IL-27 is a member of the IL-6/IL-12 cytokine family. It is composed of two subunits: Epstein–Barr virus-induced gene-3 protein and IL-27 p28 protein  and is produced by macrophages, dendritic cells, and epithelial cells . IL-27 mediates its cellular response through the IL-27 receptor, which is a heterodimer complex consisting of WSX-1 and glycoprotein 130 (gp130) [3,4]. IL-27 plays a role in the early regulation of T helper type 1 initiation, and enhances proliferation of naive CD4 T cells and naive B cells [4–11]. It, however, suppresses the development of IL-17-producing effector T cells and inducible regulatory T cells [12–15]. IL-27 is also involved in the production of IFN-γ and IgG [1,16]. We have previously reported that IL-27 is a potent anti-HIV cytokine that inhibits HIV replication in peripheral blood mononuclear cells (PBMC), CD4 T cells and monocyte-derived macrophages (MDM)  like IFN-α, IFN-β and IFN-γ (IFN).
IFN are the cytokines that suppress HIV replication in T cells and macrophages in vitro [18,19], and only IFN-α has been shown to have an anti-HIV effect in vivo [20–22]. IFN-α activates a series of genes often referred to as IFN-inducible genes (IFIG) [23–26]. IFN-α inhibits viral replication through multiple pathways including the RNA-dependent protein kinase–eukaryotic initiation factor 2a kinase (PKR/EIFZAK) pathway, the oligoadenylate synthetase (OAS)–RNaseL pathway, and the myxovirus protein (MX)–guanosine triphosphatase pathway. The purpose of the present study was to examine the mechanism(s) of IL-27-mediated HIV inhibition through an analysis of the gene expression profile.
Materials and methods
Reagents and cells
Recombinant IL-27, IFN-β and IFN-γ, neutralizing antibodies to IFN-α, IFN-β, and IFN-γ were obtained from R&D Systems (Minneapolis, Minnesota, USA). Recombinant IFN-α was obtained from Schering (Kenilworth, New Jersey, USA). HIV-1 stocks were prepared using plasmids encoding the full length of HIVNL4.3 (X4-HIV)  or HIVAD8 (R5-HIV)  as previously described . CD4 T cells and CD14 positive monocytes were isolated from PBMC of healthy donors as previously described . In this study, CD14 positive monocytes were differentiated into MDM using 25 ng/ml recombinant macrophage-colony stimulating factor (R&D Systems) in macrophage serum-free medium (Invitrogen, Carlsbad, California, USA) supplemented with 10 mmol HEPES and 5 μg/ml gentamycin (Invitrogen) for 7 days.
HIV-1 infection and replication assay
HIV-1 replication was determined as previously described . Viral replication was monitored by using a p24 antigen capture assay kit (Perkin-Elmer, Shelton, Connecticut, USA).
Quantitation of IFN genes
To quantitate a relative amount of the expression of the IFN gene, real time polymerase chain reaction (PCR) was performed using the iCycler real-time PCR detection system (Bio-Rad, Hercules, California, USA) as previously described . The amount of quantitative PCR product for IFN-α, IFN-β or IFN-γ was normalized to the amount of glyceraldehyde-3-phosphate dehydrogenase.
Enzyme-linked immunosorbent assay
The concentration of IFN in the culture supernatants from HIV-infected CD4 T cells and MDM were quantitated using an enzyme-linked immunosorbent assay kit (R&D Systems), according to the manufacture's instructions. The detection limits of enzyme-linked immunosorbent assay for IFN-α, IFN-β and IFN-γ were 12.5, 25 and 15.6 pg/ml, respectively.
Microarray DNA analysis
DNA microarray assay was performed using the Affymetrix U133A+2 human microarray (Affymetrix, Santa Clara, California, USA) as previously described . IFIG were determined using Swiss-Prot keywords. The differentially expressed genes were grouped using K-mean clustering (Parket pro); functional annotation and biological category enrichment were performed using DAVID (http://david.abcc.ncifcrf.gov).
