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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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-1β 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. Author summary: Programmed cell death is critical for regulating tissue homeostasis and host defense against infection. Pyroptosis is an inflammatory form of programmed cell death that couples cell lysis with release of inflammatory cytokines. Cell lysis is triggered by activation of particular intracellular pore forming proteins, but how regulation of cell lysis occurs is not well understood. Genetic targeting of Card19 on chromosome 13 resulted in decreased expression of the adjacent gene, Ninj1, which was recently found to regulate terminal lysis events in response to cell death-inducing stimuli. Consistently, macrophages from Card19-deficient mice were resistant to multiple forms of cell death in response to a variety of inflammatory stimuli, including canonical and non-canonical inflammasome activation, as well as triggers of cell-extrinsic apoptosis. Notably, Card19-deficient mice were more susceptible to Yersinia infection, indicating that cell lysis contributes to control of bacterial infections. Our data provide new insight into the impact of terminal cell lysis on control of bacterial infection and highlight the role of additional factors that regulate lytic cell death downstream of gasdermin cleavage. [ABSTRACT FROM AUTHOR]

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-1β 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The human non-canonical inflammasome controls caspase-4 activation and gasdermin-Ddependent 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 noncanonical inflammasome signaling. [ABSTRACT FROM AUTHOR]

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‐1β 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Summary IL-1β requires processing by caspase-1 to generate the active, pro-inflammatory cytokine. Acute IL-1β secretion from inflammasome-activated macrophages requires caspase-1-dependent GSDMD cleavage, which also induces pyroptosis. Mechanisms of IL-1β secretion by pyroptotic and non-pyroptotic cells, and the precise functions of caspase-1 and GSDMD therein, are unresolved. Here, we show that, while efficient early secretion of endogenous IL-1β from primary non-pyroptotic myeloid cells in vitro requires GSDMD, later IL-1β release in vitro and in vivo proceeds independently of GSDMD. IL-1β maturation is sufficient for slow, caspase-1/GSDMD-independent secretion of ectopic IL-1β from resting, non-pyroptotic macrophages, but the speed of IL-1β release is boosted by inflammasome activation, via caspase-1 and GSDMD. IL-1β cleavage induces IL-1β enrichment at PIP2-enriched plasma membrane ruffles, and this is a prerequisite for IL-1β secretion and is mediated by a polybasic motif within the cytokine. We thus reveal a mechanism in which maturation-induced IL-1β trafficking facilitates its unconventional secretion. [ABSTRACT FROM AUTHOR]

Neutrophil extrusion of neutrophil extracellular traps (NETs) and concomitant cell death (NETosis) provides host defense against extracellular pathogens, whereas macrophage death by pyroptosis enables defense against intracellular pathogens. We report the unexpected discovery that gasdermin D (GSDMD) connects these cell death modalities. We show that neutrophil exposure to cytosolic lipopolysaccharide or cytosolic Gram-negative bacteria (Salmonella sifA and Citrobacter rodentium) activates noncanonical (caspase-4/11) inflammasome signaling and triggers GSDMD-dependent neutrophil death. GSDMD-dependent death induces neutrophils to extrude antimicrobial NETs. Caspase-11 and GSDMD are required for neutrophil plasma membrane rupture during the final stage of NET extrusion. Unexpectedly, caspase-11 and GSDMD are also required for early features of NETosis, including nuclear delobulation and DNA expansion; this is mediated by the coordinate actions of caspase-11 and GSDMD in mediating nuclear membrane permeabilization and histone degradation. In vivo application of deoxyribonuclease I to dissolve NETs during murine Salmonella sifA challenge increases bacterial burden in wild-type but not in Casp11-/- and Gsdmd-/- mice. Our studies reveal that neutrophils use an inflammasome- and GSDMD-dependent mechanism to activate NETosis as a defense response against cytosolic bacteria. [ABSTRACT FROM AUTHOR]

The mammalian inhibitor of apoptosis proteins (IAPs) are key regulators of cell death and inflammation. A major function of IAPs is to block the formation of a cell death-inducing complex, termed the ripoptosome, which can trigger caspase-8-dependent apoptosis or caspase-independent necroptosis. Recent studies report that upon TLR4 or TNF receptor 1 (TNFR1) signaling in macrophages, the ripoptosome can also induce NLRP3 inflammasome formation and IL-1β maturation. Whether neutrophils have the capacity to assemble a ripoptosome to induce cell death and inflammasome activation during TLR4 and TNFR1 signaling is unclear. In this study, we demonstrate that murine neutrophils can signal via TNFR1-driven ripoptosome assembly to induce both cell death and IL-1β maturation. However, unlike macrophages, neutrophils suppress TLR4-dependent cell death and NLRP3 inflammasome activation during IAP inhibition via deficiencies in the CD14/TRIF arm of TLR4 signaling. [ABSTRACT FROM AUTHOR]

