Case reports
The first patient (P1) was male, with disease-onset at age 12 years. The patient presented with periorbital oedema after tonsillitis, accompanied by reduced urine output and recurrent fevers. Laboratory investigations showed proteinuria, haematuria, hypoalbuminaemia, leukopaenia, mild anaemia and hypocomplementaemia. ANA and anti-dsDNA autoantibodies were positive. Kidney biopsy showed diffuse proliferative lupus nephritis with crescent formation.
The second patient (P2) was female, with disease-onset at age 16 years. The patient had urticaria, alopecia, arthralgia, oedema and recurrent infection. Laboratory investigations showed significant proteinuria, haematuria, acute kidney injury, leukopaenia, thrombocytopenia, autoimmune haemolytic anaemia and hypocomplementaemia. ANA and anti-dsDNA autoantibodies were positive. Kidney biopsy revealed diffuse proliferative lupus nephritis with crescent formation and acute tubular injury.
The third patient (P3) was male, with disease-onset at age 19 years. The patient presented with arthritis, morning stiffness, malar rash, hair loss, photosensitivity, periorbital oedema, gross haematuria and abdominal distension. Laboratory investigations showed hypertension, elevated serum creatinine, massive proteinuria, haematuria, pancytopenia and hypocomplementaemia. ANA and anti-dsDNA autoantibodies, and rheumatoid factor were positive. Kidney biopsy revealed diffuse proliferative lupus nephritis with crescent formation and fibrinoid necrosis. Maintenance haemodialysis began at the age of 40 years.
The fourth patient (P4) was female, with disease-onset at age 49 years. The patient presented with generalized arthralgia, patchy facial erythema with itching and dry mouth. Laboratory evaluations revealed massive proteinuria, haematuria, pancytopenia and hypocomplementaemia. ANA, anti-nRNP/Smith (Sm), anti-Sm and anticardiolipin antibodies were positive. Kidney biopsy revealed membranoproliferative lupus nephritis.
The fifth patient (P5) was female, with disease-onset at age 37 years. The patient presented with patchy rashes with itching, muscle soreness and haemoptysis. Laboratory analysis indicated haematuria, proteinuria, elevated serum creatinine, anaemia and thrombocytopenia, and hypocomplementaemia. ANA and myeloperoxidase–antineutrophil cytoplasmic antibody (MPO–ANCA) were positive. Kidney biopsy showed membranoproliferative lupus nephritis with crescent formation. She began maintenance haemodialysis at the age of 43 years.
Detailed case presentations are provided in the Supplementary information.
Patients
All of the patients who met the diagnostic criteria for SLE were evaluated at Jinling Hospital. All patients enrolled in the study were evaluated under a protocol approved by the Institutional Review Boards evaluated at Jinling Hospital (2022DZKY-061-01). All patients and family members signed written informed consent.
PLD4 exonuclease activity
In HEK293T cells, plasmids encoding wild-type and various mutant PLD4 were transfected, followed by collection of protein lysates and purification through Flag-tag magnetic beads (Sigma-Aldrich, M8823). The purified protein or total cell lysates were then subjected to PLD4 enzymatic activity assay18,19 (50 mM MES pH 5.5, 150 mM NaCl, 2.5 μM substrates, 10 nM or 20 nM purified PLD4), incubated at 37 °C for different time and subsequently analysed by TBE–PAGE, with nucleic acid staining performed for 15 min before imaging.
Mice and mice treatment
Pld4-KO mice (NM-KO-200682), on the C57BL/6 background, were purchased from the Shanghai Model Organisms Center. CD45.1 mice (T054816) were purchased from GemPharmatech. All of the mice were maintained under a specific-pathogen-free environment in the Laboratory Animal Center of Zhejiang University and experimentation was approved by the Institutional Animal Care and Use Committee of Zhejiang University (ZJU20250573). No statistical method was used to calculate sample size. Sample sizes with mice were determined by the availability of animals with the correct genotypes or based on numbers used in previous publications44 where comparable sample sizes produced statistically significant results. Age- and sex-matched mice were used in each experiment (littermates). Experimenters were blinded to genotypes in the kidney histology pathology analysis. Other data collection and analyses were not performed in a blinded manner to the conditions of the experiment.
