Peter Vandenabeele

Different types of cell death are often defined by morphological criteria, without a clear reference to precise biochemical mechanisms. The Nomenclature Committee on Cell Death (NCCD) proposes unified criteria for the definition of cell death and of its different morphologies, while formulating several caveats against the misuse of words and concepts that slow down progress in the area of cell death research. Authors, reviewers and editors of scientific periodicals are invited to abandon expressions like 'percentage apoptosis' and to replace them with more accurate descriptions of the biochemical and cellular parameters that are actually measured. Moreover, at the present stage, it should be accepted that caspase-independent mechanisms can cooperate with (or substitute for) caspases in the execution of lethal signaling pathways and that 'autophagic cell death' is a type of cell death occurring together with (but not necessarily by) autophagic vacuolization. This study details the 2009 recommendations of the NCCD on the use of cell death-related terminology including 'entosis', 'mitotic catastrophe', 'necrosis', 'necroptosis' and 'pyroptosis'.

In 2009, the Nomenclature Committee on Cell Death (NCCD) proposed a set of recommendations for the definition of distinct cell death morphologies and for the appropriate use of cell death-related terminology, including ‘apoptosis’, ‘necrosis’ and ‘mitotic catastrophe’. In view of the substantial progress in the biochemical and genetic exploration of cell death, time has come to switch from morphological to molecular definitions of cell death modalities. Here we propose a functional classification of cell death subroutines that applies to both in vitro and in vivo settings and includes extrinsic apoptosis, caspase-dependent or -independent intrinsic apoptosis, regulated necrosis, autophagic cell death and mitotic catastrophe. Moreover, we discuss the utility of expressions indicating additional cell death modalities. On the basis of the new, revised NCCD classification, cell death subroutines are defined by a series of precise, measurable biochemical features.

For a long time, apoptosis was considered the sole form of programmed cell death during development, homeostasis and disease, whereas necrosis was regarded as an unregulated and uncontrollable process. Evidence now reveals that necrosis can also occur in a regulated manner. The initiation of programmed necrosis, 'necroptosis', by death receptors (such as tumour necrosis factor receptor 1) requires the kinase activity of receptor-interacting protein 1 (RIP1; also known as RIPK1) and RIP3 (also known as RIPK3), and its execution involves the active disintegration of mitochondrial, lysosomal and plasma membranes. Necroptosis participates in the pathogenesis of diseases, including ischaemic injury, neurodegeneration and viral infection, thereby representing an attractive target for the avoidance of unwarranted cell death.

One of the key challenges in cancer research is how to effectively kill cancer cells while leaving the healthy cells intact. Cancer cells often have defects in cell death executioner mechanisms, which is one of the main reasons for therapy resistance. To enable growth, cancer cells exhibit an increased iron demand compared with normal, non-cancer cells. This iron dependency can make cancer cells more vulnerable to iron-catalyzed necrosis, referred to as ferroptosis. The identification of FDA-approved drugs as ferroptosis inducers creates high expectations for the potential of ferroptosis to be a new promising way to kill therapy-resistant cancers.

Cells may die from accidental cell death (ACD) or regulated cell death (RCD). ACD is a biologically uncontrolled process, whereas RCD involves tightly structured signaling cascades and molecularly defined effector mechanisms. A growing number of novel non-apoptotic forms of RCD have been identified and are increasingly being implicated in various human pathologies. Here, we critically review the current state of the art regarding non-apoptotic types of RCD, including necroptosis, pyroptosis, ferroptosis, entotic cell death, netotic cell death, parthanatos, lysosome-dependent cell death, autophagy-dependent cell death, alkaliptosis and oxeiptosis. The in-depth comprehension of each of these lethal subroutines and their intercellular consequences may uncover novel therapeutic targets for the avoidance of pathogenic cell loss.

Regulated necrosis, termed necroptosis, is negatively regulated by caspase-8 and is dependent on the kinase activity of RIPK1 and RIPK3. Necroptosis leads to rapid plasma membrane permeabilization and to the release of cell contents and exposure of damage-associated molecular patterns (DAMPs). We are only beginning to identify the necroptotic DAMPs, their modifications, and their potential role in the regulation of inflammation. In this review, we discuss the physiological relevance of necroptosis and its role in the modulation of inflammation. For example, during viral infection, RIPK3-mediated necroptosis acts as a backup mechanism to clear pathogens. Necroptosis is also involved in apparently immunologically silent maintenance of T cell homeostasis. In contrast, the induction of necroptosis in skin, intestine, systemic inflammatory response syndrome, and ischemia reperfusion injury provoke a strong inflammatory response, which might be triggered by emission of DAMPs from necroptotic cells, showing the detrimental side of necroptosis. Regulated necrosis, termed necroptosis, is negatively regulated by caspase-8 and is dependent on the kinase activity of RIPK1 and RIPK3. Necroptosis leads to rapid plasma membrane permeabilization and to the release of cell contents and exposure of damage-associated molecular patterns (DAMPs). We are only beginning to identify the necroptotic DAMPs, their modifications, and their potential role in the regulation of inflammation. In this review, we discuss the physiological relevance of necroptosis and its role in the modulation of inflammation. For example, during viral infection, RIPK3-mediated necroptosis acts as a backup mechanism to clear pathogens. Necroptosis is also involved in apparently immunologically silent maintenance of T cell homeostasis. In contrast, the induction of necroptosis in skin, intestine, systemic inflammatory response syndrome, and ischemia reperfusion injury provoke a strong inflammatory response, which might be triggered by emission of DAMPs from necroptotic cells, showing the detrimental side of necroptosis.

Missense mutations in the CIAS1 gene cause three autoinflammatory disorders: familial cold autoinflammatory syndrome, Muckle-Wells syndrome and neonatal-onset multiple-system inflammatory disease. Cryopyrin (also called Nalp3), the product of CIAS1, is a member of the NOD-LRR protein family that has been linked to the activation of intracellular host defence signalling pathways. Cryopyrin forms a multi-protein complex termed 'the inflammasome', which contains the apoptosis-associated speck-like protein (ASC) and caspase-1, and promotes caspase-1 activation and processing of pro-interleukin (IL)-1beta (ref. 4). Here we show the effect of cryopyrin deficiency on inflammasome function and immune responses. Cryopyrin and ASC are essential for caspase-1 activation and IL-1beta and IL-18 production in response to bacterial RNA and the imidazoquinoline compounds R837 and R848. In contrast, secretion of tumour-necrosis factor-alpha and IL-6, as well as activation of NF-kappaB and mitogen-activated protein kinases (MAPKs) were unaffected by cryopyrin deficiency. Furthermore, we show that Toll-like receptors and cryopyrin control the secretion of IL-1beta and IL-18 through different intracellular pathways. These results reveal a critical role for cryopyrin in host defence through bacterial RNA-mediated activation of caspase-1, and provide insights regarding the pathogenesis of autoinflammatory syndromes.

A plethora of apoptotic stimuli converge on the mitochondria and affect their membrane integrity. As a consequence, multiple death-promoting factors residing in the mitochondrial intermembrane space are liberated in the cytosol. Pro- and antiapoptotic Bcl-2 family proteins control the release of these mitochondrial proteins by inducing or preventing permeabilization of the outer mitochondrial membrane. Once released into the cytosol, these mitochondrial proteins activate both caspase-dependent and -independent cell death pathways. Cytochrome c was the first protein shown to be released from the mitochondria into the cytosol, where it induces apoptosome formation. Other released mitochondrial proteins include apoptosis-inducing factor (AIF) and endonuclease G, both of which contribute to apoptotic nuclear DNA damage in a caspase-independent way. Other examples are Smac/DIABLO (second mitochondria-derived activator of caspase/direct IAP-binding protein with low PI) and the serine protease HtrA2/OMI (high-temperature requirement protein A2), which both promote caspase activation and instigate caspase-independent cytotoxicity. The precise mode of action and importance of cytochrome c in apoptosis in mammalian cells has become clear through biochemical, structural and genetic studies. More recently identified factors, for example HtrA2/OMI and Smac/DIABLO, are still being studied intensively in order to delineate their functions in apoptosis. A better understanding of these functions may help to develop new strategies to treat cancer.

Murine L929 fibrosarcoma cells treated with tumor necrosis factor (TNF) rapidly die in a necrotic way, due to excessive formation of reactive oxygen intermediates. We investigated the role of caspases in the necrotic cell death pathway. When the cytokine response modifier A (CrmA), a serpin-like caspase inhibitor of viral origin, was stably overexpressed in L929 cells, the latter became 1,000-fold more sensitive to TNF-mediated cell death. In addition, TNF sensitization was also observed when the cells were pretreated with Ac-YVAD-cmk or zDEVD-fmk, which inhibits caspase-1- and caspase-3-like proteases, respectively. zVAD-fmk and zD-fmk, two broad-spectrum inhibitors of caspases, also rendered the cells more sensitive, since the half-maximal dose for TNF-mediated necrosis decreased by a factor of 1,000. The presence of zVAD-fmk also resulted in a more rapid increase of TNF-mediated production of oxygen radicals. zVAD-fmk-dependent sensitization of TNF cytotoxicity could be completely inhibited by the oxygen radical scavenger butylated hydroxyanisole. These results indicate an involvement of caspases in protection against TNF-induced formation of oxygen radicals and necrosis.

Significance Cell death by regulated necrosis causes tremendous tissue damage in a wide variety of diseases, including myocardial infarction, stroke, sepsis, and ischemia–reperfusion injury upon solid organ transplantation. Here, we demonstrate that an iron-dependent form of regulated necrosis, referred to as ferroptosis, mediates regulated necrosis and synchronized death of functional units in diverse organs upon ischemia and other stimuli, thereby triggering a detrimental immune response. We developed a novel third-generation inhibitor of ferroptosis that is the first compound in this class that is stable in plasma and liver microsomes and that demonstrates high efficacy when supplied alone or in combination therapy.

Cells exposed to extreme physicochemical or mechanical stimuli die in an uncontrollable manner, as a result of their immediate structural breakdown. Such an unavoidable variant of cellular demise is generally referred to as 'accidental cell death' (ACD). In most settings, however, cell death is initiated by a genetically encoded apparatus, correlating with the fact that its course can be altered by pharmacologic or genetic interventions. 'Regulated cell death' (RCD) can occur as part of physiologic programs or can be activated once adaptive responses to perturbations of the extracellular or intracellular microenvironment fail. The biochemical phenomena that accompany RCD may be harnessed to classify it into a few subtypes, which often (but not always) exhibit stereotyped morphologic features. Nonetheless, efficiently inhibiting the processes that are commonly thought to cause RCD, such as the activation of executioner caspases in the course of apoptosis, does not exert true cytoprotective effects in the mammalian system, but simply alters the kinetics of cellular demise as it shifts its morphologic and biochemical correlates. Conversely, bona fide cytoprotection can be achieved by inhibiting the transduction of lethal signals in the early phases of the process, when adaptive responses are still operational. Thus, the mechanisms that truly execute RCD may be less understood, less inhibitable and perhaps more homogeneous than previously thought. Here, the Nomenclature Committee on Cell Death formulates a set of recommendations to help scientists and researchers to discriminate between essential and accessory aspects of cell death.

Because of its ability to efficiently inhibit in vitro cytokine production by activated macrophages, we hypothesized that interleukin (IL) 10 might be of particular interest in preventing endotoxin-induced toxicity. We therefore examined the effects of IL-10 administration before lipopolysaccharide (LPS) challenge in mice. A marked reduction in the amounts of LPS-induced tumor necrosis factor (TNF) release in the circulation was observed after IL-10 pretreatment at doses at low as 10 U. IL-10 also efficiently prevented the hypothermia generated by the injection of 100 micrograms LPS. Finally, pretreatment with a single injection of 1,000 U IL-10 completely prevented the mortality consecutive to the challenge with 500 micrograms LPS, a dose that was lethal in 50% of the control mice. We conclude that IL-10 inhibits in vivo TNF secretion and protects against the lethality of endotoxin in a murine model of septic shock.

Tumour necrosis factor (TNF) exerts two main effects: a beneficial one as an anti-infection, anti-tumour cytokine, and a detrimental one in the systemic inflammatory response syndrome (SIRS). Two receptors (TNF-R) mediate these effects, but their precise role in different cell types is far from solved. TNF induces receptor oligomerization, an event that is believed to connect the receptors to downstream signalling pathways. Recent research suggests that several TNF-R-associated proteins, including kinases, may initiate cytoplasmic signal transduction.

Apoptotic cells have long been considered as intrinsically tolerogenic or unable to elicit immune responses specific for dead cell-associated antigens. However, multiple stimuli can trigger a functionally peculiar type of apoptotic demise that does not go unnoticed by the adaptive arm of the immune system, which we named "immunogenic cell death" (ICD). ICD is preceded or accompanied by the emission of a series of immunostimulatory damage-associated molecular patterns (DAMPs) in a precise spatiotemporal configuration. Several anticancer agents that have been successfully employed in the clinic for decades, including various chemotherapeutics and radiotherapy, can elicit ICD. Moreover, defects in the components that underlie the capacity of the immune system to perceive cell death as immunogenic negatively influence disease outcome among cancer patients treated with ICD inducers. Thus, ICD has profound clinical and therapeutic implications. Unfortunately, the gold-standard approach to detect ICD relies on vaccination experiments involving immunocompetent murine models and syngeneic cancer cells, an approach that is incompatible with large screening campaigns. Here, we outline strategies conceived to detect surrogate markers of ICD in vitro and to screen large chemical libraries for putative ICD inducers, based on a high-content, high-throughput platform that we recently developed. Such a platform allows for the detection of multiple DAMPs, like cell surface-exposed calreticulin, extracellular ATP and high mobility group box 1 (HMGB1), and/or the processes that underlie their emission, such as endoplasmic reticulum stress, autophagy and necrotic plasma membrane permeabilization. We surmise that this technology will facilitate the development of next-generation anticancer regimens, which kill malignant cells and simultaneously convert them into a cancer-specific therapeutic vaccine.

Neutrophil extracellular traps (NETs) are extracellular chromatin structures that can trap and degrade microbes. They arise from neutrophils that have activated a cell death program called NET cell death, or NETosis. Activation of NETosis has been shown to involve NADPH oxidase activity, disintegration of the nuclear envelope and most granule membranes, decondensation of nuclear chromatin and formation of NETs. We report that in phorbol myristate acetate (PMA)-stimulated neutrophils, intracellular chromatin decondensation and NET formation follow autophagy and superoxide production, both of which are required to mediate PMA-induced NETosis and occur independently of each other. Neutrophils from patients with chronic granulomatous disease, which lack NADPH oxidase activity, still exhibit PMA-induced autophagy. Conversely, PMA-induced NADPH oxidase activity is not affected by pharmacological inhibition of autophagy. Interestingly, inhibition of either autophagy or NADPH oxidase prevents intracellular chromatin decondensation, which is essential for NETosis and NET formation, and results in cell death characterized by hallmarks of apoptosis. These results indicate that apoptosis might function as a backup program for NETosis when autophagy or NADPH oxidase activity is prevented.

Although mixed lineage kinase domain-like (MLKL) protein has emerged as a specific and crucial protein for necroptosis induction, how MLKL transduces the death signal remains poorly understood. Here, we demonstrate that the full four-helical bundle domain (4HBD) in the N-terminal region of MLKL is required and sufficient to induce its oligomerization and trigger cell death. Moreover, we found that a patch of positively charged amino acids on the surface of the 4HBD binds to phosphatidylinositol phosphates (PIPs) and allows recruitment of MLKL to the plasma membrane. Importantly, we found that recombinant MLKL, but not a mutant lacking these positive charges, induces leakage of PIP-containing liposomes as potently as BAX, supporting a model in which MLKL induces necroptosis by directly permeabilizing the plasma membrane. Accordingly, we found that inhibiting the formation of PI(5)P and PI(4,5)P2 specifically inhibits tumor necrosis factor (TNF)-mediated necroptosis but not apoptosis.

