Caspase-8 Collaborates with Caspase-11 to Drive Tissue Damage and Execution of Endotoxic Shock.

The execution of shock following high dose E. coli lipopolysaccharide (LPS) or bacterial sepsis in mice required pro-apoptotic caspase-8 in addition to pro-pyroptotic caspase-11 and gasdermin D. Hematopoietic cells produced MyD88- and TRIF-dependent inflammatory cytokines sufficient to initiate shock without any contribution from caspase-8 or caspase-11. Both proteases had to be present to support tumor necrosis factor- and interferon-ß-dependent tissue injury first observed in the small intestine and later in spleen and thymus. Caspase-11 enhanced the activation of caspase-8 and extrinsic cell death machinery within the lower small intestine. Neither caspase-8 nor caspase-11 was individually sufficient for shock. Both caspases collaborated to amplify inflammatory signals associated with tissue damage. Therefore, combined pyroptotic and apoptotic signaling mediated endotoxemia independently of RIPK1 kinase activity and RIPK3 function. These observations bring to light the relevance of tissue compartmentalization to disease processes in vivo where cytokines act in parallel to execute diverse cell death pathways.

RIPK1 and Caspase-8 Ensure Chromosome Stability Independently of Their Role in Cell Death and Inflammation.

Receptor-interacting protein kinase (RIPK) 1 functions as a key mediator of tissue homeostasis via formation of Caspase-8 activating ripoptosome complexes, positively and negatively regulating apoptosis, necroptosis, and inflammation. Here, we report an unanticipated cell-death- and inflammation-independent function of RIPK1 and Caspase-8, promoting faithful chromosome alignment in mitosis and thereby ensuring genome stability. We find that ripoptosome complexes progressively form as cells enter mitosis, peaking at metaphase and disassembling as cells exit mitosis. Genetic deletion and mitosis-specific inhibition of Ripk1 or Caspase-8 results in chromosome alignment defects independently of MLKL. We found that Polo-like kinase 1 (PLK1) is recruited into mitotic ripoptosomes, where PLK1's activity is controlled via RIPK1-dependent recruitment and Caspase-8-mediated cleavage. A fine balance of ripoptosome assembly is required as deregulated ripoptosome activity modulates PLK1-dependent phosphorylation of downstream effectors, such as BUBR1. Our data suggest that ripoptosome-mediated regulation of PLK1 contributes to faithful chromosome segregation during mitosis.

More than one million barriers fragment Europe's rivers.

Rivers support some of Earth's richest biodiversity1 and provide essential ecosystem services to society2, but they are often fragmented by barriers to free flow3. In Europe, attempts to quantify river connectivity have been hampered by the absence of a harmonized barrier database. Here we show that there are at least 1.2 million instream barriers in 36 European countries (with a mean density of 0.74 barriers per kilometre), 68 per cent of which are structures less than two metres in height that are often overlooked. Standardized walkover surveys along 2,715 kilometres of stream length for 147 rivers indicate that existing records underestimate barrier numbers by about 61 per cent. The highest barrier densities occur in the heavily modified rivers of central Europe and the lowest barrier densities occur in the most remote, sparsely populated alpine areas. Across Europe, the main predictors of barrier density are agricultural pressure, density of river-road crossings, extent of surface water and elevation. Relatively unfragmented rivers are still found in the Balkans, the Baltic states and parts of Scandinavia and southern Europe, but these require urgent protection from proposed dam developments. Our findings could inform the implementation of the EU Biodiversity Strategy, which aims to reconnect 25,000 kilometres of Europe's rivers by 2030, but achieving this will require a paradigm shift in river restoration that recognizes the widespread impacts caused by small barriers.

MLKL Requires the Inositol Phosphate Code to Execute Necroptosis.

