Cannabinoid Administration Attenuates the Progression of Simian Immunodeficiency Virus

Abstract

Δ⁹-Tetrahydrocannabinol (Δ⁹-THC), the primary psychoactive component in marijuana, is FDA approved to ameliorate AIDS-associated wasting. Because cannabinoid receptors are expressed on cells of the immune system, chronic Δ⁹-THC use may impact HIV disease progression. We examined the impact of chronic Δ⁹-THC administration (0.32 mg/kg im, 2 × daily), starting 28 days prior to inoculation with simian immunodeficiency virus (SIV_mac251; 100 TCID₅₀/ml, iv), on immune and metabolic indicators of disease during the initial 6 month asymptomatic phase of infection in rhesus macaques. SIV_mac251 inoculation resulted in measurable viral load, decreased lymphocyte CD4⁺/CD8⁺ ratio, and increased CD8⁺ proliferation. Δ⁹-THC treatment of SIV-infected animals produced minor to no effects in these parameters. However, chronic Δ⁹-THC administration decreased early mortality from SIV infection (p = 0.039), and this was associated with attenuation of plasma and CSF viral load and retention of body mass (p = NS). In vitro, Δ⁹-THC (10 μm) decreased SIV (10 TCID₅₀) viral replication in MT4-R5 cells. These results indicate that chronic Δ⁹-THC does not increase viral load or aggravate morbidity and may actually ameliorate SIV disease progression. We speculate that reduced levels of SIV, retention of body mass, and attenuation of inflammation are likely mechanisms for Δ⁹-THC-mediated modulation of disease progression that warrant further study.

The cannabinoids including cannabidiol, cannabinol, and Δ⁹-tetrahydrocannabinol (Δ⁹-THC) exert their effects by binding to two major subtypes of cannabinoid receptor, CB1 and CB2.¹ The CB1 receptor is preferentially expressed in the brain where it mediates neurobehavioral effects. The CB2 receptor is expressed primarily in peripheral tissues, particularly in immune cells where they have been shown to affect cytokine production, lymphocyte phenotype, function and survival, cell-mediated immunity, and balance of Th1/Th2 cells.² With the advent of highly active antiretroviral therapy (HAART), human immunodeficiency virus (HIV) infection has become a chronic disease frequently coexisting with chronic use of drugs of abuse, including marijuana.³ In addition, Dronabinol (Δ⁹-THC; Marinol) is approved by the Food and Drug Administration for treatment of HIV-associated anorexia.⁴ Although this approval has gained strong support from the lay public, little scientific evidence exists to support the efficacy of such an intervention. To date, no clinical studies have rigorously addressed the impact of chronic Δ⁹-THC use on the course and progression of HIV infection. Only one study has examined the impact of short-term (21 day) Δ⁹-THC administration (3 daily 0.9 g marijuana cigarettes; 3.95% Δ⁹-THC) on HIV viral load. The results from that study did not show substantial elevation in viral load in HIV-infected individuals receiving stable antiretroviral regimens containing nelfinavir or indinavir,⁵ and, thus, were considered to reflect the relative safety of short-term Δ⁹-THC use in this patient population. The long-term effects of cannabinoid administration on progression of HIV infection had not been previously examined.

Using a well-established nonhuman primate model of HIV disease, we examined the impact of chronic intramuscular Δ⁹-THC (provided by the National Institute on Drug Abuse, Research Technical Branch, Rockville, MD) administration on the early phase of simian immunodeficiency virus (SIV) infection in age-matched (4–6 years old) and body weight-matched healthy male Indian-derived rhesus macaques. Chronic administration of Δ⁹-THC [or 0.05 ml/kg vehicle (VEH)] was initiated prior to SIV with 0.18 mg/kg, a dose that eliminated responding in a complex operant behavioral task in almost all of the subjects. The dose was subsequently increased for each subject to 0.32 mg/kg, over a period of approximately 2 weeks, and maintained for the duration of the study. Almost all of the animals demonstrated tolerance to the behavioral effects of this dose prior to SIV inoculation (reported elsewhere).⁶ Approximately 1 month after initiating chronic Δ⁹-THC administration, eight animals were inoculated intravenously with 100 times the TCID₅₀ (50% tissue culture infective dose) of SIV_MAC251. The pathogenicity of this SIV isolate is similar to HIV.⁷ The progression of SIV disease was monitored through clinical and biochemical parameters, and viral load in plasma and cerebrospinal fluid (CSF).

