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Keywords:

  • Apoptosis;
  • necroptosis;
  • necrostatin;
  • regulated necrosis;
  • RIPK1;
  • RIPK3

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. The Detection of CICD and Necroptosis
  5. In vivo Relevance of Necroptosis—A Second-Line Defense Mechanism Against Viruses Can Result in Maladaptive Inflammatory Injury
  6. CDAMPs in Necroptosis—Strong Inducers of Innate and Adaptive Immune Responses
  7. Blocking Necroptosis in IRI
  8. Interference With Necroptosis—Open Questions
  9. Translation of Basic Insights Into Transplantation Studies and Practice
  10. Acknowledgments
  11. Disclosure
  12. References
  13. Supporting Information

Transplantation is invariably associated with ischemia–reperfusion injury (IRI), inflammation and rejection. Resultant cell death has morphological features of necrosis but programmed cell death has been synonymous with apoptosis until pathways of regulated necrosis (RN) have been described. The best-studied RN pathway, necroptosis, is triggered by perturbation of caspase-8-mediated apoptosis and depends on receptor-interacting protein kinases 1 and 3 (RIPK1/RIPK3) as well as mixed linage kinase domain like to form the necroptosome. The release of cytosolic content and cell death-associated molecular patterns (CDAMPs) can trigger innate and promote adaptive immune responses. Thus, the form of cell death can substantially influence alloimmunity and graft survival. Necroptosis is a key element of IRI, and RIPK1 interference by RN-specific inhibitors such as necrostatin-1 protects from IRI in kidney, heart and brain. Necroptosis may be a general mechanism in response to other forms of inflammatory organ injury, and will likely emerge as a promising target in solid organ transplantation. As second-generation RIPK1 and RIPK3 inhibitors become available, clinical trials for the prevention of delayed graft function and attenuation of allograft rejection-mediated injury will emerge. These efforts will accelerate upon further identification of critical necroptosis-triggering receptor(s).


Abbreviations
CAD

caspase-activated DNase

CDAMP

cell death-associated molecular pattern

cIAP

cellular inhibitor of apoptosis protein

CICD

caspase-independent cell death

CYLD

cylindromatosis

DISC

death-inducing signaling complex

Drp-1

dynamin-related protein 1

DUB

de-ubiquitinase

FADD

Fas-associated death domain containing molecule

FLIP

FLICE-like inhibitory protein

HMGB1

high-mobility group box 1

HSP

heat-shock protein

IAP

inhibitor of apoptosis signaling

IEC

intestinal epithelial cell

IRI

ischemia–reperfusion injury

lin-UB

linear ubiquitination

LUBAC

linear ubiquitin chain assembly complex

MAPK

MAP-kinase

MCAO

middle cerebral artery occlusion

MLKL

mixed lineage kinase domain like

Nec-1

necrostatin-1

NF-κb

nuclear factor-κB

NMDA

N-methyl-D-aspartate

PCD

programmed cell death

PGAM5

phosphoglycerate mutase 5

RHIM

RIPK-homology-interacting motive

RIPK

receptor-interacting protein kinase

RLR

RIG-I-like receptors

RN

regulated necrosis

TLR

Toll-like receptor

TNF

tumor necrosis factor

TNFR

tumor necrosis factor receptor

TNF-RSC

TNFR-signaling complex

TRADD

TNFR-associated factor with death domain

TRAF2

TNFR-associated factor 2

zVAD

zVAD-fmk

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. The Detection of CICD and Necroptosis
  5. In vivo Relevance of Necroptosis—A Second-Line Defense Mechanism Against Viruses Can Result in Maladaptive Inflammatory Injury
  6. CDAMPs in Necroptosis—Strong Inducers of Innate and Adaptive Immune Responses
  7. Blocking Necroptosis in IRI
  8. Interference With Necroptosis—Open Questions
  9. Translation of Basic Insights Into Transplantation Studies and Practice
  10. Acknowledgments
  11. Disclosure
  12. References
  13. Supporting Information

