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.

Viral infection induces the production of interleukin (IL)-1␤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 doublestranded 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-1␤ 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-1␤ 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-1␤ and IL-18 production.

SUMMARY NLRP12 is a member of the intracellular Nod-like receptor (NLR) family that has been suggested to down-regulate the production of inflammatory cytokines, but its physiological role in regulating inflammation has not been characterized. We generated mice deficient in Nlrp12 and studied its role in inflammatory diseases such as colitis and colorectal tumorigenesis. We show that Nlrp12-deficient mice are highly susceptible to colon inflammation and tumorigenesis, which is associated with increased production of inflammatory cytokines, chemokines and tumorigenic factors. Enhanced colon inflammation and colorectal tumor development in Nlrp12-deficient mice are due to a failure to dampen NF-κB and ERK activation in macrophages. These results reveal a critical role for NLRP12 in maintaining intestinal homeostasis and providing protection against colorectal tumorigenesis.