Fungal Zymosan and Mannan Activate the Cryopyrin Inflammasome

Some fungal species are opportunistic pathogens that can cause infection in people with compromised immune systems. Activation of caspase-1 and the subsequent secretion of mature interleukin (IL)-1β is a major signaling pathway of the innate immune system, but how yeasts induce caspase-1 activation is unknown. We show here that stimulation of macrophages and dendritic cells with heat-killed Saccharomyces cerevisiae or the purified cell wall components zymosan and mannan induced caspase-1 activation and IL-1β secretion when combined with ATP. Macrophages deficient for the inflammasome adaptor ASC were defective in caspase-1 activation and IL-1β secretion, suggesting involvement of an ASC-dependent inflammasome. Indeed, caspase-1 activation was abrogated in macrophages lacking the NOD-like (NLR) protein Cryopyrin/Nalp3 and in wild type macrophages pretreated with the pannexin-1 inhibitor probenecid. IL-1β secretion further required the Toll-like receptor (TLR) adaptors MyD88 and TRIF, and partially relied on TLR2. We previously showed that bacterial molecules such as lipopolysaccharide (LPS) and peptidoglycan induce activation of caspase-7 through the Cryopyrin inflammasome. Similarly, Cryopyrin and ASC were required for activation of caspase-7 in macrophages stimulated with zymosan or mannan and ATP. These results demonstrate that the conserved fungal components zymosan and mannan require ASC and Cryopyrin for caspase-1 activation and IL-1β secretion and suggest an important role for the Cryopyrin inflammasome during fungal infections.Pathogen recognition by the innate immune system relies on a limited number of fixed germline-encoded receptors, which have evolved to identify so-called pathogen-associated molecular patterns (PAMPs),² conserved microbial structures not shared by the host and essential for their survival (1). Examples of PAMPs are LPS from Gram-negative bacteria, peptidoglycan (PGN) from Gram-positive bacteria, and zymosan and mannan from fungi. Several structurally and functionally diverse classes of pattern-recognition receptors (PRRs) exist that induce various host defense pathways, including the Toll-like receptors (TLRs) located in the plasma membrane and intracellular organelles and the more recently identified intracellular family of NOD-like receptors (NLRs) (2).Previous studies have shown that gain-of-function mutations within the NLR protein Cryopyrin/NALP3 are associated with three autoinflammatory disorders characterized by skin rashes and prolonged episodes of fever in the absence of any apparent infection (3, 4). These hereditary periodic fever syndromes are Muckle-Wells syndrome (MWS), familial cold autoinflammatory syndrome (FACS), and neonatal-onset multisystem inflammatory disease (NOMID), and they are collectively referred to as the Cryopyrin/NALP3-associated periodic syndromes (CAPS). Subsequent studies revealed that the Cryopyrin/Nalp3 plays a crucial role in the assembly of a large (700 kDa) cytosolic protein complex termed the “inflammasome” (5–7). The bipartite adaptor protein ASC bridges the interaction between Cryopyrin/Nalp3 and caspase-1 in the inflammasome; thus allowing the recruitment and autoproteolytic activation of the cysteine protease (2). Activated caspase-1 subsequently mediates the maturation and secretion of the pro-inflammatory cytokines interleukin (IL)-1β and IL-18 (8–10). Interestingly, the Cryopyrin/Nalp3 inflammasome mediates caspase-1 activation in response to a variety of bacterial PAMPs such as LPS and PGN when combined with a second stimulus such as the P2X₇ receptor ligand ATP (11–14). Cryopyrin/Nalp3 also mediates caspase-1 activation and IL-1β secretion in macrophages stimulated with viral RNA and ATP (15) or exposed to crystalline substances including uric acid, silica and asbestos (16–18). In contrast, the related NLR protein Ipaf is required for caspase-1 activation in macrophages infected with the intracellular pathogens Salmonella, Legionella, and Shigella (19–21).Although the roles of specific inflammasomes in response to bacterial and viral PAMPs have been described, the inflammasome complexes that recognize fungal PAMPs to induce caspase-1 activation and IL-1β secretion are unknown. Here we show that heat-killed Saccharomyces cerevisiae and the purified cell wall components zymosan and mannan induced caspase-1 activation and IL-1β secretion from macrophages and dendritic cells upon co-stimulation with ATP. Macrophages deficient for the inflammasome adaptor ASC or the NLR protein Cryopyrin/Nalp3 were defective in zymosan- and mannan-induced caspase-1 activation and IL-1β secretion, whereas TNF-α secretion remained unaffected. Although macrophages lacking the TLR adaptors MyD88 or TRIF still activated caspase-1, zymosan- and mannan-induced secretion of IL-1β was significantly hampered. These results demonstrate that the conserved fungal cell wall components zymosan and mannan require ASC and Cryopyrin for caspase-1 activation and IL-1β secretion and suggest an important role for the Cryopyrin inflammasome during fungal infections.     

