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.
- Gonzalez-Scarano F, Martin-Garcia J: The neuropathogenesis of AIDS. Nat Rev Immunol 2005, 5:69–81.PubMedView Article
- Kaul M, Garden GA, Lipton SA: Pathways to neuronal injury and apoptosis in HIV-associated dementia. Nature 2001, 410:988–994.PubMedView Article
- Minagar A, Commins D, Alexander JS, Hoque R, Chiappelli F, Singer EJ, Nikbin B, Shapshak P: NeuroAIDS: characteristics and diagnosis of the neurological complications of AIDS. Mol Diagn Ther 2008, 12:25–43.PubMed
- Piacentini M, Kroemer G: Cell death pathways in retroviral infection. Cell Death Differ 2005,12(1):835–836.PubMedView Article
- Churchill MJ, Wesselingh SL, Cowley D, Pardo CA, McArthur JC, Brew BJ, Gorry PR: Extensive astrocyte infection is prominent in human immunodeficiency virus-associated dementia. Ann Neurol 2009, 66:253–258.PubMedView Article
- Perea G, Araque A: Communication between astrocytes and neurons: a complex language. J Physiol Paris 2002, 96:199–207.PubMedView Article
- Chen Y, Swanson RA: Astrocytes and brain injury. J Cereb Blood Flow Metab 2003, 23:137–149.PubMedView Article
- Bouzier-Sore AK, Merle M, Magistretti PJ, Pellerin L: Feeding active neurons: (re)emergence of a nursing role for astrocytes. J Physiol Paris 2002, 96:273–282.PubMedView Article
- Epstein LG, Gendelman HE: Human immunodeficiency virus type 1 infection of the nervous system: pathogenetic mechanisms. Ann Neurol 1993, 33:429–436.PubMedView Article
- Sabri F, Titanji K, De Milito A, Chiodi F: Astrocyte activation and apoptosis: their roles in the neuropathology of HIV infection. Brain Pathol 2003, 13:84–94.PubMedView Article
- Shi B, De Girolami U, He J, Wang S, Lorenzo A, Busciglio J, Gabuzda D: Apoptosis induced by HIV-1 infection of the central nervous system. J Clin Invest 1996, 98:1979–1990.PubMedView Article
- Petito CK, Roberts B: Evidence of apoptotic cell death in HIV encephalitis. Am J Pathol 1995, 146:1121–1130.PubMed
- Bagasra O, Lavi E, Bobroski L, Khalili K, Pestaner JP, Tawadros R, Pomerantz RJ: Cellular reservoirs of HIV-1 in the central nervous system of infected individuals: identification by the combination of in situ polymerase chain reaction and immunohistochemistry. AIDS 1996, 10:573–585.PubMedView Article
- Tornatore C, Chandra R, Berger JR, Major EO: HIV-1 infection of subcortical astrocytes in the pediatric central nervous system. Neurology 1994, 44:481–487.PubMed
- Saito Y, Sharer LR, Epstein LG, Michaels J, Mintz M, Louder M, Golding K, Cvetkovich TA, Blumberg BM: Overexpression of nef as a marker for restricted HIV-1 infection of astrocytes in postmortem pediatric central nervous tissues. Neurology 1994, 44:474–481.PubMed
- Mordelet E, Kissa K, Cressant A, Gray F, Ozden S, Vidal C, Charneau P, Granon S: Histopathological and cognitive defects induced by Nef in the brain. FASEB J 2004, 18:1851–1861.PubMedView Article
- Ranki A, Nyberg M, Ovod V, Haltia M, Elovaara I, Raininko R, Haapasalo H, Krohn K: Abundant expression of HIV Nef and Rev proteins in brain astrocytes in vivo is associated with dementia. AIDS 1995, 9:1001–1008.PubMedView Article
- Lehmann MH, Masanetz S, Kramer S, Erfle V: HIV-1 Nef upregulates CCL2/MCP-1 expression in astrocytes in a myristoylation- and calmodulin-dependent manner. J Cell Sci 2006, 119:4520–4530.PubMedView Article
- Conant K, Garzino-Demo A, Nath A, McArthur JC, Halliday W, Power C, Gallo RC, Major EO: Induction of monocyte chemoattractant protein-1 in HIV-1 Tat-stimulated astrocytes and elevation in AIDS dementia. Proc Natl Acad Sci USA 1998, 95:3117–3121.PubMedView Article
- Cinque P, Vago L, Mengozzi M, Torri V, Ceresa D, Vicenzi E, Transidico P, Vagani A, Sozzani S, Mantovani A, et al.: Elevated cerebrospinal fluid levels of monocyte chemotactic protein-1 correlate with HIV-1 encephalitis and local viral replication. AIDS 1998, 12:1327–1332.PubMedView Article
