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. Author manuscript; available in PMC: 2011 Dec 16.
Published in final edited form as: Cell Host Microbe. 2010 Dec 16;8(6):471–483. doi: 10.1016/j.chom.2010.11.007

Differential requirement for Caspase-1 autoproteolysis in pathogen-induced cell death and cytokine processing

Petr Broz a,*, Jakob von Moltke b,*, Jonathan W Jones a, Russell E Vance b, Denise M Monack a
PMCID: PMC3016200  NIHMSID: NIHMS256902  PMID: 21147462

Summary

Activation of the cysteine protease Caspase-1 is a key event in the innate immune response to infections. Synthesized as a pro-protein, Caspase-1 undergoes autoproteolysis within multi-protein complexes called inflammasomes. Activated Caspase-1 is required for proteolytic processing and release of the cytokines interleukin-1β and interleukin-18, and can also cause rapid macrophage cell death. We show that macrophage cell death and cytokine maturation in response to infection with diverse bacterial pathogens can be separated genetically and that two distinct inflammasome complexes mediate these events. Inflammasomes containing the signaling adaptor Asc form a single large ‘focus’ in which Caspase-1 undergoes autoproteolysis and processes IL-1β/IL-18. In contrast, Asc-independent inflammasomes activate Caspase-1 without autoproteolysis and do not form any large structures in the cytosol. Caspase-1 mutants unable to undergo autoproteolysis promoted rapid cell death, but processed IL-1β/18 inefficiently. Our results suggest the formation of spatially and functionally distinct inflammasomes complexes in response to bacterial pathogens.

Introduction

Caspase-1 activation is a key event in the innate immune response of macrophages to a variety of infectious and noxious stimuli. Active Caspase-1 promotes the cleavage and secretion of the pro-inflammatory interleukin-1β and interleukin-18, which are cytokines critical for coordination of immunity against various classes of pathogens. In addition, active Caspase-1 induces a pro-inflammatory form of macrophage cell death called pyroptosis.

Caspase-1 is a member of the caspase family of cysteine proteases, peptidases that use a cysteine residue as the catalytic nucleophile and that share an exquisite specificity for cleaving target proteins at sites next to aspartic acid residues (Thornberry and Lazebnik, 1998). Inappropriate activation of Caspase-1 has been linked to several autoimmune inflammatory disorders in humans, illustrating the importance of tight regulation of Caspase-1 activity (Martinon et al., 2009). Caspase-1 is synthesized as an inactive, monomeric zymogen (pro-Caspase-1) that is thought to be activated by dimerization and autoproteolytic processing (Martinon et al., 2009). Autoproteolysis of pro-Caspase-1 results in the generation of the characteristic large and small subunits (termed p20 and p10) of the catalytically active enzyme, as well as the removal of the N-terminal CARD (Caspase Activation Recruitment Domain) (Thornberry et al., 1992). However, this activation step involves prior recruitment of pro-Caspase-1 into multi-protein signaling complexes called the “inflammasomes”.

Inflammasome formation is coordinated by members of the NLR protein family (e.g., Nlrp1, Nlrp3 and Nlrc4) or the PYHIN protein family (e.g., Aim2) that function as specific sensors of a variety of pathogens and other inflammatory stimuli (Brodsky and Monack, 2009). For example, Nlrp1b is required for Caspase-1 activation in response to anthrax lethal toxin (Boyden and Dietrich, 2006). Nlrp3 responds to a large variety of structurally and chemically different molecules, but the molecular mechanism linking these molecules to Nlrp3 activation remains poorly understood (Hornung and Latz, 2010). Nlrc4 activates Caspase-1 after infection with Salmonella spp., Pseudomonas aeruginosa, Listeria monocytogenes and Legionella pneumophila (Amer et al., 2006; Franchi et al., 2006; Miao et al., 2006; Ren et al., 2006). Nlrc4 appears to detect these pathogens by recognizing molecules, such as flagellin or the T3SS rod subunit, which are secreted into the host cell cytosol by bacteria (Lightfield et al., 2008; Miao et al., 2010). Finally, Aim2 recognizes the presence of double stranded DNA in the cytoplasm and is activated during infections with certain DNA viruses and the cytosolic bacterial pathogens Francisella novicida and L. monocytogenes (Fernandes-Alnemri et al., 2010; Jones et al., 2010; Kim et al., 2010; Rathinam et al., 2010; Sauer et al.; Tsuchiya et al., 2010; Warren et al., 2010; Wu et al., 2010).

In addition to NLR/PYHIN proteins, inflammasome complexes also recruit a bipartite adaptor protein called Asc that contains both CARD and PYRIN domains (Masumoto et al., 1999). Asc serves as a linker between the PYRIN domain of the NLR/PYHIN sensors and the CARD domain of pro-Caspase-1 (Srinivasula et al., 2002). Interestingly, Nlrc4 lacks a PYRIN domain but contains a CARD domain and is thus able to directly interact with and activate pro-Caspase-1 independently of Asc (Poyet et al., 2001). Nevertheless, it has been shown that Asc greatly enhances the efficiency of Nlrc4-mediated cytokine processing (Broz et al., 2010; Mariathasan et al., 2004).

Although several different inflammasomes have been described, the precise molecular architecture and composition of inflammasomes remains largely unknown. The apoptosome, a related structure that serves as a platform for activation of Caspase-9, has been shown to be a 700-1400 kDa heptameric, wheel-shaped complex containing Apaf-1 and cytochrome C (Bao and Shi, 2007). Recently, we have shown that in response to Nlrc4, Nlrp3 or Aim2 stimuli, endogenous Asc can aggregate to form a single “Asc focus” in the cytoplasm with a diameter of 1-2 microns (Broz et al., 2010; Jones et al., 2010), vastly larger than the apoptosome. Similar Asc foci were previously observed when THP-1 cells overexpressing Asc-GFP activated Nlrp3 (Fernandes-Alnemri et al., 2007). The Asc focus appears to serve as a platform for the recruitment and activation of Caspase-1, which in turn recruits and processes pro-IL-1β to its active form (Broz et al., 2010). The formation of the Asc focus correlates with the release of mature cytokines, suggesting that it could represent the major, subcellular site of Caspase-1 activation and cytokine processing. Consistent with this hypothesis, Caspase-1 fails to undergo autoproteolysis in Asc-deficient cells, and Asc-deficient cells are severely impaired for release of mature IL-1β and IL-18 (Mariathasan et al., 2004).