Quantigene plex assay
To verify the relative expression of the IFIG, the Quantigene plex assay (Panomics, Fremont, California, USA) was performed on a customized panel of IFIG according to the manufacturer's protocol. The fluorescence intensity in the hybridized complex was measured using a Bio-Plex 200 system (Bio-Rad).
Western blot assay of STAT-1
Western blot analysis was performed using antibody to STAT-1 (Santa Cruz Biotechnology, Santa Cruz, California, USA) or phospho-STAT1 (Santa Cruz Biotechnology) as previously described . Signals on the membranes were detected using horseradish peroxidase-conjugated antigoat antibodies (Santa Cruz Biotechnology) with the ECL-plus kit (GE Healthcare, Bucks, UK).
Statistical analysis was performed using the unpaired t-test using the StarView program (Abacus Concepts, Berkeley, California, USA).
IFN-α, IFN-β and IFN-γ have no impact on IL-27-mediated HIV inhibition in CD4 T cells and monocyte-derived macrophages
To compare the mechanism of IL-27-mediated HIV inhibition with that of IFN-α, HIV-infected CD4 T cells and MDM were cultured in the presence of various amounts of each cytokine (Fig. 1a,b). IFN-α inhibited HIV replication in CD4 T cells and MDM with a 50% inhibitory concentration (IC50) of 20 ± 10 and 0.05 ± 0.03 IU/ml, respectively. IFN-α preferentially inhibited HIV replication in MDM compared with CD4 T cells (P < 0.01). A similar relationship was seen with IL-27. IL-27 inhibited HIV replication in CD4 T cells with an IC50 of 15 ± 10 ng/ml and MDM with an IC50 of 0.4 ± 0.15 ng/ml. Although 10 000 IU/ml IFN-α was capable of completely inhibiting HIV replication in CD4 T cells (data not shown), IL-27 inhibited HIV replication in the cells by almost 70–80% even in the presence of 300 ng/ml, and the antiviral effect plateaued (Fig. 1b).
To define the impact of IFN on IL-27-mediated HIV inhibition in CD4 T cells and MDM, we performed a quantitation assay. Neither the gene expression nor protein production of IFN-α and IFN-β were detected in either mock or IL-27-treated cell. The expression of the IFN-γ gene and its protein were detected in mock-treated CD4 T cells, but not in MDM. Mock-treated CD4 T cells produced 178 ± 20 pg/ml IFN-γ, but the production was not significantly increased by IL-27 treatment (220 ± 12 pg/ml; P > 0.05). Of note is the fact that IL-27 had no impact on the induction of IFN-γ from MDM. To verify the role of IFN on the anti-HIV effect further, a neutralization assay was also performed using an antibody cocktail containing neutralizing antibody to each IFN. The antibody cocktail had no impact on HIV-1 inhibition by IL-27 in CD4 T cells and MDM. As a positive control for the neutralization, an IFN cocktail was also used. The antibody cocktails partly inhibited the anti-HIV effect by the IFN cocktails in both CD4 T cells and MDM (Fig. 1c). Taken together, these results suggest that IFN do not play an important role if any in the inhibition of HIV replication by IL-27.
