RESUMO
The innate immune system functions as the first line of defense against invading bacteria and viruses. In this context, the cGAS/STING [cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) synthase/STING] signaling axis perceives the nonself DNA associated with bacterial and viral infections, as well as the leakage of self DNA by cellular dysfunction and stresses, to elicit the host's immune responses. In this pathway, the noncanonical cyclic dinucleotide 2',3'-cyclic GMP-AMP (2',3'-cGAMP) functions as a second messenger for signal transduction: 2',3'-cGAMP is produced by the enzyme cGAS upon its recognition of double-stranded DNA, and then the 2',3'-cGAMP is recognized by the receptor STING to induce the phosphorylation of downstream factors, including TBK1 (TANK binding kinase 1) and IRF3 (interferon regulatory factor 3). Numerous crystal structures of the components of this cGAS/STING signaling axis have been reported and these clarify the structural basis for their signal transduction mechanisms. In this review, we summarize recent progress made in the structural dissection of this signaling pathway and indicate possible directions of forthcoming research.
Assuntos
DNA/imunologia , Imunidade Inata , Nucleotídeos Cíclicos/imunologia , Nucleotidiltransferases/imunologia , Sistemas do Segundo Mensageiro/imunologia , Animais , Bactérias , Cristalografia por Raios X , Citosol/química , Citosol/imunologia , DNA/química , DNA/genética , Regulação da Expressão Gênica , Humanos , Fator Regulador 3 de Interferon/química , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/imunologia , Modelos Moleculares , Nucleotídeos Cíclicos/química , Nucleotídeos Cíclicos/genética , Nucleotidiltransferases/química , Nucleotidiltransferases/genética , Fosforilação , Conformação Proteica , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/imunologia , Sistemas do Segundo Mensageiro/genéticaRESUMO
Signal transduction proteins containing a pLxIS motif induce interferon (IFN) responses central to antiviral immunity. Apart from their established roles in activating the IFN regulator factor (IRF) transcription factors, the existence of additional pathways and functions associated with the pLxIS motif is unknown. Using a synthetic biology-based platform, we identified two orphan pLxIS-containing proteins that stimulate IFN responses independent of all known pattern-recognition receptor pathways. We further uncovered a diversity of pLxIS signaling mechanisms, where the pLxIS motif represents one component of a multi-motif signaling entity, which has variable functions in activating IRF3, the TRAF6 ubiquitin ligase, IκB kinases, mitogen-activated protein kinases, and metabolic activities. The most diverse pLxIS signaling mechanisms were associated with the highest antiviral activities in human cells. The flexibility of domains that regulate IFN signaling may explain their prevalence in nature.
Assuntos
Fator Regulador 3 de Interferon , Interferons , Transdução de Sinais , Fator 6 Associado a Receptor de TNF , Humanos , Interferons/metabolismo , Células HEK293 , Fator Regulador 3 de Interferon/metabolismo , Fator Regulador 3 de Interferon/genética , Fator 6 Associado a Receptor de TNF/metabolismo , Fator 6 Associado a Receptor de TNF/genética , Quinase I-kappa B/metabolismo , Quinase I-kappa B/genética , Domínios Proteicos , Animais , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Motivos de Aminoácidos , Proteínas Quinases Ativadas por Mitógeno/metabolismoRESUMO
The cyclic guanosine monophosphate (GMP)-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway plays a pivotal role in innate immune responses to viral infection and inhibition of autoimmunity. Recent studies have suggested that micronuclei formed by genotoxic stress can activate innate immune signaling via the cGAS-STING pathway. Here, we investigated cGAS localization, activation, and downstream signaling from micronuclei induced by ionizing radiation, replication stress, and chromosome segregation errors. Although cGAS localized to ruptured micronuclei via binding to self-DNA, we failed to observe cGAS activation; cGAMP production; downstream phosphorylation of STING, TBK1, or IRF3; nuclear accumulation of IRF3; or expression of interferon-stimulated genes. Failure to activate the cGAS-STING pathway was observed across primary and immortalized cell lines, which retained the ability to activate the cGAS-STING pathway in response to dsDNA or modified vaccinia virus infection. We provide evidence that micronuclei formed by genotoxic insults contain histone-bound self-DNA, which we show is inhibitory to cGAS activation in cells.
