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1
Ji, Y., L. A. Ward, and C. J. Hawkins. "Reconstitution of Human Necrosome Interactions in Saccharomyces cerevisiae." Biomolecules 11, no. 2 (2021): 153. http://dx.doi.org/10.3390/biom11020153.
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The necrosome is a large-molecular-weight complex in which the terminal effector of the necroptotic pathway, Mixed Lineage Kinase Domain-Like protein (MLKL), is activated to induce necroptotic cell death. The precise mechanism of MLKL activation by the upstream kinase, Receptor Interacting Serine/Threonine Protein Kinase 3 (RIPK3) and the role of Receptor Interacting Serine/Threonine Protein Kinase 1 (RIPK1) in mediating MLKL activation remain incompletely understood. Here, we reconstituted human necrosome interactions in yeast by inducible expression of these necrosome effectors. Functional interactions were reflected by the detection of phosphorylated MLKL, plasma membrane permeabilization, and reduced proliferative potential. Following overexpression of human necrosome effectors in yeast, MLKL aggregated in the periphery of the cell, permeabilized the plasma membrane and compromised clonogenic potential. RIPK1 had little impact on RIPK3/MLKL-mediated yeast lethality; however, it exacerbated the toxicity provoked by co-expression of MLKL with a RIPK3 variant bearing a mutated RHIM-domain. Small molecule necroptotic inhibitors necrostatin-1 and TC13172, and viral inhibitors M45 (residues 1–90) and BAV_Rmil, abated the yeast toxicity triggered by the reconstituted necrosome. This yeast model provides a convenient tool to study necrosome protein interactions and to screen for and characterize potential necroptotic inhibitors.
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Yang, Fang-Hao, Xiao-Lei Dong, Guo-Xiang Liu, et al. "The protective effect of C-phycocyanin in male mouse reproductive system." Food & Function 13, no. 5 (2022): 2631–46. http://dx.doi.org/10.1039/d1fo03741b.
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Petrie, Emma J., Richard W. Birkinshaw, Akiko Koide, et al. "Identification of MLKL membrane translocation as a checkpoint in necroptotic cell death using Monobodies." Proceedings of the National Academy of Sciences 117, no. 15 (2020): 8468–75. http://dx.doi.org/10.1073/pnas.1919960117.
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The necroptosis cell death pathway has been implicated in host defense and in the pathology of inflammatory diseases. While phosphorylation of the necroptotic effector pseudokinase Mixed Lineage Kinase Domain-Like (MLKL) by the upstream protein kinase RIPK3 is a hallmark of pathway activation, the precise checkpoints in necroptosis signaling are still unclear. Here we have developed monobodies, synthetic binding proteins, that bind the N-terminal four-helix bundle (4HB) “killer” domain and neighboring first brace helix of human MLKL with nanomolar affinity. When expressed as genetically encoded reagents in cells, these monobodies potently block necroptotic cell death. However, they did not prevent MLKL recruitment to the “necrosome” and phosphorylation by RIPK3, nor the assembly of MLKL into oligomers, but did block MLKL translocation to membranes where activated MLKL normally disrupts membranes to kill cells. An X-ray crystal structure revealed a monobody-binding site centered on the α4 helix of the MLKL 4HB domain, which mutational analyses showed was crucial for reconstitution of necroptosis signaling. These data implicate the α4 helix of its 4HB domain as a crucial site for recruitment of adaptor proteins that mediate membrane translocation, distinct from known phospholipid binding sites.
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Murphy, James M., and James E. Vince. "Post-translational control of RIPK3 and MLKL mediated necroptotic cell death." F1000Research 4 (November 19, 2015): 1297. http://dx.doi.org/10.12688/f1000research.7046.1.
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Several programmed lytic and necrotic-like cell death mechanisms have now been uncovered, including the recently described receptor interacting protein kinase-3 (RIPK3)-mixed lineage kinase domain-like (MLKL)-dependent necroptosis pathway. Genetic experiments have shown that programmed necrosis, including necroptosis, can play a pivotal role in regulating host-resistance against microbial infections. Alternatively, excess or unwarranted necroptosis may be pathological in autoimmune and autoinflammatory diseases. This review highlights the recent advances in our understanding of the post-translational control of RIPK3-MLKL necroptotic signaling. We discuss the critical function of phosphorylation in the execution of necroptosis, and highlight the emerging regulatory roles for several ubiquitin ligases and deubiquitinating enzymes. Finally, based on current evidence, we discuss the potential mechanisms by which the essential, and possibly terminal, necroptotic effector, MLKL, triggers the disruption of cellular membranes to cause cell lysis.
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Tian, Qing, Bo Qin, Yufan Gu, et al. "ROS-Mediated Necroptosis Is Involved in Iron Overload-Induced Osteoblastic Cell Death." Oxidative Medicine and Cellular Longevity 2020 (October 16, 2020): 1–22. http://dx.doi.org/10.1155/2020/1295382.
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Excess iron has been reported to lead to osteoblastic cell damage, which is a crucial pathogenesis of iron overload-related osteoporosis. However, the cytotoxic mechanisms have not been fully documented. In the present study, we focused on whether necroptosis contributes to iron overload-induced osteoblastic cell death and related underlying mechanisms. Here, we showed that the cytotoxicity of iron overload in osteoblastic cells was mainly due to necrosis, as evidenced by the Hoechst 33258/PI staining, Annexin-V/PI staining, and transmission electronic microscopy. Furthermore, we revealed that iron overload-induced osteoblastic necrosis might be mediated via the RIPK1/RIPK3/MLKL necroptotic pathway. In addition, we also found that iron overload was able to trigger mitochondrial permeability transition pore (mPTP) opening, which is a critical downstream event in the execution of necroptosis. The key finding of our experiment was that iron overload-induced necroptotic cell death might depend on reactive oxygen species (ROS) generation, as N-acetylcysteine effectively rescued mPTP opening and necroptotic cell death. ROS induced by iron overload promote necroptosis via a positive feedback mechanism, as on the one hand N-acetylcysteine attenuates the upregulation of RIPK1 and RIPK3 and phosphorylation of RIPK1, RIPK3, and MLKL and on the other hand Nec-1, siRIPK1, or siRIPK3 reduced ROS generation. In summary, iron overload induced necroptosis of osteoblastic cells in vitro, which is mediated, at least in part, through the RIPK1/RIPK3/MLKL pathway. We also highlight the critical role of ROS in the regulation of iron overload-induced necroptosis in osteoblastic cells.
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Samson, André L., Sarah E. Garnish, Joanne M. Hildebrand, and James M. Murphy. "Location, location, location: A compartmentalized view of TNF-induced necroptotic signaling." Science Signaling 14, no. 668 (2021): eabc6178. http://dx.doi.org/10.1126/scisignal.abc6178.
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Necroptosis is a lytic, proinflammatory cell death pathway, which has been implicated in host defense and, when dysregulated, the pathology of many human diseases. The central mediators of this pathway are the receptor-interacting serine/threonine protein kinases RIPK1 and RIPK3 and the terminal executioner, the pseudokinase mixed lineage kinase domain–like (MLKL). Here, we review the chronology of signaling along the RIPK1-RIPK3-MLKL axis and highlight how the subcellular compartmentalization of signaling events controls the initiation and execution of necroptosis. We propose that a network of modulators surrounds the necroptotic signaling core and that this network, rather than acting universally, tunes necroptosis in a context-, cell type–, and species-dependent manner. Such a high degree of mechanistic flexibility is likely an important property that helps necroptosis operate as a robust, emergency form of cell death.
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Speir, Mary, Joanne A. O'Donnell, Alyce A. Chen, Akshay A. D'Cruz, and Ben A. Croker. "Ptpn6 Inhibits IL-1 Release from Neutrophils By Regulation of Caspase-8- and Ripk3/Mlkl-Dependent Forms of Cell Death." Blood 132, Supplement 1 (2018): 274. http://dx.doi.org/10.1182/blood-2018-99-120197.
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Abstract Neutrophilic dermatoses are a group of inflammatory skin disorders characterized by sterile infiltrates of neutrophils. These syndromes include pyoderma gangrenosum (PG) and Sweet's syndrome (SS), and they are associated with an increased risk of inflammatory bowel disease, rheumatoid arthritis, and hematologic malignancy, particularly monocytic or myelomonocytic leukemia (AML). IL-1 was first proposed in 1987 as a factor in SS, and the presence of "fragmented neutrophil nuclei" was hypothesized to contribute to disease. IL-1 has subsequently been reported at high levels in lesions of SS, and IL-1 neutralizing therapies have met with success in some SS patients. PG is also reported to be responsive to IL-1 neutralizing therapy, including PG patients with psoriatic arthritis. Splicing variants and promoter region deletions of protein tyrosine phosphatase-6 (PTPN6, or Src homology region 2 domain-containing phosphatase-1, SHP1) are a feature of SS and PG, consistent with the findings that mutations in Ptpn6 drive spontaneous IL-1R-dependent skin inflammation in mice. Mice lacking Ptpn6 develop a cutaneous inflammatory disease that is dependent on the IL-1 receptor (IL-1R), G-CSF, and neutrophils. We hypothesized that production of IL-1 by dying neutrophils drives skin inflammation in the setting of Ptpn6 deficiency. To investigate the mechanisms controlling pathogenic IL-1 release in mice lacking Ptpn6 specifically in neutrophils (Ptpn6∆PMN), we looked directly at cytokine production from neutrophils undergoing inflammatory forms of cell death. We found that stimuli engaging Ripk3/Mlkl-dependent necroptotic forms of cell death result in transcription, processing, and release of bioactive IL-1α and IL-1β from neutrophils. Production of IL-1α and IL-1β were increased in Ptpn6∆PMN neutrophils treated to undergo necroptosis. Ptpn6∆PMN neutrophils displayed increased rates of spontaneous cell death in the presence of G-CSF or IFN-γ alone, and were hypersensitive to necroptotic stimuli. These cell death abnormalities were absent in Ptpn6∆PMN Casp8∆PMN Mlkl-/- neutrophils, demonstrating that Ptpn6 regulates both apoptosis and necroptosis to prevent IL-1 release. We next examined the contribution of apoptosis and necroptosis in the development of spontaneous cutaneous inflammatory disease in Ptpn6∆PMN mice. Loss of either the Caspase-8-dependent apoptotic pathway or the Ripk3/Mlkl-dependent necroptotic pathway was not sufficient to prevent inflammation in mice. However, combined deletion of Caspase-8 and Ripk3/Mlkl protected Ptpn6∆PMN mice (Ptpn6∆PMN Casp8∆PMN Ripk3-/- and Ptpn6∆PMN Casp8∆PMN Mlkl-/- mice). Ripk1 is known to act as a physiological negative regulator of both Caspase-8-dependent apoptosis and Ripk3/Mlkl-dependent necroptosis. We found that the absence of Ripk1 in neutrophils in Ptpn6∆PMN Ripk1∆PMN mice resulted in heightened sensitivity of neutrophils to cell death and accelerated onset of cutaneous inflammatory disease. These results reveal Ripk1 as a critical physiological negative regulator of neutrophil inflammatory cell death and IL-1 production, and cutaneous inflammation. Together, these data emphasize dual functions for Ptpn6 in negative regulation of IL-1α/β transcription, and to prevent Caspase-8- and Ripk3/Mlkl-dependent cell death and concomitant IL-1α/β processing and release. These findings implicate neutrophils as the dominant producers of IL-1 in neutrophilic dermatoses, and identify novel therapeutic targets that could be exploited to control inflammatory forms of cell death in these skin disorders. Disclosures No relevant conflicts of interest to declare.
