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Originally published online as doi:10.1189/jlb.0704424 on April 13, 2006

Published online before print April 13, 2006
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(Journal of Leukocyte Biology. 2006;79:1328-1338.)
© 2006 by Society for Leukocyte Biology

HIV regulation of the IL-7R: a viral mechanism for enhancing HIV-1 replication in human macrophages in vitro

Mingjie Zhang*, Jorg Drenkow{dagger}, Carla S. R. Lankford{ddagger}, David M. Frucht{ddagger}, Ronald L. Rabin§, Thomas R. Gingeras{dagger}, Chettemegre Venkateshan*, Franziska Schwartzkopff{ddagger}, Kathleen A. Clouse{ddagger} and Andrew I. Dayton*,1

* Laboratory of Molecular Virology, Division of Emerging and Transfusion Transmitted Diseases, Office of Blood Research & Review, and
§ Laboratory of Immunobiochemistry, DBPAP, Office of Vaccine Research and Review, Center for Biologics Evaluation & Research, U.S. Food and Drug Administration, Rockville, Maryland;
{dagger} Affymetrix, Santa Clara, California; and
{ddagger} Division of Monoclonal Antibodies, Office of Biotechnology Products, Center for Drug Evaluation and Research, Bethesda, Maryland

1Correspondence: Laboratory of Molecular Virology, Division of Emerging and Transfusion Transmitted Diseases, Office of Blood Research & Review, Center for Biologics Evaluation & Research, FDA, HFM 315, CBER, 1401 Rockville Pike, Rockville, MD 20852-1448. E-mail: Dayton{at}cber.fda.gov


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We report a novel mechanism, involving up-regulation of the interleukin (IL)-7 cytokine receptor, by which human immunodeficiency virus (HIV) enhances its own production in monocyte-derived macrophages (MDM) in vitro. HIV-1 infection or treatment of MDM cultures with exogenous HIV-1 Tat(86) protein up-regulates the IL-7 receptor (IL-7R) {alpha}-chain at the levels of steady-state RNA, protein, and functional IL-7R on the cell surface (as measured by ligand-induced receptor signaling). This IL-7R up-regulation is associated with increased amounts of HIV-1 virions in the supernatants of infected MDM cultures treated with exogenous IL-7 cytokine. The overall effect of IL-7 stimulation on HIV replication in MDM culture supernatants is typically in the range of one log and greater. The results are consistent with a model in which HIV infection produces the Tat protein, which in turn up-regulates IL-7R in a paracrine manner. This results in increased IL-7R signaling in response to the IL-7 cytokine, which ultimately promotes early events in HIV replication, including binding/entry and possibly other steps prior to reverse transcription. The results suggest that the effects of IL-7 on HIV replication in MDM should be considered when analyzing and designing clinical trials involving treatment of patients with IL-7 or Tat vaccines.

Key Words: cytokine receptors • cytokines • Tat • AIDS


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Interleukin (IL)-7 has been proposed for use as a vaccine adjuvant or to promote immune reconstitution in human immunodeficiency virus (HIV)-infected individuals [1 2 3 4 5 ], and the design of appropriate clinical trials is underway. Although extensive studies have been performed to determine the effects of IL-7 on HIV-1 replication in human and nonhuman primate T cells [2 , 4 , 6 7 8 9 10 11 ], little attention has been given to the potential impact of HIV or simian immunodeficiency virus infection on the expression of IL-7 receptor (IL-7R) in cells of the macrophage lineage. These latter cells are among the first cells infected by HIV-1 and constitute a major reservoir for viral replication and survival. The pivotal role of cells of the monocyte/macrophage lineage in HIV-1 transmission and in shuttling HIV to the brain proffers the hope that understanding the dynamics of HIV infection of macrophages will advance our comprehension of the clinical course of AIDS and lead to novel, therapeutic strategies [12 13 14 15 16 ].

Numerous cytokines have been reported to up-regulate HIV production in primary cells of the monocyte/macrophage lineage, including IL-1ß, IL-3, IL-6, IL-8, tumor necrosis factor {alpha} (TNF-{alpha}), macrophage-colony stimulating factor (M-CSF), granulocyte M-CSF (GM-CSF), growth-related oncogene-{alpha} (GRO-{alpha}), and transforming growth factor-ß (TGF-ß) [17 18 19 20 21 22 ]. Of these, IL-1ß, IL-6, IL-8, TNF-{alpha}, M-CSF, GRO-{alpha}, and TGF-ß have been implicated in direct positive-feedback loops in cells of the monocyte/macrophage lineage [23 24 25 26 27 28 29 30 31 32 33 ]. Via an indirect mechanism, nerve growth factor is up-regulated by in vitro HIV-1 infection of monocyte-derived macrophages (MDM) and protects MDM from HIV-induced apoptosis [33 34 35 ]. HIV-1 replication in MDM is also known to up-regulate the HIV-1 receptor CD4 [23 ] and the coreceptor CC chemokine receptor 5 (CCR5) [25 26 27 28 29 ], primarily via induction of M-CSF production [25 , 29 ]. Yet, increased expression of the M-CSF receptor, c-fms, in HIV-1-infected, human MDM has not been reported. However, Roulston et al. [28 ] have reported up-regulation of c-fms by chronic HIV-1 infection of an immortalized cell line having a myelomonoblastic phenotype. The work presented here is the first report of HIV-induced up-regulation of a cytokine receptor (IL-7R) with the capacity to potentiate HIV-1 replication in primary cells of the monocyte/macrophage lineage upon exposure to the relevant cytokine (IL-7).

