Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
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Originally published online as doi:10.1189/jlb.0506361 on October 24, 2006

Published online before print October 24, 2006
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(Journal of Leukocyte Biology. 2007;81:528-538.)
© 2007 by Society for Leukocyte Biology

Hypoxia inhibits Moloney murine leukemia virus expression in activated macrophages

Maura Puppo*,1, Maria Carla Bosco*,1, Maurizio Federico{dagger}, Sandra Pastorino*,2 and Luigi Varesio*,3

* Laboratory of Molecular Biology, G. Gaslini Institute, Genova, Italy; and
{dagger} National AIDS Center, Division of Pathogenesis of Retrovirus, Istituto Superiore di Sanità, Rome, Italy

3 Correspondence: Laboratorio di Biologia Molecolare, Istituto Giannina Gaslini, Padiglione 2, L.go Gerolamo Gaslini 5, 16147 Genova Quarto, Italy. E-mail: luigivaresio{at}ospedale-gaslini.ge.it


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hypoxia, a local decrease in oxygen tension, occurring in many pathological processes, modifies macrophage (M{phi}) gene expression and function. Here, we provide the first evidence that hypoxia inhibits transgene expression driven by the Moloney murine leukemia virus-long terminal repeats (MoMLV-LTR) in IFN-{gamma}-activated M{phi}. Hypoxia silenced the expression of several MoMLV-LTR-driven genes, including v-myc, enhanced green fluorescence protein, and env, and was effective in different mouse M{phi} cell lines and on distinct MoMLV backbone-based viruses. Down-regulation of MoMLV mRNA occurred at the transcriptional level and was associated with decreased retrovirus production, as determined by titration experiments, suggesting that hypoxia may control MoMLV retroviral spread through the suppression of LTR activity. In contrast, genes driven by the CMV or the SV40 promoter were up-regulated or unchanged by hypoxia, indicating a selective inhibitory activity on the MoMLV promoter. It is interesting that hypoxia was ineffective in suppressing MoMLV-LTR-controlled gene expression in T or fibroblast cell lines, suggesting a M{phi} lineage-selective action. Finally, we found that MoMLV-mediated gene expression in M{phi} was also inhibited by picolinic acid, a tryptophan catabolite with hypoxia-like activity and M{phi}-activating properties, suggesting a pathophysiological role of this molecule in viral resistance and its possible use as an antiviral agent.

Key Words: monocytes • retrovirus • gene regulation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Macrophages (M{phi}) are an important line of defense against viruses, acting as professional APC and activating the immune responses against the virus and/or the virus-infected cells [1 ]. Furthermore, M{phi} produce soluble factors, such as IFNs, to induce viral resistance in surrounding tissues [1 , 2 ]. Paradoxically, M{phi} also represent a target, reservoir, and vehicle for a number of persistent retroviral infections [3 4 5 ], thereby exerting a dual role in the pathogenesis of retroviral diseases. The nature of the host-virus relationship depends on the biology of the virus and the activation stage of M{phi} [1 , 3 4 5 ], which modify their phenotype as a result of activation [6 ], a process that is the resultant of the response to multiple signals including those derived from the immune system such as IFN-{gamma} [7 , 8 ], viral or bacterial products such as dsRNA or endotoxins [7 8 9 10 ], metabolites such as the tryptophan catabolite, picolinic acid (PA) [7 , 11 ], and the tissue environment such as changes in oxygen pressure (O2) tension [7 , 12 ].

M{phi} markedly accumulate at pathological sites, where they localize preferentially in hypoxic areas [13 ]. Hypoxia [partial O2 (pO2)<20 mmHg] is a common denominator of many pathological processes, including solid tumors, microbial infections, ischemic wounds, arthritic joints, atherosclerotic plaques, and inflammatory diseases (for a review, see ref. [14 ]), and experimental and clinical studies point toward its fundamental role in the pathogenesis of these diseases [14 15 16 ]. Hypoxia is an important regulator of gene expression, and transcription represents the primary mechanism by which mammalian cells respond to a decrease in O2 tension. The hypoxia-inducible factor-1 (HIF-1), a heterodimeric basic helix–loop–helix nuclear transcription factor composed by HIF-1ß, identical to the product of the aryl hydrocarbon receptor nuclear translocator gene, and by HIF-1{alpha}, -2{alpha}, or -3{alpha}, the oxygen-sensitive subunits, has been identified as the key mediator of hypoxia-induced gene transcription [14 , 17 , 18 ], although HIF-independent pathways were also described [12 , 19 ]. In well-oxygenated cells, HIF-{alpha} subunits are targeted for ubiquitination and rapid proteosomal degradation by the von Hippel Lindau tumor suppressor protein, whereas under hypoxia, they are stabilized post-translationally, translocate to the nucleus, where they heterodimerize with the HIF-1ß subunit, and transactivate the hypoxia responsive element (HRE) present in the promoter or enhancer elements of many O2-sensitive genes, ultimately activating their transcription (for a review, see refs. [14 , 17 ]). M{phi} respond to hypoxia with HIF-1{alpha} and HIF-2{alpha} up-regulation [18 , 20 , 21 ], and hypoxic conditions were shown to profoundly modulate M{phi} proinflammatory and immunoregulatory responses [12 , 20 21 22 23 24 25 26 ]. However, the effects of the hypoxic tissue microenvironment on retrovirus expression in M{phi} remain to be established.

