RESUMEN
Purpose: Dendritic cells (DC) initiate adaptive immune responses through the uptake and presentation of antigenic material. In preclinical studies, intratumorally injected activated DCs (aDCs; DCVax-Direct) were superior to immature DCs in rejecting tumors from mice.Experimental Design: This single-arm, open-label phase I clinical trial evaluated the safety and efficacy of aDCs, administered intratumorally, in patients with solid tumors. Three dose levels (2 million, 6 million, and 15 million aDCs per injection) were tested using a standard 3 + 3 dose-escalation trial design. Feasibility, immunogenicity, changes to the tumor microenvironment after direct injection, and survival were evaluated. We also investigated cytokine production of aDCs prior to injection.Results: In total, 39 of the 40 enrolled patients were evaluable. The injections of aDCs were well tolerated with no dose-limiting toxicities. Increased lymphocyte infiltration was observed in 54% of assessed patients. Stable disease (SD; best response) at week 8 was associated with increased overall survival. Increased secretion of interleukin (IL)-8 and IL12p40 by aDCs was significantly associated with survival (P = 0.023 and 0.024, respectively). Increased TNFα levels correlated positively with SD at week 8 (P < 0.01).Conclusions: Intratumoral aDC injections were feasible and safe. Increased production of specific cytokines was correlated with SD and prolonged survival, demonstrating a link between the functional profile of aDCs prior to injection and patient outcomes. Clin Cancer Res; 24(16); 3845-56. ©2018 AACR.
Asunto(s)
Tratamiento Basado en Trasplante de Células y Tejidos , Células Dendríticas/trasplante , Inmunoterapia , Neoplasias/terapia , Adulto , Anciano , Citocinas/genética , Citocinas/inmunología , Células Dendríticas/inmunología , Femenino , Humanos , Inyecciones Intralesiones , Interleucina-12/genética , Interleucina-8/genética , Masculino , Persona de Mediana Edad , Neoplasias/genética , Neoplasias/inmunología , Neoplasias/patología , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/inmunologíaRESUMEN
Human metapneumovirus (hMPV), a leading cause of respiratory tract infections in infants, encodes a small hydrophobic (SH) protein of unknown function. Here we show that infection of plasmacytoid dendritic cells (pDCs) with a recombinant virus lacking SH expression (rhMPV-ΔSH) enhanced the secretion of type I interferons (IFNs), which required TLR7 and MyD88 expression. HMPV SH protein inhibited TLR7/MyD88/TRAF6 signaling leading to IFN gene transcription, identifying a novel mechanism by which paramyxovirus SH proteins modulate innate immune responses.
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Células Dendríticas/virología , Interferón Tipo I/metabolismo , Metapneumovirus/genética , Proteínas Oncogénicas de Retroviridae/inmunología , Células Dendríticas/inmunología , Células HEK293 , Humanos , Inmunidad Innata , Metapneumovirus/fisiología , Factor 88 de Diferenciación Mieloide/inmunología , Transducción de Señal , Receptor Toll-Like 7/inmunologíaRESUMEN
The chemokine CXCL1 and its receptor CXCR2 play a crucial role in host immune response by recruiting and activating neutrophils for microbial killing at the tissue site. Dysregulation in this process has been implicated in collateral tissue damage causing disease. CXCL1 reversibly exists as monomers and dimers, and it has been proposed that distinct monomer and dimer activities and the monomer-dimer equilibrium regulate the neutrophil function. However, the molecular mechanisms linking the CXCL1/CXCR2 axis and the neutrophil 'beneficial' and 'destructive' phenotypes are not known. In this study, we characterized neutrophil trafficking and its consequence in the mouse lung by the CXCL1 wild type (WT), which exists as monomers and dimers, and by a nondissociating dimer. Whereas the WT, compared to the dimer, was more active at low doses, both the WT and the dimer elicited a large neutrophil efflux at high doses. Importantly, robust neutrophil recruitment elicited by the WT or dimer was not detrimental to lung tissue integrity and, further, could not be correlated to surface CXCR2 levels. We conclude that the CXCL1 monomer-dimer distribution and receptor interactions are highly coupled and regulate neutrophil trafficking and that injury in the context of disease is a consequence of inappropriate CXCR2 activation at the target tissue and not due to mechanical forces exerted by neutrophils during recruitment.
