Human subjects and ethics statement
Synovial tissues were obtained from 5 female RA patients (average age 61.6 ± 1.67 y) and 5 female OA patients (average age 64.8 ± 16.10 y) who were undergoing synovectomy or joint replacement. The diagnosis of RA conformed to the College of Rheumatology (ACR)/European League Against Rheumatism (EULAR) 2010 classification criteria . After removing fat and fibrous tissues, the synovium was cut into small pieces and incubated with 0.1% collagenase (Sigma-Aldrich) in Dulbecco’s modified Eagle’s medium (DMEM) at 37°C for 3 h. Cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS, Gibco) and maintained in a 5% CO2 incubator at 37°C. FLS were used for experiments after four to six passages. This study was performed according to the recommendations of the Declaration of Helsinki and approved by the Institutional Review Board of Chungnam National University Hospital (CNUH 2015-10-052).
Transwell migration and Matrigel invasion assay
FLS were cultured in serum-free media for 5 h. Following pre-incubation with or without NOX4 inhibitor (GLX351322, MedKoo Biosciences) for 1 h, RA FLS were stimulated with recombinant IL-17 (10 ng/ml, Peprotech) and TNF-α (10 ng/ml, Peprotech) for 1 h. For the transwell migration assay, cells were centrifuged and loaded onto transwell filters with an 8-μm pore (Corning) positioned on top of the migration chamber for 23 h. DMEM containing 10% FBS was transferred to the bottom chamber of the transwell plate as a chemoattractant. For the invasion assay, transwell filters were pre-incubated with Matrigel (Corning) at 37°C for 1 h. Then, transwells were incubated at 37°C for 3 d, fixed with 100% methanol, and stained with crystal violet (Sigma-Aldrich). Non-migrating cells on the top membrane surface were removed by washing with PBS and cotton swabs. Invaded cells were counted in five random fields per sample under an inverted microscope (magnification, x100; 0.55 numerical aperture dry objective; Scale bar, 100 µm; Olympus). For quantification, the crystal violet dye was eluted with 0.1% sodium dodecyl sulfate (SDS) and quantitated using a Sunrise absorbance reader (Tecan) at 595 nm.
Flow cytometric analysis
To analyze the intrinsic surface expressions of IL-17 or TNF receptors, FLS were stained using APC-conjugated anti-human CD217 (IL-17Ra; Invitrogen), BV421 anti-human CD120b (TNF Receptor Type Ⅱ; BD Biosciences), and mouse anti-human CD120a (TNF Receptor Type I; BD Biosciences) with FITC-conjugated anti-mouse IgG secondary antibody (BD Biosciences).
FLS cultures were incubated with serum-free media for 5 h and then stimulated with recombinant IL-17 and TNF-α for 1 h. Culture media was changed, and the cells were incubated with FITC-conjugated anti-human vascular cell adhesion protein 1 (VCAM1; BD Biosciences), PE-Cy™5-conjugated anti-human intercellular adhesion molecule 1 (ICAM1; BD Biosciences), and PE-Cy™7-conjugated anti-human neural cell adhesion molecule 1 (NCAM1; BD Biosciences). To detect ROS levels, cells were stained with MitoSOX™ Red mitochondrial superoxide indicator (Invitrogen) according to the manufacturer’s instructions. Cells were analyzed with a FACSCantoⅡ flow cytometer (BD Biosciences), and data were processed with FlowJo software (Tree Star).
Wound migration assay
When FLS cultures were approximately 90% confluent, cells were incubated with serum-free media for 5 h. FLS monolayers were wounded with pipette tips and treated with recombinant IL-17 and TNF-α for 1 h. Wound closure was monitored and photographed at 0 and 24 h with a Olympus inverted microscope (magnification, x100; 0.55 numerical aperture dry objective; Scale bar, 100 µm). To quantify the migrated cells, pictures of the initial wounded monolayers were compared with the corresponding pictures of cells at the end of the incubation.
Enzyme-linked immunosorbent assay (ELISA)
VEGF concentrations were measured using ELISA kits for human VEGF (R&D Systems) according to the manufacturers’ instructions. VEGF levels were estimated by interpolation from a standard curve generated using a Sunrise absorbance reader (Tecan) at 450 nm.
Real-time PCR and RT-PCR
Total RNA was extracted using TRI Reagent (Molecular Research Center), according to the manufacturer’s instructions. Extracted RNA was used in reverse transcription reactions with ReverTra Ace® qPCR RT Master Mix (TOYOBO) according to the manufacturer’s instructions. SYBR® Green Realtime PCR Master Mix (TOYOBO) was used for real-time PCR analysis of cDNA according to the manufacturer’s instructions. The primers were synthesized by Bioneer (see Table 1 for primer sequences). Thermal cycling conditions were as follows: initial denaturation at 95°C for 5 min, 40 cycles of 95°C for 10 s, 60°C for 15 s, and 72°C for 20 s. A melting step was performed by raising the temperature from 72°C to 95°C after the last cycle. Thermal cycling was conducted on a CFX Connect Real-Time PCR Detection System machine (Bio-Rad Laboratories). The target gene expression levels are shown as a ratio in comparison with the levels of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in in the same sample via calculation of the cycle threshold (Ct) value. The relative expression levels of target genes were calculated by the 2-ΔΔCT relative quantiﬁcation method.
For RT-PCR, the synthesized cDNA was mixed with AccuPower® RT PreMix (Bioneer) and 10 pmol of each specific PCR primer following the manufacturer’s protocol. Amplified products were separated on 1% agarose gels, stained with Midori green advance (NIPPON Genetics), and photographed under UV illumination using a GelDoc system (Bio-Rad Laboratories).
Western blot analysis
Cells were ruptured on ice using a RIPA lysis kit (ATTO Corporation), lysates were clarified by centrifugation, and samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred onto polyvinylidine (PVDF) membranes (Bio-Rad), which were then incubated with antibodies against NOX4 (1/1000 dilution; Abcam), β-actin (1/2000 dilution; Sigma-Aldrich), PI3Kd (1/1000 dilution; Cell Signaling Technology), GAPDH (1/3000 dilution; Cell Signaling Technology) overnight at 4°C. After washing with PBS-T, membranes were stained with peroxidase-conjugated goat anti-rabbit IgG (Abcam) or peroxidase-conjugated rabbit anti-mouse IgG (Abcam). Target proteins were then detected using the chemiluminescent HRP Substrate (Thermo Fisher Scientific).
Specific siRNA targeting NOX4 was purchased from Santa Cruz Biotechnology (sc-41586). Cells were transfected with lipofectamine transfection reagent (Invitrogen) and the indicated siRNA duplex targeting constructs. After incubation for 24 h, downregulation of target expression was evaluated by RT-PCR and western blot analysis.
Statistical analysis was performed using the paired Student’s t-test in SPSS 18.0. P-values <0.05 were considered statistically significant.