2.1 Caspase 5/11-mediated pyroptosis is elevated in monocytes/macrophages of SLE patients and MRL/lpr mice

To demonstrate that accelerated pyroptosis occurs in individuals with SLE, we initially examined the cleavage of GSDMD, a definitive marker of pyroptosis, in the peripheral blood mononuclear cells (PBMCs) of SLE patients. We enrolled three healthy individuals and four patients with varying Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) to assess GSDMD cleavage in PBMCs. Notably, increased cleavages of Caspase 5 and GSDMD were observed in the PBMCs of SLE patients compared with healthy controls (Figure 1A). However, a positive correlation between GSDMD-mediated pyroptosis and SLEDAI in SLE patients was not established. Multiplex immunohistochemistry (mIHC) revealed that renal biopsy samples from lupus nephritis (LN) patients showed heightened expression of GSDMD in the glomeruli, using renal peritumoral tissue from renal tumor patients as controls (Figure 1B). The Caspase 4 and Caspase 5 expressions were also significantly increased in LN (Figure S1). At the protein level, there was an increase in the cleavages of Caspase 11 and GSDMD in the kidneys of lupus mice compared with healthy control (C57BL/6 mice) (Figure 1C). Similarly, elevated expression and colocalization of GSDMD and Caspase 11 were detected in kidneys from MRL/lpr mice (Figure 1D,E). Furthermore, we aimed to identify which cell types predominantly undergo pyroptosis in SLE. Contrary to previous reports,¹⁶ our findings from immunofluorescence colocalization indicated that in the glomeruli of MRL/lpr mice, macrophages marked by F4/80, rather than podocytes marked by synaptopodin (SYNPO), were undergoing pyroptosis (Figure 1F). The CD14⁺ cells in PBMCs are circulating monocytes, which are the precursors of macrophages in tissues. Transmission electron microscopy (TEM) demonstrated multiple pores in the membranes of isolated CD14⁺ monocytes from the PBMCs of SLE patients, consistent with the morphological characteristics of pyroptosis^{17, 18} (Figure 1G). These results collectively suggest that Caspase 5/11 and GSDMD-mediated pyroptosis is significantly elevated in monocytes/macrophages of lupus mice and SLE patients.

2.2 Pyroptosis of J774A.1 and RAW264.7 cells is induced by incubation with serum from MRL/lpr mice

We hypothesized that the serum from lupus mice could induce pyroptosis in macrophages. To test this hypothesis, we analyzed morphological changes, cytotoxic reactions, and protein expression in J774A.1 cells cultured with serum [at a concentration of 20% (v/v)] from MRL/lpr mice. Morphological observations using light microscopy revealed that J774A.1 cells exhibited a pyroptosis-like phenotype, characterized by swelling, rounding, and the release of cell contents, which were not observed in cells cultured with serum from C57BL/6 mice (Figure 2A). The induction of pyroptosis was further supported by a lactate dehydrogenase (LDH) release assay, a widely accepted method for quantitatively assessing cell membrane integrity and viability. Incubation with lupus serum significantly increased LDH release from J774A.1 cells, indicating a stronger capacity to induce cell lysis compared with serum from healthy mice (Figure 2B). The dynamic processes were documented using a time-lapse microscopy assay, with videos provided in the Supporting Information. After 4 h of incubation with 20% lupus serum, J774A.1 cells began to exhibit membrane blebbing followed by cell lysis, reaching a cell death rate of 50% by the 7th hour, which was not observed in the C57BL/6 serum and PBS groups (Figure 2C). Notably, elevated cleavages of Caspase 11 and GSDMD, markers of noncanonical pyroptosis, were detected in both J774A.1 and RAW264.7 cells after incubation with 20% lupus serum (Figure 2D,E). Additionally, the levels of supernatant IL-1α and IL-1β, specific products of Caspase 11 activation,¹⁹ were also increased following incubation with lupus serum (Figure 2F). Moreover, the cleavage of GSDMD and Caspase 11 induced by lupus serum was attenuated by the Caspase 11 inhibitor wedelolactone (WDL), a natural compound that suppresses Caspase 11 expression by inhibiting NF-κB-mediated transcription,²⁰ indicating that the pyroptosis-inducing effect of lupus serum on macrophages was Caspase 11-dependent. These findings further confirm the occurrence of Caspase 11-dependent pyroptosis in the context of lupus and the presence of pyroptosis-inducing substances in the serum of lupus mice.