The cytokine IL-7 plays a central role in the immune system and is best known for its pleiotropic effects on B and T cell proliferation and development. More recently, IL-7 has been described as acting on numerous cell lineages, including differentiating dendritic cells. Originally isolated from stromal cells, IL-7 is known to be produced by a diversity of tissues and cell types, including bone marrow, spleen, thymus stromal/epithelial cells, intestinal epithelial cells, and epidermal keratinocytes [36 37 38 39 40 41 42 43 ]. IL-7R consists of a specific {alpha}-chain and a shared {gamma}c-chain and has low- and high-affinity forms [37 38 39 , 44 45 46 47 ]. In response to IL-7, the IL-7R can cause distinct signaling through a number of signal-transducing factors, including the Janus tyrosine kinase/signal transducer and activator of transcription (STAT) signal transduction pathway [38 , 39 , 44 , 45 , 48 49 50 ]. Depending on the system, IL-7 signal transduction can also involve phosphatidylinositol-3 kinase, c-myc, nuclear factor of activated T cells, activated protein-1, p56lck, p59fyn,p53lyn, c-Jun N-terminal kinase, and p38 kinase [38 , 39 , 51 ].

Broadly considered, IL-7 has at least two general effects on T and B cells: a trophic effect and an effect supporting variable/diversity/joining recombination [38 ]. In conjunction with M-CSF and IL-6, IL-7 can promote the differentiation of early murine thymocytes into macrophage-like cells [52 ]. The details of the mechanisms leading to these effects remain to be elucidated. At extremely high levels (10–100 ug/ml), IL-7 can stimulate the release of several cytokines from monocytes, including IL-1{alpha} and -ß, IL-6, and TNF-{alpha} [53 ]. The ability of IL-7 to boost T cell production and differentiation has led to its consideration as a vaccine adjuvant and therapeutic agent for immune reconstitution in HIV disease [1 2 3 4 5 ]. In HIV-1-infected patients, circulating levels of IL-7 are elevated and correlate with disease progression [54 , 55 ]. Furthermore, IL-7 increases HIV-1 replication in CD8-depleted peripheral blood mononuclear cells (PBMC) from patients chronically infected with HIV-1 [6 , 56 ]. In CD4+CD8–CD3+ thymocytes, IL-7 sustains expression of the p75 TNF receptor, allowing TNF to induce high levels of HIV-1 replication in these cells [7 ].

Here, we report a novel mechanism, whereby HIV-1 infection of MDM up-regulates expression of the IL-7R, which in the presence of IL-7 cytokine, contributes to the increased replication of HIV. This finding raises new concerns regarding the potential use of IL-7 therapy in HIV disease.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
MDM and HIV-1 infection
Monocytes isolated via leukapheresis of human donors seronegative for HIV and hepatitis B were obtained as described previously [57 ]. These monocytes were composed almost exclusively (98%) of CD33+ cells, of which 88–90% displayed a CD14+/CD33+ monocytic phenotype as determined by flow cytometry. Elutriated monocytes were cultured and differentiated to macrophages (MDM) in 1000 U/ml recombinant human (rh)M-CSF [57 ]. After differentiation, MDM were typically >99% positive for CD33 and >80% positive for CD14, as determined by flow cytometry (unpublished observations). MDM were infected 9–14 days after introduction into culture with concentrated HIV-1BAL (Strain 10-177-000, Advanced Biotechnologies, Inc., Columbia, MD) at 0.05–0.1 50% tissue culture infectious dose/ml. Cells were exposed to virus for 2 h and then cultured for varying periods of time as indicated. Viral replication was assessed by measuring HIV-1 p24 by enzyme-linked immunosorbent assay (ELISA; PerkinElmer Life Sciences, Boston, MA).

Treatment of MDM with purified recombinant HIV-1 Tat protein
Uninfected MDM, differentiated as described above, were treated with 50 ng/ml (or as otherwise indicated) HIV-1 Tat protein, Tat(86) [National Institutes of Health (NIH) AIDS Research and Reference Reagent Program, Bethesda, MD) for 48 h at 37°C. For antibody-blocking experiments, prior to addition to monocytes, Tat protein was incubated at room temperature for 1 h with polyclonal anti-Tat rabbit antiserum (NIH AIDS Reagent Program) or normal rabbit serum in a final volume of 50 ul complete medium before addition to cultures.