Contrary to what we expected, chronic cannabinoid administration did not significantly increase viral load or exacerbate immune dysfunction. Mean viral load in VEH/SIV⁺ animals was 6.56 ± 0.16 log copies at 7 days and 6.04 ± 0.6 log copies/ml at 2 months postinfection (Fig. 1A). Viral load remained elevated (6.35 ± 0.76 and 5.8 ± 0.06 log copies/ml at 3 and 6 months, respectively) throughout the duration of the study. Viral load in THC/SIV⁺ was lower (4.83 ± 0.56 log copies/ml) as compared to VEH/SIV⁺ beginning at 2 months post-SIV infection (p = NS). Viral mRNA in CSF peaked at day 14 postinfection and remained elevated up to 2 months postinfection in the VEH/SIV⁺ and THC/SIV⁺ animals (Fig. 1B). Thereafter, CSF viral load fluctuated between levels below the limit of detection and low counts in both SIV-infected groups. Across all time points, 60% of VEH/SIV⁺ CSF samples were above the limit of detection for the assay. In contrast, only 40% of CSF samples from THC/SIV⁺ animals had viral loads above the limit of detection.

FIG. 1.

Viral load detected in (A) plasma and (B) CSF (log SIV RNA copies/ml) over time in relation to SIV infection. Shown are individual viral counts for each infected animal in the vehicle-treated (VEH/SIV⁺; solid triangles) and THC-treated (THC/SIV⁺; open squares) groups as well as geometric means (lines). SIV copy number in plasma and CSF was measured by a real-time RT-PCR assay using gag primers and probe as previously described. Virus was isolated from 500 μl of plasma or 100 μl of CSF by high-speed centrifugation (20,000 × g for 60 min.) at 4°C. The limit of detection for this assay (50 copies/ml plasma and 100 copies/ml CSF) is shown in the dotted lines. The contribution of the values below the limit of detection in the CSF assay was assessed as limit of detection divided by the square root of two.²³

Complete and differential blood counts were performed using a Beckman Coulter LH755 for total leukocyte counts and Wright–Giemsa staining of blood smears for leukocyte differentials. Blood lymphocyte subsets were determined by flow cytometry as previously described.⁸ The CD4⁺ to CD8⁺ ratio decreased significantly from preinfection values (p = 0.0001) in both VEH- and THC-treated animals (Fig. 2B). The marked decrease in CD4⁺ lymphocyte counts reached statistical significance (p = 0.023) at 0.5 and 1 months post-SIV infection in VEH/SIV⁺ animals, and beginning at 2 months in THC/SIV⁺ (p = 0.037). In contrast, CD8⁺ counts showed a short-lived increase at 2 weeks postinfection, which reached statistical significance only in THC/SIV⁺ animals (p = 0.029) (Fig. 2D). CD4⁺ and CD8⁺ lymphocyte expressions of Ki67 and caspase 3 (Cas3) were measured as indices of proliferation and apoptosis. No significant changes were noted in the percent of either Ki67⁺ or Cas3⁺ on CD4⁺ lymphocytes in either of the experimental groups (data not shown). In contrast, both markers were increased significantly in CD8⁺ lymphocytes at 2 weeks postinfection, suggesting increased activation of these cells during the acute response to infection (Fig. 3A). The CD8⁺ Ki67 expression was increased 10-fold at this time point, and was significantly higher in THC/SIV⁺ animals when compared to the VEH/SIV⁺-infected animals (p = 0.049). This rise in CD8⁺ lymphocyte proliferation was transient in both groups with only values for THC/SIV⁺ animals remaining higher (p = 0.05) than controls at 1 month post-SIV infection. Values for the VEH/SIV⁺ and THC/SIV⁺ groups were not different from control beyond 1 month postinfection. The changes in the number of Ki67⁺ CD8⁺ lymphocytes were paralleled by those in number of Cas3⁺ CD8⁺ lymphocytes. Cas3⁺ CD8⁺ lymphocytes showed a transient and significant (p = 0.001) 7- to 8-fold increase that peaked at 2 weeks post-SIV infection in both VEH/SIV⁺ and THC/SIV⁺ animals (Fig. 3B). Although a similar pattern of higher levels of Cas3⁺ CD8⁺ lymphocytes was observed in the THC/SIV⁺ animals, this difference failed to reach statistical significance in comparison to the VEH/SIV⁺ animals (p = 0.06). This rise in CD8⁺ lymphocyte apoptosis was short-lived, and values after 1 month post-SIV infection were not different from uninfected control values.