Article17 January 2012free access A novel pathway combining calreticulin exposure and ATP secretion in immunogenic cancer cell death Abhishek D Garg Abhishek D Garg Cell Death Research and Therapy Unit, Department of Cellular and Molecular Medicine KU Leuven, KU Leuven, Leuven, Belgium Search for more papers by this author Dmitri V Krysko Dmitri V Krysko Molecular Signaling and Cell Death Unit, Department for Molecular Biomedical Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Tom Verfaillie Tom Verfaillie Cell Death Research and Therapy Unit, Department of Cellular and Molecular Medicine KU Leuven, KU Leuven, Leuven, Belgium Search for more papers by this author Agnieszka Kaczmarek Agnieszka Kaczmarek Molecular Signaling and Cell Death Unit, Department for Molecular Biomedical Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Gabriela B Ferreira Gabriela B Ferreira Laboratory for Experimental Medicine and Endocrinology (LEGENDO), Department of Clinical and Experimental Medicine, KU Leuven, Leuven, Belgium Search for more papers by this author Thierry Marysael Thierry Marysael Laboratory for Pharmaceutical Biology, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, Belgium Search for more papers by this author Noemi Rubio Noemi Rubio Cell Death Research and Therapy Unit, Department of Cellular and Molecular Medicine KU Leuven, KU Leuven, Leuven, Belgium Search for more papers by this author Malgorzata Firczuk Malgorzata Firczuk Department of Immunology, Centre of Biostructure Research, Medical University of Warsaw, Warsaw, Poland Department 3, Institute of Physical Chemistry, Polish Academy of Sciences, Warsaw, Poland Search for more papers by this author Chantal Mathieu Chantal Mathieu Laboratory for Experimental Medicine and Endocrinology (LEGENDO), Department of Clinical and Experimental Medicine, KU Leuven, Leuven, Belgium Search for more papers by this author Anton J M Roebroek Anton J M Roebroek Experimental Mouse Genetics, Department of Human Genetics, KU Leuven, Leuven, Belgium Search for more papers by this author Wim Annaert Wim Annaert Laboratory for Membrane Trafficking, Department of Human Genetics, KU Leuven and VIB-Center for the Biology of Disease, Leuven, Belgium Search for more papers by this author Jakub Golab Jakub Golab Department of Immunology, Centre of Biostructure Research, Medical University of Warsaw, Warsaw, Poland Department 3, Institute of Physical Chemistry, Polish Academy of Sciences, Warsaw, Poland Search for more papers by this author Peter de Witte Peter de Witte Laboratory for Pharmaceutical Biology, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, Belgium Search for more papers by this author Peter Vandenabeele Peter Vandenabeele Molecular Signaling and Cell Death Unit, Department for Molecular Biomedical Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Patrizia Agostinis Corresponding Author Patrizia Agostinis Cell Death Research and Therapy Unit, Department of Cellular and Molecular Medicine KU Leuven, KU Leuven, Leuven, Belgium Search for more papers by this author Abhishek D Garg Abhishek D Garg Cell Death Research and Therapy Unit, Department of Cellular and Molecular Medicine KU Leuven, KU Leuven, Leuven, Belgium Search for more papers by this author Dmitri V Krysko Dmitri V Krysko Molecular Signaling and Cell Death Unit, Department for Molecular Biomedical Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Tom Verfaillie Tom Verfaillie Cell Death Research and Therapy Unit, Department of Cellular and Molecular Medicine KU Leuven, KU Leuven, Leuven, Belgium Search for more papers by this author Agnieszka Kaczmarek Agnieszka Kaczmarek Molecular Signaling and Cell Death Unit, Department for Molecular Biomedical Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Gabriela B Ferreira Gabriela B Ferreira Laboratory for Experimental Medicine and Endocrinology (LEGENDO), Department of Clinical and Experimental Medicine, KU Leuven, Leuven, Belgium Search for more papers by this author Thierry Marysael Thierry Marysael Laboratory for Pharmaceutical Biology, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, Belgium Search for more papers by this author Noemi Rubio Noemi Rubio Cell Death Research and Therapy Unit, Department of Cellular and Molecular Medicine KU Leuven, KU Leuven, Leuven, Belgium Search for more papers by this author Malgorzata Firczuk Malgorzata Firczuk Department of Immunology, Centre of Biostructure Research, Medical University of Warsaw, Warsaw, Poland Department 3, Institute of Physical Chemistry, Polish Academy of Sciences, Warsaw, Poland Search for more papers by this author Chantal Mathieu Chantal Mathieu Laboratory for Experimental Medicine and Endocrinology (LEGENDO), Department of Clinical and Experimental Medicine, KU Leuven, Leuven, Belgium Search for more papers by this author Anton J M Roebroek Anton J M Roebroek Experimental Mouse Genetics, Department of Human Genetics, KU Leuven, Leuven, Belgium Search for more papers by this author Wim Annaert Wim Annaert Laboratory for Membrane Trafficking, Department of Human Genetics, KU Leuven and VIB-Center for the Biology of Disease, Leuven, Belgium Search for more papers by this author Jakub Golab Jakub Golab Department of Immunology, Centre of Biostructure Research, Medical University of Warsaw, Warsaw, Poland Department 3, Institute of Physical Chemistry, Polish Academy of Sciences, Warsaw, Poland Search for more papers by this author Peter de Witte Peter de Witte Laboratory for Pharmaceutical Biology, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, Belgium Search for more papers by this author Peter Vandenabeele Peter Vandenabeele Molecular Signaling and Cell Death Unit, Department for Molecular Biomedical Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Patrizia Agostinis Corresponding Author Patrizia Agostinis Cell Death Research and Therapy Unit, Department of Cellular and Molecular Medicine KU Leuven, KU Leuven, Leuven, Belgium Search for more papers by this author Author Information Abhishek D Garg1, Dmitri V Krysko2,3, Tom Verfaillie1, Agnieszka Kaczmarek2,3, Gabriela B Ferreira4, Thierry Marysael5, Noemi Rubio1, Malgorzata Firczuk6,7, Chantal Mathieu4, Anton J M Roebroek8, Wim Annaert9, Jakub Golab6,7, Peter de Witte5, Peter Vandenabeele2,3 and Patrizia Agostinis 1 1Cell Death Research and Therapy Unit, Department of Cellular and Molecular Medicine KU Leuven, KU Leuven, Leuven, Belgium 2Molecular Signaling and Cell Death Unit, Department for Molecular Biomedical Research, VIB, Ghent, Belgium 3Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium 4Laboratory for Experimental Medicine and Endocrinology (LEGENDO), Department of Clinical and Experimental Medicine, KU Leuven, Leuven, Belgium 5Laboratory for Pharmaceutical Biology, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, Belgium 6Department of Immunology, Centre of Biostructure Research, Medical University of Warsaw, Warsaw, Poland 7Department 3, Institute of Physical Chemistry, Polish Academy of Sciences, Warsaw, Poland 8Experimental Mouse Genetics, Department of Human Genetics, KU Leuven, Leuven, Belgium 9Laboratory for Membrane Trafficking, Department of Human Genetics, KU Leuven and VIB-Center for the Biology of Disease, Leuven, Belgium *Corresponding author. Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Leuven (KU Leuven), Campus Gasthuisberg O&N1, Herestraat 49, Box 901, 3000 Leuven, Belgium. Tel.: +32 16 345715; Fax: +32 16 345995; E-mail: [email protected] The EMBO Journal (2012)31:1062-1079https://doi.org/10.1038/emboj.2011.497 There is a Have you seen? (March 2012) associated with this Article. PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Surface-exposed calreticulin (ecto-CRT) and secreted ATP are crucial damage-associated molecular patterns (DAMPs) for immunogenic apoptosis. Inducers of immunogenic apoptosis rely on an endoplasmic reticulum (ER)-based (reactive oxygen species (ROS)-regulated) pathway for ecto-CRT induction, but the ATP secretion pathway is unknown. We found that after photodynamic therapy (PDT), which generates ROS-mediated ER stress, dying cancer cells undergo immunogenic apoptosis characterized by phenotypic maturation (CD80high, CD83high, CD86high, MHC-IIhigh) and functional stimulation (NOhigh, IL-10absent, IL-1βhigh) of dendritic cells as well as induction of a protective antitumour immune response. Intriguingly, early after PDT the cancer cells displayed ecto-CRT and secreted ATP before exhibiting biochemical signatures of apoptosis, through overlapping PERK-orchestrated pathways that require a functional secretory pathway and phosphoinositide 3-kinase (PI3K)-mediated plasma membrane/extracellular trafficking. Interestingly, eIF2α phosphorylation and caspase-8 signalling are dispensable for this ecto-CRT exposure. We also identified LRP1/CD91 as the surface docking site for ecto-CRT and found that depletion of PERK, PI3K p110α and LRP1 but not caspase-8 reduced the immunogenicity of the cancer cells. These results unravel a novel PERK-dependent subroutine for the early and simultaneous emission of two critical DAMPs following ROS-mediated ER stress. Introduction Current anticancer regimens mediate killing of tumour cells mainly by activating apoptosis, an immunosuppressive or even tolerogenic cell death process. However, it has recently emerged that a selected class of cytotoxic agents (e.g., anthracyclines) can cause tumour cells to undergo an immunogenic form of apoptosis and these dying tumour cells can induce an effective antitumour immune response (Locher et al, 2010). Immunogenic apoptosis of cancer cells displays the main biochemical hallmarks of ‘tolerogenic’ apoptosis: phosphatidylserine exposure, caspase activation, and mitochondrial depolarization. However, this type of cell death also seems to have two other important properties: (1) surface exposure or secretion of critical ‘immunogenic signals’ that fall in the category of damage-associated molecular patterns (DAMPs; Zitvogel et al, 2010a) and (2) the ability to elicit a protective immune response against tumour cells (Obeid et al, 2007; Green et al, 2009; Garg et al, 2010b; Zitvogel et al, 2010b). Several DAMPs have recently been identified as crucial for immunogenic apoptosis. These include surface calreticulin (ecto-CRT), surface HSP90 (ecto-HSP90), and secreted ATP (Spisek et al, 2007; Kepp et al, 2009). Ecto-CRT has been shown to act primarily as an ‘eat me’ signal (Gardai et al, 2005), presumably essential for priming the innate immune system, since depletion of CRT by siRNA knockdown averts the immunogenicity of cancer cell death (Obeid et al, 2007). Similarly, bortezomib-induced ecto-HSP90 exposure is crucial for immunogenic death of tumour cells and their subsequent contact with dendritic cells (DCs; Spisek et al, 2007). On the other hand, secreted ATP acts either as a ‘find me’ signal or as an activator of the NLRP3 inflammasome (Elliott et al, 2009; Ghiringhelli et al, 2009). However, while the signalling pathways governing surface exposure of CRT have been delineated to some extent (Panaretakis et al, 2009), insufficient information exists on the molecular pathway behind ATP secretion. Finally, immunogenic apoptosis is sometimes associated with disappearance of certain surface-associated molecules, for example CD47, which are referred to as ‘do not eat me’ signals (Chao et al, 2010). One common feature of all immunogenic apoptosis-inducing stimuli so far identified is induction of endoplasmic reticulum (ER) stress (Panaretakis et al, 2009; Garg et al, 2010b; Zitvogel et al, 2010b). Importantly, in the case of ecto-CRT triggered by anthracyclines, both ER stress and reactive oxygen species (ROS) production have been found to be mandatory (Panaretakis et al, 2009). However, anthracyclines suffer from dose-limiting side effects (Minotti et al, 2004; Vergely et al, 2007). Moreover, ROS production is neither a primary effect of anthracyclines nor predominantly ER directed, which makes the anthracycline-induced ‘ROS-based’ ER stress less effective and secondary in nature (Minotti et al, 2004; Vergely et al, 2007). Thus, we envisaged that one way of improving the immunogenicity of dying cancer cells is by using a therapeutic approach that can generate strong ROS-dependent ER stress as a primary effect (Garg et al, 2011). We hypothesized that photodynamic therapy (PDT; Agostinis et al, 2011) might fit the criterion of primary ER-directed ROS production. PDT can induce oxidative stress at certain subcellular sites by activating organelle-associated photosensitizers (Castano et al, 2006; Buytaert et al, 2007). Once excited by visible light and in the presence of oxygen, photosensitizers can generate organelle-localized ROS that can cause lethal damage to the cells (Agostinis et al, 2002). Additionally, this ROS-based anticancer therapy can also cause ‘emission’ of DAMPs and activate the host immune system (Korbelik et al, 2005; Garg et al, 2010a). To test this hypothesis, we used the ER-associated photosensitizer, hypericin. When it is activated by light, it causes a ROS-mediated loss-of-function of SERCA2 with consequent disruption of ER-Ca2+ homeostasis, followed by BAX/BAK-based mitochondrial apoptosis (Buytaert et al, 2006). This photo-oxidative ER stress (phox-ER stress) is accompanied by transcriptional upregulation of components of the unfolded protein response (UPR) and by changes in the expression of various genes coding for immunomodulatory proteins (Buytaert et al, 2008; Garg et al, 2010a). We report here that phox-ER stress induces immunogenic apoptosis in treated cancer cells. Early after phox-ER stress and largely preceding phosphatidylserine externalization, cancer cells mobilize CRT at the surface and secrete ATP through an overlapping PERK- and phosphoinositide 3-kinase (PI3K)-mediated mechanism, which is dissociated from caspase signalling. Intriguingly, we found that LRP1 is required for the docking of ecto-CRT. Results Phox-ER stress causes cancer cells to undergo immunogenic apoptosis At the outset, we decided to investigate whether cancer cells dying in response to phox-ER stress (Hyp-PDT based; unless otherwise mentioned) can activate human immature DCs (hu-iDCs). We used phox-ER stress (Supplementary Figure S1) mediated apoptosis-inducing conditions reported in our previous studies (Hendrickx et al, 2003; Buytaert et al, 2006) generating ∼87% of cell death of the human bladder carcinoma T24 cells within 24 h (Supplementary Figure S2). T24 cells subjected to Hyp-PDT underwent phagocytic interactions with hu-iDCs (Figure 1A). They were also phagocytosed by Mf4/4 phagocytes preferentially over untreated T24 cells (Supplementary Figure S3). Moreover, these Hyp-PDT-treated dying T24 cells induced phenotypic maturation of hu-iDCs, as indicated by surface upregulation of MHC class II (HLA-DR) and co-stimulatory CD80, CD83 and CD86 molecules (Figure 1B; Supplementary Figure S4A and B). The significant surface expression of these molecules was similar to that induced by lipopolysaccharide (LPS), a known pathogen-associated molecular pattern (PAMP) (Figure 1B; Supplementary Figure S4A and B). In contrast, freeze-thawed T24 cells undergoing accidental necrosis (AN) did not strongly stimulate DC maturation (Figure 1B; Supplementary Figure S4A and B). These findings rule out the possibility that AN might be responsible for the increased DC maturation seen against phox-ER stressed cells. Figure 1.Tumour cells dying under phox-ER stress conditions induce DC maturation and activate the adaptive immune system. (A) In-vitro phagocytosis of T24 cells treated with Hyp-PDT (red) by human immature dendritic cells (hu-iDCs) (green). The confocal fluorescence images show various phagocytic interactions between dying T24 cells and hu-iDCs, such as tethering (a), initiation of engulfment by extending the pseudopodia (b), and final stages of engulfment (c); scale bar=20 μm. (B) Human DC maturation analysis. T24 cells were left untreated (CNTR), freeze/thawed (accidental necrosis=AN), or treated with a high PDT dose. They were then co-incubated with hu-iDCs. As a positive control, hu-iDCs were stimulated with LPS for 24 h. After co-incubation, the cells were immunostained in two separate groups for CD80/CD83 positivity and CD86/HLA-DR positivity and scored by FACS analysis. Data have been normalized to the ‘CNTR T24 + hu-iDCs’ values. Fold change values are means of two independent experiments (two replicate determinations in each)±s.e.m. (*P<0.05, versus ‘CNTR T24+hu-iDCs’). (C, D) Cytokine and respiratory burst patterns exhibited by human DCs. The T24-hu-iDC co-incubation conditioned media obtained during the experiments detailed in (B) were recuperated followed by analysis for concentrations of nitrite (solubilized form of nitric oxide or NO) (C), and IL-10 (D). Absolute concentrations are the means of two independent experiments (four replicate determinations in each)±s.d. (*P<0.05 versus hu-iDC only). (E) Priming of adaptive immune system by dead/dying CT26 cells. Following immunization with PBS (CNTR) or with CT26 cells treated with tunicamycin (TUN), mitoxantrone (MTX) and the highest PDT dose, the mice were rechallenged with live CT26 tumour cells. Subsequently, the percentage of mice with tumour-free rechallenge site was determined (n represents the number of mice). Download figure Download PowerPoint To get further insight into the functional status of DCs, we evaluated the pattern of certain cytokines including the generation of nitric oxide (NO) as a marker for respiratory burst (Stafford et al, 2002). We compared DCs exposed to Hyp-PDT-treated T24 cells with those exposed to LPS or T24 cells dying following AN. We found that hu-iDCs exposed to Hyp-PDT-treated cancer cells displayed a distinguished pattern of functional activation characterized by NOhigh, IL-10absent (Figure 1C and D). This was clearly different from that induced by accidental necrotic cells (NOhigh, IL-10high) or by LPS (NOlow, IL-10low) (Figure 1C and D). Interestingly, LPS and especially accidental necrotic cells stimulated the production of IL-10 (Figure 1D), whereas Hyp-PDT-treated cells failed to stimulate the production of this immunosuppressive cytokine (Kim et al, 2006; Zitvogel et al, 2006) by hu-iDCs. To investigate the ability of cancer cells undergoing phox-ER stress to activate the adaptive immune system, we carried out in-vivo experiments in immunocompetent BALB/c mice. Before initiating the in-vivo experiments, we optimized the mouse colon carcinoma CT26 cell line for Hyp-PDT-induced apoptosis (Supplementary Figure S5) and ER stress (Supplementary Figure S1). As observed previously in other cells (Hendrickx et al, 2003; Buytaert et al, 2006), hypericin colocalized strongly with ER Tracker (Supplementary Figure S5A) and upon light irradiation induced not only appreciable cell killing (Supplementary Figure S5B) but also the main hallmarks of apoptosis, including caspase-3 and PARP cleavage (Supplementary Figure S5C). Furthermore, the CT26 cells exposed to Hyp-PDT were preferentially phagocytosed over untreated CT26 cells by murine JAWSII DCs (Supplementary Figure S6). Then, in the in-vivo study, we immunized BALB/c mice with Hyp-PDT-treated dying/dead CT26 cells. As positive and negative controls for immunogenic cell death, respectively, we used CT26 cells treated with the anthracycline, mitoxantrone (MTX) or tunicamycin (TN, an inhibitor of N-linked glycosylation) (Obeid et al, 2007). The immunized mice were then rechallenged with live CT26 tumour cells. Protection against tumour growth at the rechallenge site was interpreted as a sign of successful priming of the adaptive immune system (Figure 1E). Mice immunized with CT26 cells treated with MTX or Hyp-PDT showed robust signs of activation of the adaptive immune system: both procedures strongly prevented the tumour growth seen in the non-immunized mice. By contrast, most of the mice immunized with tunicamycin-treated CT26 cells experienced tumour growth after rechallenge (Figure 1E), which confirms the poor immunogenic properties of cancer cell death induced by this ER stress agent (Obeid et al, 2007). These data suggest that apoptotic cancer cells dying from phox-ER stress induced by Hyp-PDT activate the immune system, which is one of the important properties of immunogenic apoptosis. Cancer cells exposed to phox-ER stress surface expose or secrete/release immunogenic DAMPs We next analysed the surface exposure/release of CRT, secreted ATP and extracellular heat-shock proteins (i.e., HSP90 and HSP70) following phox-ER stress using three different Hyp-PDT doses—low, medium, and high PDT. Moreover, because of the reported effects of anthracyclines, MTX, and doxorubicin (DOXO) on immunogenic cell death (Obeid et al, 2007), we used them throughout the study for comparison. Ecto-CRT surface exposure, detected by immunofluorescence staining of T24 cells treated with Hyp-PDT or MTX, showed the characteristic surface ‘patches’ reported previously (Gardai et al, 2005; Obeid et al, 2007; Figure 2A). Cell surface biotinylation followed by immunoblot analysis of the isolated plasma membrane proteins derived from T24 cancer cells treated with Hyp-PDT revealed that phox-ER stress (Supplementary Figure S1) induced enhanced surface exposure of CRT (Figure 2B). This ecto-CRT preceded apoptosis-associated phosphatidylserine exposure (Supplementary Figure S2) under plasma membrane non-permeabilizing conditions (Figure 2C). On-cell western assay (Gonzalez-Gronow et al, 2007) confirmed these results (Supplementary Figure S7). In general, Hyp-PDT was observed to be superior to DOXO and MTX (Figure 2D and E), in terms of mobilizing CRT to the surface of cancer cells. Moreover, ecto-CRT was detectable as early as 30 min after Hyp-PDT and increased with time (Figure 2E). The 30-min threshold is much earlier than reported for anthracyclines (Obeid et al, 2007). This induction of ecto-CRT by Hyp-PDT was diminished in the presence of the 1O2 quencher L-histidine, thus revealing its ROS dependence (Buytaert et al, 2006; Supplementary Figure S8A). In contrast to anthracycline-induced ecto-CRT exposure (Panaretakis et al, 2008), ecto-CRT exposure following Hyp-PDT was not accompanied by co-translocation of ERp57 to the surface (Figure 2B). Figure 2.Phox-ER-stressed cancer cells expose calreticulin on the surface (ecto-CRT). (A) Immunofluorescence analysis of ecto-CRT. T24 cells were treated with MTX (1 μM for 4 h) and a high PDT dose (recovered 1 h post PDT) or left untreated (CNTR). Alternatively, some cells were saponin permeabilized. This was followed by staining with Sytox Green (exclusion dye), fixation, and immunostaining for CRT and counterstaining with DAPI; scale bar=20 μm. (B) Surface biotinylation analysis of ecto-CRT following phox-ER stress. T24 cells were treated with indicated doses of PDT. They were recovered at the indicated intervals after PDT treatment. Surface proteins were biotinylated followed by immunoblotting. In (B), (D), and (F), ‘+BIO' indicates controls exposed to buffer with biotin and ‘−BIO' indicates controls exposed to buffer without biotin (negative control). (C) Plasma membrane permeabilization kinetics following phox-ER stress. T24 cells were treated with PDT and the resulting conditioned media derived at the indicated times post-PDT were analysed for the presence of cytosolic LDH. Total LDH content was determined following Triton-based permeabilization of cells. Data are presented as percent LDH release; values are means of five replicate determinations±s.d. (*P<0.05, versus CNTR). (D) Phox-ER stress induces more ecto-CRT than anthracyclines. T24 cells were treated with PDT, DOXO (25 μM for 4 h), and MTX (1 μM for 4 h). They were recovered at the indicated intervals after PDT treatment. Surface proteins were biotinylated as described for (B). (E) Integrated band densitometric analysis of ecto-CRT. T24 cells were treated with DOXO (25 μM for 4 h), MTX (1 μM for 4 h), and PDT (dose and recovery time points are indicated); and surface proteins were resolved as detailed in (B). Following this, the ecto-CRT protein bands were quantified for the integrated band density via Image J software. Data have been normalized to the CNTR values. Fold change values are means of three independent determinations±s.e.m. (*P<0.05, versus CNTR). (F) Surface biotinylation analysis for KDEL sequence detection following phox-ER stress. CRT WT and KO MEFs were treated with a low PDT dose and surface biotinylated as mentioned in (B). Immunoblotting was done to detect the C-terminal KDEL sequence of various ER proteins (expected molecular weights are indicated). Download figure Download PowerPoint Likewise ecto-HSP90, certain ER proteins, such as calnexin (CNX), PERK and BiP were also undetectable on the surface of the cells under the same conditions that efficiently mobilized ecto-CRT (Supplementary Figure S8B). In addition, several other ER proteins have been reported to undergo translocation to the plasma membrane (Zai et al, 1999; Korbelik et al, 2005; Zhang et al, 2010). Therefore, we used cell surface biotinylation combined with immunoblotting to screen for surface-translocated proteins containing the KDEL ‘ER retrieval’ signal sequence, in wild-type (WT) and CRT−/− MEFs. Ecto-CRT (∼63 kDa) was the only protein with the KDEL sequence recognizable on the surface of Hyp-PDT-treated cells (Figure 2F). No KDEL-containing proteins were found in the plasma membrane fraction of cells lacking CRT (Figure 2F). On the other hand, KDEL sequences of ER resident proteins, such as GRP94, GRP78, ERp72 (ER resident protein 72), and PDI, were identifiable by their molecular weights in the intracellular protein fractions of WT and CRT−/− cells (Figure 2F). Overall, these results indicate that phox-ER stress does not lead to a general surface scrambling of ER proteins (luminal or membrane associated) but rather to a selective and rapid surface exposure of CRT in pre-apoptotic conditions. We next asked whether photo-oxidative stress mediated by other photosensitizers known to localize to other subcellular sites in addition to the ER were equally capable of surface-exposing CRT. To this end, we used photofrin (PF-PDT), a photosensitizer used in the clinic and known to induce phox-ER stress (Szokalska et al, 2009). Interestingly, while phox-ER stress mediated by Hyp-PDT strongly induced ecto-CRT, it was not so for PF-PDT (Supplementary Figure S8C) under similar apoptosis-inducing conditions as reported previously (Szokalska et al, 2009). This difference between Hyp-PDT and PF-PDT in ecto-CRT induction might be due to the more pronounced ER localization of hypericin when compared with photofrin (Buytaert et al, 2007; Szokalska et al, 2009; Luo et al, 2010). These data further underline the importance of a robust ER-directed oxidative stress in inducing ecto-CRT. Next, we addressed the possibility that apart from induction of ecto-CRT, Hyp-PDT-treated T24 cancer cells can secrete ATP into the extracellular environment. Analysis of the conditioned media showed that T24 cancer cells treated with Hyp-PDT secreted ATP (Figure 3A) under non-permeabilizing plasma membrane conditions (Figure 2C). Secretion of ATP preceded apoptosis-associated phosphatidylserine exposure (Supplementary Figure S2) and downregulation of the ‘do not eat me’ signal CD47 (Supplementary Figure S8D). Interestingly, at least at medium Hyp-PDT dose, the corresponding intracellular ATP content rose considerably in the pre-apoptotic stages (Figure 3B). Figure 3.Cancer cells exposed to phox-ER stress actively secrete ATP, passively release HSP70, HSP90, CRT, and induce IL-1β production in DCs. (A, B) ATP secretion following phox-ER stress. T24 cells were treated with PDT or left untreated (CNTR), and the conditioned media derived from these cells (1 h post PDT in serum-free media) were analysed for the presence of ATP (A). Simultaneously, the corresponding cells were permeabilization with saponin (1 h post PDT) followed by determination of ATP content in the lysate (B). Absolute concentrations are mean values of six replicate determinations±s.d. (*P<0.05, versus CNTR). (C) Cancer cells subjected to phox-ER stress stimulate IL-1β production in hu-iDCs. T24 cells were treated to undergo accidental necrosis (AN), or treated with a high PDT dose (recovered 24 h post PDT), and then co-incubated with hu-iDCs for 24 h. Simultaneously hu-iDCs were stimulated with LPS. After co-incubation, the co-incubation conditioned media (CCM) were analysed for the presence of IL-1β. Cytokine concentrations (in pg/ml) are means of two independent experiments (four replicate determinations in each) ±s.e.m. (*P<0.

