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Major cell death pathways at a glance

Linde Duprez a,b, Ellen Wirawan a,b, Tom Vanden Berghe a,b, Peter Vandenabeele a,b,*

a VIB, Department for Molecular Biomedical Research, Unit for Molecular Signaling and Cell Death, Technologiepark 927, B-9052 Ghent (Zwijnaarde),Belgium

b Ghent University, Department of Biomedical Molecular Biology, Unit for Molecular Signaling and Cell Death, Technologiepark 927, B-9052 Ghent

(Zwijnaarde), Belgium

Available online 4 September 2009

Abstract

Cell death is a crucial process during development, homeostasis and immune regulation of multicellular organisms, and its dysregulation isonilltop

and recruitment domain; Cardinal, CARD inhibitor of NF-kB activating ligands;

cFLIP, cellular FLICE-like inhibitory protein; cIAP, cellular inhibitor of apoptosis protein; CypD, cyclophilin D; Cyt c, cytochrome c; DAMP, danger/damage-

linked inhibitor of apoptosis protein; zVAD-fmk, benzyloxycarbonyl-Val-Ala-Asp-fluoro-methylketone.

* Corresponding author. VIB, Department for Molecular Biomedical Research, Unit for Molecular Signaling and Cell Death, Technologiepark 927, B-9052

Ghent (Zwijnaarde), Belgium. Tel.: 32 9 3313763; fax: 32 9 3313609.E-mail address: peter.vandenabeele@dmbr.vib-ugent.be (P. Vandenabeele).

Microbes and Infection 11 (2009) 1050e1062www.elsevier.com/locate/micinfassociated molecular pattern; DED, death effector domain; DISC, death-inducing signaling complex; Diablo, direct IAP binding protein with low pI; DIF-1,

differentiation-inducing factor-1; ER, endoplasmic reticulum; FADD, Fas-associated death domain; FIP200, focal adhesion kinase family interacting protein of

200 kD; HIN-200 protein, hematopoietic interferon-inducible nuclear protein with a 200-amino-acid repeat; HIV-1, human immunodeficiency virus-1; HtrA2, high

temperature requirement protein A2; IAP, inhibitor of apoptosis protein; ICE, interleukin-1 converting enzyme-b; IL-18, interleukin-18; IL-1b, interleukin-1b;

Ipaf, ICE-protease activating factor; I/R, ischemia/reperfusion; JNK, c-Jun N-terminal kinase; LC3, microtubule-associated protein light chain 3; LPC, lyso-

phosphatidylcholine; LPS, lipopolysaccharide; LRR, leucine-rich repeat; MAPK, mitogen activated protein kinase; MEF, mouse embryonic fibroblast; MPT,

mitochondrial permeability transition; MPTP, mitochondrial permeability transition pore; mTOR, mammalian target of rapamycin; NACHT, nucleotide binding

and oligomerization domain; Nalp, NACHT/LRR/PYD-containing protein; Nec-1, necrostatin-1; NF-kB, nuclear factor kB; NLR, NOD-like receptor; nNOS,

neuronal nitric oxide synthase; PAMP, pathogen-associated molecular pattern; PAR, poly(ADP-ribose); PARP-1, poly(ADP-ribose) polymerase-1; PE, phospha-

tidylethanolamine; PI3P, phosphatidylinositol-3-phosphate; PI3KC3, phosphatidylinositol 3-kinase class 3; Pycard, PYD and CARD domain containing protein;

PRR, pathogen recognition receptor; PS, phosphatidylserine; PYD, pyrin effector domain; RIP, receptor interacting protein; RLR, RIG-I-like receptor; RNAi, RNA

interference; ROS, reactive oxygen species; Smac, second mitochondria-derived activator of caspase; TLR, toll-like receptor; TNF, tumor necrosis factor; TNFR,

tumor necrosis factor receptor; TRADD, TNFR-associated death domain; TRAF2, TNF receptor-associated factor 2; TRAIL, TNF-related apoptosis-inducing

ligand; TRAIL-R, TRAIL-receptor; ULK, UNC-51-like kinase; UVRAG, UV radiation resistance-associated gene; VPS34, vacuolar protein sorting 34; XIAP, X-adenine nucleotide translocator; Apaf-1, apoptosis protease activating factor-1; A

Bcl-2-homology 3; Bif-1, Bax interacting factor-1; CARD, caspase activationAbbreviations: AICD, activation-induced cell death; AIM2, absent in melanoma 2; Ambra-1, activating molecule in Beclin-1-regulated autophagy; ANT,

SC, apoptotic speck protein containing a CARD; Bcl-2, B-cell lymphoma-2; BH3,Keywords: Apoptosis; Necrosis; Autophagy; Pyroptosis

1. Introduction

Different types of cell death are often defined by morpho-logical criteria, and are classified as apoptotic, necrotic,

autophagic or associated with mitotic catastrophe. Addition-ally, cell death is defined based on enzymological criteria,including the involvement of different classes of proteases(caspases, calpains, cathepsins and transglutaminases) andassociated with numerous pathologies. Cell death is often induced uphave evolved strategies to modulate host cell death. In this review, we wmajor types of programmed cell death, namely apoptosis, necrosis, au 2009 Elsevier Masson SAS. All rights reserved.1286-4579/$ - see front matter 2009 Elsevier Masson SAS. All rights reserved.doi:10.1016/j.micinf.2009.08.013pathogen infection as part of the defense mechanism, and pathogensdiscuss the molecular mechanisms and physiological relevance of fourhagic cell death and pyroptosis.

e todescribe a form of cell death associated with specific

d Inmorphological features. Since then, apoptosis has beenextensively studied and underlying signaling events are nowwell characterized. Morphologically, apoptosis is associatedwith cell shrinkage, membrane blebbing, and chromatincondensation. It is a cell-intrinsic programmed suicidemechanism that results in the controlled breakdown of the cellinto apoptotic bodies, which are subsequently recognized andengulfed by surrounding cells and phagocytes. Two mainevolutionarily conserved protein families are involved inapoptosis, namely the Bcl-2 family of proteins, which controlmitochondrial integrity [2], and the cysteinyl aspartate-specificproteases or caspases, which mediate the execution phase ofapoptosis [3].

