HIV-1 Nef increases astrocyte sensitivity towards exogenous hydrogen peroxide
© Masanetz and Lehmann; licensee BioMed Central Ltd. 2011
Received: 6 September 2010
Accepted: 22 January 2011
Published: 22 January 2011
HIV-1 infected individuals are under chronic exposure to reactive oxygen species (ROS) considered to be instrumental in the progression of AIDS and the development of HIV-1 associated dementia (HAD). Astrocytes support neuronal function and protect them against cytotoxic substances including ROS. The protein HIV-1 Nef, a progression factor in AIDS pathology is abundantly expressed in astrocytes in patients with HAD, and thus may influence its functions.
Endogenous expressed HIV-1 Nef leads to increased sensitivity of human astrocytes towards exogenous hydrogen peroxide but not towards TNF-alpha. Cell death of nef-expressing astrocytes exposed to 10 μM hydrogen peroxide for 30 min occurred within 4 h.
HIV-1 Nef may contribute to neuronal dysfunction and the development of HAD by causing death of astrocytes through decreasing their tolerance for hydrogen peroxide.
Both HIV-1 associated dementia (HAD) and a milder form of HIV-1 associated cognitive disorder, known as minor cognitive and motor disorder (MCMD) are frequent complications of the acquired immunodeficiency syndrome (AIDS) and are characterized by neuronal dysfunction and cell death caused by HIV-1 through direct and indirect mechanisms [1–4].
Recently, a sophisticated inspection of brains from HIV-1 infected patients confirmed that neurons are not infected with HIV-1 and surprisingly revealed that astrocytes, the most abundant cell type in the brain, are extensively infected. Additionally, this study elucidated that infection of astrocytes with HIV-1 correlated with the severity of neuropathology . Astrocytes play an important role in maintaining homeostasis, providing neuroprotection and regulating physiological activities within the brain [6–8]. Therefore, astrogliosis and astrocyte death occurring in HIV-infected individuals [9–12] may contribute indirectly to neuronal dysfunction.
Even though HIV-1 is integrated in the astrocyte genome, it rarely replicates in this cell type in vivo, however, regulatory proteins such as Nef are found to be abundantly expressed [13–15]. The presence of HIV-1 Nef in the brain is associated with astrogliosis and recruitment of monocytes/macrophages , correlating with the development of HAD .
Astrocytes stably transfected with HIV-1 Nef function as appropriate cellular model systems for the purpose of investigating basic mechanisms pertinent to the understanding of HAD pathogenesis. Using these cells, we previously showed that HIV-1 Nef induces CCL2/MCP-1 , thereby, providing an alternative hypothesis for the occurrence of this chemokine at high concentrations in the cerebrospinal fluid (CSF) of HIV-infected individuals with HAD [19, 20]. CCL2 plays an important role in the cerebral infiltration of monocytes/macrophages in these patients [21, 22]. Infiltrated and activated monocytes/macrophages, which are considered to be the effector cells in cellular and tissue damage in AIDS, produce cytotoxic substances such as reactive oxygen species (ROS) and inflammatory cytokines [23, 24]. Consequently, HIV-1 infected and non-infected astrocytes are subjected to an environment characterized, amongst others, by high concentrations of hydrogen peroxide and tumor necrosis factor (TNF)-alpha. Therefore, the aim of this study was to investigate the effect of HIV-1 Nef on the cellular viability of human astrocytes exposed to these particular cytotoxic substances.
Astrocytes stably transfected with HIV-1 nef are highly sensitive to hydrogen peroxide induced cell death
Hydrogen peroxide rapidly induced cell death of astrocytes stably transfected with HIV-1 nef
Astrocytes stably transfected with HIV-1 nef are as sensitive to TNF-alpha induced cell death as non-transfected cells
Chronic oxidative stress in HIV-infected patients plays an important role in AIDS progression [30, 31]. This phenomenon is explained by a depletion of endogenous antioxidant moieties and an increased production of ROS. Oxidative stress, in particular, is thought to be a cause of neuronal cell death in the brain of HIV-1 infected patients and believed to contribute to development of HAD [32, 33]. Moreover, ROS-induced astrocyte death is also thought to play a role in the occurrence of HAD [26, 27].
Here we show that a short exposure of exogenous hydrogen peroxide to nef-expressing astrocytes led to their rapid cell death. The early detection of a high number of propidium iodide/annexin V double positive cells points to necrotic cell death , which was previously suggested when astrocytes are subjected to tertiary-butyl hydroperoxide . But it can not be finally defined only from this observation what kind of cell death exactly occurred in our model. Also it depends on the concentration of hydrogen peroxide applied whether a cell dies in an apoptotic or necrotic manner . In this context it is interesting to note that astrocytes are vulnerable to hydrogen peroxide at concentrations ranging from 0.5 mM to 2.5 mM , values approximately a 1.000 fold higher than the concentration applied to induce death of nef-expressing astrocytes herein. So it remains a challenge for further studies to elucidate what HIV-1 Nef precisely alters in the cell leading to increased sensitivity to exogenous hydrogen peroxide. Intriguingly, it has been shown during the preparation of this manuscript that HIV-1 Nef in primary human astrocytes and in the brain of mice increases oxidative stress , which is in line with our finding.
