Emerging inflammasome effector mechanisms

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1 Emerging inflammasome effector mechanisms Mohamed Lamkanfi Abstract Caspase 1 activation by inflammasome complexes in response to pathogenassociated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) induces the maturation and secretion of the pro-inflammatory cytokines interleukin-1β (IL-1β) and IL-18. Recent reports have begun to identify additional inflammasome effector mechanisms that proceed independently of IL-1β and IL-18. These include the induction of pyroptotic cell death, the restriction of bacterial replication, the activation of lipid metabolic pathways for cell repair and the secretion of DAMPs and leaderless cytokines. These non-canonical functions of caspase 1 illustrate the diverse mechanisms by which inflammasomes might contribute to innate immunity, repair responses and host defence. Cryopyrinopathies A spectrum of hereditary autoinflammatory diseases that are caused by mutations in the gene encoding NLR family, pyrin domaincontaining 3 (NLRP3) that trigger continuous activation of the NLRP3 inflammasome. Based on the severity and spectrum of the symptoms which can include urticarial skin rashes, prolonged episodes of fever, sensorineural hearing loss, headaches, cognitive deficits and renal amyloidosis these diseases are classified as familial cold autoinflammatory syndrome, Muckle Wells syndrome or chronic infantile neurological cutaneous articular syndrome. Department of Biochemistry, Ghent University, and VIB Department of Medical Protein Research, Albert Baertsoenkaai 3, B 9000 Ghent, Belgium. e mail: Mohamed.Lamkanfi@ VIB UGent.be doi: /nri2936 Inflammasomes (BOX 1) are emerging as key regulators of the innate immune response, and the activity of these multiprotein complexes has been linked to inflammatory bowel diseases 1 5, vitiligo 6, gouty arthritis 7, type 1 and type 2 diabetes 8,9, and less common autoinflammatory disorders that are collectively referred to as cryopyrinopathies 10,11. Inflammasome complexes are thought to be assembled around members of the NOD-like receptor (NLR) or HIN 200 protein families 12 (FIG. 1). These receptors are thought to detect microbial pathogenassociated molecular patterns (PAMPs) and endogenous damage-associated molecular patterns (DAMPs) in intracellular compartments, similar to the role of mammalian Toll like receptors (TLRs) at the cell surface and within endosomes 13. Although it is incompletely understood how NLRs detect microbial ligands and DAMPs 14,15, it is evident that inflammasome assembly results in the activation of caspase 1 (BOX 2). This evolutionarily conserved cysteine protease is mainly known for its role in the maturation of the pro inflammatory cytokines interleukin 1β (IL 1β) and IL 18 (ReFs 16 19). IL 1β and IL 18 are related cytokines that are produced as cytosolic precursors and usually require caspase 1 mediated cleavage for full activation and secretion However, additional proteases, including caspase 8, myeloblastin (also known as proteinase 3) and granzyme A, have been shown to convert pro IL 1β into a biologically active cytokine in several established mouse models of human disease This indicates that caspase 1 is not always required for the maturation of IL 1β, and such redundancy in controlling IL 1β maturation might safeguard the host immune response against viral and bacterial pathogens that target caspase 1 activation in inflammasomes 24. Indeed, IL 1β and IL 18 were recognized early on for their ability to cause a wide variety of biological effects associated with infection, inflammation and autoimmunity 25. IL 1β regulates systemic and local responses to infection, injury and immunological challenge by generating fever, activating lymphocytes and promoting leukocyte transmigration into sites of injury or infection 25. Although IL 18 lacks the pyrogenic activity of IL 1β, it induces interferon γ (IFNγ) production by activated T cells and natural killer cells in the presence of IL 12, thereby contributing to T helper 1 (T H 1) cell polarization 25,26. In the absence of IL 12, IL 18 can promote T H 2 cell responses through the production of T H 2 cell cytokines such as IL 4, IL 5 and IL 10 (ReFs 26 28). More recently, IL 18 has also been implicated in driving T H 17 cell responses because it synergizes with IL 23 to induce IL 17 production by already committed T H 17 cells 29,30. Thus, IL 1β and IL 18 are important inflammasome effector molecules, as illustrated by the marked response to therapy with IL 1 inhibitors found in patients with cryopyrinopathies, who have increased inflammasome activation 31,32. However, not all inflammasome functions can be abrogated by neutralization of IL 1β and IL 18. For example, caspase 1 deficient mice are resistant to lipopolysaccharide (LPS) induced shock, whereas mice lacking both IL 1β and IL 18 are susceptible 33. Moreover, a recent study showed that IL 1β and IL 18 are not required for caspase 1 mediated clearance of several bacterial pathogens (namely, modified Salmonella enterica subsp. enterica serovar Typhimurium strains that constitutively express NATuRe RevIewS Immunology volume 11 MARcH

