RESUMEN
Understanding the host response to influenza A virus (IAV) infection is vital for developing intervention strategies. The primary barriers for invading respiratory pathogens are the respiratory tract epithelial cells and antimicrobial proteins generated by these cells. The antimicrobial peptide, ß-defensin-1, has antiviral activity against both enveloped and non-enveloped viruses. Significant downregulation of ß-defensin1 gene (DEFB1) expression was observed when human bronchial epithelial cells (HBEpCs) were exposed to IAV. HBEpCs overexpressing DEFB1 caused a significant reduction in IAV, that was confirmed by IAV matrix gene analysis, plaque assay, and confocal microscopy. DEFB1 expression after transfection with two micro RNAs (miRNAs), hsa-miR-186-5p and hsa-miR-340-5p, provided evidence that DEFB1 expression could be modulated by these miRNAs and hsa-miR-186-5p had a higher binding efficiency with DEFB1. Overexpression of DEFB1 in IAV-infected HBEpCs led to increased NF-κB expression. In a PCR array analysis of 84 transcription factors, either overexpressing DEFB1 or siRNA silencing of DEFB1 expression significantly modulated the expression of signal transducer and activator of transcription 3 (STAT3). In addition, Ingenuity Pathway Analysis (IPA) integrated with PCR array data showed that the JAK1/STAT3 pathway was significantly altered in cells overexpressing DEFB1, suggesting this to be one of the pathways by which defensin regulates IAV replication in HBEpCs. In conclusion, the reduction in IAV copy number in DEFB1 overexpressing cells suggests that ß-defensin-1 plays a key role in regulating IAV survival through STAT3 and is a potential target for antiviral drug development.
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MicroRNAs (miRNAs) are essential regulators of gene expression in humans and can control pathogenesis and host-virus interactions. Notably, the role of specific host miRNAs during influenza virus infections are still ill-defined. The central goal of this study was to identify novel miRNAs and their target genes in response to influenza virus infections in airway epithelium. Human airway epithelial cells exposed to influenza A virus (IAV) induced several novel miRNAs that were identified using next-generation sequencing (NGS) and their target genes by biochemical methods. NGS analysis predicted forty-two RNA sequences as possible miRNAs based on computational algorithms. The expression patterns of these putative miRNAs were further confirmed using RT-PCR in human bronchial epithelial cells exposed to H1N1, H9N1(1P10), and H9N1 (1WF10) strains of influenza virus. A time-course study showed significant downregulation of put-miR-34 in H1N1 and put-miR-35 in H9N1(1P10)-infected cells, which is consistent with the NGS data. Additionally, put-miR-34 and put-miR-35 showed a high fold enrichment in an argonaute-immunoprecipitation assay compared to the controls, indicating their ability to form a complex with argonaute protein and RNA-induced silencing complex (RISC), which is a typical mode of action found with miRNAs. Our earlier studies have shown that the replication and survival of influenza virus is modulated by certain transcription factors such as NF-ĸB. To identify the target(s) of these putative miRNAs, we screened 84 transcription factors that have a role in viral pathogenesis. Cells transfected with mimic of the put-miR-34 showed a significant decrease in the expression of Signal Transducers and Activators of Transcription 3 (STAT3), whereas the inhibitor of put-miR-34 showed a significant increase in STAT3 expression and its phosphorylation. In addition, put-miR-34 had 76% homology to the untranslated region of STAT3. NGS and PCR array data submitted to the Gene Ontology project also predicted the role of transcription factors modulated by put-miR-34. Our data suggest that put-miR-34 may be a good target for antiviral therapy.
Asunto(s)
Interacciones Huésped-Patógeno/genética , Virus de la Influenza A/genética , MicroARNs/genética , Factores de Transcripción STAT/genética , Transducción de Señal/genética , Células A549 , Bronquios/citología , Células Cultivadas , Células Epiteliales/virología , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H1N1 del Virus de la Influenza A/patogenicidad , Subtipo H3N2 del Virus de la Influenza A/genética , Subtipo H3N2 del Virus de la Influenza A/patogenicidad , Virus de la Influenza A/clasificación , Virus de la Influenza A/patogenicidad , MicroARNs/clasificación , MicroARNs/aislamiento & purificación , Replicación ViralRESUMEN
MicroRNAs (miRNAs) have remarkable stability and are key regulators of mRNA transcripts for several essential proteins required for the survival of cells and replication of the virus. Exosomes are thought to play an essential role in intercellular communications by transporting proteins and miRNAs, making them ideal in the search for biomarkers. Evidence suggests that miRNAs are involved in the regulation of influenza virus replication in many cell types. During the 2016 and 2017 influenza season, we collected blood samples from 54 patients infected with influenza and from 30 healthy volunteers to identify the potential role of circulating serum miRNAs and cytokines in influenza infection. Data comparing the exosomal miRNAs in patients with influenza B to healthy volunteers showed 76 miRNAs that were differentially expressed (p < 0.05). In contrast, 26 miRNAs were differentially expressed between patients with influenza A (p < 0.05) and the controls. Of these miRNAs, 11 were commonly expressed in both the influenza A and B patients. Interferon (IFN)-inducing protein 10 (IP-10), which is involved in IFN synthesis during influenza infection, showed the highest level of expression in both influenza A and B patients. Influenza A patients showed increased expression of IFNα, GM-CSF, interleukin (IL)-13, IL-17A, IL-1ß, IL-6 and TNFα, while influenza B induced increased levels of EGF, G-CSF, IL-1α, MIP-1α, and TNF-ß. In addition, hsa-miR-326, hsa-miR-15b-5p, hsa-miR-885, hsa-miR-122-5p, hsa-miR-133a-3p, and hsa-miR-150-5p showed high correlations to IL-6, IL-15, IL-17A, IL-1ß, and monocyte chemoattractant protein-1 (MCP-1) with both strains of influenza. Next-generation sequencing studies of H1N1-infected human lung small airway epithelial cells also showed similar pattern of expression of miR-375-5p, miR-143-3p, 199a-3p, and miR-199a-5p compared to influenza A patients. In summary, this study provides insights into the miRNA profiling in both influenza A and B virus in circulation and a novel approach to identify the early infections through a combination of cytokines and miRNA expression.
