Inflammasome Activation In Response Research Articles

37: Caspase-11 controls inflammasome activation in response to bacterial secretion systems that access the host cell cytosol

Inflammasome activation is important for antimicrobial defense because it induces cell death and regulates the secretion of IL-1 family cytokines. The inflammasome activates caspase-1 to process and secrete IL-1 β . However, the mechanisms governing IL-1 α release are less clear. Recently, a non-canonical inflammasome was described that activates caspase-11 and mediates pyroptosis and release of IL-1 α and IL-1 β through TRIF-dependent interferon production. However, it is unknown whether additional bacterial signals trigger caspase-11 activation. Many bacterial pathogens use specialized secretion systems to translocate effector proteins into the host cell cytosol. These secretion systems can also deliver flagellin into the cytosol, which triggers caspase-1 activation and pyroptosis. However, specialized secretion systems still induce inflammasome activation in the absence of flagellin, but the pathways that mediate this response are unclear. We observe rapid inflammasome activation in response to the type IV or type III secretion systems of Legionella pneumophila and Yersinia pseudotuberculosis . Unlike IL-1 β secretion, IL-1 α secretion does not require caspase-1. Instead, caspase-11 is required for both IL-1 α secretion and cell death in response to the activity of these secretion systems. Caspase-11 promotes IL-1 β release through the NLRP3/ASC inflammasome, but caspase-11-dependent release of IL-1 α is independent of NLRP3/ASC, NAIP5/NLRC4, TRIF, and type I interferon signaling. Furthermore, we find both overlapping and non-redundant roles for IL-1 α and IL-1 β in mediating neutrophil recruitment and bacterial clearance in response to pulmonary infection by L. pneumophila . Our findings demonstrate that virulent, but not avirulent, bacteria trigger a rapid caspase-11-dependent innate immune response that is critical for host defense in vivo.

The inflammasome as a target of modulation by DNA viruses.

The cellular innate immune response represents the initial reaction of a host against infecting pathogens. Host cells detect incoming microbes by way of a large and expanding array of receptors that react with evolutionarily conserved molecular patterns exhibited by microbial intruders. These receptors are responsible for initiating signaling that leads to both transcriptional activation of immunologically important genes as well as protease-dependent processing of cellular proteins. The inflammasome refers to a protein complex that functions as an activation platform for the cysteine protease caspase-1, which then processes inflammatory molecules such as IL-1β and IL-18 into functional forms. Assembly of this complex is triggered following receptor-mediated detection of pathogen-associated molecules. Receptors have been identified that are essential to inflammasome activation in response to numerous molecular patterns including virus-associated molecules such as DNA. In fact, the importance of cytoplasmic DNA as an immune stimulus is exemplified by the existence of at least nine distinct cellular receptors capable of initiating innate reactivity in response to this molecule. Viruses that employ DNA as genomic material include herpesviruses, poxviruses and adenoviruses. Each has been described as capable of inducing inflammasome-mediated activity. Interestingly, however, the cellular molecules responsible for these responses appear to vary according to host species, cell type and even viral strain. Secretion of IL-1β and IL-18 are important components of antimicrobial immunity and, as a result, pathogens have evolved factors to evade or counteract this response. This includes DNA-based viruses, many of which encode multiple redundant counteractive molecules. However, it is clear that such phenotypes are only beginning to be uncovered. The purpose of this review is to describe what is known regarding the activation of inflammasome-mediated processes in response to infection with well-examined families of DNA viruses and to discuss characterized mechanisms of manipulation and neutralization of inflammasome-dependent activity. This review aims to shed light on the biologically important phenomena regarding this virus-host interaction and to highlight key areas where important information is lacking.

Cytosolic K+ and extracellular Na+ as regulators of NLRP3 inflammasome activation and the IL‐1β secretion response of macrophages to crystalline stimuli