IL-7 treatment of MDM during growth in culture
HIV-infected MDM to be cultured with IL-7 were fed with complete medium containing rhIL-7 (BD PharMingen, San Diego, CA) at 50 ng/ml or as indicated.

Western blot analysis of IL-7R protein
Cell lysates from MDM were electrophoresed on polyacrylamide gels, blotted, and developed as described for STAT-3 signal transduction (see below). The Western blot was probed with mouse anti-human IL-7R {alpha}-chain antibody (R&D Systems, Minneapolis, MN) as the primary antibody and peroxidase-conjugated goat anti-mouse immunoglobulin G (IgG; Pierce, Rockford, IL) at a 1:15,000 dilution as the secondary antibody. Cell lysates were normalized to total protein before loading.

Flow cytometry
MDM for flow cytometry were detached from the substrate using Ca+2/Mg+2-free phosphate-buffered saline (PBS) and blocked in cold washing buffer [Ca+2/Mg+2-free PBS, 0.1% bovine serum albumin (BSA)] with normal mouse serum diluted 1:50–1:100 for 20 min. MDM were then incubated with 20–50 ul phycoerythrin (PE)- or fluorescein isothocyanate (FITC)-conjugated antibody on ice for 40 min–1 h and washed two times with cold washing buffer. If cells were infected with HIV-1, they were fixed with 1% paraformaldehyde before analysis on an LSR2 flow cytometer (BD Biosciences, San Jose, CA) and analysis with Flowjo software program (Treestar, Ashland, OR).

RNA isolation and IL-7R {alpha}-chain analysis
Total RNA was isolated from MDM using TRIzolTM reagent (Gibco-BRL, Gaithersburg, MD) and treated with DNase I prior to reverse transcription (RT) with the SuperScript preamplification system (Gibco-BRL) and amplification by polymerase chain reaction (PCR) using Supermix (Gibco-BRL). The primer pair for IL-7R {alpha}-chain is 5'-TATGTGTGAAGGTTGGAGAAAAGA-3' and 5'-TTGGTTTCTTACAAAGATGTTCCA-3'. The primer pair for ß-actin is 5'-CCTAAGGCCAACCGTGAAAAG-3' and 5'-TCTTCATGGTGCTAGGAGCCA-3'. For quantitation of RNA by RT-PCR, serial dilutions were made of the samples to be assayed before addition to standard RT-PCR reactions.

Activation of STAT-3
MDM were left untreated (no infection, no Tat protein), infected with HIV-1 for 10 days, or incubated with 50 ng/ml Tat protein as described above. MDM were rested in serum-free Dulbecco’s modified Eagle’s medium (DMEM) containing gentamicin for 4 h before induction with 100 ng/ml IL-7 for 20 min to maximize signaling. Uninduced controls were cultured in parallel. By the end of the rest period, no measurable IL-7 cytokine (less than 0.25 pg/ml by ELISA) had accumulated (data not shown) in the supernatants from rested cells. After placing plates on ice, the medium was removed, and cell lysates were prepared according to Stanley and Fauci [58 ]. The lysates were normalized for total protein concentration and then separated on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred onto nitrocellulose, and immunoblotted with rabbit anti-tyrosine 705-phosphorylated STAT-3 (Upstate Cell Signaling Solutions, Waltham, MA) as the first antibody at 1:1000 dilution and peroxidase-conjugated goat anti-rabbit IgG (Amersham, Little Chalfont, UK) at 1:5000 dilution as the secondary antibody. The signal was developed with ECL Plus Western blot detection system (Amersham), according to the manufacturer’s instructions. After exposure, blots were stripped and probed with anti-STAT-3 (Zymed, San Francisco, CA, Catalog #13-7000) to detect total STAT-3. The band corresponding to activated (phosphorylated) STAT-3 comigrated with total STAT-3.

Pseudotyping
Vesicular stomatitis virus glycoprotein (VSV-G)-pseudotyped HIV virions were obtained by electroporating 293T cells with plasmid cytomegalovirus-VSV and HIV pNL4-3 proviral clone [59 ]. Transfected cell supernatants were harvested after 48 h, filtered, normalized for p24, and used in infections as indicated.

Electroporation
MDM were detached from the substrate in Ca+2/Mg+2-free PBS and electroporated using cuvettes, reagents, and electroporation equipment from Amaxa Biosystems (Gaithersburg, MD) using the manufacturer’s recommended kit and protocol for monocyte transfection. In our system, expression of transfected genes typically reached a plateau by 4–6 h and continued for at least 2 days (unpublished observations).

