Necroptosis is a cell death pathway regulated by the receptor interacting protein kinase 3 (RIPK3) and the mixed lineage kinase domain-like (MLKL) pseudokinase. How MLKL executes plasma membrane ...rupture upon phosphorylation by RIPK3 remains controversial. Here, we characterize the hierarchical transduction of structural changes in MLKL that culminate in necroptosis. The MLKL brace, proximal to the N-terminal helix bundle (NB), is involved in oligomerization to facilitate plasma membrane targeting through the low-affinity binding of NB to phosphorylated inositol polar head groups of phosphatidylinositol phosphate (PIP) phospholipids. At the membrane, the NB undergoes a “rolling over” mechanism to expose additional higher-affinity PIP-binding sites responsible for robust association to the membrane and displacement of the brace from the NB. PI(4,5)P2 is the preferred PIP-binding partner. We investigate the specific association of MLKL with PIPs and subsequent structural changes during necroptosis.
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•MLKL activation is governed by a hierarchical transduction of structural changes•The brace undergoes oligomerization prior to plasma membrane recruitment•Low-affinity NB binding to PIP mediates plasma membrane targeting•High-affinity PIP binding to NB displaces the brace for robust membrane association
Quarato et al. show the consequences of MLKL activation for plasma membrane rupture in necroptosis. Upon activation, MLKL undergoes oligomerization mediated by the brace domain, being recruited to the plasma membrane through avidity of NB for PIP. Robust membrane association follows the initial recruitment through PIP-induced displacement of the brace.
The kinases RIPK1 and RIPK3 and the pseudo-kinase MLKL have been identified as key regulators of the necroptotic cell death pathway, although a role for MLKL within the whole animal has not yet been ...established. Here, we have shown that MLKL deficiency rescued the embryonic lethality caused by loss of Caspase-8 or FADD. Casp8−/−Mlkl−/− and Fadd−/−Mlkl−/− mice were viable and fertile but rapidly developed severe lymphadenopathy, systemic autoimmune disease, and thrombocytopenia. These morbidities occurred more rapidly and with increased severity in Casp8−/−Mlkl−/− and Fadd−/−Mlkl−/− mice compared to Casp8−/−Ripk3−/− or Fadd−/−Ripk3−/− mice, respectively. These results demonstrate that MLKL is an essential effector of aberrant necroptosis in embryos caused by loss of Caspase-8 or FADD. Furthermore, they suggest that RIPK3 and/or MLKL may exert functions independently of necroptosis. It appears that non-necroptotic functions of RIPK3 contribute to the lymphadenopathy, autoimmunity, and excess cytokine production that occur when FADD or Caspase-8-mediated apoptosis is abrogated.
•MLKL is an essential effector of necroptosis in vivo•RIPK3 exacerbates the development and progression of ALPS-like disease•RIPK3 and maybe MLKL exert additional functions beyond inducing cell death
Necroptosis is a form of regulated cell death implicated in several pathologies. MLKL was shown to be critical for necroptosis in vitro. Alvarez-Diaz et al. demonstrate that MLKL, like RIPK3, is essential for necroptosis in vivo and reveal that RIPK3 also has a role beyond cell death in promoting lymphadenopathy and autoimmune disease.
Receptor-interacting protein kinase (RIPK)-1 is involved in RIPK3-dependent and -independent signaling pathways leading to cell death and/or inflammation. Genetic ablation of ripk1 causes postnatal ...lethality, which was not prevented by deletion of ripk3, caspase-8, or fadd. However, animals that lack RIPK1, RIPK3, and either caspase-8 or FADD survived weaning and matured normally. RIPK1 functions in vitro to limit caspase-8-dependent, TNFR-induced apoptosis, and animals lacking RIPK1, RIPK3, and TNFR1 survive to adulthood. The role of RIPK3 in promoting lethality in ripk1−/− mice suggests that RIPK3 activation is inhibited by RIPK1 postbirth. Whereas TNFR-induced RIPK3-dependent necroptosis requires RIPK1, cells lacking RIPK1 were sensitized to necroptosis triggered by poly I:C or interferons. Disruption of TLR (TRIF) or type I interferon (IFNAR) signaling delayed lethality in ripk1−/−tnfr1−/− mice. These results clarify the complex roles for RIPK1 in postnatal life and provide insights into the regulation of FADD-caspase-8 and RIPK3-MLKL signaling by RIPK1.
