E-selectin-mediated rapid NLRP3 inflammasome activation regulates S100A8/S100A9 release from neutrophils via transient gasdermin D pore formation

Abstract

S100A8/S100A9 is a proinflammatory mediator released by myeloid cells during many acute and chronic inflammatory disorders. However, the precise mechanism of its release from the cytosolic compartment of neutrophils is unclear. Here, we show that E-selectin-induced rapid S100A8/S100A9 release during inflammation occurs in an NLRP3 inflammasome-dependent fashion. Mechanistically, E-selectin engagement triggers Bruton's tyrosine kinase-dependent tyrosine phosphorylation of NLRP3. Concomitant potassium efflux via the voltage-gated potassium channel K_V1.3 mediates ASC oligomerization. This is followed by caspase 1 cleavage and downstream activation of pore-forming gasdermin D, enabling cytosolic release of S100A8/S100A9. Strikingly, E-selectin-mediated gasdermin D pore formation does not result in cell death but is a transient process involving activation of the ESCRT III membrane repair machinery. These data clarify molecular mechanisms of controlled S100A8/S100A9 release from neutrophils and identify the NLRP3/gasdermin D axis as a rapid and reversible activation system in neutrophils during inflammation.

Fig. 1. E-selectin-induced S100A8/S100A9 release is GSDMD and caspase 1 dependent.

a, Schematic of the experimental design. b, Serum S100A8/S100A9 levels were analyzed by ELISA before and 2 h after intrascrotal (i.s.) TNF application to WT, Gsdmd^−/− and Casp1^−/−Casp11^−/− mice (n = 8 (WT), 7 (Gsdmd^−/−) and 4 (Casp1^−/−Casp11^−/−) mice per group); NS, not significant. c, Bone marrow neutrophils from WT, Gsdmd^−/− and Casp1^−/−Casp11^−/− mice were incubated with E-selectin or PBS for 10 min (n = 10 (WT), 6 (Gsdmd^−/−) and 5 (Casp1^−/−Casp11^−/−) mice per group). Supernatants were collected, and S100A8/S100A9 levels were analyzed. d,e, Rolling velocities of WT and Gsdmd^−/− neutrophils (d; 87 (WT) and 72 (Gsdmd^−/−) cells from n = 3 mice per group) and WT and Casp1^−/−Casp11^−/− neutrophils (e; 130 (WT) and 117 (Casp1^−/−Casp11^−/−) cells from n = 5 mice per group) were assessed in E-selectin/ICAM-1-coated flow chambers. Data are presented as mean ± s.e.m. and were analyzed by two-way repeated measures analysis of variance (RM ANOVA) with a Sidak’s multiple comparison test (b and c) or are presented as mean ± s.e.m. and cumulative distribution and were analyzed by two-tailed paired Student’s t-tests (d and e). Source data

Fig. 2. E-selectin induces NLRP3 inflammasome activation in neutrophils.

a, Schematic of the experimental design; PBMCs, peripheral blood mononuclear cells. b,c, E-selectin-induced cleavage of caspase 1 was assessed in isolated human neutrophils treated for 10 min with PBS/paquinimod/TAC242, PBS (control), E-selectin or a combination of E-selectin/paquinimod/TAC242 (b) and with PBS, E-selectin and E-selectin in the presence MCC950 (c). The amount of processed caspase 1 (casp-1 p20 and casp-1 p22) was determined in the supernatants, and the amount of procaspase 1 and GAPDH was determined in cell lysates (n = 4 independent experiments); Paq, paquinimod. d–f, Caspase 1 activity was determined in isolated human neutrophils stimulated with E-selectin or PBS (control) for 10 min loaded with FLICA dye and analyzed by confocal microscopy. Representative confocal images (d), quantification of mean FLICA⁺ area (e) and overall mean fluorescence intensity (MFI) of FLICA signal per cell (f; 76 (control) and 112 (E-selectin) cells of n = 3 independent experiments) are shown. g, Isolated human neutrophils were treated for 10 min with PBS (control), E-selectin or E-selectin in the presence of MCC950, and the amount of full-length GSDMD (FL GSDMD), GSDMD-NT and GAPDH was determined in the cell lysates (n = 4 independent experiments). h,i, Representative confocal images (h) and overall MFI of GSDMD-NT staining of human neutrophils (i) stimulated with PBS (control), E-selectin or E-selectin in the presence of MCC950 for 10 min (49 (control), 75 (E-selectin) and 62 (E-selectin + MCC950) cells of n = 3 independent experiments). Data are presented as representative western blots and mean ± s.e.m. (analyzed by one-way ANOVA with Tukey’s multiple comparison tests; b, c and g), as mean ± s.e.m. (analyzed by two-tailed paired Student’s t-tests (e and f) or one-way ANOVA with a Tukey’s multiple comparison test (i)) and as representative confocal images (d and h); scale bars, 5 µm. Source data