2.3 Increased serum LPS caused by damaged gut barrier induces pyroptosis of macrophage via TLR 4 and Caspase 11 in MRL/lpr mice

We further explored the agents inducing pyroptosis in lupus mice. Caspase 4, 5, and their murine counterpart Caspase 11, serve as specific receptors for cytoplasmic LPS. Previous study demonstrated that LPS directly interacts with Caspase 4/5/11 under the mediation of the Caspase-activation and recruitment domain of caspases, leading to their oligomerization and activation.²¹ They also found that the induction of pyroptosis by cytoplasmic LPS was prevented in Caspase 11 knockout macrophages and restored upon stable re-expression of Caspase 11.²¹ Additionally, Deng et al.²² found that High mobility group box-1 protein (HMGB1) was essential for LPS internalization and Caspase11-dependent pyroptosis. HMGB1 facilitated the internalization of LPS via the receptor for advanced glycation end-products (RAGE) in macrophages. We detected an elevated LPS and HMGB1 level in the serum of SLE patients (Figure 3A) and MRL/lpr mice (Figure 3B). We hypothesized that the increased serum LPS originated from the intestines due to compromised gut barrier integrity in lupus mice. To test intestinal permeability, fluorescein isothiocyanate (FITC)-conjugated dextran was administered into mice by gavage, and serum FITC intensity was measured.²³ The FITC–dextran penetration test indicated that MRL/lpr mice exhibited increased gut permeability compared with C57BL/6 mice (Figure 3C). The mean fluorescence intensity of serum dextran, indicative of gut leakage, positively correlated with serum LPS levels (Figure 3D), supporting our hypothesis that the serum LPS was released from the gut. Additionally, in MRL/lpr mice, the levels of Zonula Occludens protein 1 (ZO-1), an important intestinal tight junction protein, was found to be lower than those in age-matched C57BL/6 mice (Figure 3E). This decrease in ZO-1 expression aligns with the observed loss of intestinal barrier integrity in the MRL/lpr mice.

To verify whether LPS and HMGB1 could induce Caspase 11-dependent pyroptosis in macrophages. we treated RAW264.7 cells with LPS and HMGB1, both individually and in combination, and assessed the protein levels of key markers of noncanonical pyroptosis. The results indicated that HMGB1 alone did not alter the cleavage levels of Caspase 11 and GSDMD in macrophages (Figure 3F). Treatments with both LPS alone and LPS combined with HMGB1 resulted in elevated cleavages of Caspase 11 and GSDMD, suggesting that LPS is the primary activator in Caspase 11-dependent pyroptosis. In addition, Caspase 11 inhibitor WDL effectively reduced the levels of pro-Caspase 11, the cleavage of Caspase 11, and the cleavage of GSDMD induced by LPS, implying that the LPS-induced pyroptosis was Caspase 11 dependent (Figure 3F). Scanning electron microscopy was employed to evaluate the detailed surface morphology of RAW264.7 cells following treatment with 1 μg/mL LPS or an equivalent PBS as control (Figure 3G). Unlike the control group, which displayed a flat surface with abundant extracellular vesicles, LPS-treated cells exhibited multiple pores or pits on their membrane, indicative of structural compromise. Due to the varying sizes of these pores, the cell surface appeared rough, and cell volume increased following LPS treatment. This morphological change supported that LPS induced pyroptosis in RAW264.7 cells.