Analysis of viral binding/entry
MDM were pretreated with recombinant-purified Tat(86) protein at 50 ng/ml in the presence or absence of IL-7 (50 ng/ml) for 2 days. MDM (~2x106 cells per well of a six-well tissue-culture plate) were exposed to HIV-1BAL virions (450 ng p24/ml) in DMEM containing 20 mM HEPES for 2 h at 37°C. Cells were then washed three times in ice-cold PBS, set on ice for 1 h, and then removed from the plate by scraping. Virus adsorbed at the cell surface was removed by treatment with pronase (Boerhinger Mannheim, Germany, 1 mg/ml, in 300 ul) for 2 min in ice-cold DMEM containing 20 mM HEPES. Proteolysis was stopped by adding 1 ml DMEM containing 10% fetal calf serum (FCS), and cells were washed three times in RPMI supplemented with 10% FCS. Cells were lysed with 300 ul reporter lysis buffer (Promega Corp., Madison, WI) for p24 ELISA analysis (Perkin Elmer, Wellesley MA; see ref. [60 ]).

Analysis of viral RT
MDM were pretreated with Tat at 50 ng/ml in the presence or absence of IL-7 (50 ng/ml) for 2 days. HIV-1 adenosine deaminase-green fluorescent protein (ADA-GFP; M-Tropic HIV with an enhanced GFP gene replacing nef) [61 ] virions were DNase I-treated (50 ul virus stock+100 U DNase I in 100 ul reaction buffer M+BSA) at room temperature for 15 min. The reaction was stopped by addition of 5 ul 0.5 M EDTA before adding virus to cells. As a negative control, virus was heat-inactivated at 56°C for 30 min. Infection was allowed to proceed for 4–6 h, after which DNA was extracted by standard techniques. For standard PCR, samples were serially diluted as indicated and subjected to 30 cycles of PCR (92°C for 2 min followed by 92°C for 1 min, 50°C for 1 min, 72°C for 1 min, and 72°C for 10 min and then a 4°C hold) and were performed with gag region primers SK38 (5'-ATAATCCACCTATCCCAGTAGGAGAAAT-3') and SK39 (5'-TTTGGTCCTTGTCTTATGTCCAGAATGC-3'), producing a 115-base pair product (see ref. [62 ]). For TaqMan real-time PCR, HIV DNA was amplified by primers 6F (5'-CATGTTTTCAGCATTATCAGAAGGA-3') and 84R (5'-TGCTTGATGTCCCCCCACT-3') and detected by probe gag-T (5'-FAM-CCACCCCACAAGATTTAAATACCATGCTAA-Q3') using standard reagents and conditions [63 ]. HIV concentrations were normalized to ß-actin-2 genomic DNA concentrations, determined by TaqMan real-time PCR of replicate aliquots analyzed under identical conditions in the same multiwell plate. The actin primers were actin-F (5'-CACATCGTGCCCATCTACGA-3') and actin-R (5'-CTCAGTGAGGATCTTCATGAGGTAGT-3'). The actin probe was actin-P (5'-FAM- ATGCCCTCCCCCATGCCATCCTGCGT-Q3') [64 ]. Initial analyses were performed in singlicate and for each analyte, the one specimen with the highest concentration of that analyte was diluted in triplicate to generate a standard curve bracketing the range of concentrations to be analyzed. Results thus corrected for the efficiency of the reaction under the exact conditions of the assay are necessarily expressed in arbitrary units. Cells infected in parallel with heat-inactivated control virions produced negligible signals for HIV DNA.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Preliminary screening with high-density array analysis indicated that HIV-1 infection or treatment with exogenous Tat protein increased MDM IL-7R {alpha}-chain RNA steady-state levels variably between six-fold and 24-fold (data not shown). To confirm and extend these initial findings, we determined the effects of these treatments on MDM cultures from individual donors using RT-PCR. The HIV-1 "infected" and "uninfected" RNA preparations used had comparable amounts of ß-actin RNA. However, the IL-7R {alpha}-chain signal in the infected sample was between eight- and 16-fold stronger than the detectable baseline levels in the uninfected control (Fig. 1a ). This suggests, after normalization to ß-actin RNA levels, that there was approximately one log more IL-7R-{alpha} steady-state RNA in infected than in uninfected cultures. Similarly, in a separate experiment, addition of 50 ng/ml Tat protein to uninfected MDM up-regulated IL-7R steady-state RNA levels ~16-fold (Fig. 1b) above baseline levels detectable in controls. Anti-Tat antiserum specifically inhibited this increase, although not completely (last lane, Fig. 1b ). These results are typical of five experiments (five separate donors) with MDM infected by HIV-1 and five experiments (other separate donors) with MDM treated with Tat protein alone (data not shown). The donors used for infection differed from those used for Tat protein treatment.


