M csf 300 25

MBL protein was prepared as previously described (16 (link)). Recombinant human RANKL (310-01C) and M-CSF (300-25) were purchased from Peprotech (London, UK). Recombinant murine RANKL (315-11) and M-CSF (315-02) were purchased from Peprotech (London, UK). Anti-c-fos antibody (26192-1-AP), anti-GAPDH antibody (10494-1-AP), and anti-CTSK antibody (11239-1-AP) were purchased from proteintech (Chicago, IL, USA). The anti-NFATc1 antibody was purchased from Abclonal Technology (Wuhan, China). Polyclonal antibodies against p38 (8690), phospho-p38 (4511), and Histone H3 (4499) were obtained from Cell Signaling Technology (Cambridge, MA, USA). The TRAP staining kit (387A) was from Sigma-Aldrich (St. Louis, MO, USA). Safranin O-solid green cartilage staining solution (G1317), Hematoxylin-Eosin solution (G4520), Masson's trichrome staining kit (G1340), and Toluidine blue staining solution (G2543) were purchased from Solarbio (Beijing, China).

Human monocytic THP-1 cells were purchased from the China Center for Type Culture Collection (Wuhan, China) and cultured in complete RPMI-1640 medium containing 10% fetal bovine serum, 10 mM glucose, 2 mM L-glutamine and 100 U mL⁻¹ penicillin-streptomycin. THP-1 cells were differentiated into M0 macrophages by incubation with 100 ng mL⁻¹ phorbol 12-myristate 13-acetate (PMA) (P8139, Sigma-Aldrich) for 72 h. Once the cells were adherent, they were transferred to PMA-free media to obtain resting macrophages. These cells were incubated with 20 ng mL⁻¹ human macrophage colony-stimulating factor (M-CSF) (300-25, PeproTech) and then co-stimulated with 100 ng mL⁻¹ LPS (L2630, Sigma-Aldrich) plus 20 ng mL⁻¹ IFN-γ (300-02, PeproTech) or stimulated with 10 ng mL⁻¹ IL-4 (200-04, PeproTech) for 24 h to generating inflammatory or anti-inflammatory macrophages.

The following antibodies/kits were procured from eBiosciences (USA): APC Anti-Mouse/Rat-Foxp3 (FJK-16s) (17-5773), PE Anti-Human/Mouse-Rorγt (AFKJS-9) (12-6988), Foxp3/Transcription factor staining buffer (0-5523-00) and RBC lysis buffer (00-4300-54). PE/Cy7 Anti-Mouse CD8 (53-6.7) (100721), PE Anti-Mouse CD254-(TRANCE-RANKL) (IK22/5) (510005). Anti-IFN-γ (clone: XMG1.2), Anti-IL-4 (clone: 11B11), Anti-CD3e (clone: 145-2C11) and Anti-CD28 (clone: 37.51) antibodies were procured from BioLegend (USA). Following ELISA kits were brought from R&D: Mouse IL-10 (M1000B) and Mouse IL-17 (M1700) Quantikine ELISA kits. The Following ELISA kits and reagents were brought from BD (USA): Mouse IL-6 (OptEIA-555240), Mouse TNF-α (OptEIA-560478). Acid phosphatase, leukocyte (TRAP) kit (387A), 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), FITC-Phalloidin (P5282) and DAPI were bought from Sigma (USA). Macrophage-colony stimulating factor (M-CSF) (300-25) and Receptor activator of nuclear factor κB-ligand (sRANKL) (310-01), Human TGF-β1 (AF-100-21C), Murine IL-2 (AF-212-12), Murine IL-6 (AF-216-16), Murine IL-23 (200-23), were procured from PeproTech (USA). MRS broth (GM369) was purchased from HiMedia (India). α-Minimal essential media and RPMI-1640 were purchased from Gibco (USA).