Immunomodulation Mediated by Azithromycin in Experimental Periapical Inflammation

Abstract

Introduction: The purpose of the present study was to compare the immunomodulatory effect of azithromycin (AZM), ampicillin (AMP), amoxicillin (AMX), and clindamycin (CLI) in vitro and AZM on preexisting periapical lesions compared with AMP.

Methods: The susceptibility of 4 common human endodontic pathogens (Parvimonas micra, Streptococcus intermedius, Prevotella intermedia, and Fusobacterium nucleatum) to AZM, AMP, AMX, and CLI was confirmed by agar disk diffusion assay. Preexisting periapical lesions in C57BL/6J mice were treated with AZM, AMP, or phosphate-buffered saline (PBS). Periapical bone healing and the pattern of inflammatory cell infiltration were evaluated after a 10-day treatment by micro-computed tomographic and histology, respectively. Besides, the effect of antibiotics in pathogen-stimulated nuclear factor kappa B activation and the production of interleukin 1 alpha and tumor necrosis factor alpha was assessed in vitro by luciferase assay and enzyme-linked immunosorbent assay.

Results: All examined endodontic pathogens were susceptible to AZM, AMP, AMX, and CLI. AZM significantly attenuated periapical bone loss versus PBS. PBS resulted in widely diffused infiltration of mixed inflammatory cells. By contrast, AZM brought about localized infiltration of neutrophils and M2 macrophages and advanced fibrosis. Although the effect of AMP on bone was uncertain, inflammatory cell infiltration was considerably milder than PBS. However, most macrophages observed seemed to be M1 macrophages. AZM suppressed pathogen-stimulated nuclear factor kappa B activation and cytokine production, whereas AMP, AMX, and CLI reduced only cytokine production moderately.

Conclusions: This study showed that AZM led to the resolution of preexisting experimental periapical inflammation. Our data provide a perspective on host response in antibiotic selection for endodontic treatment. However, well-designed clinical trials are necessary to better elucidate the benefits of AZM as an adjunctive therapy for endodontic treatment when antibiotic therapy is recommended. Although both AZM and AMP were effective on preexisting periapical lesions, AZM led to advanced wound healing, probably depending on its immunomodulatory effect.

Keywords: Ampicillin; azithromycin; endodontic infection; immunomodulation; periapical periodontitis.

Figure 1.. Azithromycin attenuated pre-existing periapical bone loss.

(A) Representative μCT images of periapical periodontitis in the anterior-posterior direction. Pre-existing periapical lesions (PP baseline; day 10 post infection) were treated with either phosphate-buffered saline (PBS), azithromycin (AZM), or ampicillin (AMP) from day 10 to 20. The effect of the treatments was determined on day 21. The position of each CT slice was the most central part of the mandibular first molar distal root (R). Arrows indicate the margin of the periapical lesion surrounded by radiopaque alveolar bone. (B) Enumeration of periapical lesion size. * p < .05 vs. PP+PBS, vertical bar: standard deviation.

Figure 2.. Histological summary of the representative sample in each group. AZM treatment resulted in advanced periapical wound healing.

(A1-A4) H&E staining. Both AZM and AMP considerably resolved inflammation. AZM resulted in advanced periapical wound healing (mature granulation tissue) compared to AMP. (B1-B4) Neutrophils (Ly-6G). (C1-C4) Total macrophages (Mac2). (D1-D4) Proinflammatory iNOS+ cells including M1 macrophages. (E1-E4) Arg-1+ M2 macrophages. (F1-F4). Distribution of infiltrated inflammatory cells by image processing. Population and diffusion pattern of infiltrated cells were different between AZM and AMP. AZM led to localized M2 macrophage polarization, whereas diffused proinflammatory cells were observed in AMP treated mice. These histological findings were similar among samples in each group. Blue: Ly-6G+, yellow: iNOS+, green: Arg-1+. Arrows indicate the circumference of periapical periodontitis. B, alveolar bone; R, dental root. The original magnification: x100.

Figure 3.. Azithromycin exhibited a potent immunomodulatory effect compared to ampicillin in RAW264.7 cells stimulated with LPS or endodontic pathogens in vitro.

AZM inhibited LPS-stimulated NF-κB activation in a dose-dependent manner (a) and subsequent production of TNF-α (c) and IL-1α (e). As well, AZM inhibited endodontic pathogen-stimulated NF-κB activation in a dose-dependent manner (b) and subsequent production of TNF-α (d) and IL-1α f). AMP, AMX and CLI also suppressed IL-1α. These data support previous findings in other infectious diseases that AZM can down-regulate inflammation, probably its chemical structure-dependent, in addition to its antibiotic effect. * P < .05 vs. corresponding non-treated controls, N.D.: not detected; vertical bar: standard deviation; n=3 per condition.