Patients and controls
Clinical samples were the resident synovial tissue collected from patients undergoing knee arthroscopic or routine examination at Shanghai East Hospital, including six patients with RA and three patients with osteoarthritis (OA). All of the patients provided consent and met the diagnostic criteria of the American College of Rheumatology (ACR) criteria for RA (14) and OA (15). The Ethics Committee approved the study protocol of Shanghai East Hospital.
Animals
Twelve SD rats were administered intradermally at the base of the tail with a dose of bovine type II collagen (100 μg) emulsified in complete Freund's adjuvant on day 0. Then a booster injection was provided on day 21 with bovine type II collagen (100 μg) emulsified with incomplete Freund's adjuvant. The rats were then randomly divided into two groups: the CIA model group and the PADI4 inhibition group. Normal non-immunized rats were selected as the normal control group. The rats were sacrificed on the final day; joints were collected from all of the groups.
Immunohistochemical and immunofluorescence analyses
Synovial tissues from humans and joints from rats were fixed in 10% neutral buffered formalin and then embedded in paraffin. Paraffin sections were deparaffinized and rehydrated. After blocking with 3%H2O2, the sections were incubated with a goat polyclonal antibody against human CCR7 (1:500, Abcam, Cambridge, MA, US) and rabbit polyclonal antibody against human mannose receptor (CD206, 1:500, Abcam, Cambridge, MA, US) at 4°C overnight. Next, the sections were incubated with secondary antibody for 30 min at room temperature. Immunoreactive signals were visualized using DAB (diaminobenzidine 3). For double immunofluorescence staining, the sections were incubated with anti-CCR7 antibody or anti-CD206 antibody at 4°C overnight, then incubated with Donkey anti-Goat IgG Alexa Fluor 488 (1:200, Thermo Fisher, US) and Donkey anti-Rabbit IgG Alexa Fluor 594 (1:200, Thermo Fisher, US) for 30 min at room temperature. The cells were counterstained with DAPI (4′,6-diamidino-2-phenylindole) and visualized under a fluorescence microscope.
Cytokine immunoassay
Synovial fluid samples were collected, three RA, and three OA samples as controls. Undiluted media samples were plated, and analytes (IL-1, IL-6, IL-8, IL-10, IL-12 IL-13, IL-17, IFN-γ, and TNF-α) were assessed according to the protocol included with the ProcartaPlex™ Platinum Human Multiplex Assay. Each plate was read on the BioPlex 200 (BioRad).
Cell culture
Human monocytic THP-1 cells were obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) and cultured in Roswell Park Memorial Institute medium (RPMI) 1640 culture medium containing 10% heat-inactivated fetal bovine serum (FBS), 20 mg/mL penicillin, and 20 mg/mL streptomycin. Cells were treated with 100 nM PMA (Sigma-Aldrich, #P8195) for 24 h and differentiated into M0 macrophages as previously reported (16), then the cells were further cultured in a normoxic (21% O2) and a hypoxia (3% O2) incubator. M1 was obtained from M0 by being treated with 20 μg/mL IFN-γ (PeproTech) and 100 ng/mL LPS (#L8630, Sigma), while M2 was treated with 20 μg/mL IL-4 (PeproTech) for another 48 h.
Bone marrow (BM) cells were harvested from the femurs and tibias of 6- to 10-week-old C57BL/6 mice (SJA Laboratory Animal Co., Ltd). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Gibco) supplemented with 10% FBS (Wisent Biomart) and recombinant mouse M-CSF (40 ng/ml; PeproTech). After one week, BMDMs were replated and untreated (M0) macrophages were then stimulated with Escherichia coli LPS O111: B4 (100 ng/ml; Sigma) and IFN-γ (20 ng/ml; PeproTech) for 24 h (M1) or with IL-4 (20 ng/ml; PeproTech) for 24 h (M2).
Primary RA-FLSs were isolated from RA synovial tissues. After isolation from the tissue, RA-FLSs were grown further over four to six passages for our experiments. Then these cells were cocultured with THP-1 macrophages through a Transwell coculture system.
