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.

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.

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.

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.

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.

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.

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.

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 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.

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.

Cutting Edge: Blockade of Inhibitor of Apoptosis Proteins Sensitizes Neutrophils to TNF- but Not Lipopolysaccharide-Mediated Cell Death and IL-1ß Secretion.

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-1ß 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-1ß 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.

Active MLKL triggers the NLRP3 inflammasome in a cell-intrinsic manner.

Necroptosis is a physiological cell suicide mechanism initiated by receptor-interacting protein kinase-3 (RIPK3) phosphorylation of mixed-lineage kinase domain-like protein (MLKL), which results in disruption of the plasma membrane. Necroptotic cell lysis, and resultant release of proinflammatory mediators, is thought to cause inflammation in necroptotic disease models. However, we previously showed that MLKL signaling can also promote inflammation by activating the nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (NLRP3) inflammasome to recruit the adaptor protein apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC) and trigger caspase-1 processing of the proinflammatory cytokine IL-1ß. Here, we provide evidence that MLKL-induced activation of NLRP3 requires (i) the death effector four-helical bundle of MLKL, (ii) oligomerization and association of MLKL with cellular membranes, and (iii) a reduction in intracellular potassium concentration. Although genetic or pharmacological targeting of NLRP3 or caspase-1 prevented MLKL-induced IL-1ß secretion, they did not prevent necroptotic cell death. Gasdermin D (GSDMD), the pore-forming caspase-1 substrate required for efficient NLRP3-triggered pyroptosis and IL-1ß release, was not essential for MLKL-dependent death or IL-1ß secretion. Imaging of MLKL-dependent ASC speck formation demonstrated that necroptotic stimuli activate NLRP3 cell-intrinsically, indicating that MLKL-induced NLRP3 inflammasome formation and IL-1ß cleavage occur before cell lysis. Furthermore, we show that necroptotic activation of NLRP3, but not necroptotic cell death alone, is necessary for the activation of NF-κB in healthy bystander cells. Collectively, these results demonstrate the potential importance of NLRP3 inflammasome activity as a driving force for inflammation in MLKL-dependent diseases.

The murine neutrophil NLRP3 inflammasome is activated by soluble but not particulate or crystalline agonists.

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 keeping with this, alum did not trigger IL-1ß production from human PMN, and the lysosomotropic peptide Leu-Leu-OMe stimulated only weak NLRP3-dependent IL-1ß 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-1ß 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.

A novel flow cytometric method to assess inflammasome formation.

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.

An antioxidant role for catecholate siderophores in Salmonella.

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.

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.

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.

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.

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.