Involvement of IL-6 in the anti-human immunodeficiency virus activity of IFN-{tau} in human macrophages

Christine Rogez-Kreuz1,2, Benjamin Manéglier1,3, Marc Martin1, Nathalie Dereuddre-Bosquet4, Jacques Martal2, Dominique Dormont1,* and Pascal Clayette4

1 Service de Neurovirologie, Commissariat à l'Energie Atomique, Université Paris-Sud, CRSSA, EPHE, IPSC, Fontenay-aux-Roses, France
2 Unité de Biologie du Développement et de la Reproduction, Département de Physiologie Animale, Institut National de la Recherche Agronomique, Jouy-en-Josas, France
3 Unité de Pharmacologie, Département de Biologie, Centre Hospitalier de Versailles, Faculté de Médecine Paris Ile de France Ouest, Le Chesnay, France
4 SPI-BIO, CEA, 18, route du Panorama, B.P.6, 92265 Fontenay-aux-Roses Cedex, France

Correspondence to: P. Clayette; E-mail: pascal.clayette{at}cea.fr


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
IFN-{tau} is a non-cytotoxic type I IFN responsible for maternal recognition of the foetus in ruminants. IFN-{tau} has been found to inhibit HIV replication more strongly than human IFN-{alpha}, particularly in human monocyte-derived macrophages, without associated toxicity. Ovine IFN-{tau} uses the same anti-viral cellular pathways as human IFN-{alpha} in human macrophages, principally inhibiting the early steps of the biological cycle of HIV, preventing the integration of HIV DNA into the host-cell genome. In this study, we investigated the immunomodulatory properties of IFN-{tau} in human macrophages. We found that IFN-{tau} increased the production of IL-10 and IL-6, but not of IL-1ß or tumour necrosis factor {alpha}, in unstimulated, LPS-stimulated and HIV-1/Ba-L-infected macrophages. We also found that treatment with IL-6 inhibited HIV replication. Moreover, the neutralization of IL-6 activity in the cell culture supernatants of IFN-{tau}-treated macrophages led to a decrease in the anti-retroviral effects of IFN-{tau}, suggesting that IL-6 was involved in the anti-viral activity induced by IFN-{tau}. By focusing on the very early steps of the biological cycle of HIV, we showed that IL-6 co-operated with IFN-{tau} to decrease intracellular HIV RNA levels 2 h after infection.

Keywords: anti-retroviral, HIV, IFN, IFN-{tau}, IL-6, immunomodulation, macrophages


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Resistance to viral infections depends on various effector mechanisms involving cytokines and chemokines, which mediate early defence in innate and adaptive immunity. The effects of cytokines are pleiotropic and are influenced by the concentrations, presence or absence of other cytokines. Once produced, these factors may act individually or together, directly or indirectly on infected cells, activating cellular constituents of the innate system and/or promoting specific T- and B-cell adaptive responses to mediate anti-microbial effects. Human type I IFNs, in addition to having powerful anti-viral activity, play a critical role in modulating immune responses to foreign and self-antigens. They are produced early in infection and their production precedes that of most of the other innate immune response cytokines. Thus, the timing of type I IFNs production during infection suggests that the primary involvement of these molecules in host defence is priming during the initial immune response to infection (13), leading to an increase in anti-viral activity. The IFN system is induced by viral infection in most types of cell [for review (4)], particularly monocytes and macrophages, which are among the main cell targets of HIV. Moreover, exogenously administered IFNs efficiently inhibit HIV replication. In contrast to currently available anti-HIV drugs, these compounds act on several steps of the HIV biological cycle, such as viral entry (5), pro-viral DNA synthesis (6) or protein synthesis, and the maturation and budding of viral particles (7, 8). They induce three rapid cellular anti-viral pathways: (i) the 2',5'-oligoadenylate synthetase (2',5'-OAS)/RNase L pathway, which inhibits HIV replication via mechanisms involving 2',5'-oligoadenylates (911); (ii) the RNA-dependent serine–threonine kinase R (PKR) pathway, which is known to impair HIV replication in chronically infected cells (12, 13) and (iii) induction of the production of MxA protein of the GTPase superfamily (14), which acts at various points in viral replication cycles (15, 16). The anti-viral mechanism of type I IFNs and the lack of cross-resistance with existing anti-retroviral drugs render these compounds potentially valuable for HIV therapy. Nevertheless, IFN toxicity has limited IFN-{alpha} treatment because the side effects may result in low levels of compliance and treatment failure.

The trophoblast IFN-{tau} is found in ruminant ungulate species and forms part of the hormonal environment required for embryonic development (17, 18). This anti-luteolytic IFN, structurally related to IFN-{alpha} and IFN-{omega}, is secreted in large quantities by extra-embryonic trophectoderm cells during the peri-implantation period (19, 20). Like other IFNs, IFN-{tau} also displays anti-viral and anti-proliferative properties (21, 22), but this molecule is less toxic in vitro and in vivo than IFN-{alpha} or IFN-ß (2325). It could therefore be considered as a possible alternative to IFN-{alpha}. Interestingly, IFN-{tau} inhibits HIV replication more strongly than IFN-{alpha}-2a in human macrophages, without cytotoxicity (23), and the cellular molecular anti-retroviral mechanism induced by IFN-{tau} appears to be identical to that induced by human IFNs (26).

