Extrinsic and intrinsic apoptosis activate pannexin‐1 to drive NLRP 3 inflammasome assembly

Article22 March 2019free access Source DataTransparent process Extrinsic and intrinsic apoptosis activate pannexin-1 to drive NLRP3 inflammasome assembly Kaiwen W Chen Department of Biochemistry, University of Lausanne, Epalinges, Switzerland Search for more papers by this author Benjamin Demarco Department of Biochemistry, University of Lausanne, Epalinges, Switzerland Search for more papers by this author Rosalie Heilig Department of Biochemistry, University of Lausanne, Epalinges, Switzerland Search for more papers by this author Kateryna Shkarina Department of Biochemistry, University of Lausanne, Epalinges, Switzerland Search for more papers by this author Andreas Boettcher Novartis Institutes for BioMedical Research Forum 1, Basel, Switzerland Search for more papers by this author Christopher J Farady Novartis Institutes for BioMedical Research Forum 1, Basel, Switzerland Search for more papers by this author Pawel Pelczar Center for Transgenic Models, University of Basel, Basel, Switzerland Search for more papers by this author Petr Broz Corresponding Author [email protected] orcid.org/0000-0002-2334-7790 Department of Biochemistry, University of Lausanne, Epalinges, Switzerland Search for more papers by this author Kaiwen W Chen Department of Biochemistry, University of Lausanne, Epalinges, Switzerland Search for more papers by this author Benjamin Demarco Department of Biochemistry, University of Lausanne, Epalinges, Switzerland Search for more papers by this author Rosalie Heilig Department of Biochemistry, University of Lausanne, Epalinges, Switzerland Search for more papers by this author Kateryna Shkarina Department of Biochemistry, University of Lausanne, Epalinges, Switzerland Search for more papers by this author Andreas Boettcher Novartis Institutes for BioMedical Research Forum 1, Basel, Switzerland Search for more papers by this author Christopher J Farady Novartis Institutes for BioMedical Research Forum 1, Basel, Switzerland Search for more papers by this author Pawel Pelczar Center for Transgenic Models, University of Basel, Basel, Switzerland Search for more papers by this author Petr Broz Corresponding Author [email protected] orcid.org/0000-0002-2334-7790 Department of Biochemistry, University of Lausanne, Epalinges, Switzerland Search for more papers by this author Author Information Kaiwen W Chen1,‡, Benjamin Demarco1,‡, Rosalie Heilig1, Kateryna Shkarina1, Andreas Boettcher2, Christopher J Farady2, Pawel Pelczar3 and Petr Broz *,1 1Department of Biochemistry, University of Lausanne, Epalinges, Switzerland 2Novartis Institutes for BioMedical Research Forum 1, Basel, Switzerland 3Center for Transgenic Models, University of Basel, Basel, Switzerland ‡These authors contributed equally to this work *Corresponding author. Tel: + 41 21 692 5656; E-mail: [email protected] EMBO J (2019)38:e101638https://doi.org/10.15252/embj.2019101638 See also: AM Gram et al (May 2019) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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. Synopsis The apoptotic signalling cascade promotes NLRP3 inflammasome assembly by activating the channel-forming glycoprotein, pannexin-1. Extrinsic apoptosis promotes caspase-1 and -8-dependent GSDMD activation in parallel with caspase-3/7-dependent secondary necrosis. Caspase-3 suppresses GSDMD-dependent cell lysis during extrinsic apoptosis. GSDME is activated during extrinsic and intrinsic apoptosis, but it does not contribute to cell lysis in macrophages. NLRP3 assembly during extrinsic and intrinsic apoptosis is dependent on pannexin-1 but not gasdermin D or E pores. Introduction Apoptosis and pyroptosis are caspase-dependent programmed cell death pathways that promote the removal of stressed, damaged, transformed or infected cells. Consequently, abnormalities in these pathways are associated with a range of human diseases including infection, cancer, neurodegeneration and autoinflammatory disease. Pyroptosis is a form of inflammatory cell death and is best characterized as an innate immune mechanism against intracellular pathogens (Miao et al, 2010; Chen & Schroder, 2013). Pyroptosis is initiated by a multiprotein signalling complex, called the inflammasome, which assembles upon cellular stress or infection (Broz & Dixit, 2016). Inflammasome formation induces the activation of inflammatory caspases, caspase-1, caspase-4, caspase-5 and caspase-11, which cleave and activate the recently identified pore-forming protein