Biol. Chem. 2014; 395(10): 1163–1171
Review Pavel Davidovich, Conor J. Kearney and Seamus J. Martin*
Inflammatory outcomes of apoptosis, necrosis and necroptosis Abstract: Microbial infection and tissue injury are well established as the two major drivers of inflammation. However, although it is widely accepted that necrotic cell death can trigger or potentiate inflammation, precisely how this is achieved still remains relatively obscure. Certain molecules, which have been dubbed ‘damageassociated molecular patterns’ (DAMPs) or alarmins, are thought to promote inflammation upon release from necrotic cells. However, the precise nature and relative potency of DAMPs, compared to conventional pro-inflammatory cytokines or pathogen-associated molecular patterns (PAMPs), remains unclear. How different modes of cell death impact on the immune system also requires further clarification. Apoptosis has long been regarded as a non-inflammatory or even anti-inflammatory mode of cell death, but recent studies suggest that this is not always the case. Necroptosis is a programmed form of necrosis that is engaged under certain conditions when caspase activation is blocked. Necroptosis is also regarded as a highly pro-inflammatory mode of cell death but there has been little explicit examination of this issue. Here we discuss the inflammatory implications of necrosis, necroptosis and apoptosis and some of the unresolved questions concerning how dead cells influence inflammatory responses. Keywords: alarmins; apoptosis; cell death; danger; damage-associated molecular patterns; inflammation; necrosis; necroptosis. DOI 10.1515/hsz-2014-0164 Received March 13, 2014; accepted August 01, 2014; previously published online August 5, 2014 *Corresponding author: Seamus J. Martin, Cellular Biotechnology Laboratory, Saint-Petersburg State Institute of Technology, Moskovskii prospekt, St. Petersburg, Russia; and Molecular Cell Biology Laboratory, Department of Genetics, The Smurfit Institute, Trinity College, Dublin 2, Ireland, e-mail: [email protected] Pavel Davidovich: Cellular Biotechnology Laboratory, SaintPetersburg State Institute of Technology, Moskovskii prospekt, St. Petersburg, Russia Conor J. Kearney: Molecular Cell Biology Laboratory, Department of Genetics, The Smurfit Institute, Trinity College, Dublin 2, Ireland
Introduction Inflammation represents a complex set of reactions that occur in response to infection or tissue damage and are designed to mobilise the diverse forces of the immune system to eliminate the infectious agent and to repair the tissue damage (Medzhitov, 2008; Matzinger and Kamala, 2011). Because the skin and gastrointestinal tract represent the major bodily surfaces in contact with the outside world, infection is typically caused by injuries that breach these barrier tissues. Thus, injury and infection are often closely associated, with one typically leading to the other (Matzinger, 1994). For these reasons, the response to infection and tissue damage – inflammation – is highly similar. The cardinal signs of inflammation are well known and were first recognised by Celsus over 2000 years ago. The key features of inflammation include: tumour (swelling), rubor (redness), calor (heat) and dolor (pain) and these events serve to facilitate ingress of soluble (antibodies, complement, acute phase reactants) as well as cellular constituents of the immune system and to minimise further tissue injury. The swelling and redness seen during inflammatory reactions is caused by increased vascular permeability in the affected area as a consequence of cytokines (such as TNF and IL-1) that are released in response to infection. The release of vasoactive amines, such as hist amine, from resident mast cells draws attention to the injury through acting on peripheral nerve endings, thereby producing pain. Cytokines such as IL-1 that are released during inflammation along with prostaglandins can act on the hypothalamus to promote temperature elevation, which is thought to antagonise microbial replication. Inflammation can be triggered through detection of pathogen-associated molecular patterns (PAMPs) by tissue resident cells of the innate immune system, predominantly macrophages, which triggers the secretion of IL-1β and TNF, as well as a host of other cytokines and chemokines (Esche et al., 2005; Kawai and Akira, 2007). The latter factors collaborate to promote vascular permeability in the local endothelium, as well as the attraction of cells of the innate immune system, predominantly
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1164 P. Davidovich et al.: Inflammatory outcomes of apoptosis, necrosis and necroptosis neutrophils, into the locality. This sets in motion a series of events that rapidly ramps up the body’s defences towards microorganisms. Neutrophils and macrophages are highly adept at phagocytosis and killing of bacteria and the complement factors and other soluble mediators can opsonise as well as lyse infectious agents. However, although pathogen products are excellent drivers of inflammation, sterile injury (i.e., injury that fails to breach barrier tissues) that causes cell death is also a potent trigger of inflammation. The question is how?
Necrosis as a trigger for inflammation Necrotic cell death has long been recognised as a trigger for inflammation (Table 1). Despite this, precisely how necrotic cells trigger inflammation still remains relatively obscure. Matzinger introduced the concept of ‘danger signals’ almost 20 years ago to explain how necrotic cells activate cells of the immune system, in the absence of infection (Matzinger, 1994, 2002). Danger signals, now more widely known as damage-associated molecular patterns (DAMPs), are thought to be endogenous molecules with cytokine-like properties, which are not normally released
from healthy cells (Figure 1). DAMPs have the capacity to trigger activation of cells of the innate immune system upon liberation into the extracellular space during necrosis. However, despite the attractiveness of this concept for the explanation of sterile inflammation, there is still little consensus on what constitutes a danger signal. Perhaps the best candidates for danger signals are members of the extended IL-1 family that lack conventional secretory signals and are therefore not released from viable cells via conventional ER-golgi secretion pathways. The IL-1 family contains a growing number of members and now includes IL-1α, IL-1β, IL-18, IL-33, IL-36α, IL-36β, IL-36γ and several other members that remain to be fully characterised (Sims and Smith, 2010; Garlanda et al., 2013). What all of these cytokines share in common is their capacity to act as potent activators of diverse cell types, such as macrophages, mast cells, neutrophils, keratinocytes, B-cells, endothelial cells, fibroblasts and many other cell types. In all cases, IL-1 family members can promote the release of a diverse array of pro-inflammatory factors from many of these cell types thereby promoting essentially all of the hallmarks of inflammation. However, what IL-1 family cytokines also have in common is a failure to be released from viable cells. It appears that the latter property, as well as their robust pro-inflammatory activities, makes these cytokines the best candidates as the major DAMPs.
Table 1 Progress in our understanding of the immune consequences of cell death modalities. Cell death mode
Significance
Pro- or anti-inflammatory
Immune-regulatory molecule
References
Apoptosis
Necrosis
Apoptosis
Necrosis Apoptosis
Seminal study describing the phenomenon of apoptosis in biological contexts Hypothesis that endogenous molecules can promote inflammation Evidence that necrotic but not apoptotic cells activate DCs Implication of ‘danger molecule’ Inactivation of DAMPs by caspases
Presumed to avoid activation of the immune system – phagocytosis observed Pro-inflammatory
Not identified
Kerr et al., 1972
Not identified
Matzinger, 1994
Anti-inflammatory
Not identified
Gallucci et al., 1999
Pro-inflammatory Anti-inflammatory
Uric acid IL-33 inactivation by caspase-3 and -7 RIPK3-dependant RIPK3
Shi et al., 2003 Lüthi et al., 2009
Necroptosis Necroptosis
Pro-inflammatory Presumed pro-inflammatory
Necrosis Apoptosis
Cohen et al., 2010 Chen et al., 2010a, b
Pro-inflammatory Activation of pro inflammatory/proliferative signalling Anti-inflammatory?
