Caspase crosstalk: integration of apoptotic and innate immune signalling pathways

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Caspase crosstalk: integration of apoptotic and innate immune signalling pathways Emma M. Creagh School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College, Dublin, Ireland

The caspase family of cysteine proteases has been functionally divided into two groups: those involved in apoptosis and those involved in innate immune signalling. Recent findings have identified ‘apoptotic’ caspases within inflammasome complexes and revealed that ‘inflammatory’ caspases are capable of inducing cell death, suggesting that the earlier view of caspase function may have been overly simplistic. Here, I review evidence attributing nonclassical functions to many caspases and propose that caspases serve as critical mediators in the integration of apoptotic and inflammatory pathways, thereby forming an integrated signalling system that regulates cell death and innate immune responses during development, infection, and homeostasis. Apoptosis and inflammation: interlinked mechanisms on a common axis Apoptosis, or programmed cell death, is an essential process that occurs in all tissues during development, homeostasis, and disease [1]. In contrast to necrosis (see Glossary) and other forms of cell death where membrane disruption occurs, such as secondary necrosis, pyroptosis, and necroptosis, apoptosis is an immunologically silent form of cell death, whereby cells are rapidly phagocytosed and cleared without the initiation of an inflammatory response. By contrast, the inflammatory response is triggered by innate immune sensors following cellular damage, infection, or stress, and serves to clear the harmful stimulus and initiate healing. Caspases are a family of proteases that have been subdivided functionally into those involved in either apoptosis or inflammation. However, as research into both of these essential processes progresses, it has become evident that apoptosis and inflammation are inextricably linked in both lower and higher organisms (Box 1). Caspases are central to coordinating and integrating signals that result in not only apoptosis and inflammation but also other forms of programmed death, including pyroptosis and necroptosis. This view is supported by observations that proteins involved in apoptosis and inflammation contain common conserved protein domains, including caspase-associated Corresponding author: Creagh, E.M. ([email protected]). Keywords: apoptosis; caspase; innate immune signalling; inflammatory pathways. 1471-4906/ ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.it.2014.10.004

recruitment domains (CARDs) and death effector domains (DEDs), which are also present in caspases. Recent findings have revealed that classically ‘apoptotic’ caspases, particularly caspase-8, have essential roles in initiating inflammation, both directly and via inflammatory cell death pathways. Conversely, classically ‘inflammatory’ caspases are also emerging as essential drivers of cell death processes. Here, I review these recent findings and, based on this evaluation, propose that cell death and inflammation are outcomes of an integrated signalling system which is governed by caspases. Caspase activation mechanisms Caspases are cysteine proteases with primary specificity for aspartic acid (Asp) residues, cleaving their substrates after tetrapeptide sequences containing Asp in the P1 position. All caspases are synthesised as inactive single chain zymogens (procaspases), and they are all obligate heterodimers in their active forms; additional signals are required to facilitate procaspase modification and initiation of caspase activation pathways. Caspase zymogens consist of an N-terminal prodomain and a C-terminal protease domain, which has a large and a small subunit that contains the catalytic cysteine residue [1]. Initiator Glossary Apoptosome: a wheel-shaped heptameric protein complex, consisting of cytochrome c, Apaf-1, and caspase-9, which is formed during the intrinsic apoptosis pathway. The net result of apoptosome formation is the activation of caspase-9. Death-induced signalling complex (DISC): a multiprotein complex, which forms following the engagement of death receptors with their cognate ligands, recruiting adaptor proteins and caspases-8 or -10 into the complex. The net result of DISC formation is the activation of caspases-8 or -10. Extrinsic apoptosis: the pathway of DISC-mediated apoptotic cell death, activated following the engagement of death receptors of the TNF receptor family. Inflammasome: a multiprotein complex, which forms following the detection of pathogen or danger signals in the cellular cytosol. The net result of inflammasome formation is the activation of caspase-1. Intrinsic apoptosis: the pathway of mitochondrial-mediated apoptotic cell death, activated in response to cellular stresses, such as UV irradiation and chemotherapeutic agents. Necroptosis: a proinflammatory form of cell death, which is dependent on receptor-interacting protein kinases 1 and 3 (RIPK1 and RIPK3), and is inhibited by caspase-8. Necrosis: a passive, uncontrolled form of cell death characterised by cellular swelling, membrane lysis, and release of intracellular contents. Pyroptosis: an inflammatory form of programmed cell death, activated following cellular insults such as bacterial infection or exposure to toxins, and mediated by caspase-1 and/or caspase-11.

