RESUMEN
The RIG-I-like receptors (RLRs) signal innate immune defenses upon RNA virus infection, but their roles in adaptive immunity have not been clearly defined. Here, we showed that the RLR LGP2 was not essential for induction of innate immune defenses, but rather was required for controlling antigen-specific CD8(+) T cell survival and fitness during peripheral T cell-number expansion in response to virus infection. Adoptive transfer and biochemical studies demonstrated that T cell-receptor signaling induced LGP2 expression wherein LGP2 operated to regulate death-receptor signaling and imparted sensitivity to CD95-mediated cell death. Thus, LGP2 promotes an essential prosurvival signal in response to antigen stimulation to confer CD8(+) T cell-number expansion and effector functions against divergent RNA viruses, including West Nile virus and lymphocytic choriomeningitis virus.
Asunto(s)
Inmunidad Adaptativa/inmunología , Linfocitos T CD8-positivos/inmunología , Supervivencia Celular/inmunología , ARN Helicasas/inmunología , ARN Viral/inmunología , Animales , Linfocitos T CD8-positivos/metabolismo , Comunicación Celular/inmunología , Sistema Nervioso Central/inmunología , Células Dendríticas/inmunología , Humanos , Inmunidad Innata/inmunología , Interferón beta/inmunología , Coriomeningitis Linfocítica/inmunología , Virus de la Coriomeningitis Linfocítica/inmunología , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de Señal/inmunología , Fiebre del Nilo Occidental/inmunología , Virus del Nilo Occidental/inmunología , Receptor fas/inmunologíaRESUMEN
BACKGROUND & AIMS: GS-9620, an oral agonist of toll-like receptor 7 (TLR7), is in clinical development for the treatment of chronic hepatitis B (CHB). GS-9620 was previously shown to induce prolonged suppression of serum viral DNA and antigens in the woodchuck and chimpanzee models of CHB. Herein, we investigated the molecular mechanisms that contribute to the antiviral response to GS-9620 using in vitro models of hepatitis B virus (HBV) infection. METHODS: Cryopreserved primary human hepatocytes (PHH) and differentiated HepaRG (dHepaRG) cells were infected with HBV and treated with GS-9620, conditioned media from human peripheral blood mononuclear cells treated with GS-9620 (GS-9620 conditioned media [GS-9620-CM]), or other innate immune stimuli. The antiviral and transcriptional response to these agents was determined. RESULTS: GS-9620 had no antiviral activity in HBV-infected PHH, consistent with low level TLR7 mRNA expression in human hepatocytes. In contrast, GS-9620-CM induced prolonged reduction of HBV DNA, RNA, and antigen levels in PHH and dHepaRG cells via a type I interferon (IFN)-dependent mechanism. GS-9620-CM did not reduce covalently closed circular DNA (cccDNA) levels in either cell type. Transcriptional profiling demonstrated that GS-9620-CM strongly induced various HBV restriction factors - although not APOBEC3A or the Smc5/6 complex - and indicated that established HBV infection does not modulate innate immune sensing or signaling in cryopreserved PHH. GS-9620-CM also induced expression of immunoproteasome subunits and enhanced presentation of an immunodominant viral peptide in HBV-infected PHH. CONCLUSIONS: Type I IFN induced by GS-9620 durably suppressed HBV in human hepatocytes without reducing cccDNA levels. Moreover, HBV antigen presentation was enhanced, suggesting additional components of the TLR7-induced immune response played a role in the antiviral response to GS-9620 in animal models of CHB. LAY SUMMARY: GS-9620 is a drug currently being tested in clinical trials for the treatment of chronic hepatitis B virus (HBV) infection. GS-9620 has previously been shown to suppress HBV in various animal models, but the underlying antiviral mechanisms were not completely understood. In this study, we determined that GS-9620 does not directly activate antiviral pathways in human liver cells, but can induce prolonged suppression of HBV via induction of an antiviral cytokine called interferon. However, interferon did not destroy the HBV genome, suggesting that other parts of the immune response (e.g. activation of immune cells that kill infected cells) also play an important role in the antiviral response to GS-9620.
