A total of 68 patients who fulfilled the 1997 revised American College of Rheumatology classification criteria for the SLE [19] were recruited from the First Affiliated Hospital of Nanchang University from 2020.1 to 2024.6. Disease activity was assessed by the SLEDAI-2000 index [20]. Individuals with other autoimmune or rheumatic diseases, kidney diseases other than lupus nephritis, pregnancy, HIV, or malignancy were excluded from this study. 29 rheumatoid arthritis (RA) patients, and 26 Sjogren’s syndrome (SS) patients were recruited as disease controls, and 30 age- and sex-matched healthy subjects who were free of autoimmune diseases were recruited as healthy controls (HCs). Characteristics of SLE patients and controls enrolled in this study are included in Table 1. Peripheral neutrophil counts of each subject were determined. The study had approval from the Ethics Committee of the First Affiliated Hospital of Nanchang University (2014003) and complied with the Helsinki Declaration.

Whole blood was collected in EDTA vacutainer tubes. According to the manufacturer’s instructions, neutrophils were isolated from whole blood by Ficoll density gradient centrifugation. The purity of the isolated neutrophil was confirmed to be greater than 95% by flow cytometry. After isolation, neutrophils were cultured in RPMI 1640 medium (Solarbio, Beijing, China), supplemented with 10% fetal bovine serum (FBS, Gibco, Australia), at 37℃ in a humidified atmosphere of 5% CO2 and 95% air. When indicated, neutrophils were incubated with 10% fresh human serum from SLE patients or HCs, 10 µM of KLF2 inducer GGTI-298 (MedChemExpress, NJ, USA, HY-15871) or KLF2 inhibitor GGPP (Sigma Aldrich, St. Louis, MO, USA, G6025) for 4 h.

The neutrophils were incubated in a solution containing Annexin V-FITC and PI (Bestbio, Nanjing, China, BB-4101-50T) according to the manufacturer’s instructions. The apoptosis of cells was analyzed by flow cytometry using a Beckman Coulter Cytomics FC 500 Flow Cytometer. The data was analyzed using the Flow Jo software.

Cells were washed twice with cold PBS, lysed with lysis buffer (Solarbio, China, BC3710), and centrifuged at 4 ℃ 12,000 g for 30 min. The supernatant was pipetted into a new E.P. tube, and the protein concentration was measured using the BCA Protein Concentration Assay Kit (Sosarbio, Beijing, China, Solarbio, China, PC0020) according to the manufacturer’s instructions. An equal amount of protein (20 µg) for each sample and protein marker were loaded on 12% SDS-PAGE gels, and proteins were separated electrophoretically and then transferred toImmobilon®-PSQMembrane (PVDF MilliporeMassachusetts, USA, 0000278926). The PVDF membrane was then blocked with 5% non-fat skim milk for 2 h at room temperature. After blocking, the membranes were incubated overnight with the primary antibody to KLF2 (1:1000; Abcam, USA, Ab236507) and GAPDH (1:1000; Proteintech, USA, 00110344) diluted in 5% skim milk (dissolved in Tris-buffered saline with 0.1%Tween 20 detergent, TBST, Sosarbio Beijing China) at 4 °C. After three times washing with TBST, membranes were incubated in species-specific horseradish peroxidase (HRP)-coupled secondary antibody for 1 h at room temperature. Membranes were rewashed and exposed to SuperKineTM-enhanced ECL Luminous Liquid (Abbkine, Wuhan, China, ATWL22081). Protein bands were visualized using Image J software and band intensities were normalized to the internal control GAPDH.

The total RNA of neutrophils was isolated using Trizol (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. The sequences of the primers used in this study were listed in Table 2. The integrity and concentration of the RNA was assessed by agarose gel electrophoresis and a NanoDrop ND-1000 spectrophotometer (Invitrogen Bio, Waltham, MA, United States), respectively. RT-PCR were carried out with the PrimeScript™ RT kit (Takara Bio Inc., Kyoto, Japan) and SYBR Premix Ex Taq™ II (Takara Bio Inc., Kyoto, Japan) in an ABI 7500 Real-Time PCR System (Invitrogen, California, USA) with the following PCR thermocycler protocol: initial denaturation step at 95℃for 5 min, followed by 40 cycles of 95℃ for 15 s (denaturation), 60℃ for 1 min (annealing and elongation), and 72℃ for 2 min (final extension). GAPDH was used as an internal control. The data were analyzed using the 2−ΔΔCt method.

Statistical analysis and graphic presentation were carried out with GraphPad Prism 10.0 and SPSS version 20.0. A Student’s t-test was used between two groups where the samples passed the normality test; otherwise, the nonparametric Mann-Whitney test was used to analyze the data. Spearman’s method was used for correlation analysis. P < 0.05 was considered to indicate statistically significant differences.