RESUMEN
BACKGROUND: Clinically significant dysregulation of the insulin-like growth factor (IGF) family proteins occurs in HIV-infected individuals, but the details including whether the deficiencies in IGFs contribute to CNS dysfunction are unknown. METHODS: We measured the levels of IGF1, IGF2, IGFBP1, IGFBP2, and IGF2 receptor (IGF2R) in matching plasma and cerebrospinal fluid (CSF) samples of 107 HIV+ individuals from CNS HIV Antiretroviral Therapy Effects Research (CHARTER) and analyzed their associations with demographic and disease characteristics, as well as levels of several soluble inflammatory mediators (TNFα, IL-6, IL-10, IL-17, IP-10, MCP-1, and progranulin). We also determined whether IGF1 or IGF2 deficiency is associated with HIV-associated neurocognitive disorder (HAND) and whether the levels of soluble IGF2R (an IGF scavenging receptor, which we also have found to be a cofactor for HIV infection in vitro) correlate with HIV viral load (VL). RESULTS: There was a positive correlation between the levels of IGF-binding proteins (IGFBPs) and those of inflammatory mediators: between plasma IGFBP1 and IL-17 (ß coefficient 0.28, P = 0.009), plasma IGFBP2 and IL-6 (ß coefficient 0.209, P = 0.021), CSF IGFBP1 and TNFα (ß coefficient 0.394, P < 0.001), and CSF IGFBP2 and TNF-α (ß coefficient 0.14, P < 0.001). As IGFBPs limit IGF availability, these results suggest that inflammation is a significant factor that modulates IGF protein expression/availability in the setting of HIV infection. However, there was no significant association between HAND and the reduced levels of plasma IGF1, IGF2, or CSF IGF1, suggesting a limited power of our study. Interestingly, plasma IGF1 was significantly reduced in subjects on non-nucleoside reverse transcriptase inhibitor-based antiretroviral therapy (ART) compared to protease inhibitor-based therapy (174.1 ± 59.8 vs. 202.8 ± 47.3 ng/ml, P = 0.008), suggesting a scenario in which ART regimen-related toxicity can contribute to HAND. Plasma IGF2R levels were positively correlated with plasma VL (ß coefficient 0.37, P = 0.021) and inversely correlated with current CD4+ T cell counts (ß coefficient -0.04, P = 0.021), supporting our previous findings in vitro. CONCLUSIONS: Together, these results strongly implicate (1) an inverse relationship between inflammation and IGF growth factor availability and the contribution of IGF deficiencies to HAND and (2) the role of IGF2R in HIV infection and as a surrogate biomarker for HIV VL.
Asunto(s)
Infecciones por VIH/sangre , Infecciones por VIH/líquido cefalorraquídeo , Somatomedinas/metabolismo , Adulto , Terapia Antirretroviral Altamente Activa/métodos , Trastornos del Conocimiento/sangre , Trastornos del Conocimiento/líquido cefalorraquídeo , Trastornos del Conocimiento/etiología , Estudios de Cohortes , Citocinas/sangre , Citocinas/líquido cefalorraquídeo , Ensayo de Inmunoadsorción Enzimática , Femenino , Infecciones por VIH/complicaciones , Infecciones por VIH/tratamiento farmacológico , Humanos , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/sangre , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/líquido cefalorraquídeo , Péptidos y Proteínas de Señalización Intercelular/sangre , Péptidos y Proteínas de Señalización Intercelular/líquido cefalorraquídeo , Masculino , Persona de Mediana Edad , Análisis Multivariante , Examen Neurológico , Pruebas Neuropsicológicas , Progranulinas , Análisis de RegresiónRESUMEN
Treatment of cultures with toll-like receptor (TLR) ligands or cytokines has become a popular approach to investigate astrocyte neuroinflammatory responses and to simulate the neural environment in various CNS disorders. However, despite much effort, the mechanism of astrocyte activation such as their responses to the TLR ligands and IL-1 remain highly debated. We compared highly pure primary mouse and human astrocyte cultures in their ability to produce proinflammatory mediators (termed "A1") and immunoregulatory mediators (termed "A2") in response to LPS, poly IC, and IL-1 stimulation. In human astrocytes, IL-1 induced both A1 and A2 responses, poly IC induced mostly A2, and LPS induced neither. In mouse astrocytes, LPS induced mostly an A1-predominant response, poly IC induced both A1 and A2, and IL-1 neither. In addition, mouse astrocytes produce abundant IL-1 protein, whereas human astrocytes did not, despite robust IL-1 mRNA expression. Of the TLR4 receptor complex proteins, human astrocytes expressed TLR4 and MD2 but not CD14, whereas mouse astrocytes expressed all three. Mouse astrocyte CD14 (cell-associated and soluble) was potently upregulated by LPS. Silencing TLR4 or CD14 by siRNA suppressed LPS responses in mouse astrocytes. In vivo, astrocytes in LPS-injected mouse brains also expressed CD14. Our results show striking differences between human and mouse astrocytes in the use of TLR/IL-1R and subsequent downstream signaling and immune activation. IL-1 translational block in human astrocytes may be a built-in mechanism to prevent autocrine and paracrine cell activation and neuroinflammation. These results have important implications for translational research of human CNS diseases.