In addition to having anti-viral activity in their own right, type I IFNs also encourage production of the cytokine environment necessary for the establishment of a prolonged anti-viral response. Various factors are known to be produced rapidly, within hours or days of viral infection, by cells of the innate immune system such as monocytes/macrophages. HIV-1 infection and replication are continuously regulated by a complex cytokine network and several of the cytokines involved in this network have been reported to modulate HIV-1 replication in cells of the macrophage lineage (27). The direct effects of cytokines on HIV-1 may be inhibitory, stimulatory or bifunctional, depending on the dose or time of administration. Interestingly, outside of its normal physiological context, IFN-{tau} stimulates the production of IL-10 in the murine model of multiple sclerosis, preventing the occurrence of the disease (28, 29). IFN-{tau} also counteracts increased foetal resorption in this model, by increasing placental IL-4 and IL-10 levels (30). In another experimental system, based on bovine antigen-specific CD4+ T-cell lines, IFN-{tau} enhances the IL-4 and IFN-{gamma} responses (31). As IFN-{tau} has efficient anti-viral activity against HIV-1 in human macrophages, we evaluated its effects on cytokine production by macrophages, and the effects of cytokine environment on the anti-viral mechanism involved. We focused here on pro- and anti-inflammatory cytokines known to interfere with HIV-1 replication: tumour necrosis factor (TNF)-{alpha} (32, 33), IL-1ß (3436), IL-6 (34, 37) and IL-10 (3841).


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Cells
Human PBMC were isolated from healthy seronegative donors by Ficoll-hypaque density-gradient centrifugation. Monocytes were separated from PBMC by countercurrent centrifugal elutriation (42). We checked that cell preparations consisted of ≥95% monocytes by immunolabelling for CD3, CD4, CD8, CD11b, CD14 and CD19 (BD Biosciences, Mountain View, CA, USA). Monocytes were transferred to a 75-cm2 plastic flask containing DMEM-glutamax medium (Invitrogen, Groningen, The Netherlands) supplemented with 10% heat-inactivated (+56°C for 45 min) FCS (Bio West, Nuaillé, France) and 1% antibiotic mixture [penicillin, streptomycin, neomycin (PSN)—Invitrogen]. Cells were maintained at +37°C in a humidified atmosphere containing 5% CO2. Monocytes began to adhere after 1 h of culture, and then spontaneously detached from the plastic after 48 h. They were then dispensed, in medium, into 48-well plates (3 x 105 cells per well). Monocyte-derived macrophages were obtained after 7 days in culture (43).

The pro-monocytic cell line U1, derived from cells surviving acute infection of the U937 cell line, contains two integrated HIV copies per cell (44). U1 cells were provided by the National Institutes of Health (NIH) and were cultured in RPMI 1640 (Invitrogen) supplemented with 10% heat-inactivated FCS and 1% PSN antibiotic mixture.

Molecules
Ovine recombinant IFN-{tau} was produced in yeast by Transgene S.A. (Strasbourg, France), by genetic engineering techniques (45), using IFN-{tau} cDNA (46). It was purified by ion-exchange HPLC. The IFN-{tau} batch and cell culture media were shown to be endotoxin free by the limulus amoebocyte lysate test (Chromogenix, Milan, Italy). IFN-{tau} had a specific anti-viral activity of 108 IU mg–1 against vesicular stomatitis virus in Madin-Darby bovine kidney cells (47). In some experiments, macrophages were stimulated with a sub-optimal dose of 10 ng ml–1 LPS (Escherichia coli serotype 0111: B4; Sigma, St Louis, MO, USA). The anti-viral activities of human recombinant IL-6 (R&D Systems, Abingdon, UK) and IL-10 (kindly provided by Schering-Plough Research Institute, Kenilworth, NJ, USA) were tested using doses of 40–1000 pg ml–1 and 50–5000 pg ml–1, respectively. In some experiments, the biological activity of IL-6 was neutralized by adding 100 ng ml–1 of mouse monoclonal anti-human IL-6 antibody (mAb, clone 6708, R&D Systems); an irrelevant mouse Ig of the same class (IgG1, Sigma) was used at the same dose as a control. Chronically HIV-1-infected U1 cells were stimulated by incubation with 10 µM phorbol myristate acetate (PMA, Sigma) for 3 days.

Virus
Differentiated macrophages were infected with the reference macrophage-tropic HIV-1/Ba-L strain (48). This virus was amplified using PHA-P-activated umbilical blood mononuclear cells. Cell-free supernatants were ultracentrifuged at 360 000 x g for 10 min to eliminate soluble factors such as cytokines. Viral stocks were titrated using PHA-P-activated PBMC and Kärber's formula (49). In some experiments, virus was inactivated by heating at +56°C for 1 h. HIV replication was assessed in cell culture supernatants by quantifying reverse transcriptase (RT) activity, using the RetroSys® kit (Innovagen, Lund, Sweden). RT activity was measured every 3 or 4 days and, in some experiments, a cumulative total for the 25 days of culture was calculated and results are expressed as a percentage of the cumulative RT activity of the positive control.

