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.

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.

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^TM 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.

The isolated PBMCs were plated at a density of 1×10⁵ 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% CO₂ for 24 hours. The cells were then incubated with 0.2, 1, and 5 mM of H₂O₂ (Alfa aesar; Lancashire, United Kingdom) in a humidified incubator at 37°C with 5% CO₂ for 3, 10, 24, or 48 hours. The capacity of H₂O₂ at various concentrations to induce PBMC cytotoxicity at each time point was measured via trypan blue staining assay. Untreated cells served as the control.

After investigating the cytotoxic activity of H₂O₂ 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 H₂O₂ were incubated with serum samples from SLE patients and healthy controls in a humidified incubator at 37°C with 5% CO₂ 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.

Trypan blue staining assay based on a hemacytometer was applied to examine the degree of cell death and total cell numbers. The isolated PBMCs (1×10⁵ cells/ml) were cultured in RPMI medium containing the sera of SLE patients and healthy controls with or without H₂O₂ 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:

Cell death (%) = [Number of trypan blue-positive cells × 100] / Total cell number

Total cell number (cells/ml) = [Number of viable cells × dilution factor x 10⁴] / 4

To determine the degrees of both early- and late-phase apoptosis, annexin-V/PI staining assay followed by fluorescence was performed. The isolated PBMCs (1×10⁵ cells/ml) were cultured in RPMI medium that contained sera from SLE patients and healthy controls with or without H₂O₂ 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).

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.

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).

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.

Quantitative data were reported as mean ± standard deviation (SD). Comparison between the two examined groups was investigated by unpaired Student’s t 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 p-value less than 0.05 was considered statistically significant.

Table 1

Clinical parameters	Healthy controls	SLE patients
Demographics
Sex, F/M	12/0	12/0
Age (years), Mean ± SD	36.62 ± 3.48	39.38 ± 13.19
Disease status	-
Duration (years), Mean ± SD	-	11.92 ± 7.24
Rash, n (%)	-	3 (25.00%)
Arthritis, n (%)	-	7 (58.33%)
Nephritis, n (%)	-	5 (41.67%)
Thrombocytopenia, n (%)	-	2 (16.67%)
ANA (> 1:80), n (%)	-	12 (100.00%)
Drug treatment (prednisolone, azathioprine, hydroxychloroquine, etc.), n (%)	-	10 (83.33%)

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 (e.g., prednisolone) and nonsteroidal agents (e.g., azathioprine and hydroxychloroquine).