Necrosis has long been described as a consequence of physico-chemical stress and thus accidental and uncontrolled. Recently, it is becoming clear that necrotic cell death is as well controlled and programmed as caspase-dependent apoptosis, and that it may be an important cell death mode that is both pathologically and physiologically relevant. Necrotic cell death is not the result of one well-described signalling cascade but is the consequence of extensive crosstalk between several biochemical and molecular events at different cellular levels. Recent data indicate that serine/threonine kinase RIP1, which contains a death domain, may act as a central initiator. Calcium and reactive oxygen species (ROS) are main players during the propagation and execution phases of necrotic cell death, directly or indirectly provoking damage to proteins, lipids and DNA, which culminates in disruption of organelle and cell integrity. Necrotically dying cells initiate pro-inflammatory signalling cascades by actively releasing inflammatory cytokines and by spilling their contents when they lyse. Unravelling the signalling cascades contributing to necrotic cell death will permit us to develop tools to specifically interfere with necrosis at certain levels of signalling. Necrosis occurs in both physiological and pathophysiological processes, and is capable of killing tumour cells that have developed strategies to evade apoptosis. Thus detailed knowledge of necrosis may be exploited in therapeutic strategies.

Cell death is essential for a plethora of physiological processes, and its deregulation characterizes numerous human diseases. Thus, the in-depth investigation of cell death and its mechanisms constitutes a formidable challenge for fundamental and applied biomedical research, and has tremendous implications for the development of novel therapeutic strategies. It is, therefore, of utmost importance to standardize the experimental procedures that identify dying and dead cells in cell cultures and/or in tissues, from model organisms and/or humans, in healthy and/or pathological scenarios. Thus far, dozens of methods have been proposed to quantify cell death-related parameters. However, no guidelines exist regarding their use and interpretation, and nobody has thoroughly annotated the experimental settings for which each of these techniques is most appropriate. Here, we provide a nonexhaustive comparison of methods to detect cell death with apoptotic or nonapoptotic morphologies, their advantages and pitfalls. These guidelines are intended for investigators who study cell death, as well as for reviewers who need to constructively critique scientific reports that deal with cellular demise. Given the difficulties in determining the exact number of cells that have passed the point-of-no-return of the signaling cascades leading to cell death, we emphasize the importance of performing multiple, methodologically unrelated assays to quantify dying and dead cells.

Interleukin-33 (IL-33) is a member of the IL-1 family and is involved in polarization of T cells toward a T helper 2 (Th2) cell phenotype. IL-33 is thought to be activated via caspase-1-dependent proteolysis, similar to the proinflammatory cytokines IL-1β and IL-18, but this remains unproven. Here we showed that IL-33 was processed by caspases activated during apoptosis (caspase-3 and -7) but was not a physiological substrate for caspases associated with inflammation (caspase-1, -4, and -5). Furthermore, caspase-dependent processing of IL-33 was not required for ST2 receptor binding or ST2-dependent activation of the NF-κB transcription factor. Indeed, caspase-dependent proteolysis of IL-33 dramatically attenuated IL-33 bioactivity in vitro and in vivo. These data suggest that IL-33 does not require proteolysis for activation, but rather, that IL-33 bioactivity is diminished through caspase-dependent proteolysis within apoptotic cells. Thus, caspase-mediated proteolysis acts as a switch to dampen the proinflammatory properties of IL-33.

