In analogy to the Th1/Th2 functional subdivision of CD4+ T-helper lymphocytes, mononuclear phagocytes can also be transiently and reversibly polarized along with pro-inflammatory (M1) or alternatively activated, anti-inflammatory (M2) pathways [1–6]. M1 macrophages express high levels of pro-inflammatory cytokines, secrete reactive oxygen and nitrogen intermediates, and their presence has been associated to protection from viral infections and to some tumors [1–3]. In contrast, M2 polarization gears macrophages toward wound repair, tissue remodeling, and neoangiogenesis, and their presence has been correlated to tumor progression . In addition, M2 macrophages may contribute to the maintenance of a balanced microenvironment in anatomical sites under constant microbial assault such as the gut mucosa [6,8].
Both circulating monocytes [9,10] and, in particular, tissue macrophages , in addition to CD4+ T cells, are main targets of HIV-1 infection. Unlike T lymphocytes, macrophage infection by HIV-1 does not lead to their depletion, either in vivo or in vitro, rendering these cells a potential major viral reservoir even in the presence of effective combination antiretroviral therapy (cART) . Concerning the potential role of macrophage polarization in HIV-1 infection, in-vitro infected human primary monocyte-derived macrophages (MDM) polarized to either M1 or M2 showed a potent, though transient, inhibition of CCR5-dependent (R5) HIV-1 replication in comparison to unpolarized cells. This inhibitory effect was more potent in M1-MDM than in M2-MDM, although it was rapidly lost when M1-MDM were infected 3 days after removal of the polarizing cytokines, that is, interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). The M1-dependent inhibition of virus replication was associated with a strong downregulation of CD4 from the cell surface, an increased secretion of CCR5-binding chemokines, and a significant decrease in viral DNA and protein synthesis . These findings indicated that M1 polarization likely imposes a major restriction to HIV-1 infection at the level of virus entry. In partial contrast, the M2a-dependent inhibition was longer-lasting than that of M1-MDM and did not involve decreased HIV DNA synthesis or viral protein expression, suggesting interference at later steps in the viral life cycle [8,12].
In order to investigate whether postentry restriction events were triggered by M1 polarization of MDM, we infect them with an integration-competent vesicular stomatitis virus (VSV)-G pseudotyped vector controlling enhanced green fluorescence protein (eGFP) expression establishing a single round of infection. Our results support the hypothesis that, in addition to impairing HIV-1 entry, M1 polarization of macrophages creates a hostile postentry environment for HIV-1 replication in these cells.
Materials and methods
The following reagents were purchased: human, endotoxin-free recombinant cytokines (R&D Systems, Minneapolis, Minnesota, USA), bovine serum albumin (Sigma-Aldrich, St Louis, Missouri, USA), Ficoll-Hypaque (Amersham Biosciences Europe, Milan, Italy), Percoll (GE Healthcare, Piscataway, New Jersey, USA), Dulbecco's Modified Eagle's Medium (DMEM), phosphate buffered saline, fetal bovine serum (FBS), normal human serum (NHS), penicillin, streptomycin, and glutamine (Lonza, Cologne, Germany).
Vesicular stomatitis virus-G enhanced green fluorescence protein pseudotyped HIV-1 (HIV-green fluorescence protein)
The HIV-1 vector and plasmid used in this study have been described previously . HIV-GFP virus (in which nef was replaced with the eGFP reporter gene) was produced by cotransfection with a ratio of 1 : 7 of pMD2.G together with pNL4–3_GFP_R-Env-. Both plasmids were obtained from the NIH AIDS Research and Reference Program, Division of AIDS, NIAID, NIH, Bethesda, Maryland, USA. Vector containing supernatants were harvested 48 h after cell transfection, cleared by centrifugation, filtered by a 0.45 μm filter (MILLEX-HV PVDF; Millipore, Carrigtwohill, County Cork, Ireland), and stored at −80°C.
Isolation of human monocytes, differentiation into monocyte-derived macrophages, and infection with replication-competent HIV-1
Monocyte-enriched peripheral blood mononuclear cells were isolated from the buffy coats of healthy HIV-1-seronegative blood donors by Ficoll-Hypaque and then by Percoll density gradient centrifugation [8,15]. The cells were then washed, resuspended in DMEM with heat-inactivated FBS (10%) and 5% heat-inactivated NHS (complete medium), and seeded at 2.5 × 105 cells/well into 48-well plastic plates (Falcon; Becton Dickinson Labware, Lincoln Park, New Jersey, USA). Monocytes were cultivated for 7–8 additional days at 37°C in 5% CO2 to promote their full differentiation into MDM . These cells (>95% CD14+) were then stimulated or not (Control) for 18 h with either TNF-α and IFN-γ or with IL-4 to induce either M1 or M2a polarization [3,8,12]. At the end of the 18-h period, cells were washed, resuspended in complete medium, and infected with the laboratory-adapted CCR5-dependent (R5) strain HIV-1BaL (multiplicity of infection, MOI, of 0.1). Aliquots of culture supernatants were collected over a 5-week period and stored at −20°C to measure the levels of virion-associated reverse transcriptase activity .
