RESUMEN
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.
Asunto(s)
Adenosina Desaminasa , Inflamación , Proteínas de Unión al ARN , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Adenosina/metabolismo , Adenosina Desaminasa/química , Adenosina Desaminasa/deficiencia , Adenosina Desaminasa/metabolismo , Animales , Apoptosis , Enfermedades Autoinmunes del Sistema Nervioso , Caspasa 8/metabolismo , Humanos , Inflamación/metabolismo , Inflamación/prevención & control , Inosina/metabolismo , Intestinos/patología , Ratones , Necroptosis , Malformaciones del Sistema Nervioso , Edición de ARN , ARN Bicatenario , Proteínas de Unión al ARN/antagonistas & inhibidores , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Piel/patologíaRESUMEN
Human bone marrow failure (BMF) syndromes result from the loss of hematopoietic stem and progenitor cells (HSPC), and this loss has been attributed to cell death; however, the cell death triggers, and mechanisms remain unknown. During BMF, tumor necrosis factor-α (TNFα) and interferon-γ (IFNγ) increase. These ligands are known to induce necroptosis, an inflammatory form of cell death mediated by RIPK1, RIPK3, and MLKL. We previously discovered that mice with a hematopoietic RIPK1 deficiency (Ripk1HEM KO) exhibit inflammation, HSPC loss, and BMF, which is partially ameliorated by a RIPK3 deficiency; however, whether RIPK3 exerts its effects through its function in mediating necroptosis or other forms of cell death remains unclear. Here, we demonstrate that similar to a RIPK3 deficiency, an MLKL deficiency significantly extends survival and like Ripk3 deficiency partially restores hematopoiesis in Ripk1HEM KO mice revealing that both necroptosis and apoptosis contribute to BMF in these mice. Using mouse models, we show that the nucleic acid sensor Z-DNA binding protein 1 (ZBP1) is up-regulated in mouse RIPK1-deficient bone marrow cells and that ZBP1's function in endogenous nucleic acid sensing is necessary for HSPC death and contributes to BMF. We also provide evidence that IFNγ mediates HSPC death in Ripk1HEM KO mice, as ablation of IFNγ but not TNFα receptor signaling significantly extends survival of these mice. Together, these data suggest that RIPK1 maintains hematopoietic homeostasis by preventing ZBP1 activation and induction of HSPC death.
Asunto(s)
Ácidos Nucleicos , Pancitopenia , Animales , Humanos , Ratones , Apoptosis/genética , Trastornos de Fallo de la Médula Ósea , Muerte Celular/fisiología , Células Madre Hematopoyéticas/metabolismo , Necrosis/metabolismo , Ácidos Nucleicos/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismoRESUMEN
Detection of cyclic dinucleotides (cdNs) by the STING pathway potently triggers the antiviral response. McFarland et al. now show that the mouse oxidoreductase RECON acts as a sensor for some bacterial cdNs, modulating innate signaling in a manner independent of STING to promote an antibacterial state.
Asunto(s)
Proteínas de la Membrana , Transducción de Señal , Animales , RatonesRESUMEN
ZBP1 is an interferon-induced cytosolic nucleic acid sensor that facilitates antiviral responses via RIPK3. Although ZBP1-mediated programmed cell death is widely described, whether and how it promotes inflammatory signaling is unclear. Here, we report a ZBP1-induced inflammatory signaling pathway mediated by K63- and M1-linked ubiquitin chains, which depends on RIPK1 and RIPK3 as scaffolds independently of cell death. In human HT29 cells, ZBP1 associated with RIPK1 and RIPK3 as well as ubiquitin ligases cIAP1 and LUBAC. ZBP1-induced K63- and M1-linked ubiquitination of RIPK1 and ZBP1 to promote TAK1- and IKK-mediated inflammatory signaling and cytokine production. Inhibition of caspase activity suppressed ZBP1-induced cell death but enhanced cytokine production in a RIPK1- and RIPK3 kinase activity-dependent manner. Lastly, we provide evidence that ZBP1 signaling contributes to SARS-CoV-2-induced cytokine production. Taken together, we describe a ZBP1-RIPK3-RIPK1-mediated inflammatory signaling pathway relayed by the scaffolding role of RIPKs and regulated by caspases, which may induce inflammation when ZBP1 is activated below the threshold needed to trigger a cell death response.
