RESUMEN
Murine gammaherpesvirus 68 (MHV68) is a small-animal model suitable for study of the human pathogens Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus. Here, we have characterized the roles of the endosomal Toll-like receptor (TLR) escort protein UNC93B, endosomal TLR7, -9, and -13, and cell surface TLR2 in MHV68 detection. We found that the alpha interferon (IFN-α) response of plasmacytoid dendritic cells (pDC) to MHV68 was reduced in Tlr9-/- cells compared to levels in wild type (WT) cells but not completely lost. Tlr7-/- pDC responded similarly to WT. However, we found that in Unc93b-/- pDC, as well as in Tlr7-/-Tlr9-/- double-knockout pDC, the IFN-α response to MHV68 was completely abolished. Thus, the only pattern recognition receptors contributing to the IFN-α response to MHV68 in pDC are TLR7 and TLR9, but the contribution of TLR7 is masked by the presence of TLR9. To address the role of UNC93B and TLR for MHV68 infection in vivo, we infected mice with MHV68. Lytic replication of MHV68 after intravenous infection was enhanced in the lungs, spleen, and liver of UNC93B-deficient mice, in the spleen of TLR9-deficient mice, and in the liver and spleen of Tlr7-/-Tlr9-/- mice. The absence of TLR2 or TLR13 did not affect lytic viral titers. We then compared reactivation of MHV68 from latently infected WT, Unc93b-/-, Tlr7-/-Tlr9-/-, Tlr7-/-, and Tlr9-/- splenocytes. We observed enhanced reactivation and latent viral loads, particularly from Tlr7-/-Tlr9-/- splenocytes compared to levels in the WT. Our data show that UNC93B-dependent TLR7 and TLR9 cooperate in and contribute to detection and control of MHV68 infection.IMPORTANCE The two human gammaherpesviruses, Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), can cause aggressive forms of cancer. These herpesviruses are strictly host specific, and therefore the homolog murine gammaherpesvirus 68 (MHV68) is a widely used model to obtain in vivo insights into the interaction between these two gammaherpesviruses and their host. Like EBV and KSHV, MHV68 establishes lifelong latency in B cells. The innate immune system serves as one of the first lines of host defense, with pattern recognition receptors such as the Toll-like receptors playing a crucial role in mounting a potent antiviral immune response to various pathogens. Here, we shed light on a yet unanticipated role of Toll-like receptor 7 in the recognition of MHV68 in a subset of immune cells called plasmacytoid dendritic cells, as well as on the control of this virus in its host.
Asunto(s)
Células Dendríticas/inmunología , Endosomas/inmunología , Gammaherpesvirinae/patogenicidad , Infecciones por Herpesviridae/diagnóstico , Glicoproteínas de Membrana/fisiología , Células Madre Mesenquimatosas/inmunología , Receptor Toll-Like 7/fisiología , Receptor Toll-Like 9/fisiología , Animales , Células Dendríticas/metabolismo , Células Dendríticas/virología , Endosomas/metabolismo , Endosomas/virología , Femenino , Infecciones por Herpesviridae/inmunología , Infecciones por Herpesviridae/metabolismo , Infecciones por Herpesviridae/virología , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/virología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de Señal , Activación Viral , Latencia del Virus , Replicación ViralRESUMEN
UNLABELLED: Cleavage of influenza virus hemagglutinin (HA) by host cell proteases is necessary for viral activation and infectivity. In humans and mice, members of the type II transmembrane protease family (TTSP), e.g., TMPRSS2, TMPRSS4, and TMPRSS11d (HAT), have been shown to cleave influenza virus HA for viral activation and infectivity in vitro Recently, we reported that inactivation of a single HA-activating protease gene,Tmprss2, in knockout mice inhibits the spread of H1N1 influenza viruses. However, after infection of Tmprss2 knockout mice with an H3N2 influenza virus, only a slight increase in survival was observed, and mice still lost body weight. In this study, we investigated an additional trypsin-like protease, TMPRSS4. Both TMPRSS2 and TMPRSS4 are expressed in the same cell types of the mouse lung. Deletion of Tmprss4 alone in knockout mice does not protect them from body weight loss and death upon infection with H3N2 influenza virus. In contrast,Tmprss2(-/-)Tmprss4(-/-)double-knockout mice showed a remarkably reduced virus spread and lung pathology, in addition to reduced body weight loss and mortality. Thus, our results