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Necroptosis: The Release of Damage-Associated Molecular Patterns and Its Physiological Relevance

Agnieszka Kaczmarek,1,2 Peter Vandenabeele,1,2,3,* and Dmitri V. Krysko1,2,3 1Molecular Signaling and Cell Unit, Department for Molecular Biomedical Research, VIB, 9052 Ghent, Belgium 2Department of Biomedical Molecular Biology, Ghent University, 9052 Ghent, Belgium 3These authors contributed equally to this work and are co-senior authors *Correspondence: [email protected] http://dx.doi.org/10.1016/j.immuni.2013.02.003

Regulated , termed necroptosis, is negatively regulated by -8 and is dependent on the kinase activity of RIPK1 and RIPK3. Necroptosis leads to rapid plasma membrane permeabilization and to the release of cell contents and exposure of damage-associated molecular patterns (DAMPs). We are only beginning to identify the necroptotic DAMPs, their modifications, and their potential role in the regulation of inflammation. In this review, we discuss the physiological relevance of necroptosis and its role in the modu- lation of inflammation. For example, during viral infection, RIPK3-mediated necroptosis acts as a backup mechanism to clear pathogens. Necroptosis is also involved in apparently immunologically silent mainte- nance of T cell homeostasis. In contrast, the induction of necroptosis in skin, intestine, systemic inflammatory response syndrome, and ischemia provoke a strong inflammatory response, which might be triggered by emission of DAMPs from necroptotic cells, showing the detrimental side of necroptosis.

Introduction inflammatory response to diseased cells and therefore mediate Necrosis has long been described as a consequence of extreme pathogen elimination. Often, the same receptors recognize physicochemical stress, such as heat, osmotic shock, mechan- both DAMPs and pathogen-associated molecular patterns ical stress, and freeze-thawing, which kill cells quickly and (PAMPs), indicating the similarities between pathogen-induced directly. Therefore, this has been described as uncon- responses and inflammatory responses to cell stress, injury, or trolled and accidental necrosis characterized by loss of plasma death (Table 2). membrane integrity and cellular collapse, though nuclei remain Different cellular stimuli (e.g., TNF, Fas ligand, TRAIL ligand, largely intact during this process (Krysko et al., 2008a, 2008b; double-stranded RNA (dsRNA), interferon-g (IFN-g), ATP deple- Vanden Berghe et al., 2010). Loss of membrane integrity and tion, ischemia-reperfusion injury, and pathogens) have been release of intracellular content grant necrotic cells the ability to shown to induce necrosis that follows defined steps and induce an inflammatory response. These immunogenic endoge- signaling events reminiscent of a cell-death program (Vanlange- nous molecules fall under the umbrella term ‘‘damage-associ- nakker et al., 2012). This regulated necrosis, also named necrop- ated molecular patterns’’ (DAMPs) (Garg et al., 2010; Krysko tosis, can be defined as cell death mediated through a pathway et al., 2011). They include, in the case of accidental necrosis, that depends on the receptor-interacting protein kinase 1 HMGB1, IL-1a, uric acid, DNA fragments, mitochondrial content, (RIPK1)-RIPK3 complex (Figure 1) and that can be inhibited by and ATP (Tables 1 and 2)(Eigenbrod et al., 2008; Kono et al., Necrostatin-1 (Nec-1) (Galluzzi et al., 2012). RIP3K can also 2010; Krysko et al., 2008a; Sauter et al., 2000). Because the form complexes with DNA-dependent activator of interferon nomenclature of DAMPs in the literature is confusing, here we regulatory factor (DAI) and the adaptor molecule TRIF (TIR- define DAMPs as a family of molecules that are intracellular in domain-containing adaptor-inducing interferon-b; adaptor physiological conditions and can be divided into two groups: protein downstream of TLR3), leading to RIP3K-dependent pro- (1) molecules that perform noninflammatory functions in living grammed necrosis (Mocarski et al., 2012). However, these defi- cells (such as HMGB1, ATP) and acquire immunomodulatory nitions should be interpreted carefully. First, there is no unique properties when released, secreted, modified, or exposed on biochemical marker for necroptosis. Necroptotic cell death the cell surface during cellular stress, damage, or injury (Rock in vitro is confirmed by plasma membrane rupture and by lack and Kono, 2008), or (2) alarmins, i.e., molecules that possess of specific apoptotic markers such as caspase activation and -like functions (such as IL-1a and IL-33), which can be chromatin condensation. Second, it has also become clear stored in cells and released upon cell lysis, whereupon they that RIPK1 kinase activity can participate in induction contribute to the inflammatory response (Oppenheim and in conditions of cIAP (cellular inhibitors of apoptosis proteins) Yang, 2005)(Tables 1 and 2). Several DAMPs have been shown depletion or inhibition, which can result in FADD-RIPK1- to stimulate pattern-recognition receptors (PRRs) (Chen and caspase-8 ripoptosome complex formation and Nec-1-inhibited Nun˜ ez, 2010)(Table 1). The PRR family consists of Toll-like apoptosis (Feoktistova et al., 2011; Tenev et al., 2011). receptors (TLRs), RIG-I-like receptors (RLRs), nucleotide- Necroptosis can be triggered by members of the tumor binding domain and leucine-rich repeat containing molecules necrosis factor (TNF) family (e.g., TNF) (Vercammen et al., (NLRs), and C-type lectin receptors (CLRs) (Takeuchi and Akira, 1997), Toll-like receptors (TLR3 and TLR4) (Chan et al., 2003; 2010). PRRs are initial sensors of infection that coordinate the Holler et al., 2000; Laster et al., 1988), and DNA and/or RNA

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Table 1. Necrotic DAMPs, PAMPs, and Their Receptors accompanied by the release of the receptor-associated com- plex from TNFR1 and the recruitment of FADD and procas- Receptor Adaptor PAMPs DAMPs pase-8, resulting in formation of complex IIa (Figure 1). Complex TLRs II is of two types; complex IIa can trigger apoptosis, whereas TLR2 Myd88 Lipoprotein HMGB1, HSPs complex IIb, also called necrosome, initiates necroptosis. TLR3 TRIF Viral, bacterial Ribonucleoproteins, Complex IIa and complex IIb are highly regulated by c-FLIPS, (endosomal) DNA mRNA FLIPL, or viral inhibitors (vICA, CrmA, E8, etc.). When recruitment TLR4 Myd88/TRIF LPS HMGB1, HMGN1, of procaspase-8 is prevented (c-FLIPS), caspase-8 is inhibited HSPs Hyaluronan (CrmA) or genetically deleted, RIPK1 and RIPK3 accumulate Biglycan Heparin and become phosphorylated, leading to increased necrosome sulfate formation and consequently to necroptosis. The recruitment TLR6 Myd88 Diacyl U1 snRNP of RIPK3 to RIPK1 occurs through a homotypic interaction lipoprotein domain (RHIM), followed by the mutual phosphorylation of TLR7 Myd88 Viral/bacterial U1 snRNP RIPK1 and RIPK3 (Sudan et al., 2009; Zhang et al., 2009). (endosomal) ssRNA What determines the switch from complex IIa to IIb is not clear. TLR9 Myd88 Viral/bacterial mtDNA HMGB1 The switch requires the activity of the deubiquitylating enzyme (endosomal) DNA CYLD (O’Donnell et al., 2011) and it is facilitated by inhibition RNA or DNA sensors or absence of caspase-8 (Kaiser et al., 2011; Oberst et al., RIG-I MAVS dsRNA dsRNA 2011). The activity of the NAD-dependent deacetylating enzyme DAI TBK1 dsDNA dsDNA SIRT2 (Narayan et al., 2012) has been implicated in the RIPK1- DAMPs or alarmins-specific receptors mediated recruitment of RIPK3 and the formation of complex CD14/CD40/ TLR4 cofactor/ HSPs/GP96 IIb (Figure 1). Mixed lineage kinase like protein (MLKL) and CD91 TRAF6 the mitochondrial phosphatase 5 (PGAM5) are other RIPK3- CD24 Siglec HMGB1 HSPs interacting proteins that are crucial for necroptosis induction (Zhao et al., 2012). Therefore, we could define the TNF necrop- RAGE Myd88 HMGB1 S100B totic pathway as a process that involves the positive action of DNGR-1 F-actin CYLD, RIPK1, RIPK3, SIRT2, and MLKL and negative regulation P2Y P2X ATP by caspase-8 and IAPs. IL1R1 Myd88 IL1a Interestingly, besides RIPK1’s role in the decision of the cell’s ST2L/ IL1RAcP Myd88 IL33 life or death, it has been proposed as one of the possible regula- Numerous DAMPs released from cells undergoing accidental necrotic tors of the release of and alarmins in necroptotic cells cell death have been described. DAMPs and PAMPs can be recognized (Christofferson et al., 2012). The kinase activity of RIPK1 medi- by the same group of PRRs. TLRs recruit downstream the MyD88 ates the production of TNF, which might contribute to cell death adaptor (myeloid differentiation protein), except for TLR3, which signals and inflammation (Christofferson et al., 2012) and might function through the TRIF adaptor. DNA or RNA sensors can also recognize as an alarmin in myocardial injury (Gallucci and Matzinger, 2001). bacterial, viral, or self-nucleic acid. Additionally, several other receptors It has also been shown in vitro that necroptotic material is coin- that do not recognize specific PAMPs (CD24 and P2X/Y receptors, IL- gested with the extracellular fluid by antigen-presenting cells 1R) have also been reported to interact with DAMPs released from acci- dentally necrotic cells. (APCs) via macropinocytosis (Krysko et al., 2006), which occurs Abbreviations are as follows: Myd88-Myeloid differentiation primary only after loss of membrane integrity (Brouckaert et al., 2004) and response gene (88); TRIF, TIR-domain containing adaptor-inducing with slower kinetics than uptake of apoptotic cells. This implies IFN-b; MAVS, mitochondrial antiviral signaling protein; TBKA, TANK- that necroptotic cells could release more DAMPs than apoptotic binding kinase 1; ST2L, IL-1 receptor-like 1; IL1RAcP, IL-1 receptor cells, in which potential DAMPs are restrained by the preserved accessory protein. plasma membrane and apoptotic bodies. Most studies on DAMPs have been devoted to DAMPs that are released during accidental cell death (Table 2) and by secondary necrotic cells sensors (DAI and possibly RIG-1 and MDA5). Whereas TNF- following apoptosis. Relatively few studies have investigated triggered apoptosis is mediated by , TNF-facilitated whether necroptosis is accompanied by release of a distinct necroptosis requires inhibition of caspase-8 and assembly of set of DAMPs and the roles of these DAMPs in necroptosis- RIPK1-RIPK3 complex IIb, also called the necrosome (Vandena- induced inflammation. In this review, we focus on necroptosis beele et al., 2010). Additionally, after TNF triggering, RIPK1 induced by pathogens (bacteria and viruses) and discuss the ubiquitylation controls the switching between prosurvival immunological consequences of these interactions, including signaling and apoptotic and/or necroptotic cell death (Figure 1). release of DAMPs. We also discuss the role of necroptosis and Briefly, after the ligand binds to TNFR1, two complexes with the associated DAMPs in the induction of inflammation, for opposing signaling can be formed. The prosurvival complex, example in skin, intestine, systemic inflammatory response ‘‘complex I,’’ containing TRADD, TRAF2/5, RIPK1, IAPs, and syndrome (SIRS), and ischemia reperfusion injury. In this LUBAC (Haas et al., 2009), initiates the activation of NF-kB, scenario, necroptosis-induced inflammatory diseases can be JNK, and p38 MAPKs. This complex, which is highly ubiquity- seen as sterile process in genetically deficient mouse models. lated, remains associated with the TNFR1 receptor (Bertrand So the inflammation can be attributed (at least partially) to the et al., 2008). In contrast, endosomal internalization of TNFR1 is release of DAMPs. However, this sterile situation differs from

