necroptosis

ImmunityReview 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 Signaling and Cell Death Unit, Department for Molecular Biomedical Research, VIB, 9052 Ghent, Belgium 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 2Department 1Molecular Regulated necrosis, termed necroptosis, is negatively regulated by caspase-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 modulation 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 maintenance of T cell homeostasis. In contrast, the induction of necroptosis in skin, intestine, systemic inflammatory response syndrome, and ischemia reperfusion injury provoke a strong inflammatory response, which might be triggered by emission of DAMPs from necroptotic cells, showing the detrimental side of necroptosis. Introduction Necrosis has long been described as a consequence of extreme physicochemical stress, such as heat, osmotic shock, mechanical stress, and freeze-thawing, which kill cells quickly and directly. Therefore, this cell death has been described as uncontrolled and accidental necrosis characterized by loss of plasma membrane integrity and cellular collapse, though nuclei remain largely intact during this process (Krysko et al., 2008a, 2008b; Vanden Berghe et al., 2010). Loss of membrane integrity and release of intracellular content grant necrotic cells the ability to induce an inflammatory response. These immunogenic endogenous molecules fall under the umbrella term ‘‘damage-associated molecular patterns’’ (DAMPs) (Garg et al., 2010; Krysko et al., 2011). They include, in the case of accidental necrosis, HMGB1, IL-1a, uric acid, DNA fragments, mitochondrial content, and ATP (Tables 1 and 2) (Eigenbrod et al., 2008; Kono et al., 2010; Krysko et al., 2008a; Sauter et al., 2000). Because the nomenclature of DAMPs in the literature is confusing, here we define DAMPs as a family of molecules that are intracellular in physiological conditions and can be divided into two groups: (1) molecules that perform noninflammatory functions in living cells (such as HMGB1, ATP) and acquire immunomodulatory properties when released, secreted, modified, or exposed on the cell surface during cellular stress, damage, or injury (Rock and Kono, 2008), or (2) alarmins, i.e., molecules that possess cytokine-like functions (such as IL-1a and IL-33), which can be stored in cells and released upon cell lysis, whereupon they contribute to the inflammatory response (Oppenheim and Yang, 2005) (Tables 1 and 2). Several DAMPs have been shown to stimulate pattern-recognition receptors (PRRs) (Chen and ˜ ez, 2010) (Table 1). The PRR family consists of Toll-like Nun receptors (TLRs), RIG-I-like receptors (RLRs), nucleotidebinding domain and leucine-rich repeat containing molecules (NLRs), and C-type lectin receptors (CLRs) (Takeuchi and Akira, 2010). PRRs are initial sensors of infection that coordinate the inflammatory response to diseased cells and therefore mediate pathogen elimination. Often, the same receptors recognize both DAMPs and pathogen-associated molecular patterns (PAMPs), indicating the similarities between pathogen-induced responses and inflammatory responses to cell stress, injury, or death (Table 2). Different cellular stimuli (e.g., TNF, Fas ligand, TRAIL ligand, double-stranded RNA (dsRNA), interferon-g (IFN-g), ATP depletion, ischemia-reperfusion injury, and pathogens) have been shown to induce necrosis that follows defined steps and signaling events reminiscent of a cell-death program (Vanlangenakker et al., 2012). This regulated necrosis, also named necroptosis, can be defined as cell death mediated through a pathway that depends on the receptor-interacting protein kinase 1 (RIPK1)-RIPK3 complex (Figure 1) and that can be inhibited by Necrostatin-1 (Nec-1) (Galluzzi et al., 2012). RIP3K can also form complexes with DNA-dependent activator of interferon regulatory factor (DAI) and the adaptor molecule TRIF (TIRdomain-containing adaptor-inducing interferon-b; adaptor protein downstream of TLR3), leading to RIP3K-dependent programmed necrosis (Mocarski et al., 2012). However, these definitions should be interpreted carefully. First, there is no unique biochemical marker for necroptosis. Necroptotic cell death in vitro is confirmed by plasma membrane rupture and by lack of specific apoptotic markers such as caspase activation and chromatin condensation. Second, it has also become clear that RIPK1 kinase activity can participate in apoptosis induction in conditions of cIAP (cellular inhibitors of apoptosis proteins) depletion or inhibition, which can result in FADD-RIPK1caspase-8 ripoptosome complex formation and Nec-1-inhibited apoptosis (Feoktistova et al., 2011; Tenev et al., 2011). Necroptosis can be triggered by members of the tumor necrosis factor (TNF) family (e.g., TNF) (Vercammen et al., 1997), Toll-like receptors (TLR3 and TLR4) (Chan et al., 2003; Holler et al., 2000; Laster et al., 1988), and DNA and/or RNA Immunity 38, February 21, 2013 ª2013 Elsevier Inc. 209 TBKA. necroptosis-induced inflammatory diseases can be seen as sterile process in genetically deficient mouse models. IL1RAcP. except for TLR3.. DNA mRNA LPS HMGB1. 2011). In this scenario.. intestine. which is highly ubiquitylated. 2001). 2009). 2010). 2011. 2009). IL-1 receptor accessory protein. TLRs recruit downstream the MyD88 adaptor (myeloid differentiation protein). HSPs Viral. CrmA. This complex. initiates necroptosis. leading to increased necrosome formation and consequently to necroptosis. HSPs Hyaluronan Biglycan Heparin sulfate U1 snRNP U1 snRNP mtDNA HMGB1 Adaptor PAMPs DAMPs TLR6 TLR7 (endosomal) TLR9 (endosomal) RIG-I DAI CD14/CD40/ CD91 CD24 RAGE DNGR-1 P2Y P2X IL1R1 Myd88 Myd88 Myd88 Diacyl lipoprotein Viral/bacterial ssRNA Viral/bacterial DNA dsRNA dsDNA RNA or DNA sensors MAVS TBK1 TLR4 cofactor/ TRAF6 Siglec Myd88 dsRNA dsDNA HSPs/GP96 HMGB1 HSPs HMGB1 S100B F-actin ATP Myd88 IL1a IL33 DAMPs or alarmins-specific receptors ST2L/ IL1RAcP Myd88 Numerous DAMPs released from cells undergoing accidental necrotic cell death have been described.. it has been proposed as one of the possible regulators of the release of cytokines and alarmins in necroptotic cells (Christofferson et al. The activity of the NAD-dependent deacetylating enzyme SIRT2 (Narayan et al. and Their Receptors Receptor TLRs TLR2 TLR3 (endosomal) TLR4 Myd88 TRIF Myd88/TRIF Lipoprotein HMGB1. E8. RIPK1. We also discuss the role of necroptosis and the associated DAMPs in the induction of inflammation.. which might contribute to cell death and inflammation (Christofferson et al. and LUBAC (Haas et al. or viral inhibitors (vICA. Additionally. RIPK1 and RIPK3 accumulate and become phosphorylated. systemic inflammatory response syndrome (SIRS). DAMPs and PAMPs can be recognized by the same group of PRRs. IL-1 receptor-like 1. This implies that necroptotic cells could release more DAMPs than apoptotic cells. MAVS. or self-nucleic acid.. 2006). Complex II is of two types.. besides RIPK1’s role in the decision of the cell’s life or death. TRIF. Oberst et al. 2012). also called the necrosome (Vandenabeele et al. after the ligand binds to TNFR1. FLIPL. we could define the TNF necroptotic pathway as a process that involves the positive action of CYLD.’’ containing TRADD. including release of DAMPs. and MLKL and negative regulation by caspase-8 and IAPs. RIPK3. In contrast. sensors (DAI and possibly RIG-1 and MDA5). So the inflammation can be attributed (at least partially) to the release of DAMPs. IAPs. Therefore. two complexes with opposing signaling can be formed. DNA or RNA sensors can also recognize bacterial. and ischemia reperfusion injury. 