Asunto(s)
Astrocitos/efectos de los fármacos , Astrocitos/fisiología , Interleucina-1/toxicidad , Receptores de Lipopolisacáridos/fisiología , Lipopolisacáridos/toxicidad , Animales , Animales Recién Nacidos , Células Cultivadas , Feto , Humanos , Ratones , Ratones Endogámicos C57BL , Especificidad de la EspecieRESUMEN
BACKGROUND: Recent studies in experimental animals show that insulin-like growth factor 1 (IGF1) plays a trophic role during development and tissue injury and that microglia are important sources of IGF1. However, little information is available regarding the expression, regulation, and function of IGF1 and related proteins in human brain cells. In the current study, we examined the expression of IGF1 and IGF2 in human microglia in vivo and in vitro. METHODS: Expression of IGF1 and IGF2 was examined by immunohistochemistry in post-mortem human brain sections derived from HIV+ and HIV- brains. In primary cultures of human fetal microglia, IGF1 and IGF2 mRNA and protein expression was examined by Q-PCR, ELISA, and Western blot analysis. Additionally, the role of IGF1 and IGF2 in neuroprotection was examined in primary human neuronal glial cultures. RESULTS: Immunohistochemistry of human brain tissues showed that nonparenchymal cells (vessels and meninges), as well as parenchymal microglia and macrophages were positive for IGF1, in both HIV encephalitis and control brains, while IGF2 was undetectable. Cultured microglia expressed IGF1 mRNA and produced pg/ml levels of IGF1 protein; this was significantly suppressed by proinflammatory mediators, such as lipopolysaccharide (LPS), poly(I:C), and IFNγ. The Th2 cytokines IL-4 and IL-13 had no significant effect, but the cAMP analog (dibutyryl cAMP) significantly increased IGF1 production. In contrast, microglial IGF2 mRNA and protein (determined by Western blot) were upregulated by LPS. IGF1 receptor (IGF1R) immunoreactivity was predominantly expressed by neurons, and both IGF1 and IGF2 significantly protected neurons from cytokine (IL-1/IFNγ) induced death. CONCLUSIONS: Our study in human brain tissues and cells indicates that microglia are important sources of neurotrophic growth factors IGF1 and IGF2, and that microglial activation phenotypes can influence the growth factor expression. Importantly, our results suggest that chronic neuroinflammation and upregulation of proinflammatory cytokines could lead to neurodegeneration by suppressing the production of microglia-derived neuronal growth factors, such as IGF1.
Asunto(s)
Regulación de la Expresión Génica , Mediadores de Inflamación/fisiología , Factor II del Crecimiento Similar a la Insulina/biosíntesis , Factor I del Crecimiento Similar a la Insulina/biosíntesis , Microglía/metabolismo , Células Cultivadas , Técnicas de Cocultivo , Humanos , Factor I del Crecimiento Similar a la Insulina/genética , Factor II del Crecimiento Similar a la Insulina/genética , Microglía/patología , Neuroglía/metabolismo , Neuroglía/patología , Neuronas/metabolismo , Neuronas/patología , FenotipoRESUMEN
BACKGROUND: Expression of active c-Abl in adult mouse forebrain neurons in the AblPP/tTA mice resulted in severe neurodegeneration, particularly in the CA1 region of the hippocampus. Neuronal loss was preceded and accompanied by substantial microgliosis and astrocytosis. In contrast, expression of constitutively active Arg (Abl-related gene) in mouse forebrain neurons (ArgPP/tTA mice) caused no detectable neuronal loss or gliosis, although protein expression and kinase activity were at similar levels to those in the AblPP/tTA mice. METHODS: To begin to elucidate the mechanism of c-Abl-induced neuronal loss and gliosis, gene expression analysis of AblPP/tTA mouse forebrain prior to development of overt pathology was performed. Selected results from gene expression studies were validated with quantitative reverse transcription PCR , immunoblotting and bromodeoxyuridine (BrdU) labeling, and by immunocytochemistry. RESULTS: Two of the top pathways upregulated in AblPP/tTA mice with c-Abl expression for 2 weeks were cell cycle and interferon signaling. However, only the expression of interferon signaling pathway genes remained elevated at 4 weeks of c-Abl induction. BrdU incorporation studies confirm that, while the cell cycle pathway is upregulated in AblPP/tTA mice at 2 weeks of c-Abl induction, the anatomical localization of the pathway is not consistent with previous pathology seen in the AblPP/tTA mice. Increased expression and activation of STAT1, a known component of interferon signaling and interferon-induced neuronal excitotoxicity, is an early consequence of c-Abl activation in AblPP/tTA mice and occurs in the CA1 region of the hippocampus, the same region that goes on to develop severe neurodegenerative pathology and neuroinflammation. Interestingly, no upregulation of gene expression of interferons themselves was detected. CONCLUSIONS: Our data suggest that the interferon signaling pathway may play a role in the pathologic processes caused by c-Abl expression in neurons, and that the AblPP/tTA mouse may be an excellent model for studying sterile inflammation and the effects of interferon signaling in the brain.