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Huang, Ming, Shuai Zhu, Huihui Huang, et al. "Integrin-Linked Kinase Deficiency in Collecting Duct Principal Cell Promotes Necroptosis of Principal Cell and Contributes to Kidney Inflammation and Fibrosis." Journal of the American Society of Nephrology 30, no. 11 (2019): 2073–90. http://dx.doi.org/10.1681/asn.2018111162.
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BackgroundNecroptosis is a newly discovered cell death pathway that plays a critical role in AKI. The involvement of integrin-linked kinase (ILK) in necroptosis has not been studied.MethodsWe performed experiments in mice with an Ilk deletion in collecting duct (CD) principal cells (PCs), and cultured tubular epithelial cells treated with an ILK inhibitor or ILK siRNA knockdown.ResultsIlk deletion in CD PCs resulted in acute tubular injury and early mortality in mice. Progressive interstitial fibrosis and inflammation associated with the activation of the canonical TGF-β signaling cascade were detected in the kidneys of the mice lacking ILK in the CD PCs. In contrast to the minimal apoptosis detected in the animals’ injured CDs, widespread necroptosis was present in ILK-deficient PCs, characterized by cell swelling, deformed mitochondria, and rupture of plasma membrane. In addition, ILK deficiency resulted in increased expression and activation of necroptotic proteins MLKL and RIPK3, and membrane translocation of MLKL in CD PCs. ILK inhibition and siRNA knockdown reduced cell survival in cultured tubular cells, concomitant with increased membrane accumulation of MLKL and/or phospho-MLKL. Administration of a necroptosis inhibitor, necrostatin-1, blocked cell death in vitro and significantly attenuated inflammation, interstitial fibrosis, and renal failure in ILK-deficient mice.ConclusionsThe study demonstrates the critical involvement of ILK in necroptosis through modulation of the RIPK3 and MLKL pathway and highlights the contribution of CD PC injury to the development of inflammation and interstitial fibrosis of the kidney.
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Picon, Carmen, Anusha Jayaraman, Rachel James, et al. "Neuron-specific activation of necroptosis signaling in multiple sclerosis cortical grey matter." Acta Neuropathologica 141, no. 4 (2021): 585–604. http://dx.doi.org/10.1007/s00401-021-02274-7.
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AbstractSustained exposure to pro-inflammatory cytokines in the leptomeninges is thought to play a major role in the pathogenetic mechanisms leading to cortical pathology in multiple sclerosis (MS). Although the molecular mechanisms underlying neurodegeneration in the grey matter remain unclear, several lines of evidence suggest a prominent role for tumour necrosis factor (TNF). Using cortical grey matter tissue blocks from post-mortem brains from 28 secondary progressive MS subjects and ten non-neurological controls, we describe an increase in expression of multiple steps in the TNF/TNF receptor 1 signaling pathway leading to necroptosis, including the key proteins TNFR1, FADD, RIPK1, RIPK3 and MLKL. Activation of this pathway was indicated by the phosphorylation of RIPK3 and MLKL and the formation of protein oligomers characteristic of necrosomes. In contrast, caspase-8 dependent apoptotic signaling was decreased. Upregulation of necroptotic signaling occurred predominantly in macroneurons in cortical layers II–III, with little expression in other cell types. The presence of activated necroptotic proteins in neurons was increased in MS cases with prominent meningeal inflammation, with a 30-fold increase in phosphoMLKL+ neurons in layers I–III. The density of phosphoMLKL+ neurons correlated inversely with age at death, age at progression and disease duration. In vivo induction of chronically elevated TNF and INFγ levels in the CSF in a rat model via lentiviral transduction in the meninges, triggered inflammation and neurodegeneration in the underlying cortical grey matter that was associated with increased neuronal expression of TNFR1 and activated necroptotic signaling proteins. Exposure of cultured primary rat cortical neurons to TNF induced necroptosis when apoptosis was inhibited. Our data suggest that neurons in the MS cortex are dying via TNF/TNFR1 stimulated necroptosis rather than apoptosis, possibly initiated in part by chronic meningeal inflammation. Neuronal necroptosis represents a pathogenetic mechanism that is amenable to therapeutic intervention at several points in the signaling pathway.
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Chen, Jing, Renate Kos, Johan Garssen, and Frank Redegeld. "Molecular Insights into the Mechanism of Necroptosis: The Necrosome as a Potential Therapeutic Target." Cells 8, no. 12 (2019): 1486. http://dx.doi.org/10.3390/cells8121486.
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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.
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Thomas, Chloe N., Adam M. Thompson, Zubair Ahmed, and Richard J. Blanch. "Retinal Ganglion Cells Die by Necroptotic Mechanisms in a Site-Specific Manner in a Rat Blunt Ocular Injury Model." Cells 8, no. 12 (2019): 1517. http://dx.doi.org/10.3390/cells8121517.
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Closed-globe injury can cause visual loss in military and civilian populations, with retinal cell death, including retinal ganglion cell (RGC) degeneration, leading to irreversible blindness. RGC and optic nerve (ON) degeneration after eye or head injury is termed traumatic optic neuropathy (TON). There are currently no treatments for RGC loss, therefore novel therapeutics to prevent RGC death or promote axonal regeneration are a priority. We investigated necroptotic signaling mechanisms in a rat blunt ocular injury model. After bilateral blunt trauma, protein expression and retinal localization of necroptosis pathway members (receptor interacting protein kinase 1, RIPK1; receptor interacting protein kinase 3, RIPK3; and mixed lineage kinase domain like pseudokinase, MLKL) were assessed by Western blot and immunohistochemistry (IHC), and potent necroptosis inhibitor Necrostatin-1s (Nec-1s) was delivered by intravitreal injection to one eye and vehicle to the contralateral eye. RGC and photoreceptor survival were assessed by cell counting and outer nuclear layer (ONL) thickness measurements on histology. The neuroprotective effects of Nec-1s were assessed in primary retinal culture by βIII-tubulin+ RGC cell counts. MLKL protein expression were upregulated at 48 h after injury and MLKL immunolocalised to retinal binding protein with multiple splice (RBPMS)+ RGC, inner nuclear cells and ONL cells, specifically at the retinal injury site. RIPK3 expression did not increase but RIPK3 co-immunolocalised with RBPMS+ RGC in intact and injured retinae. In vitro, a Nec-1s concentration of 0.01 pg/µL was RGC neuroprotective. In the blunt ocular injury rat model, Nec-1s prevented RGC death at the center of the impact site but did not protect against ONL thinning or provide functional restitution. RGC degeneration in our blunt ocular injury model is site-specific, with necroptosis driving death at the center of the focal impact site.
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Garnish, Sarah E., and Joanne M. Hildebrand. "Rare catastrophes and evolutionary legacies: human germline gene variants in MLKL and the necroptosis signalling pathway." Biochemical Society Transactions 50, no. 1 (2022): 529–39. http://dx.doi.org/10.1042/bst20210517.
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Programmed cell death has long been characterised as a key player in the development of human disease. Necroptosis is a lytic form of programmed cell death that is universally mediated by the effector protein mixed lineage kinase domain-like (MLKL), a pseudokinase. MLKL's activating kinase, receptor interacting protein kinase 3 (RIPK3), is itself activated within context specific scaffolds of receptor interacting protein kinase 1 (RIPK1), Z-DNA Binding Protein-1 (ZBP1) or TIR domain-containing adaptor inducing interferon-β (TRIF). These core necroptosis modulating proteins have been comprehensively revealed as potent drivers and suppressors of disease in inbred mouse strains. However, their roles in human disease within the ‘real world’ of diverse genetic backgrounds, natural infection and environmental challenges remains less well understood. Over 20 unique disease-associated human germline gene variants in this core necroptotic machinery have been reported in the literature and human clinico-genetics databases like ClinVar to date. In this review, we provide an overview of these human gene variants, with an emphasis on those encoding MLKL. These experiments of nature have the potential to not only enrich our understanding of the basic biology of necroptosis, but offer important population level insights into which clinical indications stand to benefit most from necroptosis-targeted drugs.
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Cacciola, Nunzio Antonio, Angela Salzano, Nunzia D’Onofrio, et al. "Buffalo Milk Whey Activates Necroptosis and Apoptosis in a Xenograft Model of Colorectal Cancer." International Journal of Molecular Sciences 23, no. 15 (2022): 8464. http://dx.doi.org/10.3390/ijms23158464.
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Recent pharmacological research on milk whey, a byproduct of the dairy industry, has identified several therapeutic properties that could be exploited in modern medicine. In the present study, we investigated the anticancer effects of whey from Mediterranean buffalo (Bubalus bubalis) milk. The antitumour effect of delactosed milk whey (DMW) was evaluated using the HCT116 xenograft mouse model of colorectal cancer (CRC). There were no discernible differences in tumour growth between treated and untreated groups. Nevertheless, haematoxylin and eosin staining of the xenograft tissues showed clearer signs of different cell death in DMW-treated mice compared to vehicle-treated mice. Detailed biochemical and molecular biological analyses revealed that DMW was able to downregulate the protein expression levels of c-myc, phospho-Histone H3 (ser 10) and p-ERK. Moreover, DMW also activated RIPK1, RIPK3, and MLKL axis in tumour tissues from xenograft mice, thus, suggesting a necroptotic effect. The necroptotic pathway was accompanied by activation of the apoptotic pathway as revealed by increased expression of both cleaved caspase-3 and PARP-1. At the molecular level, DMW-induced cell death was also associated with (i) upregulation of SIRT3, SIRT6, and PPAR-γ and (ii) downregulation of LDHA and PPAR-α. Overall, our results unveil the potential of whey as a source of biomolecules of food origin in the clinical setting of novel strategies for the treatment of CRC.