Asunto(s)
Muerte Celular , Proteínas de Unión al ARN , Proteína Serina-Treonina Quinasas de Interacción con Receptores , Humanos , Citocinas , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Transducción de Señal , Ubiquitina , Proteínas de Unión al ARN/genética , Células HT29 , InflamaciónRESUMEN
BACKGROUND: Aicardi-Goutières syndrome (AGS) is a type I interferonopathy usually characterized by early-onset neurologic regression. Biallelic mutations in LSM11 and RNU7-1, components of the U7 small nuclear ribonucleoprotein (snRNP) complex, have been identified in a limited number of genetically unexplained AGS cases. Impairment of U7 snRNP function results in misprocessing of replication-dependent histone (RDH) pre-mRNA and disturbance of histone occupancy of nuclear DNA, ultimately driving cGAS-dependent type I interferon (IFN-I) release. OBJECTIVE: We performed a clinical, genetic, and immunological workup of 3 unrelated patients with uncharacterized AGS. METHODS: Whole exome sequencing (WES) and targeted Sanger sequencing of RNU7-1 were performed. Primary fibroblasts were used for mechanistic studies. IFN-I signature and STAT1/2 phosphorylation were assessed in peripheral blood. Cytokines were profiled on serum and cerebrospinal fluid (CSF). Histopathology was examined on brain and kidney tissue. RESULTS: Sequencing revealed compound heterozygous RNU7-1 mutations, resulting in impaired RDH pre-mRNA processing. The 3' stem-loop mutations reduced stability of the secondary U7 snRNA structure. A discrete IFN-I signature in peripheral blood was paralleled by MCP-1 (CCL2) and CXCL10 upregulation in CSF. Histopathological analysis of the kidney showed thrombotic microangiopathy. We observed dysregulated STAT phosphorylation upon cytokine stimulation. Clinical overview of all reported patients with RNU7-1-related disease revealed high mortality and high incidence of organ involvement compared to other AGS genotypes. CONCLUSIONS: Targeted RNU7-1 sequencing is recommended in genetically unexplained AGS cases. CSF cytokine profiling represents an additional diagnostic tool to identify aberrant IFN-I signaling. Clinical follow-up of RNU7-1-mutated patients should include screening for severe end-organ involvement including liver disease and nephropathy.
Asunto(s)
Enfermedades Autoinmunes del Sistema Nervioso , Malformaciones del Sistema Nervioso , ARN Nuclear Pequeño/genética , Enfermedades Autoinmunes del Sistema Nervioso/diagnóstico , Enfermedades Autoinmunes del Sistema Nervioso/genética , Quimiocina CXCL10/genética , Histonas , Humanos , Interferones , Mutación , Malformaciones del Sistema Nervioso/diagnóstico , Malformaciones del Sistema Nervioso/genética , ARN , Precursores del ARN/química , Precursores del ARN/genética , Precursores del ARN/metabolismo , Proteínas de Unión al ARN/genéticaRESUMEN
Nucleic acids are potent triggers for innate immunity. Double-stranded DNA and RNA adopt different helical conformations, including the unusual Z-conformation. Z-DNA/RNA is recognised by Z-binding domains (ZBDs), which are present in proteins implicated in antiviral immunity. These include ZBP1 (also known as DAI or DLM-1), which induces necroptosis, an inflammatory form of cell death. Using reconstitution and knock-in models, we report that mutation of key amino acids involved in Z-DNA/RNA binding in ZBP1's ZBDs prevented necroptosis upon infection with mouse cytomegalovirus. Induction of cell death was cell autonomous and required RNA synthesis but not viral DNA replication. Accordingly, ZBP1 directly bound to RNA via its ZBDs. Intact ZBP1-ZBDs were also required for necroptosis triggered by ectopic expression of ZBP1 and caspase blockade, and ZBP1 cross-linked to endogenous RNA These observations show that Z-RNA may constitute a molecular pattern that induces inflammatory cell death upon sensing by ZBP1.
