Serum samples were obtained from 40 patients with RA and 20 patients with OA. The SF samples from 24 patients with RA and 20 patients with OA were also collected. Inclusion criteria for patients with RA were as follows: (1) fulfilment of the 1987 revised criteria of the American College of Rheumatology, 1987 (formerly the American Rheumatism Association) or 2010 classification criteria for RA, and (2) age over 18 years old. Patients with RA were divided into those with active (N = 24) and inactive (N = 16) RA according to disease activity score-28 (DAS28), in which DAS28 ≥ 3.2, was judged as active state. The present study was conducted in accordance with the Declaration of Helsinki and Good Clinical Practice guidelines. Informed consent was obtained from all participants before enrollment. The experimental protocol was approved by the Konkuk University School of Medicine Human Research Ethics Committee (KUH1010186).
Enzyme-linked immunosorbent assay (ELISA) of IL-18 and IL-18BP levels in serum and synovial fluid
In brief, a 96-well plate (Eppendorf, Hamburg, Germany) was coated with monoclonal antibodies against IL-18, IL-18BP, IL-17A, and soluble RANKL (R&D Systems, Minneapolis, MN, USA) at a concentration of 4 μg/mL at 4 °C overnight. After blocking with phosphate-buffered saline/1% bovine serum albumin /0.05% Tween 20 for 2 h at room temperature (22–25 °C), the test samples and the standard recombinant IL-18, IL-18BP, IL-17A, or soluble RANKL (R&D Systems) were added to the 96-well plate and incubated at room temperature for another 2 h. The plates were washed four times with phosphate-buffered saline/Tween 20, and then incubated with 500 ng/mL biotinylated mouse monoclonal antibodies against IL-18, IL-18BP, IL-17A, or soluble RANKL (R&D Systems) for 2 h at room temperature. After washing, the streptavidin–alkaline phosphate–horseradish peroxidase conjugate (Sigma, St Louis, MA, USA) was added to the wells for 2 h, followed by another wash, and incubation with 1 mg/mL p-nitrophenyl phosphate (Sigma) dissolved in diethanolamine (Sigma) to develop the colour reaction. The reaction was stopped by the addition of 1 M NaOH, and the optical density of each well was measured at 405 nm. The lower limit of IL-18, IL-18BP, IL-17A, and soluble RANKL was 10 pg/mL. Recombinant human IL-18, IL-18BP, IL-17A, or soluble RANKL diluted in the culture medium were used as calibration standards, ranging from 10 to 2000 pg/mL. A standard curve was drawn by plotting the optical density against the log of the concentration of recombinant cytokines, and the curve was used to determine IL-18, IL-18BP, IL-17A, or soluble RANKL concentrations in the test samples.
In vitro culture of PBMCs under Th17 polarizing condition with IL-18BP
Peripheral blood mononuclear cells (PBMCs) were extracted from heparinised peripheral blood of RA patients (N = 5) using the standard Ficoll-Paque density gradient method (GE Healthcare Biosciences, Uppsala, Sweden). In addition, synovial fluid mononuclear cells (SFMCs) from patients with RA (N = 20) were stored in a liquid nitrogen tank at − 200 °C. Cells were cultured in the RPMI-1640 medium (Gibco BRL, Carlsbad, CA, USA) containing penicillin (100 U/mL), streptomycin (100 μg/mL), and 10% foetal bovine serum (Gibco BRL) that had been inactivated by heating to 55 °C for 30 min. Cell suspensions were dispensed into 48-well plates (Eppendorf). The cell culture plate was pre-incubated with an anti-CD3 antibody (1 μg/mL) for 1 h at 37 °C. PBMCs or SFMCs (1 × 106) were plated in 48-well plates (Nunc) with anti-IFN-γ (2 μg/mL) and anti-IL-4 (2 μg/mL) antibodies for 4 h. Then, an anti-CD28 antibody (1 μg/mL), IL-1β (20 ng/mL), IL-6 (20 ng/mL), and IL-23 (20 ng/mL; R&D Systems) were added and cultured for 72 h . To investigate the impact of IL-18BP (R&D Systems) on the regulation of CD4+ T cell differentiation, IL-18BP was added at various concentrations (10, 50, and 100 ng/mL).
