Alcohol abuse and AIDS are the leading public health problems in the United States, with current estimates indicating that 54.9% of adults consume alcohol at least once every month.1 Among alcohol consumers, approximately one third are binge drinker,2 5.9% are heavy drinkers,3 and 3.7% are alcohol dependent.1 By analogy, of 47,000 new HIV cases reported every year, 25,803, 8601, 1522, and 954 cases belong to alcohol consumer, binge drinker, heavy drinker, and alcohol-dependent groups, respectively. There is no conclusive evidence that acute or chronic alcohol consumption modulates the course of infection and/or disease among HIV-infected individuals, however. In several in vitro studies, alcohol has been found to increase HIV replication,4-8 whereas in another experimental design, the addition of alcohol caused no consistent effect on HIV replication.9 Likewise, results from clinical studies also remain inconclusive. Certain clinical studies have found a potential relation between alcohol use and HIV-1 infection as well as disease progression,10,11 whereas another study involving 199 HIV-positive drug users revealed that there was no correlation between chronic alcohol abuse and CD4+ T-cell loss in these individuals.12 Therefore, additional studies are required to clarify this controversy. One approach would be to initiate a clinical study and to correlate alcohol consumption with viral load over time and eventual progression to clinical disease. A potential problem with the clinical study, however, is that it requires several years before a definitive conclusion can be drawn. Alternatively, the effect of alcohol abuse can be determined in an animal model of HIV/AIDS. In a previously reported study, chronic alcohol consumption has been found to modulate virus replication in the blood of simian immunodeficiency virus (SIV)-infected macaques.13 This study used a model wherein the animals do not lose peripheral CD4+ T cells until the late stage, however, and the alcohol is administered through surgically implanted gastric catheters. Conversely, HIV-infected individuals consistently lose CD4+ T cells, which was not reflected in the previous study.
In this study, we used a self-drinking model of chronic alcohol consumption, inoculated the macaques with a mixture of neuropathogenic SIV and 2 simian-HIVs (SHIVs) and followed them for a period of 24 weeks for complete blood cell counts (CBCs), CD4 and CD8 profiles, and virus replication in the blood and cerebral compartments.
Establishment of a Model of Chronic Alcohol Consumption and Infection With Simian Immunodeficiency Virus and Simian-HIV
The 6 male rhesus macaques (Macaca mulatta) used for the study ranged in age from 1.5 to 2.5 years and in weight from 3 to 4.2 kg at the time of initiation of the study. The animals were negative for simian T-cell leukemia virus type 1 and simian retrovirus. These macaques were housed in the Animal Resource Center of the University of Puerto Rico (San Juan, PR). The experimental protocol was approved by Institutional Animal Care and Use Committee, and the research was performed in accordance with the Guide for the Care and Use of Laboratory Animals. The macaques were divided into 2 groups of 3 animals each, with the first group used for alcohol addiction and the second group serving as controls.
The alcohol addiction was established using a previously described protocol14 with little modification. These animals were deprived of water for 2 hours, after which group 1 animals were provided with a solution of 8.4% ethanol in Nutrasweet ad libitum for 1 hour for 1 week. The amount of alcohol was determined and calculated in terms of alcohol consumption in grams per kilogram of body weight. The other 3 macaques were allowed to drink Nutrasweet solution under similar condition (results not shown). To test whether increased deprivation of water and longer alcohol consumption could affect the total alcohol uptake, we deprived the animals overnight of water, followed by provision of the alcohol-Nutrasweet solution for 3 hours. We initiated this procedure on day 8 and continued each day until end of the observation period. This modified protocol substantially increased the total alcohol consumption. The alcohol consumption in the 3 animals in group 1 is shown in Figure 1 at weekly intervals.
All 6 animals were infected 7 weeks after initiation of the alcohol feeding. They were infected by intravenous injection of 2 mL of inoculum containing 104 TCID50 (tissue culture infectious dose 50%) of SHIVKU-1B,15 SHIV89.6P,16 and SIV/17E-Fr.17 The animals were bled at weeks 0, 1, 2, 3, and 4, followed by once every 2 weeks throughout the observation period of 24 weeks after infection. Cerebrospinal fluid (CSF) was also collected once every 2 weeks after infection.
