Extrinsic and intrinsic apoptosis activate pannexin-1 to drive NLRP3 inflammasome assembly

Abstract

Pyroptosis is a form of lytic inflammatory cell death driven by inflammatory caspase-1, caspase-4, caspase-5 and caspase-11. These caspases cleave and activate the pore-forming protein gasdermin D (GSDMD) to induce membrane damage. By contrast, apoptosis is driven by apoptotic caspase-8 or caspase-9 and has traditionally been classified as an immunologically silent form of cell death. Emerging evidence suggests that therapeutics designed for cancer chemotherapy or inflammatory disorders such as SMAC mimetics, TAK1 inhibitors and BH3 mimetics promote caspase-8 or caspase-9-dependent inflammatory cell death and NLRP3 inflammasome activation. However, the mechanism by which caspase-8 or caspase-9 triggers cell lysis and NLRP3 activation is still undefined. Here, we demonstrate that during extrinsic apoptosis, caspase-1 and caspase-8 cleave GSDMD to promote lytic cell death. By engineering a novel Gsdmd D88A knock-in mouse, we further demonstrate that this proinflammatory function of caspase-8 is counteracted by caspase-3-dependent cleavage and inactivation of GSDMD at aspartate 88, and is essential to suppress GSDMD-dependent cell lysis during caspase-8-dependent apoptosis. Lastly, we provide evidence that channel-forming glycoprotein pannexin-1, but not GSDMD or GSDME promotes NLRP3 inflammasome activation during caspase-8 or caspase-9-dependent apoptosis.

Keywords: NLRP3; apoptosis; gasdermin; pannexin‐1; pyroptosis.

Conflict of interest statement

A.B and C.J.F are employees of Novartis, Inc.

© 2019 The Authors.

Figures

Figure 1. Extrinsic apoptosis trigger GSDMD‐dependent and caspase‐3/7‐dependent necrosis

1. A–E

   Primary (A, B) or immortalized BMDMs (D, E) were stimulated with recombinant murine TNF (100 ng/ml) in combination with (A, D) SM or (B, E) TAK1i for 6 or 4 h, respectively. (C) Time‐lapse confocal images (hour:min) of BMDMs stimulated with recombinant murine TNF (100 ng/ml) and SM (250 nM) stained with propidium iodide (red) for 6 h. Black arrowheads indicate membrane ballooning, while white arrowheads indicate apoptotic bodies.

Data information: Data are means ± SEM of pooled data from (A‐B) five or (D‐E) eight independent experiments. Statistical analyses for normally distributed data sets were analysed using the parametric t‐test, whereas non‐normally distributed data sets were analysed using non‐parametric Mann–Whitney t‐tests. Data were considered significant when *P < 0.05, **P < 0.01, ***P < 0.001 or ****P < 0.0001. (C) Data are representative of three independent experiments.

Figure 2. Extrinsic apoptosis triggers caspase‐1/11‐independent GSDMD processing and GSDMD/E‐independent NLRP3 activation

1. A, B

   BMDMs were stimulated with TNF (100 ng/ml) in combination with TAK1i (125 nM) for the indicated time points. (A) LDH release and (B) mixed supernatant and cell extracts were analysed.

2. C

   Representation of known caspase cleavage site and molecular weight of corresponding cleavage fragment in mouse GSDMD.

3. D

   BMDMs were costimulated with TNF (100 ng/ml) and TAK1i (125 nM) for 4 h in the presence or absence of KCl (50 mM). Where indicated, cells were pre‐incubated with MCC950 (10 μM) 20–30 min prior to TNF/TAK1i stimulation.

4. E–G

   BMDMs were costimulated with TNF (100 ng/ml) and SM (E) (250 nM; 6 h), mixed supernatant and cell extracts were analysed by immunoblot, or (F) LDH release in the cell culture supernatant was quantified at the indicated time points.

Data information: Data are means ± SEM of pooled data from (A) four or (F) five independent experiments. Statistical analyses were performed using a two‐way ANOVA. Data were considered significant when *P < 0.05, **P < 0.01, ***P < 0.001 or ****P < 0.0001. All immunoblots are representative of three independent experiments.Source data are available online for this figure.

Figure EV1. Caspase‐8 triggers NLRP3/caspase‐1‐independent GSDMD processing

1. A

   BMDMs were costimulated with TNF (100 ng/ml) and SM (500 nM) for the indicated time points, and LDH release was quantified.

2. B, C

   Immortalized BMDMs were (B) costimulated with TNF (100 ng/ml) and SM (500 nM) for 6 h or (C) primed with ultrapure E. coli K12 LPS (100 ng/ml) for 3 h prior to stimulation with the SMAC‐mimetic LCL161 (1 μM) for a further 16 h. Mixed supernatant and extracts were analysed by immunoblot.

