RESUMO
Although interferon-λ [also known as type III interferon or interleukin-28 (IL-28)/IL-29] restricts infection by several viruses, its inhibitory mechanism has remained uncertain. We used recombinant interferon-λ and mice lacking the interferon-λ receptor (IFNLR1) to evaluate the effect of interferon-λ on infection with West Nile virus, an encephalitic flavivirus. Cell culture studies in mouse keratinocytes and dendritic cells showed no direct antiviral effect of exogenous interferon-λ, even though expression of interferon-stimulated genes was induced. We observed no differences in West Nile virus burden between wild-type and Ifnlr1(-/-) mice in the draining lymph nodes, spleen, or blood. We detected increased West Nile virus infection in the brain and spinal cord of Ifnlr1(-/-) mice, yet this was not associated with a direct antiviral effect in mouse neurons. Instead, we observed an increase in blood-brain barrier permeability in Ifnlr1(-/-) mice. Treatment of mice with pegylated interferon-λ2 resulted in decreased blood-brain barrier permeability, reduced West Nile virus infection in the brain without affecting viremia, and improved survival against lethal virus challenge. An in vitro model of the blood-brain barrier showed that interferon-λ signaling in mouse brain microvascular endothelial cells increased transendothelial electrical resistance, decreased virus movement across the barrier, and modulated tight junction protein localization in a protein synthesis- and signal transducer and activator of transcription 1 (STAT1)-independent manner. Our data establish an indirect antiviral function of interferon-λ in which noncanonical signaling through IFNLR1 tightens the blood-brain barrier and restricts viral neuroinvasion and pathogenesis.
Assuntos
Barreira Hematoencefálica , Interferons/farmacologia , Neurônios/virologia , Receptores de Citocinas/metabolismo , Receptores de Interferon/metabolismo , Febre do Nilo Ocidental/imunologia , Imunidade Adaptativa , Animais , Antivirais/química , Encéfalo/metabolismo , Linhagem Celular , Impedância Elétrica , Regulação da Expressão Gênica , Humanos , Imunidade Celular , Queratinócitos/citologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microcirculação , Microscopia Confocal , Neurônios/metabolismo , Permeabilidade , Proteínas Recombinantes/farmacologia , Transdução de Sinais , Replicação Viral , Febre do Nilo Ocidental/patologia , Vírus do Nilo OcidentalRESUMO
Antiviral responses must be tightly regulated to defend rapidly against infection while minimizing inflammatory damage. Type 1 interferons (IFN-I) are crucial mediators of antiviral responses and their transcription is regulated by a variety of transcription factors; principal among these is the family of interferon regulatory factors (IRFs). The IRF gene regulatory networks are complex and contain multiple feedback loops. The tools of systems biology are well suited to elucidate the complex interactions that give rise to precise coordination of the interferon response. Here we have used an unbiased systems approach to predict that a member of the forkhead family of transcription factors, FOXO3, is a negative regulator of a subset of antiviral genes. This prediction was validated using macrophages isolated from Foxo3-null mice. Genome-wide location analysis combined with gene deletion studies identified the Irf7 gene as a critical target of FOXO3. FOXO3 was identified as a negative regulator of Irf7 transcription and we have further demonstrated that FOXO3, IRF7 and IFN-I form a coherent feed-forward regulatory circuit. Our data suggest that the FOXO3-IRF7 regulatory circuit represents a novel mechanism for establishing the requisite set points in the interferon pathway that balances the beneficial effects and deleterious sequelae of the antiviral response.