2.1. Molecular mechanisms of apoptosis

In humans, twelve caspases belonging to the apoptotic andthe inflammatory subfamilies of caspases have been described.Apoptotic caspases can be further subdivided into initiator(caspase-2, -8, -9, -10) and executioner (caspase-3, -6, -7)caspases. All caspases are expressed as inactive proenzymesconsisting of an N-terminal prodomain of variable length,followed by a large subunit (p20) and a small C-terminalsubunit (p10). The long prodomain of the initiator caspasescontains proteineprotein interaction motifs belonging to thedeath domain superfamily, namely death effector domains(DEDs) and caspase activation and recruitment domains(CARDs). Initiator caspases, present in the cell as inactivemonomers, are recruited to platform molecules via theseproteineprotein interaction domains and are subsequentlyactivated by oligomerization and proximity-induced autopro-teolysis [4]. Short prodomain caspases exist as preformeddimers in the cell and their enzymatic activity requiresproteolytic maturation by the action of upstream caspases [4].In 1972, the term apoptosis was used for the first timnucleases, on functional aspects (programmed or accidental,physiological or pathological) or on immunological charac-teristics (immunogenic or non-immunogenic) [1]. The presentparadigm is that caspase-dependent apoptosis is the predom-inant cell death pathway, but (1) that caspase-independentmechanisms can cooperate with (or substitute for) caspases inthe execution of lethal signaling pathways, (2) that the majornecrotic cell death pathway is mediated through the serine/threonine kinases receptor interacting protein 1 (RIP1) and 3(RIP3), (3) that signaling involved in caspase-1-mediated celldeath (termed pyroptosis) differs from classical caspase-dependent apoptosis, and (4) that autophagic cell death isa type of cell death occurring together with (but not neces-sarily by) autophagic vacuolization. In this review, we willdiscuss the four major forms of programmed cell death at themolecular, cellular and physiological levels.

2. Apoptotic cell death

L. Duprez et al. / Microbes anIn mammalian cells, caspases are activated by the intrinsicor the extrinsic apoptotic pathway (Fig. 1). The intrinsicpathway is activated by various stimuli, such as DNA damageand cytotoxic insults, and acts through the mitochondria,which are controlled by the Bcl-2 family of proteins [2]. Inhomeostatic conditions, the anti-apoptotic Bcl-2 familymembers maintain mitochondrial integrity by preventing thepro-apoptotic multidomain Bcl-2 family members Bax andBak from causing mitochondrial damage. During cellularstress, Bcl-2-homology 3 (BH3)-only proteins are activatedand antagonize the anti-apoptotic Bcl-2 family members.Consequently, the inhibition of Bax and/or Bak is relieved,leading to their oligomerization and formation of a channelthrough which cytochrome c (cyt c) is released into thecytosol. Then, cyt c associates with Apaf-1 and ATP, forminga platform for recruitment and activation of procaspase-9, alsoknown as the apoptosome [5]. Active caspase-9 cleaves andactivates the downstream executioner caspases-3, -6 and -7,which are crucial for the execution of apoptotic cell death. Inaddition, other pro-apoptotic proteins released from themitochondria contribute to the cellular suicide mechanism. Forexample, Smac/Diablo antagonizes the action of inhibitor ofapoptosis proteins (IAPs) such as XIAP, cIAP1, and cIAP2 [6].Binding of Smac/Diablo to XIAP relieves its inhibitoryinteraction with caspase-9, -3 and -7. cIAP1 and cIAP2, unlikeXIAP, do not directly inhibit caspases. Instead, they interactwith tumor necrosis factor receptor (TNFR)-associated factor2 (TRAF2), which leads to their recruitment to TNFR1. There,they contribute to TNF-induced NF-kB activation by medi-ating the ubiquitination of RIP1 [6]. The NF-kB-mediatedexpression of anti-apoptotic genes might account for the anti-apoptotic function of cIAP1 and cIAP2.

The extrinsic pathway of apoptosis is induced upon stim-ulation of death receptors belonging to the TNFR family, suchas TNFR, Fas and TRAIL-R. Signaling by these receptors caninduce a variety of cellular responses, including proliferation,differentiation and cell death. Apoptosis is induced by theformation of a death-inducing signaling complex (DISC). Inthis complex, Fas-associated death domain (FADD) recruitsthe initiator caspases-8 and/or -10 via homotypic deathdomain interactions [7]. In contrast to signaling induced byFas and TRAIL-R, TNFR1 aggregation leads to the sequentialformation of two complexes [8]. Complex I consists ofTNFR1, TNFR-associated death domain (TRADD), TRAF2,RIP1, cIAP1 and cIAP2 and is formed at the plasmamembrane. These proteins are important mediators of TNF-induced activation of NF-kB and MAPKs. Endocytosis ofTNFR1 is followed by the formation of complex II, which isanalogous to the receptor-proximal DISC induced by FasL andTRAIL and includes TRADD, FADD, and caspase-8 and/or-10. Activation of caspase-8 and -10 leads to activation of thedownstream executioner caspases. In addition, caspase-8-mediated cleavage of the BH3-only protein Bid amplifies thedeath receptor-induced cell death program by activating themitochondrial pathway of apoptosis. Recently, it was foundthat TNF induces one of two distinct caspase-8 activationpathways, depending on the cellular condition [9]. One

1051fection 11 (2009) 1050e1062pathway is RIP1 independent and is primarily inhibited bycFLIP, a protease-dead caspase-8 homologue that competes

Bid

TRAF2

d InTRADDFADD

Procaspase-8,10

DEDDED

p10p20

DED

DED

Active caspase-8,10

Complex II - DISC

NF-kB

DEDDED

p10p20cIAP1,2Ubiquitinatione.g. TNFR1

e.g. TNF

CytosolDDART1PIR

1

Complex I

1052 L. Duprez et al. / Microbes anfor caspase-8 binding to FADD. The other pathway wasdiscovered in experiments using treatment with TNF andSmac mimetics and is absolutely dependent on kinase activeRIP1. Smac mimetics induce the autodegradation of cIAP1and cIAP2, leading to release of RIP1 from the receptorcomplex to form a caspase-8 activating platform consisting ofRIP1, FADD, and caspase-8 [9].

