Differential signalling requirements for RIPK1-dependent pyroptosis in neutrophils and macrophages.

TLR4 and TNFR1 signalling promotes potent proinflammatory signal transduction events, thus, are often hijacked by pathogenic microorganisms. We recently reported that myeloid cells retaliate Yersinia blockade of TAK1/IKK signalling by triggering RIPK1-dependent caspase-8 activation that promotes downstream GSDMD and GSDME-mediated pyroptosis in macrophages and neutrophils respectively. However, the upstream signalling events for RIPK1 activation in these cells are not well defined. Here, we demonstrate that unlike in macrophages, RIPK1-driven pyroptosis and cytokine priming in neutrophils are driven through TNFR1 signalling, while TLR4-TRIF signalling is dispensable. Furthermore, we demonstrate that activation of RIPK1-dependent pyroptosis in neutrophils during Yersinia infection requires IFN-γ priming, which serves to induce surface TNFR1 expression and amplify soluble TNF secretion. In contrast, macrophages utilise both TNFR1 and TLR4-TRIF signalling to trigger cell death, but only require TRIF but not autocrine TNFR1 for cytokine production. Together, these data highlight the emerging theme of cell type-specific regulation in cell death and immune signalling in myeloid cells.

The small molecule raptinal can simultaneously induce apoptosis and inhibit PANX1 activity.

Discovery of new small molecules that can activate distinct programmed cell death pathway is of significant interest as a research tool and for the development of novel therapeutics for pathological conditions such as cancer and infectious diseases. The small molecule raptinal was discovered as a pro-apoptotic compound that can rapidly trigger apoptosis by promoting the release of cytochrome c from the mitochondria and subsequently activating the intrinsic apoptotic pathway. As raptinal is very effective at inducing apoptosis in a variety of different cell types in vitro and in vivo, it has been used in many studies investigating cell death as well as the clearance of dying cells. While examining raptinal as an apoptosis inducer, we unexpectedly identified that in addition to its pro-apoptotic activities, raptinal can also inhibit the activity of caspase-activated Pannexin 1 (PANX1), a ubiquitously expressed transmembrane channel that regulates many cell death-associated processes. By implementing numerous biochemical, cell biological and electrophysiological approaches, we discovered that raptinal can simultaneously induce apoptosis and inhibit PANX1 activity. Surprisingly, raptinal was found to inhibit cleavage-activated PANX1 via a mechanism distinct to other well-described PANX1 inhibitors such as carbenoxolone and trovafloxacin. Furthermore, raptinal also interfered with PANX1-regulated apoptotic processes including the release of the 'find-me' signal ATP, the formation of apoptotic cell-derived extracellular vesicles, as well as NLRP3 inflammasome activation. Taken together, these data identify raptinal as the first compound that can simultaneously induce apoptosis and inhibit PANX1 channels. This has broad implications for the use of raptinal in cell death studies as well as in the development new PANX1 inhibitors.

Extracorporeal Membrane Oxygenation-Dependent Fulminant Melioidosis From Caspase 4 Mutation Reversed by Interferon Gamma Therapy.

We describe bedside-to-bench immunological and genetic elucidation of defective pyroptosis attributable to novel caspase 4 defect mediating pathogen-triggered inflammatory programmed cell death, in the setting of severe pneumonia and abscess-forming melioidosis in an overtly healthy host failing to clear Burkholderia pseudomallei infection, and how targeted adjunctive biological therapy led to a successful outcome.

Analyzing Caspase-8-Dependent GSDMD Cleavage in Response to Yersinia Infection.

Caspase-8 is best known to drive an immunologically silent form of cell death known as apoptosis. However, emerging studies revealed that upon pathogen inhibition of innate immune signalling, such as during Yersinia infection in myeloid cells, caspase-8 associates with RIPK1 and FADD to trigger a proinflammatory death-inducing complex. Under such conditions, caspase-8 cleaves the pore-forming protein gasdermin D (GSDMD) to trigger a lytic form of cell death, known as pyroptosis. Here, we describe our protocol to activate caspase-8-dependent GSDMD cleavage following Yersinia pseudotuberculosis infection in murine bone marrow-derived macrophages (BMDMs). Specifically, we describe protocols on harvesting and plating of BMDM, preparation of type 3 secretion system-inducing Yersinia, macrophage infection, lactate dehydrogenase (LDH) release assay, and Western blot analysis.

Yersinia interactions with regulated cell death pathways.

