ABSTRACT
The cytokine interleukin (IL)-1ß is a key mediator of antimicrobial immunity as well as autoimmune inflammation. Production of IL-1ß requires transcription by innate immune receptor signaling and maturational cleavage by inflammasomes. Whether this mechanism applies to IL-1ß production seen in T cell-driven autoimmune diseases remains unclear. Here, we describe an inflammasome-independent pathway of IL-1ß production that was triggered upon cognate interactions between effector CD4+ T cells and mononuclear phagocytes (MPs). The cytokine TNF produced by activated CD4+ T cells engaged its receptor TNFR on MPs, leading to pro-IL-1ß synthesis. Membrane-bound FasL, expressed by CD4+ T cells, activated death receptor Fas signaling in MPs, resulting in caspase-8-dependent pro-IL-1ß cleavage. The T cell-instructed IL-1ß resulted in systemic inflammation, whereas absence of TNFR or Fas signaling protected mice from CD4+ T cell-driven autoimmunity. The TNFR-Fas-caspase-8-dependent pathway provides a mechanistic explanation for IL-1ß production and its consequences in CD4+ T cell-driven autoimmune pathology.
Subject(s)
Autoimmunity/immunology , CD4-Positive T-Lymphocytes/immunology , Inflammation/pathology , Interleukin-1beta/metabolism , Myeloid Cells/metabolism , Animals , Caspase 1/genetics , Caspase 8/metabolism , Cells, Cultured , Dendritic Cells/immunology , Fas Ligand Protein/metabolism , Immunity, Innate/immunology , Inflammasomes/immunology , Inflammation/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , Mycobacterium tuberculosis/immunology , Receptors, Tumor Necrosis Factor, Type I/metabolism , Tumor Necrosis Factor-alpha/metabolismABSTRACT
NLRP3-inflammasome-driven inflammation is involved in the pathogenesis of a variety of diseases. Identification of endogenous inflammasome activators is essential for the development of new anti-inflammatory treatment strategies. Here, we identified that apolipoprotein C3 (ApoC3) activates the NLRP3 inflammasome in human monocytes by inducing an alternative NLRP3 inflammasome via caspase-8 and dimerization of Toll-like receptors 2 and 4. Alternative inflammasome activation in human monocytes is mediated by the Toll-like receptor adapter protein SCIMP. This triggers Lyn/Syk-dependent calcium entry and the production of reactive oxygen species, leading to activation of caspase-8. In humanized mouse models, ApoC3 activated human monocytes in vivo to impede endothelial regeneration and promote kidney injury in an NLRP3- and caspase-8-dependent manner. These data provide new insights into the regulation of the NLRP3 inflammasome and the pathophysiological role of triglyceride-rich lipoproteins containing ApoC3. Targeting ApoC3 might prevent organ damage and provide an anti-inflammatory treatment for vascular and kidney diseases.
Subject(s)
Acute Kidney Injury/immunology , Apolipoprotein C-III/immunology , Caspase 8/metabolism , Kidney Diseases/immunology , Monocytes/immunology , NLR Family, Pyrin Domain-Containing 3 Protein/immunology , Acute Kidney Injury/pathology , Adaptor Proteins, Signal Transducing , Animals , Apolipoprotein C-III/genetics , Cell Line , Disease Models, Animal , HEK293 Cells , Humans , Inflammasomes/immunology , Inflammation/genetics , Inflammation/immunology , Kidney Diseases/pathology , Membrane Proteins , Mice , Mice, Inbred C57BL , Mice, Inbred NOD , Mice, Knockout , Reactive Oxygen Species/metabolism , Toll-Like Receptor 2/metabolism , Toll-Like Receptor 4/metabolismABSTRACT
Macrophage activation is essential for effective immunity to infection but can also contribute to disease through incompletely understood mechanisms. In this issue of Immunity, Simpson et al. reveal that death of activated macrophages integrates extrinsic and intrinsic pathways of apoptosis that contribute to damaging host responses.