Necroptosis is an important form of lytic cell death triggered by injury and infection, but whether mixed lineage kinase domain-like (MLKL) is sufficient to execute this pathway is unknown. In a genetic selection for human cell mutants defective for MLKL-dependent necroptosis, we identified mutations in IPMK and ITPK1, which encode inositol phosphate (IP) kinases that regulate the IP code of soluble molecules. We show that IP kinases are essential for necroptosis triggered by death receptor activation, herpesvirus infection, or a pro-necrotic MLKL mutant. In IP kinase mutant cells, MLKL failed to oligomerize and localize to membranes despite proper receptor-interacting protein kinase-3 (RIPK3)-dependent phosphorylation. We demonstrate that necroptosis requires IP-specific kinase activity and that a highly phosphorylated product, but not a lowly phosphorylated precursor, potently displaces the MLKL auto-inhibitory brace region. These observations reveal control of MLKL-mediated necroptosis by a metabolite and identify a key molecular mechanism underlying regulated cell death.

RIPK1 and RIPK3 Kinases Promote Cell-Death-Independent Inflammation by Toll-like Receptor 4.

Macrophages are a crucial component of the innate immune system in sensing pathogens and promoting local and systemic inflammation. RIPK1 and RIPK3 are homologous kinases, previously linked to activation of necroptotic death. In this study, we have described roles for these kinases as master regulators of pro-inflammatory gene expression induced by lipopolysaccharide, independent of their well-documented cell death functions. In primary macrophages, this regulation was elicited in the absence of caspase-8 activity, required the adaptor molecule TRIF, and proceeded in a cell autonomous manner. RIPK1 and RIPK3 kinases promoted sustained activation of Erk, cFos, and NF-κB, which were required for inflammatory changes. Utilizing genetic and pharmacologic tools, we showed that RIPK1 and RIPK3 account for acute inflammatory responses induced by lipopolysaccharide in vivo; notably, this regulation did not require exogenous manipulation of caspases. These findings identified a new pharmacologically accessible pathway that may be relevant to inflammatory pathologies.

Author Correction: Design of amidobenzimidazole STING receptor agonists with systemic activity.

Change history: In this Letter, author Ana Puhl was inadvertently omitted; this error has been corrected online.An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Design of amidobenzimidazole STING receptor agonists with systemic activity.

Stimulator of interferon genes (STING) is a receptor in the endoplasmic reticulum that propagates innate immune sensing of cytosolic pathogen-derived and self DNA1. The development of compounds that modulate STING has recently been the focus of intense research for the treatment of cancer and infectious diseases and as vaccine adjuvants2. To our knowledge, current efforts are focused on the development of modified cyclic dinucleotides that mimic the endogenous STING ligand cGAMP; these have progressed into clinical trials in patients with solid accessible tumours amenable to intratumoral delivery3. Here we report the discovery of a small molecule STING agonist that is not a cyclic dinucleotide and is systemically efficacious for treating tumours in mice. We developed a linking strategy to synergize the effect of two symmetry-related amidobenzimidazole (ABZI)-based compounds to create linked ABZIs (diABZIs) with enhanced binding to STING and cellular function. Intravenous administration of a diABZI STING agonist to immunocompetent mice with established syngeneic colon tumours elicited strong anti-tumour activity, with complete and lasting regression of tumours. Our findings represent a milestone in the rapidly growing field of immune-modifying cancer therapies.

NF-κB-Independent Role of IKKα/IKKß in Preventing RIPK1 Kinase-Dependent Apoptotic and Necroptotic Cell Death during TNF Signaling.

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/IKKß 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.

MALT1 Protease Plays a Dual Role in the Allergic Response by Acting in Both Mast Cells and Endothelial Cells.

