RESUMEN
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.
Asunto(s)
ADN/inmunología , Factor 3 Regulador del Interferón/inmunología , Proteínas de la Membrana/inmunología , Proteínas Quinasas S6 Ribosómicas 90-kDa/inmunología , Adenoviridae/genética , Adenoviridae/inmunología , Animales , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Células Cultivadas , Citosol/inmunología , Citosol/metabolismo , Citosol/virología , ADN/genética , ADN/metabolismo , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Células HEK293 , Herpes Simple/inmunología , Herpes Simple/virología , Herpesvirus Humano 1/inmunología , Herpesvirus Humano 1/fisiología , Humanos , Inmunización/métodos , Immunoblotting , Factor 3 Regulador del Interferón/genética , Factor 3 Regulador del Interferón/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/inmunología , Nucleotidiltransferasas/metabolismo , Ovalbúmina/genética , Ovalbúmina/inmunología , Unión Proteica , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/inmunología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Quinasas S6 Ribosómicas 90-kDa/genética , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismoRESUMEN
The differentiation of bone marrow-derived progenitor cells into monocytes, tissue macrophages and some dendritic cell (DC) subtypes requires the growth factor CSF1 and its receptor, CSF1R. Langerhans cells (LCs) and microglia develop from embryonic myeloid precursor cells that populate the epidermis and central nervous system (CNS) before birth. Notably, LCs and microglia are present in CSF1-deficient mice but absent from CSF1R-deficient mice. Here we investigated whether an alternative CSF1R ligand, interleukin 34 (IL-34), is responsible for this discrepancy. Through the use of IL-34-deficient (Il34(LacZ/LacZ)) reporter mice, we found that keratinocytes and neurons were the main sources of IL-34. Il34(LacZ/LacZ) mice selectively lacked LCs and microglia and responded poorly to skin antigens and viral infection of the CNS. Thus, IL-34 specifically directs the differentiation of myeloid cells in the skin epidermis and CNS.
Asunto(s)
Interleucinas/metabolismo , Células de Langerhans/fisiología , Microglía/fisiología , Receptor de Factor Estimulante de Colonias de Macrófagos/metabolismo , Fiebre del Nilo Occidental/inmunología , Animales , Diferenciación Celular , Sistema Nervioso Central/metabolismo , Interleucinas/deficiencia , Interleucinas/genética , Queratinocitos/metabolismo , Células de Langerhans/citología , Células de Langerhans/inmunología , Factor Estimulante de Colonias de Macrófagos/genética , Factor Estimulante de Colonias de Macrófagos/metabolismo , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microglía/citología , Microglía/inmunología , Células Mieloides/metabolismo , Mielopoyesis , Neuronas/metabolismo , Receptor de Factor Estimulante de Colonias de Macrófagos/genética , Receptor de Factor Estimulante de Colonias de Macrófagos/inmunología , Piel/metabolismo , Fiebre del Nilo Occidental/genética , Virus del Nilo Occidental/patogenicidadRESUMEN
The 5' cap structures of higher eukaryote mRNAs have ribose 2'-O-methylation. Likewise, many viruses that replicate in the cytoplasm of eukaryotes have evolved 2'-O-methyltransferases to autonomously modify their mRNAs. However, a defined biological role for 2'-O-methylation of mRNA remains elusive. Here we show that 2'-O-methylation of viral mRNA was critically involved in subverting the induction of type I interferon. We demonstrate that human and mouse coronavirus mutants lacking 2'-O-methyltransferase activity induced higher expression of type I interferon and were highly sensitive to type I interferon. Notably, the induction of type I interferon by viruses deficient in 2'-O-methyltransferase was dependent on the cytoplasmic RNA sensor Mda5. This link between Mda5-mediated sensing of viral RNA and 2'-O-methylation of mRNA suggests that RNA modifications such as 2'-O-methylation provide a molecular signature for the discrimination of self and non-self mRNA.
