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Isolation, Transfection, and Culture of Primary Human Monocytes
Published on: December 16, 2019
HIV-Tat regulates macrophage gene expression in the context of neuroAIDS
Loreto Carvallo1, Lillie Lopez1, Jorge E Fajardo2
1Department of Pathology, Albert Einstein College of Medicine, Bronx, New York, United States of America.
Abstract:
Despite the success of cART, greater than 50% of HIV infected people develop cognitive and motor deficits termed HIV-associated neurocognitive disorders (HAND). Macrophages are the major cell type infected in the CNS. Unlike for T cells, the virus does not kill macrophages and these long-lived cells may become HIV reservoirs in the brain. They produce cytokines/chemokines and viral proteins that promote inflammation and neuronal damage, playing a key role in HIV neuropathogenesis. HIV Tat is the transactivator of transcription that is essential for replication and transcriptional regulation of the virus and is the first protein to be produced after HIV infection. Even with successful cART, Tat is produced by infected cells. In this study we examined the role of the HIV Tat protein in the regulation of gene expression in human macrophages. Using THP-1 cells, a human monocyte/macrophage cell line, and their infection with lentivirus, we generated stable cell lines that express Tat-Flag. We performed ChIP-seq analysis of these cells and found 66 association sites of Tat in promoter or coding regions. Among these are C5, CRLF2/TSLPR, BDNF, and APBA1/Mint1, genes associated with inflammation/damage. We confirmed the association of Tat with these sequences by ChIP assay and expression of these genes in our THP-1 cell lines by qRT-PCR. We found that HIV Tat increased expression of C5, APBA1, and BDNF, and decreased CRLF2. The K50A Tat-mutation dysregulated expression of these genes without affecting the binding of the Tat complex to their gene sequences. Our data suggest that HIV Tat, produced by macrophage HIV reservoirs in the brain despite successful cART, contributes to neuropathogenesis in HIV-infected people.
Insights
HIV Tat protein from macrophage reservoirs contributes to brain damage in people with HIV, even with treatment. Tat alters gene expression in macrophages, promoting inflammation and neuronal damage, key factors in HIV-associated neurocognitive disorders.
Area of Science:
- Neuroscience
- Immunology
- Virology
Background:
- Despite combination antiretroviral therapy (cART), over 50% of HIV-infected individuals develop HIV-associated neurocognitive disorders (HAND).
- Macrophages are key HIV reservoirs in the central nervous system (CNS), producing inflammatory mediators and viral proteins that drive neuropathogenesis.
- HIV Tat protein, essential for viral replication, is produced even with effective cART and plays a critical role in initiating infection and regulating viral transcription.
Purpose of the Study:
- To investigate the role of the HIV Tat protein in regulating gene expression within human macrophages.
- To identify specific genes targeted by HIV Tat in macrophages and understand Tat's contribution to HIV neuropathogenesis.
Main Methods:
- Generated stable Tat-Flag expressing THP-1 macrophage cell lines via lentiviral infection.
- Utilized Chromatin Immunoprecipitation sequencing (ChIP-seq) to identify Tat binding sites in promoter and coding regions.
- Confirmed Tat association with target genes using ChIP assay and quantified gene expression changes via qRT-PCR.
Main Results:
- Identified 66 Tat association sites, including genes like C5, CRLF2/TSLPR, BDNF, and APBA1/Mint1, linked to inflammation and neuronal damage.
- HIV Tat significantly increased the expression of C5, APBA1, and BDNF, while decreasing CRLF2 expression.
- A K50A Tat mutation altered gene expression without affecting Tat complex binding, suggesting a role for Tat's functional domains beyond DNA binding.
Conclusions:
- HIV Tat produced by macrophage reservoirs in the brain contributes to HAND pathogenesis, even during cART.
- Tat-mediated dysregulation of specific genes in macrophages promotes inflammation and neuronal damage, highlighting Tat as a therapeutic target for HAND.
