Pannexin-1-Mediated Recognition of Bacterial Molecules Activates the Cryopyrin Inflammasome Independent of Toll-like Receptor Signaling

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1 Article Pannexin-1-Mediated Recognition of Bacterial Molecules Activates the Cryopyrin Inflammasome Independent of Toll-like Receptor Signaling Thirumala-Devi Kanneganti, 1,4 Mohamed Lamkanfi, 1,4 Yun-Gi Kim, 1 Grace Chen, 2 Jong-Hwan Park, 1 Luigi Franchi, 1 Peter Vandenabeele, 3 and Gabriel Núñez 1, * 1 Department of Pathology 2 Department of Internal Medicine Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA 3 Department of Molecular Biomedical Research, Molecular Signalling and Cell Death Unit, Flanders Interuniversity Institute for Biotechnology and Ghent University, Technologiepark 927, B-9052 Zwijnaarde, Belgium 4 These authors contributed equally to this work. *Correspondence: bclx@umich.edu DOI /j.immuni SUMMARY Cryopyrin is essential for caspase-1 activation triggered by Toll-like receptor (TLR) ligands in the presence of adenosine triphosphate (ATP). However, the events linking bacterial products and ATP to cryopyrin remain unclear. Here we demonstrate that cryopyrin-mediated caspase-1 activation proceeds independently of TLR signaling, thus dissociating caspase-1 activation and IL-1b secretion. Instead, caspase-1 activation required pannexin-1, a hemichannel protein that interacts with the P2X 7 receptor. Direct cytosolic delivery of multiple bacterial products including lipopolysaccharide, but not flagellin, induced caspase-1 activation via cryopyrin in the absence of pannexin-1 activity or ATP stimulation. However, unlike Ipaf-dependent caspase-1 activation, stimulation of the pannexin- 1-cryopyrin pathway by several intracellular bacteria was independent of a functional bacterial type III secretion system. These results provide evidence for cytosolic delivery and sensing of bacterial molecules as a unifying model for caspase-1 activation and position pannexin-1 as a mechanistic link between bacterial stimuli and the cryopyrin inflammasome. INTRODUCTION Interleukin-1b (IL-1b) is a key mediator of host immune responses and plays an important role in innate and adaptive immunity as well as in the development of inflammatory disease, fever, and septic shock (Dinarello, 1996). Engagement of Toll-like receptor (TLR) signaling by various proinflammatory stimuli induces transcriptional activation of the IL-1b promoter, leading to the production of proil-1b in the cytosol of stimulated macrophages and monocytes (Dinarello, 2006). The evolutionarily conserved cysteine protease caspase-1 processes the inactive IL-1b precursor into the biologically active cytokine (Cerretti et al., 1992; Kuida et al., 1995; Li et al., 1995; Thornberry et al., 1992). Caspase-1 itself is expressed as an inactive zymogen that becomes activated in large multiprotein complexes named inflammasomes (Martinon et al., 2002). Recent studies have identified several members of the NOD-like receptor (NLR) family of proteins as critical mediators of caspase-1 activation (Franchi et al., 2006b; Ting and Davis, 2005). For instance, the NLR protein Ipaf has been implicated in the activation of caspase-1 in response to Salmonella and Legionella through the cytosolic sensing of flagellin (Amer et al., 2006; Franchi et al., 2006a; Miao et al., 2006). Cryopyrin (also called Nalp3, CIAS1, PYPAF1) is another NLR family member that plays a critical role in the regulation of caspase-1 activation (Kanneganti et al., 2006a; Mariathasan et al., 2006; Martinon et al., 2006; Sutterwala et al., 2006). The relevance of cryopyrinmediated caspase-1 activation is underscored by the finding that missense mutations within the Nlrp3 gene that encodes cryopyrin are responsible for three periodic fever syndromes (Hoffman et al., 2004). The diseaseassociated cryopyrin mutations generate activating forms of the protein, which lead to inappropriate activation of caspase-1 and hypersecretion of IL-1b (Agostini et al., 2004; Dowds et al., 2004). Recent studies have shown that cryopyrin is critical for caspase-1 activation induced by bacterial RNA, synthetic purine-like compounds, and endogenous urate crystals (Kanneganti et al., 2006a, 2006b; Martinon et al., 2006). In addition, cryopyrin regulates caspase-1 activation triggered by exogenous adenosine triphosphate (ATP) or pore-forming toxins in macrophages stimulated with several TLR agonists including lipopolysaccharide (LPS), bacterial lipopeptide, and peptidoglycan (Mariathasan et al., 2006; Sutterwala et al., 2006). Stimulation of the P2X 7 receptor with ATP induces a rapid opening of the potassium-selective channel, Immunity 26, , April 2007 ª2007 Elsevier Inc. 433

