Gram-negative bacteria that replicate in the cytosol of mammalian macrophages can activate a signaling pathway leading to caspase-1 cleavage and secretion of interleukin 1beta, a powerful host response factor. Ipaf, a cytosolic pattern-recognition receptor in the family of nucleotide-binding oligomerization domain-leucine-rich repeat proteins, is critical in such a response to salmonella infection, but the mechanism of how Ipaf is activated by the bacterium remains poorly understood. Here we demonstrate that salmonella strains either lacking flagellin or expressing mutant flagellin were deficient in activation of caspase-1 and in interleukin 1beta secretion, although transcription factor NF-kappaB-dependent production of interleukin 6 or the chemokine MCP-1 was unimpaired. Delivery of flagellin to the macrophage cytosol induced Ipaf-dependent activation of caspase-1 that was independent of Toll-like receptor 5, required for recognition of extracellular flagellin. In macrophages made tolerant by previous exposure to lipopolysaccharide, which abrogates activation of NF-kappaB and mitogen-activated protein kinases, salmonella infection still activated caspase-1. Thus, detection of flagellin through Ipaf induces caspase-1 activation independently of Toll-like receptor 5 in salmonella-infected and lipopolysaccharide-tolerized macrophages.

Missense mutations in the CIAS1 gene cause three autoinflammatory disorders: familial cold autoinflammatory syndrome, Muckle-Wells syndrome and neonatal-onset multiple-system inflammatory disease 1 . Cryopyrin (also called Nalp3), the product of CIAS1, is a member of the NOD-LRR protein family that has been linked to the activation of intracellular host defence signalling pathways 2,3 . Cryopyrin forms a multi-protein complex termed 'the inflammasome', which contains the apoptosisassociated speck-like protein (ASC) and caspase-1, and promotes caspase-1 activation and processing of pro-interleukin (IL)-1b (ref. 4). Here we show the effect of cryopyrin deficiency on inflammasome function and immune responses. Cryopyrin and ASC are essential for caspase-1 activation and IL-1b and IL-18 production in response to bacterial RNA and the imidazoquinoline compounds R837 and R848. In contrast, secretion of tumournecrosis factor-a and IL-6, as well as activation of NF-kB and mitogen-activated protein kinases (MAPKs) were unaffected by cryopyrin deficiency. Furthermore, we show that Toll-like receptors and cryopyrin control the secretion of IL-1b and IL-18 through different intracellular pathways. These results reveal a critical role for cryopyrin in host defence through bacterial RNA-mediated activation of caspase-1, and provide insights regarding the pathogenesis of autoinflammatory syndromes.To define the role of cryopyrin in inflammatory responses, we generated cryopyrin-deficient mice by homologous recombination using a targeting construct to replace exons I and II of the cryopyrin gene (Cias1), which encode the pyrin domain of cryopyrin that is essential for effector function of the protein ( Supplementary Fig. 1). Cias1 2/2 mice were fertile and appeared healthy when housed in a standard specific pathogen-free environment.We initially investigated the role of cryopyrin in caspase-1-dependent IL-1b secretion using thioglycollate-elicited peritoneal macrophages and bone marrow-derived macrophages (BMDMs) and multiple bacterial and synthetic ligands. Stimulation of peritoneal macrophages or BMDMs with several TLR2 and TLR4 agonists, including diacylated (Pam 2 CGDPKHPHSF) and triacylated (Pam 3 CSK 4 ) synthetic lipopeptides, lipoteichoic acid, highly purified lipopolysaccharide (LPS) and lipid A induced comparable levels of IL-1b in wild-type and Cias1 2/2 macrophages ( Fig. 1a and Supplementary Fig. 2). Similar results were obtained when macrophages were stimulated with bacterial ligands and treated briefly with ATP ( Supplementary Fig. 3), a signal that enhances the secretion of IL-1b in pre-stimulated macrophages 5 . Incubation of macrophages with muramyl dipeptide (MDP) did not induce secretion of IL-1b above background levels in wild-type and Cias1 2/2 macrophages, even after addition of ATP ( Fig. 1a; see also Supplementary Fig. 3). Furthermore, production of interferon-a induced by several viruses was unimpaired in macrophages and dendritic cells from Cias1 2/2 mice ( Supplementary Fig. 4).Notably, secretion o...