Three major morphologies of cell death have been described: apoptosis (type I), cell death associated with autophagy (type II) and necrosis (type III). Apoptosis and cell death associated with autophagy can be distinguished by certain biochemical events. However, necrosis is characterized mostly in negative terms by the absence of caspase activation, cytochrome c release and DNA oligonucleosomal fragmentation. A particular difficulty in defining necrosis is that in the absence of phagocytosis apoptotic cells become secondary necrotic cells with many morphological features of primary necrosis. In this review, we present a selection of techniques that can be used to identify necrosis and to discriminate it from apoptosis. These techniques rely on the following cell death parameters: (1) morphology (time-lapse and transmission electron microscopy and flow fluorocytometry); (2) cell surface markers (phosphatidylserine exposure versus membrane permeability by flow fluorocytometry); (3) intracellular markers (oligonucleosomal DNA fragmentation by flow fluorocytometry, caspase activation, Bid cleavage and cytochrome c release by western blotting); (4) release of extracellular markers in the supernatant (caspases, HMGB-1 and cytokeratin 18). Finally, we report on methods that can be used to examine interactions between dying cells and phagocytes. We illustrate a quantitative method for detecting phagocytosis of dying cells by flow fluorocytometry. We also describe a recently developed approach based on the use of fluid phase tracers and different kind of microscopy, transmission electron and fluorescence microscopy, to characterize the mechanisms used by phagocytes to internalize dying cells.

Mitochondria are 'life-essential' organelles for the production of metabolic energy in the form of ATP. Paradoxically mitochondria also play a key role in controlling the pathways that lead to cell death. This latter role of mitochondria is more than just a 'loss of function' resulting in an energy deficit but is an active process involving different mitochondrial proteins. Cytochrome c was the first characterised mitochondrial factor shown to be released from the mitochondrial intermembrane space and to be actively implicated in apoptotic cell death. Since then, other mitochondrial proteins, such as AIF, Smac/DIABLO, endonuclease G and Omi/HtrA2, were found to undergo release during apoptosis and have been implicated in various aspects of the cell death process. Members of the Bcl-2 protein family control the integrity and response of mitochondria to apoptotic signals. The molecular mechanism by which mitochondrial intermembrane space proteins are released and the regulation of mitochondrial homeostasis by Bcl-2 proteins is still elusive. This review summarises and evaluates the current knowledge concerning the complex role of released mitochondrial proteins in the apoptotic process.

Autophagy and apoptosis are two important and interconnected stress-response mechanisms. However, the molecular interplay between these two pathways is not fully understood. To study the fate and function of autophagic proteins at the onset of apoptosis, we used a cellular model system in which autophagy precedes apoptosis. IL-3 depletion of Ba/F3 cells caused caspase (casp)-mediated cleavage of Beclin-1 and PI3KC3, two crucial components of the autophagy-inducing complex. We identified two casp cleavage sites in Beclin-1, TDVD(133) and DQLD(149), cleavage at which yields fragments lacking the autophagy-inducing capacity. Noteworthy, the C-terminal fragment, Beclin-1-C, localized predominantly at the mitochondria and sensitized the cells to apoptosis. Moreover, on isolated mitochondria, recombinant Beclin-1-C was able to induce the release of proapoptotic factors. These findings point to a mechanism by which casp-dependent generation of Beclin-1-C creates an amplifying loop enhancing apoptosis upon growth factor withdrawal.

Cell death and injury often lead to release or exposure of intracellular molecules called damage-associated molecular patterns (DAMPs) or cell death-associated molecules. These molecules are recognized by the innate immune system by pattern recognition receptors - the same receptors that detect pathogen-associated molecular patterns, thus revealing similarities between pathogen-induced and non-infectious inflammatory responses. Many DAMPs are derived from the plasma membrane, nucleus, endoplasmic reticulum and cytosol. Recently, mitochondria have emerged as other organelles that function as a source of DAMPs. Here, we highlight the significance of mitochondrial DAMPs and discuss their contribution to inflammation and development of human pathologies.

Ischemia followed by reperfusion leads to severe organ injury and dysfunction. Inflammation is considered to be the most important cause of tissue injury in organs subjected to ischemia. The mechanism that triggers inflammation and organ injury after ischemia remains to be elucidated, although different causes have been postulated. We investigated the role of apoptosis in the induction of inflammation and organ damage after renal ischemia. Using a murine model, we demonstrate a relationship between apoptosis and subsequent inflammation. At the time of reperfusion, administration of the antiapoptotic agents IGF-1 and ZVAD-fmk (a caspase inactivator) prevented the early onset of not only renal apoptosis, but also inflammation and tissue injury. Conversely, when the antiapoptotic agents were administered after onset of apoptosis, these protective effects were completely abrogated. The presence of apoptosis was directly correlated with posttranslational processing of the endothelial monocyte-activating polypeptide II (EMAP-II), which may explain apoptosis-induced influx and sequestration of leukocytes in the reperfused kidney. These results strongly suggest that apoptosis is a crucial event that can initiate reperfusion-induced inflammation and subsequent tissue injury. The newly described pathophysiological insights provide important opportunities to effectively prevent clinical manifestations of reperfusion injury in the kidney, and potentially in other organs.

Caspases, a family of evolutionarily, conserved cysteinyl proteases, mediate both apoptosis and inflammation through aspartate-specific cleavage of a wide number of cellular substrates. Most substrates of apoptotic caspases have been conotated with cellular dismantling, while inflammatory caspases mediate the proteolytic activation of inflammatory cytokines. Through detailed functional analysis of conditional caspase-deficient mice or derived cells, caspase biology has been extended to cellular responses such as cell differentiation, proliferation and NF-κB activation. Here, we discuss recent data indicating that non-apoptotic functions of caspases involve proteolysis exerted by their catalytic domains as well as non-proteolytic functions exerted by their prodomains. Homotypic oligomerization motifs in the latter mediate the recruitment of adaptors and effectors that modulate NF-κB activation. The non-apoptotic functions of caspases suggest that they may become activated independently of – or without – inducing an apoptotic cascade. Moreover, the existence of non-catalytic caspase-like molecules such as human caspase-12, c-FLIP and CARD-only proteins further supports the non-proteolytic functions of caspases in the regulation of cell survival, proliferation, differentiation and inflammation.

Murine L929 fibrosarcoma cells were transfected with the human Fas (APO-1/CD95) receptor, and the role of various caspases in Fas-mediated cell death was assessed. Proteolytic activation of procaspase-3 and -7 was shown by Western analysis. Acetyl-Tyr-Val-Ala-Asp-chloromethylketone and benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethylketone++ +, tetrapeptide inhibitors of caspase-1- and caspase-3-like proteases, respectively, failed to block Fas-induced apoptosis. Unexpectedly, the broad-spectrum caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone and benzyloxycarbonyl-Asp(OMe)-fluoromethylketone rendered the cells even more sensitive to Fas-mediated cell death, as measured after 18 h incubation. However, when the process was followed microscopically, it became clear that anti-Fas-induced apoptosis of Fas-transfected L929 cells was blocked during the first 3 h, and subsequently the cells died by necrosis. As in tumor necrosis factor (TNF)-induced necrosis, Fas treatment led to accumulation of reactive oxygen radicals, and Fas-mediated necrosis was inhibited by the oxygen radical scavenger butylated hydroxyanisole. However, in contrast to TNF, anti-Fas did not activate the nuclear factor kappaB under these necrotic conditions. These results demonstrate the existence of two different pathways originating from the Fas receptor, one rapidly leading to apoptosis, and, if this apoptotic pathway is blocked by caspase inhibitors, a second directing the cells to necrosis and involving oxygen radical production.

Cryopyrin is essential for caspase-1 activation triggered by Toll-like receptor (TLR) ligands in the presence of adenosine triphosphate (ATP). However, the events linking bacterial products and ATP to cryopyrin remain unclear. Here we demonstrate that cryopyrin-mediated caspase-1 activation proceeds independently of TLR signaling, thus dissociating caspase-1 activation and IL-1beta secretion. Instead, caspase-1 activation required pannexin-1, a hemichannel protein that interacts with the P2X(7) receptor. Direct cytosolic delivery of multiple bacterial products including lipopolysaccharide, but not flagellin, induced caspase-1 activation via cryopyrin in the absence of pannexin-1 activity or ATP stimulation. However, unlike Ipaf-dependent caspase-1 activation, stimulation of the pannexin-1-cryopyrin pathway by several intracellular bacteria was independent of a functional bacterial type III secretion system. These results provide evidence for cytosolic delivery and sensing of bacterial molecules as a unifying model for caspase-1 activation and position pannexin-1 as a mechanistic link between bacterial stimuli and the cryopyrin inflammasome.

Engagement of tumor necrosis factor receptor 1 signals two diametrically opposed pathways: survival-inflammation and cell death. An additional switch decides, depending on the cellular context, between caspase-dependent apoptosis and RIP kinase (RIPK)-mediated necrosis, also termed necroptosis. We explored the contribution of both cell death pathways in TNF-induced systemic inflammatory response syndrome (SIRS). Deletion of apoptotic executioner caspases (caspase-3 or -7) or inflammatory caspase-1 had no impact on lethal SIRS. However, deletion of RIPK3 conferred complete protection against lethal SIRS and reduced the amounts of circulating damage-associated molecular patterns. Pretreatment with the RIPK1 kinase inhibitor, necrostatin-1, provided a similar effect. These results suggest that RIPK1-RIPK3-mediated cellular damage by necrosis drives mortality during TNF-induced SIRS. RIPK3 deficiency also protected against cecal ligation and puncture, underscoring the clinical relevance of RIPK kinase inhibition in sepsis and identifying components of the necroptotic pathway that are potential therapeutic targets for treatment of SIRS and sepsis.

Necroptosis, necrosis and secondary necrosis following apoptosis represent different modes of cell death that eventually result in similar cellular morphology including rounding of the cell, cytoplasmic swelling, rupture of the plasma membrane and spilling of the intracellular content. Subcellular events during tumor necrosis factor (TNF)-induced necroptosis, H(2)O(2)-induced necrosis and anti-Fas-induced secondary necrosis were studied using high-resolution time-lapse microscopy. The cellular disintegration phase of the three types of necrosis is characterized by an identical sequence of subcellular events, including oxidative burst, mitochondrial membrane hyperpolarization, lysosomal membrane permeabilization and plasma membrane permeabilization, although with different kinetics. H(2)O(2)-induced necrosis starts immediately by lysosomal permeabilization. In contrast, during TNF-mediated necroptosis and anti-Fas-induced secondary necrosis, this is a late event preceded by a defined signaling phase. TNF-induced necroptosis depends on receptor-interacting protein-1 kinase, mitochondrial complex I and cytosolic phospholipase A(2) activities, whereas H(2)O(2)-induced necrosis requires iron-dependent Fenton reactions.

The discovery of regulated cell death presents tantalizing possibilities for gaining control over the life-death decisions made by cells in disease. Although apoptosis has been the focus of drug discovery for many years, recent research has identified regulatory mechanisms and signalling pathways for previously unrecognized, regulated necrotic cell death routines. Distinct critical nodes have been characterized for some of these alternative cell death routines, whereas other cell death routines are just beginning to be unravelled. In this Review, we describe forms of regulated necrotic cell death, including necroptosis, the emerging cell death modality of ferroptosis (and the related oxytosis) and the less well comprehended parthanatos and cyclophilin D-mediated necrosis. We focus on small molecules, proteins and pathways that can induce and inhibit these non-apoptotic forms of cell death, and discuss strategies for translating this understanding into new therapeutics for certain disease contexts.

Neutrophil extracellular traps (NETs) are chromatin structures loaded with antimicrobial molecules. They can trap and kill various bacterial, fungal and protozoal pathogens, and their release is one of the first lines of defense against pathogens. In vivo, NETs are released during a form of pathogen-induced cell death, which was recently named NETosis. Ex vivo, both dead and viable neutrophils can be stimulated to release NETs composed of either nuclear or mitochondrial chromatin, respectively. In certain pathological conditions, NETs are associated with severe tissue damage or certain auto-immune diseases. This review describes the recent progress made in the identification of the mechanisms involved in NETosis and discusses its interplay with autophagy and apoptosis.

Protein kinases of the receptor interacting protein (RIP) family collaborate with death receptor proteins to regulate cell death. Recent studies (Cho et al., 2009; He et al., 2009; Zhang et al., 2009) reveal that the RIP3 kinase functions with RIP1 at the crossroads of apoptosis, necroptosis, and cell survival.

High-risk neuroblastoma is a devastating malignancy with very limited therapeutic options. Here, we identify withaferin A (WA) as a natural ferroptosis-inducing agent in neuroblastoma, which acts through a novel double-edged mechanism. WA dose-dependently either activates the nuclear factor-like 2 pathway through targeting of Kelch-like ECH-associated protein 1 (noncanonical ferroptosis induction) or inactivates glutathione peroxidase 4 (canonical ferroptosis induction). Noncanonical ferroptosis induction is characterized by an increase in intracellular labile Fe(II) upon excessive activation of heme oxygenase-1, which is sufficient to induce ferroptosis. This double-edged mechanism might explain the superior efficacy of WA as compared with etoposide or cisplatin in killing a heterogeneous panel of high-risk neuroblastoma cells, and in suppressing the growth and relapse rate of neuroblastoma xenografts. Nano-targeting of WA allows systemic application and suppressed tumor growth due to an enhanced accumulation at the tumor site. Collectively, our data propose a novel therapeutic strategy to efficiently kill cancer cells by ferroptosis.

Tumor necrosis factor (TNF) is a pleiotropic molecule with a crucial role in cellular stress and inflammation during infection, tissue damage, and cancer. TNF signaling can lead to three distinct outcomes, each of which is initiated by different signaling complexes: the gene induction or survival mode, the apoptosis mode, and the necrosis mode. The kinases receptor-interacting protein 1 (RIP1) and RIP3 are key signaling molecules in necrosis and are regulated by caspases and ubiquitination. Moreover, TNF stimulation induces the formation of a necrosome in which RIP3 is activated and interacts with enzymes that control glycolytic flux and glutaminolysis. The necrosome induces mitochondrial complex I-mediated production of reactive oxygen species (ROS) and cytotoxicity, which suggest a functional link between increased bioenergetics and necrosis. In addition, other effector mechanisms also contribute to TNF-induced necrosis, such as recruitment of NADPH (the reduced form of nicotinamide adenine dinucleotide phosphate) oxidases and subsequent ROS production at the membrane-associated TNF receptor complex I; calcium mobilization; activation of phospholipase A(2), lipoxygenases, and acid sphingomyelinases; and lysosomal destabilization. However, the link between RIP1 and RIP3 and these subcellular events remains to be established. The regulation of RIP1 and RIP3 and their downstream signaling cascades opens new therapeutic avenues for treatment of pathologies associated with cell loss, such as ischemia-reperfusion damage and neurodegeneration, and ways to stimulate alternative immunogenic cell death pathways in cancer.

