Necrosis, the death ...

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Necrosis, the death of cells and tissues, has long been recognised by anatomical pathologists.The evolutionary conservation of d"ffer'nt modes of regulated cell death may be explained by differences in their particular immunogenicity 46, 138. Following that hypothesis, apoptosis may be the least immunogenic form 46, 140. This is plausible given the billions of cells that are turned over by apoptosis in a human body every day. An immune response to this 'physiological' cellular turnover would kill the organism. In contrast, DAMP release in necrotic cell death subroutines may be similar in RN pathways 137, 141. However, the immune response to RN may be diverse, as production of pro-inflammatory cytokines might boost the immune cell triggers beyond DAMP release. In that sense, caspase-1 not only cleaves GSDMD to mediate pyroptosis, but also activates pro-IL-1? and pro-IL-18 that can subsequently be released in a GSDMD-dependent manner 84, 142. Pyroptosis, therefore, may represent a highly immunogenic type of regulated necrosis. In contrast, active transcription of IL-33 may stabilise regulatory T cells in the microenvironment that surrounds cells that die by necroptosis 143. Similarly, the production of CXCL1 may inhibit infiltrating neutrophils 144. In the case of ferroptosis, short-lived lipid peroxides may also exhibit specific effects on immune cells, but evidence for this hypothesis is very limited to date. Pathologists are expected to significantly contribute to the understanding of how different cell death pathways drive different patterns of immunity. The relevance of regulated necrosi" pathways and necroinflammation in human diseases As pointed out above in detail, necrosis is a hallmark of many human diseases.The pathophysiological importance of necroptosis Genetic evidence suggests that RIPK3-/MLKL-dependent necroptosis is of central pathophysiological importance in renal ischaemia-reperfusion injury (IRI)/tubular necrosis in mice and humans 2, and contributes to cisplatin-mediated acute kidney injury , myocardial IRI , myocardial remodelling , glucose intolerance , toxic liver diseases , atherosclerosis , diverse skin disorders and severe inflammatory response syndrome (SIRS).The pathophysiological importance of necroptosis Genetic evidence suggests that RIPK3-/MLKL-dependent necroptosis is of central pathophysiological importance in renal ischaemia-reperfusion injury (IRI)/tubular necrosis in mice 50-52 and humans 2, and contributes to cisplatin-mediated acute kidney injury 50, 53, myocardial IRI 54, myocardial remodelling 55, glucose intolerance 56, toxic liver diseases 57, atherosclerosis 58, diverse skin disorders 59-61, and severe inflammatory response syndrome (SIRS) 52, 62-65.pMLKL oligomerises and results in plasma membrane rupture by ill-defined mechanisms that are counteracted by membrane repair mechanisms, such as the ESCRT-III complex .RIPK3, besides its kinase domain, consists of a Rip homotypic interacting motif (RHIM) domain that allows its spontaneous multimerisation and formation of a hetero-amyloid supramolecular complex referred to as the necrosome.These include (i) kinase-mediated necroptosis, which depends on receptor interacting protein kinase 3 (RIPK3)-mediated phosphorylation of the pseudokinase mixed lineage kinase domain like (MLKL); (ii) gasdermin-mediated necrosis downstream of inflammasomes, also referred to as pyroptosis; and (iii) an iron-catalysed mechanism of highly specific lipid peroxidation named ferroptosis.Therefore, it is likely that other molecular pathways, such as pyroptosis or ferroptosis, are additionally involve
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Pyroptosis As pointed out in the previous section, key necroptosis molecules such as RIPK3, MLKL, and ZBP1 regulate inflammasome activation, which makes it difficult to clearly differentiate between pure necroptosis and pyroptosis, defined as gasdermin D (GSDMD)-mediated regulated necrosis.RIPK3-deficient renal and cardiac allografts are less likely to be rejected following solid organ transplantation Finally, several cancers modify the necroptosis system In all of these diseases, pMLKL immunohistochemical and immunofluorescent staining may aid diagnosis (Figure 2), if relatively easy correlative studies are performed in the future.Details are in the caption following the image Figure 2 Open in figure viewer PowerPoint Caption Details are in the caption following the image Figure 3 Open in figure viewer PowerPoint Caption Details are in the caption following the image Figure 4 Open in figure viewer PowerPoint Caption Pyroptosis As pointed out in the previous section, key necroptosis molecules such as RIPK3, MLKL, and ZBP1 regulate inflammasome activation, which makes it difficult to clearly differentiate between pure necroptosis and pyroptosis, defined as gasdermin D (GSDMD)-mediated regulated necrosis.This process is detectable by IHC or IF against cleaved caspase-3 and typically is associated with the shrinkage of a cell and plasma membrane blebbing upon exposure of phosphatidylserine ?and may represent apoptotic immune cells in several instances, e.g. neutrophils, lymphocytes or activated macrophages .This process is detectable by IHC or IF against cleaved caspase-3 and typically is associated with the shrinkage of a cell and plasma membrane blebbing upon exposure of phosphatidylserine 41, 42, and may represent apoptotic immune cells in several instances, e.g. neutrophils, lymphocytes or activated macrophages 43.Together with the assumption of the underlying cause, this classification allows the pathologist to make a diagnosis of coagulation necrosis in an anaemic myocardial infarct, of fibrinoid necrosis in a glomerulus affected by small vessel vasculitis, and of acute tubular necrosis (ATN) in a kidney biopsy (Figure 1).Together with the assumption of the underlying cause, this classification allows the pathologist to make a diagnosis of coagulation necrosis in an anaemic myocardial infarct, of fibrinoid necrosis in a glomerulus affected by small vessel vasculitis, and of acute tubular necrosis (ATN) in a kidney biopsy (Figure 1).Default upstream triggers in this complex web of interconnected pathways include Toll-like receptors (TLRs), death receptors (e.g. TNFR1), inflammasomes (e.g. the NLRP3 inflammasome), and intracellular sensing of nucleic acids (e.g. viral DNA).Together with classical cell death markers, such as TUNEL staining and detection of cleaved caspase-3 in apoptotic cells, the extension of the arsenal of necrosis markers will allow pathological detection of specific molecular pathways rather than isolated morphological descriptions.Default upstream triggers in this complex web of interconnected pathways include Toll-like receptors (TLRs), death receptors (e.g. TNFR1), inflammasomes (e.g. the NLRP3 inflammasome), and intracellular sensing of nucleic acids (e.g. viral DNA).RIPK3, besides its kinase domain, consists of a Rip homotypic interacting motif (RHIM) domain 5-7 that allows its spontaneous multimerisation and formation of a hetero-amyloid supramolecular complex 8 referred to as the necrosome.The apoptosis pathway is connected mechanistically to the pathways of necroptosis and pyroptosis, as this system is controlled by proteases (caspases), kinases (serine-threonine kinases), and the polyubiquitin system (predominantly K63 and M1 linkages).In contrast, ferroptosis represents an ancient mechanism of specific lipid peroxidation that results in plasma membrane rupture and non-cell autonomous necrosis [also referred to as synchronised regulated necrosis (SRN)].Application of these techniques will allow them to correlate the traditional