Attenuation of HIV-1 replication in macrophages by cannabinoid receptor 2 agonists

Attenuation of HIV-1 replication inmacrophages by cannabinoid receptor

2 agonistsServio H. Ramirez,*,†,1 Nancy L. Reichenbach,* Shongshan Fan,* Slava Rom,*

Steven F. Merkel,* Xu Wang,* Wen-zhe Ho,*,† and Yuri Persidsky*,†,1

*Department of Pathology and Laboratory Medicine and †Center for Substance Abuse Research, Temple University School ofMedicine, Philadelphia, Pennsylvania, USA

RECEIVED OCTOBER 23, 2012; REVISED FEBRUARY 15, 2013; ACCEPTED FEBRUARY 18, 2013. DOI: 10.1189/jlb.1012523

ABSTRACTInfiltrating monocytes and macrophages play a crucialrole in the progression of HIV-1 infection in the CNS.Previous studies showed that activation of the CB2 canattenuate inflammatory responses and affect HIV-1 in-fectivity in T cells and microglia. Here, we report thatCB2 agonists can also act as immunomodulators onHIV-1-infected macrophages. First, our findings indi-cated the presence of elevated levels of CB2 expres-sion on monocytes/macrophages in perivascular cuffsof postmortem HIV-1 encephalitic cases. In vitro analy-sis by FACS of primary human monocytes revealed astep-wise increase in CB2 surface expression in mono-cytes, MDMs, and HIV-1-infected MDMs. We nexttested the notion that up-regulation of CB2 may allowfor the use of synthetic CB2 agonist to limit HIV-1 infec-tion. Two commercially available CB2 agonists, JWH133and GP1a, and a resorcinol-based CB2 agonist, O-1966,were evaluated. Results from measurements of HIV-1RT activity in the culture media of 7 day-infected cellsshowed a significant decrease in RT activity when theCB2 agonist was present. Furthermore, CB2 activationalso partially inhibited the expression of HIV-1 pol. CB2

agonists did not modulate surface expression ofCXCR4 or CCR5 detected by FACS. We speculate thatthese findings indicate that prevention of viral entry isnot a central mechanism for CB2-mediated suppres-sion in viral replication. However, CB2 may affect theHIV-1 replication machinery. Results from a single-round infection with the pseudotyped virus revealed amarked decrease in HIV-1 LTR activation by the CB2 li-gands. Together, these results indicate that CB2 mayoffer a means to limit HIV-1 infection in macrophages.J. Leukoc. Biol. 93: 801–810; 2013.

IntroductionDespite immune recovery in individuals on combination anti-retroviral therapy, the frequency of HAND remains high forreasons that are not well understood [1]. The current under-standing is that HIV-1-associated neurodegeneration is drivenby chronic inflammatory responses in the brain, secondary toa low level of HIV-1 replication in the CNS reservoir cells(macrophages, microglia) [2, 3]. Treatment approaches target-ing inflammation and HIV-1 replication should be beneficialfor amelioration of HAND.

Cannabis has been recognized for its psychoactive propertiesfor centuries and more recently for medicinal activities. Can-nabinoids, the primary psychoactive compounds of cannabis,act through two well-characterized receptors that generate dis-tinct physiological effects when activated. The psychoactiveeffects of cannabinoids are associated with the CB1 receptor,whereas the CB2 receptor (further in the text referred as CB2)mainly mediates anti-inflammatory and immunomodulatoryactions [4]. Cannabinoids have emerged recently as potentialtreatments for HAND. For example, cannabinoid receptor li-gands are able to attenuate tissue inflammation and signifi-cantly reduce morbidity and mortality of SIV-infected ma-caques [5]. Medicinal cannabis reduces neurologic complica-tions (neuropathic pain) in HIV-infected patients [6]. It hasbeen proposed that the effects of medicinal cannabis may beassociated with activation of the nonpsychoactive CB2 receptor.In fact, up-regulation of CB2 expression has been shown onperivascular macrophages/microglia, astrocytes, and brain en-dothelial cells in HIVE [7, 8]. Furthermore, CB2 ligands sup-press HIV-1 replication in microglia, decrease microglial mi-gration toward HIV-1 Tat, protect neurons and endothelialcells against gp120 toxicity, and diminish neuroinflammationin a rodent model of HIVE [9–13]. However, thus far, the roleof CB2-selective activation in HIV-1 infection of macrophageshas not been examined comprehensively.

1. Correspondence: Dept. of Pathology and Laboratory Medicine, Temple Uni-versity School of Medicine, 3401 N. Broad St., Philadelphia, PA 19140, USA.E-mail: yuri.persidsky@tuhs.temple.edu or servio.ramirez@temple.edu

Abbreviations: �-gal��-galactosidase, �9THC�tetrahydrocannabinol,CB2�cannabinoid 2 receptor, Ct�comparative threshold,dTTP�deoxythymidine triphosphate, HAND�HIV-1-associated neurocogni-tive disorder, HIVE�HIV-1 encephalitis, Iba-1�ionized calcium-binding adap-tor molecule 1, Ki�inhibitor constant, MFI�mean fluorescence intensity,qPCR�quantitative PCR

Article

0741-5400/13/0093-801 © Society for Leukocyte Biology Volume 93, May 2013 Journal of Leukocyte Biology 801

In this study, we explore the use of highly specific CB2 re-ceptor agonists to attenuate HIV-1 infection in human macro-phages. Our results indicate that there is a significant up-regu-lation of CB2 expression upon monocyte differentiation tomacrophages that is enhanced further by HIV-1 infection. Syn-thetic CB2 receptor agonists markedly decreased HIV-1 replica-tion (measured by RT activity). CB2 activation may not appearto interfere with viral entry, as there was a lack of regulationby CB2 receptor agonists on gene or surface protein expres-sion of CXCR4 or CCR5. HIV-1 LTR activation and HIV-1 polexpression were suppressed by the agonists in infected macro-phages. These data suggest that a possible hindrance to theHIV-1 replication machinery may be the mechanism of actionbehind the inhibition of HIV-1 by the CB2 receptor.

MATERIALS AND METHODS

Reagents, cell culture, and HIV-1 infectionThe following CB2 receptor ligands were used: JWH133 [(6aR, 10aR)-3-(1,1-dimethylbutyl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo(b,d)pyran],SR144528 {5-(4-chloro-3-methylphenyl)-1-[(4-methylphenyl)methyl]-N-[(1S,2S,4R)-1,3,3-trimethyl[bicyclo(2.2.1)hept-2-yl]-1H-pyrazole-3-carboxam-ide]}, and GP1a {N-(piperidin-1-yl)-1-(2,4-dichlorophenyl)-1,4-dihydro-6-methyl-indeno[1,2-c]pyrazole-3-carboxamide} were purchased from Tocris Bioscience(Ellisville, MO, USA), and O-1966 {(1-[4-(1,1-dimethyl-heptyl)-2,6-dimethoxy-phenyl]-3-methyl-cyclohexanol)} was acquired from Organix (Woburn, MA,USA) and synthesized as described previously [14]. Unless otherwise specified,all other reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA).