2013 ª2013 Elsevier Inc. The recruitment of RIPK3 to RIPK1 occurs through a homotypic interaction domain (RHIM). and p38 MAPKs. The kinase activity of RIPK1 mediates the production of TNF. TRAF2/5.. etc. viral. Additionally.. HMGN1. SIRT2. for example in skin. resulting in formation of complex IIa (Figure 1). accompanied by the release of the receptor-associated complex from TNFR1 and the recruitment of FADD and procaspase-8. Interestingly... bacterial Ribonucleoproteins. PAMPs. In this review. also called necrosome. TIR-domain containing adaptor-inducing IFN-b. However. caspase-8 is inhibited (CrmA) or genetically deleted. which occurs only after loss of membrane integrity (Brouckaert et al. Abbreviations are as follows: Myd88-Myeloid differentiation primary response gene (88). after TNF triggering. we focus on necroptosis induced by pathogens (bacteria and viruses) and discuss the immunological consequences of these interactions.. RIPK1.. When recruitment of procaspase-8 is prevented (c-FLIPS). 2012). in which potential DAMPs are restrained by the preserved plasma membrane and apoptotic bodies. RIPK1 ubiquitylation controls the switching between prosurvival signaling and apoptotic and/or necroptotic cell death (Figure 1). 2011) and it is facilitated by inhibition or absence of caspase-8 (Kaiser et al. 2012) and might function as an alarmin in myocardial injury (Gallucci and Matzinger. 2008). initiates the activation of NF-kB. ‘‘complex I. ST2L.). TANKbinding kinase 1. whereas complex IIb. What determines the switch from complex IIa to IIb is not clear. The prosurvival complex. TNF-facilitated necroptosis requires inhibition of caspase-8 and assembly of RIPK1-RIPK3 complex IIb. The switch requires the activity of the deubiquitylating enzyme CYLD (O’Donnell et al. February 21. which signals through the TRIF adaptor. remains associated with the TNFR1 receptor (Bertrand et al. IL1R) have also been reported to interact with DAMPs released from accidentally necrotic cells. Necrotic DAMPs. several other receptors that do not recognize specific PAMPs (CD24 and P2X/Y receptors. endosomal internalization of TNFR1 is 210 Immunity 38. JNK. 2004) and with slower kinetics than uptake of apoptotic cells. Relatively few studies have investigated whether necroptosis is accompanied by release of a distinct set of DAMPs and the roles of these DAMPs in necroptosisinduced inflammation. 2009.Immunity Review Table 1. Whereas TNFtriggered apoptosis is mediated by caspases. followed by the mutual phosphorylation of RIPK1 and RIPK3 (Sudan et al. Mixed lineage kinase like protein (MLKL) and the mitochondrial phosphatase 5 (PGAM5) are other RIPK3interacting proteins that are crucial for necroptosis induction (Zhao et al. Zhang et al. It has also been shown in vitro that necroptotic material is coingested with the extracellular fluid by antigen-presenting cells (APCs) via macropinocytosis (Krysko et al. Complex IIa and complex IIb are highly regulated by c-FLIPS. Most studies on DAMPs have been devoted to DAMPs that are released during accidental cell death (Table 2) and by secondary necrotic cells following apoptosis.. 2012) has been implicated in the RIPK1mediated recruitment of RIPK3 and the formation of complex IIb (Figure 1). Briefly. mitochondrial antiviral signaling protein.. complex IIa can trigger apoptosis. this sterile situation differs from . 211 .. IL-8 Necrotic neutrophils but no other cells Y TNF. 2009) F (LN)/ T Coculture with mDCs and T cell activation assay Coculture with resident peritoneal macrophages and i. 2013 ª2013 Elsevier Inc. U-937 Human neutrophils. L5178Y-ML25 lymphoma (Yoon et al. IL-6. 2007) F/T ND (Ayna et al. IL-1b and IL-8 from LPS. injection Coculture with hDCs Coculture with LPSactivated RAW 264. February 21. 2007) (Miles et al... effective anti-tumor vaccine (A).. DC.. [ IL-6. 2012) T24 DO11.. Nlrp3 dependent IL-1a production only (B) no effect of F/T necrotic cells Infiltration of leukocytes neutrophil infiltration. CXCL1 secretion (A) neutrophil influx. IL-6. Lack of TNF and IL-6 modulation Infiltration of neutrophils into peritoneum..activated macrophages Phagocytosis but no T cell activation measured as IFN-g production Lack of IL-1b production. Although most pathogens can activate apoptotic or pyroptotic (inflammasome-mediated) cell death through TLRs or NLRs. Scheffer et al. APC. Immunological Consequences of Accidental Necrotic Cell Death In Vitro and In Vivo Cell line In vitro HeLa (A) ionomycin (B) CCCP (C) deoxy-glucose and sodium azide (D) 3 cycles of freeze/ thaw (F/T) 6 cycles F(LN)/ T 5 cycles of F/T Coculture with bone marrow macrophages (BMDM) HMGB1 [TNF (Scaffidi et al. dendritic cells (m. TNF. (C) lack of protective immune response in tumor vaccination model (El Mezayen et al. some PAMPs and DAMPs in particular cell types and conditions may be associated with necroptosis induction in vitro and in vivo (Figure 2A).10 murine T cells In vivo Bone marrow derived dendritic cells (BMDCs) B16 cells F/T HS 55 C for 25 min ND ND (Garg et al. 2012) B16. 2012) (Cvetanovic and Ucker. in which infection-induced apoptosis or necroptosis can become immunogenic as a result of the simultaneous engagement of PAMPs and DAMPs with PRR. Immunity 38. 2002) Necrosis induction Model DAMPs or Alarmins Immunological Outcome Reference THP-1. 2003).22 primary breast tumor cells 11Ba/F3 cells Coculture with THP-1 Coculture with LPSactivated macrophages HMGB1 and HSP70 ND [TNF. SP815 Tumor vaccination model ND (Goldszmid et al. 2004) 5 cycles F/T IL-1a (Eigenbrod et al. 2008) (A) pressure disruption (B) F/T ATP (Iyer et al. freeze/thaw. TLR4 dependent ND (Rider et al. 2004. human tumor cell line KS24. ND. and inducible nitric oxide synthase Only (A) but not (B) or (C): stimulation of APCs. liquid nitrogen... not defined. In this respect. (Bartholomae et al... HS.. 2011) (Allam et al... Necroptosis and DAMPs Release during Bacterial Infection Recognition of pathogens through PRRs is the first line of defense against microbial infections. antigen presenting cells. h. 2009) BD7 keratinocytes Renal cells hypoxic conditions Histones-induced necrosis (A) heat-killed (B) formaldehyde fixed (C) F/T (A) 4 cycles (B) F/T (C) osmotic shock Injection into the peritoneal cavities Histones injection into renal artery of mice Tumor vaccination model IL1-a Histone 4 TLR2. murine thymocytes. F/T. human). [ IL-12 and TNF.7 macrophages cell line Injection into the peritoneal cavities Injection into the peritoneal cavities ND (Inzkirweli et al. infectious processes. 2003) Abbreviations are as follows: LN. murine.Immunity Review Table 2. 2011) and FADD-deficient Jurkat cells (Kalai et al.. heat shock.. (B). it has been shown that stimulation of TLR3 by dsRNA (poly[I:C]) or TLR4 by LPS in combination with caspase inhibition leads to RIPK3-mediated necroptosis in murine macrophages (He et al. lack of recruitment of neutrophils to the site of injection Lack of DCs maturation Lack of NF-kB activation. L5178Y. 2008) CT26. B16.p. which in turn can recruit RIPK3 via the homotypic interaction motif (RHIM). double-stranded. it can also induce necrosome formation (dashed arrow). In contrast.. when caspase-8 is genetically deleted or inhibited. the ripoptosome activates apoptosis. also called necrosome. MLKL. this leads to ROS production and necroptosis (He et al. Interestingly.The ripoptosome is also formed after TLR4 stimulation by LPS in DCs lacking caspase-8 (h). T cell proliferation following antigen activation requires caspase-8 activation by the CARMA1-BCL-10-MALT1 complex leading to NF-kB activation. and MDA5) and endosomal sensors (TLR3. called death inducing signaling complex (DISC). TLR7-8. can also be formed following stimulation of T cell receptor. For FasL and TRAIL stimulation. MAVS (IPS-1). In the latter case. instead of mediating cell death. suggesting that HMGB1 might be released during TLR4-mediated necroptosis. February 21. caspase-8 activation occurs at the internalized receptor complex. 