Patients
Two ACPA+ and two ACPA- RA patients from Peking University Third Hospital who fulfilled the 2010 European League Against Rheumatism/American College of Rheumatology classification criteria for RA(2) were enrolled in the study. All 4 patients were newly diagnosed, treatment naive, menopausal, and non-smoker women from the northern Han population with symptom duration of less than 6 months, and none had any other chronic diseases. Three matched healthy individuals were included in the study.
PBMC collected from 40 ACPA+, 40 ACPA- RA patients, and 40 healthy controls were applied for quantitative PCR to validate candidate genes expression. Serum CXCL2 levels were detected by ELISA in a cohort consisted of 70 ACPA+, 37 ACPA- RA patients, and 40 healthy controls. The clinical and laboratory data of the patients were collected from electronic medical records. In all comparisons mentioned, the groups were age and sex matched. The present study was conducted in accordance with the Declaration of Helsinki. The Ethics Committee of Peking University Third Hospital approved the study. All procedures involving specimens obtained from humans were performed with informed consent from each patient.
RNA extraction, sequencing and analysis
PBMCs were isolated from the blood of RA patients and healthy donors via centrifugation over Ficoll-Paque Plus (GE Healthcare, Sweden). RNA was extracted from PBMCs using Trizol: chloroform, precipitated in isopropanol. Sample quality was assessed using Nanodrop. Total RNA was enriched for mRNA using poly-A selection. In brief, mRNA was fragmented, reverse transcribed, adapted with sequencing primers and sample barcodes, size selected, and PCR enriched. Libraries were sequenced on the HiSeq 2000 platform. RNA sequence reads were aligned to the human reference genome (NCBI, hg19) using TopHat2 aligners. Normalised gene counts were calculated using HTSeq, and differential gene expression between samples was identified via edgeR. Gene expression (RPKM) values were calculated using Cufflinks v2.0.2. Gene Ontology (http://geneontology.org/) associations were determined using Gorilla. Network construction was based on the Ingenuity Pathway Analysis (Qiagen, CA, USA) experimental evidence database.
Cell culture
PBMCs were isolated from the blood of RA patients as described above. CD14+ monocytes were positively selected via magnetic-activated cell sorting (Miltenyi Biotec, Germany) and an anti-CD14 antibody. The isolated CD14+ monocytes (1.5 × 105 cells per well in 48-well plates or 6 × 105 cells per well in 12-well plates) were incubated in RPMI-1640 media containing 10% fetal bovine serum (FBS). Osteoclast differentiation was induced from CD14+ monocytes treated with macrophage colony-stimulating factor (M-CSF, 50 ng/ml) and receptor activator of nuclear factor kappa-Β ligand (RANKL, 100 ng/ml). C-X-C motif chemokine ligand 2 (CXCL2, 100 ng/ml or otherwise as indicated) were added to the cultures. The medium was replaced every 3 days.
RAW264.7 macrophages (1.0 × 104 cells per well in 48-well plates or 4.5 × 104 cells per well in 12-well plates) were incubated in DMEM media containing 10% FBS. Osteoclast differentiation was induced by M-CSF (10 ng/ml) and RANKL (50 ng/ml) with or without CXCL2.
Flow cytometry
To identify the expression of C-X-C motif chemokine receptor 2 (CXCR2), CD14+ monocytes were incubated for 45 min at 4°C with anti-human CXCR2 in the dark. Cells were washed twice with staining wash buffer and centrifuged at 1000 rpm for 5 minutes to pellet the cells. Stained cells were resuspended in 200 µl of PBS then analyzed via flow cytometry. In each case, staining was compared with that of the appropriately labeled isotype control antibody.
