Nuclear Receptor Signaling Inhibits HIV-1 Replication in Macrophages through Multiple trans-Repression Mechanisms

Abstract

Sexually transmitted pathogens activate HIV-1 replication and inflammatory gene expression in macrophages through engagement of Toll-like receptors (TLRs). Ligand-activated nuclear receptor (NR) transcription factors, including glucocorticoid receptor (GR), peroxisome proliferator-activated receptor gamma (PPARγ), and liver X receptor (LXR), are potent inhibitors of TLR-induced inflammatory gene expression. We therefore hypothesized that ligand-activated NRs repress both basal and pathogen-enhanced HIV-1 replication in macrophages by directly repressing HIV-1 transcription and by ameliorating the local proinflammatory response to pathogens. We show that the TLR2 ligand PAM3CSK4 activated virus transcription in macrophages and that NR signaling repressed both basal and TLR-induced HIV-1 transcription. NR ligand treatment repressed HIV-1 expression when added concurrently with TLR ligands and in the presence of cycloheximide, demonstrating that they act independently of new cellular gene expression. We found that treatment with NR ligands inhibited the association of AP-1 and NF-κB subunits, as well as the coactivator CBP, with the long terminal repeat (LTR). We show for the first time that the nuclear corepressor NCoR is bound to HIV-1 LTR in unstimulated macrophages and is released from the LTR after TLR engagement. Treatment with PPARγ and LXR ligands, but not GR ligands, prevented this TLR-induced clearance of NCoR from the LTR. Our data demonstrate that both classical and nonclassical trans-repression mechanisms account for NR-mediated HIV-1 repression. Finally, NR ligand treatment inhibited the potent proinflammatory response induced by PAM3CSK4 that would otherwise activate HIV-1 expression in infected cells. Our findings provide a rationale for studying ligand-activated NRs as modulators of basal and inflammation-induced HIV-1 replication.

INTRODUCTION

Macrophages play critical roles in HIV-1 transmission and pathogenesis. Macrophages are productively infected with HIV-1 and are thought to be a major source of virus production in vivo (48, 66, 137, 160). The infection of specialized tissue macrophages, such as microglial cells, contributes to the organ-specific pathogenesis seen in HIV-1-infected individuals (42, 54, 60, 100, 156, 158). In addition, infected macrophages can act as a reservoir that contributes to viral persistence and, potentially, to the viral rebound seen in patients after cessation of highly active antiviral therapy (HAART) (12, 29, 86). Although resting memory CD4⁺ T cells represent the major source of the rebounding virus, a number of studies have implicated non-T cells, such as macrophages, as an alternate reservoir (4, 31, 66).

Both inflammatory and ulcerative sexually transmitted infections (STIs) as well as bacterial vaginosis have been shown to be cofactors that enhance HIV-1 transmission (44, 47, 83, 111). There are at least three mechanisms to account for this. First, infections with ulcerative STIs, such as herpes simplex viruses 1 or 2, damage the cervicovaginal epithelium and thereby expose underlying macrophages to viruses from the lumen (44, 47, 155). Second, STIs cause inflammation that leads to the recruitment of immune cells to the site of inflammation, thereby increasing the size of the HIV-1 target cell population (88, 128, 136). Third, STIs promote a favorable local environment for HIV-1 replication both by directly activating HIV-1 target cells and by inducing the release of cytokines that favor virus replication through the engagement of Toll-like receptors (TLRs) and other innate immune sensors in cells present in the mucosa (9, 10, 37, 57, 75, 122, 134, 145, 146, 161).

Nuclear receptors (NRs) are a superfamily of ligand-activated transcription factors that includes classic hormone receptors, such as glucocorticoid receptor (GR), as well as the so-called orphan receptors and adopted orphan receptors (26, 52). Included in these latter two families are peroxisome proliferator-activated receptors (PPAR) and liver X receptors (LXR). In addition to their roles as positive-acting transcription factors, recent findings have demonstrated that ligand-activated NRs are potent inhibitors of inflammation and are capable of repressing cytokine and chemokine production by TLR-activated macrophages and dendritic cells (DCs) (6, 24, 71, 117, 131). Ligand-activated NRs repress inflammatory responses in at least 3 ways. First, they antagonize the action of transcription factors that are central mediators of inflammation. Some of these include the p65 subunit of NF-κB, AP-1, STATs, and interferon regulatory factor 3 (IRF3) (24, 25, 71, 73, 87, 117, 131). Second, they interfere with activation-induced ubiquitin-mediated degradation of corepressor complexes that are bound to quiescent genes, thereby maintaining those genes in a transcriptionally silent state despite the presence of activation signals (119). Third, they interfere with signaling pathways involved in inflammation either by inhibiting p38 and extracellular signal-regulated kinase (ERK) and mitogen-activated protein kinase (MAPK) activation or by inducing the expression of IκB (6, 8, 16, 17).

In addition to their effects on inflammation, ligand-activated NRs have also been shown to directly repress HIV-1 replication and trans-infection. We, and others, have found that ligand-activated retinoic acid receptor (RAR) represses HIV-1 transcription in both cultured monocytic cell lines and primary monocyte-derived macrophages (MDMs) through the induction of an as-yet-unidentified factor (21, 57, 77, 97). Previous studies have also demonstrated that the ubiquitously expressed GR can inhibit HIV-1 replication in certain myeloid and lymphoid cell lines (78, 82, 106, 133) but not in others (79, 81, 84). In addition, ligand-activated PPAR gamma (PPARγ) has been shown to inhibit HIV-1 replication in lipopolysaccharide (LPS)- or phorbol myristate acetate (PMA)-activated macrophages (61, 142). More recently, we demonstrated that both ligand-activated PPARγ and LXR repress DC-mediated capture of HIV-1 and trans-infection of T cells through upregulation of ABCA1-dependent cholesterol efflux (56). These findings, in conjunction with their anti-inflammatory activities, led us to propose that NR ligands be considered potential therapeutic agents to be used in combination with conventional antiviral microbicides to limit HIV-1 mucosal transmission. Importantly, in that study, we also showed that PPARγ and LXR signaling repress HIV-1 replication in monocyte-derived dendritic cells (MDDCs) infected with a vesicular stomatitis virus (VSV)-pseudotyped HIV-1-based reporter virus; however, the mechanism of repression was not explored (56).

Given that NRs potently repress NF-κB- and AP-1-dependent TLR-induced inflammatory gene expression, we hypothesized that they would also inhibit TLR-induced NF-κB- and AP-1-dependent HIV-1 expression in macrophages. Here, we show that GR, PPARγ, and LXR ligands repress HIV-1 transcription in a dose-dependent manner in MDMs and MDDCs. This NR-mediated repression of HIV-1 transcription is due, at least in part, to the sequestration of specific transcription factors required for efficient HIV-1 transcription, including NF-κB and AP-1. Furthermore, PPARγ and LXR prevent the clearance of nuclear corepressor (NCoR)-containing corepressor complexes associated with the HIV-1 long terminal repeat (LTR). In addition to inhibiting HIV-1 transcription, these NRs also inhibit the production of proinflammatory cytokines and chemokines, such as tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), and IL-8, known to augment HIV-1 replication. These findings suggest that diverse NRs utilize common, overlapping mechanisms to block both HIV-1 transcription and proinflammatory cytokine production. These current findings underscore the therapeutic potential of NR ligands to limit HIV-1 transmission and pathogenesis.

MATERIALS AND METHODS

Cell isolation and culture.