Assuntos
Segregação de Cromossomos , Proteínas de Membrana , Micronúcleos com Defeito Cromossômico , Nucleotídeos Cíclicos , Nucleotidiltransferases , Transdução de Sinais , Nucleotidiltransferases/metabolismo , Nucleotidiltransferases/genética , Humanos , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Micronúcleos com Defeito Cromossômico/efeitos da radiação , Nucleotídeos Cíclicos/metabolismo , Fosforilação , Replicação do DNA/efeitos da radiação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Fator Regulador 3 de Interferon/metabolismo , Fator Regulador 3 de Interferon/genética , Imunidade Inata/efeitos da radiação , Dano ao DNA , Células HEK293 , Animais , Radiação Ionizante , Células HeLaRESUMO
The innate immune cGAS-STING pathway is activated by cytosolic double-stranded DNA (dsDNA), a ubiquitous danger signal, to produce interferon, a potent anti-viral and anti-cancer cytokine. However, STING activation must be tightly controlled because aberrant interferon production leads to debilitating interferonopathies. Here, we discover PELI2 as a crucial negative regulator of STING. Mechanistically, PELI2 inhibits the transcription factor IRF3 by binding to phosphorylated Thr354 and Thr356 on the C-terminal tail of STING, leading to ubiquitination and inhibition of the kinase TBK1. PELI2 sets a threshold for STING activation that tolerates low levels of cytosolic dsDNA, such as that caused by silenced TREX1, RNASEH2B, BRCA1, or SETX. When this threshold is reached, such as during viral infection, STING-induced interferon production temporarily downregulates PELI2, creating a positive feedback loop allowing a robust immune response. Lupus patients have insufficient PELI2 levels and high basal interferon production, suggesting that PELI2 dysregulation may drive the onset of lupus and other interferonopathies.
Assuntos
Fator Regulador 3 de Interferon , Proteínas de Membrana , Proteínas Serina-Treonina Quinases , Transdução de Sinais , Ubiquitinação , Humanos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Fosforilação , Fator Regulador 3 de Interferon/metabolismo , Fator Regulador 3 de Interferon/genética , Animais , Células HEK293 , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/metabolismo , Lúpus Eritematoso Sistêmico/virologia , Imunidade Inata , Interações Hospedeiro-Patógeno , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Camundongos , Interferons/metabolismo , Interferons/imunologia , Interferons/genética , Retroalimentação Fisiológica , Camundongos Endogâmicos C57BL , Exodesoxirribonucleases , FosfoproteínasRESUMO
The initial response to viral infection is anticipatory, with host antiviral restriction factors and pathogen sensors constantly surveying the cell to rapidly mount an antiviral response through the synthesis and downstream activity of interferons. After pathogen clearance, the host's ability to resolve this antiviral response and return to homeostasis is critical. Here, we found that isoforms of the RNA-binding protein ZAP functioned as both a direct antiviral restriction factor and an interferon-resolution factor. The short isoform of ZAP bound to and mediated the degradation of several host interferon messenger RNAs, and thus acted as a negative feedback regulator of the interferon response. In contrast, the long isoform of ZAP had antiviral functions and did not regulate interferon. The two isoforms contained identical RNA-targeting domains, but differences in their intracellular localization modulated specificity for host versus viral RNA, which resulted in disparate effects on viral replication during the innate immune response.
Assuntos
Infecções por Alphavirus/imunologia , Interferons/genética , Isoformas de Proteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Proteínas Repressoras/metabolismo , Sindbis virus/fisiologia , Infecções por Alphavirus/genética , Retroalimentação Fisiológica , Células HEK293 , Células Hep G2 , Homeostase , Humanos , Imunidade Inata , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/metabolismo , Ligação Proteica , Isoformas de Proteínas/genética , RNA/genética , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/genética , Proteínas Repressoras/genética , Replicação ViralRESUMO
Zika virus (ZIKV) can be transmitted sexually between humans. However, it is unknown whether ZIKV replicates in the vagina and impacts the unborn fetus. Here, we establish a mouse model of vaginal ZIKV infection and demonstrate that, unlike other routes, ZIKV replicates within the genital mucosa even in wild-type (WT) mice. Mice lacking RNA sensors or transcription factors IRF3 and IRF7 resulted in higher levels of local viral replication. Furthermore, mice lacking the type I interferon (IFN) receptor (IFNAR) became viremic and died of infection after a high-dose vaginal ZIKV challenge. Notably, vaginal infection of pregnant dams during early pregnancy led to fetal growth restriction and infection of the fetal brain in WT mice. This was exacerbated in mice deficient in IFN pathways, leading to abortion. Our study highlights the vaginal tract as a highly susceptible site of ZIKV replication and illustrates the dire disease consequences during pregnancy.