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Box 1. Cell death and immunity in the fly Drosophila melanogaster studies have greatly contributed to the functional understanding of caspases to date, and also serve to illustrate how cell death and immune processes are also regulated by caspases in lower organisms. There are seven Drosophila caspases, three of which (DRONC, DREDD, and STRICA) structurally resemble initiator caspases. The remaining four (DrICE, DCP-1, DAMM, and DECAY) have short prodomains and are classed as executioner caspases (reviewed in [86]). DRONC, the Drosophila orthologue of caspase-9, assembles into an apoptosome structure with the Apaf-1-related protein, DARK, resulting in the activation of executioner caspase, DrICE [87]. Apoptosomemediated DRONC activation drives stress-induced apoptosis and much of the developmentally related cell death that occurs in flies [88,89]. IAPs have a prominent role in Drosophila caspase regulation, as DIAP1 directly inhibits DRONC [90]. Apoptosis cannot proceed until a proapoptotic member of the RHG (Reaper, Hid, Grim) family, Sickle, or Jafrac-2 disrupts DIAP1-mediated caspase inhibition [91–93]. In addition to its apoptotic role, DRONC can also mediate processes, such as border cell migration, spermatid individualisation, and compensatory proliferation [94]. DREDD is the Drosophila orthologue of caspase-8. It contains two DED domains within its prodomain, through which it interacts with

caspases have long prodomains that consist of protein interaction domains: CARDs in caspases-1, -2, -4, -5, -9, 11, and -12, and DEDs in caspases-8 and -10. Caspase-10 was lost in rodents during evolution and- thus in mice, caspase-8 is the sole initiator caspase of the extrinsic death pathway [2]. These protein interaction domains facilitate the recruitment of initiator caspases into multiprotein activation complexes, such as the apoptosome, death-induced signalling complex (DISC), and inflammasomes (discussed further later). These multiprotein complexes serve to promote initiator caspase dimerisation and activation via the induced proximity mechanism [1,3]. By contrast, effector caspases (caspases-3, -6, and -7) have minimal prodomains and, once synthesised, exist as inactive homodimers until they are subsequently cleaved and activated by specific initiator/upstream caspases in a cascade-like manner. Effector caspase activation results in the cleavage of over 600 cellular substrates, some of which are involved in the apoptotic programme, while the consequences of the cleavage of other targets are still being examined [4]. Caspases during apoptosis Apoptosis, first described in 1972 by Kerr, Wyllie, and Currie [5], is activated via two distinct pathways, commonly referred to as the intrinsic and the extrinsic apoptotic pathways [3]. The intrinsic pathway is mediated by a component of the mitochondrial electron transport chain, cytochrome c, upon its release from the mitochondrial intermembrane space in response to cell stress. Once detected in the cytosol, cytochrome c triggers the rapid oligomerisation of apoptotic protease-activating factor-1 (Apaf-1), which recruits caspase-9 (via CARD/CARD interactions) into the apoptosome [6] (Figure 1). Active caspase9 then directly cleaves and activates downstream effector caspases, resulting in apoptosis. The extrinsic pathway results in initiator caspase-8 and caspase-10 activation within the DISC, via their DED-mediated interactions (Figure 1), which ultimately results in the activation of effector caspases and apoptosis. 2

the fly homologue, dFADD, to mediate immune responses. The humoral immune response in the fly is composed of two pathways: Toll and immune deficiency (IMD), both of which regulate alternative NFkB transcription factors. The IMD pathway, similar to the mammalian TNFR pathway, induces activation of Relish/NFkB transcription and the expression of anti-Gram-negative microbial peptides. Activation and cleavage of Relish (and its upstream adaptor protein, IMD) is mediated by dFADD and DREDD [95,96]. The IAP, DIAP2, is also essential for Relish activation [97]. DIAP2 promotes the proteolytic activity of DREDD towards Relish, revealing a novel, contrasting role for IAPs in the promotion of caspase activity [98]. An apoptotic role was originally predicted for DREDD following its characterisation, as Reaper- and Grim-induced killing was suppressed by heterozygosity at the dredd locus [99]. Overexpression of the Drosophila RING1 and YY-binding protein (dRYBP) induces RHDmediated apoptosis which requires dFADD and DREDD [100], although the physiological setting for this pathway has yet to be identified. However, a requirement for DREDD and Relish for neuronal cell death has been reported [101], suggesting that, similar to caspase-8, DREDD has the ability to mediate both immune and cell death pathways.