Previous evidence suggested that transgene expression driven by the Moloney murine leukemia virus-long terminal repeats (MoMLV-LTR) can be modulated in murine M{phi} by stimulation with endotoxin, TNF-{alpha}, or TGF-ß [27 ]. However, it is not fully understood how MoMLV-LTR and expression of MoMLV promoter-driven genes are affected by the pathophysiology of the tissue. We were interested in studying the regulatory role of hypoxia on the activity of the MoMLV promoter. We have demonstrated previously a cross-talk between IFN-{gamma} and hypoxia in the regulation of M{phi} activation [20 , 23 24 25 ]. Here, we provide the first evidence that hypoxia can silence the expression of MoMLV-LTR-driven genes and retrovirus production in IFN-{gamma}-activated M{phi}. Furthermore, we demonstrate the existence of a selectivity in hypoxia inhibitory activity targeted to the MoMLV promoter and M{phi} lineage restricted, which can be mimicked by PA.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell lines
The RAW-pSun1 cell line was established from the murine M{phi} cell line RAW264.7, derived from a BALB/c mouse, by infection with the recombinant retrovirus pSun1 (Fig. 1 ), a replication-defective, MoMLV-based vector generated by inserting the enhanced green fluorescent protein (EGFP) full-length cDNA into the poli-linker of the pBabe plasmid backbone [28 ] under the control of the MoMLV-LTR, as described previously [29 ]. The EGFP reporter gene, which encodes a red-shifted variant of the Aequora Victoria GFP, optimized for brighter fluorescence and higher expression in mammalian cells [31 ], was excised from the pEGFP-N1 plasmid (Clontech, Palo Alto, CA). Briefly, RAW264.7 cells in the log phase of growth were incubated for 5 h with conditioned medium from the Phoenix packaging cell line, transiently transfected with the pSun1 vector and producing infectious, recombinant, retroviral particles in the absence of helper virus, according to a protocol detailed previously [29 ]. Stable transfectants were selected by addition of 10 µg/ml puromycin (ICN, Biomedicals, Inc., Costa Mesa, CA) to the culture medium and cloned by the limiting dilution technique obtaining several cellular clones stably and randomly integrating the pSun1 provirus in the genome and actively synthesizing the viral mRNA and expressing the EGFP transgene. One of such clones, hereafter referred to as RAW-pSun1, was used for subsequent experiments.


Figure 1
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  		Figure 1. Schematic representation of the pSun1, J2, and plasmid EGFP (pEGFP)-N1 vectors. pSun1 vector: U3/R/U5, Provirus LTR; S–D, inactivated splice donor; {psi}, extended packaging site; gag, short untranslated gag sequence for increased viral incapsidation efficiency; ATG–, mutation in the gag start codon; SV40p, internal SV40 early promoter for transcription of puromycin gene; puro, puromycin-resistance gene. J2 vector: {Delta}gag, deleted MoMLV gag sequence; v-raf, raf-mil hybrid oncogene; v-myc, complete v-myc oncogene; {Delta}pol, deleted MoMLV pol sequence; env, complete MoMLV env coding sequence. pEGFP-N1 vector: CMVp, human CMV immediate early promoter; SV40pA, SV40 early mRNA polyadenylation site; SV40 ori, SV40 origin of replication; Kan/Neo, kanamycin/neomycin-resistance gene; HSV-tkpA, polyadenylation signal from the HSV thymidine kinase gene. (Table) RAW-pSun1, WGL5-pSun1, and NIH-pSun1 cell lines were generated by infection with the replication-defective pSun1 vector. RAWinf31 and MIRO cells were established by infection with pSun1 vector and superinfection with MoMLV. ANA-1 cell line was established by infection with the J2 recombinant retrovirus and MoMLV. Mannose-binding lectin-2 (MBL-2) cells were derived from a MoMLV-induced T lymphoma [30 ]. RAW-pEGFP was generated by transfection with the pEGFP-N1 vector.

 
The RAWinf31 cell line, actively producing pSun1 and MoMLV retroviruses into the supernatant, was derived from RAW-pSun1 cells by cocultivation with the MoMLV producer SNU19 cell line to rescue the pSun provirus (Fig. 1) , as detailed in ref. [29 ].

WGL5-pSun1 cell line, expressing the replication-defective pSun1 virus, and MIRO M{phi}, actively producing pSun and MoMLV retroviruses (Fig. 1) , were generated from the well-differentiated murine M{phi} cell line WGL5, derived in our laboratory from a hystiosarcoma, spontaneously arisen in Sencar mice using the same procedure used to generate RAW-pSun1 and RAWinf31, respectively, as described [29 ].

The mouse M{phi} cell line ANA-1 (Fig. 1) was established by infecting fresh bone marrow-derived cells from C57BL/6 mice with the MoMLV-based J2 recombinant retrovirus carrying the v-raf/v-myc oncogenes [32 ] and shown to display the phenotypic and functional features and morphology of well-differentiated M{phi} [20 , 23 24 25 , 32 , 33 ].

RAW-pEGFP was obtained by transfection of RAW264.7 with pEGFP-N1 vector (Fig. 1) , using the electroporation technique (240 mV/975 µF) and selection of stable transfectants in medium containing G418 at 1 mg/ml (ICN Biomedicals, Inc.) as reported [25 ].

NIH-pSun1 cells were generated by infection of mouse NIH-3T3 fibroblasts (purchased from American Type Culture Collection, Manassas, VA) with the pSun1 retrovirus-containing, Phoenix-conditioned medium (Fig. 1) , as described above.