3. D

   BMDM were costimulated with ultrapure E. coli K12 LPS (100 ng/ml) or TNF (100 ng/ml) and TAK1i (125 nM) for 2 or 4 h, and mixed supernatant and extracts were analysed by immunoblot.

4. E

   BMDMs were costimulated with TNF (100 ng/ml) and SM (500 nM) for 6 h, and mixed supernatant and extracts were analysed by immunoblot.

5. F

   BMDMs were costimulated with TNF (100 ng/ml) and TAK1i (125 nM), and LDH release was quantified at 4 h.

Data information: Data are means ± SEM of pooled data from (A) three to six or (F) four individual experiments. (A) Statistical analyses were performed using a two‐way ANOVA (F) or a non‐parametric Mann–Whitney t‐tests. Data were considered significant when *P < 0.05, **P < 0.01 and ***P < 0.001. Immunoblots are representative of two (E) or three (B–D) individual experiments.Source data are available online for this figure.

Figure EV2. Caspase‐8 cleaves GSDMD at a lower efficiency than caspase‐1

1. BMDMs were stimulated with TNF (100 ng/ml) and SM (500 nM) for 6 h, and mixed supernatant and extracts were analysed by immunoblot, representative of three independent experiments.

2. Caspase‐1 expression in HEK293T versus HeLa cells.

3. Amino acid sequence of human gasdermin D. The fluorescence lifetime substrate Ac‐Cys(Pt14)‐FLTD^GVPY‐NH2 was designed around D276 as highlighted (red); ^ indicates the Casp1/8 cleavage site.

4. The kinetic constants of the proteolysis of the FLT‐substrate Ac‐Cys(Pt14)‐FLTD^GVPY‐NH2 by Casp1/8 were determined from the time courses of product formation under initial velocity conditions. The KM value was obtained from measurements conducted at constant enzyme concentration (Casp1 = 30 nM; Casp8 = 833 nM) and different substrate concentrations as indicated.

5. Comparison of kinetic constants determined for caspase‐1/8 cleavage of (Pt14)‐LETD^Y‐NH2 and Ac‐Cys(Pt14)‐FLTD^GVPY‐NH2.

Source data are available online for this figure.

Figure 3. Caspase‐3 suppresses caspase‐8‐dependent GSDMD activation and cell lysis during extrinsic apoptosis

1. A

   HEK293T cells were transfected with doxycycline‐inducible DmrB‐caspase‐8 and the indicated GSDMD constructs. Cells were stimulated with doxycycline (10 μg/ml) for 18 h to induce DmrB‐caspase‐8 expression and exposed to B/B homodimerizer (12.5 nM) for another 2 h to activate caspase‐8. Mixed supernatant and extracts were analysed by immunoblot.

2. B, C

   Immortalized Gsdmd−/− BMDM expressing GSDMDWT and GSDMDD88A were (B) costimulated with TNF (100 ng/ml) and SM for 6 h or (C) primed for 3 h with ultrapure E. coli K12 LPS (100 ng/ml) and stimulated with LCL161 (1 μM) for 24 h, and LDH release was quantified.

3. D, E

   BMDMs were costimulated with TNF (100 ng/ml) and TAK1i for 4 h, (D) mixed supernatant and extracts were analysed by immunoblot, or (E) LDH release was quantified at the indicated time points.

Data information: All immunoblots are representative of three independent experiments. Data are means ± SEM of pooled data from (B‐C) three or (E) seven individual experiments. (B–C) Statistical analyses for normally distributed data sets were analysed using the parametric t‐test, whereas non‐normally distributed data sets were analysed using non‐parametric Mann–Whitney t‐tests. (E) Statistical analyses were performed using a two‐way ANOVA. Data were considered significant when *P < 0.05, **P < 0.01, ***P < 0.001 or ****P < 0.0001.Source data are available online for this figure.

Figure EV3. Gsdmd D88A/D88A BMDMs are more susceptible to extrinsic apoptosis

1. A, B

   BMDMs were costimulated with TNF (100 ng/ml) and SM (500 nM), (A) LDH release was quantified at the indicated time points, or (B) mixed supernatant and extracts were analysed at 5 h. (A) Data are means ± SEM of pooled data from three independent experiments. Statistical analyses were performed using a two‐way ANOVA Data were considered significant when **P < 0.01 or ****P < 0.0001. (B) Immunoblots are representative of three independent experiments.

Source data are available online for this figure.

