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PLoS ONE: Hepatitis C Virus Core Protein Induces Neuroimmune ...
by P Vivithanaporn - 2010
E-mail: chris.power@ualberta.ca ...... (2008) Clinicopathologic correlates of hepatitis C virus in brain: a pilot study. ... Find this article online; Jones G, Power C (2006) Regulation of neural cell survival by HIV-1 infection. ...
www.plosone.org/.../info%3Adoi%2F10.1371%2Fjournal.pone.0012856

"To verify HCV infection of the brain occured, autopsied brain tissues were investigated in HIV/AIDS persons without HCV infection (n = 3) and an individual with HIV/AIDS and HCV infection at death. The latter individual was a 46-year old female patient with HIV-associated dementia (HIV Dementia Scale score = 6; cranial MRI: cerebral atrophy with increased diffuse white matter signal on T2-weighted images), CD4+ T cell level = 105 cells/µl and detectable HIV and HCV viremia......These results indicated that concurrent exposure of HIV-1 and HCV encoded proteins potentiated the neuroimmune activation and neurotoxicity effects of HCV core protein.

During the last decade, there is mounting evidence to suggest HCV is neuroinvasive and HIV/HCV co-infected patients display higher rates of neuropsychological deficits. To delineate the underlying pathogenic mechanisms of HCV infection of the brain, we report for the first time that primary human astrocytes and microglia were permissive to HCV infection with cell culture-derived HCV particles. Moreover, this report demonstrates for the first time that HCV core protein activates human glia and contributes to neurotoxicity. Direct exposure of HCV core protein to primary human neurons suppressed the neuronal autophagy, leading to neurite retraction. The change in neuronal membrane potential after exposure to HCV core protein indicated that core was biologically active at the cell membrane and was able to modulate ionic conductance in neurons. In addition to direct neurotoxicity, proinflammatory cytokines and other neurotoxins released from HCV core-activated microglia into supernatants were toxic to neurons. The in vitro and in vivo aberrant immune activation and neurotoxicity mediated by HCV core protein were amplified in the presence of HIV-1 Vpr protein. These findings support the concept that the presence of HCV-encoded proteins cause neuronal damage and perhaps neurocognitive impairment in individuals co-infected with HIV and HCV.

In summary, we reported primary human microglia and astrocytes were permissive to HCV infection and HCV-encoded protein, core, was neurotoxic but also activated pro-inflammatory responses in human microglia and astrocytes. The augmented glial activation and neurotoxicity mediated by HCV core protein in the presence of HIV-1 Vpr protein or HIV-1 infection highlighted the additive effects of HCV- and HIV-encoded proteins in the pathogenesis of neurologic disease. Future studies are required to delineate the precise mechanisms by which HIV and HCV proteins interact and amplify neuropathogenesis, thereby permitting the development of potential management and therapeutic strategies."

Hepatitis C Virus Core Protein Induces Neuroimmune Activation and Potentiates Human Immunodeficiency Virus-1 Neurotoxicity

Plosone
Published September 21, 2010

Pornpun Vivithanaporn1,2, Ferdinand Maingat1, Liang-Tzung Lin3, Hong Na1, Christopher D. Richardson3, Babita Agrawal4, Éric A. Cohen5, Jack H. Jhamandas1, Christopher Power1*

1 Division of Neurology, University of Alberta, Edmonton, Alberta, Canada, 2 Department of Pharmacology, Faculty of Science, Mahidol University, Bangkok, Thailand, 3 Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia, Canada, 4 Department of Surgery, University of Alberta, Edmonton, Alberta, Canada, 5 Institut de recherches cliniques de Montréal (IRCM) and Department of Microbiology and Immunology, University of Montreal, Montreal, Quebec, Canada

Abstract

Background

Hepatitis C virus (HCV) genomes and proteins are present in human brain tissues although the impact of HIV/HCV co-infection on neuropathogenesis remains unclear. Herein, we investigate HCV infectivity and effects on neuronal survival and neuroinflammation in conjunction with HIV infection.

Methodology

Human microglia, astrocyte and neuron cultures were infected with cell culture-derived HCV or exposed to HCV core protein with or without HIV-1 infection or HIV-1 Viral Protein R (Vpr) exposure. Host immune gene expression and cell viability were measured. Patch-clamp studies of human neurons were performed in the presence or absence of HCV core protein. Neurobehavioral performance and neuropathology were examined in HIV-1 Vpr-transgenic mice in which stereotaxic intrastriatal implants of HCV core protein were performed.

