Fig 2: RNP ICs-induced release of extracellular mtDNA is significantly suppressed by mROS and GSDMD inhibition.a Immunofluorescence staining of SG, TOMM20 and 8OHdG in peripheral blood neutrophils from HV after RNP ICs treatment. Cells were pretreated with DSF (5 µM), GSK484 (10 µM), or Mito-TEMPO (10 µM) for 2 h, followed by stimulation for 12 h with RNP ICs. BF: bright field; scale bar, 15 µm. 3 μm (enlarged). b Quantitative analysis of areas of extracellular SG and mtDNA in (a). Areas of released mtDNA including regions of extracellular TOMM20/8OHdG positive staining. c Quantification of 8OHdG content in culture medium after the indicated treatment. d Quantitative analysis of mtDNA into the supernatant from the indicated treatment groups at the indicated time points by qPCR. n = 3 HVs. e Immunofluorescence staining of SG, TOMM20 and 8OHdG in LDGs after 6 h. LDGs were pretreated with DSF (5 µM), GSK484 (10 µM) or Mito-TEMPO (10 µM). Scale bar, 15 µm. 3 μm (enlarged). f Quantitative analysis of areas of extracellular SG and mtDNA in (e). g Quantification of 8OHdG content in cultured medium of LDGs. h Quantification of the released mtDNA in LDGs at the indicated time points. n = 3 SLE patients. i, j Quantification of the release of LDH into the supernatant from the indicated groups at the indicated time points by ELISA. n = 3 HVs (i) and 3 SLE patients (j). The results are pooled from three independent experiments using cells from 6 HVs (b, c). Two SLE patients were used in one experiment, and plots were pooled from three independent experiments using cells from 6 SLE patients (f, g). Representative of three independent experiments (a, d, e, h, i, j). Data are presented as mean ± SD. Significance was examined by one-way AVOVA (b, c, f, g) or unpaired two-sided Student’s t test (d, h, i, j).

Fig 3: Immune complexes mediate the activation of caspase-1 and caspase-11 by downregulating Serpinb1.a Immunoblotting of GSDMD and GSDMD-N in BM neutrophils after treatment with IFN-γ, NS from WT mice, LS from lupus mice, or IFN-γ + LS for 12 h. b Quantitative analysis of GSDMD-N/GAPDH. c Immunoblotting of GSDMD and GSDMD-N in peripheral blood neutrophils of HV. Cells were pretreated with IFN-γ. Then the cells were added with NS form HV or LS from SLE patients for 12 h. d Quantitative analysis of GSDMD-N/GAPDH levels. e Immunoblotting for GSDMD and GSDMD-N in BM neutrophils from Caspase-1−/−, Caspase-11−/− and Caspase-1−/−Caspase-11−/− mice treated with IFN-γ + LS. f Quantitative analysis of GSDMD-N/GAPDH levels. g Immunoblotting of Serpinb1, Caspase-1, Caspase-11, and GSDMD levels in BM neutrophils treated with IFN-γ + LS, or IgG deleted LS for 12 h. h Quantitative analysis of Serpinb1, Caspase-1, Caspase-11, and GSDMD-N levels. i Immunoblotting analysis of Serpinb1, Caspase-1, Caspase-11, and GSDMD levels in WT or FcγR−/− BM neutrophils after IFN-γ + LS treatment for 12 h. j Quantitative analysis of Serpinb1, Caspase-1, and Caspase-11 levels. k Immunoblotting analysis of Serpinb1, Caspase-1, and Caspase-4 levels in neutrophils isolated from the peripheral blood of HV and SLE patients. l Quantitative analysis of Serpinb1, Caspase-1, and Caspase-4 levels. m Immunoblotting analysis of Serpinb1, Caspase-1, and Caspase-11 levels in the BM neutrophils from saline or PIL mice. n Quantitative analysis of Serpinb1, Caspase-1, and Caspase-11 levels. n = 6 mice (b, f, h, j, n) or 6 donors (d, l). The immunoblotting samples shown are from the same experiment. Two blots were processed in parallel (d, l, n). Three blots were processed in parallel (b, f, h, j). Data are shown as mean ± SD. Significance was examined by one-way ANOVA (b, f, h, j), or unpaired two-sided Student’s t test (d, l, n).

