CB28CH06-Dixit ARI 12 September 2012 10:2

Inflammasomes and TheirRoles in Health and DiseaseMohamed Lamkanfi1,2 and Vishva M. Dixit3

1Department of Biochemistry, Ghent University, Ghent 9000, Belgium;2Department of Medical Protein Research, VIB, Ghent 9000, Belgium;email: mohamed.lamkanfi@vib-ugent.be3Department of Physiological Chemistry, Genentech, South San Francisco,California 94080; email: dixit.vishva@gene.com

Annu. Rev. Cell Dev. Biol. 2012. 28:137–61

First published online as a Review in Advance onSeptember 10, 2012

The Annual Review of Cell and DevelopmentalBiology is online at cellbio.annualreviews.org

This article’s doi:10.1146/annurev-cellbio-101011-155745

Copyright c© 2012 by Annual Reviews.All rights reserved

1081-0706/12/1110-0137$20.00

Keywords

NOD-like receptor, caspase, inflammation, infection, cytokine, celldeath

Abstract

Inflammasomes are a set of intracellular protein complexes that enableautocatalytic activation of inflammatory caspases, which drive host andimmune responses by releasing cytokines and alarmins into circulationand by inducing pyroptosis, a proinflammatory cell death mode. Theinflammasome type mediating these responses varies with the micro-bial pathogen or stress factor that poses a threat to the organism. Sincethe discovery that polymorphisms in inflammasome genes are linkedto common autoimmune diseases and less frequent periodic fever syn-dromes, inflammasome signaling has been dissected at the molecularlevel. In this review, we present recently gained insight on the dis-tinct inflammasome types, their activation and effector mechanisms,and their modulation by microbial virulence factors. In addition, wediscuss recently gained knowledge on the role of deregulated inflamma-some activity in human autoinflammatory, autoimmune, and infectiousdiseases.

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Contents

INTRODUCTION ANDOVERVIEW: PATHOGENRECOGNITION BYINTRACELLULAR PLATFORMPROTEINS . . . . . . . . . . . . . . . . . . . . . . . 138Pathogen Recognition: The

Foundation of the InnateImmune System . . . . . . . . . . . . . . . . 138

Intracellular and ExtracellularPattern Recognition Receptors . . 139

INFLAMMASOMES:COMPOSITION ANDSTRUCTURE . . . . . . . . . . . . . . . . . . . . 140Inflammasomes: Platforms for

Inflammatory CaspaseActivation . . . . . . . . . . . . . . . . . . . . . . 140

Inflammasome Subtypes . . . . . . . . . . . . 141INFLAMMASOME EFFECTOR

MECHANISMS. . . . . . . . . . . . . . . . . . . 142Proteolytic Maturation of proIL-1β

and proIL-18 . . . . . . . . . . . . . . . . . . . 142Pyroptosis . . . . . . . . . . . . . . . . . . . . . . . . . 143Unconventional Secretion of

Growth and InflammatoryFactors . . . . . . . . . . . . . . . . . . . . . . . . . 144

Additional Inflammasome EffectorMechanisms . . . . . . . . . . . . . . . . . . . . 145

MECHANISMS OFINFLAMMASOMEACTIVATION. . . . . . . . . . . . . . . . . . . . 145The Nlrp1 Inflammasome . . . . . . . . . . 145The Nlrp3 Inflammasome . . . . . . . . . . 146The Nlrc4 Inflammasome . . . . . . . . . . 148The Nlrp6 Inflammasome . . . . . . . . . . 149The AIM2 Inflammasome . . . . . . . . . . 149

INFLAMMASOMES INAUTOINFLAMMATIONAND AUTOIMMUNITY . . . . . . . . . 150

MODULATION OFINFLAMMASOMEACTIVATION ANDACTIVITY . . . . . . . . . . . . . . . . . . . . . . . 151

CONCLUSIONS ANDPERSPECTIVES . . . . . . . . . . . . . . . . . 153

INTRODUCTION ANDOVERVIEW: PATHOGENRECOGNITION BYINTRACELLULAR PLATFORMPROTEINS

Pathogen Recognition:The Foundation of the InnateImmune System

The human immune system consists of two dis-tinct arms that work in a concerted fashion torespond to harmful stress situations and infec-tious agents. Activation of immune responses tomicrobial pathogens and stress factors that posea threat to the organism start with activation ofthe evolutionarily more ancient innate immunearm, which temporally precedes and instructsthe more recently evolved adaptive immunesystem. The innate immune system makes useof several mechanisms to counter invasion byharmful agents. These include anatomical bar-riers such as the skin and mucous membranesthat mechanically prevent dispersion through-out the body, opsonization and removal of theinvading factor by the complement system, andpattern recognition receptors (PRRs) expressedby hematopoietic and nonhematopoietic cellssuch as macrophages, dendritic cells, and ep-ithelial cells. PRRs enable innate immune cellsto instantly detect and respond to the presenceof danger- and pathogen-associated molecularpatterns (DAMPs and PAMPs, respectively)(Kanneganti et al. 2007). PAMPs are conservedmicrobial molecules that are not producedby mammalian host cells, such as nucleic acidstructures that are unique to microorganisms,bacterial secretion systems and their effectorproteins, and microbial cell wall componentssuch as lipoproteins and lipopolysaccharides(LPSs). Such molecules often are essentialfor the infectious agent to survive in thehost’s hostile environment, which makesthem ideal for monitoring of the unwantedpresence of microbes by the host’s PRRs. Incontrast, DAMPs are a set of host-derivedmolecules that signal cellular stress, damage,or nonphysiological cell death. High-mobility

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group box 1 (HMGB1), uric acid, ATP, andheat-shock proteins hsp70 and hsp90 are a fewexamples of DAMPs that are believed to playmajor roles in eliciting inflammation and tissuerepair during infections and under conditionsof noninfectious (sterile) inflammation.

Engagement of PRRs by PAMPs andDAMPs leads to a multitude of changes in thetranscriptional and posttranslational programsof innate immune cells that bring proinflamma-tory cytokines, chemokines, and growth factorsinto circulation in a highly coordinated fashion.These molecules signal polymorphonuclearleukocytes and professional phagocytes in theperiphery to migrate to the site of infection orinjury and at the same time produce additionalsignals that aim to rapidly eliminate the threatand repair the damage elicited by the pathogenand the host’s inflammatory responses. Inaddition to these first-line responses, dendriticcells and other professional antigen-presentingcells capture and display immunogenic frag-ments of the hostile factor on their surface asa way of communicating the identity of theharmful agent to the adaptive immune system(Guermonprez et al. 2002). Through theprocess of somatic recombination, adaptive im-mune cells can generate an endless repertoireof antigen-specific receptors and highly specificantibodies against the presented molecules(Call & Wucherpfennig 2005, Di Noia &Neuberger 2007). Such targeted moleculesand receptors specifically mark the invadingagent expressing the antigen for destruction bythe complement and phagocytic activities ofthe innate immune system or by killer T cells.Thus, the combined qualities of the innate andadaptive immune arms allow highly tailoredand efficacious responses to be mounted againsta broad range of infections and harmful agents.

Intracellular and Extracellular PatternRecognition Receptors

The human immune system relies on at leastfour different PRR families to respond to

microbes and harmful particles. Members ofthe Toll-like receptor (TLR) family line theplasma membrane and endosomal membranes,where they survey the extracellular space forPAMPs and DAMPs (Kawai & Akira 2006,West et al. 2006). More recently, severalnovel PRR families that appear to guard theintracellular environment have emerged. Thisincludes the RIG-I-like receptor (RLR) as wellas the HIN200 and NOD-like receptor (NLR)families (Takeuchi & Akira 2010). Notably,many of these receptors and platform proteinsinitiate inflammatory signaling pathways thatappear partially redundant, which raises theinteresting possibility that significant crosstalk between members of the same and dif-ferent PRR families may coordinate host andinflammatory responses (Paludan et al. 2011,Takeuchi & Akira 2010). For instance, TLRsand other PRR families often recognize over-lapping sets of PAMPs, including viral RNA(recognized by TLR3 and RLRs) and micro-bial DNA (recognized by TLR9 and HIN200proteins). In addition, multiple members ofthe distinct PRR families engage the inflam-matory transcription factors nuclear factor-κB(NF-κB), activator protein 1 (AP1), and inter-feron regulatory factor (IRF) to induce secre-tion of cytokines and chemokines with inflam-matory and microbicidal properties (Takeuchi& Akira 2010, Tamura et al. 2008). Such redun-dancy may serve to tailor innate and adaptiveimmune responses to viral, bacterial, and par-asitic pathogens. Unlike most PRRs, however,certain NLR family members and the HIN200protein absent in melanoma 2 (AIM2) respondto infections and stress by assembling inflam-masomes, large cytosolic protein complexes inwhich inflammatory caspases undergo autocat-alytic activation (Kanneganti 2010, Lamkanfi &Dixit 2009). This review provides an overviewand discusses our current understanding of thecomposition of different inflammasomes, theirupstream activation and downstream effectormechanisms, and their roles in host defense anddisease.

