GSDME-Dependent Incomplete Pyroptosis Permits Selective IL-1α Release under Caspase-1 Inhibition

Abstract

Pyroptosis is a form of regulated cell death that is characterized by gasdermin processing and increased membrane permeability. Caspase-1 and caspase-11 have been considered to be essential for gasdermin D processing associated with inflammasome activation. In the present study, we found that NLRP3 inflammasome activation induces delayed necrotic cell death via ASC in caspase-1/11-deficient macrophages. Furthermore, ASC-mediated caspase-8 activation and subsequent gasdermin E processing are necessary for caspase-1-independent necrotic cell death. We define this necrotic cell death as incomplete pyroptosis because IL-1β release, a key feature of pyroptosis, is absent, whereas IL-1α release is induced. Notably, unprocessed pro-IL-1β forms a molecular complex to be retained inside pyroptotic cells. Moreover, incomplete pyroptosis accompanied by IL-1α release is observed under the pharmacological inhibition of caspase-1 with VX765. These findings suggest that caspase-1 inhibition during NLRP3 inflammasome activation modulates forms of cell death and permits the release of IL-1α from dying cells.

Keywords: Cell Biology; Functional Aspects of Cell Biology; Immunology.

Graphical abstract

Figure 1

Nigericin Induces Caspase-1/11-Independent Necrotic Cell Death via ASC (A–C) Primary peritoneal macrophages isolated from WT, Casp1/11^−/−, and Asc^–/–Casp1/11^−/− mice were rested or primed with Pam3CSK4 (100 ng/mL) for 18 h and then treated with nigericin (5 μM) for 1, 3, or 6 h. (A) The levels of LDH in the supernatants were assessed. (B) The levels of IL-1β and (C) IL-1α in the supernatants were assessed by ELISA. (D and E) Primed WT, Casp1/11^−/−, and Asc^–/–Casp1/11^−/− macrophages were labeled with Hoechst33342 and then treated with nigericin in the presence of SYTOXG. (D) Relative fluorescence units of SYTOXG were measured at 10-min intervals. (E) Images of Hoechst staining (upper left), SYTOXG staining (bottom left), merged images of Hoechst and SYTOXG (upper right), and merged images of fluorescent and bright fields (bottom right) were visualized by confocal microscopy. White arrows indicate dead Casp1/11^–/– macrophages. (F–I) Control, ASC KO, and CASP1 KO THP-1 cells were differentiated with PMA for 48 h and then treated with nigericin (5 μM). (F) The levels of LDH in the supernatants 8 h after nigericin stimulation were assessed. (G) Relative fluorescence units of SYTOXG in (G) Control, (H), ASC KO, (I) and CASP1KO THP1 cells were measured at 30-min intervals. Data represent mean ± SD of three (A and D) or four (F) independent experiments. (B, C, and G–I) Data are shown as mean ± SD of triplicate of one experiment. (B, C, E, and G–I) Data are representative of two independent experiments. (A–C, F, G, I) ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 as determined by two-way ANOVA with a post hoc test. (D) ∗∗∗p < 0.001 compared with WT Pam3 and WT Pam3+Nig, #p < 0.05, ##p < 0.01, ###p < 0.001 compared with Casp1/11^–/– Pam3 and Casp1/11^–/– Pam3+Nig as determined by two-way ANOVA with a post hoc test. n.s., not significant.

Figure 2

NLRP3 Inflammasome Activation Induces Necrotic Cell Death in the Absence of Caspase-1 (A–F) Control, ASC KO, and CASP1KO THP1 NLRP3 D303N cells were differentiated with PMA for 48 h and then treated with DOX (1 μg/mL). (A) After 6 h, lysates and supernatants were analyzed by western blot. (B) The levels of LDH in the supernatants at the indicated time points were assessed. (C and D) Cells were treated with DOX in the presence of SYTOXG. (C) Merged images of hKO1, SYTOXG, and Hoechst33342 were visualized by confocal microscopy. (D) High-magnification images of DOX-treated CASP1KO THP1 NLRP3 D303N cells. Images were visualized as merged images of fluorescence (right panels) and merged images of fluorescence and bright fields (left panels). (E–G) Relative fluorescence units of SYTOXG in (E) Control, (F) ASC KO, and (G) CASP1KO THP1 NLRP3 D303N cells were measured at 30-min intervals. Data are shown as mean ± SD of triplicate (B) or pentaplicate (E–G) of one experiment. (A–G) Data are representative of two independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 as determined by two-way ANOVA with a post hoc test.

Figure 3

Other Caspases Are Involved in Caspase-1/11-Independent Necrotic Cell Induced by NLRP3 Inflammasome Activation (A–C) Pam3CSK4-primed WT and Casp1/11^−/− macrophages were pretreated with Z-VAD (20 μM) and then treated with nigericin (5 μM). (A) The levels of LDH in the supernatants were assessed 3 h after nigericin treatment. (B) Relative fluorescence units of SYTOXG were measured at 30-min intervals. (C) Images were visualized by confocal microscopy. (D–G) Pam3CSK4-primed WT and Casp1/11^−/− macrophages were pretreated with Z-VAD and GSK′872 (3 μM) and then treated with nigericin. (D) Relative fluorescence units of SYTOXG were measured at 30-min intervals. (E) The levels of LDH in the supernatants were assessed. (F) The levels of IL-1α and (G) IL-1β in the supernatants were assessed by ELISA. (A, B, and D–F) Data are shown as mean ± SD of triplicate of one experiment. Data are representative of two (B, C, F, and G) or three (A, D, and E) independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 as determined by two-way ANOVA with a post hoc test.

