Molecular mechanisms involved ininflammasome activationClare Bryant1 and Katherine A. Fitzgerald2

1 Department of Veterinary Medicine, University of Cambridge, Cambridge, UK2 Division of Infectious Diseases and Immunology, Department of Medicine, University of Massachusetts Medical School,

Worcester, MA 01605, USA

Review

Germline-encoded pattern recognition receptors (PRRs)sense microbial or endogenous products released fromdamaged or dying cells and trigger innate immunity. Inmost cases, sensing of these signals is coupled to signaltransduction pathways that lead to transcription ofimmune response genes that combat infection or leadto cell death. Members of the NOD-like receptor (NLR)family assemble into large multiprotein complexes,termed inflammasomes. Inflammasomes do notregulate transcription of immune response genes, butactivate caspase-1, a proteolytic enzyme that cleavesand activates the secreted cytokines interleukin-1band interleukin-18. Inflammasomes also regulate pyrop-tosis, a caspase-1-dependent form of cell death that ishighly inflammatory. Here, we review exciting recentdevelopments on the role of inflammasome complexesin host defense and the discovery of a new DNA sensinginflammasome, and describe important progress madein our understanding of how inflammasomes are acti-vated. Additionally, we highlight how dysregulation ofinflammasomes contributes to human disease.

IntroductionFor eukaryotic hosts to survive infection, the immunesystem must deploy an arsenal of defense measures tocombat invading microbes. The innate immune system isthe first line of such defensemechanisms. Innate immunityfunctions to control infection and eliminate pathogens, aswell as to marshal the T- and B-cell responses of adaptiveimmunity. Considerable progress has been made in recentyears in our understanding of how microbial invaders arerecognized by the innate immune system and how thissensing translates into signaling pathways that culminatein the transcriptional regulation of immune responsegenes. These include pro-inflammatory cytokines such astumor necrosis factor, interleukin-1b, antimicrobial pep-tides, adhesion molecules and type I interferons [1,2].Several classes of germline-encoded pattern recognitionreceptors (PRRs) have now been implicated in innatedefenses. These include the Toll-like receptors (TLRs)[3], the C-type lectin receptors (CLRs) [4], the RIG-likehelicases (RLRs) [5], cytosolic DNA sensors [6–11] andmembers of the NOD-like receptor (NLR) family [12].These different PRR families, a brief description of theligands they sense and their role in the immune responseare discussed in Box 1 and Table 1. A schematic outline of

Corresponding author: Fitzgerald, K.A. (kate.fitzgerald@umassmed.edu).

0962-8924/$ – see front matter � 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.tcb.2009.0

the structural architecture of these different receptorfamilies is also shown in Figure 1.

Individual PRRs recognize products from all the majorclasses of microbial pathogens, including bacteria, viruses,yeast and parasites. Accumulating evidence reveals that,in addition to sensing microbial products, an importantrole for many of these same sensors is their ability to detectthe endogenous products (referred to as danger signals ordanger-associated molecular patterns, DAMPs) that arereleased from damaged or dying cells [13]. Tissue damageand disruption of cellular integrity are hallmarks of in-fection and inflammation. Damaged or dying cells releaseendogenous mediators such as nucleic acids, ATP and uricacid crystals, all of which trigger many of the sameresponses that are induced upon detection of microbesduring innate immunity [13]. These responses, however,are detrimental to the host, often contributing to inflam-mation. This is particularly true in the case of the NLRs. Acase in point is the NLRP3 inflammasome, where excitingnew research has uncovered a broad range of microbial,host and environmental triggers of NLRP3. Most recently,discovery of the AIM2 inflammasome has revealed a newtype of inflammasome complex which does not contaimNLR proteins and binds to double stranded DNA directly.The identification of the AIM2 inflammasome may havedirect relevance for host defense, as well as autoimmunediseases like Systemic Lupus Erythematosis (SLE). In thisreview we will discuss our current understanding ofinflammasome components and the ligands they recognizebefore describing recent developments in the field concern-ing how these pathways are turned on. Finally we willbriefly discuss the role of these proteins in human disease,and suggest how manipulation of these pathways couldhave important implications for disease protection.

The NOD like receptor (NLR) familyThe NLRs are a large family of cytosolic sensors (23members in humans, 34 members in mice), whose crucialrole in the immune system is now well accepted. The mainfunction of the NLRs appears to be to regulate the pro-duction of the proinflammatory cytokines interleukin-1b

and IL-18. IL-1b is released by many cell types and is animportant mediator of inflammation during infection[14,15]. Interleukins are a group of cytokines (secretedsignaling molecules) produced by a wide variety of cells.Interleukins promote the development and differentiationof T, B, and hematopoietic cells, and play essential roles in

6.002 Available online 26 August 2009 455

Box 1. Pattern recognition receptors

A diversity of pattern recognition receptors (PRRs) have been

uncovered in the mammalian genome over the last 10 years. PRRs

are host proteins found on the cell surface and in distinct subcellular

compartments including the endosomal–lysosomal compartment or

the cytosol. Some of the Toll-like Receptors (TLRs) as well as the C-

type lectin receptors are found on the cell surface. Addtionally, the

endosomal compartment contains some TLRs that sense nucleic

acids. Cytosolic PRRs include the RIG-like RNA helicases, the NOD

(Nucleotide Oligomerization Domain)-like receptors (NLRs) and

DNA sensors such as AIM2 and DAI. PRRs were initially shown to

discriminate self from non-self, recognizing microbial products or

pathogen-associated molecular patterns (PAMPs) to stimulate

innate immunity to fight infection. More recently, this distinction

is proving to be more complicated. It is now well appreciated that

endogenous ligands, released from dying or damaged cells also

trigger these PRRs. Sensing of endogenous danger signals or

danger-associated molecular patterns are likely to be important in

the aeitology of a wide range of diseases ranging from autoimmu-

nity to cancer. The Drosophila Toll receptor was the first PRR to be

identified, although it recognizes pathogens (fungi) indirectly

through an endogenous protein, spatzel. This discovery was

followed rapidly by the identification of mammalian TLRs and

subsequently by other PRR families. The TLRs comprise leucine-rich

repeats (LRR) important in ligand recognition, and a cytoplasmic

signaling domain, the Toll-IL1 resistance domain. NLRs, also

contain LRR domains as well as nucleotide oligomerization

domains, and signaling domains that can be either a CARD, Pyrin

or Baculovirus Inhibitor of Apoptosis Repeat (BIR) domain. DNA

sensors have also been identified recently. One of these is DAI, also

called Zbo1, a Z-DNA binding protein. A second DNA sensor AIM2 is

a member of the HIN200 family, and, in addition to the HIN200

domain, contains a pyrin domain with which it recruits downstream

effectors similar to that of the NLRPs.