2.2. Physiological relevance of apoptosis

During development, apoptotic cell death has an importantrole in organ and tissue remodeling, e.g. in development of thelens, in shaping of the inner ear, in cardiac morphogenesis, inmuscle development, and in removal of interdigital webs [10].Furthermore, establishment of both the nervous and theimmune systems are characterized by initial overproduction ofcells and subsequent elimination of excess cells by apoptosis[11]. Apoptosis is crucial in maintaining homeostasis in theadult organism, as in post-lactational mammary gland

Procaspase-3,6,7

Nucleus

Activecaspase-3,6,7

Substrate cleavage

Apoptotic cell death

p10p20p10p20

Anti-apoptoticgenes

Fig. 1. Schematic representation of extrinsic and intrinsic apoptotic signaling. Stim

TNF stimulation, complex I is formed, resulting in NF-kB activation and subsequen

is formed, in which caspase-8 is recruited and activated. Subsequent activation of t

The intrinsic pathway of apoptosis (2) is activated upon intracellular stress signals

several mitochondrial apoptotic mediators. The subsequent formation of the apop

cleavage of Bid amplifies the extrinsic pathway of apoptosis by activating the mittBid

BH3-only

Bcl-2,Bcl-XL

Cyt c

Intracellular stress signals2

Bax/Bak

fection 11 (2009) 1050e1062regression, ovarian follicular atresia and post-ovulatoryregression, and in terminating an immune response by elimi-nating of activated immune cells [11]. Because apoptotic cellsare recognized and taken up by phagocytes before they loseplasma membrane integrity, it is generally believed thatapoptotic cell death is immunologically silent and does notprovoke inflammation [12].

Apoptosis of host cells during bacterial or viral infectionfunctions as a defense mechanism by destroying the site ofpathogen replication. Pathogens have therefore evolvedseveral ways to prevent apoptosis, e.g. by protecting themitochondria and preventing cyt c release, by activating cellsurvival pathways, or by preventing caspase activation [13,14].Conversely, pathogens may also benefit from apoptosis, eitherby inducing apoptosis of infected cells, thereby favoringsystemic infection, or by killing uninfected immune cells,thereby evading the host defense [13,14].

Dysregulation of apoptosis can result in the development ofvarious pathologies. Insufficient apoptosis can lead to the

p10p20

Procaspase-9CARD

Apaf-1

Apoptosome

Active caspase-9Smac/Diablo

Pro-apoptoticproteins

Smac/Diablo

Pro-apoptoticproteins

Smac/Diablo

Pro-apoptoticproteins

e.g.

ulation of e.g. TNFR1 initiates the extrinsic pathway of apoptosis (1). Upon

t transcription of anti-apoptotic genes. After endocytosis of TNFR1, complex II

he executioner caspases leads to cleavage of their substrates and to cell death.

at the mitochondrial level. Activation of Bax and Bak induces the release of

tosome results in the activation of caspase-9. In addition, caspase-8-mediated

ochondrial pathway. See text for detailed sequence of events.

development of cancer and autoimmune diseases. Excessive orinappropriate apoptosis, on the other hand, contributes to theinjury that accompanies several diseases, such as sepsis,stroke, myocardial infarction, ischemia, neurodegenerativediseases, and diabetes.

3. Necrotic cell death

For a long time, necrosis has been considered an accidentaland uncontrolled form of cell death lacking underlyingsignaling events. This might be true for cell death resultingfrom severe physical damage, such as hyperthermia or deter-gent-induced cytolysis. However, accumulating evidencesupports the existence of caspase-independent cell deathpathways that can function even in a strictly regulated devel-opmental context, such as interdigital cell death [15]. Necroticcell death is characterized by cytoplasmic and organelleswelling, followed by the loss of cell membrane integrity andrelease of the cellular contents into the surrounding extracel-lular space.

3.1. Molecular mechanisms of necrosis

Over the past decade, it has become evident that in certainconditions, necrosis is the result of a strictly regulated

interplay of signaling events, which are initiated by a diverserange of stimuli (Fig. 2). In most cell lines, death receptorligands activate apoptosis rather than necrosis as the defaultcell death pathway. However, if caspase activation in thispathway is hampered, necrotic cell death might ensue instead,acting as a kind of back-up cell death pathway. zVAD-fmk isfrequently used as a potent inhibitor of caspases, but off-targeteffects can also contribute to caspase-independent cell death.For example, zVAD-fmk binds and blocks the adeninenucleotide translocator (ANT), inhibits other proteases such ascathepsins, and generates the highly toxic fluoroacetate due tometabolic conversion of the fluoromethylketone group [16,17].

FADD remains a crucial adaptor protein in Fas- andTRAIL-R-induced necrosis, but the importance of FADD inTNF-induced necrosis is controversial [18,19]. Recently, withthe generation of the TRADD knockout mouse, it wasdemonstrated that TRADD is essential for TNF-inducednecrosis in MEF cells [20]. RIP1 is a crucial initiator of deathreceptor-mediated necrosis [21] and the term necroptosis wasintroduced to designate programmed necrosis that depends onRIP1 [22]. The kinase activity of RIP1 is dispensable for theactivation of NF-kB and MAPKs, but is required for nec-roptosis [19,22,23]. Necrostatin-1 (Nec-1) was identified asa small molecule inhibitor of necroptosis [22], and morerecently, the RIP1 kinase activity was found to be the target of

e.g. TNFR1 TLR-4

e.g. TNF

D D

2

Ph

N

LPS

1

the

E.g. Shigella

RI

FR

erac

1053L. Duprez et al. / Microbes and Infection 11 (2009) 1050e1062Nucleus

DNA damage

?

