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.
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.
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.
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.
Limited proteolysis of gasdermin D (GSDMD) generates an N-terminal pore-forming fragment that controls pyroptosis in macrophages. GSDMD is processed via inflammasome-activated caspase-1 or -11. It is ...currently unknown whether macrophage GSDMD can be processed by other mechanisms. Here, we describe an additional pathway controlling GSDMD processing. The inhibition of TAK1 or IκB kinase (IKK) by the
effector protein YopJ elicits RIPK1- and caspase-8-dependent cleavage of GSDMD, which subsequently results in cell death. GSDMD processing also contributes to the NLRP3 inflammasome-dependent release of interleukin-1β (IL-1β). Thus, caspase-8 acts as a regulator of GSDMD-driven cell death. Furthermore, this study establishes the importance of TAK1 and IKK activity in the control of GSDMD cleavage and cytotoxicity.
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.
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•RIPK1 and RIPK3 kinases promote TRIF-dependent cytokine production by LPS in vitro•RIPK1 and RIPK3 kinase-dependent inflammation is independent of cell death•Erk1/2 mediates RIPK1 and RIPK3 kinase dependent inflammation•RIPK1 kinase and RIPK3 are mediators of LPS-induced cytokine production in vivo
Kinase activities of RIPK1 and RIPK3 are critical for necroptotic cell death. Degterev and colleagues demonstrate that RIPK1 and RIPK3 kinases also direct inflammatory gene expression induced by Toll-like receptor 4 ligand, lipopolysaccharide, in vitro, and in vivo. This regulation is independent of necroptosis and requires Erk1/2, cFos, and NF-κB.
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.
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•The four-helical bundle domain of MLKL is sufficient for necroptosis induction•MLKL binds phosphatidylinositol phosphates via positive charges in the 4HBD•Recombinant MLKL induces leakage of PIP-containing liposomes•Inhibiting the formation of PI(5)P and PI(4,5)P2 inhibits TNF-mediated necroptosis
Necroptosis is a caspase-independent form of cell death that contributes to the pathogenesis of several human diseases, including ischemia-reperfusion injury, sepsis, and viral infection. Although MLKL emerged as a specific and crucial protein for necroptosis induction, it remains poorly understood how MLKL transduces the death signal. Here, Dondelinger et al., demonstrate that MLKL is recruited to phosphatidylinositol phosphates in the plasma membrane by a positive patch in its four-helical bundle domain. Interestingly, their data suggest that MLKL is able to directly permeabilize membranes.
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.
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•IKKα/IKKβ prevent RIPK1 kinase-dependent death independently of NF-κB activation•IKKα/IKKβ directly phosphorylate RIPK1 in TNFR1 complex I•Impaired phosphorylation of RIPK1 correlates with enhanced binding to FADD/caspase-8•IKK kinase inhibition induces TNF-mediated RIPK1 kinase-dependent cell death in vivo
Dondelinger et al. describe an unexpected NF-κB-independent function of the IKK complex in protecting against TNF-induced RIPK1 kinase-dependent cell death. In TNFR1 complex I, IKKα/IKKβ directly phosphorylates RIPK1, leading to a reduction in RIPK1’s ability to bind FADD/caspase-8 and to induce apoptosis.
Herpes simplex virus (HSV)-1 and HSV-2 are significant human pathogens causing recurrent disease. During infection, HSV modulates cell death pathways using the large subunit (R1) of ribonucleotide ...reductase (RR) to suppress apoptosis by binding to and blocking caspase-8. Here, we demonstrate that HSV-1 and HSV-2 R1 proteins (ICP6 and ICP10, respectively) also prevent necroptosis in human cells by inhibiting the interaction between receptor-interacting protein kinase 1 (RIP1) and RIP3, a key step in tumor necrosis factor (TNF)-induced necroptosis. We show that suppression of this cell death pathway requires an N-terminal RIP homotypic interaction motif (RHIM) within R1, acting in concert with the caspase-8-binding domain, which unleashes necroptosis independent of RHIM function. Thus, necroptosis is a human host defense pathway against two important viral pathogens that naturally subvert multiple death pathways via a single evolutionarily conserved gene product.