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Healthcare workers concurrently may be at a higher risk of developing respiratory infections and allergic disease, such as asthma, than the general public. Increased incidence of allergic diseases is thought to be caused, in part, due to occupational exposure to chemicals that induce or augment Th2 immune responses. However, whether exposure to these chemical antimicrobials can influence immune responses to respiratory pathogens is unknown. Here, we use a BALB/c murine model to test if the Th2-promoting antimicrobial chemical triclosan influences immune responses to influenza A virus. Mice were dermally exposed to 2% triclosan for 7 days prior to infection with a sub-lethal dose of mouse adapted PR8 A(H1N1) virus (50 pfu); triclosan exposure continued until 10 days post infection (dpi). Infected mice exposed to triclosan did not show an increase in morbidity or mortality, and viral titers were unchanged. Assessment of T cell responses at 10 dpi showed a decrease in the number of total and activated (CD44hi) CD4+ and CD8+ T cells at the site of infection (BAL and lung) in triclosan exposed mice compared to controls. Influenza-specific CD4+ and CD8+ T cells were assessed using MHCI and MHCII tetramers, with reduced populations, although not reaching statistical significance at these sites following triclosan exposure. Reductions in the Th1 transcription factor T-bet were seen in both activated and tetramer+ CD4+ and CD8+ T cells in the lungs of triclosan exposed infected mice, indicating reduced Th1 polarization and providing a potential mechanism for numerical reduction in T cells. Overall, these results indicate that the immune environment induced by triclosan exposure has the potential to influence the developing immune response to a respiratory viral infection and may have implications for healthcare workers who may be at an increased risk for developing infectious diseases.
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Inmunidad Adaptativa/efectos de los fármacos , Personal de Salud , Gripe Humana/inmunología , Exposición Profesional/efectos adversos , Células TH1/efectos de los fármacos , Triclosán/efectos adversos , Administración Tópica , Animales , Modelos Animales de Enfermedad , Femenino , Humanos , Subtipo H1N1 del Virus de la Influenza A/inmunología , Gripe Humana/prevención & control , Gripe Humana/virología , Ratones , Células TH1/inmunología , Triclosán/administración & dosificaciónRESUMEN
Host-viral interaction occurring throughout the infection process between the influenza A virus (IAV) and bronchial cells determines the success of infection. Our previous studies showed that the apoptotic pathway triggered by the host cells was repressed by IAV facilitating prolonged survival of infected cells. A detailed understanding on the role of IAV in altering the cell death pathway during early-stage infection of human bronchial epithelial cells (HBEpCs) is still unclear. We investigated the gene expression profiles of IAV-infected vs. mock-infected cells at the early stage of infection with a PCR array for death receptor (DR) pathway. At early stages infection (2 h) with IAV significantly upregulated DR pathway genes in HBEpCs, whereas 6 h exposure to IAV resulted in downregulation of the same genes. IAV replication in HBEpCs decreased the levels of DR pathway genes including TNF-receptor superfamily 1, Fas-associated death domain, caspase-8, and caspase-3 by 6 h, resulting in increased survival of cells. The apoptotic cell population decreased in 6 h compared with the 2 h exposure to IAV. The PCR array data were imported into Ingenuity Pathway Analysis software, resulting in confirmation of the model showing significant modulation of the DR pathway. Our data indicate that a significant transcriptional regulation of apoptotic, necrotic, and DR genes occur at early and late hours of infection that are vital in modulating the survival of host cells and replication of IAV. These data may have provided a likely roadmap for translational approaches targeting the DR pathway to enhance apoptosis and inhibit replication of the virus.
