Published online before print August 21, 2003
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* Institute of Biochemistry G. Fornaini, University of Urbino, Via Saffi 2, 61029, Urbino, Italy;
Dept. Experimental Medicine and Biochemical Sciences, University of Rome Tor Vergata, Via Montpellier 1, 00133, Rome, Italy and
INMI L. Spallanzani, Via Portuense 292, 00149 Rome, Italy
1 Correspondence: Institute of Biological Chemistry "G. Fornaini", University of Urbino, Via Saffi 2, 61029, Urbino (PU) ITALY Email: magnani{at}uniurb.it
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Key Words: macrophages (M/M) • Fludarabine • erythrocytes (RBC)
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Persistence of residual viral replication in M/M despite prolonged treatment with HAART has been clearly demonstrated, and represents a major obstacle to HIV-1 eradication [9 10 11 12 ]. Therefore, a rational therapeutic approach directed to the selective elimination may be relevant in the therapy of HIV infection. The achievement of this option may take advantage from a deeper knowledge of the cellular mechanisms modulated by HIV infection in M/M, and of their role in the regulation of virus replication and cell survival. Among them, the alterations of cell signaling cascades, such as those involving Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathways, may be of particular relevance.
The JAK-STAT pathway is a very rapid membrane-to-nucleus signaling system that consists in a cascade of sequential tyrosine phosphorylations that activates cytoplasmic intermediates. Phosphorylated STAT proteins dimerize, translocate into the nucleus and activate cytokine-inducibile transcription of genes that control cell growth, differentiation, and maintenance of cellular homeostasis [13 14 15 ]. Seven STAT genes, STAT1-4, 5A, 5B, and 6, have been identified to date.
The modulation of STAT protein phosphorylation is likely involved in diverse pathological conditions, including immunosuppression, cancer and infections by different viruses [16 17 18 ]. Some previous reports suggested a role of the STAT proteins in HIV infection, but very little is known on the state of activation of the JAK/STAT pathway after HIV infection either in vivo or in vitro. The results are often controversial depending upon the experimental conditions and systems employed, such as cell types and viral strains [18 19 20 ]. These findings provide a strong rationale for the assessment of the expression of STAT1 in M/M experimentally infected by HIV-1. In particular, we turned our attention to M/M chronically infected by HIV (i.e., carrying HIV provirus in their genome) since their persistent viral production and long-term survival well represent the situation of tissue M/M in the body of the infected patients.
The finding that STAT1 is increased and activated in chronically infected M/M, suggests that STAT1 could be considered as a new molecular target for drug development. The drug investigated was Fludarabine (2-Fluoro-ara-AMP), a new nucleoside analog with documented activity in lymphoid malignancies. Following its entry into the cell, Fludarabine is converted to the corresponding triphosphate derivative, which specifically interferes with DNA replication and repair [21 , 22 ] through misincorporation into DNA and RNA. Other mechanisms of action, such as induction of apoptosis, and the ability to specifically inhibit STAT1 expression, have also been clearly demonstrated [23 24 25 26 ].
To avoid toxic effects exerted by Fludarabine, and to selectively deliver the drug to macrophages, we used erythrocytes (RBC); in fact, our previous reports have widely demonstrated that RBC can be successfully used for drug targeting to reticuloendothelial system (RES) [27 28 29 ]. Fludarabine encapsulated into RBC is converted to the corresponding triphosphate derivative, (its active form) [30 ], suggesting that RBC can be considered suitable carriers to deliver this drug to HIV-1-infected macrophages. Here, we report that a single 18 h treatment of HIV-1 infected macrophages with Fludarabine encapsulated into RBC selectively kills infected macrophages without affecting uninfected macrophages or inducing toxic effects on other cell types.
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Cells
Human peripheral blood mononuclear cells (PBMCs) were obtained from blood of healthy seronegative donors by Ficoll-Hypaque density gradient centrifugation.
