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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]

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]

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]

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]

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.)

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.)