IL1-alpha Mediated by JNK/Jun
Necroptosis
RIPK3-dependant
Robinson et al., 2012
Necroptosis
Presumed pro-inflammatory
MLKL
Sun et al., 2012
Apoptosis
RIPK3 required for viral control Identification of RIPK3 as a driver of necroptosis Conventional cytokine as a DAMP Demonstration that Fas engagement can promote tumour growth in the absence of apoptosis Necroptosis promotes bacterial infection Identification of MLKL downstream of RIPK3 Release of chemotactic factors by apoptotic cells
Cho et al., 2009 He et al., 2009
Pro-inflammatory
KC, MCP-1, IL-8
Cullen et al., 2013
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P. Davidovich et al.: Inflammatory outcomes of apoptosis, necrosis and necroptosis 1165
Necrosis
Release of DAMPs
Macrophage
Dendritic cell
Activation
Inflammatory cytokine production
Maturation
Migration to lymph nodes and antigen presentation
Innate immunity
LPS or DNA for activity. Interestingly, extracellular ATP is also a potent activator of LPS-primed inflammasomes, although how it achieves this is also unclear. It is also becoming apparent that necrosis is not the only mode of cell death that can influence cells of the immune system. Recent data suggest that two other modes of cell death, apoptosis and necroptosis, can also have proinflammatory consequences (Figure 2). Before we discuss the evidence for this statement, we will first outline the distinctions between necrosis, apoptosis and necroptosis.
Adaptive immunity
Figure 1 Necrosis promotes inflammation through release of ‘damage-associated molecular patterns’. Damage-associated molecular patterns (DAMPs) are a poorlydefined class of molecules that are normally confined to viable cells. However, upon release into the extracellular space as a consequence of necrosis, DAMPs (also called alarmins) promote activation of macrophages, dendritic cells and other sentinel cells of the immune system to initiate inflammation. Members of the extended IL-1 family may represent the major DAMPs.
The most intensively studied member of the IL-1 family is IL-1β and, similar to all other members of this cytokine family, the active form of IL-1β lacks a leader sequence and is not released through a classical secretory pathway. While the exact mechanism of mature IL-1β release remains controversial, caspase-1 mediated IL-1β proteolysis is closely associated with pyroptotic cell death (Miao et al., 2011). Moreover, two recent studies have shown that in addition to mature IL-1β, whole inflammasome particles are released in response to agents that promote maturation of this cytokine (Baroja-Mazo et al., 2014; Franklin et al., 2014), which is consistent with the interpretation that cell death is a primary driver of IL-1β release. However, the precise contribution of cell death as a facilitator of IL-1β release requires further clarification. In addition to members of the IL-1 family, many other DAMPs have been proposed and these include Uric acid, ATP, HMGB1, CIRP and heat shock proteins (reviewed in Chen and Nuñez, 2010). However, it appears uncertain whether some of these molecules are sufficient to promote inflammatory responses by themselves, as several of these have been found to require contaminating PAMPs such as
Apoptosis, necrosis and necroptosis: similarities and distinctions Apoptosis is a mode of cell death that is characterised by a series of morphological and biochemical alterations to the cell architecture that package a cell up for removal by cells
Caspase inhibition
Caspase activation Apoptosis
Programmed necrosis
‘Find-me’ ‘eat-me’ signals
DAMPs
DAMP receptor
Anti-inflammatory response
Pro-inflammatory response?
Necrosis
DAMPs
DAMP receptor
Pro-inflammatory response
Figure 2 Inflammatory outcomes of apoptosis, necrosis and necroptosis. Apoptosis is thought to be non-inflammatory in many settings as a result of the rapid recognition and removal of apoptotic cells before DAMP release can occur. However, certain physiological inducersof apoptosis such as TNF and Fas, can promote inflammatory cytokine release in tandem with apoptosis and such instances of apoptosis appear to be pro-inflammatory. Necrosis is thought to be pro-inflammatory because of the release of DAMPs, as outlined in Figure 1. Similarly, necroptosis (programmed necrosis), which occurs in response to TNF upon inhibition of caspase activation, is currently thought to be pro-inflammatory because of the release of DAMPs but may be considerably less pro-inflammatory than TNFstimulation alone for reasons discussed in the main text.
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1166 P. Davidovich et al.: Inflammatory outcomes of apoptosis, necrosis and necroptosis with phagocytic capacity (Taylor et al., 2008). Crucially, apoptotic cells are recognised by phagocytes and are engulfed before they leak their contents (Figure 2). Thus, apoptosis ensures that when a cell needs to be removed from a tissue, this occurs in an orderly fashion that minimises disruption to neighbouring cells. The major consideration during apoptosis is that intracellular contents do not leak into the extracellular space because this could damage surrounding cells, and trigger inflammation through release of DAMPs. Thus, apoptosis is largely concerned with avoiding disturbance to tissues in which there is ongoing homeostatic cell death. Most of the biochemical and morphological changes that typify apoptosis are the consequence of activation of a subset of the caspase family of proteases (caspases -3, -6, -7, -8 and -9). Caspases function in apoptosis similar to a controlled demolition squad, coordinating the packaging and disposal of cells in a manner that minimises damage to neighbours and the initiation of inflammation (Figure 2). In contrast to apoptosis, necrosis is generally uncontrolled and involves the sudden loss of membrane integrity, release of extracellular contents, including DAMPs, which leads to activation of the immune system and extensive inflammation (Figure 2). Necrosis is typically not associated with caspase activation (Kroemer and Martin, 2005), although the exception to this is where cell death follows aggressive activation of the inflammatory subset of caspases (caspases -1, -4 and -5), a mode of cell death termed pyroptosis. Necrotic cell death bears none of the striking features that characterise apoptotic cells, such as extensive membrane blebbing and hypercondensation and fragmentation of the nucleus. Instead, necrotic cells undergo extensive organelle and cell swelling, leading to decondensation of nuclei (Kroemer and Martin, 2005). Thus, this mode of cell death is relatively easy to distinguish from apoptosis on the basis of morphological criteria.