Since HIV-1 Nef is known to inhibit apoptosis of T-cells [29, 38, 39] and monocytes/macrophages [40, 41], it was somewhat surprising that TNF-alpha decreased the viability of U251MG-Nef cells and U251MG-parental cells equally. Additionally, this finding is in contrast to previously reported data demonstrating that HIV-1 Nef prevents TNF-alpha triggered apoptosis in astrocytic U251MG cells . This discrepancy may be due to the use of cells stably transfected with nef in our study, which could clearly well simulate the long term effect of HIV-1 Nef in chronically infected cells  than cells transiently transfected with nef. Moreover, involvement of HIV-1 Nef in cell survival is subject to generally controversy [44, 45].
HIV-1 encodes a glutathione peroxidase , which has been shown to protect the cell against exogenous and endogenous ROS . Consequently, what ever the reason why HIV-1 Nef causes an increase of sensitivity towards hydrogen peroxide, it is conceivable that the HIV-1 GPX could counteract this action of HIV-1 Nef by detoxifying hydrogen peroxide. Thereby HIV-1 GPX would prevent the cytotoxic potential of HIV-Nef, which is considered as a progression factor in AIDS [48–50] and known to induce an AIDS-like disease in a mouse model [51, 52]. Thus, this could explain the paradoxical effect that functional HIV-1 GPXs are frequently found in long-term non-progressors while non-functional HIV-1 GPXs are present in HIV-1 isolates from patients developing AIDS .
Besides other known direct and indirect effects of HIV-1 proteins, HIV-1 Nef may contribute to cellular and tissue injury frequently detected in HIV-1 infected individuals, including various AIDS-associated diseases such as HAD, by increasing the sensitivity of Nef-harboring cells to hydrogen peroxide.
Immunoblotting and immunodetection
Lysates of U251MG-parental, -pNeo and -Nef cells were prepared by directly adding 1x SDS sample loading buffer to the cells followed by sonication. Samples were separated on a 4-20% tris-glycine gradient gel (Anamed, Darmstadt, Germany) and blotted on a nitrocellulose membrane. The blotted membranes were immunostained using mouse anti-Nef 3E6 mAb provided by K. Krohn through the National Institute for Biological Standards and Control Centralised Facility for AIDS Reagents, mouse anti-GAPDH mAb MAB347 (Chemicon International, Inc., Temecula, CA) and MFP488-conjugated goat anti-mouse antibody (MoBiTec GmbH, Göttingen, Germany), and positive signals were detected by fluorescence scanning (excitation wavelength 488 nm, emission filter 520BP40) using the Typhoon 9410 Fluorescence Scanner (GE Healthcare), and analyzed using ImageQuant 5.2 software (Molecular Dynamics).
Cell viability assay
The AlamarBlue® reagent (Molecular Probes, Inc., Eugene, OR) containing the water soluble, non-toxic dye resazurin (7-Hydroxy-3H-phenoxazin-3-one 10-oxide) was used to quantify mitochondrial activity according to the manufacturer's recommendation. Briefly, 1/10th of the volume of AlamarBlue® reagent was added directly to the cells in culture medium. Using the Typhoon™ 9410 fluorescence scanner (GE Healthcare), fluorescence measurement was performed by applying an excitation wavelength of 532 nm and an emission filter of 580BP30 nm. Data were analyzed using ImageQuant™ TL software (GE Healthcare). The fluorescence intensity of medium containing only AlamarBlue® was determined simultaneously and was subtracted from all values.
Annexin V assay
Phosphatidylserine on the cell surface was detected with the Annexin V-FITC Apoptosis Detection Kit I (BD Biosciences). Briefly, cells were plated and treated in 12-well plates (Costar). Then cells were washed twice with cold PBS and incubated in the dark for 15 min in 1 × binding buffer supplemented with annexin V-FITC. Propidium iodide (PI) was added to the cell suspension immediately before analyzing with the BD FACSCanto™ flow cytometer (BD Biosciences). Data were evaluated using FlowJo© software (Tree Star).
GraphPad Prism 4 (GraphPad Software, Inc., San Diego, CA) was used for statistical analysis. The Mann-Whitney test was used to compare the groups; a P value of less than 0.05 was considered significant. Tests were performed exactly and two-tailed.
After receiving her M.Sc. in Molecular Biotechnology, SM moved to the Physiology Weihenstephan, Technical University Munich, Freising, Germany to work for her PhD.
MHL received his PhD in Biology from the Friedrich-Schiller-University of Jena, Germany and currently holds a faculty position at the Institute for Infectious Diseases and Zoonoses, Ludwig-Maximilians-Universität München, Germany.
The authors wish to thank Susanne Kramer for providing astrocytic cells, Nasim Kroegel, B.Sc., for reviewing the manuscript, and Volker Erfle for his general support. This study was supported by an internal grant from the Helmholtz Zentrum München.
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