Search

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 b r onchiolitis 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 HMGBI 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.

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.

RIP1 kinase regulates necroptosis and inflammation and may play an important role in contributing to a variety of human pathologies, including inflammatory and neurological diseases. Currently, RIP1 kinase inhibitors have advanced into early clinical trials for evaluation in inflammatory diseases such as psoriasis, rheumatoid arthritis, and ulcerative colitis and neurological diseases such as amyotrophic lateral sclerosis and Alzheimer's disease. In this paper, we report on the design of potent and highly selective dihydropyrazole (DHP) RIP1 kinase inhibitors starting from a high-throughput screen and the lead-optimization of this series from a lead with minimal rat oral exposure to the identification of dihydropyrazole 77 with good pharmacokinetic profiles in multiple species. Additionally, we identified a potent murine RIP1 kinase inhibitor 76 as a valuable in vivo tool molecule suitable for evaluating the role of RIP1 kinase in chronic models of disease. DHP 76 showed efficacy in mouse models of both multiple sclerosis and human retinitis pigmentosa.

Acute pancreatitis (AP) is a severe and potentially fatal disease caused predominantly by alcohol excess and gallstones, which lacks a specific therapy. The role of Receptor-Interacting Protein Kinase 1 (RIPK1), a key component of programmed necrosis (Necroptosis), is unclear in AP. We assessed the effects of RIPK1 inhibitor Necrostatin-1 (Nec-1) and RIPK1 modification (RIPK1K45A: kinase dead) in bile acid (TLCS-AP), alcoholic (FAEE-AP) and caerulein hyperstimulation (CER-AP) mouse models. Involvement of collateral Nec-1 target indoleamine 2,3-dioxygenase (IDO) was probed with the inhibitor Epacadostat (EPA). Effects of Nec-1 and RIPK1K45A were also compared on pancreatic acinar cell (PAC) fate in vitro and underlying mechanisms explored. Nec-1 markedly ameliorated histological and biochemical changes in all models. However, these were only partially reduced or unchanged in RIPK1K45A mice. Inhibition of IDO with EPA was protective in TLCS-AP. Both Nec-1 and RIPK1K45A modification inhibited TLCS- and FAEE-induced PAC necrosis in vitro. Nec-1 did not affect TLCS-induced Ca2+ entry in PACs, however, it inhibited an associated ROS elevation. The results demonstrate protective actions of Nec-1 in multiple models. However, RIPK1-dependent necroptosis only partially contributed to beneficial effects, and actions on targets such as IDO are likely to be important.

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.

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, RIPK1 K45A/K45A and RIPK1 D138N/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.

RIPK1 regulates cytokine signaling and cell death during infection and inflammation. Peterson et al. show that RIPK1 kinase activity triggers apoptosis in response to bacterial pathogen blockade of innate immune signaling and that this pathway of effector-triggered immunity is critical for a successful antibacterial response., Many pathogens deliver virulence factors or effectors into host cells in order to evade host defenses and establish infection. Although such effector proteins disrupt critical cellular signaling pathways, they also trigger specific antipathogen responses, a process termed “effector-triggered immunity.” The Gram-negative bacterial pathogen Yersinia inactivates critical proteins of the NF-κB and MAPK signaling cascade, thereby blocking inflammatory cytokine production but also inducing apoptosis. Yersinia-induced apoptosis requires the kinase activity of receptor-interacting protein kinase 1 (RIPK1), a key regulator of cell death, NF-κB, and MAPK signaling. Through the targeted disruption of RIPK1 kinase activity, which selectively disrupts RIPK1-dependent cell death, we now reveal that Yersinia-induced apoptosis is critical for host survival, containment of bacteria in granulomas, and control of bacterial burdens in vivo. We demonstrate that this apoptotic response provides a cell-extrinsic signal that promotes optimal innate immune cytokine production and antibacterial defense, demonstrating a novel role for RIPK1 kinase–induced apoptosis in mediating effector-triggered immunity to circumvent pathogen inhibition of immune signaling.

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-β. 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-β production in macrophages. Using genetic and pharmacologic tools, we show that catalytic activity of RIPK1 directs IFN-β synthesis induced by LPS in mice. Additionally, we report that RIPK1 kinase–dependent IFN-β 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-β production downstream of the LPS receptor TLR4. Intriguingly, we also show that RIPK1 and RIPK3 kinase–dependent synthesis of IFN-β 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-β 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.

