RESUMO
TLR3 is a sensor of double-stranded RNA that is indispensable for defense against infection with herpes simplex virus type 1 (HSV-1) in the brain. We found here that TLR3 was required for innate immune responses to HSV-1 in neurons and astrocytes. During infection with HSV-1, TLR3 recruited the metabolic checkpoint kinase complex mTORC2, which led to the induction of chemokines and trafficking of TLR3 to the cell periphery. Such trafficking enabled the activation of molecules (including mTORC1) required for the induction of type I interferons. Intracranial infection of mice with HSV-1 was exacerbated by impairment of TLR3 responses with an inhibitor of mTOR and was significantly 'rescued' by potentiation of TLR3 responses with an agonistic antibody to TLR3. These results suggest that the TLR3-mTORC2 axis might be a therapeutic target through which to combat herpes simplex encephalitis.
Assuntos
Encefalite por Herpes Simples/imunologia , Alvo Mecanístico do Complexo 2 de Rapamicina/imunologia , Receptor 3 Toll-Like/imunologia , Animais , Herpesvirus Humano 1 , Imunidade Inata/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Células NIH 3T3RESUMO
Activation of the stimulator of interferon genes (STING) pathway by microbial or self-DNA, as well as cyclic dinucleotides (CDNs), results in the induction of numerous genes that suppress pathogen replication and facilitate adaptive immunity. However, sustained gene transcription is rigidly prevented to avoid lethal STING-dependent proinflammatory disease by mechanisms that remain unknown. We demonstrate here that, after autophagy-dependent STING delivery of TANK-binding kinase 1 (TBK1) to endosomal/lysosomal compartments and activation of transcription factors interferon regulatory factor 3 (IRF3) and NF-κB, STING is subsequently phosphorylated by serine/threonine UNC-51-like kinase (ULK1/ATG1), and IRF3 function is suppressed. ULK1 activation occurred following disassociation from its repressor AMP activated protein kinase (AMPK) and was elicited by CDNs generated by the cGAMP synthase, cGAS. Thus, although CDNs may initially facilitate STING function, they subsequently trigger negative-feedback control of STING activity, thus preventing the persistent transcription of innate immune genes.
Assuntos
Imunidade Inata , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Proteína Homóloga à Proteína-1 Relacionada à Autofagia , Humanos , Proteínas de Membrana/química , Dados de Sequência Molecular , Nucleotídeos Cíclicos/metabolismo , Fosforilação , Alinhamento de SequênciaRESUMO
The cytoplasm is usually a DNA-free zone, but during fertilization, sperm DNA enters oocyte cytoplasm and could potentially trigger a response. Abe et al. (2017) identify NLRP14 as a germ-cell-specific negative regulator of DNA sensing that may be of particular importance during fertilization.
Assuntos
DNA , Fertilização/imunologia , Imunidade Inata , Oócitos , Espermatozoides , Animais , Citoplasma , Feminino , Fertilização/fisiologia , MasculinoRESUMO
Lack or loss of tumor antigenicity represents one of the key mechanisms of immune escape and resistance to T cell-based immunotherapies. Evidence suggests that activation of stimulator of interferon genes (STING) signaling in tumor cells can augment their antigenicity by triggering a type I IFN-mediated sequence of autocrine and paracrine events. Although suppression of this pathway in melanoma and other tumor types has been consistently reported, the mechanistic basis remains unclear. In this study, we asked whether this suppression is, in part, epigenetically regulated and whether it is indeed a driver of melanoma resistance to T cell-based immunotherapies. Using genome-wide DNA methylation profiling, we show that promoter hypermethylation of cGAS and STING genes mediates their coordinated transcriptional silencing and contributes to the widespread impairment of the STING signaling function in clinically-relevant human melanomas and melanoma cell lines. This suppression is reversible through pharmacologic inhibition of DNA methylation, which can reinstate functional STING signaling in at least half of the examined cell lines. Using a series of T cell recognition assays with HLA-matched human melanoma tumor-infiltrating lymphocytes (TIL), we further show that demethylation-mediated restoration of STING signaling in STING-defective melanoma cell lines can improve their antigenicity through the up-regulation of MHC class I molecules and thereby enhance their recognition and killing by cytotoxic T cells. These findings not only elucidate the contribution of epigenetic processes and specifically DNA methylation in melanoma-intrinsic STING signaling impairment but also highlight their functional significance in mediating tumor-immune evasion and resistance to T cell-based immunotherapies.
