CIA mice
All animal experiments were approved by the Institutional Animal Care and Use Committee of the Jikei University, Tokyo, Japan. Five-week-old DBA/1 J mice (n = 69) were purchased from Sankyo Labo Service (Tokyo, Japan) and immunized intradermally at the dorsal root of the tail with bovine type II collagen (200 μg/mouse; Collagen Research Center, Tokyo, Japan) emulsified in Freund’s complete adjuvant (Becton Dickinson and Company, NJ, USA) (Day 0). On Day 21, a booster injection of bovine type II collagen emulsified in Freund’s incomplete adjuvant (Becton Dickinson and Company) was administered in the same manner. The severity of arthritis was evaluated by a clinical rheumatologist (study co-author KO), who was blinded to the study groups, and expressed as the sum of the scores for all four limbs as assessed on the following scale: 0, normal; 1, swelling of digits alone or mild swelling of wrist and ankle joints; 2, clear swelling of wrist and ankle joints; and 3, severe swelling of wrist and ankle joints.
Gene expression analysis of joints
CIA mice were sacrificed under isoflurane anesthesia. The forelimbs and hindlimbs were amputated 5 mm proximal to the wrist and ankle joints, and RNA was extracted using an RNeasy Lipid Tissue Mini Kit (Qiagen, Tokyo, Japan). Real-time PCR was performed on an Applied Biosystems StepOne Plus Real-Time PCR System (Thermo Fisher Scientific, MA, USA) using TaqMan probes and primers (Thermo Fisher Scientific) for PK2 (Mm00450080_m1), PKR1 (Mm01204733_m1), PKR2 (Mm00769571_m1), IL-1 (Mm01336189_m1), IL-6 (Mm00446190_m1), TNF-α (Mm00443258_m1), VEGF (Mm01281449_m1), and β-actin (Mm00607939_s1). Gene expression levels were analyzed by the ΔΔCT method using β-actin as a reference gene.
The gene expression levels of PK2, PKR1, and PKR2 were analyzed for the CIA mice sacrificed on Days 21, 28, and 35. The gene expression levels on Days 28 and 35 were expressed relative to the arbitrary assigned value on Day 21 (=1.0). The experiment was repeated 3 times for each group of five mice (5 mice × 3 days × 3 repetitions = 45 mice). The correlation between arthritis scores and PK2, PKR1, and PKR2 gene expression levels was analyzed for all 45 mice. Separately, cytokine gene expression levels were analyzed for the PKRA7-treated and -untreated mice (n = 6 each) sacrificed on Day 35 and were expressed relative to the values obtained for untreated mice.
PKR1 and PKR2 protein analysis
For immunohistochemical staining of PKR1, isolated joints on Day 35 were perfusion-fixed, decalcified, and embedded in optimal cutting temperature compound (Sakura Finetek, CA, USA) for frozen sectioning (at a thickness of 7 μm). For PKR2 immunostaining, the tissues were embedded in optimal cutting temperature compound without fixation or decalcification and cryosectioned. Endogenous peroxidase was inactivated with a peroxidase blocking reagent (Dako, Glostrup, Denmark). Sections were incubated with a primary antibody, either rabbit anti-mouse PKR1 antibody (Covalab, Villeurbanne, France) or rabbit anti-mouse PKR2 antibody (Covalab), and then with a secondary antibody (Simple Stain Rabbit Max PO; Nichirei Bioscience, Tokyo, Japan). Simple Stain DAB Solution (Nichirei Bioscience) was used for visualization of the secondary antibody. The sections were then counterstained with hematoxylin and examined under a light microscope (Axio Imager A1, Carl Zeiss, Göttingen, Germany).
Administration of PKRA7
DMSO was used as a vehicle. Prior to administration, PKRA7 in DMSO was diluted with PBS to adjust the concentration of DMSO to 5 %. CIA mice received either 15 mg/kg/day of PKRA7 or 5 % DMSO (control) for 2 weeks from Day 21. The experiment was repeated twice in groups of 6 mice (n = 12). On Day 35, the mice were sacrificed and their limbs amputated. Isolated joints were fixed, decalcified, and embedded in paraffin, and the blocks were sectioned into slices at a thickness of 4 μm. Sections were stained with hematoxylin and eosin and examined under an Axio Imager A1 microscope. The gene expression levels of inflammatory cytokines IL-1β, IL-6, TNF-α, and VEGF were also measured by real-time PCR and compared between PKRA7-treated (n = 6) and -untreated (n = 6) CIA mice on Day 35.
Statistical analysis
The gene expression levels of PK2, PKR1, and PKR2 on Days 21, 28, and 35 were analyzed by a Kruskal-Wallis test with a Dunn’s multiple comparison test post hoc. Spearman’s rank correlation coefficients were used to examine the correlation between arthritis scores and relative gene expression. Arthritis scores for untreated and treated mice were compared by two-way analysis of variance with a Sidak’s multiple comparison test post hoc. Cytokine gene expression was analyzed by a Mann-Whitney test. All results were expressed as mean values ± SEM.