Article Figures & Data
Figures
- Figure S1.
Canonical and non-canonical inflammasome activation in Gsdmd−/− cells. (A, B) LDH release from LPS-primed WT, Asc−/−, Casp1−/−/Casp11−/−, and Gsdmd−/− iBMDMs after transfection of LPS (A) or infection with log-phase S. typhimurium, treatment with Nigericin, or infection with F. novicida (B). (C) PI influx from mock or poly(dA:dT)–transfected LPS-primed Gsdmd−/− iBMDMs and LDH release from mock or poly(dA:dT) transfected LPS-primed WT, Asc−/−, Casp1−/−/Casp11−/−, and Gsdmd−/− iBMDMs. (D) Immunoblots showing IL-1β, caspase-1, and GSDMD processing in WT, Asc−/−, Casp1−/−/Casp11−/−, and Gsdmd−/− BMDMs after transfection of poly(dA:dT). Data and blot are representative of at least three independent experiments.
- Figure 1. Canonical inflammasome activation Gsdmd-deficient macrophages results in rapid secondary necrosis.
(A, B) LDH release, PI influx, and IL-1β release from LPS-primed WT, Asc−/−, Casp1−/−/Casp11−/−, and Gsdmd−/− primary or immortalized BMDMs (BMDMs and iBMDMs) after transfection of poly(dA:dT) in the absence or presence of the indicated inhibitors. (C, D, E) DNA cleavage, PI influx, and immunoblots showing caspase-3/-7 processing from LPS-primed Gsdmd−/− BMDMs transfected with poly(dA:dT) or treated with 100 ng/ml TNF-α plus 10, 5, or 1 μM AZD5582 (extrinsic apoptosis) or 1 μM ABT-737 plus 10, 1, or 0.5 μM S63845 (intrinsic apoptosis). (F) Confocal images of LPS-primed Gsdmd−/− BMDMs transfected with poly(dA:dT) or left untreated and stained with CellTox Green (green). Scale bar = 10 μM. Graphs show mean ± SD. Data and blot are representative of at least three independent experiments.
- Figure S2. Effect of inhibitor treatment on cell death in Gsdmd−/− cells.
PI influx from poly(dA:dT)–transfected LPS-primed Gsdmd−/− iBMDMs in the presence or absence of the indicated inhibitors added to the cells 30 min prior and during the experiment at the following concentrations: 50, 25, 12.5, and 6.25 μM VX765; 100, 50, 25, and 12.5 μM Caspase-3/7–specific inhibitor I; 30, 15, 7.5, and 3.25 μM K777; 100, 50, 25, and 12.5 μM PD 150606; 100, 50, 25, and 12.5 μM Calpeptin; 60, 30, 15, and 7.5 μM 7-Cl-O-Nec1; and 100, 50, 25, and 12.5 μM GSK872. Data and blot are representative of at least three independent experiments.
- Figure S3.
AIM2 activation in Gsdmd−/− results in rapid lytic cell death. (A, B) Confocal microscopy images of poly(dA:dT) (A) or mock transfected (B) WT, Asc−/−, Casp1−/−/Casp11−/−, and Gsdmd−/− primary BMDMs. Cells were stained with CellTox Green (green) and AnnexinV (red). (A, C) Selected cells from (A). Scale bar = 10 μm.
- Figure 2. Caspase-3 drives GSDMD-independent secondary necrosis in inflammasome activated cells.
(A, B) LDH release, caspase-3/-7 activity (DEVDase activity) and immunoblots showing caspase-3/-7 processing from LPS-primed WT, Asc−/−, Casp1−/−/Casp11−/−, and Gsdmd−/− primary BMDMs after transfection of poly(dA:dT). (C) LDH release from LPS-primed WT, Asc−/−, Casp1−/−/Casp11−/−, Gsdmd−/−, Gsdmd−/−/Casp3−/−, Gsdmd−/−/Casp7−/−, and Gsdmd−/−/Casp3−/−/Casp7−/− iBMDMs after transfection of poly(dA:dT). (C, D) Confocal images of cells from (C). Insets show membrane ballooning in dying cells at 3 h post-transfection. Scale bar = 10 μm. (E) Quantification of LDH release in LPS primed WT, Asc−/−, Casp-1−/−/Casp-11−/−, Gsdmd−/−, Gsdme−/−, and Gsdmd−/−/Gsdme−/− BMDMs transfected with poly(dA:dT) for 4 h. Graphs show mean ± SD. **P ≤ 0.01, ***P ≤ 0.001, “ns,” no significance (unpaired t test). Data and blot are representative of at least three independent experiments.
- Figure S4.
Caspase-3 but not caspase-7 is required for lysis in Gsdmd−/− cells. (A) LDH release and caspase-3/-7 activity (DEVDase activity) from LPS-primed WT, Asc−/−, Casp1−/−/Casp11−/−, and Gsdmd−/− primary BMDMs after transfection of Salmonella typhimurium at MOI 10. (B) Immunoblots for caspase-3 and caspase-7 expression in lysates of Gsdmd−/−, Gsdmd−/−/Casp3−/−, Gsdmd−/−/Casp7−/−, and Gsdmd−/−/Casp3−/−/Casp7−/− iBMDMs. (C) PI influx from LPS-primed WT, Asc−/−, Casp1−/−/Casp11−/−, Gsdmd−/−, pool of Gsdmd−/−/Casp3−/− clones (n = 3), pool of Gsdmd−/−/Casp7−/− clones (n = 5), and Gsdmd−/−/Casp3−/−/Casp7−/− iBMDMs after transfection of poly(dA:dT). (D) Immunoblots showing caspase-3, caspase-7, and caspase-8 expression and LDH release after transfection poly(dA:dT) transfection from LPS-primed Gsdmd−/− iBMDMs transfected with control siRNA or siRNA targeting caspase-3, caspase-7, caspase-8 or both caspase-3/-7. Graphs show mean ± SD. **P ≤ 0.01, ***P ≤ 0.001 (unpaired t test). Data and blot are representative of at least three independent experiments.
Source data are available for this figure.
Source Data for Figure S4[LSA-2020-00735_SdataFS4.pdf]
- Figure S5.
GSDME is not required for GSDMD-independent secondary necrosis. (A) Quantification of PI uptake in LPS primed WT, Asc−/−, Casp-1−/−/Casp-11−/−, Gsdmd−/−, Gsdme−/−, and Gsdmd−/−/Gsdme−/− BMDMs transfected with poly(dA:dT) for 4 h. (B) Immunoblot for GSDMD, GSDME, caspase-1, and tubulin on pooled supernatant and lysate samples collected at indicated times. Graph shows mean ± SD. Graph and blot are representative of at least three independent experiments.
- Figure 3. Caspase-1 is required for GSDMD-independent secondary necrosis.
(A, B, C) Immunoblot showing Caspase-1 expression and caspase-3 processing, LDH release, and caspase-3/-7 activity (DEVDase activity) from LPS-primed Gsdmd−/− and Gsdmd−/−/Casp1−/− immortalized BMDMs after transfection of poly(dA:dT). (D) PI influx of WT, Asc−/−, Casp1−/−/Casp11−/−, Casp1−/−, Casp1C284A/C284A, and Gsdmd−/− primary BMDMs after transfection of poly(dA:dT). Graphs show mean ± SD. **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001 (unpaired t test). Data and blot are representative of at least three independent experiments.
- Figure S6.
Inflammasome activation in Gsdmd−/− results in rapid mitochondrial outer membrane permeabilization. (A) Confocal microscopy images showing MitoTracker Green (Green) and MitoTracker Red (Red) and DIC, 30 and 60 min post-poly(dA:dT) transfection into LPS-primed Gsdmd−/− and Asc−/− iBMDMs. (B, C, D) LDH release and Titer-Glo measurements at 0, 15, 30, and 45 min or as indicated from LPS-primed WT, Asc−/−, Casp1−/−/Casp11−/−, and Gsdmd−/− primary BMDMs after transfection with poly(dA:dT) (C) or infection with log-phase Salmonella typhimurium at MOI 10 (B, D). Scale bar = 10 μm. Graphs show mean ± SD. Data and blot are representative of at least three independent experiments.