Principal Findings

HCV-encoded RNA as well as HCV core and non-structural 3 (NS3) proteins were detectable in human microglia and astrocytes infected with HCV. HCV core protein exposure induced expression of pro-inflammatory cytokines including interleukin-1β, interleukin-6 and tumor necrosis factor-α in microglia (p<0.05) but not in astrocytes while increased chemokine (e.g. CXCL10 and interleukin-8) expression was observed in both microglia and astrocytes (p<0.05). HCV core protein modulated neuronal membrane currents and reduced both β-III-tubulin and lipidated LC3-II expression (p<0.05). Neurons exposed to supernatants from HCV core-activated microglia exhibited reduced β-III-tubulin expression (p<0.05). HCV core protein neurotoxicity and interleukin-6 induction were potentiated by HIV-1 Vpr protein (p<0.05). HIV-1 Vpr transgenic mice implanted with HCV core protein showed gliosis, reduced neuronal counts together with diminished LC3 immunoreactivity. HCV core-implanted animals displayed neurobehavioral deficits at days 7 and 14 post-implantation (p<0.05).

Conclusions


HCV core protein exposure caused neuronal injury through suppression of neuronal autophagy in addition to neuroimmune activation. The additive neurotoxic effects of HCV- and HIV-encoded proteins highlight extrahepatic mechanisms by which HCV infection worsens the disease course of HIV infection.

Introduction


Hepatitis C virus (HCV) infects approximately 180 million people worldwide [1] while 30% of individuals infected with human immunodeficiency virus type 1 (HIV-1) are co-infected with HCV due to similar routes of transmission [2]. Epidemiological studies suggest that HCV co-infection is associated with accelerated HIV disease progression, worsened clinical outcomes and increased mortality [2], [3]. HIV/HCV co-infected patients have higher HCV levels and lower likelihood of spontaneous HCV clearance, together with faster progression to liver cirrhosis [1], [2]. HCV is a member of the Flaviviridae family, which consists of several neurotropic viruses including St Louis encephalitis virus, Dengue and West Nile virus [4], [5]. HCV mono-infected and HIV/HCV co-infected individuals display neuropsychological deficits indicative of impaired cognition [5], [6], [7]. Magnetic resonance spectroscopy studies report alterations in cerebral metabolites among HCV-infected individuals correlated with neurocognitive impairment, including suppression of the neuronal marker, N-acetyl aspartate [8].

HCV transcripts and proteins have also been detected in brains from HIV/HCV co-infected patients, indicating that HCV is neuroinvasive [9], [10], [11]. The negative strand of HCV RNA, a viral replication intermediate, has been detected in the brain and cerebrospinal fluid [10], [12]. Several studies showed that HCV RNA sequences isolated from the central nervous system (CNS) were closely related to those found in peripheral blood mononuclear cells (PBMC) but were phylogenetically different from serum- or liver-derived sequences [10], [11], [12], [13], leading to the postulation that HCV enters the brain through the ‘Trojan horse’ mechanism similar to HIV-1 [4]. However, the mechanisms by which HCV exerts neuropathogenic effects remain unknown and the understanding of combined neuropathogenesis of HCV and HIV-1 is limited.

HIV-1 exerts its direct neurotoxic effects through several secreted proteins including gp120, Tat, Nef and Vpr [14]. HIV-1 Viral Protein R (Vpr) triggers neuronal apoptosis and transgenic mice expressing Vpr in brain monocytoid cells display neuronal injury as well as neurobehavioral deficits [15]. Resident brain macrophages or microglia infected with HIV-1 or exposed to HIV-encoded proteins secrete proinflammatory cytokines e.g. interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) and chemokines e.g. CXCL10 and CXCL12, which cause neuronal death and pathogenic immune responses in the brain [14]. Suppression of neuronal autophagy by retroviral infections has been highlighted as a putative mechanism leading to neuronal cell death and neurodegeneration. The increased level of p62 transcript and the reduction of light chain 3 type II (LC3-II), markers of autophagy inhibition, were detected in brain tissues of patients with HIV-associated dementia [16], [17].

Recently, HCV proteins including core, non-structural protein 3 (NS3) and NS5A were detected in macrophages/microglia and astrocytes but not in neurons nor oligodendrocytes of patients with HIV/HCV co-infection [9], [18]. Nevertheless, the underlying mechanisms by which HCV infects macrophages/microglia or astrocytes remain unclear. The HCV JFH1 clone, derived from a Japanese individual with fulminant hepatitis, replicates and produces infectious virus in Huh 7.5 hepatocytes [19], [20]. The entry of HCV into cells involves several membrane receptors including the scavenger receptor class B type I (SR-BI), the tetraspanin CD81, and the tight-junction molecules, claudin-1 and occludin [21]. Given these receptors are expressed by microglia and astrocytes in human and mouse brains [22], [23], [24], [25], [26], these cells are potentially permissive to infection by HCV.