The signaling protein MALT1 plays a key role in promoting NF-κB activation in Ag-stimulated lymphocytes. In this capacity, MALT1 has two functions, acting as a scaffolding protein and as a substrate-specific protease. MALT1 is also required for NF-κB-dependent induction of proinflammatory cytokines after FcεR1 stimulation in mast cells, implicating a role in allergy. Because MALT1 remains understudied in this context, we sought to investigate how MALT1 proteolytic activity contributes to the overall allergic response. We compared bone marrow-derived mast cells from MALT1 knockout (MALT1-/-) and MALT1 protease-deficient (MALTPD/PD) mice to wild-type cells. We found that MALT1-/- and MALT1PD/PD mast cells are equally impaired in cytokine production following FcεRI stimulation, indicating that MALT1 scaffolding activity is insufficient to drive the cytokine response and that MALT1 protease activity is essential. In addition to cytokine production, acute mast cell degranulation is a critical component of allergic response. Intriguingly, whereas degranulation is MALT1-independent, MALT1PD/PD mice are protected from vascular edema induced by either passive cutaneous anaphylaxis or direct challenge with histamine, a major granule component. This suggests a role for MALT1 protease activity in endothelial cells targeted by mast cell-derived vasoactive substances. Indeed, we find that in human endothelial cells, MALT1 protease is activated following histamine treatment and is required for histamine-induced permeability. We thus propose a dual role for MALT1 protease in allergic response, mediating 1) IgE-dependent mast cell cytokine production, and 2) histamine-induced endothelial permeability. This dual role indicates that therapeutic inhibitors of MALT1 protease could work synergistically to control IgE-mediated allergic disease.

RIP3 induces apoptosis independent of pronecrotic kinase activity.

Receptor-interacting protein kinase 3 (RIP3 or RIPK3) has emerged as a central player in necroptosis and a potential target to control inflammatory disease. Here, three selective small-molecule compounds are shown to inhibit RIP3 kinase-dependent necroptosis, although their therapeutic value is undermined by a surprising, concentration-dependent induction of apoptosis. These compounds interact with RIP3 to activate caspase 8 (Casp8) via RHIM-driven recruitment of RIP1 (RIPK1) to assemble a Casp8-FADD-cFLIP complex completely independent of pronecrotic kinase activities and MLKL. RIP3 kinase-dead D161N mutant induces spontaneous apoptosis independent of compound, whereas D161G, D143N, and K51A mutants, like wild-type, only trigger apoptosis when compound is present. Accordingly, RIP3-K51A mutant mice (Rip3(K51A/K51A)) are viable and fertile, in stark contrast to the perinatal lethality of Rip3(D161N/D161N) mice. RIP3 therefore holds both necroptosis and apoptosis in balance through a Ripoptosome-like platform. This work highlights a common mechanism unveiling RHIM-driven apoptosis by therapeutic or genetic perturbation of RIP3.

Respiratory Syncytial Virus Infection Promotes Necroptosis and HMGB1 Release by Airway Epithelial Cells.

Rationale: Respiratory syncytial virus (RSV) bronchiolitis causes significant infant mortality. Bronchiolitis is characterized by airway epithelial cell (AEC) death; however, the mode of death remains unknown.Objectives: To determine whether necroptosis contributes to RSV bronchiolitis pathogenesis via HMGB1 (high mobility group box 1) release.Methods: Nasopharyngeal samples were collected from children presenting to the hospital with acute respiratory infection. Primary human AECs and neonatal mice were inoculated with RSV and murine Pneumovirus, respectively. Necroptosis was determined via viability assays and immunohistochemistry for RIPK1 (receptor-interacting protein kinase-1), MLKL (mixed lineage kinase domain-like pseudokinase) protein, and caspase-3. Necroptosis was blocked using pharmacological inhibitors and RIPK1 kinase-dead knockin mice.Measurements and Main Results: HMGB1 levels were elevated in nasopharyngeal samples of children with acute RSV infection. RSV-induced epithelial cell death was associated with increased phosphorylated RIPK1 and phosphorylated MLKL but not active caspase-3 expression. Inhibition of RIPK1 or MLKL attenuated RSV-induced HMGB1 translocation and release, and lowered viral load. MLKL inhibition increased active caspase-3 expression in a caspase-8/9-dependent manner. In susceptible mice, Pneumovirus infection upregulated RIPK1 and MLKL expression in the airway epithelium at 8 to 10 days after infection, coinciding with AEC sloughing, HMGB1 release, and neutrophilic inflammation. Genetic or pharmacological inhibition of RIPK1 or MLKL attenuated these pathologies, lowered viral load, and prevented type 2 inflammation and airway remodeling. Necroptosis inhibition in early life ameliorated asthma progression induced by viral or allergen challenge in later life.Conclusions: Pneumovirus infection induces AEC necroptosis. Inhibition of necroptosis may be a viable strategy to limit the severity of viral bronchiolitis and break its nexus with asthma.