It is important to note that the engagement of death receptors and the recruitment of alternative adaptors [e.g., tumour necrosis factor receptor type 1-associated DEATH domain–receptor-interacting protein kinase (TRADD) and receptor-interacting serine/threonine protein kinase 1 (RIPK1) or Fas-associated death domain (FADD) and RIPK1] also signals for survival, differentiation, or other immune stimulatory activities, such as inflammation, via nuclear factor kB (NFkB), mitogen-activated protein kinase (MAPK), and c-Jun N-terminal kinase (JNK) signalling pathways [7]. Thus, differential regulation of the caspase-activating DISC complex can govern the fate of the cell. Although apoptosis is classically described as an immunologically silent mode of cell death, a study has shown that both transformed and primary cells, stimulated to undergo Fas-mediated apoptosis, produce moderate amounts of monocyte chemoattractant protein-1 (MCP-1) chemokine and IL-6 and IL-8 cytokines [8]. Fas stimulation was also shown to induce phagocyte migration in vivo, suggesting that it activates both apoptotic and proinflammatory pathways to facilitate the swift removal of dying cells. Apoptotic lymphocytes have also been shown to attenuate proinflammatory cytokine secretion from lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells (PBMCs) [9], and it has been proposed that apoptotic caspases serve to dampen inflammation by cleaving and inactivating otherwise proinflammatory cellular signals during apoptosis [10]. Therefore, certain apoptotic processes actively engage with the immune system to coordinate their efficient clearance from tissues, while minimising inflammation and its consequences on surrounding tissues. Caspases during inflammation Caspase-1: the prototypical inflammatory caspase IL-1b and IL-18 are potent proinflammatory cytokines that induce fever and interferon g (IFNg) secretion, respectively. Their production is under tight regulation: firstly, NFkB activation is required for the transcriptional upregulation

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Figure 1. Role of caspases in the initiation and execution of apoptosis. Apoptosis is initiated via intrinsic or extrinsic pathways. The intrinsic pathway is activated following cellular stress, which results in mitochondrial perturbation and release of cytochrome c (cyt.c). Cyt.c release is regulated by B cell chronic lymphocytic leukaemia/ lymphoma-2 (Bcl-2) family members (e.g., proapoptotic – Bax, Bak, and tBid; antiapoptotic – Bcl-2, Bcl-XL). Once released, cyt.c binds to apoptotic protease-activating factor-1 (Apaf-1), triggering the formation of the apoptosome complex, resulting in the activation of initiator caspase-9. The extrinsic pathway is initiated following the activation of death receptors of the tumour necrosis factor (TNF) superfamily. The trimerised receptor–ligand complex recruits procaspase-8 to the death-induced signalling complex (DISC) via the adaptor protein Fas-associated death domain (FADD), causing the rapid activation of initiator caspase-8. Activated initiator caspases-8 and -9 then process and activate the downstream effector caspases-3, 6, and 7, which are responsible for the classical phenotypic changes associated with apoptosis.

of IL-1b and, secondly, both IL-1b and IL-18 are synthesised as immature proforms, which require processing for their maturation and extracellular secretion, before they can mediate their proinflammatory functions [11]. The primary mechanism responsible for the processing of proIL-1b and proIL-18 is their cleavage by caspase-1. As with other initiator caspases, caspase-1 must itself first become activated, and this occurs within inflammasomes. In addition to the proteolytic activation of IL-1b and IL-18, inflammasome activation also results in another caspasedependent form of cell death, – pyroptosis [12] – as discussed further later. Several inflammasome complexes have been identified to date. These generally contain an inflammasome sensor protein, the adaptor protein apoptosis-associated specklike protein with a CARD (ASC), and caspase-1 [13]. The

inflammasome sensor proteins, which detect cytosolic pathogen-associated molecular patterns (PAMPs) or danger-associated molecular patterns (DAMPs), trigger inflammasome formation upon recognition of the pathogen or danger signal. Eight inflammasome sensor proteins have been identified thus far, and most of these contain Nod-like receptor proteins (NLRPs), with absent in melanoma (AIM2) and interferon-inducible protein-16 (IFI-16) being notable exceptions. Most NLRPs share a common architecture consisting of three functional domains: an Nterminal CARD or PYRIN (PYD) protein interaction domain; a central nucleotide-binding domain (NBD), which is thought to facilitate oligomerisation; and C-terminal leucine-rich repeats (LRRs), which facilitate pathogen sensing [14]. AIM2 and IFI16 are members of the PYHIN (PYD and HIN domain-containing proteins) family. AIM2 has one 3