2 Figure 1. Cryopyrin Is Essential for Caspase-1 Activation in Response to Gram-Negative and Gram-Positive Heat-Killed Bacteria and ATP (A) WT and Nlrp3 / macrophages were stimulated with the indicated heat-killed bacteria for 3 hr and then pulsed with medium (left) or ATP (right). Cell extracts were immunoblotted with caspase-1 antibody. Arrows denote procaspase-1 (p45) and its processed large subunit (p20). (B) WT macrophages were stimulated with indicated concentrations of heat-killed MonoMac-6 cells with or without ATP, and cell extracts were immunoblotted with the caspase-1 antibody. (C) Production of IL-1b, IL-6, and TNF-a by WT (filled bars) and Nlrp3 / (open bars) mice stimulated with the indicated heat-killed bacteria and then pulsed with ATP. Bars represent the mean ± SD of triplicate wells. Results are representative of three independent experiments. S.t, Salmonella typhymurium; F.t, Francisella tularensis; L.p, Legionella pneumophila; P.a, Pseudomonas aeruginosa; L.m, Listeria monocytogenes; P.g, Porphyromonas gingivalis; B.s, Bacillus subtilis; S.a, Staphylococcus aureus. followed by the gradual opening of a larger pore (Khakh and North, 2006; Surprenant et al., 1996). The larger pore is mediated by the hemichannel pannexin-1, which is recruited upon P2X 7 receptor activation (Locovei et al., 2007; Pelegrin and Surprenant, 2006a, 2006b). Notably, pannexin-1 was found to be critical for caspase-1 activation and IL-1b secretion in LPS-stimulated macrophages pulsed with ATP (Pelegrin and Surprenant, 2006b) or stimulated with the pore-forming toxins nigericin and maitotoxin (Pelegrin and Surprenant, 2006a). However, the involvement of pannexin-1 in cryopyrin-mediated caspase-1 activation and the upstream events linking bacterial stimuli to this pathway remain unknown. Moreover, although TLR signaling is required for upregulation of the IL-1b precursor, it remains unclear whether the TLR pathway is directly involved in cryopyrin-mediated caspase-1 activation. In the present report, we show that pannexin-1 activation promotes cytosolic recognition of bacterial products to activate the cryopyrin inflammasome, which proceeds independent of TLR signaling. RESULTS Cryopyrin Is Essential for Caspase-1 Activation and IL-1b Secretion Induced by Bacteria and ATP We first tested the ability of a panel of Gram-positive and Gram-negative heat-killed bacteria that included intracellular and extracellular organisms to induce caspase-1 activation. Cellular extracts prepared from macrophages incubated with heat-killed bacteria for 3 hr followed by a 30 min pulse with ATP were immunoblotted with an antibody that recognizes the p20 subunit of caspase-1. Both Gram-positive and Gram-negative bacteria induced the proteolytic activation of caspase-1 in the presence, but not in the absence, of ATP stimulation (Figure 1A). Notably, the activation of caspase-1 induced by heat-killed bacteria plus ATP was abolished in cryopyrin-deficient (Nlrp3 / ) macrophages (Figure 1A). The activation of caspase-1 induced by ATP was specific for bacterial products in that it was not observed when mouse macrophages were stimulated with ATP alone or with heat-killed human monocytic cells and ATP (Figure 1B). We next assessed the secretion of IL-1b, a process that requires the presence of active caspase-1. Consistent with results shown in Figure 1A, wild-type macrophages stimulated with heat-killed bacteria and pulsed with ATP produced IL-1b in culture supernatants, but this response was abrogated in macrophages lacking cryopyrin (Figure 1C). The requirement of cryopyrin in IL-1b secretion was specific in that wild-type and Nlrp3 / macrophages produced comparable amounts of IL-6 and TNF-a after stimulation with heat-killed bacteria and ATP (Figure 1C). Cryopyrin-Dependent Caspase-1 Activation Induced by Heat-Killed Bacteria or LPS Is Independent of TLR Signaling Previous studies showed that several TLR agonists such as LPS induce cryopyrin-dependent caspase-1 activation 434 Immunity 26, , April 2007 ª2007 Elsevier Inc.

3 Figure 2. Caspase-1 Activation by Heat-Killed Bacteria or TLR-Agonists Is Independent of TLR Signaling (A) Macrophages from WT, Tlr2 /, Tlr4 /, Ticam1 /, and Myd88 / mice were stimulated with indicated heat-killed bacteria for 3 hr and then pulsed with ATP for 30 min. Cell extracts were immunoblotted with caspase-1 antibody. (B and C) Unstimulated macrophages were either left untreated or pulsed for 30 min with LPS or LPS with ATP. Cell extracts were immunoblotted for (B) caspase-1 activation, and culture supernatants were analyzed for (C) IL-1b production. (D and E) Prior to a transient pulse of ATP, WT and Tlr4 / macrophages were either left untreated or stimulated with LPS or LA for 3 hr. Cell extracts were immunoblotted for (D) caspase-1 activation, and culture supernatants were analyzed for (E) IL-1b production. Bars represent the mean ± SD of triplicate wells. Results are representative of at least three separate experiments. after a pulse with ATP (Mariathasan et al., 2006; Sutterwala et al., 2006). However, it is unclear whether these bacterial products mediate caspase-1 activation through TLR signaling or TLR-independent pathways. To analyze the role of TLR signaling in cryopyrin-mediated caspase-1 activation, macrophages lacking TLR2 or TLR4, two major TLRs activated by bacteria, were stimulated with heatkilled bacteria and ATP, and cell extracts were immunoblotted with caspase-1 antibody. The results showed that both TLR2 and TLR4 were not required for caspase-1 activation induced by heat-killed bacteria and ATP (Figure 2A). MyD88 and TRIF are two adaptor