The most abundantly expressed HCV protein, core, is released and soluble in blood as a part of HCV morphogenesis [27] and serum levels of HCV core protein ranges from pg/ml to ng/ml [28]. HCV core protein is cytotoxic to hepatocytes through death-receptor mediated apoptosis [29], [30]; conversely, other studies have demonstrated that HCV core protein has anti-apoptotic effects in hepatocytes [29]. Recombinant HCV core protein fused to β-galactosidase induces the production of inflammatory cytokines, e.g. interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in monocytes [31] as well as the chemokine CXCL8 in monocytes [31] and lung fibroblasts [32]. Additionally, HCV core protein has been implicated in suppressing differentiation, proliferation and function of T cells, dendritic cells and macrophages [33]. Herein, we investigated cell culture-derived HCV (HCVcc) infection of astrocytes and microglia as well as the effects of HCV core protein on neurons, astrocytes and microglia in the presence or absence of HIV-1 Vpr protein. The present results demonstrated that HCV core protein caused immune activation of glial cells and was neurotoxic in an additive manner with the HIV-1 Vpr protein.

Discussion

During the last decade, there is mounting evidence to suggest HCV is neuroinvasive and HIV/HCV co-infected patients display higher rates of neuropsychological deficits. To delineate the underlying pathogenic mechanisms of HCV infection of the brain, we report for the first time that primary human astrocytes and microglia were permissive to HCV infection with cell culture-derived HCV particles. Moreover, this report demonstrates for the first time that HCV core protein activates human glia and contributes to neurotoxicity. Direct exposure of HCV core protein to primary human neurons suppressed the neuronal autophagy, leading to neurite retraction. The change in neuronal membrane potential after exposure to HCV core protein indicated that core was biologically active at the cell membrane and was able to modulate ionic conductance in neurons. In addition to direct neurotoxicity, proinflammatory cytokines and other neurotoxins released from HCV core-activated microglia into supernatants were toxic to neurons. The in vitro and in vivo aberrant immune activation and neurotoxicity mediated by HCV core protein were amplified in the presence of HIV-1 Vpr protein. These findings support the concept that the presence of HCV-encoded proteins cause neuronal damage and perhaps neurocognitive impairment in individuals co-infected with HIV and HCV.

Although HCV tropism is principally recognized in human liver cells, extrahepatic replication has been reported in PBMC [48], myocardium [49], [50] and brain cells [10], [18] by detecting HCV negative-strand RNA. Herein, detection of positive- and negative-strand RNA in different brain regions was performed in a patient with HIV and HCV co-infection and was consistent with previous reports [10], [18]. HCVcc infects and replicates in Huh 7.5 hepatocytes. However, productive infection was not detected in other hepatic cell lines (e.g. HepG2) [20], non-hepatic cell lines (e.g. HeLa, 293T and U-937) [20] and PBMC (e.g. B and T lymphocytes, monocytes and dendritic cells) [51], likely due to the lack of some entry receptors e.g. claudin-1 and their partner proteins [51] or host factors required for HCV infection e.g. liver specific microRNA miR-122 [52], [53], [54]. Herein, expression of HCV proteins, core and NS3, were immunodetected in the cytoplasm of primary human astrocytes and microglia infected with HCVcc. Although the levels of infectivity in primary astrocytes and microglia were low (less than 1%), HCV positive- and negative-strand RNA were measurable in infected astrocytes, suggesting that HCV is capable of replication in astrocytes. Both microglia and astrocytes were previously identified as HCV-infected cells in autopsied brains [9], [18] and were shown to express SR-BI, CD81, claudin-1 and occludin [22], [23], [24], [25], [26]. Although the expression levels of claudin-1 and occludin were 15 to 50 fold lower in primary human astrocytes and microglia compared with Huh 7.5 cells (Figure S2A), claudin-1 expression in glial cells was approximately 10 fold higher than in PBMC or 293T cells [51]. The higher expression of HCV entry receptors in primary astrocytes compared to microglia was also consistent with less frequently-detected HCV-immunopositive microglia after HCVcc infection. HCV replication in infected or transfected hepatocytes was reported in the membranous web structure closely associated with rough endoplasmic reticulum [55]. In the present studies, localization of HCV proteins in astrocytes was similarly associated with structures resembling endoplasmic reticulum. Further studies to determine if HCV-infected microglia or astrocytes release infectious viruses will be of interest.

HCVcc used herein belongs to the HCV genotype 2a [19] while most HCV strains in North America are genotypes 1a or 1b, which are associated with higher HCV RNA levels, poor responses to IFN-α treatment and lower mean CD4 count in HIV/HCV co-infected individuals [2]. The strain difference and other co-morbidities such as intravenous drug use might influence the infection and production of HCV in human brain, as demonstrated for HIV infection [56].