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Figure 2. Proposed roles for caspases in IL-1b-mediated inflammation. (A) The canonical Nod-like receptor protein 3 (NLRP3) inflammasome requires (1) a pattern recognition receptor (PRR)-induced priming event for nuclear factor kB (NFkB)-mediated upregulation of IL-1b and NLRP3, and (2) cytosolic pathogen-associated molecular pattern (PAMP)- or danger-associated molecular pattern (DAMP)-mediated activation of NLRP3, resulting in inflammasome complex formation and caspase-1 autoactivation. (B) The non-canonical inflammasome requires (1) priming of procaspase-11 in addition to the NLRP3 priming event, and (2) activation of caspase-11 following cytosolic lipopolysaccharide (LPS) detection, before (3) NLRP3 inflammasome formation can occur. (C) A non-canonical caspase-8 inflammasome can form following Dectin-1 stimulation, resulting in a multiprotein complex consisting of mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1), apoptosisassociated speck-like protein with a CARD (ASC), and caspase-8, leading to caspase-8-mediated IL-1b maturation. (D) Caspase-8 and Fas-associated death domain (FADD) can promote both (1) priming and (2) activation of both canonical and non-canonical inflammasomes in bone marrow-derived macrophages (BMDMs). (E) Inflammasomeindependent processing of caspase-1 via caspase-8 can occur under conditions of bacterial blockade of NFkB and mitogen-activated protein kinase (MAPK) signalling pathways. All scenarios result in caspase-1- and/or caspase-8-mediated processing of IL-1b into its mature, proinflammatory form.

DNA-binding HIN domain, which recognises nucleic acids, and a PYD domain to recruit ASC [15]. IFI16 has one PYD and two HIN domains, and signals via STING to coordinate type I IFN responses [16,17]. The NLRP3 inflammasome is so far the best characterised and understood in terms of activation and function. NLRP3 is activated upon detection of a broad range of PAMPs and DAMPs, including ATP and uric acid crystals [18,19]. Electron microscopy methods were recently used to examine the architecture of in vitro reconstituted AIM2 and NLRP3 inflammasomes [20]. A unified model of inflammasome assembly was proposed wherein stimulation of NLRP3 or AIM2 induces nucleation of ASC into helical clusters via PYD/PYD interactions. The oligomerised ASC CARDs form a platform for caspase-1 CARDs to nucleate into filaments, which, in turn, bring caspase protease domains into proximity for dimerisation, transautocleavage, and activation [20]. 4

Inflammasomes are regulated at multiple levels, and some NLRs require coreceptors for ligand recognition or inflammasome stability (e.g., NLRP1 requires Nod2; and NLRC4 requires NAIPs) [21–23]. NLRP3 is also under stringent transcriptional regulation, requiring NFkB-mediated transcriptional activation (Figure 2A) [24]. Thus, the activation of the NLRP3 inflammasome is dependent on two regulatory steps: (i) the ‘priming’ step – NFkBdependent expression of caspase-1 substrate proIL-1b and NLRP3; and (ii) intracellular PAMP/DAMP detection by NLRP3. Other regulatory processes, including ubiquitination and phosphorylation, also govern inflammasome activity. For example, it has been recently demonstrated that ASC ubiquitination and NLRP3 inflammasome assembly are dependent on the linear ubiquitin assembly complex (LUBAC) [25], and deubiquitinase enzymes have also been shown to be required for NLRP3 inflammasome activation [26]. Furthermore, Syk- and Jnk-dependent