IL-27 induces a gene expression profile in monocyte-derived macrophages similar to that of IFN-α
The gene expression profiles of IL-27 and IFN-α-treated CD4 T cells and MDM were compared using Microarray DNA analysis. IL-27 and IFN-α significantly modulated a total of 1868 genes in both cell types (Fig. 2a). A particular interest was found in clusters 1 and 2 representing a total of 405 genes in the profiles. These clusters were highly enriched in genes involved in the biotic stimulus, IFN response, defence response, immune response, response to virus, and innate immune response (see supplementary table in Fig. 2). Of the 38 IFIG on the array, IFN-α and IL-27 induced 18 and five IFIG, respectively, in CD4 T cells. In contrast, in MDM, IFN-α induced a total of 33 IFIG, and interestingly and surprisingly, IL-27 induced a total of 28 IFIG. IL-27 significantly induced the antiviral genes MX1, OAS2, and PKR/EIF2AK, by more than twofold in MDM, but not in CD4 T cells. It is reported that IFN-α is able to induce apolipoprotein B messenger RNA-editing enzyme-catalytic polypeptide-like 3G (APOBEC3G) [31,32], a recently described anti-HIV protein, in macrophages and resting CD4 T cells [33,34]. Consistent with those reports, IFN-α induced APOBEC3G in CD4 T cells and MDM. Of note is the fact that IL-27 also increased activation of APOBEC3G in only MDM but not in CD4 cells; however, IL-27 did not significantly increase the expression of 20 IFN family genes (IFN-α1, -α4, -α5, -α6, -α7, -α8, -α10, -α13, -α14, -α16, -α17, -α21, -β1, -ϵ1, -γ, -κ, -ω1, IL-28A, IL-28B, and IL-29) in either CD4 T cells or MDM cultured for either 24 h or 7 days (data not shown).
The expression levels of IFIG in MDM treated with IFN-α or IL-27 were higher than those in CD4 T cells treated with the cytokines. In MDM, IL-27 treatment induced a lower level of IFIG compared with IFN-α treatment (Fig. 2b). The expression of those genes in MDM was confirmed by the Quantigene Plex assay (Fig. 2c). IL-27 is, therefore, able to induce multiple IFIG and APOBEC3G in MDM, similar to IFN-α.
IL-27 induces activation of STAT-1 in monocyte-derived macrophages
IFN-α initiates the activation of STAT-1 followed by the induction of IFIG [35–37]. Although it is reported that IL-27 activates not only STAT-1, but also STAT-2, STAT-3, STAT-4 and STAT-5 in T cells [2,4,14,38], and activates STAT-3 in macrophages , it has not been clear whether the cytokine activates STAT-1 in macrophages. To determine whether IL-27 activates STAT-1 in CD4 T cells and MDM in our culture system, Western blot analysis was performed. The assay demonstrated that both cytokines induced the phosphorylation of STAT-1 in CD4 T cells and MDM (Fig. 2d).
We have previously reported that IL-27 is a novel anti-HIV cytokine that inhibits the replication of X4-HIV-1 and R5-HIV-1 in PBMC, CD4 T cells and MDM . Even though IL-27 suppresses the transcription of HIV, the mechanism of IL-27-mediated HIV inhibition remains unclear. In the current study, we illustrated that IL-27 preferentially inhibits HIV-1 replication in MDM compared with CD4 T cells and activates multiple IFIG in MDM like IFN-α, suggesting that IL-27 inhibits HIV-1 replication in MDM via a mechanism(s) similar to that of IFN-α. DNA microarray analysis demonstrated that IL-27 induced multiple IFIG as well as multiple genes associated with the defence response, immune response, response to virus infection and innate immune response. The mechanism of the IL-27-mediated anti-HIV effect may thus involve not only IFIG but also other factors, and both factors may additionally or synergistically play roles in the anti-HIV effect by IL-27.
IFN-α completely inhibited HIV replication in both CD4 T cells and MDM. In contrast, although IL-27 could completely inhibit HIV-1 replication in MDM, its effect plateaus at approximately 70–80% inhibition in CD4 T cells. These data suggest that IL-27 may not block HIV-1 replication in a subset of CD4 T cells. It is reported that the expression level of the IL-27 receptor is regulated during T-cell activation ; thus, the expression level of the IL-27 receptor might be affected on IL-27-mediated HIV-1 inhibition. IL-27 induces the proliferation of naive T cells but not memory T cells [4–9]; in contrast, HIV-1 preferentially replicates in memory T cells compared with naive T cells [41–44]. It is thus assumed that IL-27 might affect HIV-1 replication in memory T cells subset rather than naive T cells subset in CD4 T cells. Further study is needed to understand the regulation of HIV replication by IL-27 in CD4 T cells.