2 Box 1 Inflammasomes Inflammasomes are intracellular multiprotein complexes that mediate the proximity-induced autoactivation of caspase 1. Inflammasome-mediated caspase 1 activation has been shown to occur in macrophages, dendritic cells, epithelial cells and possibly other cell types during bacterial, viral, fungal and parasitic infections. Inflammasomes are activated in response to stimulation with damage-associated molecular patterns (DAMPs), such as uric acid and ATP, and upon exposure to crystalline substances, such as monosodium urate, silica and asbestos particles 12,24. The molecular composition of inflammasome complexes is stimulus dependent, with certain members of the NOD-like receptor (NLR) and HIN-200 receptor families functioning as the activating platform in these complexes. Genetic studies in mice indicate the existence of at least four types of inflammasome (FIG. 1). Three of these contain NLR proteins, namely NLR family, pyrin domain-containing 1B (NLRP1B), NLR family, CARD-containing 4 (NLRC4) and NLRP3. The fourth type of inflammasome contains the HIN-200 protein absent in melanoma 2 (AIM2) 12. The bipartite adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD; also known as PYCARD) probably has a key role in inflammasome assembly and caspase 1 activation by bridging the interaction between NLRs or HIN-200 proteins and caspase 1, although the precise role of ASC in the activation of the NLRP1B and NLRC4 inflammasomes is debated. NLRP1B and NLRC4 contain a caspase recruitment domain (CARD) at their carboxyl and amino termini, respectively (unlike AIM2 and NLRP3, which have a pyrin domain) and can therefore interact directly with caspase 1 when overexpressed, without requiring ASC. However, evidence of a role for ASC in the activation of the endogenous NLRC4 inflammasome is provided by the observation that robust caspase 1 activation and the production of interleukin-1β (IL-1β) and IL-18 are markedly decreased in ASC-deficient macrophages infected with viral or bacterial pathogens, or exposed to a variety of DAMPs and crystalline substances 50,63,88. Proximity-induced autoactivation A process in which two or more initiator caspases are brought sufficiently close to induce their autocatalytic activation. This process is thought to occur in large cytosolic protein complexes to which caspase zymogens are recruited by means of homotypic interactions between the caspase recruitment domain (CARD) or death effector domain (DeD) motifs in their pro-domains and several bipartite adaptor molecules. flagellin, Legionella pneumophila and Burkholderia thailandensis) 34. Similarly, caspase 1 deficient mice are more susceptible to infection with Francisella tularensis than mice lacking both IL 1β and IL 18 (ReF. 35), and this indicates that additional caspase 1 dependent mechanisms might contribute to the control of infection. In this regard, several recent publications have begun to characterize a range of new inflammasome functions and effector molecules that seem to operate independently of IL 1β and IL 18. In this article, I review these emerging non canonical inflammasome effector mechanisms and attempt to illustrate how they might contribute to immune responses. Unconventional protein secretion Secretory proteins usually contain amino terminal or internal signal peptides that target them to the translocation apparatus of the endoplasmic reticulum (er) 36,37. From the er lumen, such proteins are transported to the Golgi apparatus and then to the extracellular space in Golgi derived secretory vesicles that fuse with the plasma membrane 37. This pathway of protein export is known as the er Golgi or classical secretory pathway. However, cytoplasmic and nuclear proteins that lack an er targeting signal peptide can exit cells through erand Golgi independent pathways 38. For example, mature IL 1β was shown to be secreted independently of the er and the Golgi apparatus more than 20 years ago 39. The number of proteins that have been shown to be released by unconventional protein secretion has grown to more than 20, including fibroblast growth factor 2 (FGF2), the lectins galectin 1 and galectin 3, and possibly the DAMP high mobility group box 1 (HMGB1) 38. After their release into the extracellular space, these effectors can enhance inflammatory, cell survival and repair responses through activation of cell surface receptors, such as FGF receptor 1, the IL 1 and IL 18 receptors and the receptor for advanced glycation end products (RAGe). Although the molecular mechanism by which IL 1β, IL 18 and other proteins that lack signal peptides are secreted remains obscure, several models have been proposed to explain the release of such leaderless proteins in microvesicles that are shed from the plasma membrane, or in secretory lysosomes or exosomes 38. Interestingly, adherent