Enterococcus faecalis promotes orthodontic tooth movement in mice by M1-like macrophage polarization

Enterococcus faecalis promotes orthodontic tooth movement in mice by M1-like macrophage polarization | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Enterococcus faecalis promotes orthodontic tooth movement in mice by M1-like macrophage polarization Zhihe Zhao, Fei Yu, Wenxin Lu, Peilin Li, Jiajia Peng This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4943788/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Probiotic-mediated therapy has garnered increasing attention for its potential role in influencing bone remodeling. Recent studies have consistently shown that the microorganisms can play a role in modulating bone reconstruction. In this study, we aim to explore the impact of probiotics on accelerating orthodontic tooth movement (OTM). Potential probiotic candidates will be identified through 16S rRNA gene sequencing in a mouse model of OTM. The relative abundance of Enterococcus faecalis ( E. faecalis ) increased in the move group. To investigate the osteoimmunomodulatory impact, primary periodontal ligament cells (PDLCs) were obtained from mouse periodontal ligaments and cultured with or without conditioned media (CM) derived from macrophages post-incubation with E. faecalis . The results demonstrated that E. faecalis increased the number of M1-polarized macrophages, and a decreased osteogenic level in PDLCs treated with CM E. faecalis group. Subsequently, the microbiota of the mice has be depleted using broad-spectrum antibiotic mixture (ABX) before being administered with E. faecalis . The distance of OTM have been measured, and the alveolar bone have been analyzed using micro-CT and immunohistochemical staining. This study has explored the potential effects of E. faecalis administration on the OTM process through immunomodulation. We assessed the efficacy of E. faecalis in accelerating tooth movement, and elucidated the mechanisms by which E. faecalis modulate M1-like macrophage polarization to enhance OTM. This research will lead to an acceleration in the duration of orthodontic treatment with minimal interventions, thereby offering significant value and pioneering advancements in orthodontic treatment. Biological sciences/Microbiology/Bacteria Health sciences/Medical research Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION Probiotic-mediated therapy has sparked significant interests in various disease treatments due to its ability to eliminate harmful bacteria and regulate the host immune system. 1 , 2 Probiotics are beneficial microorganisms that play a role in maintaining a balanced and dynamic microbiota. 3 – 5 Recent studies and emerging evidence consistently demonstrate the potential of the microorganisms to modulate bone remodeling. 6 Studies have demonstrated that specific probiotic strains can increase bone density 7 and improves calcium absorption. 8 Furthermore, pre-clinical studies highlight the anti-inflammatory properties of probiotics, demonstrating their ability to mitigate bone loss in models of ovariectomy 9 and rheumatoid arthritis. 10 These findings are particularly relevant to orthodontics, where controlled bone remodeling is essential for successful tooth movement. Orthodontic treatment achieves tooth movement through bone remodeling involving alveolar bone resorption and formation, 11 and this process often requires a lengthy treatment duration. Enhancing the speed of bone reconstruction and optimizing the balance of this process are crucial for achieving accelerated orthodontic tooth movement (OTM) and shortening treatment time. Current methods for accelerating OTM, such as periodontal ligament distraction, low-intensity pulsed ultrasound, and piezopuncture, can be invasive and present challenges. Strengthening the potential of probiotics to modulate bone remodeling could offer a safer, non-invasive alternative. While probiotics are often regarded for their ability to reduce harmful bacteria, their role in orthodontic treatment remains inconclusive. The use of probiotics during fixed orthodontic treatment have been demonstrated to reduce S. mutans and Lactobacillus levels in the saliva. 12 However, a research indicates that probiotic supplementation, despite its potential benefits, did not significantly affect the development of inflammation in gingiva and decalcification in enamel. 13 While previous research has focused on inhibiting harmful bacteria, little is known about the effects of probiotics on accelerating tooth movement. The interaction between microbial and biomechanical signals on periodontal cells and tissues remains understood. 14 Modulating immune response has been recognized as one of the key mechanisms underlying probiotic action. The immune system recognizes microorganisms as foreign bodies, triggering large amounts of inflammatory cells to generate innate and adaptive responses. 15 , 16 Immunomodulatory effect is particularly important in OTM, where biomechanical forces applied to teeth trigger a complex inflammatory response in periodontal tissues. 17 This response involves immune cells that release inflammatory mediators, matrix-degrading enzymes, and osteoclast-activating molecules, that all important for bone remodeling. Particularly, macrophages have been exerts crucial roles in alveolar bone remodeling 18 and root resorption 19 , 20 during OTM. Immune cells would directly interact with bone cells, suggesting a potential avenue for probiotics to influence bone remodeling during orthodontic treatment. A large amounts of evidence suggests crucial links among probiotics, host immune response, and bone remodeling, and the precise mechanisms remain to be fully elucidated. In our study, we aims to explore how microorganisms can accelerate tooth movement, investigate the immunomodulation mechanism by which probiotics enhance OTM through macrophages. Potential probiotic candidates have been identified through 16S rRNA gene sequencing in a mouse model of OTM. To investigate the osteoimmunomodulatory impact, primary periodontal ligament cells (PDLCs) were obtained from mouse periodontal ligaments and cultured with or without conditioned media (CM) derived from macrophages post-incubation with microtia. The results demonstrated that Enterococcus faecalis ( E. faecalis ) increased the number of M1-polarized macrophages, and a decreased osteogenic level in PDLCs treated with CM E. faecalis group. Moreover, E. faecalis increased the distance in antibiotic mixture (ABX) treated mice during OTM model. Ultimately, this research endeavors to explore novel immunomodulatory mechanisms by which microorganisms can accelerate OTM, paving the way for innovative therapeutic strategies in orthodontic field. The outcomes of this research include shortened orthodontic treatment durations with minimal interventions, offering pioneering advancements in orthodontic treatment. RESULTS 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. 1 a). 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. 1 b, c). The LEfSe showed that 10 bacterial species were enriched in the move group (Fig. 1 c). The Metastats analysis consistently showed that 2 bacterial species including probiotic Enterococcus faecalis ( E. faecalis ) were enriched in the move group (Fig. 1 d). 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. 1 e). 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. 1 f).The distance of the move group was 88.66 ± 16.42 mm (Fig. 1 g). 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. 1 h). HE staining and Masson staining of distobuccal roots showed the integrity of periodontal tissue around the molars after tooth movement (Fig. 1 i,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 process 21 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. 2 a). 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. 2 b, 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. 2 e). 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. 3 a). Consistently, immunofluorescence staining results demonstrated a decreased OCN level in the CM E. faecalis group compared to the CM control group (Fig. 3 b). Qualitative assessments further indicated that CM E. faecalis dampened ALP activity and Alizarin Red level compared to the CM control group (Fig. 3 c, 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. 