Subject(s)
Interferon-gamma , Macrophage Activation , Apoptosis , Caspase 8/metabolism , Cell Death , Interferon-gamma/metabolism , Ligands , Macrophages/immunologyABSTRACT
Cell death plays an important role during pathogen infections. Here, we report that interferon-γ (IFNγ) sensitizes macrophages to Toll-like receptor (TLR)-induced death that requires macrophage-intrinsic death ligands and caspase-8 enzymatic activity, which trigger the mitochondrial apoptotic effectors, BAX and BAK. The pro-apoptotic caspase-8 substrate BID was dispensable for BAX and BAK activation. Instead, caspase-8 reduced pro-survival BCL-2 transcription and increased inducible nitric oxide synthase (iNOS), thus facilitating BAX and BAK signaling. IFNγ-primed, TLR-induced macrophage killing required iNOS, which licensed apoptotic caspase-8 activity and reduced the BAX and BAK inhibitors, A1 and MCL-1. The deletion of iNOS or caspase-8 limited SARS-CoV-2-induced disease in mice, while caspase-8 caused lethality independent of iNOS in a model of hemophagocytic lymphohistiocytosis. These findings reveal that iNOS selectively licenses programmed cell death, which may explain how nitric oxide impacts disease severity in SARS-CoV-2 infection and other iNOS-associated inflammatory conditions.
Subject(s)
COVID-19/immunology , Caspase 8/metabolism , Interferon-gamma/metabolism , Lymphohistiocytosis, Hemophagocytic/immunology , Macrophages/immunology , Mitochondria/metabolism , SARS-CoV-2/physiology , Animals , Caspase 8/genetics , Cells, Cultured , Cytotoxicity, Immunologic , Humans , Interferon-gamma/genetics , Macrophage Activation , Mice , Mice, Inbred C57BL , Mice, Knockout , Nitric Oxide Synthase Type II/metabolism , Pathogen-Associated Molecular Pattern Molecules/immunology , Signal Transduction , bcl-2 Homologous Antagonist-Killer Protein/genetics , bcl-2 Homologous Antagonist-Killer Protein/metabolism , bcl-2-Associated X Protein/genetics , bcl-2-Associated X Protein/metabolismABSTRACT
Apoptosis can potently defend against intracellular pathogens by directly killing microbes and eliminating their replicative niche. However, the reported ability of Mycobacterium tuberculosis to restrict apoptotic pathways in macrophages in vitro has led to apoptosis being dismissed as a host-protective process in tuberculosis despite a lack of in vivo evidence. Here we define crucial in vivo functions of the death receptor-mediated and BCL-2-regulated apoptosis pathways in mediating protection against tuberculosis by eliminating distinct populations of infected macrophages and neutrophils and priming T cell responses. We further show that apoptotic pathways can be targeted therapeutically with clinical-stage compounds that antagonize inhibitor of apoptosis (IAP) proteins to promote clearance of M. tuberculosis in mice. These findings reveal that any inhibition of apoptosis by M. tuberculosis is incomplete in vivo, advancing our understanding of host-protective responses to tuberculosis (TB) and revealing host pathways that may be targetable for treatment of disease.
Subject(s)
Apoptosis/immunology , Macrophages/immunology , Mycobacterium tuberculosis/immunology , Neutrophils/immunology , Tuberculosis, Pulmonary/immunology , Animals , Caspase 8/genetics , Caspase 8/metabolism , Cell Line , Dipeptides/therapeutic use , Humans , Indoles/therapeutic use , Lymphocyte Activation/immunology , Macrophages/microbiology , Mice , Mice, Inbred C57BL , Mice, Knockout , Neutrophils/microbiology , Protein Kinases/genetics , Protein Kinases/metabolism , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , T-Lymphocytes/immunology , Thiazoles/therapeutic use , Tuberculosis, Pulmonary/drug therapyABSTRACT
Caspase-8 is a master regulator of cell death pathways, although its regulation during inflammation remains elusive. Using elegant mouse genetic approaches, Schwarzer et al. and Tummers et al. revealed the importance of FADD in regulating caspase-8-mediated inflammatory responses and gut homeostasis.
Subject(s)
Gastrointestinal Microbiome , Animals , Apoptosis , Caspase 8/genetics , Caspase 8/metabolism , Cell Death , Epithelial Cells/metabolism , Fas-Associated Death Domain Protein/genetics , Fas-Associated Death Domain Protein/metabolism , Homeostasis , Inflammation , Intracellular Signaling Peptides and Proteins , Mice , Phosphate-Binding Proteins , Protein KinasesABSTRACT
Cell death pathways regulate various homeostatic processes. Autoimmune lymphoproliferative syndrome (ALPS) in humans and lymphoproliferative (LPR) disease in mice result from abrogated CD95-induced apoptosis. Because caspase-8 mediates CD95 signaling, we applied genetic approaches to dissect the roles of caspase-8 in cell death and inflammation. Here, we describe oligomerization-deficient Caspase-8F122GL123G/F122GL123G and non-cleavable Caspase-8D387A/D387A mutant mice with defective caspase-8-mediated apoptosis. Although neither mouse developed LPR disease, removal of the necroptosis effector Mlkl from Caspase-8D387A/D387A mice revealed an inflammatory role of caspase-8. Ablation of one allele of Fasl, Fadd, or Ripk1 prevented the pathology of Casp8D387A/D387AMlkl-/- animals. Removing both Fadd alleles from these mice resulted in early lethality prior to post-natal day 15 (P15), which was prevented by co-ablation of either Ripk1 or Caspase-1. Our results suggest an in vivo role of the inflammatory RIPK1-caspase-8-FADD (FADDosome) complex and reveal a FADD-independent inflammatory role of caspase-8 that involves activation of an inflammasome.