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INFLAMMASOMES:COMPOSITION ANDSTRUCTURE

Inflammasomes: Platforms forInflammatory Caspase Activation

Inflammasomes are intracellular multiproteincomplexes that mediate activation of the inflam-matory caspases-1 and -11 (Kayagaki et al. 2011,Martinon et al. 2002) (Figure 1). This processentails the recruitment of preexisting caspasezymogens into the protein complex, in whichthey undergo conformational changes associ-ated with their proximity-induced autoactiva-tion (Salvesen & Dixit 1999, Shi 2004). The

NLRP1

HumanPYD NACHT

PYD HIN200

PYD

FIIND CARD

Mouse

NLRP3/NLRP6

NLRC4

NLR

AIM2

BIRNAIP

ASC

Caspase domainCASP1/4/5/11

Platformproteins

HIN

20

0

Adaptorproteins

Effectorproteins

LRR

PYD NACHT LRR

NACHT LRR

NACHT LRR

NACHT FIIND CARDLRR

CARD

CARD

CARD

Figure 1Domain architecture of inflammasome components. A subset of NLR familymembers as well as the HIN200 protein AIM2 assemble inflammasomecomplexes. NLRs are characterized by the combined presence of a NACHTdomain followed by a variable number of LRRs. AIM2 contains anamino-terminal PYD followed by a DNA-binding HIN200 domain. MurineNlrp1b lacks the amino-terminal PYD motif found in human NLRP1. ThePYD domains of AIM2 and NLRP1, -3, and -6 recruit the bipartite adaptorprotein ASC. NLRP1 and NLRC4 may interact directly with the CARD ofcaspase-1 or may recruit caspase-1 indirectly through ASC. Human NAIP andits murine paralogs contain BIR motifs in their amino terminus. Abbreviations:AIM2, absent in melanoma 2; ASC, apoptosis-associated speck-like proteincontaining a CARD; BIR, baculovirus IAP repeat; CARD; caspase recruitmentdomain; CASP, caspase; FIIND, domain with function to find; LRR,leucine-rich repeat; NACHT, nucleotide-binding and oligomerization domain;NLR, Nod-like receptor; PYD, pyrin.

inflammatory caspases-1 and -11 belong to anevolutionarily conserved family of cysteine pro-teases that cleave their substrates behind as-partate residues (Lamkanfi et al. 2002). Theseprocessing events may cause activation or in-activation of critical signaling cascades regulat-ing programmed cell death, differentiation, andcell proliferation (Lamkanfi et al. 2006). Simi-lar to other caspases that are produced as inac-tive zymogens with large prodomains, caspases-1 and -11 are referred to as initiator caspases(together with caspase-2, -4, -5, -8, -9, -10 and-12). In contrast, caspases containing a shortprodomain are known as executioner caspases(caspase-3, -6, -7, and -14) (Figure 2). Thissegregation of initiator and executioner cas-pases also is relevant from a functional view-point because the large prodomains of initia-tor caspases typically contain interaction mo-tifs of the death domain superfamily that al-low recruitment of the zymogen into activatingprotein complexes such as the inflammasomeand the apoptosome (Lamkanfi & Dixit 2009,Riedl & Salvesen 2007). These homotypic in-teraction domains typically consist of six orseven antiparallel α-helices, the relative orien-tation of which determines their classification ascaspase recruitment domains (CARDs), pyrin(PYD), death domains, or death effector do-mains (Park et al. 2007). Initiator caspases mostoften are further segregated into inflammatory(i.e., caspase-1, -4, -5, -11, and -12) and apop-totic (i.e., caspase-2, -8, -9, and -10) caspases onthe basis of their putative roles in inflammatoryand apoptosis signaling, respectively.

Despite their functional segregation asapoptotic and inflammatory caspases, theactivation mechanisms of caspase-1 and -9 areanalogous. Both of these CARD-containinginitiator caspases are recruited into largecytosolic multiprotein complexes (the apop-tosome and the inflammasome, respectively)in which proximity-induced autoactivation isthought to result in mature caspases in whichthe catalytic domain is autoproteolyticallyseparated from the prodomain. The maturecaspase is a heterotetramer of two large andtwo small catalytic subunits, the interfaces of

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Microbiota

FlagellinPrgJ

Naip5 Cytoplasmic DNAUnknown ligand

Naip2

Nlrp3 Nlrc4Nlrp1b AIM2 Nlrp6

Nlrp1binflammasome

Nlrp3inflammasome

Microbial PAMPsEndogenous DAMPs

CrystalsUVB radiation

Nlrc4inflammasome

AIM2inflammasome

Nlrp6inflammasome

Salmonella typhimuriumPseudomonas aeruginosaLegionella pneumophila

Shigella flexneri

DNA virusesFrancisella tularensis

Listeria monocytogenesBacillus anthracis

lethal toxin

Phagosomaldestabilization,

K+ efflux,?

MKK cleavage,proteasome,phagosomal

destabilization,K+ efflux,

?

PYD NACHTPYD HIN200

BIR

LRRPYD NACHT LRR NACHT LRR

NACHT LRR

NACHT FIIND CARDLRR CARD

Figure 2Overview of stimuli and models for inflammasome activation. The NLR proteins Nlrp1b, Nlrp3, Nlrc4, and Nlrp6 as well as theHIN200 protein AIM2 assemble inflammasomes in a stimulus-specific manner. Activation of the Nlrp1b inflammasome by cytosolicBacillus anthracis lethal toxin may involve MKK processing, K+ efflux, phagosomal destabilization, and proteasomal degradation of acurrently unknown host factor. Cells exposed to microbial PAMPs, endogenous DAMPs, crystals, particulate matter, or UVB radiationmay activate the Nlrp3 inflammasome by eliciting a common cellular response (e.g., ionic fluxes and cytosolic release of lysosomalcathepsins). The Nlrc4 inflammasome is activated indirectly when the PrgJ basal body subunit of the bacterial type III secretionsystems of Salmonella, Pseudomonas, Legionella, and Shigella species interacts with Naip2. Nlrc4 also responds to bacterial flagellin, whichNaip5 detects in the cytosol of infected cells. AIM2 binds double-stranded DNA in the cytosol of cells infected with Francisellatularensis, Listeria monocytogenes, and the DNA viruses cytomegalovirus and vaccinia virus. The microbial ligands responsible foractivation of the Nlrp6 inflammasome in the gastrointestinal tract remain to be identified. Abbreviations: AIM2, absent in melanoma 2;BIR, baculovirus IAP repeat; CARD; caspase recruitment domain; DAMP, danger-associated molecular pattern; FIIND, domain withfunction to find; LRR, leucine-rich repeat; MKK, mitogen-activated protein kinase kinase; NACHT, nucleotide-binding andoligomerization domain; NLR, Nod-like receptor; PAMP, pathogen-associated molecular pattern; PYD, pyrin.

which form the two active sites at opposingends of the molecule (Salvesen & Riedl 2008).Moreover, both complexes consume ATP, andelectron micrographs of inflammasome andapoptosome particles revealed that both ofthese complexes have a double-ringed wheelstructure with sevenfold symmetry (Acehanet al. 2002, Faustin et al. 2007).

