Structural basis for the oligomerization-facilitated NLRP3 activation.

The NACHT-, leucine-rich-repeat-, and pyrin domain-containing protein 3 (NLRP3) is a critical intracellular inflammasome sensor and an important clinical target against inflammation-driven human diseases. Recent studies have elucidated its transition from a closed cage to an activated disk-like inflammasome, but the intermediate activation mechanism remains elusive. Here we report the cryo-electron microscopy structure of NLRP3, which forms an open octamer and undergoes a ~ 90° hinge rotation at the NACHT domain. Mutations on open octamer's interfaces reduce IL-1ß signaling, highlighting its essential role in NLRP3 activation/inflammasome assembly. The centrosomal NIMA-related kinase 7 (NEK7) disrupts large NLRP3 oligomers and forms NEK7/NLRP3 monomers/dimers which is a critical step preceding the assembly of the disk-like inflammasome. These data demonstrate an oligomeric cooperative activation of NLRP3 and provide insight into its inflammasome assembly mechanism.

GSDMD drives canonical inflammasome-induced neutrophil pyroptosis and is dispensable for NETosis.

Neutrophils are the most prevalent immune cells in circulation, but the repertoire of canonical inflammasomes in neutrophils and their respective involvement in neutrophil IL-1ß secretion and neutrophil cell death remain unclear. Here, we show that neutrophil-targeted expression of the disease-associated gain-of-function Nlrp3A350V mutant suffices for systemic autoinflammatory disease and tissue pathology in vivo. We confirm the activity of the canonical NLRP3 and NLRC4 inflammasomes in neutrophils, and further show that the NLRP1b, Pyrin and AIM2 inflammasomes also promote maturation and secretion of interleukin (IL)-1ß in cultured bone marrow neutrophils. Notably, all tested canonical inflammasomes promote GSDMD cleavage in neutrophils, and canonical inflammasome-induced pyroptosis and secretion of mature IL-1ß are blunted in GSDMD-knockout neutrophils. In contrast, GSDMD is dispensable for PMA-induced NETosis. We also show that Salmonella Typhimurium-induced pyroptosis is markedly increased in Nox2/Gp91Phox -deficient neutrophils that lack NADPH oxidase activity and are defective in PMA-induced NETosis. In conclusion, we establish the canonical inflammasome repertoire in neutrophils and identify differential roles for GSDMD and the NADPH complex in canonical inflammasome-induced neutrophil pyroptosis and mitogen-induced NETosis, respectively.

Bacillus anthracis induces NLRP3 inflammasome activation and caspase-8-mediated apoptosis of macrophages to promote lethal anthrax.

Lethal toxin (LeTx)-mediated killing of myeloid cells is essential for Bacillus anthracis, the causative agent of anthrax, to establish systemic infection and induce lethal anthrax. The "LeTx-sensitive" NLRP1b inflammasome of BALB/c and 129S macrophages swiftly responds to LeTx intoxication with pyroptosis and secretion of interleukin (IL)-1ß. However, human NLRP1 is nonresponsive to LeTx, prompting us to investigate B. anthracis host-pathogen interactions in C57BL/6J (B6) macrophages and mice that also lack a LeTx-sensitive Nlrp1b allele. Unexpectedly, we found that LeTx intoxication and live B. anthracis infection of B6 macrophages elicited robust secretion of IL-1ß, which critically relied on the NLRP3 inflammasome. TNF signaling through both TNF receptor 1 (TNF-R1) and TNF-R2 were required for B. anthracis-induced NLRP3 inflammasome activation, which was further controlled by RIPK1 kinase activity and LeTx-mediated proteolytic inactivation of MAP kinase signaling. In addition to activating the NLRP3 inflammasome, LeTx-induced MAPKK inactivation and TNF production sensitized B. anthracis-infected macrophages to robust RIPK1- and caspase-8-dependent apoptosis. In agreement, purified LeTx triggered RIPK1 kinase activity- and caspase-8-dependent apoptosis only in macrophages primed with TNF or following engagement of TRIF-dependent Toll-like receptors. Consistently, genetic and pharmacological inhibition of RIPK1 inhibited NLRP3 inflammasome activation and apoptosis of LeTx-intoxicated and B. anthracis-infected macrophages. Caspase-8/RIPK3-deficient mice were significantly protected from B. anthracis-induced lethality, demonstrating the in vivo pathophysiological relevance of this cytotoxic mechanism. Collectively, these results establish TNF- and RIPK1 kinase activity-dependent NLRP3 inflammasome activation and macrophage apoptosis as key host-pathogen mechanisms in lethal anthrax.