A major unresolved issue, however, is what role Asc and Caspase-1 autoproteolysis plays in the other central function of Caspase-1, namely, induction of rapid host cell death. We and others have reported previously that Asc-deficient macrophages infected with pathogens that activate Nlrc4 are severely impaired in their ability to process and release mature IL-1β, but remarkably, are still able to undergo pyroptotic cell death (Case et al., 2009; Mariathasan et al., 2004; Suzuki et al., 2007). Even more remarkably, this Nlrc4-dependent/Asc-independent cell death occurs in the absence of any detectable Caspase-1 processing, but is nevertheless abolished in Caspase-1 knockout cells. It remains unclear how cells lacking processed Caspase-1 can undergo a Caspase-1-dependent death, since autoproteolytic processing is thought to activate Caspase-1. One possibility is that Caspase-11, a proposed regulator of Caspase-1 (Wang et al., 1998), which is also absent from Caspase-1 knockout cells (Kang et al., 2000), might be critical for Nlrc4-dependent cell death. Another possibility is that the catalytic activity of Caspase-1 is not required for pyroptosis, and instead, unprocessed Caspase-1 functions to recruit other downstream death effectors. Lastly, it is possible that Nlrc4 activates Caspase-1 in a manner that does not require Asc or autoproteolysis.

In this study, we investigated the molecular basis for Caspase-1-dependent cell death. We demonstrate that sensors containing a CARD (such as Nlrc4, Nlrp1b or even a synthetic CARD-Aim2 chimera) can promote Asc-independent cell death. We also show that the Asc-independent cell death that occurs in macrophages infected with S. typhimurium, L. pneumophila and P. aeruginosa depends on the catalytic activity of Caspase-1 and does not require Caspase-11. To investigate the requirement for Caspase-1 autoproteolysis in activation of cell death, we reconstituted Caspase-1 deficient macrophages with Caspase-1 constructs unable to undergo autoproteolytic processing due to mutations in key aspartate residues. Surprisingly, signaling through both Nlrc4 and Aim2 led to pyroptotic cell death in the absence of Caspase-1 autoproteolysis, while efficient cytokine processing required autoproteolysis. In addition, we localized Caspase-1 during Asc-independent cell death. In contrast to cytokine processing that correlates with the formation of a single, large Asc/Caspase-1 focus, we observed a punctate staining for Caspase-1 in Asc-/- macrophages.

Taken together, our results suggest that two spatially and functionally distinct inflammasome-complexes can be formed in pathogen-infected cells: a large Asc/Caspase-1 focus containing active, fully cleaved Caspase-1 that is critical for cytokine processing and release, and Asc-independent inflammasomes containing unprocessed but active Caspase-1 that is able to initiate rapid cell death.

Results

Nlrc4-dependent macrophage death occurs in the absence of detectable Caspase-1 processing in Asc-deficient cells

The processing of pro-Caspase-1 into its p20 and p10 subunits is a hallmark of Caspase-1 activation. Detection of Caspase-1 processing is routinely carried out on culture supernatants, since active Caspase-1 is released via a secretion pathway that has yet to be characterized (Keller et al., 2008). Consistent with this, we confirmed previous results that found that wild-type (WT) macrophages rapidly released large amounts of processed p10 and p20 into the culture supernatant when infected with bacterial pathogens activating the Nlrc4 inflammasome, such as S. typhimurium, P. aeruginosa and L. pneumophila ((Case et al., 2009; Mariathasan et al., 2004; Suzuki et al., 2007); Fig. 1 A-C, first lane). In WT macrophages infected with these pathogens, Caspase-1 activation was accompanied by efficient secretion of mature IL-1β and pyroptotic cell death. In contrast, infections of Caspase-1-/- and Nlrc4-/- macrophages did not lead to IL-1β release or host cell death, indicating that cytokine maturation and release, and pyroptotic cell death were dependent on Caspase-1 and to a large part on Nlrc4 (Fig. 1 A-C, second and forth lane). Nlrc4-/- macrophages infected with P. aeruginosa or L. pneumophila released a small amount of IL-1β, albeit significantly less than WT macrophages, indicating that these pathogens activate other receptors in addition to Nlrc4 ((Case et al., 2009; Mariathasan et al., 2004; Suzuki et al., 2007); Fig. 1 B and C, fourth lane). To determine whether the adaptor protein Asc is important for cytokine maturation and pyroptotic cell death, we infected macrophages from Asc-deficient mice. Consistent with previous results, we found that Asc-/- macrophages infected with S. typhimurium, P. aeruginosa or L. pneumophila were severely impaired in their ability to process and secrete mature IL-1β, as shown by ELISA and western blotting. However, Asc-/- macrophages infected with these intracellular bacterial pathogens released LDH to the same extent as WT macrophages. As expected from previous reports (Case et al., 2009; Mariathasan et al., 2004; Suzuki et al., 2007), Asc-/- macrophages did not detectably process Caspase-1 (Fig. 1 A-C, third lane). Consistent with a defect in cytokine maturation, un-processed pro-IL-1β was detected in the supernatant of Asc-/- macrophages, which was probably released during pyroptotic lysis of the cells (Fig. 1 A-C, arrowheads).

Figure 1. Nlrc4-dependent macrophage death occurs in the absence of Caspase-1 cleavage in Asc-deficient cells.

