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Peroxisome proliferator-activated receptor-γ activation suppresses HIV-1 replication in an animal model of encephalitis

Potula, Raghavaa,b,d,*; Ramirez, Servio Ha,b,d,*; Knipe, Bryana,b; Leibhart, Jessicaa,b; Schall, Kathya,b; Heilman, Davida,b; Morsey, Brendaa,b; Mercer, Aarona,b; Papugani, Anila,b; Dou, Huanyua,b; Persidsky, Yuria,b,c,d

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doi: 10.1097/QAD.0b013e3283081e08
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Despite wide use of antiretroviral therapy (ART), poor penetration of antiretroviral drugs across the blood–brain barrier (BBB) undermines the control of HIV-1 replication in brain macrophages, the primary targets of HIV-1 infection in the central nervous system (CNS) [1]. Mononuclear phagocytes (brain macrophages and microglia) serve as viral reservoirs in the brain and are potential sources of HIV-1 re-seeding after ART interruptions. Given inefficient penetration of antiretroviral drugs across the BBB and chronic neuroinflammation as a cause of HIV-1-associated dementia, the nuclear receptor, peroxisome proliferators-activated receptor gamma (PPARγ) could be an attractive therapeutic target.

PPARγ is expressed in many tissues and cell types, including monocytes, macrophages [2–5] and is upregulated upon immune activation [6]. PPAR mediate transcriptional regulation of target genes by two mechanisms, transduction and transrepression. In the former, coactivators interact with nuclear receptors in a ligand-dependent manner affecting the gene transcription, whereas in the transrepression, gene transcription occurs via negative interference of PPAR with other signal-transduction pathways [3,4]. After activation by specific agonists, these receptors form dimers and translocate to the nucleus, where they act as agonist-dependent transcription factors and regulate gene expression by binding to specific promoter regions of target genes (mechanism of upregulated genes). However, for negative regulation, ligand activation of PPARγ antagonizes the activation of transcription factors [such as activator protein-1 (AP-1) and NF-κB], which results in suppression of gene expression [i.e. nitric oxide, tumor necrosis factor-α (TNFα) and interleukin-1β] [3,4]. The potential therapeutic efficacy of PPARγ ligands in neuroinflammatory and neurodegenerative diseases is highlighted by a large body of work in animal models for multiple sclerosis, Parkinson and Alzheimer diseases [7–14].

Although PPARγ agonists were shown to inhibit HIV-1 replication in macrophages and T lymphocytes in vitro[15,16], the mechanisms of PPARγ ligand effects remain elusive. The current study delineated the pathway by which PPARγ ligands block HIV-1 replication and explored the idea that PPARγ stimulation can efficiently block HIV-1 replication in vivo. Using a potent PPARγ synthetic ligand, rosiglitazone, we showed significant reduction of HIV-1 replication in human monocyte-derived macrophages (MDM). We found that the decrease in virus replication was in response to the downmodulation of HIV-1 long terminal repeat (LTR) promoter activity by PPARγ via inhibition of NF-κB. Finally, using a well developed animal model for HIV-1 encephalitis (HIVE) [17], we demonstrate efficient suppression of HIV-1 replication in brain macrophages and lymphocytes in blood suggesting that PPARγ activation can ameliorate HIV-1 CNS and systemic infection.

Materials and methods

Cell isolation and culture

Monocytes and peripheral blood lymphocytes (PBL) were obtained by countercurrent centrifugal elutriation of leukopheresis packs from HIV-1, 2 and hepatitis B seronegative donors and monocytes were cultured as described [18].

HEK 293 and TZM-bl cells were obtained from the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health, from J.C. Kappes, X. Wu, and Tranzyme (Research Triangle Park, North Carolina, USA).

Virus infection, PPAR-γ ligand treatment and HIV-1 p24 ELISA

Monocytes or MDM (after 7 days in culture in presence of macrophage-colony stimulating factor) were pretreated with PPARγ ligand (rosiglitazone, 50 μmol/l; Cayman Chemical, Ann Arbor, Michigan, USA), PPARγ antagonist (GW9662, 50 μmol/l) or 3′-azido-2′, 3′-dideoxythymidine (ZDV, 10 μmol/l) and infected for 18 h with macrophage-tropic strain, HIV-1ADA at multiplicity of infection of 0.1 [19]. MDM viability was evaluated by live/dead cell assay (Invitrogen, Carlsbad, California, USA, data not shown), and HIV-1 p24 levels were measured by ELISA (Beckman Coulter, Miami, Florida, USA) according to the manufacturer's instructions.

DNA and short hairpin (sh)RNA against PPAR-γ

HIV-1 LTR and pCEP4 transactivation activator (Tat) expression plasmid constructs were obtained from AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, National institutes of Health (NIH). shRNA were designed against human PPARγ transcript (GenBank accession number NM138711) for knockdown of PPARγ expression as described elsewhere[20]. Oligonucleotides coding for shRNA and scrambled were cloned into pRNAT-H1.1/Adeno vector and obtained from GenScript (Piscataway, New Jersey, USA). The shRNA constructs were bicistronic for green fluorescent protein (GFP) expression, allowing for assessment of transfection efficiency (data not shown).

Transfection and luciferase assay

Transient infections of HEK 293 cells was carried out using Lipofectamine 2000 (Invitrogen). Transfections of primary cells (monocytes or macrophages) were performed by Nucleofection according to the manufacturer's instruction (Amaxa Biosystem, Gaithersburg, Maryland, USA). In brief for each transfection, 9 × 106 cells were resuspended in 100 μl of Nucloefector solution either with the PPARγ response elements (PPRE, Panomics, Fremont, California, USA) linked to the luciferase reporter (1 μg), HIV-1 LTR luciferase reporter (1 μg) alone or together with PPARγ or scrambled shRNA (1 μg). The instrument programs (Y-01 for monocytes and Y-10 for macrophages) were used and the transfection efficiency determined by fluorescent-activated cell sorting (FACS) was 85–89% and 50–60% for moncytes and macrophages, respectively (data not shown). The luciferase reporter assays were performed on a fluorescence plate reader with luminometer function (M5; Molecular Devices, Sunnyvale, California, USA) according to the manufacturer's protocol.