TRAF

AP-1

NF- kB MAPK

PARP-1

vrus

-orPlavi

RIP

Ca

Fig. 2. Schematic representation of necrotic signaling. Stimulation of e.g. TN

activating transcription factors, such as NF-kB and AP-1. In addition, RIP1 intART

TRAF2

ARTDDAF

Cytosol

1PIR

1PIR3PIRD Dsignaling. Through RIP1 kinase activity, a wide range of necrotic mediators are a

ceramide. The same mediators can be activated by DNA damage or by triggeringTLR-3

ROS Ca2+ospholipases

CalpainsO

Endosome

dsRNA

Necrotic cell death

psinsCeramide

Nalp-3

P1

1 leads to the activation of RIP1, which induces a pro-survival pathway by

ts with RIP3 and both are crucial initiators of death receptor-induced necroticctivated, such as ROS, calcium, calpains, cathepsins, phospholipases, NO and

of TLR-3, TLR-4 and Nalp-3. See text for detailed sequence of events.

mitochondrial matrix [38]. MPT is accompanied by mito-g of

oxidative phosphorylation, matrix swelling, and outer mito-

d InNec-1 [24]. Furthermore, recent studies identified RIP3 asa crucial upstream activating kinase that regulates RIP1-dependent necroptosis [25e27]. TNF treatment induced theformation of a RIP1eRIP3 pro-necrotic complex and thekinase activity of both RIP1 and RIP3 was crucial for stablecomplex formation and subsequent induction of necrosis.During death receptor-induced apoptosis, RIP1 and RIP3 arecleaved by caspase-8, which suppresses their anti-apoptoticand/or pro-necrotic properties [28,29].

Besides death receptor-mediated necrosis, triggering ofpathogen recognition receptors (PRRs) can also lead tonecrotic cell death. Receptors of this family include thetransmembrane toll-like receptors (TLRs), the cytosolicNOD-like receptors (NLRs) and the RIG-I-like receptors(RLRs). They all recognize pathogen-associated molecularpatterns (PAMPs) found in bacteria or viruses, such as LPS,flagellin and double-stranded RNA (dsRNA), and stimulationof these receptors leads to the activation of innate immunityand/or cell death. In Jurkat cells and L929 cells, the recog-nition of synthetic dsRNA by TLR3 induces necrotic celldeath, which was suggested to be RIP1-dependent [30].TLR4 is expressed on macrophages and monocytes and iscritical for the recognition of LPS from Gram-negativebacteria. Impeding caspase-8 activation switches TLR4-induced cell death from apoptosis to RIP1-dependentnecrosis [31]. Pathogen-induced activation of NLRs resultsmost commonly in caspase-1-dependent cell death orpyroptosis (see below). However, a recent report showed thatthe NLR member Nalp-3 mediates necrotic cell death ofmacrophages infected with Shigella flexneri at high multi-plicity of infection [32]. RLR-induced activation of NF-kBand production of type I interferons are both dependent onFADD, RIP1 and TRADD [33,34]. Whether these proteinsare also involved in RLR-induced cell death is unknown.

Extensive DNA damage causes hyperactivation of poly-(ADP-ribose) polymerase-1 (PARP-1) and leads to necroticcell death [35]. When DNA damage is moderate, PARP-1participates in DNA repair processes. However, excessivePARP-1 activation causes depletion of NAD by catalyzingthe hydrolysis of NAD into nicotinamide and poly(ADP-ribose) (PAR), leading to ATP depletion, irreversible cellularenergy failure, and necrotic cell death. PARP-1-mediated celldeath requires the activation of RIP1 and TRAF2 [36].However, the mechanism by which these molecules sense theactivation of PARP-1 has not been elucidated.

Many mediators are involved in the execution phase ofnecrotic cell death, including reactive oxygen species (ROS),calcium (Ca2), calpains, cathepsins, phospholipases, andceramide [37]. Oxidative stress leads to damage of cellularmacromolecules, including DNA, proteins, and lipids. Asdiscussed earlier, excessive DNA damage results in hyper-activation of PARP-1 and necrotic cell death. Modification ofproteins by ROS leads to loss of the normal functions ofproteins and enhances their susceptibility to proteolyticdegradation. Other targets of ROS are the polyunsaturated

1054 L. Duprez et al. / Microbes anfatty acid residues in the membrane phospholipids, which areextremely sensitive to oxidation. In mitochondria, lipidchondrial membrane rupture [38]. If most mitochondria of thecell are disrupted, and glycolytic sources of ATP are inade-quate, the cell becomes profoundly ATP-depleted. CyclophilinD (CypD) might have an important role in MPT, as inhibitionof CypD renders cells resistant to MPT, and CypD-deficientmice are more resistant to ischemic injury than wild type mice[39,40]. Besides affecting mitochondrial respiration, Ca2

overload can activate phospholipases, proteases and neuronalnitric oxide synthase (nNOS), all of which contribute to theexecution phase of necrotic cell death. For example, calpainsare activated by elevated Ca2 levels, which then cleave theNa/Ca2 antiporter in the plasma membrane, resulting ina sustained Ca2 overload. Strong activation of calpains mayalso contribute to the release of cathepsins in the cytosol bycausing lysosomal membrane permeabilization, as proposed inthe calpainecathepsin hypothesis by Yamashima andcolleagues [41].

3.2. Physiological relevance of necrosis

Although apoptosis is indispensable for tissue remodelingduring embryogenesis, necrosis can, at least in some condi-tions, substitute for apoptosis to eliminate unwanted cells. Forexample, removal of interdigital cells during the developmentof digits in the presence of the caspase inhibitor zVAD-fmk orin Apaf-1/ mice occurs by a caspase-independent necrotic-like process [15]. Necrosis is also involved in physiologicallyrelevant signaling processes, such as ovulation, the death ofchondrocytes associated with the longitudinal growth ofbones, and cellular turnover in the small and large intestines[42]. In addition, necrotic cell death participates in activation-induced cell death (AICD) of T lymphocytes, which is animportant mechanism for reducing T cell numbers after animmune response [19]. However, it is important to point outthat in most of the above-mentioned pathways, necrotic celldeath is always observed together with apoptosis or in thechondrial inner membrane depolarization, uncouplinperoxidation affects vital mitochondrial functions. In addition,it destabilizes the plasma membrane and intracellularmembranes of endoplasmic reticulum and lysosomes, leadingto intracellular leakage of Ca2 and lysosomal proteases,respectively. Among the different ROS, hydrogen peroxide(H2O2) plays a particularly important role because it diffusesfreely across cellular membranes and can interact with iron inthe Fenton reaction [37]. This reaction is favored in thelysosomes, because they are rich in free iron and do notcontain H2O2-detoxifying enzymes. The resulting highlyreactive hydroxyl radicals are among the most potent inducersof lipid peroxidation.