The PBMCs were resuspended in RPMI 1640 medium supplemented with 20% heat-inactivated (56°C, 30 min.) fetal calf serum, penicillin (100 U/ml), streptomycin (100 µg/ml), and l-glutamine (2 mM), and then seeded into 48-well plates (1.8x106/well). M/M were separated by adherence onto plastic. After 5 days non-adherent cells were carefully removed by repeated gentle washings with warm medium, and adherent (>95% pure) M/M were cultured for additional 3 days to mature and to form a monolayer.
Human peripheral blood lymphocytes (PBLs) were obtained from PBMCs and activated with 0.5 µg/ml phytoemagglutinin (PHA) for 2 days before treatment with drug.
An African green monkey fibroblastoid kidney cell line, Vero, was used for the comparative experiments of cytotoxicity. This cell line has no phagocyting capacity.
Cell number and cytotoxicity evaluation
Number of cells present in each well was assessed by counting nuclei extracted from M/M by lysing buffer in a cell counting chamber under a phase contrast microscope, according to a previously published procedure [31
].
About 105cells/well were present at the time of infection. Viability was assayed by trypan blue exclusion method. At established time points, cells were collected by gentle scraping (for adherent cells) or by removal of cells containing supernatants (for non-adherent cells). The percentage of viable cells in treated samples was calculated in reference to the average viability of the untreated control samples at the same time.
Virus and infection
A monocytotropic strain of HIV-1, named HIV-1BaL, was used in all experiments. The virus was expanded in M/M, collected, filtered and stored at -80°C before use. The virus titer was assessed on M/M, calculated according Reed and Muench method and expressed as tissue cultures infectious dose 50% per ml (TCID 50/ml).
Human macrophage cultures were infected for 2 h with HIV-1Ba-L (300 TCID50 [50% tissue culture infective dose]/mL), a virus dose causing a linear increase of viral production up to 10-14 days followed by stabilization of HIV replication up to 50-60 days [32
33
34
]. After incubation with the virus, M/M were extensively washed to remove any residual virus particle and cultured in complete medium. When the status of chronic infection was reached (
10-14 days after infection), M/M were ready for analysis and treatment.
Assessment of virus replication
At various time points after infection, depending by the experimental procedure, virus production was assessed in the supernatants from HIV-infected M/M with a commercially available enzyme-linked immunosorbent-assay (ELISA) able to detect HIV p24gag (Abbott Laboratories, Pomezia, Italy).
HIV proviral load quantification
Based on a previously optimized PCR method [35
], the detection and quantification of HIV-1 proviral DNA content was determined in uninfected M/M, infected/untreated M/M, and infected/treated M/M by a Real Time PCR based assay using the SYBR® Green I chemistry and an ABI PRISM® Sequence Detection System (Applied Biosystem Foster City, CA).
DNA extraction was performed as described in [36 ]. The detection limit of this assay is 1 copy/reaction, and the linear dynamic range is comprised between 104 and 100 copies /reaction. Moreover an endogenous reference gene was quantified in order to normalize the variation due to the difference in the macrophage count or DNA extraction. The detailed procedure of the developed HIV-1 Real Time PCR will be described elsewhere (manuscript in preparation).
Encapsulation of Fludarabine in human erythrocytes
Human RBC were loaded with Fludarabine by a procedure of hypotonic dialysis, isotonic resealing and reannealing as described previously [30
]. Fludarabine was encapsulated in erythrocytes at a final concentration of 5.2±1.2 µmoles/ml erythrocytes. Targeting of Fludarabine-loaded RBC to macrophages was achieved by inducing band 3 clustering as described [37
].
The final concentration of Fludarabine in M/M treated with RBC was estimated to be 10 to 20 µM.
Treatments with Fludarabine
Chronically infected M/M were treated 10-12 days after infection, with free Fludarabine or Fludarabine-loaded RBC.
RBC loaded with Fludarabine were added at a ratio of 500 RBC per macrophage. After 18 h of incubation with M/M, non-ingested RBC were removed by extensive washing with culture medium. As controls, macrophage cultures were treated with "unloaded" (UL) RBC, i.e., RBC submitted to the same procedure including transient lysis and subsequent modification to increase macrophage recognition, but without addition of Fludarabine.