Intracellular NOD-like receptors in innate immunity, infection and disease

The innate immune system comprises several classes of pattern-recognition receptors, including Toll-like receptors (TLRs) and nucleotide binding and oligomerization domain-like receptors (NLRs). TLRs recognize microbes on the cell surface and in endosomes, whereas NLRs sense microbial molecules in the cytosol. In this review, we focus on the role of NLRs in host defence against bacterial pathogens. Nod1 and Nod2 sense the cytosolic presence of molecules containing meso-diaminopimelic acid and muramyl dipeptide respectively, and drive the activation of mitogen-activated protein kinase and NF-κB. In contrast, Ipaf, Nalp1b and Cryopyrin/Nalp3 promote the assembly of inflammasomes that are required for the activation of caspase-1. Mutation in several NLR members, including NOD2 and Cryopyrin, is associated with the development of inflammatory disorders. Further understanding of NLRs should provide new insights into the mechanisms of host defence and the pathogenesis of inflammatory diseases.     

Critical role for Cryopyrin/Nalp3 in activation of caspase-1 in response to viral infection and double-stranded RNA

Viral infection induces the production of interleukin (IL)-1beta and IL-18 in macrophages through the activation of caspase-1, but the mechanism by which host cells sense viruses to induce caspase-1 activation is unknown. In this report, we have identified a signaling pathway leading to caspase-1 activation that is induced by double-stranded RNA (dsRNA) and viral infection that is mediated by Cryopyrin/Nalp3. Stimulation of macrophages with dsRNA, viral RNA, or its analog poly(I:C) induced the secretion of IL-1beta and IL-18 in a cryopyrin-dependent manner. Consistently, caspase-1 activation triggered by poly(I:C), dsRNA, and viral RNA was abrogated in macrophages lacking cryopyrin or the adaptor ASC (apoptosis-associated speck-like protein containing a caspase-activating and recruitment domain) but proceeded normally in macrophages deficient in Toll-like receptor 3 or 7. We have also shown that infection with Sendai and influenza viruses activates the cryopyrin inflammasome. Finally, cryopyrin was required for IL-1beta production in response to poly(I:C) in vivo. These results identify a mechanism mediated by cryopyrin and ASC that links dsRNA and viral infection to caspase-1 activation resulting in IL-1beta and IL-18 production.     

Glyburide inhibits the Cryopyrin/Nalp3 inflammasome

Inflammasomes activate caspase-1 for processing and secretion of the cytokines interleukin-1β (IL-1β) and IL-18. Cryopyrin/NALP3/NLRP3 is an essential component of inflammasomes triggered by microbial ligands, danger-associated molecular patterns (DAMPs), and crystals. Inappropriate Cryopyrin activity has been incriminated in the pathogenesis of gouty arthritis, Alzheimer''s, and silicosis. Therefore, inhibitors of the Nalp3 inflammasome offer considerable therapeutic promise. In this study, we show that the type 2 diabetes drug glyburide prevented activation of the Cryopyrin inflammasome. Glyburide''s cyclohexylurea group, which binds to adenosine triphosphatase (ATP)–sensitive K⁺ (K_ATP) channels for insulin secretion, is dispensable for inflammasome inhibition. Macrophages lacking K_ATP subunits or ATP-binding cassette transporters also activate the Cryopyrin inflammasome normally. Glyburide analogues inhibit ATP- but not hypothermia-induced IL-1β secretion from human monocytes expressing familial cold-associated autoinflammatory syndrome–associated Cryopyrin mutations, thus suggesting that inhibition occurs upstream of Cryopyrin. Concurrent with the role of Cryopyrin in endotoxemia, glyburide significantly delays lipopolysaccharide-induced lethality in mice. Therefore, glyburide is the first identified compound to prevent Cryopyrin activation and microbial ligand-, DAMP-, and crystal-induced IL-1β secretion.     

