Emerging Inflammasome Effector Mechanisms

REVIEWS

Emerging inflammasome effector mechanisms

Mohamed Lamkanfi Abstract | Caspase 1 activation by inflammasome complexes in response to pathogen- associated 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.

(BOX 1) Cryopyrinopathies Inflammasomes are emerging as key regulators viral and bacterial pathogens that target caspase 1 activa‑ 24 A spectrum of hereditary of the innate immune response, and the activity of these tion in inflammasomes . Indeed, IL‑1β and IL‑18 were autoinflammatory diseases multiprotein complexes has been linked to inflamma‑ recognized early on for their ability to cause a wide variety that are caused by mutations tory bowel diseases1–5, vitiligo6, gouty arthritis7, type 1 of biological effects associated with infection, inflamma‑ in the gene encoding NLR and type 2 diabetes8,9, and less common autoinflamma‑ tion and autoimmunity25. IL‑1β regulates systemic and family, pyrin domain- containing 3 (NLRP3) that tory disorders that are collectively referred to as cryopy- local responses to infection, injury and immunological trigger continuous activation rinopathies10,11. Inflammasome complexes are thought to challenge by generating fever, activating lymphocytes and of the NLRP3 inflammasome. be assembled around members of the NOD-like receptor promoting leukocyte transmigration into sites of injury Based on the severity and (NLR) or HIN‑200 protein families12 (FIG. 1). These or infection25. Although IL‑18 lacks the pyrogenic activity spectrum of the symptoms — pathogen- which can include urticarial receptors are thought to detect microbial of IL‑1β, it induces interferon‑γ (IFNγ) production by skin rashes, prolonged associated molecular patterns (PAMPs) and endogenous activated T cells and natural killer cells in the presence episodes of fever, sensorineural damage-associated molecular patterns (DAMPs) in intra‑ of IL‑12, thereby contributing to T helper 1 (TH1) cell hearing loss, headaches, cellular compartments, similar to the role of mammalian polarization25,26. In the absence of IL‑12, IL‑18 can pro‑ cognitive deficits and renal Toll‑like receptors (TLRs) at the cell surface and within mote T 2 cell responses through the production of T 2 amyloidosis — these diseases H H 13 are classified as familial cold endosomes . Although it is incompletely understood cell cytokines such as IL‑4, IL‑5 and IL‑10 (ReFs 26–28). autoinflammatory syndrome, how NLRs detect microbial ligands and DAMPs14,15, it More recently, IL‑18 has also been implicated in driving Muckle–Wells syndrome or is evident that inflammasome assembly results in the TH17 cell responses because it synergizes with IL‑23 chronic infantile neurological activation of caspase 1 (BOX 2). This evolutionarily con‑ to induce IL‑17 production by already committed cutaneous articular syndrome. 29,30 served cysteine protease is mainly known for its role TH17 cells . Thus, IL‑1β and IL‑18 are important in the maturation of the pro‑inflammatory cytokines inflammasome effector molecules, as illustrated by the interleukin‑1β (IL‑1β) and IL‑18 (ReFs 16–19). marked response to therapy with IL‑1 inhibitors found IL‑1β and IL‑18 are related cytokines that are pro‑ in patients with cryopyrinopathies, who have increased duced as cytosolic precursors and usually require inflammasome activation31,32. caspase 1‑mediated cleavage for full activation and However, not all inflammasome functions can be 16–19 Department of Biochemistry, secretion . However, additional proteases, including abrogated by neutralization of IL‑1β and IL‑18. For exam‑ Ghent University, and VIB caspase 8, myeloblastin (also known as proteinase 3) ple, caspase 1‑deficient mice are resistant to lipopoly‑ Department of Medical and granzyme A, have been shown to convert pro‑IL‑1β saccharide (LPS)‑induced shock, whereas mice lacking Protein Research, Albert into a biologically active cytokine in several established both IL‑1β and IL‑18 are susceptible33. Moreover, a recent Baertsoenkaai 3, B‑9000 mouse models of human disease20–23. This indicates that study showed that IL‑1β and IL‑18 are not required for Ghent, Belgium. e‑mail: Mohamed.Lamkanfi@ caspase 1 is not always required for the maturation of caspase 1‑mediated clearance of several bacterial patho‑ VIB‑UGent.be IL‑1β, and such redundancy in controlling IL‑1β matura‑ gens (namely, modified Salmonella enterica subsp. enterica doi:10.1038/nri2936 tion might safeguard the host immune response against serovar Typhimurium strains that constitutively express