monocytes from caspase 1 deficient mice and peritoneal macrophages from mice lacking two inflammasome components namely, NLR family, pyrin domain containing 3 (NLRP3) and apoptosis associated speck like protein containing a card (ASc; also known as PYcARD) not only failed to secrete IL 1β and IL 18 after LPS stimulation 16,18,19,40, but were also partially defective in the secretion of the leaderless cytokine IL 1α 17,40. unlike IL 1β and IL 18, IL 1α does not undergo caspase 1 mediated cleavage 26. This might indicate that caspase 1 modulates IL 1α secretion indirectly by regulating the secretory pathway of this cytokine, and may point to a broader role for caspase 1 in regulating unconventional protein secretion. Indeed, pharmacological inhibition, RNA interference (RNAi) mediated downregulation and targeted deletion of caspase 1 were all recently shown to block the secretion of IL 1β, IL 1α and FGF2 by macrophages, uvairradiated fibroblasts and uvb irradiated keratinocytes, respectively 41. In addition, caspase 1 activation by either the NLRP3 inflammasome or the NLR family, cardcontaining 4 (NLRc4) inflammasome was required for secretion of the nuclear DAMP HMGB1 from activated and infected macrophages 33. Because the enzymatic activity of caspase 1 is required for the secretion of each of these leaderless proteins 33,41, caspase 1 might mediate the proteolytic activation of a secretion apparatus of unknown identity. In this context, the small GTPase RAB39A was recently characterized as a caspase 1 substrate that is involved in the secretion of IL 1β from LPS activated human THP 1 monocytes 42. However, it remains to be determined how caspase 1 mediated cleavage affects RAB39A function and whether RAB39A has a role in the secretion of additional leaderless proteins. An alternative mechanism by which caspase 1 might promote the release of leaderless proteins such as HMGB1 involves the induction of a specialized caspase 1 mediated cell death programme in activated immune cells, which is often referred to as pyroptosis (see below). elucidation of the mechanism awaits the characterization of the molecular components mediating caspase 1 dependent unconventional protein secretion and pyroptosis. Pyroptosis Most caspases (BOX 2) caspases 2, 3 and 6 10 are implicated in the induction and execution of apoptosis 43,44. This form of programmed cell death is responsible for organ shaping during embryonic development and for preserving homeostasis in adult organisms. Typical morphological features of apoptosis include plasma membrane blebbing, condensation of the nucleus, 214 MARcH 2011 volume 11

3 Figure 1 Inflammasomes: composition and stimuli. The NOD-like receptor (NLR) proteins NLR family, pyrin domain-containing 1B (NLRP1B), NLR family, CARD-containing 4 (NLRC4) and NLRP3, and the HIN-200 protein absent in melanoma 2 (AIM2) assemble inflammasomes in a stimulus-specific manner 12. NLRP1B recognizes the cytosolic presence of the Bacillus anthracis lethal toxin 64. The NLRC4 inflammasome is assembled after detection of bacterial flagellin or the basal body rod component of the bacterial type III and type IV secretion systems of Salmonella enterica subsp. enterica serovar Typhimurium, Pseudomonas aeruginosa, Legionella pneumophila and Shigella flexneri 12,58,62. NLRP3 is activated when macrophages are exposed to UV irradiation, microbial pathogenassociated molecular patterns (PAMPs), endogenous damage-associated molecular patterns (DAMPs) such as ATP, or crystals such as monosodium urate, silica and asbestos. Recognition of these PAMPs, DAMPs and crystals is thought to involve the detection of a common secondary messenger, such as K + fluxes, reactive oxygen species or lysosomal proteases 14,15. By contrast, AIM2 directly binds double-stranded DNA (dsdna) in the cytosol to induce caspase 1 activation in cells infected with Francisella tularensis, Listeria monocytogenes or DNA viruses such as cytomegalovirus and vaccinia virus 68 70, The adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD; also known as PYCARD) is probably required for the full activation of all inflammasome complexes, although its role in the NLRP1B and NLRC4 inflammasomes is still debated 45. CARD, caspase recruitment domain; LRR, leucine-rich repeat. PYD, pyrin domain. DNA fragmentation and general shrinkage of the cell volume 45. Apoptotic cells usually fail to elicit inflammatory responses because the cytoplasmic content is shielded from the extracellular environment by packaging in apoptotic bodies (FIG. 2). These apoptotic bodies are membrane bound cell fragments that are rapidly phagocytosed in vivo by neighbouring cells and resident phagocytes 46,47. The integrity of the envelope surrounding apoptotic bodies is usually preserved until after they have been engulfed by professional antigen presenting cells or neighbouring cells to prevent accidental spilling of the intracellular content into the extracellular milieu 47. Apoptotic cells can also prevent the accidental induction of