The crystalline particulate alum has widely been used as an adjuvant in vaccines. Alum and other crystalline particulates also stimulate NLRP3 (Nod‐like receptor protein 3)/caspase‐1 inflammasome assembly and secretion of mature IL‐1β via poorly understood mechanisms that involve destabilization of lysosomes and decreases in cytosolic [K+]. This study directly measures changes in cytosolic [K+] and characterizes mechanisms for K+ efflux in LPS‐primed murine bone marrow‐derived macrophages (BMDM) stimulated with either Imject Alum or the lysosomal destabilizing agent leu‐leu‐OMe. IL‐1β secretion in response to either alum or leu‐leu‐OMe was inhibited by increased extracellular [K+] or decreased extracellular [Na+]. Atomic absorbance spectroscopy was used to directly demonstrate reduced intracellular [K+] upon stimulation with alum. Alum‐stimulated K+ efflux and IL‐1β secretion was insensitive to several Trp channel antagonists. In contrast, pannexin1−/− BMDM demonstrated reduced IL‐1β release upon stimulation with alum. These observations indicate that combined efflux of intracellular K+ and influx of extracellular Na+ are necessary for optimal NLRP3 inflammasome activation in response to alum and lysosomal destabilization. Pannexin1 hemichannels may mediate in part the alum‐induced cation fluxes linked to NLRP3 inflammasome activation. (Supported by T32‐HL‐105338 and R01‐GM36387)

Neutrophil Extracellular Trap–Associated Protein Activation of the NLRP3 Inflammasome Is Enhanced in Lupus Macrophages

Neutrophil extracellular traps (NETs) represent an important defense mechanism against microorganisms. Clearance of NETs is impaired in a subset of patients with systemic lupus erythematosus, and NETosis is increased in neutrophils and, particularly, in low-density granulocytes derived from lupus patients. NETs are toxic to the endothelium, expose immunostimulatory molecules, activate plasmacytoid dendritic cells, and may participate in organ damage through incompletely characterized pathways. To better understand the role of NETs in fostering dysregulated inflammation, we examined inflammasome activation in response to NETs or to LL-37, an antibacterial protein externalized on NETs. Both NETs and LL-37 activate caspase-1, the central enzyme of the inflammasome, in both human and murine macrophages, resulting in release of active IL-1β and IL-18. LL-37 activation of the NLRP3 inflammasome utilizes P2X7 receptor-mediated potassium efflux. NET and LL-37-mediated activation of the inflammasome is enhanced in macrophages derived from lupus patients. In turn, IL-18 is able to stimulate NETosis in human neutrophils. These results suggest that enhanced formation of NETs in lupus patients can lead to increased inflammasome activation in adjacent macrophages. This leads to release of inflammatory cytokines that further stimulate NETosis, resulting in a feed-forward inflammatory loop that could potentially lead to disease flares and/or organ damage.

ATP release and purinergic signaling in NLRP3 inflammasome activation

The NLRP3 inflammasome is a protein complex involved in IL-1β and IL-18 processing that senses pathogen- and danger-associated molecular patterns (PAMPs and DAMPs). One step- or two step-models have been proposed to explain the tight regulation of IL-1β production during inflammation. Moreover, cellular stimulation triggers adenosine triphosphate (ATP) release and subsequent activation of purinergic receptors at the cell surface. Importantly some studies have reported roles for extracellular ATP, in NLRP3 inflammasome activation in response to PAMPs and DAMPs. In this mini review, we will discuss the link between active ATP release, purinergic signaling and NLRP3 inflammasome activation. We will focus on the role of autocrine or paracrine ATP export in particle-induced NLRP3 inflammasome activation and discuss how particle activators are competent to induce maturation and secretion of IL-1β through a process that involves, as a first event, extracellular release of endogenous ATP through hemichannel opening, and as a second event, signaling through purinergic receptors that trigger NLRP3 inflammasome activation. Finally, we will review the evidence for ATP as a key pro-inflammatory mediator released by dying cells. In particular we will discuss how cancer cells dying via autophagy trigger ATP-dependent NLRP3 inflammasome activation in the macrophages engulfing them, eliciting an immunogenic response against tumors.

Deubiquitination of NLRP3 by BRCC3 Critically Regulates Inflammasome Activity

NLRP3 is an important pattern recognition receptor involved in mediating inflammasome activation in response to viral and bacterial infections as well as various proinflammatory stimuli associated with tissue damage or malfunction. Upon activation, NLRP3 assembles a multimeric inflammasome complex comprising the adaptor ASC and the effector pro-caspase-1 to mediate the activation of caspase-1. Although NLRP3 expression is induced by the NF-κB pathway, the posttranscriptional molecular mechanism controlling the activation of NLRP3 remains elusive. Using both pharmacological and molecular approaches, we show that the activation of NLRP3 inflammasome is regulated by a deubiquitination mechanism. We further identify the deubiquitinating enzyme, BRCC3, as a critical regulator of NLRP3 activity by promoting its deubiquitination and characterizing NLRP3 as a substrate for the cytosolic BRCC3-containing BRISC complex. Our results elucidate a regulatory mechanism involving BRCC3-dependent NLRP3 regulation and highlight NLRP3 ubiquitination as a potential therapeutic target for inflammatory diseases.