Figure 1
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  		Figure 1. Up-regulation of IL-7R {alpha}-chain by HIV-1 infection and Tat protein. (a) RT-PCR of IL-7R-{alpha} RNA in uninfected (0) and HIV-1-infected (+) MDM. Cells were harvested on Day 10 postinfection. ß-Actin mRNA levels were measured to facilitate normalization. Serial dilutions of 1:2 (2)–1:16 (16), as indicated, were made of the RNA preparations after RT but before PCR. Results presented are typical of experiments on five different donors. Up-regulation of IL-7R RNA was not seen on Days 1 and 3 of culture (not shown). (b) RT-PCR of IL-7R-{alpha} RNA in untreated (0) and Tat-treated (+) MDM. Tat protein was added to uninfected cells on Day 14 of culture. Cells were harvested 2 days later. Results are typical of experiments using five different donors. (c) Western blot analysis of IL-7R {alpha}-chain. MDM were treated as in a and b before harvest. Results are typical of experiments on three different donors. Ab, Antibody. (d) Flow cytometry analysis of uninfected (Control) and HIV-1-infected MDM using mouse anti-human IL-7R and isotype control antiserum as indicated.

 
The increases of IL-7R {alpha}-chain mRNA levels were reflected in the expression of IL-7R {alpha}-chain protein. In the experiment depicted in Figure 1c , Western blot analysis showed that uninfected (untreated) cells had minimally detectable baseline levels of IL-7R {alpha}-chain protein, whereas HIV-1-infected MDM or MDM treated with exogenous Tat protein had clearly significant levels of IL-7R {alpha}-chain protein. Furthermore, anti-Tat antiserum blocked the effects of the exogenous Tat protein (Fig. 1c , lane 4). Flow cytometry analysis of HIV-1-infected MDM suggested that the increased IL-7R-{alpha} protein was present on the surface of a substantial proportion of the MDM population (Fig. 1d) . However, because of the high background, characteristic of immunostained MDM, it is difficult to accurately quantify the amount of the up-regulation and the size of the population affected.

To determine whether the increased amounts of IL-7R-{alpha} protein resulting from HIV infection or addition of Tat protein to the culture medium were functional and to confirm the surface expression, we measured intracellular signaling by the IL-7R in response to 20 min of stimulation by exogenous rhIL-7. Figure 2 shows the Western blot analysis of cell extracts probed with antibody to activated STAT-3 [tyrosine-phosphorylated STAT-3 (P-tyr-STAT-3)], one of several cell-signaling molecules implicated in IL-7R signaling. The anti-P-tyr-STAT-3-immunoreactive signal observed in response to the 20-min IL-7 treatment of cells infected with HIV for 10 days (Fig. 2a) or incubated with Tat protein for 48 h (Fig. 2b) was significantly greater than the baseline anti-P-tyr-STAT-3 signal from control cells, which was barely detectable in some experiments and below detectability in others. Levels of total STAT-3 remained unchanged (Fig. 2a and 2b , lower panels).


Figure 2
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  		Figure 2. Activation of STAT-3 by IL-7R signaling. Cells were left uninfected or infected with HIV for 10 days, or cells were left untreated or treated with Tat protein from Day 14 to Day 16 of culture before analysis of IL-7R signaling, where cells were rested in serum-free DMEM with gentamicin for 4 h at 37°C. During this rest period, no measurable IL-7 accumulated in the culture supernatants (less than 0.25 pg/ml by ELISA, data not shown). Cells were then harvested after 20 min of no treatment or 20 min of treatment with 100 ng/ml IL-7 for 20 min. Lysates run on Western blots were stained with rabbit antiserum specific for phosphorylated (activated) STAT-3 (anti-P-STAT-3). After exposure, blots to be "reprobed" were stripped and stained with antisera specific for total STAT-3. (a) HIV-infected MDM, probed with antisera as indicated. (b) Tat protein treatment, uninfected MDM, probed with antisera as indicated. Similar results were obtained in one additional experiment (using different donors than shown), in which the filters were stripped and reblotted with antisera to total STAT-3 as shown, and in two other additional experiments (using other, additional donors), in which the filters were not reprobed with antisera to total STAT-3. (b) Lanes 2 and 3 have been graphically switched (from their original gel positions).

 
As a first step in characterizing the physiological consequences of IL-7R signaling, we asked whether exogenous IL-7 could enhance HIV-1 expression. Table 1 shows the results of infecting MDM from five different donors in five separate experiments with exogenous IL-7 added to the cultures on Day 5 of infection 2 days before supernatant harvest compared with infected controls not treated with exogenous IL-7. Addition of 50 ng/ml IL-7 to the cultures up-regulated HIV-1 p24 antigen levels in supernatants an average of 20-fold, ranging from eightfold to 42-fold (P<0.02). Figure 3a shows a time-course of the effects of exogenous IL-7 on HIV replication in MDM. Cultures were run in triplicate. Exogenous IL-7 led to an early boost of HIV replication compared with controls, peaking at seven times control levels at Days 8 and 10. Figure 3b shows that this stimulation of HIV replication titers over a 30-fold range of IL-7 concentration (from 3 ng/ml to 100 ng/ml). These data demonstrate that IL-7 can have a significant effect on HIV-infected cells of the macrophage lineage over a broad range of concentrations.