proteins known to be essential for signaling induced through all TLRs (Kawai and Akira, 2006). To test whether other TLR pathways may be involved in caspase-1 activation, we performed the same experiment in macrophages deficient in MyD88 (Myd88 / ) or TRIF (Ticam1 / ). Caspase-1 activation was unaffected in Myd88 / and Ticam1 / macrophages, confirming that TLR signaling is not required for activation of the cryopyrin inflammasome (Figure 2A). Priming of macrophages with bacterial ligands such as LPS is typically performed for at least 3 hr to allow the induction of pro-il-b via a TLR4-dependent mechanism. We reasoned that if caspase-1 activation induced by LPS is independent of TLR4, the activation of caspase-1 and IL-1b secretion could be dissociated when macrophages are stimulated by a short pulse of LPS and ATP. Consistent with this, incubation of macrophages with LPS and ATP for 30 min induced full activation of caspase-1 (Figure 2B), but not IL-1b secretion (Figure 2C). As expected, incubation with LPS for 4 hr prior to the ATP pulse led to high amounts of secreted IL-1b (Figure 2C). To further assess the role of TLR4 in LPS-induced caspase-1 activation, wild-type and TLR4-deficient macrophages Immunity 26, , April 2007 ª2007 Elsevier Inc. 435

4 were stimulated with the TLR4 ligands LPS and synthetic lipid A in combination with ATP, and the processing of caspase-1 and IL-1b secretion were analyzed by immunoblotting and ELISA. Notably, the activation of caspase-1 induced by LPS and lipid A plus ATP was unimpaired in TLR4-deficient macrophages when compared to wild-type cells (Figure 2D). Importantly, secretion of IL-1b induced by both TLR4 and lipid A was abolished in Tlr4 / macrophages (Figure 2E), which is consistent with the requirement of TLR4 for transcriptional upregulation of pro-il-1b via NF-kB activation (Kawai et al., 2001; Seki et al., 2001). These results indicate that the induction of caspase-1 activation induced by LPS and ATP is independent of TLR4, whereas secretion of mature IL-1b additionally requires TLR-dependent upregulation of the IL-1b precursor. Thus, caspase-1 activation and IL-1b secretion triggered by LPS and ATP can be dissociated by the requirement of TLR4. Caspase-1 Activation Induced by Heat-Killed Bacteria Requires ASC, but Not Ipaf, Nod1, Nod2, or RICK Given that caspase-1 activation induced by heat-killed bacteria and LPS plus ATP required cryopyrin, but was independent of TLRs, we tested the role of ASC (apoptosisassociated speck-like protein containing a CARD, Pycard), an adaptor that is thought to link cryopyrin to caspase-1, as well as that of the NLR proteins Ipaf, Nod1, Nod2, and the kinase RICK (also known as RIP2), because these molecules have also been implicated in caspase-1 activation (Maeda et al., 2005; Thome et al., 1998; Yoo et al., 2002). We found that caspase-1 activation induced by heat-killed bacteria and ATP was abolished in macrophages deficient in ASC but proceeded normally in macrophages deficient in Ipaf (Nlrc4 / ), Nod1 (Nod1 / ), Nod2 (Nod2 / ), or RICK (Ripk2 / )(Figure 3). These results indicate that the regulation of caspase-1 activation triggered by heat-killed bacteria is highly specific and relies on cryopyrin and ASC. Functional Pannexin-1 Is Required for ATP- Dependent Caspase-1 Activation and IL-1b Secretion Recent studies have shown that pannexin-1 mediates formation of a large pore through its association with the P2X 7 receptor (Pelegrin and Surprenant, 2006a, 2006b). To test whether pannexin-1 is required for cryopyrin-dependent caspase-1 activation, macrophages were stimulated with heat-killed bacteria and pulsed with ATP in the presence of a pannexin-1-mimetic inhibitory peptide that selectively inhibits P2X 7 -mediated large pore formation, without altering other aspects of P2X 7 receptor activation (Pelegrin and Surprenant, 2006b). Notably, incubation of macrophages with the pannexin-1 inhibitory peptide, but not the control peptide, abolished caspase-1 activation induced by stimulation with heat-killed bacteria and ATP (Figure 4A). Furthermore, the pannexin-1 inhibitory peptide greatly suppressed the secretion of IL-1b triggered by stimulation of macrophages with heat-killed bacteria and ATP Figure 3. Caspase-1 Activation by Heat-Killed Bacteria Requires ASC, but Not Nod1, Nod2, RICK, or Ipaf WT, Nod1/Nod2 double knockout, Ripk2 /, Nlrc4 /, and Pycard / macrophages were stimulated with the indicated heat-killed bacteria for 3 hr and then pulsed with ATP. Cell extracts were immunoblotted for caspase-1 activation. Arrows denote procaspase-1 (p45) and its processed large subunit (p20). Results are representative of at least three separate experiments. (Figure 4B). These results indicate that the hemichannel pannexin-1 is essential for P2X 7 -mediated activation of the cryopyrin inflammasome by heat-killed bacteria. Pannexin-1 Is Required for Caspase-1 Activation Induced by Several Bacterial PAMPs but Not Flagellin Our results showed that both Gram-positive and Gramnegative heat-killed bacteria induce cryopyrin- and ASCdependent caspase-1 activation in the presence of ATP. 436 Immunity 26, , April 2007 ª2007 Elsevier Inc.