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Bronquios/patología , Células Epiteliales/metabolismo , Células Epiteliales/virología , Virus de la Influenza A/fisiología , Receptores de Muerte Celular/metabolismo , Transducción de Señal , Apoptosis/genética , Línea Celular , Células Epiteliales/patología , Regulación de la Expresión Génica , Humanos , NecrosisRESUMEN
Influenza A virus (IAV) infection remains a significant cause of morbidity and mortality worldwide. One key transcription factor that is activated upon IAV infection is nuclear factor Kappa B (NF-κB). NF-κB regulation involves the inhibitor proteins NF-κB inhibitor beta (NFKBIB), (also known as IκB ß), which form complexes with NF-κB to sequester it in the cytoplasm. In this study, microarray data showed differential expression of several microRNAs (miRNAs) on exposure to IAV. Target scan analysis revealed that miR-4776, miR-4514 and miR-4742 potentially target NFKBIB messenger RNA (mRNA). Time-course analysis of primary bronchial epithelial cells (HBEpCs) showed that miR-4776 expression is increased within 1 h of infection, followed by its downregulation 4 h post-exposure to IAV. NFKBIB upregulation of miR-4776 correlated with a decrease in NFKBIB expression within 1 h of infection and a subsequent increase in NFKBIB expression 4 h post-infection. In addition, miRNA ago-immunoprecipitation studies and the three prime untranslated region (3' UTR) luciferase assay confirmed that miR-4776 targets NFKBIB mRNA. Furthermore, uninfected HBEpCs transfected with miR-4776 mimic showed decreased expression of NFKBIB mRNA. Overexpression of NFKBIB protein in IAV infected cells led to lower levels of IAV. Taken together, our data suggest that miRNA-4776 modulates IAV production in infected cells through NFKBIB expression, possibly through the modulation of NF-κB.
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Bronquios/virología , Células Epiteliales/virología , Proteínas I-kappa B/genética , Subtipo H1N1 del Virus de la Influenza A/fisiología , MicroARNs/genética , Regiones no Traducidas 3' , Bronquios/citología , Línea Celular , Regulación hacia Abajo , Regulación de la Expresión Génica , Humanos , Proteínas I-kappa B/metabolismo , Análisis por Micromatrices , Viabilidad Microbiana , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal , Regulación hacia ArribaRESUMEN
Influenza antiviral drugs that use protein inhibitors can lose their efficacy as resistant strains emerge. As an alternative strategy, we investigated the use of small interfering RNA molecules (siRNAs) by characterizing three siRNAs (M747, M776 and M832) targeting the influenza matrix 2 gene and three (NS570, NS595 and NS615) targeting the nonstructural protein 1 and 2 genes. We also re-examined two previously reported siRNAs, M331 and M950, which target the matrix 1 and 2 genes. Treatment with M331-, M776-, M832-, and M950-siRNAs attenuated influenza titer. M776-siRNA treated cells had 29.8% less infectious virus than cells treated with the previously characterized siRNA, M950. NS570-, NS595- and NS615-siRNAs reduced nonstructural protein 1 and 2 expression and enhanced type I interferon expression by 50%. Combination siRNA treatment attenuated 20.9% more infectious virus than single siRNA treatment. Our results suggest a potential use for these siRNAs as an effective anti-influenza virus therapy.
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Regulación Viral de la Expresión Génica , Virus de la Influenza A/genética , Interferencia de ARN , Proteínas de la Matriz Viral/genética , Proteínas no Estructurales Virales/genética , Animales , Línea Celular , Perros , Orden Génico , Humanos , Virus de la Influenza A/fisiología , Gripe Humana/genética , Gripe Humana/metabolismo , Gripe Humana/virología , Interferón Tipo I/biosíntesis , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , Proteínas de la Matriz Viral/metabolismo , Proteínas no Estructurales Virales/metabolismo , Ensayo de Placa Viral , Replicación ViralRESUMEN
BACKGROUND: To prepare for a possible influenza pandemic, a better understanding of the potential for the airborne transmission of influenza from person to person is needed. OBJECTIVES: The objective of this study was to directly compare the generation of aerosol particles containing viable influenza virus during coughs and exhalations. METHODS: Sixty-one adult volunteer outpatients with influenza-like symptoms were asked to cough and exhale three times into a spirometer. Aerosol particles produced during coughing and exhalation were collected into liquid media using aerosol samplers. The samples were tested for the presence of viable influenza virus using a viral replication assay (VRA). RESULTS: Fifty-three test subjects tested positive for influenza A virus. Of these, 28 (53%) produced aerosol particles containing viable influenza A virus during coughing, and 22 (42%) produced aerosols with viable virus during exhalation. Thirteen subjects had both cough aerosol and exhalation aerosol samples that contained viable virus, 15 had positive cough aerosol samples but negative exhalation samples, and 9 had positive exhalation samples but negative cough samples. CONCLUSIONS: Viable influenza A virus was detected more often in cough aerosol particles than in exhalation aerosol particles, but the difference was not large. Because individuals breathe much more often than they cough, these results suggest that breathing may generate more airborne infectious material than coughing over time. However, both respiratory activities could be important in airborne influenza transmission. Our results are also consistent with the theory that much of the aerosol containing viable influenza originates deep in the lungs.