Necroptosis is a form of regulated cell death, which is induced by ligand binding to TNF family death domain receptors, pattern recognizing receptors and virus sensors. The common feature of these receptor systems is the implication of proteins, which contain a receptor interaction protein kinase (RIPK) homology interaction motif (RHIM) mediating recruitment and activation of receptor-interacting protein kinase 3 (RIPK3), which ultimately activates the necroptosis executioner mixed lineage kinase domain-like (MLKL). In case of the TNF family members, the initiator is the survival- and cell death-regulating RIPK1 kinase, in the case of Toll-like receptor 3/4 (TLR3/4), a RHIM-containing adaptor, called TRIF, while in the case of Z-DNA-binding protein ZBP1/DAI, the cytosolic viral sensor itself contains a RHIM domain. In this review, we discuss the different protein complexes that serve as nucleation platforms for necroptosis and the mechanism of execution of necroptosis. Transgenic models (knockout, kinase-dead knock-in) and pharmacologic inhibition indicate that RIPK1, RIPK3 or MLKL are implicated in many inflammatory, degenerative and infectious diseases. However, the conclusion of necroptosis being solely involved in the etiology of diseases is blurred by the pleiotropic roles of RIPK1 and RIPK3 in other cellular processes such as apoptosis and inflammasome activation.

Necrostatin-1 (Nec-1) is widely used in disease models to examine the contribution of receptor-interacting protein kinase (RIPK) 1 in cell death and inflammation. We studied three Nec-1 analogs: Nec-1, the active inhibitor of RIPK1, Nec-1 inactive (Nec-1i), its inactive variant, and Nec-1 stable (Nec-1s), its more stable variant. We report that Nec-1 is identical to methyl-thiohydantoin-tryptophan, an inhibitor of the potent immunomodulatory enzyme indoleamine 2,3-dioxygenase (IDO). Both Nec-1 and Nec-1i inhibited human IDO, but Nec-1s did not, as predicted by molecular modeling. Therefore, Nec-1s is a more specific RIPK1 inhibitor lacking the IDO-targeting effect. Next, although Nec-1i was ∼100 × less effective than Nec-1 in inhibiting human RIPK1 kinase activity in vitro, it was only 10 times less potent than Nec-1 and Nec-1s in a mouse necroptosis assay and became even equipotent at high concentrations. Along the same line, in vivo, high doses of Nec-1, Nec-1i and Nec-1s prevented tumor necrosis factor (TNF)-induced mortality equally well, excluding the use of Nec-1i as an inactive control. Paradoxically, low doses of Nec-1 or Nec-1i, but not Nec -1s, even sensitized mice to TNF-induced mortality. Importantly, Nec-1s did not exhibit this low dose toxicity, stressing again the preferred use of Nec-1s in vivo. Our findings have important implications for the interpretation of Nec-1-based data in experimental disease models.

Murine models are a crucial component of gut microbiome research. Unfortunately, a multitude of genetic backgrounds and experimental setups, together with inter-individual variation, complicates cross-study comparisons and a global understanding of the mouse microbiota landscape. Here, we investigate the variability of the healthy mouse microbiota of five common lab mouse strains using 16S rDNA pyrosequencing.We find initial evidence for richness-driven, strain-independent murine enterotypes that show a striking resemblance to those in human, and which associate with calprotectin levels, a marker for intestinal inflammation. After enterotype stratification, we find that genetic, caging and inter-individual variation contribute on average 19%, 31.7% and 45.5%, respectively, to the variance in the murine gut microbiota composition. Genetic distance correlates positively to microbiota distance, so that genetically similar strains have more similar microbiota than genetically distant ones. Specific mouse strains are enriched for specific operational taxonomic units and taxonomic groups, while the 'cage effect' can occur across mouse strain boundaries and is mainly driven by Helicobacter infections.The detection of enterotypes suggests a common ecological cause, possibly low-grade inflammation that might drive differences among gut microbiota composition in mammals. Furthermore, the observed environmental and genetic effects have important consequences for experimental design in mouse microbiome research.

Autophagy is a lysosomal degradation pathway that degrades damaged or superfluous cell components into basic biomolecules, which are then recycled back into the cytosol. In this respect, autophagy drives a flow of biomolecules in a continuous degradation-regeneration cycle. Autophagy is generally considered a pro-survival mechanism protecting cells under stress or poor nutrient conditions. Current research clearly shows that autophagy fulfills numerous functions in vital biological processes. It is implicated in development, differentiation, innate and adaptive immunity, ageing and cell death. In addition, accumulating evidence demonstrates interesting links between autophagy and several human diseases and tumor development. Therefore, autophagy seems to be an important player in the life and death of cells and organisms. Despite the mounting knowledge about autophagy, the mechanisms through which the autophagic machinery regulates these diverse processes are not entirely understood. In this review, we give a comprehensive overview of the autophagic signaling pathway, its role in general cellular processes and its connection to cell death. In addition, we present a brief overview of the possible contribution of defective autophagic signaling to disease.

Apoptosis is a form of programmed cell death important in the development and tissue homeostasis of multicellular organisms. Mitochondria have, next to their function in respiration, an important role in the apoptotic-signaling pathway. Malfunctioning at any level of the cell is eventually translated in the release of apoptogenic factors from the mitochondrial intermembrane space resulting in the organized demise of the cell. Some of these factors, such as AIF and endonuclease G, appear to be highly conserved during evolution. Other factors, like cytochrome c, have gained their apoptogenic function later during evolution. In this review, we focus on the role of cytochrome c, AIF, endonuclease G, Smac/DIABLO, Omi/HtrA2, Acyl-CoA-binding protein, and polypyrimidine tract-binding protein in the initiation and modulation of cell death in different model organisms. These mitochondrial factors may contribute to both caspase-dependent and caspase-independent processes in apoptotic cell death.

The lab of Jürg Tschopp was the first to report on the crucial role of receptor-interacting protein kinase 1 (RIPK1) in caspase-independent cell death. Because of this pioneer finding, regulated necrosis and in particular RIPK1/RIPK3 kinase-mediated necrosis, referred to as necroptosis, has become an intensively studied form of regulated cell death. Although necrosis was identified initially as a backup cell death program when apoptosis is blocked, it is now recognized as a cellular defense mechanism against viral infections and as being critically involved in ischemia-reperfusion damage. The observation that RIPK3 ablation rescues embryonic lethality in mice deficient in caspase-8 or Fas-associated-protein-via-a-death-domain demonstrates the crucial role of this apoptotic platform in the negative control of necroptosis during development. Here, we review and discuss commonalities and differences of the increasing list of inducers of regulated necrosis ranging from cytokines, pathogen-associated molecular patterns, to several forms of physicochemical cellular stress. Since the discovery of the crucial role of RIPK1 and RIPK3 in necroptosis, these kinases have become potential therapeutic targets. The availability of new pharmacological inhibitors and transgenic models will allow us to further document the important role of this form of cell death in degenerative, inflammatory and infectious diseases.

TNF is a master pro-inflammatory cytokine. Activation of TNFR1 by TNF can result in both RIPK1-independent apoptosis and RIPK1 kinase-dependent apoptosis or necroptosis. These cell death outcomes are regulated by two distinct checkpoints during TNFR1 signaling. TNF-mediated NF-κB-dependent induction of pro-survival or anti-apoptotic molecules is a well-known late checkpoint in the pathway, protecting cells from RIPK1-independent death. On the other hand, the molecular mechanism regulating the contribution of RIPK1 to cell death is far less understood. We demonstrate here that the IKK complex phosphorylates RIPK1 at TNFR1 complex I and protects cells from RIPK1 kinase-dependent death, independent of its function in NF-κB activation. We provide in vitro and in vivo evidence that inhibition of IKKα/IKKβ or its upstream activators sensitizes cells to death by inducing RIPK1 kinase-dependent apoptosis or necroptosis. We therefore report on an unexpected, NF-κB-independent role for the IKK complex in protecting cells from RIPK1-dependent death downstream of TNFR1.

Different lines of research have revealed that pathways activated by the endoplasmic reticulum (ER) stress response induce sterile inflammation. When activated, all three sensors of the unfolded protein response (UPR), PERK, IRE1, and ATF6, participate in upregulating inflammatory processes. ER stress in various cells plays an important role in the pathogenesis of several diseases, including obesity, type 2 diabetes, cancer, and intestinal bowel and airway diseases. Moreover, it has been suggested that ER stress-induced inflammation contributes substantially to disease progression. However, this generalization can be challenged at least in the case of cancer. In this review, we emphasize that ER stress can either aid or impede disease progression via inflammatory pathways depending on the cell type, disease stage, and type of ER stressor.

Proteome analysis of supernatant of isolated mitochondria exposed to recombinant tBid, a proapoptotic Bcl-2 member, revealed the presence of the serine protease Omi, also called HtrA2. This release was prevented in mitochondria derived from Bcl-2-transgenic mice. Release of Omi under apoptotic conditions was confirmed in vivo in livers from mice injected with agonistic anti-Fas antibodies and was prevented in livers from Bcl-2 transgenic mice. Omi release also occurs in apoptotic dying but not in necrotic dying fibrosarcoma L929 cells, treated with anti-Fas antibodies and TNF, respectively. The amino acid sequence reveals the presence of an XIAP interaction motif at the N-terminus of mature Omi. We demonstrate an interaction between endogeneous Omi and recombinant XIAP. Furthermore we show that endogenous Omi is involved in enhanced activation of caspases in cytosolic extracts.

Cell death is a crucial process during development, homeostasis and immune regulation of multicellular organisms, and its dysregulation is associated with numerous pathologies. Cell death is often induced upon pathogen infection as part of the defense mechanism, and pathogens have evolved strategies to modulate host cell death. In this review, we will discuss the molecular mechanisms and physiological relevance of four major types of programmed cell death, namely apoptosis, necrosis, autophagic cell death and pyroptosis.

Immunogenic profile of certain cancer cell death mechanisms has been transmuted by research published over a period of last few years and this change has been so drastic that a new (sub)class of apoptotic cancer cell death, redefined as 'immunogenic apoptosis' has started taking shape. In fact, it has been shown that this chemotherapeutic agent-specific immunogenic cancer cell death modality has the capabilities to induce 'anticancer vaccine effect', in vivo. These new trends have given an opportunity to combine tumour cell kill and antitumour immunity within a single paradigm, a sort of 'holy grail' of anticancer therapeutics. At the molecular level, it has been shown that the immunological silhouette of these cell death pathways is defined by a set of molecules called 'damage-associated molecular patterns (DAMPs)'. Various intracellular molecules like calreticulin (CRT), heat-shock proteins (HSPs), high-mobility group box-1 (HMGB1) protein, have been shown to be DAMPs exposed/secreted in a stress agent/factor-and cell death-specific manner. These discoveries have motivated further research into discovery of new DAMPs, new pathways for their exposure/secretion, search for new agents capable of inducing immunogenic cell death and urge to solve currently present problems with this paradigm. We anticipate that this emerging amalgamation of DAMPs, immunogenic cell death and anticancer therapeutics may be the key towards squelching cancer-related mortalities, in near future.

Successful immunogenic apoptosis in experimental cancer therapy depends on the induction of strong host anti-tumor responses. Given that tumors are often resistant to apoptosis, it is important to identify alternative molecular mechanisms that elicit immunogenic cell death. We have developed a genetic model in which direct dimerization of FADD combined with inducible expression of RIPK3 promotes necroptosis. We report that necroptotic cancer cells release damage-associated molecular patterns and promote maturation of dendritic cells, the cross-priming of cytotoxic T cells, and the production of IFN-γ in response to tumor antigen stimulation. Using both FADD-dependent and FADD-independent RIPK3 induction systems, we demonstrate the efficient vaccination potential of immunogenic necroptotic cells. Our study broadens the current concept of immunogenic cell death and opens doors for the development of new strategies in cancer therapy.

Three members of the IAP family (X-linked inhibitor of apoptosis (XIAP), cellular inhibitor of apoptosis proteins-1/-2 (cIAP1 and cIAP2)) are potent suppressors of apoptosis. Recent studies have shown that cIAP1 and cIAP2, unlike XIAP, are not direct caspase inhibitors, but block apoptosis by functioning as E3 ligases for effector caspases and receptor-interacting protein 1 (RIP1). cIAP-mediated polyubiquitination of RIP1 allows it to bind to the pro-survival kinase transforming growth factor-β-activated kinase 1 (TAK1) which prevents it from activating caspase-8-dependent death, a process reverted by the de-ubiquitinase CYLD. RIP1 is also a regulator of necrosis, a caspase-independent type of cell death. Here, we show that cells depleted of the IAPs by treatment with the IAP antagonist BV6 are greatly sensitized to tumor necrosis factor (TNF)-induced necrosis, but not to necrotic death induced by anti-Fas, poly(I:C) oxidative stress. Specific targeting of the IAPs by RNAi revealed that repression of cIAP1 is responsible for the sensitization. Similarly, lowering TAK1 levels or inhibiting its kinase activity sensitized cells to TNF-induced necrosis, whereas repressing CYLD had the opposite effect. We show that this sensitization to death is accompanied by enhanced RIP1 kinase activity, increased recruitment of RIP1 to Fas-associated via death domain and RIP3 (which allows necrosome formation), and elevated RIP1 kinase-dependent accumulation of reactive oxygen species (ROS). In conclusion, our data indicate that cIAP1 and TAK1 protect cells from TNF-induced necrosis by preventing RIP1/RIP3-dependent ROS production.

Necrotic cell death has long been considered an accidental and uncontrolled mode of cell death. But recently it has become clear that necrosis is a molecularly regulated event that is associated with pathologies such as ischemia-reperfusion (IR) injury, neurodegeneration and pathogen infection. The serine/threonine kinase receptor-interacting protein 1 (RIP1) plays a crucial role during the initiation of necrosis induced by ligand- receptor interactions. On the other hand, ATP depletion is an initiating factor in ischemia-induced necrotic cell death. Common players in necrotic cell death irrespective of the stimulus are calcium and reactive oxygen species (ROS). During necrosis, elevated cytosolic calcium levels typically lead to mitochondrial calcium overload, bioenergetics effects, and activation of proteases and phospholipases. ROS initiates damage to lipids, proteins and DNA and consequently results in mitochondrial dysfunction, ion balance deregulation and loss of membrane integrity. Membrane destabilization during necrosis is also mediated by other factors, such as acidsphingomyelinase (ASM), phospholipase A2 (PLA2) and calpains. Furthermore, necrotic cells release immunomodulatory factors that lead to recognition and engulfment by phagocytes and the subsequent immunological response. The knowledge of the molecular mechanisms involved in necrosis has contributed to our understanding of necrosis-associated pathologies. In this review we will focus on the intracellular and intercellular signaling events in necrosis induced by different stimuli, such as oxidative stress, cytokines and pathogenassociated molecular patterns (PAMPs), which can be linked to several pathologies such as stroke, cardiac failure, neurodegenerative diseases, and infections. Keywords: Necrosis, RIP1, mitochondrial Ca2+ overload, ROS, clearance of necrotic cells, phagocyte response, neurodegenerative disorders, ischemia-reperfusion injury

Although TRAIL (tumor necrosis factor (TNF)-related apoptosis inducing ligand) is a well-known apoptosis inducer, we have previously demonstrated that acidic extracellular pH (pHe) switches TRAIL-induced apoptosis to regulated necrosis (or necroptosis) in human HT29 colon and HepG2 liver cancer cells. Here, we investigated the role of RIPK1 (receptor interacting protein kinase 1), RIPK3 and PARP-1 (poly (ADP-ribose) polymerase-1) in TRAIL-induced necroptosis in vitro and in concanavalin A (Con A)-induced murine hepatitis. Pretreatment of HT29 or HepG2 with pharmacological inhibitors of RIPK1 or PARP-1 (Nec-1 or PJ-34, respectively), or transient transfection with siRNAs against RIPK1 or RIPK3, inhibited both TRAIL-induced necroptosis and PARP-1-dependent intracellular ATP depletion demonstrating that RIPK1 and RIPK3 were involved upstream of PARP-1 activation and ATP depletion. In the mouse model of Con A-induced hepatitis, where death of mouse hepatocytes is dependent on TRAIL and NKT (Natural Killer T) cells, PARP-1 activity was positively correlated with liver injury and hepatitis was prevented both by Nec-1 or PJ-34. These data provide new insights into TRAIL-induced necroptosis with PARP-1 being active effector downstream of RIPK1/RIPK3 initiators and suggest that pharmacological inhibitors of RIPKs and PARP-1 could be new treatment options for immune-mediated hepatitis.

