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 . All participants 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 ANA group (n = 48) and negative ANA group (n = 11). Another variable was renal involvement, defined as fulfilling the ACR classification criteria for renal manifestation of SLE (≥ 0.5 g 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)  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 in 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) were 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 1700 r/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 Scientific, USA). Approximately 200-800 ng of RNA was obtainted from 1mL of venous blood samples. 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 > 0.2 μg/μL were used for following reverse transcription reaction.
Real-time polymerase chain reaction 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×), 6 μL of sterile deionized water, and 2.0 μL of the synthesized cDNA. Primers were designed by Primer Premier 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′-CAACAACTCGGGGCAATCCT-3′; human 18S-rRNA, forward: 5′-TGGAAATCCCATCACCATCTTCC-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 Biosystems, CA, USA). A melt curve analysis was used to confirm the specificity of amplification.
The serum total ANA was measured by an indirect immunofluorescence assay (Euroimmun, AG) with a titer of > 1:80 scored as positive. The antibodies to 15 antigens including double-stranded DNA (dsDNA), 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 complement 1q (C1q), complement 3 (C3), complement 4 (C4), immunoglobulin G (IgG), immunoglobulin M (IgM), and immunoglobulin A (IgA) were detected by immunoturbidimetric assay (Roche, Shanghai, China), and D-dimer concentration was determined with immunoturbidimetric assay (Sysmex, Japan) according to the manufacturer’s instructions.
All data were statistically analyzed using GraphPad Prism 5 (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), and Spearman’s rank was used to analyze the correlation of the numbers of leukocyte, lymphocyte, and 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.