Research ArticleCELL DEATH

Gasdermin D plays a vital role in the generation of neutrophil extracellular traps

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Science Immunology  24 Aug 2018:
Vol. 3, Issue 26, eaar6689
DOI: 10.1126/sciimmunol.aar6689
  • Fig. 1 A chemical screen identifies a compound that inhibits NET formation and binds to GSDMD.

    (A) Screening results. NETs were quantified by image acquisition. A total of 6397 compounds reduced NET incidence to less than 50%, resulting in ≈3.4% hit rate. (B) Structure and characterization of LDC7559, showing the IC50 (in μM) for NET formation induced by PMA and cholesterol crystals, viability in PBMCs, NADPH oxidase, MPO, and NE activity; only NET formation was affected. (C to E) Human primary neutrophils were treated with 1 or 10 μM LDC7559. (C) ROS production of human primary neutrophils activated with PMA. (D) Percentage of neutrophils that phagocytosed fluorescent beads, analyzed 30 min after incubation with beads by flow cytometry. (E) NET formation upon treatment with PMA. Cell death was assessed by adding the cell-impermeable DNA dye SYTOX Green and measuring fluorescent signal over time. (F) Affinity chromatography. LDC2618 was coupled to beads and incubated with HL-60 lysates (1) without (2) or with (3) competition by the initial hit compound LDC7559. After washing, the precipitated peptides were analyzed by MS. (G) Results of MS, showing enrichment of GSDMD peptides upon pulldown with LDC2618 and competition by LDC7559 in two independent experiments. Peptides for NOX2 and MPO were identified but not enriched upon pulldown and not competed by LDC7559. (C to E) Mean ± SEM of three independent experiments. *P < 0.05, one-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons test. DMSO, dimethyl sulfoxide; ns, not significant.

  • Fig. 2 LDC7559 inhibits GSDMD and blocks IL-1β release and pyroptosis.

    (A and B) Human primary monocytes were primed with ultrapure LPS, and the inflammasome was activated with silica (A) or by transfection of poly(dA-dT) (B); IL-1β release was measured by enzyme-linked immunosorbent assay (ELISA). Values are shown as relative to non–inhibitor-treated cells due to donor variability in absolute IL-1β amounts. Cells were treated with LDC7559 at 1 or 10 μM or with caspase-1/4 (Casp-1/4) inhibitor (VX-765, 50 μM) as a control. (C) THP-1 cells were differentiated for 8 hours with PMA, transfected with LPS, and incubated overnight in the absence or presence of LDC7559 (1, 5, or 10 μM). IL-1β release was measured by ELISA. (D) Murine immortalized BMDMs were primed with Pam3CSK4 for 5 hours, transfected with LPS, and incubated overnight in the absence or presence of LDC7559 (1, 5, and 10 μM). IL-1β release was measured by ELISA. (E and F) HEK293T cells were transfected with full-length (FL), C-terminal (CT), and N-terminal (NT) human (hGSDMD; E) or murine (mGSDMD; F) GSDMD constructs. LDH release was measured 16 hours later. When indicated, LDC7559 was added at 1, 5, or 10 μM (E) or at 5 μM (F) 2 hours after transfection. (E) Caspase-1/4 inhibitor (VX-765, 50 μM) or pan-caspase inhibitor (Z-VAD-FMK, 20 μM) was added at the same time as LDC7559. (A to F) Mean ± SEM of three independent experiments or four independent experiments (A). *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA with Dunnett’s multiple comparisons test.

  • Fig. 3 GSDMD localizes to the plasma membrane and is processed during NET formation.