Drs. Antonio Rosato and Paola Zanovello (University of Padova, Italy) kindly provided the MoMLV-induced T lymphoma cell line, MBL-2, derived from a C57BL/6 mouse and producing MoMLV (Fig. 1) [30 ].

Culture conditions and reagents
M{phi} cell lines were cultured in DMEM (Euroclone, Celbio S.r.l., Milano, Italy) supplemented with 10% heat-inactivated FCS (Hyclone Laboratories, Celbio S.r.l.), 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (Celbio S.r.l.). NIH-3T3 fibroblasts were grown in DMEM supplemented with 10% calf serum (BioWhittaker, Walkersville, MD), 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. MBL-2 cell line was cultured in RPMI 1640 (Euroclone), supplemented as described for M{phi}. Cells were maintained at 37°C in a humidified incubator containing 20% O2, 5% CO2, and 75% N2. Hypoxic conditions (i.e., 1% O2) were achieved by incubating and handling the cells in an anaerobic work station incubator (BUG BOX, JOUAN, ALC International S.r.l., Cologno Monzese, Milano, Italy) flushed with a mixture of 94% N2, 5% CO2, and 1% O2 (which corresponds to a pO2 of ~7.6 mmHg) at a dynamic pressure of 35 pounds per square inch and a flow rate of 25 L/min at 37°C in a humidified atmosphere and sealed at a positive pressure to reduce atmospheric leaks. Culture medium was allowed to equilibrate for 3 h in a loosely capped flask in the hypoxic incubator before cell exposure to achieve a pO2 of 7.6 mmHg. A portable trace oxygen analyzer (Oxi 315i/set, WTW, Germany) was used to monitor O2 tension in culture medium. Recombinant mouse IFN-{gamma} (mIFN-{gamma}; sp. act. ≥107 IU/mg) was purchased from Gibco Life Technologies (Italia S.r.l., Milan, Italy). PA was purchased from Sigma-Aldrich S.r.l. (Milan, Italy). During the course of the experiments, several batches of PA were used, and all of them gave consistent and reproducible results. PA was dissolved in PBS, and the pH was adjusted to 7.4. The stock solution was then passed through a 0.2-µm filter, aliquoted, and stored at –20°C. Actinomycin D (ActD; Calbiochem-Novabiochem, VWR, Milano, Italy) was dissolved in ethanol at 1 mg/ml and used at a final concentration of 5 µg/ml or 1 µg/ml. The endotoxin content, as determined by a chromogenic Limulus amebocyte lysate test (QCL-1000, BioWhittaker), was below the detection limit of 6 pg/ml in all the reagents used.

Northern blot analysis
Cell lines were plated at 1 x 106 cells/ml in 10 or 15 cm Costar plates (Costar Corning, Celbio S.r.l.), and total cellular RNA was purified using the Tryzol Reagent (Gibco Life Technologies), according to the manufacturer’s instructions, and examined by Northern blot as described [23 , 25 ]. Briefly, 20 µg RNA was electrophoresed and transferred onto Nytran membranes (Schleicher and Schuell, Keene, NH). Filters were hybridized with [32P]deoxy-cytidine 5'-triphosphate-labeled probes (Amersham Corp., Arlington Heights, IL) and autoradiographed. The cDNA probes used for virus mRNA detection were EGFP cDNA from pEGFP-N1 for pSun1 [29 ] and env and v-myc cDNAs (Oncor, Gaithersburg, MD) for MoMLV and J2 virus, respectively [32 , 34 ]. The probes used for the detection of the inducible NO synthase (iNOS), 2'-5'oligoadenylate synthase (2'-5'OAse), and MCP-1 were described [23 , 25 ]. When needed, densitometric analysis of the autoradiographs was performed using the VersaDoc image analyzer from Bio-Rad Laboratories (Hercules, CA), and quantitative assessment of the band intensities was carried out. For each sample, expression values were normalized to the corresponding level of 28S rRNA.

Determination of pSun-1 retrovirus production
RAWinf31 (5x105) was plated in 100-mm dishes and treated with the indicated stimuli. Twenty-four hours later, pSun-1 virus-containing supernatants were harvested, centrifuged at 500 g for 10 min in a Beckman Coulter (Palo Alto, CA) GS-6R centrifuge to remove cellular debris, and then filtered through 0.45 µm filters (Millipore Corp., Bedford, MA). In some experiments, RAWinf31 conditioned medium was ultracentrifuged for 2 h at 25,000 g and 4°C in an OptimaTML-90K ultracentrifuge to remove IFN-{gamma}, and the pelleted, infectious, retroviral particles were resuspended in the original volume of fresh medium. Viral titers in the clarified and ultracentrifuged conditioned media were determined by infection of NIH-3T3 fibroblasts, according to standard protocols [35 ]. Briefly, 3T3 cells were plated in six-well plates (Costar Corning) the day before infection at a density (7.5x104 cells/well), which resulted in 40–60% confluence on the day of infection. The next day, the medium was replaced by 1 ml serial dilutions of the virus-containing supernatants in the presence of 7 µg/ml polybrene (Sigma-Aldrich S.r.l.) to promote virus binding to the cell surface, as detailed previously [29 ], and plates were incubated for 5 h at 37°C. Medium was then replaced with fresh medium to remove residual retroviral particles, and 3T3 cells were cultured for an additional 36 h. The cell number was then determined, the percentage of EGFP-transduced fibroblasts was assessed by flow cytometry as described below, and green CFU/ml (GFU/ml) were calculated as follows: Titer = (FxCinf/V)xD, where F is the percentage of EGFP+ cells; Cinf is the total number of target cells at the time of infection, V is the viral volume applied, and D is the virus dilution factor. All experiments were performed three times, and one representative set of results is shown.

