Induction of SpA in SKG mice and TAK-242 treatment
Female SKG mice were obtained from CLEA-Japan, Inc. The mice were maintained under specific pathogen-free conditions. SpA was induced in mice between 8 and 10 weeks of age by intraperitoneally (I.P) injecting 3 mg curdlan (Wako Chemicals, Osaka, Japan). Mice received either anti-IL-23 antibody or TAK-242 injection. Mice were I.P injected with 100 μg of anti-mouse anti-IL-23(P19) monoclonal antibody (BioXcell, Seoul, Korea) 1 day before curdlan injection and weekly until mice were killed. In another group of mice, TAK-242 (3 mg/kg) was given I.P 1 day before curdlan injection and three times per week. All mice were handled according to protocols that were approved by the Committee on Animals of Kyung Hee University Hospital at Gangdong.
Clinical and histologic scoring
Clinical scores were monitored weekly and scored as follows: 0, no joint swelling or redness of finger, wrist and ankle joint; 0.1, swelling or redness of one finger joint; 0.5, mild swelling or redness of wrist and ankle; and 1.0, severe swelling or redness of the wrist and ankle. Mice were monitored by the same observer who was blinded with regard to treatment. Calipers were used to measure the wrist and ankle width. Scores for the affected joints of 4 legs were summed; the maximum score was 6. The hematoxylin and eosin (H&E)–stained sections of the ileum, tail, lung and peripheral joint were scored as described previously [24]: 0 = no inflammation, 1 = few infiltrating immune cells, 2 = 1–2 small patches inflammation, 3 = inflammation and reduced joint space throughout the ankle joint, and 4 = inflammation in > 70% of the tissue.
Histology
At the experimental endpoint of 9 weeks, the peripheral joint, lung, tail and ileum from control and treated mice were fixed in 10% formalin (Sigma-Aldrich, St. Louis, MO, USA) and embedded in paraffin. Tissue specimens of joint and ileum were sectioned at 7 µm and 4 µm thickness, respectively. The sections were dewaxed using xylene (Duksan General Science, Seoul, Korea), dehydrated using an alcohol gradient, and stained with H&E (Cancer Diagnostics Inc. Durham, NC, USA). For immunohistochemistry, sections were deparaffinized, hydrated and incubated in 20 µg/mL proteinase K (Sigma-Aldrich, St. Louis, MO, USA) for 15 min at 37℃. Endogenous peroxidases were quenched in 3% hydrogen peroxide solution (Qiagen, Hilden, Germany) in methanol for 10 min. After washing with phosphate buffered saline (PBS), permeabilization with 0.4% Triton X-100 in PBS for 10 min and blocking with 1% bovine serum albumin (BSA) for 1 h at room temperature, the slides were incubated with primary antibody against IL-17A (Santa Cruz Biotechnology Inc., Dallas, TX, USA) at a 1:100 dilution for overnight at 4℃. Sections were then washed with PBS and incubated with horse anti-mouse/rabbit IgG antibody (H+L) (Universal, biotinylated, R.T.U [Vector Laboratories Inc., Burlingame, CA, USA]) for 30 min at room temperature. Next, 3,3’-diaminobenzidine peroxidase substrate solution was added, and dehydration, clearing and mounting was conducted by routine procedures. Samples were observed under a microscope and photographs were taken. Sections were analyzed, and inflammation was scored blindly by at least 3 independent researchers (CHM, HMD and LKM).
Micro-computed tomography (CT) imaging
The bodies of mice were stored in 4% formalin. The mice were washed twice with PBS and imaged at a resolution of 100 μm with a NanoSPECT/CT (Bioscan Inc., Santa Barbara, CA, USA). High resolution scans of the femurs of the mice were captured to evaluate the 3D structure. The BMDs of femur and spine were measured using the manufacturer’s 3D analysis tools.
Cell preparation from spleen
Spleens were excised from killed mice and placed immediately in RPMI 1640 media. Using the plunger end of a syringe, the spleen was mashed through the cell strainer into a 50 ml tube. After centrifugation of the tube at 1,500 rpm, 4℃ for 5 min, the samples containing white blood cells were treated with Ammonium-Chloride-Potassium Lysing Buffer (BioLegend, San Diego, CA, USA) for the lysis of red blood cells. After centrifugation at 1500 rpm, 4℃ for 5 min, the lymphocytes were stained with antibodies for analysis of T cell subtypes.
Surface and Intracellular Staining and Flow Cytometry
IFN-γ-FITC, CD4-PE-Cy™5 (BD, Franklin Lakes, NJ, USA) and IL-17-PE/Cy7 antibodies (BioLegend, San Diego, CA, USA) were used for the analysis of T cell subtypes by fluorescence-activated cell sorter (FACS) analysis. For the analysis of Th1 and Th17 cells with intracellular cytokine staining, the cells were stimulated in culture medium containing 10 ng/ml phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich), 1 ng/ml ionomycin (Sigma-Aldrich) and 1× golgistop (BD) for 4 h. The cells were treated with the intracellular fixation & permeabilization buffer set (BD) according to the manufacturer’s protocol. The stimulated cells were treated with the transcription factor staining buffer set (eBioscience, San Diego, CA, USA). Cells were analyzed using a cytometric FC 500 flow cytometer (Beckman Coulter, Brea, CA, USA). Data were analyzed by Kaluza software.
Measurement of serum cytokines
Sera from mice were obtained from heart puncture. Komabiotech Co. (Seoul, Korea) analyzed the serum level of cytokines such as IL-17A, TNF-α, IL-6, IL-2, IL-10, IFN-γ and IL-1β using the Luminex® 200™ Total System (Luminex Corporation, Austin, TX, USA).
Statistical analysis
Experimental data are expressed as mean ± standard error of the mean (SEM). Differences between three groups were analyzed using the nonparametric Kruskal–Wallis test. If a statistical difference was detected (P < 0.05), post hoc pairwise group comparisons were performed using Dunn’s test. Two-way analysis of the variance (ANOVA) was used to analyze TAK-242 effects on paw thickness and clinical score over time, following Turkey’s post hoc test. Prism software v.5 (Graphpad Software, San Diego, CA, USA) was used for statistical analysis and graphing. Differences were considered statistically significant at P<0.05.