Inflammasome Subtypes

Inflammasomes are emerging as key regula-tors of innate, adaptive, and host responses

that survey the cytosol and other intra-cellular compartments for the presenceof PAMPs and DAMPs (Kanneganti2010, Lamkanfi & Dixit 2009). Thesemultiprotein complexes have been character-ized in a variety of cells, although the focushas been mainly on epithelial cells in tissueswith mucosal surfaces and immune cells ofthe myeloid lineage. Several inflammasomecomplexes have been distinguished, each typi-cally named after the NLR or HIN200 proteinthat initiates signaling (Kanneganti 2010,Lamkanfi & Dixit 2009) (Figure 2). Recent

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gene duplication events that occurred after thebifurcation of rodents and primates gave riseto 34 NLR genes in the mouse genome (Tianet al. 2009). The corresponding gene familyin humans consists of 22 members, each con-taining a centrally located nucleotide-bindingand oligomerization domain (NACHT) motif(Figure 1). This ATPase domain is usuallyflanked at the amino terminus by CARD,PYD, or baculovirus IAP repeat (BIR)motifs, which allow NLRs to recruit adaptorproteins and downstream effectors to theirsignaling complexes. The leucine-rich repeats(LRRs) found at the carboxy terminus ofmost NLRs are generally thought—in analogyto their role in TLRs—to be responsible for de-tecting and monitoring the presence of PAMPsand DAMPs in intracellular compartments. Inaddition, LRRs are believed to modulate NLRactivity (Kanneganti et al. 2007). Biochemicaland in vivo analysis of gene-deficient micerevealed central roles for the NLR proteinsNlp1b, Nlrp3, Nlrp6, and Nlrc4 in inflamma-some signaling (Kanneganti 2010, Lamkanfi& Dixit 2009). Nlrp3 and Nlrp6 lack aCARD motif and cannot interact directly withcaspase-1. In their respective inflammasomes,the amino-terminal PYD of the bipartite adap-tor apoptosis-associated speck-like proteincontaining a CARD (ASC) interacts with theupstream NLR, whereas its carboxy-terminalCARD facilitates the recruitment of caspase-1.Consequently, ASC is essential for assemblyand activation of these PYD-containinginflammasomes (Agostini et al. 2004,Elinav et al. 2011, Kanneganti et al. 2006,Mariathasan et al. 2006, Sutterwala et al.2006). ASC probably also plays a key rolein the CARD-containing Nlrp1b and Nlrc4inflammasomes (Mariathasan et al. 2004, 2006;Sutterwala et al. 2007), although these NLRsmay also interact directly with caspase-1. In thisregard, Nlrc4 was recently suggested to assem-ble two distinct inflammasome complexes, onethat contains and one that lacks ASC (Broz et al.2010). The ASC-containing Nlrc4 inflamma-some induces caspase-1 autoproteolysis and cy-tokine maturation, whereas the complex lacking

ASC triggers caspase-1-dependent cell death inthe absence of caspase-1 autoprocessing. In ad-dition to the above NLR-containing inflamma-somes, AIM2 also assembles an inflammasome.AIM2 contains a prototypical DNA-bindingHIN200 domain that is preceded by anamino-terminal PYD motif through which itrecruits ASC and caspase-1 into the complex(Figure 1).

INFLAMMASOME EFFECTORMECHANISMS

Proteolytic Maturation of proIL-1β

and proIL-18

The best-characterized consequence ofcaspase-1 activation in the inflammasomesdescribed above is secretion of the proin-flammatory cytokines interleukin (IL)-1β andIL-18 (Figure 3). These related cytokines areproduced as inactive propeptides that need tobe processed in order to be secreted from acti-vated monocytes, macrophages, and other celltypes (Dinarello 2009, Sims & Smith 2010).Caspase-1 was originally identified as theIL-1β-converting enzyme and subsequentlydemonstrated to be required for maturation ofIL-18 as well (Cerretti et al. 1992, Ghayur et al.1997, Gu et al. 1997, Kuida et al. 1995, Li et al.1995). Consequently, caspase-1-deficient miceand macrophages fail to secrete mature IL-1β

and IL-18 under most circumstances (Ghayuret al. 1997, Gu et al. 1997, Kuida et al. 1995,Li et al. 1995), although proteases such asneutrophil serine proteinase-3 and granzymeA also mediate secretion of mature IL-1β inspecific mouse models of human disease (Gumaet al. 2009, Joosten et al. 2009, Mayer-Barberet al. 2010). This raises the interesting possi-bility that redundant mechanisms for secretionof mature IL-1β may have evolved for safe-guarding the host’s immune response againstpathogens that interfere with inflammasomeactivation and caspase-1 activity (see below).

Once secreted, IL-1β and IL-18 mediatea variety of local and systemic responses toinfection. IL-1β induces fever; promotesT cell survival, B cell proliferation, and

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antibody production; contributes to po-larization of T helper 1 (TH1), TH2, andTH17 responses; and mediates transmi-gration of leukocytes (Dinarello 2009,Sims & Smith 2010). Although IL-18 doesnot induce fever responses, it synergizeswith IL-12 to induce interferon-γ (IFNγ)production by activated T cells and nat-ural killer cells, thereby promoting TH1cell polarization (Dinarello 2009, Sims &Smith 2010). IL-18 also drives TH17 re-sponses by facilitating the production ofIL-17 from already committed TH17 cellscultured in the presence of IL-23 (Harringtonet al. 2005, Weaver et al. 2006). In the ab-sence of IL-12 and IL-23, IL-18 may promoteTH2 responses by stimulating the production ofIL-4, IL-5, and other TH2 cytokines (Dinarello2009, Hoshino et al. 2001, Nakanishiet al. 2001). In conclusion, IL-1β andIL-18 are important inflammasome effectors.This is also illustrated by the successfulapplication of IL-1 inhibitors in patientssuffering from hereditary autoinflammatorydisorders, gouty arthritis, and type II diabetes(Lachmann et al. 2009, Lamkanfi et al. 2011,Larsen et al. 2007).

Pyroptosis

Despite the importance of IL-1β and IL-18in inflammasome signaling, several linesof evidence point to a range of additionalinflammasome effector mechanisms that maycontribute to immune and host responses.For example, mice lacking IL-1β and IL-18were shown to be less susceptible to Francisellatularensis infection than those lacking caspase-1(Henry & Monack 2007). The notion thatneutralization of IL-1β and IL-18 does notabrogate all inflammasome functions is furtherillustrated by the observation that mice lackingboth IL-1β and IL-18 are susceptible to LPS-induced shock, whereas caspase-1 knockoutmice are resistant (Lamkanfi et al. 2010). More-over, caspase-1-mediated host responses toLegionella pneumophila, Burkholderia thailanden-sis, and a mutant flagellin-expressing Salmonella

Canonical

Inflammasome

Active CASP11

Active CASP1

IL-18ATP

Uric acid

HMGB1

Inflammation Pyroptosis

NoncanonicalMicrobial PAMPs,

Endogenous DAMPs,Crystals,

UVB radiation

Escherichia coliCitrobacter rodentium

Vibrio cholerae

Membranepermeabilization,

DNA fragmentation, …

IL-1β

Maturation and secretionof leaderless cytokines

Secretion of DAMPs

Figure 3Canonical and noncanonical activation of the Nlrp3 inflammasome. Cellsstimulated with ATP, silica, and uric acid crystals induce maturation andsecretion of IL-1β and IL-18, unconventional secretion of DAMPs, andpyroptotic cell death by activating caspase-1 through the canonical Nlrp3inflammasome. In contrast, noncanonical activation of caspase-1 by Escherichiacoli, Citrobacter rodentium, and Vibrio cholerae requires caspase-11 in addition tothe regular Nlrp3 inflammasome. Noncanonical activation of caspase-1 inducesmaturation and secretion of IL-1β and IL-18, whereas pyroptosis and DAMPsecretion proceed directly through caspase-11. Abbreviations: CASP, caspase;DAMP, danger-associated molecular pattern; HMGB1, high-mobility groupbox 1; IL, interleukin; NLR, Nod-like receptor; PAMP, pathogen-associatedmolecular pattern.

typhimurium strain only partially relied onIL-1β and IL-18 (Miao et al. 2010a). The laststudy characterized pyroptosis, a proinflam-matory cell death mode that requires caspase-1activity, as a critical mechanism by whichinflammasomes contribute to host responsesagainst gram-negative bacterial pathogens invivo. Pyroptosis was also implicated in clear-ance of the gram-positive pathogen Bacillus

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anthracis in vivo (Terra et al. 2010). Pyroptoticcell death has mainly been characterized inmyeloid cells infected with pathogenic bacteriasuch as Shigella flexneri, S. typhimurium, Pseu-domonas aeruginosa, L. pneumophila, B. anthracis,Staphylococcus aureus, Listeria monocytogenes, andF. tularensis (Chen et al. 1996, Hilbi et al. 1998,Jones et al. 2010, Lamkanfi & Dixit 2010,Miao et al. 2010a, Terra et al. 2010), but itmay affect cells of the central nervous systemand the cardiovascular systems under ischemicconditions as well (Bergsbaken et al. 2009).

This genetically programmed cell deathmode differs morphologically from apoptosisin that it features cytoplasmic swelling andearly plasma membrane rupture (Lamkanfi& Dixit 2010). The consequent release ofthe cytoplasmic content into the extracellularspace is thought to render pyroptosis proin-flammatory, whereas apoptosis is generallyconsidered an immunologically silent celldeath mechanism (Lamkanfi 2011, Tayloret al. 2008). However, apoptosis and pyroptosisalso share several biochemical features such asthe requirement for caspase activity (albeit thecaspases involved differ), condensation of thenuclear compartment, and oligonucleosomalfragmentation of genomic DNA (Lamkanfi& Dixit 2010). Although the biochemicalpathway by which caspase-1 activation inducespyroptosis largely remains to be elucidated,this cell death mode proceeds independently ofIL-1β and IL-18 (Lamkanfi et al. 2008; Miaoet al. 2010a; Monack et al. 1996, 2001).

