hBMSC-derived Exosomes Mitigate Th17/Treg Homeostasis in Periodontitis via miR-1246

doi:10.21203/rs.3.rs-1961434/v1

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hBMSC-derived Exosomes Mitigate Th17/Treg Homeostasis in Periodontitis via miR-1246

https://doi.org/10.21203/rs.3.rs-1961434/v1

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Background

Chronic inflammatory bone loss in periodontitis is closely related to helper T cell 17 (Th17) / regulatory T cell (Treg) imbalance. The therapeutic function of mesenchymal stem cells is mainly attributed to the paracrine exosomes. We aimed to investigate the immunomodulatory effect of human bone marrow mesenchymal stem cells (hBMSC)-derived exosomes in Th17/Treg homeostasis in periodontitis.

Methods

Peripheral blood was collected from periodontitis patients or healthy donors. The level of Th17-related biomarker interleukin-17A (IL-17A) and Treg-related forkhead box protein 3 (FOXP3) was analyzed by ELISA, and mRNA expression level of RAR-related orphan receptor C (RORC) and FOXP3 was detected by qRT-PCR. Naïve CD4⁺ T cells extracted from the peripheral blood of periodontitis patients were co-cultured with hBMSC-derived exosomes from healthy subjects. The ratio of Th17/Treg was examined by flow cytometry and the expression of inflammatory cytokines was determined by qRT-PCR. In vivo, exosomes-loaded hydrogel was injected into periodontal pockets of mice with experimental periodontitis. The periodontal inflammation and bone regeneration was evaluated by histological staining, immunofluorescence staining and micro-CT. Furthermore, the differentially expressed miRNAs in exosomes stimulated by P.g. LPS were investigated by miRNA sequencing and bioinformatics analysis. The interaction between candidate miRNA and its target gene in CD4⁺ T cells was verified by dual luciferase activity assay. Lastly, the downstream Yes-related protein 1(YAP1)/Hippo pathway was evaluated by western blotting.

Results

The ratio of Th17/Treg is significantly increased in the peripheral blood of periodontitis patients. hBMSC-derived exosomes decreased Th17/Treg ratio in CD4⁺ T cells in vitro and ameliorated inflammation and bone loss in periodontitis mice. Mechanistically, the enrichment of miR-1246 in exosomes showed the enhanced effects of down-regulating Th17/Treg ratio, which could be reversed by miR-1246 inhibitor. Furthermore, exosomal miR-1246 suppressed the expression of the target protein angiotensin converting enzyme2 (ACE2) and upregulated the expression ratio P-YAP1/YAP1 in CD4⁺ T cells.

Conclusions

hBMSC-derived exosomes could alleviate periodontal inflammation through modulating the balance of Th17/Treg via targeting ACE2 and downregulating YAP1/Hippo signaling pathway, which shed light on a novel cell-free immunotherapy for periodontal regeneration.

Periodontal disease

Stem cell

microRNA

Angiotensin-converting enzyme 2 (ACE2)

Hippo signaling pathway

Periodontitis, the main cause of tooth loss and alveolar bone destruction in adults, is an inflammatory disease with a high global burden and profound socioeconomic effects worldwide¹. Periodontitis originates from an imbalance in the host–microbe relationship in the oral cavity, which leads to dysregulation of the immuno-inflammatory response and subsequent destruction of periodontal tissue and alveolar bone^2,3. An imbalance in the ratio of T helper 17 (Th17) cells and regulatory T (Treg) cells contribute to the development of periodontitis⁴.

In response to a dysbiotic microbiome in periodontitis, expanded Th17 cells drive periodontal bone loss through excessive neutrophil recruitment and related immunopathology closely with secreted proinflammatory cytokines IL-17⁵ and orphan receptor gamma transcription (RORγt)⁶. In previous studies, we found that IL-17 promoted bone resorption via osteocyte-specific signaling pathways, and also regulated the osteogenesis of mesenchymal stem cells (MSCs)^7,8,9. Conversely, Treg cells participate in immunosuppression and tissue repair to maintain periodontal health, depending on sustained high expression of FOXP3 and interleukin-10 (IL-10)¹⁰. Rather than in periodontal tissue, an elevated Th17/Treg ratio in peripheral blood without systemic diseases is also associated with periodontal inflammation and alveolar bone destruction^2,11. However, there has been a need to fully understanding the Th17/Treg imbalance in periodontitis.