Primary human CD14⁺ monocytes were isolated from the peripheral blood mononuclear cells of healthy donors using anti-CD14 magnetic beads (Miltenyi Biotec) per the manufacturer's instructions. To generate monocyte-derived macrophages, CD14⁺ monocytes were cultured in RPMI 1640 supplemented with 10% normal human AB serum (Atlanta Biologicals), 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.29 mg/ml l-glutamine for 6 to 8 days, at the end of which the cells acquired a macrophage phenotype as assessed by flow cytometry (CD11b⁺, CD68⁺, HLA-DR^lo, CD80⁻, CD86⁻). To generate monocyte-derived dendritic cells, CD14⁺ monocytes (1.5 × 10⁶ cells/ml) were cultured in RPMI 1640 supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, 0.29 mg/ml l-glutamine, 1,000 U/ml IL-4 (PeproTech), and 1,400 U/ml granulocyte-macrophage colony-stimulating factor (GM-CSF) (PeproTech) for 6 to 8 days, at the end of which the cells acquired an immature dendritic cell phenotype as assessed by flow cytometry (CD11c⁺ DC-SIGN⁺ HLA-DR^lo CD80⁻ CD86⁻). Cells were given fresh medium supplemented with IL-4 and GM-CSF every 2 days. Primary human CD4⁺ T cells were isolated from CD14-depleted peripheral blood mononuclear cells using anti-CD4 magnetic beads (Miltenyi Biotec) per the manufacturer's instructions. CD4⁺ T cells (2 × 10⁶ cells/ml) were cultured in RPMI 1640 supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, 0.29 mg/ml l-glutamine, 50 U/ml IL-2 (R&D Systems), and 5 μg/ml phytohemagglutinin P (PHA-P) (Sigma) for 6 to 8 days, at the end of which the cells acquired a memory T cell phenotype as assessed by flow cytometry (CD3⁺ CD4⁺ CD45RO⁺ CD45RA⁻). Primary human plasmacytoid DCs (pDCs) were isolated from monocyte- and B cell-depleted peripheral blood mononuclear cells (PBMCs) using anti-BDCA4 magnetic beads (Miltenyi Biotec) per the manufacturer's instructions. pDCs were cultured in RPMI 1640 supplemented with 10% FBS, 100 U/ml penicillin, 100 mg/ml streptomycin, 0.29 mg/ml l-glutamine, and 10 ng/ml IL-3 (PeproTech) and were determined to be CD123⁺ by flow cytometry. 293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.29 mg/ml l-glutamine. MAGI-CCR5 cells were cultured in DMEM supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, 0.29 mg/ml l-glutamine, 500 μg/ml G418, 1 μg/ml puromycin, and 0.1 μg/ml hygromycin B. PM1 cells were cultured in RPMI 1640 supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.29 mg/ml l-glutamine. U1 cells, which contain two integrated HIV-1 proviral copies, and J1.1 cells, which contain a single integrated proviral copy, were cultured in RPMI 1640 supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.29 mg/ml l-glutamine.

Flow cytometry.

MDM phenotypes were assessed using antibodies to HLA-DR, CD80, CD86, CD11b, CD68, CD4, CCR5, and CXCR4. MDDC phenotypes were assessed using antibodies against HLA-DR, CD80, CD86, DC-SIGN, CD11c, CD4, CCR5, CXCR4, and CCR7. Primary CD4⁺ T cell phenotypes were assessed using antibodies to CD3, CD4, CD8, CD45RO, CD45RA, CCR5, and CXCR4. pDCs were assessed using antibodies to CD123. Flow cytometric data were acquired using a Becton-Dickenson FACScan II, and data were analyzed using FlowJo software.

Nuclear receptor ligands.

The LXR ligands used in this study were TO-901317 (Calbiochem), GW3965 (Sigma), 22(R)-hydroxycholesterol (Sigma), glycogen synthase kinase (GSK) LXRα/β agonist (Cayman Chemical), and 22(S)-hydroxycholesterol (Sigma). The GR ligands used in this study were dexamethasone (Calbiochem), cortisol (Sigma), hydrocortisone (Sigma), prednisolone (Calbiochem), RU-486 (Calbiochem), and norethisterone acetate (NEA) (Sigma). The PPARγ ligands ciglitazone, rosiglitazone, pioglitazone, troglitazone, 15-deoxyΔ^12,14-prostagladin J₂, MC-555, and GW9662 were purchased from Cayman Chemicals. Ligands were reconstituted in either dimethyl sulfoxide (DMSO) or ethanol by following the manufacturer's instructions.

Virus production.

Replication-competent R5-tropic HIV-1_Ba-L was generated by infection of PM1 cells. Single-round replication-competent HIV-1-based reporter viruses were generated by packaging a luciferase-expressing reporter virus, BruΔEnvLuc, or an enhanced green fluorescent protein (EGFP)-expressing reporter virus, BruΔEnvEGFP, with the envelope glycoprotein from VSV (VSV-G). Reporter virus stocks were generated by transfecting HEK293T cells using the calcium phosphate method. Virus was concentrated through a 20% sucrose cushion when necessary. Titers for all viruses were determined on MAGI-CCR5 cells, and p24^gag content was determined by enzyme-linked immunosorbent assay (ELISA).

Virus infections.

To assess viral replication, MDMs (2.5 × 10⁵ cells/well in 24-well plates), immature MDDCs (2.5 × 10⁵ cells/well in 96-well plates), pDCs (1 × 10⁵ cells/well in 96-well plates), or T cells (5 × 10⁵ cells/well in 96-well plates) were incubated with VSV-G-pseudotyped HIV-luciferase reporter virus at a multiplicity of infection (MOI) of 0.1 for MDMs and T cells or an MOI of 1 for MDDCs and pDCs for 4 h at 37°C. Cells were washed four to five times with phosphate-buffered saline (PBS) to remove unbound virus and cultured in growth medium. Following 48 h of culture, cells were treated with NR ligands or the vehicle as indicated in the text and figure legends. Where indicated, the cells were then treated with the TLR2/1 ligand PAM3CSK4 (100 ng/ml) for 18 h. The cells were then washed twice with PBS and lysed in PBS–0.02% Triton X-100. Protein levels in cell lysates were determined using a modified Lowry protein assay (Bio-Rad), and luciferase activity was measured using BrightGlo luciferase reagent (Promega) and an MSII luminometer. For spreading infections, MDMs were plated at a density of 2.5 × 10⁵ cells per well in 24-well plates and infected with HIV-1_Ba-L (1 ng of p24/10⁵ cells) for 4 h at 37°C. Unbound virus was removed by washing the cells with PBS four times, and the MDMs were cultured for 30 days. Virus replication was quantified by measuring p24 antigen release in cell-free culture supernatants every 3 days.

Semiquantitative RT-PCR assay.

Total cytoplasmic RNA was isolated from MDMs using the RNeasy minikit (Qiagen) according to the manufacturer's instructions. RNA (100 ng) was analyzed by reverse transcription-PCR (RT-PCR) by using the OneStep RT-PCR kit (Qiagen). RNA was reverse transcribed and amplified in a total volume of 50 μl containing 2.5 mM MgCl₂, 400 μM concentrations of each deoxynucleoside triphosphate, 10 U of RNasin RNase inhibitor (Promega), 5 μCi of [α-³²P]dATP, and 0.6 μM HIV-1-specific primers. RNA samples were reverse transcribed for 30 min at 50°C. After an initial denaturing step at 95°C for 15 min, cDNA products were amplified for 25 cycles, each consisting of a 30-s denaturing step at 94°C, a 45-s annealing step at 65°C, and a 1-min extension step at 72°C. The amplification concluded with a 10-min extension step at 72°C. Samples were resolved on 5% nondenaturing polyacrylamide gels, visualized by autoradiography, and quantified in a Molecular Dynamics PhosphorImager SI using ImageQuant software. Two sets of HIV-1 primers were used, one set specific for the R and U5 regions of the LTR that amplifies both spliced mRNAs and genomic RNA, and one set that amplifies spliced Tat, Rev, and Nef mRNAs. HIV-1 primers were sense primer 5′-GGCTAACTAGGGAACCCACTGC-3′ and antisense primer 5′-CTGCTAGAGATTTTCCACACTGAC-3′ for both spliced and unspliced RNA and sense primer 5′-TCTCTCGACGCAGGACTCGGCTTGC-3′ and antisense primer 5′-TTCTATTCCTTCGGGCCTGTCG-3′ for Tat, Rev, and Nef mRNA. β-Tubulin primers were sense primer 5′-CACCCGTCTTCAGGGCTTCTTGGTTT-3′ and antisense primer 5′-CATTTCACCATCTGGTTGGCTGGCTC-3′. RNA standards corresponding to 500, 50, and 5 ng of total cytoplasmic RNA from PAM3CSK4-activated MDMs were included in each experiment to ensure that all amplifications were within the linear range of the assay.

HIV-1 RNA stability assays.