5 We next sought to determine which bacterial pathogenassociated molecular patterns (PAMPs) are responsible for the activation of caspase-1 in macrophages. In the absence of ATP stimulation, none of the PAMPs induced caspase-1 activation (Figure 5A). In contrast, LPS, synthetic lipid A, triacylated synthetic lipopeptide (Pam3- CSK4), lipoteichoic acid, and peptidoglycan induced caspase-1 activation after a brief pulse with ATP (Figure 5A). Consistent with published results (Mariathasan et al., 2006; Martinon et al., 2006; Sutterwala et al., 2006), the activation of caspase-1 induced by PAMPs and ATP was cryopyrin dependent. Notably, flagellin, a bacterial protein that is sensed by TLR5 and the NLR protein Ipaf, did not induce caspase-1 activation after stimulation with ATP (Figure 5B), attributing specificity to cryopyrinmediated caspase-1 activation in response to bacterial PAMPs and ATP. To determine whether functional pannexin-1 is required for PAMP-induced activation of the cryopyrin inflammasome, macrophages were incubated with various PAMPs followed by ATP stimulation in the presence of the pannexin-1 inhibitory peptide. Notably, inhibition of pannexin-1 activity blocked caspase-1 activation induced by PAMPs and ATP (Figure 5C). These results indicate that the activity of the P2X 7 receptor-associated protein pannexin-1 is critical for ATP and PAMP-induced activation of the cryopyrin inflammasome. Figure 4. Functional Pannexin-1 Is Required for ATP-Dependent Caspase-1 Activation and IL-1b Secretion Macrophages were stimulated with the indicated heat-killed bacteria for 3 hr, then incubated for 30 min with medium or 500 mm 10 panx1 blocking peptide or 500 mm 14 panx1 control peptide as indicated. Finally, macrophages were pulsed with 5 mm ATP for 30 min. Cell extracts were immunoblotted for caspase-1 activation (A), and culture supernatants were analyzed for IL-1b production (B). Bars represent the mean ± SD of triplicate wells. Results are representative of at least three separate experiments. Cytosolic Delivery of Heat-Killed Bacteria or Selective PAMPs Activates the Cryopyrin Inflammasome Activation of caspase-1 through Ipaf in response to Salmonella and Legionella requires a bacterial type III or IV secretion system to deliver flagellin into the host cytosol (Amer et al., 2006; Franchi et al., 2006a; Miao et al., 2006). We hypothesized that similar to the role of Type III or IV bacterial secretion systems, ATP-induced activation of pannexin-1 may mediate intracellular uptake of bacterial components through the endosomal pathway and their delivery into the host cytosol, where they are sensed by cryopyrin. Consistent with this hypothesis, treatment of macrophages with chloroquine, an agent that blocks endosomal maturation, abolished caspase-1 activation and IL-1b secretion from macrophages stimulated with heatkilled bacteria and pulsed with ATP (see Figure S1 in the Supplemental Data available online). In line with a role for pannexin-1 in intracellular uptake of bacterial products upstream of cryopyrin signaling, the pannexin-1 inhibitory peptide blocked the ATP-induced uptake of the fluorescent dye Yopro-1 in both wild-type and cryopyrin-deficient macrophages (Figure S2). We next determined whether direct delivery of bacterial products into the cytosol bypasses the requirement for ATP-mediated pannexin-1 activation to trigger caspase-1 activation. We incubated wild-type and mutant macrophages with heatkilled bacteria without ATP in the presence or absence of streptolysin O (SLO), a pore-forming molecule derived from Streptococcus that allows delivery of exogenous molecules into the cytosol of living cells (Walev et al., 2001). The experiments revealed that SLO-mediated delivery of heat-killed bacteria in the cytosol induced caspase-1 activation in the absence of ATP stimulation (Figure 6A). Notably, this activation of caspase-1 mediated by SLO and heat-killed bacteria was abolished in cryopyrin-deficient macrophages (Figure 6A). Moreover, in contrast to macrophages stimulated with heat-killed bacteria and ATP (Figures 4 and 6C), the pannexin-1 inhibitory peptide did not interfere with caspase-1 activation induced by heat-killed bacteria and SLO (Figure 6B). Consistent with the results obtained with heat-killed bacteria, LPS- and SLO-induced activation of caspase-1 required cryopyrin, but not pannexin-1 (Figure 6D). By contrast, the activation of caspase-1 induced by cytosolic flagellin was independent of cryopyrin (Figure 6E) and functional pannexin-1 (Figure 6F), which is consistent with results Immunity 26, , April 2007 ª2007 Elsevier Inc. 437

6 Figure 5. Functional Pannexin-1 and Cryopyrin Are Required for Caspase-1 Activation Induced by Bacterial PAMPs (A and B) WT and Nlrp3 / macrophages were stimulated with the indicated bacterial PAMPs for 3 hr and then pulsed with medium or ATP. Cell extracts were immunoblotted with caspase-1 antibody. Arrows denote procaspase-1 (p45) and its processed large subunit (p20). (C) WT macrophages were stimulated with the indicated bacterial PAMPs for 3 hr and incubated for an additional 30 min with medium (left) or 10 panx1 blocking peptide (right) before pulsing with 5 mm ATP. Cell extracts were analyzed for caspase-1 activation. Results are representative of three separate experiments. presented in Figure 5. Thus, the delivery of PAMPs in the cytosol by pore-forming proteins triggers the activation of a caspase-1 inflammasome. To test whether delivery of bacterial molecules to the host cytosol can be sensed by cryopyrin through non-pore-forming mechanisms, we stimulated macrophages with heat-killed bacteria or PAMPs in the presence and absence of DOTAP, a cationic lipid formulation that mediates delivery of molecules into the cytosol of living cells (Simberg et al., 2004). Stimulation