Targeted manipulation of the gut flora is increasingly being recognized as a means to improve human health. Yet, the temporal dynamics and intra- and interindividual heterogeneity of the microbiome represent experimental limitations, especially in human cross-sectional studies. Therefore, rodent models represent an invaluable tool to study the host–microbiota interface. Progress in technical and computational tools to investigate the composition and function of the microbiome has opened a new era of research and we gradually begin to understand the parameters that influence variation of host-associated microbial communities. To isolate true effects from confounding factors, it is essential to include such parameters in model intervention studies. Also, explicit journal instructions to include essential information on animal experiments are mandatory. The purpose of this review is to summarize the factors that influence microbiota composition in mice and to provide guidelines to improve the reproducibility of animal experiments.

The HtrA family refers to a group of related oligomeric serine proteases that combine a trypsin-like protease domain with at least one PDZ interaction domain. Mammals encode four HtrA proteases, named HtrA1–4. The protease activity of the HtrA member HtrA2/Omi is required for mitochondrial homeostasis in mice and humans and inactivating mutations associated with neurodegenerative disorders such as Parkinson's disease. Moreover, HtrA2/Omi is released in the cytosol, where it contributes to apoptosis through both caspase-dependent and -independent pathways. Here, we review the current knowledge of HtrA2/Omi biology and discuss the signaling pathways that underlie its mitochondrial and apoptotic functions from an evolutionary perspective.

Since the discovery and definition of neutrophil extracellular traps (NETs) 14 years ago, numerous characteristics and physiological functions of NETs have been uncovered. Nowadays, the field continues to expand and novel mechanisms that orchestrate formation of NETs, their previously unknown properties, and novel implications in disease continue to emerge. The abundance of available data has also led to some confusion in the NET research community due to contradictory results and divergent scientific concepts, such as pro- and anti-inflammatory roles in pathologic conditions, demarcation from other forms of cell death, or the origin of the DNA that forms the NET scaffold. Here, we present prevailing concepts and state of the science in NET-related research and elaborate on open questions and areas of dispute.

The aspartate-specific cysteine protease caspase-1 is activated by the inflammasomes and is responsible for the proteolytic maturation of the cytokines IL-1β and IL-18 during infection and inflammation. To discover new caspase-1 substrates, we made use of a proteome-wide gel-free differential peptide sorting methodology that allows unambiguous localization of the processing site in addition to identification of the substrate. Of the 1022 proteins that were identified, 20 were found to be specifically cleaved after Asp in the setup incubated with recombinant caspase-1. Interestingly, caspase-7 emerged as one of the identified caspase-1 substrates. Moreover half of the other identified cleavage events occurred at sites closely resembling the consensus caspase-7 recognition sequence DEVD, suggesting caspase-1-mediated activation of endogenous caspase-7 in this setup. Consistently recombinant caspase-1 cleaved caspase-7 at the canonical activation sites Asp²³ and Asp¹⁹⁸, and recombinant caspase-7 processed a subset of the identified substrates. <i>In vivo</i>, caspase-7 activation was observed in conditions known to induce activation of caspase-1, including <i>Salmonella</i> infection and microbial stimuli combined with ATP. Interestingly <i>Salmonella</i>- and lipopolysaccharide + ATP-induced activation of caspase-7 was abolished in macrophages deficient in caspase-1, the pattern recognition receptors Ipaf and Cryopyrin, and the inflammasome adaptor ASC, demonstrating an upstream role for the caspase-1 inflammasomes in caspase-7 activation <i>in vivo</i>. In contrast, caspase-1 and the inflammasomes were not required for caspase-3 activation. In conclusion, we identified 20 new substrates activated downstream of caspase-1 and validated caspase-1-mediated caspase-7 activation <i>in vitro</i> and in knock-out macrophages. These results demonstrate for the first time the existence of a nucleotide binding and oligomerization domain-like receptor/caspase-1/caspase-7 cascade and the existence of distinct activation mechanisms for caspase-3 and -7 in response to microbial stimuli and bacterial infection.

Receptor-interacting protein kinase (RIPK) 1 and RIPK3 have emerged as essential kinases mediating a regulated form of necrosis, known as necroptosis, that can be induced by tumor necrosis factor (TNF) signaling. As a consequence, inhibiting RIPK1 kinase activity and repressing RIPK3 expression levels have become commonly used approaches to estimate the contribution of necroptosis to specific phenotypes. Here, we report that RIPK1 kinase activity and RIPK3 also contribute to TNF-induced apoptosis in conditions of cellular inhibitor of apoptosis 1 and 2 (cIAP1/2) depletion or TGF-β-activated kinase 1 (TAK1) kinase inhibition, implying that inhibition of RIPK1 kinase activity or depletion of RIPK3 under cell death conditions is not always a prerequisite to conclude on the involvement of necroptosis. Moreover, we found that, contrary to cIAP1/2 depletion, TAK1 kinase inhibition induces assembly of the cytosolic RIPK1/Fas-associated protein with death domain/caspase-8 apoptotic TNF receptor 1 (TNFR1) complex IIb without affecting the RIPK1 ubiquitylation status at the level of TNFR1 complex I. These results indicate that the recruitment of TAK1 to the ubiquitin (Ub) chains, and not the Ub chains per se, regulates the contribution of RIPK1 to the apoptotic death trigger. In line with this, we found that cylindromatosis repression only provided protection to TNF-mediated RIPK1-dependent apoptosis in condition of reduced RIPK1 ubiquitylation obtained by cIAP1/2 depletion but not upon TAK1 kinase inhibition, again arguing for a role of TAK1 in preventing RIPK1-dependent apoptosis downstream of RIPK1 ubiquitylation. Importantly, we found that this function of TAK1 was independent of its known role in canonical nuclear factor-κB (NF-κB) activation. Our study therefore reports a new function of TAK1 in regulating an early NF-κB-independent cell death checkpoint in the TNFR1 apoptotic pathway. In both TNF-induced RIPK1 kinase-dependent apoptotic models, we found that RIPK3 contributes to full caspase-8 activation independently of its kinase activity or intact RHIM domain. In contrast, RIPK3 participates in caspase-8 activation by acting downstream of the cytosolic death complex assembly, possibly via reactive oxygen species generation.

Beclin1(Atg6) is a well-known key regulator of autophagy. Although Beclin1 is enzymatically inert, it governs the autophagic process by regulating PtdIns3KC3-dependent generation of phosphatidylinositol3-phosphate (PtdIns(3)P) and the subsequent recruitment of additional Atg proteins that orchestrate autophagosome formation. Furthermore, Beclin1 is implicated in numerous biological processes, including adaptation to stress, development, endocytosis, cytokinesis, immunity, tumorigenesis, ageing and cell death. Whether all of these processes involve only the autophagy-inducing function of Beclin1 is now being seriously questioned, because Beclin1 appears to exercise several non-autophagy functions. Therefore, we should broaden our view of Beclin1 as a specialized molecule in autophagy to that of a multifunctional protein. The central role of Beclin1 in multiple signaling events obviously requires tight regulation at multiple levels. Its function is kept in check by diverse mechanisms, such as epigenetic silencing, microRNA regulation, post-translational modifications, and protein-protein interactions. Interestingly, multiple diseases are associated with deficiency or malfunction of Beclin1, which makes it a potentially valuable target for various therapies, including anti-cancer treatment. In this review, we focus on Beclin1 as a multifunctional protein, discuss the variety of mechanisms by which it is controlled, and give an overview of Beclin1-associated pathologies.

Calreticulin surface exposure (ecto-CALR), ATP secretion, maturation of dendritic cells (DCs) and stimulation of T cells are prerequisites for anticancer therapy-induced immunogenic cell death (ICD). Recent evidence suggests that chemotherapy-induced autophagy may positively regulate ICD by favoring ATP secretion. We have recently shown that reactive oxygen species (ROS)-based endoplasmic reticulum (ER) stress triggered by hypericin-mediated photodynamic therapy (Hyp-PDT) induces bona fide ICD. However, whether Hyp-PDT-induced autophagy regulates ICD was not explored. Here we showed that, in contrast to expectations, reducing autophagy (by ATG5 knockdown) in cancer cells did not alter ATP secretion after Hyp-PDT. Autophagy-attenuated cancer cells displayed enhanced ecto-CALR induction following Hyp-PDT, which strongly correlated with their inability to clear oxidatively damaged proteins. Furthermore, autophagy-attenuation in Hyp-PDT-treated cancer cells increased their ability to induce DC maturation, IL6 production and proliferation of CD4(+) or CD8(+) T cells, which was accompanied by IFNG production. Thus, our study unravels a role for ROS-induced autophagy in weakening functional interaction between dying cancer cells and the immune system thereby helping in evasion from ICD prerequisites or determinants.

p63 inhibits metastasis. Here, we show that p63 (both TAp63 and ΔNp63 isoforms) regulates expression of miR-205 in prostate cancer (PCa) cells, and miR-205 is essential for the inhibitory effects of p63 on markers of epithelial-mesenchymal transition (EMT), such as ZEB1 and vimentin. Correspondingly, the inhibitory effect of p63 on EMT markers and cell migration is reverted by anti-miR-205. p53 mutants inhibit expression of both p63 and miR-205, and the cell migration, in a cell line expressing endogenous mutated p53, can be abrogated by pre-miR-205 or silencing of mutated p53. In accordance with this in vitro data, ΔNp63 or miR-205 significantly inhibits the incidence of lung metastasis in vivo in a mouse tail vein model. Similarly, one or both components of the p63/miR-205 axis were absent in metastases or colonized lymph nodes in a set of 218 human prostate cancer samples. This was confirmed in an independent clinical data set of 281 patients. Loss of this axis was associated with higher Gleason scores, an increased likelihood of metastatic and infiltration events, and worse prognosis. These data suggest that p63/miR-205 may be a useful clinical predictor of metastatic behavior in prostate cancer.

A new concept of immunogenic cell death (ICD) has recently been proposed. The immunogenic characteristics of this cell death mode are mediated mainly by molecules called 'damage-associated molecular patterns' (DAMPs), most of which are recognized by pattern recognition receptors. Some DAMPs are actively emitted by cells undergoing ICD (e.g. calreticulin (CRT) and adenosine triphosphate (ATP)), whereas others are emitted passively (e.g. high-mobility group box 1 protein (HMGB1)). Recent studies have demonstrated that these DAMPs play a beneficial role in anti-cancer therapy by interacting with the immune system. The molecular pathways involved in translocation of CRT to the cell surface and secretion of ATP from tumor cells undergoing ICD are being elucidated. However, it has also been shown that the same DAMPs could contribute to progression of cancer and promote resistance to anticancer treatments. In this review, we will critically evaluate the beneficial and detrimental roles of DAMPs in cancer therapy, focusing mainly on CRT, ATP and HMGB1.

Cigarette smoke (CS), the primary risk factor of chronic obstructive pulmonary disease (COPD), leads to pulmonary inflammation through interleukin-1 receptor (IL-1R)I signalling, as determined using COPD mouse models. It is unclear whether interleukin (IL)-1α or IL-1β, activated by the Nlrp3/caspase-1 axis, is the predominant ligand for IL-1RI in CS-induced responses. We exposed wild-type mice (treated with anti-IL-1α or anti-IL-1β antibodies), and IL-1RI knockout (KO), Nlrp3 KO and caspase-1 KO mice to CS for 3 days or 4 weeks and evaluated pulmonary inflammation. Additionally, we measured the levels of IL-1α and IL-1β mRNA (in total lung tissue by RT-PCR) and protein (in induced sputum by ELISA) of never-smokers, smokers without COPD and patients with COPD. In CS-exposed mice, pulmonary inflammation was dependent on IL-1RI and could be significantly attenuated by neutralising IL-1α or IL-1β. Interestingly, CS-induced inflammation occurred independently of IL-1β activation by the Nlrp3/caspase-1 axis. In human subjects, IL-1α and IL-1β were significantly increased in total lung tissue and induced sputum of patients with COPD, respectively, compared with never-smokers. These results suggest that not only IL-1β but also IL-1α should be considered as an important mediator in CS-induced inflammation and COPD.

TNF receptor 1 signaling induces NF-κB activation and necroptosis in L929 cells. We previously reported that cellular inhibitor of apoptosis protein-mediated receptor-interacting protein 1 (RIP1) ubiquitination acts as a cytoprotective mechanism, whereas knockdown of cylindromatosis, a RIP1-deubiquitinating enzyme, protects against tumor necrosis factor (TNF)-induced necroptosis. We report here that RIP1 is a crucial mediator of canonical NF-κB activation in L929 cells, therefore questioning the relative cytoprotective contribution of RIP1 ubiquitination versus canonical NF-κB activation. We found that attenuated NF-κB activation has no impact on TNF-induced necroptosis. However, we identified A20 and linear ubiquitin chain assembly complex as negative regulators of necroptosis. Unexpectedly, and in contrast to RIP3, we also found that knockdown of RIP1 did not block TNF cytotoxicity. Cell death typing revealed that RIP1-depleted cells switch from necroptotic to apoptotic death, indicating that RIP1 can also suppress apoptosis in L929 cells. Inversely, we observed that Fas-associated protein via a death domain, cellular FLICE inhibitory protein and caspase-8, which are all involved in the initiation of apoptosis, counteract necroptosis induction. Finally, we also report RIP1-independent but RIP3-mediated necroptosis in the context of TNF signaling in particular conditions.

Cell death research during the last decades has revealed many molecular signaling cascades, often leading to distinct cell death modalities followed by immune responses. For historical reasons, the prototypic and best characterized cell death modes are apoptosis and necrosis (dubbed necroptosis, to indicate that it is regulated). There is mounting evidence for the interplay between cell death modalities and their redundant action when one of them is interfered with. This increase in cell death research points to the need for characterizing cell death pathways by different approaches at the biochemical, cellular and if possible, physiological level. In this review we present a selection of techniques to detect cell death and to distinguish necrosis from apoptosis. The distinction should be based on pharmacologic and transgenic approaches in combination with several biochemical and morphological criteria. A particular problem in defining necrosis is that in the absence of phagocytosis, apoptotic cells become secondary necrotic and develop morphologic and biochemical features of primary necrosis.

SARS-CoV-2 infection poses a major threat to the lungs and multiple other organs, occasionally causing death. Until effective vaccines are developed to curb the pandemic, it is paramount to define the mechanisms and develop protective therapies to prevent organ dysfunction in patients with COVID-19. Individuals that develop severe manifestations have signs of dysregulated innate and adaptive immune responses. Emerging evidence implicates neutrophils and the disbalance between neutrophil extracellular trap (NET) formation and degradation plays a central role in the pathophysiology of inflammation, coagulopathy, organ damage, and immunothrombosis that characterize severe cases of COVID-19. Here, we discuss the evidence supporting a role for NETs in COVID-19 manifestations and present putative mechanisms, by which NETs promote tissue injury and immunothrombosis. We present therapeutic strategies, which have been successful in the treatment of immunο-inflammatory disorders and which target dysregulated NET formation or degradation, as potential approaches that may benefit patients with severe COVID-19.

