Oxidative Stress in HIV Infection and Alcohol Use: Role of Redox Signals in Modulation of Lipid Rafts and ATP-Binding Cassette Transporters

Abstract

Aims: Human immunodeficiency virus (HIV) infection induces oxidative stress and alcohol use accelerates disease progression, subsequently causing immune dysfunction. However, HIV and alcohol impact on lipid rafts-mediated immune dysfunction remains unknown. In this study, we investigate the modulation by which oxidative stress induces reactive oxygen species (ROS) affecting redox expression, lipid rafts caveiloin-1, ATP-binding cassette (ABC) transporters, and transcriptional sterol regulatory element-binding protein (SREBP) gene and protein modification and how these mechanisms are associated with arachidonic acid (AA) metabolites in HIV positive alcohol users, and how they escalate immune dysfunction.

Results: In both alcohol using HIV-positive human subjects and in vitro studies of alcohol with HIV-1 gp120 protein in peripheral blood mononuclear cells, increased ROS production significantly affected redox expression in glutathione synthetase (GSS), super oxide dismutase (SOD), and glutathione peroxidase (GPx), and subsequently impacted lipid rafts Cav-1, ABC transporters ABCA1, ABCG1, ABCB1, and ABCG4, and SREBP transcription. The increased level of rate-limiting enzyme 3-hydroxy-3-methylglutaryl HMG-CoA reductase (HMGCR), subsequently, inhibited 7-dehydrocholesterol reductase (DHCR-7). Moreover, the expression of cyclooxygenase-2 (COX-2) and lipoxygenase-5 (5-LOX) mRNA and protein modification tentatively increased the levels of prostaglandin E2 synthases (PGE₂) in plasma when compared with either HIV or alcohol alone.

Innovation: This article suggests for the first time that the redox inhibition affects lipid rafts, ABC-transporter, and SREBP transcription and modulates AA metabolites, serving as an important intermediate signaling network during immune cell dysfunction in HIV-positive alcohol users.

Conclusion: These findings indicate that HIV infection induces oxidative stress and redox inhibition, affecting lipid rafts and ABC transports, subsequently upregulating AA metabolites and leading to immune toxicity, and further exacerbation with alcohol use. Antioxid. Redox Signal. 28, 324-337.

Keywords: ABC transporters; HIV; alcohol; immune cells; lipid rafts; redox expression.

FIG. 1.

Oxidative stress inducing ROS production and redox effect of GSS, SOD, and Catalase gene expression. PBMCs (3 × 10⁶ cells/ml) were treated with alcohol (0.1%), HIV-1 gp120 (50 ng/ml), and AL with HIV-1 gp120 at 24 h. The PBMCs were stained with DCF-DA for ROS production (A) and analyzed by a fluorescence microplate reader at λex: 485 nm and λem: 530 nm. RNA was extracted and reverse transcribed followed by quantitative real-time PCR for dose-response effects of GSS on HIV-1 gp120 (B), alcohol (C) and combined effect of GSS (D), SOD (E), Catalase (F), and housekeeping β-actin-specific primers. Data are expressed as mean ± SE of TAI values of three independent experiments. AL, alcohol; GSS, glutathione synthetase; HIV-1 gp120 protein, human immunodeficiency virus-1 envelop glycoprotein 120; NS, non-significant; PBMC, peripheral blood mononuclear cell; ROS, reactive oxygen species; SOD, super oxide dismutase; TAI, Transcript Accumulation Index.

FIG. 2.

Effect of AA metabolites COX-2 and 5-LOX gene expression in vitro and HIV-positive alcohol user. PBMCs (3 × 10⁶ cells/ml) were treated with alcohol (0.1%), HIV-1 gp120 (50 ng/ml), and AL with HIV-1 gp120 at 24 h in vitro. PBMCs were separated from healthy normal subjects, alcohol user, HIV-positive and HIV-positive AL users. RNA was extracted and reverse transcribed followed by quantitative real-time PCR for COX-2 (A), and 5-LOX (B) in vitro, and COX-2 (C) and 5-LOX (D) in patient's samples and housekeeping β-actin-specific primers were used as a control. Data are expressed as mean ± SE of TAI values of minimum three independent experiments in vitro and six samples/group in patients. The plasma PGE₂ level was analyzed by ELISA. Data are expressed as ng/ml plasma (E). 5-LOX, lipoxygenase-5; AA, arachidonic acid; COX-2, cyclooxygenase-2; ELISA, enzyme-linked immunosorbent assay; PGE₂, prostaglandin E2 synthases.