Assuntos
Metilação de DNA , Epigênese Genética , Melanoma/genética , Proteínas de Membrana/genética , Linfócitos T/imunologia , Linhagem Celular Tumoral , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Linfócitos do Interstício Tumoral/imunologia , Melanoma/imunologia , Proteínas de Membrana/metabolismo , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismoRESUMO
Macrophages metabolic reprogramming in response to microbial insults is a major determinant of pathogen growth or containment. Here, we reveal a distinct mechanism by which stimulator of interferon genes (STING), a cytosolic sensor that regulates innate immune responses, contributes to an inflammatory M1-like macrophage profile upon Brucella abortus infection. This metabolic reprogramming is induced by STING-dependent stabilization of hypoxia-inducible factor-1 alpha (HIF-1α), a global regulator of cellular metabolism and innate immune cell functions. HIF-1α stabilization reduces oxidative phosphorylation and increases glycolysis during infection with B. abortus and, likewise, enhances nitric oxide production, inflammasome activation and IL-1ß release in infected macrophages. Furthermore, the induction of this inflammatory profile participates in the control of bacterial replication since absence of HIF-1α renders mice more susceptible to B. abortus infection. Mechanistically, activation of STING by B. abortus infection drives the production of mitochondrial reactive oxygen species (mROS) that ultimately influences HIF-1α stabilization. Moreover, STING increases the intracellular succinate concentration in infected macrophages, and succinate pretreatment induces HIF-1α stabilization and IL-1ß release independently of its cognate receptor GPR91. Collectively, these data demonstrate a pivotal mechanism in the immunometabolic regulation of macrophages during B. abortus infection that is orchestrated by STING via HIF-1α pathway and highlight the metabolic reprogramming of macrophages as a potential treatment strategy for bacterial infections.
Assuntos
Brucella abortus/imunologia , Brucelose/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Macrófagos/metabolismo , Proteínas de Membrana/metabolismo , Animais , Brucelose/imunologia , Brucelose/microbiologia , Glicólise , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Inflamassomos/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico/metabolismo , Fosforilação Oxidativa , Espécies Reativas de Oxigênio/metabolismoRESUMO
The sensing of pathogen-associated DNA in the cytoplasm is an important trigger of host-defense responses that include the production of type I interferon. A new study suggests that the DExDc helicase DDX41 may function in dendritic cells as a DNA sensor to activate STING-dependent innate immune responses.
Assuntos
RNA Helicases DEAD-box/fisiologia , DNA Helicases/fisiologia , DNA/metabolismo , Células Dendríticas/fisiologia , Proteínas de Membrana/fisiologia , Animais , HumanosRESUMO
Spontaneous T cell responses against tumors occur frequently and have prognostic value in patients. The mechanism of innate immune sensing of immunogenic tumors leading to adaptive T cell responses remains undefined, although type I interferons (IFNs) are implicated in this process. We found that spontaneous CD8(+) T cell priming against tumors was defective in mice lacking stimulator of interferon genes complex (STING), but not other innate signaling pathways, suggesting involvement of a cytosolic DNA sensing pathway. In vitro, IFN-? production and dendritic cell activation were triggered by tumor-cell-derived DNA, via cyclic-GMP-AMP synthase (cGAS), STING, and interferon regulatory factor 3 (IRF3). In the tumor microenvironment in vivo, tumor cell DNA was detected within host antigen-presenting cells, which correlated with STING pathway activation and IFN-? production. Our results demonstrate that a major mechanism for innate immune sensing of cancer occurs via the host STING pathway, with major implications for cancer immunotherapy.