Maintaining cellular redox balance is vital for cell survival and tissue homoeostasis because imbalanced production of reactive oxygen species (ROS) may lead to oxidative stress and cell death. The antioxidant enzyme glutathione peroxidase 4 (Gpx4) is a key regulator of oxidative stress-induced cell death. We show that mice with deletion of Gpx4 in hematopoietic cells develop anemia and that Gpx4 is essential for preventing receptor-interacting protein 3 (RIP3)-dependent necroptosis in erythroid precursor cells. Absence of Gpx4 leads to functional inactivation of caspase 8 by glutathionylation, resulting in necroptosis, which occurs independently of tumor necrosis factor α activation. Although genetic ablation of Rip3 normalizes reticulocyte maturation and prevents anemia, ROS accumulation and lipid peroxidation in Gpx4-deficient cells remain high. Our results demonstrate that ROS and lipid hydroperoxides function as not-yet-recognized unconventional upstream signaling activators of RIP3-dependent necroptosis.

In human cells, the RIPK1-RIPK3-MLKL-PGAM5-Drp1 axis drives tumor necrosis factor (TNF)-induced necroptosis through mitochondrial fission, but whether this pathway is conserved among mammals is not known. To answer this question, we analyzed the presence and functionality of the reported necroptotic axis in mice. As in humans, knockdown of receptor-interacting kinase-3 (RIPK3) or mixed lineage kinase domain like (MLKL) blocks TNF-induced necroptosis in L929 fibrosarcoma cells. However, repression of either of these proteins did not protect the cells from death, but instead induced a switch from TNF-induced necroptosis to receptor-interacting kinase-1 (RIPK1) kinase-dependent apoptosis. In addition, although mitochondrial fission also occurs during TNF-induced necroptosis in L929 cells, we found that knockdown of phosphoglycerate mutase 5 (PGAM5) and dynamin 1 like protein (Drp1) did not markedly protect the cells from TNF-induced necroptosis. Depletion of Pink1, a reported interactor of both PGAM5 and Drp1, did not affect TNF-induced necroptosis. These results indicate that in these murine cells mitochondrial fission and Pink1 dependent processes, including Pink-Parkin dependent mitophagy, apparently do not promote necroptosis. Our data demonstrate that the core components of the necrosome (RIPK1, RIPK3 and MLKL) are crucial to induce TNF-dependent necroptosis both in human and in mouse cells, but the associated mechanisms may differ between the two species or cell types.

Heterogeneity between different macrophage populations has become a defining feature of this lineage. However, the conserved factors defining macrophages remain largely unknown. The transcription factor ZEB2 is best described for its role in epithelial to mesenchymal transition; however, its role within the immune system is only now being elucidated. We show here that Zeb2 expression is a conserved feature of macrophages. Using Clec4f-cre, Itgax-cre, and Fcgr1-cre mice to target five different macrophage populations, we found that loss of ZEB2 resulted in macrophage disappearance from the tissues, coupled with their subsequent replenishment from bone-marrow precursors in open niches. Mechanistically, we found that ZEB2 functioned to maintain the tissue-specific identities of macrophages. In Kupffer cells, ZEB2 achieved this by regulating expression of the transcription factor LXRα, removal of which recapitulated the loss of Kupffer cell identity and disappearance. Thus, ZEB2 expression is required in macrophages to preserve their tissue-specific identities.

Our current knowledge of the molecular mechanisms regulating the signaling pathways leading to cell survival, cell death, and inflammation has shed light on the tight mutual interplays between these processes. Moreover, the fact that both apoptosis and necrosis can be molecularly controlled has greatly increased our interest in the roles that these types of cell death play in the control of general processes such as development, homeostasis, and inflammation. In this review, we provide a brief update on the different cell death modalities and describe in more detail the intracellular crosstalk between survival, apoptotic, necroptotic, and inflammatory pathways that are activated downstream of death receptors. An important concept is that the different cell death processes modulate each other by mutual inhibitory mechanisms, serve as alternative back-up death routes in the case of a defect in the first-line cell death response, and are controlled by multiple feedback loops. We conclude by discussing future perspectives and challenges in the field of cell death and inflammation research.

Targeted mutagenesis in mice is a powerful tool for functional analysis of genes. However, genetic variation between embryonic stem cells (ESCs) used for targeting (previously almost exclusively 129-derived) and recipient strains (often C57BL/6J) typically results in congenic mice in which the targeted gene is flanked by ESC-derived passenger DNA potentially containing mutations. Comparative genomic analysis of 129 and C57BL/6J mouse strains revealed indels and single nucleotide polymorphisms resulting in alternative or aberrant amino acid sequences in 1,084 genes in the 129-strain genome. Annotating these passenger mutations to the reported genetically modified congenic mice that were generated using 129-strain ESCs revealed that nearly all these mice possess multiple passenger mutations potentially influencing the phenotypic outcome. We illustrated this phenotypic interference of 129-derived passenger mutations with several case studies and developed a Me-PaMuFind-It web tool to estimate the number and possible effect of passenger mutations in transgenic mice of interest.

Accumulation of unfolded proteins in the endoplasmic reticulum (ER) causes a state of cellular stress known as ER stress. The cells respond to ER stress by activating the unfolded protein response (UPR), a signaling network emerging from the ER-anchored receptors IRE1α, PERK and ATF6. The UPR aims at restoring ER protein-folding homeostasis, but turns into a toxic signal when the stress is too severe or prolonged. Recent studies have demonstrated links between the UPR and inflammation. Consequently, small molecule inhibitors of IRE1α and PERK have become attractive tools for the potential therapeutic manipulation of the UPR in inflammatory conditions. TNF is a master pro-inflammatory cytokine that drives inflammation either directly by promoting gene activation, or indirectly by inducing RIPK1 kinase-dependent cell death, in the form of apoptosis or necroptosis. To evaluate the potential contribution of the UPR to TNF-induced cell death, we tested the effects of two commonly used PERK inhibitors, GSK2606414 and GSK2656157. Surprisingly, we observed that both compounds completely repressed TNF-mediated RIPK1 kinase-dependent death, but found that this effect was independent of PERK inactivation. Indeed, these two compounds turned out to be direct RIPK1 inhibitors, with comparable potency to the recently developed RIPK1 inhibitor GSK'963 (about 100 times more potent than NEC-1s). Importantly, these compounds completely inhibited TNF-mediated RIPK1-dependent cell death at a concentration that did not affect PERK activity in cells. In vivo, GSK2656157 administration protected mice from lethal doses of TNF independently of PERK inhibition and as efficiently as GSK'963. Together, our results not only report on new and very potent RIPK1 inhibitors but also highlight the risk of misinterpretation when using these two PERK inhibitors in the context of ER stress, cell death and inflammation.

Abstract Lipid peroxidation (LPO) drives ferroptosis execution. However, LPO has been shown to contribute also to other modes of regulated cell death (RCD). To clarify the role of LPO in different modes of RCD, we studied in a comprehensive approach the differential involvement of reactive oxygen species (ROS), phospholipid peroxidation products, and lipid ROS flux in the major prototype modes of RCD viz. apoptosis, necroptosis, ferroptosis, and pyroptosis. LC-MS oxidative lipidomics revealed robust peroxidation of three classes of phospholipids during ferroptosis with quantitative predominance of phosphatidylethanolamine species. Incomparably lower amounts of phospholipid peroxidation products were found in any of the other modes of RCD. Nonetheless, a strong increase in lipid ROS levels was detected in non-canonical pyroptosis, but only during cell membrane rupture. In contrast to ferroptosis, lipid ROS apparently was not involved in non-canonical pyroptosis execution nor in the release of IL-1β and IL-18, while clear dependency on CASP11 and GSDMD was observed. Our data demonstrate that ferroptosis is the only mode of RCD that depends on excessive phospholipid peroxidation for its cytotoxicity. In addition, our results also highlight the importance of performing kinetics and using different methods to monitor the occurrence of LPO. This should open the discussion on the implication of particular LPO events in relation to different modes of RCD.

Abstract RIPK1 regulates cell death and inflammation through kinase-dependent and -independent mechanisms. As a scaffold, RIPK1 inhibits caspase-8-dependent apoptosis and RIPK3/MLKL-dependent necroptosis. As a kinase, RIPK1 paradoxically induces these cell death modalities. The molecular switch between RIPK1 pro-survival and pro-death functions remains poorly understood. We identify phosphorylation of RIPK1 on Ser25 by IKKs as a key mechanism directly inhibiting RIPK1 kinase activity and preventing TNF-mediated RIPK1-dependent cell death. Mimicking Ser25 phosphorylation (S &gt; D mutation) protects cells and mice from the cytotoxic effect of TNF in conditions of IKK inhibition. In line with their roles in IKK activation, TNF-induced Ser25 phosphorylation of RIPK1 is defective in TAK1- or SHARPIN-deficient cells and restoring phosphorylation protects these cells from TNF-induced death. Importantly, mimicking Ser25 phosphorylation compromises the in vivo cell death-dependent immune control of Yersinia infection, a physiological model of TAK1/IKK inhibition, and rescues the cell death-induced multi-organ inflammatory phenotype of the SHARPIN-deficient mice.

The prognosis of colon cancer (CC) is dictated by tumor-infiltrating lymphocytes, including follicular helper T (TFH) cells and the efficacy of chemotherapy-induced immune responses. It remains unclear whether gut microbes contribute to the elicitation of TFH cell-driven responses. Here, we show that the ileal microbiota dictates tolerogenic versus immunogenic cell death of ileal intestinal epithelial cells (IECs) and the accumulation of TFH cells in patients with CC and mice. Suppression of IEC apoptosis led to compromised chemotherapy-induced immunosurveillance against CC in mice. Protective immune responses against CC were associated with residence of Bacteroides fragilis and Erysipelotrichaceae in the ileum. In the presence of these commensals, apoptotic ileal IECs elicited PD-1+ TFH cells in an interleukin-1R1- and interleukin-12-dependent manner. The ileal microbiome governed the efficacy of chemotherapy and PD-1 blockade in CC independently of microsatellite instability. These findings demonstrate that immunogenic ileal apoptosis contributes to the prognosis of chemotherapy-treated CC. Local microbiome composition influences treatment efficacy of chemotherapy in colon cancer via modulation of tolerogenic versus immunogenic ileal intestinal epithelial cell death, which in turn influences follicular helper T cell priming.

Immunogenic cell death significantly contributes to the success of anti-cancer therapies, but immunogenicity of different cell death modalities widely varies. Ferroptosis, a form of cell death that is characterized by iron accumulation and lipid peroxidation, has not yet been fully evaluated from this perspective. Here we present an inducible model of ferroptosis, distinguishing three phases in the process-'initial' associated with lipid peroxidation, 'intermediate' correlated with ATP release and 'terminal' recognized by HMGB1 release and loss of plasma membrane integrity-that serves as tool to study immune cell responses to ferroptotic cancer cells. Co-culturing ferroptotic cancer cells with dendritic cells (DC), reveals that 'initial' ferroptotic cells decrease maturation of DC, are poorly engulfed, and dampen antigen cross-presentation. DC loaded with ferroptotic, in contrast to necroptotic, cancer cells fail to protect against tumor growth. Adding ferroptotic cancer cells to immunogenic apoptotic cells dramatically reduces their prophylactic vaccination potential. Our study thus shows that ferroptosis negatively impacts antigen presenting cells and hence the adaptive immune response, which might hinder therapeutic applications of ferroptosis induction.

Immune-checkpoint blockers (ICBs) have revolutionized oncology and firmly established the subfield of immuno-oncology. Despite this renaissance, a subset of cancer patients remain unresponsive to ICBs due to widespread immuno-resistance. To "break" cancer cell-driven immuno-resistance, researchers have long floated the idea of therapeutically facilitating the immunogenicity of cancer cells by disrupting tumor-associated immuno-tolerance via conventional anticancer therapies. It is well appreciated that anticancer therapies causing immunogenic or inflammatory cell death are best positioned to productively activate anticancer immunity. A large proportion of studies have emphasized the importance of immunogenic apoptosis (i.e., immunogenic cell death or ICD); yet, it has also emerged that necroptosis, a programmed necrotic cell death pathway, can also be immunogenic. Emergence of a proficient immune profile for necroptosis has important implications for cancer because resistance to apoptosis is one of the major hallmarks of tumors. Putative immunogenic or inflammatory characteristics driven by necroptosis can be of great impact in immuno-oncology. However, as is typical for a highly complex and multi-factorial disease like cancer, a clear cause versus consensus relationship on the immunobiology of necroptosis in cancer cells has been tough to establish. In this review, we discuss the various aspects of necroptosis immunobiology with specific focus on immuno-oncology and cancer immunotherapy.

Most RIPK1/3 kinase inhibitors designed to date are type II or type III kinase inhibitors, covering specific RIPK1 inhibitors, such as Nec-1s, GSK’772, GSK’547, and compound 22. Several existing multitargeting tyrosine kinase inhibitors, such as sorafenib, ponatinib, and pazopanib, show strong RIPK1 off-target effects. Kinase-independent, RIPK1-scaffold-mediated cell survival is (in)directly regulated by TAK1, IKK, MK2, and TBK1-dependent phosphorylation, implying that tyrosine kinase inhibitors that block these kinases may be not only advantageous in sensitizing cell death in cancer cells, but also disadvantageous in sensitizing RIPK1-mediated cell death, which may enhance barrier loss, infection, and inflammation. Some RIPK1 inhibitors are now in clinical trials for the treatment of rheumatoid arthritis, ulcerative colitis, and psoriasis, similar to anti-TNF (tumor necrosis factor) blocking strategies. It is thought that RIPK1 kinase inhibitors could form an alternative treatment for patients who are nonresponders or show adverse effects to anti-TNF treatment. The scaffolding function of receptor-interacting protein kinase 1 (RIPK1) regulates prosurvival signaling and inflammatory gene expression, while its kinase activity mediates both apoptosis and necroptosis; the latter involving RIPK3 kinase activity. The mutual transition between the scaffold and kinase functions of RIPK1 is regulated by (de)ubiquitylation and (de)phosphorylation. RIPK1-mediated cell death leads to disruption of epithelial barriers and/or release of damage-associated molecular patterns (DAMPs), cytokines, and chemokines, propagating inflammatory and degenerative diseases. Many drug development programs have pursued targeting RIPK1, and to a lesser extent RIPK3 kinase activity. In this review, we classify existing and novel small-molecule drugs based on their pharmacodynamic (PD) type I, II, and III binding mode. Finally, we discuss their applicability and therapeutic potential in inflammatory and degenerative experimental disease models. The scaffolding function of receptor-interacting protein kinase 1 (RIPK1) regulates prosurvival signaling and inflammatory gene expression, while its kinase activity mediates both apoptosis and necroptosis; the latter involving RIPK3 kinase activity. The mutual transition between the scaffold and kinase functions of RIPK1 is regulated by (de)ubiquitylation and (de)phosphorylation. RIPK1-mediated cell death leads to disruption of epithelial barriers and/or release of damage-associated molecular patterns (DAMPs), cytokines, and chemokines, propagating inflammatory and degenerative diseases. Many drug development programs have pursued targeting RIPK1, and to a lesser extent RIPK3 kinase activity. In this review, we classify existing and novel small-molecule drugs based on their pharmacodynamic (PD) type I, II, and III binding mode. Finally, we discuss their applicability and therapeutic potential in inflammatory and degenerative experimental disease models. interaction that alters enzymatic activity due to binding of effector molecule to an enzyme at site another than its active site. cysteine aspartic acid-specific protease that executes programmed apoptotic cell death. conserved amino acid motif (Asp-Phe-Gly) located at the beginning of the activation loop of protein kinases and that regulates kinase activity. transcription factor regulating cytokine production, immune responses, and cell survival. form of programmed necrotic or inflammatory cell death. Necroptosis is executed by mixed-lineage kinase domain-like pseudokinase (MLKL), which translocates to cell membranes upon activation and permeabilizes the plasma membrane, which leads to rupture of the cell. enzyme that can transfer a phosphate group from ATP to its target molecules (proteins), also called phosphorylation, thereby modifying the behavior of the target. study of how a drug is processed by an organism. This includes absorption, distribution, metabolism, excretion (ADME) studies. study of the biological and physiological effects of a drug on an organism. modulators of cell survival, cell death, and inflammation. domain in RIP kinases that mediates protein–protein interactions with other RHIM domain-containing proteins (ZBP1 and TRIF). It is an important domain for virus recognition. analysis of the relationship between the chemical structures of a molecule and its biological activity. environment surrounding tumor cells, including blood vessels, immune cells, fibroblasts, extracellular matrix, and signaling molecules. process of adding ubiquitin to a target protein at a lysine residue. Sequential activation of ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2), and ubiquitin ligases (E3) is needed for this process. recognizes Z-DNA or Z-RNA in the cytoplasm as an antiviral mechanism.

