Patients and controls
Twenty-seven patients with axSpA (18 AS, 9 nr-axSpA) according to the Assessment of SpondyloArthritis International Society (ASAS) classification criteria and 23 healthy age and sex-matched controls were enrolled in this study . Patients were under 45 years, with relatively recent disease onset (not exceeding 10 years of symptoms duration), naïve to synthetic and/or biological Disease Modifying Anti-Rheumatic Drugs (sDMARDs, bDMARDs) and without the administration of systemic glucocorticosteroids. Patients provided a signed informed consent and the study protocol was approved by the local bioethics committee.
ELISA and Cytometric bead Array (CBA) of bone remodeling molecules
Serum for bone remodeling molecules was obtained from axSpA patients and controls and stored at − 20 °C. RANKL, oncostatin M (OSM) (Elabscience Biotechnology Co. Ltd., Wuhan, China), (Abbexa, Cambridge, UK), M-CSF (R&D Systems, Minneapolis, MN, USA), OPG (Ray-Biotech, Norcross GA, USA) and TGFβ (Thermo Fisher Scientific Waltham, MA USA) were assessed by ELISA. We used the RANKL kit which according to the manufacturer has been developed from the human Tumor Necrosis Factor ligand superfamily member 11 protein where the sequence of the immunogen rests within the region Ile140~Asp317 and hence this kit is designed to detect free soluble RANKL. The presented M-CSF levels were assayed in a subsample of 13 patients due to limited availability of patients’ material. Samples were run in duplicates, according to the manufacturers protocols, and results were obtained using an ELISA reader (BioTek Instruments, Vinooski, VT).
Concentrations of IL-6, IL-17A, TNFα in the serum were measured using the CBA system (BD Biosciences) followed by flow cytometric analysis (FACS Canto). The CBA beads were discriminated in FL-4 and FL-5 channels, while the concentration of specified cytokines was determined by the intensity of FL-2 fluorescence, using the respective standard reference curve and FCAP Array software (BD Biosciences).
The detection levels were < 58 pg/ml for RANKL, 11 pg/ml for M-CSF and 1 pg/ml for OPG, 9,38 pg/ml for OSM, 156,3 pg/ml for TGFβ, 2,4 pg/ml for IL-6, 18,9 pg/ml for IL-17A and 3,8 pg/ml for TNFα.
Cell culture of osteoclasts for cathepsin K and RANK mRNA expression analysis
Differentiation of monocytes into osteoclasts
Peripheral blood from healthy donors was used to isolate monocytes, as described previously . Briefly, peripheral blood mononuclear cells (PBMC) were isolated by the standard Ficoll/Isopaque (Pharmacia, Uppsala, Sweden) density gradient centrifugation. Subsequent separation of monocytes form PBMC was performed with JE-5.0 elutriation system, equipped with the Sanderson separation chamber (Beckman, Palo Alto, CA, USA) . Isolation purity was over 95% as tested by staining with anti-CD14 mAb (BD Biosciences Pharmingen, San Diego, CA) and flow cytometry analysis (FACSCanto flow cytometer, Becton Dickinson, San Jose, CA, USA). Monocytes were seeded in 24-well plates at density of 2 × 105 cell/cm2 and cultured in RPMI 1640 medium (Corning) supplemented with 10% serum (from randomly chosen axial SpA patients, n = 11, or heathy controls, n = 10) and 50 μg/ml gentamycin for 14 days (with half of media change every other day). Cells were cultured with rhM-CSF (10 ng/ml) alone, or rhM-CSF and rhRANKL (10 ng/ml) (both from Peprotech, London, UK), or without addition of additional factors for the whole differentiation period. After 14 days cells were harvested for RNA isolation (Norgen, RNA purification kit).
Analysis of qRT-PCR
RNA (200 ng) from cultured cells was transcribed using NG dART RT kit (EurX, Poland). qPCR for studied genes was performed using SG qPCR Master Mix (EurX, Poland) with QuantStudio 7 cycler (Thermo Scientific). The CT values were normalised to housekeeping gene – GAPDH. Results were analysed using standard 2-ΔΔCT method. Primer sequences used in this study: GAPDH_FW: AGATCATCAGCAATGCCTCCT, GAPDH_RV: TGGTCATGAGTCCTTCCACG, Cathepsin K_FW: TTCCCGCAGTAATGACACCC, Cathepsin K_RV: GGAACCACACTGACCCTGAT, RANK_FW: TGGGACGGTGCTGTAACAAA, RANK_RV: CCAAGTATTCATCCGGGCCA.
Tartrate-resistant acid phosphate (TRAP) activity detection in monocyte-derived osteoclasts
For TRAP activity assay, monocytes were isolated and cultured as described above for 20 days (with half of cell culture medium refreshed every 3–4 days). Cells were then fixed and stained for TRAP activity using commercially available kit according to manufacturer’s protocol (Sigma-Aldrich, Acid Phosphatase, Leukocyte [TRAP] Kit). Large multinucleated cells (containing at least 3 nuclei) showing TRAP activity were considered as osteoclasts. For quantification of osteoclast differentiation efficiency, cells fulfilling osteoclast criteria (TRAP activity + at least 3 nuclei) were counted (n = 12 representative microscope images for each condition) and the percentage of osteoclasts was calculated.
X-ray of sacroiliac joints, cervical and lumbar spine
To assess structural damage radiographs of sacroiliac joints were analyzed according to modified New York (mNY) criteria; radiographs of the lateral cervical and lumbar spine were collected to calculate the mSASSS (modified Stoke Ankylosing Spondylitis Spinal Score).
Database management and analysis were performed using SAS 9.2 (SAS Institute Inc. Cary, NC, USA) and GraphPad PRISM (San Diego, CA) software packages. The variables following a non-normal distribution are presented as medians (25th–75th percentile) and those that are normally distributed as means ± standard deviations (SD). The non-normally distributed data were analyzed using nonparametric techniques (Wilcoxon’s test for comparison of unpaired continuous data). The means of normally distributed variables were compared using Student’s t-test. The proportions were compared using chi-square test. All Pvalues were two-tailed and 5% was considered as the threshold for significance.