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Journal of Virology, February 2007, p. 1492-1501, Vol. 81, No. 3
0022-538X/07/$08.00+0     doi:10.1128/JVI.01843-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Cholesterol-Depleting Statin Drugs Protect Postmitotically Differentiated Human Neurons against Ethanol- and Human Immunodeficiency Virus Type 1-Induced Oxidative Stress In Vitro{triangledown}

Edward Acheampong,1 Zahida Parveen,1* Aschalew Mengistu,1 Noel Ngoubilly,1 Brian Wigdahl,2 Albert S. Lossinsky,3 Roger J. Pomerantz,4 and Muhammad Mukhtar2*

Dorrance H. Hamilton Laboratories, Division of Infectious Diseases, Department of Medicine, Thomas Jefferson University, 1020 Locust Street, Suite 329, Philadelphia, Pennsylvania 19107,1 Department of Microbiology and Immunology, Institute for Molecular Medicine and Infectious Disease, Drexel University College of Medicine, Philadelphia, Pennsylvania 19102,2 Immunohistochemistry and Electron Microscopy Laboratories, Neural Engineering Program, Huntington Medical Research Institutes, Pasadena, California,3 Tibotec Inc., 1020 Stony Hill Road, Suite 300, Yardley, Pennsylvania 190674

Received 23 August 2006/ Accepted 7 November 2006


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ABSTRACT
 
The majority of human immunodeficiency virus type 1 (HIV-1)-infected individuals are either alcoholics or prone to alcoholism. Upon ingestion, alcohol is easily distributed into the various compartments of the body, particularly the brain, by crossing through the blood-brain barrier. Both HIV-1 and alcohol induce oxidative stress, which is considered a precursor for cytotoxic responses. Several reports have suggested that statins exert antioxidant as well as anti-inflammatory pleiotropic effects, besides their inherent cholesterol-depleting potentials. In our studies, postmitotically differentiated neurons were cocultured with HIV-1-infected monocytes, T cells, or their cellular supernatants in the presence of physiological concentrations of alcohol for 72 h. Parallel cultures were pretreated with statins (atorvastatin and simvastatin) with the appropriate controls, i.e., postmitotically differentiated neurons cocultured with uninfected cells and similar cultures treated with alcohol. The oxidative stress responses in the presence/absence of alcohol in these cultures were determined by the production of the well-characterized oxidative stress markers, 8-isoprostane-F2-{alpha}, total nitrates as an indicator for various isoforms of nitric oxide synthase activity, and heat shock protein 70 (Hsp70). An in vitro culture of postmitotically differentiated neurons with HIV-1-infected monocytes or T cells as well as supernatants from these cells enhanced the release of 8-isoprostane-F2-{alpha} in the conditioned medium six- to sevenfold (monocytes) and four- to fivefold (T cells). It was also observed that coculturing of HIV-1-infected primary monocytes over a time period of 72 h significantly elevated the release of Hsp70 compared with that of uninfected controls. Cellular supernatants of HIV-1-infected monocytes or T cells slightly increased Hsp70 levels compared to neurons cultured with uninfected monocytes or T-cell supernatants (controls). Ethanol (EtOH) presence further elevated Hsp70 in both infected and uninfected cultures. The amount of total nitrates was significantly elevated in the coculture system when both infected cells and EtOH were present. Surprisingly, pretreatment of postmitotic neurons with clinically available inhibitors of HMG-coenzyme A reductase (statins) inhibited HIV-1-induced release of stress/toxicity-associated parameters, i.e., Hsp70, isoprostanes, and total nitrates from HIV-1-infected cells. The results of this study provide new insights into HIV-1 neuropathogenesis aimed at the development of future HIV-1 therapeutics to eradicate viral reservoirs from the brain.


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INTRODUCTION
 
The AIDS-related dementia characterized by cognitive dysfunction, motor neuron disease, coordination abnormalities, and other neurological signs and symptoms develops in many human immunodeficiency virus type 1 (HIV-1)-infected individuals (17, 58). The molecular mechanisms involved in these HIV-1-associated dysfunctions of the central nervous system (CNS) remain incompletely explained and controversial (42, 45). Studies have demonstrated that although the major reservoirs for productive infection within the CNS of HIV-1-infected individuals are microglia and monocytes/macrophages, infection of other CNS-based cell types, such as microvascular endothelial cells and neurons, also appears to occur (44, 57, 92). The role of lymphocytes in AIDS dementia has also been described previously (59, 67, 68). Of note, viral replication in cerebrospinal fluid is considered to be a marker for the severity of AIDS-related neurodegeneration (16, 20). The CNS tissues are protected from the periphery by a structure known as blood-brain barrier (BBB), which limits the passage of solutes as well as infections into the brain (10). The tight junctions of brain microvascular endothelial cells strengthen the functional impermeability of BBB. Under certain pathological conditions, the BBB becomes compromised, providing access for infections/infected cells into the brain (64). The passage of HIV-1 entry into the brains of AIDS patients in vivo is not quite clear and is supposed to involve both free viruses and virally infected cells (4, 89, 91).