Cytokine production in cell culture supernatants
Cell culture supernatants were collected 8 h after LPS stimulation, HIV infection or IFN-{tau} treatment, to evaluate early cytokine production. To evaluate the production of IL-6 by macrophages throughout the 25 days of culture, cells were treated with 100 IU ml–1 IFN-{tau} before infection with HIV-1/Ba-L (m.o.i. 0.1), such that day 0 of infection corresponded to day 1 of treatment, and treatment was maintained throughout the culture. Cell culture supernatants were collected at day 0 and 1 and every 4–5 days after infection and cells were re-treated. We assessed the production of TNF-{alpha} and IL-6 in cell supernatants by means of Cayman Chemical ELISA kits (Cayman, Ann Arbor, MI, USA) and IL-1ß and IL-10 using Quantikine® immunoassay kits (R&D Systems).

Production of cytokine and cellular anti-viral factor mRNA
We assessed cytokine mRNA production by lysing cells in RNAble® (Eurobio, Les Ulis, France) to extract total RNA 8 h after LPS stimulation, HIV infection or IFN-{tau} treatment. For 2',5'-OAS mRNA levels, macrophages were treated for 24 h with 100 IU ml–1 IFN-{tau} before lysis in RNAble®. The total RNA preparation was treated with DNase, and then subjected to reverse transcription with oligo-dT primers (Eurobio). We amplified the cDNA generated from viral RNA by PCR, using specific primers for glyceraldehyde 3-phosphate dehydrogenase (GAPDH), IL-6, IL-1ß, TNF-{alpha}, IL-10 (50) and the 40-kDa (51) and 69-kDa (52) isoforms of 2',5'-OAS. Amplified cDNA signals were resolved by electrophoresis in a 1.5% agarose gel stained with ethidium bromide. The specific bands were visualized on a transilluminator (UVP Life Sciences, Cambridge, UK) and quantified by densitometry (NIH 1.2., W. Rasband, National Institutes of Health, Bethesda, MD, USA). The relative abundance of mRNA species was determined by means of a standard curve generated for each PCR amplification. Each amplification was carried out at least twice and data are expressed as a ratio of the signal obtained for each cytokine divided by the signal obtained for GAPDH from the same sample, to facilitate the comparison of RNA species between samples.

Intracellular HIV RNA quantification
Macrophages were treated with 100 IU ml–1 IFN-{tau} for 24 h and infected by incubation with the virus for 2 h. They were then washed in PBS and treated with trypsin for 2 min at +37°C to remove extracellular virus particles. Macrophages were lysed in RNAble® (Eurobio), and RNA was extracted and reverse transcription performed as described above for cytokine mRNA. We amplified the cDNA generated from viral RNA by PCR, using the U3/R primers, which were designed to amplify a sequence specific to HIV-1/Ba-L long terminal repeat (51). GAPDH cDNA was amplified in parallel for each sample, as a control. We quantified viral cDNA, using 8E5 cells (cell line contaning a single copy of HIV pro-virus per cell) to generate the standard curve. Data are expressed as the ratio of the signal obtained for HIV cDNA to that obtained for GAPDH for the same sample.

Immunolabelling of CD4 and CCR5
Macrophages were treated with 1000 pg ml–1 IL-6 for 24 h and were then detached from the plastic by incubating for 20 min at +37°C with a non-enzymatic cell dissociation solution (Sigma). Macrophages were incubated for 30 min at +4°C with PE-conjugated mAbs against CD4 and CCR5 (BD Biosciences) or their isotype-matxched controls. Cells were then washed twice with PBS and fixed in 200 µl CellFixTM (BD Biosciences). Fluorescence was analysed by flow cytometry, using an LSR apparatus (BD Biosciences). Viable cells were gated on the basis of forward- and side-light scattering patterns.

Data analysis
Data were analysed by means of Student's unpaired t-test (Statview F 4.5, SAS Institute Inc., Cary, NC, USA). Differences were considered to be significant if P ≤ 0.01 (**) or P ≤ 0.05 (*).


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Effects of IFN-{tau} on cytokine production in macrophages
IFNs promote the synthesis of several cytokines produced by macrophages that interfere with HIV infection. We therefore investigated the effects of 8 h of treatment with IFN-{tau} on the production of IL-1ß, IL-6, IL-10 and TNF-{alpha} in uninfected unstimulated macrophages, in cells stimulated with the sub-optimal dose of 10 ng ml–1 LPS and in cells infected with HIV-1/Ba-L at an m.o.i. of 0.1.

Independent of LPS stimulation or HIV infection, IFN-{tau} induced no significant change in the synthesis of IL-1ß and TNF-{alpha} (Fig. 1A). In contrast, IL-10 protein levels increased significantly in response to treatment with 10 or 100 IU ml–1 IFN-{tau} in unstimulated cells (Fig. 1A: 10 IU ml–1 IFN-{tau}, 55 ± 8 pg ml–1, and 100 IU ml–1 IFN-{tau}, 70 ± 5 pg ml–1, versus untreated control, 25 ± 16 pg ml–1; P = 0.02 and 0.002, respectively). IL-6 levels also increased in the supernatants of cells treated with IFN-{tau} (Fig. 1A: 10 IU ml–1 IFN-{tau}, 29 ± 6 pg ml–1, and 100 IU ml–1 IFN-{tau}, 38 ± 12 pg ml–1, versus untreated control, 12 ± 3 pg ml–1; P = 0.002 and <0.001, respectively).



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Fig. 1. Effects of IFN-{tau} on IL-1ß, TNF-{alpha}, IL-10 and IL-6 protein synthesis in cell culture supernatants of unstimulated (A), LPS-stimulated (10 ng ml–1) (B) or HIV-1/Ba-L-infected (m.o.i. 0.1) (C) macrophages. Results correspond to 8 h cytokine production and are expressed as means of cytokine concentration ± SD for one experiment performed in quadruplicate. The results were confirmed with samples from two other blood donors.