The term “programmed cell death” (PCD) has long been used synonymously with caspase-dependent apoptosis, while necrotic cell death and caspase-independent cell death (CICD) have been thought to occur “accidentally” that is without the involvement of an underlying genetic program. This view has recently radically changed with the discovery of signaling pathways that are capable of inducing regulated necrosis (RN) [1-3]. The best characterized of these is necroptosis, a pathway of RN that is dependent on the kinase activity of receptor-interacting protein kinase 3 (RIPK3) [4-6]. The physiological in vivo relevance of necroptosis is profound and obvious (Table S1) as the lethal phenotype of caspase-8-deficient mice, which otherwise die in utero at Day 10.5 of embryonic development, is completely reversed with the additional deletion of RIPK3 [7, 8]. The evolving understanding of the RIPK-family-dependent necroptotic pathway has led to the development of specific inhibitors of necroptosis. The RIPK1 kinase inhibitor necrostatin-1 (Nec-1) has already proven to be of benefit in preclinical models of ischemia–reperfusion injury (IRI) [9-11]. However, due to the rapid progression of necroptotic signaling, clinically feasible interference to attenuate organ injury may only be possible when necroptosis can be anticipated, such as with ischemia related to surgery as well as solid organ transplantation [12, 13]. Furthermore, there are substantial data that cell death by necrosis heavily triggers the immune system by the release of cell death-associated molecular patterns (CDAMPs) [6, 14]. It is tempting to speculate that CDAMPs, by their ability to activate both innate and adaptive immunity, promote many of the harmful immunologic responses observed in solid organ transplants. Therefore, we propose interference with pathways of RN in general, and of necroptosis specifically, to be beneficial in solid organ transplantation for two major reasons. First, the prevention of necrotic cell death minimizes the loss of functional parenchymal cells in the transplant; second, the reduction of CDAMPs would reduce pro-inflammatory responses that activate rejection pathways. In this review, we focus on the underlying pathway of necroptosis and the release of necroptosis-associated CDAMPs, and importantly the potential clinical relevance of necroptosis in several clinical models as well as IRI and transplantation.

The Detection of CICD and Necroptosis

  1. Top of page
  2. Abstract
  3. Introduction
  4. The Detection of CICD and Necroptosis
  5. In vivo Relevance of Necroptosis—A Second-Line Defense Mechanism Against Viruses Can Result in Maladaptive Inflammatory Injury
  6. CDAMPs in Necroptosis—Strong Inducers of Innate and Adaptive Immune Responses
  7. Blocking Necroptosis in IRI
  8. Interference With Necroptosis—Open Questions
  9. Translation of Basic Insights Into Transplantation Studies and Practice
  10. Acknowledgments
  11. Disclosure
  12. References
  13. Supporting Information