Box 1 | Inflammasomes inflammatory, cell survival and repair responses through activation of cell surface receptors, such as FGF recep‑ Inflammasomes are intracellular multiprotein complexes that mediate the tor 1, the IL‑1 and IL‑18 receptors and the receptor for proximity-induced autoactivation of caspase 1. Inflammasome-mediated caspase 1 advanced glycation end‑products (RAGe). activation has been shown to occur in macrophages, dendritic cells, epithelial cells and Although the molecular mechanism by which IL‑1β, possibly other cell types during bacterial, viral, fungal and parasitic infections. Inflammasomes are activated in response to stimulation with damage-associated IL‑18 and other proteins that lack signal peptides are molecular patterns (DAMPs), such as uric acid and ATP, and upon exposure to crystalline secreted remains obscure, several models have been substances, such as monosodium urate, silica and asbestos particles12,24. The molecular proposed to explain the release of such ‘leaderless pro‑ composition of inflammasome complexes is stimulus dependent, with certain members teins’ in microvesicles that are shed from the plasma of the NOD-like receptor (NLR) and HIN-200 receptor families functioning as the membrane, or in secretory lysosomes or exosomes38. activating platform in these complexes. Genetic studies in mice indicate the existence of Interestingly, adherent monocytes from caspase 1‑ at least four types of inflammasome (FIG. 1). Three of these contain NLR proteins, namely deficient mice and peritoneal macrophages from mice NLR family, pyrin domain-containing 1B (NLRP1B), NLR family, CARD-containing 4 lacking two inflammasome components — namely, (NLRC4) and NLRP3. The fourth type of inflammasome contains the HIN-200 protein NLR family, pyrin domain‑containing 3 (NLRP3) and absent in melanoma 2 (AIM2)12. The bipartite adaptor protein ASC (apoptosis-associated apoptosis‑associated speck‑like protein containing a 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 cARD (ASc; also known as PYcARD) — not only failed 16,18,19,40 NLRs or HIN-200 proteins and caspase 1, although the precise role of ASC in the activation to secrete IL‑1β and IL‑18 after LPS stimulation , of the NLRP1B and NLRC4 inflammasomes is debated. NLRP1B and NLRC4 contain a but were also partially defective in the secretion of the caspase recruitment domain (CARD) at their carboxyl and amino termini, respectively leaderless cytokine IL‑1α17,40. unlike IL‑1β and IL‑18, (unlike AIM2 and NLRP3, which have a pyrin domain) and can therefore interact directly IL‑1α does not undergo caspase 1‑mediated cleavage26. with caspase 1 when overexpressed, without requiring ASC. However, evidence of a role This might indicate that caspase 1 modulates IL‑1α for ASC in the activation of the endogenous NLRC4 inflammasome is provided by the secretion indirectly by regulating the secretory path‑ observation that robust caspase 1 activation and the production of interleukin-1β (IL-1β) way of this cytokine, and may point to a broader role for and IL-18 are markedly decreased in ASC-deficient macrophages infected with viral or caspase 1 in regulating unconventional protein secretion. bacterial pathogens, or exposed to a variety of DAMPs and crystalline substances50,63,88. Indeed, pharmacological inhibition, RNA interference (RNAi)‑mediated downregulation and targeted deletion of caspase 1 were all recently shown to block the secre‑ flagellin, Legionella pneumophila and Burkholderia thai- tion of IL‑1β, IL‑1α and FGF2 by macrophages, uvA‑ landensis)34. Similarly, caspase 1‑deficient mice are more irradiated fibroblasts and uvB‑irradiated keratinocytes, susceptible to infection with Francisella tularensis than respectively41. In addition, caspase 1 activation by either mice lacking both IL‑1β and IL‑18 (ReF. 35), and this the NLRP3 inflammasome or the NLR family, cARD‑ indicates that additional caspase 1‑dependent mecha‑ containing 4 (NLRc4) inflammasome was required for nisms might contribute to the control of infection. In secretion of the nuclear DAMP HMGB1 from activated this regard, several recent publications have begun to and infected macrophages33. Because the enzymatic characterize a range of new inflammasome functions and activity of caspase 1 is required for the secretion of each effector molecules that seem to operate independently of these leaderless proteins33,41, caspase 1 might medi‑ of IL‑1β and IL‑18. In this article, I review these emerg‑ ate the proteolytic activation of a secretion apparatus ing non‑canonical inflammasome effector mechanisms of unknown identity. In this context, the small GTPase and attempt to illustrate how they might contribute to RAB39A was recently characterized as a caspase 1 sub‑ immune responses. strate that is involved in the secretion of IL‑1β from LPS‑activated human THP‑1 monocytes42. However, it Unconventional protein secretion remains to be determined how caspase 1‑mediated cleav‑ Secretory proteins usually contain amino‑terminal or age affects RAB39A function and whether RAB39A has a internal signal peptides that target them to the translo‑ role in the secretion of additional leaderless proteins. An cation apparatus of the endoplasmic reticulum (eR)36,37. alternative mechanism by which caspase 1 might promote From the eR lumen, such proteins are transported to the release of leaderless proteins such as HMGB1 involves Proximity-induced the Golgi apparatus and then to the extracellular space the induction of a specialized caspase 1‑mediated cell autoactivation in Golgi‑derived secretory vesicles that fuse with the death programme in activated immune cells, which is A process in which two or plasma membrane37. This pathway of protein export is often referred to as pyroptosis (see below). elucidation more initiator caspases are brought sufficiently close to known as the ‘eR–Golgi’ or ‘classical’ secretory pathway. of the mechanism awaits the characterization of the induce their autocatalytic However, cytoplasmic and nuclear proteins that lack an molecular components mediating caspase 1‑dependent activation. This process is eR‑targeting signal peptide can exit cells through eR‑ unconventional protein secretion and pyroptosis. thought to occur in large and Golgi‑independent pathways38. For example, mature cytosolic protein complexes Pyroptosis to which caspase zymogens IL‑1β was shown to be secreted independently of the eR 39 are recruited by means of and the Golgi apparatus more than 20 years ago . The Most caspases (BOX 2) — caspases 2, 3 and 6–10 — are homotypic interactions number of proteins that have been shown to be released implicated in the induction and execution of apopto‑ between the caspase by unconventional protein secretion has grown to more sis43,44. This form of programmed cell death is respon‑ recruitment domain (CARD) or than 20, including fibroblast growth factor 2 (FGF2), the sible for organ shaping during embryonic development death effector domain (DeD) motifs in their pro-domains lectins galectin 1 and galectin 3, and possibly the DAMP and for preserving homeostasis in adult organisms. 38 and several bipartite adaptor high‑mobility group box 1 (HMGB1) . After their release Typical morphological features of apoptosis include molecules. into the extracellular space, these effectors can enhance plasma membrane blebbing, condensation of the nucleus,

214 | MARcH 2011 | voLuMe 11 www.nature.com/reviews/immunol © 2011 Macmillan Publishers Limited. All rights reserved 5V[RJKOWTKWO 78KTTCFKCVKQP 2CGTWIKPQUC /KETQDKCN2#/2U &0#XKTWUGU $CPVJTCEKU .RPGWOQRJKNC 'PFQIGPQWU&#/2U (VWNCTGPUKU NGVJCNVQZKP 5ƓGZPGTK %T[UVCNU .OQPQE[VQIGPGU ! 4GCEVKXGQZ[IGPURGEKGU! ! +QPȯWZ! ! %[VQUQNKE .[UQUQOCNRTQVGCUGU! FU&0#

.44U $ $ #+/ #+/ 0.42 0.42 0.4% 0.4% 0.42+ 0.42+ & & & & 2; 2; 2; 2; %#4& %#4& %#4& %#4&

! ! & & 2; 2;

#5% %#4& %#4& %#4& %#4&

2TQECURCUG

#EVKXG ECURCUG %#4& %#4&

0CVWTG4GXKGYU^+OOWPQNQI[

REVIEWS

56[RJKOWTKWO 78KTTCFKCVKQP of the intracellular content into the extracellular milieu47. 2 CGTWIKPQUC /KETQDKCN2#/2U &0#XKTWUGU Apoptotic cells can also prevent the accidental induction $CPVJTCEKU .RPGWOQRJKNC 'PFQIGPQWU&#/2U (VWNCTGPUKU of inflammation by inactivating the immunostimulatory NGVJCNVQZKP 5ƓGZPGTK %T[UVCNU .OQPQE[VQIGPGU activity of the DAMP HMGB1 through the oxidation of (ReF. 48) r+QPȯWZ! residue cys106 . Finally, the uptake of apoptotic r4GCEVKXGQZ[IGP ! bodies by macrophages and dendritic cells was recently URGEKGU! proposed to actively suppress antigen presentation and r.[UQUQOCN 49 RTQVGCUGU! %[VQUQNKE the secretion of inflammatory cytokines by these cells . FU&0# 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 spe‑

0.42+ 0.42+ 34,50–53 #+/ #+/

0.42 0.42 cialized form of cell death known as pyroptosis . 0.4% 0.4% caspase 1‑dependent cell death was first reported to & & & .44U & & occur in macrophages infected with Shigella flexneri, the 2; 2; 2; 2; %#4& %#4 causative agent of bacillary dysentery54,55. Pyroptosis was

