RESUMO
Type III interferons (IFN-lambda) are the most recently discovered members of IFN family. Synergism between different IFN types is well established, but for type I and type III IFNs no conclusive evidence has been reported so far. Possible synergism/antagonism between IFN-alpha and IFN-lambda in the inhibition of virus replication (EMCV, WNV lineage 1 and 2, CHIKV and HSV-1), and in the activation of intracellular pathways of IFN response (MxA and 2'-5' OAS) was evaluated in different cell lines (Vero E6, A549 and Wish cells). The antiviral potency of IFN-lambda1 and -l2 was lower than that of IFN-alpha. When IFN-alpha and -lambda were used together, the Combination Index (CI) for virus inhibition was greater than 1 virtually for all virus/host cell systems, indicating antagonistic effect. Antagonism between IFN-alpha and -l was also observed for the induction of mRNA for both MxA and 2'-5'OAS. Elucidating the interplay between IFN-alpha and -lambda may help to better understand innate defence mechanisms against viral infections, including the molecular mechanisms underlying the influence of IL-28B polymorphisms in the response to HCV and other viral infections.
Assuntos
2',5'-Oligoadenilato Sintetase/genética , Antivirais/antagonistas & inibidores , Interferon-alfa/antagonistas & inibidores , Interferons/farmacologia , Interleucinas/farmacologia , Animais , Chlorocebus aethiops , Vírus da Encefalomiocardite/efeitos dos fármacos , Humanos , Proteínas de Resistência a Myxovirus/genética , Células Vero , Replicação Viral/efeitos dos fármacosRESUMO
GB virus C (GBV-C) coinfection has a protective role in Human Immunodeficiency Virus (HIV) infection, and increases the duration of suppression of HIV-1 viremia in patients under Highly Active Anti-Retroviral Therapy (HAART). Since innate antiviral response may be involved in the protection, we analyzed the possible role of GBV-C as activator of innate immunity. To this aim, we measured the extent of activation of the interferon (IFN) system and of circulating Dendritic Cells (DC) in vivo, and the ability of GBV-C to activate these functions in vitro. Activation of IFN system and of circulating DC was compared in GBV-positive and -negative HIV-1 co-infected patients with HAART-driven suppression of HIV-1 viremia. Endogenous levels of IFN-gamma and RNA-dependent protein kinase (PKR) mRNA were significantly higher in peripheral blood mononuclear cells (PBMC) from GBV-C-positive when compared to GBV-C-negative patients. IFN-gamma expression was correlated with all the Interferon response genes (IRGs) and with GBV-C viremia. The frequency of circulating plasmacytoid DC (pDC) expressing the CD80 activation marker was increased in GBV-C-positive patients, and was correlated with GBV-C viral load. In vitro experiments indicated that GBV-C is able to induce IFN-gamma expression in PBMC. In addition, in PBMC cultures GBV-C induced an increase of CD80 expression by pDC, that was reduced by antibody to IFN-gamma. Our data indicate that in HIV-positive patients GBV-C coinfection promotes the activation of IFN-gamma and downstream IRG expression, as well as with the activation/maturation of circulating pDC. GBV-C-driven IFN-gamma activation is, at least in part, responsible for the increased maturation of pDC. This crosstalk may suggest a role for GBV-C coinfection in boosting the innate antiviral response to HIV infection.
Assuntos
Síndrome da Imunodeficiência Adquirida/imunologia , Células Dendríticas/fisiologia , Infecções por Flaviviridae/imunologia , Vírus GB C , Regulação da Expressão Gênica , HIV-1 , Hepatite Viral Humana/imunologia , Interferon gama/genética , Proteínas de Ligação ao GTP/genética , Humanos , Imunidade Inata , Interferon-alfa/biossíntese , Interferon gama/biossíntese , Proteínas de Resistência a Myxovirus , RNA Mensageiro/análise , Receptor de Interferon alfa e beta/genética , eIF-2 Quinase/genéticaRESUMO
The most reliable predictor of treatment efficacy in hepatitis C is HCV viremia decay at week 12 [early virological response (EVR)]. We investigated whether the ability of peripheral blood mononuclear cells (PBMC) to mount an interferon (IFN) response in vitro could be predictive of EVR. Fifteen patients treated with PEG IFNalpha + RBV, with pre-therapy frozen PBMC, were retrospectively selected. After a 3 hr PBMC exposure to IFNalpha in vitro, up-regulation of mRNA for IFN-stimulated genes (ISG) was measured by membrane super-array. ISG mRNA levels in unstimulated PBMC were low, but beta2M and CASP1 were significantly higher in EVR vs non-EVR. ISG mRNA up-regulation by IFN was more pronounced in EVR vs non-EVR. For 7 genes (IP-10, IFIT1, IFIT2, IFIT3, TRAIL, KIAA1628 and OAS2) cut-off levels were established, by ROC analysis, able to correctly classify all EVR and non-EVR. Early virological response to PEG IFNalpha +RBV is correlated with the pre-therapy ability of PBMC to activate an IFN response in vitro. If validated in a wider cohort of patients, the ability of this set of ISG to discriminate between EVR and non-EVR may be useful for pre-therapy evaluation, particularly in patients with unfavourable combinations of conventional response predictors.