Figure 1

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Bone marrow-derived macrophages of the genotypes indicated were infected with (A) S. typhimurium for 2h at an MOI of 20, (B) P. aeruginosa PAO1 for 2h at an MOI of 10, (C) L. pneumophila for 4h at an MOI of 1 and (D) F. novicida for 6h at an MOI of 100. Cell death was determined by measuring LDH release. Secretion of mature IL-1β into the culture supernatant was determined by ELISA and western blotting. Release of processed Caspase-1 p10 and p20 subunits into the culture supernatant was determined by western blotting. Corresponding cell lysates were probed for pro-IL-1β, pro-Caspase-1 and β-actin. Graphs show the mean ± standard deviation (SD) of triplicate wells and are representative of at least three independent experiments. Arrowhead indicates pro-IL-1β in the supernatant.

To determine whether activation of the Aim2 inflammasome induces cell death in the absence of Asc and Caspase-1 cleavage, macrophages from Asc-deficient mice were infected with F. novicida, which stimulates the cytosolic DNA-sensor Aim2. In contrast to S. typhimurium, P. aeruginosa and L. pneumophila, Asc-/- macrophages infected with F. novicida did not process and release IL-1β and were defective for pyroptotic cell death, as previously shown ((Case et al., 2009; Mariathasan et al., 2004; Suzuki et al., 2007); Fig. 1 D). These data confirm, in our experimental system, that Aim2-dependent cell death and Caspase-1 processing requires the adaptor molecule Asc, whereas Nlrc4-dependent cell death does not require Asc and does not appear to require Caspase-1 autoproteolysis.

A CARD domain is required for Asc-independent cell death

In contrast to Nlrc4, Aim2 contains only a PYRIN domain and no CARD domain, possibly rendering Aim2 incapable of recruiting Caspase-1 in the absence of Asc. We hypothesized that the presence of a CARD in NLR/PYHIN proteins would be sufficient to recruit and activate Caspase-1 and to promote cell death independently of Asc. To verify this hypothesis we first decided to test the only other known CARD-containing sensor, Nlrp1b, which is activated by Bacillus anthracis lethal toxin (LT)(Boyden and Dietrich, 2006). In mice, Nlrp1b is extremely polymorphic: macrophages derived from 129S1 mice express a functional allele and respond to LT by activating Caspase-1, while macrophages from C57BL/6 mice that have a non-functional allele do not respond to LT. Thus, we transduced immortalized C57BL/6 background WT and Asc-/-macrophages with the functional CARD-containing sensor Nlrp1b from 129S1 (Fig. 2A, S1A). WT macrophages expressing Nlrp1b gained the ability to process IL-1β and to undergo cell death in response to LT. In contrast, Asc-/- macrophages expressing Nlrp1b could only undergo pyroptosis, and did not efficiently process cytokines. Consistent with the Nlrc4 data (Fig. 1), transduced Asc-/- macrophages were not able to process pro-Caspase-1 to its p20 and p10 subunits. All cell lines generated in this study, expressing functional inflammasome sensors or caspase-1 (Table S1), did not exhibit any spontaneous activation in the absence of inflammasome stimuli (Fig. S1C, D).

Figure 2. CARD-containing sensors promote Asc-independent cell death.

Figure 2

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(A) Immortalized WT and Asc-/- macrophages derived from C57BL/6 mice expressing an empty vector control or the functional Nlrp1b allele from 129S1 mice were stimulated for 4h with Anthrax lethal toxin. (B, C) Immortalized WT, Aim2-/- or Asc-/- macrophages expressing an empty vector control, Aim2 WT or a CARD-Aim2 chimera were transfected with poly(dA:dT) (B) or infected with F. novicida WT or a mutant that does not activate the inflammasome (ΔFPI) (C). Cell death was determined by measuring LDH release. Secretion of IL-1β into the culture supernatant was determined by ELISA and western blotting. Release of processed Caspase-1 p20 into the culture supernatant was determined by western blotting. Corresponding cell lysates were probed for pro-IL-1β and β-actin. Graphs show the mean ± SD of triplicate wells and are representative of two independent experiments.

To further test our hypothesis that the CARD domain can activate Caspase-1-dependent death in the absence of Asc, we constructed a chimeric protein in which the Nlrc4 CARD was fused to full-length Aim2 protein. Immortalized macrophages derived from Aim2- or Asc-deficient mice were transduced with WT Aim2 or the chimeric fusion construct and monitored for host cell death and cytokine processing in response to transfections of a synthetic Aim2 ligand, poly(dA-dT)•poly(dA-dT) [hereafter referred to as poly(dA:dT)] (Fig. 2B), and to F. novicida infection (Fig. 2C). Aim2-/- macrophages transduced with WT Aim2 or the CARD-Aim2 chimera constructs gained the ability to process and release IL-1β and to undergo cell death in response to transfection with poly(dA:dT) and infection with F. novicida (Fig. 2B, C). As expected, Asc-/- macrophages transduced with the WT Aim2 construct could not cleave IL-1β or induce cell death in response to both stimuli (Fig. 2B, C). The CARD-Aim2 fusion protein, however, restored the ability of Asc-/- macrophages to undergo cell death in response to F. novicida or poly(dA:dT) transfection, while only weakly restoring the ability of Asc-/- macrophages to process and release cytokine (Fig. 2B, C). Similar to the results obtained with S. typhimurium, P. aeruginosa, and L. pneumophila infections (Fig. 1), the Asc-independent cell death promoted by the CARD-Aim2 construct occurred in the absence of detectable Caspase-1 processing (Fig. 2B, C). Interestingly, expression of the CARD-Aim2 construct lead to the production of two proteins, one with the predicted size of the CARD-Aim2 fusion and a second one, with the same size as native Aim2 (Fig. S1D). The expression of the native-sized Aim2 is likely due to the presence of an alternative translation start codon at position 286, the native Aim2 start codon. Nevertheless, only the expression of the CARD-Aim2 fusion promoted the ability to cause ASC-independent cell death. Furthermore, expression of a CARD alone did not restore cell death or cytokine processing (data not shown). Taken together, these results demonstrate that in the context of an NLR/PYHIN protein, a CARD is sufficient to promote cell death in the absence of Caspase-1 processing and independently of Asc. We hypothesize that this cell death occurs following formation of complexes between CARD-containing NLR/PYHIN proteins and pro-Caspase-1.