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Table 2. Immunological Consequences of Accidental Necrotic Cell Death In Vitro and In Vivo DAMPs Cell line Necrosis induction Model or Alarmins Immunological Outcome Reference In vitro HeLa (A) ionomycin Coculture with bone HMGB1 [TNF (Scaffidi et al., 2002) (B) CCCP marrow (C) deoxy-glucose (BMDM) and sodium azide (D) 3 cycles of freeze/ thaw (F/T) THP-1, U-937 6 cycles F(LN)/ T Coculture with THP-1 HMGB1 [TNF, IL-6, IL-8 (El Mezayen et al., 2007) and HSP70 Human neutrophils, 5 cycles of F/T Coculture with LPS- ND Necrotic neutrophils (Miles et al., 2009) murine thymocytes, activated macrophages but no other cells Y TNF, human tumor IL-6, IL-1b and IL-8 from cell line LPS- activated macrophages KS24.22 primary F (LN)/ T Coculture with mDCs ND but no T cell (Inzkirweli et al., 2007) breast tumor cells and T cell activation activation measured as assay IFN-g production 11Ba/F3 cells F/T Coculture with resident ND Lack of IL-1b production, (Ayna et al., 2012) peritoneal macrophages lack of recruitment of and i.p. injection neutrophils to the site of injection T24 F/T Coculture with hDCs ND Lack of DCs maturation (Garg et al., 2012) DO11.10 murine HS 55C for 25 min Coculture with LPS- ND Lack of NF-kB activation, (Cvetanovic and T cells activated RAW 264.7 Lack of TNF and IL-6 Ucker, 2004) macrophages cell line modulation In vivo Bone marrow 5 cycles F/T Injection into the IL-1a Infiltration of neutrophils (Eigenbrod et al., 2008) derived dendritic peritoneal cavities into peritoneum, cells (BMDCs) CXCL1 secretion B16 cells (A) pressure disruption Injection into the ATP (A) neutrophil influx, (Iyer et al., 2009) (B) F/T peritoneal cavities Nlrp3 dependent IL-1a production only (B) no effect of F/T necrotic cells BD7 keratinocytes hypoxic conditions Injection into the IL1-a Infiltration of leukocytes (Rider et al., 2011) peritoneal cavities Renal cells Histones-induced Histones injection into Histone 4 neutrophil infiltration, [ IL-6, (Allam et al., 2012) necrosis renal artery of mice TLR2, TLR4 TNF, and inducible nitric dependent oxide synthase B16, L5178Y-ML25 (A) heat-killed Tumor vaccination ND Only (A) but not (B) or (C): (Yoon et al., 2008) lymphoma (B) formalde- model stimulation of APCs, hyde fixed [ IL-12 and TNF; effective (C) F/T anti-tumor vaccine CT26, B16, L5178Y, (A) 4 cycles Tumor vaccination ND (A), (B), (C) lack of protective (Goldszmid et al., 2003), SP815 (B) F/T model immune response in tumor (Bartholomae et al., 2004; (C) osmotic shock vaccination model Scheffer et al., 2003) Abbreviations are as follows: LN, liquid nitrogen; F/T, freeze/thaw; DC, dendritic cells (m, murine, h, human); ND, not defined; APC, antigen presenting cells; HS, heat shock. infectious processes, in which infection-induced apoptosis or can activate apoptotic or pyroptotic (inflammasome-mediated) necroptosis can become immunogenic as a result of the simulta- cell death through TLRs or NLRs, some PAMPs and DAMPs in neous engagement of PAMPs and DAMPs with PRR. particular cell types and conditions may be associated with necroptosis induction in vitro and in vivo (Figure 2A). In this Necroptosis and DAMPs Release during Bacterial respect, it has been shown that stimulation of TLR3 by dsRNA Infection (poly[I:C]) or TLR4 by LPS in combination with caspase inhibition Recognition of pathogens through PRRs is the first line of leads to RIPK3-mediated necroptosis in murine macrophages defense against microbial infections. Although most pathogens (He et al., 2011) and FADD-deficient Jurkat cells (Kalai et al.,

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Figure 1. Overview of Ripoptosome Formation in Response to Different Stimuli Necroptosis can be induced by various stimuli that are recognized by specific receptors. After TNF binds its receptor, TNFR1-mediated signaling can lead to cell survival (a), apoptosis (b), or necroptosis (c). For FasL and TRAIL stimulation, caspase-8 activation occurs at the internalized receptor complex, called death inducing signaling complex (DISC), whereas TNF signaling initially forms a receptor associated complex (complex I), followed by a cytosolic complex IIa (b) and complex IIb (c). Stable complex IIb, also called necrosome, is formed in some cellular conditions such as presence of RIPK3 or reduced caspase-8 activity in which RIPK3 is recruited to RIPK1 allowing mutual phosphorylation (c). Cytosolic FADD-RIPK1-caspase-8 complex (further referred to as ripoptosome), can also be formed following stimulation of T cell receptor, TLR3 or TLR4, as well as in response to genotoxic stress. In the latter case, the ripoptosome activates apoptosis, but it cannot be ruled out that in specific conditions of caspase-8 inactivation, it can also induce necrosome formation (dashed arrow). T cell proliferation following antigen activation requires caspase-8 activation by the CARMA1-BCL-10-MALT1 complex leading to NF-kB activation. However, when caspase-8 is genetically deleted or inhibited, CYLD (as a substrate of MALT1) probably accumulates, leading to sensitization of necroptotic signaling in T cells when RIPK3 is present and the necrosome complex is formed (d). Additionally, TRIF (adaptor of TLR3 and TLR4) can recruit RIPK1, which in turn can recruit RIPK3 via the homotypic interaction motif (RHIM), thereby bridging TLR signaling to the necrosome (e). RIPK1 can also mediate proinflammatory signaling by activating the transcription factor NF-kB or AP-1-SP1 (f). Cytoplasmic (DAI-I, RIG-I, and MDA5) and endosomal sensors (TLR3, TLR7-8, and TLR9) of nucleic acids activate IRFs and NF-kB, which leads to the production of IFN-b involved in antiviral response (f). RIG-I and MDA5, through the homotypic CARD interaction domain, recruit the MAVS adaptor on mitochondria, which can further bind RIPK1 and possibly enable recruitment of RIPK3 (dashed arrows). In contrast, DAI-I contains a RHIM domain by which RIPK1 and RIPK3 can be recruited and a necrosome is formed (g).The ripoptosome is also formed after TLR4 stimulation by LPS in DCs lacking caspase-8 (h), which contains the pronecroptotic molecules RIPK1 and RIPK3, as well as MLKL and PGAM5. However, this ripoptosome, instead of mediating cell death, contributes to NALPR3 inflammsome formation and activation, leading to pro-IL1b production and its processing into active proin- flammatory cytokine IL-1. DRs, death receptors; MDA5, melanoma differentiation-associated protein 5; RIG-I, retinoic acid-inducible gene 1; INF, interferon; ds, double-stranded, ss, single-stranded; TRIF, TIR domain-containing adaptor-inducing INF-b; MAVS (IPS-1), mitochondrial antiviral-signaling protein; MLKL, mixed lineage kinase domain-like protein; PGAM5, phosphoglyceratemutase 5.

2002). Interestingly, the TLR4 signaling pathway is involved in when TLR3 is engaged, TRIF interacts with RIPK3 through the HMGB1 secretion from macrophages by a mechanism that RHIM domain; this leads to ROS production and necroptosis depends on interleukin-1 (IL-1) receptor-associated kinase 4 (He et al., 2011). Moreover, TLR3-agonist can sensitize HaCaT (IRAK4) (Wang et al., 2010), suggesting that HMGB1 might and HeLa cells to RIPK1- and RIPK3-dependent necroptosis in be released during TLR4-mediated necroptosis. Furthermore, the absence of cIAPs and inhibition of caspases (Feoktistova

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Figure 2. Pathogen-Mediated Necroptosis and Possible Modifications of DAMPs (A) Stimulation of necroptosis by bacterial PAMPs. Bacterial products can stimulate TLR3 and À4 (also possibly TLR9, dashed arrow) to trigger necroptotic cell death when caspase-8 is inhibited or genetically ablated. Additionally, stimulation of TLRs and NLRs by PAMPs can provoke inflammatory responses in infected cells (a) and interferons production (b). During S. typhimurium infection, IFN type I receptor (IFNR) engages RIPK1, leading to recruitment of RIPK3 and necrosome formation (c). Binding of IFNs to IFNR activates the JAK-STAT pathway (d) leading to IFNs production and upregulation of Ripk1 and possibly also Hmgb1, thereby providing RIPK1 molecules for necroptosome formation (e) and resulting in HMGB1 release from dying cells. PRRs can discriminate between PAMPs and DAMPs through CD24-SiglacG/10 signaling. CD24 receptor and SiglecG/10 can bind to DAMPs but not to PAMPs. This brings CD24-SiglacG/10 close to TLR, which becomes desensitized for PAMP triggering by the action of a phosphatase (SHP-1) (f). DAMPs and PAMPs become indistinguishable during infection due to removal of sialic acid residues from CD24 and both trigger inflammation (a). (B) Stimulation of necroptosis by viral infection. Viral inhibitors of caspase-8 are produced by many viruses (e.g. caspase-8 inhibitors: CrmA, vICA, BORFE2; cFLIP homologs: E8, K13, MA159) and effectively block apoptosis. The absence of active caspase-8 sensitizes an alternative cell death pathway: necroptosis (g). However, viral protein M45 has been shown to prevent ripoptosome formation by binding to the RHIM domain of RIPK3, inhibiting necroptosis and inflammation (k). Additionally, viral proteins such as NS1 and A52R have been shown to blunt PRRs signaling by inhibiting the binding and sequestration of nucleic acids. Until now, RIPK1-RIPK3-dependent necroptosis has only been observed in response to MCMV and VV viruses; and most likely, ripoptosome formation involves TLR3 signaling and TRIF–RIPK1-RIPK3 interaction (i). The cytosolic sensor of foreign DNA- DAI -directly engages RIPK1 and RIPK3 in RHIM-dependent complexes and drives the assembly a ripoptosome (j). (C) Release of DAMPs during necroptosis and their potential modifications. Many DAMPs released from accidental necrotic cells have been described (Table 2), but no specific necroptotic DAMPs have been identified. So far, several DAMPs have been associated with necroptotic conditions (C; Table 3). Also, nuclei containing HMGB1, cytokines, long genomic DNA, and histones have been observed in necrotic tissues. Damaged mitochondria (and insufficient mitophagy) are source of ROS production, mitochondrial DNA (mtDNA), and other mitochondrial molecules. Additionally, absence of caspase activation during necroptosis might modify the properties of the released DAMPs as compared to apoptosis. Caspase activity was reported to induce high levels of ROS production during apoptosis, resulting in the oxidation of HMGB1 and leading to the production of its immunologically silent form. However, oxidation of two free cysteines of HMGB1 to a disulfide bond by ROS during necroptosis can lead to generation of the proinflammatory form of HMGB1. Activity of caspase-8 leads also to production of CRT, an ‘‘eat-me’’ signal. Caspase-3 and -7 can process full-length proinflammatory IL-33 (IL-33FL) into an inactive form. Absence of caspases, which are necessary for DNA fragmentation, might induce the release of long genomic DNA (LG DNA). Caspases also participate in the generation of the ‘‘find- me’’ signal, LPC, by cleavage of PLA2. Lack of PLA2 cleavage might prevent the release of ‘‘find-me’’ signals from necroptotic cells. Additionally, the ER- associated molecule PERK is involved in the release of the ‘‘eat-me’’ signals ATP and CRT, which we speculate can be abrogated during necroptosis. MCMV, murine cytomegalovirus; vICA, viral inhibitor of caspase activation; vFLIPs, viral FLIPs; CrmA, cytokine response modifier A; E3- caspase-8 inhibitor, adenovirus; BORFE2, caspase-8 inhibitor, BHV-4 virus; HMGB1, high mobility group box 1 protein; NLRs, NOD-like receptors; ROS, reactive oxygen species; PLA2, phospholipase A2; CAD, caspase-activated DNase; ICAD, inhibitor of caspase activated DNase, LMP, lysosomal membrane permeabilization.