Fig. 3. Rapid E-selectin-induced inflammasome activation is K_V1.3 dependent.

a,b, Caspase 1 cleavage was assessed in isolated human neutrophils resuspended in normal HBSS and HBSS with high [K⁺]_ex (a) and pretreated with K_V1.3 inhibitor PAP-1 (b; 50 nM) or vehicle control and stimulated with E-selectin for 10 min. The amount of processed caspase 1 (casp-1 p20 and casp-1 p22) was determined in the supernatants, and the amounts of procaspase 1 and GAPDH were determined in cell lysates (n = 3 and 5 independent experiments, respectively). c,d, Bone marrow neutrophils from WT mice pretreated with PAP-1 (50 nM) or vehicle control (c) or from WT and Kcna3^−/− mice (d) were incubated with E-selectin or PBS (control) for 10 min (n = 4 (control), 5 (PAP-1), 4 (WT) and 6 (Kcna3^−/−) mice per group); w/o, without. e,f, Bone marrow neutrophils from WT mice were incubated with PBS, E-selectin or ATP (e) or with PBS, E-selectin, nigericin or a combination of LPS and nigericin (f) for 10 min (n = 5 (control (e), E-selectin (e), ATP (e) and nigericin (f)), 9 (control (f), LPS and nigericin (f)) and 8 (E-selectin (f)) mice per group). g, Isolated human neutrophils were stimulated with PBS, E-selectin, MAdCAM-1 or endoglycan for 10 min (n = 3 independent experiments). In c–g, supernatants were collected, and S100A8/S100A9 levels were analyzed by ELISA. h,i, IL-1β levels were analyzed by ELISA in the supernatants from WT bone marrow neutrophils stimulated with PBS, E-selectin, nigericin or a combination of LPS and nigericin for 10 min (h; n = 6 mice per group) or primed with PBS or LPS for 2.5 h and subsequently stimulated for 30 min with PBS, E-selectin or nigericin (i; n = 5 (control, LPS/nigericin) and 7 (LPS, LPS/E-selectin) mice per group). Data are presented as representative western blots and mean ± s.e.m. (a and b; data were analyzed by one-way ANOVA with a Tukey’s multiple comparison test or two-tailed paired Student’s t-tests, respectively) or mean ± s.e.m. (c–i; data were analyzed by two-way RM ANOVA with a Sidak’s multiple comparison test (c and d) or one-way ANOVA with Tukey’s multiple comparison tests (e–i)). Source data

Fig. 4. E-selectin induces NLRP3 tyrosine phosphorylation and ASC oligomerization.

a, Tyrosine phosphorylation of NLPR3 after 5 min of PBS or E-selectin stimulation was assessed via phospho-tyrosine immunoprecipitation (pY-IP) in WT bone marrow neutrophils pretreated with the BTK inhibitor ibrutinib (0.6 µM) or vehicle control (n = 5 (ibrutinib) and 6 (without ibrutinib) independent experiments). b, Bone marrow neutrophils from WT mice were incubated with ibrutinib (0.6 µM) or vehicle control and subsequently stimulated with E-selectin or PBS (control) for 10 min (n = 6 mice per group). Supernatants were collected, and S100A8/S100A9 levels were analyzed by ELISA. c, Bone marrow Kcna3^−/− neutrophils were stimulated for 5 min with PBS or E-selectin, and tyrosine phosphorylation of NLRP3 was analyzed by pY-IP (n = 3 independent experiments). d–g, Bone marrow neutrophils from WT (d and e) and Kcna3^−/− (f and g) mice were stimulated with E-selectin or PBS (control) for 10 min, and ASC oligomerization was analyzed by western blotting (n = 7 (WT) and 6 (Kcna3^−/−) mice per group) and confocal microscopy (WT: 38 (control) and 32 (E-selectin) cells; Kcna3^−/−:  41 (control) and 34 (E-selectin) cells of n = 4 mice per group). Data are presented as representative western blots and mean ± s.e.m. (a, c, d and f; data were analyzed by two-way RM ANOVA and a Sidak’s multiple comparison test (a) or two-tailed paired Student’s t-tests (c, d and f)), mean ± s.e.m. (b; data were analyzed by two-way RM ANOVA and a Sidak’s multiple comparison test) and representative micrographs and mean ± s.e.m. (e and g; 51 (WT control), 64 (WT E-selectin), 46 (Kcna3^−/− control) and 48 (Kcna3^−/− E-selectin) cells of n = 4 mice per group; data were analyzed by paired Student’s t-tests); scale bars, 5 µm. Source data