Subject(s)
Caspase 8/genetics , Disease Susceptibility , Fas-Associated Death Domain Protein/metabolism , Inflammation/etiology , Inflammation/metabolism , Necroptosis/genetics , Animals , Apoptosis/genetics , Biomarkers , Caspase 8/chemistry , Caspase 8/metabolism , Disease Models, Animal , Disease Progression , Fluorescent Antibody Technique , Gene Expression Regulation , Inflammasomes/metabolism , Inflammation/mortality , Inflammation/pathology , Lipopolysaccharides/adverse effects , Lipopolysaccharides/immunology , Mice , Mice, Knockout , Mortality , Phenotype , Protein MultimerizationABSTRACT
Pathways controlling intestinal epithelial cell (IEC) death regulate gut immune homeostasis and contribute to the pathogenesis of inflammatory bowel diseases. Here, we show that caspase-8 and its adapter FADD act in IECs to regulate intestinal inflammation downstream of Z-DNA binding protein 1 (ZBP1)- and tumor necrosis factor receptor-1 (TNFR1)-mediated receptor interacting protein kinase 1 (RIPK1) and RIPK3 signaling. Mice with IEC-specific FADD or caspase-8 deficiency developed colitis dependent on mixed lineage kinase-like (MLKL)-mediated epithelial cell necroptosis. However, MLKL deficiency fully prevented ileitis caused by epithelial caspase-8 ablation, but only partially ameliorated ileitis in mice lacking FADD in IECs. Our genetic studies revealed that caspase-8 and gasdermin-D (GSDMD) were both required for the development of MLKL-independent ileitis in mice with epithelial FADD deficiency. Therefore, FADD prevents intestinal inflammation downstream of ZBP1 and TNFR1 by inhibiting both MLKL-induced necroptosis and caspase-8-GSDMD-dependent pyroptosis-like death of epithelial cells.
Subject(s)
Caspase 8/genetics , Fas-Associated Death Domain Protein/genetics , Inflammatory Bowel Diseases/etiology , Inflammatory Bowel Diseases/metabolism , Intestinal Mucosa/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Phosphate-Binding Proteins/metabolism , Protein Kinases/metabolism , Animals , Apoptosis/genetics , Caspase 8/metabolism , Cell Death/genetics , Disease Models, Animal , Disease Susceptibility , Epithelial Cells/metabolism , Fas-Associated Death Domain Protein/metabolism , Gene Expression Profiling , Homeostasis/genetics , Immunohistochemistry , Inflammatory Bowel Diseases/pathology , Intestinal Mucosa/pathology , Intracellular Signaling Peptides and Proteins/genetics , Mice , Mice, Knockout , Phosphate-Binding Proteins/genetics , Protein Kinases/geneticsABSTRACT
In the early 2000s, receptor-interacting serine/threonine protein kinase 1 (RIPK1), a molecule already recognized as an important regulator of cell survival, inflammation and disease, was attributed an additional function: the regulation of a novel cell death pathway that came to be known as necroptosis. Subsequently, the related kinase RIPK3 and its substrate mixed-lineage kinase domain-like protein (MLKL) were also implicated in the necroptotic pathway, and links between this pathway and apoptosis were established. In this Timeline article, we outline the discoveries that have helped to identify the roles of RIPK1, RIPK3, MLKL and other regulators of necroptosis, and how they interact to determine cell fate.
