www.plosone.org 20.03.2012

Abstract

Agents that activate cannabinoid receptor pathways have been tested as treatments for cachexia, nausea or neuropathic pain in HIV-1/AIDS patients. The cannabinoid receptors (CB1R and CB2R) and the HIV-1 co-receptors, CCR5 and CXCR4, all signal via Gαi-coupled pathways. We hypothesized that drugs targeting cannabinoid receptors modulate chemokine co-receptor function and regulate HIV-1 infectivity. We found that agonism of CB2R, but not CB1R, reduced infection in primary CD4+ T cells following cell-free and cell-to-cell transmission of CXCR4-tropic virus. As this change in viral permissiveness was most pronounced in unstimulated T cells, we investigated the effect of CB2R agonism on to CXCR4-induced signaling following binding of chemokine or virus to the co-receptor. We found that CB2R agonism decreased CXCR4-activation mediated G-protein activity and MAPK phosphorylation. Furthermore, CB2R agonism altered the cytoskeletal architecture of resting CD4+ T cells by decreasing F-actin levels. Our findings suggest that CB2R activation in CD4+ T cells can inhibit actin reorganization and impair productive infection following cell-free or cell-associated viral acquisition of CXCR4-tropic HIV-1 in resting cells. Therefore, the clinical use of CB2R agonists in the treatment of AIDS symptoms may also exert beneficial adjunctive antiviral effects against CXCR4-tropic viruses in late stages of HIV-1 infection.

Cristina Maria Costantino1,2, Achla Gupta2, Alice W. Yewdall1, Benjamin M. Dale1, Lakshmi A. Devi2*, Benjamin K. Chen1*

1 Department of Infectious Diseases, Department of Medicine, Immunology Institute, Mount Sinai School of Medicine, New York, New York, United States of America, 2 Department of Pharmacology and Systems Therapeutics, Mount Sinai School of Medicine, New York, New York, United States of America

Introduction

Cannabinoid agonists are currently under investigation for the treatment of AIDS-associated cachexia, nausea, and neuropathic pain [1]–[3]. One such drug, dronabinol (Δ9-THC; Marinol®), has won Food and Drug Administration (FDA) approval for treatment of HIV-associated anorexia [4]. Additionally, the prescription of smoked or ingested cannabis (marijuana) for treatment of AIDS-related symptoms has been approved in 14 states [5]. Despite the use of cannabinoids by HIV/AIDS patients, few studies have investigated the impact of such drugs in regard to viral pathogenesis or immune regulation. Early studies conducted in the pre-HAART era suggested a positive correlation between development of opportunistic infection, progression to AIDS, and marijuana use [6], [7]. Yet recent analysis of HIV/AIDS patients enrolled a randomized, placebo-controlled clinical trial designed to study the outcome of cannabinoid administration have indicated that cannabinoid use does not result in significant immunosuppression [1]. Indeed, both smoked marijuana and dronabinol were reported to increase total CD4+ T cell number [1] and naïve T cell number [8] over a 21-day period. A decrease in viral load was also observed in these patients [1]. Similarly, in SIV infected rhesus macaques, Δ9-THC exposure reduced viral load and CD4+ T cell depletion, significantly increasing animal survival over an 11 month period [9]. Despite these findings, the mechanisms by which cannabinoid drugs can influence viral replication or pathogenicity remain unknown.

Cannabinoid agonists activate the CB1R and CB2R cannabinoid receptors. Like the HIV chemokine co-receptors CXCR4 and CCR5, CB1R and CB2R are members of the Gαi-coupled family A GPCRs [10]. CB2R is highly expressed on all CD4+ T cells [11], whereas CB1 expression is found in activated, memory subsets [12]. CB1 and CB2 have been classified as immunosuppressive receptors on CD4+ T cells [13], although antagonism of CB1R and CB2R does not enhance immune activation and knock-out mice do not exhibit differences in T cell frequency or increases in autoimmune pathogenesis [14]. The mechanism(s) by which cannabinoid agonists can modulate CD4+ T cell function remain unclear. Activation of CB2R has been shown to inhibit inflammatory cytokine production in CD4+ T cells [11], which may account for the decrease in autoimmune pathogenesis observed in therapeutic trials of cannabinoid agonists in animal models of multiple sclerosis [14], [15]. CB2R may also function as a chemotactic modulator, as CB2R activation inhibits CXCR4-induced chemotaxis in transformed lymphocytes [16]. CB2R has further been shown to regulate lymphocyte egress from the bone marrow in a role previously attributed largely to CXCR4 [17], [18]. These findings suggest that CB2R may play a role in regulating chemokine receptor signaling, specifically the activity of CXCR4. Such cross-talk between CB2R and CXCR4 may have implications for AIDS patients who take cannabinoid-derived agents for therapeutic purposes.

Although coreceptor signaling is not essential for HIV-1 infection, several recent studies have suggested that chemokine receptor signaling enhances infection of resting CD4+ T cells [19]–[21]. These cells express CXCR4, but not CCR5, whose expression is restricted to a small subset of memory CD4+ T cells [22]. In patients, the emergence of CXCR4-tropic virus usually occurs after years of infection and correlates with more rapid progression to AIDS [23], [24], [25]. Viral conversion to CXCR4-tropism increases the number of targets available to the virus [26]. Additionally, as HIV-1 can establish latency in resting T cells [27], a switch to CXCR4-tropism could enhance the establishment of a latent pools of virus within lymphoid tissues. The increased number of new targets may explain the rapid decline in CD4+ T cell numbers and increased viral load in late-stage AIDS patients with CXCR4-tropic virus [1], [24]. The late-stage patients who frequently harbor CXCR4-tropic virus are also the most likely to benefit from cannabinoid drug use. It is therefore relevant to study the potential for cannabinoid signaling to modulate CXCR4 activity and alter the course of HIV infection, Interactions between GPCRs like CB2R and CXCR4 can cause cross-desensitization, allosteric modulation, dimerization, changes in receptor localization, and alteration of physiological function among GPCR pairings [28]. Given that direct antagonism of chemokine receptor function can block viral infection [19], [29], [30], it is possible that allosteric modulation of CXCR4 through a GPCR partner may also reduce HIV-1 permissiveness. Indeed, oligomerization of the chemokine co-receptors including CXCR4 using conformationally specific monoclonal antibodies can inhibit HIV-1 entry into target cells [31], [32]. These experiments demonstrate signaling-independent modulation of coreceptor function. Allosteric agents that disrupt HIV-1 infection by modulating chemokine receptor signaling have not yet been identified. Should CB2R-induced signaling alter CXCR4 co-receptor function, this would represent first known example of a signaling-dependent GPCR interaction leading to viral inhibition.

To test this hypothesis, we examined the effect of cannabinoid receptor activation on HIV viral transmission and productive infection in CD4+ T cells using a GFP-expressing, CXCR4-tropic HIV-1 variant. We found that activation of CB2R on CD4+ T cells significantly inhibited viral infection in a CB2R -selective and dose-dependent manner. Viral inhibition was more pronounced in resting cells that were activated after infection. We investigated signaling in these cells and found that CB2R agonism significantly decreased SDF-1α-induced CXCR4 activation. Furthermore, CB2R agonism altered HIV-induced cytoskeletal rearrangement, associated with productive HIV infection in resting cells. We found that CB2R was not sufficient to block viral transfer or fusion, but did significantly diminish productive viral infection. We conclude that CB2R is a novel modulator of CXCR4-tropic HIV infection in CD4+ T cells.