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Review phosphorylation of ASC has been proposed as a critical step in the activation of the NLRP3 inflammasome in murine peritoneal macrophages [27]. Non-canonical inflammasome regulation by caspase-11 Caspase-11 is transcribed from the same chromosomal locus as caspase-1, and has been traditionally classed as an inflammatory caspase. Mice deficient in caspase-11 failed to produce mature IL-1 and were resistant to endotoxic shock, suggesting a role for caspase-11 in the regulation of caspase-1 activation [28]. Caspase-11 expression in murine tissues is usually very low; however, following LPS administration caspase-11 is robustly induced in tissues including the thymus, lung, spleen, and kidney [29]. Similarly, in innate immune cells, such as monocytes and macrophages, where caspase-1 is constitutively expressed [30], expression of caspase-11 is induced in vitro by proinflammatory stimuli including LPS, IFNb, and IFNg [31,32]. The caspase-11 promoter region contains several putative transcription factor-binding sites, including partially overlapping NFkB- and signal transducer and activation of transcription (STAT)-binding sites [31,32]. In mice, caspase-11 activation is required for NLRP3 inflammasome activation in response to infection by Gram-negative bacteria, which has been termed non-canonical NLRP3 inflammasome activation [33] (Figure 2B). Although there is no direct homologue of caspase-11 in humans, caspases-4 and -5 are considered to be its functional orthologues, and are proposed to have arisen following a gene duplication event [34]. Caspase-4 has been implicated in NLRP3 inflammasome activation in keratinocytes following UVB irradiation [35]; and transgenic mice expressing human caspase-4 display higher endotoxin sensitivity, with bone marrow-derived macrophages (BMDMs) from these mice producing mature IL-1b and IL-18 following Toll-like receptor 2 (TLR2) or TLR4 priming alone [36]. Caspase-5 was originally identified as a component of the NLRP1 inflammasome [37]. Although evidence exists to support inflammasome regulation by human caspases-4 and -5, the majority of studies surrounding non-canonical inflammasome activation have been carried out in primary murine BMDMs. Following Gram-negative infection of BMDMs, LPS-mediated upregulation of caspase-11 occurs via a TLR4–TRIF–type I IFN pathway [32]. Once upregulated, caspase-11 has been shown to become activated by cytosolic LPS (more specifically, its hexa-acyl lipid A moiety) [38,39]. A direct interaction between intracellular LPS and the CARD of caspase-11 (and human caspases-4 and -5) has recently been proposed as the activation stimulus [40]. A role for the IFN-inducible guanylate-binding proteins (GBPs) in cytoplasmic LPS-mediated caspase-11 activation has been proposed by two separate studies (Figure 2B) [41,42]. One reports that GBPs facilitate the lysis of vacuoles and subsequent release of Gram-negative bacteria (and their LPS moieties) into the cytosol, causing caspase-11 activation and triggering non-canonical inflammasome activation [41]; whereas the other study observed no effect of GBPs on vacuolar disruption [42], leaving the mechanism of GBP-mediated promotion of non-canonical inflammasome activation still in question. During Legionella

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infection, active caspase-11 is reported to regulate cofilin phosphorylation, mediating actin polymerisation and phagosome–lysosome fusion, to facilitate the release and subsequent detection of cytosolic flagellin [43]. To summarise, recent findings suggest that caspase-11 is activated during the host response against Gram-negative bacterial infection, which involves induction of GBP GTPases to facilitate the release of PAMPs from cytosolic vesicles, the direct detection of cytosolic LPS by caspase11, cytoskeletal rearrangement, and activation of the noncanonical inflammasome. Caspase-8: an unexpected mediator of inflammation Recent studies have revealed distinct roles for caspase-8, traditionally associated with the extrinsic apoptotic pathway, in the regulation of inflammation. Caspase-8 activation has been associated with the inhibition of necroptosis, and thus proposed to have an anti-inflammatory role [44]. However, caspase-8 has also been proposed to induce inflammation through several different mechanisms: (i) by direct cleavage of proIL-1b into its mature, active form [45–52]; (ii) by its incorporation and activation within inflammasome complexes [53,54]; and (iii) by direct activation of caspase-1, in an inflammasome-independent manner [55,56]. Examples of each scenario are discussed as follows. Dectin-1, a member of the C-type lectin receptor family, is expressed on the surface of phagocytes and binds to fungal surface glycans. Dectin-1 engagement results in Syk kinase-mediated signalling and the formation of a noncanonical caspase-8 inflammasome complex in primary human dendritic cells [45]. The complex is composed of mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1), caspase-8, and ASC, and has been reported to directly process and activate IL-1b [45] (Figure 2C). This study observed concomitant activation of the NLRP3 inflammasome following dectin-1 internalisation. NLRP3 activation has been implicated in dectin-1 signalling in mice [46,47], and whether cooperation exists between the caspase-8 and caspase-1 inflammasome pathways would be interesting to examine. Others have reported caspase-8-mediated processing of IL-1b following TLR3/TLR4 stimulation of 293T cells transfected with both caspase-8 and proIL-1b (and under conditions of protein synthesis inhibition); and following stimulation of immortalised murine BMDMs with LPS and the Smac mimetic compound A [inactivates inhibitor of apoptosis proteins (IAPs)] [48–50]. Furthermore, death receptor signalling, via Fas, which engages the apoptotic cell death via DISCmediated caspase-8 activation (Figure 1), has also been shown to result in caspase-8-mediated IL-1b maturation in a manner independent of caspase-1 and RIPK3 [51]. Thus, caspase-8 can function in both apoptotic and inflammatory signalling pathways. Recently, roles for caspase-8 in the priming and activation of both canonical and non-canonical NLRP3 inflammasomes have been reported. LPS-stimulated RIPK3 / FADD / and RIPK3 / Casp8 / BMDMs had decreased proIL-1b mRNA levels. At the post-translational level, canonical and non-canonical inflammasome stimuli (e.g., LPS and ATP; and Citrobacter rodentium, respectively) 5