classification of necrosis with the individual pathways of RN. Precise dissection of these pathways can be expected to improve the diagnostic precision and prediction of tissue diagnostics.Necroptosis Necroptosis is defined as the loss of plasma membrane integrity following receptor interacting kinase 3 (RIPK3)-mediated phosphorylation of the pseudokinase mixed lineage kinase domain like (MLKL/pMLKL) .In the absence of functional caspases or in the presence of viral or synthetic caspase inhibitors, caspase-8 no longer actively prevents necroptotic signalling, RIPK3 oligomerises, a 'Complex 2b' forms, and necroptosis is executed .We have recently demonstrated that antibodies against the phosphorylation site in the activation loop of human MLKL are valuable tools for the detection of activity of the necroptosis pathway in human kidney transplant biopsies 2, and we are currently generating humanised mice to further verify this antibody.Importantly, GSDMD cleavage results in the formation of a pore that consists of a 27-mer of the C-terminal portion of GSDMD that is embedded into the plasma membrane and results in a type of necrotic cell death referred to as pyroptosis 89-94.The apoptosis pathway is connected mechanistically to the pathways of necroptosis and pyroptosis, as this system is controlled by proteases (caspases), kinases (serine-threonine kinases), and the polyubiquitin system (predominantly K63 and M1 linkages).In contrast, ferroptosis represents an ancient mechanism of specific lipid peroxidation that results in plasma membrane rupture and non-cell autonomous necrosis [also referred to as synchronised regulated necrosis (SRN)].Application of these techniques will allow them to correlate the traditional classification of necrosis with the individual pathways of RN. Precise dissection of these pathways can be expected to improve the diagnostic precision and prediction of tissue diagnostics.Necroptosis Necroptosis is defined as the loss of plasma membrane integrity following receptor interacting kinase 3 (RIPK3)-mediated phosphorylation of the pseudokinase mixed lineage kinase domain like (MLKL/pMLKL) 1.In the absence of functional caspases or in the presence of viral or synthetic caspase inhibitors, caspase-8 no longer actively prevents necroptotic signalling, RIPK3 oligomerises, a 'Complex 2b' forms, and necroptosis is executed 44-46.We have recently demonstrated that antibodies against the phosphorylation site in the activation loop of human MLKL are valuable tools for the detection of activity of the necroptosis pathway in human kidney transplant biopsies 2, and we are currently generating humanised mice to further verify this antibody.Importantly, GSDMD cleavage results in the formation of a pore that consists of a 27-mer of the C-terminal portion of GSDMD that is embedded into the plasma membrane and results in a type of necrotic cell death referred to as pyroptosis 89-94.Loss of GSH, loss of GPX4 or pharmacological inhibition of the active centre of GPX4 results in a lipoxygenase-mediated, highly specific peroxidation of phosphatidylethanolamine (PE) in the plasma membrane of eukaryotic cells 121.The isolated loss of the RIPK1 RHIM domain is sufficient to cause embryonic lethality in mice, demonstrating the RIPK1RHIM-mediated autoinhibition of spontaneous RIPK3 oligomerisation .Anatomical pathologists should embrace these novel ancillary tests and the concepts behind them and test their impact on diagnostic precision, prognostication, and the prediction of response to the upcoming anti-necrotic therapies.The isolated loss of the RIPK1 RHIM domain is sufficient to cause embryonic lethality in mice, demonstrating the RIPK1RHIM-mediated autoinhibition of spontaneous RIPK3 oligomerisation 9, 10.In pathophysiological settings such as ischaemia/reperfusion injury (IRI), solid organ transplantation, myocardial infarction or stroke, ferroptosis may account for the majority of necrotic cells.In this review, we aim to summarise our current understanding of RN pathways and argue that the classical pathological picture of apoptotic cell death needs to be revisited, incorporating novel biochemical findings.This recently accumulated knowledge that is about to inform novel therapies should be incorporated into the practice of anatomical pathologists and should prompt revision of the old morphological classification of necrosis.It is, however, widely accepted that RIPK1 represents the master switch downstream of TNFR1 activation to signal towards NF-?B activation and cellular survival or to cell death by apoptosis/necroptosis and potentially pyroptosis .In the case of the absence of the polyubiquitin chains, the TNF-RSC is no longer tethered to the plasma membrane to become cytosolic (then named 'Complex 2a') and results in apoptotic death of the cell, mediated by caspases.These novel pieces of information will be extraordinarily helpful for clinicians as inhibitors of necroptosis (necrostatins), ferroptosis (ferrostatins), and inflammasomes have emerged in clinical trials.In pathophysiological settings such as ischaemia/reperfusion injury (IRI), solid organ transplantation, myocardial infarction or stroke, ferroptosis may account for the majority of necrotic cells.In this review, we aim to summarise our current understanding of RN pathways and argue that the classical pathological picture of apoptotic cell death needs to be revisited, incorporating novel biochemical findings.This recently accumulated knowledge that is about to inform novel therapies should be incorporated into the practice of anatomical pathologists and should prompt revision of the old morphological classification of necrosis.It is, however, widely accepted that RIPK1 represents the master switch downstream of TNFR1 activation to signal towards NF-?B activation and cellular survival or to cell death by apoptosis/necroptosis 26-29, and potentially pyroptosis 30-34.The ubiquitin chains, again, require a highly organised balance between assembly by the linear ubiquitin chain assembly complex (LUBAC) and OUT deubiquitinase with linear linkage specificity (OTULIN) in the case of M1 linkages 38, 39, or other E3 ligases in the case of K63 linkages 40.In the case of the absence of the polyubiquitin chains, the TNF-RSC is no longer tethered to the plasma membrane to become cytosolic (then named 'Complex 2a') and results in apoptotic death of the cell, mediated by caspases.Importantly, therefore, only necrotic-type cell death modes sufficiently explain the generation of anti-nuclear antibodies (ANAs) or anti-double-strand DNA (dsDNA) antibodies that are commonly observed in autoimmune diseases 139.Several approaches are currently being pursued to generate antibodies against the cleaved fragment of gasdermin D to reliably detect activation of pyroptosis.RIPK1 itself can be phosphorylated on multiple sites and specific antibodies against pRIPK1 have been generated, but the relative contribution of each of these phosphosites is still under scientific debate.The ubiquitin chains, again, require a highly organised balance between assembly by the linear ubiquitin chain assembly complex (LUBAC) and OUT deubiquitinase with linear linkage specificity (OTULIN) in the case of M1 linkages ?However, given our increasing knowledge about necroptosis over the last 5 years , the morphological patterns of necrotic casts found in urine (Figure 3) appear to be as diverse as the histological findings in ATN (Figure 4).Pyroptosis and the associated release of danger-associated molecular patterns (DAMPs) and pro-inflammatory cytokines render this form of regulated necrosis the most immunogenic cell death known today.Despite its unique nature of inflammasome-mediated necrosis, recent data suggest that caspase-8 and MLKL may in