4 a). Micro-CT images demonstrated a significant increase in the distance of OTM in mice treated with E. faecalis compared to the control group (Fig. 4 b, c). Interestingly, the OTM distance in the ABX control group (Fig. 4 c) was lower than in the previous normal control group (Fig. 1 g), 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. 4 d). Histological staining of distobuccal roots with HE and Masson staining showed the integrity of periodontal tissue surrounding the molars post-tooth movement (Fig. 4 e). Furthermore, the number of iNOS + cells increased on the compression side in the E. faecalis -treated group compared to the control group (Fig. 4 f). 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. 5 a, 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. 5 c). DISCUSSION This study pioneers the exploration of the impact of microorganisms on OTM, which previously dominated by antimicrobial focuses. 27 While prior research has primarily centered on eliminating pathogenic bacteria in the oral cavity during orthodontic treatment, 28 this investigation focus on the novel concept that probiotics may also exert a beneficial influence on OTM. Although orthodontic treatment itself involves a localized, “sterile” movement of teeth, 21 we hypothesize that oral-administrated E. faecalis , through intricate interactions with immune cells, could indirectly modulate the bone remodeling processes inherent to OTM. This potential influence of oral microorganisms, if confirmed, could unveil new frontiers in accelerating orthodontic treatment outcomes by harnessing the power of beneficial probiotics. Probiotics are beneficial bacteria or microorganisms that can improve overall health when consumed in adequate amounts through the diet. 3 , 4 While probiotics are often regarded for their ability to reduce harmful bacteria, their role in orthodontic treatment remains unknown. The use of probiotics during fixed orthodontic treatment have been demonstrated to reduce S. mutans and Lactobacillus levels in the saliva. 12 However, a research indicates that probiotic supplementation, despite its potential benefits, did not significantly affect the development of inflammation in gingiva and decalcification in enamel. 13 E. faecalis occur in gastrointestinal tract of animals and humans. 29 Certain strains of E. faecalis have demonstrated probiotic properties, 30 , 31 exemplified by its inclusion in the commercially available probiotic E. faecalis Symbioflor 1 (SymbioPharm, Herborn, Germany). Emerging evidence suggests that E. faecalis may influence bone remodeling processes, 32 , 33 raising possibilities for its application in orthodontics. The immunomodulatory properties of E. faecalis through M1 polarization of macrophages have been elucidated in our study. Particularly, macrophages have been exerts crucial roles in alveolar bone remodeling 18 and root resorption 19 , 20 during OTM. Our study provides evidence that enhanced production TNF-α, a pro-inflammatory cytokine contributes to impair osteogenesis of PDLCs. Consistent with our findings, E. faecalis have been demonstrates to shifts macrophage polarization to M1-like phenotype 34 . M1-like macrophages are reported to promotes alveolar bone resorption and consequent OTM after mechanical force application. 19 , 22 Moreover, E. faecalis have also been demonstrated to immunoregulates osteoclastogenesis of macrophages and upregulates expression of inflammatory cytokine iNOS. 35 Macrophages possess the capability to differentiate into osteoclasts, 36 which are responsible for bone resorption. This osteoclastogenic potential of macrophages is particularly relevant in tooth movement, as targeted bone resorption at the root surface is essential for successful tooth repositioning. Therefore, the ability of macrophages to osteoclastogenesis may constitute a key mechanism underlying the E. faecalis effects on accelerated tooth movement. The mTOR and AKT signaling pathways are key regulators of macrophage polarization, has been well-recognized. 25 , 26 Akt has been shown to modulate the activity of NF- κB, 37 a crucial transcription factor for M1 activation, in both positive and negative ways. Consistent to our reports, Akt isoforms has been proved to contribute to M1 polarization. 38 Also, mTORC1 signaling has been characterized as positive regulation in M1 macrophages, 39 and PP2A-mTOR-p70S6K/4E-BP1 axis has been demonstrated to regulate M1 polarization of pulmonary macrophages. 40 In our study, E. faecalis can drive macrophage polarization towards the M1 phenotype through immunomodulatory mechanisms, leading to the activation of the mTOR-AKT cascade. This activation promotes the production of pro-inflammatory cytokines of macrophages, ultimately downregulates osteogenic differentiation of PDLCs. In conclusion, our study has investigated the potential effects of E. faecalis administration on the accelerated OTM process through immunomodulation. We elucidated the mechanisms by which E. faecalis modulates M1-like macrophage polarization then regulates PDLCs, ultimately enhancing the distance of OTM (Fig. 5 d). Further research is needed to identify specific probiotic strains and their effects on the gastrointestinal tract microbiota to fully elucidate the potential of probiotics. This research will lead to an acceleration in the duration of orthodontic treatment with minimal interventions, thereby offering significant value in developing probiotic mouthwash for future applications. MATERIALS AND METHODS Animal Experiments Design Adult male C57BL/6 WT mice were purchased from Gempharmatech (Nanjing, China) and were housed in a specific pathogen-free (SPF) facility with a 12:12-h light-dark cycle. The use of animals in this study was approved by the Animal Care and Ethics Committee of West China School of Stomatology, Sichuan University (WCHSIRB-D-2022-611). The experimental design involved both an experimental group and a control group (n = 6:6) randomly, with both groups using nickel-titanium coil springs. After anesthesia with isoflurane, the coil springs were ligated between the maxillary incisors and the maxillary first molar with stainless steel and light-cured glass ionomer cement. The key distinction between the two groups was that no force was applied to the coil springs in the control group, whereas 40g force 19 was applied to the coil springs in the experimental group. After 7 days, all mice were sacrificed using an overdose of sodium pentobarbital. 16S rRNA gene sequencing Oral microbiota was collected from mice using oral swabs. The sample were processed by Novogene (Beijing, China). Briefly, total genomic DNA was extracted, and the 16S rRNA genes of distinct regions were amplified. Sequencing libraries were prepared using the SMRTbellTM Template Prep Kit (PacBio) according manufacturer's instructions. The library quality was assessed on the Qubit@ 2.0 Fluorometer (Thermo Scientific) and FEMTO Pulse system, and the library was sequenced on the PacBio Sequel platform. Procession of the sequencing data were performed with QIIME (Version1.9.1) and displayed with R software (Version 2.15.3). Micro-CT Mice were sacrificed 7 days after OTM model. The mandibles were then fixed in 4% polymerized formaldehyde for 48 h. The samples were scanned using a micro-CT system (Scanco Medical). To prevent dehydration, the samples under examination were kept in a moist environment during the scanning process. The scans were conducted at a resolution of 10 µm, with a voltage of 80 kV and a current of 500 mA. Morphometric analyses were carried out using software (Scanco Medical Evaluation & Visualizer, Scanco Medical). The distance between the most convex point of the maxillary first molar and the most convex mesial point of the maxillary second molar was measured using digital slide calipers on the micro-CT images. The region of interest (ROI) selected for analysis was a 100 µm cuboid extracted from the furcation area of the maxillary first molar. Various three-dimensional morphometric parameters of bone microarchitecture were then calculated for analysis. Histological analysis Following micro-CT scanning, the mandibles underwent a two-week decalcification process in PBS solution with 10% EDTA·2Na, with daily replacement of the solution. Subsequently, the maxilla were dehydrated using a gradient of ethanol, then embedded in paraffin and sliced into 5-µm-thick sections for H&E staining and Masson staining. TRAP