Figure 1. Schematic representation of the basic structure of PRRs. The basic

structural domains of different PRRs are compared in diagrammatic form. LRR=

leucine rich repeats; TIR=Toll/interleukin-1 receptor interacting domain; CLR=C-

type lectin receptor domain; ITAM=Immunoreceptor tyrosine-based activation

motif; CARD=caspaserecruitment and activation domain; Helicase=helicase

domain; PYD=pyrin domain;NACHT=nucleotide binding and oligomerisation

domain, BIR=Baculovirus inhibitor ofapoptosis repeat; HIN200=Hin 200 domain.

Z=Z-DNA binding domain.

Review Trends in Cell Biology Vol.19 No.9

both innate and adaptive immunity. Interleukin-1 b (IL-1b) was originally identified as the endogenous pyrogen.Exogenous IL-1b causes fever in experimental animals. IL-1b has multiple functions. It is an important mediator ofthe inflammatory response, and is involved in a variety ofcellular activities, including cell proliferation, differen-tiation, and apoptosis. The genes encoding IL-1b and eightother interleukin 1 family members form a cytokine genecluster on chromosome 2.

The synthesis, processing and release of IL-1b are con-trolled tightly. First, accumulation of intracellular stores ofpro-IL-1b occurs via transcriptional regulation (seeFigure 2). This is followed by cleavage of pro-IL-1b, andrelease of the mature cytokine. These events require atleast two distinct stimuli. An initial microbial stimuluspropagated through innate PRRs (e.g. through the type Itransmembrane receptors, for example, TLR4) causesaccumulation of intracellular stores of pro-IL-1b. The mol-ecular mechanisms regulating the transcription of pro-IL-1b are still being elucidated, althoughNF-kB andmitogen-activated protein (MAP) kinase signaling have both beenimplicated [16,17]. In addition to the engagement of theTLRs, other PRRs should also trigger NF-kB-dependentgene transcription; therefore, it is likely that any of thesePRRs could provide the initial NFkB signal required forthis response. The second event leading to the cleavage ofpro-IL-1b involves multiprotein complexes, commonlyreferred to as ‘inflammasomes’. As will be discussed below,inflammasomes are activated by a number of pathogens,although in most cases, the exact activating pathogenassociated molecular pattern (PAMP) has not been well

456

defined. In addition to microbial products, endogenousdanger signals released from damaged tissue and environ-mental insults also trigger inflammasome signaling.

Inflammasomes are multiprotein platforms, with a mol-ecular mass of at least 700 kDa [18]; they control theactivation of the cysteinyl aspartate protease caspase-1and the cleavage of pro-IL-1b, enabling the release of theactive mature 17-kDa cytokine [2,19]. Caspases areresponsible for crucial aspects of inflammation and celldeath and can be broadly divided into two classes based ontheir substrate specificity: those that are pro-apoptotic and

Table 1. The cytosolic PRRs and their ligands (modified from Franchi et al., 2008 [19])

Human Mouse Other names and aliases Potential ligands NLR family

CIITA* Cllta NLRA; MHC2TA; C2TA

Nlra; MHC2TA; C2TA NLRA

NAIP NLRB1; BIRC1; CLR5.1

Naip1 Birc1a

Naip2 Birc1b

Naip3 Birc1c NLRB

Naip4 Birc1d

Naip5 Birc1e Flagellin from Legionella

Naip6 Birc1f

Naip7 Birc1g

NOD1 NLRC1; CARD4; CLR7.1 GM-tripeptide NLRC

g-d-Glu-DAP(iEDAP)

d-lactyl-l-Ala-g-Glu-meso-DAP-Gly

(FK156)

heptanolyl-g-Glu-meso-DAP-Ala

(FK565)

Nod1 Nlrc1; Card4

NOD2 NLRC2; CARD15; CD; BLAU; MDP NLRC

IBD1; PSORAS1; CLR16.3

MurNAc-l-Ala-g-d-Glu-l-Lys

(M-TRILys)

Nod2 Nlrc2; Card15

NLRC3 NOD3; CLR16.2 NLRC

Nlrc3 CLR16.2

NLRC4 CARD12; CLAN; CLR2.1; IPAF Flagellin from Salmonella, Legionella,

Listeria, Pseudomonas

LRC

Nlrc4 Card12; CLAN; Ipaf

NLRC5 NOD27; CLR16.1

NIrc5

NLRP1 NALP1; DEFCAP; NAC; CARD7; MDP, Lethal Toxin ? NLRP

CLR17.1

Nlrp1a NALP1a

Nlrp1b NALP1b Lethal toxin

Nlrp1c NALP1c

NLRP2 NALP2; PYPAF2; NBS1; PAN1; NLRP

CLR19.9

Nlrp2 Pypaf2; Nbs1; Pan1

NLRP3 CIAS1; PYPAF1; Cryopyrin; NLRP

NALP3; CLR1.1 Sendai virus

Influenza virus

Adenovirus

Candida albicans

Saccharomyces cerevisiae

MDP

Nigericin

Maitotoxin

ATP

MSU

ATP

Silica

Asbestos

Alum

b-amyloid

Nlrp3 Cias1; Pypaf1; Cryopyrin;

Nalp3; Mmig1

NLRP4 NALP4; PYPAF4; PAN2; RNH2; NLRP

CLR19.5

Nlrp4a Nalp4a; Nalp-eta; Nalp9D

Nlrp4b Nalp4b; Nalp-gamma; Nalp9E

Nlrp4c Nalp4c; Nalp-alpha; Rnh2

Nlrp4d Nalp4d; Nalp-beta

Nlrp4e Nalp4e; Nalp-epsilon

Nlrp4f Nalp4f; Nalp-kappa; Nalp9F

Nlrp4g Nalp4g

NLRP5 NALP5; PYPAF8; MATER; PAN11; NLRP

CLR19.8

Nlrp5 Mater; Op1

NLRP6 NALP6; PYPAF5; PANS; CLR11.4 NLRP

Nlrp6

Review Trends in Cell Biology Vol.19 No.9

457

Table 1 (Continued )

Human Mouse Other names and aliases Potential ligands NLR family

NLRP7 NALP7; PYPAF3; NOD12; PAN7; NLRP

CLR19.4

NLRP8 NALP8; PAN4; NOD16; CLR19.2 NLRP

NLRP9 NALP9; NOD6; PAN12; CLR19.1

Nlrp9a Nalp9a; Nalp-theta NLRP

Nlrp9b Nalp9b; Nalp-delta

Nlrp9c Nalp9c; Nalp-zeta

NLRP 10 NALP10; PAN5; NOD8; PYNOD; NLRP

CLR11.1

Nlrp10 Nalp10; Pynod

NLRP 11 NALP11; PYPAF6; NOD17; NLRP

PAN10; CLR19.6

NLRP12 NALP12; PYPAF7; Monarch1; NLRP

RNO2; PAN6; CLR19.3

Nlrp12 Nalp12

NLRP13 NALP13; NOD14; PAN13; NLRP

CLR19.7

NLRP14 NALP14; NOD5; PAN8; CLR11.2 NLRP

Nlrp14 Nalp14; Nalp-iota; GC-LRR

NLRX1 NOD9; CLR11.3 NLRX

AIM2 DNA HIN200

MDNA DNA (?) HIN200

IFI16 DNA (?) HIN200

IFIX DNA (?) HIN200*Human Genome Organization Gene Nomenclature Committee (HGNC)-approved symbol.