Catalytic activity of Caspase-1 is required for Asc-independent cell death

To further investigate why processing of Caspase-1 is not required during Asc-independent cell death, we first considered the possibility that another caspase could be involved. Indeed previous reports proposed that Caspase-11 is critical for Caspase-1 activation (Wang et al., 1998) and demonstrated that Caspase-11 is absent in Caspase-1-/- mice (Kang et al., 2000). Therefore the cell death observed in Asc-/- macrophages might be a result of Caspase-11 activity, independently of Caspase-1 and Asc. To address this, we compared WT, Caspase-1-/- and Caspase-11-/- macrophages infected with S. typhimurium (Fig. S2A). WT and Caspase-11-/- macrophages were killed to the same extent in response to infections with S. typhimurium. In contrast, Caspase-1-/- macrophages were not killed. Because Caspase-1 and Caspase-11 could act redundantly, we further validated our conclusions by stably transducing immortalized Caspase-1-/- macrophages (that lack both Caspase-1 and Caspase-11) with retroviral constructs expressing Caspase-1 or Caspase-11 (Fig. S2B). Caspase-1, but not Caspase-11, was able to complement the Caspase-1-/- macrophages.

Next we hypothesized that Caspase-1 might act as an adaptor to recruit other death effectors in the absence of its own cleavage or proteolytic activity. To test this model we transduced BMDMs from Caspase-1-/- mice with retroviral constructs that contained either WT Caspase-1, or a catalytically inactive mutant Caspase-1 (Caspase-1 DEAD), in which we mutated the active Cysteine (C287A) to Alanine (Fig. 3A). Both WT Caspase-1 and Caspase-1 DEAD were expressed to comparable levels, but only WT Caspase-1 restored the ability of Caspase-1-/- macrophages to secrete IL-1β and undergo cell death in response to S. typhimurium, and L. pneumophila infections.

Figure 3. Catalytic activity of Caspase-1 is required for Asc-independent cell death.

Figure 3

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(A) Caspase-1-/- BMDMs expressing an empty vector control, WT Caspase-1 or the catalytically inactive mutation C284A (Caspase-1 DEAD) were infected with S. typhimurium for 2h at an MOI of 20 or L. pneumophila for 4h at an MOI of 1. Cell death was determined by measuring LDH release. Secretion of IL-1β into the culture supernatant was determined by ELISA and western blotting. Secretion of processed Caspase-1 p20 into the culture supernatant was determined by western blotting. Corresponding cell lysates were probed for pro-IL-1β, pro-Caspase-1 and β-actin. (B) BMDMs of the genotypes indicated were infected with S. typhimurium for 1h at an MOI of 20 or L. pneumophila for 2.5h at an MOI of 1 in the presence of Z-YVAD-FMK or DMSO (vehicle control). Graphs show the mean ± SD of triplicate wells and are representative of at least two independent experiments.

We also sought to determine if the catalytic activity of Caspase-1 is necessary for Asc-independent cell death. Caspase inhibitors have been used previously to block the catalytically active enzyme by irreversibly binding to its active site. Thus, we infected WT and Asc-/- macrophages with S. typhimurium, and L. pneumophila in the presence of a Caspase-1 inhibitor (Z-YVAD-FMK) and monitored cell death and cytokine release (Fig. 3B). Inhibitor treatment completely abolished host cell death in Asc-/- macrophages in response to both pathogens, suggesting that Asc-independent cell death required Caspase-1 activity. Interestingly, inhibitor treatment only modestly reduced host cell death in WT macrophages (Fig. 3B). This was likely due to incomplete inhibition by Z-YVAD-FMK, as our results with the Caspase-1 DEAD mutant previously confirmed a complete dependence on Caspase-1 proteolytic activity in WT cells. Taken together, our results show that Caspase-1 does not act as an adaptor, but rather that its catalytic activity is required for cell death in WT and Asc-/- macrophages in response to stimuli that activate Nlrc4.

Differential subcellular localization of Caspase-1

We showed previously that Caspase-1 is recruited to the Asc focus during S. typhimurium infection of WT macrophages (Broz et al., 2010). In addition, we reported that staining with the fluorescent activity based probe FLICA (FAM-YVAD-FMK), detects active Caspase-1 at the site of the Asc focus. Since Caspase-1 activity is absolutely required for Asc-independent cell death in macrophages, we investigated whether FLICA could detect active Caspase-1 within cells in the absence of Asc. As expected, WT macrophages infected with S. typhimurium, formed an extremely bright focus of FLICA staining that co-localizes with the Asc focus; Asc-/- or Caspase-1-/- macrophages did not contain brightly stained foci (Fig. 4A, B asterisk; (Broz et al., 2010)). However, we also detected a less intense speckled FLICA staining in the cytoplasm of infected cells. Intriguingly, this staining was observed in both WT and Asc-/- but not in Caspase-1-/- and Nlrc4-/- macrophages, indicating that it required inflammasome activation (Fig. 4C). Since the speckled staining correlated with cell death (Fig. 1, 4C), we speculated that these speckles could represent Nlrc4-pro-Caspase-1 complexes that cause the Asc-independent cell death. To test the specificity of the FLICA-staining, we co-stained FLICA-labeled macrophages with an antibody that specifically recognizes the p20 subunit of murine Caspase-1, which contains the active site that is labeled by FLICA (Fig. 4A, S3). To our surprise the Caspase-1 p20 staining and the speckled FLICA-pattern were non-overlapping, suggesting that the speckled FLICA pattern, although dependent on Caspase-1 activation, resulted from a non-specific FLICA labeling. Cells that could be stained with FLICA had lost almost all Caspase-1 p20 staining, except at the site of the Asc focus (Fig. 4A arrow). Indeed these cells were pyroptotic, since they clearly showed nuclear condensation, actin degradation and a loss of membrane integrity (Fig. 4A, B, D, unpublished data). We concluded that once pyroptosis is initiated, the cells rapidly release most of the active Caspase-1, except what is retained by the Asc focus. Pyroptotic cells may activate other proteases, which may explain the differential FLICA and Caspase-1 p20 staining pattern.

