Monocytes isolated from the peripheral blood of different donors were cultured in vitro for 7 days, when the majority of cells differentiated into macrophages. To determine whether virus particles were released from macrophages infected with the HIV-1 strains (Bal, Jago, and JRFL), the supernatants were collected at 8 days postinfection and tested for the presence of p24 viral antigen, using a commercially available ELISA (Chiron Corp, Emeryville, CA). Levels of RT activity present in HIV-1-infected macrophage culture supernatants were determined by the methods of Willey et al. (42
) with modifications. Significant RT activity and p24 antigen expression were observed at 8 days postinfection with HIV-1 R5 strains (Fig. ). Treatment of 7-day-cultured macrophages with IFN-λ1 or IFN-λ2 significantly inhibited infection of different HIV-1 R5 strains (Bal, Jago, and JRFL) as evidenced by p24 protein and RT activity (Fig. ). This inhibitory effect on HIV-1 by IFN-λ was dose and time dependent (Fig. ). IFN-λ1 or IFN-λ2 at a concentration of 100 ng/ml was shown to have anti-HIV activity similar to that of IFN-α or IFN-β at a concentration of 1,000 IU/ml (Fig. ), although it is unclear whether the concentrations used for these IFNs are comparable with regard to anti-HIV efficiency. It is apparent that the combination of IFN-λ1 and IFN-λ2 inhibited HIV replication to a greater degree than IFN-λ1 or IFN-λ2 alone. However, this combined effect of IFN-λ1 and IFN-λ2 on HIV is not statistically significant compared with either IFN-λ1 (P
= 0.13) or IFN-λ2 (P
= 0.21) alone. This inhibitory effect of IFN-λ on HIV-1 was not due to cytotoxicity, since IFN-λ at concentrations of 1,000 ng/ml or lower had no cytotoxic effect on macrophages (Fig. ). Morphologically, HIV-1 Bal-infected macrophage cultures without IFN-λ treatment demonstrated characteristic giant syncytium formation (Fig. , panel b), whereas IFN-λ-treated macrophages failed to develop HIV-1-induced giant syncytia (Fig. , panel c).
FIG. 1. Effect of IFN-λ on HIV-1 infection of macrophages. (A) Three HIV-1 R5 strains (Bal, Jago, and JRFL) were used to infect 7-day-cultured macrophages with or without IFN-λ pretreatment (100 ng/ml) for 24 h. HIV-1 RT activity and p24 production (more ...)
FIG. 2. Effect of IFN-λ on cell viability and HIV-induced syncytium formation in macrophages. (A) Cytotoxic effect of IFN-λ on macrophages. Macrophages were cultured in the presence or absence of IFN-λ1 or IFN-λ2 for 72 h. The (more ...)
Biological functions of IFN-λ are mediated through its receptors, which are composed of two chains, IL-28Rα and IL-10Rβ. IL-28Rα is IFN-λ specific, and IL-10Rβ is shared among IL-10, IL-22, and IL-26. Both IL-28Rα and IL-10Rβ are necessary to form a functional IFN-λ receptor. However, while IL-10Rβ is ubiquitously expressed, IL-28Rα expression is not detected in some cell types, mainly fibroblastic and endothelial cells, which are unresponsive to IFN-λ (39
). Monocytes freshly isolated from human blood do not express IL-28Rα but become positive when the cells differentiate into dendritic cells upon IL-4 and granulocyte-macrophage colony-stimulating factor treatment (8
). To determine if the IFN-λ receptor complex consisting of IL-10Rβ and IL-28Rα is expressed in macrophages, we examined 7-day-cultured macrophages by RT-PCR and flow cytometry assay. As shown in Fig. , the negative control cells, human diploid fibroblasts (MRC-5) and human brain capillary endothelial cells (BB19), had no detectable IL-28Rα and IL-10Rβ transcripts. In contrast, macrophages derived from monocytes isolated from five different donors expressed mRNA transcripts for both IL-28Rα and IL-10Rβ (Fig. ). The expression of these IFN-λ receptors was further confirmed by the observation that macrophages expressed IFN-λ receptor (IL-10Rβ) at the protein level (Fig. ). The identification of IFN-λ receptor expression by macrophages is essential for supporting the specific antiviral action of IFN-λ on HIV-1.
FIG. 3. Expression of IFN-λ receptor in macrophages. (A) RT-PCR analysis of IFN-λ receptor gene transcripts in macrophages. Total cellular RNA was extracted from negative control cells (MRC-5 and BB19) and macrophages derived from monocytes from (more ...)