A role for Nod-like receptors in autophagy induced by Shigella infection

Shigella infection, the cause of bacillary dysentery, induces caspase-1 activation and cell death in macrophages, but the precise mechanisms remain poorly understood. In our recent study, we presented evidence that caspase-1 activation and IL-1beta processing induced by Shigella are mediated through Ipaf, a cytosolic pattern-recognition receptor of the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family and the adaptor protein apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC). We also show that Ipaf and caspase-1 were critical for pyroptosis, a specialized form of caspase-1-dependent cell death induced in macrophages by bacterial infection, whereas ASC is dispensable. Notably, infection of macrophages with Shigella induced autophagy, which was dramatically increased by the absence of caspase-1 or Ipaf, but not ASC. Furthermore, autophagy induction was associated with transient resistance to pyroptosis. These results indicate that autophagy in macrophages is regulated by the Ipaf inflammasome, providing a novel function for NLR proteins in bacterial-host interactions.     

A role for Nod-like receptors in autophagy induced by Shigella infection

Shigella infection, the cause of bacillary dysentery, induces caspase-1 activation and cell death in macrophages, but the precise mechanisms remain poorly understood. In our recent study, we presented evidence that caspase-1 activation and IL-1β processing induced by Shigella are mediated through Ipaf, a cytosolic pattern-recognition receptor of the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family and the adaptor protein apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC). We also show that Ipaf and caspase-1 were critical for pyroptosis, a specialized form of caspase-1-dependent cell death induced in macrophages by bacterial infection, whereas ASC is dispensable. Notably, infection of macrophages with Shigella induced autophagy, which was dramatically increased by the absence of caspase-1 or Ipaf, but not ASC. Furthermore, autophagy induction was associated with transient resistance to pyroptosis. These results indicate that autophagy in macrophages is regulated by the Ipaf inflammasome, providing a novel function for NLR proteins in bacterial-host interactions.

Addendum to: Suzuki T, Franchi L, Toma C, Ashida H, Ogawa M, Yoshikawa Y, Mimuro H, Inohara N, Sasakawa C, Núñez G. Differential regulation of caspase-1 activation, pyroptosis and autophagy via Ipaf and ASC in Shigella-infected macrophages. PLoS Pathog 2007; 3:e111     

Targeted peptidecentric proteomics reveals caspase-7 as a substrate of the caspase-1 inflammasomes

The aspartate-specific cysteine protease caspase-1 is activated by the inflammasomes and is responsible for the proteolytic maturation of the cytokines IL-1 beta and IL-18 during infection and inflammation. To discover new caspase-1 substrates, we made use of a proteome-wide gel-free differential peptide sorting methodology that allows unambiguous localization of the processing site in addition to identification of the substrate. Of the 1022 proteins that were identified, 20 were found to be specifically cleaved after Asp in the setup incubated with recombinant caspase-1. Interestingly, caspase-7 emerged as one of the identified caspase-1 substrates. Moreover half of the other identified cleavage events occurred at sites closely resembling the consensus caspase-7 recognition sequence DEVD, suggesting caspase-1-mediated activation of endogenous caspase-7 in this setup. Consistently recombinant caspase-1 cleaved caspase-7 at the canonical activation sites Asp(23) and Asp(198), and recombinant caspase-7 processed a subset of the identified substrates. In vivo, caspase-7 activation was observed in conditions known to induce activation of caspase-1, including Salmonella infection and microbial stimuli combined with ATP. Interestingly Salmonella- and lipopolysaccharide + ATP-induced activation of caspase-7 was abolished in macrophages deficient in caspase-1, the pattern recognition receptors Ipaf and Cryopyrin, and the inflammasome adaptor ASC, demonstrating an upstream role for the caspase-1 inflammasomes in caspase-7 activation in vivo. In contrast, caspase-1 and the inflammasomes were not required for caspase-3 activation. In conclusion, we identified 20 new substrates activated downstream of caspase-1 and validated caspase-1-mediated caspase-7 activation in vitro and in knock-out macrophages. These results demonstrate for the first time the existence of a nucleotide binding and oligomerization domain-like receptor/caspase-1/caspase-7 cascade and the existence of distinct activation mechanisms for caspase-3 and -7 in response to microbial stimuli and bacterial infection.     