Figure 2. Mechanisms regulating IL-1b production. Generation of IL-1b requires a priming signal, often from PRRs such as TLRs that activate NF-kB and NF-kB-dependent

transcription of pro-IL-1b. The pro-IL-1b is then cleaved into the active, mature 17 kDa cytokine by caspase-1. NLR containing inflammasomes activate caspase-1. NLRs such

as NLRP3 oligomerize upon activation (by danger signals such as those shown in the box) and recruit the adapter molecule ASC that subsequently recruits and activates

caspase-1.

Review Trends in Cell Biology Vol.19 No.9

458

Review Trends in Cell Biology Vol.19 No.9

those that are pro-inflammatory. Caspase-1 is part of thislatter group that also includes caspase-4, caspase-5, cas-pase-11 and caspase-12 (reviewed in [20]). The require-ment for two distinct stimuli to regulate IL-1b productionensures that IL-1b (or the related cytokine IL-18) is notinappropriately released, since this could have deleteriousconsequences for the host. Indeed, excess production of IL-1b is associated with a number of hereditary periodic feversyndromes, as well as autoimmune and inflammatorydiseases such as gout and rheumatoid arthritis (see below)[21,22]. Recently, additional levels of crosstalk betweenPRRs and NLRP3 inflammasome activation have revealedthat NF-kB activation by TLRs as well as by cytokinereceptors such as the TNF-receptor not only control IL-1b gene transcription, but additionally turn on NLRP3transcription, thereby licensing NLRP3 inflammasomeactivation (Latz, et al. and Nunez et al., manuscripts inpress).

The NLRs have a tripartite structure, consisting of a C-terminal leucine-rich repeat domain, a central nucleotide-binding oligomerization (NOD or NACHT) domain, and avariable N-terminal protein–protein interaction domain,that can be either a caspase recruitment and activationdomain (CARD), a Pyrin domain (PYD), or a baculovirusinhibitor of apoptosis repeat domain (BIR) [19,23] (seeFigure 1 and an example of the NLRP3 inflammasomein Figure 2). The leucine-rich repeat domain has beenimplicated in ligand sensing and autoregulation of theNLRs; however, the ability of the NLRs to bind directlyto their ligands has not yet been demonstrated. The NOD(or NACHT) domain has similarity to the NB-ARCmotif ofAPAF1, a mediator of apoptosis. In APAF1, this domainmediates the dATP/ATP-dependent oligomerization ofAPAF1 upon binding to cytochrome c, leading to the apop-totic cascade. The PYD, CARD or BIR domains facilitatedownstream signaling through protein–protein inter-actions. Inflammasome complexes assemble upon acti-vation by an appropriate stimulus (discussed in detailbelow), leading to the multimerization of the adaptormolecule ASC. Subsequently, procaspase-1 is recruitedto ASC by means of interactions between the CARDdomains of ASC and that of caspase-1. These events leadto the auto-cleavage of caspase-1. The two resulting sub-units p10 and p20 assemble into the active caspase-1 thatthen cleaves IL-1b. These events are detailed in Figure 2.

The NLRP1, NLRC4 and NLRP3 inflammasomesFour inflammasome complexes have been partially charac-terized to date. The first to be identified was the NLRP1(NALP1) inflammasome, comprising NLRP1, caspase-1,caspase-5 and the adaptor proteins, ASC and CARDINAL[18]. Muramyl dipeptide (MDP), a derivative of bacterialpeptidoglycan triggers formation of the NLRP1 inflamma-some [24], and the Nlrp1b gene (one of three genes encod-ing NLRP1 inmice) has been linked to sensing of the lethaltoxin secreted by Bacillus anthracis [25].

The NLRC4 (also called IPAF or CARD12) inflamma-some consists of NLRC4, ASC, caspase-1 as well as theBIR-domain-containing protein NAIP5. NLRC4 recog-nizes bacterial flagellin from Salmonella and Pseudomo-nas species and causes activation of caspase-1 [26].

NAIP5 senses flagellin from Legionella pneumophila[27]. NLRC4 and NAIP5 appear to recognize a similarregion of flagellin (C-terminal D0 region of the protein)[27]. Sensing of flagellin in the cytosol seems to be animprobable mechanism of bacterial recognition, yet somepathogens, such as Salmonella enterica serovar Typhi-murium, directly inject flagellin into the cytosol of cells[28]. NAIP5 requires NLRC4 to activate caspase-1; how-ever NLRC4 can activate caspase-1 independently ofNAIP5 [27]. Whether ASC is required for signaling bythe NLRC4 inflammasome is somewhat controversial[29–32]. Since NLRC4 itself contains a CARD domain,it could recruit caspase-1 directly without the need forASC.Instead, ASC might modulate the activity of thisinflammasome. Interestingly, the NLRC4 inflammasomeis also activated by bacteria that are not flagellated (suchas Shigella flexneri [33]). Mycobacterium tuberculosis hasalso been shown to block NLRC4 inflammasome acti-vation. Mutants in the mycobacterial Zn2+ metallopro-tease, zmp1, activate the NLRC4 inflammasome, whilewild type bacteria fail to trigger this response [34]. Thesedata suggest that NLRC4 may recognize additionalligands besides flagellin, although the identity of thesemolecules remains to be uncovered.