The calcium-sensing receptor regulates the NLRP3 inflammasome through Ca2+ and cAMP

Mutations in the gene encoding NLRP3 cause a spectrum of autoinflammatory diseases known as cryopyrin-associated periodic syndromes (CAPS). NLRP3 is a key component of one of several distinct cytoplasmic multiprotein complexes (inflammasomes) that mediate the maturation of the proinflammatory cytokine interleukin-1β (IL-1β) by activating caspase-1. Although several models for inflammasome activation, such as K(+) efflux, generation of reactive oxygen species and lysosomal destabilization, have been proposed, the precise molecular mechanism of NLRP3 inflammasome activation, as well as the mechanism by which CAPS-associated mutations activate NLRP3, remain to be elucidated. Here we show that the murine calcium-sensing receptor (CASR) activates the NLRP3 inflammasome, mediated by increased intracellular Ca(2+) and decreased cellular cyclic AMP (cAMP). Ca(2+) or other CASR agonists activate the NLRP3 inflammasome in the absence of exogenous ATP, whereas knockdown of CASR reduces inflammasome activation in response to known NLRP3 activators. CASR activates the NLRP3 inflammasome through phospholipase C, which catalyses inositol-1,4,5-trisphosphate production and thereby induces release of Ca(2+) from endoplasmic reticulum stores. The increased cytoplasmic Ca(2+) promotes the assembly of inflammasome components, and intracellular Ca(2+) is required for spontaneous inflammasome activity in cells from patients with CAPS. CASR stimulation also results in reduced intracellular cAMP, which independently activates the NLRP3 inflammasome. cAMP binds to NLRP3 directly to inhibit inflammasome assembly, and downregulation of cAMP relieves this inhibition. The binding affinity of cAMP for CAPS-associated mutant NLRP3 is substantially lower than for wild-type NLRP3, and the uncontrolled mature IL-1β production from CAPS patients' peripheral blood mononuclear cells is attenuated by increasing cAMP. Taken together, these findings indicate that Ca(2+) and cAMP are two key molecular regulators of the NLRP3 inflammasome that have critical roles in the molecular pathogenesis of CAPS.

Inflammasome triggered by cell swelling

Mechanosensitive ion channels promote inflammasome activation in response to cell swelling.

Novel role of PKR in inflammasome activation and HMGB1 release

The inflammasome regulates release of caspase activation-dependent cytokines, including IL-1β, IL-18, and high-mobility group box 1 (HMGB1)1-5. During the course of studying HMGB1 release mechanisms, we discovered an important role of double-stranded RNA dependent protein kinase (PKR) in inflammasome activation. Exposure of macrophages to inflammasome agonists induced PKR autophosphorylation. PKR inactivation by genetic deletion or pharmacological inhibition severely impaired inflammasome activation in response to double-stranded RNA, ATP, monosodium urate, adjuvant aluminum, rotenone, live E. coli, anthrax lethal toxin, DNA transfection, and S. Typhimurium infection. PKR deficiency significantly inhibited the secretion of IL-1beta, IL-18 and HMGB1 in E. coli-induced peritonitis. PKR physically interacts with multiple inflammasome components, including NLR family pyrin domain-containing 3 (NLRP3), NLR family pyrin domain-containing 1 (NLRP1), NLR family CARD domain-containing protein 4 (NLRC4), Absent in melanoma 2 (AIM2), and broadly regulates inflammasome activation. PKR autophosphorylation in a cell free system with recombinant NLRP3, ASC and pro-casapse-1 reconstitutes inflammasome activity. These results reveal a critical role of PKR in inflammasome activation, and indicate that it should be possible to pharmacologically target this molecule to treat inflammation.

PKR is required for NLRP3 inflammasome activation and HMGB1 release (114.7)

Abstract The NLR family pyrin domain-containing 3 (NLRP3) inflammasome, an intracellular oligomeric protein complex, regulates release of caspase-1 activation-dependent cytokines, including IL-1β, IL-18, and high-mobility group box 1 (HMGB1). During the course of studying HMGB1 release mechanisms from macrophages, we unexpectedly observed that double-stranded RNA dependent protein kinase (PKR) is required for NLRP3 inflammasome activation. PKR inactivation by genetic deletion or pharmacological inhibition blocked NLRP3 inflammasome activation in response to double-stranded RNA, ATP, monosodium urate, adjuvant aluminum, and live E coli. Overexpression of PKR significantly enhanced the reconstituted NLRP3 inflammasome-induced caspase-1 activation and IL-1β maturation; whereas blocking PKR significantly inhibited the reconstituted NLRP3 inflammasome activation. PKR physically interacts with NLRP3, and PKR autophosphorylation is required for efficient PKR and NLRP3 interaction. Importantly, PKR autophosphorylation in a cell free system with recombinant NLRP3, ASC and pro-casapse-1 reconstitutes inflammasome activity. Together, these results indicate that PKR is required for inflammasome activation, and that it should be possible to target this molecule to inhibit inflammasome activity during inflammation.