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Aerosoles/análisis , Microbiología del Aire , Tos/virología , Virus de la Influenza A/fisiología , Gripe Humana/transmisión , Viabilidad Microbiana , Adulto , Espiración , Femenino , Humanos , Gripe Humana/prevención & control , Gripe Humana/virología , Masculino , Pandemias/prevención & control , ARN Viral , Espirometría , Replicación Viral , Adulto JovenRESUMEN
Intercellular cell adhesion molecule-1 (ICAM-1) is an inducible cell surface glycoprotein that is expressed on many cell types. Influenza virus infection enhanced ICAM-1 expression and messenger RNA levels. Human bronchial epithelial cells (HBEpC) and nasal epithelial cells, on exposure to different strains of influenza virus (H1N1, H3N2, and H9N1) showed significant increase in ICAM-1 gene expression (p<0.001) along with the ICAM-1 protein levels (surface and secreted). Depleting ICAM-1 in HBEpC with ICAM-1 siRNA and subsequently infecting with H1N1 showed increased viral copy numbers. Influenza virus infection in HBEpC resulted in up-regulation of NF-ĸB protein and the lack of ICAM-1 decreased NF-ĸB activity in NF-ĸB luciferase reporter assay. Addition of exogenous IL-1ß to HBEpC induced the ICAM-1 expression and decreased matrix gene copy number. Taken together, HBEpC induced ICAM-1 plays a key role in modulating the influenza virus survival possibly through the NF-ĸB pathway.
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Subtipo H1N1 del Virus de la Influenza A/inmunología , Subtipo H3N2 del Virus de la Influenza A/inmunología , Molécula 1 de Adhesión Intercelular/inmunología , Mucosa Respiratoria/inmunología , Factor de Transcripción ReIA/metabolismo , Animales , Bronquios/inmunología , Bronquios/virología , Línea Celular , Perros , Células Epiteliales/virología , Matriz Extracelular/metabolismo , Expresión Génica , Humanos , Molécula 1 de Adhesión Intercelular/biosíntesis , Molécula 1 de Adhesión Intercelular/genética , Interleucina-1beta/farmacología , Células de Riñón Canino Madin Darby , Interferencia de ARN , ARN Interferente Pequeño , Mucosa Respiratoria/citología , Mucosa Respiratoria/virología , Factor de Transcripción ReIA/biosíntesisRESUMEN
Patients with influenza release aerosol particles containing the virus into their environment. However, the importance of airborne transmission in the spread of influenza is unclear, in part because of a lack of information about the infectivity of the airborne virus. The purpose of this study was to determine the amount of viable influenza A virus that was expelled by patients in aerosol particles while coughing. Sixty-four symptomatic adult volunteer outpatients were asked to cough 6 times into a cough aerosol collection system. Seventeen of these participants tested positive for influenza A virus by viral plaque assay (VPA) with confirmation by viral replication assay (VRA). Viable influenza A virus was detected in the cough aerosol particles from 7 of these 17 test subjects (41%). Viable influenza A virus was found in the smallest particle size fraction (0.3 µm to 8 µm), with a mean of 142 plaque-forming units (SD 215) expelled during the 6 coughs in particles of this size. These results suggest that a significant proportion of patients with influenza A release small airborne particles containing viable virus into the environment. Although the amounts of influenza A detected in cough aerosol particles during our experiments were relatively low, larger quantities could be expelled by influenza patients during a pandemic when illnesses would be more severe. Our findings support the idea that airborne infectious particles could play an important role in the spread of influenza.
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Aerosoles/análisis , Microbiología del Aire , Tos/virología , Virus de la Influenza A/aislamiento & purificación , Gripe Humana/transmisión , Adolescente , Adulto , Femenino , Humanos , Masculino , Tamaño de la Partícula , ARN Viral/análisis , ARN Viral/aislamiento & purificación , Ensayo de Placa Viral , Replicación ViralRESUMEN
Influenza virus infection induces several changes in host miRNA profile, host cell death and tissue damage. Cytochrome c is a regulator of the intrinsic apoptotic pathway and is altered during viral infections. Within the first 3h of infection with influenza virus, significant down-regulation of hsa-miRNA-4276 (miRNA-4276) is followed by a 2-fold increase in cytochrome c oxidase VIC (COX6C) mRNA was found to occur in human alveolar and bronchial epithelial cells. Expression of caspase-9 also increased within the first 3h of infection, but subsequently decreased. Modulation of miR-4276 using mimic and inhibitor oligonucleotides showed significant down-regulation or up-regulation, respectively, of COX6C expression. Our data suggests that on initial exposure to influenza virus, host cells upregulate COX6C mRNA expression through silencing miR-4276 and repressed viral replication by inducing the apoptotic protein caspase-9. Taken together, these data suggest that miR-4276 may be an important regulator of the early stages of infection by influenza.