    (A) Western blot of human primary neutrophils isolated from a control donor (ctr.) or from a patient with X-linked CGD after stimulation with PMA for 2.5 hours. GSDMD was detected with an antibody directed against full-length GSDMD. In this control, donor loss of full-length GSDMD corresponded with the occurrence of a processed N terminus, which is marked by an arrow. (B) Microscopy images of human primary neutrophils activated for the indicated time points with PMA and stained with antibodies against GSDMD and NE as well as with the DNA dye DAPI; membranes were stained with DiI. GSDMD is detected in remnants of cells that had made a NET (white arrows). Images were acquired at the coverslip level; therefore, spread NETs are not seen. Scale bars, 10 μm. (C) High-resolution TIRF microscopy of human primary neutrophils undergoing NET formation. Cells were fixed at the indicated time points after PMA stimulation in the presence or absence of 5 μM LDC7559. Plasma membranes were stained with DiD. Scale bars, 10 μm. (D) Quantification of TIRF microscopy. Background signal was determined outside the cells, and GSDMD signal (at least 2× background fluorescence intensity) in the TIRF zone was analyzed. Mean ± SEM of three independent experiments is depicted. ***P < 0.001, unpaired two-tailed t test.

  • Fig. 4 NE cleaves and activates GSDMD.

    (A and B) Human neutrophils were treated with caspase inhibitors (Z-VAD-FMK, 20 μM; VX-765, 50 μM) before activation with PMA. NET formation was assessed by staining cells with SYTOX Green and measuring fluorescent signal over time. (C) GSDMD-expressing HEK293T lysates were incubated with lysates of human primary neutrophils for 20 min in the presence of absence of LDC7559 (10 μM) or NE inhibitor (NEi; 10 μM). (D to F) GSDMD-expressing HEK293T lysates were incubated with purified or recombinant proteases for 30 min. (E) Point mutations are indicated in green; caspase cleavage site D275 is marked in purple. Lysates were incubated with NE. (F) Expression of putative NE-cleaved N-terminal fragments of human GSDMD in HEK293T cells. LDH release was measured 16 hours after transfection. Mean ± SEM of three independent experiments is depicted. (G) Lysates of HEK293T cells expressing GSDMD with different four amino acid deletions were incubated with NE or caspase-4. The number on the lane of the Western corresponds to the number in the graphical depiction of the deletions at the bottom of the panel (caspase-4 site is marked in purple). FL, full-length GSDMD. (C to E and G) GSDMD was detected by Western blotting using an antibody directed against full-length GSDMD. Arrows indicate active fragment. *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA with Dunnett’s multiple comparisons test.

  • Fig. 5 GSDMD is required for NETosis.

    (A) Murine peritoneal neutrophils of WT (n = 4) and GSDMD mutant (GSDMDmut; n = 3) mice were seeded and treated with 100 nM PMA for 6 hours, and NETs were analyzed by staining with the cell-permeable DNA dye SYTO Green and the cell-impermeable DNA dye SYTOX Orange. Mean ± SEM is depicted. **P < 0.01, unpaired two-tailed t test. (B) Representative images of WT and GSDMD mutant neutrophils activated with PMA for 6 hours and stained with an anti-chromatin antibody and the DNA dye DAPI. Scale bars, 25 μm.

  • Fig. 6 GSDMD regulates NE during NETosis.

    (A) Human primary neutrophils were activated with PMA or nigericin in the presence or absence of 1 μM LDC7559 and stained with antibodies against chromatin, MPO, and the DNA dye DAPI. Scale bars, 25 μm. (B) Neutrophils were treated with 1 μM LDC7559 or the indicated doses of pyrocatechol (3.75, 7.5, 15, and 30 μM); ROS production was determined by luminometry. (C) LDH release of neutrophils 3.5 hours after PMA induction in the presence or absence of LDC7559 or increasing doses of pyrocatechol. (D) LDH release of human primary neutrophils after 3.5 hours of activation with PMA. LDC7559 (1 μM) was added at the indicated time points before or after PMA stimulation. (E) Neutrophils were activated with PMA for 2 hours and stained with an antibody against NE and with phalloidin to stain the actin cytoskeleton. Scale bars, 25 μm. (F) Neutrophils were stimulated with PMA in the presence or absence of the NE inhibitor or LDC7559, and cells were harvested at 2.5 hours. GSDMD processing was analyzed by Western blot with an antibody directed against full-length GSDMD. (G) Neutrophil lysates were harvested 2 hours after PMA stimulation and analyzed by Western blotting using an anti–histone H3 antibody to show processing. Arrow indicates processing. (B to D) Mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA with Dunnett’s multiple comparisons test.