FIG. 2.

(A) Lymphocyte count (cells × 10³/μl), (B) CD4⁺/CD8⁺ lymphocyte ratio, and (C) CD4⁺ and (D) CD8⁺ counts determined by flow cytometry in vehicle-treated uninfected (VEH/SIV⁻), THC-treated uninfected (THC/SIV⁻), vehicle-treated infected (VEH/SIV⁺), and THC-treated infected (THC/SIV⁺) rhesus macaques expressed as means over time post -SIV infection. Change from baseline was analyzed by a model that included baseline value, as a covariate, and factor effects for Study Period (SPno), THC (+/–), SIV (+/–), and all SPno, THC, and SIV interaction effects. The PROC MIXED procedure in SAS (version 9.1) was selected to perform the analyses. As these are repeated measures on the same subjects over the eight study periods, the within-subjects covariance structure was modeled as compound symmetry; the Kenward-Roger approximation was used to compute p-values for tests of significance. Values for experimental groups N = 3 in VEH/SIV⁻ and N = 4 in all others. *p < 0.05 vs. time- and treatment-matched uninfected controls.

FIG. 3.

Numbers of (A) Ki67⁺ and (B) caspase 3⁺ CD8⁺ lymphocytes as determined by flow cytometry in vehicle-treated uninfected (VEH/SIV⁻), THC-treated uninfected (THC/SIV⁻), vehicle-treated infected (VEH/SIV⁺), and THC-treated infected (THC/SIV⁺) rhesus macaques expressed as means over time post-SIV infection. Values for experimental groups N = 3 in VEH/SIV⁻ and N = 4 in all others. *p < 0.05 vs. time- and treatment-matched uninfected control. ⁺Versus time-matched VEH/SIV⁺ values.

No deaths were noted during the first 5 months post-SIV infection in VEH/SIV⁺ or THC/SIV⁺ animals (Fig. 4). Two of the VEH/SIV⁺ animals succumbed to SIV infection shortly after 5 months, and a third reached end stage at 7 months. Among the THC/SIV⁺ animals, the first animal did not reach end stage until 11 months post-SIV inoculation. A summary of the clinical indications for euthanasia and the most salient necropsy findings is presented in Table 1.

FIG. 4.

Percent survival of vehicle-treated infected (VEH/SIV⁺) and THC-treated infected (THC/SIV⁺) rhesus macaques over the initial 11 months post-SIV infection, the timepoint when the first THC/SIV⁺ animal reached end-stage criteria. Log rank statistic for the survival curve (Kaplan–Meier survival analysis) is greater than would be expected by chance (p = 0.037).

Table 1.

