Increased MLKL mRNA level in the PBMCs is correlated with autoantibody production, renal involvement, and SLE disease activity

Background Necroptosis is a form of regulated necrosis that is involved in various autoimmune diseases. Mixed lineage kinase domain-like pseudokinase (MLKL) has been identified as a key executor of necroptosis; however, the significance of MLKL in peripheral blood mononuclear cells (PBMCs) of systemic lupus erythematosus (SLE) has not been investigated. In this study, we aimed to determine the mRNA level of MLKL in PBMCs and examine its relationship with clinical features and serological parameters in SLE. Methods Real-time transcription-polymerase chain reaction (RT-PCR) analysis was used to determine the expression of MLKL mRNA in PBMCs from 59 patients with SLE, 25 patients with rheumatoid arthritis (RA), and 30 age- and sex-matched healthy controls (HC). Spearman’s correlation test was performed to assess the correlation of MLKL mRNA with clinical variables. The receiver operating characteristic (ROC) curve was created to evaluate the diagnostic value. Results Our results showed MLKL mRNA in PBMCs was upregulated in SLE patients compared to that in RA and HC individuals. SLE patients positive for antinuclear antibodies had significantly higher MLKL mRNA than antibody-negative patients. In SLE patients, MLKL mRNA was found to be upregulated in patients with lupus nephritis (LN) as compared with patients without LN, and also higher in active patients than in stable patients. MLKL mRNA level was significantly and positively correlated with c-reaction protein (CRP) (r = 0.3577, p = 0.0237), erythrocyte sedimentation rate (ESR) (r = 0.4091, p = 0.0043), serum immunoglobulin G (IgG) concentration (r = 0.3546, p = 0.0289), and the numbers of positive antinuclear antibodies (ANAs) (r = 0.3945, p = 0.0432). ROC analysis showed that MLKL mRNA in PBMCs had an area under the curve of 0.9277 (95% CI 0.8779–0.9775, p < 0.001) to discriminate SLE from controls. Conclusions These results suggest that increased MLKL mRNA level in the PBMCs of SLE patients is correlated with renal involvement and disease activity, identifying a subgroup of patients with SLE or LN who may benefit from early diagnosis and therapies targeting MLKL.


Background
SLE is a complex, heterogeneous systemic autoimmune disease that attacks various cells and tissues, resulting in chronic inflammation and persistent tissue damage [1]. A notable characteristic of SLE is the production of pathogenic autoantibodies recognizing nucleic acids or proteins binding to nucleic acids [2]. Dysregulated cell death processes and defective clearance of dying cells have been proposed to contribute to autoantigen generation and induction of autoantibodies, as well as other aberrant immune responses in SLE [3].
The phosphorylated MLKL inserts itself into the cell membrane, disrupts its integrity, and leads to cell death [9]. Various studies have revealed that necroptosis could be implicated in the pathogenesis of many inflammatory and autoimmune diseases, including SLE [6,10,11].
The diversity of the SLE might reflect differences in pathogenesis between different subgroups [12]. Approaches are needed to better understand the pathogenesis and to find new targets for various stages of the disease. Considering the role of necroptosis in the pathogenesis and development of SLE [13][14][15][16], we aimed to analyze MLKL mRNA of PBMCs and figure out whether it could serve as a biomarker for disease diagnosis and monitoring.

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 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) [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 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 Nano-Drop 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.

Statistical analysis
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.

MLKL mRNA was upregulated in the PBMCs of SLE patients
We detected MLKL mRNA levels in the PBMCs of SLE patients, RA patients, and HC individuals. The levels of MLKL mRNA were significantly higher in SLE patients than in RA patients and HC individuals (p < 0.0001, respectively, Fig. 1).
We further analyzed the expression of MLKL mRNA in the subgroups of SLE patients and found that SLE patients positive for ANAs exhibited significantly higher levels of MLKL mRNA than those with negative ANAs (p < 0.05, Fig. 2a). MLKL mRNA was also found to be obviously upregulated in LN patients when compared with patients without LN (p < 0.005, Fig. 2b), and higher in active patients than in stable patients (p < 0.05, Fig. 2c).

