Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Mar 25;15(3):e0230563.
doi: 10.1371/journal.pone.0230563. eCollection 2020.

Morphine exposure exacerbates HIV-1 Tat driven changes to neuroinflammatory factors in cultured astrocytes

Kenneth Chen  1 , Thienlong Phan  1 , Angel Lin  1 , Luca Sardo  1   2 , Anthony R Mele  3   4 , Michael R Nonnemacher  3   4 , Zachary Klase  1
Affiliations

Morphine exposure exacerbates HIV-1 Tat driven changes to neuroinflammatory factors in cultured astrocytes

Kenneth Chen et al. PLoS One. .

Abstract

Despite antiretroviral therapy human immunodeficiency virus type-1 (HIV-1) infection results in neuroinflammation of the central nervous system that can cause HIV-associated neurocognitive disorders (HAND). The molecular mechanisms involved in the development of HAND are unclear, however, they are likely due to both direct and indirect consequences of HIV-1 infection and inflammation of the central nervous system. Additionally, opioid abuse in infected individuals has the potential to exacerbate HIV-comorbidities, such as HAND. Although restricted for productive HIV replication, astrocytes (comprising 40-70% of all brain cells) likely play a significant role in neuropathogenesis in infected individuals due to the production and response of viral proteins. The HIV-1 protein Tat is critical for viral transcription, causes neuroinflammation, and can be secreted from infected cells to affect uninfected bystander cells. The Wnt/β-catenin signaling cascade plays an integral role in restricting HIV-1 infection in part by negatively regulating HIV-1 Tat function. Conversely, Tat can overcome this negative regulation and inhibit β-catenin signaling by sequestering the critical transcription factor TCF-4 from binding to β-catenin. Here, we aimed to explore how opiate exposure affects Tat-mediated suppression of β-catenin in astrocytes and the downstream modulation of neuroinflammatory genes. We observed that morphine can potentiate Tat suppression of β-catenin activity in human astrocytes. In contrast, Tat mutants deficient in secretion, and lacking neurotoxic effects, do not affect β-catenin activity in the presence or absence of morphine. Finally, morphine treatment of astrocytes was sufficient to reduce the expression of genes involved in neuroinflammation. Examining the molecular mechanisms of how HIV-1 infection and opiate exposure exacerbate neuroinflammation may help us inform or predict disease progression prior to HAND development.

