HIV-1 Tat and opioids act independently to limit antiretroviral brain concentrations and reduce blood-brain barrier integrity

Abstract

Poor antiretroviral penetration may contribute to human immunodeficiency virus (HIV) persistence within the brain and to neurocognitive deficits in opiate abusers. To investigate this problem, HIV-1 Tat protein and morphine effects on blood-brain barrier (BBB) permeability and drug brain penetration were explored using a conditional HIV-1 Tat transgenic mouse model. Tat and morphine effects on the leakage of fluorescently labeled dextrans (10-, 40-, and 70-kDa) into the brain were assessed. To evaluate effects on antiretroviral brain penetration, Tat+ and Tat- mice received three antiretroviral drugs (dolutegravir, abacavir, and lamivudine) with or without concurrent morphine exposure. Antiretroviral and morphine brain and plasma concentrations were determined by LC-MS/MS. Morphine exposure, and, to a lesser extent, Tat, significantly increased tracer leakage from the vasculature into the brain. Despite enhanced BBB breakdown evidenced by increased tracer leakiness, morphine exposure led to significantly lower abacavir concentrations within the striatum and significantly less dolutegravir within the hippocampus and striatum (normalized to plasma). P-glycoprotein, an efflux transporter for which these drugs are substrates, expression and function were significantly increased in the brains of morphine-exposed mice compared to mice not exposed to morphine. These findings were consistent with lower antiretroviral concentrations in brain tissues examined. Lamivudine concentrations were unaffected by Tat or morphine exposure. Collectively, our investigations indicate that Tat and morphine differentially alter BBB integrity. Morphine decreased brain concentrations of specific antiretroviral drugs, perhaps via increased expression of the drug efflux transporter, P-glycoprotein.

Keywords: Abacavir; Dolutegravir; Lamivudine; Morphine-3-β-glucuronide; Neuro-human immunodeficiency virus (neuroHIV); P-glycoprotein; Paracellular transport; Transcellular transport; Zonula occludens-1.

Fig. 1. Effects of HIV-1 Tat and morphine on BBB leakiness after 14-day Tat induction.

There was a significant increase in the 10 kDa (Cascade Blue®) tracer leakage into the brain in Tat+ placebo as compared to Tat− placebo mice (*p < 0.05). The 10 kDa tracer was also significantly increased in Tat− mice brains upon exposure to morphine as compared to Tat− placebo mice (*p < 0.05) (A). There was a significant main effect of morphine, resulting in reduced integrity of the BBB and increased leakage of the higher molecular weight (40 kDa and 70 kDa) tracers in morphine-exposed groups as compared to the those groups (Tat+ and Tat–together) not exposed to morphine (placebo) (^#p < 0.05; significant main effect of morphine) (B, C). Data represent the fold-change in mean fluorescence intensity ± SEM; n = 8 Tat−/placebo, n = 6 Tat+/placebo, n = 9 Tat−/morphine, and n = 7 Tat+/morphine mice.

Fig. 2

Effects of HIV-1 Tat and/or morphine exposure on horseradish peroxidase (HRP) extravasation from the vasculature into the perivascular space and/or parenchyma in the striatum (a-d). HRP antigenicity was detected by indirect immunofluorescence (red) in tissue sections counterstained with Hoechst 33342 (blue) to reveal cell nuclei, and visualized by differential interference contrast- (DIC-) enhanced confocal microscopy. HRP extravasation into the striatal perivascular space/parenchyma was especially prevalent in morphine-exposed mice (arrowheads; left-hand panels in b, d). The dotted lines (⋯⋯⋯⋯) indicate approximate edge of the capillaries/post-capillary venules; while intermittent dotted lines (⋯⋯·) indicate the approximate edge of a partly sectioned blood vessel that appears partially outside the plane of section. Representative samples from ≥ n = 4 mice per group. All images are the same magnification. Scale bar = 10 μm.

Fig. 3

Effects of HIV-1 Tat and/or morphine exposure on the cellular/subcellular localization of ZO-1 immunofluorescence within the endothelial cells of capillaries and post-capillary venules in the striatum (a-d). ZO-1 antigenicity was detected by indirect immunofluorescence (green) in tissue sections counterstained with Hoechst 33342 (blue) to reveal cell nuclei, and visualized by differential interference contrast- (DIC-) enhanced confocal microscopy. Morphine-exposure frequently resulted in a diffuse pattern of ZO-1 immunoreactivity within the cytoplasm of endothelial cells in Tat− mice (b), rather than at discrete locations associated with tight junctions as seen in placebo-treated Tat− mice (a). By contrast, ZO-1 tended to fragment into discrete foci within the endothelium of Tat+ following doxycycline induction (c). In the presence of Tat, ZO-1 displayed a fragmented distribution irrespective of morphine co-exposure (d), suggesting an overriding influence of Tat on the subcellular distribution of ZO-1. The dotted lines (⋯⋯⋯⋯) indicate the approximate edge of capillaries/post-capillary venules; the intermittent dotted lines (⋯⋯·) indicate the approximate edge of a partially sectioned blood vessel. Representative samples from ≥ n = 4 mice per group. All images are the same magnification. Scale bar = 10 μm.

Fig. 4.. Antiretroviral tissue-to-plasma ratios in striatum and hippocampus.

Irrespective of Tat exposure, morphine significantly reduced the levels of striatal and hippocampal dolutegravir (A,D) and striatal abacavir (B), but not lamivudine (C,F), compared to placebo (*p < 0.05; main effect for morphine). Data represent the tissue-to-plasma ratios ± SEM sampled from n = 9 Tat−/placebo, n = 9 Tat+/placebo, n = 6 Tat−/morphine, and n = 8 Tat+/morphine mice.

Fig. 5. Morphine plasma concentrations and tissue-to-plasma ratios in striatum and hippocampus.

Morphine concentrations were measured by LC-MS/MS in both plasma and brain tissue. There was a strong trend towards significantly increased morphine plasma concentrations in Tat+ mice as compared to their Tat− counterparts (A). Tat exposure significantly decreased the morphine tissue-to-plasma ratio in the hippocampus (C) but not in the striatum (B) (*p < 0.05). Data represent the tissue-to-plasma ratios ± SEM sampled from n = 6 Tat−, n = 9 Tat+ mice.

Fig. 6. P-glycoprotein (P-gp) expression levels in the striatum and hippocampus.

Western blots of P-glycoprotein levels in the striatum (A,C) and hippocampus (B,D) of Tat− or Tat+ mice with or without morphine co-exposure. There was a main effect of morphine in both tissue types. Exposure to morphine, irrespective of Tat status, significantly increased P-glycoprotein expression in striatum (*p < 0.05; main effect of morphine) and hippocampus (*p < 0.05; main effect of morphine) compared with the non-morphine exposed groups. Data represent the relative density of P-glycoprotein (absolute density of P-glycoprotein over the absolute density of β-actin) ± SEM, n = 4 Tat−/placebo, n = 4 Tat+/placebo, n = 4 Tat−/morphine, and n = 4 Tat+/morphine mice. As a negative control, baseline P-glycoprotein levels were also assessed in mice that were genetically Tat− or Tat+ but for which neither Tat induction nor morphine exposure had occurred. There were no statistically significant differences in regional expression of P-glycoprotein between striatum and hippocampus in Tat− mice or Tat+ mice at baseline (prior to Tat induction and morphine exposure; E, F). Data represent the relative density of P-glycoprotein ± SEM sampled from n = 4 Tat−/placebo/striatum, n = 4 Tat−/placebo/hippocampus, n = 4 Tat+/placebo/striatum, n = 4 Tat+/placebo/hippocampus.