Molecular control of necroptosis Necroptosis (also called programmed necrosis) is a form of necrosis that appears to be driven as a consequence of the deregulated activity of RIPK1 and RIPK3, leading to activation and oligomerisation of the MLKL pseudokinase within the plasma membrane. This mode of cell death is typically seen in response to engagement of certain members of the ‘death receptor’ subset of the TNF superfamily, particularly of the TNF receptor itself. However, it should be stressed that TNF and other stimuli known to promote necroptosis only do so when caspase activity is
actively inhibited using synthetic or viral-derived caspase inhibitors. Furthermore, death receptor-induced necroptosis is only observed in cells that express the kinase, RIPK3. In cells lacking RIPK3, inhibition of caspase activity completely blocks TNF-induced cell death, but in RIPK3expressing cells results in a necrotic-like mode of cell death. Necroptosis results from excessive recruitment of RIPK3 onto RIPK1, thereby promoting deregulated RIPK1 and RIPK3 kinase activity. This pivotal event is cytotoxic to cells, apparently through activation of MLKL which appears to be able to promote pore formation in plasma membranes, either directly, or through activation of the cation channel TRPM7 (Cai et al., 2014; Chen et al., 2014). Thus, necroptosis represents an alternative mode of cell death that is triggered only when the normal caspase wiring is interfered with (Figure 2). Under normal circumstances, caspase-8 restrains recruitment of RIPK3 onto RIPK1 in two ways, first by cleaving RIPK1 (Ofengeim and Yuan, 2013) and second by cleaving the deubiquitinating enzyme CYLD (O’Donnell et al., 2011). CYLD negatively regulates the degree of RIPK1 ubiquitination that occurs in response to death receptor engagement, an event that is involved in assembling signalling complexes on RIPK1 to propagate death receptor-induced NFκB activation (Mahoney et al., 2008; Wertz and Dixit, 2008). However, untrammeled CYLD activity can lead to deubiquitination of RIPK1 and excessive recruitment of RIPK3 as a consequence. Thus, by cleaving RIPK1 and CYLD, caspase-8 fine-tunes the composition of the RIPK1/RIPK3 complex, which, if perturbed, deregulates RIPK1/RIPK3 kinase activity, resulting in necroptosis. What is not clear at present is why. Moreover, the physiological relevance of this mode of cell death is currently debated but may occur upon infection with certain viruses that encode caspase inhibitors (Cho et al., 2009; Mocarski et al., 2012). Irrespective of the precise physiological relevance of necroptosis, which is under investigation at present, the major means of distinguishing conventional necrosis from necroptosis is through probing for the involvement of RIPK1, or RIPK3, where cell death occurs in the absence of caspase involvement.
Apoptosis initiated by death receptor ligands can be pro-inflammatory Engagement of the Fas (also known as CD95 or APO-1) or TRAIL receptors with their cognate ligands can result in apoptosis via a caspase-dependent pathway that is now well understood (Peter and Krammer, 2009; Falschlehner
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P. Davidovich et al.: Inflammatory outcomes of apoptosis, necrosis and necroptosis 1167
et al., 2009; Strasser et al., 2009; Dickens et al., 2012; Lavrik and Krammer, 2012). Upon engagement with their specific ligands, Fas and TRAIL receptors can promote caspase activation by recruiting specific initiator caspases (caspase-8 or -10 in humans) to the plasma membrane via the adaptor protein FADD (Wilson et al., 2009). Recruitment of several caspase molecules into the membrane receptor complex drives caspase activation through an ‘induced-proximity’ mechanism. Activate caspase-8 then propagates the death signal into the cell either by proteolytically processing effector caspases (caspases-3 and -7) downstream (called type I cells), or alternatively, by cleaving Bid, a BH3-only protein that can promote the permeabilisation of mitochondrial outer membranes and release of cytochrome c (type II cells). Upon release from mitochondria, cytochrome c acts as a co-factor for the assembly of a caspase-activating complex in the cytosol called the apoptosome, thereby propagating the caspase activation cascade (Slee et al., 1999). However, a protease dead homologue of caspase-8, called c-FLIP, can also be recruited to ‘death receptors’ and the recruitment of the latter, especially at threshold levels of death receptor stimulation, can block the recruitment of caspases-8 and -10, thereby preventing the propagation of the death signal (Lavrik et al., 2007). FLIP recruitment appears to play a key role in dictating the death or survival outcome of ‘death receptor’ engagement (Kavuri et al., 2011), although it is not clear whether the latter is central to promoting alternative outcomes of death receptor stimulation, as will be discussed below. It is also notable that the pro-inflammatory kinase RIPK1, as well as TRAF2, cIAP-1 and cIAP-2 have also been implicated as part of the Fas and TRAIL receptor signalling complexes, but the role that these molecules play within these ‘death receptor’ complexes is unclear. It is, however, important to note that RIPK1, TRAF2, cIAP-1 and cIAP-2 are all known to play important roles in promoting cytokine production in the context of TNF receptor signalling (Kovalenko and Wallach, 2006; Walczak, 2011; Kearney et al., 2013), a receptor that is well established as a driver of inflammation and cell survival signalling. Because the overwhelming majority of studies ( > 10,000 papers to date) exploring Fas and TRAIL have focused on apoptosis as the primary endpoint, this has greatly overshadowed the role of these receptors as initiators of other biological outcomes (reviewed in Peter et al., 2007). Sporadic reports have suggested that Fas or TRAIL receptor stimulation can result in the production of pro-inflammatory cytokines and chemokines (Choi et al., 2001; Park et al., 2003; Farley et al., 2006; Altemeier et al., 2007; Berg et al., 2009; Cullen et al., 2013).
Other reported consequences of Fas or TRAIL receptor engagement include: activation and maturation of dendritic cells (Rescigno et al., 2000), cell motility and invasion (Barnhart et al., 2004, Kleber et al., 2008; Hoogwater et al., 2010) cell proliferation (Chen et al., 2010a,b; Azijli et al., 2012) and metastasis (Trauzold et al., 2006). Fas and TRAIL receptor expression have also been implicated, counterintuitively, as drivers of tumour proliferation rather than apoptosis (Baader et al., 2005; Mitsiades et al., 2006; Nguyen et al., 2009; Chen et al., 2010a,b). Thus, much evidence now suggests that signalling via the Fas and TRAIL receptors can lead to endpoints other than apoptosis, but these aspects of ‘death receptor’ biology are very poorly understood. Fas and TRAIL belong to the TNFR superfamily, members of which play an important role in regulating cell life spans but also in cell-cell communication within the immune system (Croft et al., 2011). Although TNF receptor engagement can also promote apoptosis under certain conditions, few cell types die in direct response to TNF treatment unless sensitised through co-exposure to transcriptional/translational inhibitors. A key aspect of TNF function is its ability to trigger the production of numerous pro-inflammatory cytokines and chemokines (Esche et al., 2005; Brennan and McInnes, 2008). TNFinduced cytokines and chemokines, such as IL-6, IL-8, GMCSF, CXCL1 and RANTES, can instigate and amplify immune responses through triggering the production of acute phase proteins, recruitment of neutrophils, macrophages and basophils to the site of inflammation and by triggering increased production of monocytes/macrophages from bone marrow. Because the Fas and TRAIL receptor complexes share several constituents in common to the TNFR complex, as detailed above, it is perhaps not surprising that Fas or TRAIL stimulation can also promote inflammatory signalling, but this facet of Fas and TRAIL function and its biological consequences remains remarkably underexplored. As mentioned above, a small number of previous studies have reported that engagement of Fas or TRAIL receptors on macrophages, keratinocytes and T-cells can induce the production and secretion of pro-inflammatory cytokines and chemokines, particularly IL-8 and MCP-1. Indeed, we have also recently found that administration of anti-Fas antibodies into wild-type balb/c mice leads to a dramatic increase in circulating chemokines (Cullen et al., 2013). A compelling study by Wajant and colleagues also showed that several Myeloma cell lines produced a range of pro-inflammatory cytokines and chemokines in response to TRAIL stimulation (Berg et al., 2009). However, the biological impact of the production of these
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1168 P. Davidovich et al.: Inflammatory outcomes of apoptosis, necrosis and necroptosis pro-inflammatory molecules on TRAIL-positive cancers remains unknown. Similarly, the scope and biological impact of the secreted factors induced by stimulation of the Fas and TRAIL receptors remains largely unknown, especially in the context of cancer, as previous studies have not explored this issue to any depth. Furthermore, the factors that dictate the switch between ‘death receptor’-induced pro-survival and pro-inflammatory signalling also remain poorly defined. Studies by Lavrik et al. (2007), as well as Peter and colleagues (Legembre et al., 2004; Chen et al., 2010a,b), suggest that threshold levels of Fas receptor stimulation is sufficient to promote MAPK and NFκB activation, as well as cell proliferation, while avoiding the pro-apoptotic effects of Fas receptor engagement. Similar low intensity stimulation-induced proliferation has also been reported for TRAIL in a number of transformed cell types (Ehrhardt et al., 2003; Baader et al., 2005). Preferential recruitment of c-FLIPL to the Fas receptor complex is one of the factors that likely dictates this switch, but members of the linear ubiquitin complex (LUBAC) may also play a role here similar to its role in TNF signalling (Lavrik et al., 2007; Walczak, 2011) and Ashkenazi and colleagues have recently reported that TRAF2-mediated ubiquitination and degradation of caspase-8 may also be an important determinant of insensitivity to ‘death receptor’-induced apoptosis (Gonzalvez et al., 2012). However, the key regulators of ‘death receptor’-induced inflammatory cytokine production and proliferation have received little study to date.