The Western blot assays demonstrate that IL-27 induced activation of STAT-1 in both CD4 T cells and MDM. The cytokine, however, induced five and 28 IFIG in CD4 T cells and MDM, respectively; thus, it is clear that IL-27 differentially regulates the activation of IFIG in CD4 T cells and MDM. Whereas IL-27 stimulation is associated with the induction of multiple IFIG in MDM, it is unlikely that this effect is mediated by IFN. Data supporting this statement include the fact that IL-27 has no significant impact on gene activation and the production of IFN in MDM, and the IL-27 effect is not eliminated by the neutralization of IFN. In addition, further supporting this hypothesis is the fact that IL-27 did not significantly increase the activation of 20 IFN family genes in MDM. IL-27 thus appears to act similarly to IFN-α, whereas it functions independently of IFN-α induction in MDM.
As IL-27 predominantly inhibits HIV replication in MDM, achieving a better understanding of the role of IL-27 in activation, gene regulation, and HIV replication in MDM may provide new insights to control the reservoir of viral replication and aid in the development of a novel immunotherapeutic strategy for HIV-1 infection.
Plasmids encoding full-length HIV were provided by Dr M. Martin through the NIAID AIDS research and reference programme. The authors would like to thank Dr H. Imamichi for discussing and Dr J. Bharucha for critical reading of the manuscript.
Sponsorship: This project has been funded in whole with federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, under contract N01-CO-12400.
The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US government.
Conflicts of interest: None.
1. Pflanz S, Timans JC, Cheung J, Rosales R, Kanzler H, Gilbert J, et al
. IL-27, a heterodimeric cytokine composed of EBI3 and p28 protein, induces proliferation of naive CD4 (+) T cells. Immunity 2002; 16:779–790.
2. Hunter CA. New IL-12-family members: IL-23 and IL-27, cytokines with divergent functions. Nat Rev Immunol 2005; 5:521–531.
3. Pflanz S, Hibbert L, Mattson J, Rosales R, Vaisberg E, Bazan JF, et al
. WSX-1 and glycoprotein 130 constitute a signal-transducing receptor for IL-27. J Immunol 2004; 172:2225–2231.
4. Kamiya S, Owaki T, Morishima N, Fukai F, Mizuguchi J, Yoshimoto T. An indispensable role for STAT1 in IL-27-induced T-bet expression but not proliferation of naive CD4+
T cells. J Immunol 2004; 173:3871–3877.
5. Takeda A, Hamano S, Yamanaka A, Hanada T, Ishibashi T, Mak TW, et al
. Cutting edge: role of IL-27/WSX-1 signaling for induction of T-bet through activation of STAT1 during initial Th1 commitment. J Immunol 2003; 170:4886–4890.
6. Villarino A, Hibbert L, Lieberman L, Wilson E, Mak T, Yoshida H, et al
. The IL-27R (WSX-1) is required to suppress T cell hyperactivity during infection. Immunity 2003; 19:645–655.
7. Lucas S, Ghilardi N, Li J, de Sauvage FJ. IL-27 regulates IL-12 responsiveness of naive CD4+ T cells through Stat1-dependent and -independent mechanisms. Proc Natl Acad Sci U S A 2003; 100:15047–15052.
8. Owaki T, Asakawa M, Morishima N, Hata K, Fukai F, Matsui M, et al
. A role for IL-27 in early regulation of Th1 differentiation. J Immunol 2005; 175:2191–2200.
9. Owaki T, Asakawa M, Fukai F, Mizuguchi J, Yoshimoto T. IL-27 induces Th1 differentiation via p38 MAPK/T-bet- and intercellular adhesion molecule-1/LFA-1/ERK1/2-dependent pathways. J Immunol 2006; 177:7579–7587.
10. Gagro A, Servis D, Cepika AM, Toellner KM, Grafton G, Taylor DR, et al
. Type I cytokine profiles of human naive and memory B lymphocytes: a potential for memory cells to impact polarization. Immunology 2006; 118:66–77.
11. Larousserie F, Charlot P, Bardel E, Froger J, Kastelein RA, Devergne O. Differential effects of IL-27 on human B cell subsets. J Immunol 2006; 176:5890–5897.