MSCs have been regarded as a promising cell-based tissue engineering therapy and used for periodontal regeneration in clinical practice^12,13. However, the MSC therapy continues to involve many challenges, such as low tissue retention, low survival rate and loss of cell fate control¹⁴. Recently, there is growing evidence that the immunomodulatory effect of MSCs is attributed to its paracrine secretion, particularly exosomes^15,16. With a diameter of 30–200 nm, exosomes are considered as the delivery vehicle for bioactive compounds to target cells¹⁷. It has been reported that hBMSC-derived exosomes, rich in microRNAs (miRNAs) and proteins, can suppress the inflammatory responses of macrophages and promote periodontal regeneration^18,19.

miRNAs are small non-coding RNAs that modulate gene expression in various aspects of eukaryotic life, primarily via RNA-silencing mechanisms²⁰. MiRNAs in the periodontium, such as miR-17, miR-19, miR-155, and miR-301, are significantly increased in periodontitis patients or animals compared with healthy controls, playing essential roles in the progression of periodontal disease^21,22. Furthermore, miRNAs have key regulatory effects on T-cell differentiation and polarization²³. It has been reported that exosomal miRNAs from dental pulp stem cells can restore Th17/Treg homeostasis in periodontitis²⁴. However, there remains a lack of information concerning the therapeutic applications of MSC-derived exosomes and the ability of exosomal miRNAs to restore Th17/Treg homeostasis in periodontitis.

Angiotensin-converting enzyme 2 (ACE2), a vital receptor of T cells in infectious diseases²⁵, especially reported in 2019 novel coronavirus²⁶. ACE2 could be induced by miR-146a and miR-155 in periodontal inflammatory microenvironment, and further modulate host antiviral response²⁷. Moreover, Yes-related protein 1 (YAP1), a relevant protein in Hippo signaling pathway, could restrict CD4⁺ T cell activation and proliferation^29,30. However, the target of MSC-derived exosomal miRNAs on T cells in periodontitis is still unclear, and the downstream pathways have not been elucidated.

Therefore, we hypothesized that hBMSC-derived exosomes and their key miRNAs affect the Th17/Treg ratio among CD4⁺ T cells from patients with periodontitis. The target genes and related signaling pathways were sought in exosomal miRNA-regulated CD4⁺ T cells. Our findings will contribute to the development of innovative non-cellular immunotherapy for periodontitis.

Participant Recruitment

This study was approved by the Ethics Committee of Stomatological Hospital of Tongji University (Approval No. 2020-R-011) and adhered to the Declaration of Helsinki. An informed consent form was signed by each participant and/or legally authorized next of kin regarding the use of any biological sample. The diagnosis of periodontitis was defined with periodontal examinations evaluating by one periodontist: probing depth ≥ 5 mm in at least four teeth with each ≥1 site, clinical attachment level (CAL) ≥5 mm on the same site, and observed bleeding upon stimulus. All participants met the following inclusion criteria: periodontitis, aged from 18-40 years, the maintenance of systemic health. The exclusion criteria included consumption of antibiotics or anti-inflammatory medications within the last 3 months, previous periodontal treatment, any systemic diseases that might affect periodontal disease, immunodeficiency, pregnancy and previous smoking history or current smoking. The detailed information of participants was shown in Appendix Tbl. 1.

hBMSC extraction and identification

The cancellous bone marrow was extracted from discarded maxilla bone tissue of 5 orthognathic patients (3 females and 2 males,18-25 years old) with good oral health in the Stomatological Hospital of Tongji University. The bone marrow was washed with PBS once and then resuspended with minimum essential medium Alpha (α-MEM, Hyclone, Utah, USA) containing 10% fetal bovine serum (Gibco, Waltham, MA, USA) and 1% penicillin/streptomycin (Hyclone). When the confluence reached about 80%, the cells were digested by trypsin/EDTA and the passage was recorded as P1 cells. The P3 cells were identified as hBMSCs by flow cytometry analysis with the surface markers CD90, CD73, CD44 and CD105, using the Human MSC Analysis Kit (BD Biosciences, San Jose, CA).

hBMSC-derived exosome extraction and identification

When hBMSCs grew to 80% confluence, the medium was replaced with serum-free α-MEM. After 48 hours, the medium was concentrated to 1/10 of the original volume in an Amicon® Ultra 15 mL Centrifugal Filters (Membrane NMWL 100 KDa, Millipore, Bedford, MA) at 800 g for 5 min, and then transferred to a sterile container, mixed with an appropriate amount of ExoQuick precipitation solution (Invitrogen, Carlsbad, USA) and incubated at 4 °C for 16 hours. The pellet of exosomes was collected by centrifugation at 10,000 g, 4 °C for 2 hours, and re-suspended with 200 μl PBS.