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Ward, George A., Simone Jueliger, Martin Sims, et al. "Combining the IAP Antagonist Tolinapant with a DNA Hypomethylating Agent Enhances Immunogenic Cell Death in Preclinical Models of T-Cell Lymphoma." Blood 138, Supplement 1 (2021): 3986. http://dx.doi.org/10.1182/blood-2021-152176.
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Abstract Introduction: Tolinapant is a potent, non-peptidomimetic antagonist of cIAP1, cIAP2 and XIAP. In ongoing Phase 2 trial (NCT02503423), tolinapant has shown activity against highly pre-treated peripheral and cutaneous T-cell lymphoma (Samaniego et al., Hematological Oncology, 2019). Hypomethylating agents (HMAs) have also shown clinical responses in some subsets of PTCL (Lemonnier et al., Blood, 2019). Both HMAs and IAP antagonists show immunomodulatory anti-cancer potential in pre-clinical studies. A Phase 1 clinical study investigating the combination of tolinapant and ASTX727 (oral decitabine) in AML is currently in progress (NCT04155580). Here we have undertaken a biomarker-driven approach to understand the potential for induction of immunogenic forms of cell death (ICD), such as necroptosis, by rational combination of our clinical compounds in pre-clinical models of T-cell lymphoma (TCL). Methods: On-target effects of decitabine and tolinapant were measured by analysing levels of DNMT1 and cIAP1, respectively, by Western blotting in mouse and human cell lines. Levels of key apoptosis, necroptosis or pyroptosis biomarkers were also monitored by Western blotting to provide evidence of lytic cell death contributing to a potential immune response. RIPK3- or MLKL-knockout cell lines were generated by CRISPR to demonstrate involvement of necroptosis in drug-induced cell death in a T-cell lymphoma cell line (BW5147.G.1.4) in vitro. Cell death was monitored by viability (CellTiterGlo) or real-time microscopy (IncuCyte) assays. Levels of key inflammatory mediators or DAMPS were measured in tissue culture supernatants and mouse plasma by Luminex assay (Ampersand). Results: Combined treatment of tolinapant and decitabine led to depletion of cIAP1 and DNMT1 in TCL cell lines, demonstrating on-target activity of tolinapant and decitabine, respectively. The combination of tolinapant and decitabine acted synergistically in mouse and human T-cell lymphoma cell lines to reduce viability in proliferation assays. Necroptosis was induced by decitabine or tolinapant alone in mouse TCL cell lines with robust activation of the RIPK1/RIPK3/MLKL necroptosis pathway when caspase activity was inhibited, and the combination of both agents enhanced loss of viability. Furthermore, we demonstrated decitabine treatment led to re-expression of both RIPK3 and MLKL in mouse cell lines, supporting published evidence that methylation can silence these key biomarkers (Koo et al., Cell Research, 2015; Koch et al., Neoplasia, 2021). Enhanced release of chemokine, cytokine and DAMPs was demonstrated with the combination of agents in vitro and in vivo. By removal of key necroptosis pathway components using CRISPR, we confirmed the importance of this lytic cell death pathway by demonstrating that RIPK3 -/- and MLKL -/- T-cell lymphoma (BW5147.G.1.4) cell lines had reduced necroptosis potential after treatment with tolinapant or decitabine alone or in combination; and demonstrate reduced release of inflammatory mediators in vitro. Finally, our in vivo evaluation of the combination of agents in mouse syngeneic models suggested that increased anti-tumour activity and immune-potentiating systemic biomarker modulation can be achieved with a tolerated dosing regimen of both compounds. Conclusion: These data demonstrate that decitabine enhances immunogenic cell death induced by tolinapant through the re-expression of genes in the necroptotic pathway. This finding provides strong rationale to explore this combination clinically. Disclosures Sims: Astex Pharmaceuticals: Current Employment. Davis: Astex Pharmacueticals: Current Employment. Smyth: Astex Pharmaceuticals: Current Employment.
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Lyu, Ah-Ra, Tae-Hwan Kim, Sun-Ae Shin, et al. "Hearing Impairment in a Mouse Model of Diabetes Is Associated with Mitochondrial Dysfunction, Synaptopathy, and Activation of the Intrinsic Apoptosis Pathway." International Journal of Molecular Sciences 22, no. 16 (2021): 8807. http://dx.doi.org/10.3390/ijms22168807.
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Although previous studies continuously report an increased risk of hearing loss in diabetes patients, the impact of the disease on the inner ear remains unexplored. Herein, we examine the pathophysiology of diabetes-associated hearing impairment and cochlear synaptopathy in a mouse model of diabetes. Male B6.BKS(D)-Leprdb/J (db/db, diabetes) and heterozygote (db/+, control) mice were assigned into each experimental group (control vs. diabetes) based on the genotype and tested for hearing sensitivity every week from 6 weeks of age. Each cochlea was collected for histological and biological assays at 14 weeks of age. The diabetic mice exerted impaired hearing and a reduction in cochlear blood flow and C-terminal-binding protein 2 (CtBP2, a presynaptic ribbon marker) expression. Ultrastructural images revealed severely damaged mitochondria from diabetic cochlea accompanied by a reduction in Cytochrome c oxidase subunit 4 (COX4) and CR6-interacting factor 1 (CRIF1). The diabetic mice presented significantly decreased levels of platelet endothelial cell adhesion molecule (PECAM-1), B-cell lymphoma 2 (BCL-2), and procaspase-9, but not procaspase-8. Importantly, significant changes were not found in necroptotic programmed cell death markers (receptor-interacting serine/threonine-protein kinase 1, RIPK1; RIPK3; and mixed lineage kinase domain-like pseudokinase, MLKL) between the groups. Taken together, diabetic hearing loss is accompanied by synaptopathy, microangiopathy, damage to the mitochondrial structure/function, and activation of the intrinsic apoptosis pathway. Our results imply that mitochondrial dysfunction is deeply involved in diabetic hearing loss, and further suggests the potential benefits of therapeutic strategies targeting mitochondria.
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Huang, Huihui, William W. Jin, Ming Huang, et al. "Gentamicin-Induced Acute Kidney Injury in an Animal Model Involves Programmed Necrosis of the Collecting Duct." Journal of the American Society of Nephrology 31, no. 9 (2020): 2097–115. http://dx.doi.org/10.1681/asn.2019020204.
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BackgroundGentamicin is a potent aminoglycoside antibiotic that targets gram-negative bacteria, but nephrotoxicity limits its clinical application. The cause of gentamicin-induced AKI has been attributed mainly to apoptosis of the proximal tubule cells. However, blocking apoptosis only partially attenuates gentamicin-induced AKI in animals.MethodsMice treated with gentamicin for 7 days developed AKI, and programmed cell death pathways were examined using pharmacologic inhibitors and in RIPK3-deficient mice. Effects in porcine and murine kidney cell lines were also examined.ResultsGentamicin caused a low level of apoptosis in the proximal tubules and significant ultrastructural alterations consistent with necroptosis, occurring predominantly in the collecting ducts (CDs), including cell and organelle swelling and rupture of the cell membrane. Upregulation of the key necroptotic signaling molecules, mixed lineage kinase domain-like pseudokinase (MLKL) and receptor-interacting serine/threonine-protein kinase 3 (RIPK3), was detected in gentamicin-treated mice and in cultured renal tubule cells. In addition, gentamicin induced apical accumulation of total and phosphorylated MLKL (pMLKL) in CDs in mouse kidney. Inhibiting a necroptotic protein, RIPK1, with necrostatin-1 (Nec-1), attenuated gentamicin-induced necrosis and upregulation of MLKL and RIPK3 in mice and cultured cells. Nec-1 also alleviated kidney inflammation and fibrosis, and significantly improved gentamicin-induced renal dysfunction in mice. Furthermore, deletion of RIPK3 in the Ripk3−/− mice significantly attenuated gentamicin-induced AKI.ConclusionsA previously unrecognized role of programmed necrosis in collecting ducts in gentamicin-induced kidney injury presents a potential new therapeutic strategy to alleviate gentamicin-induced AKI through inhibiting necroptosis.
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Santos, Leonardo Duarte, Krist Helen Antunes, Stéfanie Primon Muraro, et al. "TNF-mediated alveolar macrophage necroptosis drives disease pathogenesis during respiratory syncytial virus infection." European Respiratory Journal 57, no. 6 (2020): 2003764. http://dx.doi.org/10.1183/13993003.03764-2020.
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Respiratory syncytial virus (RSV) is the major cause of acute bronchiolitis in infants under 2 years old. Necroptosis has been implicated in the outcomes of respiratory virus infections. We report that RSV infection triggers necroptosis in primary mouse macrophages and human monocytes in a RIPK1-, RIPK3- and MLKL-dependent manner. Moreover, necroptosis pathways are harmful to RSV clearance from alveolar macrophages. Additionally, Ripk3−/− mice were protected from RSV-induced weight loss and presented with reduced viral loads in the lungs.Alveolar macrophage depletion also protected mice from weight loss and decreased lung RSV virus load. Importantly, alveolar macrophage depletion abolished the upregulation of Ripk3 and Mlkl gene expression induced by RSV infection in the lung tissue.Autocrine tumor necrosis factor (TNF)-mediated RSV-triggered macrophage necroptosis and necroptosis pathways were also involved in TNF secretion even when macrophages were committed to cell death, which can worsen lung injury during RSV infection. In line, Tnfr1−/− mice had a marked decrease in Ripk3 and Mlkl gene expression and a sharp reduction in the numbers of necrotic alveolar macrophages in the lungs. Finally, we provide evidence that elevated nasal levels of TNF are associated with disease severity in infants with RSV bronchiolitis.We propose that targeting TNF and/or the necroptotic machinery may be valuable therapeutic approaches to reduce the respiratory morbidity caused by RSV infection in young children.
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D'Cruz, Akshay A., Meghan Bliss-Moreau, Maria Ericcson, and Ben A. Croker. "Mlkl Pores Release Neutrophil Extracellular Traps in Necroptotic Neutrophils." Blood 126, no. 23 (2015): 2200. http://dx.doi.org/10.1182/blood.v126.23.2200.2200.