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et al., 2011). In addition, stimulation of TLR9 by unmethylated infections can modify the immunological responses to DAMPs CpG triggers caspase-independent cell death of pro-B cells should also be investigated (Figure 2). The crosstalk between (Lalanne et al., 2010). However, whether TLR9-induced cell PAMP and DAMP recognition and signaling could be an impor- death involves RIPK1 and RIPK3 remains to be examined. tant issue in the etiology of sepsis (SIRS), asthma, inflammatory TLR-mediated necroptosis could also be induced in vivo and bowel disease, and autoimmune diseases. Indeed, organs such might lead to inflammation. Injection of wild-type (WT) mice as skin, intestine, and lungs are constantly exposed to both with LPS or poly(I:C) combined with zVAD-fmk, a pan-caspase commensals and pathogens, so they are more vulnerable to inhibitor, leads to necroptotic cell death of macrophages and conditions of danger and/or damage and permanent microbial increases serum concentrations of pro-inflammatory cytokines stimulation. (IL-6, TNF, MCP-1, IFN-g)(He et al., 2011). Death of macro- The DAMP-PAMP crosstalk is regulated by the CD24-SiglecG/ phages and circulating cytokine amounts were both markedly 10 axis (Chen et al., 2009), which determines whether or not inhibited when LPS and zVAD-fmk or poly(I:C) and zVAD-fmk the TLRs and/or NLRs associated with CD24 will induce was injected into RIPK3-deficient mice or dominant-negative inflammation when exposed to DAMPs (Chen et al., 2011) mutant TrifLPS2/LPS2 mice, pointing to the involvement of RIPK3 (Figure 2A). Siglec-G/10 belongs to a family of immunoglob- and TRIF in TLR-mediated necroptosis (He et al., 2011). ulin-like lectins that can recognize sialic-acid-containing struc- Although the above-mentioned studies were conducted using tures present on CD24. Moreover, DAMPs such as HMGB1, synthetic ligands or bacterial products to stimulate TLRs, several HSP70, and HSP90 interact directly with CD24 (Chen et al., models of bacterial infections have also been shown to induce 2009), and the DAMPs-CD24-Siglec G/10 complex enables necroptosis in host immune and nonmyeloid cells in vitro. phosphatases such as SHP-1 to repress TLR and NLR signaling. Epithelial cells infected with Shigella die by necrosis, as deter- Interestingly, the sialylation status of CD24 determines whether mined by double propidium iodide and Annexin V positivity or not the DAMP repression system will operate. Indeed, several and absence of TUNEL-positive staining (Carneiro et al., 2009). microbes and viruses possess sialidases as a virulence factor This cell death could not be inhibited by zVAD-fmk, and it was (Chan et al., 2012). Removal of sialic acid residues from CD24 accompanied by the secretion of KC (a neutrophil-attracting prevents the formation of the DAMP-CD24-Siglec-G/10 chemokine) into the supernatant of infected mouse embryonic complex, resulting in increased inflammatory responses to fibroblasts (MEF). It has been shown that the a-toxins of TLRs and NLRs during PAMP and DAMPs stimulation. This Clostridium septicum (Morinaga et al., 2010) and Legionella mechanism may explain the increased inflammation in diseases pneumophila (Kennedy et al., 2009) induce necrotic-like cell associated with bacterial stimulation, such as sepsis (SIRS). death in macrophages and myoblasts, respectively. Importantly, Recent studies revealed that bacterial sialidases promote in both studies the authors showed that necrotic cell death was inflammation in the cecal ligation and puncture (CLP) model of associated with the release of HMGB1 (Figure 2). Further studies sepsis. Elevated levels of sialidases lead to decreased binding are required to analyze whether this type of caspase-indepen- of CD24 with Siglec G/10, increasing the interaction between dent cell death induced by bacteria (Kennedy et al., 2009; TLRs and DAMPs (Chen et al., 2011). Moreover, stimulation of Morinaga et al., 2010) is dependent on RIPK1 and/or RIPK3 macrophages with TNF and LPS can activate the host’s cyto- and therefore could be considered as necroptosis. RIPK1- plasmic sialidases (Gee et al., 2003), and TNF-induced SIRS and/or RIPK3-dependent necroptosis is observed during has been associated with RIPK3-mediated necroptosis (Duprez Salmonella typhimurium infection (Figure 2A). In vitro and in vivo et al., 2011). Therefore, the host’s endogenous sialidases analysis showed that Salmonella induces type I interferon- released during necroptotic cell death together with bacterial dependent assembly of RIPK1 and/or RIPK3, leading to necrop- sialidases could sensitize TLRs and NLRs to DAMPs and lead tosis of macrophages accompanied by release of proinflamma- to exacerbation of inflammation in sepsis (SIRS). tory cytokines such as TNF, KC, IL-1a and IL-1b, and IFN-g (Robinson et al., 2012). This cell death was not due to endoge- Viruses: Master Regulators of Cell Death and DAMPs nous TNF production or TNFR1 stimulation, suggesting that Release? type I IFN can engage the necroptosis pathway independently Viral spread depends on the fate of infected cells. The interac- of TNF (Robinson et al., 2012). No DAMPs were analyzed in tions between viruses and eukaryotic cells have led to selection this study, but another group (Kim et al., 2009) investigated the of eukaryotic genes that propagate cell death upon infection to role of the IFN-b signaling pathway in HMGB1 release and found block viral multiplication, while some viruses encode suppres- that the IFN-b and JAK-STAT signaling pathways are critical for sors of cell death to counteract these protective mechanisms. HMBG1 release during stimulation of TLR4 by E. coli (Kim et al., Viruses suppress not only the apoptotic pathway but also the 2009). These observations collectively show that bacterial infec- necroptotic pathway, demonstrating that both programmed tion could activate TLRs and lead to production of IFNs, which in cell death mechanisms are crucial for host defense (Figure 2B). an autocrine or paracrine way could activate the necroptotic Caspase-8 is essential for T cell proliferation after antigen pathway and contribute to HMBG1 release (Figure 2A) (Robinson stimulation (Ch’en et al., 2008) and for CD4+ and CD8+ T cell- et al., 2012). mediated antiviral responses (Duttagupta et al., 2009). Interest- It is important to know whether these in vitro studies can be ingly, viruses have evolved into having many specific caspase- extrapolated to the in vivo situation and to understand whether 8 inhibitors, such as cytokine response modifier A (CrmA), viral the released HMGB1 (and very likely also other DAMPs) could inhibitor of caspase activation (v-ICA), and viral FLICE-like contribute to the inflammatory response instigated by the inhibitory proteins (FLIPs) (Mocarski et al., 2012). Here, we necroptosis induced by PAMP and bacteria. Whether microbial come to a paradoxical function of caspase-8. Initially, it was

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thought that caspase-8 inhibition is only a way to prevent induc- (DAI) was identified as an essential partner of RIPK3 in MCMV- tion of apoptosis to allow the virus to propagate in the infected induced programmed necrosis in vitro and in vivo (Upton et al., cell. However, recently it became clear that caspase-8, FLIPL, 2012). This study strongly suggests that DAI functions as and FADD negatively control necroptosis induction, probably a PRR in the pathway of RIPK3-dependent necroptosis induced by cleaving RIPK1, RIPK3, and CYLD (Dillon et al., 2012; by MCMV. O’Donnell et al., 2011) or some other target(s) downstream of Several other viruses, including WNV, HIV type 1 (HIV-1), and RIP3 kinase. This would mean that on the one hand inhibition herpes simplex virus (HSV), have been shown to induce necrotic- of caspase-8 prevents apoptosis induction while on the other like cell death. Although these viruses have not been evaluated in hand the cell would be sensitized to necroptosis induction. But RIP3K-deficient mice, cells infected in vitro with WNV develop why would viruses paradoxically activate an alternative cell a necrotic morphology followed by HMGB1 release (Chu and death program? For the virus, this could be a way to control its Ng, 2003). HIV-1-infected T cell lines, in the presence of the own pathogenicity or to modulate the release of DAMPs. For caspase inhibitor zVAD-fmk, undergo necrotic-like, mitochon- the host, necroptosis can be viewed as a ‘‘trap door’’ that opens dria-dependent cell death (Petit et al., 2002). HSV type 1 when caspase-8 is inhibited and it is involved in host defense (HSV-1) can inhibit apoptosis and elicit necroptosis in vitro; this (Mocarski et al., 2012). In this regard, evaluation of RIP3K- cell death could be partially blocked by the RIPK1 kinase deficient mice has shown that necroptosis plays an important inhibitor Nec-1 and cathepsin inhibitors (Peri et al., 2011). Addi- role in the control of vaccinia virus (VV) (Cho et al., 2009) but is tionally, dengue virus (DV) induces either apoptotic or necrotic dispensable for the control of WT murine cytomegalovirus cell death, depending on the virus titer. Interestingly, infection (MCMV) (Upton et al., 2010), mouse hepatitis virus (Lu et al., with DV causes translocation of HMGB1 from the nucleus to 2011a), and lymphocytic choriomeningitis virus (LCMV) (Ch’en the cytoplasm and its active secretion from live infected cells et al., 2011), arguing for case-specific strategies in host-virus and passive release from dying cells (Chen et al., 2008; Kamau immune responses. Infection of WT mice with VV induces tissue et al., 2009; Ong et al., 2012). The amount of HMGB1 in the extra- necroptosis and infiltration of neutrophils into visceral fat pads, cellular environment increased and was correlated with higher but these events are significantly decreased in RIPK3-deficient multiplicity of infection and a shift from apoptotic to necrotic animals (Cho et al., 2009). This reduced inflammation correlated cell death (Ong et al., 2012). Ong et al. reported that active with increased viral titers in the organs of RIPK3-deficient mice, release of HMGB1 is mediated by virus capsid protein, which and consequently, these mice were more vulnerable to the triggers acetylation and secretion of HMGB1. Once in the infection. This indicates that RIPK3 is required for protection extracellular milieu, HMGB1 acts on endothelial cells, induces against VV (Cho et al., 2009). Conspicuously, the authors vascular leakage, and increases the risk of development of reported that the inflammatory foci were concentrated in areas dengue shock syndrome (Ong et al., 2012). In addition, elevated of extensive necroptosis in WT mice, which could indicate emis- levels of the proinflammatory cytokines TNF and IL-6 were sion of DAMPs from necroptotic cells. Indeed, virus infection can observed upon infection (Kamau et al., 2009). However, it is be accompanied by emission of DAMPs such as Hsp70 and unclear whether these phenomena at higher virus titers are due HMGB1 (Lietze´ n et al., 2011; Wheeler et al., 2009). However, to necroptosis, because the involvement of RIPK1 and/or again as a result of coevolutionary selection, some viruses can RIPK3 was not analyzed in these studies. Necrotic morphology ‘‘switch off’’ the emission of DAMPs or blunt the interaction could be easily mistaken for secondary necrosis, especially of TLRs with DAMPs and/or PAMPs to prevent an immune that the type of cell death could often depend on virus titer reaction. For example, protein A52R expressed by VV inhibits (Chu and Ng, 2003). More work is required to identify DAMPs the signaling mediated by TLR2, À4, and À9, West Nile virus emitted from virus-induced necroptotic cells and to understand (WNV) nonstructural protein 1 (NS1) inhibits TLR3 signaling, their roles in induction of inflammation. and other viral proteins sequester virus dsRNA (Aravalli et al., 2008; Bowie and Unterholzner, 2008)(Figure 2B). The FADD-Caspase-8 Platform and Autophagy as Some viruses (e.g., MCMV) have evolved strategies to Mechanisms to Regulate Necroptosis in T Cells counteract several death pathways. MCMV encodes both an Several reports indicate that necroptosis could regulate T cell inhibitor of caspase-8 (Skaletskaya et al., 2001) and a RIPK3 number in peripheral tissues during T cell development. Mice inhibitor (Upton et al., 2010), and so it can efficiently block lacking caspase-8 or mice expressing mutant FADD unable to both apoptosis and necroptosis. Activation of regulated necrosis recruit caspase-8 (FADD DED deficient, FADDdd) (Lu et al., is a mechanism for combating MCMV in the absence of cas- 2011b) in T cells had fewer CD4+ and CD8+T cells in the pase-8 activity. Upton et al. showed that MCMV-induced periphery, and this was correlated with excessive cell death RIPK3-dependent programmed necrosis is a major host defense (Salmena et al., 2003). Concomitant research in double deficient pathway that can be effectively blocked by the viral RIPK3 caspase-8-RIPK3 and FADDdd-RIPK3 T cells revealed accumu- inhibitor, M45 (Figure 2B). This protein possesses a homotypic lation of excessive and abnormal lymphocytes (lymphadenop- interaction domain RHIM, which enables it to bind RIPK3. athy) in these mice, indicating that RIPK3-driven necroptosis is Consequently, MCMV can efficiently block necroptosis, but responsible for clearance of these lymphocytes during their strains lacking M45 or containing a mutation in its RHIM domain development (Ch’en et al., 2011; Kaiser et al., 2011). Additionally, induce regulated necrosis that is dependent on RIPK3 but not on necroptosis has been implicated in the elimination of excessive RIPK1 (Kaiser et al., 2011; Upton et al., 2010). But through which T cells after activation (contraction of lymphocytes). Removal receptor-adaptor system do viruses engage host RIPK3-medi- of activated T lymphocytes that had undergone clonal expansion ated necroptosis? Recently, DNA-dependent activator of IRFs in response to stimulation (infection) is essential for maintenance