Summary X-linked Inhibitor of Apoptosis (XIAP) deficiency predisposes people to pathogen-associated hyperinflammation. Upon XIAP loss, Toll-like receptor (TLR) ligation triggers RIPK3-caspase-8-mediated IL-1β activation and death in myeloid cells. How XIAP suppresses these events remains unclear. Here, we show that TLR-MyD88 causes the proteasomal degradation of the related IAP, cIAP1, and its adaptor, TRAF2, by inducing TNF and TNF Receptor 2 (TNFR2) signaling. Genetically, we define that myeloid-specific cIAP1 loss promotes TLR-induced RIPK3-caspase-8 and IL-1β activity in the absence of XIAP. Importantly, deletion of TNFR2 in XIAP-deficient cells limited TLR-MyD88-induced cIAP1-TRAF2 degradation, cell death, and IL-1β activation. In contrast to TLR-MyD88, TLR-TRIF-induced interferon (IFN)β inhibited cIAP1 loss and consequent cell death. These data reveal how, upon XIAP deficiency, a TLR-TNF-TNFR2 axis drives cIAP1-TRAF2 degradation to allow TLR or TNFR1 activation of RIPK3-caspase-8 and IL-1β. This mechanism may explain why XIAP-deficient patients can exhibit symptoms reminiscent of patients with activating inflammasome mutations. [ABSTRACT FROM AUTHOR]

Neutrophils express pattern recognition receptors (PRRs) and regulate immune responses via PRR-dependent cytokine production. An emerging theme is that neutrophil PRRs often exhibit cell type-specific adaptations in their signalling pathways. This prompted us to examine inflammasome signalling by the PRR NLRP3 in murine neutrophils, in comparison to well-established NLRP3 signalling pathways in macrophages. Here, we demonstrate that while murine neutrophils can indeed signal via the NLRP3 inflammasome, neutrophil NLRP3 selectively responds to soluble agonists but not to the particulate/ crystalline agonists that trigger NLRP3 activation in macrophages via phagolysosomal rupture. In keepingwith this, alum did not trigger IL-1β production from human PMN, and the lysosomotropic peptide Leu-Leu-OMe stimulated only weak NLRP3-dependent IL- 1β production from murine neutrophils, suggesting that lysosomal rupture is not a strong stimulus for NLRP3 activation in neutrophils. We validated our in vitro findings for poor neutrophil NLRP3 responses to particles in vivo, where we demonstrated that neutrophils do not significantly contribute to alum-induced IL-1β production in mice. In all, our studies highlight that myeloid cell identity and the nature of the danger signal can strongly influence signalling by a single PRR, thus shaping the nature of the resultant immune response. [ABSTRACT FROM AUTHOR]

The macrophage NLRC4 inflammasome drives potent innate immune responses against Salmonella by eliciting caspase-1-dependent proinflammatory cytokine production (e.g., interleukin-1β [IL-1β]) and pyroptotic cell death. However, the potential contribution of other cell types to inflammasome-mediated host defense against Salmonella was unclear. Here, we demonstrate that neutrophils, typically viewed as cellular targets of IL-1β, themselves activate the NLRC4 inflammasome during acute Salmonella infection and are a major cell compartment for IL-1β production during acute peritoneal challenge in vivo. Importantly, unlike macrophages, neutrophils do not undergo pyroptosis upon NLRC4 inflammasome activation. The resistance of neutrophils to pyroptotic death is unique among inflammasome-signaling cells so far described and allows neutrophils to sustain IL-1β production at a site of infection without compromising the crucial inflammasome-independent antimicrobial effector functions that would be lost if neutrophils rapidly lysed upon caspase-1 activation. Inflammasome pathway modification in neutrophils thus maximizes host proinflammatory and antimicrobial responses during pathogen challenge. [ABSTRACT FROM AUTHOR]

Iron acquisition is an important aspect of the host-pathogen interaction. In the case of Salmonella it is established that catecholate siderophores are important for full virulence. In view of their very high affinity for ferric iron, functional studies of siderophores have been almost exclusively focused on their role in acquisition of iron from the host. In the present study, we investigated whether the siderophores (enterobactin and salmochelin) produced by Salmonella enterica sv. Typhimurium could act as antioxidants and protect from the oxidative stress encountered after macrophage invasion. Our results show that the ability to produce siderophores enhanced the survival of Salmonella in the macrophage mainly at the early stages of infection, coincident with the oxidative burst. Using siderophore biosynthetic and siderophore receptor mutants we demonstrated that salmochelin and enterobactin protect S. Typhimurium against ROS (reactive oxygen species) in vitro and that siderophores must be intracellular to confer full protection. We also investigated whether other chemically distinct siderophores (yersiniabactin and aerobactin) or the monomeric catechol 2,3-dihydroxybenzoate could provide protection against oxidative stress and found that only catecholate siderophores have this property. Collectively, the results of the present study identify additional functions for siderophores during host-pathogen interactions. [ABSTRACT FROM AUTHOR]