identified TMPRSS4 as a second host cell protease that, in addition to TMPRSS2, is able to activate the HA of H3N2 influenza virus in vivo IMPORTANCE: Influenza epidemics and recurring pandemics are responsible for significant global morbidity and mortality. Due to high variability of the virus genome, resistance to available antiviral drugs is frequently observed, and new targets for treatment of influenza are needed. Host cell factors essential for processing of the virus hemagglutinin represent very suitable drug targets because the virus is dependent on these host factors for replication. We reported previously that Tmprss2-deficient mice are protected against H1N1 virus infections, but only marginal protection against H3N2 virus infections was observed. Here we show that deletion of two host protease genes,Tmprss2 and Tmprss4, strongly reduced viral spread as well as lung pathology and resulted in increased survival after H3N2 virus infection. Thus, TMPRSS4 represents another host cell factor that is involved in cleavage activation of H3N2 influenza viruses in vivo.
Asunto(s)
Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Subtipo H3N2 del Virus de la Influenza A/fisiología , Proteínas de la Membrana/metabolismo , Infecciones por Orthomyxoviridae/metabolismo , Infecciones por Orthomyxoviridae/virología , Serina Endopeptidasas/metabolismo , Animales , Bronquios/metabolismo , Bronquios/virología , Quimiocinas/metabolismo , Citocinas/metabolismo , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Activación Enzimática , Femenino , Eliminación de Gen , Expresión Génica , Interacciones Huésped-Patógeno , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Infecciones por Orthomyxoviridae/genética , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/mortalidad , Proteolisis , Alveolos Pulmonares/metabolismo , Alveolos Pulmonares/virología , Serina Endopeptidasas/genética , Carga Viral , Replicación ViralRESUMEN
UNLABELLED: Influenza virus infections represent a serious threat to human health. Both extrinsic and intrinsic factors determine the severity of influenza. The MX dynamin-like GTPase 1 (Mx1) gene has been shown to confer strong resistance to influenza A virus infections in mice. Most laboratory mouse strains, including C57BL/6J, carry nonsense or deletion mutations in Mx1 and thus a nonfunctional allele, whereas wild-derived mouse strains carry a wild-type Mx1 allele. Congenic C57BL/6J (B6-Mx1(r/r)) mice expressing a wild-type allele from the A2G mouse strain are highly resistant to influenza A virus infections, to both mono- and polybasic subtypes. Furthermore, in genetic mapping studies, Mx1 was identified as the major locus of resistance to influenza virus infections. Here, we investigated whether the Mx1 protective function is influenced by the genetic background. For this, we generated a congenic mouse strain carrying the A2G wild-type Mx1 resistance allele on a DBA/2J background (D2-Mx1(r/r)). Most remarkably, congenic D2-Mx1(r/r) mice expressing a functional Mx1 wild-type allele are still highly susceptible to H1N1 virus. However, pretreatment of D2-Mx1(r/r) mice with alpha interferon protected them from lethal infections. Our results showed, for the first time, that the presence of an Mx1 wild-type allele from A2G as such does not fully protect mice from lethal influenza A virus infections. These observations are also highly relevant for susceptibility to influenza virus infections in humans. IMPORTANCE: Influenza A virus represents a major health threat to humans. Seasonal influenza epidemics cause high economic loss, morbidity, and deaths each year. Genetic factors of the host strongly influence susceptibility and resistance to virus infections. The Mx1 (MX dynamin-like GTPase 1) gene has been described as a major resistance gene in mice and humans. Most inbred laboratory mouse strains are deficient in Mx1, but congenic B6-Mx1(r/r) mice that carry the wild-type Mx1 gene from the A2G mouse strain are highly resistant. Here, we show that, very unexpectedly, congenic D2-Mx1(r/r) mice carrying the wild-type Mx1 gene from the A2G strain are not fully protected against lethal influenza virus infections. These observations demonstrate that the genetic background is very important for the protective function of the Mx1 resistance gene. Our results are also highly relevant for understanding genetic susceptibility to influenza virus infections in humans.