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of T cell homeostasis, because its deregulation can lead to should then be beneficial for the cell to survive the deadly TNF immunodeficiency or autoimmunity. Defective expansion of signal’’ (Vercammen et al., 1998). The molecular mechanisms T cells after TCR activation was observed in T cells deficient in of this interplay between mitophagy and necroptosis are caspase-8 (Ch’en et al., 2011; Salmena et al., 2003) or FADD currently investigated. In that respect, Wang et al. recently (Osborn et al., 2010) as well as in FADDdd-expressing T cells showed a connection between RIPK3 kinase activity and the (Lu et al., 2011b). It has been proposed that the inability of MLKL platform, which recruits complex IIb to mitochondria. T cells to accumulate after activation was due to extensive cell This interaction results in mitochondrial fission and fragmenta- death. Indeed, Osborn et al. used Nec1 to inhibit RIPK1 activity tion (Wang et al., 2012). Taken together, decreased mitophagy and discovered that the proliferative capacity of FADD-deficient and increased rupture of mitochondria may enable the release T cells was restored when the RIPK1 kinase activity was abol- of mitochondrial DNA (or mitochondrial proteins) that serve as ished. Moreover, these authors suggested that RIPK1-driven DAMPs and promote ROS production, which may act as DAMPs necroptosis might occur naturally during the T cell development modifiers. because Nec1 treatment enabled wild-type T cells to proliferate with a higher rate than the control cells (Osborn et al., 2010). The Silent and Proinflammatory Faces of Necroptosis Similarly, the T cell accumulation defect of caspase-8 deficient It is well established that apoptotic cell death can switch to and FADDdd expressing T cells was restored by RIPK3 deletion necroptosis under certain conditions. However, the effect of (Lu et al., 2011a), indicating that necroptosis can be activated necroptotic cell death on immune responses in vitro and in vivo when active capase-8 is not available, leading to T cell depletion has been barely studied. Necroptotic material can be coingested in response to stimulation. Mice lacking caspase-8 or expressing in vitro by endocytosis-macropinocytosis (Krysko et al., 2003; FADDdd in T cells are also unable to mount a proper immune Krysko et al., 2006). Vesicles containing necroptotic material response when infected with murine hepatitis virus (MHV), might deliver DAMPs to APCs to initiate an immune response. because these cells lack cytotoxic activity. However, additional However, L929 cells subjected to TNF-induced necroptosis do deletion of RIPK3 in both cases restored the proper immune not induce proinflammatory cytokine production in macro- responses. Therefore, it seems that when caspase-8 is absent, phages. This mouse fibrosarcoma cell line is commonly used RIPK3-mediated necroptosis promotes death of ‘‘immunologi- as an in vitro model of necroptosis (Vanden Berghe et al., cally useless’’ T cells unable to mount proper immune responses 2010; Vanlangenakker et al., 2011). Similarly, B16 mouse mela- to pathogens. However, in physiological conditions the FADD- noma cells, in which necroptosis was triggered by inducible caspase-8 platform acts to prevent necroptosis because expression of FADD-death domain, were unable to induce caspase-8- and FADD-deficiency leads to the embryonic death maturation of dendritic cells (DCs) (Lohmann et al., 2009). These of mice, which can be rescue by ablation of RIPK3 (Dillon et al., observations can be interpreted in two ways: either coincubation 2012; Kaiser et al., 2011). In this respect, to the best of our of necroptotic cells with macrophages does not reflect the proin- knowledge the only instance in which caspase-8 could be flammatory capacity of necropotic cells or necroptotic cells naturally inhibited would be during infection by a virus encoding poses anti-inflammatory properties in vitro. On the other hand, caspase inhibitors, as discussed above. However, it is also investigation of necroptotic cell death in mouse models conceivable that in other pathological conditions FADD and/or discloses the proinflammatory face of this cell death mode. Cer- caspase-8 could be downregulated revealing necroptotic ulein-induced acute pancreatitis resulted in necroptotic cell signaling. death of pancreas accompanied by overexpression of RIPK3 Also other mechanisms, such as autophagy, may control the in pancreas but not in other organs (Sudan et al., 2009). Further sensitivity to necroptosis (Bray et al., 2012). T lymphocyte prolif- evidence linking RIPK1 and RIPK3-mediated necroptosis eration is impaired in the absence of Atg5, Atg7, Atg3, or Beclin-1 with inflammation arose from studies on mice with FADD and (He et al., 2012). Interestingly, RIPK1 has been proposed to be caspase-8 deficiency in keratinocytes. Mice with epidermis- a linker between necroptotic cell death and autophagy in acti- specific FADD deletion developed skin inflammation due to vated T cells. A hyperautophagic phenotype has been observed spontaneous necroptosis of keratinocytes and intense release in FADDdd- and caspase-8-deficient lymphocytes after TCR of HMGB1; this was not observed in animals double-deficient stimulation (Bell et al., 2008; Ch’en et al., 2008). Treatment with in FADD and RIPK3, demonstrating an important role of Nec1 in these models not only prevented cell death but appar- RIPK3-dependant necroptosis in inflammation (Bonnet et al., ently also reduced the extent of autophagy. However, these 2011). Studies on FADD-TNFR1 and FADD-MyD88 deficiency results should be interpreted carefully because conflicting find- revealed that both TNF and TLR signaling partially contribute ings have been reported on autophagy and its crosstalk with to progression of inflammation. TNF produced in response to apoptosis and necroptosis (Long and Ryan, 2012). Another TLR-driven MyD88 signaling amplify TNF induced necroptosis, aspect of the regulation of necroptosis by autophagy deals whereas excessive HMGB1 observed in epidermis of FADD- with the important role of mitophagy in removing damaged mito- deficient mice acts as a DAMP that triggers inflammation chondria (Youle and Narendra, 2011), because they are an through TLRs (Bonnet et al., 2011). A similar phenotype of skin important source of ROS, which contributes to necroptosis. inflammation was reported in mice lacking caspase-8 in Indeed, interference with the mechanism responsible for the keratinocytes. Although the involvement of necroptosis was removal of damaged mitochondria could sensitize for necropto- not investigated in this model, these authors observed upregula- sis. We have discussed this sensitization of necroptotic cell tion of DAMPs such as S100a9 and IL-33 (Kovalenko et al., death by CrmA overexpression already in 1998: ‘‘Elimination of 2009), which could indicate the involvement of increased such deficient but oxygen radical-producing mitochondria necroptosis in inflammation.

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Table 3. Necroptotic DAMPs in Transgenic Mice and Inflammatory Disease Models Cell Type Mouse Model Necroptosis Immune Response DAMPs References Epidermal Caspase-8 ND Skin inflammation [ S100a9, IL-33 (Kovalenko et al., 2009; keratinocytes deletion Li et al., 2010) Epidermal FADD deletion RIPK3 mediated Skin inflammation ND but MyD88 deficiency (Bonnet et al., 2011) keratinocytes delayed onset of inflammation Intestinal Caspase-8 RIPK1/RIPK3 Spontaneous ileitis; ND (Gu¨ nther et al., 2011) Epithelial Cells deletion mediated susceptibility to experimental colitis Intestinal FADD deletion RIPK3/CYLD Chronic intestinal ND but MyD88 deficiency (Welz et al., 2011) Epithelial Cells mediated inflammation partially protected from colon inflammation Disease Mode of Induction Necroptosis Immune Response DAMPs References SIRS TNF induced RIPK1/RIPK3 Systemic inflammation, Presence of mtDNA (Duprez et al., 2011) mediated [ IL-1a, IL-6 and damage in serum markers (HEX, LDH, AST, ALT, CK) SIRS Trauma patients ND Systemic inflammation, Presence of mtDNA (Zhang et al., 2010) [ IL-6, IL-8, TNF in serum Neonatal Hypoxia–ischemia RIPK1/RIPK3 Brain damage, [ IL-1b, ND but TLR2 deficiency (Northington et al., 2011) Brain Injury mediated IL-6, IL-12p35, and TNF partially prevented brain (Rosenbaum et al., 2010; damage Stridh et al., 2011) Kidney Ischemia Ischemia RIPK1 mediated Kidney damage, HMGB1-TLR4 mediated (Linkermann et al., 2012) Reperfusion [ circulating damage inflammation (Wu et al., 2010) markers (urea, creatinine) Myocardial Ischemia RIPK1/MLKL Neutrophils and ND but TLR4 deficiency (Oerlemans et al., 2012) Ischemia mediated macrophages influx, protects from myocardial I/R (Fallach et al., 2010) [ LDH and ROS Abbreviations are as follows: ND, not defined; HEX, hexosaminidase; LDH, lactate dehydrogenase; AST, aspartate transaminase; ALT, alanine trans- aminase; CK, creatine kinase; ROS, reactive oxygen species.

Additionally, patches of necrosis and acute inflammation partially protected from inflammation, suggesting that TLR stim- in the gut were described 30 years ago in patients with ulation can accelerate inflammation (Welz et al., 2011)(Table 3). Crohn’s disease (Dourmashkin et al., 1983). Only recently, As already mentioned, the implication of TLR could be partially necroptosis of Paneth cells in the terminal ileum was connected due to stimulation of TNFR1 by TNF, which is produced in with the pathogenesis of inflammatory bowel disease. Samples response to TLR-driven MyD88 signaling. In this regard, ileal collected from patients showed strong RIPK3 expression biopsies from control patients showed Paneth cell loss in the (Gu¨ nther et al., 2011), suggesting that necroptosis can contri- presence of high levels of exogenous TNF, but this was revers- bute to the development of inflammatory disorders. Specific ible by coincubation with necrostatin-1 (Gu¨ nther et al., 2011). deletion of FADD (Welz et al., 2011) or caspase-8 (Bonnet Therefore, TNF-induced necroptosis of Paneth cells might be et al., 2011; Gu¨ nther et al., 2011) in intestinal epithelial cells one of the key events in the pathogenesis of intestinal bowel (IECs) induces chronic inflammation characterized by extensive diseases. epithelial cell death. Double-deficient FADD-RIPK3 mice In a totally different context, but illustrating the effects of nec- showed no signs of inflammation, and high levels of RIPK1 roptotic cell death on the innate , it was recently and RIPK3 messenger RNA (mRNA) were observed in the intes- shown that RIPK3-dependent necroptosis causes mortality in tine of caspase-8-deficient mice, suggesting that cell death in mice subjected to experimental TNF-induced systemic inflam- the epithelium may be due to necroptosis. Interestingly, in matory response syndrome (SIRS) and CLP- induced peritoneal both cases of intestinal inflammation, Paneth cells were fewer sepsis (Duprez et al., 2011). Release of mitochondrial DAMPs or absent, suggesting that this cell type might be highly suscep- (Krysko et al., 2011) such as mitochondrial DNA (mtDNA) into tible to necroptosis. Gu¨ nther et al. (2011) showed that caspase- plasma and necroptotic cell death were reduced in RIPK3 8-deficient Paneth cells are sensitive to RIPK1-RIPK3-mediated knockout mice. Duprez et al. (2011) proposed that a combination necroptosis, leading to decreased expression of antimicrobial of several DAMPs released by necroptotic cells in this model peptides and sensitizing terminal ileum and colon to inflamma- might synergistically trigger sustained cytokine release and tion. Similarly, Welz et al. showed that FADD-deficient Paneth amplify the inflammatory response. Indeed, mtDNA can activate cells are susceptible to RIPK3-mediated necroptosis and that TLR9 and was identified as a major DAMP contributing to inflam- their production of antimicrobial peptides is impaired. Addition- matory responses in trauma patients developing sepsis (clinical ally, colon of mice lacking both FADD and MyD88 are at least SIRS) (Zhang et al., 2010).

