A total of 26 hospitalized treatment-naive DM patients were recruited from the Department of Rheumatology in the China-Japan Friendship Hospital between September 2015 and September 2016. A clinical diagnosis of DM was based on the classification criteria established by Bohan and Peter . We excluded patients suffering from infectious diseases. Patients who presented with accompanying malignancy, overlap syndromes or infection history of hepatitis virus (HAV, HBV, HCV, HDV, and HEV), and human immunodeficiency virus, were excluded. We also excluded the patients with myeloproliferative disease which were determined by peripheral blood routine and blood smear or bone marrow morphology test when necessary. Meanwhile, pregnant and lactating women were excluded. The study protocol was approved by the Ethics Committee of China-Japan Friendship. Hospital written informed consent was obtained from each participant. The study was conducted according to the Declaration of Helsinki (2000).
The physical examinations and laboratory investigations were conducted for all patients at the time of blood sample collection. The number of peripheral lymphocytes and T lymphocyte obtained from routine blood count and T lymphocyte subgroups by flow cytometry (FCM). We conducted detailed records of clinical information, including age at onset, course of disease (recorded monthly), muscle strength, rash (heliotrope rash, Gottron rash, periungual erythema, and “mechanics hand”), and interstitial lung disease (ILD). Muscle strength and severity of rash were measured using the manual muscle test (MMT8) proposed by the International Myositis Outcome Assessment Collaborative Study (IMACS) (http://www.niehs.nih.gov/research/resources/imacs/diseaseactivity/index.cfm) and the modified Cutaneous Dermatomyositis Disease Area and Severity Index (CDASI) . Clinical diagnosis of ILD was based on impaired lung function and typical high-resolution computed tomography (HRCT) features (widespread ground-glass attenuation, intralobular lines/irregular interlobular septal thickening, and honeycombing).
Jurkat cell line was a gift of the Cell Resource Center, in the Peking Union Medical College (CRC/PUMC). They were maintained in RPMI-1640 (Gibco, Carlsbad, CA, USA) in the presence of 10% (v/v) fetal bovine serum (Gibco), 100 U/ml penicillin (Gibco), 100 μg/ml streptomycin (Gibco), and 2 mmol/L l-glutamine (Gibco). The cells were cultured in suspension and maintained at 37 °C in a humidified incubator with 5% CO2.
Apoptosis and cell proliferation assays
The percentage of apoptotic T lymphocyte was detected by flow cytometry(FCM). The assay was based on the detection of labeled annexin V/7-amino-actinomycin D (7-AAD) (BD Bioscience, San Jose, CA, USA). Annexin V+/7-AAD− subset and annexin V+/7-AAD+ subset represented the early and late apoptotic population, respectively. Furthermore, CD95 (BD Bioscience) and Bcl2 (BD Bioscience) were detected as an inducer and an inhibitor of apoptosis, respectively. Bcl2 staining was conducted according to the instructions provided by the manufacturers. Human CD3+ T lymphocytes and Jurkat cells were analyzed on a BD FACS Jazz or a FACSAria III flow cytometer using conventional software provided by the manufacturer (BD Bioscience, San Jose, CA, USA). The viable cells were measured with the CCK8 assay (Dojindo Laboratories, Japan) according to the manufacturer’s instructions.
Isolation of T lymphocytes by magnetically activated cell sorting
The peripheral blood mononuclear cells (PBMCs) were isolated from blood by Ficoll density gradient centrifugation (GE Healthcare, Sweden). Human blood-derived CD3+ T lymphocytes were sorted using anti-human CD3 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s recommendations.
