The necroptotic cell death pathway operates in megakaryocytes, but not in platelet synthesis

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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Membrane permeabilization is mediated by distinct epitopes in mouse and human orthologs of the necroptosis effector, MLKL

10.1101/2021.05.03.442385 ◽

2021 ◽

Author(s):

Ashish Sethi ◽

Christopher R Horne ◽

Cheree Fitzgibbon ◽

Karyn L Wilde ◽

Katherine A Davies ◽

...

Keyword(s):

Innate Immunity ◽

Cell Death ◽

Inflammatory Diseases ◽

Upstream Regulator ◽

Cell Death Pathway ◽

Lytic Function ◽

Human Orthologs ◽

Binding Epitope ◽

Kinase Phosphorylation ◽

Terminal Effector

Necroptosis is a lytic programmed cell death pathway with origins in innate immunity that is frequently dysregulated in inflammatory diseases. The terminal effector of the pathway, MLKL, is licensed to kill following phosphorylation of its pseudokinase domain by the upstream regulator, RIPK3 kinase. Phosphorylation provokes the unleashing of MLKL's N-terminal four-helix bundle (4HB or HeLo) domain, which binds and permeabilizes the plasma membrane to cause cell death. The precise mechanism by which the 4HB domain permeabilizes membranes, and how the mechanism differs between species, remains unclear. Here, we identify the membrane binding epitope of mouse MLKL using NMR spectroscopy. Using liposome permeabilization and cell death assays, we validate K69 in the α3 helix, W108 in the α4 helix, and R137/Q138 in the first brace helix as crucial residues for necroptotic signaling. This epitope differs from the phospholipid binding site reported for human MLKL, which comprises basic residues primarily located in the α1 and α2 helices. In further contrast to human and plant MLKL orthologs, in which the α3-α4 loop forms a helix, this loop is unstructured in mouse MLKL in solution. Together, these findings illustrate the versatility of the 4HB domain fold, whose lytic function can be mediated by distinct epitopes in different orthologs.

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Human RIPK3 maintains MLKL in an inactive conformation prior to cell death by necroptosis

Nature Communications ◽

10.1038/s41467-021-27032-x ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Yanxiang Meng ◽

Katherine A. Davies ◽

Cheree Fitzgibbon ◽

Samuel N. Young ◽

Sarah E. Garnish ◽

...

Keyword(s):

Cell Death ◽

Host Defense ◽

Inflammatory Diseases ◽

Kinase Domain ◽

Human Cells ◽

Complex Assembly ◽

Inactive Conformation ◽

Upstream Regulator ◽

Widespread Interest ◽

Terminal Effector

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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RIPK3 facilitates host and pathogen interactions after oral Toxoplasma gondii infection

10.1101/2020.08.04.237362 ◽

2020 ◽

Cited By ~ 1

Author(s):

Patrick W. Cervantes ◽

Laura J. Knoll

Keyword(s):

Cell Death ◽

Toxoplasma Gondii ◽

Inflammatory Cell ◽

Immune Cell ◽

Inflammatory Responses ◽

Parasite Burden ◽

Kinase Domain ◽

Protection Mechanism ◽

Cell Death Pathway ◽

Toxoplasma Gondii Infection

AbstractToxoplasma gondii infection activates pattern recognition receptor (PRR) pathways that drive innate inflammatory responses to control infection. Necroptosis is a pro-inflammatory cell death pathway apart of the innate immune response that has evolved to control pathogenic infection. In this study we further defined the role of Z-DNA binding protein 1 (ZBP1) as a PRR and assessed its contribution to necroptosis as a host protection mechanism to T. gondii infection. We found that ZBP1 does not induce pro-inflammatory necroptosis cell death and ZBP1 null mice have reduced survival after oral T. gondii infection. In contrast, mice deleted in receptor-interacting serine/threonine-protein kinase 3 (RIPK3-/-), a central mediator of necroptosis, have significantly improved survival after oral T. gondii infection even with higher parasite burden. The physiological consequences of RIPK3 activity did not show any differences in intestine villi immunopathology but RIPK3-/- mice showed higher immune cell infiltration and edema in the lamina propria. The contribution of necroptosis to host survival was clarified with mixed lineage kinase domain like pseudokinase null (MLKL-/-) mice. We found MLKL-/- mice to succumb to oral T. gondii infection the same as wild type mice, indicating necroptosis-independent RIPK3 activity impacts host survival. These results provide new insights on the impacts of pro-inflammatory cell death pathways as a mechanism of host defense to oral T. gondii infection.