Pld4-KO mice, aged 6 weeks and with similar body weights, underwent tail vein blood sampling to measure anti-DNA and anti-RNA autoantibodies. On the basis of autoantibody levels and body weights, mice were evenly divided into two groups. One group received daily oral gavage of baricitinib (Selleck, S2851) at a dose of 30 mg per kg per day, while the other group received a solvent gavage to exclude solvent effects. The baricitinib working solution was prepared with 5% baricitinib, 50% PEG 300, 5% Tween-80 and 40% double distilled H2O.
WES and Sanger sequencing
DNA was extracted from peripheral blood using the Maxwell RSC Whole Blood DNA Extraction Kit (Promega, AS1520), with 1 μg of DNA used for WES. Data alignment was performed using the BWA, and variants were annotated using ANNOVAR (https://annovar.openbioinformatics.org/en/latest/). Variants were filtered using online databases, including gnomAD (https://gnomad.broadinstitute.org/), dbSNP (https://www.ncbi.nlm.nih.gov/snp/) and Kaviar (https://db.Systemsbiology.net/kaviar/). Subsequently, potential pathogenic variants were selected based on inheritance patterns and the biological functions of the genes. Finally, candidate mutations were confirmed by Sanger sequencing.
RNA-seq and scRNA-seq
Total RNA was extracted from PBMCs designated for RNA-seq using the QIAGEN RNeasy kit (74104). The RNA quality and purity were assessed using an Agilent Bioanalyzer RNA chip with 1 μg of total RNA. After assessment, the RNA was purified and fragmented, followed by the construction of an mRNA library using the Illumina TruSeq RNA Sample Preparation Kit V2. The RNA was then reverse-transcribed into cDNA, with an average fragment size of approximately 200 bp. Subsequent steps included end-repair, adding an A base to the 3′ ends, adaptor ligation and PCR amplification. RNA-seq was conducted on the Illumina NovaSeq platform. Sequencing data were aligned using HISAT2 in human reference genome (GRCh38), with reads counting performed by featureCounts. Differential gene expression analysis was conducted using DESeq2, and downstream heat-map visualization was performed using the R package pheatmap.
scRNA-seq used in this study was performed with samples from two sources: human PBMCs and mouse kidney cells. Human PBMCs were counted directly for library construction after separation. The mice kidney tissue was digested with collagenase IV for 120 min. After single-cell counting, the procedure was as follows: cells were uniformly mixed with gel beads using the 10x Genomics single-cell sequencer to prepare oil droplets encapsulating the cells, causing cell lysis, RNA release and reverse transcription. Adaptors were added to the cDNA from each cell, followed by PCR amplification and single-cell libraries were prepared for sequencing. The scRNA-seq analysis workflow was as follows: raw data were processed using Cell Ranger to count reads and generate expression matrices for different transcripts in each cell. The expression matrices were then quality-controlled, dimensionally reduced, annotated and visualized using the Seurat package45 in R.
CyTOF analysis
The PBMCs from both patients and healthy controls were pretreated with brefeldin A (BFA) for 4 h to block cytokine secretion and stained with 250 nM cisplatin for 5 min to label dead cells. Subsequently, cells were incubated with an Fc receptor blocking agent and a mixture of surface antibodies for 30 min at room temperature, followed by fixation and permeabilization, and further stained with intracellular detection antibodies on ice for 30 min before acquisition.
CyTOF data were initially processed using FlowJo to remove dead cells, duplicate cells and background noise. Cells were subdivided into distinct subgroups based on the expression levels of marker genes. High-dimensional data were transformed into two-dimensional representations using t-SNE dimensional reduction analysis. The data were then annotated based on marker gene expression and compared for the expression levels of target proteins.