Detection of EGFP expression
EGFP expression was analyzed by flow cytometry (FACS) and direct fluorescence microscopy. For FACS analysis, RAWinf31 and pSun-1-infected NIH-3T3 were detached from the plates, washed twice in PBS (BioWhittaker), fixed with freshly prepared 4% paraformaldehyde in PBS at 4°C for 30 min, and washed again with PBS. Single-cell suspensions were then analyzed with a FACScan (Becton Dickinson, San Jose, CA) using excitation at 588 nm and fluorescence detection at 530 ± 30 nm. For direct fluorescence microscopy, NIH-3T3 were cultured directly onto glass microscope slides, and the slide preparations were mounted in PBS/glycerol 50% and examined for EGFP fluorescence under a Zeiss Axiovert 135 microscope (Zeiss, Jena, Germany) equipped with a 450- to 490-nm long-pass excitation filter, 510 nm dichroic reflector, and 520- to 750-nm long-pass emission filter. Photomicrographs were taken with a Zeiss camera.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
To evaluate the effects of hypoxia on MoMLV-LTR activity in M{phi}, the RAW-pSun1 M{phi} cell line, expressing the replication-defective pSun-1 retrovirus (Fig. 1) , was cultured for 18 h under normoxic or hypoxic conditions in medium alone or supplemented with IFN-{gamma}, and pSun1 mRNA was evaluated by Northern blot using EGFP as a probe (Fig. 2a ). The high constitutive levels of pSun1 transcript, appearing as a 3.7-kb band of genomic RNA, were not affected by hypoxia but decreased to some extent upon M{phi} activation by IFN-{gamma}. A major inhibition of pSun1 mRNA expression was observed consistently when IFN-{gamma}-activated M{phi} were exposed to hypoxia (Fig. 2a) . Similar results were obtained with the RAWinf31 cell line (Fig. 2b) , which was derived from RAW-pSun1 cells by superinfection with the MoMLV (Fig. 1) . Hypoxia inhibitory effects could not be accounted for by a general downshift of mRNA expression occurring in activated M{phi}, as the expression of iNOS was highly induced by hypoxia in activated M{phi}, as previously reported [20 , 25 ], and the expression of 2'-5'OAse was augmented in IFN-{gamma}-activated M{phi} under normoxic and hypoxic conditions (Fig. 2a and 2b) . The reversibility of the inhibitory effects of hypoxia is demonstrated by experiments in which RAWinf31 M{phi} were treated with IFN-{gamma} under hypoxic conditions, washed, shifted to normoxia, and harvested at subsequent time-points. pSun-1 mRNA levels, which were inhibited strongly by hypoxia in activated M{phi}, were restored to control levels after 24 h of culture under normoxic conditions (Fig. 2c) .


Figure 2
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  		Figure 2. Inhibition of pSun-1 expression by hypoxia in RAW264.7-derived M{phi} cell lines. RAW-pSun1 (a) and RAWinf31 (b) cell lines were cultured under normoxic (Normo) or hypoxic (Hypo) conditions for 18 h in the presence or absence of 100 IU/ml mIFN-{gamma}, and total RNA was examined by Northern blot for pSun1 transcript using the EGFP cDNA as probe. Blots were hybridized sequentially with the 2'-5'OAse and iNOS cDNAs. Ethidium bromide (EB) staining of 28/18S rRNAs is shown as a control of RNA loading. (c) Total RNA was isolated from IFN-{gamma}-treated RAWinf31 exposed to hypoxia for 18 h followed by an additional 6 h and 24 h in the presence of normal oxygen levels (Reox) and then analyzed for pSun1 expression. EB staining of 28/18S rRNAs is shown as a control of RNA loading. Data shown are from one representative experiment of four performed.

 
The reproducibility of hypoxia inhibitory activity was confirmed in other M{phi} cell lines. Specifically, we used the WGL5-pSun1 cell line, which expresses the replication-defective pSun1 provirus, and MIRO cells, which actively produce pSun1 and MoMLV retrovirus (Fig. 1) . As shown in Figure 3 , results comparable with those observed with the RAW264.7-derived cell lines were obtained, and a major decrease of pSun1 transcript levels occurred in IFN-{gamma}-activated but not in resting M{phi} cultured under hypoxic conditions.


Figure 3
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  		Figure 3. Inhibition of pSun-1 expression by hypoxia in WGL5-derived M{phi} cell lines. WGL5-pSun1 (a) and MIRO (b) cell lines were cultured under normoxic or hypoxic conditions for 18 h in the presence or absence of 100 IU/ml mIFN-{gamma}, and total RNA was examined by Northern blot for pSun1 transcript using the EGFP cDNA as probe. EB staining of 28/18S rRNAs is shown as a control of RNA loading.

 
Kinetic experiments demonstrated that the down-regulation of pSun-1 mRNA in activated M{phi} occurred already after 3 h of exposure to hypoxia and reached maximal levels after 12 h (Fig. 4a ). Furthermore, dose response experiments (Fig. 4b) showed that as little as 1 U/ml IFN-{gamma}, which alone does not alter constitutive viral transcript levels, is sufficient to activate M{phi} to a hypoxia-sensitive stage, which results in the inhibition of viral mRNA by more than 70%.