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A toolbox for imaging RIPK1, RIPK3 and MLKL in mouse and human cells

10.1101/2020.10.26.356063 ◽

2020 ◽

Author(s):

André L. Samson ◽

Cheree Fitzgibbon ◽

Komal M. Patel ◽

Joanne M. Hildebrand ◽

Lachlan W. Whitehead ◽

...

Keyword(s):

Inflammatory Cell ◽

Immunofluorescence Microscopy ◽

Wild Type ◽

Robust Detection ◽

The Core ◽

Cell Death Pathway ◽

Mouse Cells ◽

Endogenous Proteins ◽

Human And Mouse ◽

Terminal Effector

ABSTRACTNecroptosis is a lytic, inflammatory cell death pathway that is dysregulated in many human pathologies. The pathway is executed by a core machinery comprising the RIPK1 and RIPK3 kinases, which assemble into necrosomes in the cytoplasm, and the terminal effector pseudokinase, MLKL. RIPK3-mediated phosphorylation of MLKL induces oligomerization and translocation to the plasma membrane where MLKL accumulates as hotspots and perturbs the lipid bilayer to cause death. The precise choreography of events in the pathway, where they occur within cells, and pathway differences between species, are of immense interest. However, they have been poorly characterized due to a dearth of validated antibodies for microscopy studies. Here, we describe a toolbox of antibodies for immunofluorescent detection of the core necroptosis effectors, RIPK1, RIPK3 and MLKL, and their phosphorylated forms, in human and mouse cells. By comparing reactivity with endogenous proteins in wild-type cells and knockout controls in basal and necroptosis-inducing conditions, we characterise the specificity of frequently-used commercial and recently-developed antibodies for detection of necroptosis signaling events. Importantly, our findings demonstrate that not all frequently-used antibodies are suitable for monitoring necroptosis by immunofluorescence microscopy, and methanol-is preferable to paraformaldehyde-fixation for robust detection of specific RIPK1, RIPK3 and MLKL signals.

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Necroptosis in Immuno-Oncology and Cancer Immunotherapy

Cells ◽

10.3390/cells9081823 ◽

2020 ◽

Vol 9 (8) ◽

pp. 1823 ◽

Cited By ~ 2

Author(s):

Jenny Sprooten ◽

Pieter De Wijngaert ◽

Isaure Vanmeerbeek ◽

Shaun Martin ◽

Peter Vangheluwe ◽

...

Keyword(s):

Cell Death ◽

Cancer Cells ◽

Cancer Immunotherapy ◽

Inflammatory Cell ◽

Necrotic Cell Death ◽

Immunogenic Cell Death ◽

Necrotic Cell ◽

Cell Death Pathway ◽

Immune Checkpoint Blockers ◽

Anticancer Immunity

Immune-checkpoint blockers (ICBs) have revolutionized oncology and firmly established the subfield of immuno-oncology. Despite this renaissance, a subset of cancer patients remain unresponsive to ICBs due to widespread immuno-resistance. To “break” cancer cell-driven immuno-resistance, researchers have long floated the idea of therapeutically facilitating the immunogenicity of cancer cells by disrupting tumor-associated immuno-tolerance via conventional anticancer therapies. It is well appreciated that anticancer therapies causing immunogenic or inflammatory cell death are best positioned to productively activate anticancer immunity. A large proportion of studies have emphasized the importance of immunogenic apoptosis (i.e., immunogenic cell death or ICD); yet, it has also emerged that necroptosis, a programmed necrotic cell death pathway, can also be immunogenic. Emergence of a proficient immune profile for necroptosis has important implications for cancer because resistance to apoptosis is one of the major hallmarks of tumors. Putative immunogenic or inflammatory characteristics driven by necroptosis can be of great impact in immuno-oncology. However, as is typical for a highly complex and multi-factorial disease like cancer, a clear cause versus consensus relationship on the immunobiology of necroptosis in cancer cells has been tough to establish. In this review, we discuss the various aspects of necroptosis immunobiology with specific focus on immuno-oncology and cancer immunotherapy.