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  		Table 1. IL-7 Up-regulates HIV-1 Replication in MDM (p24 Antigen pg/ml)

 

Figure 3
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  		Figure 3. Exogenous IL-7 cytokine added to MDM cultures up-regulates HIV replication. (a) MDM were infected with HIV-1BAL on Day 10 of culture, as indicated in Materials and Methods. HIV replication was monitored by determination of HIV-1 p24 antigen in supernatant, collected on the indicated days. Dashed line, HIV-1 + IL-7; solid line, HIV-1 alone; all determinations in triplicate. Error bars indicate ±1 SD. (b) HIV replication versus concentration of exogenous IL-7. Infections were performed as for Table 1 , which included the data for the "0" and "50 ng/ml" IL-7 concentrations displayed here. The ratio of "stimulated"/"unstimulated" is presented as percent of the value at 100 ng/ml IL-7. The data are representative of two experiments, each run in singlicate.

 
To determine whether IL-7 affected early stages of viral replication, we pursued two independent approaches. First, we measured genome RT products by conventional DNA PCR of cell extracts harvested 6 h after binding of HIV-1 ADA-GFP virions. The data in Figure 4a suggest that IL-7 up-regulates early viral replication 2.5- to threefold, as measured by formation of RT products. Identical results were obtained in three additional experiments on separate donors (not shown), although absolute quantitation by direct, conventional DNA PCR is problematic. Additional, identical experiments were performed on MDM from two donors using HIV-1 ADA-GFP virions and TaqMan real-time DNA-PCR quantitation, normalizing HIV concentrations to genomic ß-actin-2 DNA. Table 2 summarizes the results. The ratios of HIV DNA "+ IL-7" to HIV DNA "–IL-7" in the two donors were approximately 15-fold, suggesting five- to sixfold more up-regulation by the time of RT than indicated by the conventional DNA-PCR results. Second, we used p24 EIA to measure cell-associated virus remaining after HIV-1Bal-1 binding and light protease stripping of loosely bound virus. In the presence of Tat protein, IL-7 treatment reproducibly increased viral binding/entry by a little over twofold (Fig. 4b) . In the absence of Tat protein, IL-7 had no effect on viral binding/entry (data not shown).


Figure 4
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  		Figure 4. IL-7 stimulates early events in viral replication. MDM were pretreated with or without Tat protein and with or without IL-7 cytokine, as indicated, for 48 h before exposure to HIV-1BAL virions. (a) Ethidium bromide staining of DNA-PCR products of DNA extracted from cells lysed 6 h after binding of HIV-1BAL virions. Equivalent results from three additional donors were obtained (data not shown). (b) p24 enzyme immunoassay (EIA) analysis of p24 antigen associated with cells after stripping away loosely bound virions with pronase. Each panel represents a unique donor.

 

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  		Table 2. Real-Time DNA-PCR Analysis of HIV RT Products

 
To determine if IL-7 stimulation increased HIV replication during later stages in the viral life cycle, the T-tropic clone of HIV-1 pNL4-3 was pseudotyped with VSV-G envelope protein. Pseudotyping with VSV-G circumvents the normal HIV entry route, and a T-tropic virus, such as pNL4-3, is incapable of reinfecting MDM after the first round of infection. MDM were pretreated with Tat protein in the presence or absence of exogenous IL-7 cytokine for 2 days, infected with pseudotyped pNL4-3 virions, washed, and cultured for 48 h (in the continued presence or absence of IL-7 and Tat) before harvest for p24 analysis. After infection with VSV-pseudotyped T-tropic pNL4-3 virions, MDM were cultured for 24 h. IL-7 had only minor effects on p24 production measured in supernatants or cells (Fig. 5a ). Attempts to determine the titration of viral replication over lower concentrations of pseudotyped virions were unsuccessful, as even a tenfold dilution of infecting pseudotyped virions resulted in undetectable p24 signals in supernatant and cytoplasm.


Figure 5
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  		Figure 5. Post-entry viral replication steps are minimally affected by IL-7. (a) MDM were pretreated with or without Tat protein (50 ng/ml) and with or without IL-7 cytokine (50 ng/ml), as indicated, for 48 h before exposure to pseudotyped virions. After pseudotype infection, Tat and IL-7 concentrations were maintained until cells and supernatants were harvested after overnight culture and analyzed for p24 antigen. In all experiments, the T-tropic HIV Clone pNL4.3 was pseudotyped with VSV-G protein as indicated in Materials and Methods. HIV pNL4.3 cannot reinfect MDM after the first round of replication. Panels represent two different, unique donors. Experiments were performed in duplicate. Bars indicate values of each of the duplicate measurements. (b) MDM were co-electroporated with long-terminal repeat (LTR)-DS-Red [an expression plasmid with a red fluorescent protein (RFP) under the control of an HIV promoter], together with LTR-Tat (an expression plasmid with Tat protein under the control of the HIV promoter). Control electroporation with pUC18 was done to determine background levels of autofluorescence. The numbers above the brackets in each graph are the percent of cells transfected and ranged from 30% to 45%, depending on the donor. Similar results were obtained with three additional donors pretreated for 48 h before electroporation with Tat protein in the presence or absence of exogenous IL-7 cytokine and cultured postelectroporation in the continued presence or absence of Tat in the presence or absence of IL-7 (not shown). Panels represent two different, unique donors.

