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.

The diverse roles of RIP kinases in host-pathogen interactions.

Receptor Interacting Protein Kinases (RIPKs) are cellular signaling molecules that are critical for homeostatic signaling in both communicable and non-communicable disease processes. In particular, RIPK1, RIPK2, RIPK3 and RIPK7 have emerged as key mediators of intracellular signal transduction including inflammation, autophagy and programmed cell death, and are thus essential for the early control of many diverse pathogenic organisms. In this review, we discuss the role of each RIPK in host responses to bacterial and viral pathogens, with a focus on studies that have used pathogen infection models rather than artificial stimulation with purified pathogen associated molecular patterns. We also discuss the intricate mechanisms of host evasion by pathogens that specifically target RIPKs for inactivation, and finally, we will touch on the controversial issue of drug development for kinase inhibitors to treat chronic inflammatory and neurological disorders, and the implications this may have on the outcome of pathogen infections.

Enantiomeric profiling of a chiral benzothiazole necroptosis inhibitor.

Necroptosis is a form of programmed cell death that contributes to the pathophysiology of multiple diseases. Development of small-molecule anti-necroptosis agents has great promising clinical therapeutic relevance. The benzothiazole compounds were discovered by our group from an in-house fluorine-containing compound library as potent necroptosis inhibitors. Herein, a chiral dimethylcyclopropyl benzothiazole necroptosis inhibitor was developed and the enantiomeric profiling resulted that the (S) form was generally more potent than the (R) counterpart in 2 ~ 4-fold toward cell necroptosis, receptor-interacting protein (RIP) kinases 1 and 3. The chiral compounds could significantly inhibit the expression of the phosphorylation of RIPK1, RIPK3 and MLKL in necroptotic cells. The molecular modelling studies predicted the binding modes of the enantiomers with RIP and explained their activity differences, guiding further rational design of the chiral necroptosis inhibitors.

Axonal Degeneration Is Mediated by Necroptosis Activation.

Axonal degeneration, which contributes to functional impairment in several disorders of the nervous system, is an important target for neuroprotection. Several individual factors and subcellular events have been implicated in axonal degeneration, but researchers have so far been unable to identify an integrative signaling pathway activating this self-destructive process. Through pharmacological and genetic approaches, we tested whether necroptosis, a regulated cell-death mechanism implicated in the pathogenesis of several neurodegenerative diseases, is involved in axonal degeneration. Pharmacological inhibition of the necroptotic kinase RIPK1 using necrostatin-1 strongly delayed axonal degeneration in the peripheral nervous system and CNS of wild-type mice of either sex and protected in vitro sensory axons from degeneration after mechanical and toxic insults. These effects were also observed after genetic knock-down of RIPK3, a second key regulator of necroptosis, and the downstream effector MLKL (Mixed Lineage Kinase Domain-Like). RIPK1 inhibition prevented mitochondrial fragmentation in vitro and in vivo, a typical feature of necrotic death, and inhibition of mitochondrial fission by Mdivi also resulted in reduced axonal loss in damaged nerves. Furthermore, electrophysiological analysis demonstrated that inhibition of necroptosis delays not only the morphological degeneration of axons, but also the loss of their electrophysiological function after nerve injury. Activation of the necroptotic pathway early during injury-induced axonal degeneration was made evident by increased phosphorylation of the downstream effector MLKL. Our results demonstrate that axonal degeneration proceeds by necroptosis, thus defining a novel mechanistic framework in the axonal degenerative cascade for therapeutic interventions in a wide variety of conditions that lead to neuronal loss and functional impairment.SIGNIFICANCE STATEMENT We show that axonal degeneration triggered by diverse stimuli is mediated by the activation of the necroptotic programmed cell-death program by a cell-autonomous mechanism. This work represents a critical advance for the field since it identifies a defined degenerative pathway involved in axonal degeneration in both the peripheral nervous system and the CNS, a process that has been proposed as an early event in several neurodegenerative conditions and a major contributor to neuronal death. The identification of necroptosis as a key mechanism for axonal degeneration is an important step toward the development of novel therapeutic strategies for nervous-system disorders, particularly those related to chemotherapy-induced peripheral neuropathies or CNS diseases in which axonal degeneration is a common factor.

Necroptosis inhibition as a therapy for Niemann-Pick disease, type C1: Inhibition of RIP kinases and combination therapy with 2-hydroxypropyl-ß-cyclodextrin.