Assuntos
Encefalopatias/virologia , Encéfalo/virologia , Retardo do Crescimento Fetal/virologia , Complicações Infecciosas na Gravidez/virologia , Vagina/virologia , Replicação Viral , Infecção por Zika virus/transmissão , Zika virus/fisiologia , Aborto Habitual/virologia , Animais , Encefalopatias/imunologia , Modelos Animais de Doenças , Feminino , Retardo do Crescimento Fetal/imunologia , Fator Regulador 3 de Interferon/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Gravidez , Complicações Infecciosas na Gravidez/imunologia , Receptor de Interferon alfa e beta/genéticaRESUMO
Post-translational modifications (PTMs) of proteins are crucial to guarantee the proper biological functions in immune responses. Although protein phosphorylation has been extensively studied, our current knowledge of protein pyrophosphorylation, which occurs based on phosphorylation, is very limited. Protein pyrophosphorylation is originally considered to be a non-enzymatic process, and its function in immune signaling is unknown. Here, we identify a metabolic enzyme, UDP-N-acetylglucosamine pyrophosphorylase 1 (UAP1), as a pyrophosphorylase for protein serine pyrophosphorylation, by catalyzing the pyrophosphorylation of interferon regulatory factor 3 (IRF3) at serine (Ser) 386 to promote robust type I interferon (IFN) responses. Uap1 deficiency significantly impairs the activation of both DNA- and RNA-viruse-induced type I IFN pathways, and the Uap1-deficient mice are highly susceptible to lethal viral infection. Our findings demonstrate the function of protein pyrophosphorylation in the regulation of antiviral responses and provide insights into the crosstalk between metabolism and innate immunity.
Assuntos
Fator Regulador 3 de Interferon , Interferon Tipo I , Animais , Camundongos , Imunidade Inata , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/metabolismo , Interferon Tipo I/genética , Interferon Tipo I/metabolismo , Fosforilação , Transdução de Sinais , Galactosiltransferases/metabolismoRESUMO
The transcription regulator YAP controls organ size by regulating cell growth, proliferation and apoptosis. However, whether YAP has a role in innate antiviral immunity is largely unknown. Here we found that YAP negatively regulated an antiviral immune response. YAP deficiency resulted in enhanced innate immunity, a diminished viral load, and morbidity in vivo. YAP blocked dimerization of the transcription factor IRF3 and impeded translocation of IRF3 to the nucleus after viral infection. Notably, virus-activated kinase IKKÉ phosphorylated YAP at Ser403 and thereby triggered degradation of YAP in lysosomes and, consequently, relief of YAP-mediated inhibition of the cellular antiviral response. These findings not only establish YAP as a modulator of the activation of IRF3 but also identify a previously unknown regulatory mechanism independent of the kinases Hippo and LATS via which YAP is controlled by the innate immune pathway.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/imunologia , Fibroblastos/imunologia , Quinase I-kappa B/metabolismo , Imunidade Inata/imunologia , Lisossomos/metabolismo , Macrófagos/imunologia , Fosfoproteínas/imunologia , Infecções por Rhabdoviridae/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Sistemas CRISPR-Cas , Proteínas de Ciclo Celular , Quimiocina CCL5/genética , Quimiocina CCL5/imunologia , Quimiocina CXCL10/genética , Quimiocina CXCL10/imunologia , Imunofluorescência , Edição de Genes , Células HEK293 , Células HeLa , Humanos , Immunoblotting , Imunoprecipitação , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/imunologia , Fator Regulador 3 de Interferon/metabolismo , Interferon beta/genética , Interferon beta/imunologia , Pulmão/imunologia , Pulmão/patologia , Camundongos , Microscopia Confocal , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/imunologia , Células RAW 264.7 , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Infecções por Rhabdoviridae/patologia , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/imunologia , Vesiculovirus , Carga Viral , Proteínas de Sinalização YAPRESUMO