staining was conducted using a commercially available kit (Servicebio). The staining procedures were carried out in accordance with established protocols. Macrophages culture and infected with E. faecalis RAW264.7 macrophages were cultured in DMEM medium (Gibco) supplemented with 10% fetal bovine serum (Biological Industries), 100 U/mL penicillin-streptomycin (HyClone, Thermo Scientific). The cells were cultured in a humidified environment with 5% CO 2 at 37°C. E. faecalis was obtained from BeNa Culture Collection and cultured in sterile brain heart infusion (BHI) broth (Hopebio) under aerobic conditions. RAW264.7 macrophages were seeded overnight, and E. faecalis was added to the culture at a multiplicity of infection (MOI) of 100 for 3 h. The supernatants of macrophages were filtered through PES filters (Merck Millipore) to obtain conditioned media (CM). TNF-α concentrations in CM of macrophages were detected using mouse ELISA kit (Nanjing Jiancheng Bioengineering Institute) according to the manufacturer’s protocol. PDLCs culture and treatments Mouse PDLCs were isolated from PDL tissue and maintained in α-MEM medium with 10% FBS (Gibco) and 1% penicillin-streptomycin (HyClone, Thermo Scientific). PDLCs were treated with CM of macrophages. For qualitative assay, PDLCs were stained with BCIP/NBT ALP Color Development Kit (Beyotime) and Alizarin Red S Solution (Solarbio) according to the manufacturer’s protocol. Quantitative Real-Time-Polymerase Chain Reaction (QRT-PCR) Total RNA was extracted using TRIzol reagent (Life Technologies) and reverse transcription was carried out with the PrimeScriptTM RT reagent Kit (Takara). QRT-PCR was conducted on a LightCycler 96 system (Roche) using SYBR Green Mix (Takara). The data were analyzed using the comparative cycle threshold method (ΔΔCt) and normalized to the GAPDH housekeeping gene. Flow cytometry analysis Single-cell suspensions from macrophages were collected. Live/Dead staining (fixable viability stain 780, BD Biosciences) was used to determine the live cells. Fc receptors were blocked by treating the cells with CD16/32. CD86 antibodies (PE-Cy7, BD Biosciences) were then added as according to the manufacturer's instructions. Next, the cells were permeabilized using a flow cytometry permeabilization/wash buffer (BD Biosciences) and intracellularly stained with CD206 antibodies (AF647, BD Biosciences). The stained cells were analyzed using a Flow Cytometer (Beckman) and analyzed with Flowjo software (BD Biosciences). Immunofluorescence Staining Macrophages or PDLCs were treated by fixation with a 4% paraformaldehyde solution, followed by permeabilization using 0.1% Triton X-100, and blocking with 5% bovine serum albumin (BSA). Primary antibodies (anti-OCN from Yeasen; anti-iNOS from ABclonal Technology) were applied and incubated overnight, followed by secondary antibody (Yeasen), F-actin labeling (Yeasen), and nuclear staining (using Hoechst 33258 or DAPI from Biosharp). Immunofluorescence staining was visualized and captured using a fluorescence microscope (Leica). Antibiotic mixture (ABX) Treatments and E. faecalis Administration A mixture containing ampicillin (1 g/L), metronidazole (1 g/L), gentamicin (1 g/L), neomycin (1 g/L), and vancomycin (0.5 g/L) was administered to mice through both their drinking water and by gavage for a period of 10 days. Following this treatment, normal drinking water was provided for 2 days to eliminate the effects of the antibiotics before introducing E. faecalis laterally. The mice were randomly divided into 2 groups (n = 5:5). In experimental group, a suspension of E. faecalis (5 × 10 8 CFU/mL, 100µL 41 ) was applied to the ligature site of molar using a syringe, twice daily for 7 days during the OTM model. The control group received a PBS solution instead. After 7 days, all mice were sacrificed using an overdose of sodium pentobarbital. Immunohistochemistry Analysis Following decalcification, the maxillae were dehydrated using an ethanol gradient, embedded in paraffin, and sliced into 5-µm-thick sections. Endogenous peroxidase blocking buffer was applied, followed by washing and antigen retrieval. Subsequently, a 5% BSA solution was used for blocking, and incubated the sections with anti-iNOS (Proteintech) overnight, and then applying a secondary antibody. Immunofluorescence staining was visualized and captured using a fluorescence microscope (Leica). Western Blot Analysis Macrophages were lysed using RIPA buffer containing protease inhibitors. The extracted proteins were then separated via 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes (Millipore). After being blocked with 5% BSA, the membranes were incubated to primary antibodies specific for AKT (Cell Signaling Technology), phosphorylated AKT (p-AKT, Cell Signaling Technology), mTOR (Cell Signaling Technology), phosphorylated mTOR (p-mTOR, Cell Signaling Technology), and GAPDH (Aksomics). Statistical Analysis Data were presented as mean ± standard error of mean and analyzed using Prism 9.3 (GraphPad Software). An unpaired Student's t-test was used to compare the means of two experimental groups. P-values < 0.05 were considered statistically significant. Declarations CONFLICT OF INTERESTS The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. CONTRIBUTIONS F. Yu, contributed to conception, design, data acquisition and analysis, drafted and critically revised the manuscript; W. Lu, J. Peng and P. Li contributed to data acquisition and analysis, critically revised the manuscript; Z. Zhao contributed to conception, design, data analysis, and critically revised the manuscript. All authors gave final approval and agree to be accountable for all aspects of the work. ACKNOWLEDGEMENTS This work was supported by National Natural Science Foundation of China (32271416, 62306193), Natural Science Foundation of Sichuan Province (2023NSFSC0562), Sichuan University Postdoctoral Interdisciplinary Innovation Fund (JCXK2202), West China School/ Hospital of Stomatology, Sichuan University (RCDWJS2022-1) and IOF Research Grant ( IOF2022Y01). References Tan, L. et al. Engineered probiotics biofilm enhances osseointegration via immunoregulation and anti-infection. Sci Adv 6, (2020). Gulnaz, A. et al. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4943788","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":348263052,"identity":"46d12a65-8ec6-42c6-a9e9-8b8f1bf72285","order_by":0,"name":"Zhihe Zhao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwElEQVRIiWNgGAWjYDACCQY2IGkD4fCQoCWNdC2HSdAiP7v92YMfFeft+WckMD5428Ygb05IC+OcA+mGPWduJ864kcBsOLeNwXBnAwEtzBIJxyR4224nGEgksEnztjEkGBwgoIVNIrFN8m/bOXugFvbfRGnhkUgGGX6AcQPQFmaitEhIpLFJy5xJTpxx5mGz5JxzEoYbCGmRn5H+TPJNhZ09f3vywQ9vymzkCdqCBBgbQLYSr34UjIJRMApGAW4AABA9OKcZ8nU3AAAAAElFTkSuQmCC","orcid":"","institution":"State Key Laboratory of Oral Diseases \u0026 National Clinical Research Center for Oral Diseases, Department of Orthodontics, West China Hospital of Stomatology, Sichuan University","correspondingAuthor":true,"prefix":"","firstName":"Zhihe","middleName":"","lastName":"Zhao","suffix":""},{"id":348263053,"identity":"d0c6ab37-092a-4955-986b-2145596c6545","order_by":1,"name":"Fei Yu","email":"","orcid":"","institution":"State Key Laboratory of Oral Diseases \u0026 National Clinical Research Center for Oral Diseases, Department of Orthodontics, West China Hospital of Stomatology, Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Fei","middleName":"","lastName":"Yu","suffix":""},{"id":348263054,"identity":"f0645237-ca83-430f-a2b2-29a2612394ca","order_by":2,"name":"Wenxin Lu","email":"","orcid":"","institution":"State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, West China School of Stomatology, Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Wenxin","middleName":"","lastName":"Lu","suffix":""},{"id":348263055,"identity":"a74324d4-96b1-470e-b9f3-cbeb88c98e76","order_by":3,"name":"Peilin Li","email":"","orcid":"https://orcid.org/0000-0001-9030-4347","institution":"Sichuan Hospital of Stomatology","correspondingAuthor":false,"prefix":"","firstName":"Peilin","middleName":"","lastName":"Li","suffix":""},{"id":348263056,"identity":"efe6dcce-ad2c-48db-97cf-27bc97846aaf","order_by":4,"name":"Jiajia Peng","email":"","orcid":"","institution":"Sichuan Hospital of Stomatology","correspondingAuthor":false,"prefix":"","firstName":"Jiajia","middleName":"","lastName":"Peng","suffix":""}],"badges":[],"createdAt":"2024-08-20 09:26:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4943788/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4943788/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":65868012,"identity":"0dba2d82-734e-46af-a511-a03575106540","added_by":"auto","created_at":"2024-10-03 18:01:45","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1053818,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe relative abundance of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eEnterococcus faecalis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e in oral microbiota increase in orthodontic tooth movement model.