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•Lethality in ripk1−/− mice is rescued by ablation of ripk3 with either casp8 or fadd•TNF induces apoptosis, and TRIF or IFN induce necroptosis in ripk1−/− cells•Ripk1−/−ripk3−/−tnfr1−/− mice reach adulthood•Lethality in ripk1−/− mice is delayed by deleting tnfr1 with either trif or ifnar
Genetic ablation of RIPK-1 causes postnatal lethality, which is prevented by deletion of RIPK3 together with caspase-8, FADD, or TNFR1. The role of RIPK3 in promoting lethality in ripk1−/− mice suggests that RIPK3 activation is inhibited by RIPK1 postbirth, clarifying the complex roles for RIPK1 in postnatal life.
Elevated glucose metabolism in immune cells represents a hallmark feature of many inflammatory diseases, such as sepsis. However, the role of individual glucose metabolic pathways during immune cell ...activation and inflammation remains incompletely understood. Here, we demonstrate a previously unrecognized anti-inflammatory function of the O-linked β-N-acetylglucosamine (O-GlcNAc) signaling associated with the hexosamine biosynthesis pathway (HBP). Despite elevated activities of glycolysis and the pentose phosphate pathway, activation of macrophages with lipopolysaccharide (LPS) resulted in attenuated HBP activity and protein O-GlcNAcylation. Deletion of O-GlcNAc transferase (OGT), a key enzyme for protein O-GlcNAcylation, led to enhanced innate immune activation and exacerbated septic inflammation. Mechanistically, OGT-mediated O-GlcNAcylation of the serine-threonine kinase RIPK3 on threonine 467 (T467) prevented RIPK3-RIPK1 hetero- and RIPK3-RIPK3 homo-interaction and inhibited downstream innate immunity and necroptosis signaling. Thus, our study identifies an immuno-metabolic crosstalk essential for fine-tuning innate immune cell activation and highlights the importance of glucose metabolism in septic inflammation.
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•LPS treatment causes a decrease in HBP activity and protein O-GlcNAcylation•OGT deficiency increases activation of innate immune response and necroptosis•O-GlcNAcylation of RIPK3 on T467 inhibits RIPK3-RIPK1 and RIPK3-RIPK3 interaction
The role of individual glucose metabolic pathways in innate immunity remains largely unknown. Li et al. demonstrate that attenuated O-linked β-N-acetylglucosamine (O-GlcNAc) signaling enhances TLR-induced innate immune response and necroptosis. Mechanistically, O-GlcNAcylation of the kinase RIPK3 blocks RHIM-domain-mediated protein interaction and downstream signaling activation.
Caspase-8 or cellular FLICE-like inhibitor protein (cFLIP) deficiency leads to embryonic lethality in mice due to defects in endothelial tissues. Caspase-8−/− and receptor-interacting protein ...kinase-3 (RIPK3)−/−, but not cFLIP−/− and RIPK3−/−, double-knockout animals develop normally, indicating that caspase-8 antagonizes the lethal effects of RIPK3 during development. Here, we show that the acute deletion of caspase-8 in the gut of adult mice induces enterocyte death, disruption of tissue homeostasis, and inflammation, resulting in sepsis and mortality. Likewise, acute deletion of caspase-8 in a focal region of the skin induces local keratinocyte death, tissue disruption, and inflammation. Strikingly, RIPK3 ablation rescues both phenotypes. However, acute loss of cFLIP in the skin produces a similar phenotype that is not rescued by RIPK3 ablation. TNF neutralization protects from either acute loss of caspase-8 or cFLIP. These results demonstrate that caspase-8-mediated suppression of RIPK3-induced death is required not only during development but also for adult homeostasis. Furthermore, RIPK3-dependent inflammation is dispensable for the skin phenotype.
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•Acute deletion of caspase-8 or cFLIP in the gut or skin disrupts tissue homeostasis•Ablation of RIPK3 rescues the damaging effects of acute caspase-8, but not cFLIP, loss•RIPK3-mediated inflammation is dispensable for the skin damage by acute cFLIP loss•Neutralization of TNF rescues from the effects of acute loss of caspase-8 or cFLIP
In this study, Green and colleagues show that acute loss of caspase-8 in the gut or the skin can induce a TNF-dependent, RIPK3-mediated loss of tissue homeostasis and inflammation, demonstrating that RIPK3 function is tightly regulated in adult tissues. Strikingly, the authors show that loss of cFLIP in RIPK3-deficient background induces a similar phenotype, suggesting that loss of tissue barrier function, rather than the type of cell death (necroptosis or apoptosis), defines the onset of disease.