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Abstract Neutrophil extracellular traps (NETs) are networks of extracellular nuclear DNA and microbicidal proteins released from neutrophils in response to tissue damage and infection. Despite evidence of pathogenic roles for NETs in systemic lupus erythematosus, rheumatoid arthritis, diabetes, artherosclerosis and Alzheimer's disease, the major biochemical pathways controlling their formation remains poorly understood. Apoptosis does not contribute to NET formation but the role of regulated non-apoptotic cell death pathways such as necroptosis is not known. We have investigated the role of positive and negative regulators of necroptosis including receptor-interacting protein kinase-3 (RIPK3), mixed lineage kinase domain-like (MLKL), receptor-interacting protein kinase-1 (RIPK1) and Caspase-8. Using immunogold electron microscopy, flow cytometry, imaging flow cytometry and fluorescence microscopy, we demonstrate that necroptosis can drive NET formation via MLKL pore formation at the cell surface. This process is caspase-independent but reactive oxygen species-dependent. Genetically-modified mouse peripheral blood and bone marrow neutrophils were used to show that Caspase-8 and RIPK1 negatively regulate NET formation driven by RIPK3 and MLKL. Mice that lack MLKL are deficient in necroptosis and NET formation, and were sensitive to methicillin-resistant Staphylococcus aureus (MRSA). Neutrophil-specific Caspase-8-deficiency also leads to increased susceptibility to MRSA due to increased rates of necroptotic neutrophil death. Killing of MRSA by necroptotic neutrophils is sensitive to DNase, and is dependent on MLKL, suggesting that necroptosis-driven NET formation contributes to the bactericidal activity of neutrophils. Human peripheral blood neutrophils also generate NETs that are sensitive to pharmacological inhibitors of necroptosis, suggesting that targeting necroptosis in general may help combat autoimmune responses to DNA. This study provides a framework to investigate the role of extracellular DNA release and cell death in the setting of infection, autoimmunity and autoinflammatory disease. Disclosures No relevant conflicts of interest to declare.
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Belizário, José, Luiz Vieira-Cordeiro, and Sylvia Enns. "Necroptotic Cell Death Signaling and Execution Pathway: Lessons from Knockout Mice." Mediators of Inflammation 2015 (2015): 1–15. http://dx.doi.org/10.1155/2015/128076.
Full textAbstract:
Under stress conditions, cells in living tissue die by apoptosis or necrosis depending on the activation of the key molecules within a dying cell that either transduce cell survival or death signals that actively destroy the sentenced cell. Multiple extracellular (pH, heat, oxidants, and detergents) or intracellular (DNA damage and Ca2+overload) stress conditions trigger various types of the nuclear, endoplasmic reticulum (ER), cytoplasmatic, and mitochondrion-centered signaling events that allow cells to preserve the DNA integrity, protein folding, energetic, ionic and redox homeostasis, thus escaping from injury. Along the transition from reversible to irreversible injury, death signaling is highly heterogeneous and damaged cells may engage autophagy, apoptotic, or necrotic cell death programs. Studies on multiple double- and triple- knockout mice identifiedcaspase-8,flip, andfaddgenes as key regulators of embryonic lethality and inflammation. Caspase-8 has a critical role in pro- and antinecrotic signaling pathways leading to the activation of receptor interacting protein kinase 1 (RIPK1), RIPK3, and the mixed kinase domain-like (MLKL) for a convergent execution pathway of necroptosis or regulated necrosis. Here we outline the recent discoveries into how the necrotic cell death execution pathway is engaged in many physiological and pathological outcome based on genetic analysis of knockout mice.
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Daley-Bauer, Lisa P., Linda Roback, Lynsey N. Crosby, et al. "Mouse cytomegalovirus M36 and M45 death suppressors cooperate to prevent inflammation resulting from antiviral programmed cell death pathways." Proceedings of the National Academy of Sciences 114, no. 13 (2017): E2786—E2795. http://dx.doi.org/10.1073/pnas.1616829114.
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The complex interplay between caspase-8 and receptor-interacting protein (RIP) kinase RIP 3 (RIPK3) driving extrinsic apoptosis and necroptosis is not fully understood. Murine cytomegalovirus triggers both apoptosis and necroptosis in infected cells; however, encoded inhibitors of caspase-8 activity (M36) and RIP3 signaling (M45) suppress these antiviral responses. Here, we report that this virus activates caspase-8 in macrophages to trigger apoptosis that gives rise to secondary necroptosis. Infection with double-mutant ΔM36/M45mutRHIM virus reveals a signaling pattern in which caspase-8 activates caspase-3 to drive apoptosis with subsequent RIP3-dependent activation of mixed lineage kinase domain-like (MLKL) leading to necroptosis. This combined cell death signaling is highly inflammatory, greater than either apoptosis induced by ΔM36 or necroptosis induced by M45mutRHIM virus. IL-6 production by macrophages is dramatically increased during double-mutant virus infection and correlates with faster antiviral responses in the host. Collaboratively, M36 and M45 target caspase-8 and RIP3 pathways together to suppress this proinflammatory cell death. This study reveals the effect of antiviral programmed cell death pathways on inflammation, shows that caspase-8 activation may go hand-in-hand with necroptosis in macrophages, and revises current understanding of independent and collaborative functions of M36 and M45 in blocking apoptotic and necroptotic cell death responses.
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Lai, Ming-Zong, Yung-Hsuan Wu, Ting-Fang Chou, et al. "Regulation of necroptosis by targeting tumor suppressor death-associated protein kinase 1." Journal of Immunology 204, no. 1_Supplement (2020): 144.11. http://dx.doi.org/10.4049/jimmunol.204.supp.144.11.
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Abstract Death-associated protein kinase 1 (DAPK1, DAPk, DAPK) is known for its involvement in apoptosis and autophagy-associated cell death. Here, we identified an unexpected function of DAPK1 in suppressing necroptosis. DAPK1-deficiency renders macrophages and dendritic cells susceptible to necroptotic induction by different stimuli. We also observed an inhibitory role for DAPK1 in necroptosis in HT-29 cells, since knockdown or knockout of DAPK1 in such cells increased their sensitivity to necroptosis. Increased necroptosis was associated with enhanced formation of the RIPK1-RIPK3-MLKL complex in these DAPK1-deficient cells. We further found that DAPK1-deficiency led to decreased MAPK activated kinase 2 (MK2) activation and reduced RIPK1 S321 phosphorylation, with this latter representing a critical step controlling necrosome formation. Most TNF signaling pathways, including ERK, JNK and AKT, were not regulated by DAPK. In contrast, DAPK selectively promoted MKK3-induced p38 MAPK activation, resulting in enhanced MK2 phosphrylation. Our results reveal a novel role for DAPK1 in inhibiting necroptosis and illustrate an unexpected selectivity for DAPK1 in promoting p38 MAPK-MK2 activation. Importantly, our study suggests that modulation of necroptosis and p38/MK2-mediated inflammation may be achieved by targeting DAPK1.
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Bedient, Lori, Swechha Mainali Pokharel, Kim Roxana Chiok Casimiro, and Santanu Bose. "Lytic cell death mechanisms in human respiratory syncytial virus-infected macrophages." Journal of Immunology 204, no. 1_Supplement (2020): 93.16. http://dx.doi.org/10.4049/jimmunol.204.supp.93.16.
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Abstract Human respiratory syncytial virus (hRSV) is the most common cause of viral pneumonia in infants and children worldwide and creates significant airway disease in immunocompromised people and the elderly. Inflammation induced by hRSV infection is responsible for its hallmark manifestations of bronchiolitis and pneumonia. The cellular debris created secondary to lytic cell death of infected cells is a potent initiator of this inflammation. Macrophages are known to play a pivotal role in the early innate immune and inflammatory response to viral pathogens. While previous studies have investigated the mechanisms responsible for lytic cell death in Influenza A-infected macrophages and hRSV-infected neutrophils, the lytic cell death mechanisms associated with hRSV infection in macrophages remain unknown. In order to address this knowledge gap, we treated an hRSV-infected human THP-1 macrophage cell line with various inhibitors that block two lytic cell death pathways: pyroptosis and necroptosis. In addition, we used pyroptosis-deficient human macrophages to further clarify the lytic cell death mechanisms occurring during infection. Our studies revealed that hRSV induces lytic cell death in macrophages via both pyroptosis and necroptosis, specifically through caspase-1 and inflammasome-dependent pyroptotic mechanisms and through RIPK1-RIPK3-MLKL necroptotic mechanisms of cell death.
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Kim, Do-Yeon, Yea-Hyun Leem, Jin-Sun Park, et al. "RIPK1 Regulates Microglial Activation in Lipopolysaccharide-Induced Neuroinflammation and MPTP-Induced Parkinson’s Disease Mouse Models." Cells 12, no. 3 (2023): 417. http://dx.doi.org/10.3390/cells12030417.
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Increasing evidence suggests a pivotal role of receptor-interacting protein kinase 1 (RIPK1), an initiator of necroptosis, in neuroinflammation. However, the precise role of RIPK1 in microglial activation remains unclear. In the present study, we explored the role of RIPK1 in lipopolysaccharide (LPS)-induced neuroinflammation and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD model mice by using RIPK1-specific inhibitors necrostatin-1 (Nec-1) and necrostatin-1 stable (Nec-1s). Nec-1/Nec-1s or RIPK1 siRNA inhibited the production of proinflammatory molecules and the phosphorylation of RIPK1-RIPK3-MLKL and cell death in LPS-induced inflammatory or LPS/QVD/BV6-induced necroptotic conditions of BV2 microglial cells. Detailed mechanistic studies showed that Nec-1/Nec-1s exerted anti-inflammatory effects by modulating AMPK, PI3K/Akt, MAPKs, and NF-κB signaling pathways in LPS-stimulated BV2 cells. Subsequent in vivo studies showed that Nec-1/Nec-1s inhibited microglial activation and proinflammatory gene expression by inhibiting the RIPK1 phosphorylation in the brains of LPS-injected mice. Furthermore, Nec-1/Nec-1s exert neuroprotective and anti-inflammatory effects in MPTP-induced PD mice. We found that p-RIPK1 is mainly expressed in microglia, and thus RIPK1 may contribute to neuroinflammation and subsequent cell death of dopaminergic neurons in MPTP-induced PD model mice. These data suggest that RIPK1 is a key regulator of microglial activation in LPS-induced neuroinflammation and MPTP-induced PD mice.
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Garnish, Sarah E., Yanxiang Meng, Akiko Koide, et al. "Conformational interconversion of MLKL and disengagement from RIPK3 precede cell death by necroptosis." Nature Communications 12, no. 1 (2021). http://dx.doi.org/10.1038/s41467-021-22400-z.