Subject(s)
Apoptosis/physiology , Inflammation/pathology , Necrosis/pathology , Animals , Caspase 8/metabolism , Cell Death , Disease Models, Animal , Humans , Inflammasomes/metabolism , Inflammation/metabolism , Necrosis/physiopathology , Protein Kinases/genetics , Protein Kinases/metabolism , Receptor-Interacting Protein Serine-Threonine Kinases/genetics , Receptor-Interacting Protein Serine-Threonine Kinases/metabolismABSTRACT
Upon ligand binding, RIPK1 is recruited to tumor necrosis factor receptor superfamily (TNFRSF) and Toll-like receptor (TLR) complexes promoting prosurvival and inflammatory signaling. RIPK1 also directly regulates caspase-8-mediated apoptosis or, if caspase-8 activity is blocked, RIPK3-MLKL-dependent necroptosis. We show that C57BL/6 Ripk1(-/-) mice die at birth of systemic inflammation that was not transferable by the hematopoietic compartment. However, Ripk1(-/-) progenitors failed to engraft lethally irradiated hosts properly. Blocking TNF reversed this defect in emergency hematopoiesis but, surprisingly, Tnfr1 deficiency did not prevent inflammation in Ripk1(-/-) neonates. Deletion of Ripk3 or Mlkl, but not Casp8, prevented extracellular release of the necroptotic DAMP, IL-33, and reduced Myd88-dependent inflammation. Reduced inflammation in the Ripk1(-/-)Ripk3(-/-), Ripk1(-/-)Mlkl(-/-), and Ripk1(-/-)Myd88(-/-) mice prevented neonatal lethality, but only Ripk1(-/-)Ripk3(-/-)Casp8(-/-) mice survived past weaning. These results reveal a key function for RIPK1 in inhibiting necroptosis and, thereby, a role in limiting, not only promoting, inflammation.
Subject(s)
Genes, Lethal , Hematopoiesis , Inflammation/metabolism , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , Animals , Animals, Newborn , Caspase 8/metabolism , Cell Death , Liver/metabolism , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Myeloid Differentiation Factor 88/genetics , Myeloid Differentiation Factor 88/metabolism , Protein Kinases/genetics , Protein Kinases/metabolism , Receptor-Interacting Protein Serine-Threonine Kinases/genetics , Receptors, Tumor Necrosis Factor, Type I/metabolism , Tumor Necrosis Factors/metabolismABSTRACT
Receptor-interacting protein kinase (RIPK)-1 is involved in RIPK3-dependent and -independent signaling pathways leading to cell death and/or inflammation. Genetic ablation of ripk1 causes postnatal lethality, which was not prevented by deletion of ripk3, caspase-8, or fadd. However, animals that lack RIPK1, RIPK3, and either caspase-8 or FADD survived weaning and matured normally. RIPK1 functions in vitro to limit caspase-8-dependent, TNFR-induced apoptosis, and animals lacking RIPK1, RIPK3, and TNFR1 survive to adulthood. The role of RIPK3 in promoting lethality in ripk1(-/-) mice suggests that RIPK3 activation is inhibited by RIPK1 postbirth. Whereas TNFR-induced RIPK3-dependent necroptosis requires RIPK1, cells lacking RIPK1 were sensitized to necroptosis triggered by poly I:C or interferons. Disruption of TLR (TRIF) or type I interferon (IFNAR) signaling delayed lethality in ripk1(-/-)tnfr1(-/-) mice. These results clarify the complex roles for RIPK1 in postnatal life and provide insights into the regulation of FADD-caspase-8 and RIPK3-MLKL signaling by RIPK1.
Subject(s)
Caspase 8/metabolism , Genes, Lethal , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , Adaptor Proteins, Vesicular Transport/metabolism , Animals , Animals, Newborn , Apoptosis , Caspase 8/genetics , Cell Death , Embryo, Mammalian/cytology , Embryo, Mammalian/metabolism , Fas-Associated Death Domain Protein/metabolism , Fibroblasts/metabolism , Inflammation/metabolism , Interferons/metabolism , Mice , Mice, Inbred C57BL , Receptor-Interacting Protein Serine-Threonine Kinases/genetics , Receptors, Tumor Necrosis Factor, Type I/genetics , Receptors, Tumor Necrosis Factor, Type I/metabolism , Tumor Necrosis Factors/metabolismABSTRACT
The mechanisms of cellular energy sensing and AMPK-mediated mTORC1 inhibition are not fully delineated. Here, we discover that RIPK1 promotes mTORC1 inhibition during energetic stress. RIPK1 is involved in mediating the interaction between AMPK and TSC2 and facilitate TSC2 phosphorylation at Ser1387. RIPK1 loss results in a high basal mTORC1 activity that drives defective lysosomes in cells and mice, leading to accumulation of RIPK3 and CASP8 and sensitization to cell death. RIPK1-deficient cells are unable to cope with energetic stress and are vulnerable to low glucose levels and metformin. Inhibition of mTORC1 rescues the lysosomal defects and vulnerability to energetic stress and prolongs the survival of RIPK1-deficient neonatal mice. Thus, RIPK1 plays an important role in the cellular response to low energy levels and mediates AMPK-mTORC1 signaling. These findings shed light on the regulation of mTORC1 during energetic stress and unveil a point of crosstalk between pro-survival and pro-death pathways.
