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.

Mutations in the NALP3/CIAS1/cryopyrin gene are linked to three autoinflammatory disorders: Muckle-Wells syndrome, familial cold autoinflammatory syndrome, and chronic infantile neurologic cutaneous and articular syndrome. NALP3, with the adaptor molecule ASC, has been proposed to form a caspase-1-activating "inflammasome," a complex with pro-IL1beta-processing activity. Here, we demonstrate the effect of NALP3 deficiency on caspase-1 function. NALP3 was essential for the ATP-driven activation of caspase-1 in lipopolysaccharide-stimulated macrophages and for the efficient secretion of the caspase-1-dependent cytokines IL-1alpha, IL-1beta, and IL-18. IL-1beta has been shown to play a key role in contact hypersensitivity; we show that ASC- and NALP3-deficient mice also demonstrate an impaired contact hypersensitivity response to the hapten trinitrophenylchloride. NALP3, however, was not required for caspase-1 activation by Salmonella typhimurium, and NALP3 deficiency only partially protects mice from the lethal effects of endotoxin. These data suggest that NALP3 plays a specific role in the caspase-1 activation pathway.

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...