- Eugenin EA, Osiecki K, Lopez L, Goldstein H, Calderon TM, Berman JW: CCL2/monocyte chemoattractant protein-1 mediates enhanced transmigration of human immunodeficiency virus (HIV)-infected leukocytes across the blood-brain barrier: a potential mechanism of HIV-CNS invasion and NeuroAIDS. J Neurosci 2006, 26:1098–1106.PubMedView Article
- Gonzalez E, Rovin BH, Sen L, Cooke G, Dhanda R, Mummidi S, Kulkarni H, Bamshad MJ, Telles V, Anderson SA, et al.: HIV-1 infection and AIDS dementia are influenced by a mutant MCP-1 allele linked to increased monocyte infiltration of tissues and MCP-1 levels. Proc Natl Acad Sci USA 2002, 99:13795–13800.PubMedView Article
- Brabers NA, Nottet HS: Role of the pro-inflammatory cytokines TNF-alpha and IL-1beta in HIV-associated dementia. Eur J Clin Invest 2006, 36:447–458.PubMedView Article
- Williams KC, Hickey WF: Central nervous system damage, monocytes and macrophages, and neurological disorders in AIDS. Annu Rev Neurosci 2002, 25:537–562.PubMedView Article
- Desagher S, Glowinski J, Premont J: Astrocytes protect neurons from hydrogen peroxide toxicity. J Neurosci 1996, 16:2553–2562.PubMed
- Robb SJ, Connor JR: An in vitro model for analysis of oxidative death in primary mouse astrocytes. Brain Res 1998, 788:125–132.PubMedView Article
- Feeney CJ, Frantseva MV, Carlen PL, Pennefather PS, Shulyakova N, Shniffer C, Mills LR: Vulnerability of glial cells to hydrogen peroxide in cultured hippocampal slices. Brain Res 2008, 1198:1–15.PubMedView Article
- Kroemer G, El-Deiry WS, Golstein P, Peter ME, Vaux D, Vandenabeele P, Zhivotovsky B, Blagosklonny MV, Malorni W, Knight RA, et al.: Classification of cell death: recommendations of the Nomenclature Committee on Cell Death. Cell Death Differ 2005,12(2):1463–1467.PubMedView Article
- Geleziunas R, Xu W, Takeda K, Ichijo H, Greene WC: HIV-1 Nef inhibits ASK1-dependent death signalling providing a potential mechanism for protecting the infected host cell. Nature 2001, 410:834–838.PubMedView Article
- Baruchel S, Wainberg MA: The role of oxidative stress in disease progression in individuals infected by the human immunodeficiency virus. J Leukoc Biol 1992, 52:111–114.PubMed
- Pace GW, Leaf CD: The role of oxidative stress in HIV disease. Free Radic Biol Med 1995, 19:523–528.PubMedView Article
- Gray F, Adle-Biassette H, Chretien F, Lorin de la Grandmaison G, Force G, Keohane C: Neuropathology and neurodegeneration in human immunodeficiency virus infection. Pathogenesis of HIV-induced lesions of the brain, correlations with HIV-associated disorders and modifications according to treatments. Clin Neuropathol 2001, 20:146–155.PubMed
- Mollace V, Nottet HS, Clayette P, Turco MC, Muscoli C, Salvemini D, Perno CF: Oxidative stress and neuroAIDS: triggers, modulators and novel antioxidants. Trends Neurosci 2001, 24:411–416.PubMedView Article
- Vermes I, Haanen C, Steffens-Nakken H, Reutelingsperger C: A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J Immunol Methods 1995, 184:39–51.PubMedView Article
- Robb SJ, Connor JR: Nitric oxide protects astrocytes from oxidative stress. Ann N Y Acad Sci 2002, 962:93–102.PubMedView Article
- Hampton MB, Orrenius S: Dual regulation of caspase activity by hydrogen peroxide: implications for apoptosis. FEBS Lett 1997, 414:552–556.PubMedView Article
- Acheampong EA, Roschel C, Mukhtar M, Srinivasan A, Rafi M, Pomerantz RJ, Parveen Z: Combined effects of hyperglycemic conditions and HIV-1 Nef: a potential model for induced HIV neuropathogenesis. Virol J 2009, 6:183.PubMedView Article