Primary human monocytes derived from six donors were acquired fromthe Human Immunology Core at the University of Pennsylvania (Philadel-phia, PA, USA). The facility isolates monocytes using counterflow centrifu-gal elutriation, as described previously [15]. The monocytes were placed in24-well plates within 24 h of isolation. Monocytes were maintained inDMEM, supplemented with 10% heat-inactivated, pooled human serum,1% glutamine, 50 �g/ml gentamicin, 10 �g/ml ciprofloxacin, and 1000U/ml highly purified human rM-CSF, which was present in the medium forthe first 5 days of culture to promote differentiation and was then re-moved. Culture medium was changed every 3 days. After 7 days in suspen-sion culture, MDMs were infected with HIV-1ADA (a macrophage tropicstrain; obtained from the AIDS Research and Reference Reagent Program,National Institutes of Health, Germantown, MD, USA) at a m.o.i. of 0.1infectious virus particle/target cell. In pretreatment experiments, MDMswere exposed to the experimental treatment for 24 h before introductionof the virus. For post-treatments, the MDMs were infected with virus for 4h, washed with DMEM, and then treated with the CB2 ligand. Treatmentswere maintained for 7 days, with one-half medium replacement on Days 3,5, and 7 after HIV was added to the cells. On Days 3, 5 and 7, culture me-dium was collected and stored at �80°C until it was assayed for HIV-1 RTactivity. The reporter cell line TZM-bl was obtained through the NIH AIDSResearch and Reference Reagent Program, Division of AIDS, National Insti-tute of Allergy and Infectious Diseases; TZM-bl cells were generated by Dr.John C. Kappes, Dr. Xiaoyun Wu, and Tranzyme (Durham, NC, USA). Thecells were grown in six-well plates in DMEM, 10% FBS, 100 U penicillin,and 0.1 mg/ml streptomycin. All cell-culture reagents (media, antibiotics,etc.) were purchased from Invitrogen/Life Technologies (Carlsbad, CA,USA).

ImmunohistochemistryImmunohistochemistry was performed on serial sections from postmortemparaffin-embedded brain tissue [16]. The analyzed tissue originated fromfrontal cortical regions from four cases of HIVE and from four seroneg-ative, age-matched controls. The cases were provided by the National

NeuroAIDS Tissue Consortium (Washington, DC, USA). With the use ofstandard immunohistochemistry methods, serial sections (5 � in thickness)were generated and then baked at 65°C for 20 min, followed by deparaf-finization and rehydration. The tissue was then subjected to antigen re-trieval by incubating the sections at 100°C in 10 mM sodium citrate buffer(pH 6.0) for 20 min. Endogenous peroxidase activity was quenched bytreating with Peroxidazed 1 (Biocare Medical, Concord, CA, USA) for 15min. To block nonspecific antibody binding, the slides were incubated with1% goat serum in 1� PBS and 0.1% Triton X-100 (Sigma-Aldrich). Forimmunolabeling, the following primary antibodies were used: polyclonalantibodies against human CB2 (Thermo Scientific, Rockford, IL, USA; di-luted at 1:100 with overnight incubation), polyclonal antibodies to Iba-1(Wako Chemicals USA, Richmond, VA, USA; diluted at 1:200 with over-night incubation), and mAb to HIV-1 p24 (Clone Kal-1; Dako, Carpinteria,CA, USA; diluted at 1:10 with overnight incubation). Detection was per-formed with anti-mouse or anti-rabbit secondary antibodies (Biocare Medi-cal) in combination with the Mach 3 HRP-polymer system and the 3,3=-di-aminobenzidine substrate (Biocare Medical). Normal rabbit or mouse IgGat the same concentrations as the primary antibodies served as negativecontrols. Counterstaining of the nuclei was performed using hematoxylin(Biocare Medical). Representative images shown in the first figure origi-nate from previously published cases [16]. The control image is from CaseNumber 8, HIVE (middle) is from Case Number 2, and HIVE (bottom) isfrom Case Number 3. Clinical history of the cases used did not indicatetreatment with antiretroviral therapy.

Real-time qPCRTotal cellular RNA was isolated from macrophages (1�106 cells) on Day 7post-HIV infection using the RNaqueous 4PCR kit, according to the manu-facturer’s directions (Ambion, Austin, TX, USA). RNA purity and concen-tration were determined with a NanoDrop ND-1000 spectrophotometer(Thermo Fisher Scientific, Fair Lawn, NJ, USA). Total RNA (2 �g) was sub-jected to RT using the high-capacity cDNA RT kit, according to the manu-facturer’s directions (Applied Biosystems/Life Technologies, Carlsbad, CA,USA). The cDNA (diluted 1:20) template was then mixed with the MaximaProbe/ROX qPCR Master Mix (Fermentas, Hanover, MD, USA) and thecorresponding human TaqMan gene expression assay (Applied Biosystems/Life Technologies): CNR2: Hs00361490_m1; CXCR4: Hs00607978_s1; andCCR5: Hs99999149_s1. Maxima Probe/ROX qPCR Master Mix (2�; Fer-mentas) was used to measure HIV-1ADA Pol gene expression with the follow-ing oligonucleotide primers: 5=-GAAATTTGTACAGAAATGGAAAAGGAA-3=(forward) and 5=-TGAGTTCTCTTATTAAGTTCTCTGAAATCTAC-3= (re-verse) and FAM-5=-TTGGGCTGAAATCCATACAATACTCCAGT-3=-TAMRA(Taqman probe). All controls and samples were run in triplicate in thesame culture plate. GAPDH mRNA was measured with the Taqman geneexpression assay, Hs02758991_g, and was used as a control to normalizethe mRNA content in the samples tested. qPCR was performed on aStepOnePlus real-time PCR system (Applied Biosystems/Life Technolo-gies). Raw data were analyzed with DataAssist software (Applied Biosys-tems/Life Technologies) using the ��Ct method (relative quantification).Results are expressed in relative gene expression levels (fold) comparedwith the untreated control or the HIV-1-only condition.

Flow cytometryAnalysis of surface expression of CB2 and HIV-1 coreceptors CXCR4 andCCR5 was determined by flow cytometry. Briefly, 1 � 106 cells were placedin staining solution (2% BSA in PBS with 0.5% NaN3) containing fluoro-phore-conjugated antibodies to CXCR4 (anti-CD184-allophycocyanin; BDBiosciences, Franklin Lakes, NJ, USA) and CCR5 (anti-CD195-PE; BD Bio-sciences) for 30 min on ice. For detection of CB2 expression, the cells wereincubated for 1 h with polyclonal antibodies to human CB2 (Thermo Scien-tific), followed by detection with donkey anti-rabbit Alexa-488-conjugatedantibodies. Cells were then washed and fixed in 2% methanol-free formal-dehyde (Thermo Scientific) in 1� PBS. Acquisition and analysis of the la-beled cells were then performed using a FACSCanto II flow cytometer (BD

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Biosciences). Acquisition parameters and gating were controlled by FACS-Diva software (BD Biosciences). Data analysis was performed with FlowJosoftware (Tree Star, San Carlos, CA, USA). The data represent the fluores-cence intensity of gated populations (as determined by isotype-matchedcontrols) of at least 100,000 events recorded in a single experiment thatwas repeated at least three times.