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Movimiento Celular/inmunología , Quimiocina CXCL1/inmunología , Pulmón/inmunología , Infiltración Neutrófila , Neutrófilos/inmunología , Receptores de Interleucina-8B/inmunología , Animales , Movimiento Celular/genética , Quimiocina CXCL1/genética , Femenino , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Receptores de Interleucina-8B/genéticaRESUMEN
Human metapneumovirus (hMPV) is a major cause of respiratory tract infections in children, elderly and immunocompromised hosts, for which no vaccine or treatment are currently available. Oxidative stress and inflammatory responses represent important pathogenic mechanism(s) of hMPV infection. Here, we explored the potential protective role of dietary antioxidants in hMPV infection. Treatment of airway epithelial cells with resveratrol and quercetin during hMPV infection significantly reduced cellular oxidative damage, inflammatory mediator secretion and viral replication, without affecting viral gene transcription and protein synthesis, indicating that inhibition of viral replication occurred at the level of viral assembly and/or release. Modulation of proinflammatory mediator expression occurred through the inhibition of transcription factor nuclear factor (NF)-κB and interferon regulatory factor (IRF)-3 binding to their cognate site of endogenous gene promoters. Our results indicate the use of dietary antioxidants as an effective treatment approach for modulating hMPV induced lung oxidative damage and inflammation.
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Antioxidantes/farmacología , Suplementos Dietéticos/análisis , Metapneumovirus/efectos de los fármacos , Infecciones por Paramyxoviridae/virología , Línea Celular , Citocinas/genética , Citocinas/inmunología , Humanos , Metapneumovirus/genética , Metapneumovirus/fisiología , Estrés Oxidativo/efectos de los fármacos , Infecciones por Paramyxoviridae/tratamiento farmacológico , Infecciones por Paramyxoviridae/inmunología , Infecciones por Paramyxoviridae/metabolismo , Quercetina/farmacología , Resveratrol , Estilbenos/farmacología , Replicación Viral/efectos de los fármacosRESUMEN
Respiratory syncytial virus (RSV) and human Metapneumovirus (hMPV), viruses belonging to the family Paramyxoviridae, are the most important causes of lower respiratory tract infection in young children. Infections with RSV and hMPV are clinically indistinguishable, and both RSV and hMPV infection have been associated with aberrant adaptive immune responses. Myeloid Dendritic cells (mDCs) play a pivotal role in shaping adaptive immune responses during infection; however, few studies have examined how interactions of RSV and hMPV with individual mDC subsets (BDCA-1(+) and BDCA-3(+) mDCs) affect the outcome of anti-viral responses. To determine whether RSV and hMPV induce virus-specific responses from each subset, we examined co-stimulatory molecules and cytokines expressed by BDCA-1(+) and BDCA-3(+) mDCs isolated from peripheral blood after infection with hMPV and RSV, and examined their ability to stimulate T cell proliferation and differentiation. Our data show that RSV and hMPV induce virus-specific and subset-specific patterns of co-stimulatory molecule and cytokine expression. RSV, but not hMPV, impaired the capacity of infected mDCs to stimulate T cell proliferation. Whereas hMPV-infected BDCA-1(+) and BDCA-3(+) mDCs induced expansion of Th17 cells, in response to RSV, BDCA-1(+) mDCs induced expansion of Th1 cells and BDCA-3(+) mDCs induced expansion of Th2 cells and Tregs. These results demonstrate a virus-specific and subset-specific effect of RSV and hMPV infection on mDC function, suggesting that these viruses may induce different adaptive immune responses.
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Antígenos CD1/inmunología , Antígenos de Superficie/inmunología , Linfocitos T CD4-Positivos/inmunología , Células Dendríticas/inmunología , Glicoproteínas/inmunología , Infecciones por Paramyxoviridae/inmunología , Adulto , Linfocitos T CD4-Positivos/citología , Diferenciación Celular , Citocinas/biosíntesis , Citometría de Flujo , Humanos , Interferón-alfa/biosíntesis , TrombomodulinaRESUMEN
Human metapneumovirus (hMPV) and respiratory syncytial virus (RSV) are leading causes of upper and lower respiratory tract infections in young children and among elderly and immunocompromised patients. The pathogenesis of hMPV-induced lung disease is poorly understood. The lung macrophage population consists of alveolar macrophages (AMs) residing at the luminal surface of alveoli and interstitial macrophages present within the parenchymal lung interstitium. The involvement of AMs in innate immune responses to virus infections remains elusive. In this study, BALB/c mice depleted of AMs by intranasal instillation of dichloromethylene bisphosphonate (L-CL2MBP) liposomes were examined for disease, lung inflammation, and viral replication after infection with hMPV or RSV. hMPV-infected mice lacking AMs exhibited improved disease in terms of body weight loss, lung inflammation, airway obstruction, and hyperresponsiveness compared with AM-competent mice. AM depletion was associated with significantly reduced hMPV titers in the lungs, suggesting that hMPV required AMs for early entry and replication in the lung. In contrast, AM depletion in the context of RSV infection was characterized by an increase in viral replication, worsened disease, and inflammation, with increased airway neutrophils and inflammatory dendritic cells. Overall, lack of AMs resulted in a broad-spectrum disruption in type I IFN and certain inflammatory cytokine production, including TNF and IL-6, while causing a virus-specific alteration in the profile of several immunomodulatory cytokines, chemokines, and growth factors. Our study demonstrates that AMs have distinct roles in the context of human infections caused by members of the Paramyxoviridae family.