HIV-1-infected individuals are under chronic oxidative stress, and antioxidants have proved to ameliorate infection-associated oxidative stress. (81, 84). Oxidative stress involves disturbance in the equilibrium status of prooxidant/antioxidant systems of intact cells (11). In HIV infection, oxidative stress may be caused by both overproduction of reactive oxygen intermediates and a simultaneous deficiency of antioxidant defenses (31). Overall oxidative stress is manifested as a depletion of endogenous antioxidant moieties and an increased production of reactive oxygen species such as oxygen and nitric oxide (NO) free radicals (5, 97). Elevated expression of nitric oxide synthase (NOS), an enzyme responsible for the generation of NO free radicals in AIDS dementia, further supports the potential of oxidative stress in neuronal damage and deleterious effects on the BBB, a CNS neuroprotective layer (23, 52). The NO free radicals potentially react with oxygen free radicals, generating various cytotoxic stimuli as well as peroxinitrite, a strong oxidizing agent for various biomolecules (27, 43, 86). Recently, free radicals and peroxynitrite-mediated activation of matrix metalloproteinases, a family of proteolytic enzymes capable of degrading components of the extracellular matrix, were also described (50, 69, 77).

Prooxidative stress responses could be elevated by several cofactors, such as alcohol intake, which is more prevalent among individuals prone to or infected with HIV-1. Of importance, in vitro studies suggest deleterious effects of alcohol in HIV-1-infected individuals. For example, ethanol (EtOH) has been shown to increase tumor necrosis factor alpha-stimulated HIV-1 long terminal repeat-induced transcription in Jurkat T cells (22). Similarly, the HIV-1 regulatory protein Tat, in combination with EtOH, induced synergistic neutrophil dysfunction in transgenic mice (72). Our laboratory has shown that EtOH can cause extensive perturbations within CNS-based cells (1, 19). Furthermore, it has been shown that EtOH can increase the expression of transcription factors (66) and the secretion of heat shock proteins (62).

The present study dissects whether EtOH and HIV-1 work synergistically to induce oxidative stress and inflammatory responses in an in vitro model of CNS-based cells. We have selectively analyzed the interactions of HIV-1-infected immune cells or their cellular supernatants with postmitotically differentiated neurons by determining various oxidative stress markers such as 8-isoprostaglandin-F2-{alpha}, heat shock protein 70 (Hsp70), and total nitrate levels as an indicator for the activity of various NOS enzymes. The potential of cholesterol-depleting statin drugs in controlling the secretions of these markers in this in vitro neuronal culture system was also analyzed. Our studies suggest that the presence of HIV-1-infected cells or cellular supernatants of these cells induces oxidative stress-related neurotoxicitites. Concomitant with HIV-1-infected cells, the presence of EtOH further enhanced neurotoxicities and statins normalized these cytotoxic responses. These data provide important information relevant to the role of HIV-1-infected cells in the CNS compartment and suggest potential neuroprotective capabilities of statins in this in vitro model that need to be further validated in the in vivo setup.


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MATERIALS AND METHODS
 
Maintenance and differentiation of NT2 cells. NT2 precursor cells, obtained from the American Type Culture Collection (ATCC), have the capability of differentiating in vitro into postmitotic neurons by induction with retinoic acid (RA) (61, 99). NT2 precursor cells were grown in T75 tissue culture flasks in Dulbecco's modified Eagle's medium (DMEM) supplemented with fetal calf serum (FCS) and glutamine in a humidified environment at 37°C under 5% CO2. The differentiation of NT2 precursor cells to postmitotic neurons involved growing these cells in RA-supplemented DMEM over a period of 5 to 6 weeks. Briefly, actively growing NT2 precursor cells were passaged in T75 tissue culture flasks to a final concentration of 2.3 x 106 cells per flask. After 24 h, the cells were fed with RA-supplemented DMEM, followed by the same medium at 48-h intervals for 6 weeks. The cellular morphology was constantly observed until neuronal maturation was clearly visible microscopically. After 6 weeks, postmitotic neuron isolation was performed as described previously (61). Matured neurons were treated with mitotic inhibitor medium for selectively enriching postmitotic cells. The inhibitor medium consisted of high-glucose DMEM, 5% (vol/vol) FCS, 1% (vol/vol) penicillin-streptomycin, 5-fluoro-2'-deoxyuridine to a final concentration of 10 µM, uridine (10 µM), and cytosine ß-D-arabinofuranoside (1 µM). Finally, the neurons were removed from the mitotic inhibitor medium and grown on poly-D-lysine- and Matrigel-coated six-well plates. The purity of each differentiated neuronal batch was confirmed by immunostaining with the neuroepithelial marker microtubule-associated protein (MAP2) (Pharmingen, San Diego, CA) utilized to identify neurons (Fig. 1B).


Figure 1
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  		FIG. 1. Characterization of postmitotic neurons and other cells. Postmitotically differentiated neurons derived from neuronal cell line NT2 upon treatment with RA, followed by selective isolation. (A) Isolated neurons. (B and C) Immunofluorescence staining for the expression of cellular MAP2 and CD14 marker for postmitotic neurons and monocytes, respectively.