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Table 2

No.	Protein name	Gene name	Reference	Level in SLE	Biological process (Gene ontology)
					Apoptotic process	Regulation of apoptosis
1	A proliferation-inducing ligand	APRIL	Koyama T, et al. (2005) 22	Increased
2	Tumor necrosis factor-α-induced protein-8 like-2	TNFAIP8L2	Li D, et al. (2009) 23	Decreased
3	High mobility group box chromosomal protein 1	HMGB1	Li J, et al. (2010) 24	Increased
4	Interleukin-33	IL-33	Yang Z, et al. (2011) 25	Increased
5	RANTES (regulated upon activation, normal T cell expressed and secreted)	CCL5	Lu MM, et al. (2012) 26	Increased
6	Serum L-ficolin	FCN2	Watanabe H, et al. (2012) 27	Decreased
7	Interleukin-9	IL-9	Ouyang H, et al. (2013) 28	Increased
8	Interleukin-37	IL- 37	Ye L, et al. (2014) 29	Increased
9	Growth arrest-specific 6	Gas6	Bakr SI, et al. (2015) 30	Increased
10	Heat shock protein beta-1 (Heat shock protein 27)	HSPB1	Rai R, et al. (2015) 31	Decreased
11	Tumor necrosis factor receptor superfamily 13C (BLyS receptor 3)	TNFSF13C	Duan JH, et al. (2016) 32	Decreased
12	Tyrosine-protine kinase receptor UFO	Axl	Mok CC, et al. (2016) 33	Increased
13	Ferritin	FTH1		Increased
14	Insulin like growth factor binding protein 2	IGFBP2		Increased
15	Tumor necrosis factor receptor 2	TNFRSF1B		Increased
16	C-reactive protein	CRP	Umare et al. (2017) 34	Increased
17	Interleukin-6	IL-6		Increased
18	Thrombospondin-4	THBS4	Zhong L, et al. (2017) 35	Increased
19	Properdin	CFP		Increased
20	Collectin-11	COLEC11		Increased
21	Matrix metalloproteinase 7	MMP-7	Vira H, et al. (2017) 36	Increased
22	Leucine-rich α2-glycoprotein	LRG	Ahn SS, et al. (2018) 37	Increased
23	BTB domain and CNC homolog 2	BACH2	Zhu Z, et al. (2018) 38	Decreased
24	Serotransferrin	TF	Madda R, et al. (2018) 39	Increased
25	Clusterin	CLU		Decreased
26	Apolipoprotein A1	APOA1		Decreased
27	Apolipoprotein A2	APOA2		Decreased
28	Alpha-2-macroglobulin	A2M		Increased
29	Transthyretin	TTR		Decreased
30	Alpha-1-acid glycoprotein 1	ORM1		Increased
31	Alpha-1-acid glycoprotein 2	ORM2		Increased
32	Alpha-1-B glycoprotein	A1BG		Increased
33	Alpha-1-antitrypsin	SERPINA1		Increased
34	Alpha-1-antichymotrypsin	SERPINA3		Increased
35	Hemopexin	HPX		Decreased
36	Hemoglobin beta subunit	HBB		Increased
37	Haptoglobin	HP		Increased
38	Ceruloplasmin	CP		Increased
39	Interleukin-34	IL-34		Increased
40	Fibrinogen beta chain	FGB		Decreased
41	Apolipoprotein B	APOB		Increased
42	Aryl hydrocarbon receptor	AhR	Yu H, et al. (2019) 40	Increased
43	Fms related receptor tyrosine kinase 3 ligand	FLT3LG	Yuan X, et al. (2019) 41	Decreased
44	Cytotoxic T-lymphocyte-associated protein 4	CTLA4	Zhao L, et al. (2020) 42	Decreased
45	C-X-C chemokine receptor type 5	CXCR5	Liao J, et al. (2020) 43	Increased

The optimal concentration and time point of H₂O₂ 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 H₂O₂ 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 1 A and B). In addition, all concentrations of H₂O₂ markedly decreased the number of cells after 24-hour treatment when compared with the control (p < 0.05) (Figure 1 A and C). Interestingly, the minimum concentration of H₂O₂ 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 H₂O₂ was used as the optimal concentration needed to efficiently induce PBMC cell death in the long treatment period (24 hours) for all subsequent experiments.

Using the optimal concentration and induction time point of H₂O₂, 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 H₂O₂ induction compared with those of healthy individuals and without H₂O₂ induction (control) (p < 0.05). Furthermore, the degree of cell death in PBMCs treated with SLE patient sera and H₂O₂ was markedly higher than in PBMCs with only H₂O₂ induction (p < 0.05) (Figure 2 A and B). Meanwhile, total cell numbers of PBMCs treated with serum samples from SLE patients and healthy controls both with and without H₂O₂ were not significantly different compared to PBMCs treated with only H₂O₂ induction and the control (Figure 2 A and C).

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 H₂O₂. 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 H₂O₂ induction (control) (p < 0.05) (Figure 3). PBMCs treated with SLE patients’ sera with 0.2 mM H₂O₂ induction were observed to have elevated degrees of total, early-, and late-phase apoptosis compared with those of healthy individuals and H₂O₂ apoptosis inducer (p < 0.05) (Figure 3).

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 (HMGB1), RANTES (regulated upon activation, normal T cell expressed and secreted) (CCL5), growth arrest-specific 6 (Gas6), tyrosine protein-kinase receptor UFO (Axl), tumor necrosis factor receptor 2 (TNFRSF1B), interleukin 6 (IL-6), collectin 11 (COLEC11), aryl hydrocarbon receptor (AhR), tumor necrosis factor α-induced protein 8-like 2 (TNFAIP8L2), heat-shock protein beta 1 (heat shock protein 27, HSPB1), clusterin (CLU), fibrinogen beta chain (FGB), Fms-related receptor tyrosine kinase 3 ligand (FLT3LG), and cytotoxic T lymphocyte-associated protein 4 (CTLA4) (Figure 4).

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).