While necrosis has historically been regarded as an unregulated means of cell death that is induced by severe nonspecific and nonphysiological stress, it has recently become clear that necrosis can also occur as a highly regulated process [3, 15, 16]. The first identification of what is now generally referred to as “necroptosis” emerged from the fibrosarcoma cell line L929 in which combined stimulation with tumor necrosis factor (TNF) and the pan-caspase inhibitor zVAD-fmk (zVAD) induced necrotic cell death [17]. Normally, binding of TNF to TNF receptor 1 (TNFR1) leads to intracellular recruitment of RIPK1, TNFR-associated factor (TRAF) with death domain (TRADD), TRAF2, the cellular inhibitor of apoptosis proteins 1 and 2 (cIAP1 and cIAP2), and the linear ubiquitin chain assembly complex (LUBAC) (Figure 1) [3]. This is also referred to as formation of the TNF-R1 signaling complex (TNF-RSC). As a consequence of the E3 ubiquitin ligase activities of the two cIAPs and of LUBAC RIPK1 becomes polyubiquitinated and serves as the major hub for signals that ultimately lead to activation of nuclear factor κB (NF-κB) as well as the mitogen-activated protein kinase pathways [18, 19]. It is now clear that different types of ubiquitin linkages, including lysine 63 (K63) and linear ubiquitin (lin-Ub) linkages, cooperate in the TNF-RSC to enable activation of these pathways at the exact physiologically required level [18]. When LUBAC or cIAPs do not work properly, the consequences for the organism are severe. The LUBAC complex consists of three proteins, HOIL-1, HOIP and SHARPIN [20-23]. SHARPIN-deficient mice develop a detrimental skin phenotype and dehydration state due to the loss of the inside-out barrier, which results in death at an age of approximately 3 months, which is completely prevented by concomitant genetic ablation of TNF [20]. HOIL-1 mutations and LUBAC deficiency have recently been described to cause immunodeficiency in humans [24]. cIAP-deficiency results in an almost complete lack of functional polyubiquitination of RIPK1 and is of outstanding importance for the integrative NF-κB-signal downstream of Complex I [25]. NF-κB activation subsequently results in a strong pro-survival signal [3] (Figure 1). The TNF-RSC, which is also referred to as Complex I of TNF signaling, is destabilized by the activity of the de-ubiquitinase (DUB) known as cylindromatosis (CYLD), an enzyme that removes ubiquitins from RIPK1 and most likely other ubiquitinated components of Complex I, thereby counteracting the activities of cIAPs and LUBAC [26]. In the deubiquitinylated state, RIPK1 is released from Complex I and rendered capable of recruiting Fas-associated death domain (FADD) and procaspase-8, resulting in the formation of Complex II of TNF signaling. The formation of this complex results in activation of caspase-8, and this “initiator” caspase can then cleave the downstream apoptotic effector caspases-3, -6 and -7 [27]. Interestingly, although RIPK1 is necessary for a maximal apoptotic response, it is worth noting that active caspase-6 cleaves RIPK1 to prevent the necroptotic signal [28]. Similarly, the presence of the long version of the cellular FLICE-like inhibitory protein (cFLIPL), a caspase-8 homolog that lacks the active center cysteine typical for caspases and essential for their activity as proteases, leads to formation of caspase-8/FLIPL heterodimers. These heterodimers are still active as caspases but the activity is different from that of casapse-8 homodimers; most importantly, the activity of the caspase-8/FLIPL heterodimer prevents necroptosis [1, 8], presumably by cleaving the DUB CYLD that functions as an upstream regulator of RIPK1 deubiquitinylation, upon other mechanisms by controlling the cIAPs [29]. Collectively, and in contrast to the previous dogma, these observations indicate that the principal in vivo function of caspase-8, at least during development, is not the execution of apoptosis, but rather the prevention of necroptosis [1, 30]. It is of importance to stress that TNF-RSC is the best investigated model, but necroptosis can be triggered by a diverse array of signals, like stimulation of other death receptors and pathogen sensors like Toll-like receptors (TLRs) via the RIPK-homology-interacting motive (RHIM)-domain containing protein TRIF and RIG-I-like receptors (Figure 1). In this sense, it is worth to be mentioned that TLR2 and TLR4 have been implicated in IRI [31], as well as the Fas-FasL system in the kidney [32].

image

Figure 1. The signaling pathway of necroptosis. Death receptor trimerization results in the recruitment of intracellular adapter proteins like the tumor necrosis factor receptor (TNFR)-associated molecule with death domain (TRADD, not shown). Receptor-interacting protein kinase 1 (RIPK1) is involved in the decision process of how cells respond to death receptor ligation. In the case of TNFR1 or other death receptors, RIPK1 becomes polyubiquitinated through two independent mechanisms. First, cellular inhibitors of apoptosis signaling (cIAPs) attach Lys-63 poly-ub chains. Second, the linear ubiquitin chain assembly complex (LUBAC) adds lin-ub chains to RIPK1. These ub-chains prevent molecules like caspase-8 or RIPK3 from interacting with RIPK1 to induce cell death signals and consequently lead to survival of the cell, a process that is mediated through the nuclear factor-κB (NF-κB) in the so-called Complex I. In the case of deubiquitination of RIPK1 by DUBs like CYLD (not shown) and others, Fas-associated death domain (FADD) and procaspase-8 are recruited to the death-inducing signaling complex (DISC) and the complex eventually dissociates from the plasma membrane, now referred to as Complex II. In this process, caspase-8 becomes autocatalytically activated through forced proximity within the DISC and leads to cleavage of effector caspases like caspase-3, -6 and -7 (not shown) that ultimately execute apoptosis. The most important function of the active caspase-8 is the prevention of necroptosis signaling, which is achieved by cleavage of RIPK1 and RIPK3, thereby preventing the assembly of the so-called necroptosome, which consists of RIPK1, RIPK3 and mixed-linage kinase like domain (MLKL). Therefore, only conditions in which caspase-8 is absent, nonfunctional (e.g. mutated) or inhibited (e.g. by viral proteins or in settings of ischemia through undefined mechanisms) allow the assembly of the necroptosome, which quickly leads to necrotic type cell death, release of cell death-associated molecular patterns (CDAMPs) and severe organ damage. Prevention of necroptosis by the inhibitor necrostatin-1 (Nec-1) might serve as a therapeutic strategy in those pathologies that involve necroptosis. Importantly, the necroptosome can also be triggered by pathogen sensors like Toll-like receptors (TLRs) or RIG-I-like receptors (RLRs).