of wild-type macrophages with heat-killed bacteria or various PAMPs and DOTAP, but not with DOTAP alone, induced activation of caspase-1 in the absence of ATP (Figure S3). The activation of caspase-1 induced by heatkilled bacteria or various PAMPs was abrogated in cryopyrin-deficient macrophages (Figure S3). These results indicate that the presence of heat-killed bacteria or selective PAMPs in the cytosol results in cryopyrin-dependent caspase-1 activation regardless of the delivery mechanism. Furthermore, these results indicate that cryopyrin functions downstream of pannexin-1 and ATP to regulate caspase-1 activation in response to specific bacterial components that are detected in the cytosol. Pannexin-1 Controls Caspase-1 Activation Induced by Bacteria without a Functional Secretion System Intracellular pathogens such as Salmonella, Franciscella, and Listeria use type III secretion systems or pore-forming molecules to gain access to the host cytosol and to activate caspase-1 in infected macrophages. To test whether the ATP-regulated pathway is controlled by cryopyrin in response to infection by intracellular bacteria, wild-type and mutant macrophages were infected with various intracellular bacteria in the absence or presence of ATP. Infection with wild-type Salmonella, Franciscella, or Listeria induced caspase-1 activation in the absence of ATP, and this pathway was independent of cryopyrin and pannexin-1 (Figure 7). Salmonella, but not Franciscella or Listeria, induced caspase-1 activation through Ipaf in the absence of ATP (Figure 7). As expected, the Salmonella SipB mutant that is defective in the transfer of many type III secretion effector proteins, Listeria deficient in listeriolysin O (LLO), and the Franciscella mgla mutant did not activate caspase-1 (Figure 7). Importantly, these mutant bacteria induced caspase-1 activation in the presence of ATP (Figure 7), and this type III secretion- or pore-forming toxinindependent pathway required functional pannexin-1 and cryopyrin (Figure 7). These results demonstrate that intracellular bacteria can induce caspase-1 activation through two distinct mechanisms. In the absence of ATP stimulation, a functional type III secretion apparatus or a pore-forming protein is required. When pulsed with ATP, infected macrophages activate caspase-1 through pannexin-1 and cryopyrin even in the absence of an operational secretion system or pore-forming toxin. DISCUSSION Previous studies have shown that cryopyrin plays a critical role in ATP-driven activation of caspase-1 in response to multiple bacterial ligands (Mariathasan et al., 2006; Martinon et al., 2006; Sutterwala et al., 2006). However, the upstream events linking bacterial products and the requirement for ATP to cryopyrin remained elusive. One model postulated that TLR signaling is required for the assembly of a functional inflammasome complex (Mariathasan et al., 2006; Sutterwala et al., 2006), but a physical connection between components of TLR signaling and caspase-1 activation remains to be identified. Our results demonstrate that ATP-driven cryopyrin-mediated caspase-1 activation in response to bacterial stimuli does not require 438 Immunity 26, , April 2007 ª2007 Elsevier Inc.

7 Figure 6. Cytosolic Delivery of Heat-Killed Bacteria by the Pore-Forming Protein Streptolysin O Is Sufficient for Activation of the Cryopyrin Inflammasome (A) WT and Nlrp3 / macrophages were incubated with the indicated heat-killed bacteria for 10 min in the absence (left) or presence (right) of the poreforming protein streptolysin O (SLO, 5 mg/ml), then washed extensively and cultured for 3 hr before cell extracts were prepared and immunoblotted for caspase-1 activation. (B) Prior to stimulating cells with heat-killed bacteria and SLO as described above, WT macrophages were preincubated for 30 min with the 10 panx1 blocking peptide. The cell extracts were treated as in (A). (C) WT macrophages were stimulated with heat-killed Salmonella typhimurium (S.t.) for 2 hr. Prior to the ATP pulse, macrophages were incubated for 30 min with medium (left) or the 10 panx1 blocking peptide (right). Cell extracts were immunoblotted for caspase-1 activation. (D) WT and Nlrp3 / macrophages were left untreated (lane 1) or incubated with SLO (lane 2), purified flagellin (lane 3), or SLO and flagellin (lane 4) for 10 min, washed, and incubated for 3 hr prior to analysis of cell extracts for caspase-1 activation. (E) WT and Nlrp3 / macrophages were left untreated (lane 1), incubated with purified flagellin for 3 hr and pulsed with ATP (lane 2), stimulated with SLO and flagellin as described above (lane 3), or preincubated with the 10 panx1 blocking peptide prior to stimulation with SLO and flagellin (lane 4). Cell extracts were analyzed for caspase-1 activation. Results are representative of at least three separate experiments. TLR signaling. Caspase-1 activation was unimpaired in Tlr2 /, Tlr4 /, Myd88 /, and Ticam1 / macrophages incubated with heat-killed Gram-positive or Gramnegative bacteria and pulsed with ATP. Furthermore, the TLR4 ligands LPS and synthetic lipid A induced potent caspase-1 activation in Tlr4 / macrophages, whereas cytokine production was completely abolished. Therefore, our results demonstrate that although TLR signaling is required for cytokine production, activation of the cryopyrin inflammasome itself occurs independently of TLRs. These results are in line with several recent studies showing caspase-1 activation by intracellular bacteria in macrophages deficient in TLRs or the adaptor molecules TRIF and MyD88 (Amer et al., 2006; Franchi et al., 2006a; Miao et al., 2006; Ozoren et al., 2006). For example, caspase-1 activation in response to Salmonella, Legionella, and Listeria infection is unimpaired in macrophages deficient for TLRs or