Fig. 5. E-selectin-induced GSDMD pore formation is transient.

a, Schematic of the experimental design. b, Representative confocal images of PI uptake in isolated human neutrophils stimulated with PBS, E-selectin or E-selectin in the presence of MCC950. c,d, Time course analysis (c) and percentage of PI⁺ cells after 10 min of stimulation (d;  1,029 (PBS), 429 (E-selectin) and 315 (E-selectin/MCC950) cells of n = 3 (control and E-selectin + MCC950) and 4 (E-selectin) independent experiments); F_t, fluorescence at the respective time point; F₀, fluorescence at t₀. e, Representative confocal images of PI uptake in bone marrow neutrophils from WT and Casp1^−/−Casp11^−/− mice treated with E-selectin. f,g, Time course (f) and percentage of PI⁺ WT and Casp1^−/−Casp11^−/− neutrophils after 10 min of stimulation (g; 190 (WT) and 351 (Casp1^−/−Casp11^−/−) cells of n = 3 mice per group). h–j, Cell death of human neutrophils was assessed via LDH release after stimulation with PBS or E-selectin for 10 min (h), 30 min (i) and 180 min (j; n = 3 (10 min and 180 min) and 4 (30 min) independent experiments). k–m, PI uptake was measured in isolated human neutrophils after pretreatment of cells with E-selectin for the specified amounts of time (k). Representative confocal images of PI uptake (l) and percentage of PI⁺ neutrophils (m) are shown (274 (5 min), 313 (10 min), 358 (15 min) and 466 (20 min) cells of n = 3 independent experiments). n,o, CHMP4B expression was investigated in isolated human neutrophils stimulated with PBS or E-selectin for 15 min. Representative confocal images of CHMP4B localization (n) and number of CHMP4B puncta per cell (o; 24 (control) and 47 (E-selectin) cells of n = 4 independent experiments) are shown. Data are presented as mean ± s.e.m. (c, d, f–j, m and o; data were analyzed by one-way ANOVA with a Dunnetts’s multiple comparison test (c, d and m), two-tailed unpaired Student’s t-tests (f–j) or two-tailed paired Student’s t-tests (m and o)) and representative micrographs (b, e, l and n); scale bars, 5 µm. Source data

Fig. 6. GSDMD-dependent pore formation supports neutrophil recruitment.

a, Schematic of the experimental design. Male WT and Gsdmd^−/− mice were stimulated after intrascrotal administration of TNF (500 ng) 2 h before intravital microscopy of postcapillary venules of the mouse cremaster muscle. b–d, Neutrophil rolling (b), neutrophil rolling velocity (c) and number of adherent neutrophils per vessel surface (d) were analyzed (18 and 24 vessels, respectively, of n = 4 (WT) and 5 (Gsdmd^−/−) mice per group). e, TNF-stimulated cremaster muscles were stained with Giemsa, and the number of perivascular neutrophils was quantified (52 and 31 vessels, respectively, of n = 4 mice per group). Data are presented as mean ± s.e.m. (b–e) and as cumulative distribution (c; right) and were analyzed by two-tailed unpaired Student’s t-tests. Source data

Extended Data Fig. 1. S100A8/A9 release depends on E-selectin, GSDMD and inflammatory caspases.

a Serum S100A8/A9 levels were determined by ELISA before and 2 h after intrascrotal TNF application to WT and Sele^-/- mice (n = 3 (WT), 4 (Sele^-/-) mice/group). b-g Neutrophil and monocyte counts were analyzed in peripheral blood samples from untreated WT, Sele^-/-, Gsdmd^-/- and Casp1^-/-Casp11^-/- mice (n = 5 (WT b, c, d, e, Sele^-/-, Gsdmd^-/-), 6 (WT f,g, Casp1^-/-Casp11^-/-) mice/group). h Overall intracellular S100A8/A9 content in bone marrow neutrophils from WT, Gsdmd^-/- and Casp1^-/-Casp11^-/- mice was assessed and quantified by western blotting. Mrp14^-/- neutrophils, which exhibit a loss of both S100A8 and A9, were used as negative control (representative western blot, n = 5 mice/group). Data are presented as mean ± s.e.m. (two-tailed unpaired student’s t-test for b-g; one-way ANOVA, Tukey’s comparison for h and two-way RM ANOVA, Sidak’s multiple comparison for a) and representative Western blot for h; ns: not significant. Source data