FIG. 3.

Effect of lipid rafts Cav-1, SREBP, and HMGCR gene expression. PBMCs (3 × 10⁶ cells/ml) were treated with alcohol (0.1%), HIV-1 gp120 (50 ng/ml), and AL with HIV-1 gp120 at 24 h. RNA was extracted and reverse transcribed followed by quantitative real-time PCR for Cav-1 (A), SREBP-2 (B), and HMGCR (C), and housekeeping β-actin-specific primers. Data are expressed as mean ± SE of TAI values of minimum three independent experiments. Cav-1, Caveolin-1; HMGCR, 3-hydroxy-3-methylglutaryl HMG-CoA reductase; SREBP, sterol regulatory elementbinding protein.

FIG. 4.

Effect of ABC transporters gene expression. PBMCs (3 × 10⁶ cells/ml) were treated with alcohol (0.1%), HIV-1 gp120 (50 ng/ml), and AL with HIV-1 gp120 at 24 h. RNA was extracted and reverse transcribed followed by quantitative real-time PCR for ABCA1 (A), ABCC1 (B), ABCG1 (C), ABCG4 (D), and housekeeping β-actin-specific primers. Data are expressed as mean ± SE of TAI values of three independent experiments. ABC, ATP-binding cassette.

FIG. 5.

HIV-1 gp120 with alcohol effect of redox expression. PBMCs (3 × 10⁶ cells/ml) were treated with HIV-1 gp120 (50 ng), alcohol (0.1%), and a combination of HIV-1 gp120 with AL. Controls were maintained by drug-free medium. At the end of the incubation, equal amounts of protein lysate were resolved by 4%–15% SDS-PAGE and protein expression was analyzed by Western blot showing GSS (A), GPx (B), SOD (C), COX-2 (D), and 5-LOX (E). Data presented in (F–J) show the densitometry evaluation expressed as mean ± SE of minimum three independent experiments. GPx, glutathione peroxidase.

FIG. 6.

HIV-1 gp120 with alcohol effect of lipid rafts. PBMCs (3 × 10⁶ cells/ml) were treated with HIV-1 gp120 (50 ng), alcohol (0.1%), and a combination of HIV-1 gp120 with AL. Controls were maintained by drug-free medium. At the end of the incubation, equal amounts of protein lysate were resolved by 4%–15% SDS-PAGE and protein expression was analyzed by Western blot showing phospho-Cav-1 (cytoplasm) (A), phospho-Cav-1 (membrane) (B), HMGCR (C), DCHR-7 (G), FABP (H), and SREBP (I). Data presented in (D–F and J–L) show the densitometry evaluation expressed as mean ± SE of minimum three independent experiments. DCHR-7, 7-dehydrocholesterol reductase; FABP7, fatty acid binding protein.

FIG. 7.

HIV-1 gp120 with alcohol effect of ABC transporters. PBMCs (3 × 10⁶ cells/ml) were treated with HIV-1 gp120 (50 ng), alcohol (0.1%), and a combination of HIV-1 gp120 with AL. Controls were maintained by drug-free medium. At the end of the incubation, equal amounts of protein lysate were resolved by 4%–15% SDS-PAGE and protein expression was analyzed by Western blot showing ABCA1 (A), ABCC1 (B), ABCG1 (C), and ABCG4 (D). Data presented in (E–H) show the densitometry evaluation expressed as mean ± SE of minimum three independent experiments.

FIG. 8.

The schematic pathway of HIV infection with alcohol induced oxidative stress, and redox inhibition affected lipid rafts-mediated downstream signaling network AA metabolites and ABC transporters associated immune dysfunction and disease progression. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars