Caspase-8-dependent gasdermin D cleavage promotes antimicrobial defense but confers susceptibility to TNF-induced lethality

Abstract

Gasdermin D (GSDMD) is a pore-forming protein that promotes pyroptosis and release of proinflammatory cytokines. Recent studies revealed that apoptotic caspase-8 directly cleaves GSDMD to trigger pyroptosis. However, the molecular requirements for caspase-8-dependent GSDMD cleavage and the physiological impact of this signaling axis are unresolved. Here, we report that caspase-8-dependent GSDMD cleavage confers susceptibility to tumor necrosis factor (TNF)-induced lethality independently of caspase-1 and that GSDMD activation provides host defense against Yersinia infection. We further demonstrate that GSDMD inactivation by apoptotic caspases at aspartate 88 (D88) suppresses TNF-induced lethality but promotes anti-Yersinia defense. Last, we show that caspase-8 dimerization and autoprocessing are required for GSDMD cleavage, and provide evidence that the caspase-8 autoprocessing and activity on various complexes correlate with its ability to directly cleave GSDMD. These findings reveal GSDMD as a potential therapeutic target to reduce inflammation associated with mutations in the death receptor signaling machinery.

Fig. 1. GSDMD inactivation at D88 does not suppress pyroptosis or IL-1β secretion upon canonical inflammasome activation.

(A to F) BMDMs were primed with LPS (100 ng/ml) for 4 hours and stimulated with nigericin (5 μM), ATP (2.5 mM), log-phase S. Typhimurium [multiplicity of infection (MOI) of 10], or poly(dA:dT) (1 μg/ml). Where indicated, BMDMs were treated with TNF (100 ng/ml)/TAK1i (125 nM) for 4 hours. (A and B) LDH and (C and D) IL-1β release was measured at the indicated time points. (E and F) Mixed supernatant and cell extract from (E) two C57BL/6 and two Gsdmd^D88A/D88A mice were examined by immunoblotting 1 hour after stimulation or (F) at the indicated time points. FL, full-length; long exp., long exposure. (G and H) Bone marrow neutrophils were primed with LPS (100 ng/ml) for 4 hours and infected with log-phase S. Typhimurium (MOI of 25). (G) LDH and (H) IL-1β release was measured 3 hours after infection. (I and J) Mice were challenged with 1 × 10⁵ colony-forming units (CFU) of log-phase S. Typhimurium by intraperitoneal injection, and bacterial burden was quantified 48 hours later. (A) Data are represented as means + SEM of cell stimulation from three mice or (B to D, G, and H) means + SD of triplicate cell stimulation from three independent experiments. (I and J) Data are geometric means and pooled from two independent experiments. Immunoblots are representative of three independent experiments.

Fig. 2. Caspase-8 activation on TNF Complex IIb is most efficient in directing GSDMD cleavage.

(A) BMDMs were costimulated with TNF (100 ng/ml)/TAK1i (125 nM), TNF (100 ng/ml)/CHX (10 μg/ml), LPS (100 ng/ml)/TAK1i (125 nM), or LPS (100 ng/ml)/CHX (10 μg/ml) for 6 hours, and mixed supernatant and cell extracts were analyzed by immunoblot. (B) BMDMs were primed with TNF (100 ng/ml) or LPS (100 ng/ml) for 3 hours before stimulation with TAK1i (125 nM) or CHX (10 μg/ml) for 6 hours, and mixed supernatant and cell extracts were analyzed by immunoblot. (C) BMDMs were costimulated with TNF (100 ng/ml)/TAK1i (125 nM) or TNF (100 ng/ml)/CHX (10 μg/ml) or primed with LPS (100 ng/ml) for 16 hours and treated with FasL (100 ng/ml) for 6 hours, and mixed supernatant and cell extracts were analyzed by immunoblot. (C) Blots are cropped from the same film. Immunoblots are representative of two to three independent experiments.

Fig. 3. Differential activation of caspase-8 Complex IIa, Complex IIb, and DISC.

(A to D) BMDMs were treated with were costimulated with TNF (100 ng/ml)/TAK1i (125 nM) or TNF (100 ng/ml)/CHX (10 μg/ml) or primed with LPS (100 ng/ml) for 16 hours and treated with FasL (100 ng/ml) for the indicated time points, and mixed supernatant and cell extracts were analyzed by immunoblot or (E to G) LDH release into the cell culture supernatant was quantified. (A to D) Immunoblots are representative of two to three independent experiments. (E to G) Data are means + SEM of pooled data from (E and F) six or (G) five independent experiments. Data were normally distributed and analyzed using a parametric t test. *P < 0.05 or **P < 0.01. UT, untreated.