Environmental plasticity and colonisation history in the Atlantic salmon microbiome: A translocation experiment.

Microbial communities associated with the gut and the skin are strongly influenced by environmental factors, and can rapidly adapt to change. Historical processes may also affect the microbiome. In particular, variation in microbial colonisation in early life has the potential to induce lasting effects on microbial assemblages. However, little is known about the relative extent of microbiome plasticity or the importance of historical colonisation effects following environmental change, especially for nonmammalian species. To investigate this we performed a reciprocal translocation of Atlantic salmon between artificial and semi-natural conditions. Wild and hatchery-reared fry were transferred to three common garden experimental environments for 6 weeks: standard hatchery conditions, hatchery conditions with an enriched diet, and simulated wild conditions. We characterized the faecal and skin microbiome of individual fish before and after the environmental translocation, using a BACI (before-after-control-impact) design. We found evidence of extensive microbiome plasticity for both the gut and skin, with the greatest changes in alpha and beta diversity associated with the largest changes in environment and diet. Microbiome richness and diversity were entirely determined by environment, with no detectable effects of fish origin, and there was also a near-complete turnover in microbiome structure. However, we also identified, for the first time in fish, evidence of historical colonisation effects reflecting early-life experience, including ASVs characteristic of captive rearing. These results have important implications for host adaptation to local selective pressures, and highlight how conditions experienced during early life can have a long-term influence on the microbiome and, potentially, host health.

RIPK1 ensures intestinal homeostasis by protecting the epithelium against apoptosis.

Receptor interacting protein kinase 1 (RIPK1) has an essential role in the signalling triggered by death receptors and pattern recognition receptors. RIPK1 is believed to function as a node driving NF-κB-mediated cell survival and inflammation as well as caspase-8 (CASP8)-dependent apoptotic or RIPK3/MLKL-dependent necroptotic cell death. The physiological relevance of this dual function has remained elusive because of the perinatal death of RIPK1 full knockout mice. To circumvent this problem, we generated RIPK1 conditional knockout mice, and show that mice lacking RIPK1 in intestinal epithelial cells (IECs) spontaneously develop severe intestinal inflammation associated with IEC apoptosis leading to early death. This early lethality was rescued by antibiotic treatment, MYD88 deficiency or tumour-necrosis factor (TNF) receptor 1 deficiency, demonstrating the importance of commensal bacteria and TNF in the IEC Ripk1 knockout phenotype. CASP8 deficiency, but not RIPK3 deficiency, rescued the inflammatory phenotype completely, indicating the indispensable role of RIPK1 in suppressing CASP8-dependent apoptosis but not RIPK3-dependent necroptosis in the intestine. RIPK1 kinase-dead knock-in mice did not exhibit any sign of inflammation, suggesting that RIPK1-mediated protection resides in its kinase-independent platform function. Depletion of RIPK1 in intestinal organoid cultures sensitized them to TNF-induced apoptosis, confirming the in vivo observations. Unexpectedly, TNF-mediated NF-κB activation remained intact in these organoids. Our results demonstrate that RIPK1 is essential for survival of IECs, ensuring epithelial homeostasis by protecting the epithelium from CASP8-mediated IEC apoptosis independently of its kinase activity and NF-κB activation.