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Why would a system evolve to readily trigger mechanisms of cell death as inflammatory as programmed necrosis? Several viruses express caspase-8 inhibitors, probably to evade the host apoptotic response, a situation that obviously represents a considerable risk [33]. It follows that an alternate response should be in place to prevent evasion by pathogens. Indeed if caspase-8 loses its functions, a stable Complex IIa can no longer be formed [16], and in such cases as well as ischemic situations that will be discussed later in this review, a specialized domain in RIPK3, the RIP homotypic-interacting motif (RHIM), associates with the RHIM domain of RIPK1 [34]. This interaction rapidly recruits FADD and mixed linage kinase domain like (MLKL) [35, 36] to form the detrimental Complex IIb, also referred to as the necroptosome, which drives the necroptotic signal (Figure 1) [4, 37]. Downstream targets of the necroptosome have not clearly been identified, although one report suggests the transduction of the necroptotic signal into mitochondria via the atypical protein kinase PGAM5 by formation of a mitochondrial Complex III, which leads to processing of the mitochondrial fission factor Drp-1 [38].

In vivo Relevance of Necroptosis—A Second-Line Defense Mechanism Against Viruses Can Result in Maladaptive Inflammatory Injury

  1. Top of page
  2. Abstract
  3. Introduction
  4. The Detection of CICD and Necroptosis
  5. In vivo Relevance of Necroptosis—A Second-Line Defense Mechanism Against Viruses Can Result in Maladaptive Inflammatory Injury
  6. CDAMPs in Necroptosis—Strong Inducers of Innate and Adaptive Immune Responses
  7. Blocking Necroptosis in IRI
  8. Interference With Necroptosis—Open Questions
  9. Translation of Basic Insights Into Transplantation Studies and Practice
  10. Acknowledgments
  11. Disclosure
  12. References
  13. Supporting Information