In vivo, pyroptosis may represent a mech-anism that prevents intracellular replication ofinfectious agents by eliminating the infectedmacrophages and dendritic cells altogether. Byreleasing their intracellular content into cir-culation, pyroptotic cells may simultaneouslytarget surviving bacteria for destruction byphagocytes and neutrophils and alert otherimmune cells to imminent danger (Miao et al.2010a). Altogether, pyroptosis is emergingas an intriguing inflammasome-mediatedhost defense mechanism against intracellularpathogens.

Unconventional Secretion of Growthand Inflammatory Factors

A third emerging mechanism by which inflam-masomes may contribute to immune signalingis the secretion of leaderless cytokines andgrowth factors (Figure 3). Unlike convention-ally secreted factors, these proteins lack signalpeptides to direct them to the translocation ap-paratus of the classical endoplasmic reticulum(ER)-Golgi complex pathway (Lee et al. 2004,Trombetta & Parodi 2003). In fact, IL-1β andIL-18 were two of the first proteins recognizedto be exported independently of the ER–Golgicomplex (Rubartelli et al. 1990). Recent studieshave extended the list of unconventionallysecreted cytokines and growth factors tomore than 20 proteins, including the DAMPHMGB1, the IL-1β-related cytokine IL-1α,growth factors such as fibroblast growth factor2 (FGF2), and the lectins galectin-1 and -3(Nickel & Rabouille 2009).

The biochemical mechanism(s) by whichleaderless proteins are secreted into theextracellular space largely remains to becharacterized, but inflammasomes might playa central role in this process. In addition tothe expected defects in the secretion of matureIL-1β and IL-18, monocytes and macrophageslacking the inflammasome components Nlrp3,ASC, and caspase-1 also failed to secretenormal levels of IL-1α after LPS stimulation(Kuida et al. 1995, Sutterwala et al. 2006).Similarly, caspase-1 was required for secretionof FGF2 by macrophages, UVA-irradiatedfibroblasts, and UVB-irradiated keratinocytes(Keller et al. 2008). Finally, components ofthe Nlrp3 and Nlrc4 inflammasomes also wererequired for extracellular release of HMGB1from LPS-activated and infected macrophages(Lamkanfi et al. 2010). Unlike IL-1β andIL-18, caspase-1 does not process secretedIL-1α, FGF2, and HMGB1 (Dinarello 2009,Keller et al. 2008, Lamkanfi et al. 2010), whichsuggests that inflammasomes may indirectlyregulate unconventional protein secretion. Inthis respect, the secretion of leaderless proteinswas proposed to occur in shed microvesicles,

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secretory lysosomes, or exosomes (Nickel &Rabouille 2009), but whether caspase-1 regu-lates the trafficking of such membrane-boundparticles remains to be determined. What hasbecome clear, however, is that the release ofdifferent leaderless client proteins is not neces-sarily interdependent. For instance, althoughS. typhimurium-infected macrophages simul-taneously secrete IL-1β, IL-18, and HMGB1,secretion of the last proceeds unhampered inmacrophages lacking both IL-1β and IL-18(Lamkanfi et al. 2010). More importantly,caspase-1 enzymatic activity appears to be re-quired for the secretion of leaderless proteins.Indeed, pharmacological inhibition of caspase-1 not only prevented secretion of IL-1β andIL-18 but also affected the release of IL-1α

from LPS-activated peritoneal macrophagesand UVB-irradiated keratinocytes (Kelleret al. 2008). Similarly, HMGB1 release fromLPS-primed and S. typhimurium-infectedmacrophages was impaired by the caspase-1 in-hibitor Ac-YVAD-cmk (Lamkanfi et al. 2010).These observations suggest that caspase-1may activate a secretion apparatus of unknownidentity by cleaving a regulatory factor. Thesmall GTPase Rab39a was recently suggestedas a caspase-1 substrate that may be involved insecretion of IL-1β from LPS-activated THP-1cells (Becker et al. 2009). However, furtherstudy is required to determine whether Rab39aplays a role in secretion of other leaderless pro-teins and to examine how caspase-1-mediatedprocessing affects its functions. Alternatively,caspase-1-mediated release of leaderless pro-teins might be coupled to pyroptosis. Furthercharacterization of these processes undoubt-edly will shed more light on this matter.

Additional InflammasomeEffector Mechanisms

Apart from the effector mechanisms describedabove, inflammasomes have been implicated ininactivation of glycolysis enzymes (Shao et al.2007), activation of sterol-regulatory elementbinding protein-1 and -2 (Gonzalez et al. 2008),and activation of the executioner caspase-7

during L. pneumophila and S. typhimurium infec-tion (Akhter et al. 2009, Lamkanfi et al. 2008).Together, these mechanisms illustrate that in-flammasomes can contribute to a diverse set ofresponses that collectively may help the host toeffectively fight microbial pathogens and otherthreats (Lamkanfi 2011).

MECHANISMS OFINFLAMMASOME ACTIVATION

The Nlrp1 Inflammasome

An intriguing aspect of inflammasome biologyis that their assembly and activation proceedin a signal-specific manner (Figure 2). Forexample, the cytosolic presence of B. anthracislethal toxin specifically alerts NLRP1 (Boyden& Dietrich 2006). This toxin is the major causeof death in systemic anthrax (Dixon et al. 1999,Friedlander 2001). The protective antigensubunit of the toxin allows the metalloproteaseeffector subunit lethal factor (LF) to enterthe cytosol of infected host cells. Humansexpress NLRP1 from a single gene, whereasthe murine genome encodes three tandemparalogs (Nlrp1a, Nlrp1b, and Nlrp1c) (Boyden& Dietrich 2006). Strong genetic evidencepoints to Nlrp1b as a key susceptibility locus forLT-induced caspase-1 activation and pyropto-sis induction (Boyden & Dietrich 2006). First,macrophages from 129S1 mice are susceptibleto LF intoxication and express Nlrp1b but notNlrp1a or Nlrp1c (Boyden & Dietrich 2006).Second, Nlrp1b is highly polymorphic; fivedifferent gene variants have been identifiedin a set of 18 inbred mouse strains. Notably,susceptibility to LF-induced pyroptosis per-fectly matched these variations in Nlrp1b(Boyden & Dietrich 2006). Third, wild-typeC57BL/6 macrophages carry a dysfunctionalNlrp1b allele, but C57BL/6 mice transgenicallyexpressing a functional Nlrp1b variant from129S1 mice are susceptible to LF-inducedcaspase-1 activation and pyroptosis induction(Boyden & Dietrich 2006).

In analogy to TLRs, Nlrp1b was initiallyassumed to bind cytosolic LF directly through

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its LRR motifs. However, that LF metallopro-tease activity is required for activation of theNlrp1b inflammasome suggested that Nlrp1bindirectly senses the cytosolic presence of LFthrough the cleavage of host substrates ratherthan through direct binding of the microbialprotease (Fink et al. 2008). LF-mediated cleav-age of mitogen-activated protein (MAP) kinasekinases (MKKs) leads to impaired activationof the downstream MAP kinases p38, ERK,and JNK (Duesbery et al. 1998). Inhibition ofp38 and Akt was recently suggested to triggerATP release through connexin-43 channels,which in turn causes K+ efflux and Nlrp1bactivation downstream of the purinergic P2X7

receptor (Ali et al. 2011). Ca2+ fluxes and pro-teasome activation were also proposed to actupstream of Nlrp1b activation (Fink et al. 2008,Muehlbauer et al. 2010, Wickliffe et al. 2008).Finally, LF-induced activation of Nlrp1b wassuggested to involve cleavage of a currently un-known host factor by cathepsin B released fromdestabilized lysosomes (Newman et al. 2009).

Regardless of the precise mechanism induc-ing Nlrp1b activation, lethal toxin–mediatedactivation of the Nlrp1b inflammasome clearlyrepresents a key host defense mechanism forcontrolling infection with B. anthracis sporesin vivo (Terra et al. 2010). Both pyroptosis andsignaling downstream of the IL-1 receptor havebeen proposed to contribute to inflammasome-mediated resistance against B. anthracisinfection (Ali et al. 2011, Terra et al. 2010).Future studies should focus on further char-acterizing the mechanisms leading to Nlrp1bactivation and on determining whether Nlrp1aand Nrlp1c also assemble inflammasomes.