RT assayThe HIV-1 RT assay is a radiometric assay designed for the quantitativemeasurement of RT activity in cell culture, as described previously [17].This assay is used to determine the propagation of HIV-1 in infected MDMcells. Please note that the RT assay (as used in this study) was not in theformat used in screening for RT inhibitors, which involves challenging pu-rified HIV-1 RT directly with the test compound. Rather, the results arerepresentative of HIV-1 RT activity present in the medium of HIV-1-in-fected MDMs, untreated or treated with the indicated experimental com-pound at the given time-point. Briefly, 10 �l culture supernatant was mixedwith 50 �l reaction mixture containing 5 �g/ml poly(A) (GE Healthcare,Mickleton, NJ, USA) and oligo dT (1.57 �g/ml; USB, Cleveland, OH,USA) in 50 mM Tris, pH 7.8, 7.5 mM KCl, 2 mM DTT, 5 mM MgCl2,0.05% Nonidet P-40, and 0.5 �Ci [�-32P]dTTP (Perkin Elmer, Waltham,MA, USA) and incubated for 16 h at 37°C. Each reaction mixture was thenspotted onto a Whatman DE81 (Thermo Fisher Scientific) paper, dried,and washed five times with 2� sodium citrate buffer (200 ml/wash) andonce with 95% ethanol (150 ml) to remove unincorporated [�-32P]dTTP.Radioactivity on the air-dried filter paper was determined in a Tri-Carb2810TR liquid scintillation spectrometer (Perkin Elmer).

Single-round HIV-1 infection and �-gal assayRecombinant luciferase-encoding HIV-1 virions were pseudotyped with theenvelope from HIV-1ADA, as described previously [18]. The pseudotypedvirus was used for single-round infectivity assays in TZM-bl cells. Detectionof �-gal was performed using the �-gal staining assay (Invitrogen/LifeTechnologies). After staining, images from each experimental conditionwere acquired using an Axio Observer Z1 microscope configured with anAxioCam HR camera (Carl Zeiss MicroImaging, Thornwood, NY, USA).Analysis was performed using AxioVision (v4.7) imaging software (CarlZeiss MicroImaging). The number of stained cells was counted and aver-aged (three replicates) for each condition from at least three independentexperiments.

Cell viability assayAfter monocyte differentiation to macrophages (see above), the cells wereinfected with HIV-1ADA at a m.o.i. of 0.1. Two hours after the initial infec-tion, the indicated CB2 agonist, AZT or vehicle control was added. Sevendays postinfection, cell viability was assessed using calcein-AM (Invitrogen/Life Technologies), according to the manufacturer’s recommendations.The assay is based on the principle that intracellular esterase activity (inlive cells) converts the nonfluorescent and cell-permeable calcein-AM intopolyanionic calcein, which is retained and intensely fluorescent (excitationat �495 nm and emission at �515 nm). Calcein-AM was added at 1 �M,and the cells were incubated for 30 min, followed by three rinses with 1�

PBS. Fluorescence emitted from live cells was measured at 495 nm (excita-tion)/530 nm (emission) on an M5 fluorescence plate reader (MolecularDevices, Sunnyvale, CA, USA). All measurements were performed in tripli-cate, and the data are represented as the percent viability from the unin-fected control.

Statistical analysisResults are presented as mean � sd or sem, and P values � 0.05 are con-sidered significant. Data were analyzed using the Prism v5 software (Graph-Pad Software, La Jolla, CA, USA), and statistical significance was deter-mined by performing unpaired two-tailed Student’s t-test or ANOVA with

the Dunnett’s post-test. All in vitro studies were performed with cells fromat least three different donors with multiple replicates (at least three).

RESULTS

Up-regulation of CB2 expression in infiltratingmonocytes/macrophages in cases of HIVEIt is well established that immune cells express CB2; however,how this expression may change during HIV-1 infection re-mains unknown. Therefore, we first examined CB2 expressionin HIV-1-infected monocytes/macrophages in perivascularcuffs present in HIVE. Autopsy tissues derived from six pa-tients with HIVE, four HIV-positive controls, and five controlseronegative patients were immunostained for CB2. Clinical,pathologic, and demographic details of these cases have beenpublished before [16, 19]. Figure 1 shows representative im-ages of immunostains in two HIVE cases and one HIV-1-sero-negative control. HIVE cases demonstrated strong staining forCB2 in perivascular monocytes/macrophages and some micro-glia cells (Fig. 1B and C). Macrophages and microglial cellswere detected by immunoreactivity with antibodies to Iba-1(Fig. 1E and F). Interestingly, a number of the Iba-1-positivecells showed CB2 immunoreactivity. Immunolabeling for theHIV-1 core antigen, p24, showed variable staining in many ofthe CB2-positive cells (Fig. 1H and I). In addition to themonocytes/macrophages and microglia, microvascular endo-thelial cells featured enhanced CB2 staining in the HIVE cases(Fig. 1, compare A with B and C). In control cases, only mar-ginal staining was observed for the endothelium and residentperivascular macrophages and rarely in microglial cells (Fig.1A and D). None of the cells in control cases demonstratedHIV-1 p24 positivity. Also, HIV-1-positive cases (without signsof encephalitis) featured an increased number of activated mi-croglia and macrophages with variable intensity for CB2 reac-tivity (data not shown). These results suggest that the in-creased expression of CB2 in macrophages and microglia isassociated with HIV-1 infection and neuroinflammation.

Expression of CB2 in HIV-1-infected MDMsTo evaluate whether CB2 expression changes as a function ofdifferentiation (monocyte to macrophage) and HIV-1 infec-tion, we examined the gene and protein expression profile ofCB2 in primary human monocytes. Isolated human peripheralblood monocytes from six healthy donors were used within 24 h orallowed to differentiate for 7 days by adhesion and exposureto M-CSF. Differentiated MDMs were uninfected or infectedwith the macrophage-tropic HIV-1ADA for 7 days. qPCR andanalysis by the ��Ct method revealed a step-wise increase inCB2 expression with the following pattern: monocytes �MDMs � HIV-1-infected MDMs (Fig. 2A). Relative (fold regu-lation) expression showed MDMs with a 5.47 � 1.07 increaseover that observed for undifferentiated monocytes (set at 1).Infection of MDM with HIV-1ADA significantly increased theCB2 receptor gene expression even further, resulting in 7.41 �0.94-fold up-regulation compared with monocytes and a 1.35-fold increase over uninfected MDMs. With the use of a similarexperimental approach, we evaluated surface expression of

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CB2 by FACS analysis. With the use of antibodies for CB2,which recognize epitopes in the extracellular N-terminus seg-ment of CB2, FACS analysis showed a similar detection patternas observed in the gene-regulation profile. Figure 2B showsrepresentative histograms for detection of CB2 along with MFIvalues. A fold induction of 1.76 in MDMs compared withmonocytes was observed and a 2.28-fold increase in infectedMDMs, in contrast to uninfected MDM. Together, these resultsare complimentary in that the increase in gene regulationtranslates into an increase in CB2 protein expression. Further-more, the analysis in primary cells appears consistent with theobservations in Fig. 1, suggesting that HIV-1 infection inducesfurther elevation in CB2 expression.