CD14+ monocyte and CD3+ T-lymphocyte isolation. Peripheral blood mononuclear cells (PBMCs) from HIV-1-seronegative donors were isolated according to guidelines of the U.S. Department of Health and Human Services and modifications of a previously published technique (61). After being layered on Histopaque 1077 (Sigma-Aldrich, St. Louis, MO), PBMCs were washed and pelleted. CD3+ T lymphocytes and CD14+ monocytes were isolated from the PBMCs by direct immunomagnetic labeling, using antibodies against their respective surface markers (Miltenyi Inc.). The positive fractions were collected and cultured in RPMI 1640 medium with 10% FCS or human antibody serum in the case of CD14+ cells and penicillin plus streptomycin at 37°C in a humidified environment. The CD3+ lymphocytes were cultured in the RPMI 1640 medium supplemented with 10 to 20 ng/ml of interleukin-2 (R&D Systems Inc., MN).

Preparation of viral stocks. The viral stocks used for our studies were the X4-tropic HIV-1 strain, NL4-3, and the R5-tropic strain, YU2. These were produced by transfection of 293T cells with the respective proviral DNA as described previously (60). Briefly, 48 h after transfection of proviral DNA via the calcium phosphate transfection method, supernatants were collected, filtered through a 0.45-µm-pore-size filter, quantified by an HIV-1 p24 antigen enzyme-linked immunosorbent assay (ELISA) (NEN Life Science Products, Inc., Boston, MA), and stored at –80°C until needed.

HIV-1 infection of primary human T lymphocytes and monocytes. Two different laboratory strains, NL4-3 and YU-2, a T-cell line (X4), and monocyte tropic (RS) strain, respectively, were used for infection. HIV-1 p24 antigen (10 ng/ml) virus was utilized to infect 3.0 x 106 CD3+ T lymphocytes and CD14+ monocytes. After 4 h of infection, cells were washed thoroughly three times to remove excess viral inputs. Cells were then cultured for up to 7 days, and supernatants were collected and stored frozen until use. HIV-1 p24 antigen levels in the supernatants were measured by ELISA (PerkinElmer Life Sciences, Inc., Boston, MA). The infected cells were also collected and used in subsequent experiments.

Exposure of neurons to infected cells and supernatants. Postmitotically differentiated human neurons were seeded in six-well plates in 50% conditioned medium and 50% DMEM. The cells were allowed to attach for several days before being exposed to infected cells and supernatants from CD3+ T cells and CD14+ monocytes. Four hours after exposure to viral supernatants, the cells were washed at least three times to remove viral supernatants and cultured in 50% conditioning medium with or without EtOH at concentrations of 0.1 and 0.3% (vol/vol). These concentrations correlate with the clinical plasma levels of EtOH leading to intoxication in humans (34, 35). The statins atorvastatin and simvastatin were added at concentrations of 10 µM when needed. Following treatment with virus and EtOH, the cells were incubated for 48 h at 37°C and 5% CO2 in a humidified environment. As negative controls, cells that had not been treated with any virus, statins, or EtOH were also used.

Detection of Hsp70, 8-iso-PGF-2{alpha}, and nitric oxide. Primary human differentiated neurons were exposed to either infected cells or supernatants from CD3+ T cells and CD14+ monocytes with or without EtOH, followed by incubation for 48 h at 37°C and collection of supernatants. The levels of Hsp70, 8-isoprostaglandin-F2-{alpha} (8-iso-PGF-2{alpha}), and NO in the supernatants were measured by ELISA kits (Stressgen Biotechnologies, Victoria, BC, Canada).

Protein assays and Western blot analyses. The cellular lysates of postmitotically differentiated neurons were analyzed for the expression of Hsp70. Cells were exposed either to HIV-1-infected T cells or monocytes and statins and incubated for 48 h at 37°C with 5% CO2 in a humidified environment. Cells were then harvested and disrupted with mammalian cell lysis buffer (Pierce Biotechnologies, Rockford, IL). Protein concentrations were determined with a bicinchoninic acid protein assay kit (Pierce Biotechnologies). Approximately 25 µg of each protein preparation was resolved on sodium dodecyl sulfate-12% polyacrylamide gels (Bio-Rad) and transferred to polyvinylidene difluoride membranes (Amersham Biosciences, Piscataway, NJ) by electroblotting. Membranes were washed in phosphate-buffered saline containing 0.01% Tween 20 (Sigma-Aldrich, St. Louis, MO) and then blocked for nonspecific proteins with phosphate-buffered saline-based blocking buffer (Pierce Biotechnologies, Rockford, IL). The membranes were probed with specific monoclonal antibodies against Hsp70 (Stressgen Biotechnologies, Victoria, BC, Canada) as primary antibodies and horseradish peroxidase-labeled goat or anti-mouse immunoglobulin G (heavy plus light chains) as secondary antibodies. The protein-antibody complexes were visualized by incubating the membranes with the SuperSignal West Femto Western blotting detection system (Pierce Biotechnologies, Rockford, IL) and subsequently exposing them to BioMax MS autoradiographic film (Kodak, Rochester, NY).


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RESULTS
 
We analyzed the effects of HIV-1-infected primary monocyte and T-cell interactions with postmitotically differentiated neurons either in the presence or in the absence of EtOH. Our main focus was on three well-characterized oxidative stress markers, i.e., 8-isoprostane-F2-{alpha}, Hsp70, and total nitrate, an indirect measurement for various isoforms of NOS.