Assuntos
Hepatite C/imunologia , Hepatite C/virologia , Interferon-alfa/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Adulto , Idoso , Proteínas Reguladoras de Apoptose , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Hepatite C/genética , Humanos , Leucócitos Mononucleares/metabolismo , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas/genética , Proteínas/metabolismo , Proteínas de Ligação a RNA , Fatores de TempoRESUMO
Hepatitis C virus (HCV) persistence results from inefficiencies of both innate and adaptive immune responses to eradicate the infection. A functional impairment of circulating Vγ9Vδ2 T-cells was described but few data are available on Vγ9Vδ2 T-cells in the liver that, however, represents the battlefield in the HCV/host interaction. Aim of this work was to compare circulating and intrahepatic Vγ9Vδ2 T-cells in chronic HCV-infected patients (HCVpos) and in HCV-negative (HCVneg) subjects. Phenotypic and functional analysis was performed by flow cytometry. Anti-HCV activity was analyzed by using an in vitro autologous liver culture system. Independently from HCV infection, the liver was enriched of Vγ9Vδ2 T-cells expressing an effector/activated phenotype. In contrast, an enrichment of PD-1 expressing Vγ9Vδ2 T-cells was observed both in the peripheral blood and in the liver of HCVpos patients, probably due to a persistent antigenic stimulation. Moreover, a lower frequency of IFN-γ producing Vγ9Vδ2 T-cells was observed in the liver of HCVpos patients, suggesting a functional impairment in the cytokine production in HCVpos liver. Despite this hypo-responsiveness, intrahepatic Vγ9Vδ2 T-cells are able to exert an anti-HCV activity after specific stimulation. Altogether, our data show that HCV infection induced a dysregulation of intrahepatic Vγ9Vδ2 T cells that maintain their anti-HCV activity after specific stimulation. A study aimed to evaluate the mechanisms of the antiviral activity may be useful to identify new pathways able to improve Vγ9Vδ2 T-cells intrahepatic function during HCV infection.
Assuntos
Hepacivirus/fisiologia , Hepatite C/virologia , Fígado/imunologia , Linfócitos T/imunologia , Replicação Viral , Adulto , Idoso , Feminino , Hepacivirus/genética , Hepacivirus/isolamento & purificação , Hepatite C/imunologia , Humanos , Interferon gama/genética , Interferon gama/imunologia , Fígado/virologia , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
BACKGROUND: GB virus C (GBV-C) co-infection is associated with a better prognosis in HIV-infected persons. Since interferon activation can be one of the possible mechanisms involved in GBV-C-driven protection against HIV, we compared the endogenous activation of the interferon system in PBMC from GBV-C-positive and -negative patients infected with HIV-1. METHODS: The expression of interferon related genes was analyzed in 20 GBV-C positive and 20 GBV-C-negative HIV-infected patients, comparable in terms of CD4 cell counts and HIV viral loads. The levels of mRNA for interferon-related genes (2-5-OAS, MxA, interferon AR-1 and PKR) in PBMC were measured by real time RT-PCR, using B-actin as internal control. RESULTS: The endogenous levels of all the Interferon-related genes in HIV/GBV-C co-infected patients were higher than in HIV mono-infected subjects. The difference was statistically significant for PKR mRNA. Direct positive correlation was found between PKR and all the other interferon-related genes, suggesting a coordinated activation of the interferon system. CONCLUSIONS: Enhanced activation of the interferon system occurs in GBV-C-positive, as compared to GBV-C-negative patients harbouring HIV-1. These data may be relevant to understand the GBV-C-driven protection against HIV, suggesting that the endogenous activation of the interferon system can contribute to the control of HIV replication.
Assuntos
Infecções por Flaviviridae/complicações , Vírus GB C , Infecções por HIV/complicações , HIV-1 , Hepatite Viral Humana/complicações , Interferons/metabolismo , 2',5'-Oligoadenilato Sintetase/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Células Sanguíneas/metabolismo , Células Sanguíneas/virologia , Feminino , Infecções por Flaviviridae/imunologia , Proteínas de Ligação ao GTP/metabolismo , Infecções por HIV/imunologia , Hepatite Viral Humana/imunologia , Humanos , Masculino , Proteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Proteínas de Resistência a Myxovirus , RNA Mensageiro/metabolismo , Receptor de Interferon alfa e beta , Receptores de Interferon/metabolismo , eIF-2 Quinase/metabolismoRESUMO
Data on immune responses during human Ebola virus disease (EVD) are scanty, due to limitations imposed by biosafety requirements and logistics. A sustained activation of T-cells was recently described but functional studies during the acute phase of human EVD are still missing. Aim of this work was to evaluate the kinetics and functionality of T-cell subsets, as well as the expression of activation, autophagy, apoptosis and exhaustion markers during the acute phase of EVD until recovery. Two EVD patients admitted to the Italian National Institute for Infectious Diseases, Lazzaro Spallanzani, were sampled sequentially from soon after symptom onset until recovery and analyzed by flow cytometry and ELISpot assay. An early and sustained decrease of CD4 T-cells was seen in both patients, with an inversion of the CD4/CD8 ratio that was reverted during the recovery period. In parallel with the CD4 T-cell depletion, a massive T-cell activation occurred and was associated with autophagic/apoptotic phenotype, enhanced expression of the exhaustion marker PD-1 and impaired IFN-gamma production. The immunological impairment was accompanied by EBV reactivation. The association of an early and sustained dysfunctional T-cell activation in parallel to an overall CD4 T-cell decline may represent a previously unknown critical point of Ebola virus (EBOV)-induced immune subversion. The recent observation of late occurrence of EBOV-associated neurological disease highlights the importance to monitor the immuno-competence recovery at discharge as a tool to evaluate the risk of late sequelae associated with resumption of EBOV replication. Further studies are required to define the molecular mechanisms of EVD-driven activation/exhaustion and depletion of T-cells.