Extended Data Fig. 2. S100A8/A9 release is dependent on NLRP3 inflammasome.

a Amount of processed caspase-1 (casp-1 p20/casp-1 p22) in lysates of human neutrophils was determined and normalized to GAPDH (n = 5 independent experiments). b-c Caspase-1 activity was determined in FLICA dye-loaded, human neutrophils pretreated with VX-765 (10 µM) upon stimulation with E-selectin or PBS (control) for 10 min. Cells were analyzed by confocal microscopy. Quantification of b mean FLICA positive area and c overall mean fluorescence intensities (MFI) of FLICA signal/cell (47 (control) and 57 (E-selectin) cells of n = 4 independent experiments). d Representative super-resolution micrographs (maximum projection of 10 planes around cell centers; arrows: GSDMD-NT (green) at the plasma membrane (magenta)) and e Plasma membrane (PM) translocation index (PM vs cytosol GSDMD-NT MFI ratio) of human neutrophils treated with PBS and E-selectin (19 (control) and 19 (E-selectin) cells of n = 4 independent experiments). f WT mice were i.p. injected with NLRP3-inhibitor MCC950 or vehicle control 1 h prior to intrascrotal TNF application. Serum S100A8/A9 levels were analyzed by ELISA before and 2 h after TNF stimulation (n = 4 (WT), 5 (WT + MCC950) mice/group). g Bone marrow neutrophils from WT mice pretreated with MCC950 (1 µM, 30 min) or vehicle control were incubated with E-selectin or PBS. (n = 5 mice/group). S100A8/A9 levels were analyzed in supernatants by ELISA. Rolling velocities of h MCC950 (1 µM, 30 min) or vehicle control (65 control and 68 MCC950 treated cells from n = 3 mice/group) pretreated WT neutrophils were assessed in E-selectin/ICAM-1-coated flow chambers. Rolling velocities were determined in i MCC950 (1 µM, 30 min) or vehicle control (104 (control) and 81 (MCC950) cells from n = 3 independent experiments/group) pretreated human neutrophils. Data are presented as mean ± s.e.m., (two-tailed paired student’s t-test for a-c, e, h and i, two-way ANOVA, Sidak’s multiple comparison for f; two-way RM ANOVA, Sidak’s multiple comparison for g), as representative Western blot for a, as representative micrographs for d and as cumulative distribution for h and i. ns: not significant. Source data

Extended Data Fig. 3. E-selectin triggers rapid S100A8/A9 release.

a Isolated neutrophils were treated for 10 min with PBS, E-selectin or E-selectin in the presence of high [K⁺]_ex and the amount of full length GSDMD, GSDMD-NT and GAPDH was determined in the cell lysates (PBS control group and E-selectin stimulated group values and representative blot are the same as in Fig. 2g; n = 4 (control, E-selectin), 3 (E-selectin/ high [K⁺]_ex) independent experiments). b Overall intracellular S100A8/A9 content was assessed by Western blotting in bone marrow neutrophils of WT and Kcna3^-/- mice (n = 4 mice/group). c Isolated human neutrophils were stimulated with PBS (control) or E-selectin (1 µg ml^-1) for 1 min, 5 min or 10 min and S100A8/A9 levels in the supernatants were assessed by ELISA (n = 3 (control 10 min, E-selectin 10 min), 4 (E-selectin 1 min), 5 (control 1 min, control 5 min, E-selectin 5 min) individual experiments. Values from the 10 min time point (control and E-selectin) are the same as in Fig. 3g. Data are presented as mean ± s.e.m. (one-way ANOVA, Tukey’s comparison for a, two-tailed unpaired student’s t-test for b and two-way ANOVA, Sidak’s multiple comparison for c), as representative Western blot for a, ns: not significant. Source data

Extended Data Fig. 4. E-selectin stimulates secretion of cytosolic small alarmins.

Protein release analyzed by mass spectrometry-based proteomics of supernatants from E-selectin-activated versus PBS-treated isolated human neutrophils (n = 3 independent experiments). a Volcano plot showing differentially released proteins, cytosolic small alarmins (M_w≤50 kDa) and cytosolic alarmins (M_w>50 kDa) as indicated. b Release of indicated proteins for each donor, c release of all proteins, cytosolic alarmins (no size threshold) and cytosolic small alarmins (M_w≤50 kDa). Data are presented as volcano plot for a, as heat map for b, as box-whisker-plots for c (50% interquartile range (IQR), median center, whiskers ranging from 5-95 percentiles and dots indicating outliers). -log₁₀ transformed p-values depicted in the volcano plots were determined by Welch’s two-sided t-test. Enrichment of alarmins in c was determined by a 1D annotation enrichment with Benjamin-Hochberg FDR = 0.02, ns: not significant. Source data

Extended Data Fig. 5. E-selectin stimulation does not result in granule release.