Western blotting

Protein lysates from human monocytes or macrophages (∼9 × 106/per condition) 48 h posttransfection were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis. PPARγ or α-actin was detected by western blotting using antibodies to PPARγ (SC-7273; Santa Cruz Biotech, Santa Cruz, California, USA) at 1: 1000 and α-actin (Chemicon, Temecula, California, USA) at 1: 2000 dilutions, respectively.

PPAR-γ and NF-κB binding assay

PPARγ transcriptional activation and HIV-1 induced NF-κB p65 activation was determined using the TransAM kits (Active Motif, Carlsbad, California, USA). Nuclear extracts from macrophages or monocytes were prepared by using a nuclear extraction kit (Sigma, St Louis, Missouri, USA). For HIV-1 induced NF-κB p65 activation monocytes/macrophages were transfected (HIV-1 LTR luciferase reporter) and pretreated with rosgilitazone (50 μmol/l) and infected for 30 min (maximum activation of NF-κB p65 was observed at 30 min postinfection). The assays were performed according to the manufacturer's protocol and the transcriptional activation was quantified using a fluorescence plate reader (M5; Molecular Devices).

Generation of hu-PBL-NOD/SCID HIV-1 encephalitis mice and drug administration

Four-week-old male NOD/C.B-17 SCID mice purchased from Jackson Laboratory (Bar Harbor, Maine, USA) were maintained in sterile microisolator cages under pathogen-free conditions in accordance with ethical guidelines for care of laboratory animals at the University of Nebraska Medical Center (UNMC) and NIH. The hu-PBL-NOD/SCID (peripheral blood lymphocytes-NOD/SCID) HIVE mice were generated as described [17]. Rosiglitazone pills were purchased from the UNMC pharmacy, pulverized, mixed with carboxymethylcellose and almond paste, and administered orally (10 mg/kg/day). Drug treatment started 1 day prior to HIV-1 MDM intracerebral (i.c.) injection and continued for the entire duration of the experiment. Mice were sacrificed at 7, 14, and 21 d after i.c. injection with human MDM. In-vivo experiments were repeated twice.

Flow cytometric analysis

FACS was performed on mononuclear cells from blood and spleen at week 1–3 after i.c. injection of human MDM as described [18]. Data analysis was performed with a FACS Calibur using CellQuest software (Becton Dickinson Immunocytometry System, San Jose, California, USA). Fluorochrome-conjugated monoclonal antibodies (mAb) to human CD4, CD8, and CD3 were used and fluorescein isothiocyanate conjugated antimouse CD45 mAb were included to exclude murine cells. Control and rosiglitazone-treated MDM were labeled with anti-CXCR4 and anti-CCR5 fluorescent conjugates and sorted on a flow cytometer. Alexa Flour conjugated anti-PPARγ antibody (Santa Cruz Biotech) was used to detect PPARγ expression on CD14-positive gated macrophages. Unless otherwise mentioned, all antibodies were obtained from eBioscience (San Diego, California, USA).

Histopathology and image analysis

Mice were sacrificed at 7, 14, and 21 d after i.c. injection of human MDM, tissues were fixed in 4% phosphate-buffered paraformaldehyde and paraffin embedded. Immunohistochemistry, histopathologic evaluation and image analyses were carried out as described [17]. Primary antibodies were purchased from Dako Carpentaria, California, USA (CD68, CD8, vimentin and HIV-1 p24). The numbers of CD8, CD68 MDM, and HIV-1 p24 cells in each section were counted in a blinded fashion by three independent observers.

Statistical analysis

Results were presented as mean ± SD, and P values less than 0.05 were considered significant. Data were analyzed using Prism (Graph Pad; GraphPad Software, Inc., La Jolla, California, USA) and statistical significance for multiple comparisons was assessed by one-way analysis of variance with Newman–Keuls posttest. All in vitro assays were repeated at least twice (each condition in triplicate).


Rosiglitazone inhibits HIV-1 replication in human MDM

PPARγ activation by ligands such as thiazolidinediones (TZD) has been shown to play a role in cellular proliferation, differentiation, inflammation [21,22], and inhibition of HIV-1 replication [15]. Rosiglitazone (50 μmol/l) treated macrophages produced lower levels of HIV-1 p24 after infection (P < 0.05, Fig. 1a). Rosiglitazone (50 μmol/l) activated PPARγ as indicated by stimulation of PPARγ transcriptional activity (Fig. 1b). Given the complex biological effects of PPARγ ligand [23] and decreased PPARγ expression in HIV-1-infected patients [24], we tested whether rosiglitazone had any affect on HIV-1 coreceptor expression and whether HIV-1 infection modulated PPARγ protein expression in macrophages. Expression levels of CXCR4/CCR5 receptors (Fig. 1c) and PPARγ protein levels (Fig. 1d) were not downregulated in macrophages treated with rosiglitazone or infected with HIV-1. Similar experiments using luciferase reporter assay for PPRE further established that infection alone had no effect on PPARγ activity whereas macrophage treatment with rosiglitazone (50 μmol/l) induced a 7.5-fold increase in PPARγ reporter activity (Fig. 1e). Taken together, these data suggest that rosigiltazone induced PPARγ activation and that this stimulation may be involved in inhibition of HIV-1 infection in macrophages.

