Human peripheral blood was obtained from healthy adult donors with no history of drug abuse. Written informed consent was signed by all the study participants. The Research Ethics Committee of School of Basic Medical Sciences of Wuhan University approved this project. Purification of monocytes was performed as described previously (27). Briefly, blood was layered over lymphocyte separation medium (Alere Technologies AS, Oslo, Norway) and centrifuged at 800 g for 30 min at room temperature. The gradient of peripheral blood mononuclear cells (PBMC) were obtained above the Ficoll layer, carefully aspirated, and then transferred to gelatin-coated flasks for cellular adherence. After 45 min incubation in 5% CO₂ at 37°C, flasks were washed 8–10 times with DMEM to eliminate non-adherent cells. Cells were then exposed to 10 mM EDTA in DMEM containing 20% fetal calf serum. Freshly isolated monocytes were cultured in 48-well plates at a density of 2.5 × 10⁵ cells/well in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% FBS, 2 mM glutamine, 100 U/mL streptomycin/penicillin and 1 μM GM-CSF. The medium was replaced at 2–3 days intervals, and monocyte-derived macrophages were obtained after 7 days culture. Macrophages were stained with fluorescence-conjugated anti-human CD14 antibody and analyzed for CD14 expression by flow cytometry. The purity of the macrophages was >95% according to flow cytometry analysis (Figure S1).

The influence of methadone on cell viability was examined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. Macrophages were first seeded in 96-well plates and then treated with methadone at the indicated concentrations (0, 1, 10, 25, 50, 100, and 200 μM) for 24 h. MTT solution (20 μL, 5 mg/mL, Sigma) was added to the wells and re-incubated for 4 h at 37°C. Then medium was removed and 100 μL of dimethyl sulfoxide (Sigma) was added into each well. The optical density (OD) at 560 nm was measured by an ELISA reader. All assays were repeated three times.

Methadone was kindly provided by Drug Rehabilitation Center of Wuhan Centers for Disease Control and Prevention. The macrophage-tropic R5 strain (Bal) was maintained in our laboratory and routinely propagated in primary human macrophages. Macrophages (2.5 × 10⁵ cells/well) were treated with different concentrations (0.01, 0.1, 1.0, 5.0, and 10.0 μM) of methadone for 24 h before infection with HIV. Naltrexone (1 μM, 3B Scientific Corporation, Wuhan, China), a μ-opioid receptor antagonist, was added to the macrophage cultures for 1 h prior to methadone treatment. After drug pre-treatment, the cells were infected with HIV (Bal strain, p24, 10 ng/10⁶ cells) for 2 h at 37°C and then washed with DMEM to remove the unabsorbed virus. Fresh medium containing methadone was added to cell cultures every 3 days. The cell culture supernatants were collected to assess HIV p24 production by using p24 ELISA kit (Zeptometrix Corp., Buffalo., NY., USA) at 2, 4, 8, and 12 days post infection (dpi). Cellular RNA and lysates were collected at the indicated time points for subsequent gene and protein detection, respectively.

Total RNA from macrophages was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instruction. Total RNA (1 μg) was subjected to reverse transcription using Moloney murine leukemia virus (M-MLV) reverse transcriptase (Promega, Madison, WI, USA) with random primers for 1 h at 42°C. The reaction was terminated by incubating the reaction mixture at 72°C for 10 min and then kept at 4°C. Total DNA from macrophages was extracted with Genomic DNA kit (CWBIO, Jiangsu, China) according to the manufacturer's instruction. The resulting cDNA or DNA was then used as a template for real-time PCR quantification with the iTaq Universal SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) on a CFX Connect Real-Time PCR System (Bio-Rad). Primers sequences used in PCR reactions are shown in Table 1 and synthesized by Genecreate Biological Engineering Co (Wuhan, China). Gene expression was normalized to GAPDH and calculated using the 2^−ΔΔCt method. The U6 gene was used as an endogenous control for miRNAs detection.