The SPRY domain of Pyrin, mutated in familial Mediterranean fever patients, interacts with inflammasome components and inhibits proIL-1beta processing

The autoinflammatory disorders Muckle-Wells syndrome, familial cold urtecaria and chronic infantile neurological cutaneous and articular syndrome are associated with mutations in the NALP3 (Cryopyrin) gene, which is the central platform of the proinflammatory caspase-1 activating complex, named the inflammasome. In patients with another autoinflammatory disorder, familial Mediterranean fever (FMF), mutations in the SPRY domain of the Pyrin protein are frequently found. Recent evidence suggests that Pyrin associates with ASC, an inflammasome component, via its Pyrin domain, thereby halting the inflammatory response. This interaction, however, does not explain the effects of mutations of the SPRY domain found in FMF patients. Here we show that the Pyrin SPRY domain not only interacts with NALP3, but also with caspase-1 and its substrate pro-interleukin(IL)-1beta. Whereas a Pyrin knockdown results in increased caspase-1 activation and IL-1beta secretion, overexpression of the SPRY domain alone blocks these processes. Thus Pyrin binds to several inflammasome components thereby modulating their activity.     

A Role for Uric Acid and the Nalp3 Inflammasome in Antiphospholipid Antibody-Induced IL-1β Production by Human First Trimester Trophoblast

Women with antiphospholipid syndrome (APS) are at risk of recurrent pregnancy loss and obstetrical disorders, such as preeclampsia and intrauterine growth restriction (IUGR). Antiphospholipid antibodies (aPL) directly target the placenta by binding beta₂-glycoprotein I (β₂GPI) expressed on the trophoblast. We recently demonstrated in human first trimester trophoblast cells that anti-β₂GPI antibodies (Abs) induce the secretion of IL-1β in a Toll-like receptor 4 (TLR4)-dependent manner. IL-1β secretion requires processing of pro-IL-1β and this is mediated by the inflammasome, a complex of Nalp3, apoptosis-associated speck-like protein containing a CARD (ASC) and caspase-1. The objective of this study was to determine if aPL induce IL-1β production in trophoblast via the inflammasome. Using a human first trimester trophoblast cell line, we demonstrated that a mouse anti-β₂GPI mAb and human polyclonal aPL-IgG induce IL-1β processing and secretion, which was partially blocked upon caspase-1 inhibition. Nalp3 and ASC knockdown also attenuated anti-β₂GPI Ab-induced IL-1β secretion. Furthermore, aPL stimulated the production of uric acid in a TLR4-dependent manner; and inhibition of uric acid prevented aPL-induced IL-1β production by the trophoblast. These findings demonstrate that aPL, via TLR4 activation, induce a uric acid response in human trophoblast, which in turn activates the Nalp3/ASC inflammasome leading to IL-1β processing and secretion. This novel mechanism may account for the inflammation at the maternal-fetal interface, which causes placental dysfunction and increases the risk of adverse pregnancy outcome in patients with APS.     

Microbial pathogen-induced necrotic cell death mediated by the inflammasome components CIAS1/cryopyrin/NLRP3 and ASC

Cryopyrin (CIAS1, NLRP3) and ASC are components of the inflammasome, a multiprotein complex required for caspase-1 activation and cytokine IL-1beta production. CIAS1 mutations underlie autoinflammation characterized by excessive IL-1beta secretion. Disease-associated cryopyrin also causes a program of necrosis-like cell death in macrophages, the mechanistic details of which are unknown. We find that patient monocytes carrying disease-associated CIAS1 mutations exhibit excessive necrosis-like death by a process dependent on ASC and cathepsin B, resulting in spillage of the proinflammatory mediator HMGB1. Shigella flexneri infection also causes cryopyrin-dependent macrophage necrosis with features similar to the death caused by mutant CIAS1. This necrotic death is independent of caspase-1 and IL-1beta, and thus independent of the inflammasome. Furthermore, necrosis of primary macrophages requires the presence of Shigella virulence genes. While similar proteins mediate pathogen-induced cell death in plants, this report identifies cryopyrin as an important host regulator of programmed pathogen-induced necrosis in animals, a process we term pyronecrosis.     