some situations control the activation of caspase-1 and GSDMD cleavage and pore formation 32, 33, 107, and even that IL-1?Interestingly, expression of the inflammasomal components caspase-1 and GSDMD is not restricted to phagocytes, but are also detectable in epithelial cells, e.g. renal tubules 110-112.Details are in the caption following the image Figure 1 Open in figure viewer PowerPoint Caption Beyond apoptosis - necroptosis, pyroptosis, and ferroptosis Due to space limitations, it is beyond the scope of this review to outline all known details of the pathways of necroptosis, pyroptosis, and ferroptosis.pMLKL oligomerises and results in plasma membrane rupture by ill-defined mechanisms that are counteracted by membrane repair mechanisms, such as the ESCRT-III complex 2-4.RIPK1 itself can be phosphorylated on multiple sites 22-25 and specific antibodies against pRIPK1 have been generated, but the relative contribution of each of these phosphosites is still under scientific debate.However, given our increasing knowledge about necroptosis over the last 5 years 25, 36, 49, 71-82, the morphological patterns of necrotic casts found in urine (Figure 3) appear to be as diverse as the histological findings in ATN (Figure 4).Pyroptosis and the associated release of danger-associated molecular patterns (DAMPs) and pro-inflammatory cytokines render this form of regulated necrosis the most immunogenic cell death known today.Despite its unique nature of inflammasome-mediated necrosis, recent data suggest that caspase-8 and MLKL may in some situations control the activation of caspase-1 and GSDMD cleavage and pore formation 32, 33, 107, and even that IL-1?Interestingly, expression of the inflammasomal components caspase-1 and GSDMD is not restricted to phagocytes, but are also detectable in epithelial cells, e.g. renal tubules 110-112.The precise mechanism of how the adapter protein PEBP-1 allows lipoxygenase-mediated PE peroxidation has been elucidated recently 122.Unlike apoptosis, a regulated cell death process in which the plasma membrane does not rupture, necrosis is inevitably associated with the release of DAMPs and intracellular organelles 137, 138.Early attempts, however, to map individual phospholipids with single cell resolution have been undertaken (V Kagan, personal communication), but these are experimental techniques that will not be part of the toolbox of general pathologists in the near futureBefore these observations, when apoptosis was thought to represent the sole pathway of regulated cell death, necrosis was interpreted to occur passively due to 'overwhelming injury'.Here, we will review the pathophysiology of the various pathways of RN and we will present the novel ancillary techniques to dissect these pathways for anatomical pathologists.Necrosome formation depends on the RHIM domain of RIPK3 and is constitutively prevented by intercalation of other RHIM domain-containing proteins .A first monoclonal antibody against cleaved GSDMD is currently being established (Feng Shao, NIBS, personal communication).Before these observations, when apoptosis was thought to represent the sole pathway of regulated cell death, necrosis was interpreted to occur passively due to 'overwhelming injury'.Here, we will review the pathophysiology of the various pathways of RN and we will present the novel ancillary techniques to dissect these pathways for anatomical pathologists.Necrosome formation depends on the RHIM domain of RIPK3 and is constitutively prevented by intercalation of other RHIM domain-containing proteins 9, 10.In all of these diseases, pMLKL immunohistochemical and immunofluorescent staining may aid diagnosis (Figure 2), if relatively easy correlative studies are performed in the future.A first monoclonal antibody against cleaved GSDMD is currently being established (Feng Shao, NIBS, personal communication).Constant lipid peroxidation is prevented by glutathione peroxidase 4 (GPX4), a selenoprotein that constitutively metabolises glutathione (GSH) 114-120.Other than necroptosis and pyroptosis, ferroptosis tends to affect functional units, rather than single cells 123 in a process referred to as synchronised regulated necrosis (SRN) 46, 124-126.The exchange of a selenocysteine to a cysteine in GPX4 results in fatal epileptic seizures in mice, demonstrating a critical role for ferroptosis in the neurological system 117.Given the nature of the ferroptotic signal as SRN, a functional syncytium such as the myocardium may suggest an involvement of ferroptosis upon failure of GPX4 or other selenoproteins to function in these tissues.Pathophysiologically, this triggers an immune response because novel surfaces become accessible to both the innate and the adaptive immune system during a process referred to as necroinflammation 138.Based on the macroscopic and microscopic appearance, necrosis has been classified as coagulative, colliquative, fibrinoid, haemorrhagic and caseating, and other forms of necrosis.The outcomes of the caspase/kinase/ubiquitin system can lead to NF-?B activation, apoptosis, necroptosis or pyroptosis.Remarkably, the molecular decision depends on the kinases that are recruited to the proximal TNF-receptor signalling complex (TNF-RSC).This allows correlating pMLKL immunostaining to clinical outcome and may add another piece to our toolbox in detecting specific subroutines of cell death.Finally, RIPKs have emerged as critical regulators of inflammation and pMLKL staining may therefore be considered state-of-the-art when activity in the necroptosis pathway is investigated.Activation of inflammatory caspases classically involves the formation of inflammasomes 95, 96, a detailed description of which is beyond the scope of this review.Briefly, inflammasomes are innate immune sensors that amplify a pro-inflammatory signal through innate immune cells, predominantly macrophages 95, 96.Additionally, preclinical models investigated with GSDMD-deficient mice will be helpful to assess the role of pyroptosis.Given the molecular understanding of the nature of these pathways, specific antibodies may allow direct detection of regulated necrosis and correlation with morphological features.Based on the macroscopic and microscopic appearance, necrosis has been classified as coagulative, colliquative, fibrinoid, haemorrhagic and caseating, and other forms of necrosis.The outcomes of the caspase/kinase/ubiquitin system can lead to NF-?B activation, apoptosis, necroptosis or pyroptosis.Remarkably, the molecular decision depends on the kinases that are recruited to the proximal TNF-receptor signalling complex (TNF-RSC).This allows correlating pMLKL immunostaining to clinical outcome and may add another piece to our toolbox in detecting specific subroutines of cell death.Finally, RIPKs have emerged as critical regulators of inflammation 15, 48, 49 and pMLKL staining may therefore be considered state-of-the-art when activity in the necroptosis pathway is investigated.Activation of inflammatory caspases classically involves the formation of inflammasomes 95, 96, a detailed description of which is beyond the scope of this review.Briefly, inflammasomes are innate immune sensors that amplify a pro-inflammatory signal through innate immune cells, predominantly macrophages 95, 96.Additionally, preclinical models investigated with GSDMD-deficient mice will be helpful to assess the role of pyroptosis.Ferroptosis The vital element oxygen, especially in the presence of free iron, can become part of reactive oxygen species (ROS) that are harmful to cellular membranes.Genetic deletion of murine Gpx4 results in embryonic lethality, and inducible deletion after weaning results in lethal ATN 115.Additionally, RIPK3, MLKL, and ZBP1 can regulate inflammasomes and pyroptosis .