HIV-green fluorescence protein infection of monocyte-derived macrophages and green fluorescence protein detection by cytofluorometry
Both control and M1-MDM were infected with serial dilutions of HIV-GFP viral stock. After 6 days of culture, the MDM (7.5 × 105 cells/condition) were scraped, spun, and their pellet was resuspended in a fixing solution containing 2% paraformaldehyde (PFA). Flow cytometry for GFP expression was performed using a Gallios instrument (Beckman Coulter, Jersey City, New Jersey, USA), and the results were analyzed with the FlowJo software version 8.4.3 (Tree Star, Ashland, Oregon, USA).
PCR-based quantification of HIV-1 gag DNA, 2-long terminal repeat circles, and integrated HIV-1 DNA
Both control and M1-MDM were infected with DNAse-treated HIV-GFP (stock dilution 1 : 10) and were then cultivated at 106 cells/ml of complete medium for 2 days. Cells were then washed, lysed, and treated with proteinase K (Sigma-Aldrich) at 65°C for 2 h and then at 95°C for 15 min . An amount of lysate corresponding to 2.5 × 104 cells was used in the PCR reactions together with a HIV-1 gag gene primer pair and probe . The number of HIV-1 DNA copies was determined by interpolating a reference standard curve (showing a linear distribution, r = 0.99, between 101 and 107 copies) after normalization for human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) DNA copy number by an external standard curve, as published .
For quantitation of 2-long terminal repeat (2-LTR) DNA circles, genomic DNA was isolated from cells according to the published procedure ; the MH535 and MH536 primers and the MH603 probe were used to identify 2-LTR circles formation . All reactions were performed in a volume of 25 μl containing 1 × TaqMan Universal Master Mix (Applied Biosystems, Foster City, California, USA), and 300 nmol/l of forward primer, reverse primer, and 100 nmol/l of molecular probe. The reaction was run by the Applied Biosystems 7500 Fast Real-time PCR system and 7500 Fast System Software. The thermal program started with 2 min at 50°C, followed by a 15-min hot start at 95°C. This was followed by 40 cycles of 95°C for 15 s and 60°C for 60 s; GAPDH was used as normalization control.
The levels of integrated proviral DNA were estimated adopting the published Alu-PCR protocol, involving a nested PCR-based assay with two sets of Alu-LTR primers and a probe, with minor modifications . Genomic DNA (100 ng) was first amplified with AccuPrime Taq DNA Polymerase High Fidelity (Life Technologies Italia, Monza, Italy) in a DNA thermal cycler (Perkin Elmer, Waltham, Massachusetts, USA). Then, a real-time PCR was performed with an aliquot equivalent to 1/10th of the 25th cycle PCR product with the second-round LTR primers and TaqMan probe. For control, all samples were also amplified with a primer pair and probe targeting mitochondrial DNA. Standard curves for both target and normalizer were obtained by nested PCR, as described above, using serially diluted genomic DNA (from 500 to 0.4 ng). The levels of proviral DNA, calculated from the standard curves, were expressed as ng of integrated provirus normalized to those of a mitochondrial DNA standard curve.
Quantification of HIV-1 RNA transcripts
Total cellular RNA was obtained with the TRIzol reagent (Life Technologies) and the PureLink Micro-to-Midi Total RNA Purification System (Ambion Life science, Austin, Texas, USA). The RNA (1 μg) was incubated with DNase I (Roche Diagnostics Co.) and the cDNA synthesis was carried out using SuperScriptII and random hexamers (Life Technologies) . As negative control, the same samples were processed in the same conditions in the absence of SuperScript II.
Both unspliced (us) and multiply spliced (ms) Tat/Rev HIV-1 RNAs were quantified using 50 ng of cDNA by TaqMan assay in an ABI 7700 Prism instruments (Applied Biosystems), as published . To correct for inter-sample variations in real-time PCR efficiency and errors in sample quantification, a set of 18S PCR reactions (18S primer pair and probe from Applied Biosystems) was performed as invariant endogenous control in the assay. The usHIV-1 RNA was quantified from the cDNA with the same primer pair and probe used for the quantification of the HIV-1 DNA described above.