Ca2 overload of mitochondria causes mitochondrialpermeability transition (MPT) by the opening of largenonselective pores (the so called mitochondrial permeabilitytransition pores, MPTPs) connecting the cytosol with the

fection 11 (2009) 1050e1062presence of caspase inhibitors, suggesting that it functions asa back-up mechanism and is never the sole cell death pathway.

death also contributes to excitotoxicity, which may beener-

ative disorders [44]. More specifically, using Nec-1, it was

d Inshown that RIP1-dependent necrotic cell death or necroptosiscontributes to a wide range of pathological cell death events,such as ischemic brain injury [22] and myocardial infarction[45]. Furthermore, RIP3/ mice failed to initiate vacciniavirus-induced tissue necrosis and inflammation, resulting inmuch more viral replication and mortality [26]. Several otherreports also illustrate the occurrence of necrotic cell deathduring infection by other pathogens, such as Shigella, HIV-1,West Nile virus, and Coxsackievirus B [37]. In addition,patients carrying a disease-associated mutation in Nalp-3 showexcessive necrotic-like cell death with features similar to theShigella flexneri-induced Nalp-3-dependent necrosis [32].

In contrast to apoptosis, the recognition and uptake ofnecrotic cells by macropinocytosis is slower, less efficient andoccurs only after the loss of plasma membrane integrity [46].As a result, necrotic cells initiate a proinflammatory responseby the passive release of DAMPs (danger/damage-associatedmolecular patterns) [47]. In addition, necrotic cells activelysecrete inflammatory cytokines due to the activation of NF-kBand MAPKs [48].

4. Autophagic cell death

Autophagy is an evolutionarily conserved catabolicpathway that allows eukaryotes to degrade and recycle cellularcomponents. Proteins and organelles are sequestered inspecialized double-membrane vesicles, designated autopha-gosomes, which are typical of autophagic cells. Basal levels ofautophagy ensure the maintenance of intracellular homeo-stasis, but in addition, many studies have revealed its diversefunctions in important cellular processes, such as cellularstress, differentiation, development, longevity and immunedefense. Although a pro-survival role for autophagy is well-established, frequently debated is whether or not autophagyhas a causative role in cell death. The presence of autophagicvacuoles in dying cells has led to the introduction of auto-phagic cell death, although autophagy often accompaniesrather than causes cell death. It is plausible though thatmassive autophagic activity could result in cellular demise. Inaddition, several interconnections exist between autophagyand apoptotic or necrotic cell death [49].

4.1. Molecular mechanisms of autophagy

Several types of autophagy have been described, includingchaperone-mediated autophagy, microautophagy and macro-autophagy. Macroautophagy (hereafter referred to as autoph-agy) typically occurs in severe stress conditions and is thereforeinvolved in stroke, traumatic brain injury, and neurodegNecrotic cell death is often associated with pathologicalconditions. Necrosis has been observed during ischemia/reperfusion (I/R), which can lead to injury of organs, includingheart, brain, liver, kidney, and intestine [43]. Necrotic cell

L. Duprez et al. / Microbes anthe best characterized (Fig. 3). Macroautophagy functions by denovo formation of specialized vacuoles, the autophagosomes, inwhich the proteins are sequestered before being targeted to thelysosomes. So far, 30 autophagy-related (atg) genes have beenidentified in yeast, and several mammalian homologues havebeen isolated and functionally characterized [50].

The classical pathway of autophagic signaling acts throughmTOR (mammalian target of rapamycin), a protein kinase thatis important in controlling translation and cell-cycle progres-sion and in negative regulation of autophagy. When mTOR isinhibited in response to starvation or treatment with rapamy-cin, the ULK-Atg13-FIP200 complex is activated, leading tothe induction of autophagosome formation [51]. The synthesisof autophagic vacuoles requires vesicle nucleation, which isinitiated by the assembly of another complex, the PI3KC3(Vps34)-complex. Within this complex, Beclin-1 (Atg6)serves as a platform for binding PI3KC3 (Vps34), UVRAG,Bif-1 and Ambra-1, all of which positively regulate PI3KC3activity [52]. Interestingly, Bcl-2, amongst other anti-apoptoticBcl-2 family members, represses autophagy by binding ofBeclin-1 and thereby abrogating autophagic signaling [53].During starvation, Bcl-2 becomes phosphorylated, whichreleases Beclin-1 and stimulates autophagy [54]. Activation ofthe PI3KC3 (Vps34)-complex finally results in the generationof PI3P. Consequently, other Atg proteins that mediate vesiclemembrane elongation are recruited. Two ubiquitin-likeconjugation systems are implicated in this process. Duringa first conjugation step, an Atg12eAtg5eAtg16(L) multimercomplex that could be involved in vesicle curvature is formed[55]. During a second conjugation step, LC3 (Atg8) is lipi-dated by binding to phosphatidylethanolamine (PE). Incontrast to the cytoplasmic localization of LC3, LC3ePE(mostly referred to as LC3-II) specifically binds to the auto-phagic membranes, and for that reason it is generally used asan autophagic marker. Finally, the autophagosome fuses witha lysosome, releasing its autophagic content into the lysosomallumen for degradation by hydrolases.