Missense mutations of the tumor suppressor Neurofibromin 2 (NF2/Merlin/schwannomin) result in sporadic to frequent occurrences of tumorigenesis in multiple organs. However, the underlying pathogenicity of NF2-related tumorigenesis remains mostly unknown. Here we found that NF2 facilitated innate immunity by regulating YAP/TAZ-mediated TBK1 inhibition. Unexpectedly, patient-derived individual mutations in the FERM domain of NF2 (NF2m) converted NF2 into a potent suppressor of cGAS-STING signaling. Mechanistically, NF2m gained extreme associations with IRF3 and TBK1 and, upon innate nucleic acid sensing, was directly induced by the activated IRF3 to form cellular condensates, which contained the PP2A complex, to eliminate TBK1 activation. Accordingly, NF2m robustly suppressed STING-initiated antitumor immunity in cancer cell-autonomous and -nonautonomous murine models, and NF2m-IRF3 condensates were evident in human vestibular schwannomas. Our study reports phase separation-mediated quiescence of cGAS-STING signaling by a mutant tumor suppressor and reveals gain-of-function pathogenesis for NF2-related tumors by regulating antitumor immunity.
Assuntos
Imunidade Inata , Proteínas de Membrana/metabolismo , Mutação de Sentido Incorreto , Neoplasias/metabolismo , Neurofibromina 2/metabolismo , Nucleotidiltransferases/metabolismo , Evasão Tumoral , Animais , Feminino , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Células HEK293 , Humanos , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/metabolismo , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/metabolismo , Masculino , Melanoma Experimental/genética , Melanoma Experimental/imunologia , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/patologia , Neurofibromina 2/genética , Nucleotidiltransferases/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de SinaisRESUMO
The gene encoding PTEN is one of the most frequently mutated tumor suppressor-encoding genes in human cancer. While PTEN's function in tumor suppression is well established, its relationship to anti-microbial immunity remains unknown. Here we found a pivotal role for PTEN in the induction of type I interferon, the hallmark of antiviral innate immunity, that was independent of the pathway of the kinases PI(3)K and Akt. PTEN controlled the import of IRF3, a master transcription factor responsible for IFN-ß production, into the nucleus. We further identified a PTEN-controlled negative phosphorylation site at Ser97 of IRF3 and found that release from this negative regulation via the phosphatase activity of PTEN was essential for the activation of IRF3 and its import into the nucleus. Our study identifies crosstalk between PTEN and IRF3 in tumor suppression and innate immunity.
Assuntos
Imunidade Inata/imunologia , Fator Regulador 3 de Interferon/imunologia , Interferon Tipo I/imunologia , PTEN Fosfo-Hidrolase/imunologia , Infecções por Respirovirus/imunologia , Infecções por Rhabdoviridae/imunologia , Animais , Linhagem Celular , Linhagem Celular Tumoral , Núcleo Celular , Proliferação de Células , Citocinas/imunologia , Células Dendríticas/imunologia , Eletroforese em Gel de Poliacrilamida , Imunofluorescência , Técnicas de Transferência de Genes , Células HEK293 , Humanos , Immunoblotting , Imunoprecipitação , Fator Regulador 3 de Interferon/genética , Fator Regulador 7 de Interferon/genética , Células MCF-7 , Macrófagos/imunologia , Espectrometria de Massas , Camundongos , Microscopia Confocal , Mutagênese Sítio-Dirigida , PTEN Fosfo-Hidrolase/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Vírus Sendai , VesiculovirusRESUMO
Cytosolic DNA-mediated activation of the transcription factor IRF3 is a key event in host antiviral responses. Here we found that infection with DNA viruses induced interaction of the metabolic checkpoint kinase mTOR downstream effector and kinase S6K1 and the signaling adaptor STING in a manner dependent on the DNA sensor cGAS. We further demonstrated that the kinase domain, but not the kinase function, of S6K1 was required for the S6K1-STING interaction and that the TBK1 critically promoted this process. The formation of a tripartite S6K1-STING-TBK1 complex was necessary for the activation of IRF3, and disruption of this signaling axis impaired the early-phase expression of IRF3 target genes and the induction of T cell responses and mucosal antiviral immunity. Thus, our results have uncovered a fundamental regulatory mechanism for the activation of IRF3 in the cytosolic DNA pathway.