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Pulmón/inmunología , Macrófagos Alveolares/inmunología , Metapneumovirus/patogenicidad , Infecciones por Paramyxoviridae/inmunología , Infecciones por Virus Sincitial Respiratorio/inmunología , Virus Sincitiales Respiratorios/patogenicidad , Obstrucción de las Vías Aéreas/inmunología , Obstrucción de las Vías Aéreas/fisiopatología , Obstrucción de las Vías Aéreas/virología , Animales , Células Cultivadas , Citocinas/metabolismo , Modelos Animales de Enfermedad , Humanos , Mediadores de Inflamación/metabolismo , Pulmón/metabolismo , Pulmón/fisiopatología , Pulmón/virología , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/virología , Metapneumovirus/inmunología , Ratones Endogámicos BALB C , Infecciones por Paramyxoviridae/metabolismo , Infecciones por Paramyxoviridae/fisiopatología , Infecciones por Paramyxoviridae/virología , Neumonía/inmunología , Neumonía/fisiopatología , Neumonía/virología , Infecciones por Virus Sincitial Respiratorio/metabolismo , Infecciones por Virus Sincitial Respiratorio/fisiopatología , Infecciones por Virus Sincitial Respiratorio/prevención & control , Infecciones por Virus Sincitial Respiratorio/virología , Virus Sincitiales Respiratorios/inmunología , Factores de Tiempo , Replicación ViralRESUMEN
Human metapneumovirus (hMPV) is a leading cause of lower respiratory infection in young children, the elderly and immunocompromised patients. Repeated hMPV infections occur throughout life. However, immune evasion mechanisms of hMPV infection are largely unknown. Recently, our group has demonstrated that hMPV M2-2 protein, an important virulence factor, contributes to immune evasion in airway epithelial cells by targeting the mitochondrial antiviral-signaling protein (MAVS). Whether M2-2 regulates the innate immunity in human dendritic cells (DC), an important family of immune cells controlling antigen presenting, is currently unknown. We found that human DC infected with a virus lacking M2-2 protein expression (rhMPV-ΔM2-2) produced higher levels of cytokines, chemokines and IFNs, compared to cells infected with wild-type virus (rhMPV-WT), suggesting that M2-2 protein inhibits innate immunity in human DC. In parallel, we found that myeloid differentiation primary response gene 88 (MyD88), an essential adaptor for Toll-like receptors (TLRs), plays a critical role in inducing immune response of human DC, as downregulation of MyD88 by siRNA blocked the induction of immune regulatory molecules by hMPV. Since M2-2 is a cytoplasmic protein, we investigated whether M2-2 interferes with MyD88-mediated antiviral signaling. We found that indeed M2-2 protein associated with MyD88 and inhibited MyD88-dependent gene transcription. In this study, we also identified the domains of M2-2 responsible for its immune inhibitory function in human DC. In summary, our results demonstrate that M2-2 contributes to hMPV immune evasion by inhibiting MyD88-dependent cellular responses in human DC.
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Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Inmunidad Innata , Metapneumovirus/genética , Metapneumovirus/inmunología , Proteínas Virales/genética , Animales , Línea Celular , Quimiocinas/biosíntesis , Cricetinae , Citocinas/biosíntesis , Expresión Génica , Humanos , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/metabolismo , Unión Proteica , Transducción de Señal , Proteínas Virales/metabolismoRESUMEN
Acute respiratory tract infection (RTI) is a leading cause of morbidity and mortality worldwide and the majority of RTIs are caused by viruses, among which respiratory syncytial virus (RSV) and the closely related human metapneumovirus (hMPV) figure prominently. Host innate immune response has been implicated in recognition, protection and immune pathological mechanisms. Host-viral interactions are generally initiated via host recognition of pathogen-associated molecular patterns (PAMPs) of the virus. This recognition occurs through host pattern recognition receptors (PRRs) which are expressed on innate immune cells such as epithelial cells, dendritic cells, macrophages and neutrophils. Multiple PRR families, including Toll-like receptors (TLRs), RIG-I-like receptors (RLRs) and NOD-like receptors (NLRs), contribute significantly to viral detection, leading to induction of cytokines, chemokines and type I interferons (IFNs), which subsequently facilitate the eradication of the virus. This review focuses on the current literature on RSV and hMPV infection and the role of PRRs in establishing/mediating the infection in both in vitro and in vivo models. A better understanding of the complex interplay between these two viruses and host PRRs might lead to efficient prophylactic and therapeutic treatments, as well as the development of adequate vaccines.
