Cocaine-mediated impact on HIV infection in humanized BLT mice

Abstract

Cocaine abuse has been shown to have broad-ranging effects on human immunity. With regards to HIV infection, in vitro studies have shown that cocaine enhances infection of stimulated lymphocytes. Moreover, cohort studies in the pre- and post-HAART era have linked stimulant abuse with increased HIV pathogenesis. The latter data, however, have been undermined by a series of confounding factors underscoring the importance of controlled in vivo models to fully assess the impact of cocaine use and abuse on HIV infection and pathogenesis. Here, we have infected humanized mice with HIV-1 following acute cocaine exposure to assess the impact on infection. Stimulant exposure resulted in increased inflammatory cytokine expression, accelerated HIV infection, while blunting effector function of cytotoxic T lymphocytes. These data demonstrate cocaine's multifactorial impact on HIV infection that extends beyond high-risk behavior.

Figure 1. A schematic diagram of the experimental set up.

BLT mice were generated and treated as described in the Materials and Methods section. Cocaine treated and untreated mice were sacrificed at two weeks after infection at which we harvested blood, spleen and thymus to assess levels of HIV infection and characterize immune cells.

Figure 2. Five day acute cocaine exposure results in subtle but significant changes in CD4 T cell phenotype.

(a-c) Blood samples collected after 5 days of cocaine exposure were used for flow cytometric analysis of T cell activation markers on CD4 T cells. There were no changes on the expression of CD25, CD69 and CD38. (d) Blood samples were also tested for CCR5 expression. The data are from three mouse cohorts. Cocaine treated animals demonstrated increased levels of the receptor (Cocaine – n = 20, Non – n = 12). (e-f) Plasma from the same mice was collected and used to examine levels of Th1/Th2/Th17 cytokines (Cocaine – n = 20, Non – n = 12). For all figures, *p < 0.05, **p < 0.01, using Mann-Whitney or Kruskal-Wallis test (for 3 or more groups).

Figure 3. Acute cocaine exposure results in accelerated HIV infection of humanized mice.

(a) After 5-day treatment, mice were infected with HIV_89.6 followed by continued treatment with cocaine or saline for two more weeks. At the end of two weeks, mice were sacrificed; plasma samples were tested for levels of viral RNA. Cocaine treated mice had higher viral loads and a higher fraction of them had detectable levels of virus. (b) Samples from spleen and blood were tested for levels of viral cDNA the proportion of mice with detectable levels of viral cDNA were higher in the cocaine treated animals. (c) A higher fraction of cocaine treated animals had detectable levels of tat-rev mRNA in both spleen and blood. For all figures, *p < 0.05, **p < 0.01, using Mann-Whitney or Kruskal-Wallis test (for 3 or more groups). For proportion data, we used a Chi-square test.

Figure 4. Increased activation persists in the cocaine treated, HIV infected animals.

Splenocytes from the sacrificed mice were also used for flow cytometric analysis of lymphocyte phenotype. The COCAINE-HIV group of mice has higher levels of (a) CD4CD38 and (b) CD8CD38. (c) To assess functionality of CD8 T cells, we stimulated ex vivo splenocytes in the presence of GolgiPlug to measure levels of IFN-γ production. The COCAINE-HIV group had comparable levels to HIV despite the higher numbers of CD8CD38 T cells. For all figures, *p < 0.05, **p < 0.01, using Mann-Whitney or Kruskal-Wallis test (for 3 or more groups).

Figure 5. HIV-specific CTL are deficient in their ability to degranulate CD107a, form conjugates and kill HIV-infected cells upon treatment with cocaine.

(a) HIV-specific CTL were co-incubated with HIV-expressing U1 cells at an 1:1 effector to target ratio or were treated with 10 μg/ml PHA and 100 U/ml IL-2 for 6 hr in the presence of protein transport inhibitors and antibodies to CD107a. Cocaine treatment of CTL resulted in a two-fold reduction in CD107a degranulation when CTL were co-cultured with HIV-infected cells and 26% less CD107a degranulation when CTL were maximally stimulated with PHA and IL-2 treatment. (b and c) HIV-specific CTL were co-incubated with HIV-expressing U1 cells at the defined effector to target ratios for 4 hr and subsequently assessed for their ability to induce (b) conjugate formation with or (c) apoptosis in target U1 cells. Cocaine treatment of CTL resulted in significantly impaired conjugate formation at all E:T ratios tested, which was further underscored by the significant loss in apoptosis induction of U1 target cells. Statistical significance was assessed by two-way ANOVA analysis, *p < 0.05, **p < 0.01, ***p < 0.001.