inflammation by inactivating the immunostimulatory activity of the DAMP HMGB1 through the oxidation of residue cys106 (ReF. 48). Finally, the uptake of apoptotic bodies by macrophages and dendritic cells was recently proposed to actively suppress antigen presentation and the secretion of inflammatory cytokines by these cells 49. unlike most caspases, caspase 1 is not involved in the induction of apoptosis. Instead, caspase 1 activation in neurons, macrophages and dendritic cells drives a specialized form of cell death known as pyroptosis 34, caspase 1 dependent cell death was first reported to occur in macrophages infected with Shigella flexneri, the causative agent of bacillary dysentery 54,55. Pyroptosis was subsequently observed in macrophages and dendritic cells infected with the facultative intracellular pathogens S. Typhimurium, Pseudomonas aeruginosa and L. pneumophila 45,56,57. each of these pathogens induces caspase 1 activation through the NLRc4 inflammasome (FIG. 1). These pathogens are recognized when bacterial flagellin is transferred through specialized bacterial type III and type IV secretion systems into the host cell cytosol or upon detection of the basal body rod component of the S. flexneri or P. aeruginosa type III secretion apparatus The induction of pyroptosis is not restricted to the NLRc4 inflammasome, as Bacillus anthracis infection induces the pyroptotic cell death of mouse macrophages through the NLRP1B inflammasome 64,65. This occurs when the anthrax metalloprotease lethal factor gains access to the cytosol of susceptible macrophages 64. Notably, lethal factor induced pyroptosis was shown to confer resistance to infection with B. anthracis spores in vivo 65, highlighting the importance of pyroptosis for host defence against pathogens. The pyroptotic cell death of macrophages infected with Staphylococcus aureus requires activation of the NLRP3 inflammasome 66,67. Although the precise mechanism is still debated, activation of this inflammasome could proceed through several mutually non exclusive mechanisms, including K + efflux, the generation of reactive oxygen species, lysosomal destabilization and the translocation of microbial ligands into the host cytosol 14,15. Finally, infections with the bacterial pathogens Listeria monocytogenes and F. tularensis induce pyroptosis upon their recognition in the host cell cytosol by the absent in melanoma 2 (AIM2) inflammasome The role of caspase 1 in the immune response to F. tularensis, the causative agent of tularaemia, is illustrated by the observation that caspase 1 deficient mice have increased susceptibility to infection with this pathogen 75. Notably, mice lacking the canonical inflammasome substrates IL 1β and IL 18 are less susceptible to infection with F. tularensis than caspase 1 deficient mice 35, and this indicates that additional caspase 1 dependent mechanisms, such as pyroptosis, might make an important contribution to the control of F. tularensis infection. Indeed, although caspase 1 activity is required for pyroptosis 51,76, this form of cell death proceeds independently of IL 1β and IL 18 (ReF. 56). A recent study established the crucial in vivo role of pyroptosis in clearing a modified NATuRe RevIewS Immunology volume 11 MARcH

4 Box 2 Caspases Caspases are an evolutionarily conserved family of metazoan cysteine proteases with 11 representatives in humans: caspases 1 10 and caspase 14. Caspases cleave various cellular substrates after aspartic acid residues and have essential roles in apoptosis, inflammation, cell proliferation and cell differentiation 89. For example, caspase 3- mediated cleavage of mammalian STE20-like kinase 1 (MST1; also known as STK4) was shown to be crucial for skeletal muscle differentiation, and caspase 8-mediated cleavage of the long splice variant of cellular FLICE-like inhibitory protein (cflip L ) regulates the balance between apoptosis induction and nuclear factor-κb (NF-κB)-driven T cell proliferation 90. All caspases are synthesized as zymogens consisting of an amino-terminal pro-domain of variable length and a carboxy-terminal protease domain. Caspases can be subdivided according to the length of their pro-domain (see the figure). Initiator caspases (such as caspases 1 and 8) have large pro-domains containing homotypic protein protein interaction motifs of the death domain superfamily, specifically either a caspase recruitment domain (CARD) or a death effector domain (DED). These interaction motifs allow the recruitment of pro-caspases into multiprotein complexes (such as the inflammasomes) by homotypic interactions with adaptor molecules such as ASC (apoptosis-associated speck-like protein containing a CARD; also known as PYCARD). Within these complexes, pro-caspases undergo the conformational changes and/or autoprocessing required for their activation 91. By contrast, the effector caspases (caspases 3, 6, 7 and 14) have short pro-domains of only a few amino acids and they lack any homotypic interaction motifs. These caspases require