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•HSV-1 and HSV-2 R1 homologs block death receptor-induced necroptosis in human cells•HSV R1 homologs disrupt RIP1-RIP3 RHIM-dependent necrosome formation•Suppression of RIP3-mediated necroptosis requires both RHIM and caspase-8-binding domains•Caspase-8 inhibition blocks apoptosis but opens a necroptotic trap door
Herpes simplex virus (HSV) modulates cell death to promote infection. Guo et al. show that the HSV early protein, R1, inhibits necroptosis in human cells as a RHIM signaling competitor to disrupt RIP1-RIP3 interactions. A separate C-terminal R1 function known to inhibit caspase-8 sensitizes infected cells to necroptosis.
RIP1 (RIPK1) kinase is a key regulator of TNF-induced NF-κB activation, apoptosis, and necroptosis through its kinase and scaffolding activities. Dissecting the balance of RIP1 kinase activity and ...scaffolding function in vivo during development and TNF-dependent inflammation has been hampered by the perinatal lethality of RIP1-deficient mice. In this study, we generated RIP1 kinase-dead (Ripk1(K45A)) mice and showed they are viable and healthy, indicating that the kinase activity of RIP1, but not its scaffolding function, is dispensable for viability and homeostasis. After validating that the Ripk1(K45A) mice were specifically protected against necroptotic stimuli in vitro and in vivo, we crossed them with SHARPIN-deficient cpdm mice, which develop severe skin and multiorgan inflammation that has been hypothesized to be mediated by TNF-dependent apoptosis and/or necroptosis. Remarkably, crossing Ripk1(K45A) mice with the cpdm strain protected against all cpdm-related pathology. Together, these data suggest that RIP1 kinase represents an attractive therapeutic target for TNF-driven inflammatory diseases.
Influenza A virus (IAV) is a lytic virus in primary cultures of many cell types and in vivo. We report that the kinase RIPK3 is essential for IAV-induced lysis of mammalian fibroblasts and lung ...epithelial cells. Replicating IAV drives assembly of a RIPK3-containing complex that includes the kinase RIPK1, the pseudokinase MLKL, and the adaptor protein FADD, and forms independently of signaling by RNA-sensing innate immune receptors (RLRs, TLRs, PKR), or the cytokines type I interferons and TNF-α. Downstream of RIPK3, IAV activates parallel pathways of MLKL-driven necroptosis and FADD-mediated apoptosis, with the former reliant on RIPK3 kinase activity and neither on RIPK1 activity. Mice deficient in RIPK3 or doubly deficient in MLKL and FADD, but not MLKL alone, are more susceptible to IAV than their wild-type counterparts, revealing an important role for RIPK3-mediated apoptosis in antiviral immunity. Collectively, these results outline RIPK3-activated cytolytic mechanisms essential for controlling respiratory IAV infection.
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•RIPK3-deficient cells are resistant to IAV-induced death•RIPK3 activates both necroptosis and apoptosis upon IAV infection•RIPK3-mediated necroptosis requires MLKL, while apoptosis necessitates FADD•RIPK3-activated apoptosis compensates for loss of necroptosis in vivo
Influenza A viruses (IAV) kill the cells in which they replicate. Nogusa et al. identify a central role for the host kinase RIPK3 in triggering death of IAV-infected cells. They find that RIPK3 activates parallel, redundant pathways of necroptosis and apoptosis to destroy the infected cell and protect the host.
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 (Rip3K51A/K51A) are viable and fertile, in stark contrast to the perinatal lethality of Rip3D161N/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.
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•RIP3 kinase inhibitors block necroptosis but induce apoptosis•This apoptosis requires RHIM-dependent RIP1/FADD/cFLIPL/caspase 8 complex formation•RIP3 kinase domain suppresses RHIM signaling independent of kinase activity•RIP3 K51A kinase-dead knockin mice are viable, fertile and immunocompetent
Mandal et.al. demonstrate that small-molecule inhibitors of RIP3 kinase trigger Caspase 8-dependent apoptosis. The RIP3 kinase domain represses RHIM-dependent nucleation of a Ripoptosome-like complex. The derivation of viable RIP3 kinase-inactive knockin mice uncouples apoptosis induction from pronecrotic kinase activity.
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