We further investigated potential mediating receptors in LPS-induced pyroptosis using receptor antagonists. In experiments with RAW264.7 cells treated with LPS and HMGB1, antagonists for Toll-like receptor (TLR) 2, TLR4, and RAGE were introduced. The results revealed that the TLR4 antagonist at a concentration of 200 nM effectively inhibited the cleavage of Caspase 11 and GSDMD (Figure 3H), along with the secretion of IL-1α and IL-1β induced by LPS (Figure 3H,I). This indicates that TLR4 mediates LPS-induced Caspase 11/GSDMD-dependent pyroptosis. Additionally, The Caspase 11 inhibitor WDL effectively attenuated the enhanced cleavage of GSDMD, as well as the production of IL-1α and IL-1β (Figure 3H,I). These findings collectively demonstrate that the disruption of the intestinal barrier in MRL/lpr mice leads to the migration of LPS originating from the gut into the systemic circulation, subsequently inducing Caspase 11/GSDMD-dependent pyroptosis in macrophages through TLR4 mediation.

2.4 Caspase 1-dependent canonical pyroptosis pathway involvements in SLE

In the canonical pyroptosis pathway, inflammasomes such as Nucleotide-binding oligomerization domian like receptor family pyrin domian containing 1(Nlrp1), Absent in melanoma 2 (Aim2), Nucleotide-binding oligomerization domian like receptor family pyrin domian containing 3 (Nlrp3), and NLR family CARD domain-containing protein 4 (Nlrc4) are activated by various extracellular stimuli to initiate the Caspase 1 cleavage. Initially, we examined the inflammasome expression in PBMCs from SLE patients to investigate the potential role of canonical pyroptosis in SLE. Our findings did not reveal any elevated expressions of Nlrp1, Aim2, Nlrp3, or Nlrc4 in the PBMCs of individuals with SLE compared with the healthy ones (Figure 4A). Similarly, there were no observed elevations of Aim2 and Nlrp3 in protein levels in PBMCs of SLE patients (Figure 4B). However, significant elevated Caspase 1 cleavage was detected in PBMCs of SLE patients (Figure 1A), suggesting an enhancement in Caspase 1-mediated pyroptosis, which was independent of Nlrp1, Aim2, Nlrp3, and Nlrc4 inflammasomes.

In the kidneys of MRL/lpr mice, both the expressions and protein levels of Aim2 and Nlrp3 were significantly increased (Figure 4C,D). Correspondingly, the cleavage of Caspase 1 was also enhanced in the kidneys of MRL/lpr mice (Figure 4D), indicating an increase in Caspase 1-dependent pyroptosis in this model. However, incubation with 20% serum from MRL/lpr mice did not enhance the levels of Aim2 and Nlrp3 or the cleavage of Caspase 1 in RAW264.7 and J774A.1 cells (Figure 2D,E). Consistently, neither treatment with LPS alone nor the combination of LPS and HMGB1 increased the levels of Aim2 and Nlrp3, nor the cleavage of Caspase 1 in vitro (Figure 3F). These findings indicate that the elevated GSDMD cleavage induced by lupus serum or LPS was mediated by Caspase 11 activation rather than Caspase 1. Additionally, the cleavage of Gasdermin E (GSDME) was not detected in RAW264.7 cells incubated with lupus serum (Figure 4E).

2.5 Pyroptosis of macrophage facilitates the differentiation of B cells

We investigated the effects of pyroptotic macrophages on adaptive immunocytes to explore the link between pyroptosis and SLE pathogenesis. CD19⁺ B cells were isolated from the spleens of C57BL/6 mice and stimulated in vitro with R848 and murine IL-4, with or without pyroptotic macrophages. Autologous peritoneal macrophages were isolated and treated with LPS to induce pyroptosis. Flow cytometry plots and gating for plasma cell (PC), plasmablast, naïve B cell, memory B cell, and germinal center B (GCB) cell subsets are shown as Figure 5A. Results showed that coculture with autologous pyroptotic peritoneal macrophages facilitated the differentiation of naive B cells into plasma cells significantly (Figure 5B). when pan B cells were cocultured with macrophages alone, there was a minor rise in the proportions of PCs and plasmablasts with no change observed in the naive, memory B cells, and GCB subgroups compared with the control group. The proportion of mature B cell subsets was not significantly increased by the presence of LPS alone. However, the combination of macrophages and LPS resulted in significant increases in PCs, plasmablasts, and GCB proportions, along with decreases in naive and memory B cell proportions. There was no distinction between the LPS group and the HMGB1+LPS group. The impact of pyroptotic peritoneal macrophages on the differentiation of CD4⁺ T lymphocytes appeared ambiguous (Figure 5C,D). LPS treatment alone did not significantly affect T cell differentiation, except for a reduction in the proportion of regulatory T (Treg) cells. Interestingly, coculture with macrophages inhibited the activation of follicular helper T (Tfh) cells, Treg cells, helper T (Th) 2 cells, and the transition from central memory T (TCM) cells to effector memory T (TEM) cells. In the presence of both macrophages and LPS, the pyroptosis of macrophages induced by LPS counteracted the inhibitory effect on Tfh cell differentiation, although the effect was mild.