Enrichment of granule content (azurophilic gr., specific gr., gelatinase gr.) in supernatants of E-selectin (1 µg ml^-1) and PBS (control) stimulated human neutrophils was analyzed by mass spectrometry (n = 3 independent experiments). a Volcano plot showing differentially released proteins, cytosolic small alarmins (M_w≤50 kDa), cytosolic alarmins (M_w>50 kDa) and granules are indicated. b Release of indicated proteins for each donor, c release of all proteins, all granules, azurophilic granules, specific granules and gelatinase granules. d-g Isolated human neutrophils were stimulated with E-selectin (1 µg ml^-1), PMA (100 nM, positive control) or PBS (control) for 10 min. Surface expression of d CD63, e CD66b and f CD11b was assessed using flow cytometry (n = 4 independent experiments). g gating strategy. Data are presented as mean ± s.e.m. (one-way ANOVA, Tukey’s comparison for d-f, as volcano plot for a, as heat map for b, as box-whisker-plots for c (50% IQR, median center, whiskers ranging from 5-95 percentiles and dots indicating outliers), as representative flow cytometry dot blot image for g, and as representative histograms for d-f. -log₁₀ transformed p-values depicted in the volcano plots were determined by Welch’s two-sided t-test. Enrichment of granule content in c was determined by a 1D annotation enrichment with Benjamin-Hochberg FDR = 0.02. ns: not significant. Source data

Extended Data Fig. 6. E-selectin induces depolarization of membrane potential in neutrophils.

a-c Changes in membrane potential of isolated human neutrophil pretreated with PAP-1 (50 nM), disulfiram (30 µM) or vehicle (control) before (baseline) and after E-selectin stimulation was measured by current clamp, a representative voltage membrane traces (n = 14 (disulfiram), 17 (control), 20 (Pap-1) cells of 7 different blood donors). b Mean baseline membrane potential V_m (averaged from t_(-30)-t₍₀₎) and c changes in V_m compared to baseline (ΔV_m) upon E-selectin stimulation were analyzed. d and e Cell death of human neutrophils and f and g monocytes was assessed by LDH release after stimulation with LPS/nigericin for 30 min and 180 min (n = 3 (monocytes 180 min), 4 (neutrophils 30 min, 180 min, monocytes 30 min) independent experiments). Data are presented as mean ± s.e.m. (two-tailed unpaired student’s t-test for d-g, one-way ANOVA, Tukey’s multiple comparison for b and two-way ANOVA, Tukey’s multiple comparison for c) and as representative traces for a, ns: not significant. Source data

Extended Data Fig. 7. Receptor expression of WT and Gsdmd^-/- neutrophils.

Surface expression levels of a Mac-1/CD11b/αM, b LFA-1/CD11a/αL, c β2 integrin subunit/CD18 and d CXCR2/CD182 were determined on bone marrow neutrophils from WT and Gsdmd^-/- mice by flow cytometry (n = 3 mice/group). e Mouse neutrophils were defined as Ly6G positive cells using a PB conjugated rat anti-mouse Ly6G antibody. Data are presented as mean ± s.e.m. (two-tailed unpaired student’s t-test) and as representative histograms for a-d and as representative flow cytometry dot blot image for e. ns: not significant. Source data

Extended Data Fig. 8. Neutrophil recruitment in vivo requires NLRP3 inflammasome activity.

Male WT mice were injected i.p. with MCC950 (10 mg kg^-1) or vehicle control 1 h before i.s. application of TNF (500 ng mouse⁻¹). Two hours later, intravital microscopy of postcapillary venules of the mouse cremaster muscle was performed and a neutrophil rolling, b neutrophil rolling velocity and c number of adherent neutrophils per vessel surface was analyzed (26 and 24 vessels, respectively, of n = 5 mice/group). d TNF stimulated cremaster muscles were stained with Giemsa and number of perivascular neutrophils was quantified (35 and 25 vessels, respectively, of n = 5 mice/group). Data are presented as mean ± s.e.m. (two-tailed unpaired student’s t-test for a, b, c and d) and as cumulative distribution for b; ns: not significant. Source data