Asunto(s)
Subtipo H1N1 del Virus de la Influenza A , Proteínas de Resistencia a Mixovirus/genética , Infecciones por Orthomyxoviridae/genética , Infecciones por Orthomyxoviridae/prevención & control , Animales , Líquido del Lavado Bronquioalveolar/inmunología , Citocinas/metabolismo , Femenino , Predisposición Genética a la Enfermedad , Especificidad del Huésped , Humanos , Subtipo H1N1 del Virus de la Influenza A/patogenicidad , Subtipo H1N1 del Virus de la Influenza A/fisiología , Subtipo H3N2 del Virus de la Influenza A/patogenicidad , Gripe Humana/genética , Interferón-alfa/farmacología , Ratones , Ratones Congénicos , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Noqueados , Mutación , Infecciones por Orthomyxoviridae/virología , Replicación ViralRESUMEN
BACKGROUND: Previously, we performed a quantitative trait locus (QTL) mapping study in BXD recombinant inbred mice to identify host genetic factors that confer resistance to influenza A virus infection. We found Lst1 (leukocyte specific transcript 1) as one of the most promising candidate genes in the Qivr17-2 locus because it is non-functional in DBA/2 J mice. Several studies have proposed that LST1 plays a role in the immune response to inflammatory diseases in humans and has additional immune-regulatory functions. Here, we evaluated the relevance of LST1 for the host response to influenza A infection in B6-Lst1 (-/-) mutant mice. FINDINGS: To investigate the role of LST1, we infected B6-Lst1 (-/-) mutant and C57BL/6 N wild-type mice with a low-virulent influenza A virus (PR8M; H1N1). Lst1 deficient mice exhibited significantly increased body weight loss at days 5 and 6 after infection and slightly increased lethality compared to infected wild-type mice. Determination of viral loads, histopathological examination and analysis of immune cell composition in bronchoalveolar lavage of infected lungs did not reveal any obvious differences between KO and wild-type mice. CONCLUSIONS: The absence of Lst1 leads to a slightly more susceptible phenotype. However, deletion of Lst1 in DBA/2 J mice alone does not explain the high susceptibility of this strain to PR8M influenza infections.
Asunto(s)
Interacciones Huésped-Patógeno , Subtipo H1N1 del Virus de la Influenza A/fisiología , Proteínas de la Membrana/deficiencia , Infecciones por Orthomyxoviridae/genética , Infecciones por Orthomyxoviridae/virología , Animales , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Femenino , Péptidos y Proteínas de Señalización Intracelular , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Noqueados , Infecciones por Orthomyxoviridae/mortalidad , Fenotipo , Carga ViralRESUMEN
BACKGROUND: The host response to influenza A infections is strongly influenced by host genetic factors. Animal models of genetically diverse mouse strains are well suited to identify host genes involved in severe pathology, viral replication and immune responses. Here, we have utilized a dual RNAseq approach that allowed us to investigate both viral and host gene expression in the same individual mouse after H1N1 infection. RESULTS: We performed a detailed expression analysis to identify (i) correlations between changes in expression of host and virus genes, (ii) host genes involved in viral replication, and (iii) genes showing differential expression between two mouse strains that strongly differ in resistance to influenza infections. These genes may be key players involved in regulating the differences in pathogenesis and host defense mechanisms after influenza A infections. Expression levels of influenza segments correlated well with the viral load and may thus be used as surrogates for conventional viral load measurements. Furthermore, we investigated the functional role of two genes, Reg3g and Irf7, in knock-out mice and found that deletion of the Irf7 gene renders the host highly susceptible to H1N1 infection. CONCLUSIONS: Using RNAseq analysis we identified novel genes important for viral replication or the host defense. This study adds further important knowledge to host-pathogen-interactions and suggests additional candidates that are crucial for host susceptibility or survival during influenza A infections.