Assuntos
Linfócitos T CD8-Positivos/imunologia , DNA/imunologia , Ativação Linfocitária/imunologia , Melanoma Experimental/imunologia , Proteínas de Membrana/imunologia , Imunidade Adaptativa , Proteínas Adaptadoras de Transdução de Sinal/genética , Transferência Adotiva , Animais , Células Apresentadoras de Antígenos/citologia , Células Apresentadoras de Antígenos/imunologia , Linhagem Celular Tumoral , Proliferação de Células , Células Dendríticas/imunologia , Imunidade Inata , Fator Regulador 3 de Interferon/genética , Interferon beta/biossíntese , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/genética , Nucleotidiltransferases , Receptores de Antígenos de Linfócitos T/imunologia , Receptores Purinérgicos P2X7/genética , Receptor 4 Toll-Like/genética , Receptor Toll-Like 9/genética , Microambiente Tumoral/imunologiaRESUMO
Chromatin is traditionally viewed as a nuclear entity that regulates gene expression and silencing. However, we recently discovered the presence of cytoplasmic chromatin fragments that pinch off from intact nuclei of primary cells during senescence, a form of terminal cell-cycle arrest associated with pro-inflammatory responses. The functional significance of chromatin in the cytoplasm is unclear. Here we show that cytoplasmic chromatin activates the innate immunity cytosolic DNA-sensing cGAS-STING (cyclic GMP-AMP synthase linked to stimulator of interferon genes) pathway, leading both to short-term inflammation to restrain activated oncogenes and to chronic inflammation that associates with tissue destruction and cancer. The cytoplasmic chromatin-cGAS-STING pathway promotes the senescence-associated secretory phenotype in primary human cells and in mice. Mice deficient in STING show impaired immuno-surveillance of oncogenic RAS and reduced tissue inflammation upon ionizing radiation. Furthermore, this pathway is activated in cancer cells, and correlates with pro-inflammatory gene expression in human cancers. Overall, our findings indicate that genomic DNA serves as a reservoir to initiate a pro-inflammatory pathway in the cytoplasm in senescence and cancer. Targeting the cytoplasmic chromatin-mediated pathway may hold promise in treating inflammation-related disorders.
Assuntos
Senescência Celular/genética , Cromatina/metabolismo , Citoplasma/genética , Imunidade Inata , Inflamação/genética , Inflamação/patologia , Neoplasias/genética , Neoplasias/imunologia , Animais , Linhagem Celular Tumoral , Cromatina/imunologia , Citocinas/imunologia , Citocinas/metabolismo , Citoplasma/imunologia , Feminino , Humanos , Inflamação/imunologia , Fígado/metabolismo , Masculino , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Neoplasias/patologia , Nucleotidiltransferases/metabolismo , Proteína Oncogênica p21(ras)/genética , Proteína Oncogênica p21(ras)/imunologia , Radiação IonizanteRESUMO
Brucellosis, caused by a number of Brucella species, remains the most prevalent zoonotic disease worldwide. Brucella establish chronic infections within host macrophages despite triggering cytosolic innate immune sensors, including Stimulator of Interferon Genes (STING), which potentially limit infection. In this study, STING was required for control of chronic Brucella infection in vivo. However, early during infection, Brucella down-regulated STING mRNA and protein. Down-regulation occurred post-transcriptionally, required live bacteria, the Brucella type IV secretion system, and was independent of host IRE1-RNase activity. STING suppression occurred in MyD88-/- macrophages and was not induced by Toll-like receptor agonists or purified Brucella lipopolysaccharide (LPS). Rather, Brucella induced a STING-targeting microRNA, miR-24-2, in a type IV secretion system-dependent manner. Furthermore, STING downregulation was inhibited by miR-24 anti-miRs and in Mirn23a locus-deficient macrophages. Failure to suppress STING expression in Mirn23a-/- macrophages correlated with diminished Brucella replication, and was rescued by exogenous miR-24. Mirn23a-/- mice were also more resistant to splenic colonization one week post infection. Anti-miR-24 potently suppressed replication in wild type, but much less in STING-/- macrophages, suggesting most of the impact of miR-24 induction on replication occurred via STING suppression. In summary, Brucella sabotages cytosolic surveillance by miR-24-dependent suppression of STING expression; post-STING activation "damage control" via targeted STING destruction may enable establishment of chronic infection.