  • Fig. 7 GSDMD is required for nuclear expansion during NETosis.

    (A to D) Neutrophils were activated with PMA (A and C) or nigericin (B and D) and stained with the cell-permeable DNA dye DRAQ5 and the cell-impermeable DNA dye SYTOX Green. Images were acquired every 2 min for 6 hours. Single cells were selected semiautomatically; nuclear expansion and NET formation were quantified using an automated workflow in ImageJ and R. Bottom panels depict P values determined by unpaired two-tailed t tests at each time point, and red areas refer to P < 0.05. Mean ± SEM is depicted; n refers to the number of independent experiments. (A and B) NET formation of neutrophils activated with PMA (A) or nigericin (B). (C and D) Quantification of nuclear expansion over time in neutrophils stimulated with PMA (C) or nigericin (D). (E) Quantification of the time it took single cells to expand their nucleus upon PMA treatment in the presence or absence of LDC7559; horizontal lines depict the median. P = 1.5384 × 10−111, determined by Wilcoxon rank-sum test with continuity correction. (F) Quantification of the time it took single PMA-treated cells with an expanded nucleus to lyse; horizontal lines depict the median. LDC7559 treatment did not delay this process. P = 0.0044, determined by Wilcoxon rank-sum test with continuity correction. LDC7559 concentration was 5 μM in all experiments.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/3/26/eaar6689/DC1

    Materials and Methods

    Fig. S1. Screening strategy and titration curves.

    Fig. S2. NET-inhibiting compound LDC7559 and its derivative bind to GSDMD.

    Fig. S3. GSDMD cleavage and localization during NET formation.

    Fig. S4. TIRF microscopy of NE during NET formation.

    Fig. S5. NE processes GSDMD.

    Fig. S6. Mode of action of LDC7559.

    Fig. S7. Live-cell imaging of NET formation.

    Fig. S8. Model of GSDMD involvement in NET formation.

    Movie S1. Classification of cell states during NET formation.

    Movie S2. PMA-induced NET formation.

    Movie S3. PMA-induced NET formation in the presence of LDC7559.

    Movie S4. Nigericin-induced NET formation.

    Movie S5. Nigericin-induced NET formation in the presence of LDC7559.

    Table S1. Raw data.

    References (3234)

  • Supplementary Materials

    The PDF file includes:

    • Materials and Methods
    • Fig. S1. Screening strategy and titration curves.
    • Fig. S2. NET-inhibiting compound LDC7559 and its derivative bind to GSDMD.
    • Fig. S3. GSDMD cleavage and localization during NET formation.
    • Fig. S4. TIRF microscopy of NE during NET formation.
    • Fig. S5. NE processes GSDMD.
    • Fig. S6. Mode of action of LDC7559.
    • Fig. S7. Live-cell imaging of NET formation.
    • Fig. S8. Model of GSDMD involvement in NET formation.
    • Legends for movies S1 to S4
    • References (3234)

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.avi format). Classification of cell states during NET formation.
    • Movie S2 (.avi format). PMA-induced NET formation.
    • Movie S3 (.avi format). PMA-induced NET formation in the presence of LDC7559.
    • Movie S4 (.avi format). Nigericin-induced NET formation.
    • Movie S5 (.avi format). Nigericin-induced NET formation in the presence of LDC7559.
    • Table S1 (Microsoft Excel format). Raw data.

    Files in this Data Supplement:

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