By far the best-characterized inflammasome is thatconsisting of NLRP3 (also called NALP3 or cryopyrin;see Table 1), ASC and caspase-1. Numerous chemicallyand structurally diverse stimuli are now known to triggerthis inflammasome. These include viruses such as Sendai[35], influenza [36–38] and adenovirus strains used in genetherapy [39], fungi such as Candida albicans and Sacchar-omyces cerevisiae [40] and bacteria such as Staphylococcusaureus and Listeria monocytogenes [41]. More recent stu-dies have also indicated a role for IPAF and a third ASC-dependent pathway for caspase-1 activation in response toL. monocytogenes [42]. Microbial derivatives such as MDPand bacterial pore-forming toxins also trigger NLRP3activation. Additional ligands include extracellular ATP[43], monosodium urate crystals (MSU) [44,45], amyloid-b[46] and various environmental insults (e.g. silica, asbes-tos) [47,48]. It is unclear, however, how NLRP3 can detectsuch a diversity of stimuli. Because there is no evidencethat any of these ligands bind directly, it has beensuggested that NLRP3 activation is indirect. A major goalis to determine how NLRP3 is activated by these structu-rally diverse ligands.

Molecular mechanisms of inflammasome activationStudies aimed at addressing the upstream signals trigger-ing the indirect activation of NLRP3 by different ligandshave revealed new insights into how these PRRs function.Based on these findings, a number of mechanisms havebeen proposed. One model implicates the ATP-gated P2X7receptor (P2X7R) [43], which is an ATP-gated ion channelresponsible for potassium conductance through the cellmembrane [49]. ATP-dependent activation of NLRP3has also been linked to another type of channel, pan-nexin-1 [50–52]. Extracellular ATP, through the activationof P2X7R, opens a pore via pannexin-1 [50–52], allowingflorescent MDP to translocate from an intracellular ves-icular compartment to the cytosol to induce activation of

459

Review Trends in Cell Biology Vol.19 No.9

the NLRP3 inflammasome [53]. A second model proposedsuggests that cytosolic delivery of microbial moleculesoccurs independently of pannexin 1 [50]. The formationof pores to disrupt intracellular K+ and to deliver bacterialmolecules to the cytosol is an attractive mechanism, whichcould help clarify how a number of bacteria that do notreside in the cytosol trigger cytosolic sensors such asNLRP3. Changes in intracellular K+ has also been shownto contribute to activation of NLRP1 [54], but may [28] ormay not be important for activation of the NLRC4 inflam-masome [44,55].

Additional models of NLRP3 inflammasome activationhave been proposed in the case of particulate ligands andcrystalline materials. Particulates such as asbestos[47,48], silica [47], MSU [56], and adjuvants includingalum [48,57–60], biodegradable poly(lactide-co-glycolide)and polystyrene microparticles [57] have all been shownto activate the NLRP3 inflammasome. Activation by asbes-tos and silica has been linked to the production of reactiveoxygen species (ROS). ROS production is triggered as aresult of incomplete or ‘frustrated phagocytosis [47]’, aprocess where the particle size is too large to be engulfed.Consistent with a role for ROS generation, activation ofcaspase-1 by asbestos was attenuated in cells treated withN-acetyl cysteine (NAC), an inhibitor of ROS generation.Additionally, targeting of the NADPH oxidase subunitp22phox, (which plays a key role in ROS formation) using

Figure 3. Models of NLRP3 inflamamsome activation. The activation of NLRP3 has be

ligands. Stimulation of P2X7R by extracellular ATP induces the activation of a cation

opening of the pannexin-1 pore, which may enable the cytosolic delivery of microbial m

evidence that it may involve generation of ROS and additional studies suggest that de

cathepsin B (in the case of silica, urate crystals and fibrillar b amyloid) are critical.

460

small interfering RNA also attenuated this response [47].A second model argues against a role for ROS. In this case,silica, asbestos and b-amyloid were phagocytosed nor-mally; however, once inside the phagosomal lysosomalcompartment, the large crystals led to damage or disinteg-ration of the lysosomal membrane [46,48,57]. Crystal-induced NLRP3 activation in this case has been shownto require acidification of the phagosome and depends inpart on the lysosomal protease cathepsin B [46,48]. Inhibi-tors of cathepsin B attenuatedNLRP3 activation, althoughthe defect was only partial. In agreement with such amodel, sterile lysosomal rupture itself (in the absence ofany microbial stimulus) was sufficient to induce NLRP3activation, strongly supporting such a model [48]. Theselatter observations indicate that the NLRP3 inflamma-some may sense the disintegration of lysosomes and inter-pret this as an endogenous danger signal. These twomodels are probably not mutually exclusive, and bothmechanisms may contribute. Future studies are likely toclarify these issues. A schematic depiction of these differ-ent models of NLRP3 activation is shown in Figure 3.

Althoughwe are beginning to understand howNLRs areturned on, very little is known about the counter-regulat-ory mechanisms that are involved in curbing NLR activity.An unexpected mechanism of inflammasome inhibitionwas reported recently by Tschopp and colleagues whoshowed that effector and memory T cells block NLRP1

en shown to involve the ATP-gated P2X7R, pore-forming molecules or particulate

channel that mediates potassium efflux. In addition, P2X7R activation promotes

olecules such as MDP. How particulate ligands activate NLRP3 is less clear. There is

stabilization of the lysosomal membrane and activation of the lysosomal protease

Figure 4. The AIM2 Inflammasome. The HIN200 and pyrin domain containing

protein AIM2 protein engages dsDNA through its HIN200 domain and recruits ASC

by means of a PYD domain. The AIM2 inflammasome is the only inflammasome

known to directly bind to the ligand that triggers inflammasome formation. Upon

binding dsDNA AIM2 oligomerizes recruiting ASC, which recruits and activates

caspase-1 followed by caspase-1 dependent cleavage of pro-IL-1b and pro-IL-18 as

well as pyroptosis, an inflammatory caspase-1 dependent form of cell death.

Box 2. Pyroptosis

Cell death can occur by at least four different processes: Apoptosis,

oncosis, autophagy and caspase-1-induced cell death (pyroptosis).

Pyroptosis differs from apoptosis (caspase-dependent programmed

cell death), oncosis (toxin- or damage-driven lytic cell death

independent of caspase activity) and autophagy (self-digestion of

cells, ultimately resulting in cellular phagocytosis), in that it is an

inflammatory process stimulated by a wide range of microbes and

pathological events (such as stroke, cardiovascular disease and

cancer). The mechanisms triggering pyroptosis involve cleavage

and activation of caspase 1, rapid pore formation in the plasma

membrane to allow water to enter the cell, causing osmotic lysis and

release of pro-inflammatory cellular contents, along with the release

of the caspase 1-associated cytokines such as IL-1b and IL-18. DNA

cleavage also occurs in pyroptosis by an unidentified, caspase-1-

activated, nuclease. This process does not result in the DNA

laddering typical of apoptosis, but does cause nuclear condensation

while maintaining nuclear integrity. Pyroptosis is accompanied by

destruction of the actin cytoskeleton, with caspase-1 also degrading

the cellular inhibitor of apoptosis protein, as well as cleaving and

inactivating metabolic enzymes. Precisely how these processes

contribute to this mechanism of cell death is currently unclear (for a

detailed review on pyroptosis, see [67,69]).