 
As expected, LPS stimulation increased the production of all tested cytokines (Fig. 1B—for IL-1ß: LPS, 64 ± 6 pg ml–1, versus unstimulated, 28 ± 2 pg ml–1, P <0.001; for TNF-{alpha}: LPS, 120 ± 1 pg ml–1, versus unstimulated, 48 ± 4 pg ml–1, P < 0.001; for IL-10: LPS, 62 ± 1 pg ml–1, versus unstimulated, 25 ± 16 pg ml–1, P = 0.004; for IL-6: LPS, 31 ± 14 pg ml–1, versus unstimulated, 12 ± 3 pg ml–1, P = 0.04). IFN-{tau} treatment in LPS-stimulated cells did not increase IL-1ß and TNF-{alpha} production to levels above those achieved by LPS stimulation alone at any of the doses tested (Fig. 1B). In contrast, in LPS-stimulated macrophages, IL-10 and IL-6 levels were increased by IFN-{tau} treatment to levels higher than those in untreated and LPS-stimulated cells, and this increase was significant at 100 IU ml–1 for IL-10 (Fig. 1B: 100 IU ml–1 IFN-{tau}, 87 ± 20 pg ml–1, versus untreated LPS-stimulated, 62 ± 1 pg ml–1; P = 0.05) and at all tested doses of IFN-{tau} for IL-6 (Fig. 1B: 1 IU ml–1 IFN-{tau}, 56 ± 1 pg ml–1, 10 IU ml–1 IFN-{tau}, 106 ± 23 pg ml–1 and 100 IU ml–1 IFN-{tau}, 132 ± 31 pg ml–1, versus untreated LPS-stimulated, 31 ± 14 pg ml–1; P = 0.01, 0.001 and 0.001, respectively).

No significant change in TNF-{alpha}, IL-10 and IL-6 production by macrophages was observed during the first 8 h of HIV infection, whereas IL-1ß levels decreased (Fig. 1C—IL-1ß: infected, 10 ± 5 pg ml–1, versus uninfected, 28 ± 2 pg ml–1, P < 0.001).

IFN-{tau} had no impact on the secretion of TNF-{alpha} and IL-1ß in infected macrophages (Fig. 1C). The increase in IL-10 production observed in unstimulated cells was also observed in infected cells treated with 100 IU ml–1 IFN-{tau} (Fig. 1C: 100 IU ml–1 IFN-{tau}, 25 ± 1 pg ml–1, versus infected, 15.5 ± 4 pg ml–1, P = 0.004). IFN-{tau} also significantly increased IL-6 production in infected macrophages (Fig. 1C: 10 IU ml–1 IFN-{tau}, 35 ± 2 pg ml–1, and 100 IU ml–1 IFN-{tau}, 43 ± 12 pg ml–1, versus infected, 19 ± 10 pg ml–1, P = 0.02 for both).

We investigated whether the synthesis of IL-10 and IL-6 was modulated by IFN-{tau} at the transcriptional level. We therefore studied the synthesis of IL-10 and IL-6 mRNA in response to IFN-{tau} in macrophages, under the same conditions of stimulation and infection used in our protein study.

Independent of stimulation or infection, IL-10 mRNA synthesis was significantly and dose-dependently induced in IFN-{tau}-treated macrophages at the three doses tested (Fig. 2A—in percentage of untreated control: 1 UI ml–1 IFN-{tau}, 568 ± 8%, P < 0.01; 10 IU ml–1 IFN-{tau}, 624 ± 8%, P < 0.01; 100 IU ml–1 IFN-{tau}, 860 ± 180%, P = 0.05). As expected, LPS stimulation increased IL-10 mRNA levels (Fig. 2A: LPS-stimulated, 1044 ± 284% of unstimulated control, P = 0.02). In these experimental conditions, the synthesis of IL-10 mRNA was significantly increased by treatment with 100 IU ml–1 IFN-{tau} (Fig. 2A: 100 IU ml–1 IFN-{tau}, 1940 ± 420%, versus untreated LPS-stimulated, 1044 ± 284%, P = 0.02). In infected macrophages, as in LPS-stimulated macrophages, IFN-{tau} slightly increased IL-10 mRNA synthesis when added at a concentration of 100 IU ml–1 (Fig. 2A: 100 IU ml–1 IFN-{tau}, 824 ± 277%, versus infected control, 352 ± 139%, P = 0.02).



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Fig. 2. Effects of IFN-{tau} on IL-10 (A) and IL-6 (B) mRNA synthesis in macrophages unstimulated, stimulated with LPS (10 ng ml–1) or infected with HIV-1/Ba-L (m.o.i. 0.1). Results correspond to 8 h of mRNA synthesis and are expressed as a percentage of untreated and unstimulated control ± SD for two independent experiments performed in triplicate. (C) Signal obtained by PCR after stimulation for 2 h.