proteolytic maturation by the initiator caspases or other proteases to achieve maximum enzymatic activity. Unlike the true caspases listed above, human caspase 12 is devoid of enzymatic activity because crucial catalytic residues have been mutated. In addition, most people express a truncated form of caspase 12 that resembles the human CARD-only proteins CARD17 (also known as INCA), CARD18 (also known as ICEBERG) and CARD16 (also known as COP or pseudo-ice) 92. NOD-like receptor (NLR). The human NLR family comprises 22 members. They share a domain organization that usually includes an amino-terminal caspase recruitment domain (CARD) or pyrin domain (PYD), followed by an intermediary nucleotide-binding oligomerization domain (NOD) and carboxy-terminal leucine-rich repeat motifs. NLRs are thought to survey the host cytosol and intracellular compartments for pathogenand damage-associated molecular patterns to activate signalling pathways that contribute to the host innate immune response. S. Typhimurium strain that constitutively expressed bacterial flagellin 34. The pyroptotic cell death of infected macrophages exposed intracellular bacteria to extracellular immune surveillance and allowed their destruction by neutrophils. Pyroptosis also conferred protection against the bacterial pathogens L. pneumophila and B. thailandensis, which establishes this inflammasome effector mechanism as a crucial component of host defence against intracellular bacterial pathogens 34. Although the molecular mechanisms controlling pyroptosis are still poorly defined, this cell death mode differs morphologically from apoptosis in that pores with a diameter of 1 2 nm appear in the plasma membrane of pyroptotic cells at early time points 52. The resulting ion fluxes could explain some of the hallmarks of pyroptotic cells, including cytoplasmic swelling, osmotic lysis and the release of the intracellular content into the extracellular milieu 77 (FIG. 2). In addition to eliminating infected immune cells, this process can enhance innate and adaptive immune responses by exposing microbial antigens to surveillance by the immune system. Together with the fact that caspase 1 activation is linked with the production of mature IL 1β and IL 18 and the release of leaderless cytokines and DAMPs (such as HMGB1 and S100 proteins), these characteristics are thought to render pyroptosis (and associated inflammasome functions) an inherently pro inflammatory cell death mode. Despite the different immunological outcomes of apoptosis and pyroptosis (FIG. 2), apoptotic and pyroptotic cells share several prominent morphological and biochemical features. Nuclear condensation and oligonucleosomal DNA fragmentation are observed in both cell death modes 19,46,51,52,76. Moreover, the DNA damage sensor poly(adp ribose) polymerase 1 (PARP1) is processed into an 89 kda fragment during both apoptosis and pyroptosis 46,78. under homeostatic conditions, PARP1 participates in genomic DNA repair and DNA replication by catalysing the synthesis of poly(adp ribose) in a process that consumes NAD + and the ATP energy stores of the cell. Thus, cleavage of PARP1 during both apoptosis and pyroptosis indicates that PARP1 inactivation might be a general strategy used by cells undergoing programmed cell death, possibly to preserve energy stores in order to allow for proper dismantling of the cell. Finally, maturation of caspase 3 and caspase 7 is observed during both apoptosis and pyroptosis 51,78,79, although pyroptosisassociated DNA fragmentation, PARP1 processing and plasma membrane permeabilization are not affected in macrophages lacking either of these executioner caspases 51,55,78. This might not come as a complete surprise given that caspase 3 and caspase 7 are partially redundant and that deletion of both caspases was necessary for protection against apoptosis 80. Nevertheless, S. Typhimurium has been shown to induce pyroptosis in macrophages lacking both caspase 3 and caspase 7 (ReF. 45), and this confirms that these executioner caspases are not necessary for pyroptosis. Thus, although apoptosis and pyroptosis share the morphological and biochemical features described above (FIG. 2), the signalling pathways involved are distinct. Despite recent progress in characterizing the molecular features of pyroptotic cell death and its role in host defence against bacterial pathogens, much remains to be learned about the signalling mechanism by which caspase 1 induces pyroptosis. Inhibition of glycolysis In an attempt to identify new caspase 1 substrates that could explain the phenotype of pyroptotic cells, a caspase 1 digestome analysis was carried out, and this identified several crucial enzymes of bioenergetic pathways as potential caspase 1 targets 81. Biochemical studies confirmed that the glycolysis enzymes fructosebisphosphate aldolase, glyceraldehyde 3 phosphate dehydrogenase, α enolase and pyruvate kinase can be cleaved by recombinant caspase 1. These enzymes operate in a metabolic pathway that replenishes cellular ATP energy stores through the conversion of glucose to pyruvate. caspase 1 mediated processing of glyceraldehyde 3 phosphate dehydrogenase inhibited its enzymatic activity 81, and this indicated that caspase 1 might decrease the metabolic rate of infected cells. To address whether caspase 1 mediated processing of these metabolic enzymes occurred during infection, peritoneal macrophages of wild type and caspase 1 deficient mice were infected with S. Typhimurium. As expected, aldolase was 216 MARcH 2011 volume 11

