Research ArticleCELL DEATH

Chemical disruption of the pyroptotic pore-forming protein gasdermin D inhibits inflammatory cell death and sepsis

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Science Immunology  24 Aug 2018:
Vol. 3, Issue 26, eaat2738
DOI: 10.1126/sciimmunol.aat2738
  • Fig. 1 NSA inhibits pyroptotic cell death in immortalized murine macrophages.

    (A) Chemical structure of NSA. (B to E) Cells were primed with LPS, followed by NSA treatment 30 min before stimulation with nigericin (NG) or ATP. Pyroptotic pore formation was measured kinetically through the uptake of PI, and cell lysis was measured by LDH release. PI uptake is taken from four experiments each with technical duplicates. LDH release is from three independent experiments, each done in technical duplicate. *P < 0.05, **P < 0.01, and ***P < 0.001. (F) Measurement of pyroptotic pore formation assessed by PI uptake with simultaneous administration of NSA with nigericin stimulation. Data represent four experiments each with two technical replicates. (G) Bright-field and epifluorescent microscopy images of iBMDM cells with or without NSA treatment before and after treatment with 10 μM nigericin. Scale bars, 100 μm. All cells were primed with LPS (1 μg/ml) for 4 hours. (B to F) PI and LDH data show means ± SE. Tx, treatment.

  • Fig. 2 NSA inhibits pyroptotic cell death downstream of multiple inflammasomes in human and murine cells.

    (A) mNeon-GSDMD was stably reconstituted into Gsdmd−/− iBMDM cells. Cells were primed with LPS (1 μg/ml), treated with DMSO or NSA, and activated with 10 μM nigericin. Live cell imaging was conducted on an inverted confocal microscope 90 min after stimulation. (B) IL-1β release from iBMDM cells stimulated with LPS and nigericin with DMSO, 10 μM NSA, or 20 μM NSA. IL-1β concentration was determined by sandwich ELISA 30 and 60 min after stimulation. **P < 0.01 and ****P < 0.0001. (C) Pore formation as measured by PI uptake in iBMDM cells treated with S. Typhimurium. iBMDMs were primed for 4 hours followed by activation of the NLRC4 inflammasome with log phase S. Typhimurium. (D and E) THP-1 cells were primed with LPS, followed by NSA treatment 30 min before stimulation with nigericin (NG). Pyroptotic pore formation and cell death were assessed through PI uptake and LDH release, respectively. **P < 0.01, ***P < 0.001, and ****P < 0.0001. (B to E) PI and LDH data are means ± SE and are representative of ≥3 experimental replicates.

  • Fig. 3 NSA inhibits pyroptosis in primary macrophages.

    Primary macrophages were isolated from C57BL/6J mice. (A to D) After priming with LPS (1 μg/ml) for 4 hours, cells were treated with increasing doses of NSA for 30 min before activation of the inflammasome with either 10 μM NG or C. difficile toxin B (TCDB) (0.4 μg/ml). One hour after activation, cells were visualized using bright-field and epifluorescent microscopy. PI was used at 1 μg/ml. Cell death was measured by LDH. (A to D) Data are representative of ≥3 experimental replicates. Scale bars, 100 μm. LDH data show means ± SE. **P < 0.01, ***P < 0.001.

  • Fig. 4 NSA does not inhibit other innate immune pathways.

    (A) iBMDM cells were treated with TNF-α, zVAD-fmk, and GDC-0152 for 24 hours in the presence or absence of 20 μM NSA. Cell death was measured by LDH release. (B and C) The impact of NSA on TLR pathways was assessed using RT-PCR of inflammatory gene transcripts after stimulation of TLR1/2 with Pam3CSK or TLR4 with LPS in the presence or absence of 20 μM NSA. (D) qRT-PCR was used to measure CXCL10 and IL-6 expression after infection with S. Typhimurium. Log phase S. Typhimurium was used to infect iBMDM cells at an MOI of 50:1 with induction of IL-6 and CXCL10 genes measured after 0 and 2 hours. Relative induction was based off of GAPDH as the housekeeping reference gene. Data are taken from technical triplicates and are representative of three independent experiments. **P < 0.01. (E) Cell death as measured by LDH assay in macrophages treated for 8 hours with 100 μM etoposide and DMSO, 10 μM NSA, or 20 μM NSA. **P < 0.01. (F) Pyroptotic pore formation as assessed by PI uptake in iBMDM cells after activation of the NLRP3 inflammasome with LPS and nigericin and treatment with 20 μM NSA, TCEP, iodoacetamide, or DTT. (G) iBMDM cells expressing mCerulean-ASC that form specks upon activation of the inflammasome were stimulated with LPS and nigericin in the presence of DMSO or NSA. Formation of specks was visualized using epifluorescent microscopy. Scale bars, 100 μm. (A to F) Data are means ± SE. n.s., not significant.