Cell preparation, culture and stimulation
HEK293T and THP-1 cell lines, obtained from the American Type Culture Collection, were cultured and stimulated as follows: HEK293T cells were maintained in Dulbecco’s modified Eagle medium (Thermo Fisher Scientific, C11995500CP) supplemented with 10% FBS (Noverse, NFBS-2500A) and 1% penicillin–streptomycin (Thermo Fisher Scientific, 15140163). THP-1 cells and PBMCs were maintained in RPMI-1640 (Thermo Fisher Scientific, C11875500CP) supplemented with 10% FBS and 1% penicillin–streptomycin. PBMCs were isolated from whole blood using lymphocyte-separation medium (LSM, MPbio, 0850494) through density-gradient centrifugation according to the manufacturer’s instructions. The THP-1 PLD4-KO cell line was generated using the CRISPR–Cas9 system by infecting THP-1 wild-type cells with sgPLD4 virus packaged in HEK293T cells, followed by selection with puromycin to establish stable KO cell lines.
In the phosphorylation flow cytometry experiments on PBMCs, cells were stimulated with 5 μM CpG-DNA (ODN 2216, InvivoGen, tlrl-2216) for 20 min. For qPCR and RNA-seq experiments in PBMCs, stimulation was performed with 4 μM CpG-DNA for 24 h. For intracellular cytokine staining experiments, PBMCs were stimulated with 2 μM CpG-DNA for 24 h, with BFA added 6 h before sampling to block cytokine secretion. In the CyTOF experiments on PBMCs, cells were treated with BFA for 4 h. In the qPCR experiments on PBMCs treated with baricitinib, the baricitinib concentration was 0.5 μM for 16 h treatment. In qPCR experiments on the THP-1 PLD4 polyclonal KO cell line, cells were stimulated with 5 μM CpG-DNA for 6 h. For STING inhibition experiment, THP-1 PLD4 monoclonal KO cells were treated with 5 μM H-151 or C-176 for 48 h, then subjected to western blotting or qPCR.
Plasmids and antibodies
PCR amplification of PLD4 cDNA from healthy control PBMCs was used to construct the human PLD4 plasmid. Site-directed mutagenesis was used to generate mutant plasmids. Western blotting, flow cytometry and immunofluorescence were performed using a variety of antibodies: NF-κB p65 (Cell Signaling Technology, 8242), p-NF-κB p65 (Cell Signaling Technology, 3033), p44/42 MAPK (Cell Signaling Technology, 4696), p-p44/42 MAPK (Cell Signaling Technology, 4370), STAT1 (Cell Signaling Technology, 14994), p-STAT1 (Cell Signaling Technology, 9167), p-STAT2 (Tyr690) (Cell Signaling Technology, 88410), STAT2 (Cell Signaling Technology, 72604), β-actin (Cell Signaling Technology, 4970), GAPDH (Cell Signaling Technology, 2118), TLR7 (Cell Signaling Technology, 5632), p-STING (Ser366) (Cell Signaling Technology, 50907), STING (Cell Signaling Technology, 13647), HRP-conjugated anti-DYKDDDDK tag (Flag) (Huabio, 0912-3), CD3-APC-H7 (BD Biosciences, 560176), CD4-FITC (BD Biosciences, 555346), CD8-APC (BD Biosciences, 561952), CD14-PE-CY7 (BD Biosciences, 557742), CD19-BB700 (BD Biosciences, 566396), PE mouse anti-human IFNα (BD Biosciences, 560097), BD Pharmingen p38 MAPK (pT180/pY182) PE (BD Biosciences, 612565), BD Phosflow BV421 anti-human NF-κB p65 (pS529) (BD Biosciences, 565446), Alexa Fluor 647 anti-p-ERK1/2 (BioLegend, 369504), PLD4 (Thermo Fisher Scientific, PA5-98680), anti-C3 (Abcam, ab11862), FITC anti-human CD21 (BioLegend, 