An Apoptotic Caspase Network Safeguards Cell Death Induction in Pyroptotic Macrophages.

Pyroptosis has emerged as a key mechanism by which inflammasomes promote host defense against microbial pathogens and sterile inflammation. Gasdermin D (GSDMD)-mediated cell lysis is a hallmark of pyroptosis, but our understanding of cell death signaling during pyroptosis is fragmented. Here, we show that independently of GSDMD-mediated plasma membrane permeabilization, inflammasome receptors engage caspase-1 and caspase-8, both of which redundantly promote activation of apoptotic executioner caspase-3 and caspase-7 in pyroptotic macrophages. Impaired GSDMD pore formation downstream of caspase-1 and caspase-8 activation suffices to unmask the apoptotic phenotype of pyroptotic macrophages. Combined inactivation of initiator caspase-1 and caspase-8, or executioner caspase-3 and caspase-7, is required to abolish inflammasome-induced DEVDase activity during pyroptosis and in apoptotic Gsdmd-/- cells. Collectively, these results unveil a robust apoptotic caspase network that is activated in parallel to GSDMD-mediated plasma membrane permeabilization and safeguards cell death induction in pyroptotic macrophages.

Caspases in Cell Death, Inflammation, and Disease.

Caspases are an evolutionary conserved family of cysteine proteases that are centrally involved in cell death and inflammation responses. A wealth of foundational insight into the molecular mechanisms that control caspase activation has emerged in recent years. Important advancements include the identification of additional inflammasome platforms and pathways that regulate activation of inflammatory caspases; the discovery of gasdermin D as the effector of pyroptosis and interleukin (IL)-1 and IL-18 secretion; and the existence of substantial crosstalk between inflammatory and apoptotic initiator caspases. A better understanding of the mechanisms regulating caspase activation has supported initial efforts to modulate dysfunctional cell death and inflammation pathways in a suite of communicable, inflammatory, malignant, metabolic, and neurodegenerative diseases. Here, we review current understanding of caspase biology with a prime focus on the inflammatory caspases and outline important topics for future experimentation.

DPP8/DPP9 inhibition elicits canonical Nlrp1b inflammasome hallmarks in murine macrophages.

Activating germline mutations in the human inflammasome sensor NLRP1 causes palmoplantar dyskeratosis and susceptibility to Mendelian autoinflammatory diseases. Recent studies have shown that the cytosolic serine dipeptidyl peptidases DPP8 and DPP9 suppress inflammasome activation upstream of NLRP1 and CARD8 in human keratinocytes and peripheral blood mononuclear cells. Moreover, pharmacological inhibition of DPP8/DPP9 protease activity was shown to induce pyroptosis in murine C57BL/6 macrophages without eliciting other inflammasome hallmark responses. Here, we show that DPP8/DPP9 inhibition in macrophages that express a Bacillus anthracis lethal toxin (LeTx)-sensitive Nlrp1b allele triggered significantly accelerated pyroptosis concomitant with caspase-1 maturation, ASC speck assembly, and secretion of mature IL-1ß and IL-18. Genetic ablation of ASC prevented DPP8/DPP9 inhibition-induced caspase-1 maturation and partially hampered pyroptosis and inflammasome-dependent cytokine release, whereas deletion of caspase-1 or gasdermin D triggered apoptosis in the absence of IL-1ß and IL-18 secretion. In conclusion, blockade of DPP8/DPP9 protease activity triggers rapid pyroptosis and canonical inflammasome hallmarks in primary macrophages that express a LeTx-responsive Nlrp1b allele.

Inflammasome-Dependent Cytokines at the Crossroads of Health and Autoinflammatory Disease.