We next examined the mechanisms involved in IFN-λ-mediated anti-HIV-1 activity in macrophages. We were particularly interested in the role of IFN-λ in the modulation of the CCR5 receptor, the coreceptor for HIV-1 R5 strain entry into macrophages, and of CC chemokines (MIP-1α, MIP-1β, and RANTES), the ligands for CCR5 receptor on macrophages (1
). CC chemokines are important inhibitors of R5 strains of HIV-1 in cells of macrophage lineage (1
). Although IFN-λ had little effect on the expression of HIV-1 receptors (CD4, CCR5, and CXCR4) (Fig. ), both IFN-λ1 and IFN-λ2 effectively induced the expression of MIP-1α and MIP-1β protein in macrophages (Fig. ). The role of CC chemokines in the IFN-λ-mediated anti-HIV-1 effect was confirmed in the experiments showing that antibodies to CC chemokines (MIP-1α/β and RANTES) partially blocked IFN-λ-mediated HIV-1 inhibition in macrophages (Fig. ).
FIG. 4. Effect of IFN-λ on the expression of CD4, CCR5, and CXCR4 in macrophages. Macrophages were cultured in the presence or absence of IFN-λ (100 ng/ml) for 24 h. Total cellular RNA extracted from cell cultures was subjected to the real-time (more ...)
FIG. 5. Effect of IFN-λ on MIP-1α/β protein expression in macrophages. Macrophages derived from monocytes from three different donors (D) were incubated with or without IFN-λ1 (A) or IFN-λ2 (B) for 24 h, and the culture (more ...)
FIG. 6. Effect of antibodies to CC chemokines on IFN-λ-mediated anti-HIV-1 activity. Macrophages were incubated with or without IFN-λ1 or IFN-λ2 (100 ng/ml) and/or antibodies (Abs) (25 μg/ml each) to human CC chemokines (MIP-1α, (more ...)
Our further studies showed that IFN-λ had the ability to induce the intracellular expression of type I IFNs (Fig. ). Preincubation of macrophages with antibody to IFN-α/βR1 prior to HIV-1 infection could significantly reverse IFN-λ-mediated anti-HIV-1 activity (Fig. ). Type I IFNs (IFN-α/β) are well known for their ability to inhibit a wide range of viruses (37
), including HIV-1 (3
). IFN-α inhibits HIV-1 replication in macrophages at different stages by suppressing RT (35
) and preventing transcription of integrated provirus (26
). Similarly, IFN-β also inhibits HIV-1 replication in macrophages (9
). The importance of type I IFNs in the immunopathogenesis of HIV-1 infection is further highlighted by the in vivo observation (13
) that type I IFN production is profoundly and transiently impaired in primary HIV-1 infection. Thus, the induction of intracellular IFN-α/β expression by IFN-λ provides an additional mechanism for the anti-HIV-1 action of IFN-λ in macrophages. Although the anti-HIV-1 mechanism(s) of type I IFNs remains to be determined, several cellular factors, such as microRNAs, BST2, PKR, OAS1, and ISG15, have recently been identified as type I IFN-inducible anti-HIV-1 elements in HIV-1-infected target cells (12
). Among these factors, APOBEC3G and APOBEC3F are particularly important, as they have been recently shown to have the ability to restrict HIV-1 replication in both CD4+
T cells and macrophages. APOBEC3G can either edit the newly synthesized viral DNA or have an inhibitory effect at another site(s) of the HIV-1 life cycle (17
). Therefore, it is important to determine whether IFN-λ has the ability to enhance the expression of these newly identified innate anti-HIV-1 factors in macrophages. Our data that IFN-λ enhanced APOBEC3G and APOBEC3F expression at both the mRNA and protein levels (Fig. ) support the notion that elevated levels of APOBEC3G/3F may contribute to IFN-λ-mediated anti-HIV-1 activity in macrophages.
FIG. 7. Effect of IFN-λ on the expression of IFN-α/β in macrophages. (A) IFN-α/β mRNA expression. Macrophages were cultured in the presence or absence of IFN-λ (100 ng/ml) for 6 h. Total cellular RNA extracted from (more ...)
FIG. 8. Effect of IFN-λ on the expression of APOBEC3G/3F in macrophages. (A) APOBEC3G/3F mRNA expression. Macrophages were cultured in the presence or absence of IFN-λ (100 ng/ml) for 6 h and 24 h. Total cellular RNA extracted from cell cultures (more ...)
Collectively, our data provide the first experimental evidence at the cellular and molecular levels that IFN-λ has the ability to suppress HIV-1 infection of macrophages. There are at least two distinct mechanisms involved in IFN-λ-mediated anti-HIV-1 activity: the induction of extracellular factors, e.g., CC chemokines, which block HIV-1 entry into macrophages, and the activation of intracellular innate immunity, e.g., the induction of type I IFNs and anti-HIV-1 cellular factors (APOBEC3G and APOBEC3F). These anti-HIV-1 mechanisms of IFN-λ action offer an attractive alternative for HIV-1 therapy, as it would be difficult for HIV-1 to develop resistance to the actions at both the extracellular and intracellular levels in the context of innate immune activation. However, future studies are needed to determine the impact of IFN-λ on HIV-1 infection and replication in the ex vivo and in vivo systems. Study of the antiviral properties of IFN-λ will be essential not only for our understanding of the fundamental and biological functions of IFN-λ but also for exploring the potential to develop IFN-λ-based treatment for HIV-1 infection.