Postmitotically differentiated neurons were generated from the neuronal precursor cell line NT2 by treating them with RA for 6 weeks, followed by selective isolation of differentiated neurons (Fig. 1A). The purity of these cells was further confirmed by immunostaining with the neuroepithelial marker MAP2 (Fig. 1B). Primary isolated monocytes were immunostained with CD14 markers (Fig. 1C) and CD3+ T lymphocytes characterized with anti-CD3+ antibody (not shown).

Production of Hsp70. HIV-1-infected monocytes or T cells were cocultured with postmitotic neurons either in the presence or in the absence of EtOH. The levels of Hsp70, which is considered to be a biomarker of cellular toxicity (24), in the cellular supernatants were determined after 72 h. As revealed in Fig. 2A, coculturing of neurons and HIV-1-infected primary monocytes resulted in a fourfold increase in the production of Hsp70 compared with that of neurons and uninfected controls. The addition of 0.1 and 0.3% EtOH augmented this response; however, 0.3% EtOH alone elevated the levels of Hsp70 in uninfected controls, suggesting EtOH-associated cytotoxicity in postmitotic neurons. Neurons exposed to supernatants generated from HIV-1-infected monocytes showed elevation in the expression of Hsp70 compared with neurons exposed to uninfected monocyte supernatants (controls). The addition of 0.3% EtOH to these cultures resulted in a twofold increase in Hsp70 production. Coculturing of neurons with infected primary CD3+ T cells or neurons exposed to supernatants from HIV-1-infected primary CD3+ T cells showed similar increases in Hsp70 production (two- to threefold elevation upon exposure to either 0.1% or 0.3% EtOH) (Fig. 2B).


Figure 2
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  		FIG. 2. HIV-1-infected CD14+ monocytes and CD3+ T cells significantly induced the secretion of Hsp70 from postmitotically differentiated neurons. (A) The release of Hsp70 from postmitotic neurons cocultured with macrophage tropic HIV-1, YU2-infected CD14+ monocytes (Mono), or uninfected controls was determined. Parallel coculture setup of the same experiment with 0.1% and 0.3% EtOH was also carried out for the determination of Hsp70 release. The secretion of Hsp70 in the cocultured conditioned cellular supernatant (Sup) was quantified using the StressXpress ELISA (Stressgen, Victoria, BC, Canada). (B) The HIV-1-infected, NL4-3-infected, and uninfected CD3+ T lymphocytes and their supernatants were then cocultured with postmitotic neurons either in the presence or in the absence of 0.1% or 0.3% EtOH. Hsp70 release from conditioned cellular supernatants was quantified as described above. Results are the mean values for duplicate samples ± standard errors of the means. Experiments were carried out at least two times.

Production of 8-isoprostane-F2-{alpha}. Besides Hsp70 levels, we also determined the levels of 8-iso-PGF-2{alpha}, a major isoprostane, and a universally accepted oxidative stress marker produced by the nonenzymatic free radical-induced peroxidation of arachidonic acid present in phospholipids (55). The increased production of 8-iso-PGF-2{alpha} is usually associated with pathological conditions known to involve a heightened state of oxidative stress (33, 79). Coculturing of HIV-1-infected monocytes or T cells with postmitotically differentiated neurons with or without alcohol was performed as described above. After 72 h, the supernatants were collected and analyzed for the production of 8-iso-PGF-2{alpha}. The results of the experiments are given in Fig. 3A and B. EtOH itself at concentration levels of 0.1 to 0.3% has minimal effect on 8-iso-PGF-2{alpha} production. HIV-1-infected monocytes as well as supernatants from infected monocytes resulted in a six- to sevenfold increase in the production of 8-iso-PGF-2{alpha} in the conditioned medium. Similarly, coculturing of HIV-1-infected T cells with neurons enhanced the production of 8-iso-PGF-2{alpha} about four- to fivefold, whereas supernatants from infected T cells did not produce a significant increase in the production of this oxidative stress marker. Coculturing of neurons with EtOH and HIV-1 cells elicited an enhanced release of this stress marker.


Figure 3
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  		FIG. 3. HIV-1-infected CD14+ monocytes and CD3+ T lymphocytes significantly induced the secretion of 8-isoprostaglandin-F2-{alpha} from postmitotically differentiated neurons. (A) Postmitotically differentiated neurons were cocultured with either HIV-1-infected CD14+ monocytes (Mono) or their cellular conditioned supernatants (Sup) with and without EtOH. (B) The CD3+ T lymphocytes infected with HIV-1 NL4-3 and uninfected controls were cocultured with postmitotically differentiated neurons. Neurons were also cultured in the presence of cellular supernatants obtained from HIV-1-infected and uninfected CD3+ T cells. The levels of 8-isoprostaglandin-F2-{alpha} in the conditioned media were determined using the StressXpress ELISA (Stressgen, Victoria, BC, Canada). The effects of EtOH either in the presence or in the absence of HIV-1-infected CD3+ T cells and their cellular supernatants were also investigated. Results are the mean values for duplicate samples ± standard errors of the means. The data presented are averages for two independent experiments.