Figure 4. Differential subcellular localization of Caspase-1.

Figure 4

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(A) BMDMs of the genotypes indicated were infected with S. typhimurium for 2h at an MOI of 20, fixed and stained for DNA (Dapi), Caspase-1 p20 and with FLICA. Marked is an Asc focus stained by the Caspase-1 p20 antibody (arrow) or FLICA (asterisk). (B) Close up views of infected WT and Asc-/- macrophages stained for DNA (Dapi, blue) and with FLICA (green). (C) Percentage of cells with speckled, cytoplasmic FLICA staining. (D) WT and Asc-/- macrophages (treated with Z-YVAD-FMK or the vehicle control DMSO) or Caspase-1-/- macrophages expressing the catalytically inactive mutation C284A (Caspase-1 DEAD) were infected with S. typhimurium for 1h at an MOI of 20. Cells were fixed and stained for DNA (Dapi), Caspase-1 p20 and actin (Phalloidin). Indicated are Asc/Caspase-1 foci in the presence (arrowheads) or absence of the inhibitor (arrows). (E) Close up views of infected and uninfected WT and Asc-/- macrophages or Caspase-1-/- + Caspase-1 DEAD macrophages stained for DNA (Dapi, blue) and Caspase-1 p20 (yellow). Indicated are Asc/Caspase-1 foci (arrowheads). Cell counts in (C) were determined by counting twice 300 infected cells per sample. Images and cell counts are representative of at least two experiments. Scale bars in all images are 10 μm.

To prevent the rapid secretion of activated Caspase-1 and to determine whether Caspase-1 was recruited to structures other than the Asc focus, we repeated the experiments in the presence of Z-YVAD-FMK and in Caspase-1-/- macrophages expressing the Caspase-1 DEAD mutant (Fig. 4D, E). Under these conditions, WT macrophages and macrophages expressing the Caspase-1 DEAD mutant contained both the Asc/Caspase-1 focus as well as a punctate Caspase-1 p20 staining (Fig. 4D, E arrowheads). Asc-/- macrophages showed only the punctate staining, which was comparable to the Caspase-1 staining observed in both uninfected WT or Asc-/- macrophages (Fig. 4E). Altogether these data suggest that in the absence of Asc, Caspase-1 is not recruited into any large structure, comparable to the Asc focus, and thus might form smaller Nlrc4-pro-Caspase-1 complexes.

Caspase-1 autoproteolysis is necessary for efficient cytokine processing, but not for macrophage cell death

The above data suggest that rapid pathogen-induced cell death is mediated by Nlrc4-pro-Caspase-1 complexes, which form through homotypic CARD domain interactions (Fig. 2). Interestingly, Caspase-1 is not detectably processed in these complexes (Fig. 1 and 2). However, our data imply that Caspase-1 must nevertheless be activated in these complexes, because a catalytically inactive mutant Caspase-1 does not mediate cell death in a Caspase-1-deficient background (Fig. 3). To investigate the importance of autoproteolytic processing of Caspase-1 into its p20 and p10 subunits, we decided to mutate putative cleavage sites in the linker domain between the p20 and p10 domains (Fig. 5A, S4A). Analysis of purified human pro-Caspase-1 suggests that the full-length zymogen (p45) is autoproteolytically processed at 4 Asp-Xaa bonds (Thornberry et al., 1992). The first cleavage events at D297 and D316, remove the linker region between the p20 and p10 subunits, thus generating p10 and p35 subunits. The p35, which consists of the CARD and the p20, is then further cleaved at D122 and D103, releasing the mature p20 subunit from the CARD. The proteolytic separation of the CARD and p20 subunit, however, is not necessary for Caspase-1 activity, since mutating both D103 and D122 does not affect cell death and cytokine processing (unpublished data). Thus, for the sake of clarity, we refer to the proteolytic cleavage resulting in the separation of p20 and p10 subunits as Caspase-1 processing or autoproteolysis throughout the text. Interestingly, cleavage intermediates or alternative cleavage products like p24 and p14 subunits have been observed (Ramage et al., 1995; Thornberry et al., 1992).

Figure 5. Caspase-1 autoproteolysis is necessary for efficient cytokine processing, but not for macrophage cell death in response to S. typhimurium infections.

Figure 5

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(A) Schematic representation of the domain organization of murine pro-Caspase-1, showing an alignment of the interdomain linker of human and murine Caspase-1. Putative cleavage sites and the active cysteine are indicated. (B) Schematic representation of different mutated Caspase-1 constructs, showing mutations and expected cleavage products. (C-D) Immortalized or bone-marrow derived Caspase-1-/- macrophages expressing the indicated Caspase-1 constructs were infected with S. typhimurium for 2h at an MOI of 20. Cell death was determined by measuring LDH release. Secretion of IL-1β into the culture supernatant was determined by ELISA and western blotting. Release of Caspase-1 into the culture supernatant was determined by western blotting. Corresponding cell lysates were probed for pro-Caspase-1, pro-IL-1β and β-actin. Graphs show the mean ± SD of triplicate wells and are representative of at least two independent experiments.