To assess viral RNA stability, macrophages (2 × 10⁶ cells/well in 6-well plates) were incubated with VSV-G-pseudotyped HIV-luciferase reporter virus at an MOI of 0.1 for 4 h at 37°C. Cells were washed four to five times with PBS to remove unbound virus and cultured in growth medium. Following 48 h of culture, cells were treated with NR ligands or the vehicle for 15 min. The cells were then treated with the TLR2/1 ligand PAM3CSK4 (100 ng/ml) for 1 h. Actinomycin D (10 μg/ml) was then added to cells to block de novo RNA synthesis, and total cytoplasmic RNA was isolated at given times as described in the figure legends. Viral RNA was measured by RT-PCR using primers specific for the R and U5 regions of the LTR as described above.

Chromatin immunoprecipitation assays.

MDMs (1.2 × 10⁷) in 10-cm dishes were incubated with VSV-G-pseudotyped HIV-EGFP reporter virus at an MOI of 2 for 4 h at 37°C. Cells were washed four to five times with PBS to remove unbound virus and cultured in growth medium. Following 48 h of culture, MDMs were treated with nuclear receptor ligands for 15 min and then stimulated with PAM3CSK4 (100 ng/ml) for 1 h. Cells were then fixed in 1% formaldehyde for 10 min at room temperature, quenched with 125 mM glycine, and lysed in SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris [pH 8.1], 1 mM phenylmethylsulfonyl fluoride [PMSF], 1 μg/ml aprotinin, 1 μg/ml pepstatin A). Cellular lysates were sonicated using a cup horn (550 Sonic Dismembrator; Fisher Scientific) at a power setting of 5 with 25 20-s pulses on ice, which fragmented the chromatin to an average length of approximately 1,000 bp. Samples were diluted and immunoprecipitated with antibodies against NF-κB p65, c-fos, Sp1, NCoR, CBP, rabbit IgG, goat IgG (all from Santa Cruz Biotechnology), or acetylated histone H3 (Upstate Biotechnology). Purified DNA samples from both chromatin immunoprecipitations (ChIPs) and input controls were resuspended in 100 μl of distilled nuclease-free H₂O and analyzed by semiquantitative PCR. PCR mixtures contained 10 μl of IP sample or 1 μl of input DNA sample, 10 mm Tris-HCl (pH 8.3), 50 mm KCl, 1.5 mm MgCl₂, 100 pmol of each primer, 200 μm each dATP, dGTP, dCTP, and dTTP, 5 μCi [α-³²P]dATP, and 2.5 units of AmpliTaq Gold (Applied Biosystems) in a 50-μl reaction volume. Following an initial denaturation step at 95°C for 15 min, DNAs were amplified for 30 cycles, each consisting of a 30-s denaturing step at 94°C, a 45-s annealing step at 65°C, and a 1-min extension step at 72°C. Samples were electrophoresed on 5% nondenaturing polyacrylamide gels, visualized by autoradiography, and quantified using a Molecular Dynamics PhosphorImager SI using ImageQuant software. Primers used to amplify specifically the HIV-1 5′ LTR were 5′-TGGAAGGGCTAATTTACTCCC-3′ (sense) and 5′-CATCTCTCTCCTTCTAGCCTC-3′ (antisense). Control amplifications of a serial dilution of purified U1 genomic DNA were performed with each primer set to ensure that all amplifications were within the linear range of the reaction. Samples were visualized by autoradiography and quantified using a PhosphorImager SI (Molecular Dynamics). To calculate the relative levels of association with the LTR, PhosphorImager data of the amounts of PCR product obtained for immunoprecipitated chromatin samples were normalized against the amounts of PCR product obtained for input DNA (% input). All values represent the averages from at least three independent experiments.

Cytokine release assays.

MDMs (2.5 × 10⁵ cells/well) were treated with PAM3CSK4 (100 ng/ml) for 24 h in the presence or absence of NR ligands as described in the figure legends. Cell-free culture supernatants were collected and analyzed for TNF-α (eBioscience), IL-6 (eBioscience), and IL-8 (BioLegend) release by commercially available ELISA by following the manufacturer's instructions. Supernatants from treated MDMs were cultured with U1 or J1.1 cells (1 × 10⁶ cells/ml) to assess the ability of released cytokines to activate HIV-1 expression as measured by p24^gag ELISA.

Cell viability assays.

MDM, MDDC, and CD4⁺ T cell viability was assessed by trypan blue dye exclusion, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H- tetrazolium bromide (MTT) cytotoxicity assay (46), and lactate dehydrogenase (LDH) release using a commercial kit (Promega) per the manufacturer's instructions.

Statistical analysis.

Untreated control and ligand-treated experimental samples were compared using a one-tailed t test with two samples assuming equal variance. Experiments were performed in triplicate using cells from a minimum of three different donors unless otherwise indicated. Data are presented as the means ± standard deviations (SD) from one representative donor.

RESULTS

NR ligands inhibit HIV-1 replication in primary macrophages.

To begin to examine the ability of ligand-activated NR to restrict HIV-1 replication, we determined whether they could repress a spreading virus infection in primary MDMs. MDMs were prepared from PBMCs and were infected with the R5-tropic HIV-1_Ba-L. Two days after infection, the MDMs were treated with the GR ligand dexamethasone, the PPARγ ligand ciglitazone, or the LXR ligand TO-901317. At three-day intervals, virus levels were quantified by ELISA for p24^gag in the culture medium, and fresh NR ligands were added. In untreated cultures, virus was detected after 3 days, increased to peak levels at roughly 15 days, and thereafter decreased. The decrease was associated with extensive cell death. GR, PPARγ, and LXR ligand treatment repressed total virus production at all time points (Fig. 1). It should be noted that dexamethasone treatment induced cell death in the MDM cultures beginning at day 12, although no cell death was observed in ciglitazone- or TO-901317-treated cultures, as measured by MTT cytotoxicity and LDH release assays (data not shown). Although GR, PPARγ, and LXR signaling potently inhibited a spreading HIV-1_Ba-L infection in primary MDM cultures, it is not clear at which point in the virus life cycle the NRs exert their effects. Previous studies have demonstrated NR effects on HIV-1 transcription (61, 78, 82, 106, 133, 142), binding (56), and production of infectious virions (109, 110).

Fig. 1.

NR ligands inhibit the spreading of HIV-1 infections in MDM cultures. MDMs (2.5 × 10⁵ cells/well) were infected with HIV-1_Ba-L (1 ng/10⁵ cells). After 4 h, the cells were washed to remove unbound virus. MDMs were treated with the GR ligand dexamethasone (1 μM), the PPARγ ligand ciglitazone (30 μM), or the LXR ligand TO-901317 (1 μM). Culture medium was sampled every 3 days, and NR ligands were replaced. The data are the means (±SD) from three replicates and are representative of three independent experiments using different donors.

NR ligands inhibit TLR2/1-activated HIV-1 expression in primary macrophages and dendritic cells.

To determine at which point in the virus life cycle ligand-activated NRs repress HIV-1 expression, we used a replication-deficient HIV-1 reporter virus, BruΔEnvLuc (159), containing luciferase in the place of nef. Virus particles pseudotyped with VSV-G were generated with this vector and used to infect MDMs, MDDCs, CD4⁺ T cells, or pDCs (Fig. 2). The infected cells were treated with increasing concentrations of ligands for GR (dexamethasone), PPARγ (ciglitazone), or LXR (TO-901317) and/or with the TLR2/1 ligand PAM3CSK4. As shown in Fig. 2, PAM3CSK4 treatment activated virus expression in both MDMs and MDDCs, and as expected, MDMs supported a greater level of virus expression than MDDCs. Importantly, all NR ligands repressed both basal and TLR-induced expression in both cell types and in a dose-dependent fashion. It should be noted that no differences in cell viability were noted during the course of the experiment, as determined by MTT assay or LDH release (data not shown), indicating that, at the concentrations used, these ligands are not toxic for primary MDMs or MDDCs (56). Together with those data shown in Fig. 1, these results support our hypothesis that ligand-activated NRs inhibit HIV-1 replication.

Fig. 2.