5 Pathogen-associated molecular pattern (PAMP). A conserved pathogen molecule that is usually essential for microbial survival, and that contains either nucleic acid structures that are unique to microorganisms or cell wall components (such as lipopolysaccharide and flagellin) that are not found in mammalian cells. PAMPs are ligands for receptors of the host innate immune system. Damage-associated molecular pattern (DAMP). A molecule that is produced or released from host cells upon cellular stress, damage or non-physiological cell death. DAMPs are also referred to as alarmins and are thought to be responsible for the initiation and perpetuation of inflammatory responses and tissue repair under non-infectious (sterile) conditions. examples include high-mobility group box 1 (HMGB1), ATP, uric acid and heat-shock proteins. Unconventional protein secretion The secretion of cytoplasmic and nuclear proteins into the extracellular space through an incompletely understood mechanism that does not require the translocation apparatus of the classical endoplasmic reticulum (er) Golgi secretion pathway. Proteins that are secreted through this route include interleukin-1α (IL-1α), IL-1β, IL-18, fibroblast growth factor 2, galectin 1, galectin 3 and possibly high-mobility group box 1. Pyroptosis A specialized form of programmed cell death that requires caspase 1 activity. It is characterized by cytoplasmic swelling, early plasma membrane rupture, nuclear condensation and internucleosomal DNA fragmentation. The cytoplasmic content is released into the extracellular space, and this is thought to augment inflammatory and repair responses. Pyroptosis occurs in myeloid cells infected with pathogenic bacteria, and it might affect cells of the central nervous system and the cardiovascular system under ischaemic conditions. β processed in infected wild type macrophages, but not in those lacking caspase 1 (ReF. 81). concurrently, the rate of glycolysis in the caspase 1 deficient cells was markedly higher, further supporting an inhibitory role for caspase 1 in the regulation of glycolysis. Because myeloid cells are highly dependent on glycolysis for ATP production 82, caspase 1 mediated inactivation of glycolysis enzymes might restrict intracellular pathogen replication by quickly depleting energy sources and by preparing infected host cells to undergo pyroptosis. However, it remains to be determined whether and to what extent caspase 1 mediated inactivation of glycolysis enzymes contributes to protection against bacterial pathogenicity in vivo. Cell survival Another mechanism by which caspase 1 might protect host cells is by repairing the damage caused by bacterial pore forming toxins that are released by pathogenic bacteria 83. The pores formed by these toxins can range significantly in diameter, depending on the nature of the toxin. For example, S. aureus α toxin and Aeromonas hydrophila aerolysin typically produce holes in the host cell membrane with a diameter of only 2 nm, whereas Streptococcus pneumoniae and L. monocytogenes produce toxins that can create perforations of up to 50 nm in diameter 84. consequently, the latter toxins allow translocation of large proteins across the plasma membrane, whereas S. aureus α toxin and A. hydrophila aerolysin Figure 2 main features of pyroptosis. The molecular mechanisms underlying pyroptosis are still poorly defined. Morphologically, pyroptotic cells are characterized by the early loss of plasma membrane integrity 77, and this is accompanied by the shedding of membrane vesicles 93. Pyroptotic and apoptotic cells share several prominent features (shown in blue boxes), including nuclear condensation and internucleosomal DNA fragmentation, cleavage of the DNA damage repair enzyme poly(adp-ribose) polymerase 1 (PARP1) and activation of the executioner caspases caspase 3 and caspase 7 (ReF. 45). However, the volume of the cytoplasmic compartment of pyroptotic cells increases, whereas apoptosis is characterized by general shrinkage of the cell volume. Together with the role of caspase 1 in cytokine maturation and unconventional protein secretion, the release of the cytoplasmic content into the extracellular milieu during pyroptosis is thought to render this form of cell death inherently pro-inflammatory 45. By contrast, apoptosis is usually considered to be immunologically silent because the cytoplasmic content is packaged in apoptotic bodies and these membrane-bound cell fragments are rapidly phagocytosed in vivo by neighbouring cells