Asunto(s)
Infecciones por Coronavirus/metabolismo , Coronavirus/fisiología , ARN Helicasas DEAD-box/metabolismo , Metiltransferasas/metabolismo , Proteínas Virales/metabolismo , Animales , Línea Celular , Coronavirus/patogenicidad , Infecciones por Coronavirus/genética , Infecciones por Coronavirus/inmunología , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/inmunología , Humanos , Inmunidad Innata/genética , Interferón Tipo I/genética , Interferón Tipo I/inmunología , Interferón Tipo I/metabolismo , Helicasa Inducida por Interferón IFIH1 , Metilación , Metiltransferasas/genética , Metiltransferasas/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , ARN Viral/metabolismo , Receptor de Interferón alfa y beta/genética , Receptores de Reconocimiento de Patrones/genética , Ribosa/metabolismo , Proteínas Virales/genética , Proteínas Virales/inmunología , Virulencia/genética , Replicación Viral/genéticaRESUMEN
IgA nephropathy (IgAN) is the most prevalent primary chronic glomerular disease for which no safe disease-specific therapies currently exist. IgAN is an autoimmune disease involving the production of autoantigenic, aberrantly O-glycosylated IgA1 and ensuing deposition of nephritogenic immune complexes in the kidney. A Proliferation Inducing Ligand (APRIL) has emerged as a key B-cell-modulating factor in this pathogenesis. Using a mouse anti-APRIL monoclonal antibody (4540), we confirm both the pathogenic role of APRIL in IgAN and the therapeutic efficacy of antibody-directed neutralization of APRIL in the grouped mouse ddY disease model. Treatment with 4540 directly translated to a reduction in relevant pathogenic mechanisms including suppressed serum IgA levels, reduced circulating immune complexes, significantly lower kidney deposits of IgA, IgG and C3, and suppression of proteinuria compared to mice receiving vehicle or isotype control antibodies. Furthermore, we translated these findings to the pharmacological characterization of VIS649, a highly potent, humanized IgG2κ antibody targeting and neutralizing human APRIL through unique epitope engagement, leading to inhibition of APRIL-mediated B-cell activities. VIS649 treatment of non-human primates showed dose-dependent reduction of serum IgA levels of up to 70%. A reduction of IgA+, IgM+, and IgG+ B cells was noted in the gut-associated mucosa of VIS649-treated animals. Population-based modeling predicted a favorable therapeutic dosing profile for subcutaneous administration of VIS649 in the clinical setting. Thus, our data highlight the potential therapeutic benefit of VIS649 for the treatment of IgAN.
Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacología , Glomerulonefritis por IGA/tratamiento farmacológico , Inmunoglobulina A/inmunología , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales Humanizados/uso terapéutico , Complejo Antígeno-Anticuerpo/efectos de los fármacos , Complejo Antígeno-Anticuerpo/inmunología , Complejo Antígeno-Anticuerpo/metabolismo , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Simulación por Computador , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Epítopos de Linfocito B/inmunología , Femenino , Glomerulonefritis por IGA/inmunología , Humanos , Inmunoglobulina A/metabolismo , Inyecciones Intravenosas , Inyecciones Subcutáneas , Macaca fascicularis , Masculino , Ratones , Modelos Biológicos , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/inmunología , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/metabolismoRESUMEN
Emerging strains of influenza represent a significant public health threat with potential pandemic consequences. Of particular concern are the recently emerged H7N9 strains which cause pneumonia with acute respiratory distress syndrome. Estimates are that nearly 80% of hospitalized patients with H7N9 have received intensive care unit support. VIS410, a human antibody, targets a unique conserved epitope on influenza A. We evaluated the efficacy of VIS410 for neutralization of group 2 influenza strains, including H3N2 and H7N9 strains in vitro and in vivo. VIS410, administered at 50 mg/kg, protected DBA mice infected with A/Anhui/2013 (H7N9), resulting in significant survival benefit upon single-dose (-24 h) or double-dose (-12 h, +48 h) administration (P < 0.001). A single dose of VIS410 at 50 mg/kg (-12 h) combined with oseltamivir at 50 mg/kg (-12 h, twice daily for 7 d) in C57BL/6 mice infected with A/Shanghai 2/2013 (H7N9) resulted in significant decreased lung viral load (P = 0.002) and decreased lung cytokine responses for nine of the 11 cytokines measured. Based on these results, we find that VIS410 may be effective either as monotherapy or combined with antivirals in treating H7N9 disease, as well as disease from other influenza strains.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Subtipo H7N9 del Virus de la Influenza A/inmunología , Animales , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales Humanizados , Anticuerpos ampliamente neutralizantes , Humanos , Gripe Humana/terapia , Ratones , Ratones EndogámicosRESUMEN
Most cases of severe influenza are associated with pulmonary complications, such as acute respiratory distress syndrome (ARDS), and no antiviral drugs of proven value for treating such complications are currently available. The use of monoclonal antibodies targeting the stem of the influenza virus surface hemagglutinin (HA) is a rapidly developing strategy for the control of viruses of multiple HA subtypes. However, the mechanisms of action of these antibodies are not fully understood, and their ability to mitigate severe complications of influenza has been poorly studied. We evaluated the effect of treatment with VIS410, a human monoclonal antibody targeting the HA stem region, on the development of ARDS in BALB/c mice after infection with influenza A(H7N9) viruses. Prophylactic administration of VIS410 resulted in the complete protection of mice against lethal A(H7N9) virus challenge. A single therapeutic dose of VIS410 given 24 h after virus inoculation resulted in dose-dependent protection of up to 100% of mice inoculated with neuraminidase inhibitor-susceptible or -resistant A(H7N9) viruses. Compared to the outcomes in mock-treated controls, a single administration of VIS410 improved viral clearance from the lungs, reduced virus spread in lungs in a dose-dependent manner, resulting in a lower lung injury score, reduced the extent of the alteration in lung vascular permeability and protein accumulation in bronchoalveolar lavage fluid, and improved lung physiologic function. Thus, antibodies targeting the HA stem can reduce the severity of ARDS and show promise as agents for controlling pulmonary complications in influenza.