Research ArticleCELL DEATH

Noncanonical inflammasome signaling elicits gasdermin D–dependent neutrophil extracellular traps

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Science Immunology  24 Aug 2018:
Vol. 3, Issue 26, eaar6676
DOI: 10.1126/sciimmunol.aar6676
  • Fig. 1 Activated caspase-11 but not caspase-1 triggers neutrophil cell death.

    (A to D) Neutrophils were primed with LPS (100 ng/ml) for 4 hours before stimulation with Salmonella Typhimurium (MOI 25, log phase) or nigericin (5 μM) for the indicated times: (A and B) Caspase-1 (cleaved p20 or full-length p46) in cell supernatants (Sup.) or cell lysates (Lys.) were measured by immunoblot. (C and D) LDH release was quantified to monitor cell lysis. ns, not significant. (E to K) Neutrophils were primed with Pam3CSK4 (1 μg/ml) for 4 hours and transfected without (mock) or with ultrapure LPS (10 μg/ml) for 4 hours. LDH release (E, F, and H), GSDMD cleavage to p30 (G), or IL-1β secretion (I to K) was monitored. (L) Neutrophils were primed with Pam3CSK4 (1 μg/ml) for 4 hours and transfected without (mock) or with ultrapure LPS (10 μg/ml) for 4 hours, with and without KCl supplementation to the medium immediately before cell transfection. IL-1β release was quantified. Immunoblots are representative of data from at least three biological replicates. Graphs are means + SEM from data pooled from three (K), four (H and L), six (I), seven (E), six to eight (F and J), or eight (C and D) biological replicates. Data were analyzed for normality using the Shapiro-Wilk normality test. Statistical analyses were performed using parametric t tests (two-sided) for normally distributed data sets and nonparametric Mann-Whitney t tests (two-sided) for data sets with non-normal distributions. Data were considered significant when *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, or ****P ≤ 0.0001.

  • Fig. 2 Neutrophils express limited caspase-1, and GSDMD is more efficiently cleaved by caspase-11 than caspase-1.

    (A) Macrophages (M) and neutrophils (N) were exposed to LPS (100 ng/ml) or Pam3CSK4 (1 μg/ml) for 4 hours, and cell extracts were analyzed by immunoblot for expression of inflammasome pathway components. (B) Cytosolic lysates of HEK293T expressing GSDMD-V5 or pro–IL-1β–V5 were incubated with recombinant caspase-1 or recombinant caspase-11 for 30 min at 37°C, and substrate cleavage was assessed by anti-V5 immunoblot. (C) Neutrophils were primed with Pam3CSK4 (1 μg/ml) for 4 hours and transfected without agonist (mock) or with ultrapure LPS (10 μg/ml) or flagellin (1 μg/ml) for 4 hours. Cell lysates and supernatants were harvested and pooled for immunoblot analysis of GSDMD cleavage to p30. Blots are cropped from the same film. All data are representative of at least three biological replicates (A to C).

  • Fig. 3 LPS-activated caspase-11 induces NETosis via GSDMD.

    (A to D) Neutrophils were primed [Pam3CSK4 (1 μg/ml) for 4 hours] and then transfected with either ultrapure LPS (10 μg/ml) or flagellin (1 μg/ml) for 4 hours. Cells were stained for DNA (DAPI), MPO, and citrullinated histone H3 (H3Cit). NETosis was examined by deconvolution immunofluorescence microscopy (A to D). Images shown are representative of data from at least three biological replicates. (C) Microscopy images were blinded and quantified for NETosis, where cells with delobulated nuclei or diffuse/spread DNA were counted as cells undergoing NETosis. (D) Microscopy images were quantified using ImageJ for the percentage of cells that are positive for citrullinated histone H3. Graphs show means + SEM of data from three biological replicates (C and D). Data were analyzed for normality (Shapiro-Wilk normality test) and significance (parametric two-sided t test; ***P ≤ 0.001 or ****P ≤ 0.0001).

  • Fig. 4 The coordinated functions of GSDMD and caspase-11 drive early and late NETosis.

    (A) Murine neutrophils were primed [Pam3CSK4 (1 μg/ml) for 4 hours] and then transfected without (mock) or with ultrapure LPS (10 μg/ml) for 4 hours. NETosis was examined by deconvolution immunofluorescence microscopy, and the percentage of cells undergoing early versus late NETosis was scored by blind counting for cells with delobulated nuclei or diffuse DNA (early) versus spread extracellular DNA (late). Data are means + SEM of three biological replicates. Data were analyzed for normality (Shapiro-Wilk normality test) and significance (parametric two-sided t test; **P ≤ 0.01 or ****P ≤ 0.0001). (B) Murine neutrophils were primed [Pam3CSK4 (1 μg/ml) for 4 hours] and then transfected without (mock) or with ultrapure LPS (10 μg/ml) for 4 hours. Cells were then fractionated to give cytosolic versus nuclear extracts, which were then analyzed by immunoblot. Data are representative of three biological replicates. (C and D) Nuclei of murine neutrophils were incubated for 15 min with recombinant full-length GSDMD, with or without recombinant caspase-11 to enable GSDMD cleavage to p30. Image analysis of (C) SYTOX uptake assessed permeabilization of the nuclear membrane and (D) SYTOX volume assessed chromatin decondensation. Data are means + SEM of n > 890 cells, representative of at least three biological replicates. Statistical analyses were performed using nonparametric Mann-Whitney t tests (two-sided; ****P ≤ 0.0001). (E) Nuclear extracts were prepared from murine neutrophils and incubated with 100 nM recombinant caspase-11 for up to 2 hours. Histone H3 cleavage was assessed by immunoblot and quantified by densitometry using ImageJ. Data are representative of three biological replicates.