Fig. 1
Fig. 1:
Rosiglitazone activates PPAR-γ and suppresses HIV-1 replication in MDM. MDM were pretreated with rosiglitazone (50 μmol/l) for 18 h. Cells were infected with macrophage-tropic HIV-1ADA (at a multiplicity of infection of 0.1) for 12–18 h following which cultures were washed to remove any residual virus and replenished with fresh media. Half media exchange was performed every 2–3 days. (a) Levels of HIV-1 p24 antigen in culture supernatants. (b) Macrophages were treated with rosiglitazone (50 μmol/l) for 0–24 h and the levels of PPARγ activity was measured by the TransAM PPARγ Kit. Results are expressed as fold changes in activation compared to control. (c) Representative flow cytometer scatter plots depicting CXCR4 and CCR5 expression in macrophages. (d) Expression of PPARγ protein in control and HIV-1 infected human macrophages. (e) MDM were transfected with 1 μg of PPRE luciferase reporter plasmid. Cells were harvested 24 h following infection or after treatment with rosiglitazone (50 μmol/l) and luciferase activity of cell lysates was determined. Results are expressed as fold changes compared with basal luciferase activity. Data shown are mean ± SD of three independent experiments performed with three different MDM donors. * P < 0.05 (PPARγ ligand treated versus untreated HIV-1 infected MDM; ** P < 0.05 (Rosiglitazone treated versus HIV-1 infected MDM.

PPAR-γ activation suppresses HIV-1 LTR promoter activity

Expression of HIV-1 provirus is regulated by interaction of cellular and viral transcription factors that bind to HIV-1 LTR [25–27]. Therefore, we explored whether activation of PPARγ could suppress HIV-1 LTR activity. To test this idea, TZM-bl cells were infected with HIV-1ADA, and 48 h postinfection luciferase levels (indicating HIV-1 LTR activity) were determined. Rosiglitazone treatment (0–100 μmol/l) resulted in a dose-dependent suppression of HIV-1 LTR activity and PPARγ antagonist (GW9662, 50 μmol/l) abolished the effects of rosiglitazone (Fig. 2a). As Tat is a powerful viral transactivator and plays a central role in viral replication, we tested whether activation of PPARγ could suppress HIV-1 LTR activity. Rosiglitazone at various concentrations (0–100 μmol/l) did not significantly affect induction of HIV-1 LTR by Tat. However, Tat-independent transactivation of the HIV-1 LTR by TNFα (100 ng/ml) was suppressed (Fig. 2b). As transcriptional activation of the HIV LTR by TNFα is associated with the induction of a nuclear factor(s) binding to the NF-κB sites of the LTR, it is likely that the PPARγ-mediated LTR suppression is mediated by cellular factors rather than viral elements.

Fig. 2
Fig. 2:
PPAR-γ activation suppressed HIV-1 LTR promoter activity. (a) Promoter activity in HIV-1 infected TZM-bl cells treated either with various concentration of PPARγ ligand (rosiglitazone, 0–100 μmol/l) alone or a combination of PPARγ ligand (rosiglitazone, 0–100 μmol/l) and antagonist (GW9662, 50 μmol/l). (b) HEK 293 cells were cotransfected with HIV-1 LTR luciferase (10 ng) and Tat construct, pCEP4 Tat (50 ng). Untransfected cells as well as cells transfected with empty vectors (PUC 19) served as negative controls. Cells were infected with HIV-1ADA (at a multiplicity of infection of 0.1) for 4 h and cells lysates were collected 48 h later. In separate experiments, HIV-1 LTR luciferase transfected 293 cells were incubated with or without TNFα (100 ng) in the absence or presence of various concentrations (0–100 μmol/l) of rosiglitazone. Cells were harvested and luciferase assay was performed and normalized for renella luciferase activity. Results are expressed as fold changes compared with basal luciferase activity. Data shown are mean ± SD of five independent experiments. (c) Primary human monocytes transfected with HIV-1 LTR-luciferase in the absence or presence of rosiglitazone (50 μmol/l) were infected with HIV-1ADA (at a multiplicity of infection of 0.1) and 48 h postinfection luciferase activity was determined. The mean value of the relative promoter activity is shown mean ± SD from three independent experiments. * P < 0.05 (rosiglitazone treated versus untreated TNFα incubated HIV-1 LTR transfected 293 cells); * P < 0.01 (rosiglitazone treated versus untreated HIV-1 infected primary monocytes).

Monocytes are the cells that presumably carry HIV-1 to the brain [28] where they establish viral reservoirs that differentiate into macrophages. Therefore, we investigated the effects of PPARγ stimulation in primary human monocytes. Infection of monocytes transfected with HIV-1 LTR luciferase induced a 15-fold increase in LTR activity, and rosiglitazone treatment resulted in its five-fold inhibition (Fig. 2c). Collectively, these results suggested that PPARγ stimulation led to suppression of HIV-1 LTR activation.

PPAR-γ silencing impairs the effects of rosiglitazone on HIV-1 suppression and binding of NF-κB to HIV-1 LTR

To investigate the specificity of rosiglitazone effects on HIV-1 LTR activation, we sought to knockdown expression of PPARγ by shRNA. Multiple shRNA constructs targeting PPARγ nuclear receptors were designed and the sequence (shPPARγ-2) that provided most optimal knockdown of endogenous level of PPARγ in primary human monocytes (Fig. 3a) and macrophages (Fig. 3b) was selected. HIV-1 LTR activation was efficiently suppressed by rosiglitazone in HIV-1 infected cells and also in those that were transfected with the scramble shRNA (control). In contrast, expression of PPARγ shRNA in cells infected and treated with rosiglitazone resulted in lack of HIV-1 LTR suppression both in monocytes (Fig. 3c) and macrophages (Fig. 3d). These results indicated that the rosiglitazone mediated effects were PPARγ specific and rule out the possible involvement of other PPAR subtypes in mediating the effect of rosiglitazone on HIV-1 suppression.