Asunto(s)
Ciclo Celular/fisiología , Interferones/metabolismo , Neuronas/metabolismo , Transducción de Señal/fisiología , Animales , Bromodesoxiuridina/metabolismo , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Doxiciclina/farmacología , Hipocampo/citología , Hipocampo/efectos de los fármacos , Ratones , Ratones Transgénicos , Neurogénesis/genética , Vías Olfatorias/metabolismo , Proteínas Oncogénicas v-abl/genética , Prosencéfalo/citología , Factor de Transcripción STAT1/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genéticaRESUMEN
Tryptophan metabolism by the kynurenine pathway (KP) is important to the pathogenesis of inflammatory, infectious, and degenerative diseases. The 3-hydroxykynurenine (3-HK) branch of the KP is activated in macrophages and microglia, leading to the generation of 3-HK, 3-hydroxyanthranilic acid (3-HAA), and quinolinic acid, which are considered neurotoxic owing to their free radical-generating and N-methyl-d-aspartic acid receptor agonist activities. We investigated the role of 3-HAA in inflammatory and antioxidant gene expression and neurotoxicity in primary human fetal central nervous system cultures treated with cytokines (IL-1 with or without interferon-γ) or with Toll-like receptor ligands mimicking the proinflammatory central nervous system environment. Results were analyzed by microarray, Western blot, immunostain, enzyme-linked immunosorbent assay, and neurotoxicity assays. 3-HAA suppressed glial cytokine and chemokine expression and reduced cytokine-induced neuronal death. 3-HK also suppressed cytokine-induced neuronal death. Unexpectedly, 3-HAA was highly effective in inducing in astrocytes the expression of hemeoxygenase-1 (HO-1), an antioxidant enzyme with anti-inflammatory and cytoprotective properties. Optimal induction of HO-1 required 3-HAA and cytokines. In human microglia, 3-HAA weakly induced HO-1 and lipopolysaccharide suppressed microglial HO-1 expression. 3-HAA-mediated HO-1 expression was confirmed in cultured adult human astrocytes and in vivo after 3-HAA injection to mouse brains. Together, our results demonstrate the novel neuroprotective activity of the tryptophan metabolite 3-HAA and have implications for future therapeutic approaches for neuroinflammatory disorders.
Asunto(s)
Ácido 3-Hidroxiantranílico/farmacología , Antiinflamatorios/farmacología , Hemo-Oxigenasa 1/metabolismo , Nootrópicos/farmacología , Ácido 3-Hidroxiantranílico/metabolismo , Adulto , Animales , Astrocitos/metabolismo , Muerte Celular/efectos de los fármacos , Células Cultivadas , Quimiocinas/metabolismo , Citocinas/metabolismo , Humanos , Interferón gamma/farmacología , Interleucina-1/farmacología , Quinurenina/análogos & derivados , Quinurenina/metabolismo , Ratones , Microglía/metabolismo , Neuronas/efectos de los fármacosRESUMEN
Astrocytes, together with microglia and macrophages, participate in innate inflammatory responses in the CNS. Although inflammatory mediators such as interferons generated by astrocytes may be critical in the defense of the CNS, sustained unopposed cytokine signaling could result in harmful consequences. Interferon regulatory factor 3 (IRF3) is a transcription factor required for IFNß production and antiviral immunity. Most cells express low levels of IRF3 protein, and the transcriptional mechanism that upregulates IRF3 expression is not known. In this study, we explored the consequence of adenovirus-mediated IRF3 gene transfer (Ad-IRF3) in primary human astrocytes. We show that IRF3 transgene expression suppresses proinflammatory cytokine gene expression upon challenge with IL-1/IFNγ and alters astrocyte activation phenotype from a proinflammatory to an anti-inflammatory one, akin to an M1-M2 switch in macrophages. This was accompanied by the rescue of neurons from cytokine-induced death in glial-neuronal co-cultures. Furthermore, Ad-IRF3 suppressed the expression of microRNA-155 and its star-form partner miR-155*, immunoregulatory miRNAs highly expressed in multiple sclerosis lesions. Astrocyte miR-155/miR155* were induced by cytokines and TLR ligands with a distinct hierarchy and involved in proinflammatory cytokine gene induction by targeting suppressor of cytokine signaling 1, a negative regulator of cytokine signaling and potentially other factors. Our results demonstrate a novel proinflammatory role for miR-155/miR-155* in human astrocytes and suggest that IRF3 can suppress neuroinflammation through regulating immunomodulatory miRNA expression. © 2011 Wiley-Liss, Inc.
Asunto(s)
Astrocitos/metabolismo , Astrocitos/patología , Regulación de la Expresión Génica/genética , Factor 3 Regulador del Interferón/fisiología , MicroARNs/antagonistas & inhibidores , MicroARNs/genética , Técnicas de Cocultivo , Humanos , Factor 3 Regulador del Interferón/biosíntesis , Factor 3 Regulador del Interferón/genética , Fenotipo , Cultivo Primario de CélulasRESUMEN
BACKGROUND: Microglia are the principal cells involved in the innate immune response in the CNS. Activated microglia produce a number of proinflammatory cytokines implicated in neurotoxicity but they also are a major source of anti-inflammatory cytokines, antiviral proteins and growth factors. Therefore, an immune therapy aiming at suppressing the proinflammatory phenotype while enhancing the anti-inflammatory, growth promoting phenotype would be of great benefit. In the current study, we tested the hypothesis that interferon regulatory factor 3 (IRF3), a transcription factor required for the induction of IFNß following TLR3 or TLR4 activation, is critical to the microglial phenotype change from proinflammatory to anti-inflammatory, and that this phenotype change can be greatly facilitated by IRF3 gene transfer. METHODS: Cultures of primary human fetal microglia were transduced with IRF3 using recombinant adenovirus (Ad-IRF3) and subjected to microarray analysis, real-time PCR, immunoblotting and ELISA to determine inflammatory gene expression. Two different types of immune stimuli were tested, the TLR ligands, poly IC (PIC) and LPS, and the proinflammatory cytokines, IL-1/IFNγ. In addition, the role of the PI3K/Akt pathway was examined by use of a pharmacological inhibitor, LY294002. RESULTS: Our results show that Ad-IRF3 suppressed proinflammatory genes (IL-1α, IL-1ß, TNFα, IL-6, IL-8 and CXCL1) and enhanced anti-inflammatory genes (IL-1 receptor antagonist, IL-10 and IFNß) in microglia, regardless of the cell stimuli applied. Furthermore, Ad-IRF3 activated Akt, and LY294002 reversed the effects of Ad-IRF3 on microglial inflammatory gene expression. pAkt was critical in LPS- or PIC-induced production of IL-10 and IL-1ra. Significantly, microglial IFNß protein production was also dependent on pAkt and required both Ad-IRF3 and immunological stimuli (PIC > IL-1/IFNγ). pAkt played much less prominent and variable roles in microglial proinflammatory gene expression. This anti-inflammatory promoting role of PI3K/Akt appeared to be specific to microglia, since astrocyte proinflammatory gene expression (as well as IFNß expression) required PI3K/Akt. CONCLUSIONS: Our results show a novel anti-inflammatory role for the PI3K/Akt signaling pathway in microglia. They further suggest that IRF3 gene therapy could facilitate the microglial phenotype switch from proinflammatory ("M1-like") to anti-inflammatory and immunomodulatory ("M2-like"), in part, by augmenting the level of pAkt.