CB2 receptor agonists inhibit HIV-1Given the anti-inflammatory role that CB2 plays in immunecells and the observations that the CB2 receptor is not only

present in HIV-1-infected MDMs but also appears up-regulatedwhen compared with uninfected cells (Figs. 1 and 2), we ex-amined whether CB2 ligands may affect HIV-1 infection. Dif-ferentiated MDMs were infected with HIV-1ADA for 4 h (viralincubation period). The media were changed, and the infec-tion was allowed to continue for 7 days (see Materials andMethods). Where indicated (Fig. 3), infected MDMs were ex-posed to the potent CB2 receptor agonist JWH133; Ki � 3.4nM with �200-fold selectivity for the CB2 receptor over CB1

[20]. Performed in parallel and with the same donor, the ac-tivity of HIV-1 RT activity was measured from the culture me-dium of infected cells at the end of 7 days. JWH133 in the“pretreated” groups were introduced 24 h prior to infection,whereas in the “post-treated” groups, JWH133 was added fol-lowing the viral incubation period (Fig. 3A, pre- and post-treated). The addition of the CB2 agonist resulted in a dose-dependent decrease of RT activity. JWH133 at 1 �M reduced

Figure 1. Increased CB2 expression in brain tissue from cases of HIVE. Brain tissues from frontal cortical regions were obtained from six cases ofHIVE and from five seronegative age-matched controls (Ctr). (B and C) Representative images showing CB2-positive macrophages (arrows) foundin the perivascular spaces (P.S) of HIVE cases. Note that enhanced CB2 receptor (CB2R) immunostaining is also present in the endothelial cells.V.L, Vessel lumen. (E and F) Activated macrophages, forming perivascular cuffs and microglia in the brain parenchyma, were detected by Iba-1immunostaining. (H and I) The presence of HIV-1-infected (p24-immunopositive) macrophages/microglia. (A, D, and G) The normal controlcase lacks immunoreactivity for the HIV-1 p24 antigen (G) and minimal CB2 receptor positivity (A). Low Iba-1 staining (D) in the control caseswas limited to resting macrophages/microglial cells. Original objective magnification was 40� and 20� for inset images. Original scale bars � 50� for the 40� images and 100 � for the insert image.

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the detectable RT activity in the medium by 34.6% and 68.9%when the agonist was introduced, pre- and post-treated, respec-tively. JWH133 at 10 �M resulted in an 82.8% decrease in RTactivity in the pretreated group and similarly, when the cellswere post-treated. Administration of the CB2-inverse agonist/antagonist SR144528 had no significant effect on RT activity;however, when coadministered with JWH133, it counteractedagonist effects completely (Fig. 3A). Of note, the RT inhibitor,AZT (10 �M), served as control for suppression of HIV-1 repli-cation, thus resulting in decreased levels of RT activity in themedium of treated cells. The results also suggest that the ef-fect of the CB2 agonist on viral replication (RT assay) may notbe a result of interference with viral entry. This is surmisedfrom the post-treated group, where the agonist was added fol-lowing the 4-h virus incubation period. The results also showthat the inhibition observed in the presence of the CB2 ago-nist requires a continued and prolonged exposure, as the ef-

fect is limited at earlier time-points (Fig. 3B). Although adownward trend was observed when JWH133 was present, RTactivity at Day 3 was not reduced significantly. A similar trendwas observed at Day 5, with the only significant difference(36% inhibition) apparent at the higher concentration ofJWH133. Two additional synthetic CB2 agonists were also in-vestigated, GP1a (Ki�0.037 nM) and O-1966 (Ki�23 nM) [14,21]. As JWH133 appeared more effective when added as apost-treatment (after the introduction of HIV-1), the additionof GP1a or O-1966 was done in a similar fashion (Fig. 3C andD, respectively). The results indicated a marked decrease inthe degree of RT activity when either of the two agonists wasadded to the cells. GP1a resulted in a 87.4% (2 �M) and88.7% (10 �M) decrease in detectable RT activity. Likewise,the addition of the novel resorcinol-based compound O-1966provided a 73.3% (1 �M), 85% (5 �M), and 87.6% (10 �M)attenuation in RT activity present in the culture medium. In-terestingly, like JWH133, GP1a and O-1966 had no significanteffect on RT activity at Days 3 and 5 (data not shown). Cellviability does not appear to be a factor contributing to the de-creased level of RT activity. As reported by others [22, 23], theassessment of JWH133 on cell viability did not compromisecell survival (data not shown). GP1a and O-1966 did notchange the viability of infected MDMs at the concentrationsused above (Fig. 3E). A decrease in viability (20%) was evidentonly when GP1a was used at a much higher concentration(Fig. 3E). The results from these RT assays suggest that theup-regulation in CB2 expression in infected cells offers thepossibility to use CB2 agonists to suppress HIV-1 replication.

CB2 activation in MDMs does not affect theexpression of CXCR4 or CCR5In light of the inhibitory effect that CB2 receptor agonists haveon HIV-1 RT activity, it is possible that CB2 signaling alters theexpression of HIV-1 coreceptors, thereby preventing re-entryof HIV-1 virions into the cells. To test this possibility, expres-sion profiles for CXCR4 and CCR5 were evaluated by FACSand gene expression (Fig. 4). Figure 4A and B shows represen-tative histograms for the expression of CXCR4 and CCR5 (re-spectively) by FACS in MDMs that were treated with JWH133(10 �M), GP1a (10 �M), or O-1966 (10 �M) for 24 h. As indi-cated, there was no change in the level of these HIV-1 core-ceptors in the presence of the agonists. Interestingly, HIV-1infection (7 days) did not change gene expression for CXCR4(Fig. 4C) or CCR5 (Fig. 4D) in MDMs. Therefore, CB2-medi-ated inhibition of HIV-1 in primary human macrophages doesnot involve down-regulation of CXCR4 or CCR5.