Inflammasomes are multi-protein complexes that assemble in response to cellular infection, cellular stress or tissue damage. Inflammasomes provide signalling platforms for the activation of caspase-1, which in turn triggers lytic cell death and the maturation and secretion of the interleukins (IL), IL-1β and IL-18, which co-ordinate host-protective inflammatory responses. Recent studies also highlight emerging roles for interleukin-independent pathways in exerting microbial control. This article reviews cytokine-dependent and cytokine-independent host defence pathways engaged by inflammasomes during infection. Such inflammatory and antimicrobial mechanisms include the recruitment and activation of immune cells, the production of lipid mediators and complement proteins, the induction of the acute-phase and fever responses, the modulation of serum metallic ion content and the release of intracellular bacteria by pyroptotic cell death. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The article focuses on the study of the Caspase-8–dependent gasdermin D cleavage promotes antimicrobial defense but confers susceptibility to TNF-induced lethality. It mentions that the Gasdermin D (GSDMD) is a pore-forming protein that promotes pyroptosis and release of proinflammatory cytokines; and revealed that apoptotic caspase-8 directly cleaves GSDMD to trigger pyroptosis.

The gasdermins are a family of pore-forming proteins that have recently emerged as executors of pyroptosis, a lytic form of cell death that is induced by the innate immune system to eradicate infected or malignant cells. Mammalian gasdermins comprise a cytotoxic N-terminal domain, a flexible linker and a C-terminal repressor domain. Proteolytic cleavage in the linker releases the cytotoxic domain, thereby allowing it to form β-barrel membrane pores. Formation of gasdermin pores in the plasma membrane eventually leads to a loss of the electrochemical gradient, cell death and membrane rupture. Here we review recent work that has expanded our understanding of gasdermin biology and function in mammals by revealing their activation mechanism, their regulation and their roles in autoimmunity, host defence and cancer. We further highlight fungal and bacterial gasdermin pore formation pointing to a conserved mechanism of cell death induction., (© 2024. Springer Nature Limited.)

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., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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., (© 2022 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

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 (NLRP1 DR ) by MAP3K20/ZAKα kinase and its downstream effector, p38. Mutating a single ZAKα phosphorylation site in NLRP1 DR abrogates UVB- and ribotoxin-driven pyroptosis in human keratinocytes. Moreover, fusing NLRP1 DR 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.

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., (© 2020 Heilig et al.)

Host-protective caspase-1 activity must be tightly regulated to prevent pathology, but mechanisms controlling the duration of cellular caspase-1 activity are unknown. Caspase-1 is activated on inflammasomes, signaling platforms that facilitate caspase-1 dimerization and autoprocessing. Previous studies with recombinant protein identified a caspase-1 tetramer composed of two p20 and two p10 subunits (p20/p10) as an active species. In this study, we report that in the cell, the dominant species of active caspase-1 dimers elicited by inflammasomes are in fact full-length p46 and a transient species, p33/p10. Further p33/p10 autoprocessing occurs with kinetics specified by inflammasome size and cell type, and this releases p20/p10 from the inflammasome, whereupon the tetramer becomes unstable in cells and protease activity is terminated. The inflammasome-caspase-1 complex thus functions as a holoenzyme that directs the location of caspase-1 activity but also incorporates an intrinsic self-limiting mechanism that ensures timely caspase-1 deactivation. This intrinsic mechanism of inflammasome signal shutdown offers a molecular basis for the transient nature, and coordinated timing, of inflammasome-dependent inflammatory responses., (© 2018 Boucher et al.)

Inflammasomes are large protein complexes induced by a wide range of microbial, stress, and environmental stimuli that function to induce cell death and inflammatory cytokine processing. Formation of an inflammasome involves dramatic relocalization of the inflammasome adapter protein apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) into a single speck. We have developed a flow cytometric assay for inflammasome formation, time of flight inflammasome evaluation, which detects the change in ASC distribution within the cell. The transit of ASC into the speck is detected by a decreased width or increased height of the pulse of emitted fluorescence. This assay can be used to quantify native inflammasome formation in subsets of mixed cell populations ex vivo. It can also provide a rapid and sensitive technique for investigating molecular interactions in inflammasome formation, by comparison of wild-type and mutant proteins in inflammasome reconstitution experiments., (Copyright © 2014 by The American Association of Immunologists, Inc.)