Asunto(s)
Factor 3 Regulador del Interferón/inmunología , Microglía/inmunología , Fosfatidilinositol 3-Quinasas/inmunología , Proteínas Proto-Oncogénicas c-akt/inmunología , Transducción de Señal/inmunología , Células Cultivadas , Quimiocinas/genética , Quimiocinas/inmunología , Cromonas/farmacología , Citocinas/genética , Citocinas/inmunología , Inhibidores Enzimáticos/farmacología , Perfilación de la Expresión Génica , Técnicas de Transferencia de Gen , Humanos , Inmunidad Innata/inmunología , Factor 3 Regulador del Interferón/genética , Interferón gamma/farmacología , Interleucina-1/farmacología , Análisis por Micromatrices , Microglía/citología , Microglía/efectos de los fármacos , Morfolinas/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , TransgenesRESUMEN
Insulin-like growth factor 2 receptor (IGF2R), also known as cation-independent mannose 6-phosphate (M6P) receptor, is a transmembrane glycoprotein localized in the trans-Golgi region and is involved in targeting both M6P-bearing enzymes and IGF2 to the lysosomal compartment. During development, IGF2R plays a crucial role in removing excess growth factors from both tissue and blood. Due to the perinatal lethality of the global Igf2r knockout, the function of IGF2R in adults, particularly in the CNS, is not known. We made a novel observation that IGF2R is highly expressed in microglial nodules in human brains with HIV encephalitis. In vitro, microglial IGF2R expression was uniquely enhanced by IFNγ among the several cytokines and TLR ligands examined. Furthermore, in several in vitro models of HIV infection, including human and murine microglia, macrophages, and nonmacrophage cells, IGF2R is repeatedly shown to be a positive regulator of HIV infection. IGF2R RNAi also down-regulated the production of the IP-10 chemokine in HIV-infected human microglia. Injection of VSVg env HIV into mouse brain induced HIV p24 expression in neurons, the only cell type normally expressing IGF2R in the adult brain. Our results demonstrate a novel role for IGF2R as an inducible microglial protein involved in regulation of HIV and chemokine expression. Mice with the Csf1r- driven Igf2r knockout should be useful for the investigation of macrophage-specific IGF2R function.
Asunto(s)
Complejo SIDA Demencia/fisiopatología , VIH/fisiología , Interferón gamma/metabolismo , Microglía/metabolismo , Receptor IGF Tipo 2/metabolismo , Replicación Viral , Complejo SIDA Demencia/patología , Complejo SIDA Demencia/virología , Animales , Astrocitos/citología , Astrocitos/virología , Encéfalo/citología , Encéfalo/patología , Encéfalo/virología , Línea Celular , Células Cultivadas , VIH/genética , VIH/ultraestructura , Infecciones por VIH/patología , Infecciones por VIH/fisiopatología , Humanos , Macrófagos/citología , Macrófagos/virología , Ratones , Ratones Noqueados , Microglía/citología , Microglía/virología , Interferencia de ARN , Receptor IGF Tipo 2/genética , Virión/ultraestructuraRESUMEN
Protection against viral infections is critically dependent upon the early production of significant levels of type 1 interferons and the expression of interferon-stimulated genes that function as the effectors of innate antiviral immunity. Activation of Toll-like receptors on cells of the immune system is known to play an important role in this process. In this chapter we review evidence for a role of TLRs in innate immune responses against viral infections of the central nervous system. By far the most extensive literature pertains to TLR3. Data from various laboratories have shown that TLR3 is expressed in cells endogenous to the CNS, particularly in astrocytes and microglia. Triggering TLR3 by synthetic dsRNA, poly I:C effectively induces innate antiviral responses as well as boosts adaptive immune responses. Additional experiments show cooperative responses between TLRs (3, 7/8 and 9) in mounting an effective antiviral immune response in the periphery. Perhaps the most exciting data are from patient populations that document the critical role that specific TLRs play in specific CNS infections. Studies also suggest that inappropriate activation of the TLRs can result in a pathogenic outcome rather than a protective one. Since TLR ligands are being actively considered for their antiviral and potential adjuvant effects, this will be an important issue to address in the context of the CNS environment.