insensitive to TLR signaling (Amer et al., 2006; Franchi et al., 2006a; Miao et al., 2006; Ozoren et al., 2006). Moreover, these studies revealed that TLRindependent caspase-1 activation induced in response to S. typhimurium as well as L. pneumophila is mediated through the NLR protein Ipaf and the adaptor ASC and requires cytosolic sensing of flagellin (Amer et al., 2006; Franchi et al., 2006a; Mariathasan et al., 2004; Miao et al., 2006). Thus, similar to the cryopyrin-mediated caspase-1 activation, the induction of the Ipaf inflammasome by intracellular bacteria or purified bacterial flagellin occurs independently of TLR signaling. To further elucidate the upstream molecular mechanisms involved in activation of the cryopyrin inflammasome, we investigated the role of pannexin-1, a recently described hemichannel that associates with the P2X 7 receptor upon ATP stimulation and induces a large nonselective pore (Locovei et al., 2007; Pelegrin and Surprenant, 2006b). We found that pannexin-1 activity is critical for caspase-1 activation induced by heat-killed bacteria and multiple bacterial PAMPs in ATP-pulsed macrophages. We postulated that pannexin-1 and P2X 7 receptor mediate the passage of bacterial molecules from the endosomal compartment into the host cytosol, thus leading to cryopyrin-dependent caspase-1 activation. Consistent with this model, inhibiting endosomal maturation with chloroquine prevented ATP-induced caspase-1 activation and IL-1b release. Moreover, direct cytosolic delivery of bacterial ligands induced cryopyrin-dependent caspase-1 activation in the absence of P2X 7 stimulation and functional pannexin-1. Direct cytosolic delivery of bacterial ligands bypasses the requirement for ATP and pannexin-1 activity, so we suggest that ATP- and P2X 7 -mediated Immunity 26, , April 2007 ª2007 Elsevier Inc. 439

8 Figure 7. Pannexin-1 Controls Activation of the Cryopyrin Inflammasome Induced by Intracellular Bacteria Deficient in Pore- Forming Toxins WT, Nlrp3 /, and Nlrc4 / macrophages were infected with the indicated wild-type or mutant bacteria for 1 hr. Extracellular bacteria were washed away and macrophages were further incubated in medium containing gentamycin. Caspase-1 activation was analyzed in the absence of ATP pulse (left), after pulsing with ATP for 30 min (middle), or after pretreatment with the 10 panx1 blocking peptide followed by ATP pulse (right). Arrows denote procaspase-1 (p45) and its processed large subunit (p20). Results are representative of at least three separate experiments. activation of pannexin-1 may fulfill a role similar to that mediated by bacterial secretion systems and pore-forming toxins. Pannexin-1 is a transmembrane protein located at the cell surface and in the endoplasmic reticulum (Abeele et al., 2006; Pelegrin and Surprenant, 2006a), organelles that contribute to the formation of phagosomes. Thus, pannexin-1 could act at the plasma membrane and/or phagosomes to mediate the delivery of bacterial molecules into the cytosol. A function for pannexin-1 at the phagosome membrane is attractive in that dead bacteria and PAMPs such as LPS are known to be internalized via phagocytosis independent of TLRs (Dunzendorfer et al., 2004; Zhou et al., 2004). Such a mechanism might explain the lack of requirement for TLR signaling in caspase-1 activation induced via the pannexin- 1-cryopyrin pathway. Another possibility is that pannexin-1 functions by an indirect mechanism to promote the delivery of PAMPs into the cytosol. Although the precise mechanism by which pannexin-1 mediates pore formation in response to P2X 7 stimulation requires further analysis, our results provide a mechanistic link between upstream signals, namely bacterial products and ATP, and activation of caspase-1 in the cryopyrin inflammasome through the cytosolic delivery of bacterial ligands. A unifying model for caspase-1 activation by intracellular and extracellular bacteria is emerging, whereby the presence of PAMPs in the cytosol triggers the activation of caspase-1 via NLR family members independently of TLRs. Indeed, intracellular bacteria activate caspase-1 through a pathway that requires a functional type III or IV secretion system or a pore-forming toxin that delivers bacterial effector molecules such as flagellin into the cytosol of infected macrophages (Amer et al., 2006; Franchi et al., 2006a; Miao et al., 2006). This pathway is independent of pannexin-1 and occurs in the absence of P2X 7 activation. Consistent with this, the pathogens L. monocytogenes and Aeromonas trota have been reported to require their pore-forming toxins to induce caspase-1 activation (Gurcel et al., 2006; Mariathasan et al., 2006). In the case of Salmonella and Legionella, this pathway is controlled by Ipaf through cytosolic sensing of flagellin, whereas a yet unidentified NLR protein may sense bacterial factors released by Franciscella and Listeria in the cytosol. Intracellular and extracellular bacteria appear to activate caspase-1 through a second mechanism that requires cryopyrin. In the presence of P2X 7 activation, the latter pathway of caspase-1 activation is independent of an operational bacterial type III or IV secretion system or pore-forming proteins such as Listeria LLO. Instead, the pannexin-1-mediated pore may substitute for the bacterial type III secretion system or pore-forming toxin to trigger cryopyrin-dependent caspase-1 activation through the cytosolic delivery of PAMPs. In this model, the nature of the bacterial PAMP that is delivered into the cytosol determines the identity of the caspase-1-activating NLR protein irrespective of the delivery mechanism. For instance, cytosolic delivery of LPS through the pore-forming protein SLO or through liposomes induces activation of the 440 Immunity 26, , April 2007 ª2007 Elsevier Inc.