- Figure 4. Mitochondrial damage is caused by truncated Bid.
(A) LDH release and Titer-Glo measurements from LPS-primed WT, Asc−/−, Casp1−/−/Casp11−/−, and Gsdmd−/− primary BMDMs after transfection with poly(dA:dT). (B) Schematic cleavage profile of Bcl-2 family members generated from slice SILAC data. Bubble diameters are proportional to the number of quantified peptide matches, whereas the gradient color represents the H/L ratio, as indicated below. The green bubbles (negative log2H/L) represent protein isoforms reduced in Gsdmd−/− iBMDMs compared with Asc−/− iBMDMs at 3 h post-poly(dA:dT) transfection; red bubbles (positive log2H/L) represent protein isoforms enriched in Gsdmd−/− iBMDMs compared with Asc−/− iBMDMs. (C) Immunoblots showing Bid processing from LPS-primed WT, Asc−/−, Casp1−/−/Casp11−/−, and Gsdmd−/− primary BMDMs after transfection with poly(dA:dT). (D) LDH release from WT, Asc−/−, Casp1−/−/Casp11−/−, Gsdmd−/−, and Gsdmd−/−/Bid−/− iBMDMs after transfection with poly(dA:dT). Graphs show mean ± SD. *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001, “ns,” no significance (unpaired t test). Data and blots are representative of at least three independent experiments.
- Figure S7.
Gsdmd−/−/Bid−/− cells are resistant to canonical inflammasome activation. (A) Immunoblots showing Bid expression in Gsdmd−/− and Gsdmd−/−/Bid−/− iBMDMs. (B, C) Caspase-3/-7 activity (DEVDase activity) and PI influx in Gsdmd−/− and pool of Gsdmd−/−/Bid−/− clones (n = 5) iBMDMs after transfection with poly(dA:dT). (D) Confocal images of cells of Gsdmd−/− and Gsdmd−/−/Bid−/− iBMDMs after transfection with poly(dA:dT) for 3 h. Insets show membrane ballooning in dying cells. Scale bar = 10 μm. Graphs show mean ± SD. Data and blot are representative of at least three independent experiments.
- Figure 5. Caspase-1 drives Bid processing during GSDMD-independent secondary necrosis.
(A) Immunoblots showing caspase-1, caspase-7, caspase-3, caspase-8, and caspase-9 processing in WT, Asc−/−, Casp1−/−/Casp11−/−, and Gsdmd−/− primary BMDMs after transfection with poly(dA:dT). (B) LDH release from WT, Asc−/−, Casp1−/−/Casp11−/−, Gsdmd−/−, and Gsdmd−/−/Casp8−/− iBMDMs after transfection with poly(dA:dT). (C) Immunoblots showing caspase-3 processing in Gsdmd−/− and Gsdmd−/−/Casp8−/− iBMDMs after transfection with poly(dA:dT). (D) Immunoblots showing Bid cleavage in Gsdmd−/− and Gsdmd−/−/Casp8−/− iBMDMs after transfection with poly(dA:dT). (E) Immunoblots showing Bid cleavage in Gsdmd−/− and Gsdmd−/−/Casp1−/− iBMDMs after transfection with poly(dA:dT). (F) In vitro cleavage assay showing processing of recombinant Bid by recombinant caspase-1. Graphs show mean ± SD. * “ns,” no significance (unpaired t test). Data and blot are representative of at least three independent experiments.
- Figure S8.