354910), FITC anti-human CD24 (BioLegend, 311104), PerCP/Cyanine5.5 anti-mouse/rat/human CD27 (BioLegend, 124214), Brilliant Violet 605 anti-human CD38 (BioLegend, 303532), Brilliant Violet 421 anti-human IgG Fc (BioLegend, 410704), APC anti-human HLA-DR (BioLegend, 307610), Brilliant Violet 570 anti-mouse CD11c (BioLegend, 117331), V500 mouse anti-mouse CD45.2(104) (BD Biosciences, 562129), Brilliant Violet 650 anti-mouse CD45.1 (BioLegend,110736), BV421 hamster anti-mouse CD3e (BD Biosciences, 562600), FITC rat anti-mouse CD4 (BD Biosciences, 553046), APC-Cy7 rat anti-mouse CD8a (BD Biosciences, 557654), APC rat anti-mouse CD19 (BD Biosciences, 550992), PE rat anti-mouse CD138 (BD Biosciences, 553714), BV605 CD317 (BD Biosciences, 747606), PE-Cy7 rat anti-mouse CD45R/B220 (BD Biosciences, 552772), BUV496 CD11b (BD Biosciences, 749864), BV750 F4/80 (BD Biosciences, 747295), RB780 Ly-6C (BD Biosciences, 755871), BUV395 I-A, I-E (BD Biosciences, 569244), BUV805 CD44 (BD Biosciences, 741921), BUV563 CD62L (BD Biosciences, 741230), BV786 rat anti-mouse CD25 (BD Biosciences, 564023), UV737 rat anti-mouse CD21/CD35 (BD Biosciences, 612810), RB545 CD23 (BD Biosciences, 756344), BV480 CD95 (BD Biosciences, 746755), BUV615 CD49b (BD Biosciences, 751052), BV711 CD279 (BD Biosciences, 744547), PE-CF594 rat anti-mouse CD185 (CXCR5) (BD Biosciences, 562856), R718 Ly-6G (BD Biosciences, 567039), PERCPEF710 BCL-6 (Invitrogen, 46-5453-82), RB744 rat anti-mouse Siglec-H (BD Biosciences, 757466) and BUV661 rat anti-mouse CD98 (BD Biosciences, 752893).
Flow cytometry analysis
In experiments assessing the changes in phosphorylation levels of key proteins in inflammatory signalling pathways and intracellular inflammatory cytokines in PBMCs, cells were plated at a density of 1.5 × 106 cells per ml and stimulated with CpG-DNA for either 20 min or 24 h. After the removal of the stimulus, surface antibodies were added, and the cells were stained at room temperature for 30 min. After PFA fixation and permeabilization, intracellular antibodies were added, and the cells were stained at room temperature for 1 h before proceeding to flow cytometry analysis.
Single-cell suspensions from mouse spleens were prepared by gently triturating the tissue with the plunger end of a syringe and filtering the resultant mixture through 40 µm filters with 2% FBS in PBS (2% FBS–PBS). The filtrate was then centrifuged at 350g for 5 min. The cell pellet was resuspended in 2 ml of RBC lysis buffer and incubated at 25 °C for 3 min. The suspension was then diluted with 10 ml 2% FBS–PBS. After a second centrifugation at 350g for 5 min, the spleen cells were resuspended in PBS.
For mouse kidney single-cell suspension preparation, half of the kidney was minced into 1–2 mm3 pieces using surgical curved scissors in a dish and transferred to a 15 ml centrifuge tube. To this, 2 ml of digestion solution (1 mg ml−1 collagen IV, 200 μg ml−1 DNase I) was added, and the tissue was incubated at 37 °C for 30 min. Digestion was then halted by the addition of 10% FBS–PBS and the resultant mixture was filtered through 40 µm filters. The mixture was subsequently centrifuged and resuspended in 2 ml of RBC lysis buffer according to the spleen single-cell suspension protocol.