Exosomes stained with antibodies against CD63 and CD81 were examined by flow cytometry using a BD LSRFortessa™ Cell Analyzer (BD Biosciences, San Jose, CA), and the data were analyzed using FlowJo v7.6. For the Transmission electron microscope (TEM) analysis, samples were dried and examined under an electron microscope (FEI, Tecnai G2 F20 S-TWIN, USA). For the nanoparticle tracking analysis (NTA), the pellets collected from programmed centrifugation were resuspended in PBS, and then the size of vesicles was detected using a nanoparticle tracing assay (NanoSight, Particle Metrix, Meerbusch, German).

Analysis of the expression of inflammatory factors in peripheral blood of subjects

Serum from fasting peripheral blood of 8 healthy volunteers and 8 periodontitis patients aged 18 to 40 years were collected for ELISA analysis. Serum levels of IL-17A and FOXP3 were measured using ELISA kit (Abcam, Cambridge, UK). Moreover, the total RNA was extracted from the peripheral blood and detected by qRT-PCR. 1 μg of total RNA was reverse transcribed to single-stranded cDNA with a PrimeScript^TM RT Reagent Kit (Takara, Nojihigashi, Japan). The PCR mixture was prepared using CFX96™ Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The primers (Sangon Biotech, Shanghai, China) used in the process are shown in Appendix Tbl. 2. Human RORC and FOXP3 were normalized to ACTB.

Isolation of naïve CD4⁺ T cells from patients with periodontitis

Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of 5 periodontitis patients by FIColl-Paque (GE Healthcare, Madison, WI) gradient centrifugation. Periodontitis naïve CD4⁺ T cells were prepared from pooled mixed PBMCs through positive selection by depletion of non-T lymphocytes with the human naïve CD4⁺ T cell isolation kit (Miltenyi Biotechnologies, Teterow, German) following the manufacturer's instructions.

Internalization of exosomes by CD4⁺ T Cells

Exosomes were labelled with PKH67 Green Fluorescent Cell Linker Kit (Umibio, Shanghai, China). The stained exosomes were mixed into CD4⁺ T cells resuspended in RPMI 1640 medium and cultured for 12 hours. The nucleus was stained with DAPI. Observation and photographing were performed under a microscope (Leica, CM1900, Wetzlar, Germany).

Analysis of CD4⁺ T cells differentiation

The naïve CD4⁺ T cells from periodontitis patients were incubated with 10 µg/mL hBMSC-derived exosomes from healthy volunteers for 24 hours. Flow cytometry was used to determine the differentiated Th17 and Treg cells. The naïve CD4⁺ T cells were incubated with PE-labeled anti-CD4, APC-labeled anti-IL17A, and FITC-labeled anti-FOXP3 antibodies in blocking buffer (BioLegend, CA, USA) for 30 min. Cells were examined using a BD LSRFortessa™ Cell Analyzer (BD Biosciences) and the data were analyzed using FlowJo v7.6. Additionally, mRNA expression of RORC, FOXP3, IL10 and IL17A in CD4⁺ T cells were analyzed by qRT-PCR. The primers (Sangon Biotech, Shanghai, China) used in the process are shown in Appendix Tbl. 2.

Animals

All animal procedures were approved by the Institutional Animal Care and Use Committee of Stomatological Hospital of Tongji University (Approval No. 2020-DW-026). A prospective randomized controlled animal model design was adopted according to all the recommendations of the ARRIVE guidelines (Animal Research: Reporting In Vivo Experiments). Anesthesia and euthanasia of animals complied with the American Veterinary Medical Association (AVMA) Guidelines (2020).

A total of 12 male C57BL/6J mice (7 weeks of age, 22 to 24 g of weight, Leagene Biotech, China) were used in this study. Mice were group housed in polycarbonate cages with controlled temperature, 40% to 65% of humidity, and a 12-hour light/dark cycle. Mice were acclimatized for 1 week before the experiment, fed with a standard laboratory diet, and allowed ad libitum access to drinking water.

Murine model of periodontitis and exosome treatment

Each mouse was randomly distributed to the following treatment groups: Control group (n = 3), Periodontitis (Perio) group (with ligation, n = 3), Hydrogel group (hydrogel application after ligation, n = 3), and Hydrogel+Exosomes (Hydrogel+Exo) group (hydrogel+exosomes application after ligation, n = 3). To induce periodontitis, a 6-0 silk suture was applied to the upper second molar without causing damage to the periodontal tissue. After 7 days, the silk thread was removed when clinical signs of gingival inflammation (swelling, redness and loss of adhesion of gingival to the tooth) were observed.

