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Tim4 deficiency reduces CD301b+ macrophage and aggravates periodontitis bone loss – International Journal of Oral Science


Data collection and processing

Publicly available RNA sequencing datasets were obtained from Sequence Read Archive DataSets PRJNA914415. For all upregulated and downregulated DEGs, GO enrichment analysis and heatmap analysis were processed with the free online Dr. TOM II Platform. For scRNA-seq datasets PRJNA905945, the downstream analysis steps were performed using the R v3.5.1 software and Seurat package. Next, for quality control of each matrix, living periodontal cells defined by using the percentage of mitochondrial gene expression as an inclusion criterion (<10%) were retained, and cells with genes <200 or >5 000 were filtered out. The matrix was normalized for sequencing depth using the “NormalizeData” function. The top 2 000 variable genes of each matrix were detected applying principal component analysis. For the clustering of the whole cells, the resolution value was set to 0.8 and the integrated data were dimension reduced with pc use = 1:10. The cell clusters were annotated based on MouseRNAseqData dataset. The visualization of UMAP, heatmap, and featurePlot is done when invoking the “DimPlot”, “Doheatmap” and “FeaturePlot” functions respectively.

Ethical approval and mice model

Female C57BL/6J mice aged 8 weeks were provided by Vital River Laboratory Animal Technology Co. (Beijing, China). Tim4-knockout (Timd4−/−) mice on the C57BL/6 background were purchased from Model Organisms Center, Inc. (Shanghai, China). Mice were maintained under SPF conditions and housed in the specific pathogen-free, temperature-controlled facilities. All animal studies were performed with the approval of the Animal Care and Use Committee of the Medical Research Institute, Wuhan University (MLIC2021175) and in accordance with the ARRIVE guidelines 2.0.

The ligature-induced periodontitis model was established by tying 5-0 silk ligatures around the left and right maxillary second molars as previously described,58 and then sacrificed at different time points after ligation (specified in the figure legends).

Flow cytometry

To obtain single-cell suspensions, the periodontal soft tissues surrounding the buccal and lingual sides of the mouse maxillary molars were excised, minced and collected. The samples were then digested with 2 ml RPMI-1640 medium (HyClone, Logan, UT, USA) containing 10% fetal bovine serum (FBS, Gibco, New York, NY, USA), collagenase type II (2 mg/ml, Thermo Fisher Scientific, CA, USA) and collagenase type IV (2 mg/ml, Thermo Fisher Scientific) for 2 h at 37°C in a shaker bath. Subsequently, the digested tissues were ground and strained with 70-μm filters (Thermo Fisher Scientific) under PBS washing, and the filtrates were centrifuged with a speed of 2 500 r/min for 5 min.

Freshly isolated single cells were suspended in PBS and were first stained with Fixable Viability Stain 510 (FVS510, 1:1 000, BD Biosciences, USA) for 20 min to determine living cells. Anti-CD16/32 antibody (1:100, Biolegend, San Diego, CA, USA) was applied to block non-specific binding of immunoglobulin to the Fc receptors before surface dyeing. The fluorochrome-conjugated antibodies of surface markers were listed in Table 1. After 30 min of incubation in the dark at 4 °C, cells were resuspended in 200 μL PBS. Flow cytometry was conducted on an LSR FortessaX20 (BD Biosciences, USA) and the data analysis was performed on the software FlowJo 10.4 (FlowJo LLC, Ashland, OR, USA).

Table 1 Description of the antibodies used for flow cytometry

Histological staining

Fixed and decalcified mouse maxillae were dehydrated in 30% sucrose solution for 48 h at 4 °C and embedded in optimum cutting temperature (OCT, Sakura, America), followed by cryosection which cut samples into 8 μm thickness sections. For immunofluorescence, the sections were stained with primary antibodies against CD301b (1: 100, Invitrogen) together with Tim4 (1: 100, Sigma-Aldrich, USA), and the secondary antibodies with 488 and 593 fluorescence markers (1: 200, Invitrogen) were adopted. Images were harvested using the laser scanning confocal microscope (LSCM, Leica, Germany). The histomorphometric analysis was performed by ImageJ software (National Institutes of Health, Bethesda, MD, USA). The sections were then stained with hematoxylin and eosin (H&E, Google biotechnology, China) according to the manufacturer’s instructions.

Micro-computed tomography

Fixed mouse maxillary bones were assessed for bone parameters applying a micro-computed tomography (µCT, Skyscan1276, Bruker) system and affiliated analyzing software. The scans were performed at 55 kV and 200 mA with a resolution of 10 µm. The distance between the cementoenamel junction and alveolar bone crest (CEJ-ABC) of the maxillary second molar on the distal side was measured by Data Viewer program. The area of interradicular alveolar bone of the second molar was defined as the region of interest (ROI). The bone loss in the ROI was calculated by CTAn program for quantification, including the bone volume fraction (BV/TV) and trabecular bone number (Tb.N).