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AbstractPhosphorylation of the MLKL pseudokinase by the RIPK3 kinase leads to MLKL oligomerization, translocation to, and permeabilization of, the plasma membrane to induce necroptotic cell death. The precise choreography of MLKL activation remains incompletely understood. Here, we report Monobodies, synthetic binding proteins, that bind the pseudokinase domain of MLKL within human cells and their crystal structures in complex with the human MLKL pseudokinase domain. While Monobody-32 constitutively binds the MLKL hinge region, Monobody-27 binds MLKL via an epitope that overlaps the RIPK3 binding site and is only exposed after phosphorylated MLKL disengages from RIPK3 following necroptotic stimulation. The crystal structures identified two distinct conformations of the MLKL pseudokinase domain, supporting the idea that a conformational transition accompanies MLKL disengagement from RIPK3. These studies provide further evidence that MLKL undergoes a large conformational change upon activation, and identify MLKL disengagement from RIPK3 as a key regulatory step in the necroptosis pathway.
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Meng, Yanxiang, Katherine A. Davies, Cheree Fitzgibbon, et al. "Human RIPK3 maintains MLKL in an inactive conformation prior to cell death by necroptosis." Nature Communications 12, no. 1 (2021). http://dx.doi.org/10.1038/s41467-021-27032-x.
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AbstractThe ancestral origins of the lytic cell death mode, necroptosis, lie in host defense. However, the dysregulation of necroptosis in inflammatory diseases has led to widespread interest in targeting the pathway therapeutically. This mode of cell death is executed by the terminal effector, the MLKL pseudokinase, which is licensed to kill following phosphorylation by its upstream regulator, RIPK3 kinase. The precise molecular details underlying MLKL activation are still emerging and, intriguingly, appear to mechanistically-diverge between species. Here, we report the structure of the human RIPK3 kinase domain alone and in complex with the MLKL pseudokinase. These structures reveal how human RIPK3 structurally differs from its mouse counterpart, and how human RIPK3 maintains MLKL in an inactive conformation prior to induction of necroptosis. Residues within the RIPK3:MLKL C-lobe interface are crucial to complex assembly and necroptotic signaling in human cells, thereby rationalizing the strict species specificity governing RIPK3 activation of MLKL.
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Moujalled, Diane, Pradnya Gangatirkar, Maria Kauppi, et al. "The necroptotic cell death pathway operates in megakaryocytes, but not in platelet synthesis." Cell Death & Disease 12, no. 1 (2021). http://dx.doi.org/10.1038/s41419-021-03418-z.
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AbstractNecroptosis is a pro-inflammatory cell death program executed by the terminal effector, mixed lineage kinase domain-like (MLKL). Previous studies suggested a role for the necroptotic machinery in platelets, where loss of MLKL or its upstream regulator, RIPK3 kinase, impacted thrombosis and haemostasis. However, it remains unknown whether necroptosis operates within megakaryocytes, the progenitors of platelets, and whether necroptotic cell death might contribute to or diminish platelet production. Here, we demonstrate that megakaryocytes possess a functional necroptosis signalling cascade. Necroptosis activation leads to phosphorylation of MLKL, loss of viability and cell swelling. Analyses at steady state and post antibody-mediated thrombocytopenia revealed that platelet production was normal in the absence of MLKL, however, platelet activation and haemostasis were impaired with prolonged tail re-bleeding times. We conclude that MLKL plays a role in regulating platelet function and haemostasis and that necroptosis signalling in megakaryocytes is dispensable for platelet production.
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Preston, Simon P., Cody C. Allison, Jan Schaefer, et al. "A necroptosis-independent function of RIPK3 promotes immune dysfunction and prevents control of chronic LCMV infection." Cell Death & Disease 14, no. 2 (2023). http://dx.doi.org/10.1038/s41419-023-05635-0.
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AbstractNecroptosis is a lytic and inflammatory form of cell death that is highly constrained to mitigate detrimental collateral tissue damage and impaired immunity. These constraints make it difficult to define the relevance of necroptosis in diseases such as chronic and persistent viral infections and within individual organ systems. The role of necroptotic signalling is further complicated because proteins essential to this pathway, such as receptor interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like (MLKL), have been implicated in roles outside of necroptotic signalling. We sought to address this issue by individually defining the role of RIPK3 and MLKL in chronic lymphocytic choriomeningitis virus (LCMV) infection. We investigated if necroptosis contributes to the death of LCMV-specific CD8+ T cells or virally infected target cells during infection. We provide evidence showing that necroptosis was redundant in the pathogenesis of acute forms of LCMV (Armstrong strain) and the early stages of chronic (Docile strain) LCMV infection in vivo. The number of immune cells, their specificity and reactivity towards viral antigens and viral loads are not altered in the absence of either MLKL or RIPK3 during acute and during the early stages of chronic LCMV infection. However, we identified that RIPK3 promotes immune dysfunction and prevents control of infection at later stages of chronic LCMV disease. This was not phenocopied by the loss of MLKL indicating that the phenotype was driven by a necroptosis-independent function of RIPK3. We provide evidence that RIPK3 signaling evoked a dysregulated type 1 interferone response which we linked to an impaired antiviral immune response and abrogated clearance of chronic LCMV infection.
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Chen, Jing, Shiyu Wang, Bart Blokhuis, Rob Ruijtenbeek, Johan Garssen, and Frank Redegeld. "Cell Death Triggers Induce MLKL Cleavage in Multiple Myeloma Cells, Which may Promote Cell Death." Frontiers in Oncology 12 (July 28, 2022). http://dx.doi.org/10.3389/fonc.2022.907036.
Full textAbstract:
Necroptosis is a type of caspase-independent programmed cell death that has been implicated in cancer development. Activation of the canonical necroptotic pathway is often characterized with successive signaling events as the phosphorylation of mixed lineage kinase domain-like (MLKL) by receptor-interacting protein kinase-3 (RIPK3), followed by MLKL oligomerization and plasma membrane rupture. Here, we demonstrate that omega-3 polyunsaturated fatty acids DHA/EPA and the proteasome inhibitor bortezomib induce necroptosis in human multiple myeloma (MM) cells in a RIPK3 independent manner. In addition, it seemed to be that phosphorylation of MLKL was not essential for necroptosis induction in MM cells. We show that treatment of MM cells with these cytotoxic compounds induced cleavage of MLKL into a 35 kDa protein. Furthermore, proteolytic cleavage of MLKL was triggered by activated caspase-3/8/10, and mutation of Asp140Ala in MLKL blocked this cleavage. The pan-caspase inhibitor ZVAD-FMK efficiently prevented DHA/EPA and bortezomib induced cell death. In addition, nuclear translocation of total MLKL and the C-terminus were detected in treated MM cells. Collectively, this present study suggests that caspase-mediated necroptosis may occur under (patho)physiological conditions, delineating a novel regulatory mechanism of necroptosis in RIPK3-deficient cancer cells.
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Tovey Crutchfield, Emma C., Sarah E. Garnish, Jessica Day, et al. "MLKL deficiency protects against low-grade, sterile inflammation in aged mice." Cell Death & Differentiation, February 8, 2023. http://dx.doi.org/10.1038/s41418-023-01121-4.
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AbstractMLKL and RIPK3 are the core signaling proteins of the inflammatory cell death pathway, necroptosis, which is a known mediator and modifier of human disease. Necroptosis has been implicated in the progression of disease in almost every physiological system and recent reports suggest a role for necroptosis in aging. Here, we present the first comprehensive analysis of age-related histopathological and immunological phenotypes in a cohort of Mlkl–/– and Ripk3–/– mice on a congenic C57BL/6 J genetic background. We show that genetic deletion of Mlkl in female mice interrupts immune system aging, specifically delaying the age-related reduction of circulating lymphocytes. -Seventeen-month-old Mlkl–/– female mice were also protected against age-related chronic sterile inflammation in connective tissue and skeletal muscle relative to wild-type littermate controls, exhibiting a reduced number of immune cell infiltrates in these sites and fewer regenerating myocytes. These observations implicate MLKL in age-related sterile inflammation, suggesting a possible application for long-term anti-necroptotic therapy in humans.
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Li, Dianrong, Jie Chen, Jia Guo та ін. "A phosphorylation of RIPK3 kinase initiates an intracellular apoptotic pathway that promotes prostaglandin2α-induced corpus luteum regression". eLife 10 (24 травня 2021). http://dx.doi.org/10.7554/elife.67409.
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Receptor-interacting serine/threonine-protein kinase 3 (RIPK3) normally signals to necroptosis by phosphorylating MLKL. We report here that when the cellular RIPK3 chaperone Hsp90/CDC37 level is low, RIPK3 also signals to apoptosis. The apoptotic function of RIPK3 requires phosphorylation of the serine 165/threonine 166 sites on its kinase activation loop, resulting in inactivation of RIPK3 kinase activity while gaining the ability to recruit RIPK1, FADD, and caspase-8 to form a cytosolic caspase-activating complex, thereby triggering apoptosis. We found that PGF2α induces RIPK3 expression in luteal granulosa cells in the ovary to cause luteal regression through this RIPK3-mediated apoptosis pathway. Mice carrying homozygous phosphorylation-resistant RIPK3 S165A/T166A knockin mutations failed to respond to PGF2α but retained pro-necroptotic function, whereas mice with phospho-mimicking S165D/T166E homozygous knock-in mutation underwent spontaneous apoptosis in multiple RIPK3-expressing tissues and died shortly after birth. Thus, RIPK3 signals to either necroptosis or apoptosis depending on its serine 165/threonine 166 phosphorylation status.
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Zhang, Wenbin, Weiliang Fan, Jia Guo, and Xiaodong Wang. "Osmotic stress activates RIPK3/MLKL-mediated necroptosis by increasing cytosolic pH through a plasma membrane Na + /H + exchanger." Science Signaling 15, no. 734 (2022). http://dx.doi.org/10.1126/scisignal.abn5881.
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Necroptosis is a form of cell death triggered by stimuli such as the tumor necrosis factor family of cytokines, which induce necrotic cell death through the RIPK1-RIPK3-MLKL pathway. We report here that necroptosis is also activated by extracellular osmotic stresses. Unlike the previously identified inducers of necroptosis, osmotic stress stimulated necroptosis through the direct activation of the kinase activity of RIPK3 by an increase in cytosolic pH mediated by the Na + /H + exchanger SLC9A1. Knockout, knockdown, or chemical inhibition of SLC9A1 blocked necroptosis induced by osmotic stresses. Moreover, setting intracellular pH at above-physiological values directly activated RIPK3 and necroptosis. The activation of RIPK3 by osmotic stresses did not require its RHIM domain, the protein-interacting domain required for the activation of RIPK3 when cells respond to other previously identified necroptotic stimuli. These results thus delineate a pathway that activates necroptosis in response to osmotic stresses.
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Águeda-Pinto, Ana, Luís Q. Alves, Fabiana Neves, et al. "Convergent Loss of the Necroptosis Pathway in Disparate Mammalian Lineages Shapes Viruses Countermeasures." Frontiers in Immunology 12 (September 1, 2021). http://dx.doi.org/10.3389/fimmu.2021.747737.