Fig. 3
Fig. 3:
Decreased expression of PPAR-γ by shRNA knockdown abolished the effect of PPAR-γ ligand activation. (a) Analysis of shRNA knockdown of PPARγ protein (labeled shPPARγ-seq) in primary human monocytes expressing three different RNA hairpins directed at PPARγ (lanes 2, 4, and 5) along with control shRNA (scrambled sequence 2, lane 3). (b) Evaluation of PPARγ knockdown by flow cytometery in macrophages expressing RNA hairpins to PPARγ. Shift in greater number of macrophages transfected with shPPARγ-seq-2 toward PPARγ low-intensity mean fluorescence is indicative of decreased expression of PPARγ. Primary human monocytes (c and e) and macrophages (d and f) were transfected with either HIV-1 LTR-lucifrease alone or cotransfected with shRNA-PPARγ/(sc) shRNA-PPARγ by nucleofection. Luciferase assay was measured 48 h postinfection and expressed as the relative fold changes compared with mock-control. Nuclear extracts from cells were assayed for NF-κB p65 activation using the TransAM NF-κB p65 Chemi Kit. PPARγ ligand treatment inhibits HIV-1 induced NF-κB activation and subsequently NF-κB binding. Results are shown as mean ± SD of three independent experiments. *; P < 0.001 (Rosiglitazone treated HIV-1 infected versus Rosiglitazone treated HIV-1 infected shRNA-PPARγ), **; P < 0.05 (Rosiglitazone treated HIV-1 infected (sc) shRNA-PPARγ versus HIV-1 infected (sc) shRNA-PPARγ).

Because transrepression or negative interaction with NF-κB are suspected to underlie the effects of PPARγ stimulation [29–36] and NF-κB is one of the central transcription factors that bind the HIV-1 LTR, we investigated the effect of rosiglitazone on the DNA-binding activity of NF-κB. Rosiglitazone treatment of monocytes (Fig. 3e) or macrophages (Fig. 3f) diminished HIV-1 induced NF-κB DNA-binding activity as compared with control (P < 0.05). Transfection of shRNA vectors, to knock down endogenous PPARγ expression abolished the effect of rosiglitazone on the DNA-binding activity while irrelevant shRNA had no effect. Together, these results suggest that the PPARγ ligand could suppress HIV-1 replication via modulation of binding of NF-κB to the HIV-1 LTR promoter.

Rosiglitazone suppressed viremia in an animal model for HIV-1 infection

To determine whether PPARγ ligand can suppress HIV-1 replication in vivo, we investigated the effect of rosiglitazone in our mouse model for HIVE [17,18]. NOD/SCID mice were reconstituted with human PBL (hu-PBL-NOD/SCID) and inoculated i.c. with autologous HIV-1 infected MDM. Animals (n = 5–8 mice/group/week) were orally fed daily with rosiglitazone (10 mg/kg) or placebo and sacrificed at weeks 1, 2, and 3 after MDM inoculation. Similar percentages of CD4, CD8, and CD3 cells observed between the two groups (data not shown) assessed by flow cytometry analysis on cells derived from blood and spleen of hu-PBL-NOD/SCID HIVE mice assured comparability of the results between groups. Viremia was measured by ELISA (HIV-1 p24) and expressed in pg/ml per 1000 CD4 cells. Two weeks following the i.c. injection of HIV-1 infected MDM, both rosiglitazone and control animals (33.7 ± 9. 5 vs. 37.3 ± 15.7) had equally low levels of HIV-1 p24. At week 3, when infection spread from HIV-1-infected MDM to circulating CD4 lymphocytes control animals demonstrated high levels of viremia while rosiglitazone treated mice had a 50% reduction in HIV-1 p24 levels (P < 0.02, Fig. 4a).

Fig. 4
Fig. 4:
Viral replication in control and rosiglitazone-fed mice. (a) Viremia levels were measured by ELISA (Beckman Coulter) according to the manufacturer's instructions and expressed in pg/ml per 1000 CD4+ cells. Animals were orally fed daily with rosiglitazone (10 mg/kg) or placebo. Rosiglitazone-fed mice showed 50% reduction of viremia compared with the control group. Rosiglitazone treated mice (b and d) contained fewer HIV-1 p24 MDM as compared with controls (c and e). Vectastain Elite Kit was used to detect Primary Ab and visualized by using DAB as a substrate. Original magnification for panels A and B: 10 (insets, 200). Results are presented as mean ± SD *; P < 0.02 (rosiglitazone versus control).

Rosiglitazone suppressed HIV-1 replication in brain macrophages in hu-PBL-NOD/SCID HIV-1 encephalitis mice