When three independent groups reported in 2009 that RIPK3 is the critical mediator of necroptosis [39-41], several preclinical in vivo models were used to investigate RIPK3-deficient mice that had been reported in 2004 to have no overt spontaneous phenotype [42]. The group of Francis Chan has demonstrated that RIPK3-ko mice are susceptible to vaccinia-virus infections [41]. Whereas wild-type mice regularly survive a viral load, RIPK3-ko mice died within the first 2 weeks of exposure. Importantly, vaccinia virus expresses an inhibitor of caspase-8. While loss of functional caspase-8 triggered the necroptotic signal in the wild-type mice, the genetic absence of RIPK3 in susceptible mice prevented the necroptotic signal that would have otherwise cleared infected cells. Consistent with this, viral titers increased to extraordinarily high levels in RIPK3-ko mice whereas wild-type mice were able to suppress titers to moderate levels [41]. Interestingly murine cytomegalovirus appears to have adapted to this second-line host defense. Like vaccinia virus, it expresses an inhibitor of caspase-8, but also the viral protein M45, which contains a RHIM-domain that interferes with the RIPK1–RIPK3 interaction and the formation of Complex IIb [43, 44]. As striking as these results were, they prompted an eminent next question: Can this pathogen defense mechanism, when activated by a nonphysiological stimulus, also trigger unintended, potentially harmful consequences? RIPK3-deficient mice were demonstrated to be protected from a cerulein-induced pancreatitis [39, 40], a preclinical model of necrotic pancreatitis and a relatively common and devastating clinical problem in critically ill patients. Currently, there are no effective or specific therapeutic approaches for treatment. Unfortunately, while interference with necroptosis remains as potential therapy, clinical studies testing the RIPK1-inhibitor Nec-1 have been disappointing [45], suggesting that inhibition of RIPK1's kinase activity is not sufficient to block the damage in patients or that necrostatin does not inhibit RIPK1's kinase activity to the extent that is required in patients to reach clinical benefit. High hopes have therefore emerged for compounds that directly target RIPK3. Comparable to the results found in the model of necrotizing pancreatitis, inflammatory bowel disease generally remains undefined as to etiology but injury is promoted by robust interactions of various immune cells with intestinal epithelia. Two reports have investigated the role of necroptosis in this setting. The first report conditionally deleted FADD (FADD-deficient mice die in utero) from intestinal epithelial cells (IECs) [46]. This was shown to be due to spontaneous necroptotic cell death because concomitant genetic absence of RIPK3 completely prevented the inflammation in these mice. Conditional depletion of caspase-8 in IECs similarly activated the necroptotic pathway, resulting in bowel inflammation with features of Crohn's disease [47]. In line with the results obtained with mice deficient for FADD in IECs, injury following in mice deficient for caspase-8 in IECs was also completely prevented in mice in which RIPK3 was not present [47]. In atherosclerotic prone, LDL receptor-deficient mice, macrophages cause severe necrotic damage after mice are fed a high fat “western” diet for 16 weeks [48]. However, on a RIPK3-deficient background, this phenotype was markedly reduced. A similar protective effect has been observed in mice that are deficient in apolipoprotein E (ApoE-ko) that were crossed to a RIPK3-deficient background given a high-fat diet. Importantly, inhibition of caspase-8 and application of oxidized LDL strongly promoted invading macrophages, capacity for RIPK3-dependent necroptosis. Therefore, necrotic lesions in atherosclerosis appear to be at least partly caused by RIPK3-dependent necroptosis [48], a phenotype that was also described for ethanol-induced necrosis in hepatocytes. A recent report has confirmed a necroptotic component in this setting by utilizing RIPK3-deficient mice [49]. Not only RIPK3-ko mice were protected from hepatocyte injury and steatosis, but also expression of proinflammatory cytokines was reduced, supporting a role of necroptosis released CDAMPs in promoting immune responses (see below). Comparable to conditional depletion of caspase-8 in IECs, which resulted in a Crohn's disease-like phenotype, liver-specific knockout of caspase-8 resulted in severe nonapoptotic liver injury [50]. In addition to the above-mentioned diseases, necrotic cell death is a hallmark of retinal detachment. It has been demonstrated that in this specialized compartment, both apoptosis and necroptosis are triggered simultaneously (one cell dies by apoptosis, the next one dies by necroptosis) [51]. The same group has now confirmed that cones, but not rods, undergo necroptosis in their model of retinitis pigmentosa [52]. These data support the concept that PCD and RN are not mutually exclusive programs and they may occur in the same organ, following the same stimulus. This is similarly observed in kidneys after IRI (see below), but mechanistic explanations in kidney injury remain unclear. The complexity of multiple necrotic cell death pathways is highlighted in the complex pathophysiology of sepsis, a leading cause of mortality in intensive care patients. Endotoxin triggers massive release of TNF-α and other pro-inflammatory cytokines, which leads to nitric oxide generation and unsupportable low blood pressure. RIPK3-deficient mice are protected from lethal TNF-α-mediated shock [45, 53]. This model attracted considerable attention when it was demonstrated by Cauwels et al [54] in that the pan-caspase inhibitor zVAD, which may have been expected to provide a benefit by blocking TNF-α effect, accelerated rather than prevented death. When RIPK3 was later identified as the mediator of necroptosis and activated by caspase-8 inhibition, these results could of course be reconciled. However, conflicting results remain in the literature about the clinical utility of Nec-1 in this sepsis model [55].

CDAMPs in Necroptosis—Strong Inducers of Innate and Adaptive Immune Responses

  1. Top of page
  2. Abstract
  3. Introduction
  4. The Detection of CICD and Necroptosis
  5. In vivo Relevance of Necroptosis—A Second-Line Defense Mechanism Against Viruses Can Result in Maladaptive Inflammatory Injury
  6. CDAMPs in Necroptosis—Strong Inducers of Innate and Adaptive Immune Responses
  7. Blocking Necroptosis in IRI
  8. Interference With Necroptosis—Open Questions
  9. Translation of Basic Insights Into Transplantation Studies and Practice
  10. Acknowledgments
  11. Disclosure
  12. References
  13. Supporting Information

Necrosis and necroptotic cell death leads to the release of endogenous molecules including heat-shock proteins, high-mobility group box 1 (HMGB1), uric acid, fibronectin and others [6]. These act as CDAMPs to participate in ischemia–reperfusion-related early organ injury, for example. CDAMPs may also perpetuate adaptive immune responses by promoting inflammation through interaction with TLR and other innate receptors expressed on dendritic or other immune cells [6]. Targeting of CDAMPs may be potentially useful therapeutic strategies in blocking inflammatory organ injury with ischemia, for example. However, given the numbers of CDAMPs, this may present a considerable therapeutic challenge unless specific key CDAMPs can be identified. Alternatively, targeting critical components of the necroptosis pathway pharmacologically may be clinically feasible and have greater overall impact.

