Animals
All animal experiments in this study were conducted with the approval of Institutional Animal Care and Use Committee of the Fourth Military Medical University. C57BL/6J (#000664), Gli1-CreERT2 (#007913), ROSA26-mT/mG (#007676), and ROSA26-eGFP-DTA mice (#006331) were purchased from the Jackson Laboratory. Rab27a-flox mice (#T066643) were purchased from GemPharmatech. Gli1-CreERT2 mice were crossed with ROSA26-mT/mG mice, ROSA26-eGFP-DTA mice, Rab27afl/fl to generate Gli1CreERT2;mT/mG mice, Gli1-CreERT2;Rosa-DTA mice, Gli1-CreERT2;Rab27afl/fl mice, respectively. All mice were maintained under specific pathogen free conditions (24°C, 12 h light/dark cycles and 50% humidity), and fed with food and water at libitum. Genotype identification was conducted using PCR following established protocols from published studies48,49. In brief, mouse tail tissue was lysed in tail lysis buffer (Vigen Biotec, USA) overnight at 55°C, followed by heating at 85°C for 1 h. The supernatant was then collected for DNA amplification and gel electrophoresis. The relevant primers were shown in the Table S1. Tamoxifen (MCE, USA) was injected intraperitoneally at 100 µg/g for 4 consecutive days to induce Cre-mediated DNA recombination, and the ligature-induced periodontitis model was performed after 7 days.
Establishment of the Ligature-induced Periodontitis Model
The maxillary second molars of 6-8-week-old mice were ligatured with 5 − 0 silk sutures according to published research24. In brief, silk sutures were wrapped around the neck of the tooth with knots tying on the buccal side. For the therapeutic application of GANT61, 40 mg/kg GANT61 (MCE, USA) or equal volume of corn oil was intraperitoneally injected into the mice every other day for three times following periodontal ligation.
Immunofluorescence Staining
Immunofluorescent staining was performed according to previously published methods29. Briefly, the mice maxillary samples were separated, fixed in 4% paraformaldehyde (PFA) (Sigma, USA) at 4°C for 8 h and were decalcified in 17% ethylene diamine tetraacetic acid (EDTA) (Sigma-Aldrich, USA) at 4°C for 21 days, with the decalcified solution changed every other day. Samples were then washed thoroughly with PBS to remove the PFA, dehydrated in 30% sucrose solution overnight, embedded in optimal cutting temperature (OCT) compound (Leica, Germany) and sliced into 10 µm thick sections. For tissue samples, the sections were permeabilized in 0.5% Triton (Sigma-Aldrich, USA) at room temperature for 10 mins, blocked with goat serum at room temperature for 30 min, incubated with primary antibodies overnight at 4°C, incubated with secondary antibodies at 37°C for 1 h, and sealed with a mounting medium containing 4,’-6-diamidino-2-phenylindole (DAPI) (Abcam, USA). For cell samples, cells were seeded in round coverslip, fixed with 4% PFA for 5 min, and then underwent the same procedure as mentioned above. Images were captured using confocal scanning laser microscopes (FV3000, Olympus, Japan and LSM900, ZEISS, German) and analyzed using the ImageJ software. The SYTOX dye (Alphabio, China) was used to detect cellular nucleic acids. The antibodies used were as follows: anti-PCNA antibody (13110T, Cell Signaling Technology, USA, diluted 1:100), anti-RUNX2 antibody (12556, Cell Signaling Technology, USA, diluted 1:200), anti-Ly6G antibody (sc-53515, Santa Cruz, USA, diluted 1: 100), anti-Histone H3 (citrulline R2 + R8 + R17) (ab5103, Abcam, USA, diluted 1:300), anti-MPO (GB-15224, Servicebio, China, diluted 1:200), anti-NF-κB (sc-109, Santa Cruz, USA, diluted 1:100), Alexa Fluor 647-conjugated Goat anti-Rabbit secondary antibody (4414, Cell Signaling Technology, USA, diluted 1:200), Alexa Fluor 594-conjugated Donkey anti-Rat secondary antibody (A21209, Invitrogen, USA, diluted 1:200), Alexa Fluor 594-conjugated Donkey anti-Rabbit secondary antibody (A21207, Invitrogen, USA, diluted 1:200), Alexa Fluor 488-conjugated Donkey anti-Rabbit secondary antibody (A21206, Invitrogen, USA, diluted 1:200), and Alexa Fluor 488-conjugated Donkey anti-Rat secondary antibody (A21208, Invitrogen, USA, diluted 1:200).