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e orthodontic tooth movement (OTM) model of mouse. Nickel-titanium coil springs applied no force in the control group, while 40g force was applied in the move group. \u003cstrong\u003eb\u003c/strong\u003e Taxonomic cladogram resulting from linear discriminant analysis effect size (LEfSe) analysis of oral microbiota in different groups. \u003cstrong\u003ec\u003c/strong\u003e Linear discriminant analysis (LDA) score resulting from LEfSe analysis. \u003cstrong\u003ed \u003c/strong\u003eThe heatmap generated from Metastats analysis. \u003cstrong\u003ee\u003c/strong\u003e The relative abundance of \u003cem\u003eEnterococcus faecalis \u003c/em\u003e(\u003cem\u003eE. faecalis\u003c/em\u003e)\u003cem\u003e \u003c/em\u003ein the control group and the move group. \u003cstrong\u003ef\u003c/strong\u003e Representative micro–computerized tomography (micro-CT) images of the tooth movement. \u003cstrong\u003eg\u003c/strong\u003e The distance of OTM measured by micro-CT. \u003cstrong\u003eh\u003c/strong\u003e Quantifications of 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. The region of interest encompassed a 100 μm zone under the furcation roof in the interradicular region of the maxillary first molar. \u003cstrong\u003ei\u003c/strong\u003eRepresentative HE staining of distobuccal roots. \u003cstrong\u003ej\u003c/strong\u003e Representative Masson staining of distobuccal roots. All bars indicate mean ± standard error of the mean. * P \u0026lt; 0.05, **** P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"XXXXXX1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4943788/v1/34f892207b52f8dfb97a21a6.jpg"},{"id":65868008,"identity":"0d1a5f62-d337-4a87-b091-312480b218c4","added_by":"auto","created_at":"2024-10-03 18:01:45","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":563094,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eEnterococcus faecalis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003epromotes M1-like macrophage polarization.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003eRelative mRNA expression of M1/M2-related genes. M1-related genes: \u003cem\u003eTNFA, il1b, iNOS\u003c/em\u003e. M2-related genes:\u003cem\u003e arg1, cd206\u003c/em\u003e. \u003cstrong\u003eb,c\u003c/strong\u003e Representative flow cytometry plots of macrophages after incubation with \u003cem\u003eE. faecalis\u003c/em\u003e. Polarization of macrophages was measured by expression of CD86 (M1) and CD206 (M2). \u003cstrong\u003ed\u003c/strong\u003e Quantification of positive cells as percentages of live cells based on flow cytometry. \u003cstrong\u003ee\u003c/strong\u003e Immunofluorescence images of iNOS expression in macrophages. All bars indicate mean ± standard error of the mean. * P \u0026lt; 0.05, ** P \u0026lt; 0.01, **** P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"XXXXXX2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4943788/v1/559f22346eab297726b554c3.jpg"},{"id":65868013,"identity":"4d56e199-701d-4087-a29a-9f2a1f657981","added_by":"auto","created_at":"2024-10-03 18:01:45","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":997920,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImpact of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eEnterococcus faecalis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-primed macrophages' conditioned media (CM) on osteogenesis of periodontal ligament cells (PDLCs).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e QRT-PCR analysis of osteogenesis-related genes expression in periodontal ligament cells (PDLCs) incubated with or without conditioned media (CM) of macrophages from macrophages post-incubation with \u003cem\u003eE. faecalis\u003c/em\u003e. \u003cstrong\u003eb\u003c/strong\u003e Immunofluorescence images of OCN expression in PDLCs. \u003cstrong\u003ec \u003c/strong\u003eRepresentative ALP staining of PDLCs treated with CM control or CM E. faecalis from macrophages. \u003cstrong\u003ed\u003c/strong\u003e Representative Alizarin Red staining of BMSCs treated with CM control or CM E. faecalis from macrophages. All bars indicate mean ± standard error of the mean. * P \u0026lt; 0.05, **** P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"XXXXXX3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4943788/v1/e3fce8d8cf0db9093475902f.jpg"},{"id":65868249,"identity":"234e6cd5-00f9-4c3e-b3f6-1a2cb0e84ce4","added_by":"auto","created_at":"2024-10-03 18:09:45","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":782720,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eEnterococcus faecalis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003epromotes distance of orthodontic tooth movement in antibiotic mixture (ABX) treated mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e The mice received antibiotic mixture (ABX) treatment for 10 days followed by normal drink for 2 days. Subsequently, orthodontic tooth movement (OTM) models were established and treated with PBS or \u003cem\u003eE. faecalis\u003c/em\u003esuspension to the ligature site using a syringe, twice daily during OTM model. \u003cstrong\u003eb\u003c/strong\u003e Representative micro-CT images of the tooth movement. \u003cstrong\u003ec\u003c/strong\u003e The distance of OTM measured by micro-CT. \u003cstrong\u003ed\u003c/strong\u003eQuantifications of 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. The region of interest encompassed a 100 μm zone under the furcation roof in the interradicular region of the maxillary first molar. \u003cstrong\u003ee \u003c/strong\u003eRepresentative HE staining, Masson staining, and tartrate-resistant acid phosphatase (TRAP) staining on the compression side of distobuccal roots. \u003cstrong\u003ef\u003c/strong\u003e Representative immunohistochemical images of inducible nitric oxide synthase (iNOS) on the compression side of distobuccal roots. All bars indicate mean ± standard error of the mean. * P \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"XXXXXX4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4943788/v1/5f6aa76f533a972ed873d5ce.jpg"},{"id":65868250,"identity":"37c19556-4851-444d-b650-1e25806e295b","added_by":"auto","created_at":"2024-10-03 18:09:45","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":447811,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eEnterococcus faecalis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003epromote M1 polarization of macrophages \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003evia \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003emTOR and AKT signaling pathway.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea \u003c/strong\u003eRepresentative western blotting analysis of p-AKT, AKT, p-mTOR, mTOR, and GAPDH after treated with \u003cem\u003eE. faecalis\u003c/em\u003e at different time points. \u003cstrong\u003eb\u003c/strong\u003e Quantification of western blotting results as p-AKT/ AKT, p-mTOR/mTOR. \u003cstrong\u003ec\u003c/strong\u003e ELISA measurement of TNF-α in conditioned media of macrophages after incubation with or without \u003cem\u003eE. faecalis\u003c/em\u003e. \u003cstrong\u003ed\u003c/strong\u003e Schematic illustration of \u003cem\u003eE. faecalis\u003c/em\u003e -activated mTOR signaling pathway increasing the M1/M2 ratio in orthodontic root movement. All bars indicate mean ± standard error of the mean. * P \u0026lt; 0.05, ** P \u0026lt; 0.01, *** P \u0026lt; 0.001, **** P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"XXXXXX5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4943788/v1/accd16c2afda2c32fd26c23f.jpg"},{"id":70917550,"identity":"ffd90a99-dad5-40cd-a644-bc2b59993ed6","added_by":"auto","created_at":"2024-12-09 08:21:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4592006,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4943788/v1/58e83ba1-8f60-41f6-a7e2-2b1f5e3db271.pdf"}],"financialInterests":"(Not answered)","formattedTitle":"Enterococcus faecalis promotes orthodontic tooth movement in mice by M1-like macrophage polarization","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eProbiotic-mediated therapy has sparked significant interests in various disease treatments due to its ability to eliminate harmful bacteria and regulate the host immune system.