2002). Cytosolic FADD-RIPK1-caspase-8 complex (further referred to as ripoptosome). RIG-I and MDA5. CYLD (as a substrate of MALT1) probably accumulates. 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). 2011). TRIF interacts with RIPK3 through the RHIM domain.Immunity Review Figure 1. apoptosis (b). TIR domain-containing adaptor-inducing INF-b. DAI-I contains a RHIM domain by which RIPK1 and RIPK3 can be recruited and a necrosome is formed (g). ds. Cytoplasmic (DAI-I. whereas TNF signaling initially forms a receptor associated complex (complex I). Furthermore. TRIF. through the homotypic CARD interaction domain. TNFR1-mediated signaling can lead to cell survival (a). phosphoglyceratemutase 5. 2013 ª2013 Elsevier Inc. DRs. INF. ss.and RIPK3-dependent necroptosis in the absence of cIAPs and inhibition of caspases (Feoktistova . Moreover. However. as well as MLKL and PGAM5. retinoic acid-inducible gene 1. TLR3-agonist can sensitize HaCaT and HeLa cells to RIPK1. death receptors. mitochondrial antiviral-signaling protein. which contains the pronecroptotic molecules RIPK1 and RIPK3. TLR3 or TLR4. Additionally. 2010). Overview of Ripoptosome Formation in Response to Different Stimuli Necroptosis can be induced by various stimuli that are recognized by specific receptors. leading to sensitization of necroptotic signaling in T cells when RIPK3 is present and the necrosome complex is formed (d). as well as in response to genotoxic stress. However. the TLR4 signaling pathway is involved in HMGB1 secretion from macrophages by a mechanism that depends on interleukin-1 (IL-1) receptor-associated kinase 4 (IRAK4) (Wang et al. when TLR3 is engaged. MDA5. followed by a cytosolic complex IIa (b) and complex IIb (c). RIG-I. leading to pro-IL1b production and its processing into active proinflammatory cytokine IL-1. After TNF binds its receptor. interferon. recruit the MAVS adaptor on mitochondria. single-stranded. contributes to NALPR3 inflammsome formation and activation. but it cannot be ruled out that in specific conditions of caspase-8 inactivation.. mixed lineage kinase domain-like protein. Stable complex IIb. PGAM5. which can further bind RIPK1 and possibly enable recruitment of RIPK3 (dashed arrows). this ripoptosome. which leads to the production of IFN-b involved in antiviral response (f). and TLR9) of nucleic acids activate IRFs and NF-kB. or necroptosis (c). 212 Immunity 38. thereby bridging TLR signaling to the necrosome (e). melanoma differentiation-associated protein 5. RIPK1 can also mediate proinflammatory signaling by activating the transcription factor NF-kB or AP-1-SP1 (f). RIG-I. TRIF (adaptor of TLR3 and TLR4) can recruit RIPK1. Additionally. Lack of PLA2 cleavage might prevent the release of ‘‘find-me’’ signals from necroptotic cells. the ERassociated molecule PERK is involved in the release of the ‘‘eat-me’’ signals ATP and CRT. cytokines. inhibitor of caspase activated DNase. E3. (B) Stimulation of necroptosis by viral infection.DAI -directly engages RIPK1 and RIPK3 in RHIM-dependent complexes and drives the assembly a ripoptosome (j). Viral inhibitors of caspase-8 are produced by many viruses (e. an ‘‘eat-me’’ signal. Binding of IFNs to IFNR activates the JAK-STAT pathway (d) leading to IFNs production and upregulation of Ripk1 and possibly also Hmgb1. Pathogen-Mediated Necroptosis and Possible Modifications of DAMPs (A) Stimulation of necroptosis by bacterial PAMPs. Damaged mitochondria (and insufficient mitophagy) are source of ROS production. CAD. BORFE2. NLRs. BHV-4 virus. Absence of caspases. which we speculate can be abrogated during necroptosis. The absence of active caspase-8 sensitizes an alternative cell death pathway: necroptosis (g). Also. and most likely. 2013 ª2013 Elsevier Inc. viral proteins such as NS1 and A52R have been shown to blunt PRRs signaling by inhibiting the binding and sequestration of nucleic acids. which are necessary for DNA fragmentation. LPC. February 21. ICAD. BORFE2. CrmA. phospholipase A2.g. (C) Release of DAMPs during necroptosis and their potential modifications. LMP. Until now. caspase-activated DNase. Caspase activity was reported to induce high levels of ROS production during apoptosis. typhimurium infection. vICA. Additionally. cFLIP homologs: E8. Immunity 38. ripoptosome formation involves TLR3 signaling and TRIF–RIPK1-RIPK3 interaction (i). long genomic DNA. Caspases also participate in the generation of the ‘‘findme’’ signal. 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. dashed arrow) to trigger necroptotic cell death when caspase-8 is inhibited or genetically ablated. NOD-like receptors. caspase-8 inhibitor. by cleavage of PLA2. and other mitochondrial molecules. ROS. lysosomal membrane permeabilization. Caspase-3 and -7 can process full-length proinflammatory IL-33 (IL-33FL) into an inactive form. cytokine response modifier A. which becomes desensitized for PAMP triggering by the action of a phosphatase (SHP-1) (f). murine cytomegalovirus.Immunity Review Figure 2. thereby providing RIPK1 molecules for necroptosome formation (e) and resulting in HMGB1 release from dying cells. HMGB1. but no specific necroptotic DAMPs have been identified. viral protein M45 has been shown to prevent ripoptosome formation by binding to the RHIM domain of RIPK3. Activity of caspase-8 leads also to production of CRT. absence of caspase activation during necroptosis might modify the properties of the released DAMPs as compared to apoptosis. The cytosolic sensor of foreign DNA. might induce the release of long genomic DNA (LG DNA). viral inhibitor of caspase activation. PRRs can discriminate between PAMPs and DAMPs through CD24-SiglacG/10 signaling. high mobility group box 1 protein. Bacterial products can stimulate TLR3 and À4 (also possibly TLR9. DAMPs and PAMPs become indistinguishable during infection due to removal of sialic acid residues from CD24 and both trigger inflammation (a). Additionally. adenovirus. nuclei containing HMGB1. However. MCMV. However. RIPK1-RIPK3-dependent necroptosis has only been observed in response to MCMV and VV viruses. So far. vFLIPs.caspase-8 inhibitor. several DAMPs have been associated with necroptotic conditions (C. K13. caspase-8 inhibitors: CrmA. stimulation of TLRs and NLRs by PAMPs can provoke inflammatory responses in infected cells (a) and interferons production (b). vICA. inhibiting necroptosis and inflammation (k). Many DAMPs released from accidental necrotic cells have been described (Table 2). viral FLIPs. reactive oxygen species. leading to recruitment of RIPK3 and necrosome formation (c). Table 3). mitochondrial DNA (mtDNA). Additionally. MA159) and effectively block apoptosis. 213 . PLA2. CD24 receptor and SiglecG/10 can bind to DAMPs but not to PAMPs. resulting in the oxidation of HMGB1 and leading to the production of its immunologically silent form. IFN type I receptor (IFNR) engages RIPK1. and histones have been observed in necrotic tissues. This brings CD24-SiglacG/10 close to TLR. During S. 2011). This mechanism may explain the increased inflammation in diseases associated with bacterial stimulation. viruses have evolved into having many specific caspase8 inhibitors. 2012). Moreover.. and TNF-induced SIRS has been associated with RIPK3-mediated necroptosis (Duprez et al. 2012).. increasing the interaction between TLRs and DAMPs (Chen et al. in both studies the authors showed that necrotic cell death was associated with the release of HMGB1 (Figure 2). 2012). it was . Although the above-mentioned studies were conducted using synthetic ligands or bacterial products to stimulate TLRs. organs such as skin. Indeed. so they are more vulnerable to conditions of danger and/or damage and permanent microbial stimulation.. IL-1a and IL-1b. stimulation of TLR9 by unmethylated CpG triggers caspase-independent cell death of pro-B cells (Lalanne et al. 2009).. However. and IFN-g (Robinson et al. the sialylation status of CD24 determines whether or not the DAMP repression system will operate.. No DAMPs were analyzed in this study. and it was accompanied by the secretion of KC (a neutrophil-attracting chemokine) into the supernatant of infected mouse embryonic fibroblasts (MEF). viral inhibitor of caspase activation (v-ICA). 