Asunto(s)
Apoptosis/fisiología , Glicoproteínas/metabolismo , ARN/metabolismo , Animales , Citomegalovirus/genética , Infecciones por Citomegalovirus/metabolismo , Glicoproteínas/genética , Ratones , Ratones Transgénicos , Células 3T3 NIH , Conformación de Ácido Nucleico , ARN/química , Proteínas de Unión al ARNRESUMEN
A20 negatively regulates multiple inflammatory signalling pathways. We here addressed the role of A20 in club cells (also known as Clara cells) of the bronchial epithelium in their response to influenza A virus infection. Club cells provide a niche for influenza virus replication, but little is known about the functions of these cells in antiviral immunity. Using airway epithelial cell-specific A20 knockout (A20AEC-KO) mice, we show that A20 in club cells critically controls innate immune responses upon TNF or double stranded RNA stimulation. Surprisingly, A20AEC-KO mice are better protected against influenza A virus challenge than their wild type littermates. This phenotype is not due to decreased viral replication. Instead host innate and adaptive immune responses and lung damage are reduced in A20AEC-KO mice. These attenuated responses correlate with a dampened cytotoxic T cell (CTL) response at later stages during infection, indicating that A20AEC-KO mice are better equipped to tolerate Influenza A virus infection. Expression of the chemokine CCL2 (also named MCP-1) is particularly suppressed in the lungs of A20AEC-KO mice during later stages of infection. When A20AEC-KO mice were treated with recombinant CCL2 the protective effect was abrogated demonstrating the crucial contribution of this chemokine to the protection of A20AEC-KO mice to Influenza A virus infection. Taken together, we propose a mechanism of action by which A20 expression in club cells controls inflammation and antiviral CTL responses in response to influenza virus infection.
Asunto(s)
Cisteína Endopeptidasas/inmunología , Citotoxicidad Inmunológica/inmunología , Péptidos y Proteínas de Señalización Intracelular/inmunología , Infecciones por Orthomyxoviridae/inmunología , Mucosa Respiratoria/inmunología , Animales , Cisteína Endopeptidasas/deficiencia , Citometría de Flujo , Inmunidad Innata/inmunología , Immunoblotting , Inmunohistoquímica , Virus de la Influenza A , Péptidos y Proteínas de Señalización Intracelular/deficiencia , Activación de Linfocitos/inmunología , Ratones , Ratones Noqueados , Reacción en Cadena de la Polimerasa , Linfocitos T Citotóxicos/inmunología , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfaRESUMEN
SAMHD1 is a host restriction factor for human immunodeficiency virus 1 (HIV-1) in cultured human cells. SAMHD1 mutations cause autoimmune Aicardi-Goutières syndrome and are found in cancers including chronic lymphocytic leukaemia. SAMHD1 is a triphosphohydrolase that depletes the cellular pool of deoxynucleoside triphosphates, thereby preventing reverse transcription of retroviral genomes. However, in vivo evidence for SAMHD1's antiviral activity has been lacking. We generated Samhd1 null mice that do not develop autoimmune disease despite displaying a type I interferon signature in spleen, macrophages and fibroblasts. Samhd1(-/-) cells have elevated deoxynucleoside triphosphate (dNTP) levels but, surprisingly, SAMHD1 deficiency did not lead to increased infection with VSV-G-pseudotyped HIV-1 vectors. The lack of restriction is likely attributable to the fact that dNTP concentrations in SAMHD1-sufficient mouse cells are higher than the KM of HIV-1 reverse transcriptase (RT). Consistent with this notion, an HIV-1 vector mutant bearing an RT with lower affinity for dNTPs was sensitive to SAMHD1-dependent restriction in cultured cells and in mice. This shows that SAMHD1 can restrict lentiviruses in vivo and that nucleotide starvation is an evolutionarily conserved antiviral mechanism.