The Nlrp3 Inflammasome

The importance of inflammasome signaling tohost defense responses is not limited to B. an-thracis infection. The Nlrp3 inflammasome es-pecially has been implicated in responses to abroad spectrum of infectious agents, includingthe bacterial pathogens S. aureus, Vibrio cholerae,Escherichia coli, Neisseria gonorrhoeae, Chlamydiapneumoniae, and Citrobacter rodentium (Duncan

et al. 2009, He et al. 2010, Kayagaki et al. 2011,Shimada et al. 2011, Toma et al. 2010); the fun-gal pathogens Candida albicans and Aspergillusfumigatus (Gross et al. 2009, Hise et al. 2009,Joly et al. 2009, Said-Sadier et al. 2010); viralpathogens such as influenza A, encephalomy-ocarditis virus, and vesicular stomatitis virus(Allen et al. 2009, Ichinohe et al. 2010, Rajanet al. 2011, Thomas et al. 2009); and the para-sites Schistosoma mansoni and Dermatophagoidespteronyssinus (Dai et al. 2011, Ritter et al. 2010).The large set of pathogens activating Nlrp3suggests that this NLR senses microbes indi-rectly by monitoring the levels of a host-derivedDAMP that is produced or released as a con-sequence of cellular or tissue injury elicitedby toxins of the infectious agent (Lamkanfi &Dixit 2009) (Figure 2). Indeed, DAMPs suchas ATP, uric acid crystals, amyloid-β fibrils,and hyaluronan all activate Nlrp3 (Halle et al.2008, Mariathasan et al. 2006, Martinon et al.2006, Yamasaki et al. 2009). Crystalline par-ticles such as amyloid fibrils, alum, silica, as-bestos, and nanomaterials may simulate the ef-fects of microbial toxins and lead to Nlrp3 acti-vation through similar mechanisms (Tschopp& Schroder 2010). Given the wide array ofmolecules inducing activation of the Nlrp3 in-flammasome, its activation is tightly regulatedat multiple levels. Unlike other inflammasome-activating NLRs, Nlrp3 is expressed at verylow levels in naive macrophages and dendriticcells. Consequently, NF-κB-driven upregula-tion of Nlrp3 transcripts is a first necessity foractivation of this inflammasome (Bauernfeindet al. 2009). However, priming alone is notsufficient, because Nlrp3 inflammasome acti-vation occurs only in TLR-activated cells thatare subsequently exposed to bacterial toxins,DAMPs, or crystalline substances (Lamkanfi &Dixit 2009, Tschopp & Schroder 2010).

Although how Nlrp3 is activated remainsunclear, three putative mechanisms havebeen formulated. The first involves K+ effluxthrough the purinergic P2X7 receptor andother ion channels and pore-forming toxinssuch as nigericin, maitotoxin, and hemolysins(Franchi et al. 2007a, Perregaux & Gabel 1994,

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Petrilli et al. 2007, Walev et al. 1995). How-ever, the above ion channels and cytotoxins alsomodulate the cellular concentrations of H+,Na+, and Ca2+, which suggests that ion fluxes ingeneral may impact Nlrp3 activation (Lamkanfi& Dixit 2009). In this regard, Nlrp3 activationby phagocytosed uric acid crystals was recentlyproposed to involve a massive influx of Na+; theensuing influx of water and drop in intracellularK+ concentrations compensate for the rise inintracellular osmolarity (Schorn et al. 2011).Moreover, the influenza M2 channel deacidifiesthe Golgi complex lumen by exporting H+ ionsinto the cytosol, which in turn trigger Nlrp3activation (Ichinohe et al. 2010). However, K+

and other ion fluxes also have been implicatedin activation of the Nlrp1b (Ali et al. 2011, Finket al. 2008, Newman et al. 2009, Wickliffe et al.2008) and Nlrc4 inflammasomes (Arlehamnet al. 2010). Thus, although ion fluxes maymodulate the threshold for caspase-1 activation,they are unlikely to represent a specific signaldirectly leading to assembly of specific inflam-masomes (Lamkanfi & Dixit 2009). A secondproposal suggests that mitochondrial reactiveoxygen species (ROS) account for Nlrp3activation. This notion is based on the ob-servation that all Nlrp3-activating molecules,such as ATP, nigericin, alum, and uric acid,induce ROS production in macrophages andmonocytes (Cruz et al. 2007, Zhou et al. 2011).However, TLR signaling is also accompaniedby ROS production but nevertheless failsto activate the Nlrp3 inflammasome in theabsence of a second challenge. Concurrently,recent studies implicated mitochondrial ROSin the NF-κB-mediated upregulation of Nlrp3and proIL-1β transcripts rather than in Nlrp3inflammasome activation per se (Bauernfeindet al. 2011, Bulua et al. 2011).

The third model proposes that phagosomaldestabilization and cytosolic release of lysoso-mal cathepsins drive Nlrp3 activation. Indeed,phagocytosis of crystalline and particulatemolecules may cause damage to the lysosomalmembrane, which consequently leads to leak-age of lysosomal cathepsins into the cytosol. Inthis regard, cathepsin B–mediated processing

of a cytosolic factor was suggested to act up-stream of Nlrp3 activation by silica, alum, andamyloid-β fibrils (Halle et al. 2008, Hornunget al. 2008). Cytosolic release of cathepsin Bwas also implicated in caspase-1 activation bythe ionophore nigericin (Hentze et al. 2003),which suggests a unifying mechanism for Nlrp3activation by both particulate and nonpartic-ulate stimuli. However, the observation thatactivation of the Nlrp3 inflammasome was notaffected in cathepsin B-deficient macrophagesexposed to malarial hemozoin, uric acid crys-tals, silica, and alum suggests redundancy withother cathepsins or other pathways leading toNlrp3 activation (Dostert et al. 2009, Tschopp& Schroder 2010). In this regard, a recent studyshowed that live bacteria activate the Nlrp3inflammasome in a TIR-domain-containingadaptor-inducing interferon-β (TRIF)-depen-dent manner owing to the leakage of microbialmRNAs from damaged phagosomes into thecytosol (Sander et al. 2011). The absence ofa 3 polyadenylyl tail that is characteristic ofeukaryotic mRNAs appears critical for Nlrp3inflammasome activation by microbial RNAs.Because mRNAs are intrinsically unstable,Nlrp3 inflammasome–mediated recognitionof microbial RNAs may represent an innateimmune mechanism that distinguishes livefrom dead microbes (Sander et al. 2011).

Although further clarification of the molec-ular mechanisms leading to Nlrp3 activationis required, an intriguing role was recentlyrevealed for mouse caspase-11 (Kayagakiet al. 2011). This caspase-1-related protease isrepresented by caspases-4 and -5 in the humangenome (Lamkanfi et al. 2002). Althoughcaspase-11 was dispensable for caspase-1activation by canonical Nlrp3 activators suchas ATP and nigericin, it proved essential forcaspase-1 maturation and IL-1β secretionfrom macrophages infected with the entericbacteria E. coli, C. rodentium, and V. cholerae(Kayagaki et al. 2011) (Figure 3). Caspase-11also mediates noncanonical activation of theNlrp3 inflammasome in vivo during LPS-induced endotoxemia (Kayagaki et al. 2011,Wang et al. 1998). In keeping with this notion,

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caspase-11-deficient mice had less IL-β and IL-18 in circulation (Kayagaki et al. 2011, Wanget al. 1998). Moreover, they were markedlyresistant to lethal doses of LPS (Kayagakiet al. 2011, Wang et al. 1998). Caspase-1 wasinitially also implicated in protection againstLPS-induced lethality on the basis of theresistant phenotype of published caspase-1knockout mice (Kuida et al. 1995, Li et al.1995). However, it recently emerged that thesemice also lack caspase-11 expression owing to amutation in the caspase-11 locus of 129S mice,embryonic stem cells of which were used togenerate available caspase-1−/− mice (Kayagakiet al. 2011). Caspase-11 expression in theseapparent double knockout mice was restoredfrom an appropriate C57BL/6 bacterial artifi-cial chromosome, and subsequent studies withthese transgenic mice revealed that caspase-1deficiency alone provided only mild protectionagainst LPS-induced lethality (Kayagaki et al.2011). Concurrently, mice lacking both IL-1β

and IL-18 were demonstrated to be susceptibleto LPS-induced lethality (Lamkanfi et al. 2010).In agreement with these findings, mice lackingNlrp3 or ASC failed to produce IL-1β andIL-18 when challenged with high doses of LPSbut survived only slightly longer than wild-type mice (Kayagaki et al. 2011). Nevertheless,Nlrp3-dependent IL-1β and IL-18 productionmay provide an amplification signal giventhat Nlrp3−/− and Asc−/− mice were relativelyresistant to shock when challenged with lowerdoses of LPS (Mariathasan et al. 2004, 2006).Importantly, these observations suggest thatcaspase-11 may induce tissue damage andlethality independently of caspase-1. Indeed,LPS-induced serum levels of the DAMPIL-1α were significantly reduced in micelacking both caspases-1 and -11 and in thosedeficient only for caspase-11 (Kayagaki et al.2011). In contrast, transgenic mice lackingonly caspase-1 had high levels of IL-1α incirculation. Moreover, pyroptotic cell deathand release of IL-1α and HMGB1 frommacrophages infected with E. coli, C. rodentium,and V. cholerae required caspase-11, but notNlrp3, ASC, or caspase-1 (Kayagaki et al.