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Subtipo H7N9 del Virus de la Influenza A/efectos de los fármacos , Pulmón/efectos de los fármacos , Infecciones por Orthomyxoviridae/tratamiento farmacológico , Síndrome de Dificultad Respiratoria/prevención & control , Animales , Líquido del Lavado Bronquioalveolar/virología , Permeabilidad Capilar/efectos de los fármacos , Modelos Animales de Enfermedad , Perros , Relación Dosis-Respuesta a Droga , Relación Dosis-Respuesta Inmunológica , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Humanos , Subtipo H7N9 del Virus de la Influenza A/crecimiento & desarrollo , Pulmón/virología , Células de Riñón Canino Madin Darby , Ratones , Ratones Endogámicos BALB C , Pruebas de Neutralización , Infecciones por Orthomyxoviridae/complicaciones , Infecciones por Orthomyxoviridae/mortalidad , Infecciones por Orthomyxoviridae/virología , Síndrome de Dificultad Respiratoria/etiología , Síndrome de Dificultad Respiratoria/mortalidad , Síndrome de Dificultad Respiratoria/virología , Análisis de Supervivencia , Carga Viral/efectos de los fármacosRESUMEN
UNLABELLED: Interferon-induced protein with tetratricopeptide repeats 1 (IFIT1) is a host protein with reported cell-intrinsic antiviral activity against several RNA viruses. The proposed basis for the activity against negative-sense RNA viruses is the binding to exposed 5'-triphosphates (5'-ppp) on the genome of viral RNA. However, recent studies reported relatively low binding affinities of IFIT1 for 5'-ppp RNA, suggesting that IFIT1 may not interact efficiently with this moiety under physiological conditions. To evaluate the ability of IFIT1 to have an impact on negative-sense RNA viruses, we infected Ifit1(-/-) and wild-type control mice and primary cells with four negative-sense RNA viruses (influenza A virus [IAV], La Crosse virus [LACV], Oropouche virus [OROV], and Ebola virus) corresponding to three distinct families. Unexpectedly, a lack of Ifit1 gene expression did not result in increased infection by any of these viruses in cell culture. Analogously, morbidity, mortality, and viral burdens in tissues were identical between Ifit1(-/-) and control mice after infection with IAV, LACV, or OROV. Finally, deletion of the human IFIT1 protein in A549 cells did not affect IAV replication or infection, and reciprocally, ectopic expression of IFIT1 in HEK293T cells did not inhibit IAV infection. To explain the lack of antiviral activity against IAV, we measured the binding affinity of IFIT1 for RNA oligonucleotides resembling the 5' ends of IAV gene segments. The affinity for 5'-ppp RNA was approximately 10-fold lower than that for non-2'-O-methylated (cap 0) RNA oligonucleotides. Based on this analysis, we conclude that IFIT1 is not a dominant restriction factor against negative-sense RNA viruses. IMPORTANCE: Negative-sense RNA viruses, including influenza virus and Ebola virus, have been responsible for some of the most deadly outbreaks in recent history. The host interferon response and induction of antiviral genes contribute to the control of infections by these viruses. IFIT1 is highly induced after virus infection and reportedly has antiviral activity against several RNA and DNA viruses. However, its role in restricting infection by negative-sense RNA viruses remains unclear. In this study, we evaluated the ability of IFIT1 to inhibit negative-sense RNA virus replication and pathogenesis both in vitro and in vivo. Detailed cell culture and animal studies demonstrated that IFIT1 is not a dominant restriction factor against three different families of negative-sense RNA viruses.
Asunto(s)
Proteínas Portadoras/metabolismo , Ebolavirus/metabolismo , Virus de la Influenza A/metabolismo , Virus La Crosse/metabolismo , Infecciones por Virus ARN/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Proteínas Portadoras/genética , Línea Celular , Ebolavirus/genética , Humanos , Virus de la Influenza A/genética , Virus La Crosse/genética , Ratones , Ratones Noqueados , Infecciones por Virus ARN/genética , Proteínas de Unión al ARNRESUMEN
Cellular messenger RNA (mRNA) of higher eukaryotes and many viral RNAs are methylated at the N-7 and 2'-O positions of the 5' guanosine cap by specific nuclear and cytoplasmic methyltransferases (MTases), respectively. Whereas N-7 methylation is essential for RNA translation and stability, the function of 2'-O methylation has remained uncertain since its discovery 35 years ago. Here we show that a West Nile virus (WNV) mutant (E218A) that lacks 2'-O MTase activity was attenuated in wild-type primary cells and mice but was pathogenic in the absence of type I interferon (IFN) signalling. 2'-O methylation of viral RNA did not affect IFN induction in WNV-infected fibroblasts but instead modulated the antiviral effects of IFN-induced proteins with tetratricopeptide repeats (IFIT), which are interferon-stimulated genes (ISGs) implicated in regulation of protein translation. Poxvirus and coronavirus mutants that lacked 2'-O MTase activity similarly showed enhanced sensitivity to the antiviral actions of IFN and, specifically, IFIT proteins. Our results demonstrate that the 2'-O methylation of the 5' cap of viral RNA functions to subvert innate host antiviral responses through escape of IFIT-mediated suppression, and suggest an evolutionary explanation for 2'-O methylation of cellular mRNA: to distinguish self from non-self RNA. Differential methylation of cytoplasmic RNA probably serves as an example for pattern recognition and restriction of propagation of foreign viral RNA in host cells.
