Alcohol potentiates HIV-1 infection of human blood mononuclear phagocytes

Abstract

Background: Acute and chronic alcohol abuse impairs various functions of the immune system and thus has been implicated as a cofactor in HIV infection. The mechanisms by which alcohol affects the function of human immune cells that are the targets for HIV are unknown.

Methods: Human blood monocyte-derived macrophages (MDM) were incubated with or without alcohol (10-40 mM) for 24 hr and then infected with HIV for 24 hr. Culture supernatants were harvested for HIV reverse transcription assay. HIV entry receptor (CCR5, CD4, and CXCR4) expression was determined by reverse transcription-polymerase chain reaction and flow cytometry assays. Beta-chemokines were analyzed using enzyme-linked immunosorbent assay. Different HIV strains (Bal, SF-162, 89.6, and UG024) were used for infection experiments. In addition, ADA (macrophage-tropic strain) and murine leukemia virus envelope-pseudotyped HIV infection was carried out.

Results: Although alcohol had little effect on HIV T-lymphocyte-tropic strain infection, it significantly enhanced HIV R5 strain infection in MDM. The enhancing effect of alcohol on the HIV R5 strain was further evidenced by the observation that the R5 (ADA) strain envelope-pseudotyped HIV infection is markedly increased by alcohol, whereas murine leukemia virus envelope-pseudotyped HIV infection was not affected. Alcohol significantly up-regulated CCR5 receptor expression and inhibited the endogenous production of beta-chemokines by MDM.

Conclusion: Alcohol, through the down-regulation of beta-chemokine production and the up-regulation of CCR5 receptor expression, enhances HIV R5 strain infection of MDM and may have an important role as a cofactor in the progression of HIV disease.

Fig. 1

(A) Effect of alcohol on HIV Bal strain infection of MDM. Seven-day-cultured MDM were incubated with alcohol at the concentrations as indicated for 24 hr and then challenged with HIV Bal strain for 24 hr in the presence of alcohol. Cultures without the addition of alcohol served as the control. Fresh medium containing alcohol was added every 4 days. Day 8 culture supernatants were collected for HIV RT assay. The results shown are mean ± SEM of triplicate cultures and are representative of experiments using cells from three different donors (**p < 0.01, *p < 0.05, alcohol versus control). (B) Effect of alcohol on HIV Bal gag gene expression in MDM. MDM was treated with or without alcohol at the indicated concentrations for 24 hr and then infected with HIV Bal strain. Total cellular RNA was extracted from the cell culture 72 hr after infection and then subjected to RT-PCR. The data shown are representative of three independent experiments.

Fig. 2

Effect of alcohol on different strains of HIV infection of MDM. Different HIV strains were used to infect 7-day-cultured MDM with or without alcohol pretreatment (40 mM). HIV RT activity in the alcohol-treated and HIV-infected MDM was expressed as a percentage of that of untreated and HIV (corresponding strains)-infected MDM controls (without alcohol treatment), which were defined as 100%. R5, CCR5 tropic strains; X4, CXCR4 tropic strain; R5X4, dual tropic strain. The data shown are mean ± SEM of triplicate cultures and are representative of experiments using cells from three different donors (**p < 0.01, *p < 0.05, alcohol versus control).

Fig. 3

Effect of alcohol on pseudotyped HIV infection of MDM. Seven-day-cultured MDM were treated with alcohol at the concentrations as indicated for 24 hr and then challenged with recombinant luciferase-encoding HIV pseudotyped with either ADA Env or MLV Env for 24 hr. Luciferase activity was quantified in the cell lysates 72 hr after infection. The data are expressed as relative light units of alcohol-treated cells to that of controls incubated without alcohol. The results shown are mean ± SEM of triplicate cultures and are representative of experiments using cells from three different donors (**p < 0.01, *p < 0.05, alcohol versus control).

Fig. 4

Effect of alcohol on CCR5 mRNA expression in MDM. Seven-day-cultured MDM were incubated with or without alcohol at the indicated concentrations for 4 hr. Total cellular RNA was extracted from the cell cultures and subjected to RT-PCR (A) or real-time PCR (B). (A) The amplified PCR products (189 bp for CCR5) were visualized on an ethidium bromide–stained 3% agarose gel. β-actin was used to monitor the amount and integrity of RNA in each sample. (B) CCR5 cDNA transcribed from the total RNA was also subjected to real-time PCR for quantification of CCR5 mRNA copy numbers. CCR5 mRNA copy numbers were normalized with GAPDH mRNA. The results are expressed as CCR5 mRNA copies per 100,000 GAPDH mRNA. Data shown are mean ± SEM of duplicate cultures and are representative of experiments using cells from six different donors (**p < 0.01, *p < 0.05, alcohol versus control).

Fig. 5

Effect of alcohol on HIV entry receptor expression on MDM. Seven-day-cultured MDM were incubated with or without alcohol at 40 mM for 24 hr. (A) Expression of CCR5, CD4, CXCR4, and CD14 receptors on MDM was determined by flow cytometry. The results shown are the percentage of MDM positive for the receptors analyzed and are representative of six donors. The data shown are presented as the mean ± SEM of triplicate cultures (*p < 0.05, alcohol versus control). (B) Expression of CCR5 receptor on MDM was analyzed by flow cytometry. The shaded histogram represents control staining with isotype-match antibody (IgG 2a). The open histogram represents CCR5 expression using FITC-conjugated anti-CCR5 antibody. The results shown are percentage of CCR5-positive cells and are representative of six donors.

Fig. 6

Effect of alcohol on β-chemokine production in MDM. Seven-day-cultured MDM were incubated with or without alcohol at indicated concentrations for 24 hr. Culture supernatants were collected 24 hr posttreatment for analysis of MIP-1α (A) and MIP-1β (B) production as determined by ELISA. The data shown are presented as the mean ± SEM of triplicate cultures (**p < 0.01, *p < 0.05, alcohol versus control) and are representative of experiments using cells from six donors.