The outcome of autophagic signaling mostly reflects itspro-survival function. In homeostasis, autophagy exerts itshousekeeping role in cytoplasmic and protein turnover, whileduring stress, cells are protected from dying by elimination ofharmful organelles and protein aggregates. However, severalstudies suggest a role for autophagy in cell death [56]. Incells in which apoptotic signaling is perturbed, a necrotic-likecell death is often induced as a back-up cell death mecha-nism, which can be associated with autophagy, as monitoredby LC3-II accumulation. For example, treatment ofapoptosis-deficient Bax/Bak double knockout MEF cells withDNA-damaging or ER-stress-inducing agents causes non-apoptotic cell death, which depends on the presence ofBeclin-1 and Atg5 [57]. Another interesting study shows theinvolvement of FADD and caspase-8 in controlling auto-phagic signaling in proliferating T cells [58]. T cells lackingFADD or caspase-8 undergo extensive autophagy andsuccumb to RIP1-dependent necrotic cell death, but inhibi-tion of autophagy rescued the T cells. Moreover, treatmentwith Nec-1 repressed LC3-II accumulation and prevented T

1055fection 11 (2009) 1050e1062cells from dying [58]. Similarly, in L929 cells, caspaseinhibition with zVAD-fmk or caspase-8 RNAi also resulted in

Rapamycin

d InNutrientsGrowth factors

12

1056 L. Duprez et al. / Microbes ana RIP1-dependent autophagic cell death [59]. Cell death wascaused by severe ROS accumulation and cell damage.Surprisingly, elevated ROS after zVAD-fmk treatment wasdependent on autophagy, which selectively degraded cata-lase, a major key enzyme in the anti-oxidant defense mech-anism [60]. However, another group recently reportedcompelling data supporting a pro-survival instead of a cellkilling role for autophagy in zVAD-fmk-induced cell death[61]. Rapamycin, a potent inducer of autophagy, blockedzVAD-fmk-induced cell death, whereas chloroquine, a lyso-somal enzyme inhibitor, greatly sensitized L929 cells for this

Autophagosom

mTOR

ULKAtg13

FIP20Induction

Bcl-2Beclin-1UVRAG

Bif-1

PI PNucleation

Atg5Atg16

Atg12

+

Elongation

Fig. 3. Schematic representation of autophagic signaling. In the presence of nutr

autophagy. Nutrient or growth factor deprivation (2), or treatment with rapamycin

complex, which induces autophagosome formation. First, vesicle nucleation occurs,

PI3P leads to the recruitment of other Atg proteins and vesicle membrane elongation

and LC3 is lipidated (LC3-II). Bcl-2 is capable of inhibiting autophagy through dfection 11 (2009) 1050e1062form of cell death. The authors suggested that zVAD-fmk, inaddition to inhibiting caspases, also blocks cathepsins,resulting in decreased autophagosomal degradation andhence the appearance of typical autophagic hallmarks.

4.2. Physiological relevance of cell death associated withautophagy

Autophagic cell death has been described primarily indevelopmental processes that require extensive cell destruction

e formation

0ULK-Atg13-FIP200

complex

PI3KC3

AMBRA-1

I3P

PI3KC3

complex

LC3

LC3-II

ients and growth factors (1) mTOR is activated, leading to the inhibition of

leads to inhibition of mTOR and the activation of the ULKeAtg13eFIP200

which requires the assembly of the PI3KC3 complex. Subsequent generation of

. During this process, an Atg12eAtg5eAtg16(L) multimer complex is formed,irect binding of Beclin-1. See text for detailed sequence of events.

Atg1 support the hypothesis that autophagy is needed forproper salivary gland degradation. Moreover, transgenic

oticcell

lysis. Furthermore, caspase-1 activation initiates an inflam-

d Inoverexpression of Atg1 in salivary glands is sufficient toinduce autophagic cell death, which is independent of caspaseactivation [63].

The protist, Dictyostelium, does not contain any caspasegenes, implying that cell death in this organism is by defaultcaspase-independent [64]. It has been shown that the autoph-agy-like cell death occurs in monolayer cultures of Dictyos-telium cells subjected to starvation and differentiation-inducing factor DIF-1, mimicking the development of fruitingbodies. These fruiting bodies contain a mass of spores sup-ported by a stalk composed of extensively vacuolated deadcells. However, atg1 gene disruption blocks autophagicvacuolization, but does not suppress cell death [65], suggest-ing that cell death occurs independently of autophagicsignaling or that redundant cell death pathways exist. The non-vacuolar cell death in Atg1 mutants possesses classical char-acteristics of necrotic cell death [66].

Increasing evidence suggests a role for autophagy in celldeath during ischemia/reperfusion (I/R). For example, Atg7-deficiency in neurons protects against neonatal hypoxic/ischemic brain injury [67]. Similarly, inhibition of autophagyblocks cardiac myocyte death after I/R and the size ofmyocardial infarction is significantly decreased in Beclin-1/

mice [68]. Autophagy might also contribute to virus-inducedcell death. For example, HIV-1 induces apoptotic cell death inuninfected bystander CD4 T lymphocytes. However, celldeath is associated with typical autophagic features and inhi-bition of autophagy significantly blocks T cell death [69].

Autophagy has also been implicated in another aspect ofcell death, namely apoptotic cell clearance. Both Atg5/ andBeclin-1/ cells fail to express phosphatidylserine (PS)(eat-me signal) and secrete less lysophosphatidylcholine(LPC) (come-and-get-me signal) [70]. Interestingly, bothPS and LPC exposure require ATP, suggesting that autophagycontributes to the production of engulfment signals by main-taining cellular ATP levels. Because effective engulfment alsorequires PS exposure on the phagocytes [71], it is possible thatintact autophagy is also needed for the proper phagocyticfunction of the engulfment cells.

5. Pyroptosis

Pyroptosis is a more recently recognized form of regulatedcell death with morphological and biochemical propertiesdistinct from necrosis and apoptosis [72]. Pyroptosis has beendescribed in monocytes, macrophages and dendritic cellsand elimination. During metamorphosis in Drosophila, deathof salivary gland cells is associated with upregulation of atggenes and induction of autophagy, though this is accompaniedwith caspase activation and the appearance of typical apoptoticfeatures, including DNA fragmentation [62]. However, studiesof Atg8 mutant flies or flies overexpressing dominant negative

L. Duprez et al. / Microbes aninfected with a range of microbial pathogens, such as Salmo-nella, Francisella and Legionella, and is uniquely dependent onmatory response by the cleavage of the proinflammatorycytokines pro-IL-1b and pro-IL-18, which are released by theosmoprotection experiments [78]. The resulting osmpressure leads to water influx, cell swelling and ultimatelycaspase-1 [73]. In addition, non-infectious stimuli, such asDAMPs, can induce pyroptosis in non-macrophage cells.