Apoptosis as a pro-inflammatory mode of cell death As mentioned earlier, apoptosis has long been considered as a non-inflammatory or even anti-inflammatory mode of cell death (Table 1), despite relatively little experimental examination of this issue. While it is true that apoptosis in many contexts appears to be non-inflammatory, recent data have begun to emerge that challenge the idea that apoptotic cells are invariably non-inflammatory, particularly in the context of apoptosis induced by certain members of the TNF family (Cullen et al., 2013; Kearney et al., 2013). Specifically, engagement of the Fas (CD95/APO-1) membrane receptor, which belongs to the death receptor subset of the TNFR family, has been shown to be capable of promoting the secretion of a battery of pro-inflammatory cytokines and chemokines by apoptotic cells (Cullen et al., 2013). Similarly, when cells undergo
apoptosis in response to TNF receptor engagement, such cells can also undergo apoptosis that is associated with the production of a range of pro-inflammatory cytokines and chemokines by the dying cell (Kearney et al., 2013). The fact that Fas engagement can promote the production of pro-inflammatory cytokines and chemokine (such as IL-6, IL-8, CXCL1 and RANTES) is not surprising considering that this receptor recruits many of the same molecules, such as FADD, RIPK1 and the IKK complex, that are also recruited to the TNFR signalling complex. Despite the similarities in the Fas and TNF receptor signalling complexes, the pro-inflammatory aspects of Fas signalling have been much overlooked in the 20 or so years since the discovery of this receptor. Thus, there are certain situations where apoptotic cells can be pro-inflammatory through release of cytokines and chemokines rather than through release of DAMPs.
Necroptosis and inflammation Necroptosis, similar to necrosis, is also thought to result in the release of DAMPs into the extracellular space (Figure 2). For this reason, necroptosis is currently considered to be a highly inflammatory mode of cell death (Kacz marek et al., 2013; Moriwaki and Ka-Ming Chan, 2013). However, many of the stimuli that promote necroptosis, such as TNF and LPS, are intrinsically highly pro-inflammatory. This is important because rapid initiation of cell death in a cell that is engaged in a transcriptional inflammatory programme, as occurs upon TNF or LPS treatment, may lead to a less inflammatory outcome because of the rapid cessation of synthesis of pro-inflammatory cytokines and chemokines. Thus, whether necroptosis is more or less pro-inflammatory than the alternative depends on the relative potency of DAMPs, relative to conventional cytokines and chemokines. Following this line of reasoning, necroptosis may well transpire to be less inflammatory than TNF or LPS stimulation alone, as a result of the shutting down of TNF- or LPS-induced transcription of numerous pro-inflammatory factors. Further studies on the inflammatory outcomes of necroptosis are needed to resolve this issue.
RIPK3 and MLKL null animals as a model of necroptosis deficiency Many studies have utilised mice lacking RIPK3 as a model for necroptosis deficiency (Cho et al., 2009; He et al.,
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P. Davidovich et al.: Inflammatory outcomes of apoptosis, necrosis and necroptosis 1169
2009; Duprez et al., 2011; Linkermann et al., 2013). It has been reported that the detrimental effects of drug-induced liver damage, acute pancreatitis, the lethal effects of systemic TNF, and injury caused by ischemia-reperfusion are diminished in these animals. These observations have been widely interpreted to imply that necroptosis is a contributory factor in the damaging inflammatory responses observed in all of these situations. However, necroptosis is not typically observed in RIPK3-expressing cells unless caspase activation is inhibited and in the several of the latter studies, caspase inhibitors were not used. Therefore, the protective effects seen in RIPK3-deficient animals in various settings may be more related to a direct role for RIPK3 in promoting inflammation, rather than its role in promoting necroptosis under conditions where caspase activation is blocked. A similar argument can be made with respect to MLKL null animals, as MLKL-dependent cell death is not observed unless caspase inhibitors are employed. Thus, studies that have employed RIPK3 or MLKL null animals as ‘necroptosis-deficient’ animals may have overstated the case and these animals should instead be regarded as simply as RIPK3-deficient or MLKLdeficient. It follows logically therefore, that RIPK3 and MLKL could well be involved as signal transduction elements within inflammatory pathways, quite apart from their role in necroptosis under special conditions where caspase activation is blocked.
Conclusions Here we have argued that although the concept that cell death is a trigger for inflammation is very well established, there is much we still do not know concerning how the various modes of cell death (apoptosis, necrosis and necroptosis) influence inflammation. The precise identity of the key DAMPs is still the subject of speculation despite 20 years having elapsed since this concept was first advanced by Matzinger in 1994 (Table 1). Furthermore, it now appears that apoptosis is not always a non-inflammatory mode of cell death, particularly in the context where apoptosis is initiated by TNF, Fas and perhaps other members of the TNF family. Finally, it still remains to be determined whether necroptosis is more or less inflammatory than the alternative outcome where cell death occurs with the participation of caspases. Because excessive or persistent inflammation is a major cause of human disease, understanding how cell death contributes to inflammatory processes is likely to lead to important insights that contribute to the development of strategies to dampen or suppress inflammation in disease settings.