RESUMEN
Human metapneumovirus (hMPV) is one of the main causes of acute respiratory tract infections in children, elderly and immunocompromised patients. The mammalian Toll-like receptors (TLR) were identified as critical regulators of innate immunity to a variety of microbes, including viruses. We have recently shown that hMPV-induced cytokine, chemokine and type I interferon secretion in dendritic cells occurs via TLR4, however, its role in hMPV-induced disease is unknown. In this study, wild-type(WT) and TLR4-deficient mice (TLR4â»/â») were infected with hMPV and examined for clinical disease parameters, such as body weight loss and airway obstruction, viral clearance, lung inflammation, dendritic cell maturation, T-cell proliferation and antibody production. Our results demonstrate that absence of TLR4 in hMPV-infected mice significantly reduced the inflammatory response as well as disease severity, shown by reduced body weight loss and airway obstruction and hyperresponsiveness (AHR), compared to WT mice. Levels of cytokines and chemokines were also significantly lower in the TLR4â»/â» mice. Accordingly, recruitment of inflammatory cells in the BAL, lungs, as well as in lymph nodes, was significantly reduced in the TLR4â»/â» mice, however, viral replication and clearance, as well as T-cell proliferation and neutralizing antibody production, were not affected. Our findings indicate that TLR4 is important for the activation of the innate immune response to hMPV, however it does play a role in disease pathogenesis, as lack of TLR4 expression is associated with reduced clinical manifestations of hMPV disease, without affecting viral protection.
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Obstrucción de las Vías Aéreas/etiología , Obstrucción de las Vías Aéreas/inmunología , Inmunidad Innata , Metapneumovirus/fisiología , Receptor Toll-Like 4/metabolismo , Obstrucción de las Vías Aéreas/metabolismo , Obstrucción de las Vías Aéreas/virología , Animales , Anticuerpos Neutralizantes/biosíntesis , Anticuerpos Neutralizantes/sangre , Peso Corporal , Lavado Broncoalveolar , Proliferación Celular , Células Dendríticas/citología , Células Dendríticas/inmunología , Femenino , Ratones , Linfocitos T/citología , Linfocitos T/inmunología , Replicación ViralRESUMEN
Human metapneumovirus (hMPV) is a common cause of lung and airway infections in infants and young children. Recently, we and others have shown that hMPV infection induces Toll-like receptor (TLR)-dependent cellular signaling. However, the contribution of TLR-mediated signaling in host defenses against pulmonary hMPV infection and associated disease pathogenesis has not been elucidated. In this study, mice deficient in MyD88, a common adaptor of TLRs, was used to investigate the contribution of TLRs to in vivo pulmonary response to hMPV infection. MyD88(-/-) mice have significantly reduced pulmonary inflammation and associated disease compared with wild-type (WT) C57BL/6 mice after intranasal infection with hMPV. hMPV-induced cytokines and chemokines in bronchoalveolar lavage fluid (BALF) and isolated lung conventional dendritic cells (cDC) are also significantly impaired by MyD88 deletion. In addition, we found that MyD88 is required for the recruitment of DC, T cells, and other immune cells to the lungs, and for the functional regulation of DC and T cells in response to hMPV infection. Taken together, our data indicate that MyD88-mediated pathways are essential for the pulmonary immune and pathogenic responses to this viral pathogen.