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Review resulted in NLRP3-dependent caspase-8 processing, and deletion or inhibition of caspase-8 inhibited caspase-1 activation and IL-1b secretion [53], suggesting that both caspase-1 and caspase-8 are activated by the NLRP3 inflammasome (Figure 2D). In addition, caspase-8 has been identified within an NLRP3/NLRC4 inflammasome complex: confocal and super-resolution imaging approaches revealed that, following Salmonella infection, active forms of both caspase-1 and caspase-8 are present within the core of the multiprotein inflammasome complex, surrounded by NLRP3, NLRC4, and ASC [54]. The authors propose that this is a dynamic arrangement, with active caspases situated within the functional core, and precursor proforms localised to the exterior ASC ring. This conformation would allow for substrate specificity, permitting only bone fide substrates, such as IL-1b and IL-18, access to the active caspase core for processing. These findings are in conflict with the NLRP3 and AIM2 inflammasome crystal structures recently reported from electron microscopy methods, which suggest that caspase-1 clustering occurs on the outer ring of the complex and has yet to be shown under physiological conditions [20]. However, the study by Man et al. [54] imaged inflammasome structures, which formed in murine macroinfection, at endogenous phages following Salmonella levels of inflammasome components, and thus is a more likely structure to occur in vivo following Salmonella infection. In turn, caspase-1 can be directly activated by caspase-8 to trigger both cell death and inflammation. Following a Yersinia bacterial challenge, RIPK3 / FADD / , RIPK3 / Casp8 / , and RIPK1 / BMDMs (but not RIPK3 / BMDM) had decreased caspase-1 processing and IL-1b production, suggesting that caspase-8 is required for inflammasome-independent-mediated activation of caspase-1 and cell death [55,56]. YopJ, the Yersinia virulence factor, which inhibits NFkB and MAPK signalling, is proposed to be the activator caspase-1 and cell death via this caspase-8, FADD, and RIPK1-dependent pathway [55] (Figure 2E). Once elucidated, the mechanisms governing caspase-8 regulation in this context should provide significant insight into its general integrative mechanisms during cell death and inflammation. This section highlights the observation that caspases-1, -8, and -11 cooperate to coordinate host innate immune responses against infection or injury. The many alternative mechanisms leading to the activation of inflammation via caspases suggest that their activities may be tailored by the nature of the insult, to activate the required degree of inflammation and specificities of the cytokine response. Furthermore, inflammatory forms of cell death are also regulated by caspases, as will now be discussed. Roles of caspases in inflammatory forms of cell death Cell death has been traditionally thought to follow apoptosis, a caspase-controlled process, or to follow necrosis, an uncontrolled response to tissue damage or other severe cellular insults. In addition, two other forms of cell death – pyroptosis and necroptosis – have been identified. Both pyroptosis and necroptosis are regulated by caspases and both are proinflammatory. That caspases are responsible for initiating proinflammatory cell death mechanisms 6

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highlights just how tightly intertwined the inflammation and cell death processes are, and suggests that differential activation of caspases can dictate whether a cell survives, initiates inflammation, or dies via either immunologically silent or activating forms of cell death. Pyroptosis and IL-1 production Pyroptosis is a caspase-dependent form of cell death that differs from apoptosis morphologically, biochemically, and in its proinflammatory nature (reviewed in [12]). Whereas DNA fragmentation occurs during pyroptosis, other features associated with apoptosis do not occur. Pyroptosis is morphologically characterised by cell swelling and osmotic plasma membrane lysis. Membrane lysis results in the release of cytosolic proteins, such as lactate dehydrogenase (LDH), and LDH release assays are commonly used as an experimental readout for pyroptosis. Inflammasome-mediated activation of caspase-1 and/or caspase-11 appears to be the key driver of the process (Figure 3A) [33,57]. Salmonella strains constitutively expressing PAMPs such as flagellin, which could not be cleared by NLRC4- or Casp1deficient mice, were still efficiently cleared by IL-1/IL-18 double knockout mice, revealing the importance of pyroptosis in bacterial clearance [57]; and caspase-11 triggers caspase-1-independent pyroptosis in response to non-canonical activators, such as Escherichia coli and C. rodentium [33]. Caspase-7 has also been implicated in pyroptosis following its cleavage by NLRC4 inflammasome-activated caspase-1 during bacterial infection [58,59]. Bacterial infection can result in rapid pyroptotic cell death, which is independent of IL-1b maturation, and causes the release of bacteria from cells to initiate a rapid neutrophil-mediated phagocytic secondary response, thus pyroptosis has been proposed as a physiological process to efficiently clear intracellular bacteria [60]. However, it is unclear whether the converse is true, that is, whether IL-1b release can occur in the absence of pyroptosis. There are two schools of thought on this issue: one suggests that IL-1b release is mediated by caspase-1 via an unconventional IL-1b secretion pathway [61], while another hypothesis suggests that IL-1b is released passively by cells during pyroptosis. The latter hypothesis could not explain how sustained cytokine maturation and production would occur, as it would be accompanied by the death of the activated cell. However, recent reports have provided evidence in support of this notion: (i) neutrophils are capable of releasing inflammasome-mediated IL-1b without any concomitant pyroptosis, and thus could serve to propagate the inflammatory signal [62]; and (ii) release of active inflammasomes from pyroptotic cells can act as danger signals to local phagocytes, serving to amplify the inflammatory response by subsequently inducing inflammasome formation and further IL1b release within those phagocytes [63,64]. Therefore, it is likely that both of the above hypotheses may be valid, depending on the cell type. Caspase-1 (and caspase-11) induce IL-1b release and pyroptosis in macrophages, and IL-1b secretion in the absence of pyroptosis in neutrophils. Caspase-8-mediated regulation of necroptotic cell death Caspase-8 and caspase-10 are primary mediators of death receptor-induced apoptosis and, furthermore, as discussed