Virus vector infection
Adenovirus vectors expressing PADI4 (Genechem, China) were used in accordance with the manufacturer's instructions to transfect genes into M0. RT-PCR and Western blot were used to assess PADI4 expression.
Cocultures of RA-FLSs and M0
The coculture systems were established through a 24-well plate with an 8.0-µm Pore Polycarbonate Membrane Insert (Corning, NY, US). To study the impact of the coculture system on macrophages, macrophages cocultured with RA-FLSs were treated as coculture groups, and the macrophages cocultured macrophages as control groups. M0 were seeded in 5 × 104 cells/well, and 1 × 104 cells/well RA-FLSs were seeded in the transwell inserts in another separated well. After 6 h of incubation, cells had become firmly attached to the wall. Then, the transwell inserts with RA-FLSs were moved to the wells containing the M0 macrophage and after that cocultured for 48–72 h. The coculture system was subjected to normoxia (21% O2) and used as controls, and another set was placed under hypoxia conditions (3% O2) in the incubator.
Quantitative real-time polymerase chain reaction
Total RNA from RA-FLSs and macrophages was extracted using TRIzol™ (Takara), and reverse transcribed using the Reverse Transcription System kit (Promega Corporation, US) according to the manufacturer's instructions. A quantitative real-time polymerase chain reaction (q-PCR) was performed using Premix Ex Taq SYBR Green PCR (Takara) on an ABI PRISM 7500 (Applied Biosystems, Foster City, CA, US) according to the manufacturer's instructions. The primers used to amplify target gene products were as follows: human GAPDH 5′-ACCATGGGGAAGGTGAAG-3′, 5′-AATGAAGGGGTCATTGATGG-3'; human PADI4 5 '-TTCTCTAAGGCGGAAGCTTTT-3', 5 '-AGCAGGGAACACACCTTCTC-3'; human CCR7 5′-GCATTTGTAGTCCTGCTGCG-3′, 5′-CTCCTCAGATCTCACCCGTTG-3′; human TNF-α 5′-TCTTCTCGAACCCCGAGTGAC-3′, 5'-GGTACAGGCCCTCTGATG-3′; IL-10 5′-CACTGCTCTGTTGCCTGGTC-3′, 5′-GAAGCATGTTAGGCAGGTTGC-3'; human NOS-2 5′-CATGAGCCCCTTCATCAATGC-3′, 5′-TTGAAGTCTGTGTCCGAAGGC-3′; human TGF-β 5′-CCGAGAAGCGGTACCTGAAC-3′, 5′-CGCCAGGAATTGTTGCTGTA-3′. murine GAPDH 5′-AGGTCGGTGTGAACGGATTTG-3′, 5′-TGTAGACCATGTAGTTGAGGTCA-3′; murine PADI4 5′-TACCTGATGCTCCCATCCCA-3′, 5′-TCCTAGGTACACCCAGAGGC-3′; murine CCL2 5′-CAGCCAGATGCAGTTAACGCCCCA-3′, 5′-TGGGGTCAGCACAGACCTCTCTC-3′; murine TNF-α 5′-CCCTCACACTCAGATCATCTTCT-3′, 5′-GCTACGACGTGGGCTACAG-3′; murine CCR7 5′-CGTGGTATTCTCGCCGATGT-3′, 5′-TCTGCAAGAGAACAGAAGCCC-3′; murine IL-10 5′-GCAAGGGTGTCTCCTTCCTC-3′, 5′-CTTGTTACACTCGCCCCCTT-3′; murine Arg-1 5′-CTCCAAGCCAAAGTCCTTAGAG-3′, 5′-AGGAGCTGTCATTAGGGACATC-3′. The relative expression of each gene was determined by Ct value (ΔCt = Ct target − Ct GAPDH), and the ΔΔCt method (ΔΔCt = ΔCt sample − ΔCt control), which was used in the comparison with the control group.
Statistical analysis
Statistical analysis was performed using Prism GraphPad software (GraphPad Software, Inc., La Jolla, CA, US). Data were collected from each experiment and compared between groups using analysis of variance (ANOVA) or t-tests (two-tailed distribution) as appropriate. A P-value of ≤ 0.05 was considered to be significant.