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e Probiotics are beneficial microorganisms that play a role in maintaining a balanced and dynamic microbiota.\u003csup\u003e\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e Recent studies and emerging evidence consistently demonstrate the potential of the microorganisms to modulate bone remodeling.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e Studies have demonstrated that specific probiotic strains can increase bone density\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e and improves calcium absorption.\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e Furthermore, pre-clinical studies highlight the anti-inflammatory properties of probiotics, demonstrating their ability to mitigate bone loss in models of ovariectomy\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e and rheumatoid arthritis.\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e These findings are particularly relevant to orthodontics, where controlled bone remodeling is essential for successful tooth movement.\u003c/p\u003e \u003cp\u003eOrthodontic treatment achieves tooth movement through bone remodeling involving alveolar bone resorption and formation,\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e and this process often requires a lengthy treatment duration. Enhancing the speed of bone reconstruction and optimizing the balance of this process are crucial for achieving accelerated orthodontic tooth movement (OTM) and shortening treatment time. Current methods for accelerating OTM, such as periodontal ligament distraction, low-intensity pulsed ultrasound, and piezopuncture, can be invasive and present challenges. Strengthening the potential of probiotics to modulate bone remodeling could offer a safer, non-invasive alternative.\u003c/p\u003e \u003cp\u003eWhile probiotics are often regarded for their ability to reduce harmful bacteria, their role in orthodontic treatment remains inconclusive. The use of probiotics during fixed orthodontic treatment have been demonstrated to reduce \u003cem\u003eS. mutans\u003c/em\u003e and \u003cem\u003eLactobacillus\u003c/em\u003e levels in the saliva.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e However, a research indicates that probiotic supplementation, despite its potential benefits, did not significantly affect the development of inflammation in gingiva and decalcification in enamel.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e While previous research has focused on inhibiting harmful bacteria, little is known about the effects of probiotics on accelerating tooth movement. The interaction between microbial and biomechanical signals on periodontal cells and tissues remains understood.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eModulating immune response has been recognized as one of the key mechanisms underlying probiotic action. The immune system recognizes microorganisms as foreign bodies, triggering large amounts of inflammatory cells to generate innate and adaptive responses.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e Immunomodulatory effect is particularly important in OTM, where biomechanical forces applied to teeth trigger a complex inflammatory response in periodontal tissues.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e This response involves immune cells that release inflammatory mediators, matrix-degrading enzymes, and osteoclast-activating molecules, that all important for bone remodeling. Particularly, macrophages have been exerts crucial roles in alveolar bone remodeling\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e and root resorption\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e during OTM. Immune cells would directly interact with bone cells, suggesting a potential avenue for probiotics to influence bone remodeling during orthodontic treatment.\u003c/p\u003e \u003cp\u003eA large amounts of evidence suggests crucial links among probiotics, host immune response, and bone remodeling, and the precise mechanisms remain to be fully elucidated. In our study, we aims to explore how microorganisms can accelerate tooth movement, investigate the immunomodulation mechanism by which probiotics enhance OTM through macrophages. Potential probiotic candidates have been identified through 16S rRNA gene sequencing in a mouse model of OTM. To investigate the osteoimmunomodulatory impact, primary periodontal ligament cells (PDLCs) were obtained from mouse periodontal ligaments and cultured with or without conditioned media (CM) derived from macrophages post-incubation with microtia. The results demonstrated that \u003cem\u003eEnterococcus faecalis\u003c/em\u003e (\u003cem\u003eE. faecalis\u003c/em\u003e) increased the number of M1-polarized macrophages, and a decreased osteogenic level in PDLCs treated with CM \u003cem\u003eE. faecalis\u003c/em\u003e group. Moreover, \u003cem\u003eE. faecalis\u003c/em\u003e increased the distance in antibiotic mixture (ABX) treated mice during OTM model. Ultimately, this research endeavors to explore novel immunomodulatory mechanisms by which microorganisms can accelerate OTM, paving the way for innovative therapeutic strategies in orthodontic field. The outcomes of this research include shortened orthodontic treatment durations with minimal interventions, offering pioneering advancements in orthodontic treatment.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eOrthodontic tooth movement alters the oral microbiota\u003c/h2\u003e \u003cp\u003eTo 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.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). 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.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, c). The LEfSe showed that 10 bacterial species were enriched in the move group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). The Metastats analysis consistently showed that 2 bacterial species including probiotic \u003cem\u003eEnterococcus faecalis\u003c/em\u003e (\u003cem\u003eE. faecalis\u003c/em\u003e) were enriched in the move group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). As shown by the relative abundance of species-level microorganism, \u003cem\u003eE. faecalis\u003c/em\u003e was significantly enriched in the oral cavity in the move group \u003cem\u003eversus\u003c/em\u003e control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee). These findings altogether demonstrated that OTM promoted the accumulation of \u003cem\u003eE. faecalis\u003c/em\u003e in the oral of mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAfter 1-week application of force, micro CT of maxilla was performed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef).The distance of the move group was 88.66\u0026thinsp;\u0026plusmn;\u0026thinsp;16.42 mm (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg). The region of interest encompassed a 100 \u0026micro;m 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.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh). HE staining and Masson staining of distobuccal roots showed the integrity of periodontal tissue around the molars after tooth movement (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei,j).