A specific RIP3+ subpopulation of microglia promotes retinopathy through a hypoxia-triggered necroptotic mechanism
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE152928 | GEO | 2021/02/26
REPOSITORIES: GEO
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Project description:A specific RIP3+ subpopulation of microglia promotes retinopathy through a hypoxia-triggered necroptotic mechanism
| PRJNA641006 | ENA
Project description:Necroptosis is a lytic form of regulated cell death reported to contribute to inflammatory diseases of the gut, skin and lung, as well as ischemic-reperfusion injuries of the kidney, heart and brain. However, precise identification of the cells and tissues that undergo necroptotic cell death in vivo has proven challenging in the absence of robust protocols for immunohistochemical detection. Here, we provide automated immunohistochemistry protocols to detect core necroptosis regulators – Caspase-8, RIPK1, RIPK3 and MLKL – in formalin-fixed mouse and human tissues. We observed surprising heterogeneity in protein expression within tissues, whereby short-lived immune barrier cells were replete with necroptotic effectors, whereas long-lived cells lacked RIPK3 or MLKL expression. Local changes in the expression of necroptotic effectors occurred in response to insults such as inflammation, dysbiosis or immune challenge, consistent with necroptosis being dysregulated in disease contexts. These methods will facilitate the precise localisation and evaluation of necroptotic signaling in vivo.
2024-04-17 | GSE262762 | GEO
Project description:Retinal neovascularization is a leading cause of severe visual loss in humans, and molecular mechanisms of microglial activation-driven angiogenesis remain unknown. Using single-cell RNA sequencing, we identified a subpopulation of microglia named sMG2, which highly expressed necroptosis-related genes Rip3 and Mlkl. Genetic and pharmacological loss of function demonstrated that hypoxia-induced microglial activation committed to necroptosis through the RIP1/RIP3-mediated pathway. Specific deletion of Rip3 gene in microglia markedly decreased retinal neovascularization. Furthermore, hypoxia induced explosive release of abundant FGF2 in microglia through RIP3-mediated necroptosis. Importantly, blocking signaling components of the microglia necropotosis-FGF2 axis largely ablated retinal angiogenesis and combination therapy with simultaneously blocking VEGF produced synergistic antiangiogenic effects. Together, our data demonstrate that targeting the microglia necroptosis axis is an antiangiogenesis therapy for retinal neovascular diseases.
| S-EPMC7980367 | biostudies-literature
Project description:The activation of Mixed Lineage Kinase-Like (MLKL) by Receptor Interacting Protein Kinase-3 (RIPK3) results in plasma membrane (PM) disruption and a form of regulated necrosis, called necroptosis. Here we show that during necroptosis, MLKL-dependent calcium (Ca++) influx and phosphatidylserine (PS) exposure on the outer leaflet of the plasma membrane preceded loss of PM integrity. Activation of MLKL results in the generation of broken, PM “bubbles” with exposed PS that are released from the surface of the otherwise intact cell. Components of the ESCRT machinery are required for formation of these bubbles, and act to sustain survival of the cell when MLKL activation is limited or reversed. Under conditions of necroptotic cell death, ESCRT controls the duration of plasma membrane integrity. As a consequence of the action of ESCRT, cells undergoing necroptosis can express chemokines and other regulatory molecules, and promote antigenic cross-priming of CD8+ T cells.
2017-06-16 | GSE85660 | GEO
Project description:Necroptosis is a caspase-independent, pro-inflammatory mode of programmed cell death which relies on the activation of the terminal effector, MLKL, by the upstream protein kinase RIPK3. To mediate necroptosis, RIPK3 must stably interact with, and phosphorylate the pseudokinase domain of MLKL. While the precise molecular cue that prompts RIPK3 to activate MLKL is incompletely understood, it is known that RIPK3 is highly regulated by phosphorylation. Here, we sought to identify phosphorylation sites of RIPK3 and dissect their regulatory functions. Phosphoproteomics identified 21 phosphorylation sites in HT29 cells overexpressing human RIPK3. By comparing cells expressing wild-type and kinase-inactive D142N RIPK3, autophosphorylation sites and substrates of other cellular kinases were distinguished. Of these 21 phosphosites, extensive mutational analyses identified only pT224 and pS227 as crucial, synergistic sites for stable interaction with MLKL to promote necroptosis, while the recently reported activation loop phosphorylation at S164/T165 negatively regulate the kinase activity of RIPK3. Despite being able to phosphorylate MLKL to a similar or higher extent than the wild-type RIPK3, mutation of T224, S227, and RHIM in RIPK3 attenuated or compromised their abilities to mediate necroptosis. This finding highlights the stable recruitment of human MLKL by RIPK3 to the necrosome as an essential checkpoint in necroptosis signaling, which is independent from and precedes the phosphorylation of MLKL.
