Orthodontic tooth movement alters the oral microbiota
To assess the changes of oral microbiota in orthodontic tooth movement (OTM), the mouse model was established. To eliminate the potential impact of orthodontic springs and wires on oral hygiene and therefore microbial results, both the experimental and control groups used nickel-titanium coil springs. The difference was that no force was applied to the springs in the control group, while force was applied to the springs in the experimental group (Fig. 1a). The oral microbiota of the two groups were compared using linear discriminant analysis effect size (LEfSe) to identify the specific microorganism linked to orthodontic treatment (Fig. 1b, c). The LEfSe showed that 10 bacterial species were enriched in the move group (Fig. 1c). The Metastats analysis consistently showed that 2 bacterial species including probiotic Enterococcus faecalis (E. faecalis) were enriched in the move group (Fig. 1d). As shown by the relative abundance of species-level microorganism, E. faecalis was significantly enriched in the oral cavity in the move group versus control group (Fig. 1e). These findings altogether demonstrated that OTM promoted the accumulation of E. faecalis in the oral of mice.
After 1-week application of force, micro CT of maxilla was performed (Fig. 1f).The distance of the move group was 88.66 ± 16.42 mm (Fig. 1g). The region of interest encompassed a 100 µm cuboid zone under the furcation roof of the maxillary first molar. No significant difference was observed between the move group and the control group in trabecular bone mineral density (BMD), bone volume/tissue volume ratio (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N) and trabecular separation (Tb. Sp) based on micro-CT analysis (Fig. 1h). HE staining and Masson staining of distobuccal roots showed the integrity of periodontal tissue around the molars after tooth movement (Fig. 1i,j).
Immunomodulation on M1-like macrophage polarization by Enterococcus faecalis
In order to elucidate the role of E. faecalis, our focus is on profiling immune cells, specifically macrophages. As OTM is regarded as a "sterile" inflammatory process21 and macrophages play a significant role in the tooth movement process,18,22–24 this study emphasizes the examination of macrophages. RAW264.7 macrophages were treated with E. faecalis. Quantitative real-time polymerase chain reaction (QRT-PCR) results showed that the expression levels of TNFA, il1b, iNOS genes, which are recognized as M1 markers, were markedly increased in the E. faecalis treated group compared to the control group. The M2 makers, arg1 and cd206, were downregulated in the E. faecalis treated group compared to the control group (Fig. 2a). Consistent with QRT-PCR results, flow cytometry results also revealed elevated percentage of CD86+ macrophages and downregulated percentage of CD206+ macrophages in the E. faecalis treated group compared to the control group (Fig. 2b, c, d). Furthermore, the immunofluorescence staining results showed that the iNOS level in the E. faecalis treated group was higher compared to control group (Fig. 2e). These results suggested that E. faecalis increased the number of M1-polarized macrophages.
Enterococcus faecalis attenuated osteogenesis of periodontal ligament cells (PDLCs) via conditioned media of Enterococcus faecalis treated macrophages
To investigate the osteoimmunomodulatory impact of E. faecalis on periodontal ligament cells (PDLCs) in conjunction with macrophages, an in vitro culture system was utilized. Primary mouse PDLCs were obtained from mouse periodontal ligaments and cultured with or without conditioned media (CM) derived from macrophages post-incubation with E. faecalis. QRT-PCR analysis revealed that the expression levels of osteogenic genes (alpl, runx2, bglap, spp1) treated with CM E. faecalis were significantly lower compared to those in the CM control group (Fig. 3a). Consistently, immunofluorescence staining results demonstrated a decreased OCN level in the CM E. faecalis group compared to the CM control group (Fig. 3b). Qualitative assessments further indicated that CM E. faecalis dampened ALP activity and Alizarin Red level compared to the CM control group (Fig. 3c, d).
Enterococcus faecalis promotes orthodontic tooth movement in antibiotic mixture (ABX) treated mice
To investigate the impact of E. faecalis on OTM, PBS or E. faecalis suspension were administered to the ligature site using a syringe, twice daily to antibiotic mixture (ABX) mice during the OTM model (Fig. 4a). Micro-CT images demonstrated a significant increase in the distance of OTM in mice treated with E. faecalis compared to the control group (Fig. 4b, c). Interestingly, the OTM distance in the ABX control group (Fig. 4c) was lower than in the previous normal control group (Fig. 1g), suggesting the essential role of microbiota in orthodontic movement. Additionally, micro-CT analysis revealed no significant differences in BMD, BV/TV, Tb.Th, Tb.N and Tb. Sp between the E. faecalis and the control group (Fig. 4d). Histological staining of distobuccal roots with HE and Masson staining showed the integrity of periodontal tissue surrounding the molars post-tooth movement (Fig. 4e). Furthermore, the number of iNOS+ cells increased on the compression side in the E. faecalis-treated group compared to the control group (Fig. 4f).
Enterococcus faecalis enhances the M1 polarization of macrophages through the mTOR and AKT signaling pathways
E. faecalis has been shown to modulate macrophage activity. The mTOR and AKT signaling pathways are key regulators of macrophage polarization, has been well-recognized.25,26 Based on this understanding, our hypothesis posited that E. faecalis triggers the mTOR and AKT pathway to induce M1 polarization of macrophages. Western blotting analysis revealed a notable increase in the phosphorylation of both AKT and mTOR in macrophages exposed to E. faecalis, indicating activation of this signaling cascade (Fig. 5a, b). Furthermore, ELISA results demonstrated a significant elevation of TNF-α, a hallmark cytokine produced by M1 macrophages, in the conditioned media of macrophages treated with E. faecalis (Fig. 5c).