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Interacciones Huésped-Patógeno , Pulmón/patología , Metapneumovirus/fisiología , Factor 88 de Diferenciación Mieloide/metabolismo , Infecciones por Paramyxoviridae/patología , Animales , Líquido del Lavado Bronquioalveolar/química , Movimiento Celular , Citocinas/análisis , Células Dendríticas/inmunología , Modelos Animales de Enfermedad , Pulmón/inmunología , Pulmón/virología , Metapneumovirus/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/deficiencia , Infecciones por Paramyxoviridae/inmunología , Infecciones por Paramyxoviridae/virologíaRESUMEN
BACKGROUND: Respiratory syncytial virus (RSV) is the leading cause of respiratory infections in children, elderly, and immunocompromised individuals. Severe infection is associated with short- and long-term morbidity including pneumonia, recurrent wheezing, and abnormal pulmonary function, and several lines of evidence indicate that impaired adaptive immune responses during infection are critical in the pathophysiology of RSV-mediated disease. Myeloid Dendritic cells (mDCs) play a pivotal role in shaping antiviral immune responses in the respiratory tract; however, few studies have examined the interactions between RSV and individual mDC subsets. In this study, we examined the effect of RSV on the functional response of primary mDC subsets (BDCA-1(+) and BDCA-3(+)) isolated from peripheral blood. METHODS: BDCA-1(+) and BDCA-3(+) mDCs were isolated from the peripheral blood of healthy adults using FACS sorting. Donor-matched BDCA-1(+) and BDCA-3(+) mDCs were infected with RSV at a multiplicity of infection (MOI) of 5 for 40 hours. After infection, cells were analyzed for the expression of costimulatory molecules (CD86, CD80, and PD-L1), cytokine production, and the ability to stimulate allogenic CD4(+) T cell proliferation. RESULTS: Both BDCA-1(+) and BDCA-3(+) mDCs were susceptible to infection with RSV and demonstrated enhanced expression of CD86, and the inhibitory costimulatory molecules CD80 and PD-L1. Compared to BDCA-3(+) mDCs, RSV-infected BDCA-1(+) mDC produced a profile of cytokines and chemokines predominantly associated with pro-inflammatory responses (IL-1ß, IL-6, IL-12, MIP-1α, and TNF-α), and both BDCA-1(+) and BDCA-3(+) mDCs were found to produce IL-10. Compared to uninfected mDCs, RSV-infected BDCA-1(+) and BDCA-3(+) mDCs demonstrated a reduced capacity to stimulate T cell proliferation. CONCLUSIONS: RSV infection induces a distinct pattern of costimulatory molecule expression and cytokine production by BDCA-1(+) and BDCA-3(+) mDCs, and impairs their ability to stimulate T cell proliferation.The differential expression of CD86 and pro-inflammatory cytokines by highly purified mDC subsets in response to RSV provides further evidence that BDCA-1(+) and BDCA-3(+) mDCs have distinct roles in coordinating the host immune response during RSV infection. Findings of differential expression of PD-L1 and IL-10 by infected mDCs, suggests possible mechanisms by which RSV is able to impair adaptive immune responses.
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Antígenos de Superficie/inmunología , Linfocitos T CD4-Positivos/inmunología , Citocinas/inmunología , Células Dendríticas/inmunología , Infecciones por Virus Sincitial Respiratorio/inmunología , Infecciones por Virus Sincitial Respiratorio/patología , Adulto , Antígenos CD1 , Linfocitos T CD4-Positivos/patología , Proliferación Celular , Células Cultivadas , Células Dendríticas/patología , Femenino , Glicoproteínas , Humanos , Activación de Linfocitos/inmunología , Masculino , TrombomodulinaRESUMEN
Pancreatitis is classified into acute pancreatitis (AP) and chronic pancreatitis (CP). Apelin, a small regulatory peptide, is the endogenous ligand for the APJ receptor. Apelin and APJ are expressed in the pancreas. The aims of this study were to examine whether apelin influences the inflammatory and fibrosis responses to pancreatitis in mice and to identify mechanisms behind apelin's activities. Supramaximal cerulein induction of AP or CP caused significant (P < 0.05) elevations in pancreatic apelin and APJ expression. Levels declined during the recovery phases. In apelin gene-knockout mice with pancreatitis, pancreatic neutrophil invasion and myeloperoxidase activity were enhanced significantly, and apelin treatment suppressed both. Apelin exposure reduced CP-induced elevations of extracellular matrix-associated proteins. Apelin inhibited PDGF-simulated connective tissue growth factor production and proliferation of pancreatic stellate cells (PSCs). Serum granulocyte colony-stimulating factor and keratinocyte cytokine levels were higher in apelin gene-knockout than wild-type mice with pancreatitis. Apelin reduced AP- and CP-induced elevations in pancreatic NF-κB activation. Together, these findings imply that the pancreatic apelin-APJ system functions to curb the inflammatory and fibrosis responses during pancreatitis. Furthermore, findings suggest that apelin reduces inflammation and fibrosis by reducing neutrophil recruitment and PSC activity. Inhibition of neutrophil invasion may be mediated by reduced keratinocyte cytokine and granulocyte colony-stimulating factor secretion. Apelin-induced reductions in PSC proliferation and connective tissue growth factor production are putative mechanisms underlying apelin's inhibition of extracellular matrix production. The apelin-associated changes in NF-κB binding may be linked to apelin's regulation of pancreatic inflammatory and fibrosis responses during pancreatitis.