MLKL mRNA was positively correlated with clinical and pathological parameters of SLE
The relationships between the levels of MLKL mRNA in PBMCs and the clinical or pathological characteristics of SLE are assessed and detailed in Table 2 (Fig. 3), but not associated with C3, C4, and other serologic indicators. Taken together, we found that increased MLKL mRNA was correlated with the activity in SLE patients.

MLKL mRNA in the PBMCs was sensitive for the diagnosis of SLE
ROC analysis was employed to analyze the diagnostic efficiency of the MLKL mRNA for SLE patients. The diagnostic ability of MLKL mRNA achieved very high diagnostic accuracy 0.9277 (95% CI 0.878-0.978) with high sensitivity (81.36%) and specificity (93.3%), implying that MLKL mRNA of PBMCs may be a potential diagnosis biomarker for SLE (Fig. 4).

Discussion
In this study, we showed that MLKL mRNA level in the PBMCs of SLE patients was significantly upregulated, especially in patients with positive serum ANAs. MLKL mRNA level in the PBMCs was also significantly and positively correlated with ESR (r = 0.4091, p = 0.0043), CRP (r = 0.3571, p = 0.0237), serum IgG concentration (r = 0.3546, p = 0.0289), and the numbers of positive ANAs (r = 0.3597, p = 0.0432). So far as we know, this is the first report that MLKL mRNA level in the PBMCs is increased in SLE patients.
Once phosphorylated, MLKL translocates from the cytosol to the plasma membrane to execute necroptosis. Defective clearance of necroptotic cells has been proposed to initiate inflammatory responses by the release of danger-associated molecular patterns (DAMPs). DNA acts as a major DAMP and is sensed in endolysosomes by toll-like receptor 9 (TLR9) and in the cytoplasm by cyclic GMP-AMP (cGAMP) synthase (cGAS), inducing the production of type I and type III IFNs and eliciting strong inflammatory responses [19,20]. Several studies have demonstrated that patients with SLE have elevated circulating IFNs [21][22][23], whose signaling contributes to the steady-state expression of MLKL and the initiation of necroptosis, which not only causes tissue damage [6], but may also form a dynamic feedback loop in SLE pathogenesis.
Although SLE is a chronic inflammatory disease that can affect many organs, the kidneys are the mostly attacked [24]. LN is one of the most frequent and serious complications in SLE, and a real challenge for SLE treatment [25]. We surprisingly found that MLKL mRNA was obviously upregulated in the PBMCs of LN patients when compared with patients without LN (p < 0.005). To date, only one paper reported the correlation of necroptosis with LN, showing that PIPK3 and MLKL were activated in podocytes in renal biopsies from patients with LN [11]. Whether there is a crosstalk between the renal parenchymal cells and peripheral blood cells in necroptosis process still needs to be analyzed.
The current understanding of SLE implies autoimmunity to nuclear and cytoplasmic antigens, leading to generation large amount of ANAs [26]. Our findings are that SLE patients positive for ANAs exhibited higher MLKL mRNA levels than serum negative patients and that there are very significantly positive correlations between MLKL mRNA in the PBMCs and the numbers of positive ANAs or serum IgG concentrations, suggesting that necroptosis may play a potential role in the production of ANAs.
Conventional serologic ANAs are of limited sensitivity and/or specificity for diagnosis and monitoring in SLE [27]. Here, we reported that MLKL mRNA in the There were also several limitations. Firstly, as the patients in this study are from one hospital, whether there is a difference between patients from different areas is not known. Therefore, a multi-center cohort might be necessary for future implementation of techniques.
Secondly, the molecular mechanism that how MLKL is involved in the progression of SLE remains unclear. Lastly, which specific cell of PBMCs expressed high MLKL mRNA level needs to be explored in the future.

Conclusions
This is the first study to point out the upregulation of the MLKL mRNA in the PBMCs of SLE patients. The data presented here may provide certain evidence for the role of necroptosis in the pathogenesis and development of SLE, and also suggest new therapies by blocking signaling of necroptosis pathway in human SLE, especially . c Active patients (SLEDAI score ≥ 5, n = 27) vs stable patients (SLEDAI score < 5, n = 32). *p < 0.05; **p < 0.005  in LN patients. Importantly, the MLKL mRNA expression levels in PBMCs may be useful in identifying those subgroups of SLE patients that may benefit from necroptotic blocking therapies. Finally, we believe that these findings could be of relevance for understanding the pathogenesis and diversity of SLE.