For preparation of the exosomes-loaded hydrogel, 40 mg polyether F127 thermosensitive hydrogels (EngineeringForLife, Suzhou, China) powder was added to 200 μl exosome-PBS (1μg exosome/μl PBS) suspension. 10 μl hydrogel with or without hBMSC-derived exosomes were injected into periodontal pockets by a 26 sgauge syringe (Hamilton Company, Reno, NV, USA) every other day. Mice were anesthetized by inhalation of 4.0 % sevoflurane and maintained with 3.0 % sevoflurane. Control group and Perio group were sacrificed on day 7; Hydrogel and Hydrogel+Exo group were sacrificed on day 14.

Histomorphological analysis and immunofluorescence

The maxillae were separated, decalcified, fixed and then embedded in paraffin. Sections of 4 μm thickness were sliced out, collected on glass slides, deparaffinized and then stained with haematoxylin & eosin (H&E, KeyGen, Nanjing, China). The distance between the cementoenamel junction and alveolar bone crest (CEJ-ABC) was measured to evaluate bone loss. For immunofluorescence, the sections were then incubated at 4 °C with either anti-RORγ antibody or anti-FOXP3 antibody (Appendix Tbl. 3). Following 3 washes in PBS, the sections were then incubated with Alexa Fluor-488 or Alexa Fluor-594 secondary antibody for 1hour. Negative control sections were set by omitting the primary antibodies. The sections were mounted with the medium containing DAPI and examined under a microscope (Leica, CM1900, Wetzlar, Germany).

Micro-computerized tomography (micro-CT)

Computed tomography was used to quantify bone remodeling by using the Scanco Medical µCT50 system and related analysis software. A region of interest (ROI) was selected that contained the areas of maxillae. The key parameters were set as follows: 70 kV, 110 mA, and 10 μm increments. For quantifying trabecular bone volume/total volume (BV/TV), a total of 300 μm (30 layers) of the alveolar bone between the first and second molars were analyzed.

miRNA sequencing (miRNA-seq)

hBMSCs were stimulated with 1 μg/ml Porphyromonas gingivalis lipopolysaccharide (P.g. LPS, Sigma-Aldrich, St. Louis, MO, USA) for 24 hours and the corresponding exosomes were isolated. The control group received an equivalent dose of PBS. Small RNAs were isolated from the exosomes for miRNA-seq. The miRNA-seq libraries were prepared and sequenced with an Illumina HiSeq (Illumina, Fasteris SA, Switzerland) at Wayen Biotechnologies, Inc (Shanghai, China). Feature counts were used to calculate read counts, and DESeq2 was used to analyze the differential expression of genes. Genes with a corrected p-value ≤ 0.05 and an absolute log2 (fold-change) > 2 were considered differentially expressed.

Transfection

miR-1246 mimic, inhibitor, nc on and nc off (Ribobio, Guangzhou, China) were transfected into healthy hBMSCs for 48 hours using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's protocols. The efficiency of transfection was checked by qRT-PCR. The primers (Sangon Biotech, Shanghai, China) used in the process are shown in Appendix Tbl. 2. Human miR-1246 was normalized to U6.

Target gene prediction and dual luciferase reporter assay

miR-1246 target genes were selected using miRDB (http://mirdb.org/), miRWalk (http://mirwalk.umm.uni-heidelberg.de/) and Targetscan (https://www.targetscan.org/vert_80/). ACE2 gene, which was relevant to miR-1246, were selected for further investigation. miR-1246-binding sites on the 3’-untranslated region (UTR) of ACE2 were recognized by TargetScan online bioinformatics software (http://www.targetscan.org). Dual luciferase activity was analyzed by using a dual luciferase assay kit (Promega, Madison, WI) following the manufacturer’s instructions. In brief, ACE2 recombinant plasmids (ACE2-WT and ACE2-Mut) and mimic NC or miR-1246 were transfected into 293T cells.

Western blot

The polyvinylidene fluoride membrane onto which separated proteins were transferred was immunoblotted with primary antibodies against YAP1 (Abcam), P-YAP1 (S127) (Abcam), ACE2 (Abcam), RORγ (Affinity), FOXP3 (Affinity), and β-actin (Affinity). The antibody concentration was shown in Appendix Tbl. 3. The protein bands were visualized with enhanced chemiluminescence and imaged using an Amersham™ Imager 680 (GE Healthcare Bio-Sciences, Uppsala, Sweden).