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Péptidos y Proteínas de Señalización Intercelular/metabolismo , Pancreatitis/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Adipoquinas , Animales , Apelina , Receptores de Apelina , Ceruletida/toxicidad , Quimiocinas , Regulación de la Expresión Génica/fisiología , Factor Estimulante de Colonias de Granulocitos/genética , Factor Estimulante de Colonias de Granulocitos/metabolismo , Péptidos y Proteínas de Señalización Intercelular/genética , Interleucina-3/genética , Interleucina-3/metabolismo , Ratones , Ratones Noqueados , Pancreatitis/inducido químicamente , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores Acoplados a Proteínas G/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Human metapneumovirus (hMPV) is a recently identified RNA virus belonging to the Paramyxoviridae family. It is a common cause of respiratory tract infections in children, adults, and immunocompromised patients, for which no specific treatment or vaccine is available. Recent investigations in our lab identified hMPV glycoprotein G as an important virulence factor, as a recombinant virus lacking the G protein (rhMPV-ΔG) exhibited enhanced production of important immune and antiviral mediators, such as cytokines, chemokines and type I interferon (IFN) in airway epithelial cells, and expression of G protein alone inhibits cellular signaling dependent on retinoic induced gene (RIG)-I, a RNA helicase with a fundamental role in initiating hMPV-induced cellular responses. In this study, we have further investigated the mechanism underlying the inhibitory role of hMPV G protein on RIG-I-dependent signaling. We found that the interaction of hMPV G with RIG-I occurs primarily through the CARD domains of RIG-I N-terminus, preventing RIG-I association with the adaptor protein MAVS (mitochondrial antiviral signaling protein), recruitment of RIG-I to mitochondria, as well as the interaction between mitochondria and mitochondria-associated membrane (MAM) component of the endoplasmic reticulum (ER), which contains STINGS, an important part of the viral-induced RIG-I/MAVS signaling pathway, leading in the end to the inhibition of cytokine, chemokine and type I IFN expression. Mutagenesis analysis showed that hMPV G protein cytoplasmic domain played a major role in the observed inhibitory activity, and recombinant viruses expressing a G protein with amino acid substitution in position 2 and 3 recapitulated most of the phenotype observed with rhMPV-ΔG mutant upon infection of airway epithelial cells.
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Glicoproteínas/metabolismo , Metapneumovirus/metabolismo , Mitocondrias/metabolismo , Mucosa Respiratoria/metabolismo , Transducción de Señal , Proteínas Virales/metabolismo , Línea Celular , Retículo Endoplásmico/metabolismo , Glicoproteínas/química , Glicoproteínas/genética , Humanos , Metapneumovirus/genética , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Receptores de Ácido Retinoico/metabolismo , Mucosa Respiratoria/virología , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
Respiratory syncytial virus (RSV) is one of the most important causes of upper and lower respiratory tract infections in infants and young children, for which no effective treatment is currently available. Although the mechanisms of RSV-induced airway disease remain incompletely defined, the lung inflammatory response is thought to play a central pathogenetic role. In the past few years, we and others have provided increasing evidence of a role of reactive oxygen species (ROS) as important regulators of RSV-induced cellular signaling leading to the expression of key proinflammatory mediators, such as cytokines and chemokines. In addition, RSV-induced oxidative stress, which results from an imbalance between ROS production and airway antioxidant defenses, due to a widespread inhibition of antioxidant enzyme expression, is likely to play a fundamental role in the pathogenesis of RSV-associated lung inflammatory disease, as demonstrated by a significant increase in markers of oxidative injury, which correlate with the severity of clinical illness, in children with RSV infection. Modulation of ROS production and oxidative stress therefore represents a potential novel pharmacological approach to ameliorate RSV-induced lung inflammation and its long-term consequences.
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Infecciones por Virus Sincitial Respiratorio/metabolismo , Infecciones por Virus Sincitial Respiratorio/terapia , Humanos , Oxidación-Reducción , Especies de Nitrógeno Reactivo/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de SeñalRESUMEN
Human metapneumovirus (hMPV) is a recently identified RNA virus belonging to the Paramyxoviridae family, which includes several major human and animal pathogens. Epidemiological studies indicate that hMPV is a significant human respiratory pathogen with worldwide distribution. It is associated with respiratory illnesses in children, adults, and immunocompromised patients, ranging from upper respiratory tract infections to severe bronchiolitis and pneumonia. Interferon (IFN) represents a major line of defense against virus infection, and in response, viruses have evolved countermeasures to inhibit IFN production as well as IFN signaling. Although the strategies of IFN evasion are similar, the specific mechanisms by which paramyxoviruses inhibit IFN responses are quite diverse. In this review, we will present an overview of the strategies that hMPV uses to subvert cellular signaling in airway epithelial cells, the major target of infection, as well as in primary immune cells.
