Resident peritoneal macrophages were isolated from 6-week-old male ICR mice (Experimental Animal Center, Hallym University) as described previously . Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of RA patients by Ficoll-Hypaque density centrifugation and resuspended in RPMI-1640 containing 1% fetal bovine serum and 1% penicillin/streptomycin. FLS, isolated from synovial biopsies taken from RA patients, were cultured in DMEM containing 10% fetal bovine serum and 1% penicillin/streptomycin. For all experiments, cells were used between passages three and seven. The viability of PBMCs and FLS was evaluated after incubation for 2 h with CCK-8 (DOJINDO, Kumamoto, Japan). Optical density was read at 450 nm.
Transient transfection and luciferase assay
RAW 264.7 cells were transfected with a pcDNA3.1 control vector or a HDAC6 expression vector (pcDNA-HDAC6-FLAG) using Lipofectamine 3000 reagent (Thermo Fisher Scientific). For the reporter assays, cells were co-transfected with a pNF-κB-luc or pAP-1-luc plasmid (Stratagene, La Jolla, CA, USA) and a control (pCMV-β-galactosidase) plasmid using Lipofectamine 3000 reagent as previously described . Cell lysates were prepared and luciferase and β-galactosidase activity analyzed. The luciferase activity of each sample was normalized to that of β-galactosidase and the results were expressed as a fold change in transactivation.
Measurement of cytokines in cell culture supernatants
RA PBMCs were treated with HDAC6 inhibitors and then stimulated with LPS. RA FLS were treated with HDAC6 inhibitors and then stimulated with IL-1β. After 24 h, cell culture supernatants were collected and the amounts of TNF-α, IL-1β, IL-6, and IL-10 secreted by PBMCs and the amounts of MMP-1, MMP-3, IL-6, and IL-8 secreted by FLS were measured in enzyme-linked immunosorbent assays (ELISAs).
Cell proliferation assay
CD4+ CD25- T cells were purified from healthy PBMCs by negative selection using a CD4+ T Cell Biotin-Antibody Cocktail (Miltenyi Biotec). Induced Treg (iTreg) cells were generated from CD4+ CD25- T cells of RA patients in the presence of an anti-CD3 antibody (eBioscience, San Diego, CA, USA), an anti-CD28 antibody (BD Pharmingen, San Diego, CA, USA), IL-2 (PEPROTECH), TGF-β (PEPROTECH), and vitamin D3 (SIGMA) . CD4+CD25- T cells from healthy controls were labeled for 10 min with 5 mM carboxyfluorescein diacetate succinimidyl ester (CFSE) (Life Technologies, Eugene, OR, USA). RA iTreg cells and CFSE-labeled healthy effector T cells were co-cultured for 72 hours at a ratio of 0:1, 0.3:1, and 1:1 in the presence of Dynabeads Human T-Activator CD3/CD28 (Invitrogen Dynal AS, Life Technologies, Oslo, Norway). T cell proliferation was measured by flow cytometry.
Induction of experimental arthritis in rats
All animal experiments were approved by the Animal Care and Use Committee. Lewis rats (female, 5 weeks old) were purchased from Central Lab Animal, Inc. (Seoul, Korea). Complete Freund’s adjuvant (CFA) (Chondrex, Seattle, WA, USA) was resuspended vigorously and 100 μl injected subcutaneously into the tail base. Animals were randomized into six groups (vehicle, n = 7; CKD-506, 3 mg/kg, n = 8; CKD-506, 10 mg/kg, n = 8; CKD-506, 30 mg/kg, n = 8; CKD-506, 50 mg/kg, n = 8; and CKD-506, 100 mg/kg, n = 8). Each group received vehicle or oral CKD-506 once a day from day -1 to day 16 relative to the injection. The severity of arthritis was assessed on Days 9, 13, and 16 after injection of Complete Freund’s adjuvant. Thereafter, rats were sacrificed.
The severity of arthritis was evaluated by scoring each joint (digits, metatarsal bones, and tarsal bones) as follows: 0, no swelling or erythema; 1, slight swelling and/or erythema; 2, low to moderate edema; 3, pronounced edema with limited joint usage; and 4, excess edema with joint rigidity. The clinical scores of four joints were summed to generate a total score for each animal.
Data are presented as the mean ± SEM. Groups in vitro experiments were compared using t-tests. Clinical scores of treatment groups over time were compared using repeated measure analysis of variance (RM-ANOVA). All statistical analyses were performed in Prism software (GraphPad, La Jolla, CA, USA). P values <0.05 were considered statistically significant.