As key regulators of both innate and adaptive immunity, it is unsurprising that the activity of interleukin (IL)-1 cytokine family members is tightly controlled by decoy receptors, antagonists, and a variety of other mechanisms. Additionally, inflammasome-mediated proteolytic maturation is a prominent and distinguishing feature of two important members of this cytokine family, IL-1ß and IL-18, because their full-length gene products are biologically inert. Although vital in antimicrobial host defense, deregulated inflammasome signaling is linked with a growing number of autoimmune and autoinflammatory diseases. Here, we focus on introducing the diverse inflammasome types and discussing their causal roles in periodic fever syndromes. Therapies targeting IL-1 or IL-18 show great efficacy in some of these autoinflammatory diseases, although further understanding of the molecular mechanisms leading to unregulated production of these key cytokines is required to benefit more patients.

Single-cell analysis of pyroptosis dynamics reveals conserved GSDMD-mediated subcellular events that precede plasma membrane rupture.

Pyroptosis is rapidly emerging as a mechanism of anti-microbial host defense, and of extracellular release of the inflammasome-dependent cytokines interleukin (IL)-1ß and IL-18, which contributes to autoinflammatory pathology. Caspases 1, 4, 5 and 11 trigger this regulated form of necrosis by cleaving the pyroptosis effector gasdermin D (GSDMD), causing its pore-forming amino-terminal domain to oligomerize and perforate the plasma membrane. However, the subcellular events that precede pyroptotic cell lysis are ill defined. In this study, we triggered primary macrophages to undergo pyroptosis from three inflammasome types and recorded their dynamics and morphology using high-resolution live-cell spinning disk confocal laser microscopy. Based on quantitative analysis of single-cell subcellular events, we propose a model of pyroptotic cell disintegration that is initiated by opening of GSDMD-dependent ion channels or pores that are more restrictive than recently proposed GSDMD pores, followed by osmotic cell swelling, commitment of mitochondria and other membrane-bound organelles prior to sudden rupture of the plasma membrane and full permeability to intracellular proteins. This study provides a dynamic framework for understanding cellular changes that occur during pyroptosis, and charts a chronological sequence of GSDMD-mediated subcellular events that define pyroptotic cell death at the single-cell level.

Caspase-1 Engagement and TLR-Induced c-FLIP Expression Suppress ASC/Caspase-8-Dependent Apoptosis by Inflammasome Sensors NLRP1b and NLRC4.

The caspase activation and recruitment domain (CARD)-based inflammasome sensors NLRP1b and NLRC4 induce caspase-1-dependent pyroptosis independent of the inflammasome adaptor ASC. Here, we show that NLRP1b and NLRC4 trigger caspase-8-mediated apoptosis as an alternative cell death program in caspase-1-/- macrophages and intestinal epithelial organoids (IECs). The caspase-8 adaptor FADD was recruited to ASC specks, which served as cytosolic platforms for caspase-8 activation and NLRP1b/NLRC4-induced apoptosis. We further found that caspase-1 protease activity dominated over scaffolding functions in suppressing caspase-8 activation and induction of apoptosis of macrophages and IECs. Moreover, TLR-induced c-FLIP expression inhibited caspase-8-mediated apoptosis downstream of ASC speck assembly, but did not affect pyroptosis induction by NLRP1b and NLRC4. Moreover, unlike during pyroptosis, NLRP1b- and NLRC4-elicited apoptosis retained alarmins and the inflammasome-matured cytokines interleukin 1ß (IL-1ß) and IL-18 intracellularly. This work identifies critical mechanisms regulating apoptosis induction by the inflammasome sensors NLRP1b and NLRC4 and suggests converting pyroptosis into apoptosis as a paradigm for suppressing inflammation.

Familial Mediterranean fever mutations lift the obligatory requirement for microtubules in Pyrin inflammasome activation.