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Bronquios/citología , Complejo IV de Transporte de Electrones/metabolismo , Células Epiteliales/virología , Subtipo H3N2 del Virus de la Influenza A/fisiología , MicroARNs/metabolismo , Mucosa Respiratoria/citología , Animales , Línea Celular , Perros , Complejo IV de Transporte de Electrones/genética , Células Epiteliales/inmunología , Regulación Enzimológica de la Expresión Génica/fisiología , Humanos , Subtipo H1N1 del Virus de la Influenza A/fisiología , MicroARNs/genética , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
Understanding the host response to influenza A virus infection is essential for developing intervention approaches. We show that infection of human alveolar epithelial cells and human bronchial epithelial cells with influenza A for 3h resulted in down-regulation of host hsa-miRNA-548an (miRNA-548an) which triggered the overexpression of influenza non-structural-1A binding protein (IVNS1ABP, herein referred to as NS1ABP). Reduced NS1ABP mRNA and NS1ABP protein expression after transfection of miRNA-548an mimic or increased NS1ABP mRNA and NS1ABP protein expression after transfection of miRNA-548an inhibitor provided evidence that miRNA-548an is involved in the regulation of NS1ABP. Transfection of cells with inhibitor led to reduced apoptosis of infected cells while transfection of mimic led to increased apoptosis and reduced influenza copy number suggesting that NS1ABP has a role in viral maintenance. Thus, miRNA-548an may be an important target in controlling the early stage infection of influenza A.
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Células Epiteliales/virología , Regulación de la Expresión Génica , Virus de la Influenza A/crecimiento & desarrollo , MicroARNs/metabolismo , Proteínas Nucleares/biosíntesis , Factores de Transcripción/biosíntesis , Línea Celular , Humanos , Proteínas de Unión al ARNRESUMEN
Human rhinoviruses (HRV) are the most common agent of upper respiratory infections and an important cause of lower respiratory tract symptoms. Our previous research with other viral pathogens has shown that virus-induced airway inflammation and hyperreactivity involve neurotrophic pathways that also affect tropism and severity of the infection. The goals of this study were to analyze systematically the expression of key neurotrophic factors and receptors during HRV-16 infection of human airway epithelial cells and to test the hypothesis that neurotrophins modulate HRV infection by controlling the expression of a major cellular receptor for this virus, the intercellular adhesion molecule 1 (ICAM-1). Neurotrophins and ICAM-1 expression were analyzed at the mRNA level by real-time PCR and at the protein level by flow cytometry and immunocytochemistry. A small inhibitory RNA (siRNA) or a specific blocking antibody was utilized to suppress nerve growth factor (NGF) expression and measure its effects on viral replication and virus-induced cell death. Nasal and bronchial epithelial cells were most susceptible to HRV-16 infection at 33°C and 37°C, respectively, and a significant positive relationship was noted between expression of NGF and tropomyosin-related kinase A (TrkA) and virus copy number. ICAM-1 expression was dose dependently upregulated by exogenous NGF and significantly downregulated by NGF inhibition with corresponding decrease in HRV-16 replication. NGF inhibition also increased apoptotic death of infected cells. Our results suggest that HRV upregulates the NGF-TrkA pathway in airway epithelial cells, which in turn amplifies viral replication by increasing HRV entry via ICAM-1 receptors and by limiting apoptosis.
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Células Epiteliales/metabolismo , Molécula 1 de Adhesión Intercelular/metabolismo , Factor de Crecimiento Nervioso/metabolismo , Infecciones por Picornaviridae/metabolismo , Mucosa Respiratoria/metabolismo , Rhinovirus/fisiología , Anticuerpos Neutralizantes/farmacología , Apoptosis/efectos de los fármacos , Bronquios/metabolismo , Bronquios/patología , Bronquios/virología , Línea Celular , Células Epiteliales/patología , Células Epiteliales/virología , Regulación de la Expresión Génica , Interacciones Huésped-Patógeno , Humanos , Molécula 1 de Adhesión Intercelular/genética , Factor de Crecimiento Nervioso/farmacología , Infecciones por Picornaviridae/patología , Infecciones por Picornaviridae/virología , Mucosa Respiratoria/patología , Mucosa Respiratoria/virología , Transducción de Señal , Carga Viral/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
BACKGROUND: Early-life infection by respiratory syncytial virus (RSV) is associated with aberrant expression of the prototypical neurotrophin nerve growth factor (NGF) and its cognate receptors in human bronchial epithelium. However, the chain of events leading to this outcome, and its functional implications for the progression of the viral infection, has not been elucidated. This study sought to test the hypothesis that RSV infection modulates neurotrophic pathways in human airways by silencing the expression of specific microRNAs (miRNAs), and that this effect favors viral growth by interfering with programmed death of infected cells. METHODOLOGY: Human bronchial epithelial cells infected with green fluorescent protein-expressing RSV (rgRSV) were screened with multiplex qPCR arrays, and miRNAs significantly affected by the virus were analyzed for homology with mRNAs encoding neurotrophic factors or receptors. Mimic sequences of selected miRNAs were transfected into non-infected bronchial cells to confirm the role of each of them in regulating neurotrophins expression at the gene and protein level, and to study their influence on cell cycle and viral replication. PRINCIPAL FINDINGS: RSV caused downregulation of 24 miRNAs and upregulation of 2 (p<0.01). Homology analysis of microarray data revealed that 6 of those miRNAs exhibited a high degree of complementarity to NGF and/or one of its cognate receptors TrKA and p75(NTR). Among the selected miRNAs, miR-221 was significantly downregulated by RSV and its transfection in bronchial epithelial cells maximally inhibited gene and protein expression of NGF and TrKA, increased apoptotic cell death, and reduced viral replication and infectivity. CONCLUSIONS/SIGNIFICANCE: Our data suggest that RSV upregulates the NGF-TrKA axis in human airways by silencing miR-221 expression, and this favors viral replication by interfering with the apoptotic death of infected cells. Consequently, the targeted delivery of exogenous miRNAs to the airways may provide a new strategy for future antiviral therapies based on RNA interference.