2022-07-30 | PXD033488 | Pride
Project description:Necroptosis and ferroptosis are two pathways of regulated cell death executed in several major cardiovascular and neurological acute and degenerative diseases. While the necroptosis program relies on activation of RIP1, RIP3 kinases and MLKL, ferroptotic death is triggered by 15- Lipoxygenase (15LO) catalyzed oxidation of arachidonoyl- (AA) or adrenoyl- (AdA) phosphatidylethanolamines (PE) controlled by the phosphatidylethanolamine-binding protein 1 (PEBP1). PEBP1 displays “regulatory” promiscuity towards multiple protein partners, including RAF1 kinase. Given a distinct structural homology between RAF1 kinase and RIP3 kinase, we hypothesized that PEBP1 may interact with RIP3 and act as a switch from necroptosis to ferroptosis. Using computational, genetic and redox lipidomics approaches, we show that PEBP1 liberated from RAF1 kinase binds and sterically inhibits RIP3 thus turning-off necroptosis. Highly expressed 15LO may outcompete and bind PEBP1 to promote AA-PE/AdA-PE oxidation and ferroptosis. Using cell- based and animal models, we identified the conditions disrupting PEBP1’s interactions with RAF1 kinase to alternatively bind/inhibit RIP3 kinase or bind/activate 15LO. We further established that PEBP1 knockdown sensitizes cells to RIP3-mediated necroptosis. These newly established regulatory functions of PEBP1 serve multiple and diverse roles across various human disease states.
2020-01-16 | ST001334 | MetabolomicsWorkbench
Project description:Necroptosis is a form of regulated necrotic cell death which promotes inflammation. In cells undergoing necroptosis, the activated RIPK1 kinase mediates the formation of RIPK1/RIPK3/MLKL complex to promote MLKL oligomerization and execution of necroptosis. RIPK1 kinase activity also promotes cell-autonomous activation of proinflammatory cytokine production in necroptosis. However, the signaling pathways downstream of RIPK1 kinase in necroptosis and how RIPK1 kinase activation controls inflammatory response induced by necroptosis are still largely unknown. Here we quantitatively measured the temporal dynamics of over 7000 confident phosphosites during necroptosis using mass spectrometry. Our study defined a RIPK1-dependent phosphorylation pattern in late necroptosis that is associated with a proinflammatory component marked by p-S473 TRIM28. We show that the activation of p38 MAPK mediated by oligomerized MLKL promotes the phosphorylation of S473 TRIM28, which in turn mediates inflammation during late necroptosis. Taken together, our study illustrates a mechanism by which p38 MAPK may be activated by oligomerized MLKL to promote inflammation in necroptosis.
2021-04-01 | GSE171214 | GEO
Project description:Necroptosis is a regulated but inflammatory form of cell death. We and others have previously reported that necroptotic cells release extracellular vesicles (EVs). We found that necroptotic EVs are loaded with proteins, including the key necroptosis executor factor, phosphorylated mixed lineage kinase domain-like (pMLKL). However, the exact necroptotic EVs proteins composition and impact have not been delineated yet. To characterize their content, EVs from necroptotic and untreated U937 cells were isolated by ultracentrifugation and analyzed by mass spectrometry. A total of 3337 proteins were identified, sharing high similarity with exosome proteome databases. Unsupervised hierarchical clustering of identified proteins distinguished between necroptotic and control EVs. A total of 353 proteins was significantly upregulated in the necroptotic EVs. Among these are MLKL and caspase-8, as validated by immunoblot. Components of ESCRTIII machinery and inflammatory signaling were found to be enriched in the necroptotic EVs, as well as yet unreported components of vesicles formation and transport and necroptosis signaling pathways. Moreover, we show that necroptotic EVs can be phagocytosed by macrophages to modulate cytokine and chemokine secretion. Finally, we reveal that necroptotic EVs contain tumor neoantigen and are enriched with proteins annotated as “antigen processing and presentation” biological process. In summary, our study reveals a new layer of regulation during the early stage of necroptosis by the secretion of specific EVs that influence their microenvironment and may instigate innate and adaptive immune responses. Future investigation will shed light on new players in the necroptosis signaling and its related EVs, and will uncover the functional tasks accomplished by the cargo of these necroptotic EVs.
2021-11-11 | PXD018258 | Pride
Project description:Caspase-8 and FADD play key roles in the regulation of cell death by necroptosis. The absence of either protein results in early embryonic lethality due to the activation of the kinase RIPK3 and its phosphorylation of the necroptosis executioner, MLKL. We genetically engineered and characterized a mouse model to monitor MLKL phosphorylation in the absence of necroptosis in vivo. Ablation of caspase-8 or FADD resulted in the transcriptional upregulation in several tissues of ZBP1, a cytosolic nucleic acid sensor capable of activating RIPK3, and ZBP1 was required for spontaneous phosphorylation of MLKL. Our findings provide a novel mechanism by which the FADD-Caspase-8 complex prevents necroptosis.
2022-09-19 | GSE208744 | GEO
Project description:We developed a genetic mouse model of chronic necroptosis activation in hepatocytes (in the presence of basal NF-kB activity) that lead to hepatocellular carcinoma. Blocking Necroptosis (RIP3) or NF-kB-(IKKβ) activation in those mice blocked hepatocarcinogenesis.
2023-07-03 | GSE166875 | GEO