  • Fig. 5 A cytosolic Gram-negative bacterium induces NETosis of human neutrophils.

    Human neutrophils were primed for 2 hours with Pam3CSK4 (1 μg/ml) and then infected with C. rodentium in the presence and absence of the caspase-1/4 inhibitor VX765 (50 μM). (A) NETosis for cells infected at an MOI of 25 was measured as SYTOX uptake over a 3-hour time course. Data are means ± SEM of infections of neutrophils from six healthy donors (****P ≤ 0.0001, two-way ANOVA). (B) NETosis was imaged by confocal microscopy, with staining for MPO and extracellular chromatin relative to the DAPI DNA stain.

  • Fig. 6 Neutrophil infection with cytosolic bacteria triggers caspase-11/GSDMD-driven NETosis and in vivo host defense.

    (A to C) Neutrophils were primed for 4 hours with Pam3CSK4 (1 μg/ml) and then challenged with Salmonella Typhimurium ΔsifA (MOI 50, stationary phase) for 4 hours, and NETosis was examined by deconvolution immunofluorescence microscopy. (A) Microscopy images, representative of three biological replicates. Microscopy images were quantified for (B) NETosis and (C) the percentage of cells that were positive for citrullinated histone H3. Data are means + SEM of three biological replicates. (D) Microscopy images were also quantified for the percentage of cells infected (mean + SEM of 18 fields of view from three biological replicates) and the number of bacteria per infected cell (>100 infected cells per genotype from three biological replicates). (E) WT, Casp11−/−, and Gsdmd−/− mice were challenged with stationary-phase Salmonella Typhimurium ΔsifA (1 × 105 CFU ip), with and without in vivo 150-U DNase I application at 4 hours after infection. Bacterial burden in the spleen was quantified at 24 hours after infection. Data are geometric means and individual values of five individual mice per treatment group. Data were analyzed for normality using the Shapiro-Wilk normality test. Statistical analyses were performed using two-sided parametric t test (B to D, upper) or nonparametric Mann-Whitney t test (D, lower, and E). Data were considered significant when *P ≤ 0.05, **P ≤ 0.01, or ****P ≤ 0.0001.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/3/26/eaar6676/DC1

    Fig. S1. GSDMD mediates rapid macrophage pyroptosis.

    Fig. S2. Salmonella Typhimurium and nigericin induce IL-1β secretion from WT neutrophils.

    Fig. S3. Cytosolic LPS triggers caspase-11–dependent cell death and IL-1β production from macrophages.

    Fig. S4. Extracellular potassium blocks noncanonical NLRP3 activation and resultant release of mature IL-1β.

    Fig. S5. Cytosolic LPS but not mock or flagellin transfection triggers GSDMD-dependent cell death.

    Fig. S6. Time course analysis profiles the kinetics of caspase-11/GSDMD-induced NETosis.

    Fig. S7. NE, MPO, and PAD4 are dispensable for GSDMD cleavage and caspase-11–driven NETosis.

    Fig. S8. PMA induces NETosis of human neutrophils.

    Fig. S9. Cytosolic bacteria triggers caspase-11–dependent NETs in vivo.

    Fig. S10. Model for LPS-driven NETosis via GSDMD during cytosolic infection.

    Table S1. Raw data file.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. GSDMD mediates rapid macrophage pyroptosis.
    • Fig. S2. Salmonella Typhimurium and nigericin induce IL-1β secretion from WT neutrophils.
    • Fig. S3. Cytosolic LPS triggers caspase-11–dependent cell death and IL-1β production from macrophages.
    • Fig. S4. Extracellular potassium blocks noncanonical NLRP3 activation and resultant release of mature IL-1β.
    • Fig. S5. Cytosolic LPS but not mock or flagellin transfection triggers GSDMD-dependent cell death.
    • Fig. S6. Time course analysis profiles the kinetics of caspase-11/GSDMD-induced NETosis.
    • Fig. S7. NE, MPO, and PAD4 are dispensable for GSDMD cleavage and caspase-11–driven NETosis.
    • Fig. S8. PMA induces NETosis of human neutrophils.
    • Fig. S9. Cytosolic bacteria triggers caspase-11–dependent NETs in vivo.
    • Fig. S10. Model for LPS-driven NETosis via GSDMD during cytosolic infection.

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    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Raw data file.

    Files in this Data Supplement:

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