Familial Mediterranean fever (FMF) is the most common monogenic autoinflammatory disease worldwide. It is caused by mutations in the inflammasome adaptor Pyrin, but how FMF mutations alter signaling in FMF patients is unknown. Herein, we establish Clostridium difficile and its enterotoxin A (TcdA) as Pyrin-activating agents and show that wild-type and FMF Pyrin are differentially controlled by microtubules. Diverse microtubule assembly inhibitors prevented Pyrin-mediated caspase-1 activation and secretion of IL-1ß and IL-18 from mouse macrophages and human peripheral blood mononuclear cells (PBMCs). Remarkably, Pyrin inflammasome activation persisted upon microtubule disassembly in PBMCs of FMF patients but not in cells of patients afflicted with other autoinflammatory diseases. We further demonstrate that microtubules control Pyrin activation downstream of Pyrin dephosphorylation and that FMF mutations enable microtubule-independent assembly of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) micrometer-sized perinuclear structures (specks). The discovery that Pyrin mutations remove the obligatory requirement for microtubules in inflammasome activation provides a conceptual framework for understanding FMF and enables immunological screening of FMF mutations.

Inflammasomes as polyvalent cell death platforms.

Inflammasomes are multi-protein platforms that are organized in the cytosol to cope with pathogens and cellular stress. The pattern recognition receptors NLRP1, NLRP3, NLRC4, AIM2 and Pyrin all assemble canonical platforms for caspase-1 activation, while caspase-11-dependent inflammasomes respond to intracellular Gram-negative pathogens. Inflammasomes are chiefly known for their roles in maturation and secretion of the inflammatory cytokines interleukin-(IL)1ß and IL18, but they can also induce regulated cell death. Activation of caspases 1 and 11 in myeloid cells can trigger pyroptosis, a lytic and inflammatory cell death mode. Pyroptosis has been implicated in secretion of IL1ß, IL18 and intracellular alarmins. Akin to these factors, it may have beneficial roles in controlling pathogen replication, but become detrimental in the context of chronic autoinflammatory diseases. Inflammasomes are increasingly implicated in induction of additional regulated cell death modes such as pyronecrosis and apoptosis. In this review, we overview recent advances in inflammasome-associated cell death research, illustrating the polyvalent roles of these macromolecular platforms in regulated cell death signaling.

CARD- and pyrin-only proteins regulating inflammasome activation and immunity.

Membrane-bound and intracellular immune receptors respond to microbial pathogens by initiating signaling cascades that result in production of inflammatory cytokines and antimicrobial factors. These host responses need to be tightly regulated to prevent tissue damage and other harmful consequences of excessive inflammation. CARD-only proteins (COPs) and Pyrin-only proteins (POPs) are human- and primate-specific dominant negative inhibitors that modulate inflammatory and innate immune responses. In addition, several poxviruses encode POPs that interfere with inflammatory and host defense responses. COPs and POPs modulate inflammatory signaling at several checkpoints by sequestering key components of the inflammasome and NF-κB signaling cascades, thus hampering downstream signal transduction. Here, we review and discuss current understanding of the evolutionary history and molecular mechanisms by which roles of host- and virus-encoded COPs and POPs may regulate inflammatory and immune responses. In addition, we address their (patho)physiological roles and highlight topics for further research.

Flagellin-induced NLRC4 phosphorylation primes the inflammasome for activation by NAIP5.

The Nlrc4 inflammasome contributes to immunity against intracellular pathogens that express flagellin and type III secretion systems, and activating mutations in NLRC4 cause autoinflammation in patients. Both Naip5 and phosphorylation of Nlrc4 at Ser533 are required for flagellin-induced inflammasome activation, but how these events converge upon inflammasome activation is not known. Here, we showed that Nlrc4 phosphorylation occurs independently of Naip5 detection of flagellin because Naip5 deletion in macrophages abolished caspase-1 activation, interleukin (IL)-1ß secretion, and pyroptosis, but not Nlrc4 phosphorylation by cytosolic flagellin of Salmonella Typhimurium and Yersinia enterocolitica. ASC speck formation and caspase-1 expression also were dispensable for Nlrc4 phosphorylation. Interestingly, Helicobacter pylori flagellin triggered robust Nlrc4 phosphorylation, but failed to elicit caspase-1 maturation, IL-1ß secretion, and pyroptosis, suggesting that it retained Nlrc4 Ser533 phosphorylating-activity despite escaping Naip5 detection. In agreement, the flagellin D0 domain was required and sufficient for Nlrc4 phosphorylation, whereas deletion of the S. Typhimurium flagellin carboxy-terminus prevented caspase-1 maturation only. Collectively, this work suggests a biphasic activation mechanism for the Nlrc4 inflammasome in which Ser533 phosphorylation prepares Nlrc4 for subsequent activation by the flagellin sensor Naip5.