Kinase Activities of RIPK1 and RIPK3 Can Direct IFN-ß Synthesis Induced by Lipopolysaccharide.

The innate immune response is a central element of the initial defense against bacterial and viral pathogens. Macrophages are key innate immune cells that upon encountering pathogen-associated molecular patterns respond by producing cytokines, including IFN-ß. In this study, we identify a novel role for RIPK1 and RIPK3, a pair of homologous serine/threonine kinases previously implicated in the regulation of necroptosis and pathologic tissue injury, in directing IFN-ß production in macrophages. Using genetic and pharmacologic tools, we show that catalytic activity of RIPK1 directs IFN-ß synthesis induced by LPS in mice. Additionally, we report that RIPK1 kinase-dependent IFN-ß production may be elicited in an analogous fashion using LPS in bone marrow-derived macrophages upon inhibition of caspases. Notably, this regulation requires kinase activities of both RIPK1 and RIPK3, but not the necroptosis effector protein, MLKL. Mechanistically, we provide evidence that necrosome-like RIPK1 and RIPK3 aggregates facilitate canonical TRIF-dependent IFN-ß production downstream of the LPS receptor TLR4. Intriguingly, we also show that RIPK1 and RIPK3 kinase-dependent synthesis of IFN-ß is markedly induced by avirulent strains of Gram-negative bacteria, Yersinia and Klebsiella, and less so by their wild-type counterparts. Overall, these observations identify unexpected roles for RIPK1 and RIPK3 kinases in the production of IFN-ß during the host inflammatory responses to bacterial infection and suggest that the axis in which these kinases operate may represent a target for bacterial virulence factors.

Cell-Extrinsic TNF Collaborates with TRIF Signaling To Promote Yersinia-Induced Apoptosis.

Innate immune responses that are crucial for control of infection are often targeted by microbial pathogens. Blockade of NF-κB and MAPK signaling by the Yersinia virulence factor YopJ inhibits cytokine production by innate immune cells but also triggers cell death. This cell death requires RIPK1 kinase activity and caspase-8, which are engaged by TLR4 and the adaptor protein TRIF. Nevertheless, TLR4- and TRIF-deficient cells undergo significant apoptosis, implicating TLR4/TRIF-independent pathways in the death of Yersinia-infected cells. In this article, we report a key role for TNF/TNFR1 in Yersinia-induced cell death of murine macrophages, which occurs despite the blockade of NF-κB and MAPK signaling imposed by Yersinia on infected cells. Intriguingly, direct analysis of YopJ injection revealed a heterogeneous population of injection-high and injection-low cells, and demonstrated that TNF expression came from the injection-low population. Moreover, TNF production by this subpopulation was necessary for maximal apoptosis in the population of highly injected cells, and TNFR-deficient mice displayed enhanced susceptibility to Yersinia infection. These data demonstrate an important role for collaboration between TNF and pattern recognition receptor signals in promoting maximal apoptosis during bacterial infection, and demonstrate that heterogeneity in virulence factor injection and cellular responses play an important role in promoting anti-Yersinia immune defense.

The Mitochondrial Phosphatase PGAM5 Is Dispensable for Necroptosis but Promotes Inflammasome Activation in Macrophages.