NR ligands inhibit HIV-1 expression in MDMs and MDDCs. MDMs (2.5 × 10⁵ cells/well) (A) or MDDCs (2.5 × 10⁵ cells/well) (B) were infected with a VSV-G-pseudotyped HIV-1 reporter virus at an MOI of 0.1 or 1.0, respectively. Two days after infection, cells were treated with NR ligands at the indicated concentrations for 24 h. Cells were then treated with the TLR2/1 ligand, PAM3CSK4 (100 ng/ml) (filled histograms). Cellular extracts were prepared 18 h later and assayed for luciferase activity. The data are the means (±SD) from three different donors, each tested in triplicate. (C) CD4⁺ T cells (2.5 × 10⁵ cells/well) were infected with a VSV-G pseudotyped HIV-1 reporter virus at an MOI of 0.1. Two days after infection, cells were treated with NR ligands at the indicated concentrations for 24 h. Cellular extracts were prepared 18 h later and assayed for luciferase activity. The data are the means (±SD) from three different donors, each tested in triplicate. (D) pDCs (1 × 10⁵ cells/well) were infected with a VSV-G-pseudotyped HIV-1 reporter virus at an MOI of 1.0. Two days after infection, cells were not treated (white bar) or treated with 30 μM ciglitazone (black bar), 1 μM dexamethasone (dark-gray bar), or 1 μM TO-901317 (light-gray bar) for 24 h. Cellular extracts were prepared 18 h later and assayed for luciferase activity. The data are the means (±SD) from four different donors, each tested in duplicate. RLU, relative light units.

We also examined the effects of NR signaling on HIV-1 replication in CD4⁺ T cells and pDCs, both of which are also thought to be involved in mucosal transmission (69, 94, 105, 143). We found that LXR signaling had no significant effect on HIV-1 replication in primary CD4⁺ T cells, whereas both GR and PPARγ signaling inhibited HIV-1 replication in these cells (Fig. 2C). This inhibition is likely due to indirect effects on cell viability, as both signaling pathways induced large amounts of cell death (data not shown). Signaling through GR and PPARγ is known to induce apoptosis in T lymphocytes (58, 67, 157). Interestingly, treatment with GR, PPARγ, and LXR ligands significantly decreased HIV-1 replication in pDCs infected with an HIV-1-based reporter virus (Fig. 2D). Our data suggest that NR signaling inhibits HIV-1 replication in at least three different cell types, namely, macrophages, conventional DCs, and plasmacytoid DCs, that play important roles in HIV-1 transmission and pathogenesis.

The effects of GR, PPARγ, and LXR ligands on HIV-1 replication are not specific to TLR2/1 activation. These NR signaling pathways repress HIV-1 replication in response to other stimulators of NF-κB and AP-1 activity, including the TLR2/TLR6 ligand FSL-1, the phorbol ester PMA, and TNF-α (Fig. 3 A to C). These data suggest that NR ligand treatment represses HIV-1 replication, at least in part, through the antagonism of NF-κB and AP-1 transcription factors, independent of the manner of cellular activation. In addition, various combinations of NR ligands can repress HIV-1 replication in a synergistic fashion (Fig. 3D). The combination of the GR ligand dexamethasone and the PPARγ ligand ciglitazone, both administered at suboptimal concentrations, represses HIV-1 expression to a greater extent than either of the ligands alone. This suggests that these NR ligands may work through distinct mechanisms.

Fig. 3.

NR ligands inhibit HIV-1 replication in response to various macrophage activators. (A to C) MDMs (2.5 × 10⁵ cells/well) were infected with a VSV-G-pseudotyped HIV-1 reporter virus at an MOI of 0.1. Two days after infection, cells were treated with NR ligands (30 μM ciglitazone, 1 μM dexamethasone, or 1 μM TO-901317) for 30 min. Cells were then treated with the TLR2/6 ligand, FSL-1 (100 ng/ml) (A), PMA (10 nM) (B), or TNF-α (10 ng/ml) (C). Cellular extracts were prepared 18 h later and assayed for luciferase activity. The data are the means (±SD) from three different donors, each tested in triplicate. (D) MDMs (2.5 × 10⁵ cells/well) were infected with a VSV-G-pseudotyped HIV-1 reporter virus at an MOI of 0.1. Two days after infection, cells were treated with suboptimal concentrations of NR ligands (1 μM ciglitazone, 1 nM dexamethasone, or 1 nM TO-901317) for 30 min alone or in combination. Cells were then treated with the TLR2/1 ligand PAM3CSK4 (100 ng/ml). Cellular extracts were prepared 18 h later and assayed for luciferase activity. The data are the means (±SD) from three different donors, each tested in triplicate.

NR ligands inhibit basal and TLR-activated HIV-1 transcription in primary macrophages.

NRs are potent bifunctional modulators of gene expression, capable of either activating or repressing gene expression. It is known that certain NRs, such as RAR, inhibit HIV-1 transcription (21, 57, 61, 77, 78, 82, 106, 133, 142). We wanted to determine whether GR, PPARγ, and LXR signaling affected HIV-1 transcription. MDMs were infected with the VSV-pseudotyped BruΔEnvLuc reporter virus, treated with dexamethasone, ciglitazone, or TO-901317, and then activated with PAM3CSK4. Total cytoplasmic RNA was prepared from the infected MDMs, and viral mRNA levels were measured by semiquantitative RT-PCR. As shown in Fig. 4 A, both basal and TLR-induced HIV-1 transcript levels were decreased in MDMs treated with the three NR ligands. This decrease in HIV-1 transcript levels could be due to either an inhibition of transcription or an increase in viral RNA degradation. A previous report regarding the regulation of HIV-1 replication by PPARγ suggested that PPARγ either directly or indirectly led to the degradation of viral mRNA transcripts at late time points in infection (61). We found, however, that GR, PPARγ, and LXR ligand treatment did not decrease HIV-1 RNA stability (Fig. 4B). In fact, treatment with the PPARγ agonist ciglitazone seemed to increase HIV-1 RNA stability. Taken together, these data suggest that ligand-activated GR, PAPRγ, and LXR repress HIV-1 transcription.

Fig. 4.

NR ligands inhibit HIV-1 transcription in MDMs. (A) MDMs (2 × 10⁶ cells/well) were infected with a VSV-G-pseudotyped HIV-1 reporter virus at an MOI of 0.1. Two days after infection, cells were treated with NR ligands at the indicated concentrations for 30 min. Cells were then treated with the TLR2/1 ligand, PAM3CSK4 (100 ng/ml), for 3 h. Total cytoplasmic RNA was then prepared from the treated cultures and analyzed by semiquantitative RT-PCR for the expression of both HIV-1 (top) and α-tubulin (bottom) RNA. To ensure that all semiquantitative RT-PCR amplifications were within the linear range of the assay, 10-fold serial dilutions of total cytoplasmic RNA prepared from the PAM3CSK4-treated MDMs shown in the top panel were included as a standard (right). Shown is one experiment representative of three independent experiments using different donors. (B) MDMs (2 × 10⁶ cells/well) were infected with a VSV-G-pseudotyped HIV-1 reporter virus at an MOI of 0.1. Two days after infection, cells were treated with NR ligands at the indicated concentrations for 30 min. Cells were then treated with the TLR2/1 ligand PAM3CSK4 (100 ng/ml) for 3 h. MDMs were treated with actinomycin D (5 μg/ml) to inhibit transcription. Total cytoplasmic RNA was prepared from the treated cultures at the indicated time points following actinomycin D treatment and analyzed by semiquantitative RT-PCR for the expression of HIV-1 RNA. To ensure that all semiquantitative RT-PCR amplifications were within the linear range of the assay, 10-fold serial dilutions of total cytoplasmic RNA prepared from the PAM3CSK4-treated MDMs were included as a standard. The data are the means (±SD) from three different donors, each tested in triplicate.

NR ligands repress HIV-1 replication in a receptor-mediated manner.