RIP1 regulates necroptosis and inflammation and may play an important role in contributing to a variety of human pathologies, including immune-mediated inflammatory diseases. Small-molecule inhibitors of RIP1 kinase that are suitable for advancement into the clinic have yet to be described. Herein, we report our lead optimization of a benzoxazepinone hit from a DNA-encoded library and the discovery and profile of clinical candidate GSK2982772 (compound 5), currently in phase 2a clinical studies for psoriasis, rheumatoid arthritis, and ulcerative colitis. Compound 5 potently binds to RIP1 with exquisite kinase specificity and has excellent activity in blocking many TNF-dependent cellular responses. Highlighting its potential as a novel anti-inflammatory agent, the inhibitor was also able to reduce spontaneous production of cytokines from human ulcerative colitis explants. The highly favorable physicochemical and ADMET properties of 5, combined with high potency, led to a predicted low oral dose in humans.

Obesity is a major public health burden worldwide and is characterized by chronic low-grade inflammation driven by the cooperation of the innate immune system and dysregulated metabolism in adipose tissue and other metabolic organs. Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) is a central regulator of inflammatory cell function that coordinates inflammation, apoptosis and necroptosis in response to inflammatory stimuli. Here we show that genetic polymorphisms near the human RIPK1 locus associate with increased RIPK1 gene expression and obesity. We show that one of these single nucleotide polymorphisms is within a binding site for E4BP4 and increases RIPK1 promoter activity and RIPK1 gene expression in adipose tissue. Therapeutic silencing of RIPK1 in vivo in a mouse model of diet-induced obesity dramatically reduces fat mass, total body weight and improves insulin sensitivity, while simultaneously reducing macrophage and promoting invariant natural killer T cell accumulation in adipose tissue. These findings demonstrate that RIPK1 is genetically associated with obesity, and reducing RIPK1 expression is a potential therapeutic approach to target obesity and related diseases.

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.

[Image: see text] Herein we report the discovery of pyrazolocarboxamides as novel, potent, and kinase selective inhibitors of receptor interacting protein 2 kinase (RIP2). Fragment based screening and design principles led to the identification of the inhibitor series, and X-ray crystallography was used to inform key structural changes. Through key substitutions about the N1 and C5 N positions on the pyrazole ring significant kinase selectivity and potency were achieved. Bridged bicyclic pyrazolocarboxamide 11 represents a selective and potent inhibitor of RIP2 and will allow for a more detailed investigation of RIP2 inhibition as a therapeutic target for autoinflammatory disorders.

RIP1 regulates cell death and inflammation and is believed to play an important role in contributing to a variety of human pathologies, including immune-mediated inflammatory diseases and cancer. While small-molecule inhibitors of RIP1 kinase have been advanced to the clinic for inflammatory diseases and CNS indications, RIP1 inhibitors for oncology indications have yet to be described. Herein we report on the discovery and profile of GSK3145095 (compound 6). Compound 6 potently binds to RIP1 with exquisite kinase specificity and has excellent activity in blocking RIP1 kinase-dependent cellular responses. Highlighting its potential as a novel cancer therapy, the inhibitor was also able to promote a tumor suppressive T cell phenotype in pancreatic adenocarcinoma organ cultures. Compound 6 is currently in phase 1 clinical studies for pancreatic adenocarcinoma and other selected solid tumors.

SummaryMicrovascular endothelial cells maintain a tight barrier to prevent passage of plasma and circulating immune cells into the extravascular tissue compartment, yet endothelial cells respond rapidly to vasoactive substances, including thrombin, allowing transient paracellular permeability. This response is a cornerstone of acute inflammation, but the mechanisms responsible are still incompletely understood. Here, we demonstrate that thrombin triggers MALT1 to proteolytically cleave cylindromatosis (CYLD). Fragmentation of CYLD results in microtubule disruption and a cascade of events leading to endothelial cell retraction and an acute permeability response. This finding reveals an unexpected role for the MALT1 protease, which previously has been viewed mostly as a driver of pro-inflammatory NF-κB signaling in lymphocytes. Thus, MALT1 not only promotes immune cell activation but also acutely regulates endothelial cell biology, actions that together facilitate tissue inflammation. Pharmacologic inhibition of MALT1 may therefore have synergistic impact by targeting multiple disparate steps in the overall inflammatory response.