Caspase-8 is not required for GSDMD-independent secondary necrosis. (A) Immunoblots showing caspase-1, caspase-7, caspase-3, caspase-8, and caspase-9 processing in WT, Asc−/−, Casp1−/−/Casp11−/−, and Gsdmd−/− primary BMDMs after transfection with poly(dA:dT). (B) Immunoblots showing caspase-8 expression in Gsdmd−/− and Gsdmd−/−/Casp8−/− iBMDMs. (C) LDH release in LPS-primed Gsdmd−/− and Gsdmd−/−/Casp8−/− iBMDMs after transfection with poly(dA:dT) in the presence of 100 ng/ml TNF−α, the SMAC mimetic AZD5582 (5 μM), or AZD5582/GSK872. (D) PI influx in Gsdmd−/− and pool of Gsdmd−/−/Casp8−/− clones (n = 3) iBMDMs after transfection with poly(dA:dT). Graphs show mean ± SD. Data and blot are representative of at least three independent experiments.
- Figure S9.
SMAC release and initiator caspases-8/-9 are required for GSDMD-independent secondary necrosis. (A) Immunoblots showing Casp-9 expression in Gsdmd−/− and Gsdmd−/−/Casp9−/− iBMDMs. (B) PI uptake of Gsdmd−/− and Gsdmd−/−/Casp9−/− iBMDMs treated with 2 μM ABT737 and 2 μM S63845. (C) PI uptake in LPS-primed and poly(dA:dT)–transfected Gsdmd−/− and pool of Gsdmd−/−/Casp9−/− clones (n = 2) iBMDMs. (D) Immunoblots showing Casp-8 and Casp-9 expression in Gsdmd−/− and Gsdmd−/−/Casp8−/−/Casp9−/− iBMDMs. (E) Immunoblots showing caspase-3 processing in Gsdmd−/− and Gsdmd−/−/Bid−/− iBMDMs after transfection with poly(dA:dT). (F) Immunoblots showing caspase-3 processing in Gsdmd−/− and Gsdmd−/−/Bid−/− iBMDMs after transfection with poly(dA:dT) and treatment with AZD5582. Graphs show mean ± SD. Data and blot are representative of at least three independent experiments.
- Figure 6. SMAC release and initiator caspases-8/-9 are required for GSDMD-independent secondary necrosis.
(A) LDH release from WT, Asc−/−, Casp1−/−/Casp11−/−, Gsdmd−/−, and Gsdmd−/−/Casp9−/− iBMDMs after transfection with poly(dA:dT). (B) Immunoblots showing caspase-3 cleavage in Gsdmd−/− and Gsdmd−/−/Casp9−/− iBMDMs after transfection with poly(dA:dT). (C) LDH release from WT, Asc−/−, Casp1−/−/Casp11−/−, Gsdmd−/−, Gsdmd−/−/Casp8−/−, Gsdmd−/−/Casp9−/−, and Gsdmd−/−/Casp8−/−/Casp9−/− iBMDMs after transfection with poly(dA:dT). (D) Immunoblots showing caspase-3 cleavage in Gsdmd−/−, Gsdmd−/−/Casp8−/−, Gsdmd−/−/Casp9−/−, and Gsdmd−/−/Casp8−/−/Casp9−/− iBMDMs after transfection with poly(dA:dT). (E) Immunoblots showing caspase-3 processing from Gsdmd−/− and Gsdmd−/−/Bid−/− iBMDMs after transfection with poly(dA:dT). (F) Schematic summary of the mechanism of caspase-3 cleavage and activation. (G) PI influx in untreated or poly(dA:dT)–transfected Gsdmd−/−/Bid−/− iBMDMs in the presence or absence of the SMAC mimetic AZD5582. Graphs show mean ± SD. *P ≤ 0.05, **P ≤ 0.01 (unpaired t test). Data and blot are representative of at least three independent experiments.
- Figure 7. Model of cell death in Gsdmd-deficient myeloid cells after activation of caspase-1.
Model depicting the mechanism of canonical inflammasome activation in WT cells undergoing caspase-1– and GSDMD-dependent pyroptosis and Gsdmd−/− cells undergoing caspase-1–induced GSDMD-independent secondary necrosis.