To further assess the ability of NR ligands to repress HIV-1 replication, we treated MDMs with an array of GR, PPARγ, and LXR ligands, including FDA-approved therapeutics and putative endogenous ligands for these receptors. All of the NR agonists tested repressed HIV-1 replication in MDMs. The thiazolidinediones rosiglitazone and pioglitazone (Fig. 5 B), which are used for the systemic treatment of type II diabetes mellitus, and the glucocorticoids prednisolone and hydrocortisone (cortisol) (Fig. 5A), which are used to treat allergic, inflammatory, and autoimmune disorders, repressed HIV-1 replication in MDMs. Of particular interest, the endogenous GR ligands hydrocortisone/cortisol and cortisone (Fig. 5A), the endogenous PPARγ ligand 15-deoxyΔ^12,14-prostagladin J₂ (Fig. 5B), and the endogenous LXR ligand 22(R)-hydroxycholesterol (Fig. 5C) all repressed HIV-1 replication in MDMs at or near physiological concentrations (55, 118, 153), suggesting that NR signaling may regulate HIV-1 replication under certain circumstances in vivo. Previous studies have indicated that the PPARγ ligand 15-deoxyΔ^12,14-prostagladin J₂ has “off-target” effects, including interfering with Tat-mediated transactivation, that contribute to the inhibition of HIV-1 replication (19, 72). In order to rule out off-target effects for other ligands, NR agonist treatments were carried out in the presence of molar excesses of NR antagonists. Repression by dexamethasone, ciglitazone, and TO-901317 could be blocked by NEA, GW9662, and 22(S)-hydroxycholesterol, respectively, indicating that GR, PPARγ, and LXR ligands repress HIV-1 replication in a receptor-dependent manner (Fig. 6). As expected, repression mediated by 15-deoxyΔ^12,14-prostagladin J₂ was not completely reversed by an excess of the PPARγ antagonist GW9662, confirming that it does mediate anti-HIV-1 effects independent of PPARγ. Interestingly, the selective glucocorticoid modulator RU-486, which acts as an antagonist for GR-mediated gene transactivation but as an agonist for GR-mediated trans-repression (132), inhibited HIV-1 replication (Fig. 5), suggesting that NRs utilize a trans-repression mechanism to repress HIV-1 replication.

Fig. 5.

Endogenous NR ligands inhibit HIV-1 expression in MDMs. MDMs (2.5 × 10⁵ cells/well) were infected with a VSV-G-pseudotyped HIV-1 reporter virus at an MOI of 0.1. Two days after infection, cells were treated with the indicated ligands for GR (A), PPARγ (B), or LXR (C) for 30 min. Cells were then treated with the TLR2/1 ligand PAM3CSK4 (100 ng/ml) (filled histograms). Cellular extracts were prepared 18 h later and assayed for luciferase activity. The data are the means (±SD) of pooled data from three independent experiments, each performed in triplicate using cells from different donors. Concentrations used were cortisol, 1 μM; hydrocortisone, 1 μM; prednisolone, 1 μM; RU-486, 1 μM; rosiglitazone, 1 μM; pioglitazone, 1 μM; troglitazone, 1 μM; 15-deoxyΔ^12,14-prostaglandin J₂, 3 μM; MCC-555, 5 μM; GW3965, 1 μM; 22(R)-hydroxycholesterol, 10 μM; GSK LXR agonist, 1 μM.

Fig. 6.

NR ligand-mediated repression of HIV-1 is receptor dependent. MDMs (2.5 × 10⁵ cells/well) were infected with a VSV-G-pseudotyped HIV-1 reporter virus at an MOI of 0.1. Two days after infection, cells were treated with 1 μM dexamethasone (A), 30 μM ciglitazone or 3 μM 15-deoxyΔ^12,14 prostaglandin J₂ (B), or 1 μM TO-901317 (C) in the presence or absence of an excess of the GR antagonist NEA (10 μM), the PPARγ antagonist GW9662 (10 μM), or the LXR antagonist 22(S)-hydroxycholesterol (10 μM) for 30 min. Cells were then treated with the TLR2/1 ligand PAM3CSK4 (100 ng/ml) (filled histograms). Cellular extracts were prepared 18 h later and assayed for luciferase activity. The data are the means (±SD) of pooled data from three independent experiments, each performed in triplicate from different donors.

Ligand-activated NRs acutely repress HIV-1 expression.

As a next step in defining the mechanisms of GR-, PPARγ-, and LXR-mediated repression, we determined whether the individual ligands could inhibit HIV-1 expression acutely when added at the same time as TLR ligands or whether repression required pretreatment as shown previously for RAR (57). We found that all ligands repressed HIV-1 when the MDMs were pretreated for 24 h prior to the addition of PAM3CSK4 (Fig. 7 A). Furthermore, GR, PPARγ, and LXR ligands (dexamethasone, ciglitazone, and TO-901317) were able to repress HIV-1 expression when added either at the same time as or 6 h following PAM3CSK4 treatment, suggesting that they act acutely, independently of new cellular gene expression. To confirm this, NR ligand treatments were carried out in the presence of the translational inhibitor cycloheximide. In the presence of cycloheximide, GR, PPARγ, and LXR ligands are able to repress HIV-1 transcription (Fig. 7B), suggesting that they act independently of new cellular protein expression. These data are in contrast to the reported mechanism of RAR-mediated HIV-1 repression (57, 77) and suggest that GR, PPARγ, and LXR inhibit HIV-1 through a trans-repression mechanism.

Fig. 7.

NR-mediated repression of HIV-1 expression in MDMs is both acute and independent of new cellular protein expression. (A) MDMs (2.5 × 10⁵ cells/well) were infected with a VSV-G-pseudotyped HIV-1 reporter virus at an MOI of 0.1. Two days after infection, cells were treated with ciglitazone (30 μM), dexamethasone (1 μM), or TO-901317 (1 μM) 24 h prior to, at the same time as, or 6 h after stimulation with the TLR2/1 ligand PAM3CSK4 (100 ng/ml). Cellular extracts were prepared 18 h later and assayed for luciferase activity. The data are the means (±SD) from three different donors, with each experiment performed in triplicate. (B) Infected MDMs were treated with ciglitazone (30 μM), dexamethasone (1 μM), or TO-901317 (1 μM) 30 min prior to stimulation with the TLR2/1 ligand PAM3CSK4 (100 ng/ml). Some cultures (right) were treated with cycloheximide (CHX; 10 μg/ml) at the time of NR ligand treatment. Semiquantitative RT-PCR was used to measure the levels of HIV-1 (top) and α-tubulin (bottom) RNA accumulation. The data are representative of four independent experiments. Ten-fold serial dilutions of total cytoplasmic RNA prepared from PAM3CSK4-treated HIV-1-infected MDM cells were included as standards.

NR signaling modulates the recruitment of transcription factors to the HIV-1 LTR.

The classical trans-repression pathway utilized by most ligand-activated NRs is the binding and sequestration of transcription factors from their DNA binding sites in gene promoters or enhancers (25, 71, 73, 117, 131). In order to determine whether GR, PPARγ, and LXR could prevent the association of transcription factors with the HIV-1 LTR, we employed ChIP analysis. MDMs were infected with VSV-G-pseudotyped HIV-1-GFP reporter virus such that >50% of the cells were infected as assessed by fluorescence-activated cell sorting (FACS; data not shown). Two days after infection, the MDMs were treated with or without ciglitazone, dexamethasone, or TO-901317 for 15 min followed by PAM3CSK4 for an additional hour. The levels of the p65 subunit of NF-κB and the c-fos subunit of AP-1 bound to the LTR were measured by ChIP. We found that GR, PPARγ, and LXR ligand treatment all prevented the association of p65 and c-fos with the HIV-1 5′ LTR, although each did so to various degrees (Fig. 8 A). Ligand-activated GR prevented the association of p65 with the LTR to a greater extent than the other NRs, whereas PPARγ prevented the association of c-fos to a greater extent than GR or LXR. It was surprising that LXR inhibited p65 binding to the HIV-1 LTR since it does not appear to target NF-κB binding during trans-repression of other genes (117). Notably, NR ligand treatment does not affect the global association of transcription factors with the LTR, as the levels of bound Sp1, a transcription factor required for HIV-1 transcription, are not decreased (Fig. 8B). In fact, the level of Sp1 associated with the LTR in ciglitazone-treated MDMs is slightly increased. These data indicate that GR-, PPARγ-, and LXR-mediated repression of HIV-1 transcription is due, at least in part, to the sequestration of NF-κB p65 and AP-1 c-fos from the HIV-1 LTR.