 
Our results concerning IL-6 mRNA synthesis were consistent with those for IL-6 protein levels, with both protein and mRNA levels increasing in response to IFN-{tau} treatment. Regardless of the stimulation used, treatment of macrophages with 100 IU ml–1 IFN-{tau} resulted in a significant increase in the amount of IL-6 mRNA, beginning 2 h after treatment (Fig. 2C). After 8 h, the IFN-{tau}-induced increase in IL-6 mRNA levels was dose dependent (Fig. 2B—in percentage of unstimulated control: 100 IU ml–1 IFN-{tau}-treated unstimulated, 233 ± 42%, P = 0.01; 100 IU ml–1 IFN-{tau}-treated LPS-stimulated, 1083 ± 100%, versus LPS-stimulated control, 700 ± 200%, P < 0.01; 100 IU ml–1 IFN-{tau}-treated infected, 250 ± 33%, versus infected control, 88 ± 50%, P < 0.01).

Anti-retroviral effects of human recombinant IL-6 and IL-10
As only the synthesis of IL-10 and IL-6 seemed to be modulated by IFN-{tau} in human macrophages, we tested the effects of these two cytokines on HIV replication in our experimental conditions. Eight hours of treatment with 100 IU ml–1 IFN-{tau} in unstimulated macrophages led to the production of up to 50 pg ml–1 IL-10 and IL-6. We tested the anti-viral efficacy of various doses of IL-10 and IL-6 by treating macrophages with 50–5000 pg ml–1 IL-10 or with 40–1000 pg ml–1 IL-6 throughout the culture period. Interestingly, low concentrations of IL-10, equivalent to those in the cell supernatants of macrophages after IFN-{tau} treatment, resulted in higher levels of HIV replication (Fig. 3A: 50 pg ml–1 IL-10, 139 ± 14% of untreated control, P = 0.02). Higher doses of IL-10, 100 times higher than those quantified in the cell supernatants of IFN-{tau}-treated macrophages, are required to obtain a 72 ± 3% inhibition of HIV-1/Ba-L replication in human macrophages (Fig. 3A).



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Fig. 3. Effects of human recombinant IL-10 (A) and IL-6 (B) on HIV-1/Ba-L replication in macrophages. Results are expressed as a percentage of untreated control cumulative RT activity and are representative of three independent experiments performed in triplicate.

 
In contrast, we observed significant dose-dependent inhibition of HIV replication with IL-6 treatment. Significant inhibition of RT activity was observed at the two highest IL-6 doses (Fig. 3B: 200 pg ml–1 IL-6, 47 ± 3%, and 1000 pg ml–1 IL-6, 72 ± 11%, versus untreated control, 100 ± 17%, P < 0.01 for both doses). This suggests that IL-6 alone may decrease HIV replication in macrophages.

Effects of IFN-{tau} on IL-6 production in HIV-infected macrophages
We evaluated the long-term production of IL-6 in infected macrophages untreated or treated with IFN-{tau}. In our experimental conditions and in untreated controls, HIV replication became detectable in cell culture supernatants 5 days after infection and peaked on day 20 (Fig. 4A). We observed 85% inhibition of HIV replication on day 14 after treatment with 100 IU ml–1 IFN-{tau} (IFN-{tau}, 1854 ± 557 pg ml–1, versus infected control, 12 238 ± 2061 pg ml–1, P < 0.01). IL-6 expression did not correlate with HIV replication: IL-6 production was not modulated by infection as IL-6 concentration remained <20 pg ml–1 throughout the culture period in infected and untreated macrophages as in uninfected and untreated cells (Fig. 4B). In contrast, IL-6 production was affected by IFN-{tau} treatment, and this effect was observed within 1 day of treatment. The greatest increase in IL-6 production was observed in macrophages both treated with IFN-{tau} and infected with HIV-1/Ba-L, and reached a maximum 5 days post-infection (Fig. 4B, day 5: IFN-{tau}-treated and infected, 253 ± 24 pg ml–1, versus infected and untreated, 6 ± 3 pg ml–1, P < 0.01). Maximal IL-6 production was detected later in IFN-{tau}-treated uninfected macrophages than in IFN-{tau}-treated infected cells, with IL-6 levels being highest in these cells on day 10. (Fig. 4B, day 10: IFN-{tau}-treated uninfected, 70 ± 16 pg ml–1, versus infected and untreated, 8 ± 6 pg ml–1, P < 0.01). In IFN-{tau}-treated cells, in the presence or absence of HIV infection, IL-6 production decreased after 5–10 days, reaching levels similar to those in untreated cells by day 14.



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Fig. 4. HIV-1/Ba-L replication (A) and IL-6 production (B) in IFN-{tau}-treated macrophages. Cells were treated with 100 IU ml–1 IFN-{tau} for 24 h before infection and treatment was continued throughout the culture. Supernatants were collected on days 0, 1, 5, 10, 14, 20 and 25 of infection to quantify RT activity and IL-6 production. Results are expressed as mean ± SD of one experiment performed in triplicate with one blood donor and similar results were obtained with two other donors.

 
As IL-6 levels were higher in IFN-{tau}-treated cells than in untreated cells until day 14, we studied the potential role of this cytokine in the anti-retroviral activity and mode of action of IFN-{tau}.

