Antiretroviral drugs efavirenz, dolutegravir and bictegravir dysregulate blood-brain barrier integrity and function

Abstract

The implementation of combined antiretroviral therapy (cART) significantly reduces the mortality associated with human immunodeficiency virus (HIV) infection. However, complications such as HIV-associated neurocognitive disorders (HAND) remain a major health concern. We hypothesized that the toxicity of antiretroviral drugs (ARVs) may contribute to the pathogenesis of HAND in addition to cerebral viral infection. To address this question, we evaluated the impact of HIV integrase strand transfer inhibitors (dolutegravir and bictegravir), and a non-nucleoside reverse transcriptase inhibitor (efavirenz) on the integrity and permeability of various human and mouse blood-brain barrier (BBB) models, in vitro, ex vivo and in vivo. We observed a significant downregulation of tight junction proteins (TJP1/Tjp1, OCLN/Ocln and CLDN5/Cldn5), upregulation of proinflammatory cytokines (IL6/Il6, IL8/Il8, IL1β/Il1β) and NOS2/Nos2, and alteration of membrane-associated transporters (ABCB1/Abcb1a, ABCG2/Abcg2 and SLC2A1/Slc2a1) mRNA expression, in vitro, in human (hCMEC/D3) and primary cultures of mouse microvascular endothelial cells, and ex vivo in isolated mouse brain capillaries treated with efavirenz, dolutegravir, and/or bictegravir. We also observed a significant increase in BBB permeability in vivo following treatment with the selected ARVs in mice applying NaF permeability assay. Taken together, these results suggest that clinically recommended integrase strand transfer inhibitors such as dolutegravir may exacerbate HIV-associated cerebrovascular pathology, which may contribute to the associated short-term neuropsychiatric side effects and the high incidence of mild forms of HAND reported in the clinical setting.

Keywords: ARV toxicity; HIV-associated neurocognitive disorders; antiretroviral drugs (ARVs); bictegravir; blood-brain barrier; dolutegravir; efavirenz.

FIGURE 1

mRNA expression of TJ proteins, transporters, proinflammatory cytokines and NOS2 in hCMEC/D3 cells exposed to EFV (7,500, 10,000 ng/mL) or vehicle (DMSO) control for 6/24/48 h mRNA expression of TJP1, OCLN, CLDN5, ABCB1, ABCG2, SLC2A1, IL6, IL1β, IL8 and NOS2 in hCMEC/D3 cells exposed to EFV relative to vehicle (DMSO) control was assessed by qPCR. Results are presented as mean relative mRNA expression ±SEM normalized to the housekeeping human cyclophilin B gene from n = 4 independent experiments. One-way ANOVA with Bonferroni’s post-hoc test, *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

FIGURE 2

mRNA expression of TJ proteins, transporters, proinflammatory cytokines and Nos2 in primary cultures of mouse brain microvascular endothelial cells exposed to EFV (7,500, 10,000 ng/mL) and vehicle (DMSO) control for 6 or 24 h and in isolated mouse brain capillaries exposed to EFV (10,000 ng/mL) and vehicle (DMSO) control for 5 h (A) mRNA expression of Tjp1, Ocln, Cldn5, Abcb1a, Abcg2, Slc2a1, Il6, Il1β and Nos2 in primary cultures of mouse brain microvascular endothelial cells exposed to EFV relative to vehicle (DMSO) control was assessed by qPCR. Results are presented as mean relative mRNA expression ±SEM normalized to the housekeeping mouse cyclophilin B gene from n = 4 independent experiments. (B) mRNA expression of Tjp1, Ocln, Cldn5, Abcb1a, Abcg2, Slc2a1 and Il1β in isolated mouse brain capillaries treated for 5 h with EFV (10,000 ng/mL) relative to vehicle (DMSO) was assessed by qPCR. Results are presented as mean relative mRNA expression ±SEM normalized to the housekeeping mouse cyclophilin B gene from n = 3 independent experiments, where each experiment contained pooled brain tissues from 6 animals per group. One-way ANOVA with Bonferroni’s post-hoc test (A), unpaired two-tailed Student’s t-test (B): *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

FIGURE 3

mRNA expression of TJ proteins, transporters, proinflammatory cytokines and NOS2 in hCMEC/D3 cells exposed to DTG (2,000, 5,000 ng/mL) or vehicle (DMSO) control for 6/24/48 h mRNA expression of TJP1, OCLN, CLDN5, ABCB1, ABCG2, SLC2A1, IL6, IL1β, IL8 and NOS2 in hCMEC/D3 cells exposed to DTG relative to vehicle (DMSO) control was assessed by qPCR. Results are presented as mean relative mRNA expression ±SEM normalized to the housekeeping human cyclophilin B gene from n = 4 independent experiments. One-way ANOVA with Bonferroni’s post-hoc test, *, p < 0.05; ***, p < 0.001; ****, p < 0.0001.