\u003c/p\u003e \u003cp\u003e \u003cb\u003eImmunomodulation on M1-like macrophage polarization by\u003c/b\u003e \u003cb\u003eEnterococcus faecalis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn order to elucidate the role of \u003cem\u003eE. faecalis\u003c/em\u003e, our focus is on profiling immune cells, specifically macrophages. As OTM is regarded as a \"sterile\" inflammatory process\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e and macrophages play a significant role in the tooth movement process,\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e this study emphasizes the examination of macrophages. RAW264.7 macrophages were treated with \u003cem\u003eE. faecalis\u003c/em\u003e. Quantitative real-time polymerase chain reaction (QRT-PCR) results showed that the expression levels of \u003cem\u003eTNFA, il1b, iNOS\u003c/em\u003e genes, which are recognized as M1 markers, were markedly increased in the \u003cem\u003eE. faecalis\u003c/em\u003e treated group compared to the control group. The M2 makers, \u003cem\u003earg1\u003c/em\u003e and \u003cem\u003ecd206\u003c/em\u003e, were downregulated in the \u003cem\u003eE. faecalis\u003c/em\u003e treated group compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Consistent with QRT-PCR results, flow cytometry results also revealed elevated percentage of CD86\u003csup\u003e+\u003c/sup\u003e macrophages and downregulated percentage of CD206\u003csup\u003e+\u003c/sup\u003e macrophages in the \u003cem\u003eE. faecalis\u003c/em\u003e treated group compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, c, d). Furthermore, the immunofluorescence staining results showed that the iNOS level in the \u003cem\u003eE. faecalis\u003c/em\u003e treated group was higher compared to control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). These results suggested that \u003cem\u003eE. faecalis\u003c/em\u003e increased the number of M1-polarized macrophages.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEnterococcus faecalis\u003c/b\u003e \u003cb\u003eattenuated osteogenesis of periodontal ligament cells (PDLCs)\u003c/b\u003e \u003cb\u003evia\u003c/b\u003e \u003cb\u003econditioned media of\u003c/b\u003e \u003cb\u003eEnterococcus faecalis\u003c/b\u003e \u003cb\u003etreated macrophages\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate the osteoimmunomodulatory impact of \u003cem\u003eE. faecalis\u003c/em\u003e on periodontal ligament cells (PDLCs) in conjunction with macrophages, an \u003cem\u003ein vitro\u003c/em\u003e 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 \u003cem\u003eE. faecalis\u003c/em\u003e. QRT-PCR analysis revealed that the expression levels of osteogenic genes (\u003cem\u003ealpl, runx2, bglap, spp1\u003c/em\u003e) treated with CM \u003cem\u003eE. faecalis\u003c/em\u003e were significantly lower compared to those in the CM control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Consistently, immunofluorescence staining results demonstrated a decreased OCN level in the CM \u003cem\u003eE. faecalis\u003c/em\u003e group compared to the CM control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Qualitative assessments further indicated that CM \u003cem\u003eE. faecalis\u003c/em\u003e dampened ALP activity and Alizarin Red level compared to the CM control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec, d).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEnterococcus faecalis\u003c/b\u003e \u003cb\u003epromotes orthodontic tooth movement in antibiotic mixture (ABX) treated mice\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate the impact of \u003cem\u003eE. faecalis\u003c/em\u003e on OTM, PBS or \u003cem\u003eE. faecalis\u003c/em\u003e suspension were administered to the ligature site using a syringe, twice daily to antibiotic mixture (ABX) mice during the OTM model (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Micro-CT images demonstrated a significant increase in the distance of OTM in mice treated with \u003cem\u003eE. faecalis\u003c/em\u003e compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, c). Interestingly, the OTM distance in the ABX control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec) was lower than in the previous normal control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg), 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 \u003cem\u003eE. faecalis\u003c/em\u003e and the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). Histological staining of distobuccal roots with HE and Masson staining showed the integrity of periodontal tissue surrounding the molars post-tooth movement (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). Furthermore, the number of iNOS\u003csup\u003e+\u003c/sup\u003e cells increased on the compression side in the \u003cem\u003eE. faecalis\u003c/em\u003e-treated group compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEnterococcus faecalis\u003c/b\u003e \u003cb\u003eenhances the M1 polarization of macrophages through the mTOR and AKT signaling pathways\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eE. faecalis\u003c/em\u003e has been shown to modulate macrophage activity. The mTOR and AKT signaling pathways are key regulators of macrophage polarization, has been well-recognized.\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e Based on this understanding, our hypothesis posited that \u003cem\u003eE. faecalis\u003c/em\u003e 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 \u003cem\u003eE. faecalis\u003c/em\u003e, indicating activation of this signaling cascade (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, 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 \u003cem\u003eE. faecalis\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThis study pioneers the exploration of the impact of microorganisms on OTM, which previously dominated by antimicrobial focuses.\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e While prior research has primarily centered on eliminating pathogenic bacteria in the oral cavity during orthodontic treatment,\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e this investigation focus on the novel concept that probiotics may also exert a beneficial influence on OTM. Although orthodontic treatment itself involves a localized, \u0026ldquo;sterile\u0026rdquo; movement of teeth,\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e we hypothesize that oral-administrated \u003cem\u003eE. faecalis\u003c/em\u003e, through intricate interactions with immune cells, could indirectly modulate the bone remodeling processes inherent to OTM. This potential influence of oral microorganisms, if confirmed, could unveil new frontiers in accelerating orthodontic treatment outcomes by harnessing the power of beneficial probiotics.\u003c/p\u003e \u003cp\u003eProbiotics are beneficial bacteria or microorganisms that can improve overall health when consumed in adequate amounts through the diet.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e While probiotics are often regarded for their ability to reduce harmful bacteria, their role in orthodontic treatment remains unknown. The use of probiotics during fixed orthodontic treatment have been demonstrated to reduce \u003cem\u003eS. mutans\u003c/em\u003e and \u003cem\u003eLactobacillus\u003c/em\u003e levels in the saliva.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e However, a research indicates that probiotic supplementation, despite its potential benefits, did not significantly affect the development of inflammation in gingiva and decalcification in enamel.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eE. faecalis\u003c/em\u003e occur in gastrointestinal tract of animals and humans.\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e Certain strains of \u003cem\u003eE. faecalis\u003c/em\u003e have demonstrated probiotic properties,\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e exemplified by its inclusion in the commercially available probiotic \u003cem\u003eE. faecalis\u003c/em\u003e Symbioflor 1 (SymbioPharm, Herborn, Germany). Emerging evidence suggests that \u003cem\u003eE. faecalis\u003c/em\u003e may influence bone remodeling processes,\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e raising possibilities for its application in orthodontics.\u003c/p\u003e \u003cp\u003eThe immunomodulatory properties of \u003cem\u003eE. faecalis\u003c/em\u003e through M1 polarization of macrophages have been elucidated in our study. Particularly, macrophages have been exerts crucial roles in alveolar bone remodeling\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e and root resorption\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e during OTM. Our study provides evidence that enhanced production TNF-α, a pro-inflammatory cytokine contributes to impair osteogenesis of PDLCs. Consistent with our findings, \u003cem\u003eE. faecalis\u003c/em\u003e have been demonstrates to shifts macrophage polarization to M1-like phenotype\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. M1-like macrophages are reported to promotes alveolar bone resorption and consequent OTM after mechanical force application.