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. 2014 Jul 2:11:17.
doi: 10.1186/1742-6405-11-17. eCollection 2014.

The effects of opioids on HIV reactivation in latently-infected T-lymphoblasts

Johannes Prottengeier  1 , Eleni Koutsilieri  2 , Carsten Scheller  2
Affiliations

The effects of opioids on HIV reactivation in latently-infected T-lymphoblasts

Johannes Prottengeier et al. AIDS Res Ther. .

Abstract

Background: Opioids may have effects on susceptibility to HIV-infection, viral replication and disease progression. Injecting drug users (IDU), as well as anyone receiving opioids for anesthesia and analgesia may suffer the clinical consequences of such interactions. There is conflicting data between in vitro experiments showing an enhancing effect of opioids on HIV replication and clinical data, mostly showing no such effect. For clarification we studied the effects of the opioids heroin and morphine on HIV replication in cultured CD4-positive T cells at several concentrations and we related the observed effects with the relevant reached plasma concentrations found in IDUs.

Methods: Latently-infected ACH-2 T lymphoblasts were incubated with different concentrations of morphine and heroine. Reactivation of HIV was assessed by intracellular staining of viral Gag p24 protein and subsequent flow cytometric quantification of p24-positive cells. The influence of the opioid antagonist naloxone and the antioxidants N-acetyl-cysteine (NAC) and glutathione (GSH) on HIV reactivation was determined. Cell viability was investigated by 7-AAD staining and flow cytometric quantification.

Results: Morphine and heroine triggered reactivation of HIV replication in ACH-2 cells in a dose-dependent manner at concentrations above 1 mM (EC50 morphine 2.82 mM; EC50 morphine 1.96 mM). Naloxone did not interfere with heroine-mediated HIV reactivation, even at high concentrations (1 mM). Opioids also triggered necrotic cell death at similar concentrations at which HIV reactivation was observed. Both opioid-mediated reactivation of HIV and opioid-triggered cell death could be inhibited by the antioxidants GSH and NAC.

Conclusions: Opioids reactivate HIV in vitro but at concentrations that are far above the plasma levels of analgesic regimes or drug concentrations found in IDUs. HIV reactivation was mediated by effects unrelated to opioid-receptor activation and was tightly linked to the cytotoxic activity of the substances at millimolar concentrations, suggesting that opioid-mediated reactivation of HIV was due to accompanying effects of cellular necrosis such as activation of reactive oxygen species and NF-κB.

Keywords: ACH-2; HIV; Heroine; Morphine; Naloxone; Opioids; Reactivation.

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Figures

Figure 1
Figure 1
Opioids activate HIV replication in vitro. Latently-HIV-infected ACH-2 T lymphoblasts were cultured for 24 hours in the presence of different concentrations of morphine (A) or heroin (B). HIV replication was quantified by intracellular staining of HIV p24-antigen and flow cytometry. C, D: representative dot-plot analyses of ACH-2 cells left untreated (C) or treated with 4 mM heroin (D). The x-axis displays the forward scatter, the y-axis displays p24-expression. Pink rectangles represent the gates for p24-positive cells and the small numbers within indicate the proportion (%) of p24-positive cells from the total number of events. E: Heroin has no effects on HIV reactivation at concentrations found in plasma of IDUs (1-10 μM). F: Latently-HIV-infected ACH-2 T lymphoblasts were cultured for 24 hours in the presence of 5 mM heroin, together with different concentrations of naloxone (0 μM and 0.1μM – 1 mM). HIV replication was quantified by intracellular staining of HIV p24-antigen and flow cytometry. Data as mean ± S.E.M. from duplicates and linear regression. The slopes of both regressions are positive and significantly differ from = zero (p = 0.0008 for medium and p = 0.0152 for heroin).
Figure 2
Figure 2
Antioxidants inhibit opioid-mediated reactivation of HIV. Latently-HIV-infected ACH-2 T lymphoblasts were cultured for 24 hours with of heroin (A, C) or morphine (B, D) in the presence or absence of the antioxidants N-acetylcystein (NAC) (A, B) or glutathione (C, D). HIV replication was quantified by intracellular staining of HIV p24-antigen and flow cytometry.
Figure 3
Figure 3
Opioid-mediated reactivation of HIV is tightly linked to cellular necrosis. Latently-HIV-infected ACH-2 T lymphoblasts were cultured for 24 hours with of heroin in the presence or absence of glutathione. HIV replication was quantified by intracellular staining of HIV p24-antigen and flow cytometry (A). Cell viability was assessed by FSC/SSC-analysis in flow cytometry (B). Representative dot plots of cell viability analysis (C) and p24-expression (D) at different heroin concentrations. Pink rectangles on the right represent the gates for p24-positive cells and the small numbers within indicate the proportion (%) of p24-positive cells from the total number of events. Pink regions on the left represent the gates for living and necrotic cells and the small numbers within indicate the proportion (%) of events within the region from the total number of events.
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