Induction and evaluation of AIA
Male C57BL/6J mice (8–10 weeks old) were purchased from Vital River Laboratory (Beijing, China). Age-matched PDCD5 tg mice with the C57BL/6J background were kindly donated by Peking University Center for Human Disease Genomics . The presence of the human PDCD5 gene was confirmed by performing PCR with tail DNA. Both wild-type and transgenic mice were bred under specific pathogen-free conditions at the Experimental Animal Center, Peking University People’s Hospital (Beijing, China). The mice were anaesthetized with isoflurane and euthanized via cervical dislocation. All experimental procedures used in our study were approved by the Institutional Animal Care and Use Committee of Peking University People’s Hospital. The experimental animal were divided into four groups, each group contains 6 mice. The normal control group (NC) and the arthritis control group (AC) were used wild-type mice; while the transgenic control group (TC) and the transgenic arthritis group (TA) were used PDCD5 tg mice. CFA was purchased from Chondrex (USA) and AIA was induced as previously described . AC and TA were immunized subcutaneously in the left plantar hindpaw with a 20 μl injection of CFA containing 5 mg/ml heat-killed mycobacteria. NC and TC were injected in the same location with 20 μl of phosphate-buffered saline. Measurements of joint circumference and paw oedema were performed using a tape measure and digital callipers, respectively. The arthritis score was periodically observed to evaluate the severity of arthritis after the injection of CFA for 14 days by two independent researchers. The progression of arthritis was assessed using the following criteria, ranging from 0 to 4: 0, no erythaema or swelling; 1, joint erythaema and no swelling; 2, joint erythaema and mild swelling; 3, joint erythaema and moderate swelling; 4, severe swelling with dysfunction .
After euthanasia on day 14 following CFA injection, the tibiotarsal joints were removed. The tissue samples were fixed in 4% formalin for 48 h, decalcified in 10% EDTA solution for 4 weeks, embedded in paraffin and sectioned (5 μm). Haematoxylin and eosin (H&E) staining was applied to investigate the degree of joint inflammation and destruction. The histological scores of the tibiotarsal joints were assessed by two blinded and independent pathologists using the following criteria. The score were defined on a scale of 0–4 point: 0, no inflammation; 1, minimal inflammatory infiltration; 2, mild inflammatory infiltration and synovial hyperplasia; 3, moderate inflammatory infiltration and pannus formation; 4, severe inflammatory infiltration, cartilage and bone erosion .
Flow cytometry analysis
Spleens were harvested on the day of euthanasia to prepare single cell suspensions via passage through mesh screens. The splenocytes were washed with red blood cell lysis buffer (BioLegend, USA) and then washed twice with PBS. To perform intracellular labelling, cells (2 × 106 per sample) were incubated with Cell Stimulation Cocktail (eBioscience, USA) for 5 h at 37 °C. Subsequently, the cells were surface-stained with a FITC-labelled CD4 monoclonal antibody (eBioscience), then fixed and permeabilized using an Intracellular Fixation and Permeabilization Buffer Set (eBioscience), followed by intracellular staining with a PerCPCy5.5-labelled IFN-γ monoclonal antibody (eBioscience) and an APC-labelled IL-17A monoclonal antibody (eBioscience). To determine Treg frequency, the cells (2 × 106 per sample) were surface-stained with a FITC-labelled CD4 monoclonal antibody (eBioscience) and an APC-labelled CD25 monoclonal antibody (eBioscience). After fixation with the Intracellular Fixation and Permeabilization Buffer Set (eBioscience), the cells were stained with a PE-labelled FoxP3 monoclonal antibody (eBioscience). Isotype-matched IgG antibody (eBioscience) was used as a negative control. The stained cells were analysed using a FACSCalibur flow cytometer (BD Biosciences, USA) with FlowJo software version 7.6.
RNA extraction and real-time PCR
All extraction procedures were performed on ice using ice-cold reagents. Total RNA was isolated from spleens using a total RNA extraction kit (Tiangen Biotech, Beijing, China) and all procedures comply with the manufacturer’s instructions. The obtained mRNA was quantified by measuring the absorbance at 260 nm, and its quality was determined by measuring the 260/280 ratio. Complementary DNA synthesis was performed using a Fast Quant RT kit (Tiangen Biotech) in accordance with the manufacturer’s instructions. The mRNA expression levels of target genes in the spleen were determined by performing real-time PCR using SuperReal PreMix Plus (Tiangen Biotech) on a Bio-Radi Cycler Opnion Monitor3 System (USA). GAPDH was used as an internal control, and each reaction was performed in triplicate. The real-time PCR data were analysed using the 2−ΔΔCt method. The primers used in these assays were selected from the PubMed database. Primer sequences (5′–3′) that were used are: IFN-γ (5′-TCTGGGCTTCTCCTCCTGCGG-3′,5′-GGCGCTGGACC TGTGGGTTG-3′), IL-4 (5′-GAAGCCCTACAGACGAGCTCA-3′,5′-ACAGGAGAAGGGA CGCCAT-3′), IL-6 (5′-CCGGAGAGGAGACTTCACAG-3′,5′-GGAAATTGGGGTAGGAAGGA-3′), IL-10 (5′-ACCTGCTCCACTGCCTTGCT-3′,5′-GGTTGCCAAGCCTTATCGGA-3′), IL-17A (5′-ATCCCTCAAAGCTCAGCGTGTC-3′,5′-GGGTCTTCATTGCGGTGGAGAG-3′), TNF-α (5′-GCGGAGTCCGGGCAGGTCTA-3′,5′-GGGGGCTGGCTCTGTGAGGA-3′), GAPDH (5′-CCCAGCAAGGACACTGAGCAAG-3′,5′-GGTCTGGGATGGAAATTGTGAGGG-3′).
Cytometric bead array (CBA)
Serum samples were obtained by collecting retro-orbital plexus blood. Cytokine levels were measured using a mouse Th1/Th2/Th17 CBA kit (BD Biosciences, USA) according to the manufacturer’s instructions. Concentrations were calculated based on mean fluorescence intensity values, which were detected using a BD FACS Aria II flow cytometer (BD Biosciences, USA). The data were analysed using BD Cytometric Bead Array analysis software.
All data are expressed as the mean ± SEM. Differences between the experimental groups were tested using Student’s t test or Two-way Anova by SPSS 20.0 (USA). And p < 0.05 were considered statistically significant.