Cell migration
Monocyte migration testing was performed with 5μm pore-size polycarbonate membrane transwell tissue culture inserts (Corning, USA). Cells (5 × 104 per well) were seeded into the wells in the top chamber in 10% FBS medium, and CXCL2 was added to the lower chamber in 10% FBS medium. After incubation for 1 hour at 37°C the medium was removed and the filters were fixed with 4% paraformaldehyde for 20 minutes. The filters were then stained with 0.1% crystal violet for 15 minutes. The number of cells that had migrated from the upper surface of the filter to the lower surface of the filter was counted.
Quantitative PCR
Total RNA was extracted using TRIzol reagent (Invitrogen), and 1 μg of total RNA was reverse-transcribed into cDNA using a FastQuant RT Kit (with gDNase) (Tiangen Biotech). RT-PCR was carried out using the QuantStudio™ 5 Real-Time PCR System (Thermo Fisher Scientific) with Talent qPCR PreMix (SYBR Green) (Tiangen Biotech). Data were normalized to the expression of GAPDH. The primer nucleotide sequences for PCR were obtained from GenBank database and synthesized by Sangon Biotech (China). The primer sequence information used in the study was shown in the supplementary material, Table1.
Enzyme-linked immunosorbent assay
CXCL2 levels in sera from ACPA+ RA, ACPA- RA and healthy controls, as well as levels in the supernatants of CD14+ monocyte cultures were measured using human CXCL2 enzyme-linked immunosorbent assay kits in accordance with the manufacturer’s instructions.
TRAP staining
TRAP staining of CD14+ monocytes and RAW264.7 macrophages was performed after culturing with or without CXCL2 in the presence of M-CSF and RANKL. TRAP staining was conducted using the TRAP kit (387A-1KT, Sigma) in accordance with the manufacturer’s instructions. TRAP-positive multinucleated cells containing more than 3 nuclei were identified as osteoclasts and counted under a microscope.
F-actin ring immunofluorescence
Cells were fixed with 4% paraformaldehyde and incubated with FITC-conjugated anti-actin antibody for 1 hour at 25°C. After a PBS wash the cells were incubated with DAPI for 5 minutes at 25°C, then analyzed using an Olympus BX51 microscope (Tokyo, Japan).
Scanning electron microscopy
Cells on dentine were fixed in 4% glutaraldehyde, dehydrated via graded alcohol solutions then graded (50% to 100%) hexamethyldisilazane solutions (Sigma-Aldrich, MO, USA), then air-dried. Dentine slices were then mounted onto aluminium stubs (EMS, Hatfield, PA, USA), sputtered with gold, and examined using a scanning electron microscope (Philips SEM 505, Best, Netherlands).
Bone resorption assay
Functional evidence of osteoclast formation was determined via a lacunar resorption assay system using cell cultures on dentine slices as previously described(17). Cells were removed from the dentine slices by treatment with 0.1 M ammonium hydroxide. The dentine slices were washed in distilled water and ultrasonicated to remove adherent cells, then stained with 0.5% (v/v) toluidine blue to reveal areas of lacunar resorption, and examined via light microscopy.
Western blotting
Total protein was extracted using RIPA lysis buffer (Applygen, Beijing, China). A total of 40 μg of protein was loaded into a 10% sodium dodecyl sulphate-polyacrylamide gel and electrophoretically transferred to polyvinylidene fluoride membranes (Merck Millipore, Billerica, MA, USA). After blocking with 5% milk for 1 h, the membranes were incubated with specific antibodies at the indicated dilutions for 12–16 h at 4°C. The membranes were scanned using an Odyssey Sa Imaging System (LICOR Biosciences, Lincoln, NE, USA). The antibody information used in this study was shown in the supplementary material, Table 2.
Statistical analysis
Statistical analysis of RNA sequencing count data was conducted using edgeR with a 5% FDR. All experiments were repeated at least three times and are presented as the mean±standard deviation and the median with interquartile range. Statistical analysis were assessed via Spearman correlation, Mann-Whitney test and one-way analysis of variance followed by Tukey’s test for multiple comparisons. p values < 0.05 were considered statistically significant.