Morphine suppresses IFN signaling pathway and enhances AIDS virus infection

Abstract

Background: Opioids exert a profound influence on immunomodulation and enhance HIV infection and replication. However, the mechanism(s) of their action remains to be determined. We thus investigated the impact of morphine on the intracellular innate antiviral immunity.

Methodology/principal findings: Seven-day-cultured macrophages were infected with equal amounts of cell-free HIV Bal or SIV Delta(B670) for 2 h at 37°C after 24 h of treatment with or without morphine. Effect of morphine on HIV/SIV infection and replication was evaluated by HIV/SIV RT activity assay and indirect immunofluorescence for HIV p24 or SIV p28 antigen. The mRNA expression of cellular factors suppressed or induced by morphine treatment was analyzed by the real-time RT-PCR. We demonstrated that morphine treatment of human blood monocyte-derived macrophages significantly inhibited the expression of interferons (IFN-α, IFN-β and IFN-λ) and IFN-inducible genes (APOBEC3C/3F/3G and 3H). The further experiments showed that morphine suppressed the expression of several key elements (RIG-I and IRF-7) in IFN signaling pathway. In addition, morphine treatment induced the expression of suppressor of cytokine signaling protein-1, 2, 3 (SOCS-1, 2, 3) and protein inhibitors of activated STAT-1, 3, X, Y (PIAS-1, 3, X, Y), the key negative regulators of IFN signaling pathway.

Conclusions: These findings indicate that morphine impairs intracellular innate antiviral mechanism(s) in macrophages, contributing to cell susceptibility to AIDS virus infection.

Figure 1. Morphine enhances HIV Bal strain (A) and SIV Delta_B670 strain (B) infection of macrophages.

Seven-day-cultured macrophages were incubated with or without morphine (10⁻¹⁰ M) for 24 h before HIV or SIV infection. An opioid receptor antagonist, naltrexone (10⁻⁸ M) was added to macrophage cultures 1 h before morphine (10⁻¹⁰ M) treatment. HIV or SIV RT activity in culture supernatant was determined at day 6 postinfection. Data are expressed as HIV (A) and SIV (B) RT activity in morphine-treated cells (percentage of control) to those in untreated cells, morphine-treated cells plus naltrexone versus morphine only. The results represent the mean ± SD of three experiments using cells from three different donors. Statistical analysis was performed using one-way analysis of variance, and significance is shown with * P<0.05 (morphine vs control or morphine vs morphine + naltrexone).

Figure 2. Effects of morphine on HIV p24 protein or SIV p28 protein expression in macrophages.

Seven-day-cultured macrophages were treated with or without morphine (10⁻¹⁰ M) for 24 h and then incubated with HIV Bal strain or SIV Delta_B670 strain for 2 h in the presence or absence of morphine (10⁻¹⁰ M). HIV p24 (A) or SIV p28 (B) protein expression in macrophages at day 15 postinfection was determined by immunofluoresence staining with antibodies against HIV p24 or SIV p28 protein (green). The nuclei were stained with Hoechst 33342 (blue) (magnification, 100×; scale bar: 100 µm).

Figure 3. Dose-dependent and time-course effects of morphine on AIDS virus replication.

A and C: Dose-dependent effect of morphine on HIV or SIV replication. Seven-day-cultured macrophages were treated with or without morphine at indicated concentrations for 24 h and then incubated with HIV Bal or SIV Delta_B670 strain for 2 h in the presence or absence of morphine. Day 6 culture supernatant was collected for HIV (A) or SIV (C) RT assay. B and D: Time-course effect of morphine on HIV or SIV. Seven-day-cultured macrophages were treated with or without morphine (10⁻¹⁰ M) for 24 h prior to infection with HIV Bal strain or SIV Delta_B670 strain for 2 h and then cultured for 15 days. HIV (B) or SIV (D) RT activity was determined in cultured supernatants at indicated time points postinfection. Data are expressed as HIV or SIV RT activity in morphine-treated cells (percentage of control) compared with those in untreated cells. The results represent the mean ± SD of three independent experiments using macrophages from three different donors. Statistical analysis was performed by one-way analysis of variance (A, C) or Student's t-test (B, D), and significance is shown morphine versus control with * (P<0.05) and ** (P<0.01).

Figure 4. Effect of naltrexone on morphine-mediated down-regulation of IFNs expression.

Seven-day-cultured macrophages were incubated with or without naltrexone (10⁻⁸ M) for 1 h before treatment with or without morphine (10⁻¹⁰ M) for 3 h. Cellular RNA was subject to the real-time RT-PCR for IFN-α (A), IFN-β (B) and IFN-λ1 mRNA (C). Data are expressed as mRNA levels in morphine treated cells (percentage of control) those untreated cells and morphine treated cells. The results represent the mean ± SD of three independent experiments. Statistical analysis was performed using one-way analysis of variance, and significance is shown with *P<0.05 (morphine vs control or morphine vs morphine + naltrexone).

Figure 5. Effect of morphine on TLRs, RIG-I (A) and IRFs (B) expression.

Seven-day-cultured macrophages were treated with or without morphine (10⁻¹⁰ M) for 3 h, and then cellular RNA was subjected to the real-time RT-PCR for mRNA detection. Data are expressed as mRNA levels in morphine-treated cells (percentage of control) to those in untreated cells. The results represent the mean ± SD of three independent experiments. Statistical analysis was performed by Student's t-test and significance is shown with *P<0.05 (morphine vs control).

Figure 6. Effect of morphine on APOBEC3B/C/F/G/H mRNA expression.

Seven-day-cultured macrophages were treated with or without morphine (10⁻¹⁰ M) for 3 h, and then cellular RNA was subjected to the real-time RT-PCR for mRNA detection. Data are expressed as mRNA levels in morphine-treated cells (percentage of control) to those in untreated cells. The results represent the mean ± SD of three independent experiments. Statistical analysis was performed by Student's t-test and significance is shown with *P<0.05 (morphine versus control).

Figure 7. Effects of morphine on SOCS-1, 2, 3 (A), PIAS-1, 3, X and Y (B) expression.

Seven-day-cultured macrophages were treated with or without morphine (10⁻¹⁰ M) for 3 h, and cellular RNA was subjected to the real-time RT-PCR for mRNA detection. Data are expressed as mRNA levels in morphine-treated cells (percentage of control) to those in untreated cells. The results represent the mean ± SD of three independent experiments. Statistical analysis was performed by Student's t-test and significance is shown with *P<0.05 (morphine versus control).