%#4& %#4& ! subsequently observed in macrophages and dendritic ! cells infected with the facultative intracellular pathogens S. Typhimurium, Pseudomonas aeruginosa and L. pneu- 45,56,57 & & mophila . each of these pathogens induces caspase 1 2; 2; activation through the NLRc4 inflammasome (FIG. 1). #5% These pathogens are recognized when bacterial flagel‑ & & & & lin is transferred through specialized bacterial type III %#4 %#4 %#4 %#4 and type IV secretion systems into the host cell cytosol 2TQECURCUG or upon detection of the basal body rod component of the S. flexneri or P. aeruginosa type III secretion appara‑ tus58–63. The induction of pyroptosis is not restricted to the NLRc4 inflammasome, as Bacillus anthracis infec‑ & #EVKXG & ECURCUG tion induces the pyroptotic cell death of mouse macro‑ %#4 %#4 phages through the NLRP1B inflammasome64,65. This Figure 1 | Inflammasomes: composition and stimuli. The NOD-like receptor (NLR) occurs when the anthrax metalloprotease lethal factor proteins NLR family, pyrin domain-containing 1B (NLRP1B), NLR family, CARD-containing 4 gains access to the cytosol of susceptible macrophages64. (NLRC4) and NLRP3, and the HIN-200 protein absent in melanoma 2 (AIM2) assemble Notably, lethal factor‑induced pyroptosis was shown to inflammasomes in a stimulus-specific manner12. NLRP1B recognizes the cytosolic presence confer resistance to infection with B. anthracis spores of the Bacillus anthracis lethal toxin64. The NLRC4 inflammasome is assembled after in vivo65, highlighting the importance of pyroptosis for detection of bacterial flagellin or the basal body rod component of the bacterial type III host defence against pathogens. The pyroptotic cell death and type IV secretion systems of Salmonella enterica subsp. enterica serovar Typhimurium, 12,58,62 of macrophages infected with Staphylococcus aureus Pseudomonas aeruginosa, Legionella pneumophila and Shigella flexneri . NLRP3 is 66,67 activated when macrophages are exposed to UV irradiation, microbial pathogen- requires activation of the NLRP3 inflammasome . associated molecular patterns (PAMPs), endogenous damage-associated molecular Although the precise mechanism is still debated, acti‑ patterns (DAMPs) such as ATP, or crystals such as monosodium urate, silica and asbestos. vation of this inflammasome could proceed through Recognition of these PAMPs, DAMPs and crystals is thought to involve the detection of a several mutually non‑exclusive mechanisms, including common secondary messenger, such as K+ fluxes, reactive oxygen species or lysosomal K+ efflux, the generation of reactive oxygen species, lyso‑ proteases14,15. By contrast, AIM2 directly binds double-stranded DNA (dsDNA) in the somal destabilization and the translocation of microbial cytosol to induce caspase 1 activation in cells infected with Francisella tularensis, Listeria ligands into the host cytosol14,15. Finally, infections with monocytogenes or DNA viruses such as cytomegalovirus and vaccinia virus68–70,72–74. The the bacterial pathogens Listeria monocytogenes and adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD; also F. tularensis induce pyroptosis upon their recognition known as PYCARD) is probably required for the full activation of all inflammasome 45 in the host cell cytosol by the absent in melanoma 2 complexes, although its role in the NLRP1B and NLRC4 inflammasomes is still debated . 68–74 CARD, caspase recruitment domain; LRR, leucine-rich repeat. PYD, pyrin domain. (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 DNA fragmentation and general shrinkage of the cell to infection with this pathogen75. Notably, mice lacking volume45. Apoptotic cells usually fail to elicit inflam‑ the canonical inflammasome substrates IL‑1β and IL‑18 matory responses because the cytoplasmic content is are less susceptible to infection with F. tularensis than shielded from the extracellular environment by packag‑ caspase 1‑deficient mice35, and this indicates that ing in ‘apoptotic bodies’ (FIG. 2). These apoptotic bodies additional caspase 1‑dependent mechanisms, such as are membrane‑bound cell fragments that are rapidly pyroptosis, might make an important contribution to phagocytosed in vivo by neighbouring cells and resident the control of F. tularensis infection. Indeed, although phagocytes46,47. The integrity of the envelope surround‑ caspase 1 activity is required for pyroptosis51,76, this ing apoptotic bodies is usually preserved until after they form of cell death proceeds independently of IL‑1β have been engulfed by professional antigen‑presenting and IL‑18 (ReF. 56). A recent study established the cru‑ cells or neighbouring cells to prevent accidental spilling cial in vivo role of pyroptosis in clearing a modified

Box 2 | Caspases proteins), these characteristics are thought to render pyroptosis (and associated inflammasome functions) Caspases are an evolutionarily conserved family of metazoan cysteine proteases with an inherently pro‑inflammatory cell death mode. 11 representatives in humans: caspases 1–10 and caspase 14. Caspases cleave various Despite the different immunological outcomes of cellular substrates after aspartic acid residues and have essential roles in apoptosis, apoptosis and pyroptosis (FIG. 2), apoptotic and pyrop‑ inflammation, cell proliferation and cell differentiation89. For example, caspase 3- mediated cleavage of mammalian STE20-like kinase 1 (MST1; also known as STK4) was totic cells share several prominent morphological and shown to be crucial for skeletal muscle differentiation, and caspase 8-mediated biochemical features. Nuclear condensation and oligo‑ nucleosomal DNA fragmentation are observed in both cleavage of the long splice variant of cellular FLICE-like inhibitory protein (cFLIPL) 19,46,51,52,76 regulates the balance between apoptosis induction and nuclear factor-κB cell death modes . Moreover, the DNA damage (NF-κB)-driven T cell proliferation90. All caspases are synthesized as zymogens consisting sensor poly(ADP‑ribose) polymerase 1 (PARP1) is proc‑ of an amino-terminal pro-domain of variable length and a carboxy-terminal protease essed into an 89 kDa fragment during both apoptosis and domain. Caspases can be subdivided according to the length of their pro-domain pyroptosis46,78. under homeostatic conditions, PARP1 (see the figure). Initiator caspases (such as caspases 1 and 8) have large pro-domains participates in genomic DNA repair and DNA replica‑ containing homotypic protein–protein interaction motifs of the death domain tion by catalysing the synthesis of poly(ADP‑ribose) in a superfamily, specifically either a caspase recruitment domain (CARD) or a death effector process that consumes NAD+ and the ATP energy stores domain (DED). These interaction motifs allow the recruitment of pro-caspases into multiprotein complexes (such as the inflammasomes) by homotypic interactions with of the cell. Thus, cleavage of PARP1 during both apop‑ adaptor molecules such as ASC (apoptosis-associated speck-like protein containing a tosis and pyroptosis indicates that PARP1 inactivation CARD; also known as PYCARD). Within these complexes, pro-caspases undergo the might be a general strategy used by cells undergoing pro‑ conformational changes and/or autoprocessing required for their activation91. By grammed cell death, possibly to preserve energy stores in contrast, the effector caspases (caspases 3, 6, 7 and 14) have short pro-domains of only a order to allow for proper dismantling of the cell. Finally, few amino acids and they lack any homotypic interaction motifs. These caspases require maturation of caspase 3 and caspase 7 is observed during proteolytic maturation by the initiator caspases or other proteases to achieve maximum both apoptosis and pyroptosis51,78,79, although pyroptosis‑ enzymatic activity. Unlike the ‘true’ caspases listed above, human caspase 12 is devoid associated DNA fragmentation, PARP1 processing and of enzymatic activity because crucial catalytic residues have been mutated. In addition, plasma membrane permeabilization are not affected most people express a truncated form of caspase 12 that resembles the human in macrophages lacking either of these executioner CARD-only proteins CARD17 (also known as INCA), CARD18 (also known as ICEBERG) 51,55,78 and CARD16 (also known as COP or pseudo-ICE)92. caspases . This might not come as a complete surprise given that caspase 3 and caspase 7 are partially redundant #OKPQ %CTDQZ[N VGTOKPWU VGTOKPWU and that deletion of both caspases was necessary for pro‑ tection against apoptosis80. Nevertheless, S. Typhimurium 2TQFQOCKP 2TQVGCUGFQOCKP has been shown to induce pyroptosis in macrophages %CURCUGUCPF %#4& %CURCUGFQOCKP (ReF. 45) +PKVKCVQT lacking both caspase 3 and caspase 7 , and this ECURCUGU confirms that these executioner caspases are not neces‑ %CURCUGUCPF &'& %CURCUGFQOCKP sary for pyroptosis. Thus, although apoptosis and pyrop‑ 'ȭGEVQT tosis share the morphological and biochemical features ECURCUGU %CURCUGUCPF %CURCUGFQOCKP 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 S. Typhimurium0CVWT strainG4G thatXKGYU constitutively^+OOWPQNQI[ expressed host defence against bacterial pathogens, much remains bacterial flagellin34. The pyroptotic cell death of infected to be learned about the signalling mechanism by which macrophages exposed intracellular bacteria to extracel‑ caspase 1 induces pyroptosis. lular immune surveillance and allowed their destruction by neutrophils. Pyroptosis also conferred protection Inhibition of glycolysis against the bacterial pathogens L. pneumophila and In an attempt to identify new caspase 1 substrates B. thailandensis, which establishes this inflammasome that could explain the phenotype of pyroptotic cells, a NOD-like receptor (NLR). The human NLR family effector mechanism as a crucial component of host caspase 1 digestome analysis was carried out, and this 34 comprises 22 members. They defence against intracellular bacterial pathogens . identified several crucial enzymes of bioenergetic share a domain organization Although the molecular mechanisms controlling pathways as potential caspase 1 targets81. Biochemical that usually includes an pyroptosis are still poorly defined, this cell death mode studies confirmed that the glycolysis enzymes fructose‑ amino-terminal caspase differs morphologically from apoptosis in that pores with bisphosphate aldolase, glyceraldehyde‑3‑phosphate dehy‑ recruitment domain (CARD) or pyrin domain (PYD), a diameter of 1–2 nm appear in the plasma membrane of drogenase, α‑enolase and pyruvate kinase can be cleaved 52 followed by an intermediary pyroptotic cells at early time points . The resulting ion by recombinant caspase 1. These enzymes operate in a nucleotide-binding fluxes could explain some of the hallmarks of pyroptotic metabolic pathway that replenishes cellular ATP energy oligomerization domain (NOD) cells, including cytoplasmic swelling, osmotic lysis and stores through the conversion of glucose to pyruvate. and carboxy-terminal leucine-rich repeat motifs. the release of the intracellular content into the extracel‑ caspase 1‑mediated processing of glyceraldehyde‑3‑ 77 NLRs are thought to survey the lular milieu (FIG. 2). In addition to eliminating infected phosphate dehydrogenase inhibited its enzymatic activ‑ host cytosol and intracellular immune cells, this process can enhance innate and adap‑ ity81, and this indicated that caspase 1 might decrease compartments for pathogen- tive immune responses by exposing microbial antigens the metabolic rate of infected cells. To address whether and damage-associated to surveillance by the immune system. Together with the caspase 1‑mediated processing of these metabolic molecular patterns to activate signalling pathways that fact that caspase 1 activation is linked with the produc‑ enzymes occurred during infection, peritoneal macro‑ contribute to the host innate tion of mature IL‑1β and IL‑18 and the release of leader‑ phages of wild‑type and caspase 1‑deficient mice were immune response. less cytokines and DAMPs (such as HMGB1 and S100 infected with S. Typhimurium. As expected, aldolase was