Asunto(s)
Antivirales/farmacología , Virus de la Hepatitis B/efectos de los fármacos , Interferón Tipo I/inmunología , Pteridinas/farmacología , Receptor Toll-Like 7/agonistas , Animales , Presentación de Antígeno , Células Cultivadas , Citocinas/biosíntesis , ADN Circular/genética , ADN Circular/metabolismo , ADN Viral/genética , ADN Viral/metabolismo , Antígenos de la Hepatitis B/metabolismo , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/inmunología , Hepatitis B Crónica/tratamiento farmacológico , Hepatitis B Crónica/inmunología , Hepatitis B Crónica/virología , Hepatocitos/efectos de los fármacos , Hepatocitos/inmunología , Hepatocitos/virología , Humanos , Inmunidad Innata , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , Receptor Toll-Like 7/genéticaRESUMEN
The type I interferon (IFN) signaling response limits infection of many RNA and DNA viruses. To define key cell types that require type I IFN signaling to orchestrate immunity against West Nile virus (WNV), we infected mice with conditional deletions of the type I IFN receptor (IFNAR) gene. Deletion of the Ifnar gene in subsets of myeloid cells resulted in uncontrolled WNV replication, vasoactive cytokine production, sepsis, organ damage, and death that were remarkably similar to infection of Ifnar-/- mice completely lacking type I IFN signaling. In Mavs-/-×Ifnar-/- myeloid cells and mice lacking both Ifnar and the RIG-I-like receptor adaptor gene Mavs, cytokine production was muted despite high levels of WNV infection. Thus, in myeloid cells, viral infection triggers signaling through MAVS to induce proinflammatory cytokines that can result in sepsis and organ damage. Viral pathogenesis was caused in part by massive complement activation, as liver damage was minimized in animals lacking complement components C3 or factor B or treated with neutralizing anti-C5 antibodies. Disease in Ifnar-/- and CD11c Cre+Ifnarf/f mice also was facilitated by the proinflammatory cytokine TNF-α, as blocking antibodies diminished complement activation and prolonged survival without altering viral burden. Collectively, our findings establish the dominant role of type I IFN signaling in myeloid cells in restricting virus infection and controlling pathological inflammation and tissue injury.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Receptor de Interferón alfa y beta/metabolismo , Sepsis/metabolismo , Transducción de Señal , Fiebre del Nilo Occidental/metabolismo , Virus del Nilo Occidental/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Complemento C3/genética , Complemento C3/metabolismo , Factor B del Complemento/genética , Factor B del Complemento/metabolismo , Interferón Tipo I/genética , Interferón Tipo I/metabolismo , Ratones , Ratones Noqueados , Receptor de Interferón alfa y beta/genética , Sepsis/genética , Sepsis/patología , Sepsis/virología , Fiebre del Nilo Occidental/genética , Fiebre del Nilo Occidental/patologíaRESUMEN
The actions of the RIG-I like receptor (RLR) and type I interferon (IFN) signaling pathways are essential for a protective innate immune response against the emerging flavivirus West Nile virus (WNV). In mice lacking RLR or IFN signaling pathways, WNV exhibits enhanced tissue tropism, indicating that specific host factors of innate immune defense restrict WNV infection and dissemination in peripheral tissues. However, the immune mechanisms by which the RLR and IFN pathways coordinate and function to impart restriction of WNV infection are not well defined. Using a systems biology approach, we defined the host innate immune response signature and actions that restrict WNV tissue tropism. Transcriptional profiling and pathway modeling to compare WNV-infected permissive (spleen) and nonpermissive (liver) tissues showed high enrichment for inflammatory responses, including pattern recognition receptors and IFN signaling pathways, that define restriction of WNV replication in the liver. Assessment of infected livers from Mavs(-/-) × Ifnar(-/-) mice revealed the loss of expression of several key components within the natural killer (NK) cell signaling pathway, including genes associated with NK cell activation, inflammatory cytokine production, and NK cell receptor signaling. In vivo analysis of hepatic immune cell infiltrates from WT mice demonstrated that WNV infection leads to an increase in NK cell numbers with enhanced proliferation, maturation, and effector action. In contrast, livers from Mavs(-/-) × Ifnar(-/-) infected mice displayed reduced immune cell infiltration, including a significant reduction in NK cell numbers. Analysis of cocultures of dendritic and NK cells revealed both cell-intrinsic and -extrinsic roles for the RLR and IFN signaling pathways to regulate NK cell effector activity. Taken together, these observations reveal a complex innate immune signaling network, regulated by the RLR and IFN signaling pathways, that drives tissue-specific antiviral effector gene expression and innate immune cellular processes that control tissue tropism to WNV infection.