Figure 4
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  		Figure 4. Time- and dose-dependent, inhibitory effects of hypoxia + IFN-{gamma} on pSun-1 mRNA expression. RAWinf31 cells were cultured for different time-points under normoxic or hypoxic conditions in the presence or absence of 100 IU/ml mIFN-{gamma} (a) or for 18 h in the presence or absence of increasing concentration of mIFN-{gamma} (b), and pSun-1 mRNA levels were then assessed by Northern blot. (a) Densitometric analysis of a representative kinetics experiment is shown. Data are expressed as the relative amounts of pSun-1 mRNA detectable at different times after addition of the indicated stimuli and following normalization to the respective amounts of EB-stained 28S rRNA. (b) A Northern blot of a representative dose response experiment is shown. EB staining of 28/18S rRNAs is used as a control of RNA loading.

 
The conclusions based on mRNA data were supported by the measurement of EGFP protein expression by cytofluorimetry (Fig. 5 ). RAWinf31 M{phi} were cultured for the indicated time-points under normoxia or hypoxia in the presence or absence of IFN-{gamma}, and EGFP was then assessed. Normoxic M{phi} expressed EGFP by more than 90% at all the time-points analyzed, and hypoxia affected EGFP positivity only slightly in resting M{phi}. Treatment with IFN-{gamma} under normoxia reduced the percentage of EGFP-expressing cells in a time-dependent manner, and 61% EGFP+ cells remained after 72 h of culture. A major decrease in the percentage of EGFP+ cells was observed when cells were treated with IFN-{gamma} under hypoxic conditions for 24 h (48% EGFP+ cells), and almost complete suppression occurred after 72 h of culture (19% EGFP+ cells). We conclude that hypoxia exerts a time-dependent, inhibitory effect on viral expression in activated M{phi}.


Figure 5
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  		Figure 5. Flow cytometric analysis of EGFP expression in RAWinf31 M{phi} after stimulation with hypoxia and/or IFN-{gamma}. RAWinf31 M{phi} were cultured for 24, 48, and 72 h under normoxic or hypoxic conditions with medium alone or supplemented with 100 IU/ml mIFN-{gamma}, and EGFP fluorescence was assessed by FACS. Results from one representative experiment out of three performed are shown. The histograms represent the fluorescent profile on a log scale of pSun-1-expressing cells. The percentage of EGFP+ cells is indicated.

 
RAWinf31 expresses not only the pSun1 but also the MoMLV retrovirus (Fig. 1) . To exclude that the observed results were a result of the idiosyncratic behavior of the pSun-1 construct, MoMLV mRNA expression in RAWinf31 was examined in parallel. Furthermore, we studied the regulation by hypoxia of J2, another MoMLV backbone-based virus in ANA-1 M{phi} (Fig. 1) . MoMLV and J2 viruses have the same promoter sequences (MoMLV-LTR) as pSun1 but differ for the genes expressed, gag/env/pol and v-myc/v-raf, respectively [32 ]. RAWinf31 and ANA-1 cell lines were cultured for 18 h under normoxic or hypoxic conditions in medium alone or supplemented with IFN-{gamma}, and MoMLV and J2 mRNAs were evaluated by Northern blot using the env (Fig. 6a ) and v-myc (Fig. 6b) cDNAs as probes. Consistent with the results described for pSun1, hypoxia decreased the levels of the MoMLV transcripts (one subgenomic RNA species of 1.7 kb, corresponding to the env mRNA, and the 8.6-kb, full-length genomic RNA coding for gag, pol, and env; Fig. 6a ) and the J2 mRNA (Fig. 6b) in IFN-{gamma}-activated but not in resting M{phi}. These data demonstrate that hypoxia inhibits the expression of different MoMLV-based retroviral constructs, suggesting that the activity of MoMLV-LTR is suppressed by hypoxia in activated M{phi}.


Figure 6
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  		Figure 6. Inhibitory activity of hypoxia + IFN-{gamma} on MoMLV and J2 mRNA expression in M{phi}. Total RNA was isolated from RAWinf 31 (a) and ANA-1 M{phi} (b) treated for 18 h in medium alone or supplemented with 100 IU/ml IFN-{gamma} under normoxia or hypoxia and examined for MoMLV (a) and J2 (b) transcripts using the env and v-myc probes, respectively. EB staining of 28/18S rRNAs was assessed as a control of RNA loading.

 
Parallel experiments were carried out with the MoMLV-producing T lymphoma cell line MBL-2 [30 ] and with NIH-pSun1 fibroblasts, which express the replication-defective pSun1 virus (Fig. 1) , to determine whether hypoxia inhibitory activity on MoMLV-driven genes was also exerted on cell types other than M{phi}. As shown in Figure 7 , no modulation of the constitutive transcript levels of MoMLV in MBL-2 (Fig. 7a) of pSun1 in NIH-pSun1 (Fig. 7b) cells was detected following 18 h culture in the presence of IFN-{gamma} and/or under hypoxic conditions, as determined using the env (a) and the EGFP (b) cDNAs as probes. In contrast, the endogenous expression of 2'-5'OAse (Fig. 7a) and MCP-1 (Fig. 7b) mRNA was augmented in IFN-{gamma}-stimulated MBL-2 and NIH-pSun1 cells, respectively, and hypoxia counteracted IFN-{gamma} effects in both cell types, demonstrating cell responsiveness to the stimuli used. Consistent and reproducible results were obtained in three independent experiments (data not shown). These data demonstrate that hypoxia is ineffective in suppressing MoMLV-LTR activity in resting or activated T and fibroblast cell lines, suggesting that hypoxia inhibitory action on MoMLV is selective for M{phi}.