Figure 4

GSDME Is Processed in Caspase-1 Caspase-1/11-Independent Necrotic Cell Induced by NLRP3 Inflammasome Activation (A) Primary peritoneal macrophages isolated from WT, Casp1/11^−/−, and Asc^–/–Casp1/11^−/− mice were rested or primed with Pam3CSK4 (100 ng/mL) for 18 h and then treated with nigericin (5μM) for 3 h. Lysates and supernatants were analyzed by western blot. (B) Control, ASCKO, and CASP1 KO THP1 cells were differentiated with PMA for 48 h and treated with nigericin (5 μM) for 8 h. Lysates and supernatants were analyzed by western blot. (C) Control, ASCKO, and CASP1KO THP1 NLRP3 D303N cells were differentiated with PMA for 48 h and treated with DOX (1 μg/mL) for 18 h. Lysates and supernatants were analyzed by western blot. (D) Primed WT and Casp1/11^−/− macrophages were treated with nigericin for 3 h. Cells were lysed with Triton X-114 and separated into an aqueous phase and detergent phase. Each fraction was precipitated by acetone and analyzed by western blot. (E and F) Pam3CSK4-primed WT macrophages were stimulated with nigericin. (E) After 3 h, cells were lysed with Triton X and supernatants were analyzed by western blot. (F) After 30 min, cell lysates were cross-linked with BS3 and analyzed by western blot. (G) THP1 NLRP3 D303N/hIL1B cells were differentiated with PMA for 48 h and treated with DOX for 6 h. Lysates and supernatants were analyzed by western blot. (A–G) Data are representative of two independent experiments.

Figure 5

Caspase-8 Initiates Gasdermin E Processing during NLRP3 Inflammasome Activation (A) Pam3CSK4-primed WT and Casp1/11^−/− macrophages were pretreated with Z-VAD (20 μM) and then treated with nigericin (5 μM) for 3 h. Lysates and supernatants were analyzed by western blot. (B–D) Pam3CSK4-primed WT and Casp1/11^−/− macrophages were pretreated with DEVD and IETD (20 μM each) and then treated with nigericin. (B) After 6 h, the levels of LDH in the supernatants were assessed. (C) Relative fluorescence units of SYTOXG were measured at 10-min intervals. (D) After 3 h, lysates and supernatants were analyzed by western blot. (E and F) Control, CASP1 KO, and CASP1 and CASP8 double-KO THP1 cells were differentiated with PMA for 48 h and then treated with nigericin (5 μM) for 8 h (E) LDH release in supernatant was assessed. (F) Lysates and supernatants were analyzed by western blot. (B, C, and E) Data are shown as mean ± SD of triplicate of one experiment. Data are representative of two (A and D–F) or three (B and C) independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 as determined by two-way ANOVA with a post hoc test. n.s., not significant.

Figure 6

GSDME Serves as an Alternative Gasdermin in NLRP3 Inflammasome Activation (A–C) HeLa cells were transfected with indicated plasmids and cultured for 24 h. (A) Lysates and supernatants were analyzed by western blot. (B) LDH release in supernatant was assessed. (C) Cells were stained with SYTOXG and Hoechst33342 and analyzed by confocal microscopy. (D and E) CASP3 KO HeLa cells were transiently transfected. (D) LDH release in supernatant was assessed. (E) Lysates and supernatants were analyzed by western blot. (F) CASP1 KO and CASP1 and CASP3 double-KO THP1 cells were differentiated with PMA for 48 h and then treated with nigericin (5 μM) for 8 h. Lysates and supernatants were analyzed by western blot. (G–I) Control, GSDMD KO, GSDME KO, and GSDMD and GSDME double-KO THP1 cells were differentiated with PMA for 48 h and treated with nigericin (5 μM) for 8 h. (G) Lysates and supernatants were analyzed by western blot. (H) LDH release in supernatant was assessed. (I) Relative fluorescence units of SYTOXG were measured at 30-min intervals. (B, D, H, and I) Data are shown as mean ± SD of triplicate of one experiment. (A–H) Data are representative of two independent experiments. ∗∗p < 0.01, ∗∗∗p < 0.001 as determined by two-way ANOVA with a post hoc test.

Figure 7

Pharmacological Inhibition of Casp1 Activation Dissociates IL-1β Release and Pyroptosis (A–G) Pam3CSK4-primed WT peritoneal macrophages were pretreated with VX-765 (1–10 μM) or MCC950 (0.03–0.3 μM) for 30 min and then treated with nigericin (5 μM). Relative fluorescence units of SYTOXG in (A)VX765-treated cells and (B) MCC950-treated cells were measured at 30-min intervals. (C) The levels of LDH in the supernatants were assessed. (D and E) The levels of IL-1α and (F and G) IL-1β in the supernatants were assessed by ELISA. (A–G) Data are shown as mean ± SD of triplicate of one experiment and representative of two independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 as determined by two-way ANOVA with a post hoc test.