Assuntos
Brucella/metabolismo , Brucelose/metabolismo , Proteínas de Membrana/biossíntese , MicroRNAs/metabolismo , Animais , Brucella/genética , Brucelose/genética , Feminino , Interações Hospedeiro-Patógeno/imunologia , Macrófagos/imunologia , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , RNA Mensageiro/genética , Sistemas de Secreção Tipo IV/genética , Sistemas de Secreção Tipo IV/metabolismoRESUMO
AIMS: Lifestyle-related diseases promote atherosclerosis, a chronic inflammatory disease; however, the molecular mechanism remains largely unknown. Endogenous DNA fragments released under over-nutrient condition provoke sterile inflammation through the recognition by DNA sensors. Here, we investigated the role of stimulator of interferon genes (STING), a cytosolic DNA sensor, in atherogenesis. METHODS AND RESULTS: Apolipoprotein E-deficient (Apoe-/-) mice fed a western-type diet (WTD), a hypercholesterolaemic mouse model, showed higher STING expression and markers for DNA damage such as γH2AX, p53, and single-stranded DNA (ssDNA) accumulation in macrophages in the aorta compared with wild-type (WT) mice. The level of cGAMP, a STING agonist, in the aorta was higher in Apoe-/- mice. Genetic deletion of Sting in Apoe-/- mice reduced atherosclerotic lesions in the aortic arch, lipid, and macrophage accumulation in plaques, and inflammatory molecule expression in the aorta compared with the control. Pharmacological blockade of STING using a specific inhibitor, C-176, ameliorated atherogenesis in Apoe-/- mice. In contrast, bone marrow-specific STING expression in Apoe-/- mice stimulated atherogenesis. Expression or deletion of STING did not affect metabolic parameters and blood pressure. In vitro studies revealed that STING activation by cGAMP or mitochondrial DNA accelerated inflammatory molecule expression (e.g. TNF-α or IFN-ß) in mouse and human macrophages. Activation of nuclear factor-κB and TANK binding kinase 1 was involved in STING-associated vascular inflammation and macrophage activation. Furthermore, human atherosclerotic lesions in the carotid arteries expressed STING and cGAMP. CONCLUSION: Stimulator of interferon genes stimulates pro-inflammatory activation of macrophages, leading to the development of atherosclerosis. Stimulator of interferon genes signalling may serve as a potential therapeutic target for atherosclerosis.
Assuntos
Aterosclerose , Placa Aterosclerótica , Animais , Aterosclerose/genética , DNA , Modelos Animais de Doenças , Imunidade Inata , Inflamação , Estilo de Vida , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
The type I interferon (IFN) response initiated by detection of nucleic acids is important for antiviral defense but is also associated with specific autoimmune diseases. Mutations in the human 3' repair exonuclease 1 (Trex1) gene cause Aicardi-Goutières syndrome (AGS), an IFN-associated autoimmune disease. However, the source of the type I IFN response and the precise mechanisms of disease in AGS remain unknown. Here, we demonstrate that Trex1 is an essential negative regulator of the STING-dependent antiviral response. We used an in vivo reporter of IFN activity in Trex1-deficient mice to localize the initiation of disease to nonhematopoietic cells. These IFNs drove T cell-mediated inflammation and an autoantibody response that targeted abundant, tissue-restricted autoantigens. However, B cells contributed to mortality independently of T cell-mediated tissue damage. These findings reveal a stepwise progression of autoimmune disease in Trex1-deficient mice, with implications for the treatment of AGS and related disorders.