Assuntos
DNA/imunologia , Fator Regulador 3 de Interferon/imunologia , Proteínas de Membrana/imunologia , Proteínas Quinases S6 Ribossômicas 90-kDa/imunologia , Adenoviridae/genética , Adenoviridae/imunologia , Animais , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Células Cultivadas , Citosol/imunologia , Citosol/metabolismo , Citosol/virologia , DNA/genética , DNA/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Células HEK293 , Herpes Simples/imunologia , Herpes Simples/virologia , Herpesvirus Humano 1/imunologia , Herpesvirus Humano 1/fisiologia , Humanos , Imunização/métodos , Immunoblotting , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nucleotidiltransferases/genética , Nucleotidiltransferases/imunologia , Nucleotidiltransferases/metabolismo , Ovalbumina/genética , Ovalbumina/imunologia , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/imunologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismoRESUMO
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.
Assuntos
Caspase 8/metabolismo , Caspases/metabolismo , Infecções por Escherichia coli/enzimologia , Infecções por Escherichia coli/fisiopatologia , Choque Séptico/enzimologia , Choque Séptico/fisiopatologia , Animais , Apoptose , Proteínas Reguladoras de Apoptose/metabolismo , Caspase 8/genética , Caspases/genética , Caspases Iniciadoras , Células Cultivadas , Feminino , Inflamação/metabolismo , Inflamação/patologia , Fator Regulador 3 de Interferon/genética , Interferon beta/sangue , Interferon beta/metabolismo , Intestino Delgado/patologia , Peptídeos e Proteínas de Sinalização Intracelular , Lipopolissacarídeos/toxicidade , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas de Ligação a Fosfato , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Transdução de Sinais , Baço/patologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/sangue , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Interleukin-1 beta (IL-1ß) is a pleiotropic mediator of inflammation and is produced in response to a wide range of stimuli. During infection, IL-1ß production occurs in parallel with the onset of innate antimicrobial defenses, but the contribution of IL-1ß signaling to cell-intrinsic immunity is not defined. Here, we report that exogenous IL-1ß induces interferon regulatory factor 3 (IRF3) activation in human myeloid, fibroblast, and epithelial cells. IRF3 activation by IL-1ß is dependent upon the DNA-sensing pathway adaptor, stimulator of interferon genes (STING), through the recognition of cytosolic mtDNA by cyclic guanosine monophosphate (GMP)-AMP synthase (cGAS). IL-1ß treatment results in interferon (IFN) production and activation of IFN signaling to direct a potent innate immune response that restricts dengue virus infection. This study identifies a new function for IL-1ß in the onset or enhancement of cell-intrinsic immunity, with important implications for cGAS-STING in integrating inflammatory and microbial cues for host defense.