  • Fig. 5 NSA directly interacts with GSDMD.

    (A) LPS-primed iBMDM cells were stimulated with 10 μM nigericin in the presence or absence of NSA for 1 hour. Cleavage of full-length (FL)–GSDMD was assessed by Western blot. (B) The ability of NSA to inhibit formation of the pyroptotic pore was tested by transient expression of NTAP–p30-GSDMD in HEK-293T cells using calcium phosphate transfection. Four hours after transfection, medium was changed and DMSO or NSA was used to treat the cells. Twenty-four hours after transfection, LDH release was measured. Data are means ± SE taken from three technical replicates and are representative of three independent experiments. **P < 0.01 and ***P < 0.001. (C) Oligomerization with transient expression of GFP–p30-GSDMD expressed in HEK-293T cells was assessed by Western blot under nonreducing conditions after treatment with DMSO or 20 μM NSA. (D and E) Reciprocal in vitro binding assays of GSDMD to NSA-biotin using pulldown of biotin by streptavidin or pulldown of GSDMD with GSDMD antibody and analysis by Western blot. (F) Competitive binding assay of NSA with NSA-biotin and GSDMD. GFP-GSDMD was expressed in HEK-293T cells using calcium phosphate transfection. The in vitro competitive binding assay between NSA and NSA-biotin at a 5:1 ratio was conducted for 2 hours with analysis of binding by Western blot. (G) Surface plasmon resonance with recombinant GSDMD (immobilized) and NSA demonstrated a binding affinity of 32.0 ± 3.8 μM. IP, immunoprecipitation.

  • Fig. 6 Inhibition by NSA differs between MLKL and GSDMD.

    (A) Oligomerization of p30-GSDMD cysteine-to-alanine mutants conserved between mice and humans was tested in nonreducing conditions. (B) The cytotoxicity of p30-GSDMD cysteine-to-alanine mutants, expressed in HEK-293T cells by calcium phosphate transfection, was assessed by LDH release. (C) In vitro binding assays between GFP-GSDMD cysteine-to-alanine mutants and NSA-biotin with analysis by Western blot. (D) HEK-293T cells were transfected with either WT p30-GSDMD or C191A p30-GSDMD and either left untreated or treated with NSA. While NSA decreased WT p30-GSDMD–mediated cell death, the decrease in C191A p30-GSDMD was not statistically significant. (E) Models for the full-length human and murine GSDMDs showing the location of the conserved Cys residues that may engage NSA and are important for oligomerization. The N-terminal domains for human GSDMD and murine GSDMD are shown in ribbons and colored salmon and blue-green, respectively. The C-terminal domains are shown in surface and gray ribbons. Residues Cys191 (human GSDMD) and Cys192 (murine GSDMD) and two surrounding residues are shown as ball-and-stick models, which demonstrate that, while the surrounding residues engage in the N- and C-domain interface, the Cys residues are exposed to the solvent. Approximate locations of the interface (top) in the full-length GSDMD structures are marked in red on the bottom. (B and D) LDH data are means ± SE. Ab, antibody. *P < 0.05, **P < 0.01.

  • Fig. 7 NSA treatment extends survival in the LPS model of sepsis.

    (A to C) C57BL/6J mice (9 or 10 per group) were injected with LPS (25 mg/kg) and with NSA or DMSO (20 mg/kg) 30 min before and 10 hours after LPS administration. Volume per injection of NSA or DMSO was about 25 μl per mouse. Serum levels of IL-6 and IL-1β, measured by sandwich ELISA, were taken 6 hours after the administration of LPS. Cytokine data are means ± SE. Survival curves were analyzed by log-rank (Mantel-Cox) test demonstrating an increase in median survival of 9 hours in the NSA treatment group (P <0.0001). *P < 0.05, **P < 0.01.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/3/26/eaat2738/DC1

    Fig. S1. Etoposide-induced cell death in macrophages is independent of NSA.

    Fig. S2. Purification of human GSDMD.

    Fig. S3. Conserved cysteine residues between human and murine GSDMD in the p30-GSDMD fragment.

    Fig. S4. Single-dose NSA prolongs survival in the LPS model of sepsis.

    Fig. S5. NSA inhibits p30-GSDMD pore formation and cell death.

    Table S1. Raw data sets.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. Etoposide-induced cell death in macrophages is independent of NSA.
    • Fig. S2. Purification of human GSDMD.
    • Fig. S3. Conserved cysteine residues between human and murine GSDMD in the p30-GSDMD fragment.
    • Fig. S4. Single-dose NSA prolongs survival in the LPS model of sepsis.
    • Fig. S5. NSA inhibits p30-GSDMD pore formation and cell death.

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    Other Supplementary Material for this manuscript includes the following:

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

    Files in this Data Supplement:

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