Hyperglycemia modulates M1/M2 macrophage polarization via reactive oxygen species overproduction in ligature-induced...

Periodontitis in diabetic patients is characterized by enhanced inflammation and aggravated tissue damage in comparison with that in non-diabetic counterparts. The progression of periodontal damage under diabetic condition can be partly ascribed to hyperglycemia-induced disturbance between immune activation and inflammation resolution, where macrophages are capable of participating given their plasticity in response to different stimuli. Herein, we aimed to investigate the changes of macrophage polarization in periodontitis under diabetic condition and the underlying mechanism.

Type-1 diabetes was induced by the injection of streptozotocin (STZ, 60 mg/kg) in Sprague-Dawley rats. Rats in N-acetyl cysteine (NAC)-treated groups received NAC dissolved in drinking water (200 mg/kg/day). Experimental periodontitis was induced by ligating 3-0 silk around left maxillary second molars for 4 weeks. Alveolar bone destruction was tested by micro-computed tomography and tartrate-resistant acid phosphatase (TRAP) staining. M1/M2 macrophage polarization in periodontal tissue was detected by immunohistochemistry staining. RAW264.7 were cultured in normal glucose (5.5 mM) or high glucose environment (25 mM) with or without NAC (8 mmol/L). LPS (100 ng/ml) and IL-4 (20 ng/ml) were used to induce M1 macrophages and M2 macrophages, respectively. M1/M2 macrophage polarization was detected by qRT-PCR, immunofluorescent staining, and flow cytometry. Reactive oxygen species (ROS) accumulation was detected by fluorogenic probes. RANKL (100 ng/ml) were applied to induce osteoclastogenic differentiation of RAW264.7, and osteoclast formation was examined by TRAP staining.

Rats with diabetes displayed enhanced macrophages infiltration and M1 macrophage polarization in periodontal lesions compared with vehicle-treated rats. Under LPS or IL-4 stimulation, high glucose culture of RAW264.7 elevated ROS level and increased the expression of M1 macrophage markers (iNOS, TNF-α, and IL-6) whereas decreased the expression of M2 macrophage markers (Arg-1 and CD206). Supernatants of high glucose-treated M1/M2 macrophages enhanced osteoclast formation compared to normal glucose-cultured cells. Decreasing ROS level via NAC partially reversed the effect of high glucose on M1/M2 macrophage polarization. Meanwhile, daily intake of NAC in rodent models inhibited M1 macrophage polarization, which subsequently ameliorated alveolar bone loss and decreased osteoclast numbers in periodontitis in diabetic rats.

These findings demonstrated that hyperglycemia could polarize macrophage toward M1 macrophages via overproducing ROS under inflammatory condition, which might take responsibility for aggravated periodontal damage in periodontitis under diabetic condition. Inhibiting M1 macrophages and restoring M2 macrophages by ROS scavenger is hopefully a potential adjunct treatment strategy for diabetic periodontitis.