Additionally, we examined the effects of the Caspase 11 inhibitor WDL on the differentiation of CD19⁺ B cells and CD4⁺ T cells in the presence or absence of pyroptotic macrophages. Treatment with WDL eliminated the enhancing effect of pyroptotic macrophages on plasma cell differentiation (Figure 5E). Surprisingly, WDL also significantly inhibited plasma cell differentiation even in the absence of pyroptotic macrophages, suggesting its immunosuppressive potential, previously unreported. Furthermore, WDL exerted a strong inhibitory effect on the differentiation of the terminally differentiated effector memory T (TEMR) cell subset and a slight inhibitory effect on TEM cell subset differentiation but had no notable impact on Tfh and Treg subsets.

2.6 Caspase 11 inhibitor and antibiotics alleviate lupus symptoms in MRL/lpr mice

To determine the role of Caspase 11-dependent pyroptosis in the progression of SLE, we examined the therapeutic effects of WDL in lupus-prone mice. MRL/lpr mice were administered 10 mg/kg of WDL or a corresponding vehicle as a control starting at 10 weeks of age and were euthanized 9 weeks later. Additionally, we treated the mice with a combination of antibiotics (ampicillin, metronidazole, neomycin, and vancomycin) via gavage to reduce LPS leakage. Regarding safety, mice in both the WDL-treated and antibiotics-treated groups initially lost weight during the early and mid-stages of treatment, respectively, but subsequently regained it (Figure 6A). Both WDL and antibiotics treatments were effective in suppressing lupus activity, as evidenced by reduced anti-dsDNA antibody titers (Figure 6B), improved proteinuria levels (Figure 6C), and reduced splenomegaly (Figure 6D). Furthermore, both treatments reduced inflammatory cell infiltration, mesangial cell proliferation, and crescent formation in the kidneys of MRL/lpr mice (Figure 6E). Flow cytometry analysis of lymphocyte subsets in the spleens of these mice revealed that treatment with WDL and antibiotics increased the proportion of naïve B cells and decreased the proportion of plasmablasts, although there were no significant effects on the effector T cell subsets (Figure 6F). Collectively, these findings suggest that inhibition of Caspase 11 and the use of antibiotics can modulate immune function and alleviate lupus symptoms in MRL/lpr mice.

2.7 Caspase 11/GSDMD pathway was depressed by WDL and antibiotic treatments in MRL/lpr mice

We further explored the mechanisms underlying the effectiveness of WDL and antibiotics in treating SLE, particularly whether these treatments are associated with the inhibition of LPS and Caspase 11 as hypothesized. Immunofluorescence analysis revealed a significant increase in ZO-1 in the small intestinal epithelium of the antibiotic-treated group compared with the vehicle control group (Figure 7A), accompanied by a substantial decrease in serum LPS levels (Figure 7B). Both WDL and antibiotics significantly reduced the expression of TLR4, Caspase 11, GSDMD, and the downstream inflammatory cytokines IL-1α and IL-1β in the kidneys of the treated mice (Figure 7C). Additionally, the protein levels of Caspase 11, Caspase 11-p25, and GSDMD-p30 in the kidneys exhibited a decreasing trend following treatment with WDL and antibiotics (Figure 7D). These findings indicate that WDL and antibiotic treatments alleviate lupus symptoms by reducing Caspase 11-induced cell pyroptosis, thus confirming our initial speculation regarding their mechanism of action in SLE treatment.