CB2 agonists inhibit the activity of the HIV-1 LTRTo evaluate whether CB2 signaling may affect the activity ofthe HIV-1 LTR, single-round infection of the TZM-bl reportercell line was used. Pseudotyped envelope-deficient HIV-1 viri-ons were produced by cotransfection in 293T cells of a NL4-3HIV-1 backbone DNA construct, along with a separate DNAconstruct encoding for the HIV-1ADA envelope. Purified, pseu-dotyped viruses were then introduced to TZM-bl cells, whichstably express CXCR4 and CCR5 and also contain the HIV-1

Figure 2. Monocyte differentiation and HIV-1 infection increase CB2

receptor expression. (A) Relative expression (RQ) of the CB2 genewas determined by real-time qPCR in monocytes, MDMs, and MDMsinfected with HIV-1ADA (7 days postinfection at an m.o.i. of 0.1). Thefold change in monocytes was assigned a value of 1.0. The results arerepresented as the mean value � sd with P values shown for the indi-cated comparisons (brackets). (B) Primary human monocytes, differ-entiated MDMs, and HIV-1-infected MDMs, immunostained for CB2,were analyzed by FACS, as described (see Materials and Methods).The MFI values for surface expression are shown � sd. Gene expres-sion and FACS analysis were performed in replicates from at leastthree different donors.

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LTR-driven �-gal reporter gene. The infection was allowed tooccur for 6 h, at which point, the medium-containing virus wasremoved and changed to media containing JWH133 (1 �M)or O-1966 (1 �M). Figure 5A shows the results from the �-galassay performed at 48 h after initiation of infection. Comparedwith untreated cells, JWH133 produced a 58% decrease in thenumber of �-gal-positive cells. O-1966 had a comparable effectwith JWH133, resulting in 59% inhibition. To further validatethe inhibition of the HIV-1 LTR, qPCR studies in primaryMDMs were also performed for measurement of the level ofHIV-1 pol transcription. MDMs infected with HIV-1ADA, as de-scribed earlier, were exposed to the indicated CB2 ligands(Fig. 5B). Analysis of fold-change regulation of HIV-1 pol copynumber revealed a significant decrease at the concentrationstested for the CB2 agonists, whereas no change was observed

in the presence of the CB2 antagonist, SR144528. The de-creased fold differences between infected cells treated withagonists compared with infected but untreated cells were asfollows: JWH133, 1 �M (2.5-fold); JWH133, 5 �M (3.9-fold);O-1966, 1 �M (3.8-fold); and O-1966, 5 �M (6.3-fold). TheCB2 antagonist reversed the effects of CB2 agonists on HIV-1pol, indicating receptor-specific effects. Taken together, theseresults suggest the possibility that CB2 activation decreases viralreplication by affecting the HIV-1 LTR.

DISCUSSION

The CB2 and the CB1 receptors comprise the cannabinoid re-ceptor family. Unlike the CB1, the activation of the CB2 recep-

Figure 3. HIV-1 RT activity is decreased in in-fected MDMs treated with CB2 agonists. (A)MDMs were incubated with macrophage tropicHIV-1ADA (m.o.i.�0.1) for 4 h, and the infectionwas allowed to continue for 7 days. The CB2 ag-onist JWH133 (1 �� and 10 ��) was intro-duced 24 h before (Pre-treated) or immediatelyafter (Post-treated) the HIV-1 incubation period.The CB2 receptor inverse agonist/antagonistSR144528 (10 �M) or the RT inhibitor AZT (10�M) was used as a control. The results are rep-resented as cpm/ml to show the RT activitypresent in the medium at Day 7. Note thatJWH133 was used at 1 �M when present withSR144528 (10 �M). NT, non-treated; N.S, nostatistical significance at P � 0.05 among thegroups compared. (B) Values from RT assaysperformed on medium collected at Days 3 and 5from cells infected (inf.) as described above.

(C) Results of RT activity from medium (Day 7) of HIV-1ADA-infected MDMs treated with the CB2 agonist GP1a (2 and 10 �M), which was addedafter the virus was added for 4 h (post-treated). (D) Dose response in infected MDMs treated with the novel (resorcinol-derived) and highly selec-tive CB2 agonist, O-1966 (1, 5, and 10 �M). The agonist was added after the initial 4-h viral incubation (post-treated), and the RT assay was performedon medium from Day 7. All values are represented as the mean � sd. (E) Analysis from viability assays using the fluorescence indicator calcein-AM. Thecells were infected and treated as indicated in B and C. At the end of 7 days, the viability assay was performed as described in Materials and Methods.The results are shown as the percent mean � sd from the untreated control. The P values are provided in the figure above the noted group compari-sons (brackets). P values shown without brackets, compare each treatment with the HIV-1-only control or untreated control, as in the case of the viabil-ity results. The results were acquired from replicates of differentiated primary cells from at least three different donors.

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tor does not elicit psychoactive effects but rather promotesanti-inflammatory responses [24]. To date, the effect of CB2

on HIV-1-infected macrophages has remained largely un-known. Such information is of great interest in the context ofHIV-1 neuropathogenesis, as infection of monocytes/macro-phages is thought to be the primary cause of how HIV-1 infil-trates the CNS [25, 26]. Thus far, several studies have demon-strated CB2 expression in microglial cells, perivascular macro-phages, and T cells in SIV encephalitis [27]. More recently, astudy of HIVE from postmortem cases also showed a markedincrease in CB2 receptor expression in microglia, astrocytes,and perivascular cells [8]. Another example of the CB2 recep-tor up-regulation in HIVE can be found in the relevant mu-rine model of neuroAIDS [13]. The authors showed that theCB2 receptor, but not the CB1 or the orphan cannabinoid re-ceptor GPR55, was up-regulated in HIVE brains in an animalmodel [13]. Our results are in agreement with these previousstudies but also further add that in HIVE, microvessels andHIV-1-infected monocytes/macrophages in the perivascularspace show up-regulated levels of the CB2. The up-regulationof the CB2 on microglial cells and on infiltrating immune cellshas prompted the notion that up-regulation of the receptorand binding to endocannabinoids may act as a triggeringmechanism to attenuate inflammation. Moreover, regional in-creases in cannabinoid receptor expression have been shownto increase potency and efficacy of exogenous agonists at sitesof end-organ injury [28].

Up-regulation of CB2 is a key observation that could allowfor targeting of the CB2 receptor to 1) minimize the inflam-matory effects of HIV-1 harboring mononuclear cells and 2)reduce viral infection (the hypothesis presented in this study).Therefore, with the increasing availability of novel synthetic,highly specific CB2 agonists, we investigated whether HIV-1could be inhibited upon CB2 activation on macrophages in-fected with the M-tropic HIV-1ADA strain. We first examinedthe expression of CB2 in primary human monocytes, MDMs,and MDMs infected with HIV-1. The results for protein andgene expression showed the following increased order of CB2

receptor expression: monocytes � MDMs � HIV-1-infectedMDMs. These results, obtained from multiple donors, suggestthat CB2 expression is altered as a function of monocyte differ-entiation and activation. Up-regulation of the receptor by acti-vation appears to be a common phenomenon in inflammation[28, 29]. Earlier, we reported on the increase of CB2 receptorlevels in brain endothelial cells after exposure to IL-1�, TNF-�,and LPS [19]. Others have reported on increased CB2 recep-tor expression on activated microglia as result of Acanthamoebaculbertsoni-mediated neuroinflammation [30]. In terms of HIV-1-induced activation, our study is the first (that we are awareof) to show that HIV-1 infection up-regulates the expression ofCB2 in macrophages. Interestingly, Raborn and Cabral [31]used U937 (a monocytic cell line) to show that the HIV-1 Tathas no effect on CB2 expression. Therefore, HIV-1 accessoryproteins, such as the transactivator Tat, may not cause induc-