Niemann-Pick disease, type C1 (NPC1) is an inborn error of metabolism that results in endolysosomal accumulation of unesterified cholesterol. Clinically, NPC1 manifests as cholestatic liver disease in the newborn or as a progressive neurogenerative condition characterized by cerebellar ataxia and cognitive decline. Currently there are no FDA approved therapies for NPC1. Thus, understanding the pathological processes that contribute to neurodegeneration will be important in both developing and testing potential therapeutic interventions. Neuroinflammation and necroptosis contribute to the NPC1 pathological cascade. Receptor Interacting Protein Kinase 1 and 3 (RIPK1 and RIPK3), are protein kinases that play a central role in mediating neuronal necroptosis. Our prior work suggested that pharmacological inhibition of RIPK1 had a significant but modest beneficial effect; however, the inhibitors used in that study had suboptimal pharmacokinetic properties. In this work we evaluated both pharmacological and genetic inhibition of RIPK1 kinase activity. Lifespan in both Npc1-/- mice treated with GSK'547, a RIPK1 inhibitor with better pharmacokinetic properties, and Npc1-/-:Ripk1kd/kd double mutant mice was significantly increased. In both cases the increase in lifespan was modest, suggesting that the therapeutic potential of RIPK1 inhibition, as a monotherapy, is limited. We thus investigated the potential of combining RIPK1 inhibition with 2-hydroxypropyl-ß-cyclodextrin (HPßCD) therapy HPßCD has been shown to slow neurological disease progression in NPC1 mice, cats and patients. HPßCD appeared to have an additive positive effect on the pathology and survival of Npc1-/-:Ripk1kd/kd mice. RIPK1 and RIPK3 are both critical components of the necrosome, thus we were surprised to observe no increase survival in Npc1-/-;Ripk3-/- mice compared to Npc1-/- mice. These data suggest that although necroptosis is occurring in NPC1, the observed effects of RIPK1 inhibition may be related to its RIPK3-independent role in neuroinflammation and cytokine production.

Adhesion-induced eosinophil cytolysis requires the receptor-interacting protein kinase 3 (RIPK3)-mixed lineage kinase-like (MLKL) signaling pathway, which is counterregulated by autophagy.

BACKGROUND: Eosinophils are a subset of granulocytes that can be involved in the pathogenesis of different diseases, including allergy. Their effector functions are closely linked to their cytotoxic granule proteins. Release takes place through several different mechanisms, one of which is cytolysis, which is associated with release of intact granules, so-called clusters of free eosinophil granules. The mechanism underlying this activation-induced form of cell death in eosinophils has remained unclear. OBJECTIVE: We aimed to elucidate the molecular mechanism of eosinophil cytolysis. METHODS: Isolated blood eosinophils were incubated on glass coverslips coated with intravenous immunoglobulin and inactive complement component 3b. A morphologic characterization of the distinct stages of the proposed cascade was addressed by means of time-lapse automated fluorescence microscopy, electron microscopy, and immunohistochemistry. Experiments with pharmacologic inhibitors were performed to elucidate the sequence of events within the cascade. Tissue samples of patients with eosinophilic skin diseases or eosinophilic esophagitis were used for in vivo analyses. RESULTS: After eosinophil adhesion, we observed reactive oxygen species production, early degranulation, and granule fusion processes, leading to a distinct morphology exhibiting cytoplasmic vacuolization and, finally, cytolysis. Using a pharmacologic approach, we demonstrate the presence of a receptor-interacting protein kinase 3 (RIPK3)-mixed lineage kinase-like (MLKL) signaling pathway in eosinophils, which, after its activation, leads to the production of high levels of reactive oxygen species in a p38 mitogen-activated protein kinase and phosphatidylinositol 3'-kinase-dependent manner. All these steps are required for cytoplasmic vacuolization and subsequent cytolysis to occur. Interestingly, triggering cytolysis is associated with an induction of autophagy in eosinophils, and additional stimulation of autophagy by means of pharmacologic inhibition of the mechanistic target of rapamycin counterregulates cell death. Moreover, MLKL phosphorylation, cytoplasmic vacuolization, and cytolysis were observed in eosinophils under in vivo inflammatory conditions. CONCLUSION: We report that adhesion-induced eosinophil cytolysis takes place through RIPK3-MLKL-dependent necroptosis, which can be counterregulated by autophagy.