Subject(s)
Autophagy-Related Protein 5/genetics , Fas-Associated Death Domain Protein/genetics , Intestine, Large/metabolism , Mechanistic Target of Rapamycin Complex 1/genetics , Receptor-Interacting Protein Serine-Threonine Kinases/genetics , AMP-Activated Protein Kinases/genetics , AMP-Activated Protein Kinases/metabolism , Animals , Animals, Newborn , Autophagy-Related Protein 5/deficiency , Caspase 8/genetics , Caspase 8/metabolism , Cell Death/genetics , Fas-Associated Death Domain Protein/deficiency , Gene Expression Regulation , Glucose/antagonists & inhibitors , Glucose/pharmacology , HEK293 Cells , HT29 Cells , Humans , Intestine, Large/drug effects , Intestine, Large/pathology , Jurkat Cells , Lysosomes/drug effects , Lysosomes/metabolism , Lysosomes/pathology , Mechanistic Target of Rapamycin Complex 1/metabolism , Metformin/antagonists & inhibitors , Metformin/pharmacology , Mice , Mice, Inbred C57BL , Mice, Knockout , Phosphorylation , Receptor-Interacting Protein Serine-Threonine Kinases/deficiency , Signal Transduction , Sirolimus/pharmacology , Tuberous Sclerosis Complex 2 Protein/genetics , Tuberous Sclerosis Complex 2 Protein/metabolismABSTRACT
Interleukin 1ß (IL-1ß) is critical for the in vivo survival, expansion and effector function of IL-17-producing helper T (T(H)17) cells during autoimmune responses, including experimental autoimmune encephalomyelitis (EAE). However, the spatiotemporal role and cellular source of IL-1ß during EAE pathogenesis are poorly defined. In the present study, we uncovered a T cell-intrinsic inflammasome that drives IL-1ß production during T(H)17-mediated EAE pathogenesis. Activation of T cell antigen receptors induced expression of pro-IL-1ß, whereas ATP stimulation triggered T cell production of IL-1ß via ASC-NLRP3-dependent caspase-8 activation. IL-1R was detected on T(H)17 cells but not on type 1 helper T (T(H)1) cells, and ATP-treated T(H)17 cells showed enhanced survival compared with ATP-treated T(H)1 cells, suggesting autocrine action of T(H)17-derived IL-1ß. Together these data reveal a critical role for IL-1ß produced by a T(H)17 cell-intrinsic ASC-NLRP3-caspase-8 inflammasome during inflammation of the central nervous system.
Subject(s)
Apoptosis Regulatory Proteins/immunology , Encephalomyelitis, Autoimmune, Experimental/immunology , T-Lymphocytes/immunology , Th17 Cells/immunology , Adenosine Triphosphate/pharmacology , Animals , Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/metabolism , CARD Signaling Adaptor Proteins , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , Carrier Proteins/genetics , Carrier Proteins/immunology , Carrier Proteins/metabolism , Caspase 8/genetics , Caspase 8/immunology , Caspase 8/metabolism , Cell Survival/genetics , Cell Survival/immunology , Cells, Cultured , Encephalomyelitis, Autoimmune, Experimental/genetics , Encephalomyelitis, Autoimmune, Experimental/metabolism , Flow Cytometry , Gene Expression/immunology , Immunoblotting , Inflammasomes/genetics , Inflammasomes/immunology , Inflammasomes/metabolism , Interleukin-17/genetics , Interleukin-17/immunology , Interleukin-17/metabolism , Interleukin-1beta/genetics , Interleukin-1beta/immunology , Interleukin-1beta/metabolism , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , NLR Family, Pyrin Domain-Containing 3 Protein , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/genetics , Signal Transduction/immunology , T-Lymphocytes/drug effects , T-Lymphocytes/metabolism , Th17 Cells/drug effects , Th17 Cells/metabolismABSTRACT
Mutations of the ADAR1 gene encoding an RNA deaminase cause severe diseases associated with chronic activation of type I interferon (IFN) responses, including Aicardi-Goutières syndrome and bilateral striatal necrosis1-3. The IFN-inducible p150 isoform of ADAR1 contains a Zα domain that recognizes RNA with an alternative left-handed double-helix structure, termed Z-RNA4,5. Hemizygous ADAR1 mutations in the Zα domain cause type I IFN-mediated pathologies in humans2,3 and mice6-8; however, it remains unclear how the interaction of ADAR1 with Z-RNA prevents IFN activation. Here we show that Z-DNA-binding protein 1 (ZBP1), the only other protein in mammals known to harbour Zα domains9, promotes type I IFN activation and fatal pathology in mice with impaired ADAR1 function. ZBP1 deficiency or mutation of its Zα domains reduced the expression of IFN-stimulated genes and largely prevented early postnatal lethality in mice with hemizygous expression of ADAR1 with mutated Zα domain (Adar1mZα/- mice). Adar1mZα/- mice showed upregulation and impaired editing of endogenous retroelement-derived complementary RNA reads, which represent a likely source of Z-RNAs activating ZBP1. Notably, ZBP1 promoted IFN activation and severe pathology in Adar1mZα/- mice in a manner independent of RIPK1, RIPK3, MLKL-mediated necroptosis and caspase-8-dependent apoptosis, suggesting a novel mechanism of action. Thus, ADAR1 prevents endogenous Z-RNA-dependent activation of pathogenic type I IFN responses by ZBP1, suggesting that ZBP1 could contribute to type I interferonopathies caused by ADAR1 mutations.