Review Trends in Cell Biology Vol.19 No.9

and NLRP3 inflammasome activation [61]. Additionalmechanisms acting in the early stages of the immuneand inflammatory response are also likely to be discoveredin the next few years.

The ability of additional members of the NLR family toassemble into inflammasomes has not yet been demon-strated, although it is likely that further inflammasomecomplexes will be described. NLRP12 (also called Mon-arch-1) was originally shown to associate with ASC in vitro,consistent with its ability to form an inflammasome com-plex. However, a clear role for NLRP12 in caspase-1 clea-vage has not yet been demonstrated. NLRP12 has beenshown to function as a negative regulator of inflammatorygene expression in myeloid cells [62–64]. NLRP12 tran-scription is diminished by specific TLR stimulation andmyeloid cell maturation, consistent with its role as anegative regulator of inflammation. Loss of a functionalNLRP12 protein is also linked to hereditary periodic fever[22].

The AIM2 inflammasomeA fourth inflammasome complex was described veryrecently. Surprisingly, this complex does not contain anymembers of the NLR family, but instead contains theHIN200 and PYD domain protein Absent in melanoma-2, AIM2 [8–11]. AIM2 is a member of the HIN200 proteinfamily, a family of IFN-inducible proteins encoded bystructurally related murine (Ifi202a, Ifi202b, Ifi203,Ifi204 and D3) and human (IFI16, MNDA and AIM2)genes. The proteins encoded by genes in the family sharea unique repeat of 200-amino acids and are primarilynuclear. Unlike other proteins in the family however,AIM2 is localized in the cytoplasm [65]. AIM2 was origin-ally identified in a screen for suppressors of melanomatumorigenicity [66]. AIM2 and the related HIN200proteins fall within susceptibility loci for SLE in humansand mice.

AIM2 binds to cytosolic dsDNA via the HIN200 domainand, like some NLRs, engages the adaptor ASC through aPYD domain to recruit and activate caspase-1 (seeFigure 4). AIM2 regulates caspase-1 activation and IL-1b production in response to viral, host and bacterialdsDNA and in response to the dsDNA virus vaccinia[10]. Additional data also implicates AIM2 in IL-1b pro-duction in response to Francisella tularensis [9], a Gram-negative coccus that can cause lethal disease in humans.Interestingly, AIM2 appears to recognize dsDNA from avariety of microbial species, including that from mamma-lian cells. It is therefore tempting to speculate that AIM2may contribute to autoimmune disease (discussed in moredetail below).

Inflammasomes and pyroptosisPyroptosis, a process of caspase-1-dependent cell death(see text Box 2), is closely linked to inflammasome complexformation in certain situations [67]. In contrast to apop-tosis, which is an immunologically ‘silent’ form of celldeath, pyroptosis is a highly inflammatory form of celldeath and is often observed during infection with cytosolicpathogens. NLRP1, NLRP3, NLRC4 and AIM2 inflamma-somes all appear to trigger pyroptosis in cells of monocyte/

macrophage lineage [9,68,69]. Presumably, this will alsohold true for additional NLRs. Caspase-1 activation,particularly by the production of IL-18, is important inclearing bacterial infections [69], but pyroptosis alsoenhances inflammation, perhaps worsening the inflamma-tory process and contributing to the pathology often seen inconditions such as Muckle–Wells Syndrome and Mediter-ranean fever, where gain-of-function mutations are found[29,70]. Deletion of caspase 1 is protective in a number ofdisease models, an observation that cannot be explained

461

Review Trends in Cell Biology Vol.19 No.9

solely by the loss of IL-1b or IL-18 production [69],suggesting that pyroptosis is a probable contributor todisease pathogenesis. Understanding the function of pyr-optosis in host-defense and disease pathogenesis, anddefining the molecular mechanisms regulating pyroptosisis likely to unveil new targets for therapeutic intervention.

Additional targets of the caspase-1 pathwayAdditional substrates of caspase-1 have been identifiedrecently. Caspase-7 has emerged as one such substrate[71,72]. In vivo, caspase-7 activation was observed underconditions known to induce activation of caspase-1, in-cluding Salmonella infection and microbial stimuli com-bined with ATP. These results demonstrate the existenceof an NLR–caspase-1–caspase-7 cascade and the existenceof distinct activation mechanisms for caspase-3 and -7 inresponse to microbial stimuli and bacterial infection.Furthermore, an important role for caspase-1 in unconven-tional protein secretion has also been revealed [34]. Mam-malian cells export proteins by means of the endoplasmicreticulum/Golgi-dependent pathway. Some proteins, how-ever, are secreted via poorly characterized mechanisms,independently of the ER–Golgi. These proteins include IL-1b, IL-18, FGF-2 and caspase-1 itself. Although proIL-1a

and FGF-2 are not substrates of caspase-1, they have beenshown to associate physically with caspase-1. Caspase-1 is,therefore, not only important in generating an inflamma-tory response, but also functions to prime tissues withsoluble cytoprotective and/or repair mediators.

Inflammasomes and links to human diseaseThe importance of IL-1b and inflammasomes in inflam-mation and fever is supported by genetic evidence linkinginflammasomes to a family of hereditary periodic fevers(HPFs) – heritable disorders associated with recurrentepisodes of fever and inflammation (reviewed in [22]).These include familial cold autoinflammatory syndrome(FCAS), Muckle–Wells syndrome (MWS) and chronicinfantile cutaneous neurological articular syndrome(CINCA), also termed neonatal-onset multisystem inflam-matory disease (NOMID), and are all caused by gain offunction mutations in NLRP3 [29,73] (reviewed in [22]).Vitiligo, a skin depigmentation syndrome caused by mel-anocyte destruction, is associatedwithmuations inNLRP1[74]. Patients with vitiligo are also more susceptible to anumber of autoimmune conditions, including rheumatoidarthritis, diabetes and SLE, emphasizing the potentialimportance of the NLRP1 inflammasome [74].