12. Batten M, Li J, Yi S, Kljavin NM, Danilenko DM, Lucas S, et al
. Interleukin 27 limits autoimmune encephalomyelitis by suppressing the development of interleukin 17-producing T cells. Nat Immunol 2006; 7:929–936.
13. Stumhofer JS, Laurence A, Wilson EH, Huang E, Tato CM, Johnson JM, et al
. Interleukin 27 negatively regulates the development of interleukin 17-producing T helper cells during chronic inflammation of the central nervous system. Nat Immunol 2006; 7:937–945.
14. Yoshimura T, Takeda A, Hamano S, Miyazaki Y, Kinjyo I, Ishibashi T, et al
. Two-sided roles of IL-27: induction of Th1 differentiation on naive CD4+
T cells versus suppression of proinflammatory cytokine production including IL-23-induced IL-17 on activated CD4+
T cells partially through STAT3-dependent mechanism. J Immunol 2006; 177:5377–5385.
15. Neufert C, Becker C, Wirtz S, Fantini MC, Weigmann B, Galle PR, Neurath MF. IL-27 controls the development of inducible regulatory T cells and Th17 cells via differential effects on STAT1. Eur J Immunol 2007; 37:1809–1816.
16. Boumendjel A, Tawk L, de Malefijt WR, Boulay V, Yssel H, Pene J. IL-27 induces the production of IgG1 by human B cells. Eur Cytokine Network 2006; 17:281–289.
17. Fakruddin JM, Lempicki RA, Gorelick RJ, Yang J, Adelsberger JW, Garcia-Pineres AJ, et al
. Noninfectious papilloma virus-like particles inhibit HIV-1 replication: implications for immune control of HIV-1 infection by IL-27. Blood 2007; 109:1841–1849.
18. Meylan PR, Guatelli JC, Munis JR, Richman DD, Kornbluth RS. Mechanisms for the inhibition of HIV replication by interferons-alpha, -beta, and -gamma in primary human macrophages. Virology 1993; 193:138–148.
19. Baca-Regen L, Heinzinger N, Stevenson M, Gendelman HE. Alpha interferon-induced antiretroviral activities: restriction of viral nucleic acid synthesis and progeny virion production in human immunodeficiency virus type 1-infected monocytes. J Virol 1994; 68:7559–7565.
20. Lane HC. The role of alpha-interferon in patients with human immunodeficiency virus infection. Semin Oncol 1991; 18:46–52.
21. Poli G, Biswas P, Fauci AS. Interferons in the pathogenesis and treatment of human immunodeficiency virus infection. Antiviral Res 1994; 24:221–233.
22. Brassard DL, Grace MJ, Bordens RW. Interferon-alpha as an immunotherapeutic protein. J Leukoc Biol 2002; 71:565–581.
23. Langer JA, Cutrone EC, Kotenko S. The class II cytokine receptor (CRF2) family: overview and patterns of receptor-ligand interactions. Cytokine Growth Factor Rev 2004; 15:33–48.
24. Pestka S, Langer JA, Zoon KC, Samuel CE. Interferons and their actions. Annu Rev Biochem 1987; 56:727–777.
25. Galligan CL, Murooka TT, Rahbar R, Baig E, Majchrzak-Kita B, Fish EN. Interferons and viruses: signaling for supremacy. Immunol Res 2006; 35:27–40.
26. Samuel CE. Antiviral actions of interferons. Clin Microbiol Rev 2001; 14:778–809.
27. Adachi A, Gendelman HE, Koenig S, Folks T, Willey R, Rabson A, et al
. Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone. J Virol 1986; 59:284–291.
28. Theodore TS, Englund G, Buckler-White A, Buckler CE, Martin MA, Peden KW. Construction and characterization of a stable full-length macrophage-tropic HIV type 1 molecular clone that directs the production of high titers of progeny virions. AIDS Res Hum Retroviruses 1996; 12:191–194.