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Figure 3. Involvement of caspases in nonapoptotic cell death mechanisms. (A) Caspase-1-and/or caspase-11-mediated inflammasome activation result in the inflammatory form of cell death, pyroptosis. (B) Ripoptosome formation is triggered following genotoxic stress and loss of inhibitor of apoptosis proteins (IAPs), the exact signalling outcome is regulated by cellular FLICE inhibitory protein (cFLIP) isoforms. (C) Under conditions of caspase inhibition, engagement of T cell receptor (TCR), Toll-like receptor (TLR), tumour necrosis factor receptor (TNFR), or viral infection can induce receptor-interacting protein kinase 3 (RIPK3) oligomerisation and necroptosome formation, resulting in mixed lineage kinase domain-like (MLKL) protein phosphorylation, oligomerisation, and translocation to the plasma membrane, causing lysis.

earlier, several lines of evidence implicate caspase-8 as an inflammatory mediator. However, the prenatal lethal phenotype of the caspase-8 knockout mouse, which is at odds with phenotypes of knockout mutations of death receptors or ligands known to employ caspase-8 in their apoptotic pathway, suggests a survival role for caspase-8 during embryonic development [65]. Furthermore, identification of a human CASP8 mutation was shown to result in defective T, B, and natural killer (NK) cell activation (in addition to defective lymphocyte apoptosis) in individuals homozygous for the mutation [66]. These findings could not be explained by the apoptotic or inflammatory roles of

caspase-8 and point instead to a paradoxical prosurvival role for caspase-8. This is explained, in part, by the negative regulatory influence of caspase-8 on the receptorinteracting protein kinases, RIPK1 and RIPK3, which can form both ripoptosome and necroptosome complexes [44,67]. The ripoptosome complex contains caspase-8, FADD, and RIPK1, and can mediate cell survival, apoptosis, inflammation (and pyroptosis), or necroptosis (Figure 3B) (reviewed in [68]). The levels of cellular FLICE inhibitory protein (cFLIP) isoforms can dictate the outcome of ripoptosome formation. All cFLIP isoforms contain two DEDs and can be recruited to the DISC. Short isoforms 7