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Necroptosis in Intestinal Inflammation and Cancer: New Concepts and Therapeutic Perspectives

10.20944/preprints202009.0084.v1 ◽

2020 ◽

Author(s):

Anna Negroni ◽

Eleonora Colantoni ◽

Salvatore Cucchiara ◽

Laura Stronati

Keyword(s):

Cell Death ◽

Intestinal Epithelium ◽

Inflammatory Cell ◽

Intestinal Inflammation ◽

Inflammatory Diseases ◽

Kinase Domain ◽

Interacting Protein ◽

New Concepts ◽

Independent Form ◽

Cellular Turnover

Necroptosis is a caspases-independent form of programmed cell death exhibiting intermediate features between necrosis and apoptosis. Albeit some physiological roles during embryonic development, tissue homeostasis and innate immune response are documented, necroptosis is mainly considered a pro-inflammatory cell death. Key actors of necroptosis are the receptor-interacting-protein-kinases, RIPK1 and RIPK3, and their target, the mixed-lineage-kinase-domain-like protein, MLKL. The intestinal epithelium has one of the highest rates of cellular turnover in a process that is tightly regulated. Altered necroptosis at the intestinal epithelium leads to uncontrolled microbial translocation and deleterious inflammation. Indeed, necroptosis has been associated to chronic inflammatory diseases and cancer. Drugs that inhibit necroptosis could, therefore, be used therapeutically for the treatment of these diseases, and researches to develop such inhibitors are already underway. In this Review, we outline pathways for necroptosis and its role in chronic inflammation and cancer. We also discuss current and developing therapies that target necroptosis machinery.

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Deletion of Bcl-X in the Megakaryocyte Compartment Results in Profound Thrombocytopenia Caused by Impaired Platelet Production and Survival.

Blood ◽

10.1182/blood.v114.22.1495.1495 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1495-1495

Author(s):

Chloé James ◽

Emma C. Josefsson ◽

Michael J. White ◽

Katya J. Henley ◽

Benjamin T. Kile

Keyword(s):

Cell Death ◽

Life Span ◽

Conflicts Of Interest ◽

Platelet Factor ◽

Apoptosis Pathway ◽

Platelet Production ◽

Cell Death Pathway ◽

Platelet Survival ◽

Intrinsic Apoptosis Pathway ◽

Megakaryocyte Progenitors

Abstract Abstract 1495 Poster Board I-518 Recent studies have suggested a role for the intrinsic apoptosis pathway in megakaryocytic differentiation and platelet shedding. The intrinsic pathway is regulated by the Bcl-2 family of pro- and anti- cell death proteins. We recently demonstrated that platelet life span is controlled by an intrinsic cell death pathway, whereby the anti-apoptotic protein Bcl-xL constrains the pro-apoptotic activity of Bak to maintain platelet survival. As Bcl-xL is expressed in megakaryocytes, we investigated whether this protein is required for megakaryocyte survival, differentiation and/or platelet shedding. We specifically deleted the Bcl-x gene in the megakaryocyte lineage by crossing mice carrying a floxed allele of Bcl-x with mice carrying a platelet factor 4-regulated Cre transgene. Bcl-xfl/flCre+ mice were profoundly thrombocytopenic (26 ± 5 × 103/μl, n=14) compared with Bcl-xfl/flCre− animals (1157 ± 202 × 103/μl, n=13). Platelet life span in these mice was reduced to only 5 hours, as compared to 5 days in wild type littermates. This result confirmed that Bcl-xL is absolutely required for platelet survival. To determine whether Bcl-x deletion has an impact on platelet production, we analyzed the megakaryocyte compartment in Bcl-xfl/flCre+ and Bcl-xfl/flCre− mice. We observed that the number of megakaryocyte progenitors, and number of megakaryocytes in the bone marrow were increased in Bcl-xfl/flCre+ mice (23 ± 9 megakaryocyte progenitors vs 11 ± 5, and 51 ± 9 megakaryocytes vs 12 ± 1). This result suggested that Bcl-xL is not required for the survival of megakaryocytes or their progenitors. To determine whether Bcl-xL is required for the last step of megakaryocyte differentiation, i.e. platelet shedding, we cultured fetal liver cells with thrombopoietin. Large megakaryocytes were isolated after 3 days of differentiation on discontinuous bovine serum albumin gradient. They were cultured for 3 more days in the same media and the percentage of megakaryocytes displaying proplatelets was determined each day. Interestingly, Bcl-xfl/flCre+ megakaryocytes died much more quickly than Bcl-xfl/flCre− megakaryocytes, and almost none of those that survived were able to form proplatelets. Our study indicates that Bcl-xL is not only essential for platelet survival, but it is also required for the survival of mature megakaryocytes at the stage of platelet shedding. Disclosures: No relevant conflicts of interest to declare.