Asunto(s)
Inmunidad Celular/genética , Inmunidad Innata/genética , Tropismo Viral/genética , Fiebre del Nilo Occidental/inmunología , Virus del Nilo Occidental/inmunología , Virus del Nilo Occidental/fisiología , Animales , Redes Reguladoras de Genes/inmunología , Genes/fisiología , Humanos , Interferón Tipo I/metabolismo , Interferón Tipo I/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Receptores Inmunológicos/fisiología , Transducción de Señal/genética , Transducción de Señal/inmunología , Biología de Sistemas/métodos , Fiebre del Nilo Occidental/genéticaRESUMEN
Chronic hepatitis C virus (HCV) infection is a leading cause of liver disease. Liver inflammation underlies infection-induced fibrosis, cirrhosis and liver cancer but the processes that promote hepatic inflammation by HCV are not defined. We provide a systems biology analysis with multiple lines of evidence to indicate that interleukin-1ß (IL-1ß) production by intrahepatic macrophages confers liver inflammation through HCV-induced inflammasome signaling. Chronic hepatitis C patients exhibited elevated levels of serum IL-1ß compared to healthy controls. Immunohistochemical analysis of healthy control and chronic hepatitis C liver sections revealed that Kupffer cells, resident hepatic macrophages, are the primary cellular source of hepatic IL-1ß during HCV infection. Accordingly, we found that both blood monocyte-derived primary human macrophages, and Kupffer cells recovered from normal donor liver, produce IL-1ß after HCV exposure. Using the THP-1 macrophage cell-culture model, we found that HCV drives a rapid but transient caspase-1 activation to stimulate IL-1ß secretion. HCV can enter macrophages through non-CD81 mediated phagocytic uptake that is independent of productive infection. Viral RNA triggers MyD88-mediated TLR7 signaling to induce IL-1ß mRNA expression. HCV uptake concomitantly induces a potassium efflux that activates the NLRP3 inflammasome for IL-1ß processing and secretion. RNA sequencing analysis comparing THP1 cells and chronic hepatitis C patient liver demonstrates that viral engagement of the NLRP3 inflammasome stimulates IL-1ß production to drive proinflammatory cytokine, chemokine, and immune-regulatory gene expression networks linked with HCV disease severity. These studies identify intrahepatic IL-1ß production as a central feature of liver inflammation during HCV infection. Thus, strategies to suppress NLRP3 or IL-1ß activity could offer therapeutic actions to reduce hepatic inflammation and mitigate disease.