Measurement of NOS activity. Postmitotically differentiated neurons express various forms of NOS involved in the pathogenic process of various viral infections (29). The product of NOS, i.e., NO, is involved in both physiological and pathological events. NO is a highly reactive mammalian secretory product and, depending on its concentration in the body, NO can act as a signal molecule or induce NO-mediated oxidative stress via generation of reactive nitrogen oxide intermediates (RNOIs). The higher the concentrations of NO, the more likely that it will be rapidly converted to other RNOIs or higher nitrogen oxides, such as NO2 and N2O3, which are injurious to the cells. The activity of NOS was indirectly measured by the total nitrate/nitrite in the cellular supernatants of the neuronal medium having either HIV-1-infected cells or uninfected controls. Since both HIV-1 and EtOH have been shown to induce the production of these RNOIs, we found it imperative to study the potential of HIV-1-infected monocytes, CD3+ T cells, supernatants from infected monocytes, and T cells either alone or in combination with physiologically relevant levels of EtOH to induce the production of elevated levels of RNOIs in postmitotically differentiated neurons. Figure 4A and B depict the production of total nitrates as a measure of the concentration of RNOIs in neurons exposed to monocytes and T cells with their cellular supernatants, respectively. As can be seen from Fig. 4A, exposure of the neurons to EtOH at concentrations of 0.1 to 0.3% resulted in a moderate increase in NO production. Similarly, exposure of the neurons to HIV-1-infected monocytes as well supernatants from infected monocytes resulted in only a moderate increase in the production of NO in the conditioned medium. However, an augmented increase in NO secretion was observed when the neurons were cocultured with both HIV-1-infected monocytes/supernatants and 0.1 to 0.3% EtOH. As shown in Fig. 4B, HIV-1-infected CD3+ T-cell supernatants enhanced the levels of total nitrates, although relative amounts were smaller than in setups in which infected cells were in contact with postmitotic neurons (coculture).


Figure 4
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  		FIG. 4. HIV-1-infected CD14+ monocytes and CD3+ T lymphocytes significantly induce the secretion of total nitrates from postmitotically differentiated neurons. (A) The levels of total nitrates (an indicator of the activity of various isoforms of nitric oxide synthase) in the cellular supernatants (Sup) of postmitotic neurons cocultured with HIV-1-infected/uninfected CD14+ monocytes (Mono) in the presence or absence of various concentrations of EtOH were determined after 72 h. (B) HIV-1-infected and uninfected CD3+ T lymphocytes and their cellular supernatants were added to postmitotic neurons either in the presence or in the absence of EtOH. After 72 h, total nitrate secretion in conditioned media was quantified using the StressXpress ELISA (Stressgen, Victoria, BC, Canada). Results are the mean values for duplicate samples ± standard errors of the means. Experiments were carried out at least two times.

Inhibition of HIV-1-induced oxidative stress by atorvastatin and simvastatin. The cholesterol-depleting drugs atorvastatin and simvastatin have been shown to reduce the production of cytokines and other stress-associated molecules in vitro (82). Therefore, we wanted to assess the impact of these two cholesterol-lowering drugs on the production of Hsp70 and 8-isoprostaglandin-F2-{alpha} in postmitotically differentiated neurons cocultured with infected and uninfected monocytes and T cells. Neurons were treated overnight with 10 µM of either atorvastatin or simvastatin and then cocultured with or without alcohol and HIV-1-infected monocytes or CD3+ T cells and supernatants from HIV-1-infected monocytes or primary CD3+ T cells. Figure 5 shows the effects of atorvastatin and simvastatin on the release of HIV-1- and EtOH-associated Hsp70 release, whereas Fig. 6 represents the capability of statins to lower the oxidative stress marker 8-isoprostane-F2-{alpha}. Notably, both atorvastatin and simvastatin down-regulated the production of these oxidative stress markers by neurons in the various treatment groups to the normal or pretreatment levels. These results are very important in that if confirmed, these drugs could be used in HIV-1 patients to reduce oxidative stress.


Figure 5
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  		FIG. 5. The statins atorvastatin and simvastatin significantly block the induction of Hsp70 by HIV-1-infected CD14+ monocytes (A) and CD3+ T lymphocytes (B) from postmitotically differentiated neurons. HIV-1-infected CD14+ monocytes (Mono) and CD3+ T lymphocytes were cocultured with postmitotic neurons pretreated (24 h before coculturing) with 10 µM statins (Ator, atorvastatin; Simva, simvastatin). The levels of statins were adjusted and maintained for 72 h during the coculturing studies. Cellular supernatants were subjected to quantification of Hsp70 secretion as described above. Postmitotic neurons were washed and subjected to Western blot analyses for the determination of intracellular Hsp70 expression (see Fig. 7). Results are the mean values for duplicate samples ± standard errors of the means. Experiments were repeated at least two times.


Figure 6
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  		FIG. 6. The statins atorvastatin and simvastatin significantly block the induction of 8-isoprostaglandin-F2-{alpha} by HIV-1-infected primary CD14+ monocytes (A) and CD3+ T lymphocytes (B) from postmitotically differentiated neurons. Postmitotically differentiated neurons were cocultured with HIV-1-infected primary human monocytes (Mono) and T cells in the presence of two concentrations of EtOH (0.1 and 0.3%). A negative control consisted of postmitotic neurons cocultured with HIV-1-infected and uninfected T cells without EtOH. To study the effects of statins on isoprostane production, cells were treated with 10 µM of one of two statins, i.e., atorvastatin (Ator) or simvastatin (Simva). The data presented in the graphs are representative of two independent experiments.