Assuntos
DNA Mitocondrial/efeitos dos fármacos , Inflamação/genética , Interleucina-1beta/farmacologia , Proteínas de Membrana/genética , Nucleotidiltransferases/genética , GMP Cíclico/genética , DNA Mitocondrial/genética , Dengue/tratamento farmacológico , Dengue/genética , Dengue/virologia , Vírus da Dengue/efeitos dos fármacos , Vírus da Dengue/genética , Vírus da Dengue/patogenicidade , Interações Hospedeiro-Patógeno/genética , Humanos , Imunidade Inata/efeitos dos fármacos , Imunidade Inata/genética , Inflamação/patologia , Inflamação/virologia , Fator Regulador 3 de Interferon/genética , Interferons/biossíntese , Interleucina-1beta/genética , Células Mieloides/virologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Viral infection triggers host defenses through pattern-recognition receptor-mediated cytokine production, inflammasome activation, and apoptosis of the infected cells. Inflammasome-activated caspases are known to cleave cyclic GMP-AMP synthase (cGAS). Here, we found that apoptotic caspases are critically involved in regulating both DNA and RNA virus-triggered host defenses, in which activated caspase-3 cleaved cGAS, MAVS, and IRF3 to prevent cytokine overproduction. Caspase-3 was exclusively required in human cells, whereas caspase-7 was involved only in murine cells to inactivate cGAS, reflecting distinct regulatory mechanisms in different species. Caspase-mediated cGAS cleavage was enhanced in the presence of dsDNA. Alternative MAVS cleavage sites were used to ensure the inactivation of this critical protein. Elevated type I IFNs were detected in caspase-3-deficient cells without any infection. Casp3-/- mice consistently showed increased resistance to viral infection and experimental autoimmune encephalomyelitis. Our results demonstrate that apoptotic caspases control innate immunity and maintain immune homeostasis against viral infection.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Apoptose , Caspases/metabolismo , Fator Regulador 3 de Interferon/metabolismo , Interferon Tipo I/metabolismo , Nucleotidiltransferases/metabolismo , Viroses/enzimologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Caspase 2/genética , Caspase 2/metabolismo , Caspase 3/genética , Caspase 3/metabolismo , Caspase 7/genética , Caspase 7/metabolismo , Caspase 9/genética , Caspase 9/metabolismo , Caspases/genética , Feminino , Células HEK293 , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata , Fator Regulador 3 de Interferon/genética , Masculino , Camundongos Endogâmicos C57BL , Nucleotidiltransferases/genética , Vírus Sendai/imunologia , Vírus Sendai/patogenicidade , Transdução de Sinais , Células THP-1 , Vaccinia virus/imunologia , Vaccinia virus/patogenicidade , Viroses/genética , Viroses/imunologia , Viroses/virologiaRESUMO
The Type-I interferon (IFN-I) response is the major outcome of stimulator of interferon genes (STING) activation in innate cells. STING is more abundantly expressed in adaptive T cells; nevertheless, its intrinsic function in T cells remains unclear. Intriguingly, we previously demonstrated that STING activation in T cells activates widespread IFN-independent activities, which stands in contrast to the well-known STING-mediated IFN response. Here, we have identified that STING activation induces regulatory T cells (Tregs) differentiation independently of IRF3 and IFN. Specifically, the translocation of STING from the endoplasmic reticulum to the Golgi activates mitogen-activated protein kinase (MAPK) activity, which subsequently triggers transcription factor cAMP response element-binding protein (CREB) activation. The activation of the STING-MAPK-CREB signaling pathway induces the expression of many cytokine genes, including interleukin-2 (IL-2) and transforming growth factor-beta 2 (TGF-ß2), to promote the Treg differentiation. Genetic knockdown of MAPK p38 or pharmacological inhibition of MAPK p38 or CREB markedly inhibits STING-mediated Treg differentiation. Administration of the STING agonist also promotes Treg differentiation in mice. In the Trex1-/- autoimmune disease mouse model, we demonstrate that intrinsic STING activation in CD4+ T cells can drive Treg differentiation, potentially counterbalancing the autoimmunity associated with Trex1 deficiency. Thus, STING-MAPK-CREB represents an IFN-independent signaling axis of STING that may have profound effects on T cell effector function and adaptive immunity.
Assuntos
Diferenciação Celular , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico , Proteínas de Membrana , Linfócitos T Reguladores , Animais , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Camundongos , Transdução de Sinais , Sistema de Sinalização das MAP Quinases , Camundongos Endogâmicos C57BL , Transporte Proteico , Fator Regulador 3 de Interferon/metabolismo , Fator Regulador 3 de Interferon/genética , Camundongos Knockout , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
SARS-CoV-2 interferes with antigen presentation by downregulating major histocompatibility complex (MHC) II on antigen-presenting cells, but the mechanism mediating this process is unelucidated. Herein, analysis of protein and gene expression in human antigen-presenting cells reveals that MHC II is downregulated by the SARS-CoV-2 main protease, NSP5. This suppression of MHC II expression occurs via decreased expression of the MHC II regulatory protein CIITA. CIITA downregulation is independent of the proteolytic activity of NSP5, and rather, NSP5 delivers HDAC2 to the transcription factor IRF3 at an IRF-binding site within the CIITA promoter. Here, HDAC2 deacetylates and inactivates the CIITA promoter. This loss of CIITA expression prevents further expression of MHC II, with this suppression alleviated by ectopic expression of CIITA or knockdown of HDAC2. These results identify a mechanism by which SARS-CoV-2 limits MHC II expression, thereby delaying or weakening the subsequent adaptive immune response.