Cell death in response to infection is conserved across all kingdoms of life. In metazoans, cell death upon bacterial infection is primarily carried out by the cysteine and aspartate protease and receptor-interacting serine/threonine protein kinase families. The Gram-negative bacterial genus Yersinia includes pathogens that cause disease in humans and other animals ranging from plague to gastrointestinal infections. Pathogenic Yersiniae express a type-III secretion system (T3SS), which translocates effectors that disrupt phagocytosis and innate immune signaling to evade immune defenses and replicate extracellularly in infected tissues. Blockade of innate immune signaling, disruption of the actin cytoskeleton, and the membrane-disrupting activity of the T3SS translocon pore, are all sensed by innate immune cells. Here, we discuss recent advances in understanding the pathways that regulate Yersinia-induced cell death, and how manipulation of these cell death pathways over the course of infection promotes bacterial dissemination or host defense.

RIPK1 and RIPK3 in antibacterial defence.

Upon sensing pathogenic bacterial infection, host cells activate a multitude of inflammatory and immunogenic responses to promote bacterial clearance and restore tissue homeostasis. RIPK1 and RIPK3 are two key players in antimicrobial defence, by either driving inflammatory signalling or inducing programmed cell death activation, ranging from apoptosis, pyroptosis to necroptosis. In this review, we first discuss the mechanisms by which RIPK1 and RIPK3 promote the assembly of death-inducing complexes and how these cell death pathways are activated as host responses to counteract pathogenic bacteria. We further outline the immunological importance of cell death in antibacterial defence and highlight outstanding questions in the field.

Equally potent: Nlrp3 mutation in macrophages or neutrophils is sufficient to drive autoinflammation.

Gain-of-function mutation in NLRP3 is associated with a spectrum of autoinflammatory disorders including familial cold autoinflammatory syndrome, Muckle-Wells syndrome, and neonatal onset multisystem inflammatory disease, collectively known as cryopyrin-associated periodic syndrome (CAPS). However, the cell types mediating the pathogenesis of CAPS are not completely understood. Two studies in EMBO Reports now demonstrate that gain-of-function Nlrp3 mutation in either macrophages or neutrophils alone is sufficient to trigger systemic autoinflammation and lethality in mice.

ZAKα-driven ribotoxic stress response activates the human NLRP1 inflammasome.

Human NLRP1 (NACHT, LRR, and PYD domain-containing protein 1) is an innate immune sensor predominantly expressed in the skin and airway epithelium. Here, we report that human NLRP1 senses the ultraviolet B (UVB)- and toxin-induced ribotoxic stress response (RSR). Biochemically, RSR leads to the direct hyperphosphorylation of a human-specific disordered linker region of NLRP1 (NLRP1DR) by MAP3K20/ZAKα kinase and its downstream effector, p38. Mutating a single ZAKα phosphorylation site in NLRP1DR abrogates UVB- and ribotoxin-driven pyroptosis in human keratinocytes. Moreover, fusing NLRP1DR to CARD8, which is insensitive to RSR by itself, creates a minimal inflammasome sensor for UVB and ribotoxins. These results provide insight into UVB sensing by human skin keratinocytes, identify several ribotoxins as NLRP1 agonists, and establish inflammasome-driven pyroptosis as an integral component of the RSR.

Inflammasome and gasdermin signaling in neutrophils.

Inflammasomes and gasdermins mount potent host defense pathways against invading microbial pathogens, however, dysregulation in these pathways can drive a variety of inflammatory disorders. Neutrophils, historically regarded as effector phagocytes that drive host defense via microbial killing, are now emerging as critical drivers of immunity in vivo. Here, we summarize, the latest advancement in inflammasome, gasdermin, and cell death signaling in neutrophils. We discuss the mechanisms by which neutrophils resist caspase-1-dependent pyroptosis, the lytic function of gasdermin D and E during NETosis and Yersinia infection, and the contribution of neutrophil inflammasomes to inflammatory disorders.

Genetic targeting of Card19 is linked to disrupted NINJ1 expression, impaired cell lysis, and increased susceptibility to Yersinia infection.