2011). Clearly, these observations warrantfurther inspection of the mechanisms leadingto caspase-11 activation and the pathways bywhich it exerts its downstream functions.

The Nlrc4 Inflammasome

Unlike the Nlrp3 inflammasome, Nlrc4 iscurrently thought to respond to only two bac-terial components: flagellin and the PrgJ basalbody of bacterial type III secretion systems(Miao et al. 2010b) (Figure 2). Consequently,facultative intracellular pathogens expressingthese factors, such as S. typhimurium, S. flexneri,P. aeruginosa, B. thailandensis, and L. pneu-mophila, all activate the Nlrc4 inflammasome(Amer et al. 2006; Franchi et al. 2006, 2007b;Lamkanfi et al. 2007; Mariathasan et al. 2004;Miao et al. 2006, 2008, 2010a; Sutterwala et al.2007; Suzuki et al. 2007).

The BIR-containing NLRs Naip2 andNaip5 link Nlrc4 to recognition of PrgJ andflagellin, respectively (Kofoed & Vance 2011,Zhao et al. 2011). The murine Naip subfam-ily consists of seven NLR family members(Naip1–7), four of which (Naip-1, -2, -5, and-6) are expressed in C57BL/6 mice (Wrightet al. 2003). The observation that Naip2 andNaip5 recruit PrgJ and flagellin begs thequestion of whether detection of bacterialfactors by Naip proteins represents a generalmechanism conferring specificity to distinctinflammasomes. This appears unlikely, how-ever, given that humans encode a single NAIPprotein. Mutations in human NAIP are linkedto spinal muscular atrophy (Roy et al. 1995),but whether these mutations also increasesusceptibility to bacterial infections is notknown. Notably, unlike mouse macrophages,human monocytes and macrophages appearresistant to inflammasome activation by bacte-rial flagellin and PrgJ-like rod proteins (Zhaoet al. 2011). Instead, human NAIP activatesthe NLRC4 inflammasome upon detection ofChromobacterium violaceum CprI and homolo-gous needle subunits of the type III secretionapparatus of S. typhimurium, B. thailandensis,P. aeruginosa, and S. flexneri (Zhao et al. 2011).

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These observations raise doubt regardingthe importance of inflammasome-mediatedflagellin recognition in human infections. Theyalso suggest that Naip proteins may contributeto immunity in several ways. In this regard,naturally occurring mutations in Naip5 renderA/J mice and macrophages highly susceptibleto L. pneumophila infection but fail to preventflagellin-induced activation of the Nlrc4 in-flammasome (Lamkanfi et al. 2007, Lightfieldet al. 2008, Miao et al. 2008). These mutationsmarkedly reduce Naip5 expression levels in A/Jmacrophages relative to C57BL/6 macrophages(Wright et al. 2003). Although the pre-cise mechanism by which Naip5 regulatesL. pneumophila clearance in A/J macrophagesremains unclear, it may regulate cell death andmaturation of Legionella-containing phago-somes (Akhter et al. 2009, Fortier et al. 2007).Thus, further characterization of murine Naipproteins is required to fully understand theirroles in innate immune signaling.

The Nlrp6 Inflammasome

The roles of Nlrp6 in inflammasome signalingare less established. Nlrp6-deficient mice aremore susceptible to dextran sodium sulfate(DSS)-induced colitis and inflammation-associated colon tumorigenesis (Chen et al.2011, Elinav et al. 2011, Normand et al. 2011).Interestingly, in one study Nlrp6 deficiencycaused marked changes in the composition ofintestinal flora characterized by an increasedpresence of pathogenic Prevotellaceae andTM7 species (Elinav et al. 2011). Similarchanges in the microflora were observed inmice lacking ASC, caspase-1, and IL-18, whichsuggests that assembly of a functional Nlrp6inflammasome is required for maintenance of ahealthy colonic microflora (Elinav et al. 2011).Strikingly, the exacerbated colitis phenotypeof Asc−/− animals could be transferred tocohoused and cross-fostered wild-type mice,which suggests that the skewed microflorain Asc−/− and Nlrp6−/− mice was the maincolitogenic factor driving increased colitisseverity in these mice (Elinav et al. 2011). A

detailed biochemical characterization of thissignaling pathway awaits the identificationof specific PAMPs and DAMPs that caninduce assembly of the Nlrp6 inflammasomein isolated epithelial and hematopoietic cells.

The AIM2 Inflammasome

In addition to the NLRs above, the HIN200family member AIM2 was recently shown toassemble an inflammasome that is critical foractivating caspase-1 in macrophages infectedwith F. tularensis and in response to DNAviruses such as cytomegalovirus and vacciniavirus (Fernandes-Alnemri et al. 2010, Joneset al. 2010, Rathinam et al. 2010, Sauer et al.2010). In association with Nlrp3 and Nlrc4,the AIM2 inflammasome also contributesto caspase-1 activation by L. monocytogenes(Rathinam et al. 2010, Sauer et al. 2010).Similar to AIM2, the three remaining humanHIN200 proteins (named IFI16, MNDA, andIFIX) combine an amino-terminal PYD do-main with one or two carboxy-terminal double-stranded (ds)DNA-binding HIN200 motifs.However, the latter three HIN200 proteins arepresent in the nuclear compartment of restingmacrophages and dendritic cells, whereas AIM2is found in the cytosol (Burckstummer et al.2009). This suggests that AIM2 may recognizereplicating microbes in the cytosol of infectedmacrophages by means of a direct associationbetween its HIN200 domain and genomicmaterial of the infectious agent. Ensuingconformational changes may induce an openconformation that allows recruitment of ASCand caspase-1 through AIM2’s amino-terminalPYD. Although AIM2 may not encounter self-DNA under normal conditions, transfection ofsynthetic and mammalian dsDNA neverthelessinduced activation of the AIM2 inflammasome(Burckstummer et al. 2009, Fernandes-Alnemriet al. 2009, Hornung et al. 2009). Together withthe observation that AIM2 deficiency stimu-lates the expression of the interferon-induciblelupus susceptibility gene Ifi202 (Panchanathanet al. 2010), this suggests that inactivatingmutations in AIM2 may increase susceptibility

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to autoimmune diseases in which reactionsagainst self-DNA play an important role.

INFLAMMASOMES INAUTOINFLAMMATIONAND AUTOIMMUNITY

Recent years have seen significant progressin our understanding of how inflammasomescontribute to the molecular pathology ofmultiple autoinflammatory and autoimmunediseases. Two studies linked single-nucleotidepolymorphisms (SNPs) in the promoter andcoding regions of NLRP1 with increasedincidence of vitiligo and vitiligo-associatedAddison’s disease, respectively ( Jin et al.2007a,b). Vitiligo is a rare autoimmune diseasethat is characterized by depigmentation of theskin and hair, whereas the adrenal cortex ofpatients with Addison’s disease is attacked bythe immune system and gradually becomesimpaired in the production of glucocorticoidsand adrenal androgen. Notably, a SNP in theNLRP1 open reading frame (SNP rs12150220)also strongly linked to Addison’s disease in theabsence of vitiligo (Magitta et al. 2009, Zuraweket al. 2010). Because most identified SNPsin NLRP1 (including rs12150220) are locatedin and around the central NACHT domain,they are thought to reduce the threshold forinflammasome assembly and IL-1β production( Jin et al. 2007b). If this model holds, caspase-1inhibitors and IL-1β neutralizing therapiesmay be used for treating vitiligo and Addison’sdisease patients carrying NLRP1 SNPs.