Perturbation of the apoptosis and necroptosis pathways critically influences embryogenesis. Receptor-associated protein kinase-1 (RIPK1) interacts with Fas-associated via death domain ...(FADD)-caspase-8-cellular Flice-like inhibitory protein long (cFLIPL) to regulate both extrinsic apoptosis and necroptosis. Here, we describe Ripk1-mutant animals (Ripk1R588E RE) in which the interaction between FADD and RIPK1 is disrupted, leading to embryonic lethality. This lethality is not prevented by further removal of the kinase activity of Ripk1 (Ripk1R588E K45A REKA). Both Ripk1RE and Ripk1REKA animals survive to adulthood upon ablation of Ripk3. While embryonic lethality of Ripk1RE mice is prevented by ablation of the necroptosis effector mixed lineage kinase-like (MLKL), animals succumb to inflammation after birth. In contrast, Mlkl ablation does not prevent the death of Ripk1REKA embryos, but animals reach adulthood when both MLKL and caspase-8 are removed. Ablation of the nucleic acid sensor Zbp1 largely prevents lethality in both Ripk1RE and Ripk1REKA embryos. Thus, the RIPK1-FADD interaction prevents Z-DNA binding protein-1 (ZBP1)-induced, RIPK3-caspase-8-mediated embryonic lethality, affected by the kinase activity of RIPK1.
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•A mutation in RIPK1 (R588E) disrupts the interaction between RIPK1 and FADD•Disrupting the interaction between RIPK1 and FADD leads to embryonic lethality•The kinase activity of RIPK1 R588E prevents RIPK3-mediated embryonic lethality•Zbp1 ablation partially prevents RIPK1 R588E-mediated embryonic lethality
Rodriguez et al. show that breaking the interaction between the cell-death-regulating proteins RIPK1 and FADD results in developmental death and inflammation that are initiated by the cellular receptor ZBP1 and regulated by the kinase activity of RIPK1, shedding light on the intricate functions of this interaction.
Beneficial effects of exercise training in patients with chronic obstructive pulmonary disease (COPD) are acknowledged. However, high-intensity exercise may enhance muscle oxidative stress in severe ...COPD patients. We hypothesized that high-intensity exercise training of long duration does not deteriorate muscle redox status. In the vastus lateralis and blood of 18 severe COPD patients and 12 controls, before and after an 8-week training program, protein oxidation and nitration, antioxidant systems, and inflammatory cytokines were examined. At baseline, COPD patients showed greater muscle oxidative stress and superoxide dismutase activity and circulating inflammatory cytokines than controls. Among COPD patients, muscle and blood protein carbonylation levels were correlated. Both groups showed training-induced increase in VO2 peak and decreased blood lactate levels. After training, among the COPD patients, blood protein nitration levels were significantly reduced and muscle protein oxidation and nitration levels did not cause impairment. Muscle and blood levels of inflammatory cytokines were not modified by training in either patients or controls. We conclude that in severe COPD patients, high-intensity exercise training of long duration improves exercise capacity while preventing the enhancement of systemic and muscle oxidative stress. In addition, in these patients, resting protein oxidation levels correlate between skeletal muscle and blood compartments.
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► High-intensity exercise may enhance muscle oxidative stress in severe COPD patients ► We tested whether exercise training of long duration deteriorates muscle redox status ► In COPD patients, muscle and blood protein oxidation levels were correlated ► Training improved physiological parameters and blood protein nitration in patients ► High-intensity exercise training did not impair muscle oxidative stress in patients
Reduction of the particle size of corn increases energy digestibility and concentrations of digestible and metabolizable energy. Pelleting may also reduce particle size of grain, but it is not known ...if there are interactions between particle size reduction and pelleting. The objective of this experiment was to test the hypothesis that particle size reduction and pelleting, separately or in combination, increase N balance, apparent total tract digestibility (ATTD) of fiber and fat, and net energy (NE) in corn-soybean meal diets fed to group-housed pigs.