Cell death plays a critical role in inflammatory responses. During pyroptosis, inflammatory caspases cleave Gasdermin D (GSDMD) to release an N-terminal fragment that generates plasma membrane pores that mediate cell lysis and IL-1 cytokine release. Terminal cell lysis and IL-1ß release following caspase activation can be uncoupled in certain cell types or in response to particular stimuli, a state termed hyperactivation. However, the factors and mechanisms that regulate terminal cell lysis downstream of GSDMD cleavage remain poorly understood. In the course of studies to define regulation of pyroptosis during Yersinia infection, we identified a line of Card19-deficient mice (Card19lxcn) whose macrophages were protected from cell lysis and showed reduced apoptosis and pyroptosis, yet had wild-type levels of caspase activation, IL-1 secretion, and GSDMD cleavage. Unexpectedly, CARD19, a mitochondrial CARD-containing protein, was not directly responsible for this, as an independently-generated CRISPR/Cas9 Card19 knockout mouse line (Card19Null) showed no defect in macrophage cell lysis. Notably, Card19 is located on chromosome 13, immediately adjacent to Ninj1, which was recently found to regulate cell lysis downstream of GSDMD activation. RNA-seq and western blotting revealed that Card19lxcn BMDMs have significantly reduced NINJ1 expression, and reconstitution of Ninj1 in Card19lxcn immortalized BMDMs restored their ability to undergo cell lysis in response to caspase-dependent cell death stimuli. Card19lxcn mice exhibited increased susceptibility to Yersinia infection, whereas independently-generated Card19Null mice did not, demonstrating that cell lysis itself plays a key role in protection against bacterial infection, and that the increased infection susceptibility of Card19lxcn mice is attributable to loss of NINJ1. Our findings identify genetic targeting of Card19 being responsible for off-target effects on the adjacent gene Ninj1, disrupting the ability of macrophages to undergo plasma membrane rupture downstream of gasdermin cleavage and impacting host survival and bacterial control during Yersinia infection.

RIPK1 activates distinct gasdermins in macrophages and neutrophils upon pathogen blockade of innate immune signaling.

Injection of effector proteins to block host innate immune signaling is a common strategy used by many pathogenic organisms to establish an infection. For example, pathogenic Yersinia species inject the acetyltransferase YopJ into target cells to inhibit NF-κB and MAPK signaling. To counteract this, detection of YopJ activity in myeloid cells promotes the assembly of a RIPK1-caspase-8 death-inducing platform that confers antibacterial defense. While recent studies revealed that caspase-8 cleaves the pore-forming protein gasdermin D to trigger pyroptosis in macrophages, whether RIPK1 activates additional substrates downstream of caspase-8 to promote host defense is unclear. Here, we report that the related gasdermin family member gasdermin E (GSDME) is activated upon detection of YopJ activity in a RIPK1 kinase-dependent manner. Specifically, GSDME promotes neutrophil pyroptosis and IL-1ß release, which is critical for anti-Yersinia defense. During in vivo infection, IL-1ß neutralization increases bacterial burden in wild-type but not Gsdme-deficient mice. Thus, our study establishes GSDME as an important mediator that counteracts pathogen blockade of innate immune signaling.

Posttranslational and Therapeutic Control of Gasdermin-Mediated Pyroptosis and Inflammation.

Pyroptosis is a proinflammatory form of cell death, mediated by membrane pore-forming proteins called gasdermins. Gasdermin pores allow the release of the pro-inflammatory cytokines IL-1ß and IL-18 and cause cell swelling and cell lysis leading to release of other intracellular proteins that act as alarmins to perpetuate inflammation. The best characterized, gasdermin D, forms pores via its N-terminal domain, generated after the cleavage of full length gasdermin D by caspase-1 or -11 (caspase-4/5 in humans) typically upon sensing of intracellular pathogens. Thus, gasdermins were originally thought to largely contribute to pathogen-induced inflammation. We now know that gasdermin family members can also be cleaved by other proteases, such as caspase-3, caspase-8 and granzymes, and that they contribute to sterile inflammation as well as inflammation in autoinflammatory diseases or during cancer immunotherapy. Here we briefly review how and when gasdermin pores are formed, and then focus on emerging endogenous mechanisms and therapeutic approaches that could be used to control pore formation, pyroptosis and downstream inflammation.

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

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.

Human GBP1 binds LPS to initiate assembly of a caspase-4 activating platform on cytosolic bacteria.

The human non-canonical inflammasome controls caspase-4 activation and gasdermin-D-dependent pyroptosis in response to cytosolic bacterial lipopolysaccharide (LPS). Since LPS binds and oligomerizes caspase-4, the pathway is thought to proceed without dedicated LPS sensors or an activation platform. Here we report that interferon-induced guanylate-binding proteins (GBPs) are required for non-canonical inflammasome activation by cytosolic Salmonella or upon cytosolic delivery of LPS. GBP1 associates with the surface of cytosolic Salmonella seconds after bacterial escape from their vacuole, initiating the recruitment of GBP2-4 to assemble a GBP coat. The GBP coat then promotes the recruitment of caspase-4 to the bacterial surface and caspase activation, in absence of bacteriolysis. Mechanistically, GBP1 binds LPS with high affinity through electrostatic interactions. Our findings indicate that in human epithelial cells GBP1 acts as a cytosolic LPS sensor and assembles a platform for caspase-4 recruitment and activation at LPS-containing membranes as the first step of non-canonical inflammasome signaling.

Cross talk between intracellular pathogens and cell death.