Legionella pneumophila is an intracellular bacterium that causes an acute form of pneumonia called Legionnaires' disease. After infection of human macrophages, the Legionella-containing phagosome (LCP) avoids fusion with the lysosome allowing intracellular replication of the bacterium. In macrophages derived from most mouse strains, the LCP is delivered to the lysosome resulting in Legionella degradation and restricted bacterial growth. Mouse macrophages lacking the NLR protein Ipaf or its downstream effector caspase-1 are permissive to intracellular Legionella replication. However, the mechanism by which Ipaf restricts Legionella replication is not well understood. Here we demonstrate that the presence of flagellin and a competent type IV secretion system are critical for Legionella to activate caspase-1 in macrophages. Activation of caspase-1 in response to Legionella infection also required host Ipaf, but not TLR5. In the absence of Ipaf or caspase-1 activation, the LCP acquired endoplasmic reticulum-derived vesicles, avoided fusion with the lysosome, and allowed Legionella replication. Accordingly a Legionella mutant lacking flagellin did not activate caspase-1, avoided degradation, and replicated in wild-type macrophages. The regulation of phagosome maturation by Ipaf occurred within 2 h after infection and was independent of macrophage cell death. In vivo studies confirmed that flagellin and Ipaf play an important role in the control of Legionella clearance. These results reveal that Ipaf restricts Legionella replication through the regulation of phagosome maturation, providing a novel function for NLR proteins in host defense against an intracellular bacterium.

Apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC) is an adaptor molecule that has recently been implicated in the activation of caspase-1. We have studied the role of ASC in the host defense against the intracellular pathogen Listeria monocytogenes. ASC was found to be essential for the secretion of IL-1β/IL-18, but dispensable for IL-6, TNF-α, and IFN-β production, in macrophages infected with Listeria. Activation of caspase-1 was abolished in ASC-deficient macrophages, whereas activation of NF-κB and p38 was unaffected. In contrast, secretion of IL-1β, IL-6, and TNF-α was reduced in TLR2-deficient macrophages infected with Listeria; this was associated with impaired activation of NF-κB and p38, but normal caspase-1 processing. Analysis of Listeria mutants revealed that cytosolic invasion was required for ASC-dependent IL-1β secretion, consistent with a critical role for cytosolic signaling in the activation of caspase-1. Secretion of IL-1β in response to lipopeptide, a TLR2 agonist, was greatly reduced in ASC-null macrophages and was abolished in TLR2-deficient macrophages. These results demonstrate that TLR2 and ASC regulate the secretion of IL-1β via distinct mechanisms in response to Listeria. ASC, but not TLR2, is required for caspase-1 activation independent of NF-κB in Listeria-infected macrophages.

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Type I cells have been defined to be independent of mitochondria for the induction of Fas death receptor-mediated apoptosis, whereas Type II cells are mitochondria-dependent. Knock-out studies in mice show that thymocytes are Type I and liver cells are Type II. We have previously shown that primary human hepatocytes and HCT116 human colon carcinoma cells behave like Type II cells because TRAIL-induced apoptosis can be blocked by the caspase 9 inhibitor, Z-LEHD-FMK. On the other hand, caspase 9 inhibition does not allow survival of TRAIL-treated SW480 colon cancer cells, which is predicted for Type I cells. Investigating the differences in TRAIL-induced apoptotic pathways in HCT116 and SW480 cells revealed that although FADD, BID, and procaspase 3 protein levels are higher in SW480 cells, and although procaspase 8 and FLIP processing is more efficient at the TRAIL-DISC of SW480 cells, BID, procaspase 3, XIAP, and PARP cleavages occur more rapidly in HCT116, despite the higher levels of BCL-2 and HSP70. Cytochrome c release from the mitochondria to the cytoplasm is more efficient in HCT116 cells. These results suggest BID cleavage as a possible limiting factor in the involvement of mitochondria in TRAIL-induced cell death. Thus, regulation of BID cleavage may define if a cell is mitochondria-dependent or -independent in response to TRAIL death receptor-induced apoptosis.

Activation of the inflammasome is accompanied by rapid formation of a micrometer-sized perinuclear structure called the ASC speck, a platform for caspase-1 activity. The ASC speck is often referred to as an aggregate and shares certain features with aggresomes. It is thus an open question whether the ASC speck formation takes place via nonspecific aggregation of hydrophobic patches or specific interactions of its domains; PYD and CARD, which belong to the death fold superfamily. Bringing together structure and dynamics studies using the Gaussian network model of PYD and CARD, and molecular dynamics simulations of the wild-type and in silico mutated PYD, with the mutational analysis on the ASC structure and its separate domains in human cells, we show that the ASC speck is an organized structure with at least two levels of distinct compaction mechanisms based on the specific interactions of PYD and CARD.