Asunto(s)
Proteínas Portadoras/metabolismo , Regulación de la Expresión Génica/inmunología , Inmunidad Innata/inmunología , Interferones/inmunología , Proteínas/metabolismo , Caperuzas de ARN/metabolismo , ARN Viral/metabolismo , Células 3T3 , Proteínas Adaptadoras Transductoras de Señales , Animales , Proteínas Reguladoras de la Apoptosis , Proteínas Portadoras/genética , Células Cultivadas , Coronavirus/enzimología , Coronavirus/genética , Coronavirus/inmunología , Coronavirus/fisiología , Fibroblastos , Regulación de la Expresión Génica/genética , Humanos , Inmunidad Innata/genética , Interferones/deficiencia , Interferones/genética , Metilación , Metiltransferasas/metabolismo , Ratones , Ratones Endogámicos C57BL , Modelos Genéticos , Modelos Inmunológicos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Poxviridae/enzimología , Poxviridae/genética , Poxviridae/inmunología , Poxviridae/fisiología , Biosíntesis de Proteínas/inmunología , Proteínas/genética , Caperuzas de ARN/genética , Caperuzas de ARN/inmunología , ARN Viral/genética , ARN Viral/inmunología , Proteínas de Unión al ARN , Receptor de Interferón alfa y beta/deficiencia , Receptor de Interferón alfa y beta/genética , Tasa de Supervivencia , Replicación Viral , Virus del Nilo Occidental/enzimología , Virus del Nilo Occidental/genética , Virus del Nilo Occidental/inmunología , Virus del Nilo Occidental/fisiologíaRESUMEN
Prior studies have shown that 2'-O methyltransferase activity of flaviviruses, coronaviruses, and poxviruses promotes viral evasion of Ifit1, an interferon-stimulated innate immune effector protein. Viruses lacking 2'-O methyltransferase activity exhibited attenuation in primary macrophages that was rescued in cells lacking Ifit1 gene expression. Here, we examined the role of Ifit1 in restricting pathogenesis in vivo of wild type WNV (WNV-WT) and a mutant in the NS5 gene (WNV-E218A) lacking 2'-O methylation of the 5' viral RNA cap. While deletion of Ifit1 had marginal effects on WNV-WT pathogenesis, WNV-E218A showed increased replication in peripheral tissues of Ifit1â»/â» mice after subcutaneous infection, yet this failed to correlate with enhanced infection in the brain or lethality. In comparison, WNV-E218A was virulent after intracranial infection as judged by increased infection in different regions of the central nervous system (CNS) and a greater than 16,000-fold decrease in LD(50) values in Ifit1â»/â» compared to wild type mice. Ex vivo infection experiments revealed cell-type specific differences in the ability of an Ifit1 deficiency to complement the replication defect of WNV-E218A. In particular, WNV-E218A infection was impaired in both wild type and Ifit1â»/â» brain microvascular endothelial cells, which are believed to participate in blood-brain barrier (BBB) regulation of virus entry into the CNS. A deficiency of Ifit1 also was associated with increased neuronal death in vivo, which was both cell-intrinsic and mediated by immunopathogenic CD8⺠T cells. Our results suggest that virulent strains of WNV have largely evaded the antiviral effects of Ifit1, and viral mutants lacking 2'-O methylation are controlled in vivo by Ifit1-dependent and -independent mechanisms in different cell types.
Asunto(s)
Proteínas Portadoras/inmunología , Células Endoteliales/virología , Metiltransferasas/genética , Caperuzas de ARN/metabolismo , Virus del Nilo Occidental/genética , Virus del Nilo Occidental/patogenicidad , Proteínas Adaptadoras Transductoras de Señales , Animales , Barrera Hematoencefálica/virología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/virología , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Sistema Nervioso Central/virología , Células Endoteliales/metabolismo , Femenino , Inmunidad Innata , Interferón Tipo I/genética , Interferón Tipo I/inmunología , Macrófagos/inmunología , Macrófagos/virología , Metilación , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Caperuzas de ARN/genética , ARN Viral/metabolismo , Proteínas de Unión al ARN , Proteínas no Estructurales Virales/genética , Replicación Viral , Fiebre del Nilo Occidental/inmunología , Fiebre del Nilo Occidental/metabolismo , Fiebre del Nilo Occidental/virología , Virus del Nilo Occidental/inmunologíaRESUMEN
Type I interferon (IFN) signaling coordinates an early antiviral program in infected and uninfected cells by inducing IFN-stimulated genes (ISGs) that modulate viral entry, replication, and assembly. However, the specific antiviral functions in vivo of most ISGs remain unknown. Here, we examined the contribution of the ISG viperin to the control of West Nile virus (WNV) in genetically deficient cells and mice. While modest increases in levels of WNV replication were observed for primary viperin(-/-) macrophages and dendritic cells, no appreciable differences were detected in deficient embryonic cortical neurons or fibroblasts. In comparison, viperin(-/-) adult mice infected with WNV via the subcutaneous or intracranial route showed increased lethality and/or enhanced viral replication in central nervous system (CNS) tissues. In the CNS, viperin expression was induced in both WNV-infected and adjacent uninfected cells, including activated leukocytes at the site of infection. Our experiments suggest that viperin restricts the infection of WNV in a tissue- and cell-type-specific manner and may be an important ISG for controlling viral infections that cause CNS disease.