Isolation and culture of bone marrow-derived macrophages (BMDMs)

Bone marrow cells were extracted from the tibias and femurs of mice and cultured with high glucose Dulbecco’s modified eagle’s medium (DMEM, Hyclone, USA), in the presence of 20% FBS, 1% penicillin–streptomycin (Hyclone), and macrophage colony stimulating factor (M-CSF, 20 ng/mL, PeproTech, USA). Five days later, the mature BMDMs were eligible for further experiments.

For overexpression or knockdown of Tim4, lentivirus-carrying mouse Tim4 expression cassette or Tim4 small hairpin RNA (shRNA) lentiviral vector was designed and transfected into BMDMs, respectively. An empty lentivirus was applied as control (OE-CON or sh-CON). After transfection, BMDMs were screened with puromycin and termed as OE-Tim4 or sh-Tim4. The transfection procedures were performed according to the protocol of the transfection reagent (PT-114-15, jetPrime, France). To induce CD301b+ macrophages in vitro, BMDMs were treated with interleukin (IL)-4 (20 ng/mL, Biolegend) for 24 h.20 To verify the signaling pathway involved, specific chemical agonist Dehydrocorydaline (DHC, 20 μmol/L, MCE) and inhibitor SB203580 (SB, 10 μmol/L, Selleck) for the p38 MAPK pathway were added into the culture medium and activated for 48 h.

Real-time quantitative PCR (RT-qPCR)

Total RNA was isolated via Trizol reagent (Invitrogen) from cells or tissues in accordance with the manufacturer’s instructions (Takara, Shiga, Japan). The RNA was reverse-transcribed into cDNA using PrimeScript RT Master Mix (Takara, Japan). RT-qPCR was carried out with a LightCycler 96 System (Roche, Basal, Switzerland) applying SYBR Green reagents (Takara). Relative gene expression levels normalized to Gapdh were determined and the data were analyzed using the 2-ΔΔct method. The primers designed for target genes were shown in Table 2.

Table 2 Primers designed for qRT-PCR

Western blot

Mouse periodontal soft tissues and BMDMs were homogenized manually and lysed in radio immunoprecipitation assay (RIPA) buffer (Beyotime Biotechnology, China) containing protease inhibitor and phosphatase inhibitor. All samples were quantified and normalized by bicinchoninic acid (BCA) kit (Thermo Fisher Scientific). Protein extracts were loaded into sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF, Millipore, USA) membranes, which were then blocked in 5% skim milk for 1 h at room temperature (RT). The membranes were incubated with primary antibodies against Tim4 (1:1 000, Sigma-Aldrich), Igf1 (1:1 000, ABclonal, China), p-p38 (1:1 000, CST, USA), p38 MAPK (1:1 000, CST), and α-Tubulin (1: 5 000, ABclonal) overnight at 4 °C, followed by anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody (1: 10 000, Proteintech, China, 1:5 000) for 1 h at RT. The results were visualized with a chemiluminescence imaging system and further analyzed by ImageJ software. Relative protein expression levels normalized to α-Tubulin were evaluated.

5-Ethynyl-2′-deoxyuridine (EdU) cell proliferation assay

The EdU-488 Cell Proliferation Kit (Beyotime) was employed to detect the effect of macrophages proliferation. Briefly, a density of 1 × 104 treated BMDMs were seeded in 24-well plates in the presence of DMEM medium containing 10% FBS and 10 μmol/L EdU. After 2 h, the isolated cells were fixed with 4% paraformaldehyde (PFA) for 30 min at RT. Click Additive Solution (with Azide 488) was prepared and incubated with cells for 30 min in the dark at 4 °C, followed by PBS washing. The fluorescent-labeled cells can be visualized under the flow cytometer and the cell proliferation can be determined.

Transwell assay

The migration assay was performed using a 24-well transwell chamber with 5 μm pore inserts (Corning, USA). The bottom of the lower chamber was filled with 500 μl culture supernatants of OE-CON/OE-Tim4 as a chemoattractant, while the CD301b+ macrophages (5 × 104 cells per well) obtained by sorting BMDMs were suspended in serum-free medium and plated in the upper chamber. The established culture system was incubated for 48 h at 5% CO2 and 37 °C. After incubation, the remaining cells on the top of the membrane were wiped off and the cells through the filters on the bottom of the membrane were fixed in 4% PFA for 20 min. The inserts were stained with 1% crystal violet for 10 min. The migration results were quantified using ImageJ.

Statistical analysis

Statistical analysis was performed with GraphPad Prism 8.0 (San Diego, CA, USA) software. The differences among groups were investigated through Student’s t test and one-way analysis of variance (ANOVA). All experiments were repeated at least 3 times and the numerical data were displayed as mean ± SEM, and the statistically significance level was set at P < 0.05.



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