© 2006 Nature Publishing Group Cryopyrin activates the inflammasome in response to toxins and ATP Sanjeev Mariathasan 1 , David S. Weiss 4 , Kim Newton 1 , Jacqueline McBride 3 , Karen O’Rourke 1 , Meron Roose-Girma 2 , Wyne P. Lee 3 , Yvette Weinrauch 5 , Denise M. Monack 4 & Vishva M. Dixit 1 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)-1b and IL-18. The adaptor protein ASC is essential for inflammasome function 1,2 , 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) 5,6 . 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-1b 5,7 . Here we show that cryopyrin-deficient macrophages cannot activate caspase-1 in response to Toll-like receptor agonists plus ATP, the latter activating the P2X 7 receptor to decrease intracellular K 1 levels 8,9 . The release of IL-1b 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 2/2 macrophages, cells deficient in the gene encoding cryopyrin (Cias1 2/2 ) activated caspase-1 and secreted normal levels of IL-1b 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-1b. Therefore, cryopyrin is essential for inflammasome activation in response to signalling pathways triggered specifically by ATP, nigericin, maitotoxin, S. aureus or L. monocytogenes. Cryopyrin-deficient mice (Supplementary Fig. S1) were generated by gene targeting to investigate the role of cryopyrin in inflammatory responses to pathogen-derived molecules. Cryopyrin-deficient (Cias1 2/2 ) macrophages stimulated with the Toll-like receptor-4 (TLR4) agonist lipopolysaccharide (LPS) phosphorylated IkBa and ERK normally (Fig. 1a), and they secreted normal amounts of TNF-a (Fig. 1b), IL-12 p40 (Fig. 1c), IL-6 and IL-10 (data not shown). Similar results were obtained using the TLR2 agonists Pam 3 CSK 4 and heat-killed L. monocytogenes (HKLM) (data not shown). Our results show that cryopyrin is dispensable for NF-kB signalling by TLR2 and TLR4 in macrophages. Because mutant variants of cryopyrin are associated with diseases in which IL-1b is produced in excess 5–7 , we measured IL-1b released from Cias1 2/2 macrophages treated with TLR agonists and ATP (Fig. 1d). TLR agonists induce pro-IL-1b synthesis and ATP stimu- lates caspase-1-dependent cleavage and secretion of IL-1b 10 . In contrast to wild-type macrophages, which secreted readily detectable amounts of IL-1b and IL-18 in response to ATP plus ultra-pure LPS, Pam 3 CSK 4 , HKLM, R848 (TLR7/8 agonist), or CpG oligonucleotides (TLR9 agonist), Cias1 2/2 macrophages secreted negligible amounts of these cytokines (Fig. 1d, e). As shown previously 1,11 , Asc 2/2 macrophages exhibited a similar defect in IL-1b and IL-18 pro- duction (Fig. 1d, e). Macrophages from heterozygous Cias1 þ/2 mice secreted intermediate amounts of IL-1b and IL-18. C3H/HeJ macro- phages expressing a non-functional form of TLR4 (ref. 12) secreted IL-1b and IL-18 in response to ATP plus either Pam 3 CSK 4 or HKLM, but not LPS, demonstrating that our LPS was pure and not contaminated with other TLR agonists (Fig. 1d). To determine whether IL-1b secretion from Cias1 2/2 macrophages was defective due to impaired pro-IL-1b synthesis and/or impaired caspase-1 activation, we immunoprecipitated [ 35 S]-methionine- labelled pro-IL-1b from LPS-primed macrophages. Wild-type, Cias1 þ/2 and Cias1 2/2 macrophages produced comparable amounts of pro-IL-1b (Fig. 1f, left panel), indicating that defective IL-1b secretion from Cias1 2/2 cells was not due to impaired pro-IL-1b synthesis. Unlike their wild-type counterparts, however, Cias1 2/2 macrophages did not cleave pro-IL-1b after ATP treatment (Fig. 1f, right panel). This finding suggested that cryopyrin is essential for ATP-induced caspase-1 activation. A further indication of caspase-1 activation is its autocatalytic processing into p20 and p10 subunits. Western blotting for caspase-1 after LPS plus ATP treatment revealed the p10 and p20 subunits in wild-type but not Cias1 2/2 macro- phages (Fig. 1g). Thus, cryopyrin is essential for activation of caspase-1 in response to LPS plus ATP. Notably, ATP was necessary but not sufficient for caspase-1 activation (Fig. 1g). TLR signalling is probably needed for expression of essential inflammasome components. For example, LPS stimulation of TLR4 increases expression of caspase-11, and analyses of caspase-11-deficient mice and cells demonstrate that caspase-11 is essential for inflammasome function 13 . To test whether the role of ATP in cryopyrin- and ASC-dependent caspase-1 activation relates to its ability to stimulate the P2X 7 receptor 9 and thereby reduce intracellular K þ (ref. 8), we treated TLR-primed macrophages from wild-type, Asc 2/2 and Cias1 2/2 mice with nigericin or maitotoxin to deplete cytosolic K þ (refs 14, 15). Wild-type macrophages primed with LPS or Pam 3 CSK 4 secreted IL-1b and IL-18 in response to nigericin (Fig. 2a, b) or maitotoxin (Fig. 2c, d). By contrast, neither Asc 2/2 nor Cias1 2/2 macrophages released significant IL-1b or IL-18. Our data therefore show that ASC and cryopyrin are essential for IL-1b and IL-18 production by TLR-primed macrophages treated with agents that deplete intra- cellular K þ . Neither nigericin nor maitotoxin alone induced IL-1b release (Supplementary Fig. S2). Again, TLR signalling is probably required not only for the induction of pro-IL-1b (Fig. 1f) but also for expression of other proteins that are essential for inflammasome function. To determine whether cryopyrin is essential for inflammation LETTERS 1 Molecular Oncology Department, 2 Physiology Department, 3 Immunology Department, Genentech Inc, 1 DNA Way, South San Francisco, California 94080, USA. 4 Departments of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California 94305, USA. 5 Department of Microbiology, New York University School of Medicine, New York, New York 10016, USA. 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