Asunto(s)
Interferones/inmunología , Proteínas/inmunología , Virus del Nilo Occidental/inmunología , Virus del Nilo Occidental/patogenicidad , Animales , Células Cultivadas , Sistema Nervioso Central/virología , Células Dendríticas/virología , Fibroblastos/virología , Macrófagos/virología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/virología , Proteínas/genética , Análisis de SupervivenciaRESUMEN
SUMMARY: Influenza A viruses of avian origin represent an emerging threat to human health as the progenitors of the next influenza pandemic. In recent years, highly pathogenic avian influenza H5N1 viruses have caused unprecedented epizootics on three continents and rare but highly fatal disease among humans exposed to diseased birds. Avian viruses of the H7 and H9 subtypes have also infected humans but generally resulted in far milder disease, yet they too should be considered as possible pandemic threats. Influenza virus infection elicits a complex network of host immune responses that, in uncomplicated influenza, results in effective control of the virus and the development of long-term memory responses. However, fatal avian H5N1 virus infection in both humans and experimental mammalian models is characterized by a high viral load in the respiratory tract, peripheral leukopenia and lymphopenia, a massive infiltration of macrophages into the lung, and dysregulation of cytokine and chemokine responses. This review focuses on avian influenza viruses as a pandemic threat, their induction of host innate immune responses in mammalian species, and the contribution of these responses to the disease process.
Asunto(s)
Brotes de Enfermedades , Subtipo H5N1 del Virus de la Influenza A/fisiología , Gripe Aviar/virología , Gripe Humana/epidemiología , Gripe Humana/transmisión , Animales , Aves/virología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/virología , Citocinas/inmunología , Citocinas/metabolismo , Humanos , Factores Inmunológicos/uso terapéutico , Subtipo H5N1 del Virus de la Influenza A/inmunología , Gripe Aviar/inmunología , Gripe Humana/inmunología , Gripe Humana/virología , Macrófagos/inmunología , Macrófagos/virologíaRESUMEN
Type I interferons (IFN-α/ß) control viral infection by triggering the expression of genes that restrict transcription, translation, replication, and assembly. Many viruses induce IFN responses after recognition by cytoplasmic or endosomal RNA sensors (RIG-I-like RNA helicases [RLR] and Toll-like receptors [TLR]), which signal through the cognate adaptor signaling molecules IPS-1, TRIF, and MyD88. Recent studies have demonstrated that IPS-1-dependent induction of IFN-α/ß downstream of RLR recognition restricts West Nile virus (WNV) infection in many cell types, whereas TRIF-dependent TLR3 signaling limits WNV replication in neurons. Here, we examined the contribution of MyD88 signaling to the control of WNV by evaluating IFN induction and virus replication in genetically deficient cells and mice. MyD88(-/-) mice showed increased lethality after WNV infection and elevated viral burden primarily in the brain, even though little effect on the systemic type I IFN response was observed. Intracranial inoculation studies corroborated these findings, as WNV spread more rapidly in the central nervous system of MyD88(-/-) mice, and this phenotype preceded the recruitment of inflammatory leukocytes. In vitro, increased WNV replication was observed in MyD88(-/-) macrophages and subsets of neurons but not in myeloid dendritic cells. MyD88 had an independent effect on recruitment of monocyte-derived macrophages and T cells into the brain that was associated with blunted induction of the chemokines that attract leukocytes. Our experiments suggest that MyD88 restricts WNV by inhibiting replication in subsets of cells and modulating expression of chemokines that regulate immune cell migration into the central nervous system.
Asunto(s)
Sistema Nervioso Central/virología , Interferón Tipo I/inmunología , Factor 88 de Diferenciación Mieloide/genética , Neuronas/virología , Replicación Viral/inmunología , Fiebre del Nilo Occidental/inmunología , Virus del Nilo Occidental/inmunología , Animales , Linfocitos T CD8-positivos/inmunología , Quimiocinas/metabolismo , Cartilla de ADN/genética , Regulación de la Expresión Génica/inmunología , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/genética , Transducción de Señal/inmunología , Estadísticas no Paramétricas , Fiebre del Nilo Occidental/fisiopatologíaRESUMEN
Periodic outbreaks of highly pathogenic avian H5N1 influenza viruses and the current H1N1 pandemic highlight the need for a more detailed understanding of influenza virus pathogenesis. To investigate the host transcriptional response induced by pathogenic influenza viruses, we used a functional-genomics approach to compare gene expression profiles in lungs from 129S6/SvEv mice infected with either the fully reconstructed H1N1 1918 pandemic virus (1918) or the highly pathogenic avian H5N1 virus Vietnam/1203/04 (VN/1203). Although the viruses reached similar titers in the lung and caused lethal infections, the mean time of death was 6 days for VN/1203-infected animals and 9 days for mice infected with the 1918 virus. VN/1203-infected animals also exhibited an earlier and more potent inflammatory response. This response included induction of genes encoding components of the inflammasome. VN/1203 was also able to disseminate to multiple organs, including the brain, which correlated with changes in the expression of genes associated with hematological functions and lipoxin biogenesis and signaling. Both viruses elicited expression of type I interferon (IFN)-regulated genes in wild-type mice and to a lesser extent in mice lacking the type I IFN receptor, suggesting alternative or redundant pathways for IFN signaling. Our findings suggest that VN/1203 is more pathogenic in mice as a consequence of several factors, including the early and sustained induction of the inflammatory response, the additive or synergistic effects of upregulated components of the immune response, and inhibition of lipoxin-mediated anti-inflammatory responses, which correlated with the ability of VN/1203 to disseminate to extrapulmonary organs.
