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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.

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.

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.

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.

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.

Human NLRP1 is a multifunctional inflammasome sensor predominantly expressed in skin and airway epithelium; however its function in skin-specific immunity and its mechanisms of activation are not fully understood. Here we report that human NLRP1 is specifically activated by the ZAKɑ- driven ribotoxic stress response pathway (RSR) induced by ultraviolet B (UVB) irradiation or select microbial ribotoxins. Biochemically, RSR-triggered NLRP1 activation requires ZAKɑ- dependent hyperphosphorylation of a human-specific linker region of NLRP1 (NLRP1DR), leading to the ‘functional degradation’ of the auto-inhibitory NLRP1 N-terminal fragment. Additionally, we show that fusing NLRP1DR to the signaling domains of CARD8, which in itself is insensitive to RSR, creates a minimal inflammasome sensor for UVB and ribotoxins. In summary, these discoveries resolve the mechanisms of UVB sensing by human NLRP1, identify ZAKɑ-activating toxins as novel human NLRP1 activators, and establish NLRP1 inflammasome-dependent pyroptosis as an integral component of the ribotoxic stress response in primary human cells. UVB-induced NLRP1 activation in human keratinocytes involves a nuclear DNA-independent stress response involving photodamaged RNAZAKɑ kinase is required for UVB-triggered, but not VbP- or dsRNA-induced human NLRP1 activationZAKɑ-activating microbial ribotoxins specifically activate the NLRP1 inflammasome in multiple primary human cell typesHyperphosphorylation of a linker region (NLRP1DR) is required for RSR-dependent human NLRP1 activation

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.

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.

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 two independently-generated CRISPR/Cas9 Card19 knockout mice showed no defect in macrophage cell lysis, and expression of CARD19 in Card19lxcn macrophages did not restore cell lysis. Card19 is located on chromosome 13, adjacent to Ninj1, which was recently reported to regulate cell lysis downstream of GSDMD activation. Intriguingly, RNA-seq and western blotting revealed that Card19lxcn BMDMs are hypomorphic for NINJ1 expression, and reconstitution of Ninj1 in Card19lxcn immortalized BMDMs restored cell lysis. Card19lxcn mice exhibited significantly increased susceptibility to Yersinia infection, demonstrating that cell lysis itself plays a key role in protection against bacterial infection. Our findings identify genetic targeting of Card19 being responsible for off-target effects on the adjacent Ninj1 gene, thereby 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 SummaryProgrammed 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. We found that 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.

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.

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.

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. Refereed/Peer-reviewed

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.

Many programmed cell death pathways are essential for organogenesis, development, immunity and the maintenance of homeostasis in multicellular organisms. Pyroptosis, a highly proinflammatory form of cell death, is a critical innate immune response to prevent intracellular infection. Pyroptosis is induced upon the activation of proinflammatory caspases within macromolecular signalling platforms called inflammasomes. This article reviews our understanding of pyroptosis induction, the function of inflammatory caspases in pyroptosis execution, and the importance of pyroptosis for pathogen clearance. It also highlights the situations in which extensive pyroptosis may in fact be detrimental to the host, leading to immune cell depletion or cytokine storm. Current efforts to understand the beneficial and pathological roles of pyroptosis bring the promise of new approaches to fight infectious diseases.

NOD-like receptors (NLR) are a family of cytosolic pattern recognition receptors that include many key drivers of innate immune responses. NLRP12 is an emerging member of the NLR family that is closely related to the well-known inflammasome scaffold, NLRP3. Since its discovery, various functions have been proposed for NLRP12, including the positive regulation of dendritic cell (DC) and neutrophil migration and the inhibition of NF-κB and ERK signalling in DC and macrophages. We show here that NLRP12 is poorly expressed in murine macrophages and DC, but is strongly expressed in neutrophils. Using myeloid cells from WT and Nlrp12(-/)(-) mice, we show that, contrary to previous reports, NLRP12 does not suppress LPS- or infection-induced NF-κB or ERK activation in myeloid cells, and is not required for DC migration in vitro. Surprisingly, we found that Nlrp12 deficiency caused increased rather than decreased neutrophil migration towards the chemokine CXCL1 and the neutrophil parasite Leishmania major, revealing NLRP12 as a negative regulator of directed neutrophil migration under these conditions.