النص الأصلي

Necrosis, the death of cells and tissues, has long been recognised by anatomical pathologists. Based on the macroscopic and microscopic appearance, necrosis has been classified as coagulative, colliquative, fibrinoid, haemorrhagic and caseating, and other forms of necrosis. The recognition of cell and tissue death by these morphological patterns has been proven to be clinically useful in tissue diagnostics over the last century. Together with the assumption of the underlying cause, this classification allows the pathologist to make a diagnosis of coagulation necrosis in an anaemic myocardial infarct, of fibrinoid necrosis in a glomerulus affected by small vessel vasculitis, and of acute tubular necrosis (ATN) in a kidney biopsy (Figure 1). However, in the last two decades, pioneering biochemical research has shown that necrosis can also be a regulated process. Before these observations, when apoptosis was thought to represent the sole pathway of regulated cell death, necrosis was interpreted to occur passively due to ‘overwhelming injury’. Now we have begun to understand details about various pathways of regulated necrosis (RN), such as necroptosis, pyroptosis, and ferroptosis. The apoptosis pathway is connected mechanistically to the pathways of necroptosis and pyroptosis, as this system is controlled by proteases (caspases), kinases (serine–threonine kinases), and the polyubiquitin system (predominantly K63 and M1 linkages). Default upstream triggers in this complex web of interconnected pathways include Toll-like receptors (TLRs), death receptors (e.g. TNFR1), inflammasomes (e.g. the NLRP3 inflammasome), and intracellular sensing of nucleic acids (e.g. viral DNA). The outcomes of the caspase/kinase/ubiquitin system can lead to NF-κB activation, apoptosis, necroptosis or pyroptosis. Intriguingly, all of these outcomes can be seen as different effector mechanisms of the immune response (see below). In contrast, ferroptosis represents an ancient mechanism of specific lipid peroxidation that results in plasma membrane rupture and non-cell autonomous necrosis [also referred to as synchronised regulated necrosis (SRN)]. In pathophysiological settings such as ischaemia/reperfusion injury (IRI), solid organ transplantation, myocardial infarction or stroke, ferroptosis may account for the majority of necrotic cells. In this review, we aim to summarise our current understanding of RN pathways and argue that the classical pathological picture of apoptotic cell death needs to be revisited, incorporating novel biochemical findings. This recently accumulated knowledge that is about to inform novel therapies should be incorporated into the practice of anatomical pathologists and should prompt revision of the old morphological classification of necrosis. Here, we will review the pathophysiology of the various pathways of RN and we will present the novel ancillary techniques to dissect these pathways for anatomical pathologists. Application of these techniques will allow them to correlate the traditional classification of necrosis with the individual pathways of RN. Precise dissection of these pathways can be expected to improve the diagnostic precision and prediction of tissue diagnostics. Moreover, it will provide the evidence base for rational application of novel anti-necrotic agents to treat human disease.