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Novel cell death program leads to neutrophil extracellular traps

The Journal of Cell Biology ◽

10.1083/jcb.200606027 ◽

2007 ◽

Vol 176 (2) ◽

pp. 231-241 ◽

Cited By ~ 1610

Author(s):

Tobias A. Fuchs ◽

Ulrike Abed ◽

Christian Goosmann ◽

Robert Hurwitz ◽

Ilka Schulze ◽

...

Keyword(s):

Cell Death ◽

Nadph Oxidase ◽

Neutrophil Extracellular Traps ◽

Death Process ◽

Extracellular Traps ◽

Cell Death Pathway ◽

Antimicrobial Function ◽

Cell Death Program ◽

Cell Death Process ◽

Extracellular Structures

Neutrophil extracellular traps (NETs) are extracellular structures composed of chromatin and granule proteins that bind and kill microorganisms. We show that upon stimulation, the nuclei of neutrophils lose their shape, and the eu- and heterochromatin homogenize. Later, the nuclear envelope and the granule membranes disintegrate, allowing the mixing of NET components. Finally, the NETs are released as the cell membrane breaks. This cell death process is distinct from apoptosis and necrosis and depends on the generation of reactive oxygen species (ROS) by NADPH oxidase. Patients with chronic granulomatous disease carry mutations in NADPH oxidase and cannot activate this cell-death pathway or make NETs. This novel ROS-dependent death allows neutrophils to fulfill their antimicrobial function, even beyond their lifespan.

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Location, location, location: A compartmentalized view of TNF-induced necroptotic signaling

Science Signaling ◽

10.1126/scisignal.abc6178 ◽

2021 ◽

Vol 14 (668) ◽

pp. eabc6178

Author(s):

André L. Samson ◽

Sarah E. Garnish ◽

Joanne M. Hildebrand ◽

James M. Murphy

Keyword(s):

Cell Death ◽

Protein Kinases ◽

Host Defense ◽

Kinase Domain ◽

Dependent Manner ◽

Cell Type ◽

Mixed Lineage Kinase ◽

Cell Death Pathway ◽

Subcellular Compartmentalization ◽

High Degree

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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RIPK3 Facilitates Host Resistance to Oral Toxoplasma gondii Infection

Infection and Immunity ◽

10.1128/iai.00021-21 ◽

2021 ◽

Vol 89 (5) ◽

Author(s):

Patrick W. Cervantes ◽

Bruno Martorelli Di Genova ◽

Billy Joel Erazo Flores ◽

Laura J. Knoll

Keyword(s):

Cell Death ◽

Toxoplasma Gondii ◽

Immune Cell ◽

Inflammatory Responses ◽

Parasite Burden ◽

Kinase Domain ◽

Content Type ◽

Protection Mechanism ◽

Cell Death Pathway ◽

Toxoplasma Gondii Infection

ABSTRACT Toxoplasma gondii infection activates pattern recognition receptor (PRR) pathways that drive innate inflammatory responses to control infection. Necroptosis is a proinflammatory cell death pathway apart from the innate immune response that has evolved to control pathogenic infection. In this study, we further defined the role of Z-DNA binding protein 1 (ZBP1) as a PRR and assessed its contribution to necroptosis as a host protection mechanism to T. gondii infection. We found that ZBP1 does not induce proinflammatory necroptosis cell death, and ZBP1 null mice have reduced survival after oral T. gondii infection. In contrast, mice deleted in receptor-interacting serine/threonine-protein kinase 3 (RIPK3−/−), a central mediator of necroptosis, have significantly improved survival after oral T. gondii infection without a reduction in parasite burden. The physiological consequences of RIPK3 activity did not show any differences in intestine villus immunopathology, but RIPK3−/− mice showed higher immune cell infiltration and edema in the lamina propria. The contribution of necroptosis to host survival was clarified with mixed-lineage kinase domain-like pseudokinase null (MLKL−/−) mice. We found MLKL−/− mice succumbed to oral T. gondii infection the same as wild-type mice, indicating necroptosis-independent RIPK3 activity impacts host survival. These results provide new insights on the impacts of proinflammatory cell death pathways as a mechanism of host defense to oral T. gondii infection.

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