The same treatments were performed on not-infected macrophage cultures.
At the end of treatment, cell viability and the level of STAT proteins were assessed. Supernatants were harvested at established time points to evaluate HIV production.
In all experiments, Fludarabine (encapsulated or free) was given to cells only for 18 h and never repeated.
Western blot assay
Macrophages were washed twice with PBS (pH 7.4) and lysed in 0.5 M Tris-HCl pH 6.8, 2% SDS, 20% glycerol. Whole cell lysates were centrifuged at 6000 g for 10 min at 4°C. The protein concentration of cell extracts was determined by the Lowry protein assay.
Aliquots of 30 µg cell extracts were resolved on 7% SDS-PAGE and transferred by electroblotting on Hybond-C Extra nitrocellulose membranes (Amersham Pharmacia Biotech, Italy) for 60 min at 100 V with a Bio-Rad transblot. For the immunoassay, membranes were blocked in 5% dry milk in TBS (150 mM NaCl, 50 mM Tris pH 7.5) (Blocking Solution) for 1 h at room temperature, then incubated for 1 h at room temperature with specific antibodies diluted in Blocking Solution. Antibodies used in the different immunoblottings were the following: polyclonal antiphosphotyrosine STAT1, polyclonal anti STAT1 (New England BioLabs Inc. UK), monoclonal anti-STAT5 (Santa Cruz Biotechnology Inc., Santa Cruz, CA) and polyclonal anti-actin (Sigma Chemical Inc., Italy). Immune complexes were detected with horseradish peroxidase-conjugated goat anti-rabbit or horseradish peroxidase-conjugated goat anti-mouse antiserum (Bio-Rad,) followed by enhanced chemiluminescence reaction (ECL; Amersham Pharmacia Botech, Italy). Quantitative Western Blot analysis was performed by a chemi Doc System and Quantity One Program System (Bio-Rad).
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Figure 1. Activation of STAT1 by HIV-1 in macrophages. A: Left; Western blot analysis of STAT1, Ph-Tyr-STAT1 and actin. Right; Quantitative analyses of STAT1, Ph-Tyr-STAT1 normalized to the amount of actin level and expressed as percentage respect to control M/M (black columns). M/M were prepared as described under "Materials and Methods" section. Western blot analysis and its quantification were performed as described under "Materials and Methods" section. All experiments were run in quadruplicate. Results are the mean ± S.D. of three different experiments. B: p24 production in supernatant of M/M infected with HIV-1. At the indicated times virus production was assessed as described under "Materials and Methods" section.
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As shown in the Fig. 1 (A)
, HIV infection induces an enhancement of STAT1, just detectable 1 day after the infection, quite substantial at day 10 (> twofold respect to control), and further increased (> threefold) at day 20 (end of the experiment). Similarly, levels of phosphorylated STAT1 in HIV-infected M/M are 
threefold greater than uninfected controls at day 6 after infection. Such difference increases over time, and reaches levels up to fivefold greater than controls at day 20 after infection (end of the experiment). Thus, HIV infection of M/M is strictly related to the enhanced expression and phosphorylation of STAT1 protein, that parallel the production of HIV-proteins, as confirmed by p24 production in the supernatants of infected cultures (Fig. 1 B) .
STAT5 (another protein of the same family) was found either increased [38
] or decreased [39
] in T-lymphocytes from HIV infected patients at different stages of disease. In our experimental model a slight increase (30%) was detected only at day 1 while values similar to not infected M/M were found at the following time points studied (not shown). This suggests that STA5 is marginally (if not) related to the development of a persistent status of HIV infection in M/M.