The cytokine IL-1ß is intimately linked to many pathological inflammatory conditions. Mature IL-1ß secretion requires cleavage by the inflammasome. Recent evidence indicates that many cell death signal adaptors have regulatory roles in inflammasome activity. These include the apoptosis inducers FADD and caspase 8, and the necroptosis kinases receptor interacting protein kinase 1 (RIPK1) and RIPK3. PGAM5 is a mitochondrial phosphatase that has been reported to function downstream of RIPK3 to promote necroptosis and IL-1ß secretion. To interrogate the biological function of PGAM5, we generated Pgam5(-/-) mice. We found that Pgam5(-/-) mice were smaller compared with wild type littermates, and male Pgam5(-/-) mice were born at sub-Mendelian ratio. Despite these growth and survival defects, Pgam5(-/-) cells responded normally to multiple inducers of apoptosis and necroptosis. Rather, we found that PGAM5 is critical for IL-1ß secretion in response to NLRP3 and AIM2 inflammasome agonists. Moreover, vesicular stomatosis virus-induced IL-1ß secretion was impaired in Pgam5(-/-) bone marrow-derived macrophages, but not in Ripk3(-/-) bone marrow-derived dendritic cells, indicating that PGAM5 functions independent of RIPK3 to promote inflammasome activation. Mechanistically, PGAM5 promotes ASC polymerization, maintenance of mitochondrial integrity, and optimal reactive oxygen species production in response to inflammasome signals. Hence PGAM5 is a novel regulator of inflammasome and caspase 1 activity that functions independently of RIPK3.

RIPK1 and PGAM5 Control Leishmania Replication through Distinct Mechanisms.

Leishmaniasis is an important parasitic disease found in the tropics and subtropics. Cutaneous and visceral leishmaniasis affect an estimated 1.5 million people worldwide. Despite its human health relevance, relatively little is known about the cell death pathways that control Leishmania replication in the host. Necroptosis is a recently identified form of cell death with potent antiviral effects. Receptor interacting protein kinase 1 (RIPK1) is a critical kinase that mediates necroptosis downstream of death receptors and TLRs. Heme, a product of hemoglobin catabolism during certain intracellular pathogen infections, is also a potent inducer of macrophage necroptosis. We found that human visceral leishmaniasis patients exhibit elevated serum levels of heme. Therefore, we examined the impact of heme and necroptosis on Leishmania replication. Indeed, heme potently inhibited Leishmania replication in bone marrow-derived macrophages. Moreover, we found that inhibition of RIPK1 kinase activity also enhanced parasite replication in the absence of heme. We further found that the mitochondrial phosphatase phosphoglycerate mutase family member 5 (PGAM5), a putative downstream effector of RIPK1, was also required for inhibition of Leishmania replication. In mouse infection, both PGAM5 and RIPK1 kinase activity are required for IL-1ß expression in response to Leishmania However, PGAM5, but not RIPK1 kinase activity, was directly responsible for Leishmania-induced IL-1ß secretion and NO production in bone marrow-derived macrophages. Collectively, these results revealed that RIPK1 and PGAM5 function independently to exert optimal control of Leishmania replication in the host.

t-BuOOH induces ferroptosis in human and murine cell lines.

Reactive oxygen species (ROS)-induced apoptosis has been extensively studied. Increasing evidence suggests that ROS, for instance, induced by hydrogen peroxide (H2O2), might also trigger regulated necrotic cell death pathways. Almost nothing is known about the cell death pathways triggered by tertiary-butyl hydroperoxide (t-BuOOH), a widely used inducer of oxidative stress. The lipid peroxidation products induced by t-BuOOH are involved in the pathophysiology of many diseases, such as cancer, cardiovascular diseases, or diabetes. In this study, we exposed murine fibroblasts (NIH3T3) or human keratinocytes (HaCaT) to t-BuOOH (50 or 200 µM, respectively) which induced a rapid necrotic cell death. Well-established regulators of cell death, i.e., p53, poly(ADP)ribose polymerase-1 (PARP-1), the stress kinases p38 and c-Jun N-terminal-kinases 1/2 (JNK1/2), or receptor-interacting serine/threonine protein kinase 1 (RIPK1) and 3 (RIPK3), were not required for t-BuOOH-mediated cell death. Using the selective inhibitors ferrostatin-1 (1 µM) and liproxstatin-1 (1 µM), we identified ferroptosis, a recently discovered cell death mechanism dependent on iron and lipid peroxidation, as the main cell death pathway. Accordingly, t-BuOOH exposure resulted in a ferrostatin-1- and liproxstatin-1-sensitive increase in lipid peroxidation and cytosolic ROS. Ferroptosis was executed independently from other t-BuOOH-mediated cellular damages, i.e., loss of mitochondrial membrane potential, DNA double-strand breaks, or replication block. H2O2 did not cause ferroptosis at equitoxic concentrations (300 µM) and induced a (1) lower and (2) ferrostatin-1- or liproxstatin-1-insensitive increase in lipid peroxidation. We identify that t-BuOOH and H2O2 produce a different pattern of lipid peroxidation, thereby leading to different cell death pathways and present t-BuOOH as a novel inducer of ferroptosis.