Asunto(s)
Resistencia a la Enfermedad/genética , Genes Virales , Interacciones Huésped-Patógeno/genética , Virus de la Influenza A/genética , Infecciones por Orthomyxoviridae/genética , Infecciones por Orthomyxoviridae/virología , Análisis de Secuencia de ARN/métodos , Replicación Viral/genética , Animales , Peso Corporal , Susceptibilidad a Enfermedades , Perfilación de la Expresión Génica , Regulación Viral de la Expresión Génica , Inmunidad Innata/genética , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Análisis de Componente Principal , ARN Interferente Pequeño/metabolismo , Regulación hacia Arriba/genética , Carga Viral/genéticaRESUMEN
Annual influenza epidemics and occasional pandemics pose a severe threat to human health. Host cell factors required for viral spread but not for cellular survival are attractive targets for novel approaches to antiviral intervention. The cleavage activation of the influenza virus hemagglutinin (HA) by host cell proteases is essential for viral infectivity. However, it is unknown which proteases activate influenza viruses in mammals. Several candidates have been identified in cell culture studies, leading to the concept that influenza viruses can employ multiple enzymes to ensure their cleavage activation in the host. Here, we show that deletion of a single HA-activating protease gene, Tmprss2, in mice inhibits spread of mono-basic H1N1 influenza viruses, including the pandemic 2009 swine influenza virus. Lung pathology was strongly reduced and mutant mice were protected from weight loss, death and impairment of lung function. Also, after infection with mono-basic H3N2 influenza A virus body weight loss and survival was less severe in Tmprss2 mutant compared to wild type mice. As expected, Tmprss2-deficient mice were not protected from viral spread and pathology after infection with multi-basic H7N7 influenza A virus. In conclusion, these results identify TMPRSS2 as a host cell factor essential for viral spread and pathogenesis of mono-basic H1N1 and H3N2 influenza A viruses.
Asunto(s)
Subtipo H1N1 del Virus de la Influenza A/patogenicidad , Infecciones por Orthomyxoviridae/genética , Serina Endopeptidasas/fisiología , Animales , Células Cultivadas , Embrión de Pollo , Perros , Femenino , Células HEK293 , Interacciones Huésped-Patógeno/genética , Humanos , Subtipo H3N2 del Virus de la Influenza A/patogenicidad , Gripe Humana/genética , Gripe Humana/virología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Infecciones por Orthomyxoviridae/virología , Serina Endopeptidasas/genéticaRESUMEN
Type I IFN signaling amplifies the secretion of LPS-induced proinflammatory cytokines such as TNF-α or IL-6 and might thus contribute to the high mortality associated with Gram-negative septic shock in humans. The underlying molecular mechanism, however, is ill defined. In this study, we report the generation of mice deficient in IFN-induced protein with tetratricopeptide repeats 2 (Ifit2) and demonstrate that Ifit2 is a critical signaling intermediate for LPS-induced septic shock. Ifit2 expression was significantly upregulated in response to LPS challenge in an IFN-α receptor- and IFN regulatory factor (Irf)9-dependent manner. Also, LPS induced secretion of IL-6 and TNF-α by bone marrow-derived macrophages (BMDMs) was significantly enhanced in the presence of Ifit2. In accordance, Ifit2-deficient mice exhibited significantly reduced serum levels of IL-6 and TNF-α and reduced mortality in an endotoxin shock model. Investigation of the underlying signal transduction events revealed that Ifit2 upregulates Irf3 phosphorylation. In the absence of Irf3, reduced Ifn-ß mRNA expression and Ifit2 protein expression after LPS stimulation was found. Also, Tnf-α and Il-6 secretion but not Tnf-α and Il-6 mRNA expression levels were reduced. Thus, IFN-stimulated Ifit2 via enhanced Irf3 phosphorylation upregulates the secretion of proinflammatory cytokines. It thereby amplifies LPS-induced cytokine production and critically influences the outcome of endotoxin shock.