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The contribution of RIPK1-dependent necroptosis to multiple same cells, but it cannot be ruled out that different cells or cell organ failure has also been observed in models of ischemia- types contribute by proinflammatory cytokine production reperfusion (IR). Linkermann et al. showed that RIPK1-mediated through activation of the inflammasome, whereas other cells or necroptosis occurs during kidney ischemia-reperfusion injury cell types contribute to DAMP release through the initiation of and that it can be rescued by Nec-1 inhibitor (Linkermann necroptosis. At a certain point, both inflammasome activation et al., 2012). Similarly, increased phosphorylation of RIPK1 and and necroptosis initiation mutually affect the inflammatory RIPK3 was observed during myocardial IR, and Nec-1 prevented outcome. Altogether, these studies again place caspase-8 in necroptotic cell death and inflammation (infiltration of neutro- a central position as an important brake on inflammation that phils) (Oerlemans et al., 2012). In addition, it has been shown acts either by controlling inflammasome activation or by control- that necroptosis contributes to neuronal damage in a retina IR ling necroptosis. model (Rosenbaum et al., 2010) and in neonatal brain injury (Chavez-Valdez et al., 2012). Interestingly, it has been proposed Distinct Biochemical Pathways of Apoptosis and that TLR signaling contributes to the severity of inflammation in Necroptosis Lead to Different DAMPs Signatures IR models, suggesting that inflammation in IR models could be DAMPs can originate from any compartment of injured or mediated at least partially by DAMPs. In this regard, it has damaged cells, including plasma membrane, cytosol, nucleus, been shown that TLR4 mediates exacerbation of kidney IR via ER, and mitochondria. Distinct biochemical processes occur HMGB1 (Wu et al., 2010) and that TLR4 deficiency reduces during apoptosis and necroptosis. In apoptosis we see caspase myocardial IR (Oyama et al., 2004). activation, chromatin condensation, nuclear fragmentation, DNA All these data strongly suggest that in vivo necroptosis is asso- cleavage, contained apoptotic bodies, extracellular caspases, ciated with an inflammatory reaction (Table 3). However, there is and rapid and efficient phagocytosis (Vanden Berghe et al., only limited knowledge of the molecular mechanisms involved 2010). In necroptosis we observe no massive caspase activa- and the role of the proinflammatory DAMPs emitted during tion, no chromatin condensation, intact nuclei, spillage of cell necroptosis. Both RIPK1 and RIPK3 are required for necroptosis content, phagocytosis by macropinocytosis, lysosomal leakage, initiation (Figure 1), but necroptosis can also occur in the and an oxidative burst (Vanden Berghe et al., 2010). Therefore, absence of RIPK1 (Upton et al., 2010; Zhang et al., 2009). the compositions and signatures of apoptotic and necroptotic Therefore, it seems that RIPK3 drives necroptosis whereas DAMPs might be qualitatively and quantitatively different. RIPK1 is involved in specific cellular contexts. Recent studies Because caspases are intimately engaged in apoptosis, emphasize that it is RIPK3, through its ability to induce ROS absence of their activity during necroptosis might modulate formation, rather than necroptosis itself, that propagates the immunogenicity of released DAMPs (Martin et al., 2012) immune responses or inflammasome activation (Vince et al., (Figure 2C). In apoptosis, caspases cleave phospholipase A2 2012; Wallach et al., 2011). Moreover, RIPK3 expression (PLA2) to produce the ‘‘find-me’’ signal lysophatidylcholine depends on the cell type. Expression of RIPK3 mRNA is stronger (LPC), which enables phagocytosis (Lauber et al., 2003), and in lymphocytes, monocytes, and natural killer cells than in other caspase-activated DNase (called CAD) is responsible for inter- cell types (BioGps, http://biogps.org). Hence, it is possible that nucleosmal cleavage of genomic DNA, which might have weaker only limited types of cells undergo necroptosis in normal condi- immunostimulatory properties than high molecular weight DNA tions. But in pathological conditions, enhanced RIPK3 expres- (Martin et al., 2012). In addition, full-length IL-33 is released sion could mediate the switch from apoptotic to necroptotic during necrosis, but during apoptosis it can undergo caspase- cell death and exacerbate inflammation. On the other hand, dependent proteolysis into a nonimmunological form (Lu¨ thi a recent discovery (Kang et al., 2012) sheds new light on inflam- et al., 2009). Therefore, full-length IL-33 released in necroptotic matory diseases in transgenic mice lacking caspase-8. As conditions (Kovalenko et al., 2009) (when caspases are not discussed above, restriction of caspase-8 is associated with active) could be one of the necroptotic DAMPs (Figure 2C). Cas- skin and intestinal inflammation and excessive activation of pase activity is also thought to be responsible for modification of RIPK1 and RIPK3, which are involved in necroptosis. Therefore, the immunogenic DAMP, HMGB1, into the nonimmunogenic inflammation observed in these transgenic mice has been asso- oxidized HMGB1 form, and caspase-dependent condensation ciated with proinflammatory DAMPs released during this cell of chromatin traps HMBG1 during apoptosis (Kazama et al., death. However, lack of caspase-8 apparently also strongly 2008; Taylor et al., 2008). Furthermore, active caspase-8 is an activates the NLRP3 inflammasome in LPS-stimulated DCs, important mediator of the emission of DAMPs, such as calreticu- leading to excessive production of IL-1b. This inflammasome lin (CRT) (Panaretakis et al., 2009), that function as an ‘‘eat me’’ activation depends probably on RIPK1-RIPK3 mediated mito- signal for DCs and is required for immunogenic responses in chondrial ROS production (Vince et al., 2012, Kang et al., antitumor treatment (Krysko et al., 2012). However, in most of 2012), as mentioned above. Surprisingly, although this proin- the research conducted on necroptosis, caspase-8 was inacti- flammatory pathway involved signaling proteins engaged in initi- vated by chemical inhibition or genetic deletion. Therefore, it is ation of necroptosis, inflammasome activation occurred inde- important to point out that this ablation of caspase-8 in experi- pendently of necroptosis. Therefore mortality and inflammation mental conditions on the one hand sensitizes cells or mouse (as evaluated by increased levels of IL-1b and TNF in serum) models to necroptosis, but on the other hand it might also restrict observed in transgenic mice lacking caspase-8 in DCs occur or at least modify the emission of DAMPs during necroptosis. probably independently of necroptosis. Taken together, these Additionally, mitochondria play an important role in necrop- data point to important crosstalk between necroptosis, inflam- totic cell death (Vandenabeele et al., 2010) and DAMPs release mation, and DAMPs. This crosstalk could occur either in the (Krysko et al., 2012). Insufficient autophagy of these deteriorated

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organelles could lead to massive release of DAMPs such as Because necroptosis is associated with rapid plasma membrane mtDNA and possibly other mitochondrial proteins (Oka et al., permeabilization, DAMPs are probably released from these 2012). On the other hand, damaged mitochondria are an impor- cells, after which they perform immunomodulatory functions in tant source of ROS that not only contribute to progression of cell the intracellular milieu. However, many unanswered questions death but can also modify the oxidative status of DAMPs. and controversies still surround both necroptosis and DAMPs. HMGB1 can illustrate this redox sensitivity (Figure 2C). This The necroptotic pathway can only operate under specific molecule can become proinflammatory in its reduced form conditions and is tightly controlled. Indeed, the caspase-8-

(associated with necrosis) and immunologically silent in its FLIPL-FADD platform and autophagy are apparently gate oxidized form, which requires caspase activation and ROS keepers preventing necroptosis under physiological conditions. production (Kazama et al., 2008). HMGB1 lacks a leader peptide Moreover, caspase-8 or FADD deficiency is the only condition in for the classical ER-Golgi secretory pathway, so it resides in cells which TCR-induced necroptosis can be observed. Furthermore, in reduced form. Moreover, two mutually exclusive forms of experimental necroptosis-induced sterile inflammation also HMGB1 exert two different functions: disulfide-HMGB1 (oxida- requires inhibition or genetic ablation of caspase-8 (skin and tion of C23-C45) acts through TLR4 to induce cytokine produc- intestinal inflammation models). Therefore, besides these strict tion, whereas all-thiol-HMGB1 (all cysteines reduced) acts on the conditions of caspase-8 inactivation or ablation, what could be CXCR4 receptor to induce immune cell migration (Venereau the role of necroptosis in physiological conditions? It has been et al., 2012). Therefore, the redox status contributes to both proposed that necroptosis plays a role during development, attraction of immune cells and inflammation. Next to the role of but RIPK3-deficient mice are viable and they do not display mitochondria in DAMPs release and modification, other any obvious developmental defects. Additionally, FADD- or biochemical processes activated possibly simultaneously with caspase-8 deficient mice die as embryos at the stage of or regulating necroptosis, such as ER-stress could also affect vascular, cardiac, and hematopoietic development; this can be the quality and quantity of released DAMPs. For example, rescued by RIPK3 deletion. This observation suggests the PKR-like endoplasmic reticulum kinase (PERK) is necessary for importance of FADD-caspase-8 axis in development and indi- the emission of two important DAMPs, CRT and ATP (Garg cates that necroptosis might act as a mechanism for eliminating et al., 2012), which facilitate the uptake of dying cells by APCs. defective organisms. However, the PERK pathway can be inactivated by PERK’s The function of necroptosis in the regulation of activated pseudosubstrates or viral inhibitory proteins resembling the T cells is controversial. T cells lacking caspase-8 in a C57BL/6 way by which caspase-8 is inactivated (Galluzzi et al., 2012). In genetic context do not accumulate defective lymphocytes, these conditions, CRT and ATP would not be emitted and dying whereas caspase-8 deficient T cells in a mixed 129/C57BL/6 virus-infected cells would not induce the activation of APCs. background develop lymphoproliferative disease (Salmena and Although these possibilities have not been investigated, we Hakem, 2005), indicating that the genetic background may speculate that also proteins of ER-stress pathway could determine the efficacy of necroptosis in the T cell compartment. modulate necroptosis and necroptotic DAMPs. Additionally, A similar lymphoproliferative disease is observed in patients organelles retained inside apoptotic bodies can be released or bearing a mutated variant of caspase-8 (Chun et al., 2002). externalized during necrosis or necroptosis. For example, intact These patients accumulate abnormal lymphocytes, indicating nuclei have been detected during necroptosis in the extracellular that necroptosis does not compensate for lack of apoptosis. milieu (Krysko et al., 2003) and in tissues (Welz et al., 2011). Such Although necroptosis has not been investigated in these nuclei may contain many nuclear DAMPs, such as IL-1a (Luheshi patients, it cannot be ruled out that functional caspase-10 et al., 2009), IL-33 (Carriere et al., 2007), HMGB1 (Thomas and present in humans (but not in mice) can take over the function Stott, 2012) and largely intact genomic DNA. Other differential of defective caspase-8, suggesting that an additional antine- features of necroptosis are lysosomal membrane permeabiliza- croptotic mechanism may operate in humans. tion (LMP) (Vanden Berghe et al., 2010) and absence of transla- According to current knowledge, ‘‘naturally occurring’’ nec- tional inhibition. Because necroptotic cells continue to synthe- roptosis is observed only in infectious pathological conditions. size proteins until the last moment before plasma membrane Several bacterial and viral infections have been associated permeabilization (Saelens et al., 2005), a necroptotic environ- with necroptosis and emission of DAMPs such as HMGB1. ment is expected to be enriched in DAMPs, and proteins could The precise mechanism and the role of HMGB1 and other be modified during the cell death process by oxidation or by DAMPs in infection-associated inflammation remain unknown. lysosomal cathepsin-dependent proteolysis (Figure 2C). In this First, most probably, multiple pathways, such as inflammasome regard, it has been shown that necroptotic cells secrete the activation and induction by PAMPs during bacterial proinflammatory cytokine IL-6 (Vanden Berghe et al., 2006), or viral infection, can mediate and modify the release of which could then function as an alarmin. Therefore, the DAMPs and modulate in a paracrine way the PRRs on respond- processes activated during the different cell death types may ing cells. Second, DAMPs released from necroptotic cells profoundly affect the composition and properties of released can contribute to sensitization of necroptosis in neighboring DAMPs, and it is clear that DAMPs are important, but their mech- cells via induction of PRRs. Third, activation or modulation anisms of action are poorly understood. of ER-stress, autophagy, and IFN signaling can modify the properties of DAMPs. Especially viruses may subvert DAMPs Concluding Remarks emission, because they not only inhibit caspase-8, making cells The previous decade brought new insights into necroptotic cell more susceptible to necroptosis, but also produce viral proteins death as a programmed and actively regulated process. that interfere with other multiple cellular pathways and