NLRP3 inflammasome–mediated neutrophil recruitment and hypernociception depend on leukotriene B4 in a murine model of gout

Deposition of monosodium urate monohydrate (MSU) crystals in the joints promotes an intense inflammatory response and joint dysfunction. This study evaluated the role of the NLRP3 inflammasome and 5-lipoxygenase (5-LOX)-derived leukotriene B(4) (LTB(4) ) in driving tissue inflammation and hypernociception in a murine model of gout. Gout was induced by injecting MSU crystals into the joints of mice. Wild-type mice and mice deficient in NLRP3, ASC, caspase 1, interleukin-1β (IL-1β), IL-1 receptor type I (IL-1RI), IL-18R, myeloid differentiation factor 88 (MyD88), or 5-LOX were used. Evaluations were performed to assess neutrophil influx, LTB(4) activity, cytokine (IL-1β, CXCL1) production (by enzyme-linked immunosorbent assay), synovial microvasculature cell adhesion (by intravital microscopy), and hypernociception. Cleaved caspase 1 and production of reactive oxygen species (ROS) were analyzed in macrophages by Western blotting and fluorometric assay, respectively. Injection of MSU crystals into the knee joints of mice induced neutrophil influx and neutrophil-dependent hypernociception. MSU crystal-induced neutrophil influx was CXCR2-dependent and relied on the induction of CXCL1 in an NLRP3/ASC/caspase 1/IL-1β/MyD88-dependent manner. LTB(4) was produced rapidly after injection of MSU crystals, and this was necessary for caspase 1-dependent IL-1β production and consequent release of CXCR2-acting chemokines in vivo. In vitro, macrophages produced LTB(4) after MSU crystal injection, and LTB(4) was relevant in the MSU crystal-induced maturation of IL-1β. Mechanistically, LTB(4) drove MSU crystal-induced production of ROS and ROS-dependent activation of the NLRP3 inflammasome. These results reveal the role of the NLRP3 inflammasome in mediating MSU crystal-induced inflammation and dysfunction of the joints, and highlight a previously unrecognized role of LTB(4) in driving NLRP3 inflammasome activation in response to MSU crystals, both in vitro and in vivo.

Hemorrhagic Shock Activation of NLRP3 Inflammasome in Lung Endothelial Cells

Hemorrhagic shock (HS) due to major trauma and surgery predisposes the host to the development of systemic inflammatory response syndrome (SIRS), including acute lung injury (ALI), through activating and exaggerating the innate immune response. IL-1β is a crucial proinflammatory cytokine that contributes to the development of SIRS and ALI. Lung endothelial cells (EC) are one important source of IL-1β, and the production of active IL-1β is controlled by the inflammasome. In this study, we addressed the mechanism underlying HS activation of the inflammasome in lung EC. We show that high mobility group box 1 acting through TLR4, and a synergistic collaboration with TLR2 and receptor for advanced glycation end products signaling, mediates HS-induced activation of EC NAD(P)H oxidase. In turn, reactive oxygen species derived from NAD(P)H oxidase promote the association of thioredoxin-interacting protein with the nucleotide-binding oligomerization domain-like receptor protein NLRP3 and subsequently induce inflammasome activation and IL-1β secretion from the EC. We also show that neutrophil-derived reactive oxygen species play a role in enhancing EC NAD(P)H oxidase activation and therefore an amplified inflammasome activation in response to HS. The present study explores a novel mechanism underlying HS activation of EC inflammasome and thus presents a potential therapeutic target for SIRS and ALI induced after HS.

NAIPs: pathogen-sensing proteins

NAIP family members mediate NLRC4 inflammasome activation in response to distinct bacterial ligands.