2009).. Whether microbial 214 Immunity 38. Elevated levels of sialidases lead to decreased binding of CD24 with Siglec G/10. 2012). 2010). the host’s endogenous sialidases released during necroptotic cell death together with bacterial sialidases could sensitize TLRs and NLRs to DAMPs and lead to exacerbation of inflammation in sepsis (SIRS). The crosstalk between PAMP and DAMP recognition and signaling could be an important issue in the etiology of sepsis (SIRS). leading to necroptosis of macrophages accompanied by release of proinflammatory cytokines such as TNF. Injection of wild-type (WT) mice with LPS or poly(I:C) combined with zVAD-fmk. In vitro and in vivo analysis showed that Salmonella induces type I interferondependent assembly of RIPK1 and/or RIPK3.. while some viruses encode suppressors of cell death to counteract these protective mechanisms.. and the DAMPs-CD24-Siglec G/10 complex enables phosphatases such as SHP-1 to repress TLR and NLR signaling. Removal of sialic acid residues from CD24 prevents the formation of the DAMP-CD24-Siglec-G/10 complex. Epithelial cells infected with Shigella die by necrosis.. MCP-1. 2008) and for CD4+ and CD8+ T cellmediated antiviral responses (Duttagupta et al. and autoimmune diseases. several models of bacterial infections have also been shown to induce necroptosis in host immune and nonmyeloid cells in vitro. This cell death was not due to endogenous TNF production or TNFR1 stimulation. February 21. which determines whether or not the TLRs and/or NLRs associated with CD24 will induce inflammation when exposed to DAMPs (Chen et al. Interestingly. 2011). RIPK1and/or RIPK3-dependent necroptosis is observed during Salmonella typhimurium infection (Figure 2A). respectively. 2010) and Legionella pneumophila (Kennedy et al. such as cytokine response modifier A (CrmA). 2009). Death of macrophages and circulating cytokine amounts were both markedly inhibited when LPS and zVAD-fmk or poly(I:C) and zVAD-fmk was injected into RIPK3-deficient mice or dominant-negative mutant TrifLPS2/LPS2 mice. 2003). TNF. 2009) investigated the role of the IFN-b signaling pathway in HMGB1 release and found that the IFN-b and JAK-STAT signaling pathways are critical for HMBG1 release during stimulation of TLR4 by E. such as sepsis (SIRS). suggesting that type I IFN can engage the necroptosis pathway independently of TNF (Robinson et al. Further studies are required to analyze whether this type of caspase-independent cell death induced by bacteria (Kennedy et al. It is important to know whether these in vitro studies can be extrapolated to the in vivo situation and to understand whether the released HMGB1 (and very likely also other DAMPs) could contribute to the inflammatory response instigated by the necroptosis induced by PAMP and bacteria.Immunity Review et al.... 2012). In addition. Recent studies revealed that bacterial sialidases promote inflammation in the cecal ligation and puncture (CLP) model of sepsis. as determined by double propidium iodide and Annexin V positivity and absence of TUNEL-positive staining (Carneiro et al. TLR-mediated necroptosis could also be induced in vivo and might lead to inflammation. These observations collectively show that bacterial infection could activate TLRs and lead to production of IFNs. 2010) is dependent on RIPK1 and/or RIPK3 and therefore could be considered as necroptosis.. demonstrating that both programmed cell death mechanisms are crucial for host defense (Figure 2B). HSP70.. Importantly. stimulation of macrophages with TNF and LPS can activate the host’s cytoplasmic sialidases (Gee et al. Siglec-G/10 belongs to a family of immunoglobulin-like lectins that can recognize sialic-acid-containing structures present on CD24. intestine. Initially. 2013 ª2013 Elsevier Inc. leads to necroptotic cell death of macrophages and increases serum concentrations of pro-inflammatory cytokines (IL-6. 2011). which in an autocrine or paracrine way could activate the necroptotic pathway and contribute to HMBG1 release (Figure 2A) (Robinson et al.. 2009. Viruses suppress not only the apoptotic pathway but also the necroptotic pathway.. It has been shown that the a-toxins of Clostridium septicum (Morinaga et al.. and lungs are constantly exposed to both commensals and pathogens. 2011). IFN-g) (He et al.. Therefore.. inflammatory bowel disease. Viruses: Master Regulators of Cell Death and DAMPs Release? Viral spread depends on the fate of infected cells. but another group (Kim et al. a pan-caspase inhibitor. This cell death could not be inhibited by zVAD-fmk. and viral FLICE-like inhibitory proteins (FLIPs) (Mocarski et al. Indeed. several microbes and viruses possess sialidases as a virulence factor (Chan et al. 2011) (Figure 2A). pointing to the involvement of RIPK3 and TRIF in TLR-mediated necroptosis (He et al.. 2009). The DAMP-PAMP crosstalk is regulated by the CD24-SiglecG/ 10 axis (Chen et al.. 2009). Morinaga et al. whether TLR9-induced cell death involves RIPK1 and RIPK3 remains to be examined. 2009) induce necrotic-like cell death in macrophages and myoblasts.. and HSP90 interact directly with CD24 (Chen et al. asthma. resulting in increased inflammatory responses to TLRs and NLRs during PAMP and DAMPs stimulation. KC. Caspase-8 is essential for T cell proliferation after antigen stimulation (Ch’en et al.. Moreover. coli (Kim et al. infections can modify the immunological responses to DAMPs should also be investigated (Figure 2).. DAMPs such as HMGB1. 2011). we come to a paradoxical function of caspase-8. The interactions between viruses and eukaryotic cells have led to selection of eukaryotic genes that propagate cell death upon infection to block viral multiplication. Here. Interestingly. Although these viruses have not been evaluated in RIP3K-deficient mice.. including WNV.. depending on the virus titer. and so it can efficiently block both apoptosis and necroptosis. evaluation of RIP3Kdeficient mice has shown that necroptosis plays an important role in the control of vaccinia virus (VV) (Cho et al. MCMV can efficiently block necroptosis. especially that the type of cell death could often depend on virus titer (Chu and Ng.g... Ong et al.. probably by cleaving RIPK1. 2009. necroptosis can be viewed as a ‘‘trap door’’ that opens when caspase-8 is inhibited and it is involved in host defense (Mocarski et al. Activation of regulated necrosis is a mechanism for combating MCMV in the absence of caspase-8 activity... 2009) but is dispensable for the control of WT murine cytomegalovirus (MCMV) (Upton et al. 2012). showed that MCMV-induced RIPK3-dependent programmed necrosis is a major host defense pathway that can be effectively blocked by the viral RIPK3 inhibitor.. protein A52R expressed by VV inhibits the signaling mediated by TLR2. Necrotic morphology could be easily mistaken for secondary necrosis. 2008. Infection of WT mice with VV induces tissue necroptosis and infiltration of neutrophils into visceral fat pads. dengue virus (DV) induces either apoptotic or necrotic cell death. Indeed. 2010). 2009). and increases the risk of development of dengue shock syndrome (Ong et al. The amount of HMGB1 in the extracellular environment increased and was correlated with higher multiplicity of infection and a shift from apoptotic to necrotic cell death (Ong et al.. and FADD negatively control necroptosis induction. This study strongly suggests that DAI functions as a PRR in the pathway of RIPK3-dependent necroptosis induced by MCMV. 2011). virus infection can be accompanied by emission of DAMPs such as Hsp70 and ´ n et al. À4. 