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.

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.

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-1β expression in response to Leishmania. However, PGAM5, but not RIPK1 kinase activity, was directly responsible for Leishmania-induced IL-1β 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.

Summary 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., Graphical Abstract, Highlights • A RIPK1/FADD/Caspase-8/cFLIP complex (ripoptosome) forms during mitosis • The mitotic kinase PLK1 binds to RIPK1 and is recruited into mitotic ripoptosomes • PLK1 is cleaved by Caspase-8 in a RIPK1-dependent manner • Deletion and inhibition of RIPK1 or Caspase-8 lead to chromosomal instability, Liccardi et al. show that ripoptosome complexes assemble during physiological mitosis and regulate PLK1 activity via RIPK1-dependent recruitment and Caspase-8-mediated cleavage. These results reveal a direct role for RIPK1 and Caspase-8 in controlling chromosome segregation during mitosis.

[Image: see text] RIP2 kinase was recently identified as a therapeutic target for a variety of autoimmune diseases. We have reported previously a selective 4-aminoquinoline-based RIP2 inhibitor GSK583 and demonstrated its effectiveness in blocking downstream NOD2 signaling in cellular models, rodent in vivo models, and human ex vivo disease models. While this tool compound was valuable in validating the biological pathway, it suffered from activity at the hERG ion channel and a poor PK/PD profile thereby limiting progression of this analog. Herein, we detail our efforts to improve both this off-target liability as well as the PK/PD profile of this series of inhibitors through modulation of lipophilicity and strengthening hinge binding ability. These efforts have led to inhibitor 7, which possesses high binding affinity for the ATP pocket of RIP2 (IC(50) = 1 nM) and inhibition of downstream cytokine production in human whole blood (IC(50) = 10 nM) with reduced hERG activity (14 μM).

Summary 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-β-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.

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.

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 Yersinia 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.

The linear ubiquitin chain assembly complex (LUBAC), composed of HOIP, HOIL-1 and SHARPIN, is required for optimal TNF-mediated gene activation and to prevent cell death induced by TNF. Here, we demonstrate that keratinocyte-specific deletion of HOIP or HOIL-1 (E-KO) results in severe dermatitis causing postnatal lethality. We provide genetic and pharmacological evidence that the postnatal lethal dermatitis in HoipE-KO and Hoil-1E-KO mice is caused by TNFR1-induced, caspase-8-mediated apoptosis that occurs independently of the kinase activity of RIPK1. In the absence of TNFR1, however, dermatitis develops in adulthood, triggered by RIPK1-kinase-activity-dependent apoptosis and necroptosis. Strikingly, TRAIL or CD95L can redundantly induce this disease-causing cell death, as combined loss of their respective receptors is required to prevent TNFR1-independent dermatitis. These findings may have implications for the treatment of patients with mutations that perturb linear ubiquitination and potentially also for patients with inflammation-associated disorders that are refractory to inhibition of TNF alone.

The cytokine IL-1β is intimately linked to many pathological inflammatory conditions. Mature IL-1β 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-1β 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-1β secretion in response to NLRP3 and AIM2 inflammasome agonists. Moreover, vesicular stomatosis virus–induced IL-1β 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. Fil: Moriwaki, Kenta. University of Massachussets; Estados Unidos Fil: Farias Luz, Nivea. Universidade Federal da Bahia; Brasil. University of Massachussets; Estados Unidos Fil: Balaji, Sakthi. University of Massachussets; Estados Unidos Fil: de Rosa, Maria Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina Fil: O'Donnell, Carey L.. University of Massachussets; Estados Unidos Fil: Gough, Peter J.. Glaxo Smith Kline; Estados Unidos Fil: Bertin, John. Glaxo Smith Kline; Estados Unidos Fil: Welsh, Raymond M.. University of Massachussets; Estados Unidos Fil: Chan, Francis Ka Ming. University of Massachussets; Estados Unidos