Necroptosis
Necroptosis is defined as the loss of plasma membrane integrity following receptor interacting kinase 3 (RIPK3)-mediated phosphorylation of the pseudokinase mixed lineage kinase domain like (MLKL/pMLKL) . pMLKL oligomerises and results in plasma membrane rupture by ill-defined mechanisms that are counteracted by membrane repair mechanisms, such as the ESCRT-III complex .RIPK3, besides its kinase domain, consists of a Rip homotypic interacting motif (RHIM) domain that allows its spontaneous multimerisation and formation of a hetero-amyloid supramolecular complex referred to as the necrosome. Necrosome formation depends on the RHIM domain of RIPK3 and is constitutively prevented by intercalation of other RHIM domain-containing proteins . Currently, only four such proteins are known that contain a RHIM domain (RIPK1, RIPK3, TRIF, and ZBP1) . It was recently detected how the interplay of the RHIM domains results in formation of the necrosome. The isolated loss of the RIPK1 RHIM domain is sufficient to cause embryonic lethality in mice, demonstrating the RIPK1RHIM-mediated autoinhibition of spontaneous RIPK3 oligomerisation . Upon necrosome formation, RIPK3 becomes activated by phosphorylation (pRIPK3), which in turn allows phosphorylation of MLKL . However, it is important to realise that RIPK3 has non-necroptotic functions, such as pro-inflammatory gene transcription . Additionally, RIPK3, MLKL, and ZBP1 can regulate inflammasomes and pyroptosis .

RIPK1 itself can be phosphorylated on multiple sites and specific antibodies against pRIPK1 have been generated, but the relative contribution of each of these phosphosites is still under scientific debate. It is, however, widely accepted that RIPK1 represents the master switch downstream of TNFR1 activation to signal towards NF-κB activation and cellular survival or to cell death by apoptosis/necroptosis and potentially pyroptosis . Remarkably, the molecular decision depends on the kinases that are recruited to the proximal TNF-receptor signalling complex (TNF-RSC). Kinases such as TBK1 25, but also IKKϵ 35 are recruited to the TNF-RSC through ubiquitin chains assembled on RIPK1 . We refer to this complex as ‘Complex 1’. The ubiquitin chains, again, require a highly organised balance between assembly by the linear ubiquitin chain assembly complex (LUBAC) and OUT deubiquitinase with linear linkage specificity (OTULIN) in the case of M1 linkages ، or other E3 ligases in the case of K63 linkages . It is the loss of these ubiquitin linkages and the connected failure to recruit kinases to the TNF-RSC that represent the molecular switch between the life and death of a cell. In the case of the absence of the polyubiquitin chains, the TNF-RSC is no longer tethered to the plasma membrane to become cytosolic (then named ‘Complex 2a’) and results in apoptotic death of the cell, mediated by caspases. This process is detectable by IHC or IF against cleaved caspase-3 and typically is associated with the shrinkage of a cell and plasma membrane blebbing upon exposure of phosphatidylserine ،and may represent apoptotic immune cells in several instances, e.g. neutrophils, lymphocytes or activated macrophages . In the absence of functional caspases or in the presence of viral or synthetic caspase inhibitors, caspase-8 no longer actively prevents necroptotic signalling, RIPK3 oligomerises, a ‘Complex 2b’ forms, and necroptosis is executed .

We have recently demonstrated that antibodies against the phosphorylation site in the activation loop of human MLKL are valuable tools for the detection of activity of the necroptosis pathway in human kidney transplant biopsies 2, and we are currently generating humanised mice to further verify this antibody. In addition, a panel of pRIPK1 and pRIPK3 antibodies exist for the detection of these activated proteins in both mice and humans. Apart from pMLKL, especially for pRIPK3 there is a clear protocol available 47, albeit for mouse tissue only. This allows correlating pMLKL immunostaining to clinical outcome and may add another piece to our toolbox in detecting specific subroutines of cell death. Finally, RIPKs have emerged as critical regulators of inflammation and pMLKL staining may therefore be considered state-of-the-art when activity in the necroptosis pathway is investigated. We consider it likely that direct detection of the entire necroptosis pathway will become possible in the near future. However, interpretation of pMLKL staining will be confronted with two major limitations. First, the antibody detects one of several phosphorylation sites on MLKL, exclusively in the activation loop. Obviously, pMLKL detects activity in the necroptosis pathway, but not necessarily plasma membrane rupture. Secondly, it has not formally been demonstrated that pMLKL signal is specific in human tissues.
The pathophysiological importance of necroptosis
Genetic evidence suggests that RIPK3-/MLKL-dependent necroptosis is of central pathophysiological importance in renal ischaemia–reperfusion injury (IRI)/tubular necrosis in mice and humans 2, and contributes to cisplatin-mediated acute kidney injury , myocardial IRI , myocardial remodelling , glucose intolerance , toxic liver diseases , atherosclerosis , diverse skin disorders and severe inflammatory response syndrome (SIRS). RIPK3-deficient renal and cardiac allografts are less likely to be rejected following solid organ transplantation Finally, several cancers modify the necroptosis system In all of these diseases, pMLKL immunohistochemical and immunofluorescent staining may aid diagnosis (Figure 2), if relatively easy correlative studies are performed in the future. However, given our increasing knowledge about necroptosis over the last 5 years , the morphological patterns of necrotic casts found in urine (Figure 3) appear to be as diverse as the histological findings in ATN (Figure 4). Therefore, it is likely that other molecular pathways, such as pyroptosis or ferroptosis, are additionally involve

