Effect of mild-to-moderate smoking on viral load, cytokines, oxidative stress, and cytochrome P450 enzymes in HIV-infected individuals

Abstract

Mild-to-moderate tobacco smoking is highly prevalent in HIV-infected individuals, and is known to exacerbate HIV pathogenesis. The objective of this study was to determine the specific effects of mild-to-moderate smoking on viral load, cytokine production, and oxidative stress and cytochrome P450 (CYP) pathways in HIV-infected individuals who have not yet received antiretroviral therapy (ART). Thirty-two human subjects were recruited and assigned to four different cohorts as follows: a) HIV negative non-smokers, b) HIV positive non-smokers, c) HIV negative mild-to-moderate smokers, and d) HIV positive mild-to-moderate smokers. Patients were recruited in Cameroon, Africa using strict selection criteria to exclude patients not yet eligible for ART and not receiving conventional or traditional medications. Those with active tuberculosis, hepatitis B or with a history of substance abuse were also excluded. Our results showed an increase in the viral load in the plasma of HIV positive patients who were mild-to-moderate smokers compared to individuals who did not smoke. Furthermore, although we did not observe significant changes in the levels of most pro-inflammatory cytokines, the cytokine IL-8 and MCP-1 showed a significant decrease in the plasma of HIV-infected patients and smokers compared with HIV negative non-smokers. Importantly, HIV-infected individuals and smokers showed a significant increase in oxidative stress compared with HIV negative non-smoker subjects in both plasma and monocytes. To examine the possible pathways involved in increased oxidative stress and viral load, we determined the mRNA levels of several antioxidant and cytochrome P450 enzymes in monocytes. The results showed that the levels of most antioxidants are unaltered, suggesting their inability to counter oxidative stress. While CYP2A6 was induced in smokers, CYP3A4 was induced in HIV and HIV positive smokers compared with HIV negative non-smokers. Overall, the findings suggest a possible association of oxidative stress and perhaps CYP pathway with smoking-mediated increased viral load in HIV positive individuals.

Fig 1. Determination of viral loads in HIV-infected subjects (A) and HIV-infected macrophages (B).

The viral loads in the plasma of human subjects and macrophages were determined by analyzing HIV RNA using q-RTPCR and p24 using ELISA, respectively. The data for in vitro assay (B) represents mean of multiple analysis of p24 titer obtained from HIV-infected macrophages from three different donors. The p values (# and ** represent p≤0.1 and p≤0.01, respectively) are calculated using one way-ANOVA and presented in the graph.

Fig 2. Box and whisker plots of cytokine/chemokine levels in plasma of HIV negative non-smokers (HEALTHY), HIV positive non-smokers (HIV), HIV negative smokers (SMOKER), and HIV positive smokers (HIV SMOKER) groups.

The box represents the 25^th-75^th quartile, the whiskers represent the range of values, the median is presented as a line inside the box, and the out of range values are presented as circles or stars above and below the whiskers. The p values (* and ** represent p≤0.05 and p≤0.01, respectively) are calculated using one way-ANOVA and presented above the bars in the graph. The concentrations of RANTES, IL-6, IL-8, MCP-1 and TNF-α were shown in the figure.

Fig 3. Measurement of oxidative stress in plasma and monocyte samples of HIV negative non-smokers (HEALTHY), HIV positive non-smokers (HIV), HIV negative smokers (SMOKER), and HIV positive smokers (HIV SMOKER) groups.

The 8-OHdG contents are plotted as a bar graph for each category, and the p values ≤0.05 and ≤0.01 are represented as * and **, respectively. * represents the significance with respect to HEALTHY, # represents the significance with respect to HIV and @ represents the significance with respect to SMOKER in HIV SMOKER group.

Fig 4. Determination of antioxidant gene expression in HIV negative non-smokers (HEALTHY), HIV positive non-smokers (HIV), HIV negative smokers (SMOKER), and HIV positive smokers (HIV SMOKER) groups.

The mRNA expressions of SOD1, SOD2, catalase, and Nrf2 were measured by qRTPCR. A one way-ANOVA was employed to calculate the p value (* represent p≤0.05) with respect to HEALTHY in all the groups.

Fig 5. Determination of CYP levels in HIV negative non-smokers (HEALTHY), HIV positive non-smokers (HIV), HIV negative smokers (SMOKER), and HIV positive smokers (HIV SMOKER) groups.

The expression levels of CYP2A6, CYP3A4, and CYP2E1 mRNA were determined by qRTPCR in all the groups. A one way-ANOVA was employed to calculate the p value (** represent p≤0.01; # represents p≤0.1, borderline significance) with respect to HEALTHY in all the groups.