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Programmed cell death is a vital process in the life cycle of organisms. Necroptosis, an evolutionary form of programmed necrosis, contributes to the innate immune response by killing pathogen-infected cells. This virus-host interaction pathway is organized around two components: the receptor-interacting protein kinase 3 (RIPK3), which recruits and phosphorylates the mixed lineage kinase-like protein (MLKL), inducing cellular plasma membrane rupture and cell death. Critically, the presence of necroptotic inhibitors in viral genomes validates necroptosis as an important host defense mechanism. Here, we show, counterintuitively, that in different mammalian lineages, central components of necroptosis, such as RIPK3 and MLKL, are deleted or display inactivating mutations. Frameshifts or premature stop codons are observed in all the studied species of cetaceans and leporids. In carnivores’ genomes, the MLKL gene is deleted, while in a small number of species from afrotheria and rodentia premature stop codons are observed in RIPK3 and/or MLKL. Interestingly, we also found a strong correlation between the disruption of necroptosis in leporids and cetaceans and the absence of the N-terminal domain of E3-like homologs (responsible for necroptosis inhibition) in their naturally infecting poxviruses. Overall, our study provides the first comprehensive picture of the molecular evolution of necroptosis in mammals. The loss of necroptosis multiple times during mammalian evolution highlights the importance of gene/pathway loss for species adaptation and suggests that necroptosis is not required for normal mammalian development. Moreover, this study highlights a co-evolutionary relationship between poxviruses and their hosts, emphasizing the role of host adaptation in shaping virus evolution.
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Jacobsen, Annette V., Catia L. Pierotti, Kym N. Lowes, et al. "The Lck inhibitor, AMG-47a, blocks necroptosis and implicates RIPK1 in signalling downstream of MLKL." Cell Death & Disease 13, no. 4 (2022). http://dx.doi.org/10.1038/s41419-022-04740-w.
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AbstractNecroptosis is a form of caspase-independent programmed cell death that arises from disruption of cell membranes by the mixed lineage kinase domain-like (MLKL) pseudokinase after its activation by the upstream kinases, receptor interacting protein kinase (RIPK)-1 and RIPK3, within a complex known as the necrosome. Dysregulated necroptosis has been implicated in numerous inflammatory pathologies. As such, new small molecule necroptosis inhibitors are of great interest, particularly ones that operate downstream of MLKL activation, where the pathway is less well defined. To better understand the mechanisms involved in necroptosis downstream of MLKL activation, and potentially uncover new targets for inhibition, we screened known kinase inhibitors against an activated mouse MLKL mutant, leading us to identify the lymphocyte-specific protein tyrosine kinase (Lck) inhibitor AMG-47a as an inhibitor of necroptosis. We show that AMG-47a interacts with both RIPK1 and RIPK3, that its ability to protect from cell death is dependent on the strength of the necroptotic stimulus, and that it blocks necroptosis most effectively in human cells. Moreover, in human cell lines, we demonstrate that AMG-47a can protect against cell death caused by forced dimerisation of MLKL truncation mutants in the absence of any upstream signalling, validating that it targets a process downstream of MLKL activation. Surprisingly, however, we also found that the cell death driven by activated MLKL in this model was completely dependent on the presence of RIPK1, and to a lesser extent RIPK3, although it was not affected by known inhibitors of these kinases. Together, these results suggest an additional role for RIPK1, or the necrosome, in mediating human necroptosis after MLKL is phosphorylated by RIPK3 and provide further insight into reported differences in the progression of necroptosis between mouse and human cells.
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Wang, Xiaoliang, Damjan Avsec, Aleš Obreza, Shida Yousefi, Irena Mlinarič-Raščan, and Hans-Uwe Simon. "A Putative Serine Protease is Required to Initiate the RIPK3-MLKL—Mediated Necroptotic Death Pathway in Neutrophils." Frontiers in Pharmacology 11 (January 21, 2021). http://dx.doi.org/10.3389/fphar.2020.614928.
Full textAbstract:
Adhesion receptors, such as CD44, have been shown to activate receptor interacting protein kinase-3 (RIPK3)—mixed lineage kinase-like (MLKL) signaling, leading to a non-apoptotic cell death in human granulocyte/macrophage colony-stimulating factor (GM-CSF) – primed neutrophils. The signaling events of this necroptotic pathway, however, remain to be investigated. In the present study, we report the design, synthesis, and characterization of a series of novel serine protease inhibitors. Two of these inhibitors, compounds 1 and 3, were able to block CD44-triggered necroptosis in GM-CSF-primed neutrophils. Both inhibitors prevented the activation of MLKL, p38 mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3’—kinase (PI3K), hence blocking the increased levels of reactive oxygen species (ROS) required for cell death. Although compounds one and three partially inhibited isolated human neutrophil elastase (HNE) activity, we obtained no pharmacological evidence that HNE is involved in the initiation of this death pathway within a cellular context. Interestingly, neither serine protease inhibitor had any effect on FAS receptor-mediated apoptosis. Taken together, these results suggest that a serine protease is involved in non-apoptotic CD44-triggered RIPK3-MLKL-dependent neutrophil cell death, but not FAS receptor-mediated caspase-dependent apoptosis. Thus, a pharmacological block on serine proteases might be beneficial for preventing exacerbation of disease in neutrophilic inflammatory responses.
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Yu, Ziyu, Nan Jiang, Wenru Su, and Yehong Zhuo. "Necroptosis: A Novel Pathway in Neuroinflammation." Frontiers in Pharmacology 12 (July 12, 2021). http://dx.doi.org/10.3389/fphar.2021.701564.
Full textAbstract:
Neuroinflammation is a complex inflammatory process in the nervous system that is expected to play a significant role in neurological diseases. Necroptosis is a kind of necrosis that triggers innate immune responses by rupturing dead cells and releasing intracellular components; it can be caused by Toll-like receptor (TLR)-3 and TLR-4 agonists, tumor necrosis factor (TNF), certain microbial infections, and T cell receptors. Necroptosis signaling is modulated by receptor-interacting protein kinase (RIPK) 1 when the activity of caspase-8 becomes compromised. Activated death receptors (DRs) cause the activation of RIPK1 and the RIPK1 kinase activity-dependent formation of an RIPK1-RIPK3-mixed lineage kinase domain-like protein (MLKL), which is complex II. RIPK3 phosphorylates MLKL, ultimately leading to necrosis through plasma membrane disruption and cell lysis. Current studies suggest that necroptosis is associated with the pathogenesis of neuroinflammatory diseases, such as Alzheimer’s disease, Parkinson’s disease, and traumatic brain injury. Inhibitors of necroptosis, such as necrostatin-1 (Nec-1) and stable variant of Nec (Nec-1s), have been proven to be effective in many neurological diseases. The purpose of this article is to illuminate the mechanism underlying necroptosis and the important role that necroptosis plays in neuroinflammatory diseases. Overall, this article shows a potential therapeutic strategy in which targeting necroptotic factors may improve the pathological changes and clinical symptoms of neuroinflammatory disorders.
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Kluck, George E. G., Alexander S. Qian, Emmanuel H. Sakarya, Henry Quach, Yak D. Deng, and Bernardo L. Trigatti. "Apolipoprotein A1 Protects Against Necrotic Core Development in Atherosclerotic Plaques: PDZK1-Dependent HDL (High-Density Lipoprotein) Suppression of Necroptosis in Macrophages." Arteriosclerosis, Thrombosis, and Vascular Biology, November 10, 2022. http://dx.doi.org/10.1161/atvbaha.122.318062.
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BACKGROUND: Atherosclerosis is a chronic disease affecting artery wall and a major contributor to cardiovascular diseases. Large necrotic cores increase risk of plaque rupture leading to thrombus formation. Necrotic cores are rich in debris from dead macrophages. Programmed necrosis (necroptosis) contributes to necrotic core formation. HDL (high-density lipoprotein) exerts direct atheroprotective effects on different cells within atherosclerotic plaques. Some of these depend on the SR-B1 (scavenger receptor class B type 1) and the adapter protein PDZK1 (postsynaptic density protein/Drosophila disc-large protein/Zonula occludens protein containing 1). However, a role for HDL in protecting against necroptosis and necrotic core formation in atherosclerosis is not completely understood. METHODS: Low-density lipoprotein receptor–deficient mice engineered to express different amounts of ApoA1 (apolipoprotein A1), or to lack PDZK1 were fed a high fat diet for 10 weeks. Atherosclerotic plaque areas, necrotic cores, and key necroptosis mediators, RIPK3 (receptor interacting protein kinase 3), and MLKL (mixed lineage kinase domain-like protein) were characterized. Cultured macrophages were treated with HDL to determine its effects, as well as the roles of SR-B1, PDZK1, and the PI3K (phosphoinositide 3-kinase) signaling pathway on necroptotic cell death. RESULTS: Genetic overexpression reduced, and ApoA1 knockout increased necrotic core formation and RIPK3 and MLKL within atherosclerotic plaques. Macrophages were protected against necroptosis by HDL and this protection required SR-B1, PDZK1, and PI3K/Akt pathway. PDZK1 knockout increased atherosclerosis in LDLR KO mice, increasing necrotic cores and phospho-MLKL; both of which were reversed by restoring PDZK1 in BM-derived cells. CONCLUSIONS: Our findings demonstrate that HDL in vitro and ApoA1, in vivo, protect against necroptosis in macrophages and necrotic core formation in atherosclerosis, suggesting a pathway that could be a target for the treatment of atherosclerosis.
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Xiao, Peng, Changhua Wang, Jie Li, et al. "COP9 Signalosome Suppresses RIPK1-RIPK3–Mediated Cardiomyocyte Necroptosis in Mice." Circulation: Heart Failure 13, no. 8 (2020). http://dx.doi.org/10.1161/circheartfailure.120.006996.
