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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.

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., (© 2022 John Wiley & Sons Ltd.)

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., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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., (© 2019 The Authors.)

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., (© 2019 The Authors.)

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 Δ 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 Δ 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., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

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.)

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. 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-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 MLKL-dependent diseases., Competing Interests: D.L.V. is a consultant for Tetralogic Pharmaceuticals.

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.)

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., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

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]

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]

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]