Fig 4: Intravital renal microscopy reveals neutrophil cell death in live lupus mice.a Two photon intravital imaging analysis of infiltrated neutrophils (Ly6G-AF488) in renal blood vessels (Texas Red) of live mice (glomeruli and tubulointerstitial) after saline or pristane treatment. Scale bar, 30 μm. b Quantitative analysis of infiltrated neutrophils in glomeruli and tubulointerstitial of mice after saline or pristane treatment. n = 6 mice. c Immunofluorescence staining of Ly6G in glomeruli from Ms4a3-tdTomato (Ms4a3-Td) mice after pristane treatment. Scale bar, 30 μm. d Quantitative analysis of Ly6G+ and Ms4a3-tdTomato+ cell numbers in each glomerulus and percentage of Ms4a3-tdTomato+ Cells. Representative of three independent experiments, and each point represents one glomerulus with the mean being represented by a horizontal line. e, f Intravital imaging of the kidneys from pristane-treated Ms4a3-Td mice revealing the release of DNA (Sytox Green, green) from tdTomato+ cells (Red). Time-lapse images are shown of DNA that was released in the form of punctate particles (e) or mesh-like structures (f). Scale bar, 20 μm. 5 μm (enlarged). Representative of two independent experiments. g Quantitative analysis of PE-Ly6G+SG+ cells/10 min in kidney of PIL and Gsdmd−/− mice by intravital imaging. n = 6 mice. Representative of three independent experiments. Data are presented as mean ± SD. Significance was examined with unpaired two-sided Student’s t test (b, d, g).

Fig 5: MtDNA directly interacts with GSDMD-N.a qPCR analysis of mtDNA following GSDMD pulldown under the indicated treatment. BM neutrophils were treated with LPS + Nigericin + H2O2, LPS + Nigericin, or IFN-γ + LS. n = 6 mice. b Immunoblotting of TOMM20 and GSDMD. BM neutrophils were treated with IFN-γ + LS for 12 h, or pretreated with Mito-TEMPO. Lysates were co-immunoprecipitated with anti-GSDMD, and co-immunoprecipitates were then spotted on a nitrocellulose membrane, UV crosslinked, and probed with antibodies specific for 8OHdG, or were separated via SDS-PAGE for GSDMD and TOMM20. c Quantitative analysis of 8OHdG and TOMM20. n = 5 mice. The samples shown are from the same experiment. Three blots were processed in parallel. d Immunoblotting of TOMM20 and 8OHdG in peripheral blood neutrophils from HV or SLE patients (n = 6). e Quantitative analysis of 8OHdG and TOMM20. The samples shown are from the same experiment. Three blots were processed in parallel. Relative mtDNA enrichment was assessed via qPCR in indicated cells. 293T (f) and Gsdmd−/− MEF cells (g) were transfected with Flag-full-GSDMD, Flag-GSDMD-N or GSDMD-C, then treated with H2O2 (100 μM) for 4 h. n = 6 samples pooled from 6 independent experiments. h The cluster of four evolutionarily conserved, positively charged amino acids (red and underlined) in GSDMD-N were mutated to Ala. i X-ray crystal structure of the murine GSDMD (PDB: 6N9N). Dissociation constants (KD) of human GSDMD with human mtDNA (j) and Ox-mtDNA (k). KD of peptides from GSDMD-N with human mtDNA (l) and Ox-mtDNA (m). n, o Measurements of KD of mutant peptides from GSDMD-N with mtDNA (n) and Ox-mtDNA (o). The KD was derived from the binding response as a function of the His-tagged GSDMD or His-tagged peptides. Errors in KD represent fitting errors. Representative of three independent experiments (b, d, j–o). Data are presented as means ± SD. Significance was examined with one-way ANOVA (a, c, f, g) or unpaired two-sided Student’s t test (e).