4.1 Antibodies and reagents

A comprehensive list of the antibodies employed for immunofluorescence, western blot, flow cytometry, and mIHC can be found in Table S1. Drug information used for pyroptosis inducing or inhibition in vitro and lupus mice treatment are listed in Table S2.

4.2 Animals and experimental conditions

Female, 8 weeks C57BL/6 mice and MRL/lpr mice were procured from SLAC Laboratory Animal (Shanghai, China) and maintained in a specific pathogen-free environment at the laboratory animal center of the Institute of Dermatology, Chinese Academy of Medical Sciences. Approval for the study was obtained from the Ethics Committee of the Institute of Dermatology, Chinese Academy of Medical Sciences (No. 2022-DW-017). All experiments were performed on age-matched animals. 10 weeks old female MRL/lpr mice were divided into three groups in a random manner: the control group (n = 11), the WDL group (n = 12), and the antibiotics group (n = 12). The mice in the WDL group received a daily intraperitoneal dose of 10 mg/kg WDL for 9 weeks. Meanwhile, the mice in the control group were administered an equivalent vehicle daily. The mice in the antibiotics group were given aqueous solutions containing a combination of antibiotics (ampicillin 1 g/L, neomycin 1 g/L, metronidazole 1 g/L, and vancomycin 0.5 g/L) for 9 weeks. Biweekly, the mice were anesthetized using isoflurane to measure body weight and collect urine and blood samples. Subsequently, they were euthanized via rapid cervical dislocation to obtain kidney and spleen samples.

4.3 Clinical patient information

Serum and blood samples were obtained from six healthy individuals and nine patients diagnosed with SLE by 2017 EULAR/ACR criteria and consented to participate in our study. The clinical information is presented in Table S3. Informed consent was obtained from all participants. Kidney biopsies were obtained from an LN patient for diagnostic purposes. Renal tissue dissected adjacent to a renal tumor was obtained from a patient with a renal mass and was considered to be “normal.”

4.4 Isolation of human PBMCs and CD14⁺ cells

Blood samples from both individuals with SLE and healthy people were anticoagulated with heparin and subjected to density gradient centrifugation on Ficoll-Paque (17144003; Cytiva, USA) for 30 min at 600 × g with the brake off. The mononuclear cells at the interface of two layers were carefully harvested and suspended in PBS. Centrifugation for 10 min at 500 × g and resuspending the sediment in lysing buffer (BL503B; Biosharp, China) to lyse remaining erythrocytes. After incubating the cells at room temperature for 5 min, an equal volume of PBS was added to halt the lysis process. After Centrifugation for 10 min at 500 × g, the sediment of mononuclear cells had been washed with PBS. Isolation of CD14⁺ monocytes from PBMCs was performed utilizing MojoSort™ Human CD14⁺ Monocytes Isolation Kit (BioLegend, USA) and were examined by TEM.

4.5 Isolation of mouse peritoneal macrophage

C57BL/6 mice were euthanized, and the abdominal area was sterilized by 70% ethanol. Subsequently, 10 mL of sterile PBS was carefully administered intraperitoneally. Following gentle abdominal massage, approximately 80% of the injected volume was successfully retrieved. Peritoneal macrophages were then isolated by adjusting the peritoneal suspensions to a concentration of 3 × 10⁵ cells/mL in RPMI 1460 and seeding them into 24-well culture plates. The macrophages were cultured at 37°C for 2 h to adhere to the plastic surface. Upon completion of this incubation period, the supernatants containing nonadherent cells were gently aspirated after three times of PBS washing. The entire process was performed in a sterile environment.