Identification of bacterial muramyl dipeptide as activator of the NALP3/cryopyrin inflammasome

Activation of caspase-1 and subsequent processing and secretion of the pro-inflammatory cytokine IL-1beta is triggered upon assembly of the inflammasome complex. It is generally believed that bacterial lipopolysaccharides (LPS) are activators of the inflammasome through stimulation of Toll-like receptor 4 (TLR4). Like TLRs, NALP3/Cryopyrin, which is a key component of the inflammasome, contains Leucine-Rich-Repeats (LRRs). LRRs are frequently used to sense bacterial components, thus raising the possibility that bacteria directly activate the inflammasome. Here, we show that bacterial peptidoglycans (PGN), but surprisingly not LPS, induce NALP3-mediated activation of caspase-1 and maturation of proIL-1beta. Activation is independent of TLRs because the PGN degradation product muramyl dipeptide (MDP), which is not sensed by TLRs, is the minimal-activating structure. Macrophages from a patient with Muckle-Wells syndrome, an autoinflammatory disease associated with mutations in the NALP3/Cryopyrin gene, show increased IL-1beta secretion in the presence of MDP. The activation of the NALP3-inflammasome by MDP may be the basis of the potent adjuvant activity of MDP.     

Malarial Hemozoin Is a Nalp3 Inflammasome Activating Danger Signal

Background

Characteristic symptoms of malaria include recurrent fever attacks and neurodegeneration, signs that are also found in patients with a hyperactive Nalp3 inflammasome. Plasmodium species produce a crystal called hemozoin that is generated by detoxification of heme after hemoglobin degradation in infected red blood cells. Thus, we hypothesized that hemozoin could activate the Nalp3 inflammasome, due to its particulate nature reminiscent of other inflammasome-activating agents.

Methodology/Principal Findings

We found that hemozoin acts as a proinflammatory danger signal that activates the Nalp3 inflammasome, causing the release of IL-1β. Similar to other Nalp3-activating particles, hemozoin activity is blocked by inhibiting phagocytosis, K⁺ efflux and NADPH oxidase. In vivo, intraperitoneal injection of hemozoin results in acute peritonitis, which is impaired in Nalp3-, caspase-1- and IL-1R-deficient mice. Likewise, the pathogenesis of cerebral malaria is dampened in Nalp3-deficient mice infected with Plasmodium berghei sporozoites, while parasitemia remains unchanged.

Significance/Conclusions

The potent pro-inflammatory effect of hemozoin through inflammasome activation may possibly be implicated in plasmodium-associated pathologies such as cerebral malaria.     

Differential regulation of caspase-1 activation, pyroptosis, and autophagy via Ipaf and ASC in Shigella-infected macrophages

Shigella infection, the cause of bacillary dysentery, induces caspase-1 activation and cell death in macrophages, but the precise mechanisms of this activation remain poorly understood. We demonstrate here that caspase-1 activation and IL-1beta processing induced by Shigella are mediated through Ipaf, a cytosolic pattern-recognition receptor of the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family, and the adaptor protein apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC). We also show that Ipaf was critical for pyroptosis, a specialized form of caspase-1-dependent cell death induced in macrophages by bacterial infection, whereas ASC was dispensable. Unlike that observed in Salmonella and Legionella, caspase-1 activation induced by Shigella infection was independent of flagellin. Notably, infection of macrophages with Shigella induced autophagy, which was dramatically increased by the absence of caspase-1 or Ipaf, but not ASC. Autophagy induced by Shigella required an intact bacterial type III secretion system but not VirG protein, a bacterial factor required for autophagy in epithelial-infected cells. Treatment of macrophages with 3-methyladenine, an inhibitor of autophagy, enhanced pyroptosis induced by Shigella infection, suggesting that autophagy protects infected macrophages from pyroptosis. Thus, Ipaf plays a critical role in caspase-1 activation induced by Shigella independently of flagellin. Furthermore, the absence of Ipaf or caspase-1, but not ASC, regulates pyroptosis and the induction of autophagy in Shigella-infected macrophages, providing a novel function for NLR proteins in bacterial-host interactions.     

AIM2/ASC triggers caspase-8-dependent apoptosis in Francisella-infected caspase-1-deficient macrophages