Western blot analysis of APOBEC3G (A3G) and A3A expression
MDM were scraped from the plastic surface with a rubber policeman and lysed in buffer C (106 cells/100 μl). Cellular proteins were denatured, separated on a 10% SDS-PAGE gel, and transferred to a nitrocellulose membrane (Hybond ECL; Amersham Biosciences) by electroblotting. For A3G detection, blotting membranes were incubated with an anti-A3G rabbit polyclonal antibody, kindly provided by Dr Klaus Strebel, LMM, NIAID, NIH, at 1 : 500 dilution in 20 mmol/l TrisHCl, pH 7.6, 137 mmol/l NaCl, and 0.2% Tween 20 (tBST) buffer for 30 min at room temperature. For A3A, the membranes were directly incubated with a rabbit polyclonal antibody (1 : 500 dilution) against A3A/B (cat n. sc-86289; Santa Cruz Biotechnology Inc., Santa Cruz, California, USA) in tBST buffer for 30 min at room temperature. Antibody binding was visualized by using horseradish peroxydase (HRP)-conjugated antirabbit antibodies and electro-chemical luminescence (ECL; Amersham Biosciences).
Flow cytometric analysis of intracellular A3A expression
MDM were washed and detached from the plastic plates by scraping with a rubber policeman. The cells (1.5 × 106 per condition) were centrifuged (1500 rpm for 5 min at 4°C) and then fixed and permeabilized using a Fix&Perm kit (Life Technology); incubation with cold methanol allowed the permeabilization of both plasma and nuclear membranes. A3A expression was evaluated with the same antibody used for western blotting and a secondary antirabbit FITC-conjugated antibody. Flow cytometry was performed using a LSRII instrument (Becton Dickinson, San Jose, California, USA) and the results were analyzed with the FlowJo 8.4.3 software (Tree Star).
All statistical analysis was performed using the Prism GraphPad software v. 5.0 (GraphPad Software, www.graphpad.com). Comparison between two groups was performed using paired or unpaired, two-tailed t-test, whereas one-way analysis of variance (ANOVA) was used to compare three or more groups.
M1 polarization of monocyte-derived macrophages inhibits R5 HIV-1BaL replication and proviral transcription
We have previously reported that M1 polarization of MDM resulted in a profound inhibition of R5 HIV-1 replication, as shown in Fig. 1a. We here demonstrate that infection of M1-MDM with a replication-competent R5 HIV-1BaL strain is also characterized by an impaired proviral transcription of usRNA (full length) RNA (Fig. 1b), as measured 5 days postinfection, whereas the levels of msRNA were undetectable even by real-time PCR (data not shown).
Inhibition of HIV-green fluorescence protein expression in M1-monocyte-derived macrophages
In order to investigate whether the M1 restriction could be exclusively accounted for by a reduced entry of HIV-1 into MDM , we infected either M1-MDM or control MDM with HIV-GFP, a vector in which the VSV-G envelope protein allows fusion of the virion and cell membranes bypassing the requirement of engaging CD4 and CCR5 .
MDM cultures were established from 19 independent donors and were then either left unpolarized or were polarized to become M1 cells before infection with HIV-GFP (0.1 log10 dilution); eGFP expression was assessed by cytofluorimetric analysis 6 days postinfection. The percentage of eGFP+ positive cells of one infection representative of 19 independently performed is shown in Fig. 2a. Indeed, HIV-GFP expression was inhibited in M1-MDM in comparison to control, unpolarized cells. Similar levels of inhibition were observed when cells were infected with either 1 or 0.1 log10 dilutions of the viral stock, whereas an additional 10-fold dilution did not result in significant levels of eGFP expression in either polarized or control cells (Fig. 2b).
Inhibition of HIV-green fluorescence protein DNA synthesis, delayed proviral integration, and impaired proviral transcription in M1-monocyte-derived macrophages
We next investigated whether the inhibition of HIV-GFP single-round infection was correlated with decreased levels of HIV DNA synthesis. Indeed, 2 days after infection, M1 polarization consistently led to a marked decrease in the accumulation of HIV-1 DNA copies vs. control cells (Fig. 3a; n = 3; P = 0.055) and this effect was partially observed 7 days after infection when the levels of total DNA decreased significantly likely as a consequence of DNA degradation.