Subject(s)
Adenosine Deaminase , Interferon Type I , RNA-Binding Proteins , Adenosine Deaminase/genetics , Adenosine Deaminase/metabolism , Animals , Apoptosis , Caspase 8/metabolism , Interferon Type I/antagonists & inhibitors , Interferon Type I/immunology , Mice , Mutation , Necroptosis , RNA, Double-Stranded/metabolism , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolismABSTRACT
The RNA-editing enzyme adenosine deaminase acting on RNA 1 (ADAR1) limits the accumulation of endogenous immunostimulatory double-stranded RNA (dsRNA)1. In humans, reduced ADAR1 activity causes the severe inflammatory disease Aicardi-Goutières syndrome (AGS)2. In mice, complete loss of ADAR1 activity is embryonically lethal3-6, and mutations similar to those found in patients with AGS cause autoinflammation7-12. Mechanistically, adenosine-to-inosine (A-to-I) base modification of endogenous dsRNA by ADAR1 prevents chronic overactivation of the dsRNA sensors MDA5 and PKR3,7-10,13,14. Here we show that ADAR1 also inhibits the spontaneous activation of the left-handed Z-nucleic acid sensor ZBP1. Activation of ZBP1 elicits caspase-8-dependent apoptosis and MLKL-mediated necroptosis of ADAR1-deficient cells. ZBP1 contributes to the embryonic lethality of Adar-knockout mice, and it drives early mortality and intestinal cell death in mice deficient in the expression of both ADAR and MAVS. The Z-nucleic-acid-binding Zα domain of ADAR1 is necessary to prevent ZBP1-mediated intestinal cell death and skin inflammation. The Zα domain of ADAR1 promotes A-to-I editing of endogenous Alu elements to prevent dsRNA formation through the pairing of inverted Alu repeats, which can otherwise induce ZBP1 activation. This shows that recognition of Alu duplex RNA by ZBP1 may contribute to the pathological features of AGS that result from the loss of ADAR1 function.
Subject(s)
Adenosine Deaminase , Inflammation , RNA-Binding Proteins , Adaptor Proteins, Signal Transducing/deficiency , Adenosine/metabolism , Adenosine Deaminase/chemistry , Adenosine Deaminase/deficiency , Adenosine Deaminase/metabolism , Animals , Apoptosis , Autoimmune Diseases of the Nervous System , Caspase 8/metabolism , Humans , Inflammation/metabolism , Inflammation/prevention & control , Inosine/metabolism , Intestines/pathology , Mice , Necroptosis , Nervous System Malformations , RNA Editing , RNA, Double-Stranded , RNA-Binding Proteins/antagonists & inhibitors , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/metabolism , Skin/pathologyABSTRACT
The RNA-editing enzyme ADAR1 is essential for the suppression of innate immune activation and pathology caused by aberrant recognition of self-RNA, a role it carries out by disrupting the duplex structure of endogenous double-stranded RNA species1,2. A point mutation in the sequence encoding the Z-DNA-binding domain (ZBD) of ADAR1 is associated with severe autoinflammatory disease3-5. ZBP1 is the only other ZBD-containing mammalian protein6, and its activation can trigger both cell death and transcriptional responses through the kinases RIPK1 and RIPK3, and the protease caspase 8 (refs. 7-9). Here we show that the pathology caused by alteration of the ZBD of ADAR1 is driven by activation of ZBP1. We found that ablation of ZBP1 fully rescued the overt pathology caused by ADAR1 alteration, without fully reversing the underlying inflammatory program caused by this alteration. Whereas loss of RIPK3 partially phenocopied the protective effects of ZBP1 ablation, combined deletion of caspase 8 and RIPK3, or of caspase 8 and MLKL, unexpectedly exacerbated the pathogenic effects of ADAR1 alteration. These findings indicate that ADAR1 is a negative regulator of sterile ZBP1 activation, and that ZBP1-dependent signalling underlies the autoinflammatory pathology caused by alteration of ADAR1.