gasdermin D (GSDMD). In macrophages, cleavage of GSDMD at aspartate 276 (D276) by inflammasome-activated inflammatory caspases liberates the cytotoxic p30 N-terminal domain to generate plasma membrane pores and drive pyroptosis (He et al, 2015; Kayagaki et al, 2015; Shi et al, 2015; Aglietti et al, 2016; Ding et al, 2016; Liu et al, 2016; Sborgi et al, 2016), while GSDMD cleavage by caspase-4, caspase-11 or neutrophil elastase promotes neutrophil nuclear and plasma membrane damage that culminate in the extrusion of antimicrobial neutrophil extracellular traps (Chen et al, 2018b; Sollberger et al, 2018). A recent study demonstrated that cleavage of human GSDMD at position aspartate 87 (D87) by apoptotic caspase-3 inactivates the pyroptotic properties of GSDMD (Taabazuing et al, 2017). However, caspase-1-driven pyroptosis precedes caspase-3 activation in inflammasome-activated cells (He et al, 2015), and a low concentration of active GSDMD is sufficient to trigger pyroptosis (Sborgi et al, 2016). This indicates that caspase-3 is unlikely to suppress pyroptosis in inflammasome-activated cells, but instead may suppress GSDMD-dependent cell lysis during inflammasome-independent cell death pathways. In contrast to pyroptosis, apoptosis is traditionally believed to be an immunologically silent form of cell death that is important for the development and the removal of stressed or damaged cells. Apoptosis can be initiated by two major mechanisms, the extrinsic and intrinsic pathways. The extrinsic pathway is activated following the engagement of cell surface death receptors, such as tumour necrosis factor receptor 1 (TNFR1) or Fas (CD95). Engagement of TNFR1 initiates the formation of two distinct signalling complexes (Micheau & Tschopp, 2003; Wang et al, 2008). In healthy macrophages, TNFR1 engagement initiates the assembly of a plasma membrane-associated signalling complex, termed Complex I, which drives the expression of proinflammatory cytokines and prosurvival genes. However, when core components of Complex I such as the E3 ubiquitin ligases cellular inhibitor of apoptosis 1 (cIAP1) and cIAP2, or the kinases TAK1 or TBK1 are perturbed, TNRF1 ligation promotes the formation of a second, distinct cytosolic death-inducing complex comprising receptor-interacting serine/threonine kinase 1 (RIPK1), Fas-associated protein with death domain (FADD) and the initiator caspase, caspase-8. This secondary complex is called Complex II or commonly referred as the ripoptosome (Petersen et al, 2007; Varfolomeev et al, 2007; Feoktistova et al, 2011; Tenev et al, 2011; Vince et al, 2012; Dondelinger et al, 2013; Lafont et al, 2018; Malireddi et al, 2018). In contrast to extrinsic apoptosis, the intrinsic pathway is initiated by developmental cues, cytotoxic agents, growth factor withdrawal, infection and chemotherapeutic drugs, which alter the ratio of prosurvival and pro-apoptotic BCL-2 family proteins, leading to mitochondrial outer membrane permeabilization and activation of a distinct initiator caspase, caspase-9 (Czabotar et al, 2014). Both intrinsic and extrinsic pathways converge upon the activation of caspase-3 and caspase-7, which execute cellular demise for rapid apoptotic cell clearance and to suppress activation of proinflammatory signalling pathways. For example, caspase-3 and caspase-7 cleave the plasma membrane channel pannexin-1 at its C-terminus, which triggers membrane permeability and the release of “find-me” and “eat-me” signals to promote phagocytic clearance of apoptotic cells (Chekeni et al, 2010; Sandilos et al, 2012). While these observations suggest that apoptosis is indeed immunologically silent during homeostasis, emerging evidence from us and others indicates that exposure of innate immune cells such as macrophages and neutrophils to various therapeutics designed for cancer chemotherapy or inflammatory disorders such as SMAC mimetics, TAK1 inhibitor and BH3 mimetics promotes inflammation by driving caspase-8 or caspase-9-dependent inflammatory cell death and NLRP3 inflammasome activation (Vince et al, 2012, 2018; Lawlor et al, 2015; Wicki et al, 2016; Chauhan et al, 2018; Chen et al, 2018a; Malireddi et al, 2018). However, the mechanism by which caspase-8 or caspase-9 triggers cell lysis and NLRP3 activation is still undefined. Here, we demonstrate that extrinsic apoptosis promotes caspase-1 and caspase-8-dependent GSDMD activation and cell lysis, in parallel with caspase-3/7-mediated secondary necrosis. By generating a Gsdmd D88A knock-in mouse, we further demonstrate that the 