Figure 4. Effect of CB2 agonist on the expression of HIV-1 coreceptors, CXCR4 and CCR5. Representa-tive histograms of FACS analysis of CXCR4 (A) and CCR5 (B) expression of MDM cells exposed to CB2

receptor agonists JWH133 (10 �M), GP1a (10 �M), and O-1966 (10 �M). The data are represented asthe MFI of CXCR4 or CCR5 as a percent of the maximum intensity. (C and D) Results from real-timeqPCR analysis of CXCR4 and CCR5 gene expression analysis, respectively. Uninfected or MDMs infectedwith HIV-1ADA (m.o.i.�0.1) were cultured in the presence of JWH133 (10 �M). Total cellular RNA wasisolated from MDMs on Day 7 postinfection and converted to cDNA. The resulting cDNA was then usedas a template for real-time PCR quantification. ��Ct values normalized to the housekeeping geneGAPDH are presented as the fold change relative to that of the uninfected control.

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tion in CB2 receptor levels, but rather, cellular responses todirect viral infection do. We next examined whether the en-hanced presence of the CB2 receptor could offer a therapeuticopportunity to curb HIV-1 infection. Indeed, the CB2 agonistJWH133 demonstrated a dose-dependent inhibition (up to82%) in HIV-1 RT activity in MDMs infected for 7 days. Impor-tantly, the addition of JWH133 prior to or after HIV-1 infec-tion provided comparable levels of inhibition. These resultsare complementary with those by Costantino et al. [32], show-ing 40% inhibition in HIV-1 infection in primary CD4 Tcells using a similar concentration (1 �M) of JWH133. Similarobservations were also found in HIV-1-infected microglial cellsand CD4 T cells using WIN55,212-2 [33]. A dual agonist forCB1 and CB2 receptors, WIN55,212-2 was shown to limit HIV-1p24 expression by � 60% in microglia and CD4 T cells [33].A follow-up study from the same group using the CB2 receptoragonist JWH015 reported a 40% inhibition in HIV-1 p24 ex-pression in infected microglial cells [9], ascertaining that theHIV-1 suppression by WIN55,212-2 in microglial cells was, inpart, a result of CB2-related effects. To ensure CB2 receptorspecificity, our analysis included the reverse agonist/antagonistSR144528. Indeed, SR144528, whether coadministered withthe agonist before or after the onset of HIV-1 infection, com-pletely eliminated the effects of the agonist. In addition toJWH133, two recently discovered and potent CB2 agonists,GP1a and O-1966, were also tested for HIV-1 inhibition. GP1aand O-1966, added after HIV-1 introduction and replenishedduring a 7-day infection, revealed an inhibition of up to 88%and 85% in RT activity, respectively. Thus, the efficacy of re-ducing HIV-1 RT activity can be achieved to similar levels withCB2 receptor agonists of varied chemical composition.

To understand the mechanism behind how the agonist af-fects HIV-1 infection in macrophages, we turned to evaluationof the HIV-1 coreceptors. Previously, it has been suggestedthat one possible consequence of CB2 signaling is that it mayaffect the expression of HIV-1 coreceptors, CXCR4 and CCR5[9]. Our observations in macrophages support results by othergroups showing no effect of CB2 on CXCR4 expression inCD4 T cells [32]. Here, we provide evidence that the CB2

agonist does not alter surface protein expression of CXCR4 orits gene regulation in MDMs. Similarly, the CB2 agonist didnot change surface protein or gene expression of CCR5 inMDMs. These results are in contrast to the inhibitory effectthat WIN55,212-2 has on CCR5 expression in microglia andthat reported for GP1a in CD4 T cells (human PBLs) [9, 13].However, in the same study with GP1a, CCR5 expression inCD8 cells was unchanged. Perhaps, unlike CXCR4, CCR5could be a regulatory target of CB2 receptor signaling in cer-tain cell types but not others. Our results therefore suggestthat in MDMs, there may not be interference in HIV-1 viralentry, at least not at the level of coreceptor expression, as aresult of CB2 activation.

As the effect on the HIV-1 coreceptors did not seem to pro-vide a plausible explanation for how the CB2 agonist inhibitsHIV-1 infection, we next evaluated the activity of the HIV-1LTR. With the use of single-round infections on the reportercell line TZM-bl, which features the HIV-1 LTR-driven �-galgene, the effects of CB2 agonists were evaluated. The results

Figure 5. CB2 agonist inhibits HIV-1 LTR activity. (A) Results fromsingle-round HIV-1 infection on reporter cell line TZM-bl cells usingHIV-1-pseudotyped virus. The presence of the LTR-driven �-gal pro-vides a measure of LTR activity. TZM-bl cells were infected for 48 h inthe presence of JWH133 (1 �M) or O-1966 (1 �M). Stained cells, asrevealed by the �-gal assay, were counted in each experimental condi-tion and presented in the graph as the mean � sd from three inde-pendent experimetns. P values for the indicated comparisons (brack-ets) are also shown. Representative images (right) from each treat-ment group (dash lines) indicate the percent inhibition with JWH133or O-1966. (B) Real-time qPCR analysis for HIV-1 pol, as a second mea-sure of LTR activity, is shown. HIV-1-infected MDMs, from at leastthree different donors, were incubated in the presence of CB2 recep-tor ligands JWH133 (1 �M), O-1966 (1 �M), and SR144528 (10 �M).��Ct values are presented as the fold change relative to that of theHIV-1-only (vehicle). The results, shown as the mean � sd are repre-sentative of experiments in triplicate using cells from three differentdonors.

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indicated a clear diminution in the number of �-gal-positivecells upon treatment with JWH133 or O-1966. Moreover, re-sults of HIV-1 pol copy number substantiate the observations ofthe LTR, as relative expression of pol was reduced significantlyby all CB2 agonists tested. In light of these results, it is plausi-ble that CB2 signaling could interfere with full activation ofthe HIV-1 LTR. Admittedly, how the HIV-1 LTR may be inhib-ited by the CB2 receptor is not known at this time, but it is thesubject of ongoing studies in our laboratory. It is reasonable tospeculate that a possible explanation may come from cellulartranscription factors that share participation in proinflamma-tory responses and activation of the HIV-1 LTR. In fact, studiesusing �9THC, a partial agonist of CB1 and CB2, have showninhibition of NF-�B in the macrophage cell line Raw 264.7[34]. Furthermore, with the use of reporter assays, Bornerand colleagues [35] demonstrated inhibition of NFAT andNF-�B by �9THC or JWH015 (a CB2 receptor agonist) innaïve and stimulated Jurkat cells. Accordingly, it is conceiv-able that transcription factors involved in the transactivationof the HIV-1 LTR could be targeted for inhibition uponstimulation of the CB2.