2013 ª2013 Elsevier Inc. More work is required to identify DAMPs emitted from virus-induced necroptotic cells and to understand their roles in induction of inflammation. elevated levels of the proinflammatory cytokines TNF and IL-6 were observed upon infection (Kamau et al. undergo necrotic-like. and À9. The FADD-Caspase-8 Platform and Autophagy as Mechanisms to Regulate Necroptosis in T Cells Several reports indicate that necroptosis could regulate T cell number in peripheral tissues during T cell development. DNA-dependent activator of IRFs (DAI) was identified as an essential partner of RIPK3 in MCMVinduced programmed necrosis in vitro and in vivo (Upton et al. This protein possesses a homotypic interaction domain RHIM. 2011. Mice lacking caspase-8 or mice expressing mutant FADD unable to recruit caspase-8 (FADD DED deficient. This indicates that RIPK3 is required for protection against VV (Cho et al. 2001) and a RIPK3 inhibitor (Upton et al. But through which receptor-adaptor system do viruses engage host RIPK3-mediated necroptosis? Recently. and CYLD (Dillon et al.. HMGB1 acts on endothelial cells. FADDdd) (Lu et al. it is unclear whether these phenomena at higher virus titers are due to necroptosis. Conspicuously. MCMV) have evolved strategies to counteract several death pathways. Removal of activated T lymphocytes that had undergone clonal expansion in response to stimulation (infection) is essential for maintenance Immunity 38... This reduced inflammation correlated with increased viral titers in the organs of RIPK3-deficient mice. mouse hepatitis virus (Lu et al. Kamau et al. indicating that RIPK3-driven necroptosis is responsible for clearance of these lymphocytes during their development (Ch’en et al. mitochondria-dependent cell death (Petit et al. 215 . and lymphocytic choriomeningitis virus (LCMV) (Ch’en et al. have been shown to induce necroticlike cell death. recently it became clear that caspase-8. 2011a). Several other viruses. but these events are significantly decreased in RIPK3-deficient animals (Cho et al. 2009). HIV-1-infected T cell lines. 2003)... Kaiser et al. RIPK3. arguing for case-specific strategies in host-virus immune responses. Once in the extracellular milieu. West Nile virus (WNV) nonstructural protein 1 (NS1) inhibits TLR3 signaling. these mice were more vulnerable to the infection. 2012). and this was correlated with excessive cell death (Salmena et al. 2011). However.. and consequently.. 2012)... which could indicate emission of DAMPs from necroptotic cells. 2011) or some other target(s) downstream of RIP3 kinase. 2003)... and other viral proteins sequester virus dsRNA (Aravalli et al. HSV type 1 (HSV-1) can inhibit apoptosis and elicit necroptosis in vitro.. O’Donnell et al. M45 (Figure 2B). Concomitant research in double deficient caspase-8-RIPK3 and FADDdd-RIPK3 T cells revealed accumulation of excessive and abnormal lymphocytes (lymphadenopathy) in these mice. 2012). the authors reported that the inflammatory foci were concentrated in areas of extensive necroptosis in WT mice. this could be a way to control its own pathogenicity or to modulate the release of DAMPs. Upton et al. HMGB1 (Lietze again as a result of coevolutionary selection... 2011. HIV type 1 (HIV-1).. Interestingly. induces vascular leakage. Wheeler et al. infection with DV causes translocation of HMGB1 from the nucleus to the cytoplasm and its active secretion from live infected cells and passive release from dying cells (Chen et al. MCMV encodes both an inhibitor of caspase-8 (Skaletskaya et al. 2010). February 21. This would mean that on the one hand inhibition of caspase-8 prevents apoptosis induction while on the other hand the cell would be sensitized to necroptosis induction. Ong et al. FLIPL. For example.. necroptosis has been implicated in the elimination of excessive T cells after activation (contraction of lymphocytes). Consequently. 2008. 2010). Additionally. 2012. However. reported that active release of HMGB1 is mediated by virus capsid protein. which enables it to bind RIPK3.. However. 2011). 2002). 2009). But why would viruses paradoxically activate an alternative cell death program? For the virus.Immunity Review thought that caspase-8 inhibition is only a way to prevent induction of apoptosis to allow the virus to propagate in the infected cell. 2012). but strains lacking M45 or containing a mutation in its RHIM domain induce regulated necrosis that is dependent on RIPK3 but not on RIPK1 (Kaiser et al. In addition. 2009). some viruses can ‘‘switch off’’ the emission of DAMPs or blunt the interaction of TLRs with DAMPs and/or PAMPs to prevent an immune reaction.. this cell death could be partially blocked by the RIPK1 kinase inhibitor Nec-1 and cathepsin inhibitors (Peri et al. Upton et al... in the presence of the caspase inhibitor zVAD-fmk. For the host. Additionally. 2008) (Figure 2B).. In this regard. and herpes simplex virus (HSV). 2003). 2011. 2011b) in T cells had fewer CD4+ and CD8+T cells in the periphery. Bowie and Unterholzner. Some viruses (e. which triggers acetylation and secretion of HMGB1. cells infected in vitro with WNV develop a necrotic morphology followed by HMGB1 release (Chu and Ng. because the involvement of RIPK1 and/or RIPK3 was not analyzed in these studies. However. Another aspect of the regulation of necroptosis by autophagy deals with the important role of mitophagy in removing damaged mitochondria (Youle and Narendra. as discussed above. which contributes to necroptosis. or Beclin-1 (He et al.. Similarly. such as autophagy. T lymphocyte proliferation is impaired in the absence of Atg5. February 21. 2011). these authors observed upregulation of DAMPs such as S100a9 and IL-33 (Kovalenko et al. Wang et al. Cerulein-induced acute pancreatitis resulted in necroptotic cell death of pancreas accompanied by overexpression of RIPK3 in pancreas but not in other organs (Sudan et al. interference with the mechanism responsible for the removal of damaged mitochondria could sensitize for necroptosis. 2009). whereas excessive HMGB1 observed in epidermis of FADDdeficient mice acts as a DAMP that triggers inflammation through TLRs (Bonnet et al. Although the involvement of necroptosis was not investigated in this model. Vesicles containing necroptotic material might deliver DAMPs to APCs to initiate an immune response. Ch’en et al. However. Also other mechanisms. 2011b). RIPK3-mediated necroptosis promotes death of ‘‘immunologically useless’’ T cells unable to mount proper immune responses to pathogens. 2008. . However. 2003) or FADD (Osborn et al.and caspase-8-deficient lymphocytes after TCR stimulation (Bell et al. However. It has been proposed that the inability of T cells to accumulate after activation was due to extensive cell death... in physiological conditions the FADDcaspase-8 platform acts to prevent necroptosis because caspase-8. Defective expansion of T cells after TCR activation was observed in T cells deficient in caspase-8 (Ch’en et al.... 2010) as well as in FADDdd-expressing T cells (Lu et al. On the other hand.. these authors suggested that RIPK1-driven necroptosis might occur naturally during the T cell development because Nec1 treatment enabled wild-type T cells to proliferate with a higher rate than the control cells (Osborn et al.. The Silent and Proinflammatory Faces of Necroptosis It is well established that apoptotic cell death can switch to necroptosis under certain conditions. 2011a). it is also conceivable that in other pathological conditions FADD and/or caspase-8 could be downregulated revealing necroptotic signaling. Further evidence linking RIPK1 and RIPK3-mediated necroptosis with inflammation arose from studies on mice with FADD and caspase-8 deficiency in keratinocytes. investigation of necroptotic cell death in mouse models discloses the proinflammatory face of this cell death mode. 2012). 