Asunto(s)
Enfermedades Autoinmunes del Sistema Nervioso/metabolismo , Infecciones por VIH/fisiopatología , VIH-1/fisiología , Proteínas de Unión al GTP Monoméricas/metabolismo , Malformaciones del Sistema Nervioso/metabolismo , Transcripción Reversa/fisiología , Animales , Enfermedades Autoinmunes del Sistema Nervioso/genética , Línea Celular , Vectores Genéticos/genética , Infecciones por VIH/genética , Transcriptasa Inversa del VIH/metabolismo , VIH-1/enzimología , Interferón Tipo I/metabolismo , Ratones , Ratones Noqueados , Proteínas de Unión al GTP Monoméricas/genética , Malformaciones del Sistema Nervioso/genética , Nucleótidos/metabolismo , Transcripción Reversa/genética , Proteína 1 que Contiene Dominios SAM y HDRESUMEN
Optineurin (OPTN) is an evolutionary conserved and ubiquitously expressed ubiquitin-binding protein that has been implicated in glaucoma, Paget bone disease, amyotrophic lateral sclerosis, and other neurodegenerative diseases. From in vitro studies, OPTN was shown to suppress TNF-induced NF-κB signaling and virus-induced IRF signaling, and was identified as an autophagy receptor required for the clearance of cytosolic Salmonella upon infection. To assess the in vivo functions of OPTN in inflammation and infection, we generated OPTN-deficient mice. OPTN knockout mice are born with normal Mendelian distribution and develop normally without any signs of spontaneous organ abnormality or inflammation. However, no differences in NF-κB activation could be observed in OPTN knockout mice or fibroblasts derived from these mice upon TNF or LPS treatment. Primary bone marrow-derived macrophages from OPTN-deficient mice had slightly impaired IRF signaling and reduced IFN type I production in response to LPS or poly(I,C). Finally, OPTN-deficient mice were more susceptible to infection with Salmonella, confirming in vivo the importance of OPTN in bacterial clearance.
Asunto(s)
Proteínas del Ojo/genética , FN-kappa B/inmunología , Infecciones por Salmonella/inmunología , Salmonella typhimurium/inmunología , Factor de Necrosis Tumoral alfa/farmacología , Animales , Proteínas de Ciclo Celular , Fibroblastos/inmunología , Subtipo H3N2 del Virus de la Influenza A/inmunología , Factor 3 Regulador del Interferón/metabolismo , Interferón Tipo I/biosíntesis , Lipopolisacáridos/farmacología , Macrófagos/inmunología , Proteínas de Transporte de Membrana , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/virología , Poli I-C/farmacología , Infecciones por Salmonella/microbiología , Transducción de Señal/inmunologíaRESUMEN
SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase and a nuclease that restricts HIV-1 in noncycling cells. Germ-line mutations in SAMHD1 have been described in patients with Aicardi-Goutières syndrome (AGS), a congenital autoimmune disease. In a previous longitudinal whole genome sequencing study of chronic lymphocytic leukemia (CLL), we revealed a SAMHD1 mutation as a potential founding event. Here, we describe an AGS patient carrying a pathogenic germ-line SAMHD1 mutation who developed CLL at 24 years of age. Using clinical trial samples, we show that acquired SAMHD1 mutations are associated with high variant allele frequency and reduced SAMHD1 expression and occur in 11% of relapsed/refractory CLL patients. We provide evidence that SAMHD1 regulates cell proliferation and survival and engages in specific protein interactions in response to DNA damage. We propose that SAMHD1 may have a function in DNA repair and that the presence of SAMHD1 mutations in CLL promotes leukemia development.
Asunto(s)
Daño del ADN/genética , Mutación de Línea Germinal , Leucemia Linfocítica Crónica de Células B/genética , Proteínas de Unión al GTP Monoméricas/genética , Adulto , Enfermedades Autoinmunes del Sistema Nervioso/complicaciones , Enfermedades Autoinmunes del Sistema Nervioso/genética , Estudios de Cohortes , Hibridación Genómica Comparativa , Frecuencia de los Genes , Células HeLa , Humanos , Leucemia Linfocítica Crónica de Células B/complicaciones , Masculino , Malformaciones del Sistema Nervioso/complicaciones , Malformaciones del Sistema Nervioso/genética , Proteína 1 que Contiene Dominios SAM y HD , Adulto JovenRESUMEN
HIV-1 infects dendritic cells (DCs) without triggering an effective innate antiviral immune response. As a consequence, the induction of adaptive immune responses controlling virus spread is limited. In a recent issue of Immunity, Lahaye and colleagues show that intricate interactions of HIV capsid with the cellular cofactor cyclophilin A (CypA) control infection and innate immune activation in DCs. Manipulation of HIV-1 capsid to increase its affinity for CypA results in reduced virus infectivity and facilitates access of the cytosolic DNA sensor cGAS to reverse transcribed DNA. This in turn induces a strong host response. Here, we discuss these findings in the context of recent developments in innate immunity and consider the implications for disease control and vaccine design.