2[TQRVQUKU #RQRVQUKU Pathogen-associated /GODTCPG /GODTCPG molecular pattern UYGNNKPI TWRVWTG #RQRVQVKEDQF[ (PAMP). A conserved pathogen HQTOCVKQP molecule that is usually 2TGUGTXCVKQP /GODTCPG %CURCUGCPF essential for microbial survival, QHOGODTCPG XGUKENGHQTOCVKQP CEVKXCVKQP and that contains either nucleic KPVGITKV[ %CURCUGCPF acid structures that are unique CEVKXCVKQP to microorganisms or cell wall components (such as lipopolysaccharide and %[VQUQNKE &0# UYGNNKPI %[VQUQNKE flagellin) that are not found in */)$ HTCIOGPVCVKQP mammalian cells. PAMPs are UJTKPMCIG &0# ligands for receptors of the 0WENGCT HTCIOGPVCVKQP host innate immune system. +.β 0WENGCT EQPFGPUCVKQP EQPFGPUCVKQP Damage-associated 4GNGCUGQH molecular pattern KPVTCEGNNWNCT 2#42ENGCXCIG 2#42ENGCXCIG (DAMP). A molecule that is EQPVGPVU produced or released from host cells upon cellular stress, damage or non-physiological +. cell death. DAMPs are also referred to as ‘alarmins’ and are 2TQKPȯCOOCVQT[ +OOWPQNQIKECNN[ thought to be responsible for EGNNFGCVJ UKNGPVEGNNFGCVJ the initiation and perpetuation of inflammatory responses and Figure 2 | main features of pyroptosis. The molecular mechanisms underlying pyroptosis are still poorly defined. tissue repair under Morphologically, pyroptotic cells are characterized by the early loss of plasma membrane integrity77, and this is accompanied non-infectious (sterile) by the shedding of membrane vesicles93. Pyroptotic and apoptotic cells share several prominent0CVWT featuresG4GXKGYU (shown^+OOWPQNQI[ in blue conditions. examples include boxes), including nuclear condensation and internucleosomal DNA fragmentation, cleavage of the DNA damage repair high-mobility group box 1 enzyme poly(ADP-ribose) polymerase 1 (PARP1) and activation of the executioner caspases caspase 3 and caspase 7 (ReF. 45). (HMGB1), ATP, uric acid and However, the volume of the cytoplasmic compartment of pyroptotic cells increases, whereas apoptosis is characterized by heat-shock proteins. 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 Unconventional protein 45 secretion form of cell death inherently pro-inflammatory . By contrast, apoptosis is usually considered to be immunologically silent The secretion of cytoplasmic because the cytoplasmic content is packaged in apoptotic bodies and these membrane-bound cell fragments are rapidly and nuclear proteins into the phagocytosed in vivo by neighbouring cells and resident phagocytes47. HMGB1, high mobility group box 1; IL, interleukin. extracellular space through an incompletely understood mechanism that does not processed in infected wild‑type macrophages, but not in only render the plasma membrane permeable to small require the translocation those lacking caspase 1 (ReF. 81). concurrently, the rate of inorganic ions84. Nevertheless, depending on the concen‑ apparatus of the classical endoplasmic reticulum glycolysis in the caspase 1‑deficient cells was markedly tration of these toxins and the targeted cell type, the dam‑ (eR)–Golgi secretion pathway. higher, further supporting an inhibitory role for caspase 1 age elicited by these toxins can range from irreparable cell Proteins that are secreted in the regulation of glycolysis. Because myeloid cells are destruction to temporal perforations that can quickly be through this route include highly dependent on glycolysis for ATP production82, resealed by the host cell’s dedicated repair machinery. interleukin-1α (IL-1α), IL-1β, caspase 1‑mediated inactivation of glycolysis enzymes Repairing bacterial toxin‑induced damage to the IL-18, fibroblast growth factor 2, galectin 1, galectin 3 might restrict intracellular pathogen replication by quickly plasma membrane requires the activation of lipid bio‑ and possibly high-mobility depleting energy sources and by preparing infected host genesis pathways, the molecular machinery of which is group box 1. cells to undergo pyroptosis. However, it remains to controlled by two transcription factors known as sterol be determined whether and to what extent caspase 1‑ regulatory element‑binding protein 1 (SReBP1) and Pyroptosis (ReF. 84) A specialized form of mediated inactivation of glycolysis enzymes contributes to SReBP2 . Interestingly, activation of SReBP1 programmed cell death that protection against bacterial pathogenicity in vivo. and SReBP2 is controlled by the NLRP3 and NLRc4 requires caspase 1 activity. inflammasomes in fibroblasts that have been intoxicated It is characterized by Cell survival with S. aureus α‑toxin or A. hydrophila aerolysin, or cytoplasmic swelling, early Another mechanism by which caspase 1 might protect infected with live Aeromonas trota bacteria83. This path‑ plasma membrane rupture, nuclear condensation and host cells is by repairing the damage caused by bacte‑ way is thought to promote host cell survival because inhi‑ internucleosomal DNA rial pore‑forming toxins that are released by pathogenic bition or transcriptional downregulation of SReBP1 and fragmentation. The bacteria83. The pores formed by these toxins can range SReBP2 enhanced cell death responses83. Inflammasome‑ cytoplasmic content is released significantly in diameter, depending on the nature of the induced activation of SReBPs is thought to be indirect, into the extracellular space, and this is thought to augment toxin. For example, S. aureus α‑toxin and Aeromonas but the caspase 1 substrates that drive this pathway inflammatory and repair hydrophila aerolysin typically produce holes in the host remain to be found. It would be interesting to determine responses. Pyroptosis occurs cell membrane with a diameter of only 2 nm, whereas whether caspase 1 activates SReBPs in macrophages in myeloid cells infected with Streptococcus pneumoniae and L. monocytogenes pro‑ infected with live A. hydrophila, because these bacteria pathogenic bacteria, and it duce toxins that can create perforations of up to 50 nm were recently shown to activate caspase 1 through the might affect cells of the central 84 85 nervous system and the in diameter . consequently, the latter toxins allow trans‑ NLRP3, but not the NLRc4, inflammasome . This might cardiovascular system under location of large proteins across the plasma membrane, reveal differential signalling mechanisms induced by the ischaemic conditions. whereas S. aureus α‑toxin and A. hydrophila aerolysin three A. hydrophila cytotoxins85.