Neuronal Death After Hemorrhagic Stroke In Vitro and In Vivo Shares Features of Ferroptosis and Necroptosis.

BACKGROUND AND PURPOSE: Intracerebral hemorrhage leads to disability or death with few established treatments. Adverse outcomes after intracerebral hemorrhage result from irreversible damage to neurons resulting from primary and secondary injury. Secondary injury has been attributed to hemoglobin and its oxidized product hemin from lysed red blood cells. The aim of this study was to identify the underlying cell death mechanisms attributable to secondary injury by hemoglobin and hemin to broaden treatment options. METHODS: We investigated cell death mechanisms in cultured neurons exposed to hemoglobin or hemin. Chemical inhibitors implicated in all known cell death pathways were used. Identified cell death mechanisms were confirmed using molecular markers and electron microscopy. RESULTS: Chemical inhibitors of ferroptosis and necroptosis protected against hemoglobin- and hemin-induced toxicity. By contrast, inhibitors of caspase-dependent apoptosis, protein or mRNA synthesis, autophagy, mitophagy, or parthanatos had no effect. Accordingly, molecular markers of ferroptosis and necroptosis were increased after intracerebral hemorrhage in vitro and in vivo. Electron microscopy showed that hemin induced a necrotic phenotype. Necroptosis and ferroptosis inhibitors each abrogated death by >80% and had similar therapeutic windows in vitro. CONCLUSIONS: Experimental intracerebral hemorrhage shares features of ferroptotic and necroptotic cell death, but not caspase-dependent apoptosis or autophagy. We propose that ferroptosis or necroptotic signaling induced by lysed blood is sufficient to reach a threshold of death that leads to neuronal necrosis and that inhibition of either of these pathways can bring cells below that threshold to survival.

Genetic Inhibition of Receptor Interacting Protein Kinase-1 Reduces Cell Death and Improves Functional Outcome After Intracerebral Hemorrhage in Mice.

BACKGROUND AND PURPOSE: Recent studies using cultured cells and rodent intracerebral hemorrhage (ICH) models have implicated RIPK1 (receptor interacting protein kinase-1) as a driver of programmed necrosis and secondary injury based on use of chemical inhibitors. However, these inhibitors have off-target effects and cannot be used alone to prove a role for RIPK1. The aim of the current study was to examine the effect of genetic inhibition of the kinase domain of RIPK1 in a mouse ICH model. METHODS: We subjected 2 lines of mice with RIPK1 point mutations of the kinase domain (K45A and D138N), rendering them kinase inactive, to autologous blood ICH and measured acute cell death and functional outcome. RESULTS: Compared with wild-type controls, RIPK1K45A/K45A and RIPK1D138N/D138N had significantly less cells with plasmalemma permeability, less acute neuronal cell death, less weight loss and more rapid weight gain to baseline, and improved performance in a Morris water maze paradigm after autologous blood ICH. In addition, mice systemically administered GSK'963, a potent, specific, brain penetrant small molecule RIPK1 inhibitor, had reduced acute neuronal death at 24 hours after ICH. CONCLUSIONS: The data show that the kinase domain of RIPK1 is a disease driver of ICH, mediating both acute cell death and functional outcome, and support development of RIPK1 inhibitors as therapeutic agents for human ICH.