Assuntos
Doenças Autoimunes/fisiopatologia , Autoimunidade/imunologia , Interferons , Linfócitos/imunologia , Animais , Doenças Autoimunes/enzimologia , Doenças Autoimunes do Sistema Nervoso/fisiopatologia , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Interferons/fisiologia , Camundongos , Camundongos Knockout , Modelos Biológicos , Malformações do Sistema Nervoso/fisiopatologia , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Transdução de SinaisRESUMO
Brucella abortus is a facultative intracellular bacterium that causes brucellosis, a prevalent zoonosis that leads to abortion and infertility in cattle, and undulant fever, debilitating arthritis, endocarditis, and meningitis in humans. Signaling pathways triggered by B. abortus involves stimulator of IFN genes (STING), which leads to production of type I IFNs. In this study, we evaluated the pathway linking the unfolded protein response (UPR) and the endoplasmic reticulum-resident transmembrane molecule STING, during B. abortus infection. We demonstrated that B. abortus infection induces the expression of the UPR target gene BiP and XBP1 in murine macrophages through a STING-dependent pathway. Additionally, we also observed that STING activation was dependent on the bacterial second messenger cyclic dimeric GMP. Furthermore, the Brucella-induced UPR is crucial for induction of multiple molecules linked to type I IFN signaling pathway, such as IFN-ß, IFN regulatory factor 1, and guanylate-binding proteins. Furthermore, IFN-ß is also important for the UPR induction during B. abortus infection. Indeed, IFN-ß shows a synergistic effect in inducing the IRE1 axis of the UPR. In addition, priming cells with IFN-ß favors B. abortus survival in macrophages. Moreover, Brucella-induced UPR facilitates bacterial replication in vitro and in vivo. Finally, these results suggest that B. abortus-induced UPR is triggered by bacterial cyclic dimeric GMP, in a STING-dependent manner, and that this response supports bacterial replication. In summary, association of STING and IFN-ß signaling pathways with Brucella-induced UPR unravels a novel link between innate immunity and endoplasmic reticulum stress that is crucial for bacterial infection outcome.
Assuntos
Brucella abortus/fisiologia , Brucelose/imunologia , Interações Hospedeiro-Patógeno/imunologia , Proteínas de Membrana/imunologia , Nucleotídeos Cíclicos/imunologia , Resposta a Proteínas não Dobradas/imunologia , Animais , Brucelose/genética , Interações Hospedeiro-Patógeno/genética , Humanos , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Nucleotídeos Cíclicos/genética , Transdução de Sinais/genética , Transdução de Sinais/imunologiaRESUMO
How the cell recognizes cytosolic DNA including DNA-based microbes to trigger host-defense-related gene activation remains to be fully resolved. Here, we demonstrate that STING (stimulator of interferon genes), an endoplasmic reticulum translocon-associated transmembrane protein, acts to detect cytoplasmic DNA species. STING homodimers were able to complex with self- (apoptotic, necrotic) or pathogen-related ssDNA and dsDNA and were indispensible for HSV-1-mediated transcriptional activation of a wide array of innate immune and proinflammatory genes in addition to type I IFN. Our data indicate that STING instigates cytoplasmic DNA-mediated cellular defense gene transcription and facilitates adoptive responses that are required for protection of the host. In contrast, chronic STING activation may manifest inflammatory responses and possibly autoimmune disease triggered by self-DNA.
Assuntos
Citoplasma/metabolismo , DNA de Cadeia Simples/metabolismo , Proteínas de Membrana/metabolismo , Animais , Apoptose , Sítios de Ligação , Citoplasma/imunologia , DNA de Cadeia Simples/imunologia , Genes Reporter , Células HEK293 , Herpesvirus Humano 1/imunologia , Humanos , Imunidade Inata , Mediadores da Inflamação/metabolismo , Interferon Tipo I/genética , Interferon Tipo I/metabolismo , Luciferases de Vaga-Lume/biossíntese , Luciferases de Vaga-Lume/genética , Proteínas de Membrana/genética , Camundongos , Necrose , Multimerização Proteica , Interferência de RNA , Telomerase/genética , Telomerase/metabolismo , Transcrição Gênica , TransfecçãoRESUMO
It is uncertain how antiviral lymphocytes are activated in draining lymph nodes, the site where adaptive immune responses are initiated. Here, using intravital microscopy we show that after infection of mice with vaccinia virus (a large DNA virus) or vesicular stomatitis virus (a small RNA virus), virions drained to the lymph node and infected cells residing just beneath the subcapsular sinus. Naive CD8+ T cells rapidly migrated to infected cells in the peripheral interfollicular region and then formed tight interactions with dendritic cells, leading to complete T cell activation. Thus, antigen presentation at the lymph node periphery, not at lymphocyte exit sites in deeper lymph node venules, as dogma dictates, has a dominant function in antiviral CD8+ T cell activation.