Type III and type IV Caspase 7 activation the NLRP3 and NLRc4 inflammasomes in inducing secretion Activation of the NLRc4 inflammasome was recently proteolytic maturation of caspase 7 in activated immune Two of at least six specialized shown to restrict the intracellular replication of L. pneu- cells that were exposed to ATP and nigericin or infected secretion systems by which mophila, the causative agent of a severe form of bacte‑ with live S. Typhimurium51. In contrast to caspase 7, the Gram-negative pathogens can 61,86,87 deliver virulence factors into rial pneumonia known as Legionnaires’ disease . activation of caspase 3 was not affected in caspase 1‑ 51 eukaryotic host cells. Inflammasome activation in resistant mouse strains deficient macrophages , and this indicates that the Pathogenic bacteria such as results in the rapid caspase 1‑dependent delivery of activation of caspase 3 and caspase 7 is differentially reg‑ Shigella, Salmonella, Yersinia, L. pneumophila to lysosomes, where the bacteria are ulated during inflammation. These observations identi‑ Chlamydia and Pseudomonas degraded. By contrast, defective inflammasome activa‑ fied an alternative mechanism by which inflammasomes spp. all make use of a type III –/– –/– secretion system to infect host tion in Nlrc4 and Casp1 mice allows bacterial repli‑ might control bacterial infections. Indeed, caspase 7 cells and to modulate signalling cation in specialized intracellular vesicles that resemble activation downstream of the NLRc4 inflammasome pathways. By contrast, autophagosomes61,87. Notably, inflammasome‑mediated was subsequently reported in macrophages infected pathogens such as restriction of L. pneumophila replication proceeds with L. pneumophila79. Moreover, caspase 7‑deficient Helicobacter pylori, Legionella (ReFs 61,87) pneumophila and Bordetella independently of IL‑1β and IL‑18 , but the macrophages are less capable of restricting intracellular pertussis make use of a type IV caspase 1 substrates that are responsible for this proc‑ L. pneumophila replication, possibly owing to defects secretion system for the ess remain unclear. A proteome‑wide screen for new in the fusion of bacteria‑containing phagosomes with horizontal transfer of plasmid caspase 1 targets identified caspase 7, an effector caspase, lysosomes and the delayed induction of macrophage DNA containing antibiotic as a direct substrate of caspase 1, and biochemical studies cell death79. Importantly, caspase 1 and caspase 7 regu‑ resistance genes and to inject effector proteins into confirmed that caspase 7 is cleaved by caspase 1 after late L. pneumophila growth in the lungs of orally infected 61,79 eukaryotic host cells. the canonical activation sites Asp23 and Asp198 (ReF. 51). mice , demonstrating the importance of this inflamma‑ Importantly, studies in macrophages from Nlrp3–/–, some effector pathway in host defence against this bac‑ Glycolysis Nlrc4–/–, Asc–/– or Casp1–/– mice confirmed the role of terial pathogen. However, it remains to be determined A metabolic pathway that generates the cellular high-energy store ATP by oxidizing glucose to pyruvate. In eukaryotic cells, pyruvate is #EVKXG further oxidized into CO2 and ECURCUG %#4& %#4& H2O in a process known as ‘aerobic respiration’. This results in a net yield of 36–38 molecules of ATP per metabolized molecule of glucose. 5GETGVKQPQTTGNGCUGQH #EVKXCVKQP 5GETGVKQPQH %NGCXCIGQH %NGCXCIGQHCU 2TQECURCUG NGCFGTNGUUE[VQMKPGU QH54'$2U ITQYVJHCEVQTU 2#42CPF [GVWPKFGPVKȮGF Autophagosome CPF&#/2U IN[EQN[UKUGP\[OGU UWDUVTCVGU A double-membrane-bound vesicle that is used by eukaryotic cells to target .KRKF %JCPIGUKPEGNNWNCT /GODTCPG %CURCUG protein aggregates, damaged OGODTCPG GPGTI[UQWTEGU RGTOGCDKNK\CVKQP organelles and invading DKQIGPGUKU CPF&0# microorganisms for digestion HTCIOGPVCVKQP by lysosomal hydrolases. This catabolic process allows recycling of cellular components and is thought to +PȯCOOCVKQP 4GRCKTCPFJGCNKPI 2[TQRVQUKU 4GUVTKEVKQPQH contribute to cell death, cell .GIKQPGNNC survival during starvation, TGRNKECVKQPD[ cellular differentiation and host VCTIGVKPIVQ defence against infectious 4GOQXCNQH +OOWPG N[UQUQOGU KPVTCEGNNWNCT UWTXGKNNCPEG agents. TGRNKECVKQPPKEJGU QHOKETQDKCN EQORQPGPVU

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 0CVWTG4GXKGYU^+OOWPQNQI[ 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.