Figure 7
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  		Figure 7. Regulation of MoMLV-driven gene expression in T cells and fibroblasts. MBL-2 (a) and NIH-pSun1 (b) cells were cultured for 18 h under normoxic or hypoxic conditions in the presence or absence of mIFN-{gamma} (100 IU/ml), and total RNA was examined by Northern blot for MoMLV (a) and pSun1 (b) transcripts by evaluating env and EGFP mRNA, respectively. Blots were sequentially hybridized with the 2'-5'OAse and MCP-1 cDNAs. EB staining of 28/18S rRNAs is shown as a control of RNA loading.

 
The implication of the results obtained with the M{phi} cell lines is that retrovirus replication should be inhibited in activated M{phi} exposed to hypoxia. To test this hypothesis, pSun-1 retrovirus production by RAWinf31 exposed to normoxia or hypoxia in the presence or absence of IFN-{gamma} was assessed by virus titration. To exclude the introduction of any artifactual results as a result of the presence of IFN-{gamma} in the conditioned medium, determination of retroviral titers was carried out in parallel after removal of IFN-{gamma} by ultracentrifugation. Comparison of retroviral infectious titers in clarified versus ultracentrifuged supernatants under the different treatment conditions is presented in Figure 8 . Comparable infectious units were detectable before and after ultracentrifugation, and 4.6 ± 0.47 x 106 and 3.7 ± 0.28 x 106 GFU/ml, respectively, were obtained on average in three different experiments under normoxic conditions. Treatment with IFN-{gamma} decreased virus production to some extent, resulting in a viral titer fivefold lower than that generated by untreated cells. Hypoxia was effective in reducing virus yield in activated M{phi}, causing a 50-fold decrease of viral infectious particles after 24 h incubation (Fig. 8) . A similar pattern of results was obtained using MIRO-derived conditioned medium (data not shown), providing the first evidence that hypoxia inhibits retrovirus production by IFN-{gamma}-activated M{phi}.


Figure 8
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  		Figure 8. Inhibition of pSun-1 retrovirus production by hypoxia + IFN-{gamma}. RAWinf31 cells were cultured under normoxia or hypoxia in medium alone or supplemented with mIFN-{gamma} (100 IU/ml). Supernatants were harvested after 24 h and subjected to a titration assay for quantitation of pSun-1 infectious units, as described in Materials and Methods. Viral titers, calculated by measuring the percentage of EGFP+ NIH-3T3 target cells following infection by serial dilutions of virus-containing supernatants before (solid bars) and after (open bars) ultracentrifugation, are expressed as GFU/ml on a log scale. Data shown are from one representative experiment of three performed, which gave comparable results. A photomicrograph of EGFP+ NIH-3T3 visualized by fluorescence microscopy is shown (20x, original magnification).

 
Experiments were then performed to determine whether reduced expression of MoMLV transcripts under hypoxia resulted from inhibition of transcription or from increased mRNA degradation. The effects of hypoxia on transcription were investigated by evaluating pSun1 mRNA expression in RAWinf31 cells treated for 12 h with medium alone or supplemented with IFN-{gamma} under normoxic or hypoxic conditions in the presence or absence of the transcription inhibitor ActD (1 µg/ml). As shown in Figure. 9a , in the absence of ActD (solid bars), hypoxia + IFN-{gamma} inhibited pSun1 mRNA expression. Addition of ActD (open bars) abrogated the observed, inhibitory effects, suggesting a transcriptional mechanism of regulation of MoMLV-driven genes by hypoxia + IFN-{gamma}. The effects of hypoxia on transcript stability were then assessed (Fig. 9b) . RAWinf31 cells were incubated for 18 h with medium alone or supplemented with IFN-{gamma} under normoxic or hypoxic conditions, and pSun1 mRNA decay was determined by evaluating the remaining amounts of pSun1 transcript at various time-points after addition of 5 µg/ml ActD. As depicted in Figure 9b , neither IFN-{gamma} alone nor IFN-{gamma} + hypoxia modified pSun1 transcript stability relative to medium-treated cells, confirming that the inhibitory effects on viral expression were transcriptional.


Figure 9
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  		Figure 9. Differential effects of hypoxia + IFN-{gamma} on pSun1 mRNA transcription and stability in RAWinf31 cells, which were treated with medium alone or containing mIFN-{gamma} (100 IU/ml) under normoxic or hypoxic conditions. ActD was added 30 min before treatment (a) or after 18 h of culture for 1, 2, 4, and 6 h (b). Total RNA was isolated and analyzed for pSun1 expression by Northern blot using EGFP cDNA as a probe. (a) Densitometric analysis of pSun-1 mRNA levels in control (Ctr) and ActD-treated M{phi}. Quantitation of pSun-1 mRNA levels in hypoxia (+)-, IFN-{gamma} (+)-, or Hypo + IFN-{gamma} (+)-treated samples relative to the corresponding normoxia-treated (–) samples was obtained by densitometric analysis of the band intensity following normalization to the respective amounts of EB-stained 28S rRNA. Data are presented as a bar graph and expressed as fold changes relative to normoxia-treated samples (arbitrarily defined as equal to 1). Data shown are the mean ± SD of the results obtained in three independent experiments, and SD was less than 10% of the mean. (b, upper panel) A representative Northern blot is shown. (Lower panel) Densitometric analysis of the blot. Data are presented as the relative amounts of pSun1 mRNA remaining after addition of ActD and following normalization of the bands to the respective amounts of EB-stained 28S rRNA. Consistent and reproducible results were obtained in three independent experiments.