Western blot analyses to confirm HIV-1-induced production of Hsp70. To further confirm our findings, we analyzed the intracellular expression of Hsp70 protein levels in postmitotic neurons via Western blot analyses, and the results are depicted in Fig. 7. These analyses revealed an elevated expression of Hsp70 in cells cocultured with HIV-1-infected primary monocytes over a time period of 72 h compared to Hsp70 expression in uninfected controls. Of importance, the oxidative stress potential of HIV-1 was dramatically augmented and synergized by levels of alcohol, as depicted by the increased expression of Hsp70. Similarly, neurons exposed to supernatants generated from HIV-1-infected monocytes showed an elevated expression of this oxidative stress marker compared to controls. Coculturing of infected T cells with neurons or neurons exposed to HIV-1-infected primary CD3+ T-cell supernatants showed a similar elevated expression of Hsp70 (data not shown). Western blot analyses also indicated that clinically available inhibitors of HMG-coenzyme A reductase, atorvastatin and simvastatin, inhibited HIV-1-induced release of Hsp70 from neurons.


Figure 7
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  		FIG. 7. Statin-mediated inhibition of HIV-1- and EtOH-induced production of Hsp70 in postmitotically differentiated neurons. Postmitotically differentiated neurons were cocultured with HIV-1-infected monocytes either in the presence or in the absence of 0.3% EtOH for 72 h. The neurons were washed to remove primary monocytes. Cellular lysate protein (25 µg/lane) was loaded onto a sodium dodecyl sulfate-polyacrylamide gel and electrophoresed, followed by a transfer onto a nitrocellulose membrane. These blots were then probed with antibody specific for Hsp70. (A and B) Expression of Hsp70 under conditions without EtOH and with 0.3% EtOH, respectively. Lanes: 1, protein marker; 2, nontreated monocytes (control); 3, monocytes pretreated with simvastatin (10 µM); 4, monocytes pretreated with atorvastatin (10 µM); 5, monocytes infected with R5 HIV-1 strain YU2; 6, monocytes infected with R5 HIV-1 strain YU2 in the presence of simvastatin (10 µM); and 7, monocytes infected with R5 HIV-1 strain YU2 in the presence of atorvastatin (10 µM). Panel B represents the human ß-actin control for the various lanes in panel A, and panel D represents the control for samples shown in panel C.


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DISCUSSION
 
HIV-1 frequently infects the cellular elements of the CNS soon after seroconversion (78, 87). Several studies have shown that there is a potential interaction between alcohol and several other drugs of abuse and HIV-1 infection (26, 30, 51). Chronic consumption of alcohol can alter various functions of the immune system, including both humoral and cell-mediated processes (8, 38). It has been suggested that exposure to EtOH may both increase susceptibility to HIV infection and stimulate HIV replication in previously infected cells and also alter cytokines that increase HIV-1 replication from a quiescent state (7, 9, 22, 71-73). Exposure to EtOH can also cause devastating complications and neuronal damage in the brains of AIDS patients (49), resulting in the development of HIV-1-associated dementia (HAD) (25). HIV-1 and alcohol consumption, either alone or in combination, can induce oxidative stress with the concomitant production of Hsp70 and stimulation of glutamate receptors, such as N-methyl-D-aspartate. This may result in the elevation of lipid peroxidation, the formation of reactive oxygen species, and the production of NO and neuronal apoptosis. Russo et al. (80) showed a correlation between the mechanism of EtOH-induced oxidative stress and the expression of Hsp70 in different cerebral areas in rats fed both acutely and chronically with EtOH (80). The same study also revealed that the administration of EtOH in rats at 50 to 100 mmol/liter induces a dose-dependent increase in the production of reactive oxygen species and Hsp70, together with an impairment of respiratory chain activity.

In the present study, we also wanted to determine the synergistic effects of HIV-1 and EtOH on the induction of oxidative stress in postmitotically differentiated human neurons. We hypothesized that coculturing of HIV-1-infected cells with postmitotically differentiated neurons would induce extensive oxidative stress in neurons, which would be potentiated by EtOH. Consistent with our hypothesis, it was observed that HIV-1 infection-induced oxidative stress was synergized upon culturing these cells in the presence of physiologically relevant concentrations of EtOH. Several mechanisms could be responsible for this phenomenon. The oxidative stress marker Hsp70 was demonstrated to have augmented release from neurons when exposed to HIV-1 with CD4-positive cell lines and macrophages (15, 36, 37). The results are also in agreement with other studies that have shown that EtOH can increase the expression of transcription factors (66) and the secretion of heat shock proteins in alcohol-exposed tissue (62, 80, 94).

Heat shock proteins (HSPs) are a group of highly evolutionary conserved proteins that are present in all cells in all forms of life. The expression of these proteins is induced in response to various physical, environmental, and chemical stresses, such as heat or cold exposure, oxygen deprivation, UV irradiation, heavy metal exposure, oxidative stress, and pathological stimuli such as viral and bacterial infection (95). Physiologically, HSPs function as molecular chaperones facilitating protein folding and transport and prevent the aggregation and degradation of proteins. They are also involved in the repair of stress-induced damage (12, 15) and act as stimuli inducing proinflammatory cytokine production in human monocytes (6).