The inflammasome generates caspase-1 p20/p10, presumed to be the active protease. Boucher et al. demonstrate that the inflammasome contains an active caspase-1 species, p33/p10, and functions as a holoenzyme. Further caspase-1 self-processing generates and releases p20/p10 to terminate protease activity., 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., Graphical Abstract

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-1β. diseases. 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 MLKLinduced IL-1β 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-1β release, was not essential for MLKL-dependent death or IL-1β 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-1β 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 MLKLdependent Refereed/Peer-reviewed

The innate immune system protects against infection but also drives inflammatory disorders. Key molecular drivers of both processes are ‘inflammasomes’, multi-protein complexes that assemble in the cytosol to activate the protease, caspase-1. Active caspase-1 cleaves specific proinflammatory cytokines [e.g. interleukin (IL)-1β] into their mature, secreted forms, and initiates a form of inflammatory cell lysis called pyroptosis. Inflammasomes are assembled by select pattern recognition receptors such as NLRC4, NLRP3, AIM2, or via a non-canonical pathway involving caspase-11. Whilst inflammasomes functions have been intensely researched, the cell types mediating inflammasome signalling in distinct in vivo settings were unclear. Neutrophils are one of the first cells to arrive to a site of infection or injury, and thus have the opportunity to detect inflammasome-activating molecules in vivo, but their ability to signal by inflammasome pathways had not been closely examined. This thesis offers a detailed investigation of NLRC4, NLRP3 and caspase-11 inflammasome signalling in neutrophils during in vitro or in vivo challenge with whole microbe, purified microbial components, or adjuvant. This thesis demonstrates that acute Salmonella infection triggered NLRC4-dependent caspase-1 activation and IL-1β processing in neutrophils, and neutrophils were a major cellular compartment for IL-1β production during acute Salmonella challenge in vivo. Importantly, neutrophils did not undergo pyroptotic cell death upon NLRC4 activation, allowing these cells to sustain IL-1β production at a site of infection without compromising their crucial inflammasome-independent antimicrobial effector functions. Since IL-1β drives neutrophil recruitment and activation, the finding that neutrophils themselves produce IL- 1β suggests neutrophils perform novel and important auto-regulatory functions during infection. Neutrophil NLRP3 pathways were next examined. In macrophages, diverse molecules can trigger NLRP3 activation. Some of these agonists are insoluble molecules (particles or crystals), which activate NLRP3 through lysosomal rupture. Interestingly, the neutrophil NLRP3 inflammasome selectively responded to soluble, but not insoluble, NLRP3 agonists, and lysosomal rupture was a weak stimulus for neutrophil NLRP3 activation. This finding was replicated in vivo, where neutrophils did not significantly contribute to IL-1β production during alum-induced peritonitis. Prior studies in macrophages demonstrated that NLRP3 activation triggers the oligomerisation of the inflammasome adaptor ASC, which then recruits two caspases, pro- caspase-1 and -8. Inflammasome-activated caspase-8 initiates macrophage apoptosis in parallel to caspase-1-dependent pyroptosis. This thesis shows that like macrophages, neutrophil NLRP3 activation triggered ASC oligomerisation, caspase-1 cleavage and IL-1β secretion, but unlike macrophages, caspase-1 activation did not induce cell lysis, suggesting that resistance to pyroptosis is a universal feature of neutrophil inflammasome pathways. Neutrophils also appeared unable to undergo NLRP3/ASC/caspase-8- dependent apoptosis, likely due to inefficient caspase-8 self-processing. This finding prompted examination of other caspase-8/NLRP3 pathways in neutrophils. Chemical inhibition of the inhibitor of apoptosis proteins (IAPs) in LPS-primed macrophages leads to RIPK3 activation and formation of a caspase-8 activating platform, the ripoptosome, which promotes apoptosis and can cleave pro-IL-1β into its mature form. If caspase-8 activity is suppressed, RIPK3 forms a different molecular complex, the necrosome, to induce necroptosis. In macrophages, ripoptosome and necrosome signalling induces NLRP3 inflammasome activation and downstream caspase-1 activity. Surprisingly, blockade of IAP function was a weak stimulus for cell death in LPS-stimulated neutrophils, and did not trigger caspase-8-dependent IL-1β maturation or NLRP3/caspase-1 activation, suggesting that NLRP3 regulation by RIPK3/IAPs is differentially regulated in macrophages versus neutrophils. Caspase-11 functions are important for host defence against cytoplasmic Gram-negative bacteria, but also contribute to pathological immune responses in murine models of septic shock. Like caspase-1, caspase-11 induces pyroptosis. But caspase-11 appears unable to directly cleave cytokines, and instead triggers pro-IL-1β/18 processing through activation of the NLRP3 inflammasome. Because neutrophilia is a hallmark of sepsis, and neutrophils produce NLRP3-dependent IL-1β without concomitant cell death, the possibility that neutrophils contribute to septic shock was next investigated. Neutrophil exposure to intracellular LPS in vitro indeed triggered caspase-11-dependent IL-1β production but not cell death. Nlrp3-/- mice, like Casp11-/- mice, were protected from lethal endotoxin shock, suggesting that caspase-11/NLRP3-dependent cytokine production may contribute to LPS lethality. Importantly, prior neutrophil depletion protected wild type but not Casp11-/- mice from endotoxic shock. Although further investigations are required, these data suggest that iv neutrophils may be a sustained cellular source of caspase-11/NLRP3-dependent, pathogenic IL-1β during murine endotoxic shock. In summary, this thesis demonstrates that neutrophils signal via specialised inflammasome pathways to maximise host responses during pathogen challenge in vivo. The finding that neutrophils themselves secrete IL-1β to drive their own recruitment, activation and survival is in line with an emerging theme that neutrophils perform important immunoregulatory functions during infection. Although neutrophils are often regarded as pro-apoptotic cells, this thesis reports that, surprisingly, neutrophils resist all known forms of inflammasome- induced cell death. Presumably this allows neutrophils time to exert their important functions in host defence, but may also contribute to sustained cytokine production and thus pathology in inflammatory diseases such as endotoxic shock. The interplay between inflammation and neutrophil cell death warrants further examination to understand the underlying mechanisms of neutrophil-driven inflammatory disorders.