Asunto(s)
Interferón Tipo I/metabolismo , Lipopolisacáridos/inmunología , Proteínas/inmunología , Choque Séptico/inmunología , Factor de Necrosis Tumoral alfa/biosíntesis , Animales , Proteínas Reguladoras de la Apoptosis , Citocinas/biosíntesis , Modelos Animales de Enfermedad , Femenino , Regulación de la Expresión Génica , Inflamación/inmunología , Inflamación/metabolismo , Ratones , Ratones Noqueados , Proteínas/genética , ARN Mensajero/genética , Proteínas de Unión al ARN , Transducción de SeñalRESUMEN
The interferon (IFN) pathway plays an essential role in the innate immune response following viral infections and subsequent shaping of adaptive immunity. Infections with influenza A viruses (IAV) activate the IFN pathway after the recognition of pathogen-specific molecular patterns by respective pattern recognition receptors. The IFN regulatory factors IRF3 and IRF7 are key players in the regulation of type I and III IFN genes. In this study, we analyzed the role of IRF3 and IRF7 for the host response to IAV infections in Irf3-/-, Irf7-/-, and Irf3-/-Irf7-/- knockout mice. While the absence of IRF3 had only a moderate impact on IFN expression, deletion of IRF7 completely abolished IFNα production after infection. In contrast, lack of both IRF3 and IRF7 resulted in the absence of both IFNα and IFNß after IAV infection. In addition, IAV infection of double knockout mice resulted in a strong increase of mortality associated with a massive influx of granulocytes in the lung and reduced activation of the adaptive immune response.
Asunto(s)
Granulocitos/inmunología , Inflamación/inmunología , Virus de la Influenza A/inmunología , Factor 3 Regulador del Interferón/metabolismo , Factor 7 Regulador del Interferón/metabolismo , Pulmón/inmunología , Infecciones por Orthomyxoviridae/inmunología , Inmunidad Adaptativa , Animales , Células Cultivadas , Granulocitos/virología , Factor 3 Regulador del Interferón/genética , Factor 7 Regulador del Interferón/genética , Interferón-alfa/metabolismo , Pulmón/virología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de SeñalRESUMEN
Influenza A infection is a serious threat to human and animal health. Many of the biological mechanisms of the host-pathogen-interactions are still not well understood and reliable biomarkers indicating the course of the disease are missing. The mouse is a valuable model system enabling us to study the local inflammatory host response and the influence on blood parameters under controlled circumstances. Here, we compared the lung and peripheral changes after PR8 (H1N1) influenza A virus infection in C57BL/6J and DBA/2J mice using virus variants of different pathogenicity resulting in non-lethal and lethal disease. We monitored hematological and immunological parameters revealing that the granulocyte to lymphocyte ratio in the blood represents an early indicator of severe disease progression already two days after influenza A infection in mice. These findings might be relevant to optimize early diagnostic options of severe influenza disease and to monitor successful therapeutic treatment in humans.