Recently, a greater capacity of necrotic cells to promote immune responses has been noted, in contrast to a greater tolerogenic influence of apoptotic cells [6]. Indeed, in apoptotic cells, an important function of caspases is to process molecules to a nonimmunogenic state. HMGB1 appears to be converted into a less immunogenic oxidized form following apoptosis in contrast to necroptosis [56]. Another prominent example is DNA, which is classically degraded by the caspase-activated DNase to provide smaller DNA fragments that are less immunogenic [57]. Another strong CDAMP is IL-33, a cytokine that is readily released from necroptotic cells but is degraded in the presence of active capases [58, 59]. In contrast, there is no attenuation of immune responses following rapid loss of plasma membrane integrity in necrotic cell death [6]. Indeed, in mice deficient for caspase-8 or FADD in keratinocytes, which show increased necroptosis in the skin, high levels of HMGB1 have been reported [60]. In models biased to necrosis, the detrimental impact of inflammation on tissue is evident [6]. This exaggerated response is likely to be magnified in solid organ transplants subject to IRI and alloimmunity, particularly if apoptosis is blocked and necroptosis is enhanced. Thus apoptosis and necroptosis are counterbalanced, regulated forms of cell death that differentially direct inflammation. However, these are now individually amenable to specific inhibition, which will provide both new mechanistic insight and novel therapeutic opportunities.

Blocking Necroptosis in IRI

  1. Top of page
  2. Abstract
  3. Introduction
  4. The Detection of CICD and Necroptosis
  5. In vivo Relevance of Necroptosis—A Second-Line Defense Mechanism Against Viruses Can Result in Maladaptive Inflammatory Injury
  6. CDAMPs in Necroptosis—Strong Inducers of Innate and Adaptive Immune Responses
  7. Blocking Necroptosis in IRI
  8. Interference With Necroptosis—Open Questions
  9. Translation of Basic Insights Into Transplantation Studies and Practice
  10. Acknowledgments
  11. Disclosure
  12. References
  13. Supporting Information

The first model in which the RIPK1-inhibitor (Nec-1) was shown to be protective was cerebral IRI [9], even prior to confirming that the kinase domain of RIPK1 was the therapeutic target [10]. The clear reduction of infarct size in a model of middle cerebral artery occlusion in mice that was achieved by Nec-1 was the beginning of a series of neurologic investigations that included models of controlled cortical impact, traumatic brain injury, N-methyl-D-aspartate (NMDA)-induced excitotoxicity in cortical neurons, glutamate-induced necroptosis in HT-22 cells and, most importantly, neonatal hypoxia–ischemia models in which Nec-1 not only protects from oxidative damage but also strikingly prevented the subsequent deteriorating immune cell infiltration [9, 61-64]. The group of Northington has also demonstrated the influence of Nec-1 on mitochondrial dysfunction in neurons [61]. Like mice deficient in FADD, caspase-8 or FLIP, RIPK1-deficient mice are not viable, but unlike the FADD-, caspase-8- and FLIP-deficient mice, RIPK1-deficient mice do not die in utero but perinatally instead [65]. In contrast to FADD- and caspase-8-deficient mice, cFLIP- and RIPK1-deficient mice are not rescued by RIPK3 deficiency, and cFLIP-deficient mice only survive when FADD and RIPK3 are concomitantly deleted on a triple knockout background [66]. Interestingly, FADD-deficient embryos that are crossed to RIPK1-deficient mice survive the checkpoint at Day 10.5 in utero and die perinatally, as do RIPK1-deficient mice [65]. It is not straightforward to identify the precise role of RIPK1 given this genetic complexity, except through the use of the specific inhibitor Nec-1. Few other kinase inhibitors have undergone such in-depth specificity analysis [10, 67]. Within the entire kinome, only two other kinases are affected by high concentrations of Nec-1 to more than 35% [67]. RIPK1-kinase dead “knock-in” mice are currently being generated. In addition, inhibitors of RIPK3's kinase activity are currently under development, yet there are no published reports to date. These agents, along with RIPK3-kinase dead knock-in mice, will be informative in IRI and transplant studies.