SummaryRIPK1 and RIPK3, two closely related RIPK family members, have emerged as important regulators of pathologic cell death and inflammation. In the current work, we report that the Bcr-Abl inhibitor and anti-leukemia agent ponatinib is also a first-in-class dual inhibitor of RIPK1 and RIPK3. Ponatinib potently inhibited multiple paradigms of RIPK1- and RIPK3-dependent cell death and inflammatory tumor necrosis factor alpha (TNF-α) gene transcription. We further describe design strategies that utilize the ponatinib scaffold to develop two classes of inhibitors (CS and PN series), each with greatly improved selectivity for RIPK1. In particular, we detail the development of PN10, a highly potent and selective “hybrid” RIPK1 inhibitor, capturing the best properties of two different allosteric RIPK1 inhibitors, ponatinib and necrostatin-1. Finally, we show that RIPK1 inhibitors from both classes are powerful blockers of TNF-induced injury in vivo. Altogether, these findings outline promising candidate molecules and design approaches for targeting RIPK1- and RIPK3-driven inflammatory pathologies.

Caspase 8, the initiator caspase for death receptor–induced apoptosis, functions as a negative regulator of receptor interacting protein kinase 3 (RIPK3), an essential factor for TNF-, TLR3-, and TLR4-induced necroptosis. In certain situations, caspase 8 can also participate in pro–IL-1β processing. However, the biochemical complex that mediates caspase 8–mediated processing is not defined. In this study, we show that RIPK3 is crucial for caspase 1– and caspase 8–mediated pro–IL-1β and pro–IL-18 processing in bone marrow–derived dendritic cells (BMDCs) in response to LPS stimulation. Caspase 8–mediated pro–IL-1β processing requires intact RIPK1, RIPK3, TRIF, and FADD. In response to LPS, a complex that contains RIPK1, RIPK3, FADD, and caspase 8 is formed. Surprisingly, RIPK3-specific kinase inhibitors strongly enhanced caspase 8 activation and pro–IL-1β processing in LPS-stimulated BMDCs. However, studies in BMDCs expressing the kinase-inactive RIPK3-K51A mutant or RIPK1-K45A mutant showed that the kinase activity of neither RIPK1 nor RIPK3 is required for LPS-induced caspase 8 activation and IL-1β secretion. Hence, RIPK3 is an unexpected positive regulator of caspase 8 activity that promotes IL-1β maturation in BMDCs.

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.

GSK2982772 is a highly selective inhibitor of receptor‐interacting protein kinase 1 (RIPK1) being developed to treat chronic inflammatory diseases. This first‐in‐human study evaluated safety, tolerability, pharmacokinetics (PK), and exploratory pharmacodynamics (PD) of GSK2982772 administered orally to healthy male volunteers. This was a Phase I, randomized, placebo‐controlled, double‐blind study. In Part A, subjects received single ascending doses of GSK2982772 (0.1‐120 mg) or placebo in a crossover design during each of 4 treatment periods. In Part B, subjects received repeat doses of GSK2982772 (20 mg once daily [QD] to up to 120 mg twice daily [BID]) or placebo for 14 days. Part C was an open‐label relative bioavailability study comparing 20‐mg tablets vs capsules. Safety, tolerability, pharmacokinetics (PK), RIPK1 target engagement (TE), and pharmacodynamics (PD) were assessed. The most common adverse events (AEs) were contact dermatitis and headache. Most AEs were mild in intensity, and there were no deaths or serious AEs. The PK of GSK2982772 was approximately linear over the dose range studied (up to 120 mg BID). There was no evidence of drug accumulation upon repeat dosing. Greater than 90% RIPK1 TE was achieved over a 24‐hour period for the 60‐mg and 120‐mg BID dosing regimens. Single and repeat doses of GSK2982772 were safe and well tolerated. PK profiles showed dose linearity. The high levels of RIPK1 TE support progression into Phase II clinical trials for further clinical development.