Flow Cytometry Analysis
Mice were sacrificed and the periodontal tissue around the upper second molar was collected. After being washed by cold PBS, the tissue was digested with 1 mg/mL collagenase I (Gibco, USA) for 30 min at 37°C. The supernatants were collected and filtered through a 100 µm cell strainer, centrifuged at 500 g for 5 min, and resuspended with 100 µL PBS to obtain single-cell suspension. Then, the cells were stained with CD45 Monoclonal Antibody (30-F11), PE (12-0451-82, Invitrogen, USA, 0.125 µg/test), CD11b Monoclonal Antibody (M1/70), Brilliant Violet™ 421 (404-0112-80, Invitrogen, USA, 0.125 µg/test) and Ly6G Monoclonal Antibody (1A8-Ly6g), APC (17-9668-82, Invitrogen, USA, 0.5 µg/test) for 30 min on ice in the dark. The cells were washed twice and PI (SL7091, Coolaber, China, diluted 1:100) was added 5 min before the detection. Then samples were detected on a flow cytometer (Cytomics FC 500, Beckman-Coulter, USA). The results were analyzed using FlowJo_V10 software.
Micro-CT Analysis
At the endpoints of each experiment, the mice were sacrificed and the maxillae were dissected and fixed with 4% PFA solution overnight at 4℃. The fixed maxillae were scanned using micro-CT (Siemens Inveon, Germany and PerkinElmer, USA) with a resolution of 18 µm, a voltage of 50 kV and a current of 80 µA. Micro-CT data was reconstructed and analyzed by VGStudio MAX software (Volume Graphics, Germany). The CEJ-ABC distance on the buccal surface of the second molar and the BV/TV in the interdental regions between first and second molars were analyzed.
Histological Analysis
The isolated maxillae were fixed in 4% PFA solution overnight at 4℃ and decalcified in 17% EDTA (Sigma-Aldrich, USA) at 4°C for 21 days, with the decalcified solution changed every other day. The decalcified maxillae were rinsed with running water thoroughly, dehydrated using graded ethanol, embedded in paraffin and sliced into 4 µm thick sections. The sections were deparaffinized, and underwent H&E staining (Leica, Germany) and TRAP staining (Sigma–Aldrich, USA), according to the manufacturer’s instructions. Images were acquired using a Leica microscope.
Sorting and Culture of Gli1 + Cells
4-week-old Gli1-CreERT2;mT/mG mice received intraperitoneal injections of tamoxifen for four consecutive days to induce the expression of GFP in Gli1+ cells. Seven days later, the mice were sacrificed, and the femurs and tibias were dissected and washed with ice-cold sterile PBS. Then, the bone marrow was flushed with α-MEM (Invitrogen, USA) containing 20% fetal bovine serum (FBS) (Tianhang, China), 2 mm L-glutamine (Invitrogen, USA), and 1% penicillin/streptomycin (Invitrogen, USA), with the remaining bones being cut into small pieces. The cell suspension was centrifuged at 500 g for 5 min, and then the cell pellet and bone fragments were seeded in cell culture flasks (Thermo, USA) at 37°C with 5% CO2. Cell medium was changed every three days and cell passaging was initiated when the cells reached approximately 80%-90% confluence. The third passage of cells was used for FACS of GFP positive cells. Specifically, cells were digested with 0.25% trypsin (MP Biomedicals, USA), followed by collection through centrifugation at 500 g for 5 min. After resuspension in PBS containing 2% FBS at a density of 1 × 106 cells/mL, GFP-positive cells were subsequently sorted using a flow cytometer (BD, USA)21. The sorted cells were washed by sterile PBS for twice and then cultured in α-MEM (Invitrogen, USA) containing 20% FBS (Tianhang, China), 2 mm L-glutamine (Invitrogen, USA), and 1% penicillin/streptomycin (Invitrogen, USA) at 37°C with 5% CO2.