whether inflammasome‑mediated activation of caspase 7 might instruct the adaptive immune system regarding also restricts the replication of S. Typhimurium and other the systemic risk posed by the invading microbial agent. bacterial pathogens, and whether this inflammasome At an early phase of infection, activation of caspase 7 effector pathway is activated during viral infection. downstream of the inflammasome might halt pathogen replication in intracellular replication niches, while at Conclusions and perspectives the same time inflammasome‑mediated activation of It has become evident in recent years that inflamma‑ SReBPs could repair pathogen‑induced damage to the somes have important roles in innate immune signalling plasma membrane. when bacterial (or viral) loads fur‑ and host defence. In particular, our knowledge of how ther increase, inflammasomes can induce pyroptosis inflammasome complexes of distinct composition are and instruct macrophages and dendritic cells to initi‑ assembled in a stimulus‑dependent manner has grown ate the unconventional secretion or passive release of significantly. Now, the different effector mechanisms (in pro‑inflammatory cytokines, growth factors, DAMPs addition to IL‑1β and IL‑18 secretion) by which inflam‑ and microbial antigens to alert the immune system of masomes might contribute to immunity and host defence an imminent threat and to initiate repair responses. are also starting to emerge. As described above, recent In this context, the crucial role of pyroptosis in host studies have highlighted a range of new inflammasome defence was recently demonstrated in mice infected functions and effector mechanisms (FIG. 3). caspase 1 with the Gram‑negative bacterial pathogens L. pneu- has been shown to control the secretion of leaderless mophila and B. thailandensis34. Pyroptosis also confers cytokines and proteins such as IL‑1α and FGF2, as well resistance against Gram‑positive pathogens such as as the release of endogenous DAMPs such as HMGB1. B. anthracis in vivo65, but its role in clearing viral and Moreover, excessive caspase 1 activation in damaged neu‑ fungal infections remains to be determined. In addi‑ rons and infected myeloid cells induces pyroptotic cell tion, much remains to be learned regarding exactly how death. Furthermore, caspase 1 dampens the metabolic caspase 1 induces pyroptosis and how inflammasomes rate of infected cells by cleaving key enzymes of the glyco‑ regulate the unconventional secretion of leaderless pro‑ lysis pathway and regulates lipid metabolic pathways for teins. what are the crucial components of the molecular cell repair. Finally, activation of the executioner caspase 7 machinery driving the secretion of leaderless proteins downstream of inflammasomes contributes to restriction and how are they regulated by inflammasomes? Are of Legionella replication in infected macrophages. unconventional protein secretion and pyroptosis intrin‑ Interestingly, most of these emerging functions of sically linked or can they be uncoupled? what is the caspase 1 seem to operate independently of the canoni‑ relative contribution of each of these inflammasome cal substrates IL‑1β and IL‑18, and this indicates that effector mechanisms to innate and adaptive immune inflammasomes contribute to innate immune responses responses during microbial infections? Answering in a variety of ways. Indeed, these effector mechanisms these and other questions will undoubtedly illuminate probably function together to mount a fast and effec‑ intriguing new mechanisms by which inflammasomes tive innate immune response against the pathogen and contribute to host defence and immunity.

1. Villani, A. C. et al. Common variants in the NLRP3 11. Hoffman, H. M., Mueller, J. L., Broide, D. H., 21. Irmler, M. et al. Granzyme A is an interleukin 1β- region contribute to Crohn’s disease susceptibility. Wanderer, A. A. & Kolodner, R. D. Mutation of a converting enzyme. J. Exp. Med. 181, 1917–1922 Nature Genet. 41, 71–76 (2009). new gene encoding a putative pyrin-like protein (1995). 2. Zaki, M. H. et al. The NLRP3 inflammasome protects causes familial cold autoinflammatory syndrome 22. Maelfait, J. et al. Stimulation of Toll-like receptor 3 against loss of epithelial integrity and mortality during and Muckle–Wells syndrome. Nature Genet. 29, and 4 induces interleukin-1β maturation by caspase-8. experimental colitis. Immunity 32, 379–391 (2010). 301–305 (2001). J. Exp. Med. 205, 1967–1973 (2008). 3. Allen, I. C. et al. The NLRP3 inflammasome functions 12. Lamkanfi, M. & Dixit, V. M. The inflammasomes. 23. Mayer-Barber, K. D. et al. Caspase-1 independent as a negative regulator of tumorigenesis during colitis- PLoS Pathog. 5, e1000510 (2009). IL-1β production is critical for host resistance to associated cancer. J. Exp. Med. 207, 1045–1056 13. Kawai, T. & Akira, S. TLR signaling. Cell Death Differ. Mycobacterium tuberculosis and does not require (2010). 13, 816–825 (2006). TLR signaling in vivo. J. Immunol. 184, 3326–3330 4. Bauer, C. et al. Colitis induced in mice with dextran 14. Tschopp, J. & Schroder, K. NLRP3 inflammasome (2010). sulfate sodium (DSS) is mediated by the NLRP3 activation: the convergence of multiple signalling 24. Kanneganti, T. D. Central roles of NLRs and inflammasome. Gut 59, 1192–1199 (2010). pathways on ROS production? Nature Rev. Immunol. inflammasomes in viral infection. Nature Rev. 5. Dupaul-Chicoine, J. et al. Control of intestinal 10, 210–215 (2010). Immunol. 10, 688–698 (2010). homeostasis, colitis, and colitis-associated colorectal 15. Lamkanfi, M. & Dixit, V. M. Inflammasomes: 25. Sims, J. E. & Smith, D. E. The IL-1 family: regulators of cancer by the inflammatory caspases. Immunity guardians of cytosolic sanctity. Immunol. Rev. 227, immunity. Nature Rev. Immunol. 10, 89–102 (2010). 32, 367–378 (2010). 95–105 (2009). 26. Dinarello, C. A. Immunological and inflammatory 6. Jin, Y. et al. NALP1 in vitiligo-associated multiple 16. Gu, Y. et al. Activation of interferon-γ inducing factor functions of the interleukin-1 family. Annu. Rev. autoimmune disease. N. Engl. J. Med. 356, mediated by interleukin-1β converting enzyme. Immunol. 27, 519–550 (2009). 1216–1225 (2007). Science 275, 206–209 (1997). 27. Hoshino, T. et al. Cutting edge: IL-18-transgenic mice: 7. Martinon, F., Petrilli, V., Mayor, A., Tardivel, A. & 17. Kuida, K. et al. Altered cytokine export and apoptosis in vivo evidence of a broad role for IL-18 in Tschopp, J. Gout-associated uric acid crystals activate in mice deficient in interleukin-1β converting enzyme. modulating immune function. J. Immunol. 166, the NALP3 inflammasome. Nature 440, 237–241 Science 267, 2000–2003 (1995). 7014–7018 (2001). (2006). 18. Ghayur, T. et al. Caspase-1 processes IFN-γ-inducing 28. Nakanishi, K., Yoshimoto, T., Tsutsui, H. & Okamura, H. 8. Magitta, N. F. et al. A coding polymorphism in NALP1 factor and regulates LPS-induced IFN-γ production. Interleukin-18 regulates both Th1 and Th2 responses. confers risk for autoimmune Addison’s disease and Nature 386, 619–623 (1997). Annu. Rev. Immunol. 19, 423–474 (2001). type 1 diabetes. Genes Immun. 10, 120–124 (2009). 19. Li, P. et al. Mice deficient in IL-1β-converting enzyme 29. Weaver, C. T., Harrington, L. E., Mangan, P. R., 9. Larsen, C. M. et al. Interleukin-1-receptor antagonist are defective in production of mature IL-1β and Gavrieli, M. & Murphy, K. M. Th17: an effector CD4 in type 2 diabetes mellitus. N. Engl. J. Med. 356, resistant to endotoxic shock. Cell 80, 401–411 (1995). T cell lineage with regulatory T cell ties. Immunity 24, 1517–1526 (2007). 20. Joosten, L. A. et al. Inflammatory arthritis in caspase 1 677–688 (2006). 10. Agostini, L. et al. NALP3 forms an IL-1β-processing gene-deficient mice: contribution of proteinase 3 to 30. Harrington, L. E. et al. Interleukin 17-producing CD4+ inflammasome with increased activity in Muckle–Wells caspase 1-independent production of bioactive effector T cells develop via a lineage distinct from the autoinflammatory disorder. Immunity 20, 319–325 interleukin-1β. Arthritis Rheum. 60, 3651–3662 T helper type 1 and 2 lineages. Nature Immunol. (2004). (2009). 6, 1123–1132 (2005).