Inflammasome activity also contributes to gout, asbes-tosis, silicosis and Alzheimer’s disease. The accumulationof uric acid crystals in joints triggers inflammation. Recentevidence has revealed that MSU triggers inflammation bymeans of the NLRP3 inflammasome [56] ASC-knockoutmice show reduced neutrophil migration in a urate crystal-induced inflammatory model [56]. In a pilot study, inter-ference with IL-1b signaling using IL-1 receptorantagonists led to remission of gout symptoms in patients,supporting the concept that the NLRP3 pathway is amen-able to therapeutic targeting [75]. Silicosis and asbestosisare characterized by pulmonary fibrosis, which that leadsto lung cancer. Inhalation of silica and asbestos induce

462

inflammation in animalmodels and in the lungs of patientswith asbestos-related lung disease [76]. Asbestos and silicaactivate NLRP3 [47,48,77] and, once inhaled, the inflam-matory response and subsequent development of pulmon-ary fibrosis is dependent on the NLRP3 inflammasome[77].Autoimmune diseases such SLE have been linked toaberrant sensing of DNA. Patients with SLE form immunecomplexes to dsDNA by means of autoreactive antibodie-s.Elevated IL-1b levels have been found in patients withSLE. The identification of AIM2 as a sensor of cytoplasmicDNA, its role in IL-1b production and the link betweenAIM2 and related family members in the aeitology of SLErender AIM2 an attractive target for intervention in thisdisease. Moreover, chronic arthritis caused by mammalianDNA that escapes from degradation has also been shown tobe associated with increased levels of IL-1b and IL-18[78,79]. Defining the role of AIM2 in these events thereforeholds significant therapeutic promise for infectious as wellas autoimmune diseases such as SLE. Finally, the role ofAIM2 as a putative tumor suppressor might also implicatethe AIM2 inflammasome in cancer. The putative tumor-suppressive activity of AIM2 might relate to its ability toinduce caspase-1-dependent cell death (also called pyrop-tosis) in cells that co-express ASC and caspase-1, althoughadditional work is needed to further address these possi-bilities. The discovery of the AIM2 inflammasome there-fore represents a new target for the treatment of chronicinflammatory diseases.

Therapeutic intervention of inflammasome pathwaysThe genetic association of NLRP3 and other NLR geneswith human disease and the role of NLR and AIM2 inflam-masomes in sensing endogenous danger signals andenvironmental insults suggests that inflammasomesrepresent important therapeutic targets. The potentialimportance of modulating inflammasome activity is high-lighted by the fact that many of the autoinflammatorydiseases are refractory to most therapies other than immu-nosuppressive agents that have many side effects. Arecombinant IL-1b receptor antagonist known as ‘Ana-kinra’ is showing promise in clinical trials for a numberof diseases, including rheumatoid arthritis, various her-editary autoimmune conditions and gout [80]. Anotherapproach is to develop NLR antagonists. This couldrepresent an attractive approach if the NLRs bind directlyto their ligands; however at least in the case of NLRP3, thisseems unlikely. A paucity of structural data on the leucine-rich repeats of the NLRs renders structure-based drugdesign difficult at present. Small molecule inhibitors ofthe NOD domain/ATP binding site of the NLRs representan additional target.

Concluding remarksRemarkable progress in the past few years has greatlyincreased our understanding of how the NLRs function,and the importance of these PRRs and associated com-ponents of inflammasomes in host defense and diseasepathogenesis. NLR-containing inflammasomes are import-ant components of host defense against viruses, fungi andbacteria, but also appear to contribute to disease pathologyin inflammatory and autoimmune conditions. While it is

Box 3. Future Questions

A large number of questions about cytosolic PRRs remain

unanswered, and are of great importance, particularly with respect

to the development of future therapeutic compounds. There are 22

NLRs in humans and yet we have only identified ‘ligands’ for very

few of them. One of the key questions in the field of NLR biology is

’what are the functions of these apparent orphan receptors?’ A

further complicating problem is that NLRs such as NLRP3 appear to

have a wide range of ligands, suggesting that there may be a

common upstream mechanism that activates NLRP3, i.e. the true

ligand for NLRP3. How ligands gain access to cytosolic NLRs and

DNA sensors is another poorly understood area. In many cases, it is

unclear what the physiological roles are for these receptors. For

example, NLRC4 recognises flagellin, but does not appear to affect

the course of infection in response to pathogens such as Salmonella

enterica serovar Typhimurium. In some cases, such as caspase

1-driven pyroptosis, a physiological process has been identified, but

the role of this process in disease pathogenesis is unclear.

Review Trends in Cell Biology Vol.19 No.9

clear that priming by microbial products such as LPS isessential for IL-1b gene transcription, the activatingstimulus responsible for these events in sterile inflam-mation is less clear. Recent work from Nunez and col-leagues reveals that TNF and IL-1 could both primecells for inflammasome activation by particulate ligands,in the absence of microbial stimuli (Nunez, personal com-munication).

Despite the amazing progress outlined here in our un-derstanding of how the NLRs function, many key questionsremain unanswered. Whether or not any of these PRRsdirectly recognize their ligands is an outstanding question.A second key question is how NLRP3 is activated, and theunderlyingmechanisms of phagosomal destabilization and/or ROS generation remain to be clarified. A focus on the IL-1b pathway as the signature response activated by thesereceptorshas clearly ledto important insights into theroleofNLRs indefense and disease; however, additional pathwaysmust exist downstream of these receptors. These pathwaysare likely to be uncovered in time. There are 22 NLRmembers in humans, yet the function of most of these isunknown. Deciphering the ligands of these receptors is anarea ripe for investigation. There is much potential foridentifying new ligands, new pathways and new targetsfor therapeutic intervention in infectious and inflammatorydiseases (Box 3). Understanding how inflammasomes areregulated and how their downstream pathways contributeto inflammation is therefore important for the developmentof therapeutic agents, to enhance the innate immuneresponse, if needed, or to limit it in situations where cyto-kines are detrimental to host survival.

References1 Kawai, T. and Akira, S. (2008) Toll-like receptor and RIG-I-like

receptor signaling. Ann. N. Y. Acad. Sci. 1143, 1–202 Meylan, E. et al. (2006) Intracellular pattern recognition receptors in

the host response. Nature 442, 39–443 Takeda, K. and Akira, S. (2005) Toll-like receptors in innate immunity.