Data Analysis

Results are representative of independent (n ≥ 3) experiments; Data were presented as mean ± standard deviation. 2-tailed Student’s t test was used for comparison between 2 groups and one-way analysis of variance test was used for multiple comparison. P < 0.05 was considered statistical significance. All statistical analyses were performed using GraphPad Prism software (version 9.0).

Identification of hBMSCs and exosomes

hBMSCs with fibroblast-like morphology were isolated from maxillae of periodontally healthy subjects during orthognathic surgery (Fig. 1A). The osteogenic and adipogenic characteristics of hBMSCs were confirmed by alizarin red and oil red O staining, respectively (Fig. 1B, C). The purity of hBMSCs was detected by flow cytometry analysis with the surface markers CD90 (92.4%), CD73 (92.3%), CD44 (64.1%), and CD105 (91.9%) (Fig. 1D). hBMSC-derived exosomes exhibited round or tea receptacle microvesicle structure ranging in diameter from 90 to 150 nm (Fig. 1 E, F), and expressed surface markers CD63 and CD81 (Fig. 1G).

Peripheral Levels of Th17- and Treg-related Biomarkers were disrupted in Periodontitis Patients

Th17 and Treg biomarkers were measured in peripheral blood from periodontitis patients and healthy subjects. The mRNA expression level of RORC was significantly upregulated in periodontitis patients than that of healthy volunteers; in contrast, the FOXP3 mRNA expression level was significantly downregulated in periodontitis patients (Fig. 2A). Similarly, ELISA showed that the level of Th17-related IL-17A in periodontitis patients was significantly increased as compared with healthy controls, while Treg-related FOXP3 was significantly decreased (Fig. 2B) (P < 0.001). These results implied that periodontitis tips the balance of immune homeostasis by enhance the function of Th17 cells and impair Treg cells.

Periodontally Healthy hBMSC-derived Exosomes Restored the Th17/Treg Ratio in Naïve CD4⁺ T Cells Derived from Peripheral Blood of Periodontitis Patients in vitro

Naïve CD4⁺ T cells were derived from PBMCs of periodontitis patients (Fig. 2C, Appendix Fig. 1) PKH67-labeled exosomes were visualized in CD4⁺ T cells after treatment for 12 hours. (Fig. 2D). We next analyzed CD4⁺T-cell subpopulation after 24 hours incubation with hBMSC-derived exosomes. Flow cytometry assay revealed that exosmes remarkably reduced the proportion of Th17 cells by 60% (P < 0.01), while increased the proportion of Treg cells by 10% (P < 0.05), leading to the significant downregulation of the Th17/Treg ratio (P < 0.05) (Fig. 2E, F). Consistently, stimulation with hBMSC-derived exosomes led to more than 50% reduction in the mRNA expression levels of RORC and IL17A in periodontitis patient-derived CD4⁺ T cells (P < 0.05), while the mRNA expression levels of IL10 and FOXP3 were significantly increased (P < 0.01) (Fig. 2G). Overall, these data suggested that hBMSC-derived exosomes of healthy origins could correct the imbalance in Th17/Treg differentiation from periodontitis patient-derived CD4⁺ T cells in vitro.

Healthy hBMSC-derived Exosomes Reduced the Th17/Treg Ratio and Promoted the Bone Regeneration in vivo

Since hBMSC-derived exosomes showed beneficial immune modulation effects in vitro, we tested whether hBMSC-derived exosomes could promote periodontal recovery in vivo. The schematic diagram of animal treatment was shown in Fig 3 A and B. After 7-day treatment, H&E staining indicated that the bone resorption level (CEJ-ABC) was conspicuously reduced in the group that received exosome-loaded hydrogel than that received pure hydrogel (P < 0.01, Fig. 3C). Furthermore, micro-CT analysis showed that exosome-loaded hydrogel treatment substantially promoted the alveolar bone height and bone volume/tissue volume (BV/TV) ratio, compared to treatment with hydrogel alone (Fig. 3C). By immunofluorescence staining, we found that the expression level of Th17-related RORγ increased in periodontitis group, which was inhibited by injection of hydrogel and even further repressed by exosome-loaded hydrogel (P < 0.05, Fig. 3D). Consistently, inhibition of the Treg-related FOXP3 level in inflamed periodontal tissue was reversed after hydrogel treatment and the level was further increased by exosome-loaded hydrogel treatment (P < 0.001, Fig. 3D). These results suggested that hBMSC-derived exosomes promoted the recovery of alveolar bone in periodontitis in vivo.

