Wound healing in periodontal disease induces macrophage polarization characterized by different arginine-metabolizing enzymes.

Abstract

Macrophages play important roles from the initiation of inflammation to wound healing. Two phenotypes of macrophages, namely pro-inflammatory type macrophages (M1-MΦ) and anti-inflammatory type macrophages (M2-MΦ), have been reported. Two contrasting metabolic enzymes that use arginine as a substrate, inducible nitric oxide synthase (iNOS), and arginase-1 (Arg-1), have been identified as M1-MΦ and M2-MΦ markers, respectively. The purpose of this study was to elucidate the temporal dynamics of the macrophage phenotype during the progression and healing phases of experimental periodontitis in mice. A total of 63 C57BL/6J mice were divided into the following 3groups: control (C), periodontitis (P), and healing (H). To induce periodontitis, a silk ligature was placed around the maxillary bilateral second molars of mice in the periodontitis and healing groups. In the healing group, the ligature was removed 3days after ligation to induce tissue healing. Maxillary tissue was collected on day 0 for the control group, days 1, 3, 5, and 7 for the periodontitis group (P1, P3, P5, and P7), and days 5 and 7 for the healing group (H5 and H7: 3days with the ligation + 2days or 4days following ligature removal). The left side of the maxilla was subjected to bone structure analysis using micro-computed tomography and gene expression analysis using polymerase chain reaction. On the right side, immunohistochemistry was performed to histopathologically evaluate the localization of macrophages by phenotype in the periodontal tissue. In the alveolar bone structure analysis, the linear distance of bone height increased significantly in the P5 and P7groups, whereas bone volume fraction and bone mineral density decreased over time after ligature placement; in the healing group (H5 and H7), these parameters improved significantly compared with the periodontitis group (P5 and P7). Expression of genes encoding pro-inflammatory cytokines and iNOS increased in the periodontitis group, and expression of anti-inflammatory cytokine genes and Arg-1 increased in the healing group. Furthermore, the iNOS/Arg-1 expression ratio increased with ligation, whereas the ratio in the healing groups (H5 and H7) significantly decreased compared with the periodontitis groups (P5 and P7). Immunofluorescence staining revealed a significant increase in the number of iNOS-positive macrophages in the periodontitis group and decrease in the healing group. In contrast, the number of Arg-1-positive macrophages decreased in the periodontitis group and increased in the healing group. The results of the present study suggest that wound healing in periodontal disease induces macrophage polarization from M1-MΦ to M2-MΦ characterized by iNOS and Arg-1.