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ABSTRACT: Glioblastoma multiforme (GBM) is the most common, highly malignant primary tumor of the brain with poor prognosis. Even with the improved therapy regimen including temozolomide, the average survival rate is less than 2 years. Additional approaches to therapy targeting multiple aspects of glioma progression are in need. In the present work, we have tested the possibility that upregulation of the transcription factor interferon regulatory factor 3 (IRF3) can inhibit glioma invasiveness, proliferation and production of pro-inflammatory and pro-angiogenic factors in cultures of malignant glioma cell lines (U271, U87 and SNB-19). IRF3 is an essential transcription factor involved in TLR3/4-mediated signaling and generation of type I interferons. Although IRF3 has been suggested as a potential tumor suppressor gene, its role in glioma remains uninvestigated. In this study, we find that human glioma immune activation is potently elicited by a cytokine combination, IL-1/IFNγ (or poly IC), but not by bacterial lipopolysaccharide (LPS), similar to primary human astrocytes. GBM biopsy specimens show little detectable IRF3 immunoreactivity, and in vitro adenovirus-mediated IRF3 gene transfer in glioma cells modulates IL-1/IFNγ-induced cytokine and chemokine genes, resulting in upregulation of IFNβ and IP-10 (IRF3-stimulated genes) and downregulation of proinflammatory and angiogenic genes including IL-8, TNFα and VEGF (IRF3-represssed genes). Cytokines (IL-1β and TNFα) also induce the expression of miR-155 and miR-155*, the microRNAs crucial in immunity and inflammation-induced oncogenesis and this is dose-dependently suppressed by IRF3. Importantly, IRF3 also inhibits glioma proliferation, migration and invasion. Together, these data suggest that IRF3 can suppress glioma progression. Agents that promote IRF3 activation and expression (such as IRF3 gene transfer) could be explored as potential future therapy.
Journal of Neuro-Oncology 03/2013; · 3.21 Impact Factor
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ABSTRACT: 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 IFNgamma. 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/IFNgamma) 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.
Journal of Neuroinflammation 03/2013; 10(1):37. · 3.83 Impact Factor
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ABSTRACT: 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 IFNβ 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.
Journal of Neuroimmune Pharmacology 06/2012; · 4.57 Impact Factor
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ABSTRACT: 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.
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.
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.
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.
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.
PLoS ONE 01/2012; 7(4):e35115. · 4.09 Impact Factor
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ABSTRACT: 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 IFNβ 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.
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.
Our results show that Ad-IRF3 suppressed proinflammatory genes (IL-1α, IL-1β, TNFα, IL-6, IL-8 and CXCL1) and enhanced anti-inflammatory genes (IL-1 receptor antagonist, IL-10 and IFNβ) 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 IFNβ 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 IFNβ expression) required PI3K/Akt.
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.
Journal of Neuroinflammation 12/2011; 8:187. · 3.83 Impact Factor
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ABSTRACT: 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 IFNβ 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.
Glia 12/2011; 59(12):1911-22. · 4.82 Impact Factor
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Daniela Krause,
Hyeon-Sook Suh,
Leonid Tarassishin,
Qiao Ling Cui,
Bryce A Durafourt,
Namjong Choi,
Avital Bauman,
Melissa Cosenza-Nashat,
Jack P Antel,
Meng-Liang Zhao, Sunhee C Lee
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ABSTRACT: 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.
American Journal Of Pathology 09/2011; 179(3):1360-72. · 4.89 Impact Factor
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ABSTRACT: Cannabinoids have been proposed for treating various neurodegenerative disorders and as adjunct therapy for HIV+ patients with neurologic sequelae. The expression of cannabinoid receptors (CB1 and CB2) has been reported in neurodegenerative diseases and in simian immunodeficiency virus encephalitis, yet the receptor expression in the central nervous system of HIV+ individuals is not known.
An anti-CB1 antibody and two anti-CB2 antibodies were employed for immunohistochemistry in the cerebral cortex and white matter of HIV encephalitis (HIVE) and HIV-associated comorbidities, as well as control brains (HIV- and HIV+).
By quantitative image analysis, we observed that CB1 was increased in HIVE brains and those with comorbidities, while CB2 was significantly increased in the white matter of HIVE. Morphologically, CB1 was present in neurones, and both CB1 and CB2 were present in meningeal macrophages and subpial glia in all brains. In HIVE, CB1 was found in white matter microglia and perivascular cells, while CB2 was increased in microglia, astrocytes and perivascular macrophages. Double immunofluorescence with cell-specific markers and immunoblots on primary cultured microglia and astrocytes substantiated the glial localization of the cannabinoid receptors and specificity of the antibodies.