Inhibition of M/M viability by Fludarabine
The effect of Fludarabine given as a single dose for 18 h (and then removed) on cell viability was assessed in resting M/M and actively replicating primary lymphocytes. As shown in Fig. 2 (A)
, toxic doses 50% (TD50) of Fludarabine was 60 µM in uninfected M/M, while 1 µM Fludarabine was already sufficient to determine an 50% reduction of viability in M/M infected by HIV. Infection did not modulate the effect of Fludarabine in actively replicating lymphocytes; indeed, TD50 of Fludarabine was 1 µM both in infected and uninfected lymphocytes (data not shown). Therefore, Fludarabine induces a remarkable effect on viability of infected M/M at concentrations >50-fold lower than in non-infected M/M.
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Figure 2. Inhibition of M/M viability by Fludarabine. A; effect of Fludarabine on viability of M/M infected with HIV-1 (filled square) or not infected (open square). Viability was assayed by trypan blue exclusion method. The percentage of viable cells in Fludarabine-treated samples was calculated in reference to the viability of the respective untreated control samples (uninfected or infected M/M). B; above: Western blot analysis of STAT1 on total cell extracts of M/M uninfected or infected with HIV-1 untreated or treated with 10 µM Fludarabine; below: quantitative analysis of residual STAT1 levels in untreated M/M (black columns) and M/M treated for 18 h with 10 µM Fludarabine (white columns). STAT1 levels were normalized to the actin content and expressed as percentage respect to control untreated M/M. Results are the mean ± S.D. of three different experiments.
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Overall results strongly indicate that STAT1 is modulated during infection of M/M, and that the treatment with Fludarabine markedly decreases the viability of infected M/M, and consequently virus release from these cells.
Inhibition of M/M viability by Fludarabine-loaded RBC
Despite such results may have potential applications in practice, their suitability is limited by the in vivo toxicity of Fludarabine upon replicating cells (such as activated lymphocytes). To avoid this side phenomenon, we encapsulated Fludarabine onto RBC. As previously reported [30
], resident enzymes of human RBC are able to convert most of Fludarabine to the di- and triphosphate (active derivative). The membrane of drug-loaded RBC was then modified to induce band 3 clustering (band 3 is a transmembrane anion channel protein abundantly present on the RBC membrane). Once band 3 is in clusters, the RBC are opsonized by autologous IgG and complement up to C3b. The opsonized, drug-loaded RBC are then selectively recognized by the Fc and C3b receptors present on the M/M membrane and actively phagocytized. Thus, RBC loaded with Fludarabine and modified as above are able to deliver their drug content only to M/M.
After 48 h from a single 18 h treatment with Fludarabine-loaded RBC (L-RBC), the effect upon viability of uninfected M/M is negligible (not shown). In contrast, viability of HIV-infected M/M exposed to the same treatment is decreased up to 75% compared with M/M infected but not treated (Fig. 3A
). In parallel, a decrease of virus production from infected M/M was detected, with p24 levels 85% lower than those found in untreated M/M (Fig. 3 B)
. Consistent with previous observations [37
, 40
], a limited inhibition of HIV release was achieved by UL-RBC in the first days after RBC exposure, probably related to the production of superoxide anion induced by opsonized RBC or haemoglobin [40
, 41
]. By contrast, the inhibition of HIV replication by L-RBC is consistent, or even better, than the HIV inhibition obtained by free Fludarabine (Fig. 3 B)
.
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Figure 3. Inhibition of M/M viability by Fludarabine-loaded RBC. A; effect of Fludarabine-loaded RBC (L-RBC) on HIV-1 infected M/M viability evaluated 2 days after the end of treatment. M/M were infected and treated with Fludarabine free or encapsulated into RBC as described under "Materials and Methods" section. B; p24 production (evaluated 2 days after the end of treatment) in HIV-infected M/M treated with L-RBC, UL-RBC and 10 µM Fludarabine.