 
To confirm the minimal effect of IL-7 on viral replication events subsequent to entry and to determine if IL-7 had transcriptional effects that might be masked by other effects on viral replication, we developed the technology of electroporating DNA into primary MDM. In our system, the electroporation technique used typically supports levels of expression, which plateau starting at 4–6 h postelectroporation and continue for at least 2 days (unpublished observations). LTR-RFP, a plasmid construct with the RFP DS-Red, under the transcriptional control of the HIV promoter (including transactivation-responsive region), was used to measure the efficiency of HIV promoter transcription by flow cytometry analysis. A plasmid construct with Tat under the transcriptional control of the HIV promoter was coelectroporated into cells with LTR-RFP, and the cells were cultured overnight in the presence or absence of exogenous IL-7 cytokine before flow cytometric analysis for RFP (DS-Red). When these plasmids were electroporated into the monocytoid line U937, IL-7 caused a modest increase in transcription from the HIV LTR (data not shown). However, when they were electroporated into MDM, IL-7 had no significant effects on the efficiency of transcription from the HIV promoter (Fig. 5b) . Similar results were obtained with three additional donors pretreated for 48 h before electroporation with the Tat protein in the presence or absence of exogenous IL-7 cytokine and cultured postelectroporation in the continued presence or absence of Tat and IL-7, except that lower transfection efficiencies and lower levels of expression per cell were attained (data not shown). The lack of IL-7 effects at lower levels of expression of transfected indicator reporter constructs (in essence, an unintended titration) suggests that the results presented in Figure 5b are not a result of saturation of intracellular systems.

To determine if IL-7 (in the presence of Tat) alters the expression of cellular genes involved in HIV-binding/entry, we measured the effects of IL-7 on Tat-treated MDM expression of CD4 and CCR5 using flow cytometry. IL-7 had no demonstrable effects on the expression of CD4 or CCR5 (Fig. 6 ).


Figure 6
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  		Figure 6. Influence of IL-7 on expression of cell-surface molecules involved in HIV-binding/entry. MDM were treated with 50 ng/ml Tat in the presence or absence of 50 ng/ml IL-7 cytokine for 2 days and analyzed by flow cytometry with commercial antisera specific for (a) human CD4 or (b) human CCR5. Each result is representative of two donors.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The observations that HIV infection or treatment with the HIV-1 Tat protein up-regulates IL-7R-{alpha} in human MDM have proven remarkably consistent, if quantitatively variable. The increase in steady-state RNA is typically in the range of tenfold and greater and is accompanied by similar increases in IL-7R-{alpha} protein (Fig. 1) . To address the question of whether the increased IL-7R-{alpha} protein is functional, we asked if it were associated with an increase in intracellular signaling in response to IL-7. The data demonstrate clearly that under conditions resulting in up-regulated IL-7R expression, whether by HIV infection (Fig. 2a) or by Tat protein (Fig. 2b) , the anti-P-tyr-STAT-3-immunoreactive signals are much higher in response to acute (20 min) IL-7 stimulation than they are under conditions that do not support up-regulation of IL-7R expression (uninfected or untreated controls). Thus, the newly expressed (i.e., up-regulated) IL-7R is indeed functional. Although the anti-P-tyr-STAT-3-immunoreactive signal is clearly elevated in MDM by IL-7R signaling, involvement of other intracellular signal-transduction molecules in MDM IL-7R signaling remains to be determined. Similarly, we have yet to definitively ascribe any particular downstream event to STAT-3 signaling per se, including the up-regulation of HIV replication and the enhancement of HIV virion binding/entry. Although the specific effects identified could be mediated by different signal transduction mechanisms, the increased activation of STAT-3 by IL-7, when increased amounts of IL-7R are present, confirms that the increased levels of IL-7R are functional and appropriately displayed for IL-7 signal transduction and qualifies STAT-3 as a candidate mediator for the observed effects.

The enhancement of HIV-1 p24 antigen production by IL-7 is also remarkably consistent if quantitatively variable in infected MDM from different donors and supports the existence of a viral mechanism that promotes its own replication in the presence of IL-7 cytokine. IL-7 increases supernatant p24 antigen levels typically in the range of tenfold and greater, even when present only from Day 5 to Day 7 postinfection. It is not known what cell types are responsible for the increased, circulating levels of IL-7 observed with clinical progression in AIDS [54 55 56 , 65 ], but our data raise the possibility that these higher levels may increase HIV replication in MDM. Regardless of the magnitude of the contribution of MDM to plasma viral load, the potential increase in viral replication in the brain as a result of IL-7-stimulated macrophages could be significant.