Assuntos
Apresentação de Antígeno , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , Linfonodos/imunologia , Ativação Linfocitária , Animais , Células Apresentadoras de Antígenos/imunologia , Movimento Celular , Células Dendríticas/imunologia , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Camundongos , Camundongos Transgênicos , Vaccinia virus/imunologia , Vesiculovirus/imunologiaRESUMO
Although Toll-like receptor 9 (TLR9) has been implicated in cytokine and type I interferon (IFN) production during malaria in humans and mice, the high AT content of the Plasmodium falciparum genome prompted us to examine the possibility that malarial DNA triggered TLR9-independent pathways. Over 6000 ATTTTTAC ("AT-rich") motifs are present in the genome of P. falciparum, which we show here potently induce type I IFNs. Parasite DNA, parasitized erythrocytes and oligonucleotides containing the AT-rich motif induce type I IFNs via a pathway that did not involve the previously described sensors TLR9, DAI, RNA polymerase-III or IFI16/p204. Rather, AT-rich DNA sensing involved an unknown receptor that coupled to the STING, TBK1 and IRF3-IRF7 signaling pathway. Mice lacking IRF3, IRF7, the kinase TBK1 or the type I IFN receptor were resistant to otherwise lethal cerebral malaria. Collectively, these observations implicate AT-rich DNA sensing via STING, TBK1 and IRF3-IRF7 in P. falciparum malaria.
Assuntos
Sequência Rica em At/genética , DNA de Protozoário/genética , Malária Falciparum/imunologia , Oligonucleotídeos/genética , Plasmodium falciparum/fisiologia , Animais , DNA de Protozoário/metabolismo , Perfilação da Expressão Gênica , Humanos , Imunidade Inata/genética , Fator Regulador 3 de Interferon/metabolismo , Fator Regulador 7 de Interferon/metabolismo , Interferon Tipo I/genética , Interferon Tipo I/metabolismo , Malária Falciparum/parasitologia , Malária Falciparum/fisiopatologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Oligonucleotídeos/metabolismo , Plasmodium falciparum/patogenicidade , Proteínas Serina-Treonina Quinases/metabolismo , Receptor de Interferon alfa e beta/genética , Transdução de Sinais/genéticaRESUMO
Immunity against microbes depends on recognition of pathogen-associated molecular patterns by innate receptors. Signaling pathways triggered by Brucella abortus DNA involves TLR9, AIM2, and stimulator of IFN genes (STING). In this study, we observed by microarray analysis that several type I IFN-associated genes, such as IFN-ß and guanylate-binding proteins (GBPs), are downregulated in STING knockout (KO) macrophages infected with Brucella or transfected with DNA. Additionally, we determined that STING and cyclic GMP-AMP synthase (cGAS) are important to engage the type I IFN pathway, but only STING is required to induce IL-1ß secretion, caspase-1 activation, and GBP2 and GBP3 expression. Furthermore, we determined that STING but not cGAS is critical for host protection against Brucella infection in macrophages and in vivo. This study provides evidence of a cGAS-independent mechanism of STING-mediated protection against an intracellular bacterial infection. Additionally, infected IFN regulatory factor-1 and IFNAR KO macrophages had reduced GBP2 and GBP3 expression and these cells were more permissive to Brucella replication compared with wild-type control macrophages. Because GBPs are critical to target vacuolar bacteria, we determined whether GBP2 and GBPchr3 affect Brucella control in vivo. GBPchr3 but not GBP2 KO mice were more susceptible to bacterial infection, and small interfering RNA treated-macrophages showed reduction in IL-1ß secretion and caspase-1 activation. Finally, we also demonstrated that Brucella DNA colocalizes with AIM2, and AIM2 KO mice are less resistant to B. abortus infection. In conclusion, these findings suggest that the STING-dependent type I IFN pathway is critical for the GBP-mediated release of Brucella DNA into the cytosol and subsequent activation of AIM2.