Lymphocyte Subset Staining and Real-Time Reverse Transcriptase Polymerase Chain Reaction
Circulating CD4+ T-cell levels were determined by staining for CD3, CD4, and CD8 surface markers using a previously described method.18 Approximately 105 peripheral blood mononuclear cells (PBMCs) were stained with 10 μL of antibody mix against CD3, CD4, and CD8. The unbound antibodies were removed by washing the cells with cold phosphate-buffered saline (PBS). The cells were fixed with 0.5% paraformaldehyde and analyzed using a FACSCalibur fluorescence-activated cell sorter (BD Biosciences, San Jose, CA). Absolute CD4 and CD8 counts were obtained by multiplying the percentage of lymphocyte subset with absolute number of lymphocytes per microliter of blood from the CBC.
The viral load in plasma and CSF was determined in duplicate by using a real-time reverse transcriptase (RT) polymerase chain reaction (PCR) assay.19 The RNA copy number was determined by comparison with an external standard curve consisting of in vitro transcripts representing bases 211 to 2101 of the SIVmac239 genome. This assay has sensitivity of ≤80 copies/mL of plasma or CSF.
All 3 animals were successfully adapted to chronic alcohol consumption. The alcohol consumption in these animals was recorded every day. For the sake of clarity, however, the consumption is shown in grams of alcohol per kilogram of body weight of the animal once every week (see Fig. 1). A 3-hour alcohol drinking period increased the total alcohol consumption; however all 3 animals showed significant variation in daily alcohol consumption.
Mean CD4+ T-cell counts and the CD4/CD8 ratios are shown in Figure 2A and B, respectively. The week 0 mean CD4+ T-cell counts were 1235 ± 241 and 1366 ± 313 cells/μL of blood in the alcohol and control groups, respectively. Both groups showed precipitous loss in circulating CD4+ T-cell numbers. The loss in the alcohol group was significantly higher at week 1 after infection (P = 0.03), however. The maximum CD4 loss was observed 3 weeks after infection, after which both groups showed moderate CD4 recovery. Generally, the control group showed a pattern of higher CD4+ T-cell counts throughout the observation period. The CD4/CD8 ratios in both groups also followed a similar pattern, with the control group showing a relatively higher ratio than that seen in the alcohol group.
The viral loads in the plasma and CSF are shown in Figure 2C and D, respectively. The virus replicated at high levels in the control and alcohol groups. The 2 groups showed comparable peak viral loads at week 2 after infection (1.35 × 107 ± 7.37 × 106 and 9.08 × 106 ± 6.36 × 106 RNA copy Eq/mL of plasma in the alcohol and control groups, respectively). Furthermore, both groups showed a gradual decline in the plasma viral loads over the next 10 weeks. The week 12 viral loads in the alcohol and control groups were 1.46 × 105 ± 1.27 × 105 and 2.28 × 104 ± 1.22 × 104 RNA copy Eq/mL of plasma, respectively, suggesting that the viral set point in the control group was 6-fold lower than that in the alcohol group. In the alcohol group, the extent of virus replication in the blood increased after the set point, whereas in the control group, there was no particular trend observed in the virus replication in the blood. The virus replication in the alcohol group was 31-, 44-, 69-, and 85-fold higher at weeks 18, 20, 22, and 24, respectively, than that in the control group.
The CSF virus loads in the alcohol and control groups are shown in Figure 2D. The alcohol and control groups showed comparable viral loads for first 8 weeks after infection. Thereafter, the CSF viral load increased in the alcohol group, whereas the control group animals showed a relatively low level of virus in the cerebral compartment. The virus replication in the alcohol group was 5-, 18-, and 36-fold higher at weeks 10, 12, and 14, respectively. The virus became undetectable in the control animals at week 18 after infection and continued to remain undetectable in this group throughout the observation period. At the end of observation period, the mean CSF viral load in the alcohol group was 1.42 × 105 RNA copy Eq/mL of CSF.
Alcohol has been shown to affect the immunologic status of the host, which might modulate viral replication and affect AIDS progression. Alcohol-mediated T helper (Th)1/Th2 switch,20 increased nuclear factor (NF)-κB levels,21,22 and increased tumor necrosis factor (TNF) levels occur, which, in turn, increases NF-κB levels.23,24 This was eventually tested in a chronic binge model of ethanol consumption in macaques that were infected with SIV. The virus replication in these animals was found to be significantly higher compared with that in control monkeys.13 Although there is no information available on the effect of alcohol on SIV/HIV-specific immune responses, the influence of alcohol has been studied for its ability to modulate glycoprotein 120 (gp120)-induced immune responses in rats. Alcohol consumption has been found to suppress the production of gp120-specific antibodies and chemokines that are normally produced after immunization with recombinant gp120.25,26 Furthermore in vitro alcohol treatment has been found to suppress TNFα production in blood cells27 and alveolar macrophages,28 neutrophil phagocytosis,29 and CD11b expression on polymorphonuclear leukocytes29 collected from SIV-infected macaques. Taken together, all these findings suggest a potential detrimental role for alcohol abuse on AIDS pathogenesis.