Subject(s)
Adenosine Deaminase , Immune System Diseases , Inflammation , Mutation , RNA-Binding Proteins , Adenosine Deaminase/genetics , Adenosine Deaminase/metabolism , Animals , Caspase 8/genetics , Caspase 8/metabolism , Cell Death , Gene Deletion , Immune System Diseases/genetics , Immune System Diseases/metabolism , Immune System Diseases/pathology , Inflammation/genetics , Inflammation/metabolism , Inflammation/pathology , Mammals/genetics , Protein Kinases/deficiency , Protein Kinases/genetics , RNA, Double-Stranded/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Receptor-Interacting Protein Serine-Threonine Kinases/deficiency , Receptor-Interacting Protein Serine-Threonine Kinases/genetics , Signal TransductionABSTRACT
Necroptosis induction in vitro often requires caspase-8 (Casp8) inhibition by zVAD because pro-Casp8 cleaves RIP1 to disintegrate the necrosome. It has been unclear how the Casp8 blockade of necroptosis is eliminated naturally. Here, we show that pro-Casp8 within the necrosome can be inactivated by phosphorylation at Thr265 (pC8T265). pC8T265 occurs in vitro in various necroptotic cells and in the cecum of TNF-treated mice. p90 RSK is the kinase of pro-Casp8. It is activated by a mechanism that does not need ERK but PDK1, which is recruited to the RIP1-RIP3-MLKL-containing necrosome. Phosphorylation of pro-Casp8 at Thr265 can substitute for zVAD to permit necroptosis in vitro. pC8T265 mimic T265E knockin mice are embryonic lethal due to unconstrained necroptosis, and the pharmaceutical inhibition of RSK-mediated pC8T265 diminishes TNF-induced cecum damage and lethality in mice by halting necroptosis. Thus, phosphorylation of pro-Casp8 at Thr265 by RSK is an intrinsic mechanism for passing the Casp8 checkpoint of necroptosis.
Subject(s)
3-Phosphoinositide-Dependent Protein Kinases/metabolism , Caspase 8/metabolism , Necroptosis , Ribosomal Protein S6 Kinases, 90-kDa/metabolism , Signal Transduction , Animals , Cecum/injuries , Cecum/pathology , Cell Line , Embryo, Mammalian/metabolism , Embryonic Development/drug effects , Extracellular Signal-Regulated MAP Kinases/metabolism , Humans , Mice, Inbred C57BL , Mutation/genetics , Necroptosis/drug effects , Organ Specificity , Phosphorylation/drug effects , Phosphothreonine/metabolism , Protein Kinases/metabolism , Ribosomal Protein S6 Kinases, 90-kDa/antagonists & inhibitors , Signal Transduction/drug effects , Tumor Necrosis Factor-alpha/pharmacologyABSTRACT
Cytosolic caspase-8 is a mediator of death receptor signaling. While caspase-8 expression is lost in some tumors, it is increased in others, indicating a conditional pro-survival function of caspase-8 in cancer. Here, we show that tumor cells employ DNA-damage-induced nuclear caspase-8 to override the p53-dependent G2/M cell-cycle checkpoint. Caspase-8 is upregulated and localized to the nucleus in multiple human cancers, correlating with treatment resistance and poor clinical outcome. Depletion of caspase-8 causes G2/M arrest, stabilization of p53, and induction of p53-dependent intrinsic apoptosis in tumor cells. In the nucleus, caspase-8 cleaves and inactivates the ubiquitin-specific peptidase 28 (USP28), preventing USP28 from de-ubiquitinating and stabilizing wild-type p53. This results in de facto p53 protein loss, switching cell fate from apoptosis toward mitosis. In summary, our work identifies a non-canonical role of caspase-8 exploited by cancer cells to override the p53-dependent G2/M cell-cycle checkpoint.