We first generated retroviral constructs, expressing murine pro-Caspase-1 mutated at cleavage sites defined previously in human Caspase-1. The constructs were tested by transducing immortalized Caspase-1-/- macrophage cell lines as well as bone-marrow derived Caspase-1-/- macrophages and infecting them with either S. typhimurium or L. pneumophila (Fig. 5, S4). In immortalized macrophages, the expression levels of the mutant Caspase-1 constructs were comparable, but lower than WT Caspase-1 (Fig. 5C). WT Caspase-1, as well as the mutant constructs, were expressed to similar levels when transduced into Caspase-1-/- bone-marrow derived macrophages (Fig. 5D, S4). However, the mutant constructs migrated at a slightly lower size than expected, likely due to the loss of charged residues. We first tested retroviral construct 47 (C47), expressing pro-Caspase-1 in which the aspartates D296, D313 and D314 were mutated to asparagines (Fig. 5B). To our surprise, the C47 mutant protein was still processed and restored the ability of Caspase-1-/- macrophages to cleave IL-1β and induce cell death in response to S. typhimurium or L. pneumophila infections (Fig. 5C, S4B, C). Although the C47 mutant construct was processed, the p20 and p33 (CARD-p20) subunits were slightly larger (Fig. 5C, S4B, C, asterisk), suggesting that the construct is processed at an alternative cleavage site. Alignment of pro-Caspase-1 from human and mouse revealed that murine Caspase-1 contained 3 additional aspartates between D296 and D314 (D297 and D316 in humans), of which at least two (D304, D308) could also serve as Caspase-1 cleavage sites based on the SVM algorithm (www.casbase.org; Fig. 5A, S4A). To define the cleavage sites between the p20 and p10 subunits, we constructed additional putative cleavage site mutations in the C47 mutant: C51, C52, C53 containing the D300N, D304N and D308N mutations, respectively (Table S1, Fig. 5B). We then infected macrophages transduced with the different constructs with S. typhimurium or L. pneumophila, and analyzed Caspase-1 processing by western blotting for the p20 subunit in culture supernatants. Mutating D308 in addition to D296, D313 and D314 (construct C53) completely abolished the generation of p20 and p10 subunits (Fig. 5C, D and S4B, C), while mutations in D300 and D304 had no effect (unpublished data). Instead, supernatants from macrophages transduced with the C53 construct contained two bands, labeled p32 and p34 that were detected by both the p20 and the p10 antibodies (arrowheads, unpublished data). We speculated that these cleavage products are the result of cleavage at residues D103 or D122 that leads to the removal of the CARD from the rest of the protein. This would be consistent with the observation that alternative cleavage at D103 and D119 can produce two different large subunits of human Caspase-1 (p20, p24) (Ramage et al., 1995; Thornberry et al., 1992). The apparent size of these bands is slightly smaller than expected, presumably due to the change in charge of the mutated constructs. To prove that the p32/p34 subunits are a result of processing at residues D103 and D122, we mutated these residues in the C53 background, generating mutant construct C71 (Fig. 5B). Indeed, mutating D103, D122 in addition to D296, D308, D313 and D314 generated a completely uncleavable Caspase-1, since both the p34 and p32 bands disappeared and only full-length pro-Caspase-1 was released into the supernatant (Fig. 5D).

To analyze the importance of Caspase-1 processing for cytokine maturation and release, and host cell death, we infected the transduced macrophages with S. typhimurium or L. pneumophila. Caspase-1-/- macrophages transduced with WT Caspase-1 or C47 processed and released IL-1β and mediated cell death (Fig. 5C, S4). In contrast, the unprocessed Caspase-1 construct C53 mediated very low levels of IL-1β secretion into the supernatant when infected with S. typhimurium or L. pneumophila (Fig. 5C, D and S4). Surprisingly, transduction of Caspase-1-/- macrophages with the C53 construct partially rescued the cell death deficiency, causing >50% of WT Caspase-1 cell death in response to both pathogens. Importantly, cell death of macrophages expressing the C53 construct was dependent on Caspase-1 catalytic activity, since a mutation of the active cysteine C284 in the C53 background completely abolished activity (Fig. S4D).

It is, however, possible that low levels of cleavage at residues D300 and D304 (undetectable by western blotting) might be enough to mediate cell death in C53-transduced macrophages. Thus, we examined macrophages transduced with constructs C59 and C60 that contain additional D300N and D300N, D304N mutations, respectively (Fig. 5C, S4). Both C59 and C60 constructs had similar processing patterns to each other and were indistinguishable from C53, with p32/p34 subunits. Importantly, the unprocessed C59 and C60 constructs did not restore the ability of Caspase-1-deficient macrophages to process and release IL-1β, but they did mediate host cell death in response to S. typhimurium and L. pneumophila infections (Fig. S4B, C). Importantly, cell death in these mutants and C53 did not depend on the formation of the p32/p34 subunits, since the completely uncleavable mutant C71 (Fig. 5D, fourth lane) promoted cell death and only inefficiently processed IL-1β. This result confirmed that even if Caspase-1 cannot be processed, due to mutations of cleavage sites (Fig. 5B) or due to the absence Asc (Fig. 1), it can nevertheless efficiently promote pyroptotic cell death in response to bacterial infections. Overall, these results demonstrate that while Caspase-1 processing is not required for Nlrc4-dependent macrophage death in response to infection with S. typhimurium or L. pneumophila ((Amer et al., 2006; Mariathasan et al., 2004); Fig. 1), it is important for efficient cytokine processing.

Caspase-1 processing is not required for macrophage death in response to Aim2 stimuli

Our data suggest that Nlrc4 can induce, through homotypic CARD domain interactions, Caspase-1-dependent cell death without a requirement for complete autoproteolytic processing of Caspase-1. We wondered, however, whether other receptors, such as Aim2, that require Asc for their interaction with Caspase-1, are capable of activating Caspase-1 autoproteolytic mutants. To test this, we infected immortalized Caspase-1-/- macrophages expressing the Caspase-1 mutant constructs described above (Fig. 5B) with F. novicida, which has been previously shown to activate Aim2 ((Fernandes-Alnemri et al., 2010; Jones et al., 2010; Rathinam et al., 2010), Fig 1D). Surprisingly, macrophages expressing Caspase-1 autoproteolytic mutants did not efficiently release IL-1β when infected with F. novicida, while they were killed to similar levels compared to WT macrophages (Fig. 6). Similarly, transfections of macrophages expressing Caspase-1 autoproteolytic mutant constructs with poly(dA:dT) or poly(dG:dC) did not result in cytokine processing but mediated levels of host cell death that were similar to those seen in WT macrophages (Fig. S5). Together our results demonstrate that Caspase-1 processing is not required for cell death in response to a variety of inflammasome stimuli that activate different inflammasome receptors.