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Background: Mechanisms governing the induction of heart failure by the impairment of autophagy and the ubiquitin-proteasome system and the molecular pathways to cardiomyocyte necrosis remain incompletely understood. COPS8 is an essential subunit of the COP9 (COnstitutive Photomorphogenesis 9) signalosome, a key regulator of ubiquitination. Mice with cardiomyocyte-restricted knockout of Cops8 (Cops8-cko) show autophagic and ubiquitin-proteasome system malfunction and massive cardiomyocyte necrosis followed by acute heart failure and premature death, providing an excellent animal model to address the mechanistic gaps specified above. This study was conducted to determine the nature and underlying mechanisms of the cardiomyocyte necrosis in Cops8-cko mice. Methods and Results: Compared with littermate control mice, myocardial protein levels of key factors in the necroptotic pathway (RIPK1 [receptor-interacting protein kinase 1], RIPK3, MLKL [mixed lineage kinase-like], the RIPK1-bound RIPK3), protein carbonyls, full-length Casp8 (caspase 8), and BCL2, as well as histochemical staining of superoxide anions were significantly higher but the cleaved Casp8 and the Casp8 activity were significantly lower in Cops8-cko mice. In vivo cardiomyocyte uptake of Evan’s blue dye was used as an indicator of necrosis. Cops8-cko mice treated with a RIPK1 kinase inhibitor (Nec-1 [Necrostatin-1]) showed less Evans blue dye uptake (0.005% versus 0.20%; P <0.0001) and longer median lifespan (32.5 versus 27 days; P <0.01) than those treated with vehicle control. RIPK3 haploinsufficiency showed similar rescuing effects on Cops8-cko but Cyclophilin D deficiency did the opposite. Conclusions: Cardiac Cops8/COP9 signalosome malfunction causes RIPK1-RIPK3 dependent, but mitochondrial permeability transition pore independent, cardiomyocyte necroptosis in mice and the COP9 signalosome plays an indispensable role in suppressing cardiomyocyte necroptosis.
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Jayaraman, Anusha, Thein Than Htike, Rachel James, Carmen Picon, and Richard Reynolds. "TNF-mediated neuroinflammation is linked to neuronal necroptosis in Alzheimer's disease hippocampus." Acta Neuropathologica Communications 9, no. 1 (2021). http://dx.doi.org/10.1186/s40478-021-01264-w.
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AbstractThe pathogenetic mechanisms underlying neuronal death and dysfunction in Alzheimer’s disease (AD) remain unclear. However, chronic neuroinflammation has been implicated in stimulating or exacerbating neuronal damage. The tumor necrosis factor (TNF) superfamily of cytokines are involved in many systemic chronic inflammatory and degenerative conditions and are amongst the key mediators of neuroinflammation. TNF binds to the TNFR1 and TNFR2 receptors to activate diverse cellular responses that can be either neuroprotective or neurodegenerative. In particular, TNF can induce programmed necrosis or necroptosis in an inflammatory environment. Although activation of necroptosis has recently been demonstrated in the AD brain, its significance in AD neuron loss and the role of TNF signaling is unclear. We demonstrate an increase in expression of multiple proteins in the TNF/TNF receptor-1-mediated necroptosis pathway in the AD post-mortem brain, as indicated by the phosphorylation of RIPK3 and MLKL, predominantly observed in the CA1 pyramidal neurons. The density of phosphoRIPK3 + and phosphoMLKL + neurons correlated inversely with total neuron density and showed significant sexual dimorphism within the AD cohort. In addition, apoptotic signaling was not significantly activated in the AD brain compared to the control brain. Exposure of human iPSC-derived glutamatergic neurons to TNF increased necroptotic cell death when apoptosis was inhibited, which was significantly reversed by small molecule inhibitors of RIPK1, RIPK3, and MLKL. In the post-mortem AD brain and in human iPSC neurons, in response to TNF, we show evidence of altered expression of proteins of the ESCRT III complex, which has been recently suggested as an antagonist of necroptosis and a possible mechanism by which cells can survive after necroptosis has been triggered. Taken together, our results suggest that neuronal loss in AD is due to TNF-mediated necroptosis rather than apoptosis, which is amenable to therapeutic intervention at several points in the signaling pathway.
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Altman, Aaron M., Michael J. Miller, Jamil Mahmud, Nicholas A. Smith, and Gary C. Chan. "Human Cytomegalovirus-Induced Autophagy Prevents Necroptosis of Infected Monocytes." Journal of Virology 94, no. 22 (2020). http://dx.doi.org/10.1128/jvi.01022-20.
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ABSTRACT Key to the viral dissemination strategy of human cytomegalovirus (HCMV) is the induction of monocyte survival, where monocytes are normally short-lived cells. Autophagy is a cellular process that preserves cellular homeostasis and promotes cellular survival during times of stress. We found that HCMV rapidly induced autophagy within infected monocytes. The early induction of autophagy during HCMV infection was distinctly required for the survival of HCMV-infected monocytes, as repression of autophagosome formation led to cellular death of infected cells but had no effect on the viability of uninfected monocytes. The inhibition of caspases was insufficient to rescue cell viability of autophagy-repressed infected monocytes, suggesting that autophagy was not protecting cells from apoptosis. Accordingly, we found that HCMV blocked the activation of caspase 8, which was maintained in the presence of autophagy inhibitors. Necroptosis is an alternative form of cell death triggered when apoptosis is impeded and is dependent on RIPK3 phosphorylation of MLKL. Although we found that HCMV activated RIP3K upon infection, MLKL was not activated. However, inhibition of autophagy removed the block in RIPK3 phosphorylation of MLKL, suggesting that autophagy was protecting infected monocytes from undergoing necroptosis. Indeed, survival of autophagy-inhibited HCMV-infected monocytes was rescued when MLKL and RIPK3 were suppressed. Taken together, these data indicate that HCMV induces autophagy to prevent necroptotic cell death in order to ensure the survival of infected monocytes and thus facilitate viral dissemination within the host. IMPORTANCE Human cytomegalovirus (HCMV) infection is endemic throughout the world, with a seroprevalence of 40 to 100% depending on geographic location. HCMV infection is generally asymptomatic, but can cause severe inflammatory organ diseases in immunocompromised individuals. The broad array of organ diseases caused by HCMV is directly linked to the systematic spread of the virus mediated by monocytes. Monocytes are naturally programmed to undergo apoptosis, which is rapidly blocked by HCMV to ensure the survival and dissemination of infected monocytes to different organ sites. In this work, we demonstrate infected monocytes also initiate necroptosis as a “trap door” death pathway in response to HCMV subversion of apoptosis. HCMV then activates cellular autophagy as a countermeasure to prevent the execution of necroptosis, thereby promoting the continued survival of infected monocytes. Elucidating the mechanisms by which HCMV stimulates monocyte survival is an important step to the development of novel anti-HCMV drugs that prevent the spread of infected monocytes.
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Karunakaran, Denuja, My-Anh Nguyen, Michele Geoffrion та ін. "RIPK1 Expression Associates with Inflammation in Early Atherosclerosis in Humans and Can be Therapeutically Silenced to Reduce NF-κB Activation and Atherogenesis in Mice". Circulation, 23 листопада 2020. http://dx.doi.org/10.1161/circulationaha.118.038379.
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Background: Chronic activation of the innate immune system drives inflammation and contributes directly to atherosclerosis. Previously, we showed that macrophages in the atherogenic plaque undergo RIPK3-MLKL-dependent programmed necroptosis in response to sterile ligands such as oxidized LDL and damage-associated patterns (DAMPs) and necroptosis is active in advanced atherosclerotic plaques. Upstream of the RIPK3-MLKL necroptotic machinery lies RIPK1, which acts as a master switch that controls whether the cell undergoes NFκB-dependent inflammation, caspase-dependent apoptosis or necroptosis in response to extracellular stimuli. We therefore set out to investigate the role of RIPK1 in the development of atherosclerosis, which is largely driven by NFκB-dependent inflammation at early stages. We hypothesize that, unlike RIPK3 and MLKL, RIPK1 primarily drives NFκB-dependent inflammation in early atherogenic lesions and knocking down RIPK1 will reduce inflammatory cell activation and protect against the progression of atherosclerosis. Methods: We examined expression of RIPK1 protein and mRNA in both human and mouse atherosclerotic lesions, and using loss-of-function approaches in vitro in macrophages and endothelial cells to measure inflammatory responses. We administered weekly injections of RIPK1 anti-sense oligonucleotides (ASO) to Apoe -/- mice fed a cholesterol-rich (Western) diet for 8 weeks. Results: We find RIPK1 expression is abundant in early-stage atherosclerotic lesions in both humans and mice. Treatment with RIPK1 ASOs led to a reduction in aortic sinus and en face lesion areas (47.2% or 58.8% decrease relative to control, p<0.01) and plasma inflammatory cytokines (IL-1α, IL-17A, p<0.05) compared to controls. RIPK1 knockdown in macrophages decreased inflammatory genes (NFκB, TNFα, IL-1α) and in vivo LPS- and atherogenic diet-induced NF-κB activation. In endothelial cells, knockdown of RIPK1 prevented NF-κB translocation to the nucleus in response to TNFα, where accordingly there was a reduction in gene expression of IL1B, E-selectin and monocyte attachment. Conclusions: We have identified RIPK1 as a central driver of inflammation in atherosclerosis by its ability to activate the NF-κB pathway and promote inflammatory cytokine release. Given the high levels of RIPK1 expression in human atherosclerotic lesions, our study suggests RIPK1 as a future therapeutic target to reduce residual inflammation in patients at high risk of coronary artery disease.
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Fan, Guo-Chang, Dongze Qin, Xiaohong Wang, Liwang Yang, Wei Huang, and Yigang Wang. "Abstract 325: miR-223 Negatively Regulate Ischemia/Reperfusion-induced Cardiac Necroptosis." Circulation Research 117, suppl_1 (2015). http://dx.doi.org/10.1161/res.117.suppl_1.325.
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It is well known that myocardial ischemia/reperfusion (I/R) causes myocyte apoptosis and necrosis. For many years, apoptosis was considered to be the only form of gene-regulated cell death, whereas necrosis was thought as a passive accidental cell death. Recent studies, however, clearly indicate that necrosis can be controlled by multiple genes, and RIPK1/3-regulated necrosis, called necroptosis, has gained well attention. We and others previously showed that miR-223, an anti-inflammatory miRNA, was greatly up-regulated in the infarcted heart. To test whether miR-223 regulates I/R-induced cardiac necroptosis, transgenic (TG) mice with cardiac-specific overexpression of miR-223 and miR-223 knockout (KO) mice were used and underwent global no-flow I/R (30min/1h). We observed that TG hearts displayed the better recovery of contractile function (+dP/dt: 92±4%), compared with wild-type (WT) hearts (65±3%). This improvement was accompanied with a 2.4-fold decrease in lactate dehydrogenase (LDH), a marker of necrosis, released from TG hearts, comparable to WTs. By contrast, KO-hearts showed the worse recovery of contractile function (+dP/dt: 41± 3%) than WTs (+dP/dt: 70±4%), and increased LDH release (3-fold). Notably, both TUNEL-staining and DNA fragmentation analysis for cardiac apoptosis showed no difference between groups. Western-blotting assays showed that protein levels of RIPK1, RIPK3 and MLKL, three known mediators in the necroptotic pathway, were reduced in TG hearts, whereas they were increased in KOs, compared to respective WT controls upon I/R. Furthermore, pre-injection of NEC-1s (1.65mg/kg), a specific inhibitor of necroptosis, into miR-223-KO mice, significantly improved cardiac function recovery during I/R, compared to saline-injected KOs. To elucidate the mechanisms underlying the miR-223-mediated cardiac necroptosis, we performed a series of experiments (bioinformatics, luciferase report assay, and western-blotting). Our results showed that miR-223 negatively regulated the expression of TNFR1 and death receptor 6 (DR6), two activators of the necroptotic pathway. Put together, this study indicates that miR-223 could control I/R-induced cardiac necroptosis via targeting the DR6/TNFR1-RIP1/3-MLKL pathway.