The effects of CB2 activation have been well characterized asa means to reduce inflammatory responses in various cellstypes, particularly those of the immune system [24]. To date,however, no study has exclusively focused on whether CB2 re-ceptor activation may affect HIV-1 expression in macrophages.The study presented in this report invites the possibility of us-ing CB2 receptor agonists as a means to limit HIV-1 replicationin infected macrophages. We show that synthetic CB2 agonistsprovide a similar effect on HIV-1 infection, whether appliedbefore or after the onset of infection. The findings also seemto implicate attenuation of the HIV-1 replication machineryrather than a hindrance to viral entry upon addition of theagonists. It should be noted that aside from affecting HIV-1infection in primary macrophages, as reported here, CB2 re-ceptor agonists have pleiotropic effects in macrophages thatmay further lessen the burden of HIV-1. These effects includea decrease in overall proinflammatory response and possibleinhibition in monocyte/macrophage migration into the CNS[13, 31]. Although immunoregulatory, it is also important topoint out that activation of the cannabinoid receptor system isnot immunosuppressive. Systemic administration of GP1a (in amouse model of HIVE) and the now U.S. Food and Drug Ad-ministration-approved use of �9THC for AIDS-related wastinghas not been shown to have adverse consequences on immunefunction [36, 37]. Therefore, the role of the CB2 receptor inHIV-1 infection merits further investigation and testing ofnovel, synthetic CB2 agonists.

AUTHORSHIP

N.L.R., S.F., S.R., and S.F.M. carried out the experiments inthis study. S.H.R. and Y.P. designed experiments, analyzeddata, and wrote the manuscript. X.W. and W-z.H. contributedvital reagents and materials.

ACKNOWLEDGMENTS

This study was supported (in part) by research funding fromthe U.S. National Institutes of Health (RO1MH065151 andR37AA015913 to Y.P.). We thank Dr. Ronald Tuma for kindlyproviding the O-1966 compound.

REFERENCES

1. Ellis, R. J., Badiee, J., Vaida, F., Letendre, S., Heaton, R. K., Clifford, D.,Collier, A. C., Gelman, B., McArthur, J., Morgello, S., McCutchan, J. A.,Grant, I. (2011) CD4 nadir is a predictor of HIV neurocognitive impair-ment in the era of combination antiretroviral therapy. AIDS 25, 1747–1751.

2. Kraft-Terry, S. D., Buch, S. J., Fox, H. S., Gendelman, H. E. (2009) Acoat of many colors: neuroimmune crosstalk in human immunodefi-ciency virus infection. Neuron 64, 133–145.

3. Thompson, K. A., Cherry, C. L., Bell, J. E., McLean, C. A. (2011) Braincell reservoirs of latent virus in presymptomatic HIV-infected individuals.Am. J. Pathol. 179, 1623–1629.

4. Miller, A. M., Stella, N. (2008) CB2 receptor-mediated migration of im-mune cells: it can go either way. Br. J. Pharmacol. 153, 299–308.

5. Molina, P. E., Amedee, A., LeCapitaine, N. J., Zabaleta, J., Mohan, M.,Winsauer, P., Vande Stouwe, C. (2011) Cannabinoid neuroimmunemodulation of SIV disease. J. Neuroimmune Pharmacol. 6, 516–527.

6. Ellis, R. J., Toperoff, W., Vaida, F., van den Brande, G., Gonzales, J.,Gouaux, B., Bentley, H., Atkinson, J. H. (2009) Smoked medicinal can-nabis for neuropathic pain in HIV: a randomized, crossover clinicaltrial. Neuropsychopharmacology 34, 672–680.

7. Persidsky, Y., Ho, W., Ramirez, S. H., Potula, R., Abood, M. E., Unter-wald, E., Tuma, R. (2011) HIV-1 infection and alcohol abuse: neurocog-nitive impairment, mechanisms of neurodegeneration and therapeuticinterventions. Brain Behav. Immun. 25 (Suppl. 1), S61–S70.

8. Cosenza-Nashat, M. A., Bauman, A., Zhao, M. L., Morgello, S., Suh,H. S., Lee, S. C. (2011) Cannabinoid receptor expression in HIV en-cephalitis and HIV-associated neuropathologic comorbidities. Neuro-pathol. Appl. Neurobiol. 37, 464–483.

9. Rock, R. B., Gekker, G., Hu, S., Sheng, W. S., Cabral, G. A., Martin,B. R., Peterson, P. K. (2007) WIN55,212-2-mediated inhibition of HIV-1expression in microglial cells: involvement of cannabinoid receptors. J.Neuroimmune Pharmacol. 2, 178–183.

10. Fraga, D., Raborn, E. S., Ferreira, G. A., Cabral, G. A. (2011) Cannabi-noids inhibit migration of microglial-like cells to the HIV protein Tat. J.Neuroimmune Pharmacol. 6, 566–577.

11. Lu, T. S., Avraham, H. K., Seng, S., Tachado, S. D., Koziel, H., Makri-yannis, A., Avraham, S. (2008) Cannabinoids inhibit HIV-1 Gp120-medi-ated insults in brain microvascular endothelial cells. J. Immunol. 181,6406–6416.

12. Hu, S., Sheng, W. S., Rock, R. B. (2011) Immunomodulatory propertiesof � opioids and synthetic cannabinoids in HIV-1 neuropathogenesis. J.Neuroimmune Pharmacol. 6, 528–539.

13. Gorantla, S., Makarov, E., Roy, D., Finke-Dwyer, J., Murrin, L. C., Gen-delman, H. E., Poluektova, L. (2010) Immunoregulation of a CB2 re-ceptor agonist in a murine model of neuroAIDS. J. Neuroimmune Pharma-col. 5, 456–468.

14. Wiley, J. L., Beletskaya, I. D., Ng, E. W., Dai, Z., Crocker, P. J., Mahade-van, A., Razdan, R. K., Martin, B. R. (2002) Resorcinol derivatives: anovel template for the development of cannabinoid CB(1)/CB(2) andCB(2)-selective agonists. J. Pharmacol. Exp. Ther. 301, 679–689.

15. Wahl, L. M., Katona, I. M., Wilder, R. L., Winter, C. C., Haraoui, B.,Scher, I., Wahl, S. M. (1984) Isolation of human mononuclear cell sub-sets by counterflow centrifugal elutriation (CCE). I. Characterization ofB-lymphocyte-, T-lymphocyte-, and monocyte-enriched fractions by flowcytometric analysis. Cell Immunol. 85, 373–383.

16. Ramirez, S. H., Fan, S., Dykstra, H., Reichenbach, N., Del Valle, L.,Potula, R., Phipps, R. P., Maggirwar, S. B., Persidsky, Y. (2010) Dyad ofCD40/CD40 ligand fosters neuroinflammation at the blood-brain bar-rier and is regulated via JNK signaling: implications for HIV-1 encephali-tis. J. Neurosci. 30, 9454–9464.