To examine whether rosiglitazone treatment can affect HIV-1 replication in brain macrophages, serial brain sections were immunostained for human CD68 (a marker for human MDM), HIV-1 p24 (viral antigen), and human CD8 (T lymphocytes indicating an antiviral responses). A prominent reduction of HIV-1 p24 MDM was noted in rosiglitazone treated mice (Fig. 4c) at week 1 as compared with controls (Fig. 4b). Similarly, at week 2 rosiglitazone treated mice (Fig. 4e) featured fewer HIV-1 p24 MDM (Fig. 4d). The level of virus replication, elimination of virus-infected macrophages and CD8 cell infiltration were quantitatively analyzed by counting CD68, HIV-1 p24, and CD8 lymphocytes in brain tissue sections in a blinded fashion by three independent observers. As shown in (Fig. 5a), the mean numbers of CD68 MDM in the rosiglitazone and control groups were similar at weeks 1–3. The percentage of infected macrophages was significantly lower in rosiglitazone as compared with controls at week 1 and 2 (P < 0.05, Fig. 5b), and a similar trend was found at week 3. At week 1, fewer HIV-1 p24 MDM were found in rosiglitazone mice as compared with controls (130 ± 34.8 vs. 47.2 ± 21.5, P < 0.05, Fig. 5c). A similar trend existed at week 2; however, differences failed to reach statistical significance (47.9 ± 17.2 vs. 22 ± 7.8, P > 0.05). Comparable amounts of CD8 T cells were detected at weeks 1 and 2 in rosiglitazone and control mice (Fig. 5d). These finding suggest that oral administration of rosiglitazone was effective in diminishing viral replication in brain macrophages and blood CD4 lymphocytes of hu-PBL-NOD/SCID HIVE mice.

Fig. 5
Fig. 5:
PPARγ ligand suppresses HIV-1 replication in brain macrophages in hu-PBL-NOD rosiglitazone/SCID HIV-1 encephalitis mice. Serial brains sections were immunostained for human macrophages (CD68+), HIV-1 p24 MDM (viral antigen), and human lymphocytes (CD8). Coronal brain sections were cut first to identify the injection site containing MDM. Serial 5-μm-thick sections of the brain (30–100) covering the entire area of human MDM injection were cut for each mouse brain and three to seven matched slides (10 sections apart) were analyzed. Mean numbers of stained cells per section within the injected hemisphere were calculated for each mouse (three to seven sections per mouse), and a total of five to eight mice per group were examined. Number of CD68 MDM (a), percentage of infected MDM (b), number of infected MDM (c), and CD8 lymphocytes (d) were counted in brain tissue sections of control and rosiglitazone-fed hu-PBL-NOD/SCID HIVE mice. Rosiglitazone-fed mice demonstrated fewer HIV-1 p24 MDM as compared with controls at week 1 (c). PPARγ treatment significantly decreased the percentage of HIV-1 p24 MDM rosiglitazone-fed mice at weeks 1 and 2 (b). Values represent mean ± SD per 5-μm section (n = 5–8 mice/group). *,**,# P < 0.05 (rosiglitazone versus control).


Several lines of evidence indicate that PPARγ natural and synthetic ligands such as TZDs control brain inflammation by inhibiting microglial activation, and synthesis of nitric oxide, prostaglandins, chemokines, and inflammatory cytokines [12,14,37,38]. CNS inflammation during HIV-1 infection is driven in part by HIV-1 replication, which utilizes cellular transcription factors. Hence, we sought to evaluate whether PPARγ, via inhibition of transcription factors important in proinflammatory responses, could attenuate HIV-1 replication. PPARγ ligand treatment resulted in a significant decrease of virus infection in primary human MDM, the major virus reservoir in the brain. TZD are rapidly absorbed by oral administration and efficiently permeate the BBB [39]. Capitalizing on this ability of TZD, we evaluated the effect of PPARγ activation on HIV-1 infection in a mouse model of HIVE. Animals fed with PPARγ ligand, rosiglitazone showed a 50% reduction of viremia and diminished HIV-1 replication in human MDM inoculated into mice brains.

PPARγ, a member of the family of nuclear factors is expressed in abundance in macrophages and lymphocytes [4,40]. In addition to upregulation of target genes, PPARγ also forms repressor complexes (transrepression) to downregulate genes induced by other transcription factors (reviewed in [41,42]). It is through transrepression that PPARγ ligands act as anti-inflammatory agents by inhibition of AP-1, NF-κB, and STAT-1 either by direct interaction or sequestration of essential cofactors [4,36,43,44]. Although previously Hayes et al. [15] speculated that transcriptional and posttranscriptional effects could be responsible for inhibited virus replication in macrophages by PPARγ ligands, the mechanism of virus suppression remained elusive. Our study found that activation of PPARγ receptor with the ligand rosiglitazone suppressed HIV-1 LTR activity both in a HIV-1 LTR stable transfected cell line and in primary mononuclear cells. Moreover, the replication of HIV-1 was inhibited by rosiglitazone in human macrophages. We also showed that the effect of the ligand was specific to PPARγ, as rosiglitazone failed to attenuate the induction of HIV-1 LTR promoter activity (HIV-1 infection) in monocytes expressing PPARγ specific shRNA or in the presence of antagonist.

The role of NF-κB in activation of HIV-1 transcription was analyzed [45] which is of particular importance as it is stimulated by several cytokines involved in immune and inflammatory responses [25]. NF-κB is one of the several transcription factors that strongly induces HIV-1 LTR. PPARγ activation in HIV-1 infected monocytes reduced the NF-κB-DNA binding activity similar to findings in other studies where PPARγ stimulation downregulated NF-κB–DNA binding in a variety of cell types [46,47]. Consequently, ligand treatment had no effect on NF-κB binding in infected monocytes with diminished PPARγ receptor expression. There is a possibility that PPARγ could inhibit LTR activity by interfering with HIV-1 protein, Tat. Tat is a key regulatory protein of HIV-1 and its ability to stimulate HIV transcription from the LTR [48] via RNA rather than DNA promoter elements is unique among transcriptional activators. Although HIV-1 LTR contains binding sites for multiple cellular transcription factors, it is primarily targeted by HIV-1 Tat. Interestingly, PPARγ had no effect on HIV-1 Tat-mediated induction of the LTR, which explains the partial inhibition of HIV-1 replication (via NF-κB inhibition). These findings indicated that suppression of HIV-1 replication in monocytes/macrophages is achieved via suppression of NF-κB activation.