RIPK1 protects hepatocytes from Kupffer cells-mediated TNF-induced apoptosis in mouse models of PAMP-induced hepatitis.

BACKGROUND & AIMS: The severity of liver diseases is exacerbated by the death of hepatocytes, which can be induced by the sensing of pathogen associated molecular patterns (PAMPs) derived from the gut microbiota. The molecular mechanisms regulating these cell death pathways are poorly documented. In this study, we investigated the role of the receptor interacting protein kinase 1 (RIPK1), a protein known to regulate cell fate decisions, in the death of hepatocytes using two in vivo models of PAMP-induced hepatitis. METHODS: Hepatitis was induced in mice by independent injections of two different bacterial PAMPs: lipopolysaccharide (LPS) and unmethylated CpG oligodeoxynucleotide (CpG-DNA) motifs. The role of RIPK1 was evaluated by using mice specifically lacking RIPK1 in liver parenchymal cells (Ripk1LPC-KO). Administration of liposome-encapsulated clodronate served to investigate the role of Kupffer cells in the establishment of the disease. Etanercept, a tumor necrosis factor (TNF)-decoy receptor, was used to study the contribution of TNF-α during LPS-mediated liver injury. RESULTS: Whereas RIPK1 deficiency in liver parenchymal cells did not trigger basal hepatolysis, it greatly sensitized hepatocytes to apoptosis and liver damage following a single injection of LPS or CpG-DNA. Importantly, hepatocyte death was prevented by previous macrophage depletion or by TNF inhibition. CONCLUSIONS: Our data highlight the pivotal function of RIPK1 in maintaining liver homeostasis in conditions of macrophage-induced TNF burst in response to PAMPs sensing. LAY SUMMARY: Excessive death of hepatocytes is a characteristic of liver injury. A new programmed cell death pathway has been described involving upstream death ligands such as TNF and downstream kinases such as RIPK1. Here, we show that in the presence of LPS liver induced hepatic injury was due to secretion of TNF by liver macrophages, and that RIPK1 acts as a powerful protector of hepatocyte death. This newly identified pathway in the liver may be helpful in the management of patients to predict their risk of developing acute liver failure.

Activation of the pseudokinase MLKL unleashes the four-helix bundle domain to induce membrane localization and necroptotic cell death.

Necroptosis is considered to be complementary to the classical caspase-dependent programmed cell death pathway, apoptosis. The pseudokinase Mixed Lineage Kinase Domain-Like (MLKL) is an essential effector protein in the necroptotic cell death pathway downstream of the protein kinase Receptor Interacting Protein Kinase-3 (RIPK3). How MLKL causes cell death is unclear, however RIPK3-mediated phosphorylation of the activation loop in MLKL trips a molecular switch to induce necroptotic cell death. Here, we show that the MLKL pseudokinase domain acts as a latch to restrain the N-terminal four-helix bundle (4HB) domain and that unleashing this domain results in formation of a high-molecular-weight, membrane-localized complex and cell death. Using alanine-scanning mutagenesis, we identified two clusters of residues on opposing faces of the 4HB domain that were required for the 4HB domain to kill cells. The integrity of one cluster was essential for membrane localization, whereas MLKL mutations in the other cluster did not prevent membrane translocation but prevented killing; this demonstrates that membrane localization is necessary, but insufficient, to induce cell death. Finally, we identified a small molecule that binds the nucleotide binding site within the MLKL pseudokinase domain and retards MLKL translocation to membranes, thereby preventing necroptosis. This inhibitor provides a novel tool to investigate necroptosis and demonstrates the feasibility of using small molecules to target the nucleotide binding site of pseudokinases to modulate signal transduction.

HDAC6 inhibition attenuates renal injury by reducing IL-1ß secretion and RIP kinase mediated necroptosis in acute oxalate nephropathy.