\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eMoreover, \u003cem\u003eE. faecalis\u003c/em\u003e have also been demonstrated to immunoregulates osteoclastogenesis of macrophages and upregulates expression of inflammatory cytokine iNOS.\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e Macrophages possess the capability to differentiate into osteoclasts,\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e which are responsible for bone resorption. This osteoclastogenic potential of macrophages is particularly relevant in tooth movement, as targeted bone resorption at the root surface is essential for successful tooth repositioning. Therefore, the ability of macrophages to osteoclastogenesis may constitute a key mechanism underlying the \u003cem\u003eE. faecalis\u003c/em\u003e effects on accelerated tooth movement.\u003c/p\u003e \u003cp\u003eThe mTOR and AKT signaling pathways are key regulators of macrophage polarization, has been well-recognized.\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e Akt has been shown to modulate the activity of NF- κB,\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e a crucial transcription factor for M1 activation, in both positive and negative ways. Consistent to our reports, Akt isoforms has been proved to contribute to M1 polarization.\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e Also, mTORC1 signaling has been characterized as positive regulation in M1 macrophages,\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e and PP2A-mTOR-p70S6K/4E-BP1 axis has been demonstrated to regulate M1 polarization of pulmonary macrophages.\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e In our study, \u003cem\u003eE. faecalis\u003c/em\u003e can drive macrophage polarization towards the M1 phenotype through immunomodulatory mechanisms, leading to the activation of the mTOR-AKT cascade. This activation promotes the production of pro-inflammatory cytokines of macrophages, ultimately downregulates osteogenic differentiation of PDLCs.\u003c/p\u003e \u003cp\u003eIn conclusion, our study has investigated the potential effects of \u003cem\u003eE. faecalis\u003c/em\u003e administration on the accelerated OTM process through immunomodulation. We elucidated the mechanisms by which \u003cem\u003eE. faecalis\u003c/em\u003e modulates M1-like macrophage polarization then regulates PDLCs, ultimately enhancing the distance of OTM (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). Further research is needed to identify specific probiotic strains and their effects on the gastrointestinal tract microbiota to fully elucidate the potential of probiotics. This research will lead to an acceleration in the duration of orthodontic treatment with minimal interventions, thereby offering significant value in developing probiotic mouthwash for future applications.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eAnimal Experiments Design\u003c/h2\u003e \u003cp\u003eAdult male C57BL/6 WT mice were purchased from Gempharmatech (Nanjing, China) and were housed in a specific pathogen-free (SPF) facility with a 12:12-h light-dark cycle. The use of animals in this study was approved by the Animal Care and Ethics Committee of West China School of Stomatology, Sichuan University (WCHSIRB-D-2022-611). The experimental design involved both an experimental group and a control group (n\u0026thinsp;=\u0026thinsp;6:6) randomly, with both groups using nickel-titanium coil springs. After anesthesia with isoflurane, the coil springs were ligated between the maxillary incisors and the maxillary first molar with stainless steel and light-cured glass ionomer cement. The key distinction between the two groups was that no force was applied to the coil springs in the control group, whereas 40g force\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e was applied to the coil springs in the experimental group. After 7 days, all mice were sacrificed using an overdose of sodium pentobarbital.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e16S rRNA gene sequencing\u003c/h2\u003e \u003cp\u003eOral microbiota was collected from mice using oral swabs. The sample were processed by Novogene (Beijing, China). Briefly, total genomic DNA was extracted, and the 16S rRNA genes of distinct regions were amplified. Sequencing libraries were prepared using the SMRTbellTM Template Prep Kit (PacBio) according manufacturer's instructions. The library quality was assessed on the Qubit@ 2.0 Fluorometer (Thermo Scientific) and FEMTO Pulse system, and the library was sequenced on the PacBio Sequel platform. Procession of the sequencing data were performed with QIIME (Version1.9.1) and displayed with R software (Version 2.15.3).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMicro-CT\u003c/h2\u003e \u003cp\u003eMice were sacrificed 7 days after OTM model. The mandibles were then fixed in 4% polymerized formaldehyde for 48 h. The samples were scanned using a micro-CT system (Scanco Medical). To prevent dehydration, the samples under examination were kept in a moist environment during the scanning process. The scans were conducted at a resolution of 10 \u0026micro;m, with a voltage of 80 kV and a current of 500 mA. Morphometric analyses were carried out using software (Scanco Medical Evaluation \u0026amp; Visualizer, Scanco Medical). The distance between the most convex point of the maxillary first molar and the most convex mesial point of the maxillary second molar was measured using digital slide calipers on the micro-CT images. The region of interest (ROI) selected for analysis was a 100 \u0026micro;m cuboid extracted from the furcation area of the maxillary first molar. Various three-dimensional morphometric parameters of bone microarchitecture were then calculated for analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eHistological analysis\u003c/h2\u003e \u003cp\u003eFollowing micro-CT scanning, the mandibles underwent a two-week decalcification process in PBS solution with 10% EDTA\u0026middot;2Na, with daily replacement of the solution. Subsequently, the maxilla were dehydrated using a gradient of ethanol, then embedded in paraffin and sliced into 5-\u0026micro;m-thick sections for H\u0026amp;E staining and Masson staining. TRAP staining was conducted using a commercially available kit (Servicebio). The staining procedures were carried out in accordance with established protocols.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMacrophages culture and infected with\u003c/b\u003e \u003cb\u003eE. faecalis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eRAW264.7 macrophages were cultured in DMEM medium (Gibco) supplemented with 10% fetal bovine serum (Biological Industries), 100 U/mL penicillin-streptomycin (HyClone, Thermo Scientific). The cells were cultured in a humidified environment with 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C. \u003cem\u003eE. faecalis\u003c/em\u003e was obtained from BeNa Culture Collection and cultured in sterile brain heart infusion (BHI) broth (Hopebio) under aerobic conditions. RAW264.7 macrophages were seeded overnight, and \u003cem\u003eE. faecalis\u003c/em\u003e was added to the culture at a multiplicity of infection (MOI) of 100 for 3 h. The supernatants of macrophages were filtered through PES filters (Merck Millipore) to obtain conditioned media (CM). TNF-α concentrations in CM of macrophages were detected using mouse ELISA kit (Nanjing Jiancheng Bioengineering Institute) according to the manufacturer\u0026rsquo;s protocol.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003ePDLCs culture and treatments\u003c/h2\u003e \u003cp\u003eMouse PDLCs were isolated from PDL tissue and maintained in α-MEM medium with 10% FBS (Gibco) and 1% penicillin-streptomycin (HyClone, Thermo Scientific). PDLCs were treated with CM of macrophages. For qualitative assay, PDLCs were stained with BCIP/NBT ALP Color Development Kit (Beyotime) and Alizarin Red S Solution (Solarbio) according to the manufacturer\u0026rsquo;s protocol.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative Real-Time-Polymerase Chain Reaction (QRT-PCR)\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted using TRIzol reagent (Life Technologies) and reverse transcription was carried out with the PrimeScriptTM RT reagent Kit (Takara). QRT-PCR was conducted on a LightCycler 96 system (Roche) using SYBR Green Mix (Takara). The data were analyzed using the comparative cycle threshold method (ΔΔCt) and normalized to the GAPDH housekeeping gene.