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Patton, Timothy, Zhe Zhao, Xin Yi Lim, et al. "RIPK3 controls MAIT cell accumulation during development but not during infection." Cell Death & Disease 14, no. 2 (2023). http://dx.doi.org/10.1038/s41419-023-05619-0.
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AbstractCell death mechanisms in T lymphocytes vary according to their developmental stage, cell subset and activation status. The cell death control mechanisms of mucosal-associated invariant T (MAIT) cells, a specialized T cell population, are largely unknown. Here we report that MAIT cells express key necroptotic machinery; receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like (MLKL) protein, in abundance. Despite this, we discovered that the loss of RIPK3, but not necroptotic effector MLKL or apoptotic caspase-8, specifically increased MAIT cell abundance at steady-state in the thymus, spleen, liver and lungs, in a cell-intrinsic manner. In contrast, over the course of infection with Francisella tularensis, RIPK3 deficiency did not impact the magnitude of the expansion nor contraction of MAIT cell pools. These findings suggest that, distinct from conventional T cells, the accumulation of MAIT cells is restrained by RIPK3 signalling, likely prior to thymic egress, in a manner independent of canonical apoptotic and necroptotic cell death pathways.
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Rodriguez, Diego A., Giovanni Quarato, Swantje Liedmann, et al. "Caspase-8 and FADD prevent spontaneous ZBP1 expression and necroptosis." Proceedings of the National Academy of Sciences 119, no. 41 (2022). http://dx.doi.org/10.1073/pnas.2207240119.
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The absence of Caspase-8 or its adapter, Fas-associated death domain (FADD), results in activation of receptor interacting protein kinase-3 (RIPK3)- and mixed-lineage kinase-like (MLKL)–dependent necroptosis in vivo. Here, we show that spontaneous activation of RIPK3, phosphorylation of MLKL, and necroptosis in Caspase-8– or FADD-deficient cells was dependent on the nucleic acid sensor, Z-DNA binding protein-1 (ZBP1). We genetically engineered a mouse model by a single insertion of FLAG tag onto the N terminus of endogenous MLKL ( Mlkl FLAG/FLAG ), creating an inactive form of MLKL that permits monitoring of phosphorylated MLKL without activating necroptotic cell death. Casp8 −/− Mlkl FLAG/FLAG mice were viable and displayed phosphorylated MLKL in a variety of tissues, together with dramatically increased expression of ZBP1 compared to Casp8 +/+ mice. Studies in vitro revealed an increased expression of ZBP1 in cells lacking FADD or Caspase-8, which was suppressed by reconstitution of Caspase-8 or FADD. Ablation of ZBP1 in Casp8 −/− Mlkl FLAG/FLAG mice suppressed spontaneous MLKL phosphorylation in vivo. ZBP1 expression and downstream activation of RIPK3 and MLKL in cells lacking Caspase-8 or FADD relied on a positive feedback mechanism requiring the nucleic acid sensors cyclic GMP-AMP synthase (cGAS), stimulator of interferon genes (STING), and TBK1 signaling pathways. Our study identifies a molecular mechanism whereby Caspase-8 and FADD suppress spontaneous necroptotic cell death.
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Jonczyk, Agnieszka Walentyna, Katarzyna Karolina Piotrowska-Tomala та Dariusz Jan Skarzynski. "Effects of prostaglandin F2α (PGF2α) on cell-death pathways in the bovine corpus luteum (CL)". BMC Veterinary Research 15, № 1 (2019). http://dx.doi.org/10.1186/s12917-019-2167-3.
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Abstract Background Prostaglandin F2α (PGF2α) may differentially affect viability of luteal cells by inducing either proliferation or cell death (via apoptosis or necroptosis). The diverse effects of PGF2α may depend on its local vs. systemic actions. In our study, we determined changes in expression of genes related to: (i) apoptosis: caspase (CASP) 3, CASP8, BCL2 associated X (BAX), B-cell lymphoma 2 (BCL2) and (ii) necroptosis: receptor-interacting protein kinase (RIPK) 1, RIPK3, cylindromatosis (CYLD), and mixed lineage kinase domain-like (MLKL) in the early and mid-stage corpus luteum (CL) that accompany local (intra-CL) vs. systemic (i.m.) analogue of PGF2α (aPGF2α) actions. Cows at day 4 (n = 24) or day 10 (n = 24) of the estrous cycle were treated by injections as follows: (1) systemic saline, (2) systemic aPGF2α (25 mg; Dinoprost), (3) local saline, (4) local aPGF2α (2.5 mg; Dinoprost). After 4 h, CLs were collected by ovariectomy. Expression levels of mRNA and protein were investigated by RT-q PCR, Western blotting and immunohistochemistry, respectively. Results We found that local and systemic administration of aPGF2α in the early-stage CL resulted in decreased expression of CASP3 (P < 0.01), but CASP8 mRNA expression was up-regulated (P < 0.05). However, the expression of CASP3 was up-regulated after local aPGF2α treatment in the middle-stage CL, whereas systemic aPGF2α administration increased both CASP3 and CASP8 expression (P < 0.01). Moreover, we observed that both local and systemic aPGF2α injections increased RIPK1, RIPK3 and MLKL expression in the middle-stage CL (P < 0.05) while CYLD expression was markedly higher after i.m. aPGF2α injections (P < 0.001). Moreover, we investigated the localization of necroptotic factors (RIPK1, RIPK3, CYLD and MLKL) in bovine CL tissue after local and systemic aPGF2α injections in the bovine CL. Conclusion Our results demonstrated for the first time that genes related to cell death pathways exhibit stage-specific responses to PGF2α administration depending on its local or systemic actions. Locally-acting PGF2α plays a luteoprotective role by inhibiting apoptosis and necroptosis in the early CL. Necroptosis is a potent mechanism responsible for structural CL regression during PGF2α-induced luteolysis in cattle.
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Puertas-Neyra, Kevin, Nadia Galindo-Cabello, Leticia A. Hernández-Rodríguez, et al. "Programmed Cell Death and Autophagy in an in vitro Model of Spontaneous Neuroretinal Degeneration." Frontiers in Neuroanatomy 16 (February 11, 2022). http://dx.doi.org/10.3389/fnana.2022.812487.
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Retinal neurodegenerative diseases are the leading causes of visual impairment and irreversible blindness worldwide. Although the retinal response to injury remains closely similar between different retinal neurodegenerative diseases, available therapeutic alternatives are only palliative, too expensive, or very specific, such as gene therapy. In that sense, the development of broad-spectrum neuroprotective therapies seems to be an excellent option. In this regard, it is essential to identify molecular targets involved in retinal degeneration, such as cell death mechanisms. Apoptosis has been considered as the primary cell death mechanism during retinal degeneration; however, recent studies have demonstrated that the only use of anti-apoptotic drugs is not enough to confer good neuroprotection in terms of cell viability and preservation. For that reason, the interrelationship that exists between apoptosis and other cell death mechanisms needs to be characterized deeply to design future therapeutic options that simultaneously block the main cell death pathways. In that sense, the study aimed to characterize the programmed cell death (in terms of apoptosis and necroptosis) and autophagy response and modulation in retinal neurodegenerative diseases, using an in vitro model of spontaneous retinal neurodegeneration. For that purpose, we measured the mRNA relative expression through qPCR of a selected pool of genes involved in apoptosis (BAX, BCL2, CASP3, CASP8, and CASP9), necroptosis (MLKL, RIPK1, and RIPK3), and autophagy (ATG7, BCLIN1, LC3B, mTOR, and SQSTM1); besides, the immunoexpression of their encoding proteins (Casp3, MLKL, RIPK1, LC3B, and p62) were analyzed using immunohistochemistry. Our results showed an increase of pro-apoptotic and pro-necroptotic related genes and proteins during in vitro retinal neurodegeneration. Besides, we describe for the first time the modulation between programmed cell death mechanisms and autophagy in an in vitro retinal neurodegeneration model. This study reinforces the idea that cell death mechanisms are closely interconnected and provides new information about molecular signaling and autophagy along the retinal degeneration process.
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Molnár, Tamás, Anett Mázló, Vera Tslaf, Attila Gábor Szöllősi, Gabriella Emri, and Gábor Koncz. "Current translational potential and underlying molecular mechanisms of necroptosis." Cell Death & Disease 10, no. 11 (2019). http://dx.doi.org/10.1038/s41419-019-2094-z.
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Abstract Cell death has a fundamental impact on the evolution of degenerative disorders, autoimmune processes, inflammatory diseases, tumor formation and immune surveillance. Over the past couple of decades extensive studies have uncovered novel cell death pathways, which are independent of apoptosis. Among these is necroptosis, a tightly regulated, inflammatory form of cell death. Necroptosis contribute to the pathogenesis of many diseases and in this review, we will focus exclusively on necroptosis in humans. Necroptosis is considered a backup mechanism of apoptosis, but the in vivo appearance of necroptosis indicates that both caspase-mediated and caspase-independent mechanisms control necroptosis. Necroptosis is regulated on multiple levels, from the transcription, to the stability and posttranslational modifications of the necrosome components, to the availability of molecular interaction partners and the localization of receptor-interacting serine/threonine-protein kinase 1 (RIPK1), receptor-interacting serine/threonine-protein kinase 3 (RIPK3) and mixed lineage kinase domain-like protein (MLKL). Accordingly, we classified the role of more than seventy molecules in necroptotic signaling based on consistent in vitro or in vivo evidence to understand the molecular background of necroptosis and to find opportunities where regulating the intensity and the modality of cell death could be exploited in clinical interventions. Necroptosis specific inhibitors are under development, but >20 drugs, already used in the treatment of various diseases, have the potential to regulate necroptosis. By listing necroptosis-modulated human diseases and cataloging the currently available drug-repertoire to modify necroptosis intensity, we hope to kick-start approaches with immediate translational potential. We also indicate where necroptosis regulating capacity should be considered in the current applications of these drugs.