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-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.

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.

Toll-like receptor 4 (TLR4) is activated by bacterial lipopolysaccharide (LPS) to mount innate immune responses. The TLR4-induced release of pro- and anti-inflammatory cytokines generates robust inflammatory responses, which must then be restrained to avoid disease. New mechanisms for the critical regulation of TLR-induced cytokine responses are still emerging. Here we find TLR4 complexes localized in LPS-induced dorsal ruffles on the surface of macrophages. We discover that the small GTPase Rab8a is enriched in these ruffles and recruits phosphatidylinositol 3-kinase (PI3Kγ) as an effector by interacting directly through its Ras-binding domain. Rab8a and PI3Kγ function to regulate Akt signalling generated by surface TLR4. Rab8a and PI3Kγ do not affect TLR4 endocytosis, but instead regulate mammalian target of rapamycin signalling as a mechanism for biasing the cytokine profile to constrain inflammation in innate immunity.

SummaryThe 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.

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.

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-dihydroxy-benzoate 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.

The macrophage NLRC4 inflammasome drives potent innate immune responses against Salmonella , by eliciting caspase-1-dependent pro-inflammatory cytokine production (e.g. interleukin (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 amongst 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 pro-inflammatory and antimicrobial responses during pathogen challenge. Cell Reports (in press).

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.

Injection of effector proteins to block host innate immune signalling is a common strategy used by many pathogenic organisms to establish an infection. Pathogenic Yersinia species for example inject the acetyltransferase YopJ into target cells to inhibit NF-κB and MAPK signalling. To counteract this, detection of YopJ activity in myeloid cells promotes the assembly of a RIPK1-caspase-8 death-inducing platform that confers antibacterial defence. While recent studies revealed that caspase-8 cleaves the pore-forming protein, gasdermin D (GSDMD) to trigger pyroptosis in macrophages, whether RIPK1 activates additional substrates downstream of caspase-8 to promote host defence 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-1β release, which is critical for anti-Yersinia defence. During in vivo infection, IL-1β neutralisation 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 signalling.

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 ΔsifA 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 ΔsifA 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.