Macrophages were washed with ice-cold PBS three times and lysed in RIPA with 1% protease inhibitor PMSF. Protein concentrations were determined by BCA protein assay. Proteins were resuspended in SDS-PAGE Sample Loading Buffer (Biosharp, Hefei, Anhui, China; BL502A) and heated for 5 min at 95°C. The equivalent amounts of protein (50 μg) for each sample were then resolved by 12% SDS-PAGE and transferred to PVDF membranes. The blots were subsequently immunoblotted with the following antibodies: anti-APOBEC3G (1:500; Santa Cruz Biotechnology, Santa Cruz, California, USA; sc-48820), anti-APOBEC3F (1:200; Santa Cruz; sc-46725), anti-MxB (1:1000; Santa Cruz; sc-271527), and anti-GAPDH (1:5000; Sungene biotech, Tianjin, China; KM9002). The blots were incubated with the HRP-Goat anti-Rabbit secondary antibody (1:5000; Cell Signaling Technology, Shanghai, China; 7074s) or HRP-Goat anti-mouse secondary antibody (1:5000; Cell Signaling Technology, Shanghai; 7076), respectively. The bound antibodies were detected using Electro-Chemi-Luminescence Substrate Kit (Wuhan Servicebio Technology Co, Wuhan, Hubei, China), and the signal intensities of protein bands were analyzed by ImageJ software (Dr. Wayne Basband, National Institutes of Health, Bethesda, MD, USA). Human MIP-1β, IFN-β, IFN-λ2 (4A Biotech, Beijing, China) and viral particles p24 (Zeptometrix Corp. Buffalo. NY. USA) in the supernatants were detected using ELISA kits according to the manufacturer's instruction.

Student's t-test was used to evaluate the significance of difference between groups, and multiple comparisons were performed by regression analysis and one-way analysis of variance. Statistical analyses were performed with Prism Software, and all data were presented as mean ± SD. Statistical significance was defined as p < 0.05.

Macrophages were treated with different concentrations of methadone (10–200 μM), and drug cytotoxicity was examined by MTT assay. Methadone treatment had little effect on viability of macrophages at concentrations lower than 10 μM and the median cytotoxic concentration (CC₅₀) of methadone was 437.56 μM (Table 2). Thus, we utilized methadone at concentrations of 10 μM or lower throughout the following experiments.

To evaluate effect of methadone on HIV infection/replication, macrophages were incubated in the presence or absence of different concentrations of methadone (0.01, 0.1, 1.0, 5.0, and 10.0 μM) for 24 h and then challenged with HIV Bal strain for 2 h. As shown in Figure 1A, methadone significantly enhanced HIV GAG gene expression in macrophages in a dose-dependent manner at 8 dpi. In addition, methadone-treated macrophages showed higher levels of p24 protein compared to untreated cells (Figure 1B) at 8 dpi. Methadone (1 μM) significantly enhanced HIV GAG gene expression and p24 protein in macrophages in a time-dependent manner (Figures 1C,D). Pretreatment of macrophages with naltrexone, a μ-opioid receptor antagonist, blocked the methadone-induced upregulation of HIV p24 expression (Figure 1E) at 8 dpi, which indicated that this effect of methadone on HIV infection was acting through the opioid receptors.

Previous studies have demonstrated CCR5, the key coreceptor for HIV entry into the target cells, was elevated in methadone-treated macrophages compared to untreated cells (23). Here we also verified this phenomenon of methadone enhancement on CCR5 at both RNA and protein levels (Figure S2). Thus, we tested how methadone affected the early steps of HIV replication (virus entry and reverse transcription). Strong Stop DNA, the early product of reverse transcription synthesized shortly after viral entry, was used for the assessment of viral entry (28). Late-RT DNA was applied to monitor formation of reverse transcription products right after HIV entered the cell (29). As shown in Figure 1F, methadone enhanced HIV-1 Strong Stop DNA and reverse transcription DNA at 24 hpi, which implied that the critical step of HIV-1 infection elevated by methadone treatment occurred right after virus entry and initiation of reverse transcription.