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Introduction
Necrosis, the death of cells and tissues, has long been recognised by anatomical pathologists. Based on the macroscopic and microscopic appearance, necrosis has been classified as coagulative, colliquative, fibrinoid, haemorrhagic and caseating, and other forms of necrosis. The recognition of cell and tissue death by these morphological patterns has been proven to be clinically useful in tissue diagnostics over the last century. Together with the assumption of the underlying cause, this classification allows the pathologist to make a diagnosis of coagulation necrosis in an anaemic myocardial infarct, of fibrinoid necrosis in a glomerulus affected by small vessel vasculitis, and of acute tubular necrosis (ATN) in a kidney biopsy (Figure 1). However, in the last two decades, pioneering biochemical research has shown that necrosis can also be a regulated process. Before these observations, when apoptosis was thought to represent the sole pathway of regulated cell death, necrosis was interpreted to occur passively due to ‘overwhelming injury’. Now we have begun to understand details about various pathways of regulated necrosis (RN), such as necroptosis, pyroptosis, and ferroptosis. The apoptosis pathway is connected mechanistically to the pathways of necroptosis and pyroptosis, as this system is controlled by proteases (caspases), kinases (serine–threonine kinases), and the polyubiquitin system (predominantly K63 and M1 linkages). Default upstream triggers in this complex web of interconnected pathways include Toll-like receptors (TLRs), death receptors (e.g. TNFR1), inflammasomes (e.g. the NLRP3 inflammasome), and intracellular sensing of nucleic acids (e.g. viral DNA). The outcomes of the caspase/kinase/ubiquitin system can lead to NF-κB activation, apoptosis, necroptosis or pyroptosis. Intriguingly, all of these outcomes can be seen as different effector mechanisms of the immune response (see below). In contrast, ferroptosis represents an ancient mechanism of specific lipid peroxidation that results in plasma membrane rupture and non-cell autonomous necrosis [also referred to as synchronised regulated necrosis (SRN)]. In pathophysiological settings such as ischaemia/reperfusion injury (IRI), solid organ transplantation, myocardial infarction or stroke, ferroptosis may account for the majority of necrotic cells. In this review, we aim to summarise our current understanding of RN pathways and argue that the classical pathological picture of apoptotic cell death needs to be revisited, incorporating novel biochemical findings. This recently accumulated knowledge that is about to inform novel therapies should be incorporated into the practice of anatomical pathologists and should prompt revision of the old morphological classification of necrosis. Here, we will review the pathophysiology of the various pathways of RN and we will present the novel ancillary techniques to dissect these pathways for anatomical pathologists. Application of these techniques will allow them to correlate the traditional classification of necrosis with the individual pathways of RN. Precise dissection of these pathways can be expected to improve the diagnostic precision and prediction of tissue diagnostics. Moreover, it will provide the evidence base for rational application of novel anti-necrotic agents to treat human disease.

Details are in the caption following the image
Figure 1
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Beyond apoptosis – necroptosis, pyroptosis, and ferroptosis
Due to space limitations, it is beyond the scope of this review to outline all known details of the pathways of necroptosis, pyroptosis, and ferroptosis. Our brief presentation of these RN pathways is meant to provide a general overview of cell death signalling.

Necroptosis
Necroptosis is defined as the loss of plasma membrane integrity following receptor interacting kinase 3 (RIPK3)-mediated phosphorylation of the pseudokinase mixed lineage kinase domain like (MLKL/pMLKL) 1. pMLKL oligomerises and results in plasma membrane rupture by ill-defined mechanisms that are counteracted by membrane repair mechanisms, such as the ESCRT-III complex 2-4. RIPK3, besides its kinase domain, consists of a Rip homotypic interacting motif (RHIM) domain 5-7 that allows its spontaneous multimerisation and formation of a hetero-amyloid supramolecular complex 8 referred to as the necrosome. Necrosome formation depends on the RHIM domain of RIPK3 and is constitutively prevented by intercalation of other RHIM domain-containing proteins 9, 10. Currently, only four such proteins are known that contain a RHIM domain (RIPK1, RIPK3, TRIF, and ZBP1) 11, 12. It was recently detected how the interplay of the RHIM domains results in formation of the necrosome. The isolated loss of the RIPK1 RHIM domain is sufficient to cause embryonic lethality in mice, demonstrating the RIPK1RHIM-mediated autoinhibition of spontaneous RIPK3 oligomerisation 9, 10. Upon necrosome formation, RIPK3 becomes activated by phosphorylation (pRIPK3), which in turn allows phosphorylation of MLKL 13, 14. However, it is important to realise that RIPK3 has non-necroptotic functions, such as pro-inflammatory gene transcription 15. Additionally, RIPK3, MLKL, and ZBP1 can regulate inflammasomes and pyroptosis 16-21.

RIPK1 itself can be phosphorylated on multiple sites 22-25 and specific antibodies against pRIPK1 have been generated, but the relative contribution of each of these phosphosites is still under scientific debate. It is, however, widely accepted that RIPK1 represents the master switch downstream of TNFR1 activation to signal towards NF-κB activation and cellular survival or to cell death by apoptosis/necroptosis 26-29, and potentially pyroptosis 30-34. Remarkably, the molecular decision depends on the kinases that are recruited to the proximal TNF-receptor signalling complex (TNF-RSC). Kinases such as TBK1 25, but also IKKϵ 35 are recruited to the TNF-RSC through ubiquitin chains assembled on RIPK1 36, 37. We refer to this complex as ‘Complex 1’. The ubiquitin chains, again, require a highly organised balance between assembly by the linear ubiquitin chain assembly complex (LUBAC) and OUT deubiquitinase with linear linkage specificity (OTULIN) in the case of M1 linkages 38, 39, or other E3 ligases in the case of K63 linkages 40. It is the loss of these ubiquitin linkages and the connected failure to recruit kinases to the TNF-RSC that represent the molecular switch between the life and death of a cell. In the case of the absence of the polyubiquitin chains, the TNF-RSC is no longer tethered to the plasma membrane to become cytosolic (then named ‘Complex 2a’) and results in apoptotic death of the cell, mediated by caspases. This process is detectable by IHC or IF against cleaved caspase-3 and typically is associated with the shrinkage of a cell and plasma membrane blebbing upon exposure of phosphatidylserine 41, 42, and may represent apoptotic immune cells in several instances, e.g. neutrophils, lymphocytes or activated macrophages 43. In the absence of functional caspases or in the presence of viral or synthetic caspase inhibitors, caspase-8 no longer actively prevents necroptotic signalling, RIPK3 oligomerises, a ‘Complex 2b’ forms, and necroptosis is executed 44-46.