Int. Immunol. 17, 1–144 Huysamen, C. and Brown, G.D. (2009) The fungal pattern recognition

receptor, Dectin-1, and the associated cluster of C-type lectin-likereceptors. FEMS Microbiol. Lett. 290, 121–128

5 Yoneyama, M. and Fujita, T. (2007) RIG-I family RNA helicases:cytoplasmic sensor for antiviral innate immunity. Cytokine GrowthFactor Rev. 18, 545–551

6 Takaoka, A. and Taniguchi, T. (2008) Cytosolic DNA recognition fortriggering innate immune responses. Adv. Drug. Deliv. Rev. 60, 847–

8577 Takaoka, A. et al. (2007) DAI (DLM-1/ZBP1) is a cytosolic DNA sensor

and an activator of innate immune response. Nature 448, 501–5058 Burckstummer, T. et al. (2009) An orthogonal proteomic-genomic

screen identifies AIM2 as a cytoplasmic DNA sensor for theinflammasome. Nat. Immunol. 10, 266–272

9 Fernandes-Alnemri, T. et al. (2009) AIM2 activates the inflammasomeand cell death in response to cytoplasmic DNA. Nature 458, 509–513

10 Hornung, V. et al. (2009) AIM2 recognizes cytosolic dsDNA and forms acaspase-1-activating inflammasome with ASC. Nature 458, 514–518

11 Roberts, T.L. et al. (2009) HIN-200 proteins regulate caspase activationin response to foreign cytoplasmic DNA. Science 323, 1057–1060

12 Martinon, F. et al. (2009) The inflammasomes: guardians of the body.Annu. Rev. Immunol. 27, 229–265

13 Kono, H. and Rock, K.L. (2008) How dying cells alert the immunesystem to danger. Nat. Rev. Immunol. 8, 279–289

14 Dinarello, C.A. (2005) Interleukin-1beta. Crit. Care Med 33, S460–S46215 Dinarello, C.A. (2002) The IL-1 family and inflammatory diseases.

Clin. Exp. Rheumatol. 20, S1–S1316 Hiscott, J. et al. (1993) Characterization of a functional NF-kappa B

site in the human interleukin 1 beta promoter: evidence for a positiveautoregulatory loop. Mol. Cell Biol. 13, 6231–6240

17 Heitmeier, M.R. et al. (1998) Double-stranded RNA-induced induciblenitric-oxide synthase expression and interleukin-1 release by murinemacrophages requires NF-kappaB activation. J. Biol. Chem. 273,15301–15307

18 Martinon, F. et al. (2002) The inflammasome: a molecular platformtriggering activation of inflammatory caspases and processing of proIL-beta. Mol. Cell 10, 417–426

19 Franchi, L. et al. (2009) Function of Nod-like receptors in microbialrecognition and host defense. Immunol. Rev. 227, 106–128

20 Siegel, R.M. (2006) Caspases at the crossroads of immune-cell life anddeath. Nat. Rev. Immunol. 6, 308–317

21 McDermott, M.F. and Tschopp, J. (2007) From inflammasomes tofevers, crystals and hypertension: how basic research explainsinflammatory diseases. Trends. Mol. Med. 13, 381–388

22 Masters, S.L. et al. (2009) Horror autoinflammaticus: the molecularpathophysiology of autoinflammatory disease (*). Annu. Rev. Immunol.27, 621–668

23 Martinon, F. and Tschopp, J. (2005) NLRs join TLRs as innate sensorsof pathogens. Trends. Immunol. 26, 447–454

24 Faustin, B. et al. (2007) Reconstituted NALP1 inflammasome revealstwo-step mechanism of caspase-1 activation. Mol. Cell 25, 713–724

25 Boyden, E.D. and Dietrich, W.F. (2006) Nalp1b controls mousemacrophage susceptibility to anthrax lethal toxin. Nat. Genet. 38,240–244

26 Miao, E.A. et al. (2007) TLR5 and Ipaf: dual sensors of bacterialflagellin in the innate immune system. Semin. Immunopathol. 29,275–288

27 Lightfield, K.L. et al. (2008) Critical function for Naip5 ininflammasome activation by a conserved carboxy-terminal domain offlagellin. Nat. Immunol. 9, 1171–1178

28 Sun, Y.H. et al. (2007) Injection of flagellin into the host cell cytosol bySalmonella enterica serotype Typhimurium. J. Biol. Chem. 282,33897–33901

29 Agostini, L. et al. (2004) NALP3 forms an IL-1beta-processinginflammasome with increased activity in Muckle-Wellsautoinflammatory disorder. Immunity 20, 319–325

30 Franchi, L. et al. (2006) Cytosolic flagellin requires Ipaf for activation ofcaspase-1 and interleukin 1beta in salmonella-infected macrophages.Nat. Immunol. 7, 576–582

31 Mariathasan, S. et al. (2004) Differential activation of theinflammasome by caspase-1 adaptors ASC and Ipaf. Nature 430,213–218

32 Miao, E.A. et al. (2006) Cytoplasmic flagellin activates caspase-1 andsecretion of interleukin 1beta via Ipaf. Nat. Immunol. 7, 569–575

33 Suzuki, T. et al. (2007) Differential regulation of caspase-1 activation,pyroptosis, and autophagy via Ipaf and ASC in Shigella-infectedmacrophages. PLoS Pathog. 3, e111

34 Master, S.S. et al. (2008) Mycobacterium tuberculosis preventsinflammasome activation. Cell Host. Microbe. 3, 224–232

463

Review Trends in Cell Biology Vol.19 No.9

35 Kanneganti, T.D. et al. (2006) Critical role for Cryopyrin/Nalp3 inactivation of caspase-1 in response to viral infection and double-stranded RNA. J. Biol. Chem. 281, 36560–36568

36 Thomas, P.G. et al. (2009) The intracellular sensor NLRP3 mediateskey innate and healing responses to influenza A virus via theregulation of caspase-1. Immunity 30, 566–575

37 Allen, I.C. et al. (2009) The NLRP3 inflammasome mediates in vivoinnate immunity to influenza A virus through recognition of viral RNA.Immunity 30, 556–565

38 Ichinohe, T. et al. (2009) Inflammasome recognition of influenza virusis essential for adaptive immune responses. J. Exp. Med. 206, 79–87

39 Muruve, D.A. et al. (2008) The inflammasome recognizes cytosolicmicrobial and host DNA and triggers an innate immune response.Nature 452, 103–107

40 Gross, O. et al. (2009) Syk kinase signalling couples to the Nlrp3inflammasome for anti-fungal host defence. Nature 459, 433–436

41 Mariathasan, S. et al. (2006) Cryopyrin activates the inflammasome inresponse to toxins and ATP. Nature 440, 228–232

42 Warren, S.E. et al. (2008) Multiple Nod-like receptors activate caspase 1during Listeria monocytogenes infection. J. Immunol. 180, 7558–7564

43 Kahlenberg, J.M. et al. (2005) Potentiation of caspase-1 activation bythe P2X7 receptor is dependent on TLR signals and requires NF-kappaB-driven protein synthesis. J. Immunol. 175, 7611–7622

44 Petrilli, V. et al. (2007) The inflammasome: a danger sensing complextriggering innate immunity. Curr. Opin. Immunol. 19, 615–622

45 Shi, Y. et al. (2003) Molecular identification of a danger signal thatalerts the immune system to dying cells. Nature 425, 516–521

46 Halle, A. et al. (2008) The NALP3 inflammasome is involved in theinnate immune response to amyloid-beta. Nat. Immunol. 9, 857–865