 
We then examined the possibility that hypoxia was selective in targeting MoMLV-LTR for inhibition by testing a construct in which EGFP expression was driven by the CMV promoter in RAWpEGFP M{phi} (Fig. 1) . EGFP expression was up-regulated by hypoxia in resting and IFN-{gamma}-activated M{phi} (Fig. 10a ), demonstrating that CMV response to hypoxia is different from that of MoMLV-LTR. Furthermore, the expression of the puromycin and neomycin genes, which are part of the pSun1 and the pEGFP-N1 constructs, respectively, and are driven by the SV40 promoter (Fig. 1) , was constitutive and not modulated by hypoxia in resting and activated M{phi} (data not shown). We conclude that hypoxia exerts different regulatory effects on various viral promoters and that its inhibitory activity is targeted to a group of promoters of which MoMLV-LTR is the prototype.


Figure 10
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  		Figure 10. Differential modulation of LTR- and CMV-driven genes by hypoxia or PA in IFN-{gamma}-treated M{phi}. RAWpEGFP (a) and RAWinf31 (b) were cultured under hypoxic conditions or with 4 mM PA for 18 h in the presence or absence of 100 IU/ml IFN-{gamma}. Total RNA was isolated after 18 h and hybridized sequentially with EGFP and 2'-5'OAse cDNAs. EB staining of 28/18 S rRNAs was used as a control of RNA loading. Representative Northern blots are shown.

 
PA is a biologically active tryptophan metabolite with M{phi}-activating properties, which mimics hypoxia in inducing gene expression [20 , 36 ]. We studied whether PA modulated viral promoter-driven gene expression. The results demonstrate that PA exerted stimulatory effects on CMV-driven EGFP (Fig. 10a) , whereas it inhibited MoMLV-LTR-driven gene expression (Fig. 10b) in IFN-{gamma}-activated M{phi} without affecting the expression of 2'-5'OAse (Fig. 10b) , thereby mimicking the effects of hypoxia.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Hypoxia can modulate the expression of endogenous genes in M{phi} as well as M{phi} reactivity in a positive and negative manner [12 , 20 , 21 , 23 , 24 , 26 ]. In this study, we provide the first evidence that hypoxia can inhibit the expression of viral (env, v-myc) or reporter (EGFP) MoMLV-driven transgenes and decrease retrovirus replication in IFN-{gamma}-activated but not in resting M{phi} through the suppression of LTR activity.

Hypoxia inhibitory effects were exerted consistently in distinct M{phi} cell lines, established from different strains of mice by various immortalization procedures and independently infected with distinct MoMLV-based, replication-defective or competent retroviruses. Some variation in the levels of MoMLV-driven gene expression was detectable among the various cellular systems and proviruses used in response to IFN-{gamma} or hypoxia alone, which can be attributed to a different sensitivity of distinct cell lines to stimulatory agents. However, the demonstration that all the M{phi} cell lines tested were susceptible to the inhibitory effects of hypoxia + IFN-{gamma} supports the general validity of our observation. Furthermore, we demonstrated that hypoxia did not affect the expression of MoMLV-driven genes in resting or activated T and fibroblast cell lines, suggesting a M{phi} lineage-restricted action of hypoxia on MoMLV-LTR. These findings are consistent with previous reports by us and others showing a cell type-specific modulation of hypoxia-regulated genes [23 ].

The influence of the hypoxic microenvironment on virus expression has been reported previously in other cell systems. Oxygen deprivation was shown to negatively affect viral replication and gene expression [37 38 39 ] or stimulate virus production and spread in vitro and in vivo within hypoxic areas of tumors [40 41 42 43 ], depending on the virus analyzed. This study provides the first evidence that retroviruses are susceptible to inhibition by decreased O2 concentrations. It is interesting that we demonstrated that genes driven by other viral promoters, such as CMV-driven EGFP or SV40-driven puromycn or neomycin genes, were up-regulated or unmodified by hypoxia in resting or activated M{phi}, indicating the existence of a selectivity in hypoxia inhibitory activity targeted to the MoMLV promoter. Taken together, these observations suggest that different viruses have evolved specific strategies to deal with the hypoxic stress and support the conclusion that the hypoxic microenvironment can exert different and cell lineage-dependent, regulatory effects on distinct viral promoters.

Inhibition of MoMLV-driven gene expression levels was dependent, not only on the O2 concentration but also on the extent and duration of the hypoxic exposure. In fact, we observed a time-dependent reduction of pSun1 viral mRNA in activated M{phi}, which occurred already within 3 h of exposure to hypoxia, reached maximal levels after 12 h, and was paralleled by decreased protein expression, as determined by cytofluorimetric analysis of EGFP. The delayed and progressive disappearance of EGFP protein, as compared with the mRNA, could probably be accounted for by the persistence of some mRNA levels sufficient for protein synthesis combined with EGFP high stability and slow turnover rate [31 ] and with the nonsynchronous cell cycle distribution of the M{phi} population at the time of treatment. MoMLV-LTR inhibition was a transient event, which we showed, could be reversed by cell reoxygenation, restoring viral mRNA to levels comparable with those detected in normoxic cells. These results suggest that MoMLV-driven transgene expression in M{phi} may vary dynamically in vivo with the degree of local oxygenation, which is quite heterogeneous and rapidly fluctuating within inflammatory and tumor lesions [44 ].