2011). 2006). 1998). 2009). recently showed a connection between RIPK3 kinase activity and the MLKL platform.. Similarly. This interaction results in mitochondrial fission and fragmentation (Wang et al.. 2011).. TNF produced in response to TLR-driven MyD88 signaling amplify TNF induced necroptosis.. used Nec1 to inhibit RIPK1 activity and discovered that the proliferative capacity of FADD-deficient T cells was restored when the RIPK1 kinase activity was abolished. Taken together. 2011). The molecular mechanisms of this interplay between mitophagy and necroptosis are currently investigated. Treatment with Nec1 in these models not only prevented cell death but apparently also reduced the extent of autophagy. Interestingly. these results should be interpreted carefully because conflicting findings have been reported on autophagy and its crosstalk with apoptosis and necroptosis (Long and Ryan. In this respect. 2011). demonstrating an important role of RIPK3-dependant necroptosis in inflammation (Bonnet et al. In that respect. indicating that necroptosis can be activated when active capase-8 is not available. Vanlangenakker et al. We have discussed this sensitization of necroptotic cell death by CrmA overexpression already in 1998: ‘‘Elimination of such deficient but oxygen radical-producing mitochondria 216 Immunity 38. this was not observed in animals double-deficient in FADD and RIPK3.. the T cell accumulation defect of caspase-8 deficient and FADDdd expressing T cells was restored by RIPK3 deletion (Lu et al. RIPK1 has been proposed to be a linker between necroptotic cell death and autophagy in activated T cells. 2008).. the effect of necroptotic cell death on immune responses in vitro and in vivo has been barely studied. Krysko et al... 2013 ª2013 Elsevier Inc. A hyperautophagic phenotype has been observed in FADDdd. it seems that when caspase-8 is absent. were unable to induce maturation of dendritic cells (DCs) (Lohmann et al.. additional deletion of RIPK3 in both cases restored the proper immune responses. This mouse fibrosarcoma cell line is commonly used as an in vitro model of necroptosis (Vanden Berghe et al. 2010). 2010.. because these cells lack cytotoxic activity. However. Mice lacking caspase-8 or expressing FADDdd in T cells are also unable to mount a proper immune response when infected with murine hepatitis virus (MHV). should then be beneficial for the cell to survive the deadly TNF signal’’ (Vercammen et al. 2011. L929 cells subjected to TNF-induced necroptosis do not induce proinflammatory cytokine production in macrophages. which can be rescue by ablation of RIPK3 (Dillon et al. Moreover. 2012). B16 mouse melanoma cells.. Atg3. because they are an important source of ROS. 2012). leading to T cell depletion in response to stimulation. Osborn et al. Studies on FADD-TNFR1 and FADD-MyD88 deficiency revealed that both TNF and TLR signaling partially contribute to progression of inflammation. which may act as DAMPs modifiers. 2003. in which necroptosis was triggered by inducible expression of FADD-death domain. decreased mitophagy and increased rupture of mitochondria may enable the release of mitochondrial DNA (or mitochondrial proteins) that serve as DAMPs and promote ROS production. Atg7. Indeed. A similar phenotype of skin inflammation was reported in mice lacking caspase-8 in keratinocytes. to the best of our knowledge the only instance in which caspase-8 could be naturally inhibited would be during infection by a virus encoding caspase inhibitors. which recruits complex IIb to mitochondria. Salmena et al. because its deregulation can lead to immunodeficiency or autoimmunity. 2012).. may control the sensitivity to necroptosis (Bray et al.. Kaiser et al. Indeed.. Necroptotic material can be coingested in vitro by endocytosis-macropinocytosis (Krysko et al.and FADD-deficiency leads to the embryonic death of mice. 2012.. which could indicate the involvement of increased necroptosis in inflammation.Immunity Review of T cell homeostasis. 2009). Mice with epidermisspecific FADD deletion developed skin inflammation due to spontaneous necroptosis of keratinocytes and intense release of HMGB1. These observations can be interpreted in two ways: either coincubation of necroptotic cells with macrophages does not reflect the proinflammatory capacity of necropotic cells or necroptotic cells poses anti-inflammatory properties in vitro. Therefore. However. 2011) (Linkermann et al. AST. IL-6 and damage markers (HEX.. 2009. ALT... Indeed. IL-6. Welz et al. 2010) (Bonnet et al. mtDNA can activate TLR9 and was identified as a major DAMP contributing to inflammatory responses in trauma patients developing sepsis (clinical SIRS) (Zhang et al. Samples collected from patients showed strong RIPK3 expression ¨ nther et al. Additionally. but illustrating the effects of necroptotic cell death on the innate immune system. leading to decreased expression of antimicrobial peptides and sensitizing terminal ileum and colon to inflammation. Release of mitochondrial DAMPs (Krysko et al. necroptosis of Paneth cells in the terminal ileum was connected with the pathogenesis of inflammatory bowel disease. 2012) (Wu et al. 2011) (Table 3).. Interestingly.. suggesting that this cell type might be highly suscep¨ nther et al. Similarly. 2013 ª2013 Elsevier Inc. As already mentioned.. creatinine) Neutrophils and macrophages influx. ROS.. Double-deficient FADD-RIPK3 mice showed no signs of inflammation. 2011). LDH. 2011). but this was revers¨ nther et al. [ circulating damage markers (urea. in both cases of intestinal inflammation.. aspartate transaminase.. which is produced in response to TLR-driven MyD88 signaling. suggesting that necroptosis can contri(Gu bute to the development of inflammatory disorders. creatine kinase. Gu (IECs) induces chronic inflammation characterized by extensive epithelial cell death. 2011) SIRS Neonatal Brain Injury Kidney Ischemia Reperfusion Myocardial Ischemia Trauma patients Hypoxia–ischemia ND RIPK1/RIPK3 mediated RIPK1 mediated Presence of mtDNA in serum ND but TLR2 deficiency partially prevented brain damage HMGB1-TLR4 mediated inflammation ND but TLR4 deficiency protects from myocardial I/R (Zhang et al. Paneth cells were fewer or absent... 2010) (Oerlemans et al. suggesting that cell death in the epithelium may be due to necroptosis.. CK) Systemic inflammation. 2010) (Northington et al. (2011) proposed that a combination of several DAMPs released by necroptotic cells in this model might synergistically trigger sustained cytokine release and amplify the inflammatory response. 2012) (Fallach et al. IL-33 ND but MyD88 deficiency delayed onset of inflammation ND References (Kovalenko et al.. [ IL-1a. Gu 8-deficient Paneth cells are sensitive to RIPK1-RIPK3-mediated necroptosis. it was recently shown that RIPK3-dependent necroptosis causes mortality in mice subjected to experimental TNF-induced systemic inflammatory response syndrome (SIRS) and CLP.. susceptibility to experimental colitis Chronic intestinal inflammation Immune Response Systemic inflammation... 2011) Mode of Induction TNF induced References (Duprez et al. 2011) (Gu ND but MyD88 deficiency partially protected from colon inflammation DAMPs Presence of mtDNA in serum (Welz et al. February 21.. In this regard. the implication of TLR could be partially due to stimulation of TNFR1 by TNF. 2010.. Only recently. suggesting that TLR stimulation can accelerate inflammation (Welz et al. HEX. Stridh et al.Immunity Review Table 3. and high levels of RIPK1 and RIPK3 messenger RNA (mRNA) were observed in the intestine of caspase-8-deficient mice.. patches of necrosis and acute inflammation in the gut were described 30 years ago in patients with Crohn’s disease (Dourmashkin et al. 2011) or caspase-8 (Bonnet ¨ nther et al. [ IL-1b. colon of mice lacking both FADD and MyD88 are at least partially protected from inflammation. ileal biopsies from control patients showed Paneth cell loss in the presence of high levels of exogenous TNF. lactate dehydrogenase. 