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Células Epiteliales/metabolismo , MicroARNs/genética , Factor de Crecimiento Nervioso/genética , Virus Sincitiales Respiratorios/genética , Adolescente , Adulto , Anciano , Apoptosis/genética , Bronquios/citología , Células Cultivadas , Células Epiteliales/virología , Femenino , Perfilación de la Expresión Génica , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Interacciones Huésped-Patógeno , Humanos , Masculino , Microscopía Confocal , Persona de Mediana Edad , Factor de Crecimiento Nervioso/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Receptor trkA/genética , Receptor trkA/metabolismo , Virus Sincitiales Respiratorios/metabolismo , Virus Sincitiales Respiratorios/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección , Replicación Viral/genética , Adulto JovenRESUMEN
Respiratory syncytial virus (RSV) is the most common respiratory pathogen in infants and young children. The pathophysiology of this infection in the respiratory system has been studied extensively, but little is known about its consequences in other systems. We studied whether RSV infects human bone marrow stromal cells (BMSCs) in vitro and in vivo, and investigated whether and how this infection affects BMSC structure and hematopoietic support function. Primary human BMSCs were infected in vitro with recombinant RSV expressing green fluorescent protein. In addition, RNA from naive BMSCs was amplified by PCR, and the products were sequenced to confirm homology with the RSV genome. The BMSC cytoskeleton was visualized by immunostaining for actin. Finally, we analyzed infected BMSCs for the expression of multiple cytokines and chemokines, evaluated their hematopoietic support capacity, and measured their chemotactic activity for both lymphoid and myeloid cells. We found that BMSCs support RSV replication in vitro with efficiency that varies among cell lines derived from different donors; furthermore, RNA sequences homologous to the RSV genome were found in naive primary human BMSCs. RSV infection disrupted cytoskeletal actin microfilaments, altered cytokine/chemokine expression patterns, decreased the ability of BMSCs to support B cell maturation, and modulated local chemotaxis. Our data indicate that RSV infects human BMSCs in vitro, and this infection has important structural and functional consequences that might affect hematopoietic and immune functions. Furthermore, we have amplified viral RNA from naive primary BMSCs, suggesting that in vivo these cells provide RSV with an extrapulmonary target.