4.6 Mouse CD4⁺ T cells and pan B cells isolation and activation

Spleens were extracted from C57BL/6 mice and CD4⁺ T cells and CD19⁺ B cells were isolated separately using magnetic cell sorting systems (MojoSort™ Mouse CD4 T Cell Isolation Kit and MojoSort™ Mouse Pan B Cell Isolation Kit; BioLegend). The isolated cells were cultured in RPMI 1640 culture medium with 10% FBS, 1% penicillin, 1% streptomycin, and 2-mercaptoethanol at 37°C in a 5% CO₂-humidified incubator. CD4⁺ T cells (1.5 × 10⁶ cells) were seeded in a 24-well plate and then stimulated with anti-mouse CD28 antibody (2 μg/mL) and plate-bound anti-mouse CD3 antibody (5 μg/mL). For CD19⁺ B cells, R848 (100 ng/mL) and murine IL-4 (30 ng/mL) were used to stimulate naïve B cells (1 × 10⁶ cells were seeded in a 24-well plate). These cells were treated separately or combined as follows: cocultured with peritoneal macrophages isolated as processed above, LPS (1 μg/mL), HMGB 1 (400 ng/mL), and WDL (5 μg/mL).

4.7 Gut barrier permeability determination in vivo

Mice that had been fasted for 8 h were orally gavaged with 4 kDa fluorescent dextran (R-FD-001; Xinqiao Biotechnology, Hangzhou, China) at a dose of 300 mg/kg to assess intestinal permeability in vivo. After 2 h, serum was obtained from the blood of mice by centrifugation. Fluorescence intensity of FITC in serum was then analyzed using a microplate reader with excitation at 485 nm and emission at 535 nm.

4.8 Assessment of renal injury

After execution, the left kidneys of mice were fixed in 10% formalin for 24 h. Subsequently, the kidneys were embedded in paraffin and cut into 4 μm-thick sections using a section cutter. Following this, the H&E staining of sections were performed following the manufacturer's guidelines. Renal injury was measured by the Austin score as previously described.³⁵

4.9 Incubation of macrophages with serum and microscopy imaging

J774A.1 or RAW264.7 cells were cultivated by DMEM culture medium (with 10% FBS, 1% penicillin, 1% streptomycin) with 20% PBS or C57BL/6 mice serum or MRL/lpr mice serum at 37°C in a 5% CO₂-humidified incubator. To examine the morphology of pyroptotic macrophages, cells were seeded in 15-mm culture dishes and treated as above. Static bright-field images were captured using a Nikon Eclipse Ti2. The Time-lapse microscopy assay was recorded by DeltaVision Elite Cell Imaging System (GE Healthcare Life Sciences, USA).

4.10 Immunofluorescence

Murine kidney and small intestine sections from frozen OCT embedded tissues (5 μm) were subjected to three washes with PBS and then blocked for 30 min using Blocking Buffer (P0260; Beyotime, China). Subsequently, the sections were exposed to specified primary antibodies overnight at 4°C. Following this, they underwent a gentle PBS wash three times before being treated with secondary antibodies at 37°C for 1 h. Finally, the sections were stained with DAPI staining solution (C1005; Beyotime) at room temperature for 15 min. Images were captured with Olympus BX53 microscopy and data were analyzed with cellSense software (Olympus).

4.11 Multiplex immunohistochemistry

mIHC staining was conducted following the guidelines provided by the manufacturer (PerkinElmer; Opal® Kit). The procedure involved the sequential application of the following antibodies and fluorescent dyes: Caspase 4/Opal620, Caspase 5/Opal520, GSDMD/Opal590. Sections of the paraffinized tissue were cut at 4 μm thickness and heated at 60°C for 1 h, then rehydrated in xylene, 100% alcohol, 90% alcohol, 70% alcohol successively. Epitope retrieval solution was preheated for antigen unmasking, followed by inactivation of endogenous peroxidase through a 15-min incubation in blocking buffer (P0100A; Beyotime). Subsequently, the sections underwent preincubation with the kit's blocking buffer and were then exposed to specified primary antibodies at 37°C for 2 h. Then, the sections were treated with HRP-conjugated secondary antibodies (ab205722; Abcam) at appropriate concentration for 30 min at 37°C. Then, the opal dyes were applied to bond the primary antibodies. Slides were imaged and scanned using Mantra software. The images were batch-analyzed by Inform software (PerkinElmer).