Our results suggested that PPARγ ligand activation in HIV-1 infected monocytes downregulated NF-κB DNA activity through a transrepression that involves p65 subunit of NF-κB and PPARγ resulting in decreased HIV-1 LTR transcription and HIV-1 virus replication. Nevertheless, the possibility of PPARγ ligand-mediated suppression of HIV-1 virus independent of NF-κB transrepression cannot be completely ruled out. For instance, PPAR ligands can activate kinases that lead to phosphorylation cascades that, in turn, trigger transcriptional changes leading to several levels of downstream regulation (reviewed by Diradourian et al.[49] and Gardner et al.[50]). They also can alter ligand binding, corepressor/coactivator recruitment, or heterodimerization with retinoid X receptor α, all of which influencing PPAR-mediated transcription (reviewed in Peraza et al.[51]). Whether these interactions play any role in suppression of HIV-1 still remains to be elucidated.

Beneficial effects of PPARγ stimulation were demonstrated in a variety of animal models of inflammatory and neurodegenerative disorders (reviewed by Moraes et al.[52], Bernardo and Minghetti [37]). Our in vivo model of HIVE recapitulates adaptive and innate immune responses, immunopathological features of HIV-1 CNS infection (including neuroinflammation and neuronal demise), and viremia, [17,53]. These features are relevant for the chronic immune activation seen in HIV-1 infection driven by activation of NF-κB and increased viral transcription [54–56].

In vivo results supported the in vitro observations showing that PPARγ receptor stimulation reduced viremia and suppressed HIV-1 infection in MDM in the brain. As CNS macrophages are a major viral reservoir poorly accessible by ART [57], these findings are of particular interest since effects of PPARγ ligand on HIV-1 replication might not be prevailed by viral mutations. The dose of rosiglitazone used in our animal studies did not affect PBL engraftment levels and was within the range used for patient treatment. Antigen-specific CD8 T lymphocytes serve a prominent role in the control of HIV-1 infection of brain macrophages in the hu-PBL-NOD/SCID mouse model of HIVE [17,18]. PPARγ treatment did not affect the number of brain infiltrating CD8 T lymphocytes. Whether PPARγ stimulation provides additional immunomodulatory or anti-inflammatory effects awaits future investigation.

In summary, our results suggest that the suppressive mechanism of PPARγ ligand involves transrepression of NF-κB and decreased HIV-1 LTR promoter transcription. In-vitro and in-vivo data strongly support potential use of PPARγ for treatment of HIVE and suppression of HIV-1 replication in lymphocytes and MDM thus offering new therapeutic intervention in brain and systemic infection.


The authors thank Drs. Sanjay Maggirwar and Tsuneya Ikezu for critical reading of the manuscript. The authors appreciate the excellent administrative support from Ms Robin Taylor and Ms Pamela Ferrick.

This work was supported by NIH grants RO1 AA15913, PO1 NS043985, RO1 MH65151 to Y.P

The authors have no conflicting financial interests.

Author contributions: R.P. and S.H.R. designed the study and wrote the paper. B.K., J.L., K.S., D.H., B.M., A.M., A.P., and H.D. provided all technical help. Y.P. helped designing the experiments, writing the paper and supervised the study.