To quantify IL-17A+, RANKL+, and CD25+ forkhead box P3 (FOXP3)+ cells in CD4+ T cells, PBMCs cultured under Th17 polarisation conditions with various doses of IL-18BP were immunostained using a PerCP-conjugated anti-CD4 antibody (BD Biosciences, San Jose, CA, USA), then fixed and permeabilised using a Cytofix/Cytoperm Plus kit (BD Biosciences). Following the manufacturer’s instructions, PBMCs were stained with phycoerythrin-conjugated anti-IL-17A (eBiosciences, San Diego, CA, USA), or phycoerythrin-conjugated anti-RANKL (eBiosciences), or allophycocyanin-conjugated anti-CD25 (BD Biosciences) with phycoerythrin-conjugated anti-FOXP3 (BioLegend, San Diego, CA, USA) antibodies. All cells were detected using a FACSCalibur flow cytometer (BD Pharmingen, Franklin Lakes, NJ, USA).
CD14+ monocytes were prepared from PBMCs using microbeads (Miltenyi Biotec, Auburn, CA, USA). Human CD14+ monocytes were seeded in 48-well plates at 5 × 104 cells/well in 1 mL of medium. Monocytes were cultured in α-minimum essential medium supplemented with 10% heat-inactivated foetal bovine serum and 25 ng/mL recombinant human macrophage colony-stimulating factor (rhM-CSF, R&D system) for 3 days. Monocytes were then cultured under RANKL (30 ng/mL) + M-CSF (25 ng/mL) or IL-17A (50 ng/mL) + M-CSF (25 ng/mL) for 10 to 14 days. IL-18BP (0, 10, 50, or 100 ng/mL) was also added three days after rhM-CSF stimulation to evaluate its effect on osteoclastogenesis. After 10–14 days of RANKL + M-CSF + IL-18BP or IL-17A + M-CSF + IL-18BP stimulation, tartrate-resistant acid phosphatase (TRAP)-positive cells were identified using a TRAP staining kit (Cosmo Bio, Tokyo, Japan), as described previously [20, 21].
RNA preparation and quantification of gene expression levels using real-time quantitative polymerase chain reaction
Total RNA was extracted using an easy-spin™ Total RNA Extraction Kit (Intron Biotechnology, Seongnam, Republic of Korea) according to the manufacturer’s instructions. RNA samples were then quantified, aliquoted, and stored at − 80 °C until analysis. Total RNA (500 ng) was reverse transcribed into cDNA using an AccuPower CycleScript RT PreMix cDNA synthesis kit (Bioneer, Daejeon, Republic of Korea) according to the manufacturer’s instructions. RT-qPCR was conducted in a total volume of 20 μL containing 7.2 μL of PCR-grade distilled water, 0.4 μL of forward and reverse primers, and 10 μL of the SYBR Green I Master mix (Roche Diagnostics, Mannheim, Germany). PCR conditions were as follows: 95 °C for 10 min, followed by 35 cycles of 95 °C for 10 s, 55 °C for 10 s, and 72 °C for 10 s. All primers were synthesised by Bioneer Corp. (Daejeon, Republic of South Korea). The relative mRNA expression levels were normalised to β-actin mRNA levels.
All data are expressed as the mean ± standard error of the mean. Statistical analysis was performed using one-way analysis of variance followed by the post hoc Bonferroni’s multiple comparison test. Spearman’s correlation test was used to determine the correlation between serum levels of IL-18 and IL-18BP. Statistical significance was set at P < 0.05. All statistical analyses were performed using Prism 9.0 (GraphPad Software Inc., San Diego, CA, USA).