Acknowledgments: This work was supported by grants of the Russian government for state support of scientific research, as well as Science Foundation Ireland.
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Review Pavel Davidovich, Conor J. Kearney and Seamus J. Martin*
Inflammatory outcomes of apoptosis, necrosis and necroptosis Abstract: Microbial infection and tissue injury are well established as the two major drivers of inflammation. However, although it is widely accepted that necrotic cell death can trigger or potentiate inflammation, precisely how this is achieved still remains relatively obscure. Certain molecules, which have been dubbed ‘damageassociated molecular patterns’ (DAMPs) or alarmins, are thought to promote inflammation upon release from necrotic cells. However, the precise nature and relative potency of DAMPs, compared to conventional pro-inflammatory cytokines or pathogen-associated molecular patterns (PAMPs), remains unclear. How different modes of cell death impact on the immune system also requires further clarification. Apoptosis has long been regarded as a non-inflammatory or even anti-inflammatory mode of cell death, but recent studies suggest that this is not always the case. Necroptosis is a programmed form of necrosis that is engaged under certain conditions when caspase activation is blocked. Necroptosis is also regarded as a highly pro-inflammatory mode of cell death but there has been little explicit examination of this issue. Here we discuss the inflammatory implications of necrosis, necroptosis and apoptosis and some of the unresolved questions concerning how dead cells influence inflammatory responses. Keywords: alarmins; apoptosis; cell death; danger; damage-associated molecular patterns; inflammation; necrosis; necroptosis. DOI 10.1515/hsz-2014-0164 Received March 13, 2014; accepted August 01, 2014; previously published online August 5, 2014 *Corresponding author: Seamus J. Martin, Cellular Biotechnology Laboratory, Saint-Petersburg State Institute of Technology, Moskovskii prospekt, St. Petersburg, Russia; and Molecular Cell Biology Laboratory, Department of Genetics, The Smurfit Institute, Trinity College, Dublin 2, Ireland, e-mail: [email protected] Pavel Davidovich: Cellular Biotechnology Laboratory, SaintPetersburg State Institute of Technology, Moskovskii prospekt, St. Petersburg, Russia Conor J. Kearney: Molecular Cell Biology Laboratory, Department of Genetics, The Smurfit Institute, Trinity College, Dublin 2, Ireland
Introduction Inflammation represents a complex set of reactions that occur in response to infection or tissue damage and are designed to mobilise the diverse forces of the immune system to eliminate the infectious agent and to repair the tissue damage (Medzhitov, 2008; Matzinger and Kamala, 2011). Because the skin and gastrointestinal tract represent the major bodily surfaces in contact with the outside world, infection is typically caused by injuries that breach these barrier tissues. Thus, injury and infection are often closely associated, with one typically leading to the other (Matzinger, 1994). For these reasons, the response to infection and tissue damage – inflammation – is highly similar. The cardinal signs of inflammation are well known and were first recognised by Celsus over 2000 years ago. The key features of inflammation include: tumour (swelling), rubor (redness), calor (heat) and dolor (pain) and these events serve to facilitate ingress of soluble (antibodies, complement, acute phase reactants) as well as cellular constituents of the immune system and to minimise further tissue injury. The swelling and redness seen during inflammatory reactions is caused by increased vascular permeability in the affected area as a consequence of cytokines (such as TNF and IL-1) that are released in response to infection. The release of vasoactive amines, such as hist amine, from resident mast cells draws attention to the injury through acting on peripheral nerve endings, thereby producing pain. Cytokines such as IL-1 that are released during inflammation along with prostaglandins can act on the hypothalamus to promote temperature elevation, which is thought to antagonise microbial replication. Inflammation can be triggered through detection of pathogen-associated molecular patterns (PAMPs) by tissue resident cells of the innate immune system, predominantly macrophages, which triggers the secretion of IL-1β and TNF, as well as a host of other cytokines and chemokines (Esche et al., 2005; Kawai and Akira, 2007). The latter factors collaborate to promote vascular permeability in the local endothelium, as well as the attraction of cells of the innate immune system, predominantly
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1164 P. Davidovich et al.: Inflammatory outcomes of apoptosis, necrosis and necroptosis neutrophils, into the locality. This sets in motion a series of events that rapidly ramps up the body’s defences towards microorganisms. Neutrophils and macrophages are highly adept at phagocytosis and killing of bacteria and the complement factors and other soluble mediators can opsonise as well as lyse infectious agents. However, although pathogen products are excellent drivers of inflammation, sterile injury (i.e., injury that fails to breach barrier tissues) that causes cell death is also a potent trigger of inflammation. The question is how?
Necrosis as a trigger for inflammation Necrotic cell death has long been recognised as a trigger for inflammation (Table 1). Despite this, precisely how necrotic cells trigger inflammation still remains relatively obscure. Matzinger introduced the concept of ‘danger signals’ almost 20 years ago to explain how necrotic cells activate cells of the immune system, in the absence of infection (Matzinger, 1994, 2002). Danger signals, now more widely known as damage-associated molecular patterns (DAMPs), are thought to be endogenous molecules with cytokine-like properties, which are not normally released
from healthy cells (Figure 1). DAMPs have the capacity to trigger activation of cells of the innate immune system upon liberation into the extracellular space during necrosis. However, despite the attractiveness of this concept for the explanation of sterile inflammation, there is still little consensus on what constitutes a danger signal. Perhaps the best candidates for danger signals are members of the extended IL-1 family that lack conventional secretory signals and are therefore not released from viable cells via conventional ER-golgi secretion pathways. The IL-1 family contains a growing number of members and now includes IL-1α, IL-1β, IL-18, IL-33, IL-36α, IL-36β, IL-36γ and several other members that remain to be fully characterised (Sims and Smith, 2010; Garlanda et al., 2013). What all of these cytokines share in common is their capacity to act as potent activators of diverse cell types, such as macrophages, mast cells, neutrophils, keratinocytes, B-cells, endothelial cells, fibroblasts and many other cell types. In all cases, IL-1 family members can promote the release of a diverse array of pro-inflammatory factors from many of these cell types thereby promoting essentially all of the hallmarks of inflammation. However, what IL-1 family cytokines also have in common is a failure to be released from viable cells. It appears that the latter property, as well as their robust pro-inflammatory activities, makes these cytokines the best candidates as the major DAMPs.
Table 1 Progress in our understanding of the immune consequences of cell death modalities. Cell death mode
Significance
Pro- or anti-inflammatory
Immune-regulatory molecule
References
Apoptosis
Necrosis
Apoptosis
Necrosis Apoptosis
Seminal study describing the phenomenon of apoptosis in biological contexts Hypothesis that endogenous molecules can promote inflammation Evidence that necrotic but not apoptotic cells activate DCs Implication of ‘danger molecule’ Inactivation of DAMPs by caspases
Presumed to avoid activation of the immune system – phagocytosis observed Pro-inflammatory
Not identified
Kerr et al., 1972
Not identified
Matzinger, 1994
Anti-inflammatory
Not identified
Gallucci et al., 1999
Pro-inflammatory Anti-inflammatory
Uric acid IL-33 inactivation by caspase-3 and -7 RIPK3-dependant RIPK3
Shi et al., 2003 Lüthi et al., 2009
Necroptosis Necroptosis
Pro-inflammatory Presumed pro-inflammatory
Necrosis Apoptosis
Cohen et al., 2010 Chen et al., 2010a, b
Pro-inflammatory Activation of pro inflammatory/proliferative signalling Anti-inflammatory?