The pathophysiological importance of necroptosis
Genetic evidence suggests that RIPK3-/MLKL-dependent necroptosis is of central pathophysiological importance in renal ischaemia–reperfusion injury (IRI)/tubular necrosis in mice 50-52 and humans 2, and contributes to cisplatin-mediated acute kidney injury 50, 53, myocardial IRI 54, myocardial remodelling 55, glucose intolerance 56, toxic liver diseases 57, atherosclerosis 58, diverse skin disorders 59-61, and severe inflammatory response syndrome (SIRS) 52, 62-65. RIPK3-deficient renal and cardiac allografts are less likely to be rejected following solid organ transplantation 66, 67. Finally, several cancers modify the necroptosis system 60, 68-70. In all of these diseases, pMLKL immunohistochemical and immunofluorescent staining may aid diagnosis (Figure 2), if relatively easy correlative studies are performed in the future. However, given our increasing knowledge about necroptosis over the last 5 years 25, 36, 49, 71-82, the morphological patterns of necrotic casts found in urine (Figure 3) appear to be as diverse as the histological findings in ATN (Figure 4). Therefore, it is likely that other molecular pathways, such as pyroptosis or ferroptosis, are additionally involved.

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Figure 2
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Pyroptosis
As pointed out in the previous section, key necroptosis molecules such as RIPK3, MLKL, and ZBP1 regulate inflammasome activation, which makes it difficult to clearly differentiate between pure necroptosis and pyroptosis, defined as gasdermin D (GSDMD)-mediated regulated necrosis. Pyroptosis may occur downstream of inflammasomes or independently of inflammasome activation. GSDMD is a cytosolic protein that contains a caspase cleavage site specific for inflammatory caspases (caspase-1, caspase-4, caspase-5, and caspase-11) 83-85. Importantly, these caspases and their cleavage sites are different from the well-studied apoptotic caspases (caspase-8, caspase-10, caspase-3, caspase-6, caspase-7, and caspase-9). Apoptotic caspases in general were believed to be unable to cleave GSDMD, but this view has changed very recently, at least for caspase-8 86-88. Importantly, GSDMD cleavage results in the formation of a pore that consists of a 27-mer of the C-terminal portion of GSDMD that is embedded into the plasma membrane and results in a type of necrotic cell death referred to as pyroptosis 89-94. However, besides GSDMD, inflammatory caspases also cleave pro-IL-1β and pro-IL-18 to generate active pro-inflammatory cytokines. Upon GSDMD pore formation, IL-1β and IL-18 may be secreted through the pore or, upon pyroptosis execution, passively access the interstitial space.

Activation of inflammatory caspases classically involves the formation of inflammasomes 95, 96, a detailed description of which is beyond the scope of this review. Briefly, inflammasomes are innate immune sensors that amplify a pro-inflammatory signal through innate immune cells, predominantly macrophages 95, 96. Pyroptosis and the associated release of danger-associated molecular patterns (DAMPs) and pro-inflammatory cytokines render this form of regulated necrosis the most immunogenic cell death known today. It is of vital importance to defend intracellular bacteria and to increase the set point of body temperature in the hypothalamus during sepsis (referred to as fever) 1, 33, 34, 83, 97-106. Despite its unique nature of inflammasome-mediated necrosis, recent data suggest that caspase-8 and MLKL may in some situations control the activation of caspase-1 and GSDMD cleavage and pore formation 32, 33, 107, and even that IL-1β secretion may occur independently of GSDMD 108. However, as with necroptosis, the terminal execution steps of pyroptosis are also regulated by the ESCRT machinery 87. It needs to be pointed out that there is also evidence for the involvement of gasdermins in apoptosis 109.

The pathophysiological importance of pyroptosis
Pyroptosis and inflammasomes have best been studied in professional phagocytes, and the role in host defence has been pointed out above. Pathophysiologically, the role of pyroptosis is much less clear. Interestingly, expression of the inflammasomal components caspase-1 and GSDMD is not restricted to phagocytes, but are also detectable in epithelial cells, e.g. renal tubules 110-112. However, to the best of our knowledge, no convincing evidence suggests a pathophysiological role of pyroptosis in epithelial tissues. It is therefore important to characterise the role of the pyroptosis machinery in epithelial cells. One previously unrecognised approach is the immunohistochemical detection of cleaved caspases and cleaved gasdermins in tissues. A first monoclonal antibody against cleaved GSDMD is currently being established (Feng Shao, NIBS, personal communication). Additionally, preclinical models investigated with GSDMD-deficient mice will be helpful to assess the role of pyroptosis. Obviously, fever syndromes have been investigated as far as mouse models are available, e.g. a mouse model of familial Mediterranean fever 113. Apart from genetic testing, the detection of cleaved GSDMD may be diagnostic for such patients. It is currently under investigation if pyroptosis is of any relevance in ischaemic and toxic injury.

Ferroptosis
The vital element oxygen, especially in the presence of free iron, can become part of reactive oxygen species (ROS) that are harmful to cellular membranes. Constant lipid peroxidation is prevented by glutathione peroxidase 4 (GPX4), a selenoprotein that constitutively metabolises glutathione (GSH) 114-120. Loss of GSH, loss of GPX4 or pharmacological inhibition of the active centre of GPX4 results in a lipoxygenase-mediated, highly specific peroxidation of phosphatidylethanolamine (PE) in the plasma membrane of eukaryotic cells 121. The precise mechanism of how the adapter protein PEBP-1 allows lipoxygenase-mediated PE peroxidation has been elucidated recently 122.