As with NLRP1, gain-of-function muta-tions in and around the NLRP3 NACHTdomain have been associated with a spec-trum of hereditary autoinflammatory diseasesthat are collectively referred to as cryopyrin-associated periodic syndromes (CAPS). Theprimary symptoms of CAPS patients are ur-ticarial skin rashes and prolonged episodes offever, but arthralgia, sensorineural hearing loss,headaches, elevated spinal fluid pressure, cog-nitive deficits, and renal amyloidosis also maybe observed (Feldmann et al. 2002, Hoffmanet al. 2001). Apart from the bony overgrowth

seen in some CAPS patients, excessive produc-tion of IL-1β and IL-18 by mononuclear cellsmay explain most of these symptoms. Indeed,the contribution of excessive IL-1β levels wasrecently confirmed in mice expressing CAPS-associated Nlrp3 variants (Brydges et al. 2009,Meng et al. 2009). Moreover, IL-1 neutraliz-ing therapies proved highly beneficial in CAPSpatients (Hawkins et al. 2003; Hoffman et al.2004, 2008; Lachmann et al. 2009).

Notably, another set of SNPs in theNLRP3 promoter have been associated withincreased susceptibility to Crohn’s diseasein humans. These polymorphisms causeddecreased NLRP3 expression and reducedIL-1β production in cells stimulated with TLRagonists (Villani et al. 2009). In addition, poly-morphisms in IL-18 correlated with increasedsusceptibility to Crohn’s disease (Tamura et al.2002). Further insight into the roles of Nlrp3and IL-18 in protection against intestinalinflammation came from the analysis of gene-deficient mice. Nlrp3−/− mice presented withincreased body weight loss, rectal bleeding, di-arrhea, and mortality when subjected to DSS-and 2,4,6-trinitrobenzene sulfonate–inducedcolitis, which confirms that Nlrp3 expression isrequired for protection against gastrointestinalinflammation (Allen et al. 2010, Hirota et al.2010, Zaki et al. 2010a). The critical role ofinflammasome signaling in protection againstcolon inflammation was confirmed in micelacking ASC and caspase-1 (Allen et al. 2010,Dupaul-Chicoine et al. 2010, Zaki et al. 2010a)as well as in animals lacking IL-1β and IL-18or their cognate receptors (Lebeis et al. 2009,Salcedo et al. 2010, Takagi et al. 2003). Micelacking components of the Nlrp3 inflamma-some also suffered from increased dysplasia andtumor formation in the azoxymethane/DSStumorigenesis model (Allen et al. 2010, Zakiet al. 2010b). Furthermore, mice lacking Nlrc4were protected from tumor formation (Huet al. 2010), which points to a key role forinflammasome signaling in regulating guthomeostasis and colon tumorigenesis.

Finally, inflammasome signaling might con-tribute to multiple sclerosis, as it was shown

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to exacerbate disease progression in the exper-imental autoimmune encephalomyelitis (EAE)mouse model. Indeed, mice lacking Nlrp3 andASC were protected from EAE developmentbecause of reduced TH1 and TH17 responses(Gris et al. 2010, Shaw et al. 2010). This pro-tective phenotype was attributed to defectivecaspase-1 activation and IL-18 secretion be-cause caspase-1−/− and il-18−/− mice were alsoprotected (Furlan et al. 1999, Gris et al. 2010,Shaw et al. 2010). Further insight into howinflammasomes regulate neuronal inflamma-tion may pave the way for the development ofnovel therapeutic options for this debilitatingdisease.

MODULATION OFINFLAMMASOME ACTIVATIONAND ACTIVITY

Inflammasome activation contributes signifi-cantly to host and inflammatory responses, butthe association of gain-of-function mutationsin NLRP3, NLRP1, and other inflammasomecomponents with autoimmune and autoin-flammatory disorders illustrates that excessiveinflammasome activity can be harmful. There-fore, inflammasome activation and activity aretightly regulated to avoid sterile inflammation.Inflammasome components such as NLRP3,caspase-11, and proIL-1β are expressed atrelatively low levels, and priming with NF-κB-activating inflammatory cytokines, TLRligands, and other PAMPs is required for theirmRNAs to be induced (Bauernfeind et al. 2011,Bulua et al. 2011, Kayagaki et al. 2011). Inaddition, type I interferon signaling is requiredfor efficient activation of the AIM2 inflamma-some by F. tularensis, although it is dispensablefor activation of this inflammasome by mousecytomegalovirus (Fernandes-Alnemri et al.2010, Henry et al. 2007, Jones et al. 2010,Rathinam et al. 2010). Because AIM2 levelswere not altered in F. tularensis-infected Irf3−/−

and Ifnar−/− cells (Fernandes-Alnemri et al.2010), type I interferon signals were proposedto enhance phagosomal digestion and cytosolicrelease of microbial DNA (Fernandes-Alnemri

et al. 2010). Further regulatory checkpointsinvolve human CARD-only proteins (COPs),such as ICEBERG, COP, INCA, and caspase-12S, and PYD-only proteins (POPs), such ashuman cPOP1 and -2 (Lamkanfi & Dixit 2011).These molecules interfere with inflammasomeassembly by scavenging ASC and caspase-1.Recent work also demonstrated that autophagynegatively regulates inflammasome activation,possibly by promoting accumulation of dys-functional mitochondria and the release ofmitochondrial DNA into the cytosol (Nakahiraet al. 2011, Saitoh et al. 2008). Finally, theenzymatic activity of caspase-1 is directlyregulated by the serpin proteinase inhibitor 9(PI-9) and its two rodent homologs (Lamkanfi& Dixit 2011).

The different checkpoint mechanismsabove illustrate the importance of preventingunwarranted and disproportional activationof inflammasome effector pathways. It is thusnot surprising that pathogens evolved differentvirulence mechanisms to modulate inflamma-some activation to their benefit (Figure 4).A strategy often used by viruses is to mimicthe mechanisms used by host cells to evadeinflammasome activation. This theme is bestillustrated by the cowpox virus PI-9 homologcytokine response modifier A (CrmA) andsimilar serpins encoded by the orthopoxvirusesvaccinia, ectromelia, and rabbitpox. In additionto the CrmA homologs SPI-1 and SPI-2, vac-cinia produces soluble IL-18-binding proteins(vIL-18BPs) that prevent activation of the IL-18 receptor as well as an IL-1β-neutralizingscavenger receptor named virus-encodedIL-1β receptor (vIL-1βR) (Lamkanfi & Dixit2011). Myxoma virus M013L and Shopefibroma virus S013L also provide examplesof how viral mimicry contributes to viremia.These viral POPs inhibit IL-1β production byinterfering with its transcription while simul-taneously scavenging ASC through their PYDdomains to prevent proIL-1β maturation ininflammasomes (Rahman et al. 2009). Further-more, Kaposi’s sarcoma-associated herpesvirusexpresses Orf63, a NLRP1 homolog that con-tributes to virulence by preventing assembly

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IL-1R IL-18R

ASC

CASP1 Inflammasome

Nlrp3

NF-κB

TLR

ProIL-1β, proIL-18

EndosomeEndosome

KSHV Orf63

vPOPs:Myxoma virus M013LShope fibroma virus S013L

VacciniaEctromeliaCowpox vIL-18BPVaccinia vIL-1βR

IL-18

IL-1β

Active CASP11

Influenza NS1Mycobacterium tuberculosis zmp1Yersinia enterocolitica YopE, YopTYersinia pseudotuberculosis YopKPseudomonas aeruginosa ExoS, ExoUFrancisella tularensis mviN

Legionella pneumophilaPoxviruses, other pathogens

Cowpox CrmA homologs:Rabbitpox CrmAMyxoma virus Serp2Vaccinia virus SP1/2

PYD CARD

PYD NACHT LRR

Caspase domainCARD

Figure 4Virulence factors modulating inflammasome signaling. Certain viruses and bacterial pathogens express proteins that inhibitinflammasome assembly and activity. Cowpox CrmA and homologous serpins of myxoma and vaccinia virus bind and inhibit theenzymatic activity of caspase-1 directly. Orthopoxviruses also produce scavenger receptors that bind secreted IL-1β and IL-18. Inaddition, they express vPOPs that prevent inflammasome assembly by scavenging ASC. Similarly, KSHV Orf63 is a Nlrp1 decoyprotein that prevents inflammasome assembly. Poxviruses, Legionella pneumophila, and other pathogens inhibit transcription of ASC,proIL-1β, and proIL-18 mRNA. Certain virulence factors encode enzymatic activities that modulate inflammasome activation.Examples are influenza NS1 protein; the Mycobacterium tuberculosis putative Zn2+ metalloprotease zmp1; the Yersinia effectors YopE,YopT, and YopK; the Pseudomonas aeruginosa virulence factors ExoS and ExoU; and Francisella tularensis mviN. Abbreviations: ASC,apoptosis-associated speck-like protein containing a CARD (caspase recruitment domain); CASP, caspase; CrmA, cytokine responsemodifier A; IL, interleukin; KSHV, Kaposi’s sarcoma-associated herpesvirus; LRR, leucine-rich repeat; NACHT, nucleotide-bindingand oligomerization domain; NLR, Nod-like receptor; PYD, pyrin; TLR, Toll-like receptor; vPOP, PYD-only protein.

of the NLRP1 and NLRP3 inflammasomes(Gregory et al. 2011). In addition to using pro-teins mimicking host regulatory mechanisms,viruses have devised new ways to regulateinflammasome function. Human influenzaA/PR/8/34 (H1N1) virus NS1 and baculovirus

p35 are two examples of potent inflammasomeinhibitors that lack apparent human paralogs(Lamkanfi & Dixit 2011).