BACKGROUND: Pathogenesis of acute kidney injury is driven by necro-inflammation, which is comprised of IL-1ß mediated inflammation and RIP-1 mediated tubular necroptosis. HDAC6 is reported to regulate both inflammation and cell death. In the present study, we explored the role of HDAC6 in the lysosomal exocytosis of IL-1ß and RIP-1 mediated necroptosis in the context of oxalate nephropathy. METHODS: Raw 264.7 macrophages and NRK52E stimulated with oxalate crystals and LPS with or without HDAC6 inhibitor for in vitro experiments. Acute oxalate nephropathy was induced in C57BL/6 mice by injecting sodium oxalate (75 mg/kg). For the drug intervention study, Tubastain A (TSA) was given an hour before injection of sodium oxalate. Mice were sacrificed 24 hrs after the oxalate injection, blood and kidney were harvested. Blood samples were analyzed for BUN and IL-1ß levels. Renal tissues were analyzed for histology, immunohistochemistry, RNA, and protein expression. RESULTS: HDAC6 and IL-1ß upregulated in crystal stimulated macrophages and acute oxalate nephropathy. Pre-treatment of macrophages with TSA reduced IL-1ß in supernatant without affecting the expression of pro-IL-1ß and mature IL-1ß in cell lysate. The effect of TSA on IL-1ß secretion was influenced by tubulin acetylation. Renal epithelial cell NRK52E stimulated with crystals showed upregulation of necroptosis pathway markers and concentration-dependent cell death. TSA inhibited RIP-1, RIP3, and MLKL expression along with p-MLKL in stimulated epithelial cells. TSA treatment of oxalate nephropathy mice showed decreased inflammation and tubular cell death by regulating IL-1ß and necroptosis and reduced renal injury. CONCLUSION: This study highlights the role of HDAC6 in regulating the tubulin-mediated secretion of IL-1ß and RIP kinase mediated necroptosis in acute oxalate nephropathy.

Molecular Insights into the Mechanism of Necroptosis: The Necrosome As a Potential Therapeutic Target.

Necroptosis, or regulated necrosis, is an important type of programmed cell death in addition to apoptosis. Necroptosis induction leads to cell membrane disruption, inflammation and vascularization. It plays important roles in various pathological processes, including neurodegeneration, inflammatory diseases, multiple cancers, and kidney injury. The molecular regulation of necroptotic pathway has been intensively studied in recent years. Necroptosis can be triggered by multiple stimuli and this pathway is regulated through activation of receptor-interacting protein kinase 1 (RIPK1), RIPK3 and pseudokinase mixed lineage kinase domain-like (MLKL). A better understanding of the mechanism of regulation of necroptosis will further aid to the development of novel drugs for necroptosis-associated human diseases. In this review, we focus on new insights in the regulatory machinery of necroptosis. We further discuss the role of necroptosis in different pathologies, its potential as a therapeutic target and the current status of clinical development of drugs interfering in the necroptotic pathway.

Complex roles of necroptosis in cancer.

Necroptosis is a tightly regulated form of necrosis that requires the activation of receptor-interacting protein (RIP) kinases RIPK1 and RIPK3, as well as the RIPK3 substrate mixed lineage kinase domain-like protein (MLKL). Because of membrane rupture, necroptotic cells release damage-associated molecular patterns (DAMPs) that evoke immune responses. Necroptosis is emerging as an important cellular response in the modulation of cancer initiation, progression, and metastasis. Necroptosis of cancer cells is considered to be an immunogenic cell death capable of activating anti-tumor immunity. Necroptosis also participates in the promotion of myeloid cell-induced adaptive immune suppression and thus contributes to oncogenesis. In addition, necroptosis of endothelial cells and tumor cells is conducive to tumor metastasis. In this review, we summarize the current knowledge of the complex role of necroptosis in cancer and discuss the potential of targeting necroptosis components for cancer therapies.

LRRK2 and the "LRRKtosome" at the Crossroads of Programmed Cell Death: Clues from RIP Kinase Relatives.

Since its cloning and identification in 2004, considerable gains have been made in the understanding of the basic functionality of leucine-rich repeat kinase 2 (LRRK2), including its kinase and GTPase activities, its protein interactors and subcellular localization, and its expression in the CNS and peripheral tissues. However, the mechanism(s) by which expression of mutant forms of LRRK2 lead to the death of dopaminergic neurons of the ventral midbrain remains largely uncharacterized. Because of its complex domain structure, LRRK2 exhibits similarities with multiple protein families including ROCO proteins, as well as the RIP kinases. Cellular models in which mutant LRRK2 is overexpressed in neuronal-like cell lines or in primary neurons have found evidence of apoptotic cell death involving components of the extrinsic as well as intrinsic death pathways. However, since the expression of LRRK2 is comparatively quite low in ventral midbrain dopaminergic neurons, the possibility exists that non-cell autonomous signaling also contributes to the loss of these neurons. In this chapter, we will discuss the different neuronal death pathways that may be activated by mutant forms of LRRK2, guided in part by the behavior of other members of the RIP kinase protein family.