2011) Caspase-8 deletion FADD deletion RIPK1/RIPK3 mediated RIPK3/CYLD mediated Necroptosis RIPK1/RIPK3 mediated Spontaneous ileitis. In a totally different context. Additionally. Duprez et al.. IL-12p35. IL-8. (2011) showed that caspasetible to necroptosis. LDH. ible by coincubation with necrostatin-1 (Gu Therefore. 2011. showed that FADD-deficient Paneth cells are susceptible to RIPK3-mediated necroptosis and that their production of antimicrobial peptides is impaired.. Li et al. hexosaminidase.. ALT. reactive oxygen species. 1983). not defined. 2011) (Rosenbaum et al. TNF Brain damage. Specific deletion of FADD (Welz et al.. [ IL-6. [ LDH and ROS ¨ nther et al. 217 . 2010). 2011) in intestinal epithelial cells et al. alanine transaminase. Necroptotic DAMPs in Transgenic Mice and Inflammatory Disease Models Cell Type Epidermal keratinocytes Epidermal keratinocytes Intestinal Epithelial Cells Intestinal Epithelial Cells Disease SIRS Mouse Model Caspase-8 deletion FADD deletion Necroptosis ND RIPK3 mediated Immune Response Skin inflammation Skin inflammation DAMPs [ S100a9. 2011) such as mitochondrial DNA (mtDNA) into plasma and necroptotic cell death were reduced in RIPK3 knockout mice. 2010) Ischemia Ischemia RIPK1/MLKL mediated Abbreviations are as follows: ND. 2011).induced peritoneal sepsis (Duprez et al. AST.. Immunity 38. CK. TNF-induced necroptosis of Paneth cells might be one of the key events in the pathogenesis of intestinal bowel diseases. and TNF Kidney damage. However. Caspase activity is also thought to be responsible for modification of the immunogenic DAMP. the compositions and signatures of apoptotic and necroptotic DAMPs might be qualitatively and quantitatively different. leading to excessive production of IL-1b. which might have weaker immunostimulatory properties than high molecular weight DNA (Martin et al. 2012) sheds new light on inflammatory diseases in transgenic mice lacking caspase-8. and natural killer cells than in other cell types (BioGps. Wallach et al.. Therefore mortality and inflammation (as evaluated by increased levels of IL-1b and TNF in serum) observed in transgenic mice lacking caspase-8 in DCs occur probably independently of necroptosis.org). rather than necroptosis itself. 2010). Distinct biochemical processes occur during apoptosis and necroptosis. Insufficient autophagy of these deteriorated .. However. 2011). 2008. and DAMPs. which are involved in necroptosis. nuclear fragmentation.. monocytes. 2003). 2012.. and rapid and efficient phagocytosis (Vanden Berghe et al. Zhang et al. through its ability to induce ROS formation. enhanced RIPK3 expression could mediate the switch from apoptotic to necroptotic cell death and exacerbate inflammation. 2009). full-length IL-33 is released during necrosis. 2008).. Therefore. suggesting that inflammation in IR models could be mediated at least partially by DAMPs.. 2010. In apoptosis we see caspase activation. it has been shown that necroptosis contributes to neuronal damage in a retina IR model (Rosenbaum et al. absence of their activity during necroptosis might modulate the immunogenicity of released DAMPs (Martin et al. inflammation. inflammasome activation occurred independently of necroptosis. including plasma membrane. 2012). same cells. but it cannot be ruled out that different cells or cell types contribute by proinflammatory cytokine production through activation of the inflammasome. Linkermann et al. But in pathological conditions. Furthermore.. lack of caspase-8 apparently also strongly activates the NLRP3 inflammasome in LPS-stimulated DCs. Altogether. lysosomal leakage. 2012). Expression of RIPK3 mRNA is stronger in lymphocytes. such as calreticulin (CRT) (Panaretakis et al. into the nonimmunogenic oxidized HMGB1 form. that propagates immune responses or inflammasome activation (Vince et al. Therefore. and caspase-dependent condensation of chromatin traps HMBG1 during apoptosis (Kazama et al. All these data strongly suggest that in vivo necroptosis is associated with an inflammatory reaction (Table 3). Therefore. full-length IL-33 released in necroptotic conditions (Kovalenko et al.. In necroptosis we observe no massive caspase activation. but during apoptosis it can undergo caspase¨ thi dependent proteolysis into a nonimmunological form (Lu et al. ER. restriction of caspase-8 is associated with skin and intestinal inflammation and excessive activation of RIPK1 and RIPK3. As discussed above. contained apoptotic bodies. 2004).Immunity Review The contribution of RIPK1-dependent necroptosis to multiple organ failure has also been observed in models of ischemiareperfusion (IR). showed that RIPK1-mediated necroptosis occurs during kidney ischemia-reperfusion injury and that it can be rescued by Nec-1 inhibitor (Linkermann et al.. Taylor et al. increased phosphorylation of RIPK1 and RIPK3 was observed during myocardial IR. 2012). 2012). 2013 ª2013 Elsevier Inc.. caspase-8 was inactivated by chemical inhibition or genetic deletion. a recent discovery (Kang et al. Similarly. However. Additionally. active caspase-8 is an important mediator of the emission of DAMPs. chromatin condensation. DNA cleavage... but on the other hand it might also restrict or at least modify the emission of DAMPs during necroptosis. both inflammasome activation and necroptosis initiation mutually affect the inflammatory outcome. it is possible that only limited types of cells undergo necroptosis in normal conditions. This crosstalk could occur either in the 218 Immunity 38.... it seems that RIPK3 drives necroptosis whereas RIPK1 is involved in specific cellular contexts. In addition. but necroptosis can also occur in the absence of RIPK1 (Upton et al. 2009) (when caspases are not active) could be one of the necroptotic DAMPs (Figure 2C). these studies again place caspase-8 in a central position as an important brake on inflammation that acts either by controlling inflammasome activation or by controlling necroptosis. whereas other cells or cell types contribute to DAMP release through the initiation of necroptosis. it is important to point out that this ablation of caspase-8 in experimental conditions on the one hand sensitizes cells or mouse models to necroptosis. intact nuclei. Therefore. Kang et al. caspases cleave phospholipase A2 (PLA2) to produce the ‘‘find-me’’ signal lysophatidylcholine (LPC). 2012. 2010) and DAMPs release (Krysko et al. Therefore. which enables phagocytosis (Lauber et al. mitochondria play an important role in necroptotic cell death (Vandenabeele et al. spillage of cell content. and mitochondria. http://biogps. This inflammasome activation depends probably on RIPK1-RIPK3 mediated mitochondrial ROS production (Vince et al. At a certain point. Surprisingly. and Nec-1 prevented necroptotic cell death and inflammation (infiltration of neutrophils) (Oerlemans et al. it has been proposed that TLR signaling contributes to the severity of inflammation in IR models. Both RIPK1 and RIPK3 are required for necroptosis initiation (Figure 1). 2012). 2010). and an oxidative burst (Vanden Berghe et al. 2010) and that TLR4 deficiency reduces myocardial IR (Oyama et al.. Taken together. 2010) and in neonatal brain injury (Chavez-Valdez et al. nucleus. Hence.. as mentioned above. there is only limited knowledge of the molecular mechanisms involved and the role of the proinflammatory DAMPs emitted during necroptosis... Because caspases are intimately engaged in apoptosis. February 21.. Moreover. 2009)... 