Several unique features characterise ferroptosis. Other than necroptosis and pyroptosis, ferroptosis tends to affect functional units, rather than single cells 123 in a process referred to as synchronised regulated necrosis (SRN) 46, 124-126. The mechanisms by which SRN is mediated from a cell to its neighbours are currently a subject of intensive research. In contrast to necroptosis 127, mitochondria are partially involved in ROS production during ferroptosis 128, but other sources of ROS may exist and have not been identified.

The pathophysiological importance of ferroptosis
Research on ferroptosis has attracted attention because of several diseases associated with changes that appear typical of ferroptosis. Genetic deletion of murine Gpx4 results in embryonic lethality, and inducible deletion after weaning results in lethal ATN 115. The exchange of a selenocysteine to a cysteine in GPX4 results in fatal epileptic seizures in mice, demonstrating a critical role for ferroptosis in the neurological system 117. In fact, several neurodegenerative disorders have been associated with features of ferroptosis, such as Alzheimer’s disease 129 and Parkinson’s disease 130. The roles of ferroptosis in stroke 131 and haemorrhagic stroke 132, 133 have also been demonstrated.

A role for ferroptosis Is currently under investigation in cardiovascular diseases such as myocardial infarction, following hints towards a critical role of iron and glutaminolysis 134. Given the nature of the ferroptotic signal as SRN, a functional syncytium such as the myocardium may suggest an involvement of ferroptosis upon failure of GPX4 or other selenoproteins to function in these tissues.

Visceral organs such as the liver and kidney were demonstrated to be protected from IRI upon treatment with the specific ferrostatins liproxstatin-1 (LPX-1) 115 and 16-86 50, respectively. Along these lines, perfusion supplements that contain these small molecules should be investigated upon solid organ transplantation.

Finally, cancers are more or less sensitive to ferroptosis, depending on the relative level of GPX4. Outstanding examples are diffuse large B-cell lymphomas and clear cell carcinomas of the kidney 119. In addition, a link between the tumour suppressor p53 and ferroptosis has been proposed 135 and is currently under intensive investigation. Clearly, the control of ferroptosis sensitivity is under control of the complex p53 system 136.

Necroinflammation
Cell necrosis is defined by loss of plasma membrane integrity. Unlike apoptosis, a regulated cell death process in which the plasma membrane does not rupture, necrosis is inevitably associated with the release of DAMPs and intracellular organelles 137, 138. Pathophysiologically, this triggers an immune response because novel surfaces become accessible to both the innate and the adaptive immune system during a process referred to as necroinflammation 138. Importantly, therefore, only necrotic-type cell death modes sufficiently explain the generation of anti-nuclear antibodies (ANAs) or anti-double-strand DNA (dsDNA) antibodies that are commonly observed in autoimmune diseases 139.

The evolutionary conservation of d”ffer’nt modes of regulated cell death may be explained by differences in their particular immunogenicity 46, 138. Following that hypothesis, apoptosis may be the least immunogenic form 46, 140. This is plausible given the billions of cells that are turned over by apoptosis in a human body every day. An immune response to this ‘physiological’ cellular turnover would kill the organism. In contrast, DAMP release in necrotic cell death subroutines may be similar in RN pathways 137, 141. However, the immune response to RN may be diverse, as production of pro-inflammatory cytokines might boost the immune cell triggers beyond DAMP release. In that sense, caspase-1 not only cleaves GSDMD to mediate pyroptosis, but also activates pro-IL-1β and pro-IL-18 that can subsequently be released in a GSDMD-dependent manner 84, 142. Pyroptosis, therefore, may represent a highly immunogenic type of regulated necrosis. In contrast, active transcription of IL-33 may stabilise regulatory T cells in the microenvironment that surrounds cells that die by necroptosis 143. Similarly, the production of CXCL1 may inhibit infiltrating neutrophils 144. In the case of ferroptosis, short-lived lipid peroxides may also exhibit specific effects on immune cells, but evidence for this hypothesis is very limited to date. Pathologists are expected to significantly contribute to the understanding of how different cell death pathways drive different patterns of immunity.

The relevance of regulated necrosi” pathways and necroinflammation in human diseases
As pointed out above in detail, necrosis is a hallmark of many human diseases. However, to date, very limited knowledge has been generated regarding the molecular nature of the necrosis pathways that mediate this pathological picture. TUNEL staining is non-specific and detects a positive signal in all dead or dying cells that contain fragmented DNA. Thus, for apoptosis detection, immunohistochemistry or immunofluorescence of cleaved caspase-3 is recommended.

The distinction of RN pathways by standard light microscopy on routine staining is not possible. As highlighted in Figure 5, some tools to identify specific modes of regulated necrosis are already available and others are being developed. The first human evidence for the direct detection of necroptosis took advantage of an antibody against pMLKL (Figure 5A). With this antibody, the quality of the immunohistochemistry appears sufficiently convincing and it is conceivable that necrotic cells stained positive 2. The first evidence was demonstrated in human kidney biopsies with ATN. Several approaches are currently being pursued to generate antibodies against the cleaved fragment of gasdermin D to reliably detect activation of pyroptosis. In theory, this should be equally as possible as the generation of antibodies against cleaved caspase-3 (see above). However, to date, no such antibody is commercially available.
Figure5
The detection of ferroptosis is much more difficult. It is difficult, although not impossible 145, to raise monoclonal antibodies against structures such as peroxidised phosphatidylethanolamine (ox-PE), the only accepted direct marker of ferroptosis. Therefore, mass spectrometry in general and quantitative lipidomics in particular are currently required to detect ferroptosis. Because of the nature of these methods, cellular localisation is not possible. Early attempts, however, to map individual phospholipids with single cell resolution have been undertaken (V Kagan, personal communication), but these are experimental techniques that will not be part of the toolbox of general pathologists in the near future

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