Immunofluorescence
For immunofluorescence staining of mouse kidney tissue cryosections, the sections were first fixed and permeabilized, followed by blocking with freshly prepared 10% normal goat serum (NGS, Beyotime, C0265)/PBS for 1 h. Mouse antibodies diluted in 10% NGS/PBS (IgG 1:200; C3 1:200) were applied to the sections and incubated overnight at 4 °C. Secondary antibodies diluted at 1:500 in 10% NGS/PBS were then incubated with the sections at room temperature for 1 h. Finally, the sections were imaged using a Zeiss 710 inverted microscope.
Western blotting and immunoprecipitation
For western blotting, cells were lysed on ice for 20 min in NP-40 lysis buffer containing protease and phosphatase inhibitors (Thermo Fisher Scientific, 78442), followed by centrifugation at 12,000g for 10 min at 4 °C. The supernatant was mixed with SDS sample buffer, heated at 95 °C for 5 min, then subjected to separation by SDS–PAGE.
For immunoprecipitation of endogenous PLD4 in PBMCs, homemade PLD4 antibody whole immune serum was used at a 1:50 ratio to immunoprecipitate 500 µg of PBMC protein. The immunocomplexes were then eluted with 0.2 M glycine at pH 2.5 and subsequently neutralized using 1.0 M Tris-HCl at pH 8.0.
RT–qPCR
qPCR with reverse transcription (RT–qPCR) was performed using ABclonal’s 2× Universal SYBR Green Fast qPCR Mix (RK21203), Vazyme HiScript IV All-in-One Ultra RT SuperMix for qPCR(R433), ChamQ Blue Universal SYBR qPCR Master Mix (Q312-02) and Roche’s LightCycler480 qPCR system to measure mRNA expression levels in various cell or tissue samples. Fluorescence signal intensities from the collected samples and primers were used to obtain Ct values. Relative expression levels were normalized to the reference gene GAPDH/Gapdh and calculated using the ΔΔCt method.
ELISA
This study primarily conducted two types of enzyme-linked immunosorbent assay (ELISA): one for the detection of cytokines in cell lines and another for coating DNA/RNA to detect autoantibodies in mouse plasma, with similar steps for both. Initially, an appropriate amount of coating buffer was used to dilute coating antibodies or antigens (1 μg DNA or RNA), which were then added to the wells of an ELISA plate and incubated overnight at 4 °C. Subsequently, the plate was blocked with diluent buffer to reduce non-specific binding and incubated at room temperature for 1 h. Samples and standards were then diluted in diluent buffer and incubated at room temperature for 2 h. Detection antibodies were diluted in diluent buffer and further incubated for 1 h at room temperature. This was followed by dilution of HRP conjugate (Beyotime, A0216) in diluent buffer and a further 0.5 h incubation at room temperature. TMB substrate (Beyotime, P0209) was added and incubated for 30 min. Finally, stop solution (Beyotime, P0215) was added before measuring the absorbance at 450 nm using a microplate reader.
Bone marrow chimeras
B6-CD45.1(Ptprc-p.K302E) were taken lethally irradiated (7 Gly), then reconstituted with mixed bone marrow (5 × 106 cells, 1:1 ratio of B6-CD45.1 and either wild-type or Pld4-dificient (B6-CD45.2)). After 8 weeks of reconstitution, mice were euthanized, and plasma was collected for ELISA analysis, while kidneys and spleens were collected for flow cytometry analysis.
Statistical analysis
Data analysis and graphing were performed using GraphPad Prism 8, R v.3.5.2 and PyMOL (v.3.1)46. Data for statistical tests were derived from three or more independent experiments. Mouse, cell and human-derived data are presented as mean ± s.e.m. For comparisons between two groups, unpaired t-tests were used for significance analysis; for comparisons involving more than two groups, ANOVA was used; P < 0.05 was considered to be statistically significant.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
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