Asunto(s)
Infecciones del Sistema Nervioso Central/inmunología , Infecciones del Sistema Nervioso Central/virología , Receptor Toll-Like 3/inmunología , Virosis/inmunología , Animales , Humanos , Virosis/virologíaRESUMEN
CD45 is a membrane tyrosine phosphatase that modulates the function of the hematopoietic cells. In vitro, agonist antibodies to CD45RO or CD45RB isoforms have been shown to suppress microglial activation, but whether microglia in vivo express these isoforms in HIV encephalitis (HIVE) is unknown. Brain sections from control and HIVE were immunostained for CD45 isoforms using exon-specific antibodies (RA, RB, RC and RO). RA and RC were limited to rare lymphocytes, while RB expression was robust in microglia and inflammatory cells. RO was low in control microglia, but increased in HIVE. RO was also localized to macrophages and CD8+ T cells. Targeting CD45 in vivo with isoform-specific antibodies remains a therapeutic option for neuroinflammatory diseases.
Asunto(s)
Complejo SIDA Demencia/metabolismo , Encéfalo/patología , VIH-1/inmunología , Antígenos Comunes de Leucocito/biosíntesis , Microglía/metabolismo , Complejo SIDA Demencia/etiología , Complejo SIDA Demencia/inmunología , Encéfalo/inmunología , VIH-1/metabolismo , Humanos , Inmunohistoquímica , Linfocitos/inmunología , Linfocitos/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Persona de Mediana Edad , Isoformas de Proteínas/biosíntesisRESUMEN
Progranulin (PGRN) is a highly unusual molecule with both neuronal and microglial expression with two seemingly unrelated functions, i.e., as a neuronal growth factor and a modulator of neuroinflammation. Haploinsufficiency due to loss of function mutations lead to a fatal presenile dementing illness (frontotemporal lobar degeneration), indicating that adequate expression of PGRN is essential for successful aging. PGRN might be a particularly relevant factor in the pathogenesis of HIVencephalitis (HIVE) and HIV-associated neurocognitive disorders (HAND). We present emerging data and a review of the literature which show that cells of myeloid lineage such as macrophages and microglia are the primary sources of PGRN and that PGRN expression contributes to pathogenesis of CNS diseases. We also present evidence that PGRN is a macrophage antiviral cytokine. For example, PGRN mRNA and protein expression are significantly upregulated in brain specimens with HIVE, and in HIV infected microglia in vitro. Paradoxically, our preliminary CHARTER data analyses indicate that lower PGRN levels in CSF trended towards an association with HAND, particularly in those without detectable virus. Based upon these findings, we introduce the hypothesis that PGRN plays dual roles in modulating antiviral immunity and neuronal dysfunction in the context of HIV infection. In the presence of active viral replication, PGRN expression is increased functioning as an anti-viral factor as well as a neuroprotectant. In the absence of active HIV replication, ongoing inflammation or other stressors suppress PGRN production from macrophages/microglia contributing to neurocognitive dysfunction. We propose.
Asunto(s)
Complejo SIDA Demencia/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Microglía/metabolismo , Complejo SIDA Demencia/patología , Animales , Trastornos del Conocimiento/metabolismo , Trastornos del Conocimiento/virología , Humanos , ProgranulinasRESUMEN
BACKGROUND: Compelling data exist that show that normal levels of progranulin (PGRN) are required for successful CNS aging. PGRN production is also modulated by inflammation and infection, but no data are available on the production and role of PGRN during CNS HIV infection. METHODS: To determine the relationships between PGRN and HIV disease, neurocognition, and inflammation, we analyzed 107 matched CSF and plasma samples from CHARTER, a well-characterized HIV cohort. Levels of PGRN were determined by ELISA and compared to levels of several inflammatory mediators (IFNγ, IL-6, IL-10, IP-10, MCP-1, TNFα, IL-1ß, IL-4 and IL-13), as well as clinical, virologic and demographic parameters. The relationship between HIV infection and PGRN was also examined in HIV-infected primary human microglial cultures. RESULTS: In plasma, PGRN levels correlated with the viral load (VL, p<0.001). In the CSF of subjects with undetectable VL, lower PGRN was associated with neurocognitive impairment (pâ=â0.046). CSF PGRN correlated with CSF IP-10, TNFα and IL-10, and plasma PGRN correlated with plasma IP-10. In vitro, microglial HIV infection increased PGRN production and PGRN knockdown increased HIV replication, demonstrating that PGRN is an innate antiviral protein. CONCLUSIONS: We propose that PGRN plays dual roles in people living with HIV disease. With active HIV replication, PGRN is induced in infected macrophages and microglia and functions as an antiviral protein. In individuals without active viral replication, decreased PGRN production contributes to neurocognitive dysfunction, probably through a diminution of its neurotrophic functions. Our results have implications for the pathogenesis, biomarker studies and therapy for HIV diseases including HIV-associated neurocognitive dysfunction (HAND).