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30% in M/M treated with L-RBC compared with UL-RBC. The result was further substantiated by immunofluorescence analysis, showing, 48 h after treatment, a percentage of M/M expressing viral proteins of 10-12%, compared with 50% in infected, untreated M/M (data not shown). This further demonstrates that the drop of virus production is mediated by a decrease of the number of infected M/M more than by a generic inhibitory effect upon virus production. Again, STAT1 levels have been measured by quantitative Western Blot analysis on cell extracts obtained from uninfected M/M and HIV-infected M/M, either treated with L-RBC or UL-RBC. STAT1 is strongly reduced in infected M/M after treatment with Fludarabine encapsulated in RBC, while is not influenced in uninfected M/M (Fig. 4 ). Thus, Fludarabine encapsulated within RBC has a remarkable effect upon the expression of STAT1, while UL-RBC do not produce STAT1 variations in any of the experimental conditions we analyzed.
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Figure 4. Inhibition of STAT1 expression by Fludarabine-loaded RBC. Above: Western blot analysis of STAT1 on total cell extracts of M/M uninfected or infected with HIV-1 treated with L-RBC or UL-RBC. Below: Quantitative analyses of STAT1 levels in untreated M/M (gray columns) and in M/M after treatment with UL-RBC (black columns) or L-RBC (white columns). STAT1 levels were normalized to the amount of actin level and expressed as percentage respect to control untreated M/M Western blot analysis and its quantification were performed as described under "Materials and Methods" section. All experiments were run in quadruplicate. Results are the mean ± S.D. of three different experiments.
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To assess whether the effect of Fludarabine is reversible, virus production from chronically-infected M/M was evaluated also at later time points after Fludarabine removal. Figure 5 shows the production of p24 gag (% calculated with respect to infected but not treated M/M) up to 25 days after a single 18 h treatment with L-RBC. Again, UL-RBC caused a temporary lowering of p24 production in chronically HIV-infected M/M (not shown). The effect is transient, however, with a nadir of p24 production at day 1, followed by a rapid return to levels in the range of those of controls. By contrast, the inhibition of HIV p24 production induced by Fludarabine (either free or loaded onto RBC) was even increased over time, reaching >98% at day 5; this level remained stable up to day 25, despite the fact that the initial short-term treatment with L-RBC was not repeated (not shown). Thus, the effect of Fludarabine is not reversible in resting, chronically-infected M/M.
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Figure 5. Long-term inhibition of HIV-1 production by Fludarabine-loaded RBC and free Fludarabine. Mock treatment (dark gray columns), UL-RBC (light gray columns), L-RBC (white columns), and free Fludarabine at the concentration of 10 µM (black columns) were given to M/M 14 days after infection, and carefully removed 18 h afterwards. Cells were maintained in drug-free medium up to the end of the experiments (for a total of 25 days after treatment, that is 39 days after infection). Percentage of inhibition is expressed respect to HIV-1 infected and not treated M/M (dark gray columns). Virus production was assessed at the indicated times (after the end of treatments). Results are the mean ± S.D. of three different experiments.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We found that in vitro HIV-1 infection can induce a selective up-modulation of STAT1 in human primary M/M, that is reversed by treatment with Fludarabine, a selective inhibitor of STAT1 [23 , 24 ]. These results are similar to those showing that HIV-1 induces activation of STAT1 on human M/M in a single-cycle HIV infection using HIV-1 preparations pseudotyped with the membrane glycoprotein from VSV [19 ], but in contrast with recent results showing that HIV-1 can induce activation of multiple STATs (and not only of STAT1) [20 ].
The factors related to HIV infection of M/M that lead to the activation of JAK-STAT1 pathway are not completely known. In an attempt to clarify this point, we tested the production of IFN-gamma and IFN 
, two cytokines known to activate STAT proteins [42
43
44
] in supernatants of M/M infected with HIV. The quantitative "sandwich" high sensitivity immunoassay techniques (Amersham Pharmacia Biotech) used in our experiments did not demonstrate detectable levels of IFN or gamma (data not shown); therefore STAT activation does not seem to be correlated with the production of these cytokines in our experimental conditions. IFN-gamma belongs to a group of cytokines exerting pleiotropic effects on HIV-1 infection; exposure of infected M/M to IFN gamma leads to a reduction in viral replication while exposure of M/M to IFN gamma prior to infection results in subsequent enhancement of viral production. Therefore, its lacks of involvment upon the activation of STAT under condition of HIV infection is not fully surprising. So activation of STAT1 in our experimental model could occur through other cytokine signaling, or through cytokine-indipendent means.