The RNA-editing enzyme adenosine deaminase acting on RNA 1 (ADAR1) limits the accumulation of endogenous immunostimulatory double-stranded RNA (dsRNA)1. In humans, reduced ADAR1 activity causes the severe inflammatory disease Aicardi-Goutières syndrome (AGS)2. In mice, complete loss of ADAR1 activity is embryonically lethal3-6, and mutations similar to those found in patients with AGS cause autoinflammation7-12. Mechanistically, adenosine-to-inosine (A-to-I) base modification of endogenous dsRNA by ADAR1 prevents chronic overactivation of the dsRNA sensors MDA5 and PKR3,7-10,13,14. Here we show that ADAR1 also inhibits the spontaneous activation of the left-handed Z-nucleic acid sensor ZBP1. Activation of ZBP1 elicits caspase-8-dependent apoptosis and MLKL-mediated necroptosis of ADAR1-deficient cells. ZBP1 contributes to the embryonic lethality of Adar-knockout mice, and it drives early mortality and intestinal cell death in mice deficient in the expression of both ADAR and MAVS. The Z-nucleic-acid-binding Zα domain of ADAR1 is necessary to prevent ZBP1-mediated intestinal cell death and skin inflammation. The Zα domain of ADAR1 promotes A-to-I editing of endogenous Alu elements to prevent dsRNA formation through the pairing of inverted Alu repeats, which can otherwise induce ZBP1 activation. This shows that recognition of Alu duplex RNA by ZBP1 may contribute to the pathological features of AGS that result from the loss of ADAR1 function.

Rationale: Necroptosis, mediated by RIPK3 (receptor-interacting protein kinase 3) and MLKL (mixed lineage kinase domain-like), is a form of regulated necrosis that can drive tissue inflammation and destruction; however, its contribution to chronic obstructive pulmonary disease (COPD) pathogenesis is poorly understood. Objectives: To determine the role of necroptosis in COPD. Methods: Total and active (phosphorylated) RIPK3 and MLKL were measured in the lung tissue of patients with COPD and control subjects without COPD. Necroptosis-related mRNA and proteins as well as cell death were examined in lungs and pulmonary macrophages of mice with cigarette smoke (CS)-induced experimental COPD. The responses of Ripk3-/- and Mlkl-/- mice to acute and chronic CS exposure were compared with those of wild-type mice. The combined inhibition of apoptosis (with the pan-caspase inhibitor quinoline-Val-Asp-difluorophenoxymethylketone [qVD-OPh]) and necroptosis (with deletion of Mlkl in mice) was assessed. Measurements and Main Results: The total MLKL protein in the epithelium and macrophages and the pRIPK3 and pMLKL in lung tissue were increased in patients with severe COPD compared with never-smokers or smoker control subjects without COPD. Necroptosis-related mRNA and protein levels were increased in the lungs and macrophages in CS-exposed mice and experimental COPD. Ripk3 or Mlkl deletion prevented airway inflammation upon acute CS exposure. Ripk3 deficiency reduced airway inflammation and remodeling as well as the development of emphysematous pathology after chronic CS exposure. Mlkl deletion and qVD-OPh treatment reduced chronic CS-induced airway inflammation, but only Mlkl deletion prevented airway remodeling and emphysema. Ripk3 or Mlkl deletion and qVD-OPh treatment reduced CS-induced lung-cell death. Conclusions: Necroptosis is induced by CS exposure and is increased in the lungs of patients with COPD and in experimental COPD. Inhibiting necroptosis attenuates CS-induced airway inflammation, airway remodeling, and emphysema. Targeted inhibition of necroptosis is a potential therapeutic strategy in COPD.

The gasdermin (GSDM) family has evolved as six gene clusters (GSDMA-E and Pejvakin, PJVK), and GSDM proteins are characterized by a unique N-terminal domain (N-GSDM). With the exception of PJVK, the N-GSDM domain is capable of executing plasma membrane permeabilization. Depending on the cell death modality, several protease- and kinase-dependent mechanisms directly regulate the activity of GSDME and GSDMD, the two most widely expressed and best-studied GSDMs. We provide an overview of all GSDMs in terms of biological function, tissue expression, activation, regulation, and structure. In-depth phylogenetic analysis reveals that GSDM genes show many gene duplications and deletions, suggesting that strong evolutionary forces and a unique position of the PJVK gene are associated with the occurrence of complex inner-ear development in vertebrates.

Direct targeting of the downstream mitogen-activated protein kinase (MAPK) pathway to suppress extracellular-regulated kinase (ERK) activation in KRAS and BRAF mutant colorectal cancer (CRC) has proven clinically unsuccessful, but promising results have been obtained with combination therapies including epidermal growth factor receptor (EGFR) inhibition. To elucidate the interplay between EGF signalling and ERK activation in tumours, we used patient-derived organoids (PDOs) from KRAS and BRAF mutant CRCs. PDOs resemble in vivo tumours, model treatment response and are compatible with live-cell microscopy. We established real-time, quantitative drug response assessment in PDOs with single-cell resolution, using our improved fluorescence resonance energy transfer (FRET)-based ERK biosensor EKAREN5. We show that oncogene-driven signalling is strikingly limited without EGFR activity and insufficient to sustain full proliferative potential. In PDOs and in vivo, upstream EGFR activity rigorously amplifies signal transduction efficiency in KRAS or BRAF mutant MAPK pathways. Our data provide a mechanistic understanding of the effectivity of EGFR inhibitors within combination therapies against KRAS and BRAF mutant CRC. Ponsioen et al. use a FRET‐based ERK biosensor EKAREN5 in patient‐derived organoids to show that EGFR activity amplifies signal transduction efficiency in KRAS or BRAF mutant MAPK pathways.

The most common cause of death in the intensive care unit (ICU) is the development of multiorgan dysfunction syndrome (MODS). Besides life-supporting treatments, no cure exists, and its mechanisms are still poorly understood. Catalytic iron is associated with ICU mortality and is known to cause free radical-mediated cellular toxicity. It is thought to induce excessive lipid peroxidation, the main characteristic of an iron-dependent type of cell death conceptualized as ferroptosis. Here we show that the severity of multiorgan dysfunction and the probability of death are indeed associated with plasma catalytic iron and lipid peroxidation. Transgenic approaches underscore the role of ferroptosis in iron-induced multiorgan dysfunction. Blocking lipid peroxidation with our highly soluble ferrostatin-analogue protects mice from injury and death in experimental non-septic multiorgan dysfunction, but not in sepsis-induced multiorgan dysfunction. The limitations of the experimental mice models to mimic the complexity of clinical MODS warrant further preclinical testing. In conclusion, our data suggest ferroptosis targeting as possible treatment option for a stratifiable subset of MODS patients.

Regulated cell death (RCD) relies on activation and recruitment of pore-forming proteins (PFPs) that function as executioners of specific cell death pathways: apoptosis regulator BAX (BAX), BCL-2 homologous antagonist/killer (BAK) and BCL-2-related ovarian killer protein (BOK) for apoptosis, gasdermins (GSDMs) for pyroptosis and mixed lineage kinase domain-like protein (MLKL) for necroptosis. Inactive precursors of PFPs are converted into pore-forming entities through activation, membrane recruitment, membrane insertion and oligomerization. These mechanisms involve protein-protein and protein-lipid interactions, proteolytic processing and phosphorylation. In this Review, we discuss the structural rearrangements incurred by RCD-related PFPs and describe the mechanisms that manifest conversion from autoinhibited to membrane-embedded molecular states. We further discuss the formation and maturation of membrane pores formed by BAX/BAK/BOK, GSDMs and MLKL, leading to diverse pore architectures. Lastly, we highlight commonalities and differences of PFP mechanisms involving BAX/BAK/BOK, GSDMs and MLKL and conclude with a discussion on how, in a population of challenged cells, the coexistence of cell death modalities may have profound physiological and pathophysiological implications.

Immunogenic cell death (ICD) refers to an immunologically distinct process of regulated cell death that activates, rather than suppresses, innate and adaptive immune responses. Such responses culminate into T cell-driven immunity against antigens derived from dying cancer cells. The potency of ICD is dependent on the immunogenicity of dying cells as defined by the antigenicity of these cells and their ability to expose immunostimulatory molecules like damage-associated molecular patterns (DAMPs) and cytokines like type I interferons (IFNs). Moreover, it is crucial that the host's immune system can adequately detect the antigenicity and adjuvanticity of these dying cells. Over the years, several well-known chemotherapies have been validated as potent ICD inducers, including (but not limited to) anthracyclines, paclitaxels, and oxaliplatin. Such ICD-inducing chemotherapeutic drugs can serve as important combinatorial partners for anti-cancer immunotherapies against highly immuno-resistant tumors. In this Trial Watch, we describe current trends in the preclinical and clinical integration of ICD-inducing chemotherapy in the existing immuno-oncological paradigms.

A hallmark of apoptosis is the fragmentation of nuclear DNA. Although this activity involves the caspase-3-dependent DNAse CAD (caspase-activated DNAse), evidence exists that DNA fragmentation can occur independently of caspase activity. Here we report on the ability of truncated Bid (tBid) to induce the release of a DNAse activity from mitochondria. This DNAse activity was identified by mass spectrometry as endonuclease G, an abundant 30 kDa protein released from mitochondria under apoptotic conditions. No tBid-induced endonuclease G release could be observed in mitochondria from Bcl-2-transgenic mice. The in vivo occurrence of endonuclease G release from mitochondria during apoptosis was confirmed in the liver from mice injected with agonistic anti-Fas antibody and is completely prevented in Bcl-2 transgenic mice. These data indicate that endonuclease G may be involved in CAD-independent DNA fragmentation during cell death pathways in which truncated Bid is generated.

Interleukin-10 (IL-10) is a potent inhibitor of lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF) production and has been shown to protect mice from endotoxin shock. As IFN-gamma is another important mediator of LPS toxicity, we studied the effects of IL-10 on LPS-induced IFN-gamma synthesis in vitro and in vivo. First, we found that the addition of recombinant human IL-10 (rhIL-10) (10 U/ml) to human whole blood markedly suppressed LPS-induced IFN-gamma release while neutralization of endogenously synthesized IL-10 resulted in increased IFN-gamma levels. The ability of rIL-10 to inhibit LPS-induced IFN-gamma synthesis was also observed in vivo in mice. Indeed, administration of 1000 U recombinant mouse IL-10 (rmIL-10) 30 min before and 3 h after challenge of BALB/c mice with 100 micrograms LPS resulted in a threefold decrease in peak IFN-gamma serum levels. We then examined the production and the role of IL-10 during murine endotoxemia. We found that LPS injection causes the rapid release of IL-10, peak IL-10 serum levels being observed 90 min after LPS challenge. Neutralization of endogenously produced IL-10 by administration of 2 mg JES5-2A5 anti-IL-10 monoclonal antibody (mAb) 2 h before LPS challenge resulted in a marked increase in both TNF and IFN-gamma serum levels while irrelevant isotype-matched mAb had no effect. The enhanced production of inflammatory cytokines in anti-IL-10 mAb-treated mice was associated with a 60% lethality after injection of 500 micrograms LPS, while all mice pretreated with control mAb survived. We conclude that the rapid release of IL-10 during endotoxemia is a natural antiinflammatory response controlling cytokine production and LPS toxicity.

Recent data show that a strong relation exists in certain cells between mitochondria and caspase activation in apoptosis. We further investigated this relation and tested whether treatment with the permeability transition (PT)‐inducing agent atractyloside of Percoll‐purified mitochondria released a caspase‐processing activity. Following detection of procaspase‐11 processing, we further purified this caspase‐processing protease and identified it as cathepsin B. The purified cathepsin B, however, was found to be derived from lysosomes which were present as minor contaminants in the mitochondrial preparation. Besides procaspase‐11, caspase‐1 is also readily processed by cathepsin B. Procaspase‐2, ‐6, ‐7, ‐14 are weak substrates and procaspase‐3 is a very poor substrate, while procaspase‐12 is no substrate at all for cathepsin B. In addition, cathepsin B induces nuclear apoptosis in digitonin‐permeabilized cells as well as in isolated nuclei. All newly described activities of cathepsin B, namely processing of caspase zymogens and induction of nuclear apoptosis, are inhibited by the synthetic peptide caspase inhibitors z‐VAD.fmk, z‐DEVD.fmk and to a lesser extent by Ac‐YVAD.cmk.

Caspases are a family of cysteine proteases which play a crucial role in apoptosis and inflammation. The involvement of caspases in these processes can be demonstrated by their irreversible inhibition with fluoromethyl ketone and chloromethyl ketone derivatives of peptides resembling the cleavage site of known caspase substrates. These inhibitors irreversibly alkylate the cysteine residue in the active site of caspases. In this study we show that a biotinylated fluoromethyl ketone peptide inhibitor of caspases (z‐VAD.fmk) also efficiently affinity‐labeled cathepsin B and cathepsin H. In addition, the caspase inhibitors z‐VAD.fmk, z‐DEVD.fmk and Ac‐YVAD.cmk also efficiently inhibited cathepsin B activity in vitro and in tissue culture cells at concentrations that are generally used to demonstrate the involvement of caspases.

Myeloic cells express a peculiar surface receptor for extracellular ATP, called the P2Z/P2X 7 purinoreceptor, which is involved in cell death signalling. Here, we investigated the role of caspases, a family of proteases implicated in apoptosis and the cytokine secretion. We observed that extracellular ATP induced the activation of multiple caspases including caspase‐1, ‐3 and ‐8, and subsequent cleavage of the caspase substrates PARP and lamin B. Using caspase inhibitors, it was found that caspases were specifically involved in ATP‐induced apoptotic damage such as chromatin condensation and DNA fragmentation. In contrast, inhibition of caspases only marginally affected necrotic alterations and cell death proceeded normally whether or not nuclear damage was blocked. Our results therefore suggest that the activation of caspases by the P2Z receptor is required for apoptotic but not necrotic alterations of ATP‐induced cell death.