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Evasión Inmune , Inmunidad Innata , Metapneumovirus/inmunología , Metapneumovirus/patogenicidad , Humanos , Interferones/antagonistas & inhibidores , Proteínas Virales/metabolismo , Factores de Virulencia/metabolismoRESUMEN
Human metapneumovirus (hMPV) is a leading cause of respiratory infections in pediatric populations globally, with no prophylactic or therapeutic measures. Recently, a recombinant hMPV lacking the M2-2 protein (rhMPV-ΔM2-2) demonstrated reduced replication in the respiratory tract of animal models, making it a promising live vaccine candidate. However, the exact nature of the interaction between the M2-2 protein and host cells that regulates viral infection/propagation is largely unknown. By taking advantage of the available reverse genetics system and ectopic expression system for viral protein, we found that M2-2 not only promotes viral gene transcription and replication but subverts host innate immunity, therefore identifying M2-2 as a novel virulence factor, in addition to the previously described hMPV G protein. Since we have shown that the RIG-I/MAVS pathway plays an important role in hMPV-induced signaling in airway epithelial cells, we investigated whether M2-2 antagonizes the host cellular responses by targeting this pathway. Reporter gene assays and coimmunoprecipitation studies indicated that M2-2 targets MAVS, an inhibitory mechanism different from what we previously reported for hMPV G, which affects RIG-I- but not MAVS-dependent gene transcription. In addition, we found that the domains of M2-2 responsible for the regulation of viral gene transcription and antiviral signaling are different. Our findings collectively demonstrate that M2-2 contributes to hMPV immune evasion through the inhibition of MAVS-dependent cellular responses.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Evasión Inmune/genética , Metapneumovirus/metabolismo , Transducción de Señal/fisiología , Transcripción Genética/fisiología , Proteínas Virales/metabolismo , Factores de Virulencia/metabolismo , Análisis de Varianza , Animales , Western Blotting , Chlorocebus aethiops , Cartilla de ADN/genética , Humanos , Inmunoprecipitación , Metapneumovirus/genética , Metapneumovirus/inmunología , Plásmidos/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética/genética , Células Vero , Proteínas Virales/genética , Factores de Virulencia/genéticaRESUMEN
Chemokines exert their function by binding the GPCR class of receptors on leukocytes and cell surface GAGs in target tissues. Most chemokines reversibly exist as monomers and dimers, but very little is known regarding the molecular mechanisms by which the monomer-dimer equilibrium modulates in vivo function. For the chemokine CXCL8, we recently showed in a mouse lung model that monomers and dimers are active and that the monomer-dimer equilibrium of the WT plays a crucial role in regulating neutrophil recruitment. In this study, we show that monomers and dimers are also active in the mouse peritoneum but that the role of monomer-dimer equilibrium is distinctly different between these tissues and that mutations in GAG-binding residues render CXCL8 less active in the peritoneum but more active in the lung. We propose that tissue-specific differences in chemokine gradient formation, resulting from tissue-specific differences in GAG interactions, are responsible for the observed differences in neutrophil recruitment. Our observation of differential roles played by the CXCL8 monomer-dimer equilibrium and GAG interactions in different tissues is novel and reveals an additional level of complexity of how chemokine dimerization regulates in vivo recruitment.
Asunto(s)
Quimiotaxis de Leucocito/fisiología , Glicosaminoglicanos/metabolismo , Interleucina-8/química , Pulmón/inmunología , Neutrófilos/fisiología , Peritoneo/inmunología , Sustitución de Aminoácidos , Animales , Quimiotaxis de Leucocito/efectos de los fármacos , Dimerización , Femenino , Humanos , Interleucina-8/farmacología , Interleucina-8/fisiología , Pulmón/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Especificidad de Órganos , Peritoneo/efectos de los fármacos , Unión Proteica , Conformación Proteica , Proteínas Recombinantes de Fusión/farmacología , Organismos Libres de Patógenos EspecíficosRESUMEN
Human metapneumovirus (hMPV), a leading cause of respiratory tract infections in infants, inhibits type I interferon (IFN) signaling by an unidentified mechanism. In this study, we showed that infection of airway epithelial cells with hMPV decreased cellular level of Janus tyrosine kinase (Jak1) and tyrosine kinase 2 (Tyk2), due to enhanced proteosomal degradation and reduced gene transcription. In addition, hMPV infection also reduced the surface expression of type I IFN receptor (IFNAR). These inhibitory mechanisms are different from the ones employed by respiratory syncytial virus (RSV), which does not affect Jak1, Tyk2 or IFNAR expression, but degrades downstream signal transducer and activator of transcription proteins 2 (STAT2), although both viruses are pneumoviruses belonging to the Paramyxoviridae family. Our study identifies a novel mechanism by which hMPV inhibits STAT1 and 2 activation, ultimately leading to viral evasion of host IFN responses.