5.1. Molecular mechanisms of pyroptosis

Caspase-1, previously known as Interleukin-1 (IL-1b) Con-verting Enzyme (ICE), was the first mammalian caspase to beidentified. As a member of the inflammatory caspases, it is notinvolved in apoptotic cell death [74]; vice versa, the apoptoticcaspases usually do not contribute to pyroptosis [75]. Like allcaspases, caspase-1 is present in the cytosol as an inactivezymogen. In analogy to activation of caspase-9 in the apopto-some, caspase-1 is activated in a complex called the inflamma-some. This molecular platform includes NLR family membersthat recruit caspase-1 through adaptor molecules, such as ASC/Pycard and is formed through homotypic interactions betweenthese inflammasome components (Fig. 4). Until now, fourinflammasomes have been characterized and named after theirNLR (Nalp-1, Nalp-3 and Ipaf) or HIN-200 protein (AIM2)[73,76]. Assembly of the inflammasome occurs when NLRs aretriggered by intracellular bacterial, viral or host danger signals.For example, Nalp-1 recognizes cytosolic delivery of Bacillusanthracis lethal toxin, Ipaf recognizes cytosolic flagellin, andNalp-3 responds to multiple DAMPs and PAMPs [73] (Fig. 4).Most NLRs consist of three distinct domains: an N-terminalCARD domain or pyrin effector domain (PYD), a centralnucleotide binding and oligomerization domain (NACHT), andseveral C-terminal leucine-rich repeats (LRRs). In addition,Nalp-1 has a C-terminal extension that harbors a CARD domain.In contrast to human Nalp-1, the mouse orthologue Nalp-1b doesnot contain anN-terminal PYD domain. Upon stimulation, NLRsundergo oligomerization through homotypic NACHT domaininteractions. Subsequently, the NLRs associate with the adaptorprotein ASC through homotypic PYD interactions. In addition,Nalp-3 associates with the adaptor Cardinal in its inflammasome.These adaptormolecules then recruit caspase-1 through CARDeCARD interactions, resulting in its oligomerization and prox-imity-induced activation.

Recently, the AIM2 inflammasome was identified [76].Through its HIN domain, AIM2 can directly bind to dsDNA,resulting in the activation of caspase-1 and maturation of pro-IL-1b. The source of the cytoplasmic dsDNA appears unim-portant for AIM2 activation because viral, bacterial, mamma-lian and synthetic dsDNA could all activate caspase-1 [76].Double stranded DNA-dependent cell death depends on AIM2,ASC and caspase-1 and shows features of pyroptosis [77].

Active caspase-1 is the central executor of pyroptotic celldeath and acts mainly by inducing the formation of discretelysized ion-permeable pores in the plasma membrane, asanalyzed by the use of membrane impermeable dyes and

1057fection 11 (2009) 1050e1062cell upon their activation [79]. However, this inflammatoryresponse is not required for the execution of cell death [80].

d InFlagellin

Bacillus anthracis

lethal toxin,MDP

1058 L. Duprez et al. / Microbes anAlthough caspase-1 activation is inherently associated with aninflammatory response, it is still unclear whether it is inevi-tably linked to pyroptotic cell death. Which molecularmechanisms are implicated in the bifurcation between cas-pase-1-dependent inflammation and caspase-1-mediatedpyroptosis is also an open question.

Cytosol

Active cas

Pro-IL-1

Pro-IL-18

IL-1

IL-18

Inflammation

Pyroptotic ce

Pore formin plasma m

LRRs

NACHT

Caspase-1

Ipaf

inflammasome

Ipaf

01p02p

01p02p

CARD

PYD

ASC?

Caspase-1

Nalp-1b

inflammasome

Nalp-1b

ASC

FIIND

CARD

01p02p

01p02p

Fig. 4. Schematic representation of pyroptotic signaling. During pyroptosis, recogni

inflammasomes by triggering oligomerization of Nalp-1b, Nalp-3, Ipaf or AIM2

formation in the plasma membrane and concomitant pyroptotic cell death. In additio

an inflammatory response. Furthermore, caspase-1 activation leads to the cleavage

muramyl dipeptide; MSU, monosodium urate; PPD, calcium pyrophosphate dehydDAMPs: PAMPs:MSU, PPD,silica, alumasbestosamyloid-

LPS,peptidoglycan,

viral and bacterialRNA, DNA

fection 11 (2009) 1050e1062The digestome of caspase-1 comprises more than 40 cas-pase-1 substrates, among which are chaperones, cytoskeletaland translation machinery proteins, proteins involved inimmunity, and a series of unexpected proteins along theglycolysis pathway [81]. Whether these caspase-1-dependentcleavage events contribute to pyroptosis is unknown. In

pase-1

ll death

ationembrane

Cleavage ofother substrates:

chaperonescytoskeletal proteins

translational machinery proteinsglycolytic proteins

caspase-7...

dsDNA

AIM2

Cytosolic dsDNA

AIM2

inflammasome

Caspase-1

ASC

Caspase-1

Nalp-3

inflammasome

Nalp-3

ASC

01p02p

01p02p

02p01p01p

02p

tion of bacterial, viral or host danger signals induces the formation of different

. Via the adaptor ASC, caspase-1 is recruited and activated, leading to pore

n, caspase-1-mediated maturation of IL-1b and IL-18 results in the induction of

of a range of other substrates. See text for detailed sequence of events. (MDP,

rate).

replication and exposing the pathogen to other antimicrobialene-

ficial for certain pathogens, as in the elimination of immune

d Incells. Pathogens have therefore evolved strategies to efficientlyinhibit or to induce pyroptotic host cell death. For example,poxviruses encode PYD-containing proteins that interact withASC in the inflammasome, thereby inhibiting caspase-1 acti-vation and promoting infection [84].