Since CC chemokines (MIP-1α, MIP-1β, and RANTES) are the natural ligands for CCR5 (30), we investigated the effect of methadone on CC chemokines expression in macrophages. Macrophages were incubated with methadone for 24 h, and the intracellular gene expression of the CC chemokines (MIP-1α, MIP-1β, and RANTES) and protein level of the MIP-1β in the whole cell lysate were quantified by real-time RT-PCR and ELISA assay, respectively. As shown in Figure 2, although methadone had little effects on MIP-1α and RANTES (Figures 2A,B), methadone effectively inhibited the expression of MIP-1β in macrophages (75.5 ± 2.5% reduction in mRNA level; and 14.3 ± 2.8% reduction in protein level). Pretreatment of macrophages with naltrexone prior to methadone treatment eliminated the suppressive effect of methadone on MIP-1β expression at both mRNA and protein levels (the 4th column, Figures 2C,D), whereas the cell culture treated with naltrexone alone were not affected as to the CC chemokine expression (the 3rd column, Figures 2C,D). It is worth noting that methadone had the similar effect in HIV-infected macrophages regarding the inhibitory effect on MIP-1β expression at 8 dpi (the fifth and sixth columns, Figures 2C,D).

Since IFNs play an important role in host cell innate immunity against viral infections (31–34), we examined the effect of methadone on IFN expression in macrophages. Methadone treatment of macrophages significantly suppressed the expression of IFN-β (36.9 ± 0.7% reduction in mRNA level, Figure 3A; 53.8 ± 10% reduction in protein level, Figure 3C) and IFN-λ2 (50.8 ± 2.1% reduction in mRNA level, Figure 3A; 49.5 ± 5.3% reduction in protein level, Figure 3C), respectively. Pretreatment of macrophages with naltrexone dismissed the inhibitory effect of methadone on IFN-β and IFN-λ2 expression at both mRNA and protein levels, and naltrexone alone had little effect on IFN-β and IFN-λ2. However, methadone had little effect on the expression of IFN-α and IFN-γ in macrophages in our observation (Figure 3B). Moreover, we also found that methadone treatment of macrophages suppressed the expression of IFN-β and IFN-λ2 in the context of HIV infection (the fifth and sixth columns, Figures 3A,B).

Host restriction factors are an important and potent class of intracellular components to block to viral replication and initiate the innate immune response to viral infection. These host proteins are often induced by interferon signaling and antagonized by viral factors (35, 36). We further investigated the effect of methadone on the expression of APOBEC3F, APOBEC3G, and MxB in macrophages. We found macrophages treated with methadone showed significant reduction on the expressions of APOBEC3F (47.4 ± 15.6% reduction), APOBEC3G (40 ± 21.5% reduction), and MxB (60.7 ± 6.5% reduction) compared to untreated cells (Figure 4A). Western blot analysis further confirmed the methadone action on the protein production of APOBEC3G, APOBEC3F, and MxB, with 32.3 ± 3.8%, 43.9 ± 11%, and 61.8 ± 20% of downregulation, respectively (Figure 4B). The inhibitory effects of methadone can be blocked by pretreatment of macrophages with naltrexone (Figure 4). It is important to note that during HIV infection, methadone treatment of macrophages reduced the expression of APOBEC3G, APOBEC3F, and MxB in both mRNA and protein levels (Figures 4D–F).

Previous studies have shown that certain cellular miRNAs contribute to restriction of HIV replication in CD4+ T lymphocytes (37), monocytes and macrophages (38). Thus, we assessed the effect of methadone on the expression of the HIV restriction miRNAs (miRNA-28, miRNA-125b, miRNA-150, and miRNA-155) in macrophages. We demonstrated that methadone reduced the expression of these miRNAs compared to untreated cells (Figure 5). Pretreatment of macrophages with naltrexone diminished the suppressive effect of methadone on the HIV restriction miRNAs, whereas naltrexone alone had little effect on these miRNAs (Figure 5).

We already revealed the inhibitory effect of methadone in IFN production as described above, which raises an interesting question whether this IFN downregulation is directly linked to host restriction factors and miRNAs modulation. We utilized exogenous IFN-β treatment on methadone-treated cells and found that IFN-β restored the decreased gene expression of IFN-β caused by methadone (Figure 6A). Consequently, reduced ISGs due to methadone treatment, including APOBEC3G, APOBEC3F, and MxB, could be recovered by IFN-β addition at the mRNA (Figures 6C–E) and protein level (Figure 6B). A pack of HIV restriction miRNAs (miR-125b, miR-150, and miR-155) also reached elevated levels after IFN-β treatment (Figures 6F–H). Therefore, we concluded that IFN-β addition reverted the inhibitory effect of methadone on restriction factors (APOBEC3G, APOBEC3F, and MxB) and miRNAs (miR-125b, miR-150, and miR-155).