[Histone demethylase JMJD3 inhibits alveolar bone loss by regulating macrophage polarization in periodontitis]

Objective: To investigate the expression of histone demethylase, Jumonji domain-containing protein 3 (JMJD3), in inflammatory periodontal tissues and its potential mechanism for the regulation of periodontitis. Methods: The results of single-cell sequencing of periodontal tissues published in the Gene Expression Omnibus (GEO) database in 2022 were analyzed. Nine gingival samples each from healthy and inflamed periodontal patients were collected during periodontal surgery or tooth extractions for immunohistochemical staining and real-time fluorescence quantitative PCR (RT-qPCR). Mice periodontitis models were constructed, and the experimental groups were: healthy control+saline group, silk ligation+saline group, silk ligation+GSK-J4(inhibitor of JMJD3) group. Lipopolysaccharide (LPS) derived from Porphyromonas gingivalis (Pg) (Pg-LPS) was used to mimic the periodontal inflammatory microenvironment. The macrophages were treated with small interfering RNA (siRNA) targeting Jmjd3 and the JMJD3 inhibitor GSK-J4. siRNA transfection experiments were grouped into the following: the NC group (negative control sequence transfection group), the siRNA-Jmjd3 group, the NC+LPS group, siRNA-Jmjd3+LPS group. Inhibitor experiments were grouped as dimethyl sulfoxide (DMSO) group, GSK-J4 group, DMSO+LPS group, GSK-J4+LPS group. Western blotting and immunofluorescence staining were used to explore the effects of JMJD3 on macrophage polarization and periodontal inflammation in the in vivo and in vitro settings. Results: RT-qPCR results showed that JMJD3 expression in gingival tissues of periodontitis patients (1.97±0.91) was significantly higher than that in healthy gingival tissues (1.00±0.33) (t=2.45, P=0.048). RT-qPCR results of in vitro experiments showed that either siRNA knockdown of JMJD3 or inhibition of JMJD3 using GSK-J4 promoted M1 polarization and inhibited M2 polarization in macrophages under inflammatory environment: the expression of arginase Ⅰ (Arg 1) in the NC+LPS group (0.90±0.06) was significantly higher than that in the siRNA-Jmjd3+LPS group (0.61±0.11) (P<0.01); the expression of interleukin (Il)-6, Il-1β, and tumor necrosis factor alpha (Tnf-α) in the NC+LPS group (8.50±0.16, 5.56±0.20, 3.44±0.16) were significantly lower than those in the siRNA-Jmjd3+LPS group (14.63±0.48, 8.55±0.10, 11.72±0.58) (P<0.01). The expression of Arg1, chitinase-like 3 (Ym1), Il-10 in the DMSO+LPS group (0.82±0.01, 0.35±0.16, 1.47±0.11) were significantly higher (P<0.01) than the GSK-J4+LPS group (0.55±0.03, 0.22±0.21, 0.51±0.11); the expression of Il-6, Il-1β, and Tnf-α in the DMSO+LPS group (2.03±0.13, 3.63±0.14, 4.06±0.03) were significantly lower than the GSK-J4+LPS group (2.69±0.16, 15.04±1.15, 4.36±0.10) (P<0.01). The results of the in vivo experiments revealed that inhibition of JMJD3 exacerbated bone loss in experimental periodontitis mice, increased macrophage M1 polarization, and decreased M2 polarization in inflamed periodontal tissues. The buccal cemento-enamel junction (CEJ)-alveolar bone crest (ABC), palatal CEJ-ABC, as well as the ratio of M1/M2 type macrophages were significantly lower in the silk ligation+saline group [(0.26±0.03), (0.24±0.01) mm, 0.35±0.10] than in the silk ligation+GSK-J4 group [(0.34±0.04), (0.30±0.05) mm, 2.50±0.58] (t=3.65, P=0.006; t=2.67, P=0.049; t=7.31, P=0.004; respectively). Conclusions: Single-cell sequencing as well as the in vitro and in vivo experiments verified that JMJD3 expression was upregulated in periodontitis periodontal tissues. JMJD3 may exert a protective role in periodontitis by regulating macrophage polarization, thereby inhibiting alveolar bone destruction associated with the periodontitis.