MiR-1246 of hBMSC-derived Exosomes Regulated Th17/Treg Homeostasis

As hBMSC-derived exosomes are critical for T-cell responses in periodontitis, we next analyzed which miRNA is the vital factor. Exosomal miRNA profiles with or without stimulation of P. gingivalis lipopolysaccharide (P.g. LPS) were examined by miRNA-seq. 48 significantly upregulated and 101 significantly downregulated miRNAs were revealed in the P.g.LPS-stimulated group. Heatmap, volcano plot, and principal component analysis assessments demonstrated the reduction of miR-1246 expression (Fig. 4A-C, Appendix Tbl. 4). The schematic diagram of miR-1246 transfection was shown in Fig 3F and the transfection efficiency was shown in Fig 3F. Furthermore, periodontitis patient-derived naïve CD4⁺ T cells were treated with exosomes from hBMSCs or miR-1246 mimic/ inhibitor-transfected hBMSCs. Flow cytometric analysis revealed that the reduced Th17/Treg ratio treated with normal exosomes was further suppressed by miR-1246 mimic transfection. Furthermore, the opposite treatment results of exosomes with miR-1246 inhibitor confirmed that the regulatory effect was derived from exosomal miR-1246 (P < 0.01) (Fig. 4F, G). Thus, we concluded that miR-1246 in hBMSC-derived exosomal played an essential role in the regulation of Th17/Treg homeostasis.

hBMSC-derived Exosomal miR-1246 Targeted ACE2 on CD4⁺ T Cells and Regulated the YAP1 Signaling Pathway

To investigate the function of exosomal miR-1246, potential target genes of miR-1246 were sought in miRDB, miRWALK, and TargetScan; 216 intersections were identified in a Venn diagram (Fig. 5A, Appendix Tbl. 5). As one of the relevant gene in periodontitis and CD4⁺ T cell differentiation, ACE2 could bind to miR-1246 in its 3′-untranslated region (UTR) (Fig. 5B). The dual‐luciferase reporter assay confirmed that the activity of the ACE2 3′-UTR region decreased upon transfection with has-miR-1246 (P < 0.01), suggesting that has-miR-1246 acts on wildtype (but not mutant) ACE2 3′-UTR (Fig. 5C). For protein level verification by western blot, the expression of ACE2 in CD4⁺ T cells was reduced by exosomes overexpressing miR-1246, whereas reversed by exosomes containing miR-1246 inhibitor (Fig. 5F, G).

Furthermore, we revealed the interaction network of ACE2 by STRING analysis (Fig. 5D) and found relevant top 5 pathways by Kyoto Encyclopedia of Genes and Genomes (KEGG) based on miRNA-seq datasets (Fig. 5E, Appendix Tbl. 6). As the Hippo signaling pathway was strongly suggested as a potential downstream pathway of hBMSC-derived exosomal miRNAs, we analyzed this pathway and revealed that YAP1 expression was inhibited while P-YAP1 (S127) was activated by exosomes. Moreover, the P-YAP1 (S127)/YAP1 expression ratio in CD4⁺ T cells was upregulated by hBMSC-derived exosomes and further increased by exosomes overexpressing miR-1246, whereas it was downregulated in the miR-1246 inhibitor group (Fig. 5F, G). Collectively, these data suggested that the YAP1/Hippo signaling pathway is regulated by exosomal miR-1246.

Impaired bone repair and reconstruction in inflamed periodontium remains a major clinical challenge for bone regeneration in periodontitis patients³¹. The imbalance between Th17 and Treg cells in periodontal tissue is closely related to the progression of inflammation¹⁰. An elevated Th17/Treg ratio in peripheral blood is also associated with periodontal inflammation and alveolar bone destruction^11,32. Consistently, our study revealed that the Th17/Treg ratio in peripheral blood was substantially greater in periodontitis patients than in healthy subjects. All the periodontitis patients in this study were excluded from any systemic disease and potentially harmful routines (such as smoking) that might affect Th17/Treg balance in peripheral blood. The improvement of immunoregulation by correcting the Th17/Treg ratio offers a new approach for periodontitis treatment. Overall, we discovered that hBMSC-derived exosomes effectively reduced the Th17/Treg ratio in periodontitis, both in vitro and in vivo.