Asunto(s)
Células Sanguíneas/patología , Células Sanguíneas/virología , Infecciones por Orthomyxoviridae/sangre , Trastornos Respiratorios/sangre , Trastornos Respiratorios/virología , Animales , Recuento de Células Sanguíneas , Células Cultivadas , Modelos Animales de Enfermedad , Perros , Femenino , Granulocitos/patología , Interacciones Huésped-Patógeno/inmunología , Subtipo H1N1 del Virus de la Influenza A/inmunología , Subtipo H1N1 del Virus de la Influenza A/patogenicidad , Linfocitos/patología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Infecciones por Orthomyxoviridae/complicaciones , Infecciones por Orthomyxoviridae/mortalidad , Infecciones por Orthomyxoviridae/patología , Trastornos Respiratorios/mortalidad , Trastornos Respiratorios/patología , Índice de Severidad de la EnfermedadRESUMEN
Interferons represent one of the first and essential host defense mechanisms after infection, and the activation of the IFN-pathway results in the transcriptional activation of hundreds of interferon-stimulated genes. The alpha-inducible protein 27 like 2A (Ifi27l2a) gene (human synonym: ISG12) is strongly up-regulated in the lung after influenza A infection in mice and has been shown in gene expression studies to be highly correlated to other activated genes. Therefore, we investigated the role of Ifi27l2a for the host defense to influenza A infections in more detail. RT-PCR analyses in non-infected mice demonstrated that Ifi27l2a was expressed in several tissues, including the lung. Detailed analyses of reporter gene expression in lungs from Ifi27l2a-LacZ mice revealed that Ifi27l2a was expressed in macrophages and lymphocytes but not in alveolar cells or bronchiolar epithelium cells. The number of macrophages and lymphocyte strongly increased in the lung after infection, but no significant increase in expression levels of the LacZ reporter gene was found within individual immune cells. Also, no reporter gene expression was found in bronchiolar epithelial cells, alveolar cells or infiltrating neutrophils after infection. Thus, up-regulation of Ifi27l2a in infected lungs is mainly due to the infiltration of macrophages and lymphocytes. Most surprisingly, deletion of Ifi27l2a in mouse knock-out lines did not result in increased susceptibility to infections with H1N1 or H7N7 influenza A virus compared to wild type C57BL/6N mice, suggesting a less important role of the gene for the host response to influenza infections than for bacterial infections.
Asunto(s)
Gripe Humana/genética , Pulmón/metabolismo , Proteínas/genética , Activación Transcripcional/genética , Animales , Eliminación de Gen , Regulación de la Expresión Génica , Humanos , Subtipo H1N1 del Virus de la Influenza A/patogenicidad , Subtipo H7N7 del Virus de la Influenza A/patogenicidad , Gripe Humana/patología , Interferones/genética , Interferones/inmunología , Interferones/metabolismo , Pulmón/inmunología , Pulmón/patología , Linfocitos/inmunología , Linfocitos/metabolismo , Linfocitos/patología , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/patología , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , RatonesRESUMEN
Current model DBA/2J (D2J) mice lack CD94 expression due to a deletion spanning the last coding exon of the Klrd1 gene that occurred in the mid- to late 1980s. In contrast, DBA/2JRj (D2Rj) mice, crosses derived from DBA/2J before 1984, and C57BL/6J (B6) mice lack the deletion and have normal CD94 expression. For example, BXD lines (BXD1-32) generated in the 1970s by crossing B6 and D2J do not segregate for the exonic deletion and have high expression, whereas BXD lines 33 and greater were generated after 1990 are segregating for the deletion and have highly variable Klrd1 expression. We performed quantitative trait locus analysis of Klrd1 expression by using BXD lines with different generation times and found that the expression difference in Klrd1 in the later BXD set is driven by a strong cis-acting expression quantitative trait locus. Although the Klrd1/CD94 locus is essential for mousepox resistance, the genetic variation among D2 substrains and the later set of BXD strains is not associated with susceptibility to the Influenza A virus PR8 strain. Substrains with nearly identical genetic backgrounds that are segregating functional variants such as the Klrd1 deletion are useful genetic tools to investigate biological function.