31. Hoffman, H. M. et al. Efficacy and safety of rilonacept 53. Lamkanfi, M. et al. Glyburide inhibits the Cryopyrin/ apoptosis in infected macrophages. Proc. Natl Acad. (interleukin-1 trap) in patients with cryopyrin- Nalp3 inflammasome. J. Cell Biol. 187, 61–70 (2009). Sci. USA 93, 9833–9838 (1996). associated periodic syndromes: results from two 54. Chen, Y., Smith, M. R., Thirumalai, K. & Zychlinsky, A. 77. Fernandes-Alnemri, T. et al. The pyroptosome: a sequential placebo-controlled studies. Arthritis A bacterial invasin induces macrophage apoptosis by supramolecular assembly of ASC dimers mediating Rheum. 58, 2443–2452 (2008). binding directly to ICE. EMBO J. 15, 3853–3860 inflammatory cell death via caspase-1 activation. 32. Lachmann, H. J. et al. Use of canakinumab in the (1996). Cell Death Differ. 14, 1590–1604 (2007). cryopyrin-associated periodic syndrome. 55. Hilbi, H. et al. Shigella-induced apoptosis is 78. Malireddi, R. K., Ippagunta, S., Lamkanfi, M. & N. Engl. J. Med. 360, 2416–2425 (2009). dependent on caspase-1 which binds to IpaB. Kanneganti, T. D. Cutting edge: proteolytic inactivation 33. Lamkanfi, M. et al. Inflammasome-dependent J. Biol. Chem. 273, 32895–32900 (1998). of poly(ADP-ribose) polymerase 1 by the Nlrp3 and release of the alarmin HMGB1 in endotoxemia. 56. Monack, D. M., Detweiler, C. S. & Falkow, S. Nlrc4 inflammasomes. J. Immunol. 185, 3127–3130 J. Immunol. 185, 4385–4392 (2010). Salmonella pathogenicity island 2-dependent (2010). This paper describes the role of the NLRP3 and macrophage death is mediated in part by the host This paper describes the inflammasome‑dependent NLRC4 inflammasomes in extracellular release of cysteine protease caspase-1. Cell. Microbiol. cleavage of PARP1 during pyroptosis. HMGB1. 3, 825–837 (2001). 79. Akhter, A. et al. Caspase-7 activation by the Nlrc4/Ipaf 34. Miao, E. A. et al. Caspase-1-induced pyroptosis is This paper reports that pyroptosis proceeds inflammasome restricts Legionella pneumophila an innate immune effector mechanism against independently of IL‑1β and IL‑18. infection. PLoS Pathog. 5, e1000361 (2009). intracellular bacteria. Nature Immunol. 11, 57. van der Velden, A. W., Velasquez, M. & Starnbach, This paper describes the role of caspase 7 1136–1142 (2010). M. N. Salmonella rapidly kill dendritic cells via a activation by the NLRC4 inflammasome in This paper describes the crucial role of caspase-1-dependent mechanism. J. Immunol. 171, restricting Legionella replication. caspase 1‑mediated cell death as a host defence 6742–6749 (2003). 80. Lakhani, S. A. et al. Caspases 3 and 7: key mediators mechanism against bacterial pathogens. 58. Suzuki, T. et al. Differential regulation of caspase-1 of mitochondrial events of apoptosis. Science 311, 35. Henry, T. & Monack, D. M. Activation of the activation, pyroptosis, and autophagy via Ipaf and 847–851 (2006). inflammasome upon Francisella tularensis infection: ASC in Shigella-infected macrophages. PLoS Pathog. 81. Shao, W., Yeretssian, G., Doiron, K., Hussain, S. N. & interplay of innate immune pathways and virulence 3, e111 (2007). Saleh, M. The caspase-1 digestome identifies the factors. Cell. Microbiol. 9, 2543–2551 (2007). 59. Miao, E. A. et al. Cytoplasmic flagellin activates glycolysis pathway as a target during infection and This paper describes the increased susceptibility caspase-1 and secretion of interleukin 1β via Ipaf. septic shock. J. Biol. Chem. 282, 36321–36329 of caspase 1‑deficient mice to infection with Nature Immunol. 7, 569–575 (2006). (2007). Francisella tularensis compared with mice lacking 60. Franchi, L. et al. Cytosolic flagellin requires Ipaf for This paper describes glycolysis enzymes as IL‑1β and IL‑18. activation of caspase-1 and interleukin 1β in substrates of caspase 1. 36. Trombetta, E. S. & Parodi, A. J. Quality control and salmonella-infected macrophages. Nature Immunol. 82. Cramer, T. et al. HIF-1α is essential for myeloid cell- protein folding in the secretory pathway. Annu. Rev. 7, 576–582 (2006). mediated inflammation. Cell 112, 645–657 (2003). Cell. Dev. Biol. 19, 649–676 (2003). 61. Amer, A. et al. Regulation of Legionella phagosome 83. Gurcel, L., Abrami, L., Girardin, S., Tschopp, J. & 37. Lee, M. C., Miller, E. A., Goldberg, J., Orci, L. & maturation and infection through flagellin and host van der Goot, F. G. Caspase-1 activation of lipid Schekman, R. Bi-directional protein transport between Ipaf. J. Biol. Chem. 281, 35217–35223 (2006). metabolic pathways in response to bacterial pore- the ER and Golgi. Annu. Rev. Cell. Dev. Biol. 20, 62. Miao, E. A. et al. Innate immune detection of the forming toxins promotes cell survival. Cell 126, 87–123 (2004). type III secretion apparatus through the NLRC4 1135–1145 (2006). 38. Nickel, W. & Rabouille, C. Mechanisms of regulated inflammasome. Proc. Natl Acad. Sci. USA 107, This paper reports that inflammasomes activate unconventional protein secretion. Nature Rev. Mol. 3076–3080 (2010). lipid metabolic pathways. Cell Biol. 10, 148–155 (2009). 63. Sutterwala, F. S. et al. Immune recognition of 84. Gonzalez, M. R., Bischofberger, M., Pernot, L., 39. Rubartelli, A., Cozzolino, F., Talio, M. & Sitia, R. Pseudomonas aeruginosa mediated by the IPAF/ van der Goot, F. G. & Freche, B. Bacterial pore-forming A novel secretory pathway for interleukin-1β, a protein NLRC4 inflammasome. J. Exp. Med. 204, toxins: the (w)hole story? Cell. Mol. Life Sci. 65, lacking a signal sequence. EMBO J. 9, 1503–1510 3235–3245 (2007). 493–507 (2008). (1990). 64. Boyden, E. D. & Dietrich, W. F. Nalp1b controls mouse 85. McCoy, A. J. et al. Cytotoxins of the human pathogen 40. Sutterwala, F. S. et al. Critical role for NALP3/CIAS1/ macrophage susceptibility to anthrax lethal toxin. Aeromonas hydrophila trigger, via the NLRP3 Cryopyrin in innate and adaptive immunity through Nature Genet. 