M1 polarization led to a significant reduction of both episomal 2-LTR DNA circles and of integrated proviral DNA, as measured by Alu-PCR, in comparison to control cells 2 days postinfection (P = 0.027 and P = 0.039, respectively, n = 3; Fig. 3b and c). However, these differences were not observed when the levels of both DNA forms were quantified 7 days postinfection (Fig. 3b and c). Unlike what was observed with quantification of gag DNA, both 2-LTR and integrated proviral DNA did not decrease in control MDM; however, it should be underscored that these DNA forms represent only a fraction of the total HIV DNA (Fig. 3a). Thus, M1 polarization of MDM seems to result in a transient inhibition of HIV-1 DNA synthesis and delayed kinetics of proviral integration, but not in an absolute impairment of retroviral integration. This interpretation was supported by the observation that R5 HIV-1 infection of M1-MDM was still inhibited when an HIV integrase inhibitor (Indole) was added to the culture 3 days after infection, when infection of control MDM was no longer inhibited by this agent (data not shown).
As for HIV-1 DNA synthesis, and in analogy to what was observed with replication-competent R5 HIV-1 (Fig. 1b), inhibition of HIV-GFP msRNA was significantly reduced 2 days postinfection (P = 0.033; Fig. 3d), although this inhibitory effect was greatly lost 7 days after infection.
Selective upregulation of A3A, but not of A3G, in M1-monocyte-derived macrophages
Searching for potential molecular determinants of the postentry restriction the HIV life cycle imposed by M1 polarization of MDM, we focused on the APOBEC family of restriction factors that encompasses A3G, a very potent inhibitor of HIV-1 replication in different cell types, including macrophages .
Therefore, we performed western blot analysis for A3G expression in both control and M1-MDM lysates prepared at the time of HIV-1 infection (i.e. 18 h postpolarization). However, M1-MDM expressed levels of A3G comparable to those of both control and M2a-MDM in all the five donors tested (Fig. 4a). In contrast, whereas both control and M2a-MDM showed barely detectable levels of A3A, M1-MDM expressed significant levels of this protein that were comparable to those of freshly isolated monocytes (Fig. 4a and b). We further confirmed this observation by an intracellular staining and fluorescence activated cell sorter (FACS) analysis of control, M1-MDM, and M2a-MDM for A3A expression. Approximately, 50% of M1-MDM vs. 10% of control and M2a-MDM were positive for A3A expression (Fig. 4c). However, as western blotting did not reveal detectable levels of A3A expression in either control or M2a-MDM (Fig. 4a and b), it is likely that the weak positivity shown by control and M2a-MDM was due to the described cross-reactivity of the antibody for A3B (data not shown).
A3A expression correlates with HIV-1 restriction in M1-monocyte-derived macrophages
In order to substantiate the potential role of A3A in M1-induced restriction of virus replication, western blotting was performed with lysates of both control and M1-MDM prepared 18, 72, and 168 h postpolarization. M1-MDM, but not control, MDM expressed detectable levels of A3A 18 and 72 h after polarization, whereas its expression was almost undetectable in either cell type 168 h postpolarization (Fig. 5a). No differences in the expression of A3G between control and M1-MDM were observed at all time points (data not shown).
The same MDM were also infected with HIV-GFP at the same time points after polarization (i.e., 18, 72, and 168 h) and the percentage of eGFP+ cells was determined by cytofluorimetric analysis 6 days after the infection. The percentage of eGFP+ M1-MDM was significantly lower than that of control MDM when the infection was performed both 18 and 72 h after polarization (Fig. 5b). In contrast and in concomitance with the disappearance of A3A expression, substantially higher levels of eGFP, approaching control levels, were expressed by M1-MDM infected 168 h postpolarization (Fig. 5b).
In the present study, we have demonstrated that HIV-1 replication was significantly impaired in M1-MDM, even when the infection occurred in a CD4/CCR5-independent manner by an HIV-GFP vector leading to a single-round replication, as revealed by eGFP expression. As observed with R5 HIV-1, the M1-dependent restriction of single-round infection was associated with a decreased synthesis of HIV DNA, both total gag and 2-LTR episomal circles, delayed proviral integration, and reduced transcription of msRNA after 2 days of infection. A clear-cut upregulation of A3A to levels observed in freshly isolated monocytes, but not of A3G, was observed in M1-MDM. Disappearance of A3A expression after 7 days of culture was paralleled by a greatly restored capacity of M1-MDM to sustain HIV-1 replication.