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processes (such as TLRs signaling, caspases inhibition, ER outcome of autophagic signaling in proliferating T cells. Proc. Natl. Acad. stress modulation). Sci. USA 105, 16677–16682. Numerous DAMPs have been associated with accidental Bertrand, M.J., Milutinovic, S., Dickson, K.M., Ho, W.C., Boudreault, A., necrosis, but it seems that the modes of necrosis induction as Durkin, J., Gillard, J.W., Jaquith, J.B., Morris, S.J., and Barker, P.A. (2008). cIAP1 and cIAP2 facilitate cancer cell survival by functioning as E3 ligases well as the cell type affect the immunogenic outcome (Tables 1 that promote RIP1 ubiquitination. Mol. Cell 30, 689–700. and 2). Accordingly, several DAMPs (including HMGB1, S100 proteins, IL33, and mtDNA; Table 3) have been associated with Bonnet, M.C., Preukschat, D., Welz, P.S., van Loo, G., Ermolaeva, M.A., Bloch, W., Haase, I., and Pasparakis, M. (2011). The adaptor protein FADD protects necroptosis-induced inflammatory diseases, but their roles in epidermal keratinocytes from necroptosis in vivo and prevents skin inflamma- immune responses remain to be investigated. It is also conceiv- tion. Immunity 35, 572–582. able that other cellular processes are simultaneously activated Bowie, A.G., and Unterholzner, L. (2008). Viral evasion and subversion of with necroptosis such as autophagy/mitophagy, ER stress, pattern-recognition receptor signalling. Nat. Rev. Immunol. 8, 911–922. ROS production, and lysosomal leakage regulate the quality of DAMPs in vivo. Therefore, it is of great importance to investigate Bray, K., Mathew, R., Lau, A., Kamphorst, J.J., Fan, J., Chen, J., Chen, H.Y., Ghavami, A., Stein, M., DiPaola, R.S., et al. (2012). Autophagy suppresses necroptotic DAMPs in their natural state because processes RIP kinase-dependent necrosis enabling survival to mTOR inhibition. PLoS such as oxidation and proteolysis could affect their immunolog- ONE 7, e41831. ical properties. Brouckaert, G., Kalai, M., Krysko, D.V., Saelens, X., Vercammen, D., Ndlovu, Identification of new and/or specific necroptotic DAMPs will M.N., Haegeman, G., D’Herde, K., and Vandenabeele, P. (2004). Phagocytosis help to identify and characterize new markers of necroptosis of necrotic cells by macrophages is phosphatidylserine dependent and does not induce inflammatory cytokine production. Mol. Biol. Cell 15, 1089–1100. and understand their roles in inflammatory diseases. Studying the potential involvement of necroptosis in inflammatory disor- Carneiro, L.A., Travassos, L.H., Soares, F., Tattoli, I., Magalhaes, J.G., Bozza, ders is of particular interest because it will provide new targets M.T., Plotkowski, M.C., Sansonetti, P.J., Molkentin, J.D., Philpott, D.J., and Girardin, S.E. (2009). Shigella induces mitochondrial dysfunction and cell for clinical application and contribute to better treatment of infec- death in nonmyleoid cells. Cell Host Microbe 5, 123–136. tious and inflammatory diseases, and to development of improved strategies for induction of in Carriere, V., Roussel, L., Ortega, N., Lacorre, D.A., Americh, L., Aguilar, L., Bouche, G., and Girard, J.P. (2007). IL-33, the IL-1-like cytokine ligand for cancer. ST2 receptor, is a chromatin-associated nuclear factor in vivo. Proc. Natl. Acad. Sci. USA 104, 282–287.

ACKNOWLEDGMENTS Ch’en, I.L., Beisner, D.R., Degterev, A., Lynch, C., Yuan, J., Hoffmann, A., and Hedrick, S.M. (2008). Antigen-mediated T cell expansion regulated by parallel We thank A. Bredan for editing the manuscript. This work was supported pathways of death. Proc. Natl. Acad. Sci. USA 105, 17463–17468. by project grants from the Fund for Scientific Research Flanders (FWO- Vlaanderen, G.0728.10, 3G067512, 3G060713, and 3G0A5413 to D.V.K.) Ch’en, I.L., Tsau, J.S., Molkentin, J.D., Komatsu, M., and Hedrick, S.M. (2011). and by an individual research grant from FWO-Vlaanderen (31507110 to Mechanisms of necroptosis in T cells. J. Exp. Med. 208, 633–641. D.V.K.). D.V.K. is a postdoctoral fellow and A.K. is a doctoral fellow, both Chan, F.K., Shisler, J., Bixby, J.G., Felices, M., Zheng, L., Appel, M., Orenstein, paid by fellowships from FWO-Vlaanderen. A.K. is also a recipient of an Emma- J., Moss, B., and Lenardo, M.J. (2003). A role for nuel van der Schueren scholarship from the Flemish league against cancer. receptor-2 and receptor-interacting protein in programmed necrosis and anti- Research in the Vandenabeele group is supported by European grants (FP6 viral responses. J. Biol. Chem. 278, 51613–51621. ApopTrain, MRTNCT-035624; FP7 EC RTD Integrated Project, Apo-Sys, FP7-200767; Euregional PACT II), Belgian grants (Interuniversity Attraction Chan, J., Watson, J.N., Lu, A., Cerda, V.C., Borgford, T.J., and Bennet, A.J. Poles, IAP 6/18, IAP 7/32), Flemish grants (Research Foundation Flanders, (2012). Bacterial and viral sialidases: contribution of the conserved active FWO G.0875.11, FWO G.0973.11, and FWO G.0A45.12N), Ghent University site glutamate to catalysis. Biochemistry 51, 433–441. grants (MRP, GROUP-ID consortium), grants from Flanders Institute for Biotechnology (VIB), and grants from the Foundation against Cancer (F94). Chavez-Valdez, R., Martin, L.J., and Northington, F.J. (2012). Programmed P.V. holds a Methusalem grant (BOF09/01M00709) from the Flemish Govern- Necrosis: A Prominent Mechanism of Cell Death following Neonatal Brain ment. The figures in this review were produced using Servier Medical Art Injury. Neurol. Res. Int. 2012, 257563. (www.servier.com). Chen, G.Y., and Nun˜ ez, G. (2010). Sterile inflammation: sensing and reacting to damage. Nat. Rev. Immunol. 10, 826–837. REFERENCES Chen, L.C., Yeh, T.M., Wu, H.N., Lin, Y.Y., and Shyu, H.W. (2008). Dengue virus Allam, R., Scherbaum, C.R., Darisipudi, M.N., Mulay, S.R., Hagele, H., Lichtne- infection induces passive release of high mobility group box 1 protein by kert, J., Hagemann, J.H., Rupanagudi, K.V., Ryu, M., Schwarzenberger, C., epithelial cells. J. Infect. 56, 143–150. et al. (2012). Histones from Dying Renal Cells Aggravate Kidney Injury via TLR2 and TLR4. J Am Soc Nephrol. 23, 1375–1388. Chen, G.Y., Tang, J., Zheng, P., and Liu, Y. (2009). CD24 and Siglec-10 selectively repress tissue damage-induced immune responses. Science 323, Aravalli, R.N., Hu, S., and Lokensgard, J.R. (2008). Inhibition of toll-like 1722–1725. receptor signaling in primary murine microglia. J. Neuroimmune Pharmacol. 3, 5–11. Chen, G.Y., Chen, X., King, S., Cavassani, K.A., Cheng, J., Zheng, X., Cao, H., Yu, H., Qu, J., Fang, D., et al. (2011). Amelioration of sepsis by inhibiting siali- Ayna, G., Krysko, D.V., Kaczmarek, A., Petrovski, G., Vandenabeele, P., and dase-mediated disruption of the CD24-SiglecG interaction. Nat. Biotechnol. Fe´ su¨ s, L. (2012). ATP release from dying autophagic cells and their phagocy- 29, 428–435. tosis are crucial for inflammasome activation in macrophages. PLoS ONE 7, e40069. Cho, Y.S., Challa, S., Moquin, D., Genga, R., Ray, T.D., Guildford, M., and Chan, F.K. (2009). Phosphorylation-driven assembly of the RIP1-RIP3 Bartholomae, W.C., Rininsland, F.H., Eisenberg, J.C., Boehm, B.O., Lehmann, complex regulates programmed necrosis and virus-induced inflammation. P.V., and Tary-Lehmann, M. (2004). T cell immunity induced by live, necrotic, Cell 137, 1112–1123. and apoptotic tumor cells. J. Immunol. 173, 1012–1022. Christofferson, D.E., Li, Y., Hitomi, J., Zhou, W., Upperman, C., Zhu, H., Bell, B.D., Leverrier, S., Weist, B.M., Newton, R.H., Arechiga, A.F., Luhrs, K.A., Gerber, S.A., Gygi, S., and Yuan, J. (2012). A novel role for RIP1 kinase in medi- Morrissette, N.S., and Walsh, C.M. (2008). FADD and caspase-8 control the ating TNFa production. Cell Death Dis 3, e320.

220 Immunity 38, February 21, 2013 ª2013 Elsevier Inc. Immunity Review

Chu, J.J., and Ng, M.L. (2003). The mechanism of cell death during West Haas, T.L., Emmerich, C.H., Gerlach, B., Schmukle, A.C., Cordier, S.M., Nile virus infection is dependent on initial infectious dose. J. Gen. Virol. 84, Rieser, E., Feltham, R., Vince, J., Warnken, U., Wenger, T., et al. (2009). 3305–3314. Recruitment of the linear ubiquitin chain assembly complex stabilizes the TNF-R1 signaling complex and is required for TNF-mediated gene induction. Chun, H.J., Zheng, L., Ahmad, M., Wang, J., Speirs, C.K., Siegel, R.M., Dale, Mol. Cell 36, 831–844. J.K., Puck, J., Davis, J., Hall, C.G., et al. (2002). Pleiotropic defects in lympho- cyte activation caused by caspase-8 mutations lead to human immunodefi- He, S., Liang, Y., Shao, F., and Wang, X. (2011). Toll-like receptors activate ciency. Nature 419, 395–399. programmed necrosis in macrophages through a receptor-interacting kinase-3-mediated pathway. Proc. Natl. Acad. Sci. USA 108, 20054–20059. Cvetanovic, M., and Ucker, D.S. (2004). Innate immune discrimination of apoptotic cells: repression of proinflammatory transcription is He, M.X., McLeod, I.X., Jia, W., and He, Y.W. (2012). Macroautophagy in T coupled directly to specific recognition. J. Immunol. 172, 880–889. lymphocyte development and function. Front Immunol 3, 22.