IL1-alpha Mediated by JNK/Jun
Necroptosis
RIPK3-dependant
Robinson et al., 2012
Necroptosis
Presumed pro-inflammatory
MLKL
Sun et al., 2012
Apoptosis
RIPK3 required for viral control Identification of RIPK3 as a driver of necroptosis Conventional cytokine as a DAMP Demonstration that Fas engagement can promote tumour growth in the absence of apoptosis Necroptosis promotes bacterial infection Identification of MLKL downstream of RIPK3 Release of chemotactic factors by apoptotic cells
Cho et al., 2009 He et al., 2009
Pro-inflammatory
KC, MCP-1, IL-8
Cullen et al., 2013
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P. Davidovich et al.: Inflammatory outcomes of apoptosis, necrosis and necroptosis 1165
Necrosis
Release of DAMPs
Macrophage
Dendritic cell
Activation
Inflammatory cytokine production
Maturation
Migration to lymph nodes and antigen presentation
Innate immunity
LPS or DNA for activity. Interestingly, extracellular ATP is also a potent activator of LPS-primed inflammasomes, although how it achieves this is also unclear. It is also becoming apparent that necrosis is not the only mode of cell death that can influence cells of the immune system. Recent data suggest that two other modes of cell death, apoptosis and necroptosis, can also have proinflammatory consequences (Figure 2). Before we discuss the evidence for this statement, we will first outline the distinctions between necrosis, apoptosis and necroptosis.
Adaptive immunity
Figure 1 Necrosis promotes inflammation through release of ‘damage-associated molecular patterns’. Damage-associated molecular patterns (DAMPs) are a poorlydefined class of molecules that are normally confined to viable cells. However, upon release into the extracellular space as a consequence of necrosis, DAMPs (also called alarmins) promote activation of macrophages, dendritic cells and other sentinel cells of the immune system to initiate inflammation. Members of the extended IL-1 family may represent the major DAMPs.
The most intensively studied member of the IL-1 family is IL-1β and, similar to all other members of this cytokine family, the active form of IL-1β lacks a leader sequence and is not released through a classical secretory pathway. While the exact mechanism of mature IL-1β release remains controversial, caspase-1 mediated IL-1β proteolysis is closely associated with pyroptotic cell death (Miao et al., 2011). Moreover, two recent studies have shown that in addition to mature IL-1β, whole inflammasome particles are released in response to agents that promote maturation of this cytokine (Baroja-Mazo et al., 2014; Franklin et al., 2014), which is consistent with the interpretation that cell death is a primary driver of IL-1β release. However, the precise contribution of cell death as a facilitator of IL-1β release requires further clarification. In addition to members of the IL-1 family, many other DAMPs have been proposed and these include Uric acid, ATP, HMGB1, CIRP and heat shock proteins (reviewed in Chen and Nuñez, 2010). However, it appears uncertain whether some of these molecules are sufficient to promote inflammatory responses by themselves, as several of these have been found to require contaminating PAMPs such as
Apoptosis, necrosis and necroptosis: similarities and distinctions Apoptosis is a mode of cell death that is characterised by a series of morphological and biochemical alterations to the cell architecture that package a cell up for removal by cells
Caspase inhibition
Caspase activation Apoptosis
Programmed necrosis
‘Find-me’ ‘eat-me’ signals
DAMPs
DAMP receptor
Anti-inflammatory response
Pro-inflammatory response?
Necrosis
DAMPs
DAMP receptor
Pro-inflammatory response
Figure 2 Inflammatory outcomes of apoptosis, necrosis and necroptosis. Apoptosis is thought to be non-inflammatory in many settings as a result of the rapid recognition and removal of apoptotic cells before DAMP release can occur. However, certain physiological inducersof apoptosis such as TNF and Fas, can promote inflammatory cytokine release in tandem with apoptosis and such instances of apoptosis appear to be pro-inflammatory. Necrosis is thought to be pro-inflammatory because of the release of DAMPs, as outlined in Figure 1. Similarly, necroptosis (programmed necrosis), which occurs in response to TNF upon inhibition of caspase activation, is currently thought to be pro-inflammatory because of the release of DAMPs but may be considerably less pro-inflammatory than TNFstimulation alone for reasons discussed in the main text.
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1166 P. Davidovich et al.: Inflammatory outcomes of apoptosis, necrosis and necroptosis with phagocytic capacity (Taylor et al., 2008). Crucially, apoptotic cells are recognised by phagocytes and are engulfed before they leak their contents (Figure 2). Thus, apoptosis ensures that when a cell needs to be removed from a tissue, this occurs in an orderly fashion that minimises disruption to neighbouring cells. The major consideration during apoptosis is that intracellular contents do not leak into the extracellular space because this could damage surrounding cells, and trigger inflammation through release of DAMPs. Thus, apoptosis is largely concerned with avoiding disturbance to tissues in which there is ongoing homeostatic cell death. Most of the biochemical and morphological changes that typify apoptosis are the consequence of activation of a subset of the caspase family of proteases (caspases -3, -6, -7, -8 and -9). Caspases function in apoptosis similar to a controlled demolition squad, coordinating the packaging and disposal of cells in a manner that minimises damage to neighbours and the initiation of inflammation (Figure 2). In contrast to apoptosis, necrosis is generally uncontrolled and involves the sudden loss of membrane integrity, release of extracellular contents, including DAMPs, which leads to activation of the immune system and extensive inflammation (Figure 2). Necrosis is typically not associated with caspase activation (Kroemer and Martin, 2005), although the exception to this is where cell death follows aggressive activation of the inflammatory subset of caspases (caspases -1, -4 and -5), a mode of cell death termed pyroptosis. Necrotic cell death bears none of the striking features that characterise apoptotic cells, such as extensive membrane blebbing and hypercondensation and fragmentation of the nucleus. Instead, necrotic cells undergo extensive organelle and cell swelling, leading to decondensation of nuclei (Kroemer and Martin, 2005). Thus, this mode of cell death is relatively easy to distinguish from apoptosis on the basis of morphological criteria.