The role of Hsp70 in HIV-1 infection is unknown; however, some studies have suggested that the enhancement of Hsp70 expression during infection may contribute to reduced susceptibility of cells to virus-mediated cytotoxicity, suggesting that Hsp70 may contribute to antiviral immunity (15). Other studies have also suggested that increased Hsp70 expression could be due to oxidative stress secondary to HIV infection (2). Recently, Stebbing et al. (88) reported that there is up-regulation in the expression of the HSP receptor CD91 in monocytes isolated from HIV-1-seropositive long-term nonprogressors. This observation is important in that CD8 antiviral factor, a key component of the soluble factor for CD91, has been shown to suppress HIV-1 replication and is up-regulated in its expression in CD8 T lymphocytes from such long-term nonprogressors (98). This suggests that Hsp70 may play a role in inhibiting viral replication. Hsp70 may also confer a survival advantage to cells. For example, Russo et al. (80) has reported a drastic reduction in the cellular metabolism in Hsp70-deprived astrocytes, particularly when these cells were also EtOH treated. Furthermore, transfection of control astrocytes with Hsp70 antisense induced moderate oxidative damage and a drastic decrease in the viability of EtOH-treated cells, with the mitochondrial functionality being particularly affected (80). Thus, in the present study, it appears that the increased production of Hsp70 confers a survival advantage to the neurons, providing a protective and stabilizing effect on stress-induced cell injury as well as preventing oxidative stress and nuclear damage. The increased Hsp70 production may also be an attempt by the cells to suppress viral replication. Whether similar mechanisms are involved in the pathogenesis of AIDS dementia in vivo still needs to be explored.

In our in vitro experimental setup, postmitotically differentiated human neurons showed enhanced production of NO that was further augmented in the presence of EtOH. Studies have shown that HIV infection is accompanied by the simultaneous activation of free radical species in CNS cells (41, 83). Particularly, HIV-1-induced oxidative stress modulates various isoforms of NOS as well as dysregulates the BBB cellular machinery (41, 83). Enhanced activities of various NOSs generate hyperactive NO free radicals that enter into a chain reaction by interacting with oxygen free radicals which are cogenerated with NO. The end product of these interactions is the production of the strong oxidizing and nitrating agent peroxynitrite, which forms various deleterious products (13, 90). We observed an increase in the production of total nitrates, an indirect indication of the hyperactivity of NOS, in cells exposed to either HIV-1-infected cells or supernatants. This in vitro evaluation of the synergistic effects of HIV-1 infection and exposure to EtOH of differentiated neurons demonstrated that HIV-1 infection induces oxidative stress by markedly stimulating the production of NO via activating NOSs and associated inflammatory pathways in the CNS. Consistent with the production of Hsp70, exposure to EtOH in addition to HIV-1-infected cells/supernatants markedly increased the production of total nitrates, an indication of the hyperactivity of NOSs. This also suggests that overproduction of nitrates, possibly via the release of proinflammatory substances by HIV-infected CD14+ monocytes/macrophages and CD3+ T cells, may contribute to the pathophysiological mechanisms underlying the development of HAD. In agreement, similar elevated expression levels of various isoforms of NOS, the enzymes responsible for the generation of NO free radicals in AIDS dementia, have been reported. Existing evidence suggests that NO and oxygen radicals, such as superoxide, play key roles in the pathogenesis of various infectious diseases (3, 76). Unlike in microbial infections, where oxygen radicals and NO have antimicrobial activity, in viral infections, a high NO production from hyperactive NOS exacerbates the infection by interacting with oxygen radicals and reactive oxygen intermediates to produce reactive NO species, such as peroxynitrite. Through the oxidation and nitration of various biomolecules, the reactive nitrogen species cause oxidative injury to cells and tissues. The oxides of nitrogen affect the host's immune response, with immunopathological consequences. For example, it has been reported that NO suppresses type 1 helper T-cell-dependent immune responses, leading to type 2 helper T-cell-biased immunological host responses. Thus, NO-induced immunosuppression may contribute to HIV-1 pathogenesis by enhancing the expansion of quasispecies populations of virus. Taken together, the results of the present study further support the potential involvement of neuronal interactions with HIV-1-infected cells to induce deleterious oxidative stress that could potentially disrupt the neuroprotective BBB (65, 85).

In addition to the increased production of NO, 8-isoprostane-F2-{alpha} was identified as an additional source of cellular oxidative stress induced in differentiated neurons by HIV-1 and EtOH. Isoprostanes are prostaglandin-like end products of arachidonic acid peroxidation that are produced by a free radical-catalyzed mechanism (56). The isoprostanes consist of stereoisomers and regioisomers of the common prostaglandins and were first reported for prostaglandin-F2. Later, isoprostanes of the E2 and D2 series were discovered (55), and 8-iso-PGF-2{alpha} is the best characterized isoprostane. 8-Iso-PGF-2{alpha} is produced mainly as a nonenzymatic free radical-induced peroxidation of arachidonic acid present in phospholipids (54, 55). It can also be produced as a product of cyclooxygenase 1 enzymes in human platelets and cyclooxygenase 2 isoforms in human monocytes (74, 75).