FIGURE 4

mRNA expression of TJ proteins, transporters, proinflammatory cytokines and Nos2 in primary cultures of mouse brain microvascular endothelial cells exposed to DTG (2000, 5,000 ng/mL) and vehicle (DMSO) control for 6 or 24 h and in isolated mouse brain capillaries exposed to DTG (5,000 ng/mL) and vehicle (DMSO) control for 5 h (A) mRNA expression of Tjp1, Ocln, Cldn5, Abcb1a, Abcg2, Slc2a1, Il6, Il1β and Nos2 in primary cultures of mouse brain microvascular endothelial cells exposed to DTG relative to vehicle (DMSO) control was assessed by qPCR. Results are presented as mean relative mRNA expression ±SEM normalized to the housekeeping mouse cyclophilin B gene from n = 4 independent experiments. (B) mRNA expression of Tjp1, Ocln, Cldn5, Abcb1a, Abcg2, Slc2a1 and Il1β in isolated mouse brain capillaries treated for 5 h with DTG (5,000 ng/mL) relative to vehicle (DMSO) was assessed by qPCR. Results are presented as mean relative mRNA expression ±SEM normalized to the housekeeping mouse cyclophilin B gene from n = 3 independent experiments, where each experiment contained pooled brain tissues from 6 animals per group. One-way ANOVA with Bonferroni’s post-hoc test (A), unpaired two-tailed Student’s t-test (B): *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

FIGURE 5

mRNA expression of TJ proteins, transporters, proinflammatory cytokines and NOS2 in hCMEC/D3 cells exposed to BTG (3,000, 6,000 ng/mL) or vehicle (DMSO) control for 6 or 24 h mRNA expression of TJP1, OCLN, CLDN5, ABCB1, ABCG2, SLC2A1, IL6, IL1β, IL8 and NOS2 in hCMEC/D3 cells exposed to BTG relative to vehicle (DMSO) control was assessed by qPCR. Results are presented as mean relative mRNA expression ±SEM normalized to the housekeeping human cyclophilin B gene from n = 4 independent experiments. One-way ANOVA with Bonferroni’s post-hoc test, *, p < 0.05; **, p < 0.01.

FIGURE 6

mRNA expression of TJ proteins, transporters, proinflammatory cytokines and Nos2 in primary cultures of mouse brain microvascular endothelial cells exposed to BTG (3,000, 6,000 ng/mL) and vehicle (DMSO) control for 6 or 24 h and in isolated mouse brain capillaries exposed to BTG (6,000 ng/mL) and vehicle (DMSO) control for 5 h (A) mRNA expression of Tjp1, Ocln, Cldn5, Abcb1a, Abcg2, Slc2a1, Il6, Il1β and Nos2 in primary cultures of mouse brain microvascular endothelial cells exposed to BTG relative to vehicle (DMSO) control was assessed by qPCR. Results are presented as mean relative mRNA expression ±SEM normalized to the housekeeping mouse cyclophilin B gene from n = 4 independent experiments. (B) mRNA expression of Tjp1, Ocln, Cldn5, Abcb1a, Abcg2, Slc2a1 and Il1β in isolated mouse brain capillaries treated for 5 h with BTG (6,000 ng/mL) relative to vehicle (DMSO) was assessed by qPCR. Results are presented as mean relative mRNA expression ±SEM normalized to the housekeeping mouse cyclophilin B gene from n = 3 independent experiments, where each experiment contained pooled brain tissues from 6 animals per group. One-way ANOVA with Bonferroni’s post-hoc test (A), unpaired two-tailed Student’s t-test (B): *, p < 0.05; ***, p < 0.001.

FIGURE 7

Quantification of NaF level in the mouse brain. Mice were treated for 14 days with either vehicle (regular PB pellet), DTG (5 mg/kg/day), BTG (5 mg/kg/day) or EFV (10 mg/kg/day). Diffusion of sodium fluorescein (NaF) from plasma into the brain parenchyma was used as the indicator of BBB permeability. PB = peanut butter. Data are mean ± SEM, expressed as fold change compared to vehicle, n = 5 per group. One-way ANOVA with Bonferroni’s post-hoc test: *, p < 0.05; ****, p < 0.0001.

FIGURE 8

mRNA expression of TJ proteins, membrane associated transporters and proinflammatory cytokine in mouse brain capillaries. The mRNA expression of Tjp1, Ocln, Cldn5, Abcb1a, Abcg2, Slc2a1 or Il1β genes was assessed by qPCR in brain capillaries isolated from mice treated with DTG (5 mg/kg/day), BTG (5 mg/kg/day) or vehicle (regular PB pellet) for 14 days. Results are presented as mean relative mRNA expression ±SEM normalized to the housekeeping mouse cyclophilin B gene from n = 6 animals per group. One-way ANOVA with Bonferroni’s post-hoc test: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.