Recruitment of subjects

The participants included 12 SLE patients, aged between 20 and 50 years, who were recruited as the subjects of this study. All the patients had previously been diagnosed with SLE according to the criteria of the European League Against Rheumatism (EULAR)/American College of Rheumatology (ACR). The inclusion criteria were as follows: aged between 20 and 50 years and diagnosed with SLE according to EULAR/ACR criteria within 1 month prior to enrolment in the study. Patients who received immunosuppressive drugs, anti-inflammatory drugs, or had other diseases related to SLE, such as systemic inflammatory diseases or autoimmune diseases (e.g., rheumatoid arthritis, Sjogren's syndrome), were excluded from the study. Healthy age- and sex-matched individuals who had no illnesses and no cognitive or chronic diseases, namely systemic inflammatory diseases or autoimmune diseases, were recruited as controls. The sample size of the patients and controls in this study was calculated based on the SLE disease prevalence in Thailand.

Collection of blood and isolation of serum samples

Blood samples were collected from the median cubital vein of individual participants using standard venipuncture with aseptic technique. The collection of blood samples from each SLE patient (N=12) was performed using vacutainer tubes containing clot activator (Becton Dickinson; Franklin Lakes, NJ). The collection of blood samples from each healthy control subject (N=12) was conducted using both vacutainer tubes containing clotting factor and heparin anticoagulant solution (Becton Dickinson; Broken bow, NE) for further isolation of serum and peripheral blood mononuclear cells (PBMCs), respectively.

To isolate the serum samples, clotted blood derived from SLE patients and healthy control subjects was centrifuged at 1,800 g for 5 minutes. Then, individual serum samples were carefully collected, aliquoted, and stored at ‑80 °C until use.

Isolation of PBMCs by Ficoll-Hypaque density gradient centrifugation

PBMCs were isolated from the peripheral blood of healthy control subjects using Ficoll-Hypaque density gradient centrifugation. Briefly, anticoagulated blood was diluted with an equal volume of phosphate-buffered saline (PBS) (pH 7.4). The diluted blood was then continuously layered over Lyphoprep solution with a density of 1.077 g/mL (Stemcell Technologies; Vancouver, Canada). This solution was promptly centrifuged at 400 g at 25°C for 30 minutes. Subsequently, the PBMC interface between the Ficoll-Paque solution and plasma was carefully collected and washed once with PBS via centrifugation at 300 g at 25°C for 5 minutes. The PBMC pellet was then resuspended in a red blood cell (RBC)-lysing buffer (Sigma; St. Louis, MO) and incubated at 25°C for 10 minutes with gentle mixing to lyse any contaminating RBCs. Thereafter, the decontaminated PBMCs were washed with PBS twice via centrifugation at 300 g at 25°C for 5 minutes, and measurement of cell viability and PBMC number was performed via hemocytometer-based trypan blue staining assay.

Induction of cytotoxicity by HO

The isolated PBMCs were plated at a density of 1×10 cells/ml in RPMI medium supplemented with 100 U/ml penicillin G and 100 mg/ml streptomycin (Sigma; St. Louis, MO), and maintained in a humidified incubator at 37°C with 5% CO for 24 hours. The cells were then incubated with 0.2, 1, and 5 mM of HO (Alfa aesar; Lancashire, United Kingdom) in a humidified incubator at 37°C with 5% CO for 3, 10, 24, or 48 hours. The capacity of HO at various concentrations to induce PBMC cytotoxicity at each time point was measured via trypan blue staining assay Untreated cells served as the control.

Treatment of the cells with serum samples

After investigating the cytotoxic activity of HO for the selection of optimal concentration and incubation time, the PBMCs were treated with serum samples. Briefly, cells from healthy individuals that were induced or not induced with 0.2 mM of HOwere incubated with serum samples from SLE patients and healthy controls in a humidified incubator at 37°C with 5% CO for 24 hours. Next, the effect of the SLE patients’ sera on promoting cell death and apoptosis was determined and compared with the healthy control subjects via trypan blue and annexin-V/PI-staining assay, respectively.