38, 240–244 (2006). inflammasome, caspase-1 activation in macrophages. its regulation of caspase-1. Immunity 24, 317–327 65. Terra, J. K. et al. Cutting edge: resistance to Bacillus Eur. J. Immunol. 40, 2797–2803 (2010). (2006). anthracis infection mediated by a lethal toxin sensitive 86. Lamkanfi, M. et al. The Nod-like receptor family 41. Keller, M., Ruegg, A., Werner, S. & Beer, H. D. allele of Nalp1b/Nlrp1b. J. Immunol. 184, 17–20 member Naip5/Birc1e restricts Legionella Active caspase-1 is a regulator of unconventional (2010). pneumophila growth independently of caspase-1 protein secretion. Cell 132, 818–831 (2008). 66. Munoz-Planillo, R., Franchi, L., Miller, L. S. & activation. J. Immunol. 178, 8022–8027 (2007). This paper characterizes the role of caspase 1 in Nunez, G. A critical role for hemolysins and bacterial 87. Zamboni, D. S. et al. The Birc1e cytosolic pattern- the secretion of IL‑1α and FGF2. lipoproteins in Staphylococcus aureus-induced recognition receptor contributes to the detection and 42. Becker, C. E., Creagh, E. M. & O’Neill, L. A. activation of the Nlrp3 inflammasome. J. Immunol. control of Legionella pneumophila infection. Nature Rab39a binds caspase-1 and is required for 183, 3942–3948 (2009). Immunol. 7, 318–325 (2006). caspase-1-dependent interleukin-1β secretion. 67. Mariathasan, S. et al. Cryopyrin activates the 88. Broz, P. et al. Redundant roles for inflammasome J. Biol. Chem. 284, 34531–34537 (2009). inflammasome in response to toxins and ATP. receptors NLRP3 and NLRC4 in host defense 43. Salvesen, G. S. & Riedl, S. J. Caspase mechanisms. Nature 440, 228–232 (2006). against Salmonella. J. Exp. Med. 207, 1745–1755 Adv. Exp. Med. Biol. 615, 13–23 (2008). 68. Sauer, J. D. et al. Listeria monocytogenes triggers (2010). 44. Riedl, S. J. & Shi, Y. Molecular mechanisms of caspase AIM2-mediated pyroptosis upon infrequent 89. Fischer, U., Janicke, R. U. & Schulze-Osthoff, K. regulation during apoptosis. Nature Rev. Mol. Cell bacteriolysis in the macrophage cytosol. Many cuts to ruin: a comprehensive update of Biol. 5, 897–907 (2004). Cell Host Microbe 7, 412–419 (2010). caspase substrates. Cell Death Differ. 10, 76–100 45. Lamkanfi, M. & Dixit, V. M. Manipulation of host cell 69. Fernandes-Alnemri, T. et al. The AIM2 inflammasome (2003). death pathways during microbial infections. Cell Host is critical for innate immunity to Francisella tularensis. 90. Lamkanfi, M., Festjens, N., Declercq, W., Microbe 8, 44–54 (2010). Nature Immunol. 11, 385–393 (2010). Vanden Berghe, T. & Vandenabeele, P. Caspases in cell 46. Strasser, A., O’Connor, L. & Dixit, V. M. Apoptosis 70. Rathinam, V. A. et al. The AIM2 inflammasome is survival, proliferation and differentiation. Cell Death signaling. Annu. Rev. Biochem. 69, 217–245 (2000). essential for host defense against cytosolic bacteria Differ. 14, 44–55 (2007). 47. Taylor, R. C., Cullen, S. P. & Martin, S. J. Apoptosis: and DNA viruses. Nature Immunol. 11, 395–402 91. Boatright, K. M. et al. A unified model for apical controlled demolition at the cellular level. Nature Rev. (2010). caspase activation. Mol. Cell 11, 529–541 (2003). Mol. Cell Biol. 9, 231–241 (2008). 71. Jones, J. W. et al. Absent in melanoma 2 is required 92. Martinon, F. & Tschopp, J. Inflammatory caspases 48. Kazama, H. et al. Induction of immunological for innate immune recognition of Francisella and inflammasomes: master switches of inflammation. tolerance by apoptotic cells requires caspase- tularensis. Proc. Natl Acad. Sci. USA 107, Cell Death Differ. 14, 10–22 (2007). dependent oxidation of high-mobility group box-1 9771–9776 (2010). 93. Pizzirani, C. et al. Stimulation of P2 receptors causes protein. Immunity 29, 21–32 (2008). 72. Wu, J., Fernandes-Alnemri, T. & Alnemri, E. S. release of IL-1β-loaded microvesicles from human 49. Savill, J., Dransfield, I., Gregory, C. & Haslett, C. Involvement of the AIM2, NLRC4, and NLRP3 dendritic cells. Blood 109, 3856–3864 (2007). A blast from the past: clearance of apoptotic cells inflammasomes in caspase-1 activation by Listeria regulates immune responses. Nature Rev. Immunol. monocytogenes. J. Clin. Immunol. 30, 693–702 Acknowledgements 2, 965–975 (2002). (2010). This work was supported by European Union Framework 50. Mariathasan, S. et al. Differential activation of the 73. Warren, S. E. et al. Cutting edge: cytosolic bacterial Program 7 (Marie Curie grant 256432) and by the Fund for inflammasome by caspase-1 adaptors ASC and Ipaf. DNA activates the inflammasome via Aim2. Scientific Research – Flanders. Nature 430, 213–218 (2004). J. Immunol. 185, 818–821 (2010). 51. Lamkanfi, M. et al. Targeted peptidecentric 74. Tsuchiya, K. et al. Involvement of absent in melanoma 2 Competing interests statement proteomics reveals caspase-7 as a substrate of the in inflammasome activation in macrophages infected The author declares no competing financial interests. caspase-1 inflammasomes. Mol. Cell. Proteomics with Listeria monocytogenes. J. Immunol. 185, 7, 2350–2363 (2008). 1186–1195 (2010). This paper describes caspase 7 as a downstream 75. Mariathasan, S., Weiss, D. S., Dixit, V. M. & FURTHER INFORMATION effector of the NLRP3 and NLRC4 inflammasomes. Monack, D. M. Innate immunity against Francisella Mohamed Lamkanfi’s homepage: http://www.vib.be/en/ 52. Fink, S. L. & Cookson, B. T. Caspase-1-dependent tularensis is dependent on the ASC/caspase-1 axis. research/scientists/Pages/Mohamed-Lamkanfi-Lab.aspx pore formation during pyroptosis leads to osmotic J. Exp. Med. 202, 1043–1049 (2005). lysis of infected host macrophages. Cell. Microbiol. 76. Monack, D. M., Raupach, B., Hromockyj, A. E. & All lInks Are ACtIve In the onlIne pdf 8, 1812–1825 (2006). Falkow, S. Salmonella typhimurium invasion induces