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry analysis\u003c/h2\u003e \u003cp\u003eSingle-cell suspensions from macrophages were collected. Live/Dead staining (fixable viability stain 780, BD Biosciences) was used to determine the live cells. Fc receptors were blocked by treating the cells with CD16/32. CD86 antibodies (PE-Cy7, BD Biosciences) were then added as according to the manufacturer's instructions. Next, the cells were permeabilized using a flow cytometry permeabilization/wash buffer (BD Biosciences) and intracellularly stained with CD206 antibodies (AF647, BD Biosciences). The stained cells were analyzed using a Flow Cytometer (Beckman) and analyzed with Flowjo software (BD Biosciences).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence Staining\u003c/h2\u003e \u003cp\u003eMacrophages or PDLCs were treated by fixation with a 4% paraformaldehyde solution, followed by permeabilization using 0.1% Triton X-100, and blocking with 5% bovine serum albumin (BSA). Primary antibodies (anti-OCN from Yeasen; anti-iNOS from ABclonal Technology) were applied and incubated overnight, followed by secondary antibody (Yeasen), F-actin labeling (Yeasen), and nuclear staining (using Hoechst 33258 or DAPI from Biosharp). Immunofluorescence staining was visualized and captured using a fluorescence microscope (Leica).\u003c/p\u003e \u003cp\u003e \u003cb\u003eAntibiotic mixture (ABX) Treatments and\u003c/b\u003e \u003cb\u003eE. faecalis\u003c/b\u003e \u003cb\u003eAdministration\u003c/b\u003e\u003c/p\u003e \u003cp\u003eA mixture containing ampicillin (1 g/L), metronidazole (1 g/L), gentamicin (1 g/L), neomycin (1 g/L), and vancomycin (0.5 g/L) was administered to mice through both their drinking water and by gavage for a period of 10 days. Following this treatment, normal drinking water was provided for 2 days to eliminate the effects of the antibiotics before introducing \u003cem\u003eE. faecalis\u003c/em\u003e laterally. The mice were randomly divided into 2 groups (n\u0026thinsp;=\u0026thinsp;5:5). In experimental group, a suspension of \u003cem\u003eE. faecalis\u003c/em\u003e (5 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e CFU/mL, 100\u0026micro;L\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e) was applied to the ligature site of molar using a syringe, twice daily for 7 days during the OTM model. The control group received a PBS solution instead. After 7 days, all mice were sacrificed using an overdose of sodium pentobarbital.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry Analysis\u003c/h2\u003e \u003cp\u003eFollowing decalcification, the maxillae were dehydrated using an ethanol gradient, embedded in paraffin, and sliced into 5-\u0026micro;m-thick sections. Endogenous peroxidase blocking buffer was applied, followed by washing and antigen retrieval. Subsequently, a 5% BSA solution was used for blocking, and incubated the sections with anti-iNOS (Proteintech) overnight, and then applying a secondary antibody. Immunofluorescence staining was visualized and captured using a fluorescence microscope (Leica).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eWestern Blot Analysis\u003c/h2\u003e \u003cp\u003eMacrophages were lysed using RIPA buffer containing protease inhibitors. The extracted proteins were then separated via 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes (Millipore). After being blocked with 5% BSA, the membranes were incubated to primary antibodies specific for AKT (Cell Signaling Technology), phosphorylated AKT (p-AKT, Cell Signaling Technology), mTOR (Cell Signaling Technology), phosphorylated mTOR (p-mTOR, Cell Signaling Technology), and GAPDH (Aksomics).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eData were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of mean and analyzed using Prism 9.3 (GraphPad Software). An unpaired Student's t-test was used to compare the means of two experimental groups. P-values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCONFLICT OF INTERESTS\u003c/h2\u003e \u003cp\u003eThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCONTRIBUTIONS\u003c/h2\u003e \u003cp\u003eF. Yu, contributed to conception, design, data acquisition and analysis, drafted and critically revised the manuscript; W. Lu, J. Peng and P. Li contributed to data acquisition and analysis, critically revised the manuscript; Z. Zhao contributed to conception, design, data analysis, and critically revised the manuscript. All authors gave final approval and agree to be accountable for all aspects of the work.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eACKNOWLEDGEMENTS\u003c/h2\u003e \u003cp\u003eThis work was supported by National Natural Science Foundation of China (32271416, 62306193), Natural Science Foundation of Sichuan Province (2023NSFSC0562), Sichuan University Postdoctoral Interdisciplinary Innovation Fund (JCXK2202), West China School/ Hospital of Stomatology, Sichuan University (RCDWJS2022-1) and IOF Research Grant ( IOF2022Y01).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eTan, L. \u003cem\u003eet al.\u003c/em\u003e Engineered probiotics biofilm enhances osseointegration via immunoregulation and anti-infection. 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Periodontol. 91, 1367\u0026ndash;1374 (2020).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4943788/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4943788/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eProbiotic-mediated therapy has garnered increasing attention for its potential role in influencing bone remodeling. Recent studies have consistently shown that the microorganisms can play a role in modulating bone reconstruction. In this study, we aim to explore the impact of probiotics on accelerating orthodontic tooth movement (OTM). Potential probiotic candidates will be identified through 16S rRNA gene sequencing in a mouse model of OTM. The relative abundance of \u003cem\u003eEnterococcus faecalis\u003c/em\u003e (\u003cem\u003eE. faecalis\u003c/em\u003e) increased in the move group. To investigate the osteoimmunomodulatory impact, primary periodontal ligament cells (PDLCs) were obtained from mouse periodontal ligaments and cultured with or without conditioned media (CM) derived from macrophages post-incubation with \u003cem\u003eE. faecalis\u003c/em\u003e. The results demonstrated that \u003cem\u003eE. faecalis\u003c/em\u003e increased the number of M1-polarized macrophages, and a decreased osteogenic level in PDLCs treated with CM \u003cem\u003eE. faecalis\u003c/em\u003e group. Subsequently, the microbiota of the mice has be depleted using broad-spectrum antibiotic mixture (ABX) before being administered with \u003cem\u003eE. faecalis\u003c/em\u003e. The distance of OTM have been measured, and the alveolar bone have been analyzed using micro-CT and immunohistochemical staining. This study has explored the potential effects of \u003cem\u003eE. faecalis\u003c/em\u003e administration on the OTM process through immunomodulation. We assessed the efficacy of \u003cem\u003eE. faecalis\u003c/em\u003e in accelerating tooth movement, and elucidated the mechanisms by which \u003cem\u003eE. faecalis\u003c/em\u003e modulate M1-like macrophage polarization to enhance OTM. This research will lead to an acceleration in the duration of orthodontic treatment with minimal interventions, thereby offering significant value and pioneering advancements in orthodontic treatment.\u003c/p\u003e","manuscriptTitle":"Enterococcus faecalis promotes orthodontic tooth movement in mice by M1-like macrophage polarization","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-03 18:01:40","doi":"10.21203/rs.3.rs-4943788/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9d867ecb-704c-4dab-a420-d0916d7b7ecf","owner":[],"postedDate":"October 3rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":36944463,"name":"Biological sciences/Microbiology/Bacteria"},{"id":36944464,"name":"Health sciences/Medical research"}],"tags":[],"updatedAt":"2024-12-09T08:13:26+00:00","versionOfRecord":[],"versionCreatedAt":"2024-10-03 18:01:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4943788","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4943788","identity":"rs-4943788","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}