Asunto(s)
Proteínas Portadoras/metabolismo , Hepatitis C Crónica/inmunología , Inflamasomas/inmunología , Interleucina-1beta/biosíntesis , Macrófagos del Hígado/metabolismo , Caspasa 1/metabolismo , Línea Celular , Quimiocinas/biosíntesis , Citocinas/biosíntesis , Activación Enzimática , Hepacivirus/inmunología , Humanos , Interleucina-1beta/sangre , Interleucina-1beta/genética , Macrófagos del Hígado/inmunología , Hígado/inmunología , Hígado/metabolismo , Hígado/virología , Hepatopatías/inmunología , Hepatopatías/virología , Factor 88 de Diferenciación Mieloide/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR , Fagocitosis , ARN Mensajero/biosíntesis , Transducción de Señal , Tetraspanina 28 , Receptor Toll-Like 7/metabolismoRESUMEN
Many viruses induce type I interferon responses by activating cytoplasmic RNA sensors, including the RIG-I-like receptors (RLRs). Although two members of the RLR family, RIG-I and MDA5, have been implicated in host control of virus infection, the relative role of each RLR in restricting pathogenesis in vivo remains unclear. Recent studies have demonstrated that MAVS, the adaptor central to RLR signaling, is required to trigger innate immune defenses and program adaptive immune responses, which together restrict West Nile virus (WNV) infection in vivo. In this study, we examined the specific contribution of MDA5 in controlling WNV in animals. MDA5(-/-) mice exhibited enhanced susceptibility, as characterized by reduced survival and elevated viral burden in the central nervous system (CNS) at late times after infection, even though small effects on systemic type I interferon response or viral replication were observed in peripheral tissues. Intracranial inoculation studies and infection experiments with primary neurons ex vivo revealed that an absence of MDA5 did not impact viral infection in neurons directly. Rather, subtle defects were observed in CNS-specific CD8(+) T cells in MDA5(-/-) mice. Adoptive transfer into recipient MDA5(+/+) mice established that a non-cell-autonomous deficiency of MDA5 was associated with functional defects in CD8(+) T cells, which resulted in a failure to clear WNV efficiently from CNS tissues. Our studies suggest that MDA5 in the immune priming environment shapes optimal CD8(+) T cell activation and subsequent clearance of WNV from the CNS.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Sistema Nervioso Central/inmunología , ARN Helicasas DEAD-box/inmunología , Receptores de Reconocimiento de Patrones/inmunología , Fiebre del Nilo Occidental/inmunología , Virus del Nilo Occidental/inmunología , Animales , Linfocitos T CD8-positivos/virología , Sistema Nervioso Central/virología , ARN Helicasas DEAD-box/deficiencia , ARN Helicasas DEAD-box/metabolismo , Modelos Animales de Enfermedad , Helicasa Inducida por Interferón IFIH1 , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de Reconocimiento de Patrones/metabolismo , Análisis de Supervivencia , Carga Viral , Fiebre del Nilo Occidental/virología , Virus del Nilo Occidental/aislamiento & purificaciónRESUMEN
West Nile virus (WNV) is an emerging flavivirus capable of infecting the central nervous system (CNS) and mediating neuronal cell death and tissue destruction. The processes that promote inflammation and encephalitis within the CNS are important for control of WNV disease but, how inflammatory signaling pathways operate to control CNS infection is not defined. Here, we identify IL-1ß signaling and the NLRP3 inflammasome as key host restriction factors involved in viral control and CNS disease associated with WNV infection. Individuals presenting with acute WNV infection displayed elevated levels of IL-1ß in their plasma over the course of infection, suggesting a role for IL-1ß in WNV immunity. Indeed, we found that in a mouse model of infection, WNV induced the acute production of IL-1ß in vivo, and that animals lacking the IL-1 receptor or components involved in inflammasome signaling complex exhibited increased susceptibility to WNV pathogenesis. This outcome associated with increased accumulation of virus within the CNS but not peripheral tissues and was further associated with altered kinetics and magnitude of inflammation, reduced quality of the effector CD8(+) T cell response and reduced anti-viral activity within the CNS. Importantly, we found that WNV infection triggers production of IL-1ß from cortical neurons. Furthermore, we found that IL-1ß signaling synergizes with type I IFN to suppress WNV replication in neurons, thus implicating antiviral activity of IL-1ß within neurons and control of virus replication within the CNS. Our studies thus define the NLRP3 inflammasome pathway and IL-1ß signaling as key features controlling WNV infection and immunity in the CNS, and reveal a novel role for IL-1ß in antiviral action that restricts virus replication in neurons.