4.12 Quantitative RT-PCR

To conduct quantitative real-time PCR (RT-qPCR) analysis, total RNA was isolated using RNA-easy Isolation Reagent (R701-01; Vanzyme, China) and subsequently underwent cDNA synthesis utilizing the HiScript III RT SuperMix (R323-01; Vanzyme). RT-qPCR was conducted by utilizing SYBR Green Supermix (Q711-02; Vanzyme). β-Actin was used as the reference for normalizing the relative expression levels of mRNA. The sequences of qPCR primers for TLR 2 (5′-GCTCCTGCGAACTCCTATC-3′ and 5′-GCCGGACTCATCGTACTCC-3′), TLR 4 (5′-CCTGACACCAGGAAGCTTGAA-3′ and 5′-TCTGATCCATGCATTGGTAGGT-3′), Caspase 11 (5′-GTGGTGAAAGAGGAGCTTACAGC-3′ and 5′-GCACCAGGAATGTGCTGTCTGA-3′), GSDMD (5′-CCATCGGCCTTTGAGAAA GTG-3′ and 5′-ACACATGAATAACGGGGTTTCC-3′), IL-1α (5′-CGCTTGAGTCGGCAAAGAAA-3′ and 5′-GAGAGAGATGGTCAATGGCAGA-3′), IL-1β (5′-TGGACCTTCCAGGATGAGGACA-3′ and 5′-GTTCATCTCGGAGCCTGTAGTG-3′) and actin (5′-GTGACGTTGACATCCGTAAAGA-3′ and 5′-GCCGGACTCATCGTACTCC-3′).

4.13 Western blot

After lysis of cells and tissues in cell lysis buffer (P0013B; Beyotime) supplemented with protease and phosphatase inhibitor cocktail (P1045; Beyotime), the proteins were extracted by 12,000 × g centrifugation for 18 min. BCA protein assay kit was employed to determine the protein concentration in the supernatant. After denaturation of protein by heating, equal amounts of total protein were loaded and separated by SDS-PAGE and then electrophoretically transferred onto a NC membrane. The membranes were blocked and incubated overnight with primary antibody at 4°C. After washing in TBST, the membranes were exposed to HRP-conjugated antibodies for 1 h at room temperature. After washing in TBST, the bands were visualized by the Tanon 5200 system (TANON SCIENCE & TECHONLOGY, China).

4.14 Flow cytometry data acquisition and analysis

Cells were treated with Fc blocker diluted in PBS for 15 min. After that, the cells were incubated with specific flow cytometry antibodies along with Aqua (BioLegend) for 45 min at 4°C to stain membrane molecules. Then, the cells were treated with Cytofix/Cytoperm™ Fixation (BD Biosciences) for intracellular staining. Following incubation with antibodies for intracellular staining, the cells underwent two PBS washes and were subsequently analyzed using flow cytometry. Data acquisition was performed with Cytek Aurora (Cytek Biosciences) and analyzed utilizing FlowJo software (Tree Star, Inc.).

4.15 ELISAs

Serum was obtained from blood samples by centrifugation at 2000 × g for 5 min. ELISAs kits (Cusabio, Wuhan, China) were employed to detect the levels of anti-dsDNA antibody, IL-1β, IL-1α, and HMGB1. The procedures conducted in accordance with the provided instructions.

4.16 Cytotoxicity assay

Following the specified treatments, the supernatant was gathered and the LDH assay was carried out utilizing the LDH Cytotoxicity Assay Kit (C0016; Beyotime) under the instruction of kit's guidelines. The cytotoxicity percentages was calculated based on the maximum LDH release observed in cells treated with 1% Triton X-100.

4.17 Analysis of proteinuria

Mouse urine samples were tested by Urine protein content Assay Kit (D799855; Sangon Biotech, China) and Creatinine (Cr) Assay kit (C011-2-1, Nanjing Jiancheng Bioengineering Institute, China).

Proteinuria was measured by the urine protein/creatinine ratio (PCR).

4.18 Statistics analysis

GraphPad Prism 9.0 was utilized for the statistical analysis. The data are presented as mean ± SD (standard deviation), with specific statistical information provided in the figure legend. A two-sample t-test was conducted to assess variances between the two groups, with statistical significance defined as p < 0.05. The correlation analysis between the two samples was conducted using Pearson correlation analysis.