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Células de la Médula Ósea/virología , Células del Estroma/virología , Adulto , Secuencia de Bases , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Células Cultivadas , Quimiocinas/metabolismo , Quimiotaxis , Niño , Citocinas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Células Epiteliales/virología , Humanos , Datos de Secuencia Molecular , Células Precursoras de Linfocitos B , ARN Viral/genética , Infecciones por Virus Sincitial Respiratorio/inmunología , Infecciones por Virus Sincitial Respiratorio/virología , Virus Sincitiales Respiratorios/fisiología , Homología de Secuencia de Ácido Nucleico , Células del Estroma/inmunología , Células del Estroma/metabolismo , Replicación ViralRESUMEN
BACKGROUND: Overall expression of neurotrophins in the respiratory tract is upregulated in infants infected by the respiratory syncytial virus (RSV), but it is unclear where (structural vs. inflammatory cells, upper vs. lower airways) and why, these changes occur. We analyzed systematically the expression of neurotrophic factors and receptors following RSV infection of human nasal, tracheal, and bronchial epithelial cells, and tested the hypothesis that neurotrophins work as innate survival factors for infected respiratory epithelia. METHODOLOGY: Expression of neurotrophic factors (nerve growth factor, NGF; brain-derived neurotrophic factor, BDNF) and receptors (trkA, trkB, p75) was analyzed at the protein level by immunofluorescence and flow cytometry and at the mRNA level by real-time PCR. Targeted siRNA was utilized to blunt NGF expression, and its effect on virus-induced apoptosis/necrosis was evaluated by flow cytometry following annexin V/7-AAD staining. PRINCIPAL FINDINGS: RSV infection was more efficient in cells from more distal (bronchial) vs. more proximal origin. In bronchial cells, RSV infection induced transcript and protein overexpression of NGF and its high-affinity receptor trkA, with concomitant downregulation of the low-affinity p75(NTR). In contrast, tracheal cells exhibited an increase in BDNF, trkA and trkB, and nasal cells increased only trkA. RSV-infected bronchial cells transfected with NGF-specific siRNA exhibited decreased trkA and increased p75(NTR) expression. Furthermore, the survival of bronchial epithelial cells was dramatically decreased when their endogenous NGF supply was depleted prior to RSV infection. CONCLUSIONS/SIGNIFICANCE: RSV infection of the distal airway epithelium, but not of the more proximal sections, results in overexpression of NGF and its trkA receptor, while the other p75(NTR) receptor is markedly downregulated. This pattern of neurotrophin expression confers protection against virus-induced apoptosis, and its inhibition amplifies programmed cell death in the infected bronchial epithelium. Thus, pharmacologic modulation of NGF expression may offer a promising new approach for management of common respiratory infections.
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Bronquios/patología , Supervivencia Celular/fisiología , Factor de Crecimiento Nervioso/fisiología , Infecciones por Virus Sincitial Respiratorio/patología , Secuencia de Bases , Células Cultivadas , Cartilla de ADN , Células Epiteliales/citología , Citometría de Flujo , Técnicas de Silenciamiento del Gen , Humanos , Microscopía Fluorescente , Factor de Crecimiento Nervioso/genética , Reacción en Cadena de la Polimerasa , ARN Interferente PequeñoRESUMEN
Exposure to arsenic (As) is a risk factor for the development of diabetes, vascular diseases and cancer. Several theories have been proposed to account for the mechanisms potentially responsible for As toxicity and carcinogenesis. Currently, we have investigated whether the eukaryotic translation initiation factor 4E (eIF4E), the mRNA cap binding and rate limiting factor required for translation, is a target for As-induced cytotoxicity and cell death. We have also investigated the potential cellular mechanisms underlying the As-induced de-regulation of expression of eIF4E that are most likely responsible for the cytotoxicity and cell death induced by As. Exposure of four different human cell lines - HCT15 (colorectal adenocarcinoma), PLC/PR/5 (hepatocellular carcinoma), HeLa (cervical adenocarcinoma) and Chang (likely derived from HeLa cells) to sodium arsenite (NaAsO2) for time intervals up to 24 h resulted in a concentration-dependent cytotoxicity and cell death. All the NaAsO2-treated cells exhibited significant inhibition of eIF4E gene (protein). The potential involvement of eIF4E gene expression in the NaAsO2-induced cytotoxicity and cell death was investigated by silencing the cellular expression of the eIF4E gene by employing a small interfering RNA (SiRNA) specifically targeting the eIF4E gene's expression. The SiRNA-mediated silencing of eIF4E gene expression also resulted in significant cytotoxicity and cell death suggesting that the toxicity noticed among the NaAsO2-treated cells was probably due to the chemically induced inhibition of eIF4E gene expression. The potential involvement of inhibition of eIF4E gene expression in the NaAsO2-induced cytotoxicity and cell death was further investigated by employing transgenic cell lines overexpressing the eIF4E gene. Overexpression of the eIF4E gene in the Chinese hamster ovary cell line was protective against the NaAsO2-induced cytotoxicity and cell death. Additional studies conducted to understand the potential mechanisms responsible for NaAsO2-induced inhibition of eIF4E gene expression demonstrated that exposure to NaAsO2 resulted in transcriptional down-regulation of the eIF4E gene only in HCT-15 and HeLa cells, while in the NaAsO2-treated and PLC/PR/5 and Chang cells, the eIF4E mRNA expression level was comparable to those of the corresponding control cells. Cellular levels of ubiquitin and the process of ubiquitination were significantly higher in the NaAsO2-treated cells compared with the control cells. Immunoprecipitation of lysates obtained from the NaAsO2-treated cells and the subsequent western blot analysis of the immunoprecipitated protein(s) using the eIF4E antibody detected the presence of eIF4E protein in the immunoprecipitate suggesting possible ubiquitination of eIF4E protein in the NaAsO2-treated cells. Pre-exposure of the NaAsO2-treated cells to proteasome inhibitors blocked the inhibition of eIF4E gene expression as well as the resulting cytotoxicity and cell death. Furthermore, exposure of cells to NaAsO2 resulted in a significant inhibition of expression of the cell cycle and growth regulating gene, cyclin D1. Whether or not the inhibition of cyclin D1 in the NaAsO2-treated cells is mediated through the inhibition of eIF4E was tested by silencing the expression of eIF4E gene in the cells. Transfection of cells with SiRNA specifically targeting eIF4E gene expression resulted in a significant inhibition of cyclin D1 gene suggesting that the observed inhibition of cyclin D1 gene in the NaAsO2-treated cells is most likely mediated through inhibition of eIF4E gene. Taken together, our results indicate that the exposure of cells to NaAsO2 resulted in cytotoxicity and cell death, at least in part, due to the inhibition of eIF4E gene expression leading to diminished cellular levels of critical genes such as cyclin D1.