Asunto(s)
Cognición , Infecciones por VIH/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Adulto , Femenino , VIH/fisiología , Infecciones por VIH/sangre , Infecciones por VIH/líquido cefalorraquídeo , Infecciones por VIH/fisiopatología , Humanos , Inflamación/metabolismo , Péptidos y Proteínas de Señalización Intercelular/sangre , Péptidos y Proteínas de Señalización Intercelular/líquido cefalorraquídeo , Masculino , Microglía/metabolismo , Microglía/virología , Análisis Multivariante , Progranulinas , Carga Viral , Replicación ViralRESUMEN
Interferon regulatory factor 3 (IRF3) is a transcription factor critical in the induction of antiviral immunity. IRF3 is activated following stimulation of cell membrane or cytosolic nucleic acid sensors and is essential in the induction of the IFNß gene. Most cells constitutively express IRF3 in vitro, but little is known about the regulation of expression of IRF3 in vivo. Immunohistochemical analysis of selected human and mouse tissues demonstrated that IRF3 expression is highly organ- and cell-type specific, showing high expression in certain epithelial cells. In the CNS, while ependymal cells are strongly positive, brain parenchyma has little detectable IRF3 immunoreactivity. The importance of IRF3 in antiviral immunity has been demonstrated by the requirement for IRF3 in suppressing viral replication, but also by the demonstration that virus degrades IRF3 protein in infected cells. Furthermore, HIV-infected microglia in human CNS show abnormal IRF3+ aggregates, indicative of aberrant protein processing in vivo. In addition to antiviral immunity, IRF3 also plays a critical role in the modulation of neuroinflammation. A combination of dominant-negative and over-expression strategies in vitro as well as transgenic expression of IRF3 in vivo demonstrated that IRF3 plays a major role in modulating glial cytokine expression, i.e., suppression of proinflammatory cytokines and promotion of anti-inflammatory or immunoregulatory cytokines. These observations together suggest that IRF3 is a crucial regulator of immune responses against pathogen- and damage-associated molecules. We review recent literature on the molecular pathways of IRF3 activation and function of IRF3 and discuss their implications for CNS diseases.
Asunto(s)
Antiinflamatorios no Esteroideos , Antivirales , Enfermedades del Sistema Nervioso Central/tratamiento farmacológico , Enfermedades del Sistema Nervioso Central/inmunología , Factor 3 Regulador del Interferón/farmacología , Animales , Enfermedades del Sistema Nervioso Central/prevención & control , Encefalitis/inmunología , Encefalitis/patología , Humanos , Factor 3 Regulador del Interferón/químicaRESUMEN
BACKGROUND: The essential role of progranulin (PGRN) as a neurotrophic factor has been demonstrated by the discovery that haploinsufficiency due to GRN gene mutations causes frontotemporal lobar dementia. In addition to neurons, microglia in vivo express PGRN, but little is known about the regulation of PGRN expression by microglia. GOAL: In the current study, we examined the regulation of expression and function of PGRN, its proteolytic enzyme macrophage elastase (MMP-12), as well as the inhibitor of PGRN proteolysis, secretory leukocyte protease inhibitor (SLPI), in human CNS cells. METHODS: Cultures of primary human microglia and astrocytes were stimulated with the TLR ligands (LPS or poly IC), Th1 cytokines (IL-1/IFNγ), or Th2 cytokines (IL-4, IL-13). Results were analyzed by Q-PCR, immunoblotting or ELISA. The roles of MMP-12 and SLPI in PGRN cleavage were also examined. RESULTS: Unstimulated microglia produced nanogram levels of PGRN, and PGRN release from microglia was suppressed by the TLR ligands or IL-1/IFNγ, but increased by IL-4 or IL-13. Unexpectedly, while astrocytes stimulated with proinflammatory factors released large amounts of SLPI, none were detected in microglial cultures. We also identified MMP-12 as a PGRN proteolytic enzyme, and SLPI as an inhibitor of MMP-12-induced PGRN proteolysis. Experiments employing PGRN siRNA demonstrated that microglial PGRN was involved in the cytokine and chemokine production following TLR3/4 activation, with its effect on TNFα being the most conspicuous. CONCLUSIONS: Our study is the first detailed examination of PGRN in human microglia. Our results establish microglia as a significant source of PGRN, and MMP-12 and SLPI as modulators of PGRN proteolysis. Negative and positive regulation of microglial PGRN release by the proinflammatory/Th1 and the Th2 stimuli, respectively, suggests a fundamentally different aspect of PGRN regulation compared to other known microglial activation products. Microglial PGRN appears to function as an endogenous modulator of innate immune responses.
Asunto(s)
Astrocitos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Metaloproteinasa 12 de la Matriz/metabolismo , Microglía , Astrocitos/enzimología , Astrocitos/metabolismo , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Regulación de la Expresión Génica , Humanos , Inmunidad Innata/genética , Péptidos y Proteínas de Señalización Intercelular/genética , Metaloproteinasa 12 de la Matriz/genética , Microglía/enzimología , Microglía/metabolismo , Progranulinas , Proteolisis , ARN Interferente Pequeño , Inhibidor Secretorio de Peptidasas Leucocitarias/metabolismo , Transducción de SeñalRESUMEN
Histone deacetylase inhibitors (HDACi) have been proposed as therapies for certain cancers and as an anti-reservoir therapy for HIV+ individuals with highly active anti-retroviral therapy, yet their roles in glial inflammatory and innate antiviral gene expression have not been defined. In this study, we examined the effects of two non-selective HDACi, trichostatin A and valproic acid, on antiviral and cytokine gene expression in primary human microglia and astrocytes stimulated with TLR3 or TLR4 ligand. HDACi potently suppressed the expression of innate antiviral molecules such as IFNß, interferon-simulated genes, and proteins involved in TLR3/TLR4 signaling. HDACi also suppressed microglial and astrocytic cytokine and chemokine gene expression, but with different effects on different groups of cytokines. These results have important implications for the clinical use of HDACi.