Figure 4. RIPK3 promotes caspase‐3 activation and pannexin‐1 activity to drive NLRP3 assembly during extrinsic apoptosis

1. A–E

   BMDMs were costimulated with TNF (100 ng/ml) and TAK1i (125 nM) for 4 h, (A, B) LDH release was quantified, or (C, D, E) mixed supernatant and extracts were analysed by immunoblot. Where indicated, cells were treated with the inhibitors Nec‐1s (50 μM), MCC950 (10 μM), GSK'872 (1 μM), probenecid (1 mM) 20–30 min prior to cell stimulation. KCl (50 mM) was added together with TNF and TAK1i. (C) To induce necroptosis, BMDMs were primed for 3 h with ultrapure E. coli K12 LPS (100 ng/ml) and Q‐VD‐OPh (10 μM) was added at the last 20–30 min of priming and stimulated with SM (500 nM) for 4 h. (E) To activate the NLRP3 inflammasome, BMDMs were primed with ultrapure E. coli K12 LPS (100 ng/ml) for 4 h and stimulated with nigericin (10 μM) for 1 h.

2. F

   BMDMs were primed for 3 h with ultrapure E. coli K12 LPS (100 ng/ml) and stimulated with SM (0.5 μM) for a further 4 h. Probenecid (1 mM) was added 20–30 min prior to cell stimulation, and mixed supernatant and extracts were analysed by immunoblot.

3. G, H

   BMDMs were stimulated with TNF (100 ng/ml) and SM (0.5 μM) for 6 h, (G) mixed supernatant and extracts were analysed by immunoblot, or (H) LDH release was quantified.

Data information: All immunoblots are representative of three independent experiments. (A, B, H) Data are means ± SEM of pooled data from (A, H) four or (B) three independent experiments. Statistical analyses for normally distributed data sets were analysed using the parametric t‐test, whereas non‐normally distributed data sets were analysed using non‐parametric Mann–Whitney t‐tests. Data were considered significant when *P < 0.05, **P < 0.01 or ****P < 0.0001.Source data are available online for this figure.

Figure EV4. Extrinsic and intrinsic apoptosis promote NLRP3 assembly through pannexin‐1

1. A–C

   BMDMs were stimulated with TNF (100 ng/ml) or E. coli K12 LPS (100 ng/ml) and TAK1i (125 nM) for 4 h, mixed supernatant and extracts were analysed by immunoblot (A), or (B‐C) LDH release was quantified.

2. D

   Unprimed BMDMs were stimulated with ABT‐737 (500 nM) and S63845 (500 nM) for 6 h, and mixed supernatant and extracts were analysed by immunoblot.

Data information: Immunoblots are representative of three independent experiments. Data are mean ± SEM of pooled data from (B, C) four independent experiments.Source data are available online for this figure.

Figure 5. Intrinsic apoptosis drives gasdermin‐independent cell lysis but promotes NLPR3 assembly through pannexin‐1 activity

1. A, B

   BMDMs were stimulated with an increasing dose of S63845 in the presence of ABT‐737 (0.5 μM), and LDH release was quantified at 6 h.

2. C–F

   BMDMs were stimulated with ABT‐737 (0.5 μM) and S63845 (0.5 μM), LDH release was quantified (C, E), or mixed supernatant and extracts were analysed by immunoblot at 6 h (D, F).

3. G, H

   BMDMs were primed with ultrapure E. coli K12 LPS (100 ng/ml) for 3 h and further stimulated with ABT‐737 (1 μM) and S63845 (1 μM) for 24 h, mixed supernatant and extracts were analysed by immunoblot (G), and IL‐1β in cell‐free supernatant was quantified by ELISA (H).

Data information: All immunoblots are representative of three independent experiments. Data are means ± SEM of pooled data from (A) four, (B) five or (C, E, H) three independent experiments.Source data are available online for this figure.

Figure EV5. Schematic for ripoptosome‐ and apoptosome‐mediated cell death and NLRP3 activation

TNFR1 signalling in the presence of SM or TAK1i promotes the assembly of the ripoptosome, a caspase‐8 activating platform. Caspase‐8 triggers direct GSDMD activation to induce cell lysis, which is amplified by the NLRP3 inflammasome. The cytotoxic function of caspase‐8 is suppressed by caspase‐3‐mediated inactivation of GSDMD. Caspase‐3 cleaves GSDME, but GSDME does not contribute to cell death in BMDM. Finally, RIPK3 promotes caspase‐3 activation downstream of the ripoptosome to promote pannexin‐1 activity, potassium efflux and NLRP3 activation. By contrast, GSDMD does not promote cell lysis during mitochondrial apoptosis. The related gasdermin family member, GSDME, is processed into its active form during mitochondrial apoptosis, but does not promote cell lysis. Consistent with TNF‐induced apoptosis, pannexin‐1 but not gasdermins promote NLRP3 activation during mitochondrial apoptosis.