N-GSDMD trafficking to neutrophil organelles facilitates IL-1β release independently of plasma membrane pores and pyroptosis

Abstract

Gasdermin-D (GSDMD) in inflammasome-activated macrophages is cleaved by caspase-1 to generate N-GSDMD fragments. N-GSDMD then oligomerizes in the plasma membrane (PM) to form pores that increase membrane permeability, leading to pyroptosis and IL-1β release. In contrast, we report that although N-GSDMD is required for IL-1β secretion in NLRP3-activated human and murine neutrophils, N-GSDMD does not localize to the PM or increase PM permeability or pyroptosis. Instead, biochemical and microscopy studies reveal that N-GSDMD in neutrophils predominantly associates with azurophilic granules and LC3⁺ autophagosomes. N-GSDMD trafficking to azurophilic granules causes leakage of neutrophil elastase into the cytosol, resulting in secondary cleavage of GSDMD to an alternatively cleaved N-GSDMD product. Genetic analyses using ATG7-deficient cells indicate that neutrophils secrete IL-1β via an autophagy-dependent mechanism. These findings reveal fundamental differences in GSDMD trafficking between neutrophils and macrophages that underlie neutrophil-specific functions during inflammasome activation.

Fig. 1. Plasma membrane permeability and Ca²⁺—dependent plasma membrane repair in NLRP3-activated human and murine macrophages and neutrophils.

a–c NLRP3-activated (LPS/nigericin) bone marrow derived macrophages from C57BL/6 and Gsdmd ^−/− mice showing a time course of propidium iodide (PI) uptake (a), representative PI positive cells after 45 min (b), and LDH release after 90 min (c). 5 mM glycine was added to the extracellular media for PI imaging of C57BL/6 and Gsdmd^−/− macrophages during nigericin stimulation to avoid cell lysis but was not added to neutrophil cultures. d Time course of PI uptake by LPS/nigericin activated WT and Gsdmd^−/− THP-1 human macrophages. e–g NLRP3-activated bone marrow neutrophils from C57BL/6 and Gsdmd ^−/− mice showing PI uptake (e, f) and LDH release after 90 min incubation (g). h, i PI uptake by LPS/nigericin and LPS/ATP—activated peripheral blood neutrophils from two healthy human donors. (IL-1β production is shown in Supplemental Fig. 3). j–m PI uptake and LDH release (45 min) by murine macrophages (j, k) and neutrophils (l, m) stimulated with nigericin in either Ca²⁺ supplemented or Ca²⁺ free media. Time course of PI uptake (a, d, g, h, i, j, l) are mean ± SD of 4 independent experiments (n = 4); LDH data (c, g, k, m) are mean ± SD of biological replicates for each condition from 5 or more independent experiments (n = 5 data points). p-Values were derived by two-way ANOVA using Sidak’s multiple comparisons test; p < 0.05 is significant, n.s.: not significant. Panels B, F: scale bar = 100 µm. Source data for panels a, c, d, e, g, h, i, j, k, l, m are provided in the separate Source Data file.

Fig. 2. Subcellular localization of N-GSDMD in neutrophils versus macrophages.