Dillon, C.P., Oberst, A., Weinlich, R., Janke, L.J., Kang, T.B., Ben-Moshe, T., Holler, N., Zaru, R., Micheau, O., Thome, M., Attinger, A., Valitutti, S., Bodmer, Mak, T.W., Wallach, D., and Green, D.R. (2012). Survival function of the J.L., Schneider, P., Seed, B., and Tschopp, J. (2000). Fas triggers an alterna- FADD-CASPASE-8-cFLIP(L) complex. Cell Rep 1, 401–407. tive, caspase-8-independent cell death pathway using the kinase RIP as effector molecule. Nat. Immunol. 1, 489–495. Dourmashkin, R.R., Davies, H., Wells, C., Shah, D., Price, A., O’Morain, C., and Levi, J. (1983). Epithelial patchy necrosis in Crohn’s disease. Hum. Pathol. 14, Inzkirweli, N., Gu¨ ckel, B., Sohn, C., Wallwiener, D., Bastert, G., and Lindner, M. 643–648. (2007). Antigen loading of dendritic cells with apoptotic tumor cell-prepara- tions is superior to that using necrotic cells or tumor lysates. Anticancer Res. Duprez, L., Takahashi, N., Van Hauwermeiren, F., Vandendriessche, B., 27(4B), 2121–2129. Goossens, V., Vanden Berghe, T., Declercq, W., Libert, C., Cauwels, A., and Vandenabeele, P. (2011). RIP kinase-dependent necrosis drives lethal Iyer, S.S., Pulskens, W.P., Sadler, J.J., Butter, L.M., Teske, G.J., Ulland, T.K., systemic inflammatory response syndrome. Immunity 35, 908–918. Eisenbarth, S.C., Florquin, S., Flavell, R.A., Leemans, J.C., and Sutterwala, F.S. (2009). Necrotic cells trigger a sterile inflammatory response through Duttagupta, P.A., Boesteanu, A.C., and Katsikis, P.D. (2009). Costimulation the Nlrp3 inflammasome. Proc. Natl. Acad. Sci. USA 106, 20388–20393. signals for memory CD8+ T cells during viral infections. Crit. Rev. Immunol. 29, 469–486. Kaiser, W.J., Upton, J.W., Long, A.B., Livingston-Rosanoff, D., Daley-Bauer, L.P., Hakem, R., Caspary, T., and Mocarski, E.S. (2011). RIP3 mediates the Eigenbrod, T., Park, J.H., Harder, J., Iwakura, Y., and Nu´ n˜ ez, G. (2008). Cutting embryonic lethality of caspase-8-deficient mice. Nature 471, 368–372. edge: critical role for mesothelial cells in necrosis-induced inflammation through the recognition of IL-1 alpha released from dying cells. J. Immunol. Kalai, M., Van Loo, G., Vanden Berghe, T., Meeus, A., Burm, W., Saelens, X., 181, 8194–8198. and Vandenabeele, P. (2002). Tipping the balance between necrosis and apoptosis in human and murine cells treated with interferon and dsRNA. Cell El Mezayen, R., El Gazzar, M., Seeds, M.C., McCall, C.E., Dreskin, S.C., and Death Differ. 9, 981–994. Nicolls, M.R. (2007). Endogenous signals released from necrotic cells augment inflammatory responses to bacterial endotoxin. Immunol. Lett. 111, Kamau, E., Takhampunya, R., Li, T., Kelly, E., Peachman, K.K., Lynch, J.A., 36–44. Sun, P., and Palmer, D.R. (2009). Dengue virus infection promotes transloca- tion of high mobility group box 1 protein from the nucleus to the cytosol in Fallach, R., Shainberg, A., Avlas, O., Fainblut, M., Chepurko, Y., Porat, E., and dendritic cells, upregulates cytokine production and modulates virus replica- Hochhauser, E. (2010). Cardiomyocyte Toll-like receptor 4 is involved in heart tion. J. Gen. Virol. 90, 1827–1835. dysfunction following septic shock or myocardial ischemia. J. Mol. Cell. Cardiol. 48, 1236–1244. Kang, T.B., Yang, S.H., Toth, B., Kovalenko, A., and Wallach, D. (2012). Caspase-8 Blocks Kinase RIPK3-Mediated Activation of the NLRP3 Inflamma- Feoktistova, M., Geserick, P., Kellert, B., Dimitrova, D.P., Langlais, C., Hupe, some. Immunity 38, 27–40. M., Cain, K., MacFarlane, M., Ha¨ cker, G., and Leverkus, M. (2011). cIAPs block Ripoptosome formation, a RIP1/caspase-8 containing intracellular cell death Kazama, H., Ricci, J.E., Herndon, J.M., Hoppe, G., Green, D.R., and Ferguson, complex differentially regulated by cFLIP isoforms. Mol. Cell 43, 449–463. T.A. (2008). Induction of immunological tolerance by apoptotic cells requires caspase-dependent oxidation of high-mobility group box-1 protein. Immunity Gallucci, S., and Matzinger, P. (2001). Danger signals: SOS to the immune 29, 21–32. system. Curr. Opin. Immunol. 13, 114–119. Kennedy, C.L., Smith, D.J., Lyras, D., Chakravorty, A., and Rood, J.I. (2009). Galluzzi, L., Vitale, I., Abrams, J.M., Alnemri, E.S., Baehrecke, E.H., Blagos- Programmed cellular necrosis mediated by the pore-forming alpha-toxin klonny, M.V., Dawson, T.M., Dawson, V.L., El-Deiry, W.S., Fulda, S., et al. from Clostridium septicum. PLoS Pathog. 5, e1000516. (2012). Molecular definitions of cell death subroutines: recommendations of the Nomenclature Committee on Cell Death 2012. Cell Death Differ. 19, Kim, J.H., Kim, S.J., Lee, I.S., Lee, M.S., Uematsu, S., Akira, S., and Oh, K.I. 107–120. (2009). Bacterial endotoxin induces the release of high mobility group box 1 via the IFN-beta signaling pathway. J. Immunol. 182, 2458–2466. Garg, A.D., Nowis, D., Golab, J., Vandenabeele, P., Krysko, D.V., and Agosti- nis, P. (2010). Immunogenic cell death, DAMPs and anticancer therapeutics: Kono, H., Karmarkar, D., Iwakura, Y., and Rock, K.L. (2010). Identification of an emerging amalgamation. Biochim. Biophys. Acta 1805, 53–71. the cellular sensor that stimulates the inflammatory response to sterile cell death. J. Immunol. 184, 4470–4478. Garg, A.D., Krysko, D.V., Verfaillie, T., Kaczmarek, A., Ferreira, G.B., Marysael, T., Rubio, N., Firczuk, M., Mathieu, C., Roebroek, A.J., et al. (2012). A novel Kovalenko, A., Kim, J.C., Kang, T.B., Rajput, A., Bogdanov, K., Dittrich-Brei- pathway combining calreticulin exposure and ATP secretion in immunogenic holz, O., Kracht, M., Brenner, O., and Wallach, D. (2009). Caspase-8 deficiency cancer cell death. EMBO J. 31, 1062–1079. in epidermal keratinocytes triggers an inflammatory skin disease. J. Exp. Med. 206, 2161–2177. Gee, K., Kozlowski, M., and Kumar, A. (2003). Tumor necrosis factor-alpha induces functionally active hyaluronan-adhesive CD44 by activating sialidase Krysko, D.V., Brouckaert, G., Kalai, M., Vandenabeele, P., and D’Herde, K. through p38 mitogen-activated protein kinase in lipopolysaccharide-stimu- (2003). Mechanisms of internalization of apoptotic and necrotic L929 cells lated human monocytic cells. J. Biol. Chem. 278, 37275–37287. by a macrophage cell line studied by electron microscopy. J. Morphol. 258, 336–345. Goldszmid, R.S., Idoyaga, J., Bravo, A.I., Steinman, R., Mordoh, J., and Wainstok, R. (2003). Dendritic cells charged with apoptotic tumor cells induce Krysko, D.V., Denecker, G., Festjens, N., Gabriels, S., Parthoens, E., D’Herde, long-lived protective CD4+ and CD8+ T cell immunity against B16 melanoma. K., and Vandenabeele, P. (2006). Macrophages use different internalization J. Immunol. 171, 5940–5947. mechanisms to clear apoptotic and necrotic cells. Cell Death Differ. 13, 2011–2022. Gu¨ nther, C., Martini, E., Wittkopf, N., Amann, K., Weigmann, B., Neumann, H., Waldner, M.J., Hedrick, S.M., Tenzer, S., Neurath, M.F., and Becker, C. (2011). Krysko, D.V., Vanden Berghe, T., D’Herde, K., and Vandenabeele, P. (2008a). Caspase-8 regulates TNF-a-induced epithelial necroptosis and terminal ileitis. Apoptosis and necrosis: detection, discrimination and phagocytosis. Methods Nature 477, 335–339. 44, 205–221.

Immunity 38, February 21, 2013 ª2013 Elsevier Inc. 221 Immunity Review

Krysko, D.V., Vanden Berghe, T., Parthoens, E., D’Herde, K., and Vandena- Mocarski, E.S., Upton, J.W., and Kaiser, W.J. (2012). Viral infection and the beele, P. (2008b). Methods for distinguishing apoptotic from necrotic cells evolution of caspase 8-regulated apoptotic and necrotic death pathways. and measuring their clearance. Methods Enzymol. 442, 307–341. Nat. Rev. Immunol. 12, 79–88.

Krysko, D.V., Agostinis, P., Krysko, O., Garg, A.D., Bachert, C., Lambrecht, Morinaga, Y., Yanagihara, K., Nakamura, S., Hasegawa, H., Seki, M., Izumi- B.N., and Vandenabeele, P. (2011). Emerging role of damage-associated kawa, K., Kakeya, H., Yamamoto, Y., Yamada, Y., Kohno, S., and Kamihira, molecular patterns derived from mitochondria in inflammation. Trends Immu- S. (2010). Legionella pneumophila induces cathepsin B-dependent necrotic nol. 32, 157–164. cell death with releasing high mobility group box1 in macrophages. Respir. Res. 11, 158. Krysko, D.V., Garg, A.D., Kaczmarek, A., Krysko, O., Agostinis, P., and Vande- nabeele, P. (2012). Immunogenic cell death and DAMPs in cancer therapy. Narayan, N., Lee, I.H., Borenstein, R., Sun, J., Wong, R., Tong, G., Fergusson, Nat. Rev. Cancer 12, 860–875. M.M., Liu, J., Rovira, I.I., Cheng, H.L., et al. (2012). The NAD-dependent de- acetylase SIRT2 is required for programmed necrosis. Nature 492, 199–204. Lalanne, A.I., Moraga, I., Hao, Y., Pereira, J.P., Alves, N.L., Huntington, N.D., Freitas, A.A., Cumano, A., and Vieira, P. (2010). CpG inhibits pro-B cell Northington, F.J., Chavez-Valdez, R., Graham, E.M., Razdan, S., Gauda, E.B., expansion through a cathepsin B-dependent mechanism. J. Immunol. 184, and Martin, L.J. (2011). Necrostatin decreases oxidative damage, inflamma- 5678–5685. tion, and injury after neonatal HI. J. Cereb. Blood Flow Metab. 31, 178–189.

Laster, S.M., Wood, J.G., and Gooding, L.R. (1988). Tumor necrosis factor O’Donnell, M.A., Perez-Jimenez, E., Oberst, A., Ng, A., Massoumi, R., Xavier, can induce both apoptic and necrotic forms of cell lysis. J. Immunol. 141, R., Green, D.R., and Ting, A.T. (2011). Caspase 8 inhibits programmed 2629–2634. necrosis by processing CYLD. Nat. Cell Biol. 13, 1437–1442.

Lauber, K., Bohn, E., Kro¨ ber, S.M., Xiao, Y.J., Blumenthal, S.G., Lindemann, Oberst, A., Dillon, C.P., Weinlich, R., McCormick, L.L., Fitzgerald, P., Pop, C., R.K., Marini, P., Wiedig, C., Zobywalski, A., Baksh, S., et al. (2003). Apoptotic Hakem, R., Salvesen, G.S., and Green, D.R. (2011). Catalytic activity of the cells induce migration of phagocytes via caspase-3-mediated release of a lipid caspase-8-FLIP(L) complex inhibits RIPK3-dependent necrosis. Nature 471, attraction signal. Cell 113, 717–730. 363–367.

Li, C., Lasse, S., Lee, P., Nakasaki, M., Chen, S.W., Yamasaki, K., Gallo, R.L., Oerlemans, M.I., Liu, J., Arslan, F., den Ouden, K., van Middelaar, B.J., and Jamora, C. (2010). Development of atopic dermatitis-like skin disease Doevendans, P.A., and Sluijter, J.P. (2012). Inhibition of RIP1-dependent from the chronic loss of epidermal caspase-8. Proc. Natl. Acad. Sci. USA necrosis prevents adverse cardiac remodeling after myocardial ischemia- 107, 22249–22254. reperfusion in vivo. Basic Res. Cardiol. 107, 270.

Lietze´ n, N., Ohman, T., Rintahaka, J., Julkunen, I., Aittokallio, T., Matikainen, Oka, T., Hikoso, S., Yamaguchi, O., Taneike, M., Takeda, T., Tamai, T., Oyabu, S., and Nyman, T.A. (2011). Quantitative subcellular proteome and secretome J., Murakawa, T., Nakayama, H., Nishida, K., et al. (2012). Mitochondrial DNA profiling of influenza A virus-infected human primary macrophages. PLoS that escapes from autophagy causes inflammation and heart failure. Nature Pathog. 7, e1001340. 485, 251–255. Ong, S.P., Lee, L.M., Leong, Y.F., Ng, M.L., and Chu, J.J. (2012). Dengue virus Linkermann, A., Bra¨ sen, J.H., Himmerkus, N., Liu, S., Huber, T.B., Kunzendorf, infection mediates HMGB1 release from monocytes involving PCAF acetylase U., and Krautwald, S. (2012). Rip1 (receptor-interacting protein kinase 1) medi- complex and induces vascular leakage in endothelial cells. PLoS ONE 7, ates necroptosis and contributes to renal ischemia/reperfusion injury. Kidney e41932. Int. 81, 751–761. Oppenheim, J.J., and Yang, D. (2005). Alarmins: chemotactic activators of Lohmann, C., Muschaweckh, A., Kirschnek, S., Jennen, L., Wagner, H., and immune responses. Curr. Opin. Immunol. 17, 359–365. Ha¨ cker, G. (2009). Induction of tumor cell apoptosis or necrosis by conditional expression of cell death proteins: analysis of cell death pathways and in vitro Osborn, S.L., Diehl, G., Han, S.J., Xue, L., Kurd, N., Hsieh, K., Cado, D., Robey, immune stimulatory potential. J. Immunol. 182, 4538–4546. E.A., and Winoto, A. (2010). Fas-associated death domain (FADD) is a negative regulator of T-cell receptor-mediated necroptosis. Proc. Natl. Acad. Sci. USA Long, J.S., and Ryan, K.M. (2012). New frontiers in promoting tumour cell 107, 13034–13039. death: targeting apoptosis, necroptosis and autophagy. Oncogene 31, 5045–5060. Oyama, J., Blais, C., Jr., Liu, X., Pu, M., Kobzik, L., Kelly, R.A., and Bourcier, T. (2004). Reduced myocardial ischemia-reperfusion injury in toll-like receptor Lu, J.V., Weist, B.M., van Raam, B.J., Marro, B.S., Nguyen, L.V., Srinivas, P., 4-deficient mice. Circulation 109, 784–789. Bell, B.D., Luhrs, K.A., Lane, T.E., Salvesen, G.S., and Walsh, C.M. (2011a). Complementary roles of Fas-associated death domain (FADD) and receptor Panaretakis, T., Kepp, O., Brockmeier, U., Tesniere, A., Bjorklund, A.C., interacting protein kinase-3 (RIPK3) in T-cell homeostasis and antiviral Chapman, D.C., Durchschlag, M., Joza, N., Pierron, G., van Endert, P., et al. immunity. Proc. Natl. Acad. Sci. USA 108, 15312–15317. (2009). Mechanisms of pre-apoptotic calreticulin exposure in immunogenic cell death. EMBO J. 28, 578–590. Lu, J.V., Weist, B.M., van Raam, B.J., Marro, B.S., Nguyen, L.V., Srinivas, P., Bell, B.D., Luhrs, K.A., Lane, T.E., Salvesen, G.S., and Walsh, C.M. (2011b). Peri, P., Nuutila, K., Vuorinen, T., Saukko, P., and Hukkanen, V. (2011). Cathep- Complementary roles of Fas-associated death domain (FADD) and receptor sins are involved in virus-induced cell death in ICP4 and Us3 deletion interacting protein kinase-3 (RIPK3) in T-cell homeostasis and antiviral mutant herpes simplex virus type 1-infected monocytic cells. J. Gen. Virol. immunity. Proc. Natl. Acad. Sci. USA 108, 15312–15317. 92, 173–180.