1. Persidsky Y, Gendelman HE. Mononuclear phagocyte immunity and the neuropathogenesis of HIV-1 infection. J Leukoc Biol 2003; 74:691–701.
2. Staels B, Fruchart JC. Therapeutic roles of peroxisome proliferator-activated receptor agonists. Diabetes 2005; 54:2460–2470.
3. Jiang C, Ting AT, Seed B. PPAR-gamma agonists inhibit production of monocyte inflammatory cytokines. Nature 1998; 391:82–86.
4. Ricote M, Li AC, Willson TM, Kelly CJ, Glass CK. The peroxisome proliferator-activated receptor-gamma is a negative regulator of macrophage activation. Nature 1998; 391:79–82.
5. Denning GM, Stoll LL. Peroxisome proliferator-activated receptors: potential therapeutic targets in lung disease? Pediatr Pulmonol 2006; 41:23–34.
6. Ricote M, Huang J, Fajas L, Li A, Welch J, Najib J, et al. Expression of the peroxisome proliferator-activated receptor gamma (PPARgamma) in human atherosclerosis and regulation in macrophages by colony stimulating factors and oxidized low density lipoprotein. Proc Natl Acad Sci U S A 1998; 95:7614–7619.
7. Klotz L, Schmidt M, Giese T, Sastre M, Knolle P, Klockgether T, Heneka MT. Proinflammatory stimulation and pioglitazone treatment regulate peroxisome proliferator-activated receptor gamma levels in peripheral blood mononuclear cells from healthy controls and multiple sclerosis patients. J Immunol 2005; 175:4948–4955.
8. Kiaei M, Kipiani K, Chen J, Calingasan NY, Beal MF. Peroxisome proliferator-activated receptor-gamma agonist extends survival in transgenic mouse model of amyotrophic lateral sclerosis. Exp Neurol 2005; 191:331–336.
9. Hume DA, Fairlie DP. Therapeutic targets in inflammatory disease. Curr Med Chem 2005; 12:2925–2929.
10. Dehmer T, Heneka MT, Sastre M, Dichgans J, Schulz JB. Protection by pioglitazone in the MPTP model of Parkinson's disease correlates with I kappa B alpha induction and block of NF kappa B and iNOS activation. J Neurochem 2004; 88:494–501.
11. Niino M, Iwabuchi K, Kikuchi S, Ato M, Morohashi T, Ogata A, et al. Amelioration of experimental autoimmune encephalomyelitis in C57BL/6 mice by an agonist of peroxisome proliferator-activated receptor-gamma. J Neuroimmunol 2001; 116:40–48.
12. Kielian T, Drew PD. Effects of peroxisome proliferator-activated receptor-gamma agonists on central nervous system inflammation. J Neurosci Res 2003; 71:315–325.
13. Camacho IE, Serneels L, Spittaels K, Merchiers P, Dominguez D, De Strooper B. Peroxisome-proliferator-activated receptor gamma induces a clearance mechanism for the amyloid-beta peptide. J Neurosci 2004; 24:10908–10917.
14. Sastre M, Klockgether T, Heneka MT. Contribution of inflammatory processes to Alzheimer's disease: molecular mechanisms. Int J Dev Neurosci 2006; 24:167–176.
15. Hayes MM, Lane BR, King SR, Markovitz DM, Coffey MJ. Peroxisome proliferator-activated receptor gamma agonists inhibit HIV-1 replication in macrophages by transcriptional and posttranscriptional effects. J Biol Chem 2002; 277:16913–16919.
16. Skolnik PR, Rabbi MF, Mathys JM, Greenberg AS. Stimulation of peroxisome proliferator-activated receptors alpha and gamma blocks HIV-1 replication and TNFalpha production in acutely infected primary blood cells, chronically infected U1 cells, and alveolar macrophages from HIV-infected subjects. J Acquir Immune Defic Syndr 2002; 31:1–10.
17. Potula R, Poluektova L, Knipe B, Chrastil J, Heilman D, Dou H, et al. Inhibition of Indoleamine 2,3-dioxygenase (IDO) enhances elimination of virus-infected macrophages in animal model of HIV-1 encephalitis. Blood 2005; 106:2383–2390.
18. Potula R, Haorah J, Knipe B, Leibhart J, Chrastil J, Heilman D, et al. Alcohol abuse enhances neuroinflammation and impairs immune responses in an animal model of human immunodeficiency virus-1 encephalitis. Am J Pathol 2006; 168:1335–1344.
19. Gendelman HE, Orenstein JM, Martin MA, Ferrua C, Mitra R, Phipps T, et al. Efficient isolation and propagation of human immunodeficiency virus on recombinant colony-stimulating factor 1-treated monocytes. J Exp Med 1988; 167:1428–1441.
20. Ramirez SH, Heilman D, Morsey B, Potula R, Haorah J, Persidsky Y. Activation of peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) suppresses Rho GTPases in human brain microvascular endothelial cells and inhibits adhesion and transendothelial migration of HIV-1 infected monocytes. J Immunol 2008; 180:1854–1865.
21. Klappacher GW, Glass CK. Roles of peroxisome proliferator-activated receptor gamma in lipid homeostasis and inflammatory responses of macrophages. Curr Opin Lipidol 2002; 13:305–312.
22. Rosen ED, Spiegelman BM. PPARgamma: a nuclear regulator of metabolism, differentiation, and cell growth. J Biol Chem 2001; 276:37731–37734.
23. Lemay DG, Hwang DH. Genome-wide identification of peroxisome proliferator response elements using integrated computational genomics. J Lipid Res 2006; 47:1583–1587.
24. Bastard JP, Caron M, Vidal H, Jan V, Auclair M, Vigouroux C, et al. Association between altered expression of adipogenic factor SREBP1 in lipoatrophic adipose tissue from HIV-1-infected patients and abnormal adipocyte differentiation and insulin resistance. Lancet 2002; 359:1026–1031.
25. Rohr O, Marban C, Aunis D, Schaeffer E. Regulation of HIV-1 gene transcription: from lymphocytes to microglial cells. J Leukoc Biol 2003; 74:736–749.
26. Wu Y. HIV-1 gene expression: lessons from provirus and nonintegrated DNA. Retrovirology 2004; 1:13.
27. Pereira LA, Bentley K, Peeters A, Churchill MJ, Deacon NJ. A compilation of cellular transcription factor interactions with the HIV-1 LTR promoter. Nucleic Acids Res 2000; 28:663–668.
28. Williams KC, Corey S, Westmoreland SV, Pauley D, Knight H, deBakker C, et al. Perivascular macrophages are the primary cell type productively infected by simian immunodeficiency virus in the brains of macaques: implications for the neuropathogenesis of AIDS. J Exp Med 2001; 193:905–915.
29. Genolet R, Wahli W, Michalik L. PPARs as drug targets to modulate inflammatory responses? Curr Drug Targets Inflamm Allergy 2004; 3:361–375.