Cell death and total cell number measurement by trypan blue staining assay

Trypan blue staining assay based on a hemacytometer was applied to examine the degree of cell death and total cell numbers. The isolated PBMCs (1×10 cells/ml) were cultured in RPMI medium containing the sera of SLE patients and healthy controls with or without HO induction in a 96-well plate. The cells were then collected and washed twice with PBS (pH 7.4). After resuspension in PBS, the suspension was mixed with 0.4% trypan blue solution (Gibco; Grand Island, NY) and the cells were counted using a hemacytometer counting chamber; cells that were stained by trypan blue represented dead cells. The percentage of cell death and total cell number were calculated using the following formulae:

× x

Cell apoptosis measurement by annexin-V/PI staining assay

To determine the degrees of both early- and late-phase apoptosis, annexin-V/PI staining assay followed by fluorescence was performed. The isolated PBMCs (1×10 cells/ml) were cultured in RPMI medium that contained sera from SLE patients and healthy controls with or without HO induction in a 24-well plate. The cells were collected and washed twice with PBS (pH 7.4). The cell pellet was resuspended with annexin-V-FLUOS labeling solution (annexin-V-fluorescein and PI) (Roche Diagnostics; Rotkreuz, Switzerland) and incubated at 25°C for 15 minutes. The stained cells were later observed under an upright fluorescence microscope (Optika; Ponteranica, Italy) with a digital camera. The fluorescence intensity of fluorescein and PI of 100 individual cells in each condition were measured using Proview software version x64 (Optika; Ponteranica, Italy).

Data retrieval of serum and plasma proteins

The proteomics data of serum or plasma proteins from SLE patients were collected from all published articles cited on PubMed, MEDLINE, and ScienceDirect databases between 2005 and 2020 using the search terms “serum/plasma protein of SLE” or “serum/plasma proteomic data of SLE.” All the serum or plasma proteins in SLE patients were reported at abnormal levels (higher or lower) compared with healthy individuals in the collected proteomics data, which reported based on name, gene symbol, and molecular weight.

Biological prediction by bioinformatics tools

All the altered serum or plasma proteins in SLE patients collected from the published articles predicted the biological functions involved in the process of cell apoptosis using the global network analysis relative to the gene ontology (GO) terms. The gene symbols of all collected proteins were submitted with medium stringency and confidence level (score ≥ 0.400) to Search Tool for the Retrieval of Interacting Gene/Proteins (STRING) version 11.0 (https://string-db.org).

Interleukin 6 level measurement by enzyme-linked immunosorbent assay

The levels of IL-6 in the sera of SLE patients and healthy controls were measured using a human IL-6 duoSet ELISA kit (R & D systems; Minneapolis, MN) following the manufacturer’s instructions. Briefly, 100 µl of the working concentration of capture antibody was added to a 96-well MaxiSorp™ flat-bottom plate (Thermo Fisher Scientific; Waltham, MA) and incubated overnight at 25°C. The plate was washed twice with washing buffer (0.05% Tween-PBS) and blocked with 1% BSA-PBS at 25°C for 1 hour. The plate was then washed twice with washing buffer. Subsequently, 100 µl of various standard concentrations of IL-6 and serum samples from SLE patients (N=12) and healthy controls (N=12) were added to each well of the microplate. The plate was incubated at 25°C for 2 hours and washed twice with washing buffer. Thereafter, 100 µl of the working concentration of the detection antibody was added to the plate, incubated at 25°C for 2 hours, and washed twice with washing buffer. To each well of the plate, we subsequently added 100 µl of the working dilution of streptavidin-HRP, incubated at 25°C in the dark for 20 minutes, and washed twice with washing buffer. Thereafter, 100 µl of substrate solution (TMB) was added to each well and incubated at 25°C in the dark for 10 minutes. Finally, 50 µl of stop solution was added to each well and the absorbance was examined at a wavelength of 450 nm using a microplate reader.

Statistical analysis

Quantitative data were reported as mean ± standard deviation (SD). Comparison between the two examined groups was investigated by unpaired Student’s test, whereas multiple comparisons were performed using one-way analysis of variance (ANOVA) with Tukey’s post-hoc test using SPSS statistical software, version 25 (IBM; North Castle, NY). A pvalue less than 0.05 was considered statistically significant.

Demographic data of the patients

The study participants consisted of 12 SLE patients and healthy controls. All the SLE patients had been previously diagnosed according to the EULAR/ACR criteria for SLE. In addition, 12 non-pregnant Thai women with no illness, history of illness, or clinical manifestations of cognitive or chronic diseases, especially systemic inflammatory diseases or autoimmune diseases, were selected as matching comparative controls. The demographic information and disease status of the healthy controls and SLE patients at the time of the study are summarized in Table 1. The data demonstrated that there were no significant differences in terms of sex and age between the healthy control group (all female, 33 – 41 years old) and the SLE test group (all female, 26 – 53 years old). In all the SLE patients, the mean (range) duration of disease was approximately 12 years (4 – 20 years). Among the 12 patients, 3 (25%) had clinical manifestation with rash, 7 (58.33%) had arthritis, 5 (41.67%) had nephritis, and 2 (16.67%) had thrombocytopenia. Moreover, all the patients (100%) had laboratory results with critical antinuclear antibody (ANA) titer of more than 80, and 10 patients (83.33%) had received drug treatments, including both steroids (, prednisolone) and nonsteroidal agents (, azathioprine and hydroxychloroquine).

