Study cohorts
We enrolled 59 patients with SLE and 25 patients with RA admitted to the Department of Rheumatology and Immunology of The Third Affiliated Hospital, Southern Medical University, China, from July 2019 to December 2019. Thirty age- and sex-matched HC individuals with no history of SLE or other immune disorders were enrolled at the Health Management Center in the same hospital. All the subjects had no infections. The diagnosis of SLE was according to the 1997-revised American College of Rheumatology (ACR) classification criteria[17]. All participants were provided written informed consent for blood draw and MLKL mRNA testing. Serum samples were obtained from all participants during the study.
Analyzing subgroups of SLE is increasingly important to better understand the pathogenesis of disease and provide more tailored medic protocols. Then we sorted SLE patients into different groups based on serological features, renal involvement and disease activity. Firstly, SLE patients were divided into two groups: positive ANAs group (n=48) and negative ANAs group (n=11). Another variable was renal involvement, defined as fulfilling the ACR classification criteria for renal manifestation of SLE (≥0.5 grams of proteinuria per day or 3+ protein on urine dipstick analysis) or having evidence of LN on kidney biopsy. SLE patients were divided into two groups: LN patients (n=23) and non-LN (n=36) patients. Lastly, SLE patients were evaluated using the SLE Disease Activity Index (SLEDAI) [18] and divided into 2 groups: stable patients (SLEDAI score<5, n=32) and active patients (SLEDAI score≥5, n=27), according to the physicians’ evaluation.
Isolation of PBMCs and RNA extraction
Considering that autoreactive PBMCs, mainly lymphocytes, may participate the autoimmune inflammatory process, we chose PBMCs as a source for determining MLKL mRNA level in SLE patients. The venous blood samples (4–5 ml) was collected in an EDTA-K2 tube from all the participants before breakfast, and PBMCs separated within 2 h by Ficoll (TBD science, Tianjin, China) gradient centrifugation for 30 min at 1700r/min. PBMCs were then transferred into 1 ml TRIzol Reagent in 1.5 ml centrifuge tubes and stored at –80°C until RNA extraction.
Total RNA was extracted from PBMCs by using TRIzol Reagent (Invitrogen, CA, USA) according to the manufacturer’s protocol and quantified with the NanoDrop ND-1000 (Thermo Scientifc, USA). Approximately 200-800 ng of RNA obtained from 1 ml of venous. Samples were used only if the ratio of the absorbance at 260 nm to that at 280 nm (A 260 /A 280 ) was between 1.8 and 2.1. RNA samples with concentrations >0.2μg/μl were used for following reverse transcription reaction.
Real-time polymerase chain reaction (RT-PCR) validations
According to the manufacturer’s recommendations, 20 μl of final reaction mixture was used containing 10 μl of SYBR Green PCR Master Mix (Takara, Dalian, China), 0.8 μl of sense primer, 0.8 μl of antisense primer, 0.4 µl ROX Reference Dye (50×), 6µl of sterile deionizedwater and 2.0 µl of the synthesized cDNA. Primers were designed by Primer Primer 5.0 and synthesized by Sangon Biotech (Sangon, Shanghai, China). Primers, targeting MLKL and human 18S-rRNA were used. MLKL, forward: 5’-GCCACTGGAAAGATCCCGTT-3’, reverse: 5’-CAACAACTC
GGGGCAATCCT-3’; human18S-rRNA, forward: 5’-TGGAAATCCCATCACCATC
TTCC-3’, reverse: 5-GGTTCACACCCATGACG-3’. The relative expression level of MLKL was normalized to the internal control 18s-rRNA expression and calculated by the comparative CT (△△CT) method. Amplification was performed in 40 cycles (30 s at 95°C, 5 s at 95°C, 34 s at 60°C) by ABI Step One Plus Real-Time PCR system (Applied Biosystem, CA, USA). A melt curve analysis was used to confirm the specificity of amplification.
Serological assays
The serum total ANA was measured by an indirect immunofluorescence assay (Euroimmun, AG) with a titre of >1:80 scored as positive. The antibodies to 15 antigens including double-stranded DNA (ds-DNA), smith antigen (Sm), and nucleosome (Nuc), SSA/60, SSA/52, SSB/La, ribonucleoprotein (rRNP), centromereprotein B(CENPB), ribosome P protein (Rib-p), histone (His), proliferating cell nuclear antigen (PCNA), Scl-70, Jo-1 and mitochondria (M2) were detected by chemiluminescent immunoassay (CLIA) (HOB, Suzhou, China). Serum complement1q (C1q), complement3 (C3), complement4 (C4), immunoglobulin G (IgG), immunoglobulin M (IgM), immunoglobulin A (IgA) were detected by immunoturbidimetric assay (Roche, Shanghai, China), D-Dimer concentration was determined with immunoturbidimetric assay (Sysmex, Guangzhou, Japan) according to the manufacturer’s instructions.
Statistical analysis
All data were statistically analyzed using Graph Pad Prism5 (version 5.0) software. Quantitative data were expressed as the mean±SD. Data with a Gaussian distribution was analyzed using an unpaired t test or one-way analysis of variance (ANOVA), Spearman’s rank was used to analyze the correlation of the numbers of leucocyte , lymphocyte, monocyte, with the numbers of positive ANA, CRP, ESR and D-Dimer (D-D) levels. The area under the curve (AUC) was used to assess the specificity and sensitivity of using MLKL mRNA as a novel diagnostic tool for the detection of SLE. P values less than 0.05 were considered statistically significant.