Asunto(s)
Astrocitos/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Inhibidores de Histona Desacetilasas/farmacología , Inmunidad Innata/genética , Inflamación/genética , Microglía/efectos de los fármacos , Astrocitos/metabolismo , Western Blotting , Células Cultivadas , Citocinas/biosíntesis , Citocinas/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Humanos , Ácidos Hidroxámicos/farmacología , Microglía/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ácido Valproico/farmacologíaRESUMEN
In the CNS, microglia are the primary targets of HIV infection. In this study, we investigated the effect of activation of the innate antiviral receptors TLR3 and TLR4 on HIV infection of primary human microglia, as well as microglial cell signaling and gene expression. Ligands for both TLR3 and TLR4 potently inhibited HIV replication in microglia through a pathway requiring IRF3. Surprisingly, a remarkably similar pattern of cell signaling and gene expression was observed in TLR3- and TLR4-activated microglia, suggesting a relatively minor role for MyD88 following TLR4 activation in these cells. HIV did not activate IRF3 but rather decreased IRF3 protein, indicating that HIV does not activate TLR3 or RIG-like helicases in microglia. Taken together, these results indicate that activation of TLR3 or TLR4 will elicit antiviral immunity, in addition to inducing proinflammatory responses. We suggest that a balanced expression between inflammatory and innate immune genes might be achieved by IRF3 over-expression.
Asunto(s)
Infecciones por VIH/inmunología , Factor 3 Regulador del Interferón/inmunología , Microglía/inmunología , Receptor Toll-Like 3/inmunología , Receptor Toll-Like 4/inmunología , Células Cultivadas , Perfilación de la Expresión Génica , VIH/fisiología , Humanos , Interferón beta/inmunología , Microglía/virología , Análisis de Secuencia por Matrices de Oligonucleótidos , Interferencia de ARN , Replicación ViralRESUMEN
Indoleamine 2,3-dioxygenase (IDO) is the first and rate-limiting enzyme in the kynurenine pathway of tryptophan catabolism and has been implicated in neurotoxicity and suppression of the antiviral T-cell response in HIV encephalitis (HIVE). Here we show that the Toll-like receptor 3 (TLR3) ligand poly(I:C) (PIC) induces the expression of IDO in human astrocytes. PIC was less potent than gamma interferon (IFN-gamma) but more potent than IFN-beta in inducing IDO. PIC induction of IDO was mediated in part by IFN-beta but not IFN-gamma, and both NF-kappaB and interferon regulatory factor 3 (IRF3) were required. PIC also upregulated TLR3, thereby augmenting the primary (IFN-beta) and secondary (IDO and viperin) response genes upon subsequent stimulation with PIC. In HIVE, the transcripts for TLR3, IFN-beta, IDO, and viperin were increased and IDO immunoreactivity was detected in reactive astrocytes as well as macrophages and microglia. PIC caused suppression of intracellular replication of human immunodeficiency virus pseudotyped with vesicular stomatitis virus G protein and human cytomegalovirus in a manner dependent on IRF3 and IDO. The involvement of IDO was demonstrated by partial but significant reversal of the PIC-mediated antiviral effect by IDO RNA interference and/or tryptophan supplementation. Importantly, the cytokine interleukin-1 abolished IFN-gamma-induced IDO enzyme activity in a nitric oxide-dependent manner without suppressing protein expression. Our results demonstrate that IDO is an innate antiviral protein induced by double-stranded RNA and suggest a therapeutic utility for PIC in human viral infections. They also show that IDO activity can be dissociated from protein expression, indicating that the local central nervous system cytokine and nitric oxide environment determines IDO function.
Asunto(s)
Astrocitos/inmunología , Encefalitis Viral/inmunología , Infecciones por VIH/inmunología , VIH-1/inmunología , Indolamina-Pirrol 2,3,-Dioxigenasa/inmunología , Inductores de Interferón/farmacología , Poli I-C/farmacología , Receptor Toll-Like 3/inmunología , Replicación Viral/inmunología , Astrocitos/enzimología , Astrocitos/virología , Células Cultivadas , Citocinas/inmunología , Citocinas/farmacología , Citomegalovirus/genética , Citomegalovirus/inmunología , Infecciones por Citomegalovirus/enzimología , Infecciones por Citomegalovirus/genética , Infecciones por Citomegalovirus/inmunología , Encefalitis Viral/enzimología , Encefalitis Viral/genética , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/genética , Regulación Enzimológica de la Expresión Génica/inmunología , Infecciones por VIH/enzimología , Infecciones por VIH/genética , VIH-1/genética , Humanos , Indolamina-Pirrol 2,3,-Dioxigenasa/biosíntesis , Indolamina-Pirrol 2,3,-Dioxigenasa/genética , Factor 3 Regulador del Interferón/inmunología , Factor 3 Regulador del Interferón/metabolismo , Quinurenina/genética , Quinurenina/inmunología , Quinurenina/metabolismo , Ligandos , Macrófagos/enzimología , Macrófagos/inmunología , Macrófagos/virología , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Glicoproteínas de Membrana/metabolismo , Microglía/enzimología , Microglía/inmunología , Microglía/virología , FN-kappa B/inmunología , FN-kappa B/metabolismo , Óxido Nítrico/inmunología , Óxido Nítrico/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Proteínas/inmunología , Proteínas/metabolismo , Receptor Toll-Like 3/agonistas , Receptor Toll-Like 3/metabolismo , Triptófano/inmunología , Triptófano/metabolismo , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunología , Proteínas del Envoltorio Viral/metabolismoRESUMEN
Macrophages and microglia are productively infected by HIV-1 and play a pivotal role in the pathogenesis of AIDS dementia. Although macrophages and microglia express CD45, a transmembrane protein tyrosine phosphatase, whether modulation of its activity affects human immunodeficiency virus type 1 (HIV-1) replication is unknown. Here, we report that of the five human CD45 isoforms, microglia express CD45RB and CD45RO (RB > RO) and treatment of microglia with a CD45 agonist antibody alphaCD45RO (UCHL-1) inhibits HIV-1 replication. alphaCD45RO prevented HIV-1 negative factor (Nef)-induced autophosphorylation of hematopoietic cell kinase (Hck), a myeloid lineage-specific Src kinase. Recombinant CD45 protein also inhibited HIV-1-induced Hck phosphorylation in microglia. Antennapedia-mediated delivery of Hck Src homology domain 3 (SH3), a domain that binds to the Nef PxxP motif with high affinity, reduced HIV-1-induced Hck phosphorylation and HIV-1 production in microglia. HIV-1-induced LTR transactivation was observed in U38 cells stably overexpressing wild-type Hck but not kinase-inactive Hck. In microglia, alphaCD45RO reduced activation of transcription factors (NF-kappaB and CCAAT enhancer binding protein) necessary for LTR transactivation in macrophages. These results establish that in myeloid lineage cells, Nef interacts with the Hck SH3 domain, resulting in autophosphorylation of Hck and an increase in HIV-1 transcription. alphaCD45RO-mediated inhibition of HIV-1 replication in microglia identifies the CD45 protein tyrosine phosphatase as a potential therapeutic target for HIV-1 infection/AIDS dementia.