RNA isolation and real-time quantitative polymerase chain reaction analysis
Total RNA was extracted from cells using TRIzol (Thermo, Carlsbad, CA, USA). The RNA samples were reverse-transcribed using the TransScript First-Strand cDNA Synthesis SuperMix (TaKaRa, Dalian, China). Quantitative real-time reverse transcriptase PCR was performed using SYBR Green (TaKaRa, Dalian, China) in an ABI 7500 system (Applied Biosystems, Singapore). The PCR conditions were carried out according to the manufacturer’s instructions. Each sample was analyzed in triplicate. The relative levels of the mRNA expression of each gene of interest were normalized based on the mRNA levels of the housekeeping gene GAPDH. The expression levels were calculated according to the formula 2 − ΔCT. The primer sequences for the genes were as follows: human RIG-I, Forward:5′-CTTGGCATGTTACACAGCTGAC-3′, Reverse: 5-TTGGCTTGGGATGTGGTCTAC-3′, human GAPDH, Forward: 5′-GAGAAGGCTGGGGCTCATTTGCA-3′. Reverse:5′-TTGGCCAGGGGTGCTAAGCAGT-3′. Primers were synthesized by Tsingke Biological Technology (Beijing, China).
Western blotting analysis
Western blotting was performed as described previously . Briefly, protein extracts were isolated from each group of cells using the NP-40 protein lysis buffer, which contained protease inhibitor cocktail (Roche, Germany). Total proteins were separated by 10% or 15% SDS-PAGE and were transferred on polyvinylidene difluoride membranes (Millipore, Bedford, Mass, USA) using a semi-dry Gel Transfer Device (Bio-Rad, Hercules, Calif, USA). The membranes were blocked in 5% non-fat milk and probed with primary antibodies and HRP-conjugated secondary antibodies. Antigen-antibody complexes were visualized using the chemiluminescent ECL (Thermo, USA) detection system and analyzed with a ChemDoc XRS+ image analyzer (Bio-Rad, USA). β-actin was used as an internal control. The intensities of the bands were measured by Image J 1.43U software (NIH Image, Bethesda, MD, USA). The antibodies included anti-RIG-I (Cell Signaling Technology, Danvers, MA, USA), anti-Fas (Abcam, Cambridge, UK), anti-cleaved caspase 3 (Cell Signaling Technology, USA), and anti-β-actin (Sigma, St Louis, MO, USA).
RIG-I overexpression in Jurkat cells
PUNO1-RIG-I plasmid (Invivogene, San Diego, USA) was transfected into Jurkat cells by Lipofectamine 2000 transfection reagent (Thermo, USA), whereas empty plasmid PUNO1-MCS (Invivogene, San Diego, USA) was used as the control. RIG-I protein expression levels were measured by Western blot analysis. Stable RIG-I overexpressing cells were established by blasticidin selection for 5–7 days.
The RIG-I-specific ligand pppRNA (Invivogene, San Diego, USA) was transfected at a concentration of 0.5 μg/ml using Lipofectamine 2000 transfection reagent (Thermo, USA) and pppRNA control was used as control. The analysis was performed at 12- and 24-h time periods.
RIG-I gene knockout in Jurkat cells
The CRISPR/Cas9 gene editing system was used for RIG-I knockout in Jurkat cells. The pSpCas9(BB)-2A-GFP (PX458) plasmid was a gift from Feng Zhang (Addgene plasmid # 48138). The experiment was performed as described previously . Briefly, two sgRNAs were designed using Zhang Lab’s CRISPR DESIGN tools (https://zlab.bio/guide-design-resources). The annealed sgRNAs were cloned into the PX458 plasmid and transfected into Jurkat cells by electroporation (Lonza, Germany). The fluorescent cells were sorted by the BD Aria III FACS system (BD, USA). Finally, two clones were obtained and the gene mutations were confirmed by Sanger sequencing (Tsingke Biological Technology) and Western blot analysis. The sgRNAs used were as follows: sgRNA1 site: GGGTCTTCCGGATATAATCC TGG; sgRNA2 site: GGATTATATCCGGAAGACCC TGG. Oligo DNA was synthesized by Tsingke Biological Technology (Beijing, China). The sequencing results are shown in the supplementary data.
The analysis was performed using PASW statistics 18. The comparisons between groups were performed using independent tests for continuous variables and the Pearson chi-square test for categorical variables. The paired t test was applied in the follow-up study. Mann-Whitney tests were applied for data of abnormal distribution. For independent samples(K ≥ 3), we used the Kruskal-Wallis H test and adjust p value for comparison between two groups with Bonferroni method. A correlation analysis for the variables of interest was performed using Pearson correlation. Continuous variables were presented as mean ± SD unless otherwise stated. Two-sided values of p < 0.05 were considered statistically significant.