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Review (cFLIPS/cFLIPR) inhibit caspase-8 activation, whereas at certain concentrations the long isoform (cFLIPL) can heterodimerise with caspase-8 to promote its activity. Engagement of death receptors, such as Fas/CD95R, but particularly tumour necrosis factor receptor 1 (TNFR1), results in necroptosis in the presence of caspase inhibitors [69,70]. This RIPK-dependent form of cell death has also been observed under caspase-inhibitory conditions during other processes, including T cell activation and TLR ligation (Figure 3C) [71,72]. Caspase-8 has been shown to play a major role in restricting the RIPK1/RIPK3-mediated induction of necrotic cell death, thought to be via processing of activated RIPK1 and RIPK3 molecules [73,74]. As necroptosis initiates inflammation, caspase-8 can be described as having an anti-inflammatory role in this context. In support of this, spontaneous inflammation has been shown to develop in the mouse skin when caspase-8 was deleted from basal cells of the epidermis, and deletion of caspase-8 from enterocytes was also shown to trigger chronic inflammation of the intestine [75,76]. The precise mechanisms regulating the interplay among caspase-8driven apoptosis, inflammation, and inhibition of RIPK3 necroptosis are still being uncovered, but the most recent evidence suggests that RIPK1 can regulate both FADD– caspase-8 apoptosis and RIPK3 necroptosis signalling pathways [77,78]. In addition, recent data suggesting that RIPK3 can activate inflammasome-dependent IL-1b production, in the absence of cell death, suggest an additional role for necroptosis during inflammation [79]. An integrated signalling system that regulates cell death and innate immune responses This article proposes an integrated signalling system, mediated by caspases, that regulates both cell death and inflammatory responses in cells. Evidence discussed in the article reveals the critical roles of caspases in driving the activation and execution of cell death and inflammatory processes, and highlights caspases that are involved in the coordination of both processes. Two families of proteins, which were initially identified as apoptotic regulators (Figure 1), the IAPs and the B cell chronic lymphocytic leukaemia/lymphoma-2 (Bcl-2) proteins, can also regulate inflammation. Through their ubiquitin ligase activities, IAPs have been found to regulate multiple aspects of innate immune regulation, including LPS-mediated TLR4 signalling via MyD88, NOD1/2 signalling, and antiviral RIG-1 responses [80]. The antiapoptotic Bcl-2 proteins, Bcl-2 and Bcl-XL, have been demonstrated to negatively regulate the NLRP1 inflammasome by binding NLRP1, preventing inflammasome formation [81]; and there is debate over whether Bcl-2 can also inhibit NLRP3 inflammasome activation [82,83]. The ability of IAPs and Bcl-2 family members to influence both apoptotic and inflammatory processes strengthens the proposal that these processes are common to a singular pathway regulated by caspases, which serve to tailor the response to result in an apoptotic, inflammatory, or inflammatory cell death outcome. The ‘decision’ for caspases to result in a specific outcome may be dictated by the cellular insult and the nature of death/inflammatory receptors that become engaged. 8

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One outcome of the pathway is apoptosis, which is triggered in response to mitochondrial stress or death receptor engagement. During apoptosis, effector caspases, initiated following caspase-9 or caspase-8 activation, induce the efficient deconstruction of the cell and packaging of its contents into apoptotic bodies that signal for their own removal by phagocytosis, without the initiation of inflammation. Detection of tissue infection or damage through the recognition of PAMPs/DAMPs by receptors of innate immune cells initiates their NFkB- and IFNmediated ‘priming’ and the transcriptional upregulation of procaspase-11 and other components, such as NLRP3 and GBPs. The second outcome of the caspase-mediated pathway is inflammation, which is triggered in response to intracellular detection of PAMPs/DAMPs and is coordinated by caspases-1, -8, and -11 to produce mature IL-1b and IL-18. The inflammatory outcome is often closely followed by the third and final outcome of inflammatory cell death, which is also coordinated by the trio of caspases-1, -8, and 11 during bacterial infection. The combined outcome of inflammation and cell death facilitates enhanced clearance of virulent bacterial infections from the host system. The caspase-mediated pathway also serves to limit necrotic forms of cell death, such as RIPK3-mediated necroptosis, as they illicit an uncontrolled host response, causing significant tissue damage. Concluding remarks This review proposes caspases as the central regulators of apoptotic and inflammatory processes. Additional studies to support this proposal have shown nonapoptotic functions for caspases-3 and -7 in processes, such as proliferation, cell cycle, and inflammation, and proapoptotic functions for caspase-1 [84,85]. This review highlights the ability of distinct caspases to regulate three alternative cell death pathways: apoptosis, pyroptosis, and necroptosis, which reveals the pleiotropic effects of caspases in their ability to induce or suppress inflammation and cell death processes. It proposes that the previous segregation of caspases into apoptotic or inflammatory groups is no longer possible, and places caspases at the regulatory core of the apoptosis–inflammation signalling pathway. The recent findings discussed here raise many questions. It is still not known what signals dictate the ability of caspases to divert from apoptotic to inflammatory outcomes. Could transcriptional- and post-translational-mediated control of the levels of key components of signalling pathways (as seen for NLRP3) be sufficient to dictate the cellular response? Do additional substrates for caspases-1, -8, and -11, which could activate pyroptosis and inflammation, exist? Finally, is cell death an inevitable consequence of IL-1b release in macrophages, and what cell types remain viable following inflammasome activation? Many complexities of the caspase-mediated signalling pathway have yet to be deciphered. Acknowledgements Thanks to Aisling Dunne for critical review of the manuscript. The Creagh laboratory is supported by grants from the Science Foundation Ireland (SFI) and the Programme for Research in Third-Level Institutions (PRTLI) in Ireland, which is cofunded by the European Regional Development Fund and the Higher Education Authority (HEA).

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