Asunto(s)
Inflamación/patología , Subtipo H5N1 del Virus de la Influenza A/inmunología , Subtipo H5N1 del Virus de la Influenza A/patogenicidad , Lipoxinas/inmunología , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/patología , Transducción de Señal , Animales , Femenino , Regulación de la Expresión Génica , Inflamación/inmunología , Subtipo H1N1 del Virus de la Influenza A/inmunología , Subtipo H1N1 del Virus de la Influenza A/patogenicidad , Pulmón/patología , Pulmón/virología , Ratones , Infecciones por Orthomyxoviridae/mortalidad , Infecciones por Orthomyxoviridae/virología , Análisis de Supervivencia , VirulenciaRESUMEN
Interferon regulatory factors (IRF)-3 and IRF-7 are master transcriptional factors that regulate type I IFN gene (IFN-alpha/beta) induction and innate immune defenses after virus infection. Prior studies in mice with single deletions of the IRF-3 or IRF-7 genes showed increased vulnerability to West Nile virus (WNV) infection. Whereas mice and cells lacking IRF-7 showed reduced IFN-alpha levels after WNV infection, those lacking IRF-3 or IRF-7 had relatively normal IFN-b production. Here, we generated IRF-3(-/-)x IRF-7(-/-) double knockout (DKO) mice, analyzed WNV pathogenesis, IFN responses, and signaling of innate defenses. Compared to wild type mice, the DKO mice exhibited a blunted but not abrogated systemic IFN response and sustained uncontrolled WNV replication leading to rapid mortality. Ex vivo analysis showed complete ablation of the IFN-alpha response in DKO fibroblasts, macrophages, dendritic cells, and cortical neurons and a substantial decrease of the IFN-beta response in DKO fibroblasts and cortical neurons. In contrast, the IFN-beta response was minimally diminished in DKO macrophages and dendritic cells. However, pharmacological inhibition of NF-kappaB and ATF-2/c-Jun, the two other known components of the IFN-beta enhanceosome, strongly reduced IFN-beta gene transcription in the DKO dendritic cells. Finally, a genetic deficiency of IPS-1, an adaptor involved in RIG-I- and MDA5-mediated antiviral signaling, completely abolished the IFN-beta response after WNV infection. Overall, our experiments suggest that, unlike fibroblasts and cortical neurons, IFN-beta gene regulation after WNV infection in myeloid cells is IPS-1-dependent but does not require full occupancy of the IFN-beta enhanceosome by canonical constituent transcriptional factors.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Regulación de la Expresión Génica/inmunología , Interferón beta/biosíntesis , Células Mieloides/virología , Fiebre del Nilo Occidental/genética , Proteínas Adaptadoras Transductoras de Señales/inmunología , Animales , Western Blotting , Encéfalo/inmunología , Encéfalo/virología , Ensayo de Inmunoadsorción Enzimática , Expresión Génica , Inmunidad Innata , Factor 3 Regulador del Interferón/genética , Factor 3 Regulador del Interferón/inmunología , Factor 7 Regulador del Interferón/genética , Factor 7 Regulador del Interferón/inmunología , Interferón beta/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/inmunología , Neuronas/virología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Replicación Viral/fisiología , Fiebre del Nilo Occidental/inmunología , Virus del Nilo Occidental/inmunologíaRESUMEN
BACKGROUND: Highly pathogenic avian influenza viruses of the H5N1 subtype continue to cross the species barrier to infect humans and cause severe disease. It has been suggested that an exaggerated immune response contributes to the pathogenesis of H5N1 virus infection in mammals. In particular, H5N1 virus infections are associated with a high expression of the proinflammatory cytokines, including interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-α). METHODS: We investigated the compounding affects of both cytokines on the outcome of H5N1 virus disease by using triple mutant mice deficient in 3 signaling receptors, TNF-R1, TNF-R2, and IL-1-RI. RESULTS: Triple mutant mice exhibited reduced morbidity and a significant delay in mortality following lethal challenge with a lethal H5N1 virus, whereas no such differences were observed with the less virulent A/PR/8/34 (H1N1) virus. H5N1-infected triple mutant mice displayed diminished cytokine production in lung tissue and a quantifiable decrease of macrophages and neutrophils in the lungs postinfection. Moreover, morphometric analysis of airway sections revealed less extensive inflammation in H5N1-infected triple mutant mice, compared with infected wild-type mice. CONCLUSIONS: The combined signaling from the TNF or IL-1 receptors promotes maximal lung inflammation that may contribute to the severity of disease caused by H5N1 virus infection.