29. Brann TW, Dewar RL, Jiang MK, Shah A, Nagashima K, Metcalf JA, et al
. Functional correlation between a novel amino acid insertion at codon 19 in the protease of human immunodeficiency virus type 1 and polymorphism in the p1/p6 Gag cleavage site in drug resistance and replication fitness. J Virol 2006; 80:6136–6145.
30. Oguariri RM, Brann TW, Imamichi T. Hydroxyurea and interleukin-6 synergistically reactivate HIV-1 replication in a latently infected promonocytic cell line via SP1/SP3 transcription factors. J Biol Chem 2007; 282:3594–3604.
31. Sheehy AM, Gaddis NC, Choi JD, Malim MH. Isolation of a human gene that inhibits HIV-1 infection and is suppressed by the viral Vif protein. Nature 2002; 418:646–650.
32. Chiu YL, Greene WC. Multifaceted antiviral actions of APOBEC3 cytidine deaminases. Trends Immunol 2006; 27:291–297.
33. Peng G, Lei KL, Jin W, Greenwell-Wild T, Wahl SM. Induction of APOBEC3 family proteins, a defensive maneuver underlying interferon-induced anti-HIV-1 activity. J Exp Med 2006; 203:41–46.
34. Chen K, Huang J, Zhang C, Huang S, Nunnari G, Wang FX, et al
. Alpha interferon potently enhances the antihuman immunodeficiency virus type 1 activity of APOBEC3G in resting primary CD4 T cells. J Virol 2006; 80:7645–7657.
35. Ghislain JJ, Fish EN. Application of genomic DNA affinity chromatography identifies multiple interferon-alpha-regulated Stat2 complexes. J Biol Chem 1996; 271:12408–12413.
36. Li X, Leung S, Qureshi S, Darnell JE Jr, Stark GE. Formation of STAT1-STAT2 heterodimers and their role in the activation of IRF-1 gene transcription by interferon-alpha. J Biol Chem 1996; 271:5790–5794.
37. Ghislain JJ, Wong T, Nguyen M, Fish EN. The interferon-inducible Stat2:Stat1 heterodimer preferentially binds in vitro to a consensus element found in the promoters of a subset of interferon-stimulated genes. J Interferon Cytokine Res 2001; 21:379–388.
38. Hibbert L, Pflanz S, de Waal Malefyt R, Kastelein RA. IL-27 and IFN-alpha signal via Stat1 and Stat3 and induce T-Bet and IL-12Rbeta2 in naive T cells. J Interferon Cytokine Res 2003; 23:513–522.
39. Holscher C, Holscher A, Ruckerl D, Yoshimoto T, Yoshida H, Mak T, et al
. The IL-27 receptor chain WSX-1 differentially regulates antibacterial immunity and survival during experimental tuberculosis. J Immunol 2005; 174:3534–3544.
40. Villarino AV, Larkin J III, Saris CJ, Caton AJ, Lucas S, Wong T, et al
. Positive and negative regulation of the IL-27 receptor during lymphoid cell activation. J Immunol 2005; 174:7684–7691.
41. Spina CA, Prince HE, Richman DD. Preferential replication of HIV-1 in the CD45RO memory cell subset of primary CD4 lymphocytes in vitro
. J Clin Invest 1997; 99:1774–1785.
42. Chun TW, Chadwick K, Margolick J, Siliciano RF. Differential susceptibility of naive and memory CD4+ T cells to the cytopathic effects of infection with human immunodeficiency virus type 1 strain LAI. J Virol 1997; 71:4436–4444.
43. Riley JL, Levine BL, Craighead N, Francomano T, Kim D, Carroll RG, et al
. Naive and memory CD4 T cells differ in their susceptibilities to human immunodeficiency virus type 1 infection following CD28 costimulation: implications for transmission and pathogenesis. J Virol 1998; 72:8273–8280.
44. Ostrowski MA, Chun TW, Justement SJ, Motola I, Spinelli MA, Adelsberger J, et al
. Both memory and CD45RA+/CD62L+ naive CD4(+) T cells are infected in human immunodeficiency virus type 1-infected individuals. J Virol 1999; 73:6430–6435.