Molecular control of necroptosis Necroptosis (also called programmed necrosis) is a form of necrosis that appears to be driven as a consequence of the deregulated activity of RIPK1 and RIPK3, leading to activation and oligomerisation of the MLKL pseudokinase within the plasma membrane. This mode of cell death is typically seen in response to engagement of certain members of the ‘death receptor’ subset of the TNF superfamily, particularly of the TNF receptor itself. However, it should be stressed that TNF and other stimuli known to promote necroptosis only do so when caspase activity is
actively inhibited using synthetic or viral-derived caspase inhibitors. Furthermore, death receptor-induced necroptosis is only observed in cells that express the kinase, RIPK3. In cells lacking RIPK3, inhibition of caspase activity completely blocks TNF-induced cell death, but in RIPK3expressing cells results in a necrotic-like mode of cell death. Necroptosis results from excessive recruitment of RIPK3 onto RIPK1, thereby promoting deregulated RIPK1 and RIPK3 kinase activity. This pivotal event is cytotoxic to cells, apparently through activation of MLKL which appears to be able to promote pore formation in plasma membranes, either directly, or through activation of the cation channel TRPM7 (Cai et al., 2014; Chen et al., 2014). Thus, necroptosis represents an alternative mode of cell death that is triggered only when the normal caspase wiring is interfered with (Figure 2). Under normal circumstances, caspase-8 restrains recruitment of RIPK3 onto RIPK1 in two ways, first by cleaving RIPK1 (Ofengeim and Yuan, 2013) and second by cleaving the deubiquitinating enzyme CYLD (O’Donnell et al., 2011). CYLD negatively regulates the degree of RIPK1 ubiquitination that occurs in response to death receptor engagement, an event that is involved in assembling signalling complexes on RIPK1 to propagate death receptor-induced NFκB activation (Mahoney et al., 2008; Wertz and Dixit, 2008). However, untrammeled CYLD activity can lead to deubiquitination of RIPK1 and excessive recruitment of RIPK3 as a consequence. Thus, by cleaving RIPK1 and CYLD, caspase-8 fine-tunes the composition of the RIPK1/RIPK3 complex, which, if perturbed, deregulates RIPK1/RIPK3 kinase activity, resulting in necroptosis. What is not clear at present is why. Moreover, the physiological relevance of this mode of cell death is currently debated but may occur upon infection with certain viruses that encode caspase inhibitors (Cho et al., 2009; Mocarski et al., 2012). Irrespective of the precise physiological relevance of necroptosis, which is under investigation at present, the major means of distinguishing conventional necrosis from necroptosis is through probing for the involvement of RIPK1, or RIPK3, where cell death occurs in the absence of caspase involvement.
Apoptosis initiated by death receptor ligands can be pro-inflammatory Engagement of the Fas (also known as CD95 or APO-1) or TRAIL receptors with their cognate ligands can result in apoptosis via a caspase-dependent pathway that is now well understood (Peter and Krammer, 2009; Falschlehner
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P. Davidovich et al.: Inflammatory outcomes of apoptosis, necrosis and necroptosis 1167
et al., 2009; Strasser et al., 2009; Dickens et al., 2012; Lavrik and Krammer, 2012). Upon engagement with their specific ligands, Fas and TRAIL receptors can promote caspase activation by recruiting specific initiator caspases (caspase-8 or -10 in humans) to the plasma membrane via the adaptor protein FADD (Wilson et al., 2009). Recruitment of several caspase molecules into the membrane receptor complex drives caspase activation through an ‘induced-proximity’ mechanism. Activate caspase-8 then propagates the death signal into the cell either by proteolytically processing effector caspases (caspases-3 and -7) downstream (called type I cells), or alternatively, by cleaving Bid, a BH3-only protein that can promote the permeabilisation of mitochondrial outer membranes and release of cytochrome c (type II cells). Upon release from mitochondria, cytochrome c acts as a co-factor for the assembly of a caspase-activating complex in the cytosol called the apoptosome, thereby propagating the caspase activation cascade (Slee et al., 1999). However, a protease dead homologue of caspase-8, called c-FLIP, can also be recruited to ‘death receptors’ and the recruitment of the latter, especially at threshold levels of death receptor stimulation, can block the recruitment of caspases-8 and -10, thereby preventing the propagation of the death signal (Lavrik et al., 2007). FLIP recruitment appears to play a key role in dictating the death or survival outcome of ‘death receptor’ engagement (Kavuri et al., 2011), although it is not clear whether the latter is central to promoting alternative outcomes of death receptor stimulation, as will be discussed below. It is also notable that the pro-inflammatory kinase RIPK1, as well as TRAF2, cIAP-1 and cIAP-2 have also been implicated as part of the Fas and TRAIL receptor signalling complexes, but the role that these molecules play within these ‘death receptor’ complexes is unclear. It is, however, important to note that RIPK1, TRAF2, cIAP-1 and cIAP-2 are all known to play important roles in promoting cytokine production in the context of TNF receptor signalling (Kovalenko and Wallach, 2006; Walczak, 2011; Kearney et al., 2013), a receptor that is well established as a driver of inflammation and cell survival signalling. Because the overwhelming majority of studies ( > 10,000 papers to date) exploring Fas and TRAIL have focused on apoptosis as the primary endpoint, this has greatly overshadowed the role of these receptors as initiators of other biological outcomes (reviewed in Peter et al., 2007). Sporadic reports have suggested that Fas or TRAIL receptor stimulation can result in the production of pro-inflammatory cytokines and chemokines (Choi et al., 2001; Park et al., 2003; Farley et al., 2006; Altemeier et al., 2007; Berg et al., 2009; Cullen et al., 2013).
Other reported consequences of Fas or TRAIL receptor engagement include: activation and maturation of dendritic cells (Rescigno et al., 2000), cell motility and invasion (Barnhart et al., 2004, Kleber et al., 2008; Hoogwater et al., 2010) cell proliferation (Chen et al., 2010a,b; Azijli et al., 2012) and metastasis (Trauzold et al., 2006). Fas and TRAIL receptor expression have also been implicated, counterintuitively, as drivers of tumour proliferation rather than apoptosis (Baader et al., 2005; Mitsiades et al., 2006; Nguyen et al., 2009; Chen et al., 2010a,b). Thus, much evidence now suggests that signalling via the Fas and TRAIL receptors can lead to endpoints other than apoptosis, but these aspects of ‘death receptor’ biology are very poorly understood. Fas and TRAIL belong to the TNFR superfamily, members of which play an important role in regulating cell life spans but also in cell-cell communication within the immune system (Croft et al., 2011). Although TNF receptor engagement can also promote apoptosis under certain conditions, few cell types die in direct response to TNF treatment unless sensitised through co-exposure to transcriptional/translational inhibitors. A key aspect of TNF function is its ability to trigger the production of numerous pro-inflammatory cytokines and chemokines (Esche et al., 2005; Brennan and McInnes, 2008). TNFinduced cytokines and chemokines, such as IL-6, IL-8, GMCSF, CXCL1 and RANTES, can instigate and amplify immune responses through triggering the production of acute phase proteins, recruitment of neutrophils, macrophages and basophils to the site of inflammation and by triggering increased production of monocytes/macrophages from bone marrow. Because the Fas and TRAIL receptor complexes share several constituents in common to the TNFR complex, as detailed above, it is perhaps not surprising that Fas or TRAIL stimulation can also promote inflammatory signalling, but this facet of Fas and TRAIL function and its biological consequences remains remarkably underexplored. As mentioned above, a small number of previous studies have reported that engagement of Fas or TRAIL receptors on macrophages, keratinocytes and T-cells can induce the production and secretion of pro-inflammatory cytokines and chemokines, particularly IL-8 and MCP-1. Indeed, we have also recently found that administration of anti-Fas antibodies into wild-type balb/c mice leads to a dramatic increase in circulating chemokines (Cullen et al., 2013). A compelling study by Wajant and colleagues also showed that several Myeloma cell lines produced a range of pro-inflammatory cytokines and chemokines in response to TRAIL stimulation (Berg et al., 2009). However, the biological impact of the production of these
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1168 P. Davidovich et al.: Inflammatory outcomes of apoptosis, necrosis and necroptosis pro-inflammatory molecules on TRAIL-positive cancers remains unknown. Similarly, the scope and biological impact of the secreted factors induced by stimulation of the Fas and TRAIL receptors remains largely unknown, especially in the context of cancer, as previous studies have not explored this issue to any depth. Furthermore, the factors that dictate the switch between ‘death receptor’-induced pro-survival and pro-inflammatory signalling also remain poorly defined. Studies by Lavrik et al. (2007), as well as Peter and colleagues (Legembre et al., 2004; Chen et al., 2010a,b), suggest that threshold levels of Fas receptor stimulation is sufficient to promote MAPK and NFκB activation, as well as cell proliferation, while avoiding the pro-apoptotic effects of Fas receptor engagement. Similar low intensity stimulation-induced proliferation has also been reported for TRAIL in a number of transformed cell types (Ehrhardt et al., 2003; Baader et al., 2005). Preferential recruitment of c-FLIPL to the Fas receptor complex is one of the factors that likely dictates this switch, but members of the linear ubiquitin complex (LUBAC) may also play a role here similar to its role in TNF signalling (Lavrik et al., 2007; Walczak, 2011) and Ashkenazi and colleagues have recently reported that TRAF2-mediated ubiquitination and degradation of caspase-8 may also be an important determinant of insensitivity to ‘death receptor’-induced apoptosis (Gonzalvez et al., 2012). However, the key regulators of ‘death receptor’-induced inflammatory cytokine production and proliferation have received little study to date.