Assuntos
Antígenos de Histocompatibilidade Classe II , Histona Desacetilase 2 , Proteínas Nucleares , Regiões Promotoras Genéticas , SARS-CoV-2 , Transativadores , Humanos , Apresentação de Antígeno/genética , Células Apresentadoras de Antígenos/metabolismo , Células Apresentadoras de Antígenos/imunologia , COVID-19/virologia , COVID-19/imunologia , COVID-19/genética , COVID-19/metabolismo , Cisteína Endopeptidases/metabolismo , Cisteína Endopeptidases/genética , Regulação para Baixo/genética , Células HEK293 , Antígenos de Histocompatibilidade Classe II/metabolismo , Antígenos de Histocompatibilidade Classe II/genética , Histona Desacetilase 2/metabolismo , Histona Desacetilase 2/genética , Fator Regulador 3 de Interferon/metabolismo , Fator Regulador 3 de Interferon/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Regiões Promotoras Genéticas/genética , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , SARS-CoV-2/imunologia , Transativadores/metabolismo , Transativadores/genética , Proteínas não Estruturais Virais/metabolismo , Proteínas não Estruturais Virais/genéticaRESUMO
Tripartite-motif protein-56 (TRIM56) positively regulates the induction of type I interferon response via the TLR3 pathway by enhancing IRF3 activation and depends on its C-terminal residues 621-750 for interacting with the adaptor TRIF. However, the precise underlying mechanism and detailed TRIM56 determinants remain unclear. Herein, we show ectopic expression of murine TRIM56 also enhances TLR3-dependent interferon-ß promoter activation, suggesting functional conservation. We found that endogenous TRIM56 and TRIF formed a complex early (0.5-2 h) after poly-I:C stimulation and that TRIM56 overexpression also promoted activation of NF-κB by poly-I:C but not that by TNF-α or IL-1ß, consistent with a specific effect on TRIF prior to the bifurcation of NF-κB and IRF3. Using transient transfection and Tet-regulated cell lines expressing various TRIM56 mutants, we demonstrated the Coiled-coil domain and a segment spanning residues â¼434-610, but not the B-box or residues 355-433, were required for TRIM56 augmentation of TLR3 signaling. Moreover, alanine substitution at each putative phosphorylation site, Ser471, Ser475, and Ser710, abrogated TRIM56 function. Concordantly, mutants bearing Ser471Ala, Ser475Ala, or Ser710Ala, or lacking the Coiled-coil domain, all lost the capacity to enhance poly-I:C-induced establishment of an antiviral state. Furthermore, the Ser710Ala mutation disrupted the TRIM56-TRIF association. Using phospho-specific antibodies, we detected biphasic phosphorylation of TRIM56 at Ser471 and Ser475 following TLR3 stimulation, with the early phase occurring at â¼0.5 to 1 h, prior to IRF3 phosphorylation. Together, these data reveal novel molecular details critical for the TRIM56 augmentation of TLR3-dependent antiviral response and highlight important roles for TRIM56 scaffolding and phosphorylation.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular , Imunidade Inata , Receptor 3 Toll-Like , Proteínas com Motivo Tripartido , Animais , Humanos , Camundongos , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/imunologia , Células HEK293 , Fator Regulador 3 de Interferon/metabolismo , Fator Regulador 3 de Interferon/genética , NF-kappa B/metabolismo , Fosforilação , Poli I-C/farmacologia , Domínios Proteicos , Transdução de Sinais , Receptor 3 Toll-Like/metabolismo , Receptor 3 Toll-Like/genética , Proteínas com Motivo Tripartido/metabolismo , Proteínas com Motivo Tripartido/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genéticaRESUMO