Fig. 8.

NR ligands prevent the recruitment of NF-κB and AP-1 subunits to the HIV-1 5′ LTR. (A) MDMs (12 × 10⁶ cells/plate) were infected with a VSV-G-pseudotyped HIV-1 GFP reporter virus at an MOI of 2. Two days after infection, cells were treated with NR ligands for 30 min and then treated with the TLR2/1 ligand PAM3CSK4 (100 ng/ml) for 1 h. MDMs were then fixed with formaldehyde, lysed, and sonicated to generate fragments approximately 1,000 bp in length. Samples were then immunoprecipitated with antibodies against NF-κB p65, AP-1 c-fos, or rabbit IgG (isotype control) and processed for semiquantitative PCR. A portion of each lysate was saved as a control to normalize the amount of input DNA between experimental samples. Amplifications of 10-fold dilutions of total DNA isolated from U1 cells were performed in parallel to ensure that the PCRs were in the linear range of the assay. Shown are results representative of four independent experiments using different donors. Shown to the right are the levels of immunoprecipitation relative to input DNA (% input) for p65 and c-fos. The data are the means (±SD) from four independent experiments using different donors. (B) MDMs (12 × 10⁶ cells/plate) were infected and treated with NR and TLR ligands as described in the legend to panel A. MDMs were then fixed with formaldehyde, lysed, sonicated to generate fragments approximately 1,000 bp in length, and immunoprecipitated with antibodies against Sp1 or rabbit IgG (isotype control) and processed for semiquantitative PCR as described in the legend to panel A. Shown are results representative of four independent experiments using different donors. Shown to the right are the levels of immunoprecipitation relative to input DNA (% input) for Sp1. The data are the means (±SD) from four independent experiments using different donors.

NR signaling modulates the recruitment of coactivators and corepressors to the HIV-1 LTR.

The coactivator/histone acetyltransferase (HAT) p300/CBP has been shown to activate HIV-1 expression through the acetylation of transcription factors and histones associated with the LTR (15, 32, 64). Other studies have shown that ligand-activated NRs prevent the association of HATs with certain genes (73). As such, we wanted to determine whether NR signaling in MDMs had an effect on the recruitment of coactivators such as p300/CBP and the subsequent acetylation of histones associated with the LTR. We found that NR ligand treatment strongly repressed the association of CBP with the HIV-1 5′ LTR (Fig. 9). In addition, downstream events such as the acetylation of histone H3 at the LTR are blocked in MDMs treated with NR ligand (Fig. 9).

Fig. 9.

NR ligands alter the recruitment of coactivator and corepressor complexes to the HIV-1 5′ LTR. MDMs (12 × 10⁶ cells/plate) were infected with a VSV-G-pseudotyped HIV-1 GFP reporter virus at an MOI of 2. Two days after infection, cells were treated with NR ligands for 30 min and then treated with the TLR2/1 ligand PAM3CSK4 (100 ng/ml) for 1 h. MDMs were then fixed with formaldehyde, lysed, and sonicated to generate fragments approximately 1,000 bp in length. Samples were then immunoprecipitated with antibodies against NCoR, CBP, acetylated histone H3, goat IgG (isotype control), or rabbit IgG (isotype control) and processed for semiquantitative PCR. A portion of each lysate was saved as a control to normalize the amount of input DNA between experimental samples. Amplifications of 10-fold dilutions of total DNA isolated from U1 cells were performed in parallel to ensure that the PCRs were in the linear range of the assay. Shown are results representative of four independent experiments using different donors. Shown to the right are the levels of immunoprecipitation relative to input DNA (% input) for NCoR, CBP, and acH3. The data are the means (±SD) from four independent experiments using different donors.

Both PPARγ and LXR have been shown to repress inflammatory gene expression by interfering with activation-induced ubiquitin-mediated degradation of NCoR/silencing mediator of retinoid and thyroid hormone receptors (SMRT)-containing corepressor complexes that are bound to quiescent genes (50, 51, 70, 119). Interestingly, a recent study has shown that variations in NCoR2 are associated with HIV-1 transmission rates (28). Therefore, we wanted to determine whether NCoR was associated with the HIV-1 LTR in infected MDMs and whether GR, PPARγ, and LXR ligand treatment could prevent its clearance following TLR activation. We found that NCoR is associated with the LTR in infected, unactivated MDMs and that it is effectively cleared from the LTR within 1 h after activation with the TLR2 ligand PAM3CSK4 (Fig. 9). Furthermore, PPARγ and LXR ligand treatment prevented the clearance of NCoR from the LTR in PAM3CSK4-activated MDMs (Fig. 9). GR ligand treatment, on the other hand, had little to no effect on NCoR clearance. These data suggest that PPARγ and LXR utilize multiple trans-repressive mechanisms, namely, the sequestration of transcription factors and the prevention of corepressor clearance, to repress HIV-1 transcription in MDMs.

NR ligands inhibit TLR2/1-induced cytokine expression in MDMs.

TLR signaling in macrophages influences HIV-1 pathogenesis not only by directly activating virus replication but also by inducing the release of proinflammatory cytokines that further activate virus replication. Numerous reports have shown that NR signaling is a potent inhibitor of proinflammatory gene expression (6, 25, 71, 117, 119, 131). It was therefore important to determine whether NR ligands could inhibit the release of proinflammatory cytokines important for HIV-1 transmission and replication from TLR-activated MDMs. Both uninfected and HIV-1-infected MDMs were treated with PAM3CSK4 in the presence or absence of NR ligands. TNF-α, IL-6, and IL-8 levels were then measured in cell-free supernatants. We found that PAM3CSK4 induced a potent proinflammatory response by directing the release of TNF-α, IL-8, and IL-6 (Fig. 10 and data not shown). Importantly, NR ligand treatment inhibited this proinflammatory response. Interestingly, the different NR ligands inhibited TNF-α, IL-6, and IL-8 expression differentially. For example, TO-901317 (LXR) inhibited IL-8 ∼37-fold but inhibited TNF-α less potently. In contrast, dexamethasone (GR) strongly inhibited IL-8 (∼29-fold) and completely shut down TNF-α production. These results are important given that TNF-α and IL-6 are potent activators of HIV-1 expression (45, 68, 123) and that IL-8 has been shown to increase HIV-1 replication in cervical tissue explants (112). To determine whether the decrease in proinflammatory cytokine secretion corresponded with a decreased ability to activate HIV-1 replication in infected cells, cell culture supernatants from NR- and/or PAM3CSK4-treated MDMs were cultured with latently infected HIV-1 cell lines. Cell culture supernatants from TLR-activated MDMs caused a significant increase in HIV-1 production and release from latently infected U1 and J1.1 cells (data not shown). In comparison, cell culture supernatants from MDMs treated with GR, PPARγ, or LXR ligands prior to TLR activation were inhibited in their ability to stimulate HIV-1 production from these cells. Supernatants from NR-treated MDMs in the absence of TLR stimulation had little to no effect on HIV-1 production in the latently infected cells (data not shown). This indicates that NR signaling, in addition to directly repressing virus expression in infected cells, is capable of dampening the proinflammatory cytokine response that activates HIV-1 expression in an autocrine or paracrine fashion.

Fig. 10.

NR ligand treatment prevents TLR-induced proinflammatory cytokine production. MDMs (2.5 × 10⁵ cells/well) were treated with NR ligands at the indicated concentrations for 30 min prior to stimulation with the TLR2/1 ligand PAM3CSK4 (100 ng/ml) (filled histograms). Cell culture supernatant was harvested 18 h later and assayed for TNF-α, IL-6 (data not shown), and IL-8 (data not shown) by ELISA. The data are the means (±SD) from three different donors, each tested in triplicate.

DISCUSSION

Ligand-activated NRs repress NF-κB- and AP-1-driven proinflammatory gene expression in macrophages through a number of trans-repression mechanisms. Here, we show that GR, PPARγ, and LXR ligands repress HIV-1 transcription in a dose-dependent manner in MDMs through the sequestration of NF-κB and AP-1 and, in the case of PPARγ and LXR, through the retention of NCoR-containing corepressor complexes at the HIV-1 LTR. In addition to directly inhibiting HIV-1 transcription, these NRs also inhibit the production of TNF-α, IL-6, and IL-8, which have been demonstrated to augment HIV-1 replication. Our findings suggest that GR, PPARγ, and LXR utilize common, overlapping mechanisms to block both HIV-1 transcription and proinflammatory cytokine production.