Fig. 4. Caspase-8 dimerization and autoprocessing is required for GSDMD cleavage.

(A) HEK 293T cells were transfected with GSDMD together with WT caspase-8 (Casp8^WT), caspase-8 catalytic mutant (Casp8^{cat. mut.}), or uncleavable caspase-8 (Casp8^uncl.). Vector control was transfected such that each set of constructs received equivalent amounts of DNA. Twenty-four hours after transfection, transfected cells were treated with AP20187 (dimerizer) for a further 6 hours. Cell extracts (XT) and precipitated supernatants (S/N) were analyzed by immunoblotting for GSDMD and caspase-8. (B to G) WT or caspase-8 uncleavable (D387A) Casp8^D387A/D387A BMDMs were stimulated with TNF (100 ng/ml)/TAK1i (125 nM) or TNF (100 ng/ml)/CHX (10 μg/ml) or primed with LPS (100 ng/ml) for 16 hours and treated with FasL (100 ng/ml). Where indicated, cells were treated with GSK’872 (1 μM) 20 to 30 min before stimulation. LDH release was measured at (B and C) 4 hours or (D) 6 hours after stimulation. (E to G) Mixed supernatant (S/N) and cell extracts were examined by immunoblotting. (G) Blots are cropped from the same film. (E to G) Immunoblots are representative of three independent experiments. (B to D) Data are means + SEM of pooled data from four independent experiments. Data were normally distributed and analyzed using a parametric t test. *P < 0.05 or **P < 0.01.

Fig. 5. GSDMD drives TNF-induced lethality independently of inflammasomes.

(A to K) Mice were challenged intravenously with TNF (500 μg/kg). (A) Body temperature and (B) survival were monitored over time. (C) Plasma LDH was quantified at 4 hours after TNF challenge. A₄₉₀, absorbance at 490 nm. (D to K) Plasma cytokines were measured 3 hours after TNF challenge. CCL2, C-C motif chemokine ligand 2; KC, keratinocyte-derived chemokine; PBS, phosphate-buffered saline; ns, not significant; G-CSF, granulocyte colony-stimulating factor. (L) Mice were challenged intravenously with Fc-FAS (50 μg/kg) and monitored for lethality. The number of mice (n) used in each experiment are indicated. (A and B) Data are means + SEM pooled from three independent experiments, and (A) statistical analysis was performed using a two-way analysis of variance (ANOVA). (B) Survival curves were compared using log-rank Mantel-Cox test. (C to K) Data are geometric means from four to five mice per genotype and analyzed using a parametric t test. *P < 0.05; **P < 0.01.

Fig. 6. GSDMD-dependent cell death promotes anti-Yersinia defense in vivo.

(A) Unprimed or LPS-primed (100 ng/ml; 3 hours) BMDMs were infected with Y. pseudotuberculosis (Yptb) at an MOI of 10 and for 3 hours, and mixed supernatant and cell extracts were examined by immunoblotting. (B) Unprimed or (C) LPS-primed (100 ng/ml; 3 hours) BMDMs were infected with Y. pseudotuberculosis at the indicated MOI for 3 hours, and LDH release was quantified. (D) Unprimed BMDMs were infected with Y. pseudotuberculosis at the indicated MOI, and IL-1β release was measured 16 hours after infection. Where indicated, cells were treated with MCC950 (10 μM) 20 to 30 min before infection. (E) LPS-primed (100 ng/ml; 3 hours) BMDMs were challenged with Y. pseudotuberculosis at an MOI of 20, and IL-1β release was measured at the indicated time points. (F) BMDMs were infected with Y. pseudotuberculosis with the indicated MOI for 4 hours, and precipitated supernatant and cell extracts were analyzed by immunoblot. (G to J) Mice were challenged orally with 2 × 10⁸ CFU of Y. pseudotuberculosis, (G) immunofluorescence of infected spleen were analyzed (white arrows indicate large clusters surrounded by pyogranulomatous reaction), and (H and I) bacterial burden was quantified 5 days after infection. DAPI, 4′,6-diamidino-2-phenylindole. (J) Mouse survival was monitored for 12 days. (A and F) Immunoblots are representative of two to three independent experiments. (B, C, and E) Data are means + SEM of pooled data from three independent experiments. (D) Data are means + SD of triplicate cell stimulation and are a representative from four independent experiments. (H and I) Data are geometric means and pooled from two independent experiments. (G) Images are representative of four to five mice. (B and C) Data were normally distributed and analyzed using a parametric t test. (J) Survival curves were compared using log-rank Mantel-Cox test. (H and I) Data were analyzed using Mann-Whitney t tests. *P < 0.05, **P < 0.01, or ***P < 0.001.