Asunto(s)
Subtipo H5N1 del Virus de la Influenza A , Enfermedades Pulmonares/genética , Infecciones por Orthomyxoviridae/genética , Infecciones por Orthomyxoviridae/mortalidad , Receptores de Interleucina-1/genética , Receptores del Factor de Necrosis Tumoral/genética , Animales , Encéfalo/virología , Recuento de Células , Citocinas/deficiencia , Citocinas/inmunología , Inflamación/genética , Inflamación/inmunología , Inflamación/mortalidad , Enfermedades Pulmonares/inmunología , Enfermedades Pulmonares/mortalidad , Enfermedades Pulmonares/fisiopatología , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación/genética , Neutrófilos/inmunología , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/fisiopatología , Transducción de Señal , Carga ViralRESUMEN
Sterile alpha and HEAT/Armadillo motif (SARM) is a highly conserved Toll/interleukin-1 receptor (TIR)-containing adaptor protein that is believed to negatively regulate signaling of the pathogen recognition receptors Toll-like receptor 3 (TLR3) and TLR4. To test its physiological function in the context of a microbial infection, we generated SARM(-/-) mice and evaluated the impact of this deficiency on the pathogenesis of West Nile virus (WNV), a neurotropic flavivirus that requires TLR signaling to restrict infection. Although SARM was preferentially expressed in cells of the central nervous system (CNS), studies with primary macrophages, neurons, or astrocytes showed no difference in viral growth kinetics. In contrast, viral replication was increased specifically in the brainstem of SARM(-/-) mice, and this was associated with enhanced mortality after inoculation with a virulent WNV strain. A deficiency of SARM was also linked to reduced levels of tumor necrosis factor alpha (TNF-alpha), decreased microglia activation, and increased neuronal death in the brainstem after WNV infection. Thus, SARM appears to be unique among the TIR adaptor molecules, since it functions to restrict viral infection and neuronal injury in a brain region-specific manner, possibly by modulating the activation of resident CNS inflammatory cells.
Asunto(s)
Proteínas del Dominio Armadillo/fisiología , Tronco Encefálico/patología , Proteínas del Citoesqueleto/fisiología , Microglía/fisiología , Factor de Necrosis Tumoral alfa/biosíntesis , Virus del Nilo Occidental/patogenicidad , Animales , Proteínas del Dominio Armadillo/análisis , Proteínas del Dominio Armadillo/deficiencia , Tronco Encefálico/virología , Muerte Celular , Proteínas del Citoesqueleto/análisis , Proteínas del Citoesqueleto/deficiencia , Ratones , Ratones Noqueados , Neuronas/patología , Neuronas/virología , Distribución Tisular , Receptores Toll-LikeRESUMEN
Although highly pathogenic avian influenza H5N1 viruses have yet to acquire the ability to transmit efficiently among humans, the increasing genetic diversity among these viruses and continued outbreaks in avian species underscore the need for more effective measures for the control and prevention of human H5N1 virus infection. Additional small animal models with which therapeutic approaches against virulent influenza viruses can be evaluated are needed. In this study, we used the guinea pig model to evaluate the relative virulence of selected avian and human influenza A viruses. We demonstrate that guinea pigs can be infected with avian and human influenza viruses, resulting in high titers of virus shedding in nasal washes for up to 5 days postinoculation (p.i.) and in lung tissue of inoculated animals. However, other physiologic indicators typically associated with virulent influenza virus strains were absent in this species. We evaluated the ability of intranasal treatment with human alpha interferon (alpha-IFN) to reduce lung and nasal wash titers in guinea pigs challenged with the reconstructed 1918 pandemic H1N1 virus or a contemporary H5N1 virus. IFN treatment initiated 1 day prior to challenge significantly reduced or prevented infection of guinea pigs by both viruses, as measured by virus titer determination and seroconversion. The expression of the antiviral Mx protein in lung tissue correlated with the reduction of virus titers. We propose that the guinea pig may serve as a useful small animal model for testing the efficacy of antiviral compounds and that alpha-IFN treatment may be a useful antiviral strategy against highly virulent strains with pandemic potential.