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Arsenitos/toxicidad , Factor 4E Eucariótico de Iniciación/metabolismo , Compuestos de Sodio/toxicidad , Animales , Células CHO , Supervivencia Celular/efectos de los fármacos , Cricetinae , Cricetulus , Ciclina D1/genética , Ciclina D1/metabolismo , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Factor 4E Eucariótico de Iniciación/biosíntesis , Factor 4E Eucariótico de Iniciación/genética , Regulación de la Expresión Génica/efectos de los fármacos , Células HeLa , Humanos , Interferencia de ARN , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transfección , Ubiquitina/metabolismoRESUMEN
Whether translation initiation factor 4E (eIF4E), the mRNA cap binding and rate-limiting factor required for translation, is a target for cytotoxicity and cell death induced by cadmium, a human carcinogen, was investigated. Exposure of human cell lines, HCT15, PLC/PR/5, HeLa, and Chang, to cadmium chloride resulted in cytotoxicity and cell death, and this was associated with a significant decrease in eIF4E protein levels. Similarly, specific silencing of the expression of the eIF4E gene, caused by a small interfering RNA, resulted in significant cytotoxicity and cell death. On the other hand, overexpression of the eIF4E gene was protective against the cadmium-induced cytotoxicity and cell death. Further studies revealed the absence of alterations in the eIF4E mRNA level in the cadmium-treated cells despite their decreased eIF4E protein level. In addition, exposure of cells to cadmium resulted in enhanced ubiquitination of eIF4E protein while inhibitors of proteasome activity reversed the cadmium-induced decrease of eIF4E protein. Exposure of cells to cadmium, as well as the specific silencing of eIF4E gene, also resulted in decreased cellular levels of cyclin D1, a critical cell cycle and growth regulating gene, suggesting that the observed inhibition of cyclin D1 gene expression in the cadmium-treated cells is most likely due to decreased cellular level of eIF4E. Taken together, our results demonstrate that the exposure of cells to cadmium chloride resulted in cytotoxicity and cell death due to enhanced ubiquitination and consequent proteolysis of eIF4E protein, which in turn diminished cellular levels of critical genes such as cyclin D1.
Asunto(s)
Cloruro de Cadmio/farmacología , Factor 4E Eucariótico de Iniciación/metabolismo , Animales , Células CHO , Cadmio/química , Cadmio/metabolismo , Cloruro de Cadmio/química , Muerte Celular , Línea Celular Tumoral , Cricetinae , Ciclina D1/metabolismo , Relación Dosis-Respuesta a Droga , Silenciador del Gen , Células HeLa , Hemo Oxigenasa (Desciclizante)/biosíntesis , Hemo-Oxigenasa 1 , Humanos , Inmunoprecipitación , Proteínas de la Membrana , Metalotioneína/biosíntesis , Unión Proteica , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes/química , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Transgenes , Ubiquitina/químicaRESUMEN
Several studies have demonstrated the overexpression of certain eukaryotic translation factors in human cancer cell lines and in malignant tissues. In this study, with human cancer cell lines derived from lungs, breast, prostate, and skin, we have examined the expression profile of 36 translation factors consisting of 27 initiation factors, 8 elongation factors, and 1 termination factor. Translation initiation factors 2C2 and 4E1 and translation elongation factors 1A2 and 1delta were found overexpressed (2- to 2000-fold) in many of the cancer cell lines compared to their corresponding normal cell lines. Among the translation factors analyzed, translation elongation factor 1A2 exhibited the most significant alteration in expression: 10- to 2000-fold overexpression was noticed in nine out of ten cancer cell lines analyzed. Whether the overexpression of translation elongation factor 1A2 can be used as a potential tumor marker was tested with the cancer profiling array (BD Biosciences, Palo Alto, CA) consisting of 241 paired cDNA samples generated from 13 different cancer/noncancer tissue types. Overexpression of translation elongation factor 1A2 was noticed in several tumor tissue samples, most notably in the human colon cancer samples which exhibited at least a twofold overexpression among 35% of the samples analyzed. Besides colon, tumor samples derived from lungs, kidney, rectum, and ovary also exhibited more than a twofold overexpression of translation elongation factor 1A2 in at least 20% of the samples analyzed. These results indicate that human carcinogenesis is often associated with alterations in the expression of various translation factors especially the overexpression of eukaryotic translation elongation factor 1A2.