Asunto(s)
Cápside/inmunología , ADN Complementario/metabolismo , Células Dendríticas , Infecciones por VIH/inmunología , VIH-1/inmunología , VIH-2/inmunología , Nucleotidiltransferasas/metabolismo , HumanosRESUMEN
The innate immune response provides the first line of defense against viruses and other pathogens by responding to specific microbial molecules. Influenza A virus (IAV) produces double-stranded RNA as an intermediate during the replication life cycle, which activates the intracellular pathogen recognition receptor RIG-I and induces the production of proinflammatory cytokines and antiviral interferon. Understanding the mechanisms that regulate innate immune responses to IAV and other viruses is of key importance to develop novel therapeutic strategies. Here we used myeloid cell specific A20 knockout mice to examine the role of the ubiquitin-editing protein A20 in the response of myeloid cells to IAV infection. A20 deficient macrophages were hyperresponsive to double stranded RNA and IAV infection, as illustrated by enhanced NF-κB and IRF3 activation, concomitant with increased production of proinflammatory cytokines, chemokines and type I interferon. In vivo this was associated with an increased number of alveolar macrophages and neutrophils in the lungs of IAV infected mice. Surprisingly, myeloid cell specific A20 knockout mice are protected against lethal IAV infection. These results challenge the general belief that an excessive host proinflammatory response is associated with IAV-induced lethality, and suggest that under certain conditions inhibition of A20 might be of interest in the management of IAV infections.
Asunto(s)
Proteínas de Unión al ADN/deficiencia , Virus de la Influenza A/patogenicidad , Péptidos y Proteínas de Señalización Intracelular/deficiencia , Macrófagos Alveolares/enzimología , Neutrófilos/enzimología , Ubiquitina-Proteína Ligasas/deficiencia , Animales , Cisteína Endopeptidasas , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Modelos Animales de Enfermedad , Regulación Viral de la Expresión Génica , Virus de la Influenza A/fisiología , Factor 3 Regulador del Interferón/biosíntesis , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Pulmón/enzimología , Pulmón/inmunología , Pulmón/virología , Macrófagos Alveolares/inmunología , Macrófagos Alveolares/virología , Ratones , Ratones Noqueados , FN-kappa B/biosíntesis , Neutrófilos/inmunología , Neutrófilos/virología , ARN Bicatenario/genética , ARN Bicatenario/metabolismo , ARN Mensajero/metabolismo , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Recent progress in human and mouse genetics has transformed our understanding of the molecular mechanisms by which recognition of self double-stranded RNA (self-dsRNA) causes immunopathology. Novel mouse models recapitulate loss-of-function mutations in the RNA editing enzyme ADAR1 that are found in patients with Aicardi-Goutières syndrome (AGS) - a monogenic inflammatory disease associated with increased levels of type I interferon. Extensive analyses of the genotype-phenotype relationships in these mice have now firmly established a causal relationship between increased intracellular concentrations of endogenous immunostimulatory dsRNA and type I interferon-driven immunopathology. Activation of the dsRNA-specific immune sensor MDA5 perpetuates the overproduction of type I interferons, and chronic engagement of the interferon-inducible innate immune receptors PKR and ZBP1 by dsRNA drives immunopathology by activating an integrated stress response or by inducing excessive cell death. Biochemical and genetic data support a role for the p150 isoform of ADAR1 in the cytosol in suppressing the spontaneous, pathological response to self-dsRNA.
Asunto(s)
Interferón Tipo I , ARN Bicatenario , Humanos , Ratones , Animales , Adenosina Desaminasa/genética , Adenosina Desaminasa/metabolismoRESUMEN
The involvement of necroptosis in the control of influenza A virus (IAV) infection has been reported in multiple studies. Downstream of the nucleic acid sensor ZBP1, RIPK3 kinase activity is critically involved in the induction of necroptotic cell death by phosphorylating MLKL, while RIPK3 as a scaffold can induce apoptosis. Paradoxically, RIPK3-deficiency of mice may result in increased or decreased susceptibility to IAV infection. Here, we critically review the published reports on the involvement of RIPK3 in IAV infection susceptibility and try to identify differences in experimental settings that could explain seemingly conflicting outcomes. Analysis of the experimental reports revealed differences in the IAV challenge dose, the IAV inoculum preparation, IAV titer assessment, as well as the route of inoculation between studies. Furthermore, differences were noticed in the inclusion of littermate controls, which show high variance in viral sensitivity. Our evaluation argues for a standardized setup for IAV infection experiments including the preparation of the IAV virus, the use of different IAV infectious doses description and the proper experimental genetic controls of the mouse strains to increase inter-laboratory consistency in this field. Workflow for IAV infection studies in vivo: Viral preparation and titer assessment should be as standardized as possible with the use of a universal repository (such as BEI resources). Infection studies in genetically modified mice and littermate controls should include dose-response experimentation, following a defined infection route and inoculation volume. Data are generated by consistent analysis methods.