Burn patients suffer from immunological dysfunction for which there are currently no successful interventions. Similar to previous observations, we find that burn shock patients (≥15% Total Burn Surface Area (TBSA) injury) have elevated levels of the innate immune cytokines Interleukin-6 (IL-6) and Monocyte Chemoattractant Protein-1 (MCP-1)/CC-motif Chemokine Ligand 2(CCL2) early after hospital admission (0–48 Hours Post-hospital Admission (HPA). Functional immune assays with patient Peripheral Blood Mononuclear Cells (PBMCs) revealed that burn shock patients (≥15% TBSA) produced elevated levels of MCP-1/CCL2 after innate immune stimulation ex vivo relative to mild burn patients. Interestingly, treatment of patient PBMCs with the Nuclear Factor-Erythroid-2-Related Factor 2 (NRF2) agonist, CDDO-Me(bardoxolone methyl), reduced MCP-1 production but not IL-6 or Interleukin-10 (IL-10) secretion. In enriched monocytes from healthy donors, CDDO-Me(bardoxolone methyl) also reduced LPS-induced MCP1/CCL2 production but did not alter IL-6 or IL-10 secretion. Similar immunomodulatory effects were observed with Compound 7, which activates the NRF2 pathway through a different and non-covalent Mechanism Of Action (MOA). Hence, our findings with CDDO-Me(bardoxolone methyl) and Compound 7 are likely to reflect a generalizable aspect of NRF2 activation. These observed effects were not specific to LPS-induced immune responses, as NRF2 activation also reduced MCP-1/CCL2 production after stimulation with IL-6. Pharmacological NRF2 activation reduced Mcp-1/Ccl2 transcript accumulation without inhibiting either Il-6 or Il-10 transcript levels. Hence, we describe a novel aspect of NRF2 activation that may contribute to the beneficial effects of NRF2 agonists during disease. Our work also demonstrates that the NRF2 pathway is retained and can be modulated to regulate important immunomodulatory functions in burn patient immune cells.

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.

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 (Ripk1K45A) 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 Ripk1K45A 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 Ripk1K45A 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.

SummaryAlthough 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.

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.

Toll-like receptor (TLR) signaling is triggered by pathogen-associated molecular patterns that mediate well established cytokine-driven pathways, activating NF-κB together with IRF3/IRF7. In addition, TLR3 drives caspase 8-regulated programmed cell death pathways reminiscent of TNF family death receptor signaling. We find that inhibition or elimination of caspase 8 during stimulation of TLR2, TLR3, TLR4, TLR5, or TLR9 results in receptor interacting protein (RIP) 3 kinase-dependent programmed necrosis that occurs through either TIR domain-containing adapter-inducing interferon-β (TRIF) or MyD88 signal transduction. TLR3 or TLR4 directly activates programmed necrosis through a RIP homotypic interaction motif-dependent association of TRIF with RIP3 kinase (also called RIPK3). In fibroblasts, this pathway proceeds independent of RIP1 or its kinase activity, but it remains dependent on mixed lineage kinase domain-like protein (MLKL) downstream of RIP3 kinase. Here, we describe two small molecule RIP3 kinase inhibitors and employ them to demonstrate the common requirement for RIP3 kinase in programmed necrosis induced by RIP1-RIP3, DAI-RIP3, and TRIF-RIP3 complexes. Cell fate decisions following TLR signaling parallel death receptor signaling and rely on caspase 8 to suppress RIP3-dependent programmed necrosis whether initiated directly by a TRIF-RIP3-MLKL pathway or indirectly via TNF activation and the RIP1-RIP3-MLKL necroptosis pathway.

[This corrects the article DOI: 10.1371/journal.pone.0184164.].

Macrophage class A scavenger receptor types I and II (SR-AI and II) mediate the uptake of oxidized LDL in atherosclerotic lesions. The recently described type III receptor (SR-AIII), which lacks amino acids encoded by exon 10 of the SR-A gene, is unable to mediate the uptake of ligands and acts as a dominant negative regulator in the trimeric SR-A molecule. To find out whether SR-AIII might play a role in the regulation of SR-A activity in the arterial wall, we studied its expression in normal and atherosclerotic aortic intima-medias of Watanabe heritable hyperlipidemic (WHHL) and cholesterol-fed New Zealand white (NZW) rabbits. SR-A mRNA was amplified by reverse transcription–polymerase chain reaction (RT–PCR) with a SR-AIII-specific primer pair and with a primer pair suitable for both SR-AI and III. Very low SR-AI expression and no SR-AIII expression was found in the lesion-free aortic intima-medias of WHHL rabbits and control NZW rabbits. WHHL rabbit fatty streaks contained abundant SR-AI expression and low-level SR-AIII expression. In contrast, the numerous fatty streaks and fatty plaques appearing in the aortas of cholesterol-fed (14 weeks) NZW rabbits, and the fatty plaques of WHHL rabbits contained clearly detectable SR-AIII expression in addition to the abundant SR-AI expression. In addition, SR-AIII mRNA was detected in NZW and WHHL rabbit livers. The results suggest that in advanced atherosclerotic lesions, cells may protect themselves from the excessive uptake of oxidized lipoproteins by generating SR-A molecules which cannot bind modified LDL.