Regarding the mechanism of enhanced HIV-1 p24 antigen supernatant levels in response to exogenous IL-7 cytokine, syncytia do not appear to contribute to these effects, inasmuch as there is no visible syncytia formation in the culture system used here (data not shown). Similarly, IL-7 stimulation appears to have minimal effects on viral replication subsequent to the early steps in the replication cycle bypassed by pseudotyping (Fig. 5a) . Effects on the efficiency of transcription from the viral promoter are minor or nonexistent (Fig. 5b) . However, IL-7 treatment does cause increases in the early steps in viral replication prior to RT (Fig. 4 , a and b, and Table 2 ). Direct binding experiments suggest IL-7 up-regulates viral binding/entry (as defined by resistance to light pronase stripping in the cold) by a little over twofold (Fig. 4b) . DNA PCR, on serially diluted samples, also suggests a two- to threefold increase in early viral replication (Fig. 4a) , which could only account for the log increase in viral replication we measure in culture if multiple replication cycles occur during the time of IL-7 exposure or if direct cell-to-cell transmission is quantitatively more affected by IL-7 than the virion-to-cell transmission measured in our experiments on viral entry and RT. In T cell systems, direct cell-to-cell transmission of HIV is two to three logs more efficient than transmission via free virions [66 ]. Direct cell-to-cell transmission of HIV from in vitro-infected MDM to PBMC can also be efficient (Dimiter Dimitrov, NIH, Bethesda, MD, personal communication). However, real-time DNA PCR of viral replication products suggests IL-7 enhances viral replication by approximately 15-fold, which would account for the predominance of the increased viral replication we detect. Steps late in the entry process (after resistance to pronase), such as uncoating, could account for the additional replication by the time of RT (as determined by real-time PCR), but little is known about cellular (or even viral) contributions to such events. The lack of an effect of IL-7 when HIV is introduced through pseudotyping would weigh against the possibility of IL-7 increasing RT efficiency per se (unless it did so indirectly through modification of some unidentified, previous step) or the efficiency of subsequent steps in the viral lifecycle. Curiously, flow cytometry analysis of MDM treated with Tat showed no effect on the levels of CD4 or CCR5 attributable to IL-7 cytokine (Fig. 6) , leaving the mechanism of up-regulation of viral binding/entry unresolved. However, cellular determinants other than CD4 and CCR5 can influence the efficiency of HIV binding/uptake (e.g., annexin II [67 ] and proteoglycans [68 ]), and it is conceivable that modest alterations in a number of cellular factors could contribute, individually or collectively, to the effects of IL-7R signaling reported here.

To the extent that viral infection interferes with infection by subsequent virions, up-regulation of HIV infection by up-regulation of binding/entry and other steps in early replication underscores the importance of paracrine (bystander) effects of the Tat protein. A likely in vivo scenario would be HIV-infected cells producing extracellular Tat, which subsequently acts on uninfected macrophages to up-regulate the IL-7R. This could then have pleiotropic, downstream effects on macrophages, including stimulation of early viral replication.

We speculate that the viral enhancement mechanism described here may play a role in the clinical course of HIV infection by mechanisms other than enhancement of viral replication. Chronic overexpression of IL-7R in critical immune reservoirs may lead to significant immune dysregulation as well as contributing to a variety of documented effects of Tat protein on host cells (see refs. [57 , 69 70 71 72 ] and references cited therein).

We have demonstrated that HIV-1 infection up-regulates the IL-7R {alpha}-chain, which in turn, increases the replication of HIV (as measured by p24 antigen) by cultured, primary human MDM exposed to exogenous IL-7 cytokine. Our data also suggest that this up-regulation can be mediated, at least in part, through the HIV-1 Tat protein and involves enhancement of early replication, including increased virion binding/entry. Our observations of the response of HIV to IL-7R stimulation in this culture system suggest that IL-7R signaling might be an attractive, adjunctive, therapeutic target were IL-7R signaling not so central to the development and functioning of the immune system. Stimulation of the IL-7 pathway could prime the MDM "reservoir" cells, facilitating the spread of infection. Despite reports that IL-7 administration does not increase in vivo viral load in some model systems [4 ], the design of clinical protocols involving administration of IL-7 to HIV-infected patients should consider the effects of IL-7R up-regulation and signaling, not only in T cells [9 ] but also in macrophages.


   ACKNOWLEDGEMENTS
 
This work was supported in part by a grant from the NIH Intramural AIDS Targeted Antiviral Program (IATAP). Opinions expressed in this publication reflect the professional views of the authors and should not be viewed as official policy of the U.S. Food and Drug Administration or the government of the United States. We thank Indira Hewlett, Eric Freed, D. Dimitrov, and Mike Norcross for advice; K-T. Jeang for advice and for providing pHIV-1 ADA-GFP; Tie-Hua Ng and Rolf Taffs for help with the statistics; and Klaus Strebel for providing pCMV-VSV.

Received July 27, 2004; revised February 7, 2006; accepted February 9, 2006.


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 RESULTS
 DISCUSSION
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