RESUMEN
The T cell population size is stringently controlled before, during, and after immune responses, as improper cell death regulation can result in autoimmunity and immunodeficiency. RIPK1 is an important regulator of peripheral T cell survival and homeostasis. However, whether different peripheral T cell subsets show a differential requirement for RIPK1 and which programmed cell death pathway they engage in vivo remains unclear. In this study, we demonstrate that conditional ablation of Ripk1 in conventional T cells (Ripk1ΔCD4) causes peripheral T cell lymphopenia, as witnessed by a profound loss of naive CD4+, naive CD8+, and FoxP3+ regulatory T cells. Interestingly, peripheral naive CD8+ T cells in Ripk1ΔCD4 mice appear to undergo a selective pressure to retain RIPK1 expression following activation. Mixed bone marrow chimeras revealed a competitive survival disadvantage for naive, effector, and memory T cells lacking RIPK1. Additionally, tamoxifen-induced deletion of RIPK1 in CD4-expressing cells in adult life confirmed the importance of RIPK1 in post-thymic survival of CD4+ T cells. Ripk1K45A mice showed no change in peripheral T cell subsets, demonstrating that the T cell lymphopenia was due to the scaffold function of RIPK1 rather than to its kinase activity. Enhanced numbers of Ripk1ΔCD4 naive T cells expressed the proliferation marker Ki-67+ despite the peripheral lymphopenia and single-cell RNA sequencing revealed T cell-specific transcriptomic alterations that were reverted by additional caspase-8 deficiency. Furthermore, Ripk1ΔCD4Casp8 ΔCD4 and Ripk1ΔCD4Tnfr1-/- double-knockout mice rescued the peripheral T cell lymphopenia, revealing that RIPK1-deficient naive CD4+ and CD8+ cells and FoxP3+ regulatory T cells specifically die from TNF- and caspase-8-mediated apoptosis in vivo. Altogether, our findings emphasize the essential role of RIPK1 as a scaffold in maintaining the peripheral T cell compartment and preventing TNFR1-induced apoptosis.
Asunto(s)
Apoptosis , Proteína Serina-Treonina Quinasas de Interacción con Receptores , Receptores Tipo I de Factores de Necrosis Tumoral , Linfocitos T Reguladores , Animales , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Ratones , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Ratones Endogámicos C57BL , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/inmunología , Ratones Noqueados , Caspasa 8/metabolismo , Linfopenia/patología , Linfopenia/inmunologíaRESUMEN
The envelope glycoprotein of vesicular stomatitis virus (VSV-G) enables viral entry into hosts as distant as insects and vertebrates. Because of its ability to support infection of most, if not all, human cell types VSV-G is used in viral vectors for gene therapy. However, neither the receptor nor any specific host factor for VSV-G has been identified. Here we demonstrate that infection with VSV and innate immunity via Toll-like receptors (TLRs) require a shared component, the endoplasmic reticulum chaperone gp96. Cells without gp96 or with catalytically inactive gp96 do not bind VSV-G. The ubiquitous expression of gp96 is therefore essential for the remarkably broad tropism of VSV-G. Cells deficient in gp96 also lack functional TLRs, which suggests that pathogen-driven pressure for TLR-mediated immunity maintains the broad host range of VSV-G by positively selecting for the ubiquitous expression of gp96.
Asunto(s)
Retículo Endoplásmico/metabolismo , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/fisiología , Virus de la Estomatitis Vesicular Indiana/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Línea Celular , Islas de CpG , Herpesvirus Humano 1/metabolismo , Humanos , Inmunidad Innata , Glicoproteínas de Membrana/química , Chaperonas Moleculares/química , Mutagénesis , Retroviridae/metabolismo , Receptores Toll-Like/metabolismo , Transferrina/química , Transferrina/metabolismoRESUMEN
Nucleic acid sensing is a central process in the immune system, with far-reaching roles in antiviral defense, autoinflammation, and cancer. Z-DNA binding protein 1 (ZBP1) is a sensor for double-stranded DNA and RNA helices in the unusual left-handed Z conformation termed Z-DNA and Z-RNA. Recent research established ZBP1 as a key upstream regulator of cell death and proinflammatory signaling. Recognition of Z-DNA/RNA by ZBP1 promotes host resistance to viral infection but can also drive detrimental autoinflammation. Additionally, ZBP1 has interesting roles in cancer and other disease settings and is emerging as an attractive target for therapy.