The benefit of blocking necroptosis in other organ injury has also now been described. The cardioprotective effect of Nec-1 was first discussed in 2007 [68]. From studies that investigated microRNA-155, which targets RIPK1 [69], it was suggested that RIPK1 functioned as an inducer of necrosis of cardiomyocytes but it was only in 2012 when Oerlemans et al [70] showed a protective effect for Nec-1 in myocardial IRI, focusing on the prevention of adverse cardiac remodeling.

In line with the above-mentioned reports, we found hallmarks of RN in intravital microscopy applied to ischemia–reperfusion pretreated mice (Video S1). A protective effect of Nec-1 in kidney IRI was directly compared with the effect of the apoptosis-blocker pan-caspase inhibitor zVAD or caspase-8 deficiency in addition to RIPK3-deficiency [13, 71]. In contrast to previous reports, we could not detect a protective effect on renal IRI exerted by zVAD. In determining the therapeutic window, we applied Nec-1 before as well as 15 and 30 min following reperfusion. The protective effect almost completely disappeared when Nec-1 was applied 30 min after reperfusion, and strongly reduced when Nec-1 was applied 15 min following reperfusion [13]. In line with several reports, this highlights the rapid necroptotic signaling in kidney IRI (compare Video S1) and that interference might have to be timed very close to reperfusion or, ideally, before reperfusion. While these data collectively support the therapeutic targeting of the necroptosis pathway, the stringent timing suggests that unfortunately only few clinical conditions will be amenable to therapy by Nec-1. Most clinical scenarios such as stroke or myocardial infarction (with the exception of recanalization of completely occluded coronary vessels) will have limited response to Nec-1 as the necroptosome is likely to have formed already [12]. Therefore, interference with necroptosis is more likely to be of clinical benefit in situations in which the reperfusion damage can be anticipated, such as cardiac surgery and, intriguingly, solid organ transplantation.

Interference With Necroptosis—Open Questions

  1. Top of page
  2. Abstract
  3. Introduction
  4. The Detection of CICD and Necroptosis
  5. In vivo Relevance of Necroptosis—A Second-Line Defense Mechanism Against Viruses Can Result in Maladaptive Inflammatory Injury
  6. CDAMPs in Necroptosis—Strong Inducers of Innate and Adaptive Immune Responses
  7. Blocking Necroptosis in IRI
  8. Interference With Necroptosis—Open Questions
  9. Translation of Basic Insights Into Transplantation Studies and Practice
  10. Acknowledgments
  11. Disclosure
  12. References
  13. Supporting Information

To evaluate the clinical potential of necroptosis inhibition, further studies are obviously required. Although Nec-1 studies in human trials were proposed several years ago, none have currently emerged. This may be due to conflicting results using Nec-1 in TNF-mediated shock [13, 55] or related to the expected release of more specific inhibitors of RIPK1's kinase activity such as Nec-1s, a second-generation compound that was recently described [55]. It may be of greater benefit to develop specific inhibitors of RIPK3's kinase activity if it is indeed the kinase activity of RIPK3 that transduces the necroptotic signal, rather than the RHIM domain. RIPK3 kinase inhibitors are also under development but have not been published so far. Finally, an additional potential target is the recently identified necroptosis pathway component MLKL, a kinase-domain-containing pseudokinase. Again, it needs to be determined, which domain of MLKL is required for necroptosis signaling.

The most intriguing challenge remains in unequivocally identifying the necroptosis-inducing death receptor. Whereas in vitro data have most often referred to TNFR1 as the primary inducer of necroptosis, this has not been confirmed in vivo. In this regard, TNFR1/2-double-deficient mice are not protected from renal IRI [32]. Many other receptors, including other death receptors, TLRs and intracellular molecules, like the RHIM-domain containing proteins DAI and TRIF, are potential candidates. Intuitively, interference with activation of the receptor responsible for induction of pathological necroptosis directly would likely provide the most efficacious approach to blocking the clinical consequences of aberrant necroptosis.