Six corn-soybean meal-based diets were used in a 3 × 2 factorial design with 3 particle sizes of corn (i.e., 700, 500, or 300 μm) and 2 diet forms (i.e., meal or pelleted). Pigs were allowed ad libitum access to feed and water. Twenty-four castrated male pigs (initial weight: 29.52 kg; standard diviation: 1.40) were allotted to the 6 diets using a 6 × 6 Latin square design with 6 calorimeter chambers (i.e., 4 pigs/chamber) and 6 periods. Oxygen consumption and CO
and CH
productions were measured during fed and fasting states and fecal and urine samples were collected.
Regardless of particle size of corn, the ATTD of gross energy (GE), N, and acid-hydrolyzed ether extract (AEE), and the concentration of NE were greater (P < 0.05) in pelleted diets than in meal diets. Regardless of diet form, the ATTD of GE, N, and AEE, and the concentration of NE were increased (linear; P < 0.05) by reducing the particle size of corn, but the increase was greater in meal diets than in pelleted diets (interaction; P < 0.05).
Both pelleting and reduction of corn particle size increased nutrient digestibility and NE, but increases were greater in meal diets than in pelleted diets.
Influenza A virus (IAV) is a lytic RNA virus that triggers receptor-interacting serine/threonine-protein kinase 3 (RIPK3)-mediated pathways of apoptosis and mixed lineage kinase domain-like ...pseudokinase (MLKL)-dependent necroptosis in infected cells. ZBP1 initiates RIPK3-driven cell death by sensing IAV RNA and activating RIPK3. Here, we show that replicating IAV generates Z-RNAs, which activate ZBP1 in the nucleus of infected cells. ZBP1 then initiates RIPK3-mediated MLKL activation in the nucleus, resulting in nuclear envelope disruption, leakage of DNA into the cytosol, and eventual necroptosis. Cell death induced by nuclear MLKL was a potent activator of neutrophils, a cell type known to drive inflammatory pathology in virulent IAV disease. Consequently, MLKL-deficient mice manifest reduced nuclear disruption of lung epithelia, decreased neutrophil recruitment into infected lungs, and increased survival following a lethal dose of IAV. These results implicate Z-RNA as a new pathogen-associated molecular pattern and describe a ZBP1-initiated nucleus-to-plasma membrane “inside-out” death pathway with potentially pathogenic consequences in severe cases of influenza.
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•Replicating influenza A virus (IAV) produces Z-RNAs•IAV Z-RNAs are sensed by host ZBP1 in the nucleus•ZBP1 activates MLKL in the nucleus, triggering nuclear envelope rupture•MLKL-induced nuclear rupture and necroptosis drive IAV disease severity
Z-RNAs produced by influenza viruses in the nucleus of infected cells are detected by host ZBP1, which activates RIPK3 and MLKL to lead to nuclear envelope rupture and necroptosis, ultimately resulting in neutrophil recruitment and activation in infected tissue.
Autonomic modulation is a valuable therapeutic option for the management of ventricular arrhythmias. Bilateral cardiac sympathetic denervation (BCSD) has shown promising results in the acute, ...intermediate, and long-term management of polymorphic and monomorphic ventricular tachycardia (VT) in patients with structural heart disease. Cardiomyopathy (CM) due to Chagas disease (CD), and associated VT, is thought to be in part due to autonomic neuronal destruction and dysfunction.
The purpose of this study was to assess whether BCSD is a safe and effective treatment modality in patients with CD and VT storm or refractory VT.
A retrospective analysis of data from patients with chagasic CM who underwent BCSD between 2009 and 2015 at 2 international centers was performed.
Of 75 patients who underwent BCSD for VT storm or refractory VT in the setting of CM, 7 (9.3%) patients had CD as the etiology of CM. All patients had monomorphic VT. Median follow-up was 7 months (range 1-46 months). All patients either underwent previous unsuccessful catheter ablation or were not candidates for ablation. The median number of implantable cardioverter-defibrillator (ICD) shocks 1 month before BCSD was 4 (range 2-30) and decreased to 0 (range 0-2) during available follow-up after BCSD. When antitachycardia pacing therapies were included in the analysis, the median number of ICD therapies (shocks + antitachycardia pacing) still decreased to 1 (range 0-3).
In patients with chagasic CM presenting with refractory monomorphic VT, early evidence suggests that BCSD reduces appropriate ICD therapy and may represent a valuable treatment option.
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