Asunto(s)
Sistema Nervioso Central/inmunología , Interleucina-1beta/inmunología , Transducción de Señal/inmunología , Replicación Viral/inmunología , Fiebre del Nilo Occidental/inmunología , Virus del Nilo Occidental/fisiología , Animales , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/inmunología , Proteínas Portadoras/metabolismo , Sistema Nervioso Central/metabolismo , Sistema Nervioso Central/virología , Femenino , Humanos , Inflamasomas/genética , Inflamasomas/inmunología , Inflamasomas/metabolismo , Interferón Tipo I/genética , Interferón Tipo I/inmunología , Interferón Tipo I/metabolismo , Interleucina-1beta/sangre , Interleucina-1beta/genética , Masculino , Ratones , Ratones Mutantes , Proteína con Dominio Pirina 3 de la Familia NLR , Neuronas/inmunología , Neuronas/metabolismo , Neuronas/virología , Fiebre del Nilo Occidental/sangre , Fiebre del Nilo Occidental/genéticaRESUMEN
CD8(+) cytotoxic T lymphocytes play a major role in defense against intracellular pathogens, and their functions are specified by antigen recognition and innate cytokines. IL-12 and IFN-α/ß are potent "signal 3" cytokines that are involved in both effector and memory cell development. Although the majority of effector cells are eliminated as inflammation resolves, some survive within the pool of memory cells and retain immediate effector function. In this study, we demonstrate that IL-12 instructs a unique program of effector cell differentiation that is distinct from IFN-α/ß. Moreover, effector memory (T(EM)) cells within peripheral blood display many common attributes of cells differentiated in vitro in response to IL-12, including proinflammatory cytokine secretion and lytic activity. A pattern of IL-12-induced genes was identified that demarcate T(EM) from central memory cells, and the ontologies of these genes correlated precisely with their effector functions. Further, we uncovered a unique program of gene expression that was acutely regulated by IL-12 and reflected in stable gene expression patterns within T(EM), but not T central memory cells in vivo. Thus, this study directly links a selective set of IL-12-induced genes to the programming of effector functions within the stable population of human CD8(+) T(EM) cells in vivo.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Regulación de la Expresión Génica , Interferón Tipo I/inmunología , Interleucina-12/inmunología , Linfocitos T CD8-positivos/metabolismo , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Citocinas/inmunología , Humanos , Memoria Inmunológica , Receptores CCR7/genética , Receptores CCR7/inmunología , Receptores CXCR3/genética , Receptores CXCR3/inmunologíaRESUMEN
4-1BB (CD137) is an activation-induced costimulatory receptor that regulates immune responses of activated CD8 T and natural killer cells, by enhancing proliferation, survival, cytolytic activity, and IFNγ production. The ability to induce potent antitumor activity by stimulating 4-1BB on tumor-specific cytotoxic T cells makes 4-1BB an attractive target for designing novel immuno-oncology therapeutics. To minimize systemic immune toxicities and enhance activity at the tumor site, we have developed a novel bispecific antibody that stimulates 4-1BB function when co-engaged with the tumor-associated antigen 5T4. ALG.APV-527 was built on the basis of the ADAPTIR bispecific platform with optimized binding domains to 4-1BB and 5T4 originating from the ALLIGATOR-GOLD human single-chain variable fragment library. The epitope of ALG.APV-527 was determined to be located at domain 1 and 2 on 4-1BB using X-ray crystallography. As shown in reporter and primary cell assays in vitro, ALG.APV-527 triggers dose-dependent 4-1BB activity mediated only by 5T4 crosslinking. In vivo, ALG.APV-527 demonstrates robust antitumor responses, by inhibiting growth of established tumors expressing human 5T4 followed by a long-lasting memory immune response. ALG.APV-527 has an antibody-like half-life in cynomolgus macaques and was well tolerated at 50.5 mg/kg. ALG.APV-527 is uniquely designed for 5T4-conditional 4-1BB-mediated antitumor activity with potential to minimize systemic immune activation and hepatotoxicity while providing efficacious tumor-specific responses in a range of 5T4-expressing tumor indications as shown by robust activity in preclinical in vitro and in vivo models. On the basis of the combined preclinical dataset, ALG.APV-527 has potential as a promising anticancer therapeutic for the treatment of 5T4-expressing tumors.