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 upon caspase-8 activation. In contrast, we find no evidence that supports GSDME, a closely related gasdermin family protein, in driving cell lysis during extrinsic or intrinsic apoptosis. Lastly, we demonstrate that channel-forming glycoprotein pannexin-1, but not GSDMD or GSDME promotes NLRP3 inflammasome activation during extrinsic and intrinsic apoptosis. Results Caspase-8 activation promotes GSDMD-dependent cell lysis and caspase-3/7-dependent secondary necrosis Because caspase-1-dependent pyroptosis precedes caspase-3 activation, caspase-3 is unlikely to suppress pyroptosis in inflammasome-activated cells (He et al, 2015). Since caspase-3 activation is a hallmark of apoptosis, we reasoned that caspase-3-dependent cleavage and inactivation of GSDMD at position D87 in humans or D88 in mice might counteract the lytic function of GSDMD during apoptosis. First, we investigated whether GSDMD promotes cell lysis during extrinsic apoptosis by stimulating wild-type (WT) or Gsdmd−/− bone marrow-derived macrophages (BMDMs) with TNF in combination with the SMAC-mimetic AZD 5582 (hereafter referred as SM) or the TAK1 inhibitor 5z-7-oxozeaenol (hereafter referred as TAK1i), to induce the assembly of the caspase-8-activating ripoptosome complex (Vince et al, 2012; Dondelinger et al, 2013; Lawlor et al, 2015; Chen et al, 2018a). Surprisingly, caspase-8 activation triggered GSDMD-dependent cell lysis (Fig 1A and B) and induced hallmarks of pyroptosis including membrane ballooning and uptake of the membrane-impermeable dye propidium iodide (PI) in WT macrophages (Fig 1C; black arrowhead; Movie EV1). By contrast, Gsdmd-deficient cells retained membrane integrity and displayed classical apoptotic morphology such as cell shrinkage and the release of apoptotic bodies (Fig 1C; white arrowhead; Movie EV2). However, Gsdmd deficiency did not completely protect macrophages from caspase-8-dependent cell lysis (Fig 1A and B), and Gsdmd-deficient apoptotic bodies incorporate PI over time (Fig 1C; white arrowhead). Therefore, we investigate whether GSDMD-independent cell lysis is driven by caspase-3 and/or caspase-7-dependent secondary necrosis. To investigate this possibility, we deleted caspase-3, caspase-7 or caspase-3 and caspase-7 in Gsdmd−/− immortalized BMDMs (iBMDMs) using CRISPR/Cas9 technology and stimulated these cells with TNF and SM or TAK1i. Indeed, Gsdmd−/−Casp3−/−, Gsdmd−/−Casp7−/− and Gsdmd−/− Casp3/7−/− iBMDMs were resistant to cell lysis following caspase-8 activation (Fig 1D and E). Taken together, our data indicate that extrinsic apoptosis promotes GSDMD-dependent cell lysis in parallel with caspase-3/7-dependent secondary necrosis. Figure 1. Extrinsic apoptosis trigger GSDMD-dependent and caspase-3/7-dependent necrosis 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. Download figure Download PowerPoint GSDMD or GSDME pores do not promote NLRP3 assembly during apoptosis Since caspase-8 activation may indirectly promote GSDMD cleavage by activating the NLRP3 inflammasome (Vince et al, 2012; Lawlor et al, 2015), we next investigated cell death and GSDMD processing in WT versus inflammasome-deficient BMDMs. Indeed, we observed that Nlrp3, Asc, Caspase-1/11 deficiency similarly reduced TNF and SM- or TAK1i-induced cell death (Figs 2A and EV1A). Unexpectedly, Gsdmd−/− BMDMs were even more protected than Nlrp3−/− and Caspase-1/11−/− BMDMs following TNF and TAK1i stimulation (Fig 2A), suggesting that GSDMD may be activated by caspase-1-dependent and caspase-1-independent pathways. Consistent with that, we observed robust GSDMD processing into the active p30 and the appearance of an inactive p20 fragment, resulting from the inactivating cleavage by caspase-3 at position D88, in WT, Nlrp3 and Caspase-1/11-deficient primary or immortalized BMDM after stimulation with TNF or LPS in combination with SM or TAK1i (Figs 2B and C, and EV1B and C). Caspase-8 triggers NLRP3 activation through a potassium efflux-dependent mechanism (Conos et al, 2017), suggesting that caspase-8 likely triggers NLRP3 assembly by inducing plasma membrane damage. We therefore hypothesize that caspase-1/11-independent GSDMD activation promotes plasma membrane pore formation, potassium efflux and NLRP3 activation, analogous to NLRP3 activation by the non-canonical inflammasome (Kayagaki et al, 2011; Ruhl & Broz, 2015). However, TNF or LPS costimulated with SM or TAK1i induced comparable levels of caspase-1 p20 autoprocessing, a hallmark of inflammasome activation, between WT and Gsdmd−/− macrophages (Figs 2B, D, E and G, and EV1D and E), and extracellular potassium similarly reduced caspase-1 processing in WT and Gsdmd−/− BMDMs (Fig 2D and E). Gasdermin E (GSDME), another pyroptotic effector from the gasdermin protein family, is activated by caspase-3 during apoptosis and proposed to mediate secondary necrosis in BMDMs (Rogers et al, 2017; Wang et al, 2017). Consistent with previous reports, we observed robust GSDME processing into the active p30 fragment during apoptosis; however, Gsdme deficiency did not reduce cell death or caspase-1 processing in WT or Gsdmd−/− macrophages (Figs 2F and G, and EV1F), indicating that caspase-8 triggers potassium efflux and NLRP3 activation through GSDMD- or GSDME-independent pores. Figure 2. Extrinsic apoptosis triggers caspase-1/11-independent GSDMD processing and GSDMD/E-independent NLRP3 activation 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. C. Representation of known caspase cleavage site and molecular weight of corresponding cleavage fragment in mouse GSDMD. 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. 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. Source Data for Figure 2 [embj2019101638-sup-0005-SDataFig2.eps] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Caspase-8 triggers NLRP3/caspase-1-independent GSDMD processing A. BMDMs were costimulated with TNF (100 ng/ml) and SM (500 nM) for the indicated time points, and LDH release was quantified. 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. 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. 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. 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. Download figure Download PowerPoint Caspase-3 counteracts caspase-8-dependent GSDMD activation during extrinsic apoptosis Since we observed that GSDMD processing is ablated in Ripk3−/−Casp8−/− during extrinsic apoptosis (Fig EV2A), and caspase-1 and caspase-8 recognize and cleave overlapping sequences in substrates such as pro-IL-1β (Maelfait et al, 2008; Vince et al, 2012), we hypothesized that caspase-8 could directly trigger GSDMD activation. For this, we engineered a doxycycline-inducible caspase-8 construct, in which we replaced the DED domains of caspase-8 with a DmrB domain to control caspase-8 dimerization upon addition of the B/B homodimerizer drug. Next, we ectopically expressed DmrB-caspase-8 with WT full-length GSDMD, a caspase-1/11-uncleavable D276A mutant, a caspase-3-uncleavable D88A mutant and a D88A D276A double mutant in HEK293T cells which are naturally deficient in caspase-1 (Fig EV2B). Indeed, addition of doxycycline and B/B homodimerizer to induce caspase-8 expression and dimerization triggered cleavage of WT GSDMD into the active p30 fragment and the appearance of the inactive p43 and p20 fragments, corresponding to cleavage of full-length GSDMD or further processing of active p30 at position D88 by caspase-3 (Figs 3A and 2C). Consistent with that, we observed an accumulation of active p30 and complete disappearance of the inactive p43 and p20 fragment in the D88A mutant. Conversely, caspase-8 dimerization triggered an accumulation of the inactive p43 but not the appearance of active p30 and inactive p20 when D276 was mutated, indicating that caspase-8 and caspase-1 can process GSDMD at the same residue (Fig 3A); consistent with a recent report that caspase-8 triggers direct GSDMD during Yersinia infection (Orning et al, 2018). To verify in vitro that human caspase-1 and caspase-8 cleave GSDMD at the same residue, we designed a GSDMD-based substrate derived from the caspase-1 cleavage site in GSDMD and monitored its cleavage using a fluorometric assay (Fig EV2C). Indeed, human caspase-8 processed the GSDMD-based substrate, though 30-fold less efficiently than caspase-1 (Fig EV2D and E). Since caspase-3 is activated downstream of caspase-8 during apoptosis, we next investigated whether the direct caspase-8-dependent GSDMD activation is counteracted by caspase-3-dependent GSDMD inactivation at position D88. For this, we reconstituted immortalized Gsdmd−/− BMDMs with either WT GSDMD (GSDMDWT) or a caspase-3-uncleavable D88A mutant (GSDMDD88A) by lentiviral transduction and monitored cell death after TNF and SM or LPS and LCL161 (SMAC mimetic)-induced caspase-8 activation. Remarkably, GSDMDD88A-expressing iBMDMs were significantly more susceptible than GSDMDWT-expressing controls to both TNF- and LPS-induced apoptosis (Fig 3B and C). To investigate whether GSDMD inactivation at D88 is observed in primary macrophages, we generated a GsdmdD88A