Clinical Indications for Euthanasia and Necropsy Findings^a

Subject ID	Time days post-SIV	Clinical findings	Necropsy findings
VEH/SIV+
A2L046	148	Head pressing, ataxia, inactivity, lack of a palpebral reflex and decreased papillary light reflex in the right eye, serum albumin <3 mg/dl, and dehydration, unresponsive to treatment	Cytomegalovirus meningitis with cerebral edema and uncal herniationAcute bilateral bronchopneumonia and pulmonary edema
A1R047	166	Unilateral right facial nerve paralysis, lethargic, ventral recumbence, dyspnea	Giant cell pneumonia with Mycobacterium sp.Pneumocystis jiroveci, meningoencephalitis, and cerebral vasculitis
A1R058	220	Decreased platelet count, hematuria, hematochezia, pyuria; collapsed and did not respond to CPR	Pulmonary thromboembolism
A2R065	503	None	Pulmonary congestion, hepatic steatosis
THC/SIV+
A1R044	343	Chronic progressive intermittent diarrhea, nonresponsive to treatment, chronic intermittent rectal prolapse, gradual body weight loss (>25%), serum albumin <3 mg/dl	Mycobacterium sp. involving lungs, liver, spleen, kidneys, gastrointestinal tract, and mesenteric lymph nodes with granulomatous inflammation
A1R022	351	None	Pulmonary edema, congestion, mild hepatic microsteatosis
A1R005	461	None	Steatohepatitis, pulmonary granulomatous inflammation, chronic pancreatitis
A2L094	503	None	Mild cerebral edema, mild acute steatohepatitis

^aAnimals were studied during the pretreatment period (prior to initiating the Δ⁹-THC administration) and following SIV infection during the “asymptomatic phase of infection” prior to any of them reaching criteria for euthanasia. This period was defined as the period of time following SIV infection during which none of the animals showed clinical signs of disease including weight loss greater than 5% of basal, secondary infections that required antibiotic treatment, neurological manifestations of disease, severe diarrhea, and dehydration. Animals were sacrificed and necropsy performed when they met predefined clinical indications including the presence of three or more of the following: weight loss greater than 15%, hypoalbuminemia (<3 mg/dl) in the presence of edema, anemia and thrombocytopenia, 3 days of complete anorexia, major organ failure or medical conditions unresponsive to treatment (including respiratory distress, intractable diarrhea, or persistent vomiting, secondary infections that required antibiotic treatment), neurological manifestations of disease, severe diarrhea, and dehydration. Survival was tracked throughout the first year postinfection, at which time all animals still on study were euthanized and necropsy was performed.

Notably different patterns in weight gain were observed between the initial 3 month post-SIV infection period and the subsequent 3- to 6-month post-SIV period (Fig. 5). This was particularly true for the VEH/SIV⁺ animals, which showed a tendency for greater (p = NS) weight loss than the THC/SIV⁺ animals. Echocardiography (Toshiba Aplio at 8.5 MHz) performed prior to (baseline) and 3 and/or 6 months after SIV infection in the two SIV-infected groups (VEH and THC) failed to show significant alterations in cardiac systolic or diastolic function (data not shown).

FIG. 5.

Body weight change (kg) during the initial 3 months post-SIV infection (left panels) and during the subsequent 3- to 6-month period of infection. Top panels depict change in body weight in vehicle-treated uninfected (VEH/SIV⁻), THC-treated uninfected (THC/SIV⁻), vehicle-treated infected (VEH/SIV⁺), and THC-treated infected (THC/SIV⁺) rhesus macaques expressed as means ± SEM over time post-SIV infection. Because of the individual variability, data are shown for each animal during the initial 3 months post-SIV infection and during the later 3–6 months post-SIV infection in the lower panels, showing the trends in body weight changes. The symbols in the line graphs represent individual animals in the specified treatment group and carry over across time.