Because of its dependence on caspase-1 activity, pyroptosisis associated with the initiation of a proinflammatory response,which is further amplified by the release of the cytoplasmiccontent upon cell lysis. Since NLR-mediated activation ofcaspase-1 affects several cellular pathways, it is difficult todistinguish the precise role of caspase-1 in the cell deathprocess itself. Caspase-1-deficient mice are more susceptiblethan wild type mice to infection, for example by Salmonella,and these mice are more susceptible than IL-1b and IL-18double knockout mice [85]. On the other hand, caspase-1-deficiency confers protection against Escherichia coli-inducedsepsis, again in contrast to wild type mice and IL-1b and IL-18double knockout mice [80]. These observations suggest thatthe phenotype associated with caspase-1-deficiency is not onlydue to lack of these proinflammatory cytokines but thatpyroptosis itself or other caspase-1-dependent events areinvolved in the control of infection.

Inappropriate or overwhelming activation of caspase-1 ismechanisms. On the other hand, host cell death can be baddition, by using a proteomics approach to search for novelcaspase-1 targets, caspase-7 was identified as a caspase-1substrate [75]. Also, it was reported that caspase-7 processinginduced by Salmonella or Legionella infection requiredinflammasome signaling [75,82]. Caspase-7/ macrophagesinfected with Legionella showed defective delivery of theorganism to the lysosome, delayed cell death and substantialLegionella replication [82]. However, a role for caspase-7 inpyroptosis should not be generalized, as caspase-7/

macrophages infected with Salmonella were not protectedfrom cell death [75].

Cells dying by pyroptosis have biochemical and morpho-logical features of both apoptotic and necrotic cells [73].Pyroptotic cells lose their mitochondrial membrane potentialand plasma membrane integrity and release their cytoplasmiccontents into the extracellular milieu. As in apoptosis,pyroptotic cells undergo DNA fragmentation and nuclearcondensation. However, this caspase-1-dependent nuclease-mediated cleavage of DNA does not exhibit the oligonucleo-somal fragmentation pattern characteristic of apoptosis [83].In addition, the DNA damage and concomitant PARP-1 acti-vation associated with pyroptotic cell death are not requiredfor cell lysis to occur [78].

5.2. Physiological relevance of pyroptosis

As a cellular suicide program, pyroptosis is part of the hostdefense system for fighting off pathogens. Death of the hostcell destroys the pathogenic niche, thereby limiting microbial

L. Duprez et al. / Microbes anassociated with the pathogenesis of several diseases, such asmyocardial infarction, cerebral ischemia, neurodegenerativediseases, inflammatory bowel disease and endotoxic shock[73]. These diseases are all characterized by inflammation andcell death. Caspase-1-deficiency, or its pharmacological inhi-bition, results in protection against inflammation and celldeath associated with these diseases [86]. These studiessuggest that the level of caspase-1 activation after recognitionof danger signals plays a role in the host response [73]. Lowlevels of caspase-1 initiate cell survival responses, controlintracellular bacterial growth and stimulate inflammatorycytokine production. In contrast, higher levels of caspase-1may lead to the induction of pyroptosis, and inappropriate oroverwhelming caspase-1 activation contributes to severalinflammatory conditions.

6. Concluding remarks

It has become clear over the past decade that apoptosis is notthe only cell death program available to mammalian organismsfor removal of unwanted cells. Apoptotic cell death is mediatedby caspases, which are responsible for its typical morphologicalfeatures. Accidental necrosis results from physicochemicaldamage without involvement of underlying signaling events, butnecrosis can also be the result of a programmed interplay ofsignaling events leading to plasma membrane rupture. Apoptosisand necrosis differ in their inflammatory outcome. Whereasapoptosis is immunologically silent, necrosis results in the releaseof the cytoplasmic contents with consequent inflammatoryresponses. Autophagic cell death can have characteristics of bothapoptotic and necrotic cell death. Whether autophagy representsa distinct form of cell death or rather triggers or accompaniesanother form of cell death is debatable and needs further inves-tigation. Pyroptosis is part of the host defense system to fight offpathogens and has morphological and biochemical propertiesdistinct from necrosis and apoptosis. Because pyroptosis isassociated with caspase-1-mediated maturation of pro-IL-1b andpro-IL-18 and ultimately results in release of the cytoplasmiccontent, it also elicits an immune response. As discussedthroughout this review, all four forms of programmed cell deathare involved in several pathological conditions. Better under-standing of signaling events critical in these cell death pathwayswould not only give insight into disease pathogenesis, but couldalso lead to the development of new therapeutic strategies fortreatment of cell death-related diseases.

Acknowledgements

We thank A. Bredan for editing the manuscript. Thisresearch has been supported by Flanders Institute forBiotechnology (VIB), European grants (EC Marie CurieTraining and Mobility Program, FP6 ApopTrain, MRTN-CT-035624; EC RTD Integrated Project, FP6 Epistem, LSHB-CT-2005-019067; EC RTD Integrated Project, Apo-Sys,FP7-200767), Belgian grants (Interuniversity Attraction Poles,IAP 6/18), Flemish grants (Fonds Wetenschappelijke Onder-zoek Vlaanderen, 3G.0218.06), and Ghent University grants

1059fection 11 (2009) 1050e1062(BOF-GOA - 12.0505.02 and 01GC0205). Peter Vandenabeeleis full professor at Ghent University and holder of

d Ina Methusalem grant from the Flemish Government, TomVanden Berghe is supported by an FWO postdoctoral fellow-ship, Ellen Wirawan is supported by GOA project (01GC0205)and Linde Duprez obtained a predoctoral fellowship from theFWO.

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1062 L. Duprez et al. / Microbes and Infection 11 (2009) 1050e1062

Major cell death pathways at a glanceIntroductionApoptotic cell deathMolecular mechanisms of apoptosisPhysiological relevance of apoptosis

Necrotic cell deathMolecular mechanisms of necrosisPhysiological relevance of necrosis

Autophagic cell deathMolecular mechanisms of autophagyPhysiological relevance of cell death associated with autophagy

PyroptosisMolecular mechanisms of pyroptosisPhysiological relevance of pyroptosis

Concluding remarksAcknowledgementsReferences