This is the first demonstration of increased SIV/SHIV replication in the self-drinking model of alcohol consumption that remains closest to alcohol abuse observed in human beings. We inoculated rhesus macaques with a mixture of 2 SHIVs and a neuropathogenic SIV. The choice of the challenge virus was based on the ability of the mixture of these 3 viruses to induce uniform disease in a relatively short time.18 In other studies in which we used a single virus in the challenge inoculum, some of the animals required 2 years before they developed clinical AIDS.30,31 Another reason for using the mixture of 3 viruses is based on the fact that we wanted to determine the effect of alcohol abuse on virus replication in the cerebral compartment in a model system wherein all animals lose circulating CD4+ T cells. The 2 SHIVs used in this study are known to cause precipitous CD4+ T-cell loss in the blood, and SIV/17E-Fr is known to cause AIDS-related neurologic disorders. These animals were followed for a period of 24 weeks. Our differential PCR analysis18 showed that whereas all 3 viruses replicated in the blood, only SIV/17E-Fr crossed the blood-brain barrier (results not shown). Therefore, it was safe to conclude that the CSF viral load reflected replication of only SIV/17E-Fr.
Our study showed that first-week and peak viral replication in the blood was comparable in the alcohol and control groups, whereas a previously reported study found >60-fold increased viral replication in the alcohol group 1 week after infection.13 The exact reason for this is unknown, but possible reasons may include choice of the model (self-drinking vs. delivery by intragastric catheters) and challenge virus. Our study included 3 viruses, whereas the previous study included only 1 virus. Our study also provided evidence that alcohol abuse may be a possible cause of continued virus replication in the cerebral compartment. Furthermore, this study also provided evidence that alcohol abuse might inflict higher CD4+ T-cell loss in the animals.
The increased virus replication in the alcohol-dependent macaques can be explained by the alcohol-mediated apoptosis and impaired proliferative responses in T cells. In several previous studies, alcohol has been found to decrease the CD4+ and CD8+ T-cell numbers in blood as well as in the thymus, lymph node, and spleen,32-34 and this decrease was attributed to increased apoptosis.35-38 In this study, although mean CD4+ T-cell counts in the control and alcohol groups were more or less similar on the day of virus inoculation, individual animals in the alcohol group showed a relative decrease in the CD4+ T-cell number (data not shown). Our future studies should determine whether this loss is attributable to increased apoptosis. The increased viral replication in the cerebral compartment can also be attributed to alcohol-mediated increased NF-κB activity,39 which, in turn, could activate viral long terminal repeat (LTR) leading to enhanced replication.40 Another possible mechanism for the increased replication in the brain comes from another study in which alcohol was found to confer an additive effect to Tat-induced central nervous system (CNS) pathologic findings,41 thereby perhaps providing a better environment for virus replication. Furthermore, chronic alcohol consumption is known to enhance the production of vascular endothelial growth factor, E-selectin, and intracellular adhesion molecule-1,42,43 which have been reported to be associated with the development of AIDS-associated dementia.44 Long-term follow-up of these animals should determine whether SHIV/SIV-infected alcohol-dependent animals develop SIV-associated encephalitis. Another possible reason for increased virus replication might include alcohol-mediated overexpression of CXCR46,45 which could facilitate higher virus infection by making more coreceptor available for viral entry.
In view of the significant difference in CD4+ T-cell loss and higher virus replication in the blood and brain compartment between the alcohol and control groups, it is likely that alcohol-dependent animals may develop crippled virus-specific immune responses; as a result, the onset of clinical disease would be faster than that in the control animals. The possibility of reduced virus-specific immune responses can be attributed to the several previously reported studies wherein alcohol has been found to cause (1) inhibition of lymphocyte proliferation,46 (2) reduced Th1 cytokine production,47-49 (3) increased Th2 cytokine production,20,50 (4) impaired B-cell function,51 and (5) impairment of delayed type hypersensitivity responses.52 Furthermore, Th2 cytokine has been shown to favor HIV and SIV replication.53,54 A long-term follow-up of these animals may reveal whether alcohol addiction causes accelerated clinical disease in these animals.
The authors thank Janice E. Clements, Johns Hopkins University School of Medicine, Baltimore, MD; Norm L. Letvin, Harvard Medical School, Boston, MA; and Edward B. Stephens for providing SIV/17E-Fr, SHIV89.6P, and SHIVKU, respectively.
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