HIV-1 gp120 and tobacco smoke synergistically disrupt the integrity of the blood-brain barrier

Abstract

In the United States, the Centers for Disease Control and Prevention (CDC) terms HIV and tobacco use among the ten most important public health challenges we face today. In the last decade, there has been a remarkable decrease in the number of deaths due to HIV/AIDS, especially after the widespread availability and use of combination antiretroviral therapy (cART). However, people living with HIV/AIDS have a heightened risk of chronic complications and comorbidities, including neurological disorders. Around 40-60 % of HIV-infected individuals progress to NeuroAIDS, a group of disorders caused primarily by HIV-mediated damage to the central and peripheral nervous systems, despite receiving cART. The detrimental effects of chronic smoking on the cerebrovascular system are also well studied and reported. Addictive behavior, such as smoking, is more common in HIV patients compared to the general population. In this context, given the existing immune suppression, smoking can pose a significant risk for the progression of the disease to NeuroAIDS by disrupting the integrity of the blood-brain barrier (BBB). Here we show that co-treatment with Tobacco Smoke Extract (TSE) and HIV-1 gp120 (HIV envelope glycoprotein) in primary cultures of human brain microvascular endothelial cells promoted heightened cellular stress responses compared to control and individual treatments. Our findings suggest that a potential synergistic effect between smoke exposure and gp120 can worsen the loss of BBB viability, possibly exacerbating NeuroAIDS progression.

Keywords: Alternative; Cerebrovascular; Chronic; Exposure; Inflammation; Ischemia; Oxidative stress; Smoking; Toxicity; Vaping.

Fig. 1.

Effects of TS and gp120 exposure on cell viability and oxidative stress. A: MTT cytotoxicity assay for cell viability following TS - gp120 treatments for 24 h. B: DCFDA assay as an indicator for reactive oxygen species (ROS) in cells. n = 3 biological replicates/group. *p < 0.05, ** p < 0.01, **p < 0.001, ****p < 0.0001 vs. control; #p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. TS + gp120 co-treatment. n.s. = non statistical significance.

Fig. 2.. Effects of TSE-gp120 exposure on NRF2 levels.

A. Western Blot analysis of NRF2 protein. B. qPCR results show NRF2 mRNA levels. n = 3 biological replicates/group. *p < 0.05, ** p < 0.01, ****p < 0.0001 vs. control; ⁺p < 0.05, ⁺⁺⁺p < 0.001 vs. gp120^{; #}p < 0.05, ^####p < 0.0001 vs. TS + gp120 co-treatment.

Fig. 3.

A: NF-κB transcription assessed by qPCR showed substantial increase in NF-κB mRNA levels. B: Western Blot analysis showed elevated levels of NF-κB protein in the TSE-gp120 treated group. n = 3 biological replicates/group. ***p < 0.001, ****p < 0.0001 vs. control); ^##p < 0.01, ^###p < 0.001 vs. TS + gp120 co-treatment. n.s. = non statistical significance.

Fig. 4.

A: Co-treatment of TSE-gp120 diminished basal oxygen consumption rate (OCR) in co-treated group when compared with individual treatments and control. B: Reduced mitochondrial membrane potential in TSE-gp120 combination treatment. n = 3 biological replicates/group. *p < 0.05, **p < 0.01, ****p < 0.0001 vs. control; ^##p < 0.01, ^###p < 0.001, ^####p < 0.0001 vs. TS + gp120 co-treatment. n.s. = non statistical significance.

Fig. 5.. Effects of TSE-Gp120 exposure on tight junction proteins and BBB integrity.

Western blotting analysis showing the downregulation of ZO-1 (A) along with the corresponding Immunofluorescence images showing protein expression and distribution (B). Western blotting analysis showing the downregulation of Occludin (C) along with the corresponding Immunofluorescence images showing protein expression and distribution (D). TEER (E) and fluorescein permeability (F) demonstrating a significant decrease of BBB integrity. *p < 0.05, ** p < 0.01, ***p < 0.001, ****p < 0.0001 vs. control; ⁺p < 0.05, ⁺⁺p < 0.01 vs. gp120; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, ^####p < 0.0001 vs. TS + gp120 co-treatment. n.s. = non statistical significance.