Apoptosis as a pro-inflammatory mode of cell death As mentioned earlier, apoptosis has long been considered as a non-inflammatory or even anti-inflammatory mode of cell death (Table 1), despite relatively little experimental examination of this issue. While it is true that apoptosis in many contexts appears to be non-inflammatory, recent data have begun to emerge that challenge the idea that apoptotic cells are invariably non-inflammatory, particularly in the context of apoptosis induced by certain members of the TNF family (Cullen et al., 2013; Kearney et al., 2013). Specifically, engagement of the Fas (CD95/APO-1) membrane receptor, which belongs to the death receptor subset of the TNFR family, has been shown to be capable of promoting the secretion of a battery of pro-inflammatory cytokines and chemokines by apoptotic cells (Cullen et al., 2013). Similarly, when cells undergo
apoptosis in response to TNF receptor engagement, such cells can also undergo apoptosis that is associated with the production of a range of pro-inflammatory cytokines and chemokines by the dying cell (Kearney et al., 2013). The fact that Fas engagement can promote the production of pro-inflammatory cytokines and chemokine (such as IL-6, IL-8, CXCL1 and RANTES) is not surprising considering that this receptor recruits many of the same molecules, such as FADD, RIPK1 and the IKK complex, that are also recruited to the TNFR signalling complex. Despite the similarities in the Fas and TNF receptor signalling complexes, the pro-inflammatory aspects of Fas signalling have been much overlooked in the 20 or so years since the discovery of this receptor. Thus, there are certain situations where apoptotic cells can be pro-inflammatory through release of cytokines and chemokines rather than through release of DAMPs.
Necroptosis and inflammation Necroptosis, similar to necrosis, is also thought to result in the release of DAMPs into the extracellular space (Figure 2). For this reason, necroptosis is currently considered to be a highly inflammatory mode of cell death (Kacz marek et al., 2013; Moriwaki and Ka-Ming Chan, 2013). However, many of the stimuli that promote necroptosis, such as TNF and LPS, are intrinsically highly pro-inflammatory. This is important because rapid initiation of cell death in a cell that is engaged in a transcriptional inflammatory programme, as occurs upon TNF or LPS treatment, may lead to a less inflammatory outcome because of the rapid cessation of synthesis of pro-inflammatory cytokines and chemokines. Thus, whether necroptosis is more or less pro-inflammatory than the alternative depends on the relative potency of DAMPs, relative to conventional cytokines and chemokines. Following this line of reasoning, necroptosis may well transpire to be less inflammatory than TNF or LPS stimulation alone, as a result of the shutting down of TNF- or LPS-induced transcription of numerous pro-inflammatory factors. Further studies on the inflammatory outcomes of necroptosis are needed to resolve this issue.
RIPK3 and MLKL null animals as a model of necroptosis deficiency Many studies have utilised mice lacking RIPK3 as a model for necroptosis deficiency (Cho et al., 2009; He et al.,
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2009; Duprez et al., 2011; Linkermann et al., 2013). It has been reported that the detrimental effects of drug-induced liver damage, acute pancreatitis, the lethal effects of systemic TNF, and injury caused by ischemia-reperfusion are diminished in these animals. These observations have been widely interpreted to imply that necroptosis is a contributory factor in the damaging inflammatory responses observed in all of these situations. However, necroptosis is not typically observed in RIPK3-expressing cells unless caspase activation is inhibited and in the several of the latter studies, caspase inhibitors were not used. Therefore, the protective effects seen in RIPK3-deficient animals in various settings may be more related to a direct role for RIPK3 in promoting inflammation, rather than its role in promoting necroptosis under conditions where caspase activation is blocked. A similar argument can be made with respect to MLKL null animals, as MLKL-dependent cell death is not observed unless caspase inhibitors are employed. Thus, studies that have employed RIPK3 or MLKL null animals as ‘necroptosis-deficient’ animals may have overstated the case and these animals should instead be regarded as simply as RIPK3-deficient or MLKLdeficient. It follows logically therefore, that RIPK3 and MLKL could well be involved as signal transduction elements within inflammatory pathways, quite apart from their role in necroptosis under special conditions where caspase activation is blocked.
Conclusions Here we have argued that although the concept that cell death is a trigger for inflammation is very well established, there is much we still do not know concerning how the various modes of cell death (apoptosis, necrosis and necroptosis) influence inflammation. The precise identity of the key DAMPs is still the subject of speculation despite 20 years having elapsed since this concept was first advanced by Matzinger in 1994 (Table 1). Furthermore, it now appears that apoptosis is not always a non-inflammatory mode of cell death, particularly in the context where apoptosis is initiated by TNF, Fas and perhaps other members of the TNF family. Finally, it still remains to be determined whether necroptosis is more or less inflammatory than the alternative outcome where cell death occurs with the participation of caspases. Because excessive or persistent inflammation is a major cause of human disease, understanding how cell death contributes to inflammatory processes is likely to lead to important insights that contribute to the development of strategies to dampen or suppress inflammation in disease settings.
Acknowledgments: This work was supported by grants of the Russian government for state support of scientific research, as well as Science Foundation Ireland.
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