17. Ghorpade, A., Nukuna, A., Che, M., Haggerty, S., Persidsky, Y., Carter,E., Carhart, L., Shafer, L., Gendelman, H. E. (1998) Human immunode-ficiency virus neurotropism: an analysis of viral replication and cytopath-icity for divergent strains in monocytes and microglia. J. Virol. 72, 3340–3350.

18. Guo, C. J., Li, Y., Tian, S., Wang, X., Douglas, S. D., Ho, W. Z. (2002)Morphine enhances HIV infection of human blood mononuclearphagocytes through modulation of �-chemokines and CCR5 receptor. J.Investig. Med. 50, 435–442.

19. Ramirez, S. H., Hasko, J., Skuba, A., Fan, S., Dykstra, H., McCormick,R., Reichenbach, N., Krizbai, I., Mahadevan, A., Zhang, M., Tuma, R.,Son, Y. J., Persidsky, Y. (2012) Activation of cannabinoid receptor 2 at-

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www.jleukbio.org Volume 93, May 2013 Journal of Leukocyte Biology 809

tenuates leukocyte-endothelial cell interactions and blood-brain barrierdysfunction under inflammatory conditions. J. Neurosci. 32, 4004–4016.

20. Huffman, J. W., Liddle, J., Yu, S., Aung, M. M., Abood, M. E., Wiley,J. L., Martin, B. R. (1999) 3-(1=,1=-dimethylbutyl)-1-deoxy-�8-THC andrelated compounds: synthesis of selective ligands for the CB2 receptor.Bioorg. Med. Chem. 7, 2905–2914.

21. Murineddu, G., Lazzari, P., Ruiu, S., Sanna, A., Loriga, G., Manca, I.,Falzoi, M., Dessi, C., Curzu, M. M., Chelucci, G., Pani, L., Pinna, G. A.(2006) Tricyclic pyrazoles. 4. Synthesis and biological evaluation of ana-logues of the robust and selective CB2 cannabinoid ligand 1-(2=,4=-di-chlorophenyl)-6-methyl-N-piperidin-1-yl-1,4-dihydroindeno[1,2-c]pyrazole-3-carboxamide. J. Med. Chem. 49, 7502–7512.

22. Montecucco, F., Burger, F., Mach, F., Steffens, S. (2008) CB2 cannabi-noid receptor agonist JWH-015 modulates human monocyte migrationthrough defined intracellular signaling pathways. Am. J. Physiol. HeartCirc. Physiol. 294, H1145–H1155.

23. Preet, A., Qamri, Z., Nasser, M. W., Prasad, A., Shilo, K., Zou, X.,Groopman, J. E., Ganju, R. K. (2011) Cannabinoid receptors, CB1 andCB2, as novel targets for inhibition of non-small cell lung cancer growthand metastasis. Cancer Prev. Res. (Phila). 4, 65–75.

24. Pini, A., Mannaioni, G., Pellegrini-Giampietro, D., Passani, M. B., Mas-troianni, R., Bani, D., Masini, E. (2012) The role of cannabinoids in in-flammatory modulation of allergic respiratory disorders, inflammatorypain and ischemic stroke. Curr. Drug Targets 13, 984–993.

25. Roberts, T. K., Buckner, C. M., Berman, J. W. (2010) Leukocyte transmi-gration across the blood-brain barrier: perspectives on neuroAIDS. Front.Biosci. 15, 478–536.

26. Persidsky, Y., Ramirez, S. H., Haorah, J., Kanmogne, G. D. (2006)Blood-brain barrier: structural components and function under physio-logic and pathologic conditions. J. Neuroimmune Pharmacol. 1, 223–236.

27. Benito, C., Kim, W. K., Chavarria, I., Hillard, C. J., Mackie, K., Tolon,R. M., Williams, K., Romero, J. (2005) A glial endogenous cannabinoidsystem is upregulated in the brains of macaques with simian immunode-ficiency virus-induced encephalitis. J. Neurosci. 25, 2530–2536.

28. Miller, L. K., Devi, L. A. (2011) The highs and lows of cannabinoid re-ceptor expression in disease: mechanisms and their therapeutic implica-tions. Pharmacol. Rev. 63, 461–470.

29. Benito, C., Tolon, R. M., Pazos, M. R., Nunez, E., Castillo, A. I., Ro-mero, J. (2008) Cannabinoid CB2 receptors in human brain inflamma-tion. Br. J. Pharmacol. 153, 277–285.

30. Cabral, G. A., Marciano-Cabral, F. (2005) Cannabinoid receptors in mi-croglia of the central nervous system: immune functional relevance. J.Leukoc. Biol. 78, 1192–1197.

31. Raborn, E. S., Cabral, G. A. (2010) Cannabinoid inhibition of macro-phage migration to the trans-activating (Tat) protein of HIV-1 is linkedto the CB(2) cannabinoid receptor. J. Pharmacol. Exp. Ther. 333, 319–327.

32. Costantino, C. M., Gupta, A., Yewdall, A. W., Dale, B. M., Devi, L. A.,Chen, B. K. (2012) Cannabinoid receptor 2-mediated attenuation ofCXCR4-tropic HIV infection in primary CD4 T cells. PLoS One 7,e33961.

33. Peterson, P. K., Gekker, G., Hu, S., Cabral, G., Lokensgard, J. R. (2004)Cannabinoids and morphine differentially affect HIV-1 expression inCD4() lymphocyte and microglial cell cultures. J. Neuroimmunol. 147,123–126.

34. Jeon, Y. J., Yang, K. H., Pulaski, J. T., Kaminski, N. E. (1996) Attenua-tion of inducible nitric oxide synthase gene expression by � 9-tetrahy-drocannabinol is mediated through the inhibition of nuclear factor-�B/Rel activation. Mol. Pharmacol. 50, 334–341.

35. Borner, C., Smida, M., Hollt, V., Schraven, B., Kraus, J. (2009) Cannabi-noid receptor type 1- and 2-mediated increase in cyclic AMP inhibits Tcell receptor-triggered signaling. J. Biol. Chem. 284, 35450–35460.

36. Abrams, D. I., Hilton, J. F., Leiser, R. J., Shade, S. B., Elbeik, T. A.,Aweeka, F. T., Benowitz, N. L., Bredt, B. M., Kosel, B., Aberg, J. A.,Deeks, S. G., Mitchell, T. F., Mulligan, K., Bacchetti, P., McCune, J. M.,Schambelan, M. (2003) Short-term effects of cannabinoids in patientswith HIV-1 infection: a randomized, placebo-controlled clinical trial.Ann. Intern. Med. 139, 258–266.

37. Soldan, S. S., Jacobson, S. (2010) Viral infections of the central nervoussystem: pathogenesis to therapeutics. J. Neuroimmune. Pharmacol. 5, 267–270.

KEY WORDS:neuroinflammation � JWH133 � CXCR4 � CCR5 � encephalitis � CB2

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