The controlled release of mitochondrial content into the cytosol has emerged as one of the key steps in mitochondrial signaling. In particular, the release of mitochondrial DNA (mtDNA) into the cytosol has been shown to activate interferon beta (IFN-ß) gene expression to execute the innate immune response. In this report, we show that human adenovirus type 5 (HAdV-C5) infection induces the release of mtDNA into the cytosol. The release of mtDNA is mediated by the viral minor capsid protein VI (pVI), which localizes to mitochondria. The presence of the mitochondrial membrane proteins Bak and Bax are needed for the mtDNA release, whereas the viral E1B-19K protein blocked pVI-mediated mtDNA release. Surprisingly, the pVI-mediated mtDNA release did not increase but inhibited the IFN-ß gene expression. Notably, the pVI expression caused mitochondrial leakage of the HSP60 protein. The latter prevented specific phosphorylation of the interferon regulatory factor 3 (IRF3) needed for IFN-ß gene expression. Overall, we assign a new mitochondria and IFN-ß signaling-modulating function to the HAdV-C5 minor capsid protein VI. IMPORTANCE: Human adenoviruses (HAdVs) are common pathogens causing various self-limiting diseases, including conjunctivitis and the common cold. HAdVs need to interfere with multiple cellular signaling pathways during the infection to gain control over the host cell. In this study, we identified human adenovirus type 5 (HAdV-C5) minor capsid protein VI as a factor modulating mitochondrial membrane integrity and mitochondrial signaling. We show that pVI-altered mitochondrial signaling impedes the cell's innate immune response, which may benefit HAdV growth. Overall, our study provides new detailed insights into the HAdV-mitochondria interactions and signaling. This knowledge is helpful when developing new anti-viral treatments against pathogenic HAdV infections and improving HAdV-based therapeutics.
Assuntos
Adenovírus Humanos , Proteínas do Capsídeo , DNA Mitocondrial , Interferon beta , Mitocôndrias , Transdução de Sinais , Humanos , Adenovírus Humanos/fisiologia , Adenovírus Humanos/genética , Adenovírus Humanos/metabolismo , Proteínas do Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Mitocôndrias/metabolismo , DNA Mitocondrial/metabolismo , DNA Mitocondrial/genética , Interferon beta/metabolismo , Interferon beta/genética , Imunidade Inata , Fator Regulador 3 de Interferon/metabolismo , Fator Regulador 3 de Interferon/genética , Infecções por Adenovirus Humanos/virologia , Infecções por Adenovirus Humanos/metabolismo , Membranas Mitocondriais/metabolismo , Células HEK293 , Fosforilação , Citosol/metabolismo , Citosol/virologiaRESUMO
Zika virus (ZIKV) is a Flavivirus responsible for recent epidemics in Pacific Islands and in the Americas. In humans, the consequences of ZIKV infection range from asymptomatic infection to severe neurological disease such as Guillain-Barré syndrome or fetal neurodevelopmental defects, suggesting, among other factors, the influence of host genetic variants. We previously reported similar diverse outcomes of ZIKV infection in mice of the Collaborative Cross (CC), a collection of inbred strains with large genetic diversity. CC071/TauUnc (CC071) was the most susceptible CC strain with severe symptoms and lethality. Notably, CC071 has been recently reported to be also susceptible to other flaviviruses including dengue virus, Powassan virus, West Nile virus, and to Rift Valley fever virus. To identify the genetic origin of this broad susceptibility, we investigated ZIKV replication in mouse embryonic fibroblasts (MEFs) from CC071 and two resistant strains. CC071 showed uncontrolled ZIKV replication associated with delayed induction of type-I interferons (IFN-I). Genetic analysis identified a mutation in the Irf3 gene specific to the CC071 strain which prevents the protein phosphorylation required to activate interferon beta transcription. We demonstrated that this mutation induces the same defective IFN-I response and uncontrolled viral replication in MEFs as an Irf3 knock-out allele. By contrast, we also showed that Irf3 deficiency did not induce the high plasma viral load and clinical severity observed in CC071 mice and that susceptibility alleles at other genes, not associated with the IFN-I response, are required. Our results provide new insight into the in vitro and in vivo roles of Irf3, and into the genetic complexity of host responses to flaviviruses.