a GSDMD western blot (Abcam EPR 19828) from total cell lysate + supernatant of LPS-primed, NLRP3-stimulated (45 mins) bone marrow derived macrophages and bone marrow neutrophils from C57BL/6 mice (neutrophil and macrophage lysates were run side by side on the same gels). All neutrophil lysates were generated in the presence of 5 mM DFP (serine protease inhibitor) in addition to a standard protease inhibitor cocktail. b LPS-primed neutrophils incubated with the pan-caspase inhibitor zVAD (1.5 µM) for 30 min before adding nigericin or ATP. c LPS - primed THP-1 macrophages (PMA-differentiated) and human donor neutrophils were stimulated 45 min with 10 μM nigericin or 3 mM ATP. Western blots of total cell lysates from THP-1 macrophages (100 μg) and human neutrophils (200 μg) were probed with either Santa Cruz clone H-6 that recognizes pro- and cleaved GSDMD, or with EPR 20829-408 (Abcam) that specifically recognizes p31 N-GSDMD. β-actin was the loading control. Molecular weight markers in kDa are shown on the right. d–g NLRP3-activated THP-1 human macrophages and human neutrophils immunostained with the N-GSDMD specific antibody (Abcam EPR 20829-408). d, e Representative Imagestream images of WT THP-1 cells stimulated with LPS only (no GSDMD cleavage) (d) or with LPS/nigericin of WT THP-1 macrophages (e) and co-stained with wheat germ agglutinin (WGA) to identify the plasma membrane and N-GSDMD colocalization. (Supplemental Fig. 7 shows Imagestream of Gsdmd^−/− THP-1 macrophages stimulated with LPS/nigericin.) f, g WGA and N-GSDMD staining in NLRP3-activated (LPS + ATP) peripheral blood neutrophils captured by Imagestream (f) or confocal microscopy (Huygens deconvolution) images (g). Yellow and green arrows in the enlarged images show plasma membrane (PM) and N-GSDMD, respectively. Western blots are representative of 3 independent experiments (n = 3), and Imagestream and confocal microscopy images are representative of neutrophils from three donors (n = 3). Source data for all western blots in Panels A, B, C are provided as uncropped blots in Supplementary Information, Supplementary Fig. 15.

Fig. 3. Localization of neutrophil N-GSDMD to azurophilic granules and release of active neutrophil elastase to the cytosol.

a Schematic of subcellular fractionation of organelles, plasma membrane (PM) and cytosolic fractions. b Subcellular fractionation of cell homogenate of LPS + ATP-stimulated neutrophils and macrophages. P0.7 (nuclei, undisrupted cells): pellet from 0.7k g; P10 (organelle fraction): pellet from 10k g; P100 (plasma membrane fraction): pellet from 100k g; and S100 (cytosolic fraction): supernatant from 100k g. Tom 20, Cadherin and GAPDH are markers for mitochondria, PM and cytosolic fractions, respectively. c P10 organelle fractions of NLRP3-activated C57BL/6 macrophages and neutrophils. Neutrophil elastase (ELA2), and myeloperoxidase (MPO) indicate azurophilic granules; LC3I and LC3II are markers for autophagosomes; Tom-20 and ATBP1 are mitochondrial markers, and GAPDH was the loading control. d, e LPS + ATP stimulated human neutrophils immunostained with antibodies to N-GSDMD (EPR 20829-408) and MPO. Representative images were acquired by Imagestream (d) or by super-resolution microscopy (e). Arrows show overlap of N-GSDMD (green) and MPO (red) (d), and proximity is indicated by arrowheads of N-GSDMD (green) and MPO (red) in three representative neutrophils, and by arrows in enlarged images (lower panels) of super resolution microscopy images (e). Scale bar = 2 µm. Quantification of N-GSDMD proximity to MPO (f); each data point represents one neutrophil (median of 12 foci measured per cell), and percent of puncta where N-GSDMD is <50 nm, 50–100 nm, or >100 nm from MPO. g, h Time course of NE release into cytosol, and secondary GSDMD processing by cytosolic NE in LPS/ATP activated bone marrow neutrophils. Neutrophil elastase in cytosolic fractions and whole cell lysates of C57BL/6 and Gsdmd^−/− neutrophils (g), and GSDMD in whole cell lysates of neutrophils from C57BL/6 and neutrophil elastase-deficient (NE^−/−) mice (probed with EPR19828) (h). All neutrophil lysates were generated in the presence of DFP and standard protease inhibitors. Western blots are representative of at least 3 independent experiments (n = 3). Molecular weight markers in KDa are indicated to the right. Images are representative of four different human donor neutrophils (n = 4). Source data for all western blots in Panels b, c, g, h are provided as uncropped blots in Supplementary Information, Supplementary Figs. 16 and 17. Source data for panel f is provided in the separate Source Data file.