Some reports have demonstrated that the HIV gp120 envelope protein alone is sufficient to induce STAT1 [20 , 45 ]. Therefore, the correlation between the gradual enhancement of STAT1 and of HIV replication up to day 20 of culture suggests that both phenomena are strictly associated, and that sufficient virus replication is required for the progressive activation of STAT. This is further supported by other studies showing that HIV-Nef, an important regulatory protein released upon HIV infection, is associated to STAT induction [46 ] and that infections by other viruses (also able to establish chronic infection, such as Hepatitis B virus) could generate an alterated pattern of STAT activation [16 , 47 , 48 ].
The role of the activation of STAT1 upon HIV infection is interesting, in view of the association between Fludarabine-induced STAT1 inhibition and the dramatic decrease of cell viability. This result postulates a role of STAT1 as survival factor of M/M promoting their role as persistent viral reservoirs. This turns to be an evident advantage for the virus, since survival of the infected cells (such as the case of M/M) allows a greater rate of virus replication than that allowed by cells undergoing a virus related cytopathic effect. Therefore, identification of survival factors can be crucial in designing treatment strategies to selectively eliminate persistently infected cells; in particular, STAT1 may be a useful target for Fludarabine, whose effect is mainly mediated by a specific loss of STAT1 in vivo as well as in vitro [23 ], although we cannot exclude a less specific effect of Fludarabine upon HIV infected M/M suffering substantial alterations of their homeostasis (that may in turn easily trigger a cascade of events leading to their death.).
Many reports indicate that macrophages infected in vitro with HIV endogenously produce macrophage-colony stimulating factor (M-CSF) with kinetics paralleling virus replication, which can lead to enhanced spreading of the infection. Similarly, the inhibition of HIV replication leads to a concomitant reduction of M-CSF [49 ]. Interestingly, M-CSF activates several members belonging to the STAT family [50 , 51 ] and in particular causes the activation of STAT1 in bone marrow macrophages [52 ]. Under these circumstances, we cannot exclude an interplay of STAT-1 and M-CSF in macrophages infected by HIV-1, and that this can be perturbed by Fludarabine. Future experiments can be devoted to assess this point.
A potential limitation of the therapeutic approach described in this paper is given by the aspecific toxic effect of Fludarabine upon actively replicating cells (such as lymphocytes), more sensitive than resting cells (such as M/M) to the DNA-polymerase inhibition induced by this drug. Under these conditions, a therapeutic approach foreseeing the use of Fludarabine could not be considered without the utilization of a system for a selective delivery of the drug to resting cells. By taking advantage of macrophage phagocyting capacity, we used RBC as carriers to selectively delivery Fludarabine to M/M. Application of RBC in drug delivery has been exploited extensively [27 28 29 ]. Also the results reported in this paper clearly support this approach, by showing an even increased efficacy of Fludarabine if loaded within RBC upon HIV-infected M/M, without any cytopathic effect upon non phagocyting, actively-replicating cells. Such effect is remarkable also with a single exposure to Fludarabine-loaded RBC, is sustained over time (no rebound of virus replication after Fludarabine removal), and specific (non phagocyting cells are not affected by such experimental approach).
In conclusion, these results show that the activation of JAK-STAT pathway is associated with survival of M/M to HIV infection, and that Fludarabine loaded within RBC heavily and selectively contributes to the induction of cell death. This opens the way to experimental therapeutic approaches aimed to selectively eliminate persistently-infected cells by using RBC as specific carriers. Preliminary results, conducted in small-scale clinical trials, demonstrate the safety and efficacy of this approach for diseases sustained by M/M dysfunctions [27 ]. Further studies are then warranted in animal models infected by retrovirus before considering such therapeutic strategy for HIV-infected patients.
Received April 16, 2003; revised July 16, 2003; accepted July 24, 2003.
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