M1 polarized macrophages activate host defense programs against intracellular pathogens, including viruses, upregulate the expression of major histocompatibility complex (MHC) class I and class II antigens, and secrete complement factors that facilitate phagocytosis . Conversely, M2a-MDM are specialized in wound healing and tissue repair [6,7]. Unlike CD4+ T cells, M1/M2 macrophage polarization is reversible within a few days after cytokine stimulation, as we also demonstrated in the context of in-vitro HIV-1 infection . In particular, a more potent inhibition of R5 HIV-1 replication vs. M2-MDM was observed in M1-MDM. This observation was correlated to a profound downregulation of CD4 from the cell surface, increased secretion of CCR5-binding chemokines, and decreased synthesis of HIV-1 DNA [8,12]. This restriction profile strongly suggested that M1 polarization impedes R5 HIV-1 entry; however, our present study indicates that additional, postentry steps in the HIV-1 life cycle are also impaired upon M1 polarization of MDM. In fact, a reduced HIV-1 DNA synthesis was also observed with an HIV-GFP vector bypassing the requirement for CD4-CCR5-dependent entry. This effect was associated with a delayed integration of HIV-GFP proviral DNA that, however, reached levels comparable to those of control MDM 7 days after infection. These observations suggest that M1 polarization causes a decreased efficiency of the reverse transcription and proviral integration processes. In this regard, a potential role in M1 restriction could be played by Pin1, a TNF-inducible  prolyl-isomerase enzyme that catalyzes a conformational modification of integrase and was previously shown to regulate HIV-1 integration in activated T lymphocytes .
Regarding the potential role of the APOBEC cytidine deaminase family of intracellular proteins, both IFN-α and IFN-γ are known to upregulate the expression of this protein in a variety of cells, including human MDM, resulting in an improved resistance to HIV-1 infection . In particular, A3G exerts a potent anti-HIV activity by different mechanisms [28–30], including the impairment of the reverse transcription step; as countermeasure, HIV-1 encodes the accessory protein Vif that targets A3G for proteasomal degradation [29,31]. In MDM, siRNA-mediated silencing of A3G resulted in significant blockade of IFN-induced anti-HIV activity . However, M1-polarized MDM demonstrated only a modest upregulation of A3G levels without any evident correlation with HIV-GFP kinetics of expression. In contrast, we observed a clear-cut association between the pattern of HIV-1 restriction in M1-MDM and the expression of A3A.
A3A shares sequence and functional homology with A3G and it is a potent inhibitor of adeno-associated virus and parvoviruses infections  as well as of retrotransposons . A potential role of cell-associated A3A has been hypothesized as responsible for the infrequent editing of HIV-1 reverse transcripts , although others concluded that this protein alone was not a restriction factor for HIV-1 . Unlike A3G, nascent virions did not package A3A due to lack of its interaction with Vif .
A3A was recently shown to be a myeloid-specific protein upregulated by HIV-1 infection of MDM and interfering with the early phases of HIV-1 infection . In this regard, both IFN or CpG DNA stimulation of macrophages upregulated A3A expression leading to cytidine to uridine deamination of foreign double-stranded DNA and DNA degradation [37,38]. A3A is constitutively expressed in monocytes, cells characterized by a significantly restricted HIV-1 replication, whereas its expression was lost during macrophage differentiation and it was rescued by IFN-α stimulation . Thus, A3A can be considered a potential anti-HIV restriction factor expressed by monocytes and M1-polarized MDM. Whether A3A and A3G (nonetheless expressed in both M1-MDM and control cells) could physically or functionally interact is currently unknown, although both A3A-Vpr and AG-3A chimeras inhibited efficiently HIV-1 replication [35,40].
In conclusion, M1 polarization of macrophages in vitro induces a transient state of relative resistance to HIV-1 infection, involving reduced entry and reverse transcription, delayed integration of proviral DNA, and transcriptional repression. These results highlight the plasticity of macrophages in response to environmental signals, such as cytokines, that, in the case of M1 polarization, may lead to multistep restriction of HIV-1 replication while enhancing their antigen-presenting capacity, thus potentiating the adaptive immune response to the infection. Our study supports the concept that investigating macrophage polarization in vivo may not only allow a better understanding of the immune response to HIV (and to other viral infections), but also contribute to the development of more effective strategies for preventing infections by means of vaccines and microbicides.
L.C., E.S., G.D.C., and F.G. performed this study as partial fulfilment of their PhD in Cellular and Molecular Biology (L.C.) and in Molecular Medicine, Section of Basic and Applied Immunology, respectively, of the Università Vita-Salute San Raffaele, School of Medicine, Milan, Italy.
This study was supported by the CARIPLO grant 2008-2230 (to G.P.) and by the grants n. 40H76, 40H18 ,and 40H11 (to G.P., E.V., and M.A., respectively) of the Program of AIDS research 2009–2010 of the Ministry of Health, Italy.
Conflicts of interest
The authors declare that there are no conflicts of interest.
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