In recent years, MSCs have emerged as a promising therapy for periodontal regeneration in studies³³. Stem cell-derived exosomes provide a new cell-free treatment for periodontitis. hBMSC-derived exosomes suppressed the inflammatory responses of macrophages to P. gingivalis¹⁸ and promoted periodontal regeneration¹⁹. In our in vivo study, we demonstrated that hBMSC-derived exosomes conditioned medium in promoting periodontal regeneration in a mouse model. In our in vitro study, hBMSC-derived exosomes significantly correct the Th17/Treg ratio, is relevant with the findings of Zheng et al³⁴. Notably, compared with the periodontal ligament stem cell-derived exosomes in Zheng’s study (Th17 decreased by 25%), it seems that hBMSC-derived exosomes could inhibited the differentiation of Th17 more efficiently (Th17 decreased by 75%).

In our in vivo study, mice with periodontitis underwent injection of exosome-loaded hydrogels. The hydrogel (F127) has been proved safe and effective in the periodontal repair³⁵. Consistently, we found the therapeutic effect when this hydrogel used alone. Notably, exosomes showed great synergistic effect in periodontitis of mice models when loaded into hydrogel. Periodontal homeostasis was restored by improvement of alveolar bone height, the reduced level of Th17-related RORγ and increased level of Treg-related FOXP3.

P. gingivalis-associated miRNAs influence the innate immune response, whereas P.g. LPS may affect the nature of host miRNAs and their mRNA targets³⁶. In the present study, P.g.LPS stimulation led to 48 upregulated and 101 downregulated miRNAs in hBMSC-derived exosomes. The expression difference of miR-144-5p and miR-6073 was the most obvious, but none report about their immune regulation based on our literature review. Moreover, miR-1246 was obviously down-regulated according to miRNA-seq. miR-1246 has been reported to regulate Th17/Treg in Henoch-Schonlein purpura³⁷ and hepatic ischemia/reperfusion injury³⁸. Hence, we further investigated the role of miR-1246 in the differentiation of CD4⁺ T cells in periodontitis. Notably, Zhang et al. sequenced exosomal miRNAs derived from dental pulp stem cells cultured in 2D and 3D systems; they identified miR-1246 with the most differential expression²⁴. In the present study, we sequenced exosomal miRNA with or without P.g.LPS stimulation because the periodontitis microenvironment usually involves P.g.LPS stimulation. Additionally, miR-1246 in exosomes from the stem cells of human deciduous exfoliated teeth has been reported to inhibit angiogenesis and oral squamous cell carcinoma²⁶. Our findings extend the knowledge of miR-1246-specific regulatory effects in periodontal immunity. The transfection analysis confirmed that miR-1246-overexpressing exosomes could further promote the mitigation of the Th17/Treg ratio, which could be reversed by miR-1246 inhibitors. Thus, we have provided an innovative miR-1246-mimic modified hBMSC-derived exosome that can exert an enhanced immunomodulatory effect on Th17/Treg imbalance in vitro.

Through bioinformatics analysis and dual luciferase assays, we identified ACE2 as the target gene of hBMSC-derived exosomal miR-1246 in CD4⁺ T cells. The level of soluble ACE2 in saliva was positively correlated with the severity of periodontitis in a recent clinical study³⁹. Furthermore, exosomes containing miR-1246 inhibitor reduced the expression of ACE2 in Escherichia coli LPS (E.c. LPS)-stimulated pulmonary endothelial cells⁴⁰. In the present study, miR-1246-overexpressing exosomes significantly inhibited ACE2 expression in periodontitis patient-derived CD4⁺ T cells. This disparity might be related to differences between E.c. LPS and P.g. LPS, and between pulmonary endothelial cells and periodontitis patient-derived T cells. Consistent with our findings, inhibition of miR-1246 increased ACE2 expression in small airway epithelium of smokers⁴¹. In addition to ACE2, exosomal miR-1246 has various target genes, such as nuclear factor of activated T cells 5 of mesenchymal stem cells in inflamed periodontium ²⁴ and glycogen synthase kinase 3 beta of human umbilical cord blood mesenchymal stem cells in hepatic ischemia/reperfusion injury^38,39. However, further studies are needed to characterize other possible target genes regarding exosomal miR-1246.

Considering the mechanism of exosomal miR-1246, we explored the potential signaling pathway in CD4⁺ T cells. We found that YAP1 expression was downregulated by hBMSC-derived exosomes and further suppressed by exosomes overexpressing miR-1246, whereas the opposite trend was observed for P-YAP1 expression. When the Hippo pathway is inactive, YAP1 is unphosphorylated and located in the nucleus; when the Hippo pathway is active, phosphorylated YAP1(S127) remains in the cytoplasm and mediates the expression of multiple downstream genes⁴². Our results suggested that lower expression of total YAP1 is associated with a lower Th17/Treg ratio, which is consistent with the finding of Zhou et al. that abundant expression of YAP enhanced Th17 cell differentiation in patients with asthma exacerbation⁴³.