Anti-retroviral role of IL-6 in HIV-infected macrophages
Neutralization of the biological activity of IL-6 led to a significant increase in HIV replication in cultures of HIV-infected cells treated with IFN-{tau} (Fig. 5A: IFN-{tau} alone, 16 ± 1% of untreated control, versus IFN-{tau} + anti-IL-6 antibody, 60 ± 12% of untreated control, P < 0.01). HIV replication was unaffected by the addition of antibodies alone to cell culture medium or by the addition of the isotype-matched control IgG1 to IFN-{tau} treatment (Fig. 5A). Thus, the anti-retroviral activity of IFN-{tau} seems to require the biological activity of IL-6. We used the promonocytic U1 cell line, chronically infected with HIV, to determine whether these effects of IL-6 were involved in the inhibition of the early and/or late steps of the HIV biological cycle. The stimulation of U1 cells with PMA for 3 days strongly induced viral transcription, resulting in the efficient production of virus particles [Fig. 5B: PMA-stimulated, 100 ± 4% (4719 ± 171 pg ml–1 RT), versus unstimulated, 30 ± 2% (318 ± 107 pg ml–1 RT), P < 0.01]. The addition of HIV protease inhibitors, such as indinavir (IDV), together with PMA made it possible to maintain HIV replication at basal levels. This result is consistent with the known mode of action of IDV, which prevents the maturation of viral neo-particles. In contrast, 10 µM azidothymidine, a dose efficient in models of non-chronically infected cells, did not inhibit HIV replication in U1 cells, demonstrating the absence of re-infection cycles in this experimental model. The addition of 1000 pg ml–1 IL-6 increased HIV replication by 233 ± 16% (Fig. 5B), suggesting that IL-6 increased HIV replication by positive interference in the late steps of the HIV biological cycle. IFN-{tau} alone had a dose-dependent inhibitory effect on HIV replication in the U1 cell line (Fig. 6B). Although this IFN molecule is most efficient during the early stages of the HIV cycle in primary cultures of macrophages, it also induces anti-viral mechanisms, such as the PKR pathway, that interfere with late steps in the HIV biological cycle (26). Neutralization of the biological activity of IL-6 had no deleterious or beneficial effects on the anti-retroviral efficacy of IFN-{tau} in chronically infected cells (Fig. 5B), indicating that the involvement of IL-6 in the anti-retroviral effects of IFN-{tau} does not concern late steps of the biological cycle of HIV.



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Fig. 5. Involvement of IL-6 in the anti-viral mechanism of IFN-{tau}: effects of IL-6 or the neutralization of IL-6 activity on HIV replication in primary macrophages (A) and in PMA-stimulated U1 cells (B). Doses of antibodies: 100 ng ml–1. (A) Macrophages were treated with 100 IU ml–1 IFN-{tau} with or without anti-IL-6 antibodies or their isotype control IgG1 24 h before infection and throughout the culture. Results are expressed as means ± SD of one experiment performed in triplicate with one blood donor and similar results were obtained with two other donors. (B) U1 cells were stimulated with PMA (10 µM) for 3 days and treated with IDV (10 µM), with IL-6 (1000 pg ml–1) or with 100 IU ml–1 IFN-{tau}, with or without anti-IL-6 antibodies or their isotype control IgG1. This experiment performed in triplicate is representative of three independent experiments.

 


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Fig. 6. Involvement of IL-6 in the anti-viral mechanism of IFN-{tau} in macrophages. Doses: IFN-{tau}, 100 IU ml–1; IL-6, 1000 pg ml–1; antibodies, 100 ng ml–1. (A) Effects of IL-6 or the neutralization of IL-6 activity on the intracellular accumulation of HIV RNA 2 h after infection. Cells were treated for 24 h before infection with HIV-1/Ba-L (m.o.i. 0.1) for 2 h. Treatment with HSA–CD4 control was simultaneous to infection. PCR amplification from a sample obtained from one blood donor and quantification in percent inhibition ± SD for three different blood donors. (B) Effects of 24 h of IL-6 treatment on macrophage CD4 expression. (C) Effects of IL-6 on induction of the 40- and 69-kDa isoforms of 2',5'-OAS by IFN-{tau}. Two dilutions of cDNA were subjected to amplification. (D) Effects of a 24-h treatment with IL-6 on expression of MIP-1{alpha} by human macrophages.

 
Effect of IL-6 on the early steps of the HIV biological cycle
We showed in a previous study that IFN-{tau} was most efficient at inhibiting the early steps of the biological cycle of HIV in human macrophages, before the integration of the HIV pro-viral DNA into the host-cell genome (26).

We therefore investigated the effects of IL-6 in very early steps of the HIV biological cycle by studying the effects of treatment for 24 h with IL-6, IFN-{tau} and/or neutralizing anti-IL-6 antibodies, before infection, on the intracellular levels of HIV RNA a short time after infection. Macrophages were incubated for 2 h with HIV-1/Ba-L, treated with trypsin to eliminate extracellular viruses and lysed and treated with DNase to ensure the degradation of any viral DNA in the viral particle. As demonstrated by PCR amplification of the viral U3R sequence present in HIV genomic RNA (Fig. 6A, top), amplification from the cDNA present in cells infected with inactivated viruses was not possible. As heat-inactivated viruses are unable to enter target cells, this suggests that our method eliminated all extracellular viruses and only allowed the amplification of intracellular viral RNA sequences. Moreover, 83 ± 3% inhibition was achieved with treatment with human serum albumin (HSA)–CD4 molecules, a fusion protein of HSA and the first two domains of CD4 (Fig. 6A, bottom), which prevents gp-120–CD4 interactions, thereby preventing cellular infection. The administration of either IFN-{tau} or IL-6 alone for 24 h before infection decreased the number of intracellular copies of HIV RNA present 2 h after infection. HIV genomic RNA levels were 81 ± 8% and 73 ± 2% lower, respectively, in IFN-{tau}- and IL-6-treated cells (Fig. 6A, bottom). If the biological activity of IL-6 was neutralized, IFN-{tau} was 60% less efficient at decreasing the amount of intracellular HIV RNA: IFN-{tau} inhibition of HIV RNA synthesis was reduced to 12 ± 2%, whereas it was 81 ± 8% in the absence of neutralizing antibodies. Thus, IL-6 seems to play a role in the anti-viral mechanism of IFN-{tau} very early in the biological cycle of HIV.