The inflammasome is a signalling platform leading to caspase-1 activation. Caspase-1 causes pyroptosis, a necrotic-like cell death. AIM2 is an inflammasome sensor for cytosolic DNA. The adaptor molecule ASC mediates AIM2-dependent caspase-1 activation. To date, no function besides caspase-1 activation has been ascribed to the AIM2/ASC complex. Here, by comparing the effect of gene inactivation at different levels of the inflammasome pathway, we uncovered a novel cell death pathway activated in an AIM2/ASC-dependent manner. Francisella tularensis, the agent of tularaemia, triggers AIM2/ASC-dependent caspase-3-mediated apoptosis in caspase-1-deficient macrophages. We further show that AIM2 engagement leads to ASC-dependent, caspase-1-independent activation of caspase-8 and caspase-9 and that caspase-1-independent death is reverted upon caspase-8 inhibition. Caspase-8 interacts with ASC and active caspase-8 specifically colocalizes with the AIM2/ASC speck thus identifying the AIM2/ASC complex as a novel caspase-8 activation platform. Furthermore, we demonstrate that caspase-1-independent apoptosis requires the activation of caspase-9 and of the intrinsic pathway in a typical type II cell manner. Finally, we identify the AIM2/ASC-dependent caspase-1-independent pathway as an innate immune mechanism able to restrict bacterial replication in vitro and control IFN-γ levels in vivo in Casp1(KO) mice. This work underscores the crosstalk between inflammasome components and the apoptotic machinery and highlights the versatility of the pathway, which can switch from pyroptosis to apoptosis.     

Anthrax lethal toxin-induced inflammasome formation and caspase-1 activation are late events dependent on ion fluxes and the proteasome

Anthrax lethal toxin (LT) is cytotoxic to macrophages from certain inbred mouse strains. The gene controlling macrophage susceptibility to LT is Nalp1b . Nalp1b forms part of the inflammasome, a multiprotein complex involved in caspase-1 activation and release of interleukin (IL)-1β and IL-18. We confirm the role of caspase-1 in LT-mediated death by showing that caspase inhibitors differentially protected cells against LT, with the degree of protection corresponding to each compound's ability to inhibit caspase-1. Caspase-1 activation and cytokine processing and release were late events inhibited by elevated levels of KCl and sucrose, by potassium channel blockers, and by proteasome inhibitors, suggesting that inflammasome formation requires a protein-degradation event and occurs downstream of LT-mediated potassium efflux. In addition, IL-18 and IL-1β release was dependent on cell death, indicating that caspase-1-mediated cytotoxicity is independent of these cytokines. Finally, inducing NALP3-inflammasome formation in LT-resistant macrophages did not sensitize cells to LT, suggesting that general caspase-1 activation cannot account for sensitivity to LT and that a Nalp1b-mediated event is specifically required for death. Our data indicate that inflammasome formation is a contributing, but not initiating, event in LT-mediated cytotoxicity and that earlier LT-mediated events leading to ion fluxes are required for death.     

Inflammatory caspases: linking an intracellular innate immune system to autoinflammatory diseases

Caspases not only play an essential role during apoptotic cell death, but a subfamily of them-the inflammatory caspases-are associated with immune responses to microbial pathogens. Activation of inflammatory caspases, such as caspase-1 and caspase-5, occurs upon assembly of an intracellular complex, designated the inflammasome. This results in the cleavage and activation of the proinflammatory cytokines IL-1beta and IL-18. Mutations in one of the scaffold proteins of the inflammasome, NALP3/Cryopyrin, are associated with autoinflammatory disorders underscoring the importance of regulating inflammatory caspase activation.     

AIM2 and NLRP3 inflammasomes activate both apoptotic and pyroptotic death pathways via ASC

Inflammasomes are protein complexes assembled upon recognition of infection or cell damage signals, and serve as platforms for clustering and activation of procaspase-1. Oligomerisation of initiating proteins such as AIM2 (absent in melanoma-2) and NLRP3 (NOD-like receptor family, pyrin domain-containing-3) recruits procaspase-1 via the inflammasome adapter molecule ASC (apoptosis-associated speck-like protein containing a CARD). Active caspase-1 is responsible for rapid lytic cell death termed pyroptosis. Here we show that AIM2 and NLRP3 inflammasomes activate caspase-8 and -1, leading to both apoptotic and pyroptotic cell death. The AIM2 inflammasome is activated by cytosolic DNA. The balance between pyroptosis and apoptosis depended upon the amount of DNA, with apoptosis seen at lower transfected DNA concentrations. Pyroptosis had a higher threshold for activation, and dominated at high DNA concentrations because it happens more rapidly. Gene knockdown showed caspase-8 to be the apical caspase in the AIM2- and NLRP3-dependent apoptotic pathways, with little or no requirement for caspase-9. Procaspase-8 localised to ASC inflammasome ‘specks'' in cells, and bound directly to the pyrin domain of ASC. Thus caspase-8 is an integral part of the inflammasome, and this extends the relevance of the inflammasome to cell types that do not express caspase-1.     