The class A macrophage scavenger receptors (SR- A) are macrophage-specific trimeric integral membrane gly- coproteins that have been implicated in many macrophage- associated physiological and pathological processes including atherosclerosis, Alzheimer's disease, and host defense. There are two forms of the receptor that have been previously cloned, and both are generated by alternative splicing of a single gene. Here we report the cloning of a third, alterna- tively spliced isoform of the human SR-A gene (type III hSR- A). The novel isoform is expressed in the human monocytic leukemia cell line THP-1 and also in primary human mono- cyte derived macrophages. When expressed in CHO-K1 cells, type III hSR-A does not internalize AcLDL despite having the domain shown to mediate this function in type I and II hSR-A. We show that type III protein has altered intracellular process- ing and is trapped within the endoplasmic reticulum, making it unable to perform endocytosis. Type III protein acts as a dominant negative isoform by reducing modified LDL uptake in CHO cells stably expressing either type I or type II SR-A. The demonstration that a naturally occurring splice variant of SR-A mRNA can act as a dominant negative isoform suggests a novel mechanism for regulation of scavenger receptor activity in macrophages.— Gough, P. J., D. R. Greaves, and S. Gor- don. A naturally occurring isoform of the human macro- phage scavenger receptor (SR-A) gene generated by alterna- tive splicing blocks modified LDL uptake. J. Lipid Res. 1998. 39: 531-543.

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.

Receptor interacting protein kinase 3 (RIPK3) induces necroptosis, a type of regulated necrosis, through its kinase domain and RIP homotypic interaction motif (RHIM). In addition, RIPK3 has been shown to regulate NLRP3 inflammasome and NF-κB activation. However, the relative contribution of these signaling pathways to RIPK3-dependent inflammation in distinct immune effectors is unknown. To interrogate these questions, we generated RIPK3-GFP reporter mice. We found that colonic CD11c+CD11b+CD14+ mononuclear phagocytes (MNPs) expressed the highest level of RIPK3 in the lamina propria. Consequently, deletion of the RIPK3 RHIM in CD11c+ cells alone was sufficient to impair DSS-induced IL-23 and IL-1β expression, leading to severe intestinal inflammation. In contrast, mice expressing kinase inactive RIPK3 were not hypersensitive to DSS. Thus, a key physiological function of RIPK3 is to promote reparative cytokine expression through intestinal CD11c+ MNPs in a kinase- and necroptosis-independent manner.

The recent discovery of the role of receptor interacting protein 1 (RIP1) kinase in tumor necrosis factor (TNF)-mediated inflammation has led to its emergence as a highly promising target for the treatment of multiple inflammatory diseases. We screened RIP1 against GSK's DNA-encoded small-molecule libraries and identified a novel highly potent benzoxazepinone inhibitor series. We demonstrate that this template possesses complete monokinase selectivity for RIP1 plus unique species selectivity for primate versus nonprimate RIP1. We elucidate the conformation of RIP1 bound to this benzoxazepinone inhibitor driving its high kinase selectivity and design specific mutations in murine RIP1 to restore potency to levels similar to primate RIP1. This series differentiates itself from known RIP1 inhibitors in combining high potency and kinase selectivity with good pharmacokinetic profiles in rodents. The favorable developability profile of this benzoxazepinone template, as exemplified by compound 14 (GSK'481), makes it an excellent starting point for further optimization into a RIP1 clinical candidate.

Optimisation of a series of biaryl sulphonamides resulted in the identification of compound 14 [corrected] which demonstrated dose-dependent and strain-specific inhibition of monocyte recruitment in a thioglycollate-induced peritonitis model of inflammation. [Formula: see text]. [corrected].