Negative regulation of the NLRP3 inflammasome by A20 protects against arthritis.

Rheumatoid arthritis is a chronic autoinflammatory disease that affects 1-2% of the world's population and is characterized by widespread joint inflammation. Interleukin-1 is an important mediator of cartilage destruction in rheumatic diseases, but our understanding of the upstream mechanisms leading to production of interleukin-1ß in rheumatoid arthritis is limited by the absence of suitable mouse models of the disease in which inflammasomes contribute to pathology. Myeloid-cell-specific deletion of the rheumatoid arthritis susceptibility gene A20/Tnfaip3 in mice (A20(myel-KO) mice) triggers a spontaneous erosive polyarthritis that resembles rheumatoid arthritis in patients. Rheumatoid arthritis in A20(myel-KO) mice is not rescued by deletion of tumour necrosis factor receptor 1 (ref. 2). Here we show, however, that it crucially relies on the Nlrp3 inflammasome and interleukin-1 receptor signalling. Macrophages lacking A20 have increased basal and lipopolysaccharide-induced expression levels of the inflammasome adaptor Nlrp3 and proIL-1ß. As a result, A20-deficiency in macrophages significantly enhances Nlrp3 inflammasome-mediated caspase-1 activation, pyroptosis and interleukin-1ß secretion by soluble and crystalline Nlrp3 stimuli. In contrast, activation of the Nlrc4 and AIM2 inflammasomes is not altered. Importantly, increased Nlrp3 inflammasome activation contributes to the pathology of rheumatoid arthritis in vivo, because deletion of Nlrp3, caspase-1 and the interleukin-1 receptor markedly protects against rheumatoid-arthritis-associated inflammation and cartilage destruction in A20(myel-KO) mice. These results reveal A20 as a novel negative regulator of Nlrp3 inflammasome activation, and describe A20(myel-KO) mice as the first experimental model to study the role of inflammasomes in the pathology of rheumatoid arthritis.

Activation of the NLRP1b inflammasome independently of ASC-mediated caspase-1 autoproteolysis and speck formation.

Despite its clinical importance in infection and autoimmunity, the activation mechanisms of the NLRP1b inflammasome remain enigmatic. Here we show that deletion of the inflammasome adaptor ASC in BALB/c mice and in C57BL/6 macrophages expressing a functional NLRP1b prevents anthrax lethal toxin (LeTx)-induced caspase-1 autoproteolysis and speck formation. However, ASC(-/-) macrophages undergo normal LeTx-induced pyroptosis and secrete significant amounts of interleukin (IL)-1ß. In contrast, ASC is critical for caspase-1 autoproteolysis and IL-1ß secretion by the NLRC4, NLRP3 and AIM2 inflammasomes. Notably, LeTx-induced inflammasome activation is associated with caspase-1 ubiquitination, which is unaffected in ASC-deficient cells. In vivo, ASC-deficient mice challenged with LeTx produce significant levels of IL-1ß, IL-18 and HMGB1 in circulation, although caspase-1 autoproteolysis is abolished. As a result, ASC(-/-) mice are sensitive to rapid LeTx-induced lethality. Together, these results demonstrate that ASC-driven caspase-1 autoprocessing and speck formation are dispensable for the activation of caspase-1 and the NLRP1b inflammasome.

FADD and caspase-8 mediate priming and activation of the canonical and noncanonical Nlrp3 inflammasomes.

The Nlrp3 inflammasome is critical for host immunity, but the mechanisms controlling its activation are enigmatic. In this study, we show that loss of FADD or caspase-8 in a RIP3-deficient background, but not RIP3 deficiency alone, hampered transcriptional priming and posttranslational activation of the canonical and noncanonical Nlrp3 inflammasome. Deletion of caspase-8 in the presence or absence of RIP3 inhibited caspase-1 and caspase-11 activation by Nlrp3 stimuli but not the Nlrc4 inflammasome. In addition, FADD deletion prevented caspase-8 maturation, positioning FADD upstream of caspase-8. Consequently, FADD- and caspase-8-deficient mice had impaired IL-1ß production when challenged with LPS or infected with the enteropathogen Citrobacter rodentium. Thus, our results reveal FADD and caspase-8 as apical mediators of canonical and noncanonical Nlrp3 inflammasome priming and activation.