HCV infection has been correlated with cognitive impairment with changes indicative of neuronal damage by magnetic resonance spectroscopy [8]. Herein, we demonstrated that the direct exposure of HCV core protein or exposure to supernatants from HCV core-exposed microglia were toxic to primary human neurons together with neurobehavioral deficits and neuronal loss in HCV core-implanted animals. Although the toxic concentration of HCV core for primary human neurons in our in vitro experiments was higher than the reported serum levels in HCV-infected patients, it was consistent with previous studies [31], [32], [57]. It is plausible that HCV core concentrations at the surface of infected and proximate (target) cells are higher than in serum; moreover, chronic and repeated HCV exposures might yield augmented neurotoxic effects. Remarkably, the concentration of HCV core required to activate microglia and astrocytes was one log less than the concentration needed for direct neurotoxicity.

HIV-1 Vpr is highly neurotoxic without substantial proinflammatory properties [58] and its expression in brain monocytic cells contributes to synaptic injury and neurobehavioral abnormalities in transgenic mice [15]. At subtoxic concentrations, simultaneous exposure of HCV core and HIV-1 Vpr proteins to neurons exerted additive effects, resulting in increased IL-6 expression in glial cells and neuronal injury. Likewise, implantation of HCV core into the striatum of HIV-1 Vpr transgenic mice resulted in marked glial activation coupled with neuronal loss. It is likely that Vpr and HCV core act through different mechanisms to yield cumulative neurotoxic effects as Vpr's putative receptor is the nuclear glucocorticoid receptor [59] while HCV core's receptor might be a cell membrane protein [31], [32], [57], [60]. Additionally, it is conceivable that HCV core protein might also potentiate the neurotoxic or neuroinflammatory effects of other HIV-1 proteins e.g. Tat, Nef or gp120. This possibility might highlight the importance of interactions between HCV and HIV and warrants further investigation.

Our data showed that core protein from HCV genotype 1b triggered in vitro and in vivo activation of microglia and astrocytes, which is consistent with a recent magnetic resonance spectroscopy study that revealed an increased level of myo-inositol, a hallmark of gliosis, in frontal white matter of HCV-infected individuals [61]. Increased serum TNF-α and IL-1β levels in HCV-infected individuals were correlated with severity of neuropsychiatric dysfunction [62]. In vitro HCV infection in primary human macrophages resulted in increased TNF-α and CXCL8 [63]. Increased expression of CXCL10 and CXCL8 in astrocytes was also reported in brains of individuals with HIV encephalitis [64]. A recent study demonstrated that HIV-infected macrophages induced CXCL8 production from astrocytes through IL-1β and TNF-α [65]. The chemoattractant CXCL10 plays an important role in leukocyte infiltration into various tissues including CNS [66]. CXCL8 recruits not only neutrophils but also monocytes and lymphocytes [67]. It is plausible that the increased expression of CXCL10 and CXCL8 by HCV core-exposed microglia and astrocytes might enhance recruitment of T helper cells and monocytes in the brain, leading to deleterious effects. Of interest, we also observed that HCV core exposure to microglia induced the transcript levels of indoleamine 2,3-dioxygenase (IDO) (data not shown), which has been linked to neuronal injury [68], [69]. Additionally, we also demonstrated that supernatants from HCV core-treated microglia were neurotoxic. In these experiments, the concentration of HCV core protein was subtoxic to neurons but was sufficient to induce the expression of neurotoxic factors. It is also possible that there was carry-over of HCV core protein in supernatants which might play a role in the overall neurotoxicity. However, these results indicate that exposure of HCV core protein to glial cells could lead to deleterious effects on neurons.

Several studies have reported that recombinant HCV core activated cell surface receptors e.g. Toll-like receptor 2 (TLR2) on monocytes [31] and dendritic cells [70] or the putative HCV core receptor, gC1qR, on T lymphocytes [57], [60] and lung fibroblasts [32]. Microglia/macrophages are known to express both receptors [71], [72] while astrocytes express gC1qR but not TLR2 [72], [73] and there was a 30% increase in the gC1qR transcript level in HIV-infected brains (Figure S2B). Inhibition of either TLR2 or gC1qR by blocking antibodies or RNA silencing will be of interest. Nonetheless, HCV core might also be endocytosed by the microglia or astrocytes, and subsequently act through intracellular or nuclear receptors.

In summary, we reported primary human microglia and astrocytes were permissive to HCV infection and HCV-encoded protein, core, was neurotoxic but also activated pro-inflammatory responses in human microglia and astrocytes. The augmented glial activation and neurotoxicity mediated by HCV core protein in the presence of HIV-1 Vpr protein or HIV-1 infection highlighted the additive effects of HCV- and HIV-encoded proteins in the pathogenesis of neurologic disease. Future studies are required to delineate the precise mechanisms by which HIV and HCV proteins interact and amplify neuropathogenesis, thereby permitting the development of potential management and therapeutic strategies.

 
 
 
 
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