Figure 6. Caspase-1 processing is not required for macrophage death in response to F. novicida infections.

Figure 6

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Immortalized Caspase-1-/- macrophages expressing the indicated Caspase-1 constructs were infected with F. novicida for 9h at an MOI of 20. (A) Western blots for processed Caspase-1 p20 in culture supernatants. (B) ELISA for mature, secreted IL-1β in culture supernatants. (C) Cell death as determined by measuring LDH release. Arrows, arrowheads and asterisk indicate pro-Caspase-1 and different Caspase-1 auto-processing products. Results are representative of at least three independent experiments, error bars represent mean ± SD of triplicate wells.

Discussion

Mammalian genomes encode multiple NLR/PYHIN proteins, each with the potential to assemble inflammasomes and activate Caspase-1 independently in response to diverse signals. However, little is known about the molecular mechanisms of how inflammasomes assemble and activate Caspase-1. A pivotal Caspase-1 activation step is thought to be the autoproteolytic separation of its p20 and p10 subunits. In addition to NLR/PYHIN proteins and Caspase-1, inflammasomes also contain Asc, an adaptor whose major function is believed to be to recruit Caspase-1 to PYRIN domain-containing proteins. Nlrc4, however, lacks a PYRIN domain, but has a CARD that has been shown to interact directly with the CARD of Caspase-1 in the absence of Asc (Poyet et al., 2001). Since Asc is nevertheless required for some aspects of Nlrc4-mediated Caspase-1 activation, the role of Asc remained unclear (Case et al., 2009; Mariathasan et al., 2004; Suzuki et al., 2007).

In this study, we have clarified the molecular mechanisms that lead to Caspase-1 activation and defined the role of Asc in Caspase-1 autoproteolysis, pro-inflammatory cytokine processing and host cell death. By using mutant Caspase-1 alleles defective for autoproteolytic processing, we were able to genetically and functionally distinguish two inflammasome complexes (Fig. 7): 1) “death-inflammasomes”, that do not require autoproteolysis of Caspase-1, and which mediate macrophage death but only inefficient cytokine release; and 2) one large Asc focus, which is formed when Asc aggregates with NLR/PYHIN proteins and Caspase-1, and which mediates very efficient processing and release of pro-inflammatory cytokines. Our data suggest that Asc-/- macrophages only assemble the “death-inflammasomes” in response to Nlrc4 or Nlrp1b stimuli. However, we can only speculate about the situation in WT macrophages. Both types of inflammasome complexes could be formed and remain spatially and functionally distinct. Alternatively, the “death-inflammasomes” might just be intermediates and serve as precursors or nuclei for the formation of the Asc focus. Future work investigating inflammasome assembly in single cells, using fluorescently-tagged components and time-lapse imaging, might provide more insight into the molecular dynamics and kinetics of inflammasome-complex formation.

Figure 7. Formation of spatially and functionally specialized inflammasome-complexes by CARD-containing receptors.

Figure 7

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1) Activation of CARD-containing sensors (Nlrc4, Nlrp1b or CARD-Aim2) by their respective stimuli induces dimerization of the sensor and recruitment of pro-Caspase-1 through CARD-CARD interactions. Proximity-driven dimerization in this “Death-complex” does not lead to autoproteolysis, however it activates pro-Caspase-1 sufficiently to promote cell death, but only inefficient cytokine processing. 2) Asc foci are formed when Asc is recruited in addition to pro-Caspase-1 to the activated sensor. Proximity driven dimerization of Asc promotes the recruitment of further Asc molecules by CARD-CARD and PYRIN-PYRIN interactions, leading to rapid oligomerization of Asc, thus forming the focus. In the Asc focus, conformational changes of the pro-Caspase-1 dimer allow for autoproteolytic processing into the p10 and p20 subunits. Only fully processed Caspase-1 is able to efficiently promote cytokine maturation. 3) Alternatively, the “Death-complexes” could serve as precursors or nuclei for Asc oligomerization and be absorbed by the Asc focus once it forms.

Our results clarify the precise and essential molecular functions of Asc. Not only does Asc appear to function as a vital adaptor for the recruitment of caspase-1 to PYRIN-containing receptors, such as Aim2, but our data indicate that Asc also seems to be crucial for induction of caspase-1 processing. This processing, rather than merely Asc itself, in turn appears to be essential for efficient cytokine maturation. Indeed, although Asc is present in macrophages expressing completely uncleavable Caspase-1 (Fig. 5D, construct C71), and Asc foci are still formed in response to infection, cytokines are not efficiently processed. However, these cells release full-length pro-Caspase-1 and undergo cell death. This demonstrates that the pro-Caspase-1 zymogen can promote pyroptosis when activated, but requires Asc to be processed to its p20 and p10 subunits to promote cytokine maturation.

Caspase-1 processing has widely been used as the hallmark of Caspase-1 activation. However, our data imply that Caspase-1 can exist in two activation states, unprocessed and fully processed, depending on the composition of the inflammasome. How does this compare to other initiator caspases, such as Caspase-2, -8, -9, D. melanogaster Dronc and C. elegans CED-3? Similar to Caspase-1, these other initiator caspases have been shown to require recruitment to multiprotein activation platforms, like the PIDDosome for Caspase-2, the DISC-complex for Caspase-8, the apoptosome for Caspase-9, the DARK-apoptosome for Dronc, and the CED-4 tetramer for CED-3 (Bao and Shi, 2007). All of these initiator caspases are expressed as inactive monomers, in contrast to effector caspases that are expressed as inactive dimers. According to the proximity-driven dimerization model, the activation step is the dimerization that is induced in the activation platform (Boatright et al., 2003). The importance of the autoproteolytic processing that occurs simultaneously with the dimerization is less clear. Experimental evidence using non-cleavable Caspase-9 suggests that Caspase-9 does not require processing for activity (Stennicke et al., 1999). However, processing seems to be important to stabilize the dimers formed by Caspase-2, -8 and Dronc, as auto-cleaved forms of these caspases preferentially form dimers and only these dimers have sufficient catalytical activity to induce cell death (for a review see (Bao and Shi, 2007)). Consistent with previous studies of apoptotic caspases, we suggest that Caspase-1 activation also proceeds by proximity-induced dimerization. However, our data suggest that Caspase-1 activation and autoproteolysis are distinct events and depend on the exact composition of the multiprotein activation platform. The recruitment of Asc to the inflammasome probably changes the conformation of the pro-Caspase-1 dimer in a way that allows for autoproteolysis, thus stabilizing the dimer and enhancing activity or even changing substrate specificity. Human Caspase-1 has been crystallized in its processed forms as well as a zymogen (Elliott et al., 2009), but the structure of Caspase-1 in complex with different receptors and/or Asc remains an exciting task for crystallographers to pursue.