Fig. 4. ATG7 and GSDMD - dependent IL-1β release from NLRP3 inflammasome - activated neutrophils or macrophages.

a–c Bone marrow neutrophils and macrophages from Atg7^f/f (WT) and Atg7^M∆ (Atg7 KO) mice. a Time course of IL-1β secretion (a) and western blot of lysates and TCA precipitated supernatants of naïve (no LPS) or LPS primed neutrophils after stimulation with ATP or nigericin (b). c Time course of IL-1β secretion by bone marrow derived macrophages from Atg7^f/f (WT) and Atg7^M∆ (Atg7 KO) mice. d–f Bone marrow neutrophils and BM derived macrophages from C57BL/6 (WT) and Gsdmd^−/− mice showing IL-1β secretion from neutrophils (d), western blots of pro- and cleaved IL-1β and caspase-1 in cell lysates and supernatants (Sup; e) and IL-1β secretion by macrophages (f). For all experiments, cells were LPS primed for 3 h followed by 45 min stimulation with 10 µM nigericin or 3 mM ATP. Complete media + 2% FBS was used for all cell stimulations. Western blot data are representative of at least 3 biological repeats (n = 3); ELISA data are combined from 3 independent experiments (n = 3) with 4 replicates per condition. p-values were derived by two-way ANOVA and Sidak’s multiple comparisons test; p < 0.05: significant, n.s.: not significant. Source data for all western blots in Panels b, e are provided as uncropped blots in Supplementary Information, Supplementary Fig. 18. Source data for panels a, c, d, e are provided in the separate Source Data file.

Fig. 5. Localization of neutrophil N-GSDMD to LC3⁺ autophagosomes.

a, b LPS + ATP activated human peripheral blood neutrophils immunostained with antibodies to N-GSDMD (green) and LC3 (red). Representative images were acquired by Imagestream showing overlapping N-GSDMD and LC3 staining (a) and by super resolution STORM images (b) showing close proximity (indicated by arrowheads) of N-GSDMD (green) and LC3 (red). Enlarged images of boxed areas are shown in the panels on the right. Scale bar = 2 µm. c Quantification of N-GSDMD puncta proximity to LC3 puncta. Each data point represents one neutrophil (median of 8 foci measured per cell), and percent of puncta where N-GSDMD is <50 nm, 50–100 nm, or >100 nm from LC3. Imagestream data are representative of at least 3 independent experiments from human blood donors (n = 3). Source data for panel C is provided in the separate Source Data file.

Fig. 6. Geldanamycin inhibition of NLRP3 inflammasome-induced IL-1β release.

a–d Human peripheral blood neutrophils (n = 12 donors) or bone marrow neutrophils from C57BL/6 mice were LPS primed (3 h), followed by 45 min stimulation with nigericin or ATP in the presence or absence of the HSP-90 inhibitor geldanamycin (Gel, 10 μM). Secretion of IL-1β (a, c), CXCL8 (b), or CXCL2 (d) was quantified by ELISA. e Western blots of ASC oligomers, caspase-1 and GSDMD from LPS/nigericin-stimulated murine bone marrow neutrophils ±10 μM geldanamycin. Western blot data are representative of 3 independent experiments. Molecular weight markers in kDa are indicated on the right. For human neutrophils, each data point represents 1 donor and p-values were based on paired t tests from 12 donors (n = 12). For murine neutrophils, means±SD from at least 5 independent experiments are shown as individual data points (n = 5). p-values for murine neutrophils were derived by unpaired t test using Tukey post analysis; p < 0.05: significant, n.s.: not significant. Source data for all western blots in panel e are provided as uncropped blots in Supplementary Information, Supplementary Fig. 19. Source data for panels a, c, b, d are provided in the separate Source Data file.

Fig. 7. Proposed model for GSDMD signaling in inflammasome activated neutrophils and macrophages.

Comparison of NLRP3 inflammasome signaling and downstream GSDMD trafficking in neutrophils and macrophages (see text for detailed discussion).