The central inflammasome adaptor protein ASC activates the inflammasome after transition from a soluble to an insoluble state

Apoptosis-associated speck-like protein containing a caspase recruitment domain (CARD) (ASC) is a 22 kDa protein that functions as the central adaptor for inflammasome assembly. ASC forms insoluble specks in monocytes undergoing pyroptosis, and the polymerization of ASC provides a template of CARDs that leads to proximity-mediated autoactivation of caspase-1 in canonical inflammasomes. However, specks are insoluble protein complexes, and solubility is typically important for protein function. Therefore, we sought to define whether ASC specks comprise active inflammasome complexes or are simply the end stage of exhausted ASC polymers. Using a THP-1 cell–lysing model of caspase-1 activation that is ASC dependent, we compared caspase-1 activation induced by preassembled insoluble ASC specks and soluble monomeric forms of ASC. Unexpectedly, after controlling for the concentration dependence of ASC oligomerization, we found that only insoluble forms of ASC promoted caspase-1 autocatalysis. This link to insolubility was recapitulated with recombinant ASC. We show that purified recombinant ASC spontaneously precipitated and was functional, whereas the maltose-binding protein–ASC fusion to ASC (promoting enhanced solubility) was inactive until induced to insolubility by binding to amylose beads. This functional link to insolubility also held true for the Y146A mutation of the CARD of ASC, which avoids insolubility and caspase-1 activation. Thus, we conclude that the role of ASC insolubility in inflammasome function is inextricably linked to its pyrin domain–mediated and CARD-mediated polymerizations. These findings will support future studies into the molecular mechanisms controlling ASC solubility.     

Multiple Nod-like receptors activate caspase 1 during Listeria monocytogenes infection

Listeria monocytogenes escapes from the phagosome of macrophages and replicates within the cytosolic compartment. The macrophage responds to L. monocytogenes through detection pathways located on the cell surface (TLRs) and within the cytosol (Nod-like receptors) to promote inflammatory processes aimed at clearing the pathogen. Cytosolic L. monocytogenes activates caspase 1, resulting in post-translational processing of the cytokines IL-1beta and IL-18 as well as caspase 1-dependent cell death (pyroptosis). We demonstrate that the presence of L. monocytogenes within the cytosolic compartment induces caspase 1 activation through multiple Nod-like receptors, including Ipaf and Nalp3. Flagellin expression by cytosolic L. monocytogenes was detected through Ipaf in a dose-dependent manner. Concordantly, detection of flagellin promoted bacterial clearance in a murine infection model. Finally, we provide evidence that suggests cytosolic L. monocytogenes activates caspase 1 through a third pathway, which signals through the adaptor protein ASC. Thus, L. monocytogenes activates caspase 1 in macrophages via multiple pathways, all of which detect the presence of bacteria within the cytosol.     

Multiple Binding Sites on the Pyrin Domain of ASC Protein Allow Self-association and Interaction with NLRP3 Protein

A key process underlying an innate immune response to pathogens or cellular stress is activation of members of the NOD-like receptor family, such as NLRP3, to assemble caspase-1-activating inflammasome complexes. Activated caspase-1 processes proinflammatory cytokines into active forms that mediate inflammation. Activation of the NLRP3 inflammasome is also associated with common diseases including cardiovascular disease, diabetes, chronic kidney disease, and Alzheimer disease. However, the molecular details of NLRP3 inflammasome assembly are not established. The adaptor protein ASC plays a key role in inflammasome assembly. It is composed of an N-terminal pyrin domain (PYD) and a C-terminal caspase recruitment domain, which are protein interaction domains of the death fold superfamily. ASC interacts with NLRP3 via a homotypic PYD interaction and recruits procaspase-1 via a homotypic caspase recruitment domain interaction. Here we demonstrate that ASC PYD contains two distinct binding sites important for self-association and interaction with NLRP3 and the modulatory protein POP1. Modeling of the homodimeric ASC PYD complex formed via an asymmetric interaction using both sites resembles a type I interaction found in other death fold domain complexes. This interaction mode also permits assembly of ASC PYDs into filaments. Furthermore, a type I binding mode is likely conserved in interactions with NLRP3 and POP1, because residues critical for interaction of ASC PYD are conserved in these PYDs. We also demonstrate that ASC PYD can simultaneously self-associate and interact with NLRP3, rationalizing the model whereby ASC self-association upon recruitment to NLRP3 promotes clustering and activation of procaspase-1.