Extraction and Culture of Neutrophils
Mice was sacrificed, and the femurs and tibias were dissected. Bone marrow was flushed out with ice-cold sterile PBS using syringe, followed by erythrocyte lysis in RBC lysis buffer (Yuanye Bio-Technology, China). 3 mL Histopaque-1119 (Sigma, USA) was added to the bottom of the 15 mL centrifugation tube, with 3 mL Histopaque-1077 (Sigma, USA) in the middle, and the cell suspension on the top. After centrifugation at 800 g for 30 min, neutrophils at the interface of the Histopaque-1119 and Histopaque-1077 layers were collected and washed by ice-cold sterile PBS for twice. Then, the neutrophils were cultured in Roswell Park Memorial Institute (RPMI) 1640 (Gibco, USA) supplemented with 10% FBS (Tianhang, China) and 1% penicillin/streptomycin (Invitrogen, USA) or resuspended in Hanks balanced salt solution (HBSS).
Collection and Treatment of Cell Supernatants
Gli1+ cells were stimulated with LPS (Sigma, USA, 2 µg/mL) or equal volume of PBS as control for 24 h. Then, the medium was changed into α-MEM (Invitrogen, USA) containing 20% FBS (Tianhang, China) and the cell supernatants were collected after 24 h which was used to treat neutrophils as “Stimulated supernatants” or “Supernatants”.
Collection and Treatment of EVs Derived from LPS Pre-stimulated Gli1 + Cells
Gli1+ cell was stimulated with LPS (Sigma, USA, 2 µg/mL) or equal volume of PBS as control for 24 h, and the medium was changed into α-MEM (Invitrogen, USA) containing 20% EV-free FBS (Tianhang, China). For “EVs” group, cell supernatants were collected after 24 h and the EVs were isolated by gradient centrifugation. Briefly, cell supernatants were centrifuged at 800 g for 10 min to remove cells, 2000 g for 10 min to remove cell debris, and 16000 g for 30 min to pellet EVs29, which were characterized by NTA and TEM, and then used to treat neutrophils. For “Stimulated supernatants-EVs” group, the stimulated supernatants were collected and depleted of EVs by ultracentrifugation29. For “GW4869” group, Gli1+ cell was co-stimulated with LPS (Sigma, USA, 2 µg/mL) and GW4869 (MCE, USA, 10 µM) for 24 h. Then, the medium was changed into α-MEM (Invitrogen, USA) containing 20% EV-free FBS (Tianhang, China) and the cell supernatants were collected after 24 h.
Transwell Migration Assay
Different cell supernatants or α-MEM (Invitrogen, USA) were added to the lower chamber. Then, the neutrophils were resuspended in 100 µL RPMI 1640 (Gibco, USA) and seeded to the upper chamber of a transwell plate (5 × 105/well). After 30 min, the medium of the lower chamber was collected and the number of cells that had migrated into the lower chamber was determined by cell counter. In addition, the transwell membrane's lower surface was fixed with 4% PFA for 15 min and then stained with 0.1% crystal violet (Servicebio, China) for 30 min. Migrated cells were observed and captured under a microscope in randomly selected visual fields.
NTA
To evaluate the size distribution of EVs, NTA was conducted as the protocol described in a previous study21. In brief, EVs were resuspended in PBS at a final concentration of 6.6 × 107 particles/mL and then analyzed using a PMX ZetaView instrument (Particle Metrix, Germany).
SEM
First, neutrophils were seeded in round coverslip, then they were fixed, dehydrated, and dried to eliminate moisture. After drying, they were coated with a thin conductive layer, typically gold or platinum. The prepared samples were then mounted on SEM stubs and placed in a vacuum chamber. Finally, the samples were examined under a scanning electron microscope (ThermoFisher Quattro S, ThermoFisher, Czech Republic).