Validation of HO induction

The optimal concentration and time point of HO to induce PBMC cell death was initially defined for further evaluation of its effect on SLE patients’ sera. The data showed that the degree of cell death was increased by approximately 50% in PMBCs treated with all concentrations (0.2, 1, and 5 mM) of HO for 24 and 48 hours, and cell death was also markedly increased in a dose-dependent manner compared with those without the treatment that served as controls in this study (p < 0.05) (Figure 1A and B). In addition, all concentrations of HO markedly decreased the number of cells after 24-hour treatment when compared with the control (p < 0.05) (Figure 1A and C). Interestingly, the minimum concentration of HO used in our study (0.2 mM) was able to increase cell death and decrease the total number of PBMCs following 24 hours of treatment, which was suggested as the long induction period (Figure 1). Therefore, 0.2 mM of HO was used as the optimal concentration needed to efficiently induce PBMC cell death in the long treatment period (24 hours) for all subsequent experiments.

Effect of SLE patients’ sera on cell death

Using the optimal concentration and induction time point of HO, the effects of SLE patients’ and healthy individuals’ sera on PBMC death were investigated. Following 24 hours of treatment, cell death was significantly enhanced in PBMCs treated with serum samples from SLE patients both with and without 0.2-mM HO induction compared with those of healthy individuals and without HO induction (control) (p < 0.05). Furthermore, the degree of cell death in PBMCs treated with SLE patient sera and HO was markedly higher than in PBMCs with only HO induction (p < 0.05) (Figure 2A and B). Meanwhile, total cell numbers of PBMCs treated with serum samples from SLE patients and healthy controls both with and without HO were not significantly different compared to PBMCs treated with only HO induction and the control (Figure 2A and C).

Effect of SLE patients’ sera on cellular apoptosis

To further determine whether SLE patients’ sera could induce cell death via an apoptotic process, the effect of serum samples from SLE patients compared with healthy controls in each phase and total PBMC apoptosis was investigated using the optimal concentration and induction time point of HO. Following 24 hours of treatment, the degree of total, early-, and late-phase cell apoptosis in PBMCs treated with SLE patients’ sera had significantly increased compared with that of healthy individuals and without HO induction (control) (p < 0.05) (Figure 3). PBMCs treated with SLE patients’ sera with 0.2 mM HO induction were observed to have elevated degrees of total, early-, and late-phase apoptosis compared with those of healthy individuals and HO apoptosis inducer (p < 0.05) (Figure 3).

Systematic analysis of serum and plasma proteins related to cell apoptosis

All the serum and plasma proteins that were altered in SLE patients were retrieved from the previous proteomics studies using literature cited in PubMed, MEDLINE, and ScienceDirect databases from 2005 to 2021. The data showed that 45 proteins collected in the serum or plasma of SLE patients were present in abnormal levels compared with healthy individuals; the levels of 32 proteins were increased and 13 proteins were reduced, as detailed in Table 2.

For biological analysis of apoptotic processes, all the collected sera and proteins were submitted to the STRING tool and the functional categories of these proteins were subsequently predicted by global network analysis relative to GO terms. The integrative proteome network created by STRING software reported that 14 altered proteins had biological roles involved in the apoptotic process (red node) or regulation of apoptosis (blue node), which consisted of high mobility group box chromosomal protein 1 (), RANTES (regulated upon activation, normal T cell expressed and secreted) (), growth arrest-specific 6 (), tyrosine protein-kinase receptor UFO (), tumor necrosis factor receptor 2 (), interleukin 6 (), collectin 11 (), aryl hydrocarbon receptor (), tumor necrosis factor α-induced protein 8-like 2 (), heat-shock protein beta 1 (heat shock protein 27, ), clusterin (), fibrinogen beta chain (), Fms-related receptor tyrosine kinase 3 ligand (), and cytotoxic T lymphocyte-associated protein 4 () (Figure 4).

Confirmation of increased IL-6 levels in the sera of SLE patients

The level of IL-6 in the sera of SLE patients was determined and compared to healthy controls via ELISA assay with capture and detection antibodies specific to human IL-6. The results revealed increased levels of IL-6 (142.54 ± 25.41 pg/ml) in the sera of SLE patients compared with healthy controls (21.87 ± 1.61 pg/ml) (p < 0.05) (Figure 5).