Histone modifications in herpesvirus infections.

In eukaryotic cells, gene expression is not only regulated by transcription factors but also by several epigenetic mechanisms including post-translational modifications of histone proteins. There are numerous histone modifications described to date and methylation, acetylation, ubiquitination and phosphorylation are amongst the best studied. In parallel, certain viruses interact with the very same regulatory mechanisms, hereby manipulating the normal epigenetic landscape of the host cell, to fit their own replication needs. This review concentrates on herpesviruses specifically and how they interfere with the histone-modifying enzymes to regulate their replication cycles. Herpesviruses vary greatly with respect to the cell types they infect and the clinical diseases they cause, yet they share various common features including their capacity to encode viral proteins which affect and interfere with the normal functions of histone-modifying enzymes. Studying the epigenetic manipulation/dysregulation of herpesvirus-host interactions not only generates novel insights into the pathogenesis of these viruses but may also have important therapeutic implications.

Effects of interferon on immediate-early mRNA and protein levels in sensory neuronal cells infected with herpes simplex virus type 1 or pseudorabies virus.

Most alphaherpesviruses are able to establish latency in sensory neurons and reactivate upon specific stimuli to cause recurrent symptoms. We have previously shown that interferon (IFN) is capable of inducing a quiescent HSV-1 and PRV infection that strongly resembles in vivo latency in primary cultures of TG neurons. This IFN-induced latency-like quiescence was found to correlate with suppression of the immediate-early protein ICP4 in HSV-1 and its ortholog IE180 in PRV. Here, we mechanistically investigated the IFN-mediated suppression of ICP4 and IE180 in sensory neuronal cells. RT-qPCR showed that mRNA levels of either HSV ICP4 or PRV IE180 at 4 hpi were mildly but not significantly different in IFN-treated samples versus control samples, whereas a strong reduction was observed at 8 hpi and 12 hpi. However, at 4 hpi, HSV ICP4 but not PRV IE180 protein expression was already markedly reduced in IFN-treated samples. In line with this difference in IFN-mediated suppression of HSV ICP4 versus PRV IE180 protein levels, we found that IFN resulted in an increase in phosphorylation of the translation initiation factor eIF2α in HSV-infected but not in PRV-infected cells. The latter finding indicates that PRV efficiently circumvents IFN-mediated translation inhibition by interfering with phosphorylation of eIF2α.

Interferon alpha induces establishment of alphaherpesvirus latency in sensory neurons in vitro.

BACKGROUND: Several alphaherpesviruses, including herpes simplex virus 1 (HSV-1) and pseudorabies virus (PRV), establish lifelong latency in neurons of the trigeminal ganglion (TG). Although it is thought that efficient establishment of alphaherpesvirus latency is based on a subtle interplay between virus, neurons and the immune system, it is not clear which immune components are of major importance for the establishment of latency. METHODOLOGY/PRINCIPAL FINDINGS: Here, using an in vitro model that enables a natural route of infection, we show that interferon alpha (IFNalpha) has the previously uncharacterized capacity to induce a quiescent HSV-1 and PRV infection in porcine TG neurons that shows strong similarity to in vivo latency. IFNalpha induced a stably suppressed HSV-1 and PRV infection in TG neurons in vitro. Subsequent treatment of neurons containing stably suppressed virus with forskolin resulted in reactivation of both viruses. HSV and PRV latency in vivo is often accompanied by the expression of latency associated transcripts (LATs). Infection of TG neurons with an HSV-1 mutant expressing LacZ under control of the LAT promoter showed activation of the LAT promoter and RT-PCR analysis confirmed that both HSV-1 and PRV express LATs during latency in vitro. CONCLUSIONS/SIGNIFICANCE: These data represent a unique in vitro model of alphaherpesvirus latency and indicate that IFNalpha may be a driving force in promoting efficient latency establishment.