PubMed Disclaimer

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Tat101WT suppressed β-catenin signaling activity in U87MG astrocytes and primary fetal astrocytes (PFAs).
U87MGs (A) and PFAs from Donor 1 (B) were co-transfected with the TCF/LEF β-catenin luciferase reporter plasmid and increasing concentrations of the Flag-Tat101 WT expression vector [A) 0.35ng, 3.5ng, 35ng; B) 3.5ng, 35ng] 24 hours post-transfection, cells were treated with LiCl (50mM), a β-catenin activator. Luciferase activity was measured 24 hours post-treatment. Data shown is the average of technical triplicates from one experiment ± SD. ** p<0.01 by two-tailed student’s t test.
Fig 2
Fig 2. Morphine potentiates Tat mediated suppression of β-catenin signaling.
U87MGs (A) and PFAs from Donor 1 (B) were co-transfected with the TCF/LEF β-catenin luciferase reporter plasmid and increasing concentrations of the Flag-Tat101 WT expression vector [A) 0.35ng, 3.5ng, 35ng; B) 3.5ng, 35ng]. 24 hours post-transfection, cells were treated with LiCl (50mM) in the presence or absence of Morphine (50nM or 500nM), as indicated. Luciferase activity was measured 24 hours post-treatment. Untreated control data from Fig 1 is replotted here for reference. Data shown is the average of technical triplicates from one experiment ± SD. ** p<0.01 by two-tailed student’s t test.
Fig 3
Fig 3. Expression of HIV-1 Tat in PFAs results in loss of active β-catenin protein levels.
PFAs (Donor #1) were transfected with empty vector (pUC19) or the Flag-Tat101WT (0.35ng) expression vector. 24 hours post-transfection, cells were treated with or without Morphine (500nM). Cells were collected and lysed for SDS-PAGE and western blotting 24 hours post-treatment (A) Proteins extracts were immunoblotted for total β-catenin, active β-catenin, and GAPDH. (B) Densitometry was performed on the resulting immunoblot and used to determine the ratio of active to total β-catenin for each condition. Data shown is from one experiment.
Fig 4
Fig 4. Tat-mediated suppression of β-catenin activity in PFAs is donor-dependent.
PFAs from Donor 1 and Donor 2 were co-transfected with the TOPFlash β-catenin reporter plasmid and the indicated Flag-Tat expression vectors [A),Tat86WT and Tat101WT; 12.5ng, 125ng]; [B,C), Tat101WT, Tat101C31R, Tat101W11F, Tat101W11Y; 12.5ng]. 24 hours post transfection, cells were treated with or without BIO (1mM), a β-catenin activator and, where indicated, cells were treated with or without Morphine (500nM). Luciferase activity was measured 24-hours post-treatment. Data shown is the average of technical triplicates from one experiment ± SD. * p < 0.05 and **** p < 0.001 between low and high Tat or between mutant and WT were calculated by two-way ANOVA with Tukey’s multiple comparisons test. Inter-donor comparisons are marked with “*” and intra-donor comparisons are marked with “*”.
Fig 5
Fig 5. Treatment of astrocytes with morphine decreases expression of neuroinflammatory genes.
U87MG astrocytes (A and B) and PFAs (C, D and E) from three donors were transfected with either mock plasmid (pUC19) or the Flag-Tat101WT expression vector. 24 hours post-transfection, cells were treated with or without Morphine (500nM). 48 hours post-treatment, RNA was harvested and RT-qPCR was performed using primers specific for TrkB (A and C), NLRP-1 (B and D), and BDNF (E) mRNA. Fold change values were calculated by normalizing relative mRNA gene expression (Ct) to GAPDH (ΔCt) relative to the control (ΔΔCt). Data is plotted as the fold change of the gene over the conditions (2ΔΔCt). Data shown is from one experiment performed in parallel with three individual PFA donors. For PFAs, significance between conditions and control were calculated by two-way ANOVA with Tukey’s multiple comparisons test.
Fig 6
Fig 6. Tat mutants have variable, donor-dependent effects on expression of neuroinflammatory genes in PFAs.
PFAs from three donors were transfected with the indicated Tat expression vectors, Flag-Tat101WT, Flag-Tat101C31R, Flag-Tat101W11F, or Flag-Tat101W11Y. 48 hours post-transfection, RNA was harvested and RT-qPCR was performed using primers specific for TrkB (A), NLRP-1 (B), and BDNF (C) mRNA. Fold change values were calculated by normalizing relative mRNA gene expression (Ct) to GAPDH (ΔCt) relative to the control (ΔΔCt). Data is plotted as the fold change of the gene over the conditions (2ΔΔCt). Data shown is from one experiment performed in parallel with three individual PFA donors. Significance between mutant and WT were calculated by two-way ANOVA with Tukey’s multiple comparisons test.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Fletcher C.V., et al., Persistent HIV-1 replication is associated with lower antiretroviral drug concentrations in lymphatic tissues. Proceedings of the National Academy of Sciences of the United States of America, 2014. 111(6): p. 2307–12. 10.1073/pnas.1318249111 - DOI - PMC - PubMed
    1. Lee E., et al., The impact of antiretroviral therapy duration on the HIV-1 infection of T-cells within anatomic sites. J Virol, 2019. - PMC - PubMed
    1. Jacobs J.L., et al., Persistent HIV-1 Viremia on Antiretroviral Therapy: Measurement and Mechanisms. Front Microbiol, 2019. 10: p. 2383 10.3389/fmicb.2019.02383 - DOI - PMC - PubMed
    1. Balcom E.F., et al., HIV-1 persistence in the central nervous system: viral and host determinants during antiretroviral therapy. Curr Opin Virol, 2019. 38: p. 54–62. 10.1016/j.coviro.2019.06.004 - DOI - PubMed
    1. Davis L.E., et al., Early viral brain invasion in iatrogenic human immunodeficiency virus infection. Neurology, 1992. 42(9): p. 1736–9. 10.1212/wnl.42.9.1736 - DOI - PubMed

Publication types

MeSH terms