Luheshi, N.M., McColl, B.W., and Brough, D. (2009). Nuclear retention of IL-1 Petit, F., Arnoult, D., Lelie` vre, J.D., Moutouh-de Parseval, L., Hance, A.J., alpha by necrotic cells: a mechanism to dampen sterile inflammation. Eur. J. Schneider, P., Corbeil, J., Ameisen, J.C., and Estaquier, J. (2002). Productive Immunol. 39, 2973–2980. HIV-1 infection of primary CD4+ T cells induces mitochondrial membrane per- meabilization leading to a caspase-independent cell death. J. Biol. Chem. 277, Lu¨ thi, A.U., Cullen, S.P., McNeela, E.A., Duriez, P.J., Afonina, I.S., Sheridan, 1477–1487. C., Brumatti, G., Taylor, R.C., Kersse, K., Vandenabeele, P., et al. (2009). Suppression of interleukin-33 bioactivity through proteolysis by apoptotic Rider, P., Carmi, Y., Guttman, O., Braiman, A., Cohen, I., Voronov, E., White, caspases. Immunity 31, 84–98. M.R., Dinarello, C.A., and Apte, R.N. (2011). IL-1a and IL-1b recruit different myeloid cells and promote different stages of sterile inflammation. J. Immunol. Martin, S.J., Henry, C.M., and Cullen, S.P. (2012). A perspective on mamma- 187, 4835–4843. lian caspases as positive and negative regulators of inflammation. Mol. Cell 46, 387–397. Robinson, N., McComb, S., Mulligan, R., Dudani, R., Krishnan, L., and Sad, S. (2012). Type I interferon induces necroptosis in macrophages during infection Miles, K., Clarke, D.J., Lu, W., Sibinska, Z., Beaumont, P.E., Davidson, D.J., with Salmonella enterica serovar Typhimurium. Nat. Immunol. 13, 954–962. Barr, T.A., Campopiano, D.J., and Gray, M. (2009). Dying and necrotic neutro- phils are anti-inflammatory secondary to the release of alpha-defensins. Rock, K.L., and Kono, H. (2008). The inflammatory response to cell death. J. Immunol. 183, 2122–2132. Annu. Rev. Pathol. 3, 99–126.

222 Immunity 38, February 21, 2013 ª2013 Elsevier Inc. Immunity Review

Rosenbaum, D.M., Degterev, A., David, J., Rosenbaum, P.S., Roth, S., Grotta, Vandenabeele, P., Galluzzi, L., Vanden Berghe, T., and Kroemer, G. (2010). J.C., Cuny, G.D., Yuan, J., and Savitz, S.I. (2010). Necroptosis, a novel form of Molecular mechanisms of necroptosis: an ordered cellular explosion. Nat. caspase-independent cell death, contributes to neuronal damage in a retinal Rev. Mol. Cell Biol. 11, 700–714. ischemia-reperfusion injury model. J. Neurosci. Res. 88, 1569–1576. Vanlangenakker, N., Bertrand, M.J., Bogaert, P., Vandenabeele, P., and Saelens, X., Festjens, N., Parthoens, E., Vanoverberghe, I., Kalai, M., van Kup- Vanden Berghe, T. (2011). TNF-induced necroptosis in L929 cells is tightly peveld, F., and Vandenabeele, P. (2005). Protein synthesis persists during regulated by multiple TNFR1 complex I and II members. Cell Death Dis 2, e230. necrotic cell death. J. Cell Biol. 168, 545–551. Vanlangenakker, N., Vanden Berghe, T., and Vandenabeele, P. (2012). Many Salmena, L., and Hakem, R. (2005). Caspase-8 deficiency in T cells leads to stimuli pull the necrotic trigger, an overview. Cell Death Differ. 19, 75–86. a lethal lymphoinfiltrative immune disorder. J. Exp. Med. 202, 727–732. Venereau, E., Casalgrandi, M., Schiraldi, M., Antoine, D.J., Cattaneo, A., De Salmena, L., Lemmers, B., Hakem, A., Matysiak-Zablocki, E., Murakami, K., Marchis, F., Liu, J., Antonelli, A., Preti, A., Raeli, L., et al. (2012). Mutually Au, P.Y., Berry, D.M., Tamblyn, L., Shehabeldin, A., Migon, E., et al. (2003). exclusive redox forms of HMGB1 promote cell recruitment or proinflammatory Essential role for caspase 8 in T-cell homeostasis and T-cell-mediated immu- cytokine release. J. Exp. Med. 209, 1519–1528. nity. Genes Dev. 17, 883–895. Vercammen, D., Vandenabeele, P., Beyaert, R., Declercq, W., and Fiers, W. Sauter, B., Albert, M.L., Francisco, L., Larsson, M., Somersan, S., and (1997). Tumour necrosis factor-induced necrosis versus anti-Fas-induced Bhardwaj, N. (2000). Consequences of cell death: exposure to necrotic tumor apoptosis in L929 cells. Cytokine 9, 801–808. cells, but not primary tissue cells or apoptotic cells, induces the maturation of immunostimulatory dendritic cells. J. Exp. Med. 191, 423–434. Vercammen, D., Brouckaert, G., Denecker, G., Van de Craen, M., Declercq, W., Fiers, W., and Vandenabeele, P. (1998). Dual signaling of the Fas receptor: Scaffidi, P., Misteli, T., and Bianchi, M.E. (2002). Release of chromatin protein initiation of both apoptotic and necrotic cell death pathways. J. Exp. Med. 188, HMGB1 by necrotic cells triggers inflammation. Nature 418, 191–195. 919–930.

Scheffer, S.R., Nave, H., Korangy, F., Schlote, K., Pabst, R., Jaffee, E.M., Vince, J.E., Wong, W.W., Gentle, I., Lawlor, K.E., Allam, R., O’Reilly, L., Mason, Manns, M.P., and Greten, T.F. (2003). Apoptotic, but not necrotic, tumor K., Gross, O., Ma, S., Guarda, G., et al. (2012). Inhibitor of apoptosis proteins cell vaccines induce a potent immune response in vivo. Int. J. Cancer 103, limit RIP3 kinase-dependent interleukin-1 activation. Immunity 36, 215–227. 205–211. Wallach, D., Kovalenko, A., and Kang, T.B. (2011). ‘Necrosome’-induced Skaletskaya, A., Bartle, L.M., Chittenden, T., McCormick, A.L., Mocarski, E.S., inflammation: must cells die for it? Trends Immunol. 32, 505–509. and Goldmacher, V.S. (2001). A cytomegalovirus-encoded inhibitor of Wang, F., Lu, Z., Hawkes, M., Yang, H., Kain, K.C., and Liles, W.C. (2010). Fas apoptosis that suppresses caspase-8 activation. Proc. Natl. Acad. Sci. USA (CD95) induces rapid, TLR4/IRAK4-dependent release of pro-inflammatory 98, 7829–7834. HMGB1 from macrophages. J. Inflamm. (Lond.) 7, 30. Stridh, L., Smith, P.L., Naylor, A.S., Wang, X., and Mallard, C. (2011). Regula- Wang, Z., Jiang, H., Chen, S., Du, F., and Wang, X. (2012). The mitochondrial tion of toll-like receptor 1 and -2 in neonatal mice brains after hypoxia- phosphatase PGAM5 functions at the convergence point of multiple necrotic ischemia. J. Neuroinflammation 8, 45. death pathways. Cell 148, 228–243.

Sudan, H., Lai, W., Lin, M., Tao, W., Fenghe, D., Liping, Z., and Xiaodong, W. Welz, P.S., Wullaert, A., Vlantis, K., Kondylis, V., Ferna´ ndez-Majada, V., (2009). Receptor Interacting Protein Kinase-3 Determines Cellular Necrotic Ermolaeva, M., Kirsch, P., Sterner-Kock, A., van Loo, G., and Pasparakis, M. Response to TNF-a. Cell 137, 1100–1111. (2011). FADD prevents RIP3-mediated epithelial cell necrosis and chronic intestinal inflammation. Nature 477, 330–334. Takeuchi, O., and Akira, S. (2010). Pattern recognition receptors and inflam- mation. Cell 140, 805–820. Wheeler, D.S., Chase, M.A., Senft, A.P., Poynter, S.E., Wong, H.R., and Page, K. (2009). Extracellular Hsp72, an endogenous DAMP, is released by virally Taylor, R.C., Cullen, S.P., and Martin, S.J. (2008). Apoptosis: controlled demo- infected airway epithelial cells and activates neutrophils via Toll-like receptor lition at the cellular level. Nat. Rev. Mol. Cell Biol. 9, 231–241. (TLR)-4. Respir. Res. 10, 31.

Tenev, T., Bianchi, K., Darding, M., Broemer, M., Langlais, C., Wallberg, F., Wu, H., Ma, J., Wang, P., Corpuz, T.M., Panchapakesan, U., Wyburn, K.R., Zachariou, A., Lopez, J., MacFarlane, M., Cain, K., and Meier, P. (2011). The and Chadban, S.J. (2010). HMGB1 contributes to kidney ischemia reperfusion Ripoptosome, a signaling platform that assembles in response to genotoxic injury. J. Am. Soc. Nephrol. 21, 1878–1890. stress and loss of IAPs. Mol. Cell 43, 432–448. Yoon, T.J., Kim, J.Y., Kim, H., Hong, C., Lee, H., Lee, C.K., Lee, K.H., Hong, S., Thomas, J.O., and Stott, K. (2012). H1 and HMGB1: modulators of chromatin and Park, S.H. (2008). Anti-tumor immunostimulatory effect of heat-killed structure. Biochem. Soc. Trans. 40, 341–346. tumor cells. Exp. Mol. Med. 40, 130–144.

Upton, J.W., Kaiser, W.J., and Mocarski, E.S. (2010). Virus inhibition of RIP3- Youle, R.J., and Narendra, D.P. (2011). Mechanisms of mitophagy. Nat. Rev. dependent necrosis. Cell Host Microbe 7, 302–313. Mol. Cell Biol. 12, 9–14.

Upton, J.W., Kaiser, W.J., and Mocarski, E.S. (2012). DAI/ZBP1/DLM-1 Zhang, D.W., Shao, J., Lin, J., Zhang, N., Lu, B.J., Lin, S.C., Dong, M.Q., and complexes with RIP3 to mediate virus-induced programmed necrosis that is Han, J. (2009). RIP3, an energy metabolism regulator that switches TNF- targeted by murine cytomegalovirus vIRA. Cell Host Microbe 11, 290–297. induced cell death from apoptosis to necrosis. Science 325, 332–336.

Vanden Berghe, T., Kalai, M., Denecker, G., Meeus, A., Saelens, X., and Zhang, Q., Raoof, M., Chen, Y., Sumi, Y., Sursal, T., Junger, W., Brohi, K., Vandenabeele, P. (2006). Necrosis is associated with IL-6 production but Itagaki, K., and Hauser, C.J. (2010). Circulating mitochondrial DAMPs cause apoptosis is not. Cell. Signal. 18, 328–335. inflammatory responses to injury. Nature 464, 104–107.

Vanden Berghe, T., Vanlangenakker, N., Parthoens, E., Deckers, W., Devos, Zhao, J., Jitkaew, S., Cai, Z., Choksi, S., Li, Q., Luo, J., and Liu, Z.G. (2012). M., Festjens, N., Guerin, C.J., Brunk, U.T., Declercq, W., and Vandenabeele, Mixed lineage kinase domain-like is a key receptor interacting protein 3 down- P. (2010). Necroptosis, necrosis and secondary necrosis converge on similar stream component of TNF-induced necrosis. Proc. Natl. Acad. Sci. USA 109, cellular disintegration features. Cell Death Differ. 17, 922–930. 5322–5327.

Immunity 38, February 21, 2013 ª2013 Elsevier Inc. 223