Translation of Basic Insights Into Transplantation Studies and Practice

  1. Top of page
  2. Abstract
  3. Introduction
  4. The Detection of CICD and Necroptosis
  5. In vivo Relevance of Necroptosis—A Second-Line Defense Mechanism Against Viruses Can Result in Maladaptive Inflammatory Injury
  6. CDAMPs in Necroptosis—Strong Inducers of Innate and Adaptive Immune Responses
  7. Blocking Necroptosis in IRI
  8. Interference With Necroptosis—Open Questions
  9. Translation of Basic Insights Into Transplantation Studies and Practice
  10. Acknowledgments
  11. Disclosure
  12. References
  13. Supporting Information

The potential applicability of blocking necroptosis in transplantation is obvious, but has not been extensively tested to date. We have described the opposite effects of caspase-8 silencing by shRNA and genetic deletion of RIPK3 expression in donor kidneys in allotransplantation. Importantly, caspase-8-suppression-related apoptosis inhibition enhanced necrosis and reduced renal allograft survival whereas RIPK3-deficient allografts had greater renal function and rejection-free survival compared with wild-type controls [72]. These data are clearly encouraging for clinical transplantation, as it should be sufficient to saturate donor organs with necroptosis-inhibiting drugs prior to implantation, using perfusion solution delivery. Further work is required to test the ability of these small molecules to alter necroptosis at cold perfusion temperatures, to elucidate the mechanisms by which RIP3 blocking benefits transplanted organs and to confirm that reduced release of CDAMPs attenuates immune-mediated organ injury as speculated here. Transplanted organs are invariably injured by both the procurement process as well as a wide variety of cytotoxic injuries, which are now appreciated to involve necroptosis. The ability to block necroptosis by robust and specific pharmacologic agents may represent a paradigm-changing approach to IRI and alloimmune injury in transplantation. Importantly, ideal continuous administration of Nec-1, as it might be possible in intensive care medicine, could serve as a compound that prevents necroptosis, which occurs hours after reperfusion.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. The Detection of CICD and Necroptosis
  5. In vivo Relevance of Necroptosis—A Second-Line Defense Mechanism Against Viruses Can Result in Maladaptive Inflammatory Injury
  6. CDAMPs in Necroptosis—Strong Inducers of Innate and Adaptive Immune Responses
  7. Blocking Necroptosis in IRI
  8. Interference With Necroptosis—Open Questions
  9. Translation of Basic Insights Into Transplantation Studies and Practice
  10. Acknowledgments
  11. Disclosure
  12. References
  13. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. The Detection of CICD and Necroptosis
  5. In vivo Relevance of Necroptosis—A Second-Line Defense Mechanism Against Viruses Can Result in Maladaptive Inflammatory Injury
  6. CDAMPs in Necroptosis—Strong Inducers of Innate and Adaptive Immune Responses
  7. Blocking Necroptosis in IRI
  8. Interference With Necroptosis—Open Questions
  9. Translation of Basic Insights Into Transplantation Studies and Practice
  10. Acknowledgments
  11. Disclosure
  12. References
  13. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
ajt12448-sm-0001-SuppVideo-s1.mp43193KVideo S1: Necrosis in ischemia–reperfusion injury. Time lapse in vivo multiphoton microscopy imaging of mouse kidney tubuli after a 30-min ischemia period. At the start of the time lapse imaging 15 mg propidium iodide were injected via a carotid artery catheter. Recording of one image per minute, total recording time 60 min. The vasculature is labeled using 70 kDa Texas red dextrane, autofluorescence of tubular cells appears green. Note the swelling of one nucleus (arrow), the rupture of the membrane of this cell (as was referred to as “an ordered cellular explosion” [30]) and the following release of intracellular CDAMPs into the tubular lumen. In addition, note the rapid closure of the tubular defect starting from neighbor cells from the basolateral compartment. Similar swelling was seen in approximately 15% of tubular cells within the first hour of reperfusion.
ajt12448-sm-0001-SuppData-s1.doc131KTable S1: Evidence for the in vivo relevance of necroptosis. Physiological and pathophysiological aspects are separated. Note the involvement in diverse models of ischemia/reperfusion injury.

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