Asunto(s)
Anticuerpos Biespecíficos , Neoplasias , Anticuerpos de Cadena Única , Humanos , Anticuerpos Biespecíficos/farmacología , Anticuerpos Biespecíficos/uso terapéutico , Antígenos de Neoplasias , Linfocitos T , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral , Ligando 4-1BB/metabolismoRESUMEN
T helper 2 cells regulate inflammatory responses to helminth infections while also mediating pathological processes of asthma and allergy. IL-4 promotes Th2 development by inducing the expression of the GATA3 transcription factor, and the Th2 phenotype is stabilized by a GATA3-dependent autoregulatory loop. In this study, we found that type I IFN (IFN-alpha/beta) blocked human Th2 development and inhibited cytokine secretion from committed Th2 cells. This negative regulatory pathway was operative in human but not mouse CD4(+) T cells and was selective to type I IFN, as neither IFN-gamma nor IL-12 mediated such inhibition. IFN-alpha/beta blocked Th2 cytokine secretion through the inhibition of GATA3 during Th2 development and in fully committed Th2 cells. Ectopic expression of GATA3 via retrovirus did not overcome IFN-alpha/beta-mediated inhibition of Th2 commitment. Thus, we demonstrate a novel role for IFN-alpha/beta in blocking Th2 cells, suggesting its potential as a promising therapy for atopy and asthma.
Asunto(s)
Factor de Transcripción GATA3/metabolismo , Interferón-alfa/farmacología , Interferón beta/farmacología , Células Th2/efectos de los fármacos , Animales , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Factor de Transcripción GATA3/genética , Expresión Génica/efectos de los fármacos , Humanos , Interleucina-4/metabolismo , Ratones , Ratones Transgénicos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Th2/citología , Células Th2/metabolismoRESUMEN
Multiple innate signals regulate the genesis of effector and memory CD8+ T cells. In this study, we demonstrate that the innate cytokines interleukin (IL)-12 and interferon (IFN)-alpha/beta regulate distinct aspects of effector and memory human CD8+ T-cell differentiation. IL-12 exclusively promoted the development of IFN-gamma- and tumor necrosis factor (TNF)-alpha-secreting T effector memory (T(EM)) cells, whereas IFN-alpha drove the development of T central memory (T(CM)) cells. The development of T(EM) and T(CM) was linked to cell division. In rapidly dividing cells, IL-12 programmed T(EM) through induction of the IL-12 receptor beta2. In contrast, IFN-alpha regulated T(CM) development by slowing the progression of cell division in a subpopulation of cells that selectively expressed elevated IFN-alpha/beta receptor-2. The strength of signal delivered through T-cell receptor (TCR) engagement regulated the responsiveness of cells to IL-12 and IFN-alpha. In the presence of both IL-12 and IFN-alpha, these cytokine signals were amplified as the strength of the TCR signal was increased, promoting the simultaneous development of both T(CM) and T(EM). Together, our results support a novel model in which IL-12 and IFN-alpha act in a nonredundant manner to regulate the colinear generation of both effector and memory cells.