Sexually transmitted infections (STIs) directly activate virus replication in macrophages and dendritic cells and induce an inflammatory environment conducive to HIV-1 replication through the engagement of pattern recognition receptors, such as the TLRs. Given that TLR signaling leads to the activation and nuclear translocation of NF-κB, AP-1, and IRFs, transcription factors that are known to interact with the LTR and positively regulate virus expression (5, 13, 68, 91, 92, 124, 151), it is not surprising that a number of TLR ligands activate HIV-1 expression in immune cells (3, 11, 34, 36, 114, 145, 161). In agreement with other reports (3, 11, 36, 98, 114, 145, 161), we show that signaling through TLR2 activates HIV-1 expression in macrophages and DCs (Fig. 2 and 3). Importantly, we also show that TLR-induced HIV-1 expression and proinflammatory cytokine production are potently repressed by ligand-activated NRs, including GR, PPARγ, and LXR (Fig. 2 and 10), suggesting that cross talk between TLRs and NRs influences HIV-1 replication and pathogenesis. STI pathogens that are cofactors for HIV-1 transmission, including Neisseria gonorrhoeae, Candida albicans, and Chlamydia trachomatis, express TLR2 ligands (22, 43, 62, 89, 116, 126, 140, 161). Importantly, engagement of TLR2 has been shown to promote the release of inflammatory cytokines and chemokines from immune cells (75) and to directly activate HIV-1 replication in macrophages, DCs, and CD4⁺ T cells (10, 57, 59, 108, 115, 125, 134, 139, 161). Therefore, the use of the synthetic TLR2 ligand PAM3CSK4 recapitulates the contribution of other STI cofactors on HIV-1 replication in macrophages.

The ligands for GR include cortisol and other compounds produced by the body in response to stress (18). The ligands for PPARs include fatty acids involved in lipid metabolism, while the ligands for LXRs include oxysterols and other fatty acids involved in cholesterol metabolism (26, 52). Importantly, NRs, including GR, PPARγ, and LXR, are expressed in HIV-1 target cells, such as macrophages (26, 52). Currently, several GR and PPARγ agonists are licensed for use in humans. GR agonists, approved for the treatment of inflammatory and autoimmune disorders, are used widely, although they do have significant side effects associated with systemic administration. The thiazolidendiones, a class of PPARγ agonists approved for the systemic treatment of type II diabetes mellitus, have a very good safety profile and are generally well tolerated. LXR agonists, on the other hand, are not approved for clinical use in humans. The LXR agonist TO-901317 was tested in a clinical trial for the treatment of atherosclerosis and hyperlipidemia; however, the trial was discontinued due to unexpected neurotoxicity after systemic administration (33, 74). In a topical preparation, however, the systemic adverse effects associated with NR ligand administration might be limited.

NRs are bifunctional modulators of gene expression, capable of both positively and negatively regulating gene expression. The switch between gene activation and repression properties is complex, gene specific, and often context dependent. In the context of gene activation, ligand-activated NRs bind to specific DNA response elements in promoters or enhancers and activate gene expression by recruiting coactivator complexes to these genes (ligand-dependent transactivation) (30, 52, 53). In contrast, NRs negatively regulate gene expression in two general ways. First, certain NRs bind to their cognate response elements in the absence of ligand and mediate repression through the recruitment of corepressor complexes (ligand-independent repression) (121, 130). Second, ligand-activated NRs act directly to negatively regulate gene expression by antagonizing the activities of transcription factors in a mechanism termed trans-repression (ligand-dependent trans-repression) (70, 87, 90, 95, 117, 119, 129, 135). Our data are consistent with the latter mechanism of repression of HIV-1 replication. This conclusion is based on three lines of evidence. First, the selective glucocorticoid receptor modulator RU-486, which antagonizes GR-mediated transactivation but promotes GR-mediated trans-repression (132), represses TLR-induced HIV-1 expression in infected MDMs (Fig. 5). Second, NR-mediated repression of HIV-1 replication is achieved when the NR ligand is added at the same time as or even after MDM activation (Fig. 7). Third, ligand-dependent GR-, PPARγ-, and LXR-mediated repression does not require new cellular protein expression, as their repressive effects are not abrogated in the presence of cycloheximide (Fig. 7). Together, these data provide support for the hypothesis that NR-mediated repression is mediated through a trans-repression mechanism.

It is unlikely that either transactivation or ligand-dependent active repression through DNA-bound NRs is involved in repression of HIV-1. Here, we show that NR-mediated repression of HIV-1 requires neither lengthy treatment (Fig. 7A) nor new cellular protein synthesis (Fig. 7B), indicating that NR-mediated transactivation is not involved in repression of HIV-1. Although our time course and cycloheximide data do not exclude a role for active repression by DNA-bound NRs, which is an acute process that utilizes preexisting cellular complexes, it is unlikely to play a role in the repression we observe. Class II NRs, such as PPARγ, inhibit cellular gene expression when bound to DNA in the absence of ligand (52), while the repression we observe is ligand dependent. An alternate possibility is that ligand-activated GR homodimers and PPARγ/RXR heterodimers can actively repress transcription by binding to glucocorticoid response elements (GREs) or PPAR response elements (PPREs) within the promoters of certain genes, as has been described for RAR (80). Binding sites for both GR and PPARγ have been described in the HIV-1 LTR. There are several GR binding sites located in the U5, R, and U3 regions and one putative PPARγ binding site located in the U5 region (reviewed in reference 120). Studies using truncated LTR constructs in transient-transfection assays have mapped the cis-acting sequences required for the repressive effects of GR and PPARγ ligands to the proximal enhancer and core promoter regions of the HIV-1 LTR (T. Hanley and G. Viglianti, unpublished data), which includes binding sites for NF-κB and Sp1, among other transcription factors (reviewed in reference 120), but no sequences similar to known PPAREs. Although this region does contain a GRE and GR has been shown to bind to the core promoter region of the HIV-1 LTR in vitro (107), glucocorticoids have not been shown to alter HIV-1 transcription through this GRE (107). Taken together, these data suggest that it is unlikely that repression results from ligand-associated GRs binding to the HIV-1 LTR.

Our data are consistent with the hypothesis that GR, PPARγ, and LXR repress HIV-1 transcription in macrophages via a trans-repression mechanism. There are several reported mechanisms for NR-mediated trans-repression. These include (i) direct interactions between NR and other transcription factors, such as NF-κB, AP-1, STATs, and IRFs (70, 87, 90, 95, 117, 119, 129, 135); (ii) regulation of the activity of kinases, such as MAPKs and IκB kinase (IKK) (6, 8, 16, 17); (iii) competition for coactivator complexes, such as p300/CBP (73); (iv) interactions with corepressor complexes that contain NCoR/SMRT (121, 130); and (v) prevention of ubiquitin-mediated corepressor clearance (50, 51, 70, 119). In this report, we provide evidence that NR signaling inhibits HIV-1 replication through multiple nonredundant trans-repression mechanisms. GR-, PPARγ-, and LXR-mediated repression of HIV-1 transcription in MDMs seems to be mediated in part by the prevention of NF-κB and AP-1 binding to the HIV-1 LTR through what is most likely a squelching mechanism (Fig. 8), suggesting that the canonical NF-κB and AP-1 trans-repression pathways are involved in the NR-mediated repression we observe in MDMs. GR has been shown to directly interact with components of both NF-κB and AP-1 and to prevent them from interacting with other promoters in vivo (90, 95, 117, 129, 135). PPARγ has been shown to interact with NF-κB p65 (7, 27), suggesting that binding to and preventing p65 from interacting with the LTR is a possible mechanism of PPARγ-mediated trans-repression. LXR has been shown to bind to STATs and prevent their association with gene promoters (87); however, it has never before been shown to interact with NF-κB as a means of repressing gene expression. The effect of LXR ligand treatment on NF-κB recruitment to the LTR may be due to either a direct interaction between LXR and NF-κB that prevents NF-κB from associating with the LTR or an as-yet-unidentified indirect mechanism that interferes with this association. The exact nature of this putative indirect effect is being actively pursued.

