Immunomodulation of Macrophages in Periapical Wound Healing

Background: A periapical lesion is an infection-induced inflammation around the apex of the tooth caused by bacterial invasion and ultimately results in the destruction of bone in the area. To date, the relationship between pathogens and the host immune system in the development of periapical lesions have been extensively investigated. However, a detailed understanding of the host factors that determine successful periapical wound healing is currently lacking. The long-term goal of the present study is to identify the molecular network that regulates wound healing and tissue regeneration in dentoalveolar infections in order to develop rational therapeutic approaches. Although a rodent model of periapical lesion was well established in Forsyth, adequate models for periapical wound healing are currently unavailable. Since the nature of apical periodontitis is infection-induced inflammation, the aim of this study was to systematically examine the effect of azithromycin treatment compared to ampicillin as a wound-healing treatment approach in mouse periapical lesions. Azithromycin is a macrolide antibiotic that specifically accumulates in macrophages exhibiting an anti-inflammatory effect via alteration of macrophage phenotype from pro-inflammatory M1 to anti-inflammatory M2 macrophages. Material and Methods: Dental pulps of mandibular first molars in C57BL/6J mice (6 weeks of age) were surgically exposed and inoculated with a cocktail of common human endodontic pathogens, which are sensitive to employed antibiotics. Mice received daily doses of azithromycin (8 mg/kg), ampicillin (20 mg/kg) or phosphate-buffered saline (PBS) from day 10 to 20 post infection. No root canal treatments were provided. Mandibles were isolated on days 10 (disease baseline) and 21. Right hemi mandibles were subjected to micro-computed tomography (μCT) to determine the extent of periapical bone loss and subsequent histological and immunohistochemistry analysis. Left hemi mandibles were used for RNA extraction for gene expression analysis through quantitative RT-PCR. Moreover, primary peritoneal macrophages stimulated with LPS and treated with either azithromycin or ampicillin were evaluated for pro-inflammatory cytokines expression (using ELISA) and nitric oxide production (iNOS assay). The effect of azithromycin in the level of NF-kB gene expression was assessed through luciferase reporter assay using Luciferase Stable RAW 264.7 cells. Results: The μCT results indicated that azithromycin treatment suppressed the extent of periapical bone loss compared to PBS treatment with a significant difference (p<0.05) in the average size of periapical lesions. In addition, the extent of bone loss on Day 21 in azithromycin group was significantly “recovered” compared with the baseline disease group (p<0.05). In histological observations, azithromycin-treated animals showed less neutrophil infiltration and a polarization of M2 macrophages in periapical lesions, while PBS treatment resulted in moderate neutrophil infiltration and an M1/M2-mixed macrophage profile. mRNA expression of pro-inflammatory cytokine IL-6 and chemokine CXCL2, as well as colony stimulating factor 2 (CSF2) had a significant down regulation in the azithromycin group when compared to baseline disease (p<0.05). Up regulation of IL-4-mRNA was also observed. In vitro experiments confirmed the down regulation of IL-1α and IL-1β (p>0.05) and nitric oxide production (p<0.05) in LPS stimulated peritoneal macrophages treated with azithromycin. Finally, the luciferase activity was decreased when cells were stimulated with azithromycin 50uM. Conclusions: These results taken together suggest that the suppression of periapical bone loss by azithromycin appears to be dependent on its immunomodulatory properties. This report is the first finding on pro-resolving M2 macrophage polarization during periapical wound healing. This research can shed light to the development of a mouse model of periapical wound healing. In addition, this dual role of azithromycin, microbicidal and anti-inflammatory effects, could pose as a great adjuvant on the root canal therapy being highly translational.