Asunto(s)
VIH-1/metabolismo , Antígenos Comunes de Leucocito/inmunología , Microglía/citología , Proteínas Tirosina Quinasas/fisiología , Proteínas Proto-Oncogénicas c-hck/metabolismo , Replicación Viral/fisiología , Complejo SIDA Demencia , Línea Celular , VIH-1/enzimología , VIH-1/genética , VIH-1/fisiología , Humanos , Antígenos Comunes de Leucocito/metabolismo , Microglía/enzimología , Microglía/inmunología , Inhibidores de Proteínas Quinasas/farmacología , Proteína Tirosina Fosfatasa no Receptora Tipo 1 , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/metabolismoRESUMEN
TLR3 functions as a viral nucleic acid sentinel activated by dsRNA viruses and virus replication intermediates within intracellular vesicles. To explore the spectrum of genes induced in human astrocytes by TLR3, we used a microarray approach and the analog polyriboinosinic polyribocytidylic acid (pIC) as ligand. As expected for TLR activation, pIC induced a wide array of cytokines and chemokines known for their role in inflammatory responses, as well as up-regulation of the receptor itself. The data also showed activation of a broad spectrum of antiviral response genes. To determine whether pIC induced an antiviral state in astrocytes, a pseudotyped HIV viral particle, vesicular stomatitis virus g-env-HIV-1, was used. pIC significantly abrogated HIV-1 replication, whereas IL-1, which also potently activates astrocytes, did not. One of the most highly up-regulated genes on microarray was the protein viperin/cig5. We found that viperin/cig5 expression was dependent on IFN regulatory factor 3 and NF-kappaB signaling, and that repetitive stimulation with pIC, but not IL-1, further increased expression. Viperin induction could also be substantially inhibited by neutralizing Abs to IFN-beta, as could HIV-1 replication. To explore a role for viperin in IFN-beta-mediated inhibition of HIV-1, we used an RNA interference (RNAi) approach. RNAi directed against viperin, but not a scrambled RNAi, significantly inhibited viperin expression, and also significantly reversed pIC-induced inhibition of HIV-1 replication. We conclude that viperin contributes to the antiviral state induced by TLR3 ligation in astrocytes, supporting a role for astrocytes as part of the innate immune response against infection in the CNS.
Asunto(s)
Antivirales/inmunología , Astrocitos/inmunología , Astrocitos/virología , Poli I-C/inmunología , Proteínas/inmunología , Receptor Toll-Like 3/metabolismo , Western Blotting , Quimiocinas/metabolismo , Feto , VIH-1/inmunología , Humanos , Inmunohistoquímica , Interferón beta/inmunología , Interleucina-1/inmunología , Análisis de Secuencia por Matrices de Oligonucleótidos , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , ARN Interferente Pequeño , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección , Virus de la Estomatitis Vesicular Indiana/genética , Virus de la Estomatitis Vesicular Indiana/inmunología , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
Increasing evidence suggests that CD45, a transmembrane protein tyrosine phosphatase, is an important modulator of macrophage activation. Microglia, resident brain macrophages, express CD45 and proliferate under pathologic conditions. In this study, we examined the role of CD45 in modulating GM-CSF-induced proliferation and signal transduction in primary human microglial cultures. Soluble, but not immobilized anti-CD45RO induced tyrosine phosphatase activity and inhibited GM-CSF-induced microglial proliferation. Microglial proliferation was also inhibited by PP2 (Src inhibitor), LY294002 (PI3K inhibitor), and U0126 (MEK inhibitor). GM-CSF induced phosphorylation of Jak2, Stat5, Hck (the myeloid-restricted Src kinase), Akt, Stat3, and Erk MAPKs in microglia. Of these, anti-CD45RO inhibited phosphorylation of Hck and Akt, and PP2 inhibited phosphorylation of Hck and Akt. In a macrophage cell line stably overexpressing wild-type or kinase-inactive Hck, GM-CSF increased proliferation of the control (empty vector) and wild-type but not kinase-inactive cells, and this was inhibited by anti-CD45RO. Together, these results demonstrate that, in macrophages, Hck tyrosine kinase is activated by GM-CSF, and that Hck plays a pivotal role in cell proliferation and survival by activating the PI3K/Akt pathway. Ab-mediated activation of macrophage and microglial CD45 tyrosine phosphatase may have therapeutic implications for CNS inflammatory diseases.