Asunto(s)
Linfocitos T CD8-positivos/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Memoria Inmunológica/efectos de los fármacos , Interferón-alfa/farmacología , Interleucina-12/farmacología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/fisiología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Humanos , Memoria Inmunológica/inmunología , Interferón-alfa/fisiología , Interleucina-12/fisiología , Receptor de Interferón alfa y beta/metabolismo , Receptores de Interleucina-12/metabolismoRESUMEN
IL-2 is a hallmark cytokine secreted by central memory CD4(+) T cells (T(CM)). Although naive cells rapidly secrete IL-2 in response to Ag stimulation, IL-12 inhibits IL-2 secretion in daughter cells as they differentiate into Th1 cells. In this study, we uncover a unique role for IFN-alpha in regulating IL-2 secretion by human T(CM) cells. IFN-alpha synergized with IL-12 to enhance a subset of cells that secreted high and sustained levels of IL-2. These IL-2-secreting cells displayed phenotypic and functional characteristics of T(CM) and were capable of generating IFN-gamma-secreting effectors upon secondary activation. T-bet has been implicated in negatively regulating IL-2 secretion in murine T cells; however, T-bet expression did not inhibit IFN-alpha-dependent IL-2 secretion in human T(CM) cells. Thus, our results highlight a unique role for IFN-alpha in regulating the development of IL-2-secreting human T(CM) cells.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Memoria Inmunológica , Interferón-alfa/fisiología , Interleucina-2/metabolismo , Proteínas de Dominio T Box/fisiología , Subgrupos de Linfocitos T/metabolismo , Adulto , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Diferenciación Celular/inmunología , Células Cultivadas , Humanos , Interferón beta/fisiología , Interleucina-12/fisiología , Transducción de Señal/inmunología , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/inmunología , Células TH1/citología , Células TH1/inmunologíaRESUMEN
Induction of interferon beta (IFN-ß), IFN-stimulated genes (ISGs), and inflammatory responses is critical for control of viral infection. We recently identified an essential linkage of stimulation of the inflammatory cytokine interleukin-1ß (IL-1ß) and induction of ISGs that function as host restriction pathways against the emerging flavivirus West Nile virus (WNV) in vivo Here we utilized ex vivo global transcriptome analysis of primary dendritic cells, known targets of WNV replication, to define gene signatures required for this IL-1ß-driven antiviral response. Dendritic cells that were deficient in IL-1 receptor signaling showed dysregulation of cell-intrinsic defense genes and loss of viral control during WNV infection. Surprisingly, we found that in wild-type cells, IL-1ß treatment, in the absence of infection, drove the transcription of IFN-ß and ISGs at late times following treatment. Expression of these antiviral innate immune genes was dependent on the transcription factor IFN regulatory factor 3 (IRF3) and appears to reflect a general shift in IL-1ß signaling from an early inflammatory response to a late IFN-mediated response. These data demonstrate that inflammatory and antiviral signals integrate to control viral infection in myeloid cells through a process of IL-1ß-to-IRF3 signaling crosstalk. Strategies to exploit these cytokines in the activation of host defense programs should be investigated as novel therapeutic approaches against individual pathogens.IMPORTANCE West Nile virus is an emerging mosquito-borne flavivirus that can result in serious illness, neuropathology, and death in infected individuals. Currently, there are no vaccines or therapies for human use against West Nile virus. Immune control of West Nile virus infection requires inflammatory and antiviral responses, though the effect that each arm of this response has on the other is unclear. The significance of our research is in defining how virus-induced inflammatory responses regulate critical antiviral immune programs for effective control of West Nile virus infection. These data identify essential mechanisms of immune control that can inform therapeutic efforts against West Nile virus, with potential efficacy against other neuroinvasive viruses.
