Cynomolgus monkeys weighing 2.5–3.0 kg at the start of the experiment were purchased from GENIA (Seongnam, South Korea). Animals were housed in single cages in a controlled temperature room (21–29°C) and light (12-h light-dark cycle) conditions. The monkeys had free access to a gamma-ray-sterilized diet (5048, LabDiet, St. Louis, MO, USA) and autoclaved R/O water. All animal research procedures were conducted in accordance with the Laboratory Animals Welfare Act, the Guide for the Care and Use of Laboratory Animals, and the Guidelines and Policies for Non-human Primate (NHP) Experiments provided by the Institutional Animal Care and Use Committee (ORIENT-IACUC-16255).
Macaca fascicularis B*01 gene analysis
Total mRNA was isolated from B cells of each Macaca fascicularis and converted to cDNA. The cDNA was subjected to polymerase chain reaction (PCR) to identify the CIA-related B*01 gene. The following B-specific primers were used: MBS (sense) 5′- AAT TCA TGG CGC CCC GAA CCC TCC TCC TGC -3′ and (anti-sense) 5′- CTA GAC CAC ACA AGA CAG TTG TCT CAG-3′.
Induction of arthritis
CIA was induced in Macaca fascicularis. Type II collagen (CII) was dissolved overnight in 0.1 N acetic acid (4 mg/mL) with gentle rotation at 4°C. Female Macaca fascicularis were immunized intradermally at the base of the tail with 100 µg of chicken CII (Chondrex Inc., Redmond, WA, USA) in complete Freund’s adjuvant (Chondrex Inc.). Macaca fascicularis were boosted with 400 µg of CII emulsified with incomplete Freund’s adjuvant (Chondrex Inc.) and injected intradermally into 10 sites on the back, 21 days after the primary immunization.
The arthritis score was created by grading for disease severity. We used four scoring methods. First, clinical scoring was conducted as follows: 0, no disease symptoms; 0.5, fever; 1, apathy, lessened mobility and loss of appetite; 2, weight loss, warm extremities, and treatable pain without sinus tarsi syndrome (STS); 3, redness of joints, normal flexibility of extremities; 4, severe STS of joints, joint stiffness; and 5, untreatable pain, immobility of joints, weight loss > 25%. Second, phenotype scoring was as follows: 0, no evidence of swelling; 1, over 0.5 cm swelling of the proximal interphalangeal (PIP) joint compared with week 0 (< 0.5 cm2); 2, score 1 evidence in two PIP joints; 3, score 1 evidence in three PIP joints; and 4, score 1 evidence in four PIP joints. Third, behavior scoring was as follows: 0, no evidence of behavior; 1, drag or limp in a hind leg; 2, slipping over in cage; and 3, one leg is not working. Fourth, pain scoring was as follows: 0, no evidence of pain; 1, tremor in paws; 2, tremor in paws and losing grip; and 3, severe tremor in paws and a crouching position.
The PIP joints were collected from each group at 8 weeks after the first immunization. The tissues were fixed in 10% neutral buffered formalin solution, decalcified using Decalcifying Solution-Lite (Sigma, St. Louis, MO, USA), and embedded in paraffin. Sections of 4- to 5-μm thickness were cut, dewaxed using xylene, dehydrated in an alcohol gradient, and stained with hematoxylin and eosin (H&E) and safranin O.
Paraffin-embedded sections were incubated at 4°C with the following primary monoclonal antibodies: anti-IL-1β, anti-IL-6 (Novus Biologicals, Littleton, CO, USA), anti-IL-17, and anti-TNF-α (Abcam, Cambridge, UK). The samples were then incubated with horseradish peroxidase-conjugated secondary antibody for 30 min. The reaction product was developed using 3,3-diaminobenzidine chromogen (Dako, Carpinteria, CA, USA). Histological assessments were conducted by three independent blinded observers. Images were captured using a DP71 digital camera (Olympus, Shinjuku, Tokyo, Japan) attached to a photomicroscope at a magnification of 200×. Positively stained cells were counted using Adobe Photoshop software (Adobe, San Jose, CA, USA), and the mean values were calculated.
Flow cytometry analysis
Peripheral blood mononuclear cells (PBMCs) were isolated at 8 weeks after induction of arthritis. To analyze the population of T helper cells, the isolated PBMCs were stained with anti-CD4 peridinin chlorophyll protein complex (PerCP; BD Biosciences, San Diego, CA, USA), anti-IFNγ-fluorescein isothiocyanate (FITC; BD Biosciences), and anti-IL-17A eFluor 660 (eBioscience, San Diego, CA, USA). For regulatory T cells, the PBMCs were stained with anti-CD4 PerCP (BD Biosciences), anti-CD25-allophycocyanin (APC; BD Biosciences), and anti-Foxp3-PE (BD Biosciences). Cells were permeabilized and fixed with CytoPerm/CytoFix (BD Biosciences) according to the manufacturer’s protocol. Flow cytometry was performed using a FACSCalibur cytometer (BD Biosciences).
In vivo micro-CT imaging and analysis
Micro-CT imaging and analysis were performed using an animal scanner (SkyScan 1176, Billerica, MA, USA). The animals were imaged at settings of 60 kVp and 417 μA using an aluminum 0.5-mm thick filter. The pixel size was 35.76 μm, and the rotation step was 0.4°. Cross-sectional images were reconstructed using a filtered back-projection algorithm (NRecon Software, Bruker Micro CT, Kontich, Belgium). For each scan, a stack of 286 cross-sections was reconstructed at 4000 × 2670 pixels. Bone volume and bone surfaces were analyzed at the proximal interphalangeal and wrist joints.
Enzyme-linked immunosorbent assay (ELISA)
The concentrations of IL-6 in the blood serum of Macaca fascicularis were measured using a sandwich ELISA (DuoSet; R&D Systems, Lille, France).
Hematology and serum biochemistry
Hematology values were determined using a Cell-Dyn® 3700 hematologic analyzer (Abbott Diagnostics, Wiesbaden, Germany), and serum biochemistry was evaluated using a Hitachi clinical analyzer 7180 (Hitachi Ltd., Tokyo, Japan). The hematology analysis included hemoglobin (Hb; g/dL), and biochemistry analysis included C-reactive protein (CRP; mg/dL).
The results are expressed as means ± standard deviation and were obtained from three separate experiments. Statistical significance was determined according to the Mann–Whitney U-test or an ANOVA with Bonferroni’s post-hoc test, performed using GraphPad Prism (version 5.01, GraphPad Software, San Diego, CA, USA). A p-value < 0.05 was considered to indicate statistical significance.