TLR2 Signaling Contributes to Rapid Inflammasome Activation during F. novicida Infection

BackgroundEarly detection of microorganisms by the innate immune system is provided by surface-expressed and endosomal pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs). Detection of microbial components by TLRs initiates a signaling cascade leading to the expression of proinflammatory cytokines including IL-6 and IL-1β. Some intracellular bacteria subvert the TLR response by rapidly escaping the phagosome and entering the cytosol. However, these bacteria may be recognized by the inflammasome, a multi-protein complex comprised of a sensor protein, ASC and the cysteine protease caspase-1. Inflammasome activation leads to release of the proinflammatory cytokines IL-1β and IL-18 and death of the infected cell, an important host defense that eliminates the pathogen's replicative niche. While TLRs and inflammasomes are critical for controlling bacterial infections, it is unknown whether these distinct host pathways cooperate to activate defenses against intracellular bacteria.Methodology/Significant FindingsUsing the intracellular bacterium Francisella novicida as a model, we show that TLR2−/− macrophages exhibited delayed inflammasome activation compared to wild-type macrophages as measured by inflammasome assembly, caspase-1 activation, cell death and IL-18 release. TLR2 also contributed to inflammasome activation in response to infection by the cytosolic bacterium Listeria monocytogenes. Components of the TLR2 signaling pathway, MyD88 and NF-κB, were required for rapid inflammasome activation. Furthermore, TLR2−/− mice exhibited lower levels of cell death, caspase-1 activation, and IL-18 production than wild-type mice upon F. novicida infection.Conclusions/SignificanceThese results show that TLR2 is required for rapid inflammasome activation in response to infection by cytosolic bacterial pathogens. In addition to further characterizing the role of TLR2 in host defense, these findings broaden our understanding of how the host integrates signals from spatiotemporally separated PRRs to coordinate an innate response against intracellular bacteria.

The NLRP3 Inflammasome Detects Encephalomyocarditis Virus and Vesicular Stomatitis Virus Infection

Inflammasomes are cytosolic protein complexes that regulate caspase-1 activation and the secretion of interleukin-1β (IL-1β) and IL-18. Several different inflammasome complexes have been identified, but the NLRP3 inflammasome is particularly notable because of its central role in diseases of inflammation. Recent work has demonstrated an essential role for the NLRP3 inflammasome in host defense against influenza virus. We show here that two other RNA viruses, encephalomyocarditis virus (EMCV) and vesicular stomatitis virus (VSV), activate the NLRP3 inflammasome in dendritic cells and macrophages through a mechanism requiring viral replication. Inflammasome activation in response to both viruses does not require MDA5 or RIG-I signaling. Despite the ability of the NLRP3 inflammasome to detect EMCV and VSV, wild-type and caspase-1-deficient mice were equally susceptible to infection with both viruses. These findings indicate that the NLRP3 inflammasome may be a common pathway for RNA virus detection, but its precise role in the host response may be variable.

Mitochondrial ROS fuel the inflammasome

Mitochondrial ROS fuel the inflammasome

The AIM2 inflammasome is essential for host defense against cytosolic bacteria and DNA viruses

Inflammasomes regulate the activity of capase-1 and maturation of interleukin-1β and interleukin-18. Recently, AIM2 was shown to bind DNA and engage ASC to form a caspase-1 activating inflammasome. Using Aim2-deficient mice, we reveal a central role for AIM2 in regulating caspase-1-dependent maturation of IL-1β and IL-18, as well as pyroptosis in response to synthetic dsDNA. AIM2 is essential for inflammasome activation in response to Fransicella tularensis, vaccinia virus, mouse cytomegalovirus and plays a partial role in sensing Listeria monocytogenes. Moreover, production of IL-18 and NK cell-dependent IFN-γ production, events critical in early control of virus replication were dependent on AIM2 during mCMV infection in vivo. Collectively, these observations reveal the importance of AIM2 in sensing both bacterial and viral pathogens and triggering innate immunity.

Abstract 5558: Inflammasome Activation in Cardiac Fibroblasts is Essential for Myocardial Ischemia-reperfusion Injury