Some bacteria appear to use strategiesaimed at preventing host recognition al-together by preventing their uptake and by

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masking their ligands. For example, the Yersiniapseudotuberculosis effector YopK prevents acti-vation of the Nlrp3 and Nlrc4 inflammasomesby masking the bacterial type III secretionsystem (Lamkanfi & Dixit 2011). In addition,Yersinia enterocolitica YopE and YopT interferewith Rho GTPases to prevent cytoskeletalreorganizations and inflammasome assembly.Pathogens such as L. pneumophila downregulatetranscription of ASC to prevent inflammasomeactivation and to promote their replicationin human monocytes (Abdelaziz et al. 2011).Other bacterial virulence factors encodeenzymatic activity to interfere with inflam-masome activation. For example, P. aeruginosaexoenzyme U (ExoU) is a phospholipase thatinhibits Nlrc4 inflammasome-driven secretionof IL-1β and IL-18, whereas the effector ExoSinhibits caspase-1 activation through its ADP-ribosyl transferase activity (Galle et al. 2008,Sutterwala et al. 2007). Finally, F. tularensisdampens AIM2 inflammasome-mediatedIL-1β secretion and macrophage pyroptosiswith its putative lipid II flippase mviN, whereasMycobacterium tuberculosis inhibits activation ofthe Nlrp3 inflammasome using the putativeZn2+ metalloprotease zmp1 (Master et al.2008, Ulland et al. 2010).

CONCLUSIONS ANDPERSPECTIVES

Step by step, our understanding of inflamma-somes has made a giant leap in the past decade.

The appreciation that caspase-1 activationis not regulated by a single pathway, butinstead is governed by a multitude of cytosolicprotein complexes that are engaged in a highlyregulated manner, has revolutionized ourunderstanding of innate immune processes.Moreover, it has fueled our understanding ofthe mechanisms underlying autoinflammatorydisorders such as CAPS and familial Mediter-ranean fever. However, many importantquestions remain to be answered, includinghow host cells decide which inflammasome toactivate under particular conditions and howinflammasome signaling is intertwined withother innate and adaptive immune pathways.Undoubtedly, the roles of caspase-1 andcaspase-11 and their relative contributionsto infectious and autoinflammatory disordersare additional focal topics for inflammasomeresearch in coming years. In addition, theprecise mechanisms by which these inflam-matory caspases initiate pyroptotic cell deathand mediate unconventional protein secretionrequire further dissection. Answering these andother questions will surely expand the scopeof ailments to which aberrant inflammasomesignaling contributes. As the field movesforward, we expect to see increased applicationto human disease models. In addition to strate-gies targeting inflammatory caspases, clinicaltranslation of this newly gained knowledge mayunveil novel promising targets for therapeuticintervention in infectious, autoinflammatory,and autoimmune diseases.

DISCLOSURE STATEMENT

V.M.D. is an employee of Genentech, Inc.

ACKNOWLEDGMENTS

The authors apologize to those whose citations were omitted owing to space limitations. Wethank Dr. Lieselotte Vande Walle for help with graphics. M.L. is supported by European UnionMarie-Curie grant 256432, ERC Grant 281600, and grants G030212N, 1.2.201.10.N.00, and1.5.122.11.N.00 from the Fund for Scientific Research–Flanders.

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Annual Reviewof Cell andDevelopmentalBiology

Volume 28, 2012 Contents

A Man for All Seasons: Reflections on the Life and Legacyof George PaladeMarilyn G. Farquhar � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 1

Cytokinesis in Animal CellsRebecca A. Green, Ewa Paluch, and Karen Oegema � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �29

Driving the Cell Cycle Through MetabolismLing Cai and Benjamin P. Tu � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �59

Dynamic Reorganization of Metabolic Enzymesinto Intracellular BodiesJeremy D. O’Connell, Alice Zhao, Andrew D. Ellington,

and Edward M. Marcotte � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �89

Mechanisms of Intracellular ScalingDaniel L. Levy and Rebecca Heald � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 113

Inflammasomes and Their Roles in Health and DiseaseMohamed Lamkanfi and Vishva M. Dixit � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 137

Nuclear Organization and Genome FunctionKevin Van Bortle and Victor G. Corces � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 163

New Insights into the Troubles of AneuploidyJake J. Siegel and Angelika Amon � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 189

Dynamic Organizing Principles of the Plasma Membrane thatRegulate Signal Transduction: Commemorating the FortiethAnniversary of Singer and Nicolson’s Fluid-Mosaic ModelAkihiro Kusumi, Takahiro K. Fujiwara, Rahul Chadda, Min Xie,

Taka A. Tsunoyama, Ziya Kalay, Rinshi S. Kasai, and Kenichi G.N. Suzuki � � � � � � � � 215

Structural Basis of the Unfolded Protein ResponseAlexei Korennykh and Peter Walter � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 251

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The Membrane Fusion Enigma: SNAREs, Sec1/Munc18 Proteins,and Their Accomplices—Guilty as Charged?Josep Rizo and Thomas C. Sudhof � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 279

Diversity of Clathrin Function: New Tricks for an Old ProteinFrances M. Brodsky � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 309

Multivesicular Body MorphogenesisPhyllis I. Hanson and Anil Cashikar � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 337

Beyond Homeostasis: A Predictive-Dynamic Frameworkfor Understanding Cellular BehaviorPeter L. Freddolino and Saeed Tavazoie � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 363

Bioengineering Methods for Analysis of Cells In VitroGregory H. Underhill, Peter Galie, Christopher S. Chen,

and Sangeeta N. Bhatia � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 385

Emerging Roles for Lipid Droplets in Immunityand Host-Pathogen InteractionsHector Alex Saka and Raphael Valdivia � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 411

Second Messenger Regulation of Biofilm Formation:Breakthroughs in Understanding c-di-GMP Effector SystemsChelsea D. Boyd and George A. O’Toole � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 439

Hormonal Interactions in the Regulation of Plant DevelopmentMarleen Vanstraelen and Eva Benkova � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 463

Hormonal Modulation of Plant ImmunityCorne M.J. Pieterse, Dieuwertje Van der Does, Christos Zamioudis,

Antonio Leon-Reyes, and Saskia C.M. Van Wees � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 489

Functional Diversity of LamininsAnna Domogatskaya, Sergey Rodin, and Karl Tryggvason � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 523

LINE-1 Retrotransposition in the Nervous SystemCharles A. Thomas, Apua C.M. Paquola, and Alysson R. Muotri � � � � � � � � � � � � � � � � � � � � � � � 555

Axon Degeneration and Regeneration: Insights from Drosophila Modelsof Nerve InjuryYanshan Fang and Nancy M. Bonini � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 575

Cell Polarity as a Regulator of Cancer Cell Behavior PlasticitySenthil K. Muthuswamy and Bin Xue � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 599

Planar Cell Polarity and the Developmental Control of Cell Behaviorin Vertebrate EmbryosJohn B. Wallingford � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 627

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The Apical Polarity Protein Network in Drosophila Epithelial Cells:Regulation of Polarity, Junctions, Morphogenesis, Cell Growth,and SurvivalUlrich Tepass � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 655

Gastrulation: Making and Shaping Germ LayersLila Solnica-Krezel and Diane S. Sepich � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 687

Cardiac Regenerative Capacity and MechanismsKazu Kikuchi and Kenneth D. Poss � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 719

Paths Less Traveled: Evo-Devo Approaches to Investigating AnimalMorphological EvolutionRicardo Mallarino and Arhat Abzhanov � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 743

Indexes

Cumulative Index of Contributing Authors, Volumes 24–28 � � � � � � � � � � � � � � � � � � � � � � � � � � � 765

Cumulative Index of Chapter Titles, Volumes 24–28 � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 768

Errata

An online log of corrections to Annual Review of Cell and Developmental Biology articlesmay be found at http://cellbio.annualreviews.org/errata.shtml

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