and resident phagocytes 47. HMGB1, high mobility group box 1; IL, interleukin. only render the plasma membrane permeable to small inorganic ions 84. Nevertheless, depending on the concentration of these toxins and the targeted cell type, the damage elicited by these toxins can range from irreparable cell destruction to temporal perforations that can quickly be resealed by the host cell s dedicated repair machinery. Repairing bacterial toxin induced damage to the plasma membrane requires the activation of lipid biogenesis pathways, the molecular machinery of which is controlled by two transcription factors known as sterol regulatory element binding protein 1 (SReBP1) and SReBP2 (ReF. 84). Interestingly, activation of SReBP1 and SReBP2 is controlled by the NLRP3 and NLRc4 inflammasomes in fibroblasts that have been intoxicated with S. aureus α toxin or A. hydrophila aerolysin, or infected with live Aeromonas trota bacteria 83. This pathway is thought to promote host cell survival because inhibition or transcriptional downregulation of SReBP1 and SReBP2 enhanced cell death responses 83. Inflammasomeinduced activation of SReBPs is thought to be indirect, but the caspase 1 substrates that drive this pathway remain to be found. It would be interesting to determine whether caspase 1 activates SReBPs in macrophages infected with live A. hydrophila, because these bacteria were recently shown to activate caspase 1 through the NLRP3, but not the NLRc4, inflammasome 85. This might reveal differential signalling mechanisms induced by the three A. hydrophila cytotoxins 85. NATuRe RevIewS Immunology volume 11 MARcH

6 Type III and type IV secretion Two of at least six specialized secretion systems by which Gram-negative pathogens can deliver virulence factors into eukaryotic host cells. Pathogenic bacteria such as Shigella, Salmonella, Yersinia, Chlamydia and Pseudomonas spp. all make use of a type III secretion system to infect host cells and to modulate signalling pathways. By contrast, pathogens such as Helicobacter pylori, Legionella pneumophila and Bordetella pertussis make use of a type IV secretion system for the horizontal transfer of plasmid DNA containing antibiotic resistance genes and to inject effector proteins into eukaryotic host cells. Glycolysis A metabolic pathway that generates the cellular high-energy store ATP by oxidizing glucose to pyruvate. In eukaryotic cells, pyruvate is further oxidized into CO 2 and H 2 O in a process known as aerobic respiration. This results in a net yield of molecules of ATP per metabolized molecule of glucose. Autophagosome A double-membrane-bound vesicle that is used by eukaryotic cells to target protein aggregates, damaged organelles and invading microorganisms for digestion by lysosomal hydrolases. This catabolic process allows recycling of cellular components and is thought to contribute to cell death, cell survival during starvation, cellular differentiation and host defence against infectious agents. Caspase 7 activation Activation of the NLRc4 inflammasome was recently shown to restrict the intracellular replication of L. pneumophila, the causative agent of a severe form of bacterial pneumonia known as Legionnaires disease 61,86,87. Inflammasome activation in resistant mouse strains results in the rapid caspase 1 dependent delivery of L. pneumophila to lysosomes, where the bacteria are degraded. By contrast, defective inflammasome activation in Nlrc4 / and Casp1 / mice allows bacterial replication in specialized intracellular vesicles that resemble autophagosomes 61,87. Notably, inflammasome mediated restriction of L. pneumophila replication proceeds independently of IL 1β and IL 18 (ReFs 61,87), but the caspase 1 substrates that are responsible for this process remain unclear. A proteome wide screen for new caspase 1 targets identified caspase 7, an effector caspase, as a direct substrate of caspase 1, and biochemical studies confirmed that caspase 7 is cleaved by caspase 1 after the canonical activation sites Asp23 and Asp198 (ReF. 51). Importantly, studies in macrophages from Nlrp3 /, Nlrc4 /, Asc / or Casp1 / mice confirmed the role of the NLRP3 and NLRc4 inflammasomes in inducing proteolytic maturation of caspase 7 in activated immune cells that were exposed to ATP and nigericin or infected with live S. Typhimurium 51. In contrast to caspase 7, the activation of caspase 3 was not affected in caspase 1 deficient macrophages 51, and this indicates that the activation of caspase 3 and caspase 7 is differentially regulated during inflammation. These observations identified an alternative mechanism by which inflammasomes might control bacterial infections. Indeed, caspase 7 activation downstream of the NLRc4 inflammasome was subsequently reported in macrophages infected with L. pneumophila 79. Moreover, caspase 7 deficient macrophages are less capable of restricting intracellular L. pneumophila replication, possibly owing to defects in the fusion of bacteria containing phagosomes with lysosomes and the delayed