30. Okamoto H, Iwamoto T, Kotake S, Momohara S, Yamanaka H, Kamatani N. Inhibition of NF-kappaB signaling by fenofibrate, a peroxisome proliferator-activated receptor-alpha ligand, presents a therapeutic strategy for rheumatoid arthritis. Clin Exp Rheumatol 2005; 23:323–330.
31. Hisada S, Shimizu K, Shiratori K, Kobayashi M. Peroxisome proliferator-activated receptor gamma ligand prevents the development of chronic pancreatitis through modulating NF-kappaB-dependent proinflammatory cytokine production and pancreatic stellate cell activation. Rocz Akad Med Bialymst 2005; 50:142–147.
32. Fan W, Yanase T, Morinaga H, Mu YM, Nomura M, Okabe T, et al. Activation of peroxisome proliferator-activated receptor-gamma and retinoid X receptor inhibits aromatase transcription via nuclear factor-kappaB. Endocrinology 2005; 146:85–92.
33. Chen F, Harrison LE. Ciglitazone induces early cellular proliferation and NF-kappaB transcriptional activity in colon cancer cells through p65 phosphorylation. Int J Biochem Cell Biol 2005; 37:645–654.
34. Vanden Berghe W, Vermeulen L, Delerive P, De Bosscher K, Staels B, Haegeman G. A paradigm for gene regulation: inflammation, NF-kappaB and PPAR. Adv Exp Med Biol 2003; 544:181–196.
35. Chen F, Wang M, O'Connor JP, He M, Tripathi T, Harrison LE. Phosphorylation of PPARgamma via active ERK1/2 leads to its physical association with p65 and inhibition of NF-kappabeta. J Cell Biochem 2003; 90:732–744.
36. Delerive P, De Bosscher K, Besnard S, Vanden Berghe W, Peters JM, Gonzalez FJ, et al. Peroxisome proliferator-activated receptor alpha negatively regulates the vascular inflammatory gene response by negative cross-talk with transcription factors NF-kappaB and AP-1. J Biol Chem 1999; 274:32048–32054.
37. Bernardo A, Minghetti L. PPAR-gamma agonists as regulators of microglial activation and brain inflammation. Curr Pharm Des 2006; 12:93–109.
38. Park EJ, Park SY, Joe EH, Jou I. 15d-PGJ2 and rosiglitazone suppress Janus kinase-STAT inflammatory signaling through induction of suppressor of cytokine signaling 1 (SOCS1) and SOCS3 in glia. J Biol Chem 2003; 278:14747–14752.
39. Maeshiba Y, Kiyota Y, Yamashita K, Yoshimura Y, Motohashi M, Tanayama S. Disposition of the new antidiabetic agent pioglitazone in rats, dogs, and monkeys. Arzneimittelforschung 1997; 47:29–35.
40. Ricote M, Valledor AF, Glass CK. Decoding transcriptional programs regulated by PPARs and LXRs in the macrophage: effects on lipid homeostasis, inflammation, and atherosclerosis. Arterioscler Thromb Vasc Biol 2004; 24:230–239.
41. Desvergne B, Wahli W. Peroxisome proliferator-activated receptors: nuclear control of metabolism. Endocr Rev 1999; 20:649–688.
42. Mandard S, Muller M, Kersten S. Peroxisome proliferator-activated receptor alpha target genes. Cell Mol Life Sci 2004; 61:393–416.
43. Wang N, Verna L, Chen NG, Chen J, Li H, Forman BM, Stemerman MB. Constitutive activation of peroxisome proliferator-activated receptor-gamma suppresses pro-inflammatory adhesion molecules in human vascular endothelial cells. J Biol Chem 2002; 277:34176–34181.
44. Yang XY, Wang LH, Chen T, Hodge DR, Resau JH, DaSilva L, Farrar WL. Activation of human T lymphocytes is inhibited by peroxisome proliferator-activated receptor gamma (PPARgamma) agonists. PPARgamma co-association with transcription factor NFAT. J Biol Chem 2000; 275:4541–4544.
45. Hiscott J, Kwon H, Genin P. Hostile takeovers: viral appropriation of the NF-kappaB pathway. J Clin Invest 2001; 107:143–151.
46. Chinetti G, Griglio S, Antonucci M, Torra IP, Delerive P, Majd Z, et al. Activation of proliferator-activated receptors alpha and gamma induces apoptosis of human monocyte-derived macrophages. J Biol Chem 1998; 273:25573–25580.
47. Chung SW, Kang BY, Kim SH, Pak YK, Cho D, Trinchieri G, Kim TS. Oxidized low-density lipoprotein inhibits interleukin-12 production in lipopolysaccharide-activated mouse macrophages via direct interactions between peroxisome proliferator-activated receptor-gamma and nuclear factor-kappa B. J Biol Chem 2000; 275:32681–32687.
48. Barboric M, Peterlin BM. A new paradigm in eukaryotic biology: HIV Tat and the control of transcriptional elongation. PLoS Biol 2005; 3:e76.
49. Diradourian C, Girard J, Pegorier JP. Phosphorylation of PPARs: from molecular characterization to physiological relevance. Biochimie 2005; 87:33–38.
50. Gardner OS, Dewar BJ, Graves LM. Activation of mitogen-activated protein kinases by peroxisome proliferator-activated receptor ligands: an example of nongenomic signaling. Mol Pharmacol 2005; 68:933–941.
51. Peraza MA, Burdick AD, Marin HE, Gonzalez FJ, Peters JM. The toxicology of ligands for peroxisome proliferator-activated receptors (PPAR). Toxicol Sci 2006; 90:269–295.
52. Moraes LA, Piqueras L, Bishop-Bailey D. Peroxisome proliferator-activated receptors and inflammation. Pharmacol Ther 2006; 110:371–385.
53. Poluektova LY, Munn DH, Persidsky Y, Gendelman HE. Generation of cytotoxic T cells against virus-infected human brain macrophages in a murine model of HIV-1 encephalitis. J Immunol 2002; 168:3941–3949.
54. Liu J, Perkins ND, Schmid RM, Nabel GJ. Specific NF-kappa B subunits act in concert with Tat to stimulate human immunodeficiency virus type 1 transcription. J Virol 1992; 66:3883–3887.
55. West MJ, Lowe AD, Karn J. Activation of human immunodeficiency virus transcription in T cells revisited: NF-kappaB p65 stimulates transcriptional elongation. J Virol 2001; 75:8524–8537.
56. Hunt G, Tiemessen CT. Occurrence of additional NF-kappaB-binding motifs in the long terminal repeat region of South African HIV type 1 subtype C isolates. AIDS Res Hum Retroviruses 2000; 16:305–306.
57. Sacktor N, McDermott MP, Marder K, Schifitto G, Selnes OA, McArthur JC, et al. HIV-associated cognitive impairment before and after the advent of combination therapy. J Neurovirol 2002; 8:136–142.

animal model of HIV-1 encephalitis; brain macrophages; peroxisome proliferator-activated receptor-γ; rosiglitazone; suppression of HIV-1 replication

© 2008 Lippincott Williams & Wilkins, Inc.