Assuntos
Brucella abortus/imunologia , Brucelose/imunologia , Brucelose/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Inflamassomos/metabolismo , Proteínas de Membrana/metabolismo , Transdução de Sinais , Animais , Brucella abortus/genética , Brucelose/genética , Brucelose/microbiologia , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , Citocinas/metabolismo , Proteínas de Ligação ao GTP/genética , Expressão Gênica , Perfilação da Expressão Gênica , Granuloma/metabolismo , Granuloma/microbiologia , Granuloma/patologia , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata , Mediadores da Inflamação , Fator Regulador 3 de Interferon/metabolismo , Interferon Tipo I/genética , Interferon Tipo I/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Modelos Biológicos , NF-kappa B/metabolismoRESUMO
The ability to control lentivirus replication may be determined, in part, by the extent to which individual viral proteins are targeted by the immune system. Consequently, defining the antigens that elicit the most protective immune responses may facilitate the design of effective HIV-1 vaccines. Here we vaccinated four groups of rhesus macaques with a heterologous vector prime/boost/boost/boost (PBBB) regimen expressing the following simian immunodeficiency virus (SIV) genes: env, gag, vif, rev, tat, and nef (Group 1); env, vif, rev, tat, and nef (Group 2); gag, vif, rev, tat, and nef (Group 3); or vif, rev, tat, and nef (Group 4). Following repeated intrarectal challenges with a marginal dose of the neutralization-resistant SIVmac239 clone, vaccinees in Groups 1-3 became infected at similar rates compared to control animals. Unexpectedly, vaccinees in Group 4 became infected at a slower pace than the other animals, although this difference was not statistically significant. Group 1 exhibited the best post-acquisition virologic control of SIV infection, with significant reductions in both peak and chronic phase viremia. Indeed, 5/8 Group 1 vaccinees had viral loads of less than 2,000 vRNA copies/mL of plasma in the chronic phase. Vaccine regimens that did not contain gag (Group 2), env (Group 3), or both of these inserts (Group 4) were largely ineffective at decreasing viremia. Thus, vaccine-induced immune responses against both Gag and Env appeared to maximize control of immunodeficiency virus replication. Collectively, these findings are relevant for HIV-1 vaccine design as they provide additional insights into which of the lentiviral proteins might serve as the best vaccine immunogens.
Assuntos
Infecções por HIV/imunologia , HIV-1/imunologia , Reto/virologia , Vacinas contra a SAIDS/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/imunologia , Produtos do Gene gag do Vírus da Imunodeficiência Humana/imunologia , Animais , Anticorpos Antivirais/imunologia , Modelos Animais de Doenças , Infecções por HIV/virologia , HIV-1/genética , HIV-1/fisiologia , Humanos , Macaca mulatta , Reto/imunologia , Vacinas contra a SAIDS/administração & dosagem , Vacinas contra a SAIDS/genética , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/genética , Replicação Viral , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genéticaRESUMO
Zika virus (ZIKV) has become a serious public health concern because of its link to brain damage in developing human fetuses. Recombinant vesicular stomatitis virus (rVSV) was shown to be a highly effective and safe vector for the delivery of foreign immunogens for vaccine purposes. In this study, we generated rVSVs (wild-type and attenuated VSV with mutated matrix protein [VSVm] versions) that express either the full length ZIKV envelope protein (ZENV) alone or include the ZENV precursor to the membrane protein upstream of the envelope protein, and our rVSV-ZIKV constructs showed efficient immunogenicity in murine models. We also demonstrated maternal protective immunity in challenged newborn mice born to female mice vaccinated with VSVm-ZENV containing the transmembrane domain. Our data indicate that rVSVm may be a suitable strategy for the design of effective vaccines against ZIKV.