induction of macrophage cell death 79. Importantly, caspase 1 and caspase 7 regulate L. pneumophila growth in the lungs of orally infected mice 61,79, demonstrating the importance of this inflammasome effector pathway in host defence against this bacterial pathogen. However, it remains to be determined Figure 3 Caspase 1 effector mechanisms. Pathogen invasion of macrophages and dendritic cells triggers the assembly of inflammasome complexes and caspase 1 activation. Active caspase 1 induces inflammation by mediating the extracellular secretion or release of leaderless cytokines such as interleukin-1β (IL-1β), IL-18 and IL-1α, and possibly damage-associated molecular patterns (DAMPs) such as high mobility group box 1 (HMGB1), through an as yet unknown mechanism. Caspase 1 also promotes repair and healing responses by inducing lipid membrane biogenesis through the activation of sterol regulatory element binding proteins (SREBPs) and through the secretion or release of growth factors such as fibroblast growth factor 2. The latter contributes to repair through ligation of cell surface receptors on target cells. Caspase 1 cleaves poly(adp-ribose) polymerase 1 (PARP1) and glycolysis enzymes, possibly to prepare host cells to undergo pyroptosis. This specialized cell death programme removes intracellular niches for microbial replication and eliminates infected immune cells. Moreover, it might help to tune immune responses by releasing microbial components into the extracellular milieu, where they can be detected by the immune system. It is probable that caspase 1 cleaves additional as yet unidentified substrates that are responsible for early membrane permeabilization and oligonucleosomal DNA fragmentation during pyroptosis. Finally, inflammasome-mediated activation of caspase 7 (an effector caspase) restricts bacterial replication in Legionella-infected macrophages by targeting the infectious agent to lysosomes. CARD, caspase recruitment domain. 218 MARcH 2011 volume 11

7 whether inflammasome mediated activation of caspase 7 also restricts the replication of S. Typhimurium and other bacterial pathogens, and whether this inflammasome effector pathway is activated during viral infection. Conclusions and perspectives It has become evident in recent years that inflammasomes have important roles in innate immune signalling and host defence. In particular, our knowledge of how inflammasome complexes of distinct composition are assembled in a stimulus dependent manner has grown significantly. Now, the different effector mechanisms (in addition to IL 1β and IL 18 secretion) by which inflammasomes might contribute to immunity and host defence are also starting to emerge. As described above, recent studies have highlighted a range of new inflammasome functions and effector mechanisms (FIG. 3). caspase 1 has been shown to control the secretion of leaderless cytokines and proteins such as IL 1α and FGF2, as well as the release of endogenous DAMPs such as HMGB1. Moreover, excessive caspase 1 activation in damaged neurons and infected myeloid cells induces pyroptotic cell death. Furthermore, caspase 1 dampens the metabolic rate of infected cells by cleaving key enzymes of the glycolysis pathway and regulates lipid metabolic pathways for cell repair. Finally, activation of the executioner caspase 7 downstream of inflammasomes contributes to restriction of Legionella replication in infected macrophages. Interestingly, most of these emerging functions of caspase 1 seem to operate independently of the canonical substrates IL 1β and IL 18, and this indicates that inflammasomes contribute to innate immune responses in a variety of ways. Indeed, these effector mechanisms probably function together to mount a fast and effective innate immune response against the pathogen and might instruct the adaptive immune system regarding the systemic risk posed by the invading microbial agent. At an early phase of infection, activation of caspase 7 downstream of the inflammasome might halt pathogen replication in intracellular replication niches, while at the same time inflammasome mediated activation of SReBPs could repair pathogen induced damage to the plasma membrane. when bacterial (or viral) loads further increase, inflammasomes can induce pyroptosis and instruct macrophages and dendritic cells to initiate the unconventional secretion or passive release of pro inflammatory cytokines, growth factors, DAMPs and microbial antigens to alert the immune system of an imminent threat and to initiate repair responses. In this context, the crucial role of pyroptosis in host defence was recently demonstrated in mice infected with the Gram negative bacterial pathogens L. pneumophila and B. thailandensis 34. 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Competing interests statement The author declares no competing financial interests. FURTHER INFORMATION Mohamed Lamkanfi s homepage: research/scientists/pages/mohamed-lamkanfi-lab.aspx All links Are ACtIve In the online pdf 220 MARcH 2011 volume 11