Assuntos
Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica/imunologia , Inflamação/imunologia , Inflamação/patologia , Fator Regulador 7 de Interferon/metabolismo , Vesiculovirus/imunologia , Animais , Feminino , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/deficiência , Fatores de Transcrição Forkhead/genética , Deleção de Genes , Inflamação/genética , Fator Regulador 7 de Interferon/deficiência , Fator Regulador 7 de Interferon/genética , Interferon Tipo I/imunologia , Pulmão/imunologia , Pulmão/patologia , Pulmão/virologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Reprodutibilidade dos TestesRESUMO
The T-box transcription factor T-bet is important for the differentiation of naive CD4(+) T helper cells (Th cells) into the Th1 phenotype. Much is known about T-bet's role as a transcriptional activator, but less is known about the mechanisms by which T-bet functionally represses alternative Th cell genetic programs. In this study, we first identify Socs1, Socs3, and Tcf7 (TCF-1) as gene targets that are negatively regulated by T-bet. Significantly, T-bet's role in the repression of these genes is through a direct interaction with their promoters. Consistent with this, we identified two T-bet DNA-binding elements in the Socs1 promoter that are functionally used to down-regulate transcription in primary Th1 cells. Importantly, T-bet's novel role in transcriptional repression is because of its ability to physically associate with, and functionally recruit, the transcriptional repressor Bcl-6 to a subset of promoters. Furthermore, T-bet functionally recruits Bcl-6 to the Ifng locus in late stages of Th1 differentiation to repress its activity, possibly to prevent the overproduction of IFN-γ, which could result in autoimmunity. Collectively, these data establish a novel mechanism for T-bet-mediated gene repression in which two lineage-defining transcription factors, one a classical activator and one a repressor, collaborate to promote and properly regulate Th1 development.
Assuntos
Regulação para Baixo/imunologia , Proteínas Proto-Oncogênicas c-bcl-6/imunologia , Proteínas Repressoras/imunologia , Elementos de Resposta/imunologia , Proteínas com Domínio T/imunologia , Células Th1/imunologia , Animais , Autoimunidade/fisiologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Linhagem Celular , Regulação para Baixo/genética , Loci Gênicos/genética , Loci Gênicos/imunologia , Fator 1-alfa Nuclear de Hepatócito , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas c-bcl-6/genética , Proteínas Proto-Oncogênicas c-bcl-6/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Elementos de Resposta/genética , Proteína 1 Supressora da Sinalização de Citocina , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina/genética , Proteínas Supressoras da Sinalização de Citocina/imunologia , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Fator 1 de Transcrição de Linfócitos T/genética , Fator 1 de Transcrição de Linfócitos T/imunologia , Fator 1 de Transcrição de Linfócitos T/metabolismo , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Células Th1/citologia , Células Th1/metabolismo , Transcrição Gênica/genética , Transcrição Gênica/imunologiaRESUMO
BACKGROUND: The process of cellular differentiation is governed by complex dynamical biomolecular networks consisting of a multitude of genes and their products acting in concert to determine a particular cell fate. Thus, a systems level view is necessary for understanding how a cell coordinates this process and for developing effective therapeutic strategies to treat diseases, such as cancer, in which differentiation plays a significant role. Theoretical considerations and recent experimental evidence support the view that cell fates are high dimensional attractor states of the underlying molecular networks. The temporal behavior of the network states progressing toward different cell fate attractors has the potential to elucidate the underlying molecular mechanisms governing differentiation. RESULTS: Using the HL60 multipotent promyelocytic leukemia cell line, we performed experiments that ultimately led to two different cell fate attractors by two treatments of varying dosage and duration of the differentiation agent all-trans-retinoic acid (ATRA). The dosage and duration combinations of the two treatments were chosen by means of flow cytometric measurements of CD11b, a well-known early differentiation marker, such that they generated two intermediate populations that were poised at the apparently same stage of differentiation. However, the population of one treatment proceeded toward the terminally differentiated neutrophil attractor while that of the other treatment reverted back toward the undifferentiated promyelocytic attractor. We monitored the gene expression changes in the two populations after their respective treatments over a period of five days and identified a set of genes that diverged in their expression, a subset of which promotes neutrophil differentiation while the other represses cell cycle progression. By employing promoter based transcription factor binding site analysis, we found enrichment in the set of divergent genes, of transcription factors functionally linked to tumor progression, cell cycle, and development. CONCLUSION: Since many of the transcription factors identified by this approach are also known to be implicated in hematopoietic differentiation and leukemia, this study points to the utility of incorporating a dynamical systems level view into a computational analysis framework for elucidating transcriptional mechanisms regulating differentiation.