The detection of isoprostanes in vivo is used as a quantitative index for the existence of oxidative stress and also as a biomarker for certain types of diseases, such as cancer (33, 79). In accordance with this, elevated levels of isoprostanes have been detected in situations associated with increased free radical generation, in HIV-1-infected individuals, in postmenopausal women, and in chronic cigarette smokers (33, 53). Similarly, increased production of arachidonic acid from different sources associated with various pathological conditions has been reported (47). Arachidonic acid released from macrophages has been reported to impair the ability of astrocytes to clear the neurotransmitter glutamate from the CNS, resulting in the activation of astrocytes and excitotoxicity (39). In such instances, the activated astrocytes are further stimulated by {alpha}-chemokine stromal cell-derived factor 1{alpha} in association with cytokines to release glutamate in addition to the free radical nitric oxide (NO)* (39). (NO)* may react with the superoxide anion (O2*–) to form the neurotoxic molecule peroxynitrite (ONOO–). In addition, NO may also activate extracellular matrix metalloproteinases, which may degrade the components of the extracellular matrix of the CNS (32, 39). Considering its free radical-dependent formation, the release of the isoprostane 8-iso-PGF-2{alpha} during HIV-1 infection may play an important role in oxidative stress-related CNS dysfunction. Indeed, HAD has been reported to be the result of HIV-1-induced oxidative stress and the accompanying overproduction of several toxic factors, including prostaglandins, CD95 ligand, and free radicals (18, 70, 81, 93). It is important to note that since NO is able to combine with superoxide anions to generate peroxynitrite, the nitrogen free radicals are responsible for the production of relevant peroxides in HIV-related damage in CNS-based cells (52). Thus, it is likely that abnormal release of peroxynitrite may play a role in the apoptotic cell death which occurs in CNS-based cells during HIV-1 infection (14).

In our in vitro studies, cholesterol-depleting statins seem to ameliorate HIV-1- and EtOH-induced cytotoxic oxidative responses. Statins are clinically available inhibitors of HMG-coenzyme A reductase that play prominent roles in various biological processes both in vivo and in vitro. For example, statins have been reported to inhibit the secretion of matrix metalloproteinases and cytokines (40, 48); they have also been reported to inhibit the activation of macrophages and T lymphocytes (63, 96). Recently, it was reported that statins are able to inhibit HIV-1 replication in PBMCs (21). Similarly, Giguère and Tremblay (28) reported that in vitro, lovastatin and simvastatin are able to inhibit HIV-1-induced intercellular adhesion molecule 1-lymphocyte function-associated antigen 1 interactions. Due to their innovative profiles, statins could potentially be used to alter the development of HAD, which involves the induction of oxidative stress by infected monocytes and T cells via the Trojan horse pathway. We evaluated the potential of statins to inhibit HIV-1-induced oxidative stress in the CNS-based cells. We exposed neurons to 10 µM atorvastatin and simvastatin, before and after exposure to HIV-1-infected CD14+ monocytes and CD3+ T lymphocytes and supernatants. We observed that these compounds inhibited HIV-1-induced oxidative stress in neurons by decreasing the production of Hsp70 and 8-isoprostaglandin-2{alpha}. The results of the present study confirmed earlier observations that statins can down-modulate several inflammatory responses and down-regulate the production of inflammatory cytokines (46). Thus, statins appear highly promising for potential adjunct neuroprotective therapy in addition to normal antiretroviral treatment for the prevention and treatment of HAD. As well, the use of statins may represent the basis for alternative and efficient strategies in the treatment of HAD.

In conclusion, this study has demonstrated that the presence of HIV-1-infected immune cells and/or exposure to EtOH can induce oxidative stress by enhancing the production of cytotoxic markers such as 8-iso-PGF-2{alpha}, Hsp70, and NOS activity responsible for the generation of oxidative stress precursors in human differentiated neurons. Compared to treatments with HIV-1 or EtOH alone, coculturing of neurons with HIV-1 and EtOH further enhanced the production of specific oxidative stress markers in neurons. Such up-regulation of prooxidative and proinflammatory pathways is proof of the concept that HIV-1 and EtOH could potentially induce oxidative stress in HIV-1-infected individuals who abuse alcohol.


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ACKNOWLEDGMENTS
 
We thank Cassandra Roschel for her excellent assistance in the preparation of the manuscript.

This work was supported mainly by the Pfizer Atorvastatin Research Award (ARA) to M.M. and in part by U.S. Public Health Service grants MH074375-01A1 to M.M., MH074359-01 to Z.P., and DA019807 to B.W.


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FOOTNOTES
 
* Corresponding author. Mailing address for Muhammad Mukhtar: Drexel University College of Medicine, Department of Microbiology and Immunology, 245 N. 15th Street, Room 18107, Philadelphia, PA 19107. Phone: (215) 762-3719. Fax: (215) 762-1955. E-mail: muhammad.mukhtar{at}drexelmed.edu. Mailing address for Zahida Parveen: Dorrance H. Hamilton Laboratories, Division of Infectious Diseases, Department of Medicine, Thomas Jefferson University, 1020 Locust Street, Suite 329, Philadelphia, PA 19107. Phone: (215) 503-9097. Fax: (215) 923-1956. E-mail: zahida.parveen{at}jefferson.edu. Back

{triangledown} Published ahead of print on 15 November 2006. Back


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Journal of Virology, February 2007, p. 1492-1501, Vol. 81, No. 3
0022-538X/07/$08.00+0     doi:10.1128/JVI.01843-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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