Asunto(s)
Células Dendríticas/metabolismo , Interleucina-1beta/metabolismo , Animales , Línea Celular , Células Cultivadas , Cricetinae , Ensayo de Inmunoadsorción Enzimática , Flavivirus/patogenicidad , Inmunidad Innata/fisiología , Inflamasomas/metabolismo , Factor 3 Regulador del Interferón/genética , Factor 3 Regulador del Interferón/metabolismo , Interleucina-1beta/genética , Ratones , Ratones Endogámicos C57BL , Células Mieloides/metabolismo , Células Mieloides/virología , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/metabolismo , Transducción de Señal/genética , Transducción de Señal/fisiología , Virus del Nilo Occidental/patogenicidadRESUMEN
The structural maintenance of chromosome 5/6 complex (Smc5/6) is a restriction factor that represses hepatitis B virus (HBV) transcription. HBV counters this restriction by expressing HBV X protein (HBx), which targets Smc5/6 for degradation. However, the mechanism by which Smc5/6 suppresses HBV transcription and how HBx is initially expressed is not known. In this study we characterized viral kinetics and the host response during HBV infection of primary human hepatocytes (PHH) to address these unresolved questions. We determined that Smc5/6 localizes with Nuclear Domain 10 (ND10) in PHH. Co-localization has functional implications since depletion of ND10 structural components alters the nuclear distribution of Smc6 and induces HBV gene expression in the absence of HBx. We also found that HBV infection and replication does not induce a prominent global host transcriptional response in PHH, either shortly after infection when Smc5/6 is present, or at later times post-infection when Smc5/6 has been degraded. Notably, HBV and an HBx-negative virus establish high level infection in PHH without inducing expression of interferon-stimulated genes or production of interferons or other cytokines. Our study also revealed that Smc5/6 is degraded in the majority of infected PHH by the time cccDNA transcription could be detected and that HBx RNA is present in cell culture-derived virus preparations as well as HBV patient plasma. Collectively, these data indicate that Smc5/6 is an intrinsic antiviral restriction factor that suppresses HBV transcription when localized to ND10 without inducing a detectable innate immune response. Our data also suggest that HBx protein may be initially expressed by delivery of extracellular HBx RNA into HBV-infected cells.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Virus de la Hepatitis B/inmunología , Hepatitis B/inmunología , Inmunidad Innata/inmunología , Proteínas Nucleares/metabolismo , Transactivadores/metabolismo , Animales , Antígenos Nucleares/genética , Antígenos Nucleares/metabolismo , Autoantígenos/genética , Autoantígenos/metabolismo , Proteínas de Ciclo Celular/genética , Células Cultivadas , Proteínas Cromosómicas no Histona , Citocinas/genética , Citocinas/metabolismo , Hepatitis B/metabolismo , Hepatitis B/virología , Hepatocitos/citología , Hepatocitos/metabolismo , Humanos , Masculino , Ratones , Ratones SCID , Proteínas Nucleares/genética , Proteína de la Leucemia Promielocítica/genética , Proteína de la Leucemia Promielocítica/metabolismo , Transactivadores/genética , Proteínas Reguladoras y Accesorias Virales , Replicación ViralRESUMEN
During inflammatory immune responses, the innate cytokine IL-12 promotes CD4+ Th-1 development through the activation of the second messenger STAT4 and the subsequent expression of T-bet. In addition, type I IFN (IFN-alphabeta), secreted primarily during viral and intracellular bacterial infections, can promote STAT4 activation in human CD4+ T cells. However, the role of IFN-alphabeta in regulating Th1 development is controversial, and previous studies have suggested a species-specific pathway leading to Th1 development in human but not mouse CD4+ T cells. In this study, we found that although both IFN-alpha and IL-12 can promote STAT4 activation, IFN-alpha failed to promote Th1 commitment in human CD4+ T cells. The difference between these innate signaling pathways lies with the ability of IL-12 to promote sustained STAT4 tyrosine phosphorylation, which correlated with stable T-bet expression in committed Th1 cells. IFN-alpha did not promote Th1 development in human CD4+ T cells because of attenuated STAT4 phosphorylation, which was insufficient to induce stable expression of T-bet. Further, the defect in IFN-alpha-driven Th1 development was corrected by ectopic expression of T-bet within primary naive human CD4+ T cells. These results indicate that IL-12 remains unique in its ability to drive Th1 development in human CD4+ T cells and that IFN-alpha lacks this activity due to its inability to promote sustained T-bet expression.