However, consistently with our previous work (26), IL-6, like IFN-{tau}, had no effect on expressions of the HIV receptor CD4 (Fig. 6B) and of the co-receptor CCR5 (data not shown) on macrophages.

Genomic viral RNA can also be rapidly degraded by activation of the 2',5'-OAS/RNase L pathway induced by IFN-{tau}. We investigated the involvement of IL-6 in the transcriptional induction of two different isoforms of 2',5'-OAS, the synthesis of which is activated by IFN-{tau} (26). IL-6 alone did not induce the synthesis of 2',5'-OAS RNAs (Fig. 6C). In contrast, simultaneous treatment with IFN-{tau} and IL-6 slightly increased induction over the levels observed with IFN-{tau} alone (Fig. 6C). However, neutralization of the biological activity of IL-6 did not affect the ability of IFN-{tau} to induce the synthesis of either of the two isoforms of 2',5'-OAS (Fig. 6C). As IFN-{tau} was shown to increase the synthesis of ß-chemokines in human macrophages (26), the effects of IL-6 on the expression of macrophage inflammatory protein 1{alpha} (MIP-1{alpha}) were evaluated. The table presented in Fig. 6(D) shows a slight dose-dependent increase in MIP-1{alpha} expression in response to IL-6 treatment. This increase is, however, not very biologically significant.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Type I IFNs, and IFN-{alpha} in particular, play a very important role in host anti-viral defences, directly inhibiting the intracellular life cycle of the virus or regulating the cellular immune response during viral infections (1). The number of circulating natural IFN-{alpha}-producing cells in HIV-infected patients' blood has been shown to be negatively correlated with HIV viral load (52). This suggests that these cells, and thus IFN-{alpha}, play a key role in the maintenance of the cytokine profile required for protection against opportunistic infection and Kaposi's sarcoma. Type I IFNs, with their wide variety of anti-viral, anti-lymphoproliferative and immunomodulatory functions, have proven clinically effective in >16 years of use. IFN-{alpha} therapy has been suggested as a treatment for HIV infection. Unlike highly active anti-retroviral therapy, IFN-{alpha} has immunotherapeutic properties in addition to its anti-viral activity, facilitating restoration of the host-cell response. However, the side effects of IFN-{alpha} have limited its clinical use.

We have previously shown that ovine IFN-{tau} has similar anti-viral properties to human IFN-{alpha} and induces MIP-1{alpha}, MIP-1ß and RANTES in human macrophages (26). This molecule therefore has potential for use in anti-retroviral and immunomodulatory therapies. IFN-{tau} not only fulfils a necessary immunomodulatory function during pregnancy in ruminants but also has beneficial effects against many detrimental inflammatory processes, these effects being mediated by the induction of IL-10 synthesis in murine models, in particular (24, 30). We showed here that IFN-{tau} significantly increases IL-6 production in human macrophages and that this cytokine co-operates with IFN-{tau} to prevent chronic infection of macrophages, by decreasing the amount of HIV genomic RNA available for reverse transcription and integration into the host-cell genome. IFN-{tau} induces IL-10 synthesis in various murine models (28, 30). We confirmed these effects in our model of human macrophages in primary culture. However, the biological significance of the increase in IL-10 protein production in response to IFN-{tau} is unclear. Many studies have investigated the effects of HIV infection and IL-10 production on each other, and the results obtained have often conflicted. Bergamini et al. (53) and Dereuddre-Bosquet et al. (54) showed that HIV infection did not modulate IL-10 production by macrophages. In contrast, other in vivo and in vitro studies have reported an increase in IL-10 production in the course of HIV infection (55, 56). Several studies have demonstrated that IL-10 has anti-retroviral activity (39, 40, 55), but IL-10 has also been shown to increase HIV replication (41, 57). As in other studies, we found that the effects of IL-10 on HIV replication depend on the dose of IL-10. As reported by Weissman et al. (41), low doses of IL-10 increase HIV replication. Although endogenously produced IL-10 and exogenous recombinant IL-10 cannot be directly compared, it is unlikely that the low levels of IL-10 found in cell supernatants of IFN-{tau}-treated cells are involved in the inhibition of HIV replication observed with IFN-{tau}.

In contrast, IFN-{tau} treatment led to a reproducible increase in IL-6 mRNA and protein synthesis. This result is consistent with what has been observed for IFN-{alpha}: the administration of IFN-{alpha} to patients with viral hepatitis increases plasma IL-6 concentration, principally by increasing IL-6 mRNA levels in PBMC (5860). Plasma IL-6 concentration is also increased in HIV-infected patients and can have a deleterious effect on the progression of AIDS. Marfaing-Koka et al. (61) showed that increases in IL-6 production in HIV-infected patients at a late stage of the infection did not stimulate HIV replication in vivo, but might contribute to metabolic and immunological disturbances associated with the disease, such as the induction of B-cell lymphoma (62). However, in HIV-infected thrombocytopaenic individuals receiving IFN-{alpha} treatment, the increase in IL-6 production makes it possible to restore platelet production, as IL-6 is one of the main factors controlling thrombocytopoiesis (60). Thus, increases in IL-6 levels in HIV-infected patients are probably more a consequence than a cause of disease progression, and IL-6 production induced by IFN treatment may not have the deleterious effects observed with in vivo HIV-induced IL-6.