Asunto(s)
Regulación hacia Abajo , Células Epiteliales/virología , Interferón beta/metabolismo , Janus Quinasa 1/metabolismo , Metapneumovirus/fisiología , Transducción de Señal , TYK2 Quinasa/metabolismo , Células Epiteliales/citología , Células Epiteliales/enzimología , Células Epiteliales/metabolismo , Humanos , Interferón beta/farmacología , Complejo de la Endopetidasa Proteasomal/metabolismo , Subunidades de Proteína/metabolismo , Proteolisis , Receptor de Interferón alfa y beta/metabolismo , Factor de Transcripción STAT2/metabolismo , Transcripción Genética , Replicación ViralRESUMEN
Human metapneumovirus (hMPV) is a major cause of upper and lower respiratory infections in children and adults. Recent work from our group demonstrated that hMPV G glycoprotein is an important virulence factor, responsible for inhibiting innate immune responses in airway epithelial cells. Myeloid dendritic cells (DCs) are potent APCs and play a major role in initiating and modulating the innate and adaptive immune responses. In this study, we found that TLR4 plays a major role in hMPV-induced activation of monocyte-derived DCs (moDCs), as downregulation of its expression by small interfering RNA significantly blocked hMPV-induced chemokine and type I IFN expression. Similar results were found in bone marrow-derived DCs from TLR4-deficient mice. moDCs infected with a virus lacking G protein expression produced higher levels of cytokines and chemokines compared with cells infected with wild-type virus, suggesting that G protein plays an inhibitory role in viral-induced cellular responses. Specifically, G protein affects TLR4-dependent signaling, as infection of moDCs with recombinant hMPV lacking G protein inhibited LPS-induced production of cytokine and chemokines significantly less than did wild-type virus, and treatment of moDCs with purified G protein resulted in a similar inhibition of LPS-dependent signaling. Our results demonstrate that hMPV G protein plays an important role in inhibiting host innate immune responses, likely affecting adaptive responses too.
Asunto(s)
Células Dendríticas/inmunología , Células Dendríticas/virología , Glicoproteínas/fisiología , Mediadores de Inflamación/fisiología , Metapneumovirus/inmunología , Transducción de Señal/inmunología , Receptor Toll-Like 4/antagonistas & inhibidores , Receptor Toll-Like 4/fisiología , Proteínas Virales/fisiología , Inmunidad Adaptativa , Adulto , Animales , Línea Celular , Células Cultivadas , Quimiocinas/metabolismo , Citocinas/metabolismo , Células Dendríticas/metabolismo , Humanos , Inmunidad Innata , Mediadores de Inflamación/metabolismo , Macaca mulatta , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Monocitos/inmunología , Monocitos/metabolismo , Monocitos/virología , Receptor Toll-Like 4/deficienciaRESUMEN
Lower respiratory tract infections caused by the paramyxoviruses human metapneumovirus (hMPV) and respiratory syncytial virus (RSV) are characterized by short-lasting virus-specific immunity and often long-term airway morbidity, both of which may be the result of alterations in the Ag-presenting function of the lung which follow these infections. In this study, we investigated whether hMPV and RSV experimental infections alter the phenotype and function of dendritic cell (DC) subsets that are recruited to the lung. Characterization of lung DC trafficking demonstrated a differential recruitment of plasmacytoid DC (pDC), conventional DC (cDC), and IFN-producing killer DC to the lung and draining lymph nodes after hMPV and RSV infection. In vitro infection of lung DC indicated that in pDC, production of IFN-alpha, TNF-alpha, and CCL5 was induced only by hMPV, whereas CCL3 and CCL4 were induced by both viruses. In cDC, a similar repertoire of cytokines was induced by hMPV and RSV, except for IFN-beta, which was not induced by RSV. The function of lung pDC was altered following hMPV or RSV infection in vivo, as we demonstrated a reduced capacity of lung pDC to produce IFN-alpha as well as other cytokines including IL-6, TNF-alpha, CCL2, CCL3, and CCL4 in response to TLR9 stimulation. Moreover, we observed an impaired capacity of cDC from infected mice to present Ag to CD4(+) T cells, an effect that lasted beyond the acute phase of infection. Our findings suggest that acute paramyxovirus infections can alter the long-term immune function of pulmonary DC.