9 cryopyrin inflammasome, whereas that of flagellin triggers cryopyrin-independent caspase-1 activation that relies on the NLR protein Ipaf (Amer et al., 2006; Franchi et al., 2006b). Furthermore, the delivery mechanism may also contribute to the specificity of the caspase-1-signaling pathway. For instance, LPS activates caspase-1 in ATPstimulated macrophages, whereas flagellin does not. In contrast, both LPS and flagellin induce caspase-1 activation when delivered into the cytosol via SLO or liposomes. Although the mechanism for this differential effect remains to be elucidated, one possibility is that the size or structure of the bacterial PAMP is important for this specificity. Because caspase-1 activation induced by Salmonella is largely dependent on flagellin and Ipaf (Franchi et al., 2006a; Miao et al., 2006; Molofsky et al., 2006), the Ipaf inflammasome appears to be the major pathway activated by Salmonella, at least in vitro. However, intracellular bacteria such as Salmonella and Francisella are killed by several antimicrobial mechanisms in the host and may release PAMPs such as LPS and peptidoglycan, which induce caspase-1 activation through cryopyrin (Mariathasan et al., 2006; Sutterwala et al., 2006). The mechanism by which NLR family members such as Ipaf or cryopyrin sense bacterial molecules in the cytosol remains poorly understood. There is currently no evidence for a direct physical interaction of mammalian NLR family members with their microbial agonists. In plants, the microbial recognition mediated by NLR homologs is largely indirect, in that NLRs sense alterations in host factors induced by the cytosol activity of bacterial elicitors (Mackey et al., 2002, 2003). Thus, the sensing of PAMPs via cryopyrin might be indirect. This possibility is in agreement with the fact that cryopyrin induces caspase-1 activation in response to a panel of bacterial ligands with widely distinct molecular structures. Therefore, the presence of PAMPs in the cytosol might be recognized by one or more host factors that induce the activation and assembly of the cryopyrin inflammasome. Alternatively, cryopyrin may respond to endogenous signals invoked by the cytosolic presence of PAMPs. In this regard, cryopyrin is known to induce caspase-1 activation in response to endogenous monosodium urate (MSU) and calcium pyrophosphate dehydrate (CPPD) crystals in the absence of ATP (Martinon et al., 2006). MSU and CPPD crystals have well-known roles in the inflammatory syndromes gout and pseudogout, respectively (Liu-Bryan and Liote, 2005). It would be informative to determine whether pannexin-1 has a role in CPPD and MSU crystal-induced activation of caspase-1. Although the analysis of the in vivo function of pannexin-1 in infection and immunity awaits the generation of pannexin-1-deficient mice, these animals can be expected to have a wider immunological phenotype than that displayed by P2X 7 / mice (Labasi et al., 2002; Solle et al., 2001). Indeed, whereas both pannexin-1 and the P2X 7 receptor are required for ATP-induced activation of caspase-1, only pannexin-1 is essential for caspase-1 activation induced by the bacterially derived potassium ionophore nigericin and the shellfish toxin maitotoxin (Pelegrin and Surprenant, 2006b; Solle et al., 2001). Intriguingly, in contrast to human monocytes, addition of exogenous ATP is required for robust LPS-induced IL-1b secretion in primary mouse macrophages cultured in vitro. The high concentrations of ATP that are required for in vitro P2X 7 receptor activation and production of high levels of IL-1b secretion are not found normally in the extracellular milieu, although they could be reached in the context of cell lysis or injury (Ferrari et al., 2006). Thus, further work is required to delineate the signaling pathways that induce pannexin-1-dependent activation of caspase-1. EXPERIMENTAL PROCEDURES Mice and Macrophages Nlrp3 /, Nlrc4 /, Nod1/Nod2 double knockout, Pycard /, Ripk2 /, Tlr2 /, Tlr4 /, Ticam1 /, and Myd88 / mice have been described (Franchi et al., 2006a; Kanneganti et al., 2006b; Ozoren et al., 2006; Park et al., 2007). C57BL6/J6 mice were purchased from Jackson Laboratories. Mice were housed in a pathogen-free facility. Bone marrow-derived macrophages were prepared as described before (Kanneganti et al., 2006a). The animal studies were conducted under approved protocols by the University of Michigan Committee on Use and Care of Animals. Bacteria Listeria wild-type strain 10403S and mutant Listeria containing an inframe deletion of the hly gene (LLO, DP-L2161) were a gift of M. O Riordan (University of Michigan). S. enterica serovar typhimurium strain SL1344 and the isogenic orga mutant strain BJ66 (SipB ) were kindly provided by D. Monack (Stanford University). The flib /flic Salmonella strain was a generous gift of A. Aderem (University of Washington). Single colonies were inoculated into 3 ml of BHI medium and grown overnight at 30 C with shaking. On the day of the infection, a 1/10 dilution of the overnight culture was prepared and allowed to grow at 37 C with shaking to A600 = 0.5, which corresponds to 10 9 CFU/ml. Infection of macrophages was allowed to proceed for 30 min at 37 C. Macrophages were then washed and incubated for 2 hr in IMDM containing gentamycin (33 mg/ml) to kill extracellular bacteria. In experiments with nonmotile mutants, plates were spun at 1000 rpm for 10 min before incubation to ensure a similar uptake in the absence of motility. Microbial Ligands and Heat-Killed Bacteria Purified bacterial ligands and heat-killed Legionella penumophila, Porphoromonas gingivalis, and Staphylococcus aureus were purchased from Invivogen. Heat-killed Salmonella typhymurium, Francisella tularensis, Pseudomonas aeruginosa, Listeria monocytogenes, and Bacillus subtilis were prepared by washing bacterial cultures three times in cold PBS, resuspending them in cold PBS at a concentration of cells/ml, and heating them for 45 min at 95 C. Heat-killed bacteria were used to stimulate macrophages at a concentration of 10 8 cells/ml, and the ligands were used at a concentration of 5 mg/ml. In some experiments, macrophages were pretreated with chloroquine (Sigma, 250 mm) prior to stimulation with heat-killed bacteria. Streptolysin O (Sigma) or DOTAP liposomes (Roche) were used in some experiments to deliver microbial ligands or heat-killed bacteria into the cytosol of macrophages as described previously (Amer et al., 2006; Franchi et al., 2006b). Peptides The Pannexin-1 mimetic blocking peptide 10 panx1 (WRQAAFVDSY) and the control peptide 14panx1 (SGILRNDSTVPDQF) were synthesized by Sigma-Genosys and have been used as described before (Pelegrin and Surprenant, 2006b). Immunity 26, , April 2007 ª2007 Elsevier Inc. 441

10 Immunoblotting Extracts were prepared from cells and culture supernatants in lysis buffer solution (150 mm NaCl, 10 mm Tris-HCl [ph 7.4], 5 mm EDTA, 1 mm EGTA, and 0.1% Nonidet P40) supplemented with 2 mm dithiothreitol and a protease cocktail inhibitor tablet (Roche). Samples were clarified, denaturated with SDS buffer, and boiled for 5 min. Lysates were separated by SDS-PAGE and transferred to PVDF membranes by semidry blotting in a buffer containing 25 mm Tris-HCl (ph 8.0), 190 mm glycine, and 20% methanol. Membranes were incubated with an antibody against caspase-1 (Lamkanfi et al., 2004), followed by a horseradish peroxidase-conjugated secondary antibody against rabbit immunoglobulin (Jackson ImmunoResearch Laboratories). Immunoreactive proteins were visualized with the enhanced chemiluminescence method (Pierce). Measurements of Cytokines Macrophages were stimulated for 3 hr with heat-killed bacteria or microbial ligands. Subsequently, cells were either pulsed for 30 min with 5 mm ATP (Roche) or left untreated until culture supernatants were collected. Secretion of IL-1b, TNF-a, and IL-6 was determined by enzyme-linked immunoabsorbent assay (ELISA) (R&D Systems). YoPro-1 Assays and Microscopy YoPro-1 (Invitrogen, 2 mm) was present 10 min before macrophages were left untreated or stimulated with 5 mm ATP for 5 min, and fluorescence microscopy was carried out under a 203 objective on a Zeiss Axiophot-2 microscope equipped with a Zeiss Axiocam CCD digital camera. Fluoresence signals in digital black-and-white fluorographs were colored green with Adobe Photoshop. Supplemental Data Three Supplemental Figures can be found with this article online at ACKNOWLEDGMENTS We thank A. Coyle, E. Grant, and J. Bertin (Millennium Pharmaceuticals) and S. Akira (Osaka University) for generous supply of mutant mice and J. Whitfield from the Cellular Immunology Core Facility of the University of Michigan Cancer Center for technical support. T.-D.K. was supported by Training Grant 5/T32/HL from National Institutes of Health. Research at P.V. s lab is supported by grants from IAP6/18, GOA , FWO 2G , Belgian Foundation against Cancer SCIE This work was supported by National Institutes of Health Grants AI and A/ to G.N. The authors declare that they have no competing financial interests. Received: January 29, 2007 Revised: March 1, 2007 Accepted: March 12, 2007 Published online: April 12, 2007 REFERENCES Abeele, F.V., Zholos, A., Bidaux, G., Shuba, Y., Thebault, S., Beck, B., Flourakis, M., Panchin, Y., Skryma, R., and Prevarskaya, N. (2006). Ca(2+)-independent phospholipase A(2)-dependent gating of TRPM8 by lysophospholipids. J. Biol. Chem. 281, Agostini, L., Martinon, F., Burns, K., McDermott, M.F., Hawkins, P.N., and Tschopp, J. (2004). 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