Patients and controls
57 patients with RA (Table1), 28 osteoarthritis (OA) patients, as well as 49 age- and sex-matched healthy control (HC) were enrolled in this study. All the patients met the 2010 American College of Rheumatology (ACR) revised criteria for RA  and 1986 ACR criteria for OA . The study was approved by the Institutional Medical Ethics Review Board of Peking University People's Hospital. Moreover, all participants provided informed consent.
Clinical and laboratory indices of RA
The following data of patients with RA were recorded: gender, age, duration, swollen joint count (SJC), tender joint count (TJC) and laboratory parameters including white blood cells (WBC), red blood cells (RBC), hemoglobin (Hb), platelets (PLT), immunoglobulin (Ig) A, IgG, IgM, anti-cyclic citrullinated peptide antibody (anti-CCP antibody), erythrocyte sedimentation rate (ESR), C-reactive protein (CRP). Disease activity scores were calculated using the 28-joint Disease Activity Score-erythrocyte sedimentation rate (DAS28-ESR) in patients with RA. DAS28-ESR > 5.1 was considered a high disease activity according to the recommendations from the European League Against Rheumatism (EULAR).
Antibodies and Reagents
Recombinant human macrophage colony-stimulating factor (rhM-CSF) (Cat# 300-25) was obtained from PerproTech GmbH (Rocky Hill, CT). Recombinant human RANKL (rhRANKL) (Cat# 390-TN), recombinant human Gas6 (rhGas6) (Cat# 885-GSB), human anti-Tyro3TK antibody (Cat# MAB859, Clone# 96201) proved to demonstrate blocking activity , human Tyro3TK PE-conjugated antibody (Cat# FAB859P), Mouse IgG2b PE-conjugated antibody (Cat# IC0041P) were purchased from R&D Systems (Minneapolis, MN). Human TruStain FcX™ (Fc Receptor Blocking Solution) (Cat# 422302) was purchased from BioLegend (San Diego, CA). Human CD14 FITC-conjugated antibody (Cat# 11-0141-81) and human CD16 APC-conjugated antibody (Cat# 17-0168-42) were purchased from eBioscience (San Diego, CA). Leukocyte Acid Phosphatase Kit (Cat# 387A) was purchased from Sigma-Aldrich (St Louis, MO). α-minimum essential medium (α-MEM) (Cat# C11965500BT), 1% penicillin/streptomycin, and fetal bovine serum were purchased from Invitrogen (Carlsbad, CA).
Flow cytometry analysis and sorting
Peripheral blood mononuclear cells (PBMCs) were isolated from fresh EDTA blood samples using Ficoll density-gradient centrifugation. Before staining with antibodies, single-cell suspensions were incubated with human Fc Receptor Blocking Solution for 10 min at room temperature to block the FcR-involved unwanted staining without interfering with antibody-mediated specific staining.
To detect the expression of Tyro3TK on CD14+CD16+ and CD14+CD16- monocytes, cells were stained with CD14 FITC-conjugated antibody, CD16 APC-conjugated antibody, and Tyro3TK PE-conjugated antibody. Corresponding negative isotype and fluorochrome-matched control (FMO) staining were also performed. The cells were then analyzed on FACS Aria II.
For CD14+CD16+ and CD14+CD16- monocytes sorting, cells were stained with CD14 FITC-conjugated antibody and CD16 APC-conjugated antibody. Then the stained cells were sorted with FACS Aria II. The purified CD14+CD16+ and CD14+CD16- monocytes were further analyzed after sorting, the purity of which used for experiments was ~90%.
qPCR analysis of Tyro3TK expression
Total RNA was isolated from purified CD14+CD16- monocytes using RNeasy mini kit (Qiagen, Hilden), then reverse transcribed into the oligo (dT)-primed cDNA by Revert Aid First Strand kit (Fermentas, Glen Burnie, MD). Real-time quantitative PCR (qPCR) was performed to analyze the expression of Tyro3TK mRNA in CD14+CD16- monocytes from RA patients and HC according to the manufacturer's instructions. The sequences of the primers used in this study were as follows: the forward GAPDH primer was 5′-AAGGTGAAGGTCGGAGTCAA-3′, the reverse GAPDH primer was 5′-AATGAAGGGGTCATTGATGG-3′, the forward Tyro3TK primer was 5′-CAGCCGGTGAAGCTCAACT-3′, the reverse Tyro3TK primer was 5′-TGGCACACCTTCTACCGTGA-3′.
In vitro osteoclast differentiation
CD14+CD16+ and CD14+CD16- monocytes from freshly isolated PBMCs were purified by FACS sorting. Then the cells were cultivated 17 days separately in 96-well plates (5×104 cells/200 μl per well) in α-MEM with 1% PenStrep, 10% heat-inactivated fetal bovine serum, 30 ng/ml rhM-CSF and 50 ng/ml rhRANKL. Different concentrations of rhGas6 and/or human anti-Tyro3TK antibody were added as indicated. The medium was changed with fresh medium every 6 days. Osteoclast differentiation was evaluated by staining cells for TRAP using a Leukocyte Acid Phosphatase kit (Sigma-Aldrich) according to the manufacturer's instructions. TRAP-positive multinucleated cells were counted by an inverted fluorescence microscope (Olympus IX71-141, Tokyo, Japan).
All data were analyzed on the statistical software program SPSS 24.0 for windows (SPSS, Chicago, IL). Differences between groups were evaluated by Student's t-test, non-parametric Mann-Whitney U test, one-way ANOVA test, Kruskal-Wallis H test, and Spearman's correlation test. P value less than 0.05 was considered statistically significant (*P < 0.05, ** P < 0.01, *** P < 0.001, ns, not significant).