In our in vitro model of primary cultures of human macrophages, consistent with other in vitro studies (63), neither early infection nor productive infection with HIV-1/Ba-L increased the production of IL-6 by macrophages. However, if cells were treated for 24 h with IFN-{tau} before infection, then much larger increases in IL-6 levels were observed after 5 days of infection. Several other studies have highlighted the higher levels of IL-6 production in response to stimuli such as LPS in HIV-infected macrophages than in uninfected macrophages (63, 64). Moreover, previous studies performed with IFN-{alpha} and IL-6 have shown that IFN-{alpha} priming synergistically increases the production of IL-6 in cells in response to dsRNA (6567). HIV genomic RNA is single stranded but carries dsRNA structures such as the TAR sequence, which may be involved in this synergy in macrophages.

The anti-viral effects of IL-6 are also unclear. We and others (34) have shown that IL-6 increases HIV replication in pro-monocytic HIV-infected U1 cells. These observations are not inconsistent with the finding that IL-6 inhibits HIV replication in primary cultures of human macrophages. Poli et al. (34) showed that IL-6 increases HIV replication by transcriptional and post-transcriptional mechanisms, affecting the late steps of the biological cycle of HIV. However, IL-6 does not seem to increase HIV replication in HIV-infected patients (61). We showed here that pre-infection treatment with human recombinant IL-6 decreased HIV infection in primary macrophages by interfering with the early steps of the biological cycle of HIV, protecting cells against HIV RNA accumulation. Zaitseva et al. demonstrated that treatment with IL-6 or IFN-{gamma} 1 or 2 days before infection affected the susceptibility of macrophages to HIV-1 infection (68). These two cytokines increased the susceptibility of primary macrophages to infection with T-cell tropic CXCR4 strains, but not with CCR5 strains such as HIV-1/Ba-L. IFN-{gamma} even decreased macrophage infection with HIV-1/Ba-L, whereas IL-6 had no effect at the high concentrations (20 ng ml–1) used. As also observed here, Zaitseva et al. showed that IL-6 had no effect on CD4 and CCR5 membrane expression, but HIV-inhibitory levels of ß-chemokines (MIP-1{alpha} and MIP-1ß) were detected in the cell culture supernatants of IL-6-treated cells (68). The doses of IL-6 used in this study did not allow a great increase in the expression of MIP-1{alpha} by macrophages: IL-6 may contribute to induction of ß-chemokines in response to IFN-{tau} in human macrophages, but is not responsible for it, as levels of MIP-1{alpha} observed after IFN-{tau} treatment are higher than those observed for this IL-6 (26).

IFN-{tau} induces the 2',5'-OAS/RNase L pathway, which inhibits HIV replication by degrading viral RNA. IL-6, in combination with type I IFNs, has been implicated in the induction of 2',5'-OAS gene expression (69, 70). In this study, IL-6 alone had no effect, but IFN-{tau} and IL-6 co-treatment slightly increased 2',5'-OAS mRNA levels compared to IFN-{tau} alone. However, the neutralization of IL-6 activity in IFN-{tau}-treated cells did not decrease the induction obtained with 24 h of IFN-{tau} treatment. In murine macrophages, the addition of IL-6 potentiates the stimulation of 2',5'-OAS activity by IFN-{alpha} (71), whereas IL-6 alone had no effect on 2',5'-OAS activity. A similar mechanism may operate in human macrophages: IL-6 induced by IFN-{tau} may potentiate 2',5'-OAS activity, which together with the induction of ß-chemokines, may be involved in the anti-retroviral mechanism of IFN-{tau}.

This study provides new insight into the mechanism of action of IFN-{tau}, extending beyond the anti-retroviral action of this molecule to its immunomodulatory functions. The anti-viral and immunomodulatory properties of ovine recombinant IFN-{tau} cross the species barrier and resemble those of human IFN, except that the anti-viral pathway triggered is not associated with toxicity. This property renders IFN-{tau} a potentially interesting candidate for the treatment of HIV-infected or hepatitis C virus/HIV-co-infected patients.


    Acknowledgements
 
We thank L'Haridon from Institut National de la Recherche Agronomique (Jouy-en-Josas, France) for IFN anti-viral titration. We obtained U1/HIV-1 from Thomas Folks of the AIDS Research and Reference Reagent Program, Division of AIDS, NIAIS, NIH. This work was supported by Ensemble contre le SIDA—SIDACTION.


    Abbreviations
 
GAPDH   glyceraldehyde 3-phosphate dehydrogenase
HAS   human serum albumin
IDV   indinavir
MIP   macrophage inflammatory protein
NIH   National Institutes of Health
2',5'-OAS   2',5'-oligoadenylate synthetase
PKR   serine–threonine kinase R
PMA   phorbol myristate acetate
PSN   penicillin, streptomycin, neomycin
RT   reverse transcriptase
TNF   tumour necrosis factor

    Notes
 
* Dedicated to Professor Dominique Dormont who died in November 2003 Back

Transmitting editor: S. Romagnani

Received 14 November 2003, accepted 17 May 2005.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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