- Wolf D, Witte V, Laffert B, Blume K, Stromer E, Trapp S, d'Aloja P, Schurmann A, Baur AS: HIV-1 Nef associated PAK and PI3-kinases stimulate Akt-independent Bad-phosphorylation to induce anti-apoptotic signals. Nat Med 2001, 7:1217–1224.PubMedView Article
- Greenway AL, McPhee DA, Allen K, Johnstone R, Holloway G, Mills J, Azad A, Sankovich S, Lambert P: Human immunodeficiency virus type 1 Nef binds to tumor suppressor p53 and protects cells against p53-mediated apoptosis. J Virol 2002, 76:2692–2702.PubMedView Article
- Choi HJ, Smithgall TE: HIV-1 Nef promotes survival of TF-1 macrophages by inducing Bcl-XL expression in an extracellular signal-regulated kinase-dependent manner. J Biol Chem 2004, 279:51688–51696.PubMedView Article
- Olivetta E, Federico M: HIV-1 Nef protects human-monocyte-derived macrophages from HIV-1-induced apoptosis. Exp Cell Res 2006, 312:890–900.PubMedView Article
- Robichaud GA, Poulin L: HIV type 1 nef gene inhibits tumor necrosis factor alpha-induced apoptosis and promotes cell proliferation through the action of MAPK and JNK in human glial cells. AIDS Res Hum Retroviruses 2000, 16:1959–1965.PubMedView Article
- Kramer-Hammerle S, Hahn A, Brack-Werner R, Werner T: Elucidating effects of long-term expression of HIV-1 Nef on astrocytes by microarray, promoter, and literature analyses. Gene 2005, 358:31–38.PubMedView Article
- Schindler M, Munch J, Kirchhoff F: Human immunodeficiency virus type 1 inhibits DNA damage-triggered apoptosis by a Nef-independent mechanism. J Virol 2005, 79:5489–5498.PubMedView Article
- Laforge M, Petit F, Estaquier J, Senik A: Commitment to apoptosis in CD4(+) T lymphocytes productively infected with human immunodeficiency virus type 1 is initiated by lysosomal membrane permeabilization, itself induced by the isolated expression of the viral protein Nef. J Virol 2007, 81:11426–11440.PubMedView Article
- Zhao L, Cox AG, Ruzicka JA, Bhat AA, Zhang W, Taylor EW: Molecular modeling and in vitro activity of an HIV-1-encoded glutathione peroxidase. Proc Natl Acad Sci USA 2000, 97:6356–6361.PubMedView Article
- Cohen I, Boya P, Zhao L, Metivier D, Andreau K, Perfettini JL, Weaver JG, Badley A, Taylor EW, Kroemer G: Anti-apoptotic activity of the glutathione peroxidase homologue encoded by HIV-1. Apoptosis 2004, 9:181–192.PubMedView Article
- Dyer WB, Geczy AF, Kent SJ, McIntyre LB, Blasdall SA, Learmont JC, Sullivan JS: Lymphoproliferative immune function in the Sydney Blood Bank Cohort, infected with natural nef/long terminal repeat mutants, and in other long-term survivors of transfusion-acquired HIV-1 infection. AIDS 1997, 11:1565–1574.PubMedView Article
- Hofmann-Lehmann R, Vlasak J, Williams AL, Chenine AL, McClure HM, Anderson DC, O'Neil S, Ruprecht RM: Live attenuated, nef-deleted SIV is pathogenic in most adult macaques after prolonged observation. AIDS 2003, 17:157–166.PubMedView Article
- Kestler HW, Ringler DJ, Mori K, Panicali DL, Sehgal PK, Daniel MD, Desrosiers RC: Importance of the nef gene for maintenance of high virus loads and for development of AIDS. Cell 1991, 65:651–662.PubMedView Article
- Simard MC, Chrobak P, Kay DG, Hanna Z, Jothy S, Jolicoeur P: Expression of simian immunodeficiency virus nef in immune cells of transgenic mice leads to a severe AIDS-like disease. J Virol 2002, 76:3981–3995.PubMedView Article
- Hanna Z, Kay DG, Rebai N, Guimond A, Jothy S, Jolicoeur P: Nef harbors a major determinant of pathogenicity for an AIDS-like disease induced by HIV-1 in transgenic mice. Cell 1998, 95:163–175.PubMedView Article
- Kohleisen B, Shumay E, Sutter G, Foerster R, Brack-Werner R, Nuesse M, Erfle V: Stable expression of HIV-1 Nef induces changes in growth properties and activation state of human astrocytes. AIDS 1999, 13:2331–2341.PubMedView Article
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