Another distinguishing feature of inflammasome complexes compared to apoptosomes, is that the different inflammasome complexes seem to have distinct functions, i.e. cell death and cytokine processing, which are also spatially distinct. We propose that the “death-inflammasomes” could be a functional analog of the apoptosome complexes, since they are most probably small, do not localize in a large focus, and specialize in performing only one task, i.e. induction of cell death. The Asc focus, however, does not seem to have an equivalent in apoptotic signaling pathways. One could speculate that induction of host cell death might have been the primordial function of the inflammasome, providing the host with the vital features of eliminating the intracellular niche of the infected cell, and exposing intracellular pathogens to more robust extracellular immune defenses. Cytokine processing in the Asc focus could have developed as a secondary feature, enabling the infected cell to attract other immune cells like neutrophils to the site of the infection, where they would encounter the newly exposed intracellular pathogens. Most leukocytes have been shown to express both Asc and CARD-containing receptors, but variations in the expression levels could also allow some cell types to respond by either producing cytokines or inducing pyroptotic cell death. High expression levels of Asc might sequester Caspase-1, possibly preventing premature cell death and the resulting spread of pathogen, and allowing prolonged pro-inflammatory cytokine production. Intriguingly, several host- as well as pathogen-derived Pyrin-only proteins (cPOPs and vPOPs) have been shown to inhibit or regulate the inflammasome by sequestering Asc, which could also result in Caspase-1-dependent cell death in the absence of cytokine processing (for a review see (Taxman et al., 2010)). Thus, regulating the availability of Asc could be a mechanism to tip the balance between Caspase-1 mediated cell death and cytokine processing.

Experimental Procedures

Generation of immortalized macrophage cell lines

C57BL/6 WT, Aim2-/-, Asc-/- and Caspase -1-/- bone marrow was infected with the v-myc/v-raf expressing J2 retrovirus (Blasi et al., 1985), and differentiated in 10% L929-MCSF supernatant. After 30d in culture, L929-MCSF supernatant was removed from the media and the surviving immortalized macrophage lines were cultured in RPMI with 10% FCS.

Retroviral transductions

Genes encoding Caspase-1, Aim2, Nlrc4, Flag-Nlrp1b or CARD-Aim2 were cloned into a replication-defective mouse stem cell retroviral construct (pMSCV2.2). Site directed mutagenesis was performed using QuickChange (Stratagene). For transduction of primary bone marrow cells, retroviral particles were generated with Phoenix-Eco packaging cells and used to transduce bone marrow cells after 48h and 72h of culture in medium with 10% L929-MCSF supernatant. Cells were typically infected 4d after the first transduction. Immortalized macrophages were transduced with vesicular stomatitis virus pseudotyped virus packaged in GP2 cells, and sorted by FACS to enrich to >80% GFP+ cells.

Cell culture and infections

Bone marrow was isolated from femurs of 6-8 week old C57BL/6 mice, differentiated and cultured as described previously (Broz et al., 2010). For Caspase-1 inhibitor treatments, the macrophages were cultured in 100 μM Z-YVAD-FMK in DMSO or DMSO 1h before the infection and throughout the infection. For infections with S. typhimurium, P. aeruginosa and F. novicida macrophages were prestimulated with 0.1 μM LPS for 16h, for L. pneumophila infections with 0.5 μg/ml PAM3CSK4 for 4h. S. typhimurium and P. aeruginosa were grown overnight in LB at 37°C, sub-cultured for 3-4h. F. novicida were grown overnight in Tryptic Soy Broth (TSB). L. pneumophila (Lp02 or Lp02ΔflaA) were grown overnight in BYE to post-log phase (OD > 3.8).

Highlights.

• CARD-domain containing, pathogen sensing receptors promote Asc-independent cell death

• Macrophage cell death does not require Caspase-1 autoproteolytic processing

• Asc is required for Caspase-1 autoproteolysis

• Caspase-1 autoproteolysis mutants promote rapid cell death, but process cytokines inefficiently

Supplementary Material

01

Acknowledgments

We thank members of the Monack and Vance Labs for help and discussions, and Eric Boyden for discussions and the mouse Nlrp1b cDNA. We thank Drs. Kim Newton and Vishva Dixit for Caspase-11-/- mice. We thank Dr. Kate Fitzgerald for the J2 virus used to immortalize macrophages. This work was supported by NIH NIAID P01 AI063302 (DMM) and R01 AI075039 (REV). DMM and REV are recipients of Investigators in the Pathogenesis of Infectious Disease Awards from the Burroughs Wellcome Fund. REV is also supported by an Investigator award from the Cancer Research Institute. PB holds a Stanford ITI Young Investigator Award and was supported by postdoctoral fellowships from the Swiss National Science Foundation (SNSF) and the Human Frontiers in Science Program (HFSP). The authors acknowledge no competing financial interests. Authors contributions: PB, JvM, REV and DMM designed the experiments. PB, JvM and JWJ performed the experiments. All authors analyzed data and wrote the paper.

Footnotes

Full methods are available online in Supplementary Materials.

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