Infections with bacterial pathogens often results in the initiation of programmed cell death as part of the host innate immune defense, or as a bacterial virulence strategy. Induction of host cell death is controlled by an elaborate network of innate immune and cell death signaling pathways and manifests in different morphologically and functionally distinct forms of death, such as apoptosis, necroptosis, NETosis and pyroptosis. The mechanism by which host cell death restricts bacterial replication is highly cell-type and context depended, but its physiological importance is highlighted the diversity of strategies bacterial pathogens use to avoid induction of cell death or to block cell death signaling pathways. In this review, we discuss the latest insights into how bacterial pathogens elicit and manipulate cell death signaling, how different forms of cell death kill or restrict bacteria and how cell death and innate immune pathway cross talk to guard against pathogen-induced inhibition of host cell death.

Caspase-1 cleaves Bid to release mitochondrial SMAC and drive secondary necrosis in the absence of GSDMD.

Caspase-1 drives a lytic inflammatory cell death named pyroptosis by cleaving the pore-forming cell death executor gasdermin-D (GSDMD). Gsdmd deficiency, however, only delays cell lysis, indicating that caspase-1 controls alternative cell death pathways. Here, we show that in the absence of GSDMD, caspase-1 activates apoptotic initiator and executioner caspases and triggers a rapid progression into secondary necrosis. GSDMD-independent cell death required direct caspase-1-driven truncation of Bid and generation of caspase-3 p19/p12 by either caspase-8 or caspase-9. tBid-induced mitochondrial outer membrane permeabilization was also required to drive SMAC release and relieve inhibitor of apoptosis protein inhibition of caspase-3, thereby allowing caspase-3 auto-processing to the fully active p17/p12 form. Our data reveal that cell lysis in inflammasome-activated Gsdmd-deficient cells is caused by a synergistic effect of rapid caspase-1-driven activation of initiator caspases-8/-9 and Bid cleavage, resulting in an unusually fast activation of caspase-3 and immediate transition into secondary necrosis. This pathway might be advantageous for the host in counteracting pathogen-induced inhibition of GSDMD but also has implications for the use of GSDMD inhibitors in immune therapies for caspase-1-dependent inflammatory disease.

Pannexin-1 promotes NLRP3 activation during apoptosis but is dispensable for canonical or noncanonical inflammasome activation.

Inflammasomes are multimeric protein complex that assemble in the cytosol upon microbial infection or cellular stress. Upon activation, inflammasomes drive the maturation of proinflammatory cytokines, IL-1ß and IL-18, and also activate the pore-forming protein, gasdermin D to initiate a form of lytic cell death known as "pyroptosis". Pannexin-1 is channel-forming glycoprotein that promotes membrane permeability and ATP release during apoptosis; and was implicated in canonical NLRP3 or noncanonical inflammasome activation. Here, by utilizing three different pannexin-1 channel inhibitors and two lines of Panx1-/- macrophages, we provide genetic and pharmacological evidence that pannexin-1 is dispensable for canonical or noncanonical inflammasome activation. In contrast, we demonstrate that pannexin-1 cleavage and resulting channel activity during apoptosis promotes NLRP3 inflammasome activation.

Beyond inflammasomes: emerging function of gasdermins during apoptosis and NETosis.

Programmed cell death is a key mechanism involved in several biological processes ranging from development and homeostasis to immunity, where it promotes the removal of stressed, damaged, malignant or infected cells. Abnormalities in the pathways leading to initiation of cell death or removal of dead cells are consequently associated with a range of human diseases including infections, autoinflammatory disease, neurodegenerative disease and cancer. Apoptosis, pyroptosis and NETosis are three well-studied modes of cell death that were traditionally believed to be independent of one another, but emerging evidence indicates that there is extensive cross-talk between them, and that all three pathways can converge onto the activation of the same cell death effector-the pore-forming protein Gasdermin D (GSDMD). In this review, we highlight recent advances in gasdermin research, with a particular focus on the role of gasdermins in pyroptosis, NETosis and apoptosis, as well as cell type-specific consequences of gasdermin activation. In addition, we discuss controversies surrounding a related gasdermin family protein, Gasdermin E (GSDME), in mediating pyroptosis and secondary necrosis following apoptosis, chemotherapy and inflammasome activation.

Pannexin-1 channels bridge apoptosis to NLRP3 inflammasome activation.

Apoptosis can promote inflammation by triggering activation of the NLRP3 inflammasome (NLR family, pyrin domain containing 3). However, the molecular mechanisms regulating these processes are ill-defined. We recently reported that pannexin-1 is required to promote NLRP3 inflammasome assembly. We further demonstrate that differential cleavage of gasdermin D (GSDMD) by apoptotic caspases regulates inflammatory cell lysis. Here, we discuss our findings and perspectives for future studies.

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

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.