C,D) GSDMD, N-GSDMD and pro-IL-1β were assessed by Western blot. (E) Flow cytometry of macrophages stained by PI. (F) Analysis of LDH release to measure cell lysis. (G) Secretion of IL-1β in the supernatant by ELISA. Values are mean ± SD. * p < 0.05. ** p < 0.01. *** p < 0.001. PI, propidium iodide; LDH, lactate dehydrogenase.

Figure 4

High glucose promoted ROS generation in macrophages stimulated by LPS. (A–D) THP-1 macrophages were cultivated in NG or HG medium for 24 h, and were then stimulated with or without LPS (1 μg/mL) for 24 h. (A) THP-1 macrophages were stained with mitoSOX and analyzed by flow cytometry. (B) Transmission electron microscope (TEM) images (upper panel) of THP-1 macrophages (scale bar: 500 nm). The red arrow represents dysfunctional mitochondria and the green arrow represents autolysosome. JC-1 staining (lower panel) to detect mitochondrial membrane potential (MMP) in THP-1 macrophages (scale bar: 400 μm). (C) Quantitative analysis of JC-1 using red/green ratio. (D) Macrophages were stained with MitoTracker Green and LysoTracker Red. The colocalization of mitochondria and lysosome dots were observed using confocal microscopy (scale bar: 10 μm). ** p < 0.01. *** p < 0.001. TEM, transmission electron microscope; MMP, mitochondrial membrane potential.

Figure 5

HG activates the mTOR pathway and decreases autophagic flux in THP-1 macrophages. (A–C) THP-1 macrophages were cultivated in NG or HG medium for 24 h and were then stimulated with or without LPS (1 μg/mL) for 24 h. For the last 2 h, cells were treated with or without bafilomycin (Baf, 100 nM). LC3B-II and p62 protein levels were measured by Western blot and quantified using ImageJ analysis. LC3B-II and p62 induction by bafilomycin demonstrates autophagic flux. (D,E) Macrophages were transfected with mRFP-GFP-LC3 adenovirus for 6 h (MOI = 200) and were then stimulated as illustrated above. (D) Representative microscopic images of LC3 puncta (scale bar: 10 μm). (E) Quantification based on counting LC3 puncta per cell. (F–I) Detection of mTOR pathway expression of THP-1 macrophages stimulated as in Figure 3A (F,G) and Figure 3C (H,I). Values are mean ± SD. * p < 0.05. ** p < 0.01. *** p < 0.001.

Figure 6

Modulation of autophagy and ROS-inflammasome pathway reduced IL-1β secretion and pyroptosis. (A–D) Macrophages were pretreated with rapamycin (200 nM) 2 h before LPS stimulation. Proteins related to mTOR pathway, autophagy (A), and pyroptosis (B) were assessed by Western blot. (C,D) Representative microscopic images and quantification of LC3 puncta (scale bar: 10 μm). (E) MCC950 (5 μM) and mitoTEMPO (10 μM) were added to the media 2 h before LPS stimulation and were maintained until assays were performed. GSDMD, N-GSDMD, and pro-IL-1β were measured. (F) LDH in the supernatant to measure cell lysis. (G) Measurement of IL-1β by ELISA. Data in (D,F,G) are shown as mean ± SD. * p < 0.05. ** p < 0.01. *** p < 0.001. LDH, lactate dehydrogenase.

Figure 7

Diagram illustrates that hyperglycemia could promote macrophage pyroptosis via autophagy impairment and ROS-inflammasome pathway and aggravate inflammatory diseases such as periodontitis.