Our study indicates that cannabinoid receptor expression occurs in glia in HIVE brains, and this may have ramifications for the potential use of cannabinoid ligands in HIV-infected patients.
Neuropathology and Applied Neurobiology 03/2011; 37(5):464-83. · 3.80 Impact Factor
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ABSTRACT: 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.
American Journal Of Pathology 10/2010; 177(5):2446-58. · 4.89 Impact Factor
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ABSTRACT: 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 IFNβ, 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.
Journal of Neuroimmune Pharmacology 02/2010; 5(4):521-32. · 4.57 Impact Factor
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ABSTRACT: 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.
Virology 08/2009; 392(2):246-59. · 3.35 Impact Factor
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ABSTRACT: Astrocytes are coupled via gap junctions (GJs) comprising connexin 43 (Cx43) (Gja1) and Cx30 (Gjb6), which facilitate intercellular exchange of ions. Astrocyte connexins also form heterotypic GJs with oligodendrocytic somata and lamellae. Loss of oligodendrocyte gap junctions results in oligodendrocyte and myelin pathology. However, whether loss of astrocyte GJs affects oligodendrocytes and myelin is not known. To address this question, mice with astrocyte-targeted deletion of Cx43 and global loss of Cx30 [double knock-out (dKO)] were studied using Western blotting, immunohistochemistry, electron microscopy, and functional assays. Commencing around postnatal day 23 and persisting into old age, we found widespread pathology of white matter tracts comprising vacuolated oligodendrocytes and intramyelinic edema. In contrast, gray matter pathology was restricted to the CA1 region of the hippocampus, and consisted of edematous astrocytes. No differences were observed in synaptic density or total NeuN(+) cells in the hippocampus, or olig2(+) cells in the corpus callosum. However, in dKO mice, fewer CC1-positive mature oligodendrocytes were detected, and Western blotting indicated reduced myelin basic protein. Pathology was not noted in mice expressing a single allele of either Cx43 or Cx30. When compared with single connexin knock-outs, dKO mice were impaired in sensorimotor (rotarod, balance beam assays) and spatial memory tasks (object recognition assays). We conclude that loss of astrocytic GJs can result in white matter pathology that has functional consequences.
Journal of Neuroscience 07/2009; 29(24):7743-52. · 7.11 Impact Factor
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ABSTRACT: 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.
Current topics in microbiology and immunology 02/2009; 336:63-81. · 4.93 Impact Factor
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ABSTRACT: Microglia are involved in neurodegeneration, are prime targets for anti-inflammatory therapy and are potential biomarkers of disease progression. For example, positron emission tomography imaging employing radioligands for the mitochondrial translocator protein of 18 kDa (TSPO, formerly known as the peripheral benzodiazepine receptor) is being scrutinized to detect neuroinflammation in various diseases. TSPO is presumably present in activated microglia, but may be present in other neural cells.
We sought to elucidate the protein expression in normal human central nervous system, several neurological diseases (HIV encephalitis, Alzheimer's disease, multiple sclerosis and stroke) and simian immunodeficiency virus encephalitis by performing immunohistochemistry with two anti-TSPO antibodies.
Although the overall parenchymal staining was minimal in normal brain, endothelial and smooth muscle cells, subpial glia, intravascular monocytes and ependymal cells were TSPO-positive. In disease states, elevated TSPO was present in parenchymal microglia, macrophages and some hypertrophic astrocytes, but the distribution of TSPO varied depending on the disease, disease stage and proximity to the lesion or relation to infection. Staining with the two antibodies correlated well in white matter, but one antibody also stained cortical neurones. Quantitative analysis demonstrated a significant increase in TSPO in the white matter of HIV encephalitis compared with brains without encephalitis. TSPO expression was also increased in simian immunodeficiency virus encephalitis.
This report provides the first comprehensive immunohistochemical analysis of the expression of TSPO. The results are useful for informing the usage of positron emission tomography as an imaging modality and have an impact on the potential use of TSPO as an anti-inflammatory pharmacological target.