47 Dostert, C. et al. (2008) Innate immune activation through Nalp3inflammasome sensing of asbestos and silica. Science 320, 674–677

48 Hornung, V. et al. (2008) Silica crystals and aluminum salts activatethe NALP3 inflammasome through phagosomal destabilization. Nat.Immunol. 9, 847–856

49 Ferrari, D. et al. (2006) The P2X7 receptor: a key player in IL-1processing and release. J. Immunol. 176, 3877–3883

50 Kanneganti, T.D. et al. (2007) Pannexin-1-mediated recognition ofbacterial molecules activates the cryopyrin inflammasomeindependent of Toll-like receptor signaling. Immunity 26, 433–443

51 Pelegrin, P. et al. (2008) P2X7 receptor differentially couples to distinctrelease pathways for IL-1beta in mouse macrophage. J. Immunol. 180,7147–7157

52 Pelegrin, P. and Surprenant, A. (2006) Pannexin-1 mediates large poreformation and interleukin-1beta release by the ATP-gated P2X7receptor. EMBO J. 25, 5071–5082

53 Marina-Garcia, N. et al. (2008) Pannexin-1-mediated intracellulardelivery of muramyl dipeptide induces caspase-1 activation viacryopyrin/NLRP3 independently of Nod2. J. Immunol. 180, 4050–4057

54 Hsu, L.C. et al. (2008) A NOD2-NALP1 complex mediates caspase-1-dependent IL-1beta secretion in response to Bacillus anthracisinfection and muramyl dipeptide. Proc. Natl. Acad. Sci. U. S. A.105, 7803–7808

55 Franchi, L. et al. (2007) Differential requirement of P2X7 receptor andintracellular K+ for caspase-1 activation induced by intracellular andextracellular bacteria. J. Biol. Chem. 282, 18810–18818

56 Martinon, F. et al. (2006) Gout-associated uric acid crystals activate theNALP3 inflammasome. Nature 440, 237–241

57 Sharp, F.A. et al. (2009) Uptake of particulate vaccine adjuvants bydendritic cells activates the NALP3 inflammasome. Proc. Natl. Acad.Sci. U. S. A. 106, 870–875

58 Eisenbarth, S.C. et al. (2008) Crucial role for the Nalp3 inflammasomein the immunostimulatory properties of aluminium adjuvants. Nature453, 1122–1126

464

59 Franchi, L. and Nunez, G. (2008) The Nlrp3 inflammasome is criticalfor aluminium hydroxide-mediated IL-1beta secretion but dispensablefor adjuvant activity. Eur. J. Immunol. 38, 2085–2089

60 Li, H. et al. (2008) Cutting edge: inflammasome activation by alumand alum’s adjuvant effect are mediated by NLRP3. J. Immunol. 181,17–21

61 Guarda, G. et al. (2009) T cells dampen innate immune responsesthrough inhibition of NLRP1 and NLRP3 inflammasomes.Nature 460,269–273

62 Lich, J.D. and Ting, J.P. (2007) Monarch-1/PYPAF7 and otherCATERPILLER (CLR, NOD, NLR) proteins with negativeregulatory functions. Microbes. Infect 9, 672–676

63 Lich, J.D. et al. (2007) Monarch-1 suppresses non-canonical NF-kappaB activation and p52-dependent chemokine expression inmonocytes. J. Immunol. 178, 1256–1260

64 Williams, K.L. et al. (2005) The CATERPILLER protein monarch-1 isan antagonist of toll-like receptor-, tumor necrosis factor alpha-, andMycobacterium tuberculosis-induced pro-inflammatory signals. J.Biol. Chem. 280, 39914–39924

65 Choubey, D. et al. (2000) Cytoplasmic localization of the interferon-inducible protein that is encoded by the AIM2 (absent in melanoma)gene from the 200-gene family. FEBS Lett. 474, 38–42

66 Cresswell, K.S. et al. (2005) Biochemical and growth regulatoryactivities of the HIN-200 family member and putative tumorsuppressor protein, AIM2. Biochem. Biophys. Res. Commun 326,417–424

67 Ting, J.P. et al. (2008) NLRs at the intersection of cell death andimmunity. Nat. Rev. Immunol. 8, 372–379

68 Kufer, T.A. and Sansonetti, P.J. (2007) Sensing of bacteria: NOD alonely job. Curr. Opin. Microbiol. 10, 62–69

69 Bergsbaken, T. et al. (2009) Pyroptosis: host cell death andinflammation. Nat. Rev. Microbiol. 7, 99–109

70 Dowds, T.A. et al. (2003) Regulation of cryopyrin/Pypaf1 signaling bypyrin, the familial Mediterranean fever gene product. Biochem.Biophys. Res. Commun. 302, 575–580

71 Akhter, A. et al. (2009) Caspase-7 activation by the Nlrc4/Ipafinflammasome restricts Legionella pneumophila infection. PLoSPathog. 5, e1000361

72 Lamkanfi, M. et al. (2008) Targeted peptidecentric proteomics revealscaspase-7 as a substrate of the caspase-1 inflammasomes. Mol. CellProteomics 7, 2350–2363

73 Dowds, T.A. et al. (2004) Cryopyrin-induced interleukin 1beta secretioninmonocytic cells: enhanced activity of disease-associatedmutants andrequirement for ASC. J. Biol. Chem. 279, 21924–21928

74 Jin, Y. et al. (2007) NALP1 in vitiligo-associated multiple autoimmunedisease. N. Engl. J. Med. 356, 1216–1225

75 So, A. et al. (2007) A pilot study of IL-1 inhibition by anakinra in acutegout. Arthritis. Res. Ther. 9, R28

76 Mossman, B.T. and Churg, A. (1998) Mechanisms in the pathogenesisof asbestosis and silicosis. Am. J. Respir. Crit. Care Med. 157, 1666–

168077 Cassel, S.L. et al. (2008) The Nalp3 inflammasome is essential for the

development of silicosis. Proc. Natl. Acad. Sci. U. S. A. 105, 9035–904078 Kawane, K. et al. (2006) Chronic polyarthritis caused by mammalian

DNA that escapes from degradation in macrophages.Nature 443, 998–

100279 Nagata, S. (2007) Autoimmune diseases caused by defects in clearing

dead cells and nuclei expelled from erythroid precursors. Immunol.Rev. 220, 237–250

80 Kalliolias, G.D. and Liossis, S.N. (2008) The future of the IL-1 receptorantagonist anakinra: from rheumatoid arthritis to adult-onset Still’sdisease and systemic-onset juvenile idiopathic arthritis. Expert. Opin.Investig. Drugs 17, 349–359