Subject(s)
Caspase 8/metabolism , Cell Nucleus/enzymology , Cell Proliferation , G2 Phase Cell Cycle Checkpoints , Neoplasms/enzymology , Tumor Suppressor Protein p53/metabolism , Ubiquitin Thiolesterase/metabolism , Antineoplastic Agents/pharmacology , Apoptosis , Caspase 8/genetics , Cell Nucleus/drug effects , Cell Nucleus/genetics , Cell Nucleus/pathology , Cell Proliferation/drug effects , Drug Resistance, Neoplasm , Female , G2 Phase Cell Cycle Checkpoints/drug effects , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Neoplastic , HCT116 Cells , HeLa Cells , Humans , MCF-7 Cells , Male , Neoplasms/drug therapy , Neoplasms/genetics , Neoplasms/pathology , PC-3 Cells , Protein Stability , Signal Transduction , Tumor Cells, Cultured , Tumor Suppressor Protein p53/genetics , Ubiquitin Thiolesterase/geneticsABSTRACT
Genetic lesions in X-linked inhibitor of apoptosis (XIAP) pre-dispose humans to cell death-associated inflammatory diseases, although the underlying mechanisms remain unclear. Here, we report that two patients with XIAP deficiency-associated inflammatory bowel disease display increased inflammatory IL-1ß maturation as well as cell death-associated caspase-8 and Gasdermin D (GSDMD) processing in diseased tissue, which is reduced upon patient treatment. Loss of XIAP leads to caspase-8-driven cell death and bioactive IL-1ß release that is only abrogated by combined deletion of the apoptotic and pyroptotic cell death machinery. Namely, extrinsic apoptotic caspase-8 promotes pyroptotic GSDMD processing that kills macrophages lacking both inflammasome and apoptosis signalling components (caspase-1, -3, -7, -11 and BID), while caspase-8 can still cause cell death in the absence of both GSDMD and GSDME when caspase-3 and caspase-7 are present. Neither caspase-3 and caspase-7-mediated activation of the pannexin-1 channel, or GSDMD loss, prevented NLRP3 inflammasome assembly and consequent caspase-1 and IL-1ß maturation downstream of XIAP inhibition and caspase-8 activation, even though the pannexin-1 channel was required for NLRP3 triggering upon mitochondrial apoptosis. These findings uncouple the mechanisms of cell death and NLRP3 activation resulting from extrinsic and intrinsic apoptosis signalling, reveal how XIAP loss can co-opt dual cell death programs, and uncover strategies for targeting the cell death and inflammatory pathways that result from XIAP deficiency.
Subject(s)
Inflammasomes , NLR Family, Pyrin Domain-Containing 3 Protein , Humans , Apoptosis , Caspase 1/genetics , Caspase 1/metabolism , Caspase 3/metabolism , Caspase 7/metabolism , Caspase 8/genetics , Caspase 8/metabolism , Cell Death , Inflammasomes/metabolism , Interleukin-1beta/genetics , Interleukin-1beta/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Pyroptosis/physiology , X-Linked Inhibitor of Apoptosis Protein/genetics , X-Linked Inhibitor of Apoptosis Protein/metabolismABSTRACT
The execution of shock following high dose E. coli lipopolysaccharide (LPS) or bacterial sepsis in mice required pro-apoptotic caspase-8 in addition to pro-pyroptotic caspase-11 and gasdermin D. Hematopoietic cells produced MyD88- and TRIF-dependent inflammatory cytokines sufficient to initiate shock without any contribution from caspase-8 or caspase-11. Both proteases had to be present to support tumor necrosis factor- and interferon-ß-dependent tissue injury first observed in the small intestine and later in spleen and thymus. Caspase-11 enhanced the activation of caspase-8 and extrinsic cell death machinery within the lower small intestine. Neither caspase-8 nor caspase-11 was individually sufficient for shock. Both caspases collaborated to amplify inflammatory signals associated with tissue damage. Therefore, combined pyroptotic and apoptotic signaling mediated endotoxemia independently of RIPK1 kinase activity and RIPK3 function. These observations bring to light the relevance of tissue compartmentalization to disease processes in vivo where cytokines act in parallel to execute diverse cell death pathways.