knock-in mouse. GsdmdD88A/D88A animals were born at expected Mendelian ratio, healthy and developed a normal immune system (Appendix Fig S1A–F). Indeed, we observed an accumulation of the cytotoxic GSDMD p30 fragment and the disappearance of the inactive p20 fragment upon TNF and TAK1i or SM stimulation of GsdmdD88A/D88A cells, which correlated with a 1.5-fold enhancement in cell death (Figs 3D and E, and EV3A and B). Although the cytotoxic GSDMD p30 fragment was significantly enriched in GsdmdD88A/D88A cells compared to WT littermates, caspase-1 processing was unchanged between GsdmdD88A/D88A and WT littermate controls (Figs 3D and EV3B), reiterating that GSDMD pores do not promote caspase-8-dependent NLRP3 activation. Click here to expand this figure. Figure EV2. Caspase-8 cleaves GSDMD at a lower efficiency than caspase-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. Caspase-1 expression in HEK293T versus HeLa cells. 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. 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. 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. Download figure Download PowerPoint Figure 3. Caspase-3 suppresses caspase-8-dependent GSDMD activation and cell lysis during extrinsic apoptosis 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. 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. 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. Source Data for Figure 3 [embj2019101638-sup-0006-SDataFig3.eps] Download figure Download PowerPoint Click here to expand this figure. Figure EV3. GsdmdD88A/D88A BMDMs are more susceptible to extrinsic apoptosis 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. Download figure Download PowerPoint The channel-forming membrane protein pannexin-1 promotes NLRP3 activation during extrinsic apoptosis Since the RIPK1 kinase inhibitor Nec-1s suppressed caspase-8-mediated cell death (Wang et al, 2008; Feoktistova et al, 2011; Tenev et al, 2011) (Fig 4A), and RIPK1 signalling is often associated with RIPK3, we next investigated the role of RIPK3 in promoting cell death and NLRP3 activation during TNF-induced apoptosis. RIPK3 did not contribute to TNF and TAK1i-induced cell lysis and GSDMD activation (Fig 4B and C); however, caspase-1 processing was remarkably reduced in Ripk3−/− compared to WT macrophages. Extracellular potassium and the NLRP3-specific inhibitor MCC950 reduced GSDMD and processing in WT but not Ripk3−/− cells, confirming that RIPK3 is indeed upstream of NLRP3 during TNF-induced apoptosis (Fig 4C). RIPK3 kinase activity can drive NLRP3 activation by promoting MLKL pore formation and potassium efflux (Conos et al, 2017; Gutierrez et al, 2017). However, TNF and TAK1i did not trigger RIPK3-dependent MLKL phosphorylation (Fig 4C) and MLKL did not contribute to cell death or caspase-1 processing (Appendix Fig S2A and B). In agreement with a RIPK3 kinase-independent role for driving NLRP3 activation, the RIPK3 kinase inhibitor GSK'872 did not suppress cell lysis (Appendix Fig S2C) but paradoxically promoted caspase-1 processing (Fig 4C), most probably through altering the ripoptosome conformation (Mandal et al, 2014; Newton et al, 2014; Moriwaki et al, 2015). Consistent with previous reports, our data provide support for the hypothesis that it is the RIPK3 scaffolding function that promotes ripoptosome-mediated caspase-3 activation (Fig 4D) (Vince et al, 2012; Dondelinger et al, 2013). Since both caspase-3 activation and caspase-1 activation were reduced in Ripk3−/− cells, we hypothesized that ripoptosome-induced caspase-3 activity drives potassium efflux and NLRP3 activation. Pannexin-1, a channel-forming glycoprotein, is activated by caspase-3/7-dependent cleavage at its C-terminus during apoptosis (Chekeni et al, 2010; Sandilos et al, 2012). This cleavage event promotes the removal of its inhibitory C-terminal domain, resulting in the opening of the pannexin-1 channel, membrane permeability, ATP release and potassium efflux (Chekeni et al, 2010; Yang et al, 2015). We therefore investigated whether the ripoptosome promotes pannexin-1 activity for NLRP3 assembly by using two well-established pannexin-1 inhibitors, probenecid (Silverman et al, 2008) and the antibiotic trovafloxacin (Poon et al, 2014). Remarkably, probenecid and trovafloxacin strongly reduced caspase-1 activation during TNF-induced apoptosis (Fig 4E; Appendix Fig S3A). By contrast, both inhibitors had no effect on caspase-1 processing following nigericin or poly(dAdT) stimulation to activate NLRP3 or AIM2 inflammasome, respectively (Fig 4E, App