Study limitation

Our study has limitations. To begin with, the recovery process of periodontitis is complex, including the regeneration of soft and hard tissues, however, the soft tissues was not systematically studied in our research. Secondly, the specific activity of miR-1246 hasn’t been confirmed in vivo in present study. Thus, considering the long-term and complex recovery process of periodontitis, we will focus on the immunoregulatory mechanisms of exosomal miRNA in periodontal tissue in the future.

The roles of hBMSC-derived exosomes in Th17/Treg cell differentiation are summarized in Fig. 6. In conclusion, our study revealed that hBMSC-derived exosomes can effectively correct Th17/Treg imbalance and promote the alleviation of inflammation in periodontitis. To our knowledge, this is the first study to suggest that ACE2 is an important target gene for exosomal miR-1246 during the differentiation of CD4⁺ T cells. Furthermore, the immunomodulatory role of exosomal miR-1246 was closely associated with the YAP1/Hippo pathway in CD4⁺ T cells. Hence, our study provides insights into promising non-cellular therapies for correcting Th17/Treg imbalance in periodontitis.

Th17

Helper T cell 17

Treg

Regulatory T cell

hBMSCs

Human bone marrow mesenchymal stem cells

IL-17A

Interleukin-17A

FOXP3

Forkhead box protein 3

RORC

RAR-related orphan receptor C

YAP1

Yes-related protein 1

ACE2

Angiotensin converting enzyme2

RORγt

Orphan receptor gamma transcription

MiRNA

MicroRNA

CAL

Clinical attachment level

α-MEM

Minimum essential medium Alpha

TEM

Transmission electron microscope

NTA

Nanoparticle tracking analysis

ELISA

Enzyme linked immunosorbent assay

PBMCs

Peripheral blood mononuclear cells

H&E

Hematoxylin-eosin staining

CEJ-ABC

The distance between the cementoenamel junction and alveolar bone crest

P.g. LPS

Porphyromonas gingivalis lipopolysaccharide

BV/TV

Bone volume/tissue volume

KEGG

Kyoto Encyclopedia of Genes and Genomes

Author Contributions

Y.X. Xia, contributed to conception, design, data acquisition, analysis, and interpretation, drafted the manuscript; T.F. Cheng, contributed to data acquisition and interpretation, drafted the manuscript; M. Zhou contributed to the recruitment of participants, clinic periodontal examinations, and critically revised the manuscript; F.W. Kang contributed to conception, design, and critically revised the manuscript; C.S. Liao 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

We appreciate Mrs. H. Shan for performing the recruitment of participants in the study, all the medical and technical staff of Shanghai Engineering Research Center of Tooth Restoration and Regeneration, and the public instrument platform of School of Medicine, Tongji University. We thank S.J. Li, T Xu, J.F. He, L.L. Pan and M.M. Bie for helping make this study possible. We also thank Figdraw (www.figdraw.com) for providing painting materials for Fig. 6. We also thank Ryan Chastain-Gross, Ph.D., from Liwen Bianji (Edanz) (www.liwenbianji.cn/) for editing the English text of a draft of this manuscript.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article. This study was funded by the National Natural Science Foundation of China (82001008, 81901013), Science and Technology Commission of Shanghai Municipality (Pujiang Program, 20PJ1414400), Chinese Stomatological Association (Young Talent Project of Orthodontics, COS-B2021-05) and Shanghai Municipal Health Commission (Rising Star Young Physician Talent Project, 20RS88; Youth Scientific Research Project, 20204Y0097).

Ethics approval and consent to participate

This study was approved by the Ethics Committee of Stomatological Hospital of Tongji University (Approval No. 2020-R-011) and adhered to the Declaration of Helsinki. Written informed consent was obtained from all donors of hBMSCs and PBMCs. All animal procedures were approved by the Institutional Animal Care and Use Committee of Stomatological Hospital of Tongji University (Approval No. 2020-DW-026).

Consent for publication

Consent for publication was obtained from every participant.

Competing interests

The authors declare that they have no competing interests.

Availability of data and materials

All data generated or analyzed during this study are available from the corresponding author on reasonable request.

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hBMSC-derived Exosomes Mitigate Th17/Treg Homeostasis in Periodontitis via miR-1246

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