Background: Inflammatory responses play a key role in the pathophysiology of myocardial ischemia-reperfusion (I/R) injury. We previously investigated the role of ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain), an adaptor protein that forms inflammasome whose activation leads to caspase-1-dependent interleukin (IL)-1 β maturation, in myocardial I/R injury and found that ASC deficiency prevented IL-1 β secretion and inflammatory cell infiltration, resulting in improving myocardial injury and remodeling. In this study, we investigated the mechanisms by which I/R initiates activation of inflammasome in the heart. Methods and Results: To examine what kind of cells are responsible for inflammasome activation in the heart, primary cardiomyocytes (CM) and cardiac fibroblasts (CF) from the wild-type mice were prepared. Although ASC was clearly expressed in both CM and CF, IL-1 β secretion was detected in only CF after stimulation with relatively high dose of lypopolysaccharide (LPS: 100 ng/mL). The intracellular pro-IL-1 β accumulation in CF was similar between wild-type (WT) and ASC deficient (ASC −/− ) mice; however, IL-1 β processing and secretion were significantly inhibited in ASC −/− -CF, compared with that in WT-CF (p<0.01). Hypoxia/reoxygenation (H/R: 6 h of H following 18 h R) dramatically induced IL-1 β secretion in CF after low dose (10 ng/mL) LPS priming. To determine the mechanisms by H/R might activate inflammasome, CF were treated with ATP (10 mM), HMGB1 (10 mg/mL), and K+ ionophore nigericin (1mM) after LPS priming. Marked IL-1 β secretion was detected only in nigericin-treated CF. Further, H/R-induced IL-1 β secretion was significantly inhibited by the treatment with antioxidant N-acetyl-L-cysteine (NAC: 10 mM), K + channel blockers (glimepiride 10 mM, glibenclamide 10 mM, TEA 10 mM) or high extracellular K + (130 mM), indicating that inflammasome activation in response to H/R might be mediated through K+ efflux and ROS production. Conclusion: These findings demonstrate that initial activation of inflammasome in response to I/R occurs in CF, but not in CM, in the heart, and suggest that inflammasome in the CF could be a potential target for the treatment of myocardial I/R injury.

Type I interferon signaling is required for activation of the inflammasome during Francisella infection

Francisella tularensis is a pathogenic bacterium whose virulence is linked to its ability to replicate within the host cell cytosol. Entry into the macrophage cytosol activates a host-protective multimolecular complex called the inflammasome to release the proinflammatory cytokines interleukin (IL)-1β and -18 and trigger caspase-1–dependent cell death. In this study, we show that cytosolic F. tularensis subspecies novicida (F. novicida) induces a type I interferon (IFN) response that is essential for caspase-1 activation, inflammasome-mediated cell death, and release of IL-1β and -18. Extensive type I IFN–dependent cell death resulting in macrophage depletion occurs in vivo during F. novicida infection. Type I IFN is also necessary for inflammasome activation in response to cytosolic Listeria monocytogenes but not vacuole-localized Salmonella enterica serovar Typhimurium or extracellular adenosine triphosphate. These results show the specific connection between type I IFN signaling and inflammasome activation, which are two sequential events triggered by the recognition of cytosolic bacteria. To our knowledge, this is the first example of the positive regulation of inflammasome activation. This connection underscores the importance of the cytosolic recognition of pathogens and highlights how multiple innate immunity pathways interact before commitment to critical host responses.

Cryopyrin activates the inflammasome in response to toxins and ATP

A crucial part of the innate immune response is the assembly of the inflammasome, a cytosolic complex of proteins that activates caspase-1 to process the proinflammatory cytokines interleukin (IL)-1beta and IL-18. The adaptor protein ASC is essential for inflammasome function, binding directly to caspase-1 (refs 3, 4), but the triggers of this interaction are less clear. ASC also interacts with the adaptor cryopyrin (also known as NALP3 or CIAS1). Activating mutations in cryopyrin are associated with familial cold autoinflammatory syndrome, Muckle-Wells syndrome and neonatal onset multisystem inflammatory disease, diseases that are characterized by excessive production of IL-1beta. Here we show that cryopyrin-deficient macrophages cannot activate caspase-1 in response to Toll-like receptor agonists plus ATP, the latter activating the P2X7 receptor to decrease intracellular K+ levels. The release of IL-1beta in response to nigericin, a potassium ionophore, and maitotoxin, a potent marine toxin, was also found to be dependent on cryopyrin. In contrast to Asc-/- macrophages, cells deficient in the gene encoding cryopyrin (Cias1-/-) activated caspase-1 and secreted normal levels of IL-1beta and IL-18 when infected with Gram-negative Salmonella typhimurium or Francisella tularensis. Macrophages exposed to Gram-positive Staphylococcus aureus or Listeria monocytogenes, however, required both ASC and cryopyrin to activate caspase-1 and secrete IL-1beta. Therefore, cryopyrin is essential for inflammasome activation in response to signalling pathways triggered specifically by ATP, nigericin, maitotoxin, S. aureus or L. monocytogenes.