Regulation of gene transcription is the primary mechanism by which mammalian cells respond to hypoxia [14 , 17 ]. Here, we present data showing that inhibition of transcription represents at least one level of control of pSun1 mRNA expression by hypoxia in IFN-{gamma}-treated M{phi}, thus raising the issue of the regulatory pathway(s) involved. Hypoxia transactivates the HRE to modulate gene transcription, and PA shares this ability with hypoxia [20 ]. However, HRE sequences are not present in the MoMLV promoter, and there is no evidence that HRE transactivation is involved in inhibition of gene expression. O2 signaling pathways involving a number of transcription factors and cis-acting regulatory elements other than HIF-1/HRE were described [19 ], and a few hypoxia-inducible transcription repressors have been identified recently [45 ]. The MoMLV-LTR contains various positive and negative regulatory elements, which bind nuclear factors of mammalian cells [27 ] and could be the target of hypoxia or PA. Inhibition of LTR activity may occur via suppression of transcriptional activators or induction/stabilization of transcriptional repressors, e.g., OTZ18 [46 ] or ZAP [47 ], which are known to affect infection and replication of different retroviruses from MoMLV to HIV, and studies are currently underway to identify the regulatory element(s) and transacting factor(s) implicated in hypoxia- or PA-dependent inhibition.

IFN-{gamma}-activated M{phi} and hypoxic environment are present concomitantly in pathological tissues [12 , 13 ]. The silencing of MoMLV-LTR-driven genes by O2 levels similar to those found in vivo caused a decrease of MoMLV virus production by infected M{phi}, suggesting that hypoxia may be biologically relevant in the local control of viral spread. This conclusion is supported by the observation that low concentrations of IFN-{gamma} are sufficient to activate M{phi} to a hypoxia-responsive stage, and such levels of IFN-{gamma} can be found in vivo in a variety of pathological situations and can be induced easily by a retroviral infection [2 , 48 ]. It is interesting that IFN-{gamma} activity against the vesicular stomatitis virus was reported to be increased under hypoxia in other cell types [49 ], suggesting that the local O2 availability may affect the sensitivity to IFN-{gamma} of different viruses. IFN-{gamma} is known to induce some degree of protection against viral infection [2 , 50 ] by triggering the JAK-STAT pathway, eventually leading to the induction of 2'-5'OAse/RNase L, dsRNA-dependent protein kinase R and Mx protein pathways [2 ]. We observed some inhibition of MoMLV production by IFN-{gamma} alone in M{phi} cell lines, which correlated with a major induction of 2'-5'OAse. Hypoxia or PA did not or only slightly modified the expression of 2'-5'OAse in IFN-{gamma}-activated M{phi}, suggesting that other mechanisms may be responsible for their inhibitory activity on retrovirus expression. NO is produced by activated M{phi} [11 ] and can contribute to the antiviral effect of IFN-{gamma} [2 , 50 ]. However, we observed a similar reduction of retroviral mRNA under hypoxic conditions, when NO cannot be produced [51 ], and in response to PA, when high levels of NO are produced [36 ], indicating that this mechanism cannot account for hypoxia and PA silencing effects.

PA is active on several pathways, and a role for iron chelation in the response to PA was documented [11 , 33 ]. PA has been detected in vivo in human milk, pancreatic juice, and serum [52 ], and in vitro studies suggest that PA regulates the inflammatory responses at multiple levels [11 , 20 , 33 ]. It may be possible that PA is a physiological mediator of antiviral resistance, which substitutes hypoxia in a normoxic environment. We have reported that PA can act synergistically with IFN-{gamma} in modulating the M{phi} phenotype [11 , 20 ] and in decreasing the production of retrovirus by activated M{phi} [34 ]. The results presented in this study support and extend these observations, indicating that PA antiviral activity is targeted to the MoMLV-LTR and suggest a potential, pharmacological use of this molecule as an antiviral agent.

In conclusion, we identified a novel silencing mechanism by which retrovirus-mediated gene expression in M{phi} may be attenuated in vivo without cell death, deletion of transgene, or methylation of retroviral sequences. The biological relevance of this pathway in M{phi} is evident and becomes effective when the cell migrates into pathological lesions, where it is exposed to activating cytokines reprogrammed by the hypoxic environment.


   ACKNOWLEDGEMENTS
 
This work was supported by grants from the Italian Association for Cancer Research (AIRC), San Paolo Company, Fondazione Italiana per la Lotta al Neuroblastoma, Associazione Italiana per la Glicogenosi, Italian Health Ministry, and Ministero Istruzione Universita’ e Ricerca (FIRB-MIUR). M. P. is a recipient of an Italian Neuroblastoma Foundation Fellowship (Progetto Carriera).


   FOOTNOTES
 
1 These authors contributed equally to this work. Back

2 Current address: Neuro-Oncology Branch, NCI-NIH, Bldg. 36, Rm. 3B05, 9000 Rockville Pike, Bethesda, MD 20892, USA. Back

Received May 30, 2006; revised September 28, 2006; accepted October 2, 2006.


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