TEM
For cell samples, neutrophils were initially fixed in a 2.5% glutaraldehyde solution to preserve their cellular structures. This was followed by post-fixation with 1–2% osmium tetroxide to further stabilize lipids and proteins. Dehydration was carried out through a graded series of ethanol solutions, starting from 30% and progressing to 100%, to gradually remove water from the samples. After dehydration, the samples were infiltrated with a resin. The infiltration was performed by gradually increasing the concentration of resin in ethanol until only pure resin remained. The samples were then embedded in fresh resin and polymerized, usually by heating at 60°C for 24–48 h. Once the resin blocks were hardened, ultrathin sections (approximately 60–90 nm thick) were cut. These sections were collected on copper grids and contrasted with heavy metals, such as uranyl acetate and lead citrate, to enhance the electron density and improve image contrast. Finally, the prepared grids were examined under a transmission electron microscope (HT7800, HITACHI, Japan). For EV samples, EVs were first resuspended in 2% PFA, then dropped onto 200-mesh copper grids. After removing excess liquid with filter paper, the samples were stained with uranyl acetate (Sigma-Aldrich, USA) for 2 min at room temperature. Following a distilled water rinse and drying process, images were captured using a transmission electron microscope (HT7800, HITACHI, Japan).
Apoptosis Assay
Neutrophils were cultured in different medium for 8 h, followed by apoptosis detection using the PE Annexin V Apoptosis Detection Kit I (BD Biosciences, 559763). Briefly, the cells were washed with cold PBS and resuspended in 100 µL of 1× binding buffer. Subsequently, 5 µL of PE Annexin V and 5 µL of 7-amino-Actinomycin D (7-AAD) solution were added to the cell suspension, which was then incubated at room temperature for 30 min. After incubation, an additional 400 µL of binding buffer was added. The samples were analyzed using a flow cytometer
(Cytomics FC 500, Beckman-Coulter, USA) within 1 h. The results were analyzed using FlowJo_V10 software. The apoptotic rate of neutrophils was calculated as the combined percentage of Annexin V+/7-AAD− and Annexin V+/7-AAD+ cells.
Oxidative Stress Evaluation
Cell ROS production was detected with the ROS assay kit (Beyotime Biotechnology, China). Briefly, neutrophils receiving different treatments were stained with DCFH-DA (10 µМ) for 20 min at 37°C, and the fluorescence was examined by flow cytometry (Cytomics FC 500, Beckman-Coulter, USA) and fluorescence microscopy (Olympus, Japan).
RNA Sequencing
Neutrophils were cultured in LPS pre-stimulated Gli1+ cell supernatants or control medium for 8 h and then washed with PBS for 3 times. Total RNA was extracted using the MIsZOL Reagent (Mishushengwu, China) according to the manufacturer’s protocol. RNA purity and quantification were evaluated using the NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). RNA integrity was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Then the libraries were constructed using VAHTS Universal V6 RNA-seq Library Prep Kit according to the manufacturer’s instructions. The transcriptome sequencing and analysis were conducted by OE Biotech Co., Ltd. (Shanghai, China). The libraries were sequenced on a llumina Novaseq 6000 platform and 150 bp paired-end reads were generated. Raw reads of fastq format were firstly processed using fastp and the low quality reads were removed to obtain the clean reads. The clean reads were mapped to the reference genome using HISAT2. FPKM of each gene was calculated and the read counts of each gene were obtained by HTSeq-count. PCA analysis were performed using R (v 3.2.0) to evaluate the biological duplication of samples. Differential expression analysis was performed using the DESeq2. Q value < 0.05 and foldchange > 2 or foldchange < 0.5 was set as the threshold for DEGs. Volcano mapping and Hierarchical clustering diagram was drawn using R (v 3.2.0) according to DGEs. Based on the hypergeometric distribution, GO and KEGG pathway enrichment analysis of upregulated genes in stimulated supernatants group were performed to screen the significant enriched term using R (v 3.2.0), respectively. GSEA was performed using GSEA software.
qRT-PCR
Total RNAs were extracted using MIsZOL Reagent (Mishushengwu, China) following the manufacturer’s instructions and purified through phenol-chloroform extraction. Complementary DNA (cDNA) was prepared by TaqMan Reverse Transcription Reagents (Takara) as previously described50. The primer sequences can be found in Table S2. Genes were amplified and analyzed by qRT PCR using Bio-Rad SYBR Green Premix Ex Taq (Takara) in a Roche LightCycler 96 system.
Statistical Analysis
Data were expressed as the mean ± standard deviation (SD). GraphPad Prism 8.0 was used for statistical analysis. Two groups were compared by two-tailed Student’s t test, and multiple groups were compared by one-way ANOVA with Turkey's or Dunnet's post-hoc test. P < 0.05 (*, P < 0.05; **, P < 0.01; ***, P < 0.001) were considered to have a statistical difference.