Assuntos
Vetores Genéticos , Imunidade Inata/imunologia , Proteínas do Envelope Viral/imunologia , Vacinas Virais/imunologia , Infecção por Zika virus/imunologia , Animais , Linhagem Celular , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Feminino , Imunofluorescência , Humanos , Immunoblotting , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase em Tempo Real , Vírus da Estomatite Vesicular Indiana/imunologiaRESUMO
Baboons naturally infected with simian T lymphotropic virus (STLV) are a potentially useful model system for the study of vaccination against human T lymphotropic virus (HTLV). Here we expanded the number of available full-length baboon STLV-1 sequences from one to three and related the T cell responses that recognize the immunodominant Tax protein to the tax sequences present in two individual baboons. Continuously growing T cell lines were established from two baboons, animals 12141 and 12752. Next-generation sequencing (NGS) of complete STLV genome sequences from these T cell lines revealed them to be closely related but distinct from each other and from the baboon STLV-1 sequence in the NCBI sequence database. Overlapping peptides corresponding to each unique Tax sequence and to the reference baboon Tax sequence were used to analyze recognition by T cells from each baboon using intracellular cytokine staining (ICS). Individual baboons expressed more gamma interferon and tumor necrosis factor alpha in response to Tax peptides corresponding to their own STLV-1 sequence than in response to Tax peptides corresponding to the reference baboon STLV-1 sequence. Thus, our analyses revealed distinct but closely related STLV-1 genome sequences in two baboons, extremely low heterogeneity of STLV sequences within each baboon, no evidence for superinfection within each baboon, and a ready ability of T cells in each baboon to recognize circulating Tax sequences. While amino acid substitutions that result in escape from CD8+ T cell recognition were not observed, premature stop codons were observed in 7% and 56% of tax sequences from peripheral blood mononuclear cells from animals 12141 and 12752, respectively.IMPORTANCE It has been estimated that approximately 100,000 people suffer serious morbidity and 10,000 people die each year from the consequences associated with human T lymphotropic virus (HTLV) infection. There are no antiviral drugs and no preventive vaccine. A preventive vaccine would significantly impact the global burden associated with HTLV infections. Here we provide fundamental information on the simian T lymphotropic virus (STLV) naturally transmitted in a colony of captive baboons. The limited viral sequence heterogeneity in individual baboons, the identity of the viral gene product that is the major target of cellular immune responses, the persistence of viral amino acid sequences that are the major targets of cellular immune responses, and the emergence in vivo of truncated variants in the major target of cellular immune responses all parallel what are seen with HTLV infection of humans. These results justify the use of STLV-infected baboons as a model system for vaccine development efforts.
Assuntos
Produtos do Gene tax/química , Produtos do Gene tax/genética , Infecções por HTLV-I/virologia , Vírus Linfotrópico T Tipo 1 de Símios/isolamento & purificação , Linfócitos T/imunologia , Substituição de Aminoácidos , Animais , DNA Viral/genética , Produtos do Gene tax/imunologia , Genoma Viral , Infecções por HTLV-I/imunologia , Infecções por HTLV-I/transmissão , Sequenciamento de Nucleotídeos em Larga Escala , Imunidade Celular , Interferon gama/biossíntese , Interferon gama/imunologia , Papio anubis , Filogenia , Reação em Cadeia da Polimerase , Vírus Linfotrópico T Tipo 1 de Símios/imunologia , Linfócitos T/virologia , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/imunologiaRESUMO
GLI1 oncogene has been implicated in the pathobiology of several neoplasms including diffuse large B-cell lymphoma (DLBCL). However, mechanisms underlying GLI1-increased activity in DLBCL are poorly characterized. Herein, we demonstrate that IKKß phosphorylates GLI1 in DLBCL. IKKß activation increased GLI1 protein levels and transcriptional activity, whereas IKKß silencing decreased GLI1 levels and transcriptional activity. Tumor necrosis factor-α (TNFα) mediated IKKß activation-impaired GLI1 binding with the E3 ubiquitin ligase-ITCH, leading to decreased K48-linked ubiquitination/degradation of GLI1. We found 8 IKKß-dependent phosphorylation sites that mediate GLI1 stability. Mutating or deleting these residues facilitated GLI1-ITCH interaction and decreased the protective effect of TNFα on GLI1 stability. IKKß-GLI1 crosstalk is significant because combined inhibition of both molecules resulted in synergistic suppression of DLBCL viability in vivo and in vitro. By linking IKKß-mediated nuclear factor-κB activity with GLI1, we identified a crosstalk between these 2 pathways that can inform the design of novel therapeutic strategies in DLBCL.