Cannabinoids, including Δ⁹-THC, have been shown to have immunomodulatory effects^9,10 on cytokine production and lymphocyte phenotype, function, and survival^11,12,13 as well as cell-mediated immunity.¹⁴ Similar immunosuppressant effects on lymphocyte¹⁵ and alveolar macrophage¹⁶ function have been reported in nonhuman primates. Furthermore, the potential of cannabinoids to regulate the activation and balance of human Th1/Th2 cells by a CB2 receptor-dependent pathway has been supported by findings from several studies.^17,18 More recent studies have provided evidence that the synthetic cannabinoid WIN 55,212-2 can potently inhibit HIV-1 expression in CD4⁺ lymphocytes and microglial cell cultures in a time- and concentration-dependent manner.¹⁹ Moreover, Rock et al.²⁰ demonstrated the involvement of CB2 receptors in cannabinoid antiviral activity in microglial cells. To examine the possibility that the improved survival of SIV-infected rhesus could have been due to direct suppression of viral replication, we examined the in vitro effects of 1 and 10 μM Δ⁹-THC on viral replication in MT4-R5 cells (human T cell line)²¹ infected with SIV_MAC251 (10 TCID₅₀). Pretreatment with 10 μM Δ⁹-THC (3.2 μg/ml) resulted in a significant decrease in cell-associated viral load in cells collected at 3 and 6 days post-SIV inoculation (Fig. 6). Several possible mechanisms could be involved in this suppression of viral replication, including effects on cell cycle, interaction of cannabinoids with viral coreceptor, and viral integration into the host genome. Moreover, the contribution of the specific cannabinoid receptor subtypes to the overall effects also remains to be examined.

FIG. 6.

MT4-R5 cells (human T cell line) were seeded at a cell density of 5 × 10⁵/ml in RPMI 1640 containing 10% heat-inactivated fetal calf serum, 2 mM glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 1 μg/ml puromycin in T25 flasks and maintained at 37°C with 5% CO₂ in the presence of Δ⁹-THC (1 or 10 μM) or vehicle for 11 days. Culture media was replaced at 3–4 day intervals. After 11 days, 1.5 × 10⁶ MT4-R5 cells from each treatment group were inoculated with 10 TCID₅₀ SIV_mac in fresh media containing Δ⁹-THC or vehicle, plated in 96-well dishes at a density of 1 × 10⁵ cells/well, and maintained at 37°C with 5% CO₂. Uninfected cultures of Δ⁹-THC- and vehicle-treated cells were maintained identically for use as controls. One-half of the replicate cultures were harvested 3 days post-SIV-infection, with the remainder harvested 6 days after infection. Supernatant and cell pellets were fractionated by centrifugation at 700 × g for 5 min at room temperature. Cell viability was monitored through cell counts using Trypan blue. DNA was prepared and the quantity of proviral SIV DNA in the cell pellets was determined by real-time PCR. DNA viral load (log SIV DNA copies/well) was quantified in cells harvested at 3 and 6 days postinoculation. Expression of CB1 and CB2 receptors was confirmed in these cells prior to using them in the study. Values are mean ± SEM of eight replicate wells. Results were analyzed with two-way ANOVA followed by pairwise multiple comparison procedures (Holm–Sidak method) *p ≤ 0.008.

In summary, this study is the first to report in vivo experimental data demonstrating that chronic Δ⁹-THC initiated prior to, and continued throughout the asymptomatic phase of SIV infection, does not impair the host's ability to control viral load, and does not increase morbidity and mortality from the infection. Although the small groups and natural variation in SIV disease are limitations of the study, the vehicle-treated group is representative of typical rhesus infections with SIV_mac251. Δ⁹-THC treatment clearly did not increase disease progression, and indeed resulted in generalized attenuation of the classic markers of SIV disease (set point viral load/viral level in general). The mechanisms underlying the decreased mortality in cannabinoid-treated SIV-infected animals remain to be elucidated. However, based on our results and reports in the literature, we speculate that retention of body mass, attenuation of viral replication, and an overall immunosuppressant effect of cannabinoids may contribute to the amelioration of SIV disease progression seen in our study. The cellular mechanisms, including the potential role of the recently identified interaction between the HIV coreceptor CXCR4 and the cannabinoid system²² as potential sites of pharmacoimmunomodulation, remain to be determined.

Acknowledgments

The authors would like to thank Joseph Moerschbaecher, PhD, for his helpful discussions on chronic THC administration and dosing and Dr. Catalin Filipeanu for cannabinoid receptor expression measurements. Additionally, the authors would like to acknowledge the technical expertise of Connie Porretta, Jean Carnal, Nedra Lacour, Peter Lewis, Merlin Johnson, Jessica Howard, and Jaime Hubbell. Supported by NIDA-020419-01.

Author Disclosure Statement

No competing financial interests exist.