The coactivator/HAT p300/CBP has been shown to be important for HIV-1 expression (15, 32, 64). p300/CBP is recruited to the promoters of many genes through its interaction with transcription factors, such as NF-κB, AP-1, and Sp1, as well as HIV-1 Tat (23, 49, 65, 73, 99, 103), and is thought to acetylate transcription factors and/or histones associated with nucleosomes at transcriptional start sites. In general, histone acetylation is correlated with transcriptional activation and is thought to both modify chromatin structure to allow greater access of the transcription machinery to promoters and provide binding sites for bromodomain-containing regulatory factors that recognize acetylated lysine residues (113). A number of studies have provided evidence that histone acetylation is involved in chromatin remodeling and the subsequent activation of HIV-1 transcription (96, 138, 152), though histone acetylation is not obligatorily linked to transcriptional activation (77). Dexamethasone, ciglitazone, and TO-901317 treatment of MDMs results in the lack of CBP recruitment to the LTR (Fig. 9), but it is unclear whether this is due to the lack of NF-κB p65 and AP-1 (two proteins known to recruit CBP to promoters) associated with the LTR or a direct interaction between CBP and NRs.

In contrast to GR, which seems to employ a single trans-repression mechanism, PPARγ- and LXR-mediated repression of HIV-1 exhibits the characteristics of multiple trans-repression mechanisms. In addition to decreasing the association of NF-κB p65, AP-1, and CBP with the LTR (Fig. 8), PPARγ and LXR also prevent the TLR-induced loss of NCoR from the LTR (Fig. 9). It is unclear whether the PPARγ- and LXR-mediated retention of NCoR at the 5′ LTR is due to NR-mediated prevention of clearance or due to lack of coactivator-mediated displacement. Previous studies have shown that cellular activation leads to the recruitment of coactivator complexes that displace corepressor complexes from gene promoters (93, 127). A number of recent studies have shown that PPARγ and LXR prevent the signal-dependent clearance of corepressor complexes from the promoters of a subset of inflammatory response genes (50, 51, 70, 119). It will be interesting to determine whether PPARγ and LXR utilize these same mechanisms to prevent HIV-1 transcription in infected cells. Previous reports examining the effects of NR signaling on proinflammatory gene expression suggest that GR, PPARγ, and LXR control distinct but overlapping subsets of genes through divergent trans-repression mechanisms (71, 117). For example, LXR signaling represses TLR4-induced expression of inducible nitric oxide synthetase (iNOS), cyclooxygenase 2 (COX-2), and IL-6 in murine macrophages, while PPARγ signaling represses IL-1β, granulocyte colony-stimulating factor (G-CSF), monocyte chemotactic protein 1 (MCP-1), MCP-3, and MIP-1α expression (71). Importantly, the different ligand-activated NRs can act combinatorially to repress a broad array of inflammatory genes (117). We show here that these NRs utilize divergent mechanisms to control HIV-1 gene transcription and that they can act combinatorially to further repress virus expression.

We show for the first time that NCoR binds to the HIV-1 LTR in unstimulated macrophages but that it is cleared from the LTR in response to cellular stimulation through TLR2 (Fig. 9). Our findings suggest that NCoR may play an important role in the negative regulation of HIV-1 transcription. Interestingly, a recent high-throughput, genome-wide scan has identified single-nucleotide polymorphisms (SNPs) in NCoR2, also called SMRT, as factors that positively correlate with HIV-1 transmission rates (28). It will be important to determine whether these NCoR2 SNPs alter its function in such a way that NCoR2 cannot associate with the LTR to regulate HIV-1 transcription.

In the present study, we have shown that GR, PPARγ, and LXR signaling contribute to the repression of HIV-1 transcription in macrophages. Macrophages are hypothesized to serve as long-term reservoirs for HIV-1 and may contribute to the inability of current antiviral strategies to eradicate the virus. Our data demonstrate that MDMs cultured in the presence of physiological concentrations of cortisol, 15-deoxyΔ^12,14-prostagladin J₂, and 22(R)-hydroxycholesterol (Fig. 6) are refractory to HIV-1 replication. These natural ligands are present in serum at nanomolar to micromolar concentrations; however, they can be elevated in localized tissues or under certain physiological conditions (55, 118, 153) and may therefore contribute to the establishment of long-term-infected populations of cells. Perhaps strategies that look to manipulate NR signaling pathways will be useful for further repressing HIV-1 expression in these cells, thereby contributing to a decrease in the size of this important viral reservoir. Studies conducted with synthetic ligands that selectively target NR-dependent trans-repression of gene expression, thereby eliminating the potential negative side effects of NR-dependent gene transactivation, are currently being developed (38, 63, 144) and might prove useful clinically for this purpose.

Ligand-activated GR, PPARγ, and LXR are potent repressors of proinflammatory gene expression (6, 25, 71, 117, 131). They are capable of repressing the TLR2-induced expression of TNF-α, IL-6, and IL-8 in both MDMs (Fig. 10) and MDDCs (56) (data not shown). This repression of proinflammatory cytokines has functional significance for HIV-1 replication, as latently infected monocytes or T cells cultured in medium derived from NR ligand-treated, TLR-activated MDMs express lower levels of virus than those cells cultured with medium from TLR-activated MDMs (data not shown). It is important to note that these ligand-activated NRs repress HIV-1 expression in response to stimulation with concentrations of TNF-α, IL-6, and IL-8 expected at local sites where HIV-1-infected macrophages reside in vivo (Fig. 10) (14, 101). It is unlikely that the effects on HIV-1 replication in U1 and J1.1 cells are due to carryover of NR ligands for three reasons. First, supernatants from NR-treated MDMs that were not activated with PAM3CSK4 did not affect HIV-1 expression in U1 or J1.1 cells (data not shown). Second, the half-lives of PPARγ and LXR agonists in culture are relatively short (20, 104, 147), suggesting that they would be metabolized before they had a chance to act directly on the latently infected cells. Third, NR ligands are highly lipophilic and readily diffuse across the macrophage plasma membrane, where they are bound by either their cognate receptors or by hormone binding protein(s) and retained intracellularly.

Due to the lack of an effective vaccine for HIV, the development of other methods for limiting viral transmission has taken on greater importance. Over the past decade, a great deal of effort has been devoted to developing anti-HIV microbicides that can effectively prevent virus transmission (35, 76, 85, 102, 154). With the exception of the CAPRISA 004 trial (2), most microbicide trials conducted to date have failed due to a number of factors, including poor adherence (141) and unexpected inflammation that increased the risk of transmission (1, 39–41, 148–150). There is a need, therefore, to identify and develop new microbicidal agents that are effective at both blocking virus transmission and preventing the inflammation that has derailed past microbicide trails. One way to achieve this goal is to include anti-inflammatory drugs as combination therapies with anti-HIV agents that target viral entry, reverse transcription, and/or maturation. Our data suggest that NR ligands might serve a role as potential microbicide candidates due to their ability to target multiple steps during virus transmission. We recently reported that PPARγ and LXR signaling inhibits DC-mediated HIV-1 capture and transfer to T cells by activating expression of the cholesterol transporter ABCA1 (56). An ABCA1-dependent decrease in cell-associated cholesterol disrupted the ability of DCs to capture virus particles and subsequently transfer them to CD4⁺ T cells. These effects were dependent on the transcriptional transactivation activities of the ligand-activated NR. In the present study, we demonstrate that ligand-activated GR, PPARγ, and LXR repress both basal and TLR-activated HIV-1 transcription through nonredundant trans-repression mechanisms. They do so by preventing the association of NF-κB p65 and AP-1 with the HIV-1 LTR, as well as by preventing the clearance of NCoR from the LTR. These ligand-activated NRs also potently repress the expression of proinflammatory cytokines that have been shown to augment HIV-1 replication. Collectively, our findings demonstrate that the bifunctional activities of ligand-activated NRs can be exploited to inhibit multiple pathways contributing to HIV-1 replication and transmission and underscore the clinical potential of NR ligands to be used in conjunction with conventional antiviral therapies to limit virus replication and transmission.