Synovial tissues and FLS were derived from patients with RA and OA who underwent surgical knee joint replacement (Department of Joint Surgery, Honghui Hospital, Xi’an Jiaotong University, China). All the patients’ data are summarized in the supplemental Table 1. All participants gave their informed consent in writing prior to inclusion in the study. The study was approved by the Medical Ethics Committee of Xi’an Jiaotong University (No. 2016-261 and No.2017-666).
Histology and immunofluorescence
For routine histopathological analysis, paraffin-embedded synovial tissue sections from RA and OA patients were deparaffinized and stained with hematoxylin and eosin (H&E). For immunofluorescence staining, 6 µm thick tissue sections were incubated overnight at 4°C with the following primary antibodies diluted in PBS: mouse monoclonal antibody against SLC7A5 (1:100, Santa Cruz, sc-374232) and rabbit polyclonal antibody to Vimentin (1:100, Bioss, bs-23064R). Next morning, the samples were washed three times in PBS and incubated for 45 min at room temperature with secondary antibodies, i.e. FITC AffiniPure goat anti-mouse IgG (H+L) (1:400, Earthox, E031210-01) and Cy3 AffiniPure goat anti-rabbit IgG (H+L) (1:400, Earthox, E031620-01). DAPI (4′,6-diamidino-2-phenylindole) was used to detect the nucleus (1:100000, Sigma-Aldrich, D9542). Immunofluorescent staining procedure was followed with slight modifications, as previously described . The immunofluorescent images were captured with a fluorescence microscope (Olympus, Japan) and analyzed by Image J software.
Cytokines and inhibitor treatment
Cell were treated with IL-1β (20 ng/mL), TNF-α (20 ng/mL), IFN-γ (20 ng/mL), IL-6 (20 ng/mL) and IL-17A (20 ng/mL) (Genscript, China) for 24 hours and total protein analysis was performed using Western blotting assay.
The samples were incubated with JNK inhibitor SP600125 (10 μM, Selleckchem, s1460), NF-κB inhibitor BAY11-7085 (10 μM, Selleckchem, s7352) or P38 inhibitor SB203580 (10 μM, MEC, HY10256A) for 4 hours, followed by the addition of 20 ng/mL IL-1β for 24 hours, to stimulate the cells. Expression at mRNA and protein levels was determined by RT-qPCR and Western blotting, respectively.
Blocking assay of SLC7A5
SLC7A5 antibody (20 μg/mL, a mouse anti- SLC7A5 monoclonal antibody, IgG1, Santa cruz, USA) was administrated to the FLS, following the procedure as detailed in our previous paper . Briefly, FLS were seeded in 12 well plates at a density of 4×104/mL and incubated with SLC7A5 antibody or isotype-matched IgG1 (CST, #5415, USA) for 24 hours. The cells were then treated with IL-1β for 18 hours and collected to detect the mRNA and protein levels of MMP3 and MMP13.
Total protein lysates from synovial tissues and cells were extracted by using the RIPA solution (Beyotime, China) with a cocktail of protease and phosphatase inhibitors (Roche). The total protein concentration of each sample was determined by a BCA Protein Assay kit (Thermo Scientiﬁc, USA). Subsequently, 20 μg from cell lysates were separated by 6% or 8% SDS-PAGE gels and transferred to the polyvinylidene fluoride membrane (EMD Millipore, Billerica, MA, USA). The membrane was incubated with primary antibodies at 4˚C overnight. The list of primary antibodies is depicted in supplemental table S5. After washing, the membrane was further incubated with a horseradish peroxidase‑conjugated goat anti-rabbit or goat anti-mouse IgG secondary antibody (0.4 μg/ml, Abcam, USA) for 2 h at room temperature. Signal intensity was determined by Supersignal® West Pico Kit (Thermo Scientiﬁc) using the enhanced chemi-luminescence detection system (EMD Millipore). The band density was measured by Image J software normalized to β-actin.
RNA isolation and RT-qPCR
Total RNA from the synovial tissues and cells was isolated using the TRI Reagent™ solution (Thermo Scientific, USA), and reverse transcribed to cDNA using the First Strand cDNA Synthesis Kit (Thermo Scientific, USA) according to the manufacturer's instructions. RT-qPCR was performed by using iQ5 optical system software (Bio-Rad Laboratories, USA) with Fast Start Universal SYBR Green Master (ROX) (Roche, USA) for relative quantification of the target genes at mRNA level. Gene expression analyses were calculated by 2-ΔΔCt method.
Small interfering RNAs (siRNAs) targeting SLC7A5 (si1:5’- CATTATACAGCGGCCTCTTT-3’, si2: 5’- TAGATCCCAACTTCTCATTT-3’) and the Negative control (NC, 5’-GCGACGAUCUGCCUAAGAUTT-3’) were purchased from Oligobio (Beijing, China). Cells were transfected with 75 nmol/L of either SLC7A5 siRNA or NC siRNA using Lipofectamine™ 2000 Transfection Reagent (Thermo Scientific, USA) according to the manufacturer’s guidelines. The cells were collected for RNA or protein isolation 24-48 hours post transfection, where indicated to detect the treatment effects and the signal pathways.
Cytokine profiling assay
RA FLS were seeded in 6 well plates (2×105 cells/mL) and incubated overnight in DMEM medium containing 5% FBS. Subsequently, the cells were transfected with siRNA, and 4h later the medium was replaced by containing 0.2% FBS and incubated for 48 hours. Supernatants were collected, centrifuged (at 2000 rpm for 10 min at 4 ˚C), and aliquots were stored at -80˚C before further analyses.
Cytokine expression in siRNA treated RA FLS supernatants was detected by using RayBio® C-Series human cytokine antibody array (AAH-CYT-5). Dot ELISA based membrane coated with 80 human cytokines (listed in the supplemental Table S2) was incubated with RA FLS supernatants pooled from 4 donors, transfected with si-SLC7A5 or si-NC for 48h. The detection and analysis of the cytokine array were performed by RayBiotech Company according to the manufacturer’s instructions. Dot immunoblot signals from the membrane array were captured and the raw intensity was calculated as shown in the supplemental Table S3.
Amino acid deficiency and supplement assay
Lab self-made DMEM were followed by the Dulbecco's Modified Eagle's Medium (DME) Formulation recipe in Sigma-aldrich website. The single amino acid deficient medium was prepared at the laboratory based on the Dulbecco's Modified Eagle's Medium (DMEM) formulation from Sigma Aldrich lacking either phenylalanine (Phe), or tryptophan (Trp). For the amino acid supplement assay, additional 1 mM phenylalanine (Phe), tryptophan (Trp), or Kynurenine (Kyn) were added into the DMEM medium. The FLS were cultured in single amino acid deficiency medium initially for 8 hours before the addition of IL-1β into the treatment group medium and incubated for another 16 hours. The cells were collected for mTOR activity and MMPs expression analyses.
Data were expressed as mean ± standard error of mean and SPSS software was used for statistical analyses. One-way ANOVA among the groups, and Student’s t-test or Mann-Whitney-Wilcoxon test between the two groups were used to determine significant differences according to distribution of the data (normal distribution was validated using Shapiro-Wilk test). p less than 0.05 was considered statistically significant.