2012) (Figure 2C). it has been shown that TLR4 mediates exacerbation of kidney IR via HMGB1 (Wu et al. extracellular caspases... In this regard. in most of the research conducted on necroptosis. although this proinflammatory pathway involved signaling proteins engaged in initiation of necroptosis. inflammation observed in these transgenic mice has been associated with proinflammatory DAMPs released during this cell death. that function as an ‘‘eat me’’ signal for DCs and is required for immunogenic responses in antitumor treatment (Krysko et al. these data point to important crosstalk between necroptosis. 2012). In addition. In apoptosis. 2012). Recent studies emphasize that it is RIPK3. RIPK3 expression depends on the cell type. and caspase-activated DNase (called CAD) is responsible for internucleosmal cleavage of genomic DNA. cytosol. Interestingly. HMGB1. no chromatin condensation.. Distinct Biochemical Pathways of Apoptosis and Necroptosis Lead to Different DAMPs Signatures DAMPs can originate from any compartment of injured or damaged cells. 2009). On the other hand. phagocytosis by macropinocytosis.. For example. 2009). IL-33 (Carriere et al. First. In this regard. a necroptotic environment is expected to be enriched in DAMPs. 2005). intact nuclei have been detected during necroptosis in the extracellular milieu (Krysko et al. T cells lacking caspase-8 in a C57BL/6 genetic context do not accumulate defective lymphocytes. so it resides in cells in reduced form. Because necroptosis is associated with rapid plasma membrane permeabilization. but also produce viral proteins that interfere with other multiple cellular pathways and Immunity 38. indicating that the genetic background may determine the efficacy of necroptosis in the T cell compartment.. and hematopoietic development. whereas all-thiol-HMGB1 (all cysteines reduced) acts on the CXCR4 receptor to induce immune cell migration (Venereau et al. it has been shown that necroptotic cells secrete the proinflammatory cytokine IL-6 (Vanden Berghe et al. after which they perform immunomodulatory functions in the intracellular milieu. Second. the caspase-8FLIPL-FADD platform and autophagy are apparently gate keepers preventing necroptosis under physiological conditions. 2007). Therefore. organelles retained inside apoptotic bodies can be released or externalized during necrosis or necroptosis. but RIPK3-deficient mice are viable and they do not display any obvious developmental defects. HMGB1 can illustrate this redox sensitivity (Figure 2C). However. 2002). 219 . On the other hand. These patients accumulate abnormal lymphocytes.. ‘‘naturally occurring’’ necroptosis is observed only in infectious pathological conditions. 2012). caspase-8 or FADD deficiency is the only condition in which TCR-induced necroptosis can be observed. activation or modulation of ER-stress. this can be rescued by RIPK3 deletion. it cannot be ruled out that functional caspase-10 present in humans (but not in mice) can take over the function of defective caspase-8. This molecule can become proinflammatory in its reduced form (associated with necrosis) and immunologically silent in its oxidized form. Third.. 2011). In these conditions. indicating that necroptosis does not compensate for lack of apoptosis. Therefore. Moreover.. CRT and ATP (Garg et al. Such nuclei may contain many nuclear DAMPs. and IFN signaling can modify the properties of DAMPs. 2008). The function of necroptosis in the regulation of activated T cells is controversial.. Other differential features of necroptosis are lysosomal membrane permeabilization (LMP) (Vanden Berghe et al.. For example. such as IL-1a (Luheshi et al. Several bacterial and viral infections have been associated with necroptosis and emission of DAMPs such as HMGB1. whereas caspase-8 deficient T cells in a mixed 129/C57BL/6 background develop lymphoproliferative disease (Salmena and Hakem. According to current knowledge. 2013 ª2013 Elsevier Inc. experimental necroptosis-induced sterile inflammation also requires inhibition or genetic ablation of caspase-8 (skin and intestinal inflammation models). such as ER-stress could also affect the quality and quantity of released DAMPs.. A similar lymphoproliferative disease is observed in patients bearing a mutated variant of caspase-8 (Chun et al. Especially viruses may subvert DAMPs emission. DAMPs are probably released from these cells. 2010) and absence of translational inhibition. which requires caspase activation and ROS production (Kazama et al. but their mechanisms of action are poorly understood. PKR-like endoplasmic reticulum kinase (PERK) is necessary for the emission of two important DAMPs. Although necroptosis has not been investigated in these patients. cardiac. we speculate that also proteins of ER-stress pathway could modulate necroptosis and necroptotic DAMPs. many unanswered questions and controversies still surround both necroptosis and DAMPs. Indeed. HMGB1 lacks a leader peptide for the classical ER-Golgi secretory pathway. The precise mechanism and the role of HMGB1 and other DAMPs in infection-associated inflammation remain unknown.. which could then function as an alarmin. Although these possibilities have not been investigated. the PERK pathway can be inactivated by PERK’s pseudosubstrates or viral inhibitory proteins resembling the way by which caspase-8 is inactivated (Galluzzi et al. The necroptotic pathway can only operate under specific conditions and is tightly controlled. Additionally. multiple pathways. Moreover. the processes activated during the different cell death types may profoundly affect the composition and properties of released DAMPs.Immunity Review organelles could lead to massive release of DAMPs such as mtDNA and possibly other mitochondrial proteins (Oka et al. Because necroptotic cells continue to synthesize proteins until the last moment before plasma membrane permeabilization (Saelens et al.. 2012). 2012). autophagy. damaged mitochondria are an important source of ROS that not only contribute to progression of cell death but can also modify the oxidative status of DAMPs.. However. 2005).. FADD. which facilitate the uptake of dying cells by APCs. Next to the role of mitochondria in DAMPs release and modification. Concluding Remarks The previous decade brought new insights into necroptotic cell death as a programmed and actively regulated process. 2012). other biochemical processes activated possibly simultaneously with or regulating necroptosis. such as inflammasome activation and pyroptosis induction by PAMPs during bacterial or viral infection. can mediate and modify the release of DAMPs and modulate in a paracrine way the PRRs on responding cells. DAMPs released from necroptotic cells can contribute to sensitization of necroptosis in neighboring cells via induction of PRRs. 2012) and largely intact genomic DNA.. two mutually exclusive forms of HMGB1 exert two different functions: disulfide-HMGB1 (oxidation of C23-C45) acts through TLR4 to induce cytokine production. February 21.. HMGB1 (Thomas and Stott. besides these strict conditions of caspase-8 inactivation or ablation. the redox status contributes to both attraction of immune cells and inflammation. most probably. Furthermore. making cells more susceptible to necroptosis. because they not only inhibit caspase-8. This observation suggests the importance of FADD-caspase-8 axis in development and indicates that necroptosis might act as a mechanism for eliminating defective organisms. 2006). and proteins could be modified during the cell death process by oxidation or by lysosomal cathepsin-dependent proteolysis (Figure 2C). suggesting that an additional antinecroptotic mechanism may operate in humans.or caspase-8 deficient mice die as embryos at the stage of vascular. Therefore. Additionally. 2003) and in tissues (Welz et al. and it is clear that DAMPs are important. 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