Neuropathology and Applied Neurobiology 01/2009; 35(3):306-28. · 3.80 Impact Factor
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ABSTRACT: Cerebral malaria complicated by cognitive sequelae is a major cause of morbidity in humans infected with Plasmodium falciparum. To model cognitive function after malaria, we created a rodent model of cerebral malaria by infecting C57BL/6 mice with Plasmodium berghei strain ANKA. After 7 days, an object-recognition test of working memory revealed a significant impairment in the visual memory of infected mice. This impairment was observed in the absence of confounding effects of infection. The cognitive dysfunction correlated with hemorrhage and inflammation. Furthermore, microglial activity and morphological changes detected throughout the brains of infected mice were absent from the brains of control mice, and this correlated with the measured cognitive defects. Similar testing methods in human studies could help identify subjects at risk for an adverse cognitive outcome. This murine model should facilitate the study of adjunctive methods to ameliorate adverse neurological outcomes in cerebral malaria.
The Journal of Infectious Diseases 05/2008; 197(11):1621-7. · 6.41 Impact Factor
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ABSTRACT: It is well known that infection by the human immunodeficiency virus (HIV) dysregulates cell physiology, but little information is available on the consequences of HIV infection in primary macrophages and microglia. The authors examined the relationship between cell proliferation and HIV infection in primary cultures of microglia and in human central nervous system (CNS). In cultures infected with HIV (ADA and BaL), granulocyte-macrophage colony-stimulating factor (GM-CSF)-mediated cell proliferation was reduced in productively infected (p24+) cells as compared to p24- cells. The reduction was observed with both Ki67 and BrdU labeling, suggesting a G1/S block. The reduction was insignificant when microglia were infected with a Vpr- mutant virus. In human CNS, proliferating (Ki67+) cells were rare but were increased in the HIV+ and HIV encephalitis (HIVE) groups compared to the HIV- group. A positive correlation between GM-CSF immunoreactivity and Ki67 counts, implicating GM-CSF as a growth factor in human CNS was found. The relationship between total macrophage (CD68+) proliferation and infected macrophage (p24+) proliferation was assessed in HIVE by double labeling. Whereas 1.2% of total CD68+ cells were Ki67+, only 0.5% of HIV p24+ cells were Ki67+ (P < .05). Furthermore, staining for CD45RB (as opposed to CD68) facilitated the identification of Ki67+ microglia, indicating that CD68 could underestimate proliferating microglia. The authors conclude that although there is increased expression of GM-CSF and increased cell proliferation in the CNS of HIV-seropositive individuals, cell proliferation in the productively infected population is actually suppressed. These data suggest that there might be a viral gain in the suppressed host cell proliferation.
Journal of NeuroVirology 12/2007; 13(6):536-48. · 2.31 Impact Factor
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ABSTRACT: 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.
Journal of Virology 10/2007; 81(18):9838-50. · 5.40 Impact Factor
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ABSTRACT: 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.
The Journal of Immunology 11/2006; 177(7):4735-41. · 5.79 Impact Factor
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ABSTRACT: Loss of blood-brain barrier (BBB) integrity is believed to be an early and significant event in lesion pathogenesis in the inflammatory demyelinating disease multiple sclerosis (MS), and understanding mechanisms involved may lead to novel therapeutic avenues for this disorder. Well-differentiated endothelium forms the basis of the BBB, while astrocytes control the balance between barrier stability and permeability via production of factors that restrict or promote vessel plasticity. In this study, we report that the proinflammatory cytokine IL-1beta, which is prominently expressed in active MS lesions, causes a shift in the expression of these factors to favor plasticity and permeability. The transcription factor, hypoxia inducible factor-1 (HIF-1), plays a significant role in this switch. Using a microarray-based approach, we found that in human astrocytes, IL-1beta induced the expression of genes favoring vessel plasticity, including HIF-1alpha and its target, vascular endothelial growth factor-A (VEGF-A). Demonstrating relevance to MS, we showed that HIF-1alpha and VEGF-A were expressed by reactive astrocytes in active MS lesions, while the VEGF receptor VEGFR2/flk-1 localized to endothelium and IL-1 to microglia/macrophages. Suggesting functional significance, we found that expression of IL-1beta in the brain induced astrocytic expression of HIF-1alpha, VEGF-A, and BBB permeability. In addition, we confirmed VEGF-A to be a potent inducer of BBB permeability and angiogenesis, and demonstrated the importance of IL-1beta-induced HIF-1alpha in its regulation. These results suggest that IL-1beta contributes to BBB permeability in MS via reactivation of the HIF-VEGF axis. This pathway may represent a potential therapeutic target to restrict lesion formation.
The Journal of Immunology 11/2006; 177(8):5574-84. · 5.79 Impact Factor
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ABSTRACT: 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.
Brain Pathology 11/2006; 16(4):256-65. · 3.99 Impact Factor