Asunto(s)
ADN de Forma Z , Ácidos Nucleicos , Proteínas de Unión al ARN/metabolismo , Muerte Celular , ARN/metabolismoRESUMEN
Dendritic cells (DCs) mature in an immunogenic or tolerogenic manner depending on the context in which an antigen is perceived, preserving the balance between immunity and tolerance. Whereas the pathways driving immunogenic maturation in response to infectious insults are well-characterized, the signals that drive tolerogenic maturation during homeostasis are still poorly understood. We found that the engulfment of apoptotic cells triggered homeostatic maturation of type 1 conventional DCs (cDC1s) within the spleen. This maturation process could be mimicked by engulfment of empty, nonadjuvanted lipid nanoparticles (LNPs), was marked by intracellular accumulation of cholesterol, and was highly specific to cDC1s. Engulfment of either apoptotic cells or cholesterol-rich LNPs led to the activation of the liver X receptor (LXR) pathway, which promotes the efflux of cellular cholesterol, and repressed genes associated with immunogenic maturation. In contrast, simultaneous engagement of TLR3 to mimic viral infection via administration of poly(I:C)-adjuvanted LNPs repressed the LXR pathway, thus delaying cellular cholesterol efflux and inducing genes that promote T cell-mediated immunity. These data demonstrate that conserved cellular cholesterol efflux pathways are differentially regulated in tolerogenic versus immunogenic cDC1s and suggest that administration of nonadjuvanted cholesterol-rich LNPs may be an approach for inducing tolerogenic DC maturation.
Asunto(s)
Células Dendríticas , Transducción de Señal , Receptores X del Hígado/metabolismo , Transducción de Señal/genética , Homeostasis , ColesterolRESUMEN
Cell death forms an essential component of the antiviral immune response. Viral infection elicits different forms of host cell death, including the lytic and inflammatory cell death modes necroptosis or pyroptosis. The induction of both types of cell death not only eliminates virus-infected cells but also contributes to the development of innate and adaptive immunity through the release of inflammatory mediators. The importance of necroptosis and pyroptosis in host defence is evident from the numerous viral evasion mechanisms that suppress these cell death pathways. Here, we review the emerging principles by which viruses antagonise host cell necroptosis and pyroptosis to promote their spread and block host immunity.
Asunto(s)
Necroptosis , Virosis , Apoptosis , Muerte Celular , Humanos , PiroptosisRESUMEN
The kinase Receptor-interacting serine/threonine protein kinase 3 (RIPK3) and its substrate mixed lineage kinase domain-like (MLKL) are critical regulators of necroptosis, an inflammatory form of cell death with important antiviral functions. Autophosphorylation of RIPK3 induces phosphorylation and activation of the pore-forming executioner protein of necroptosis MLKL. Trafficking and oligomerization of phosphorylated MLKL at the cell membrane results in cell lysis, characteristic of necroptotic cell death. The nucleic acid sensor ZBP1 is activated by binding to left-handed Z-form double-stranded RNA (Z-RNA) after infection with RNA and DNA viruses. ZBP1 activation restricts virus infection by inducing regulated cell death, including necroptosis, of infected host cells. Immunofluorescence microscopy permits the visualization of different signaling steps downstream of ZBP1-mediated necroptosis on a per-cell basis. However, the sensitivity of standard fluorescence microscopy, using current commercially available phospho-specific antibodies against human RIPK3 and MLKL, precludes reproducible imaging of these markers. Here, we describe an optimized staining procedure for serine (S) phosphorylated RIPK3 (S227) and MLKL (S358) in human HT-29 cells infected with herpes simplex virus 1 (HSV-1). The inclusion of a tyramide signal amplification (TSA) step in the immunofluorescent staining protocol allows the specific detection of S227 phosphorylated RIPK3. Moreover, TSA greatly increases the sensitivity of the detection of S358 phosphorylated MLKL. Together, this method enables the visualization of these two critical signaling events during the induction of ZBP1-induced necroptosis.