Asunto(s)
Factores Inmunológicos/uso terapéutico , Subtipo H1N1 del Virus de la Influenza A/patogenicidad , Subtipo H5N1 del Virus de la Influenza A/patogenicidad , Interferón-alfa/uso terapéutico , Infecciones por Orthomyxoviridae/prevención & control , Esparcimiento de Virus/inmunología , Administración Intranasal , Animales , Anticuerpos Antivirales/sangre , Modelos Animales de Enfermedad , Cobayas , Factores Inmunológicos/administración & dosificación , Subtipo H1N1 del Virus de la Influenza A/inmunología , Subtipo H5N1 del Virus de la Influenza A/inmunología , Interferón-alfa/administración & dosificación , Pulmón/virología , Cavidad Nasal/virología , Infecciones por Orthomyxoviridae/patología , Infecciones por Orthomyxoviridae/virología , VirulenciaRESUMEN
Widespread distribution of highly pathogenic avian H5N1 influenza viruses in domesticated and wild birds continues to pose a threat to public health, as interspecies transmission of virus has resulted in increasing numbers of human disease cases. Although the pathogenic mechanism(s) of H5N1 influenza viruses has not been fully elucidated, it has been suggested that the ability to evade host innate responses, such as the type I interferon response, may contribute to the virulence of these viruses in mammals. We investigated the role that type I interferons (alpha/beta interferon [IFN-alpha/beta]) might play in H5N1 pathogenicity in vivo, by comparing the kinetics and outcomes of H5N1 virus infection in IFN-alpha/beta receptor (IFN-alpha/betaR)-deficient and SvEv129 wild-type mice using two avian influenza A viruses isolated from humans, A/Hong Kong/483/97 (HK/483) and A/Hong Kong/486/97 (HK/486), which exhibit high and low lethality in mice, respectively. IFN-alpha/betaR-deficient mice experienced significantly more weight loss and more rapid time to death than did wild-type mice. HK/486 virus caused a systemic infection similar to that with HK/483 virus in IFN-alpha/betaR-deficient mice, suggesting a role for IFN-alpha/beta in controlling the systemic spread of this H5N1 virus. HK/483 virus replicated more efficiently than HK/486 virus both in vivo and in vitro. However, replication of both viruses was significantly reduced following pretreatment with IFN-alpha/beta. These results suggest a role for the IFN-alpha/beta response in the control of H5N1 virus replication both in vivo and in vitro, and as such it may provide some degree of protection to the host in the early stages of infection.
Asunto(s)
Subtipo H5N1 del Virus de la Influenza A/efectos de los fármacos , Subtipo H5N1 del Virus de la Influenza A/fisiología , Interferón Tipo I/farmacología , Infecciones por Orthomyxoviridae/metabolismo , Replicación Viral/efectos de los fármacos , Animales , Línea Celular Tumoral , Pollos , Humanos , Ratones , Ratones Noqueados , Infecciones por Orthomyxoviridae/genética , ARN Polimerasa Dependiente del ARN/genética , ARN Polimerasa Dependiente del ARN/metabolismo , Receptor de Interferón alfa y beta/deficiencia , Receptor de Interferón alfa y beta/genética , Receptor de Interferón alfa y beta/metabolismo , Especificidad por Sustrato , Factores de Tiempo , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Influenza A infections cause significant seasonal morbidity and mortality as well as periodic pandemic infections. Currently, no approved therapies exist for patients hospitalized with influenza. The efficacy of VIS410, a broadly neutralizing human immunoglobulin IgG1 monoclonal antibody engineered to bind to the stem region of group 1 and 2 influenza A hemagglutinins, was explored in experimental human influenza infection. Healthy volunteers were inoculated with influenza A/California/07/2009 (H1N1) and received a single dose of VIS410 or placebo 24 h later. Subjects were monitored for symptoms, viral shedding, and safety, including cytokine measurements. The primary efficacy endpoint was the area under the curve (AUC) of viral load (VL) in the VIS410 group versus placebo. VIS410 treatment was associated with a 76% reduction in median VL AUC as measured by qRT-PCR (p = 0.024). Similar VIS410 antiviral activity was observed by virus culture, with a 91% reduction in median VL AUC by TCID50 (p = 0.019) compared to placebo-treated volunteers. Influenza symptoms were generally mild or moderate, with a trend toward faster resolution in VIS410-treated subjects. Treatment with VIS410 was generally safe, with an increase in gastrointestinal events that were largely mitigated by pre-treatment with oral diphenhydramine (50 mg) in combination with 600 mg of ibuprofen. Transient elevation of specific cytokines (IL-8 and TNFα) were associated with gastrointestinal adverse events. Treatment with VIS410 did not interfere with the endogenous immune response to influenza A. These data indicate that VIS410 may provide therapeutic benefit in influenza A infection. TRIAL REGISTRATION: ClinicaTtrials.gov Identification NCT02468115; https://clinicaltrials.gov/ct2/show/NCT02468115?term=NCT02468115&rank=1).
Asunto(s)
Anticuerpos Monoclonales Humanizados/uso terapéutico , Antivirales/uso terapéutico , Anticuerpos ampliamente neutralizantes/uso terapéutico , Gripe Humana/tratamiento farmacológico , Adulto , Anticuerpos Antivirales , Citocinas/inmunología , Relación Dosis-Respuesta a Droga , Determinación de Punto Final , Femenino , Humanos , Inmunidad , Inmunoglobulina G/uso terapéutico , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Subtipo H1N1 del Virus de la Influenza A/inmunología , Masculino , Persona de Mediana Edad , ARN Viral , Resultado del Tratamiento , Carga Viral , Adulto JovenRESUMEN
The replicative properties of influenza virus hemagglutinin (HA) mutants with altered receptor binding characteristics were analyzed following intranasal inoculation of mice. Among the mutants examined was a virus containing a Y98F substitution at a conserved position in the receptor binding site that leads to a 20-fold reduction in binding. This mutant can replicate as well as wild-type (WT) virus in MDCK cells and in embryonated chicken eggs but is highly attenuated in mice, exhibiting titers in lungs more than 1,000-fold lower than those of the WT. The capacity of the Y98F mutant to induce antibody responses and the structural locations of HA reversion mutations are examined.