Methamphetamine induces transcriptional changes in cultured HIV-infected mature monocytes that may contribute to HIV neuropathogenesis

Abstract

HIV-associated neurocognitive impairment (HIV-NCI) persists in 15-40% of people with HIV (PWH) despite effective antiretroviral therapy. HIV-NCI significantly impacts quality of life, and there is currently no effective treatment for it. The development of HIV-NCI is complex and is mediated, in part, by the entry of HIV-infected mature monocytes into the central nervous system (CNS). Once in the CNS, these cells release inflammatory mediators that lead to neuroinflammation, and subsequent neuronal damage. Infected monocytes may infect other CNS cells as well as differentiate into macrophages, thus contributing to viral reservoirs and chronic neuroinflammation. Substance use disorders in PWH, including the use of methamphetamine (meth), can exacerbate HIV neuropathogenesis. We characterized the effects of meth on the transcriptional profile of HIV-infected mature monocytes using RNA-sequencing. We found that meth mediated an upregulation of gene transcripts related to viral infection, cell adhesion, cytoskeletal arrangement, and extracellular matrix remodeling. We also identified downregulation of several gene transcripts involved in pathogen recognition, antigen presentation, and oxidative phosphorylation pathways. These transcriptomic changes suggest that meth increases the infiltration of mature monocytes that have a migratory phenotype into the CNS, contributing to dysregulated inflammatory responses and viral reservoir establishment and persistence, both of which contribute to neuronal damage. Overall, our results highlight potential molecules that may be targeted for therapy to limit the effects of meth on HIV neuropathogenesis.

Keywords: HIV; methamphetamine; migration; monocytes; neuroinflammation; viral reservoirs.

Figure 1

RNA sequencing analyses demonstrate differential gene expression in HIV-infected mature monocytes treated with meth compared to untreated HIV-infected cells. (A, B) Principal component analysis (PCA) plot of sample clustering based on gene expression patterns before (A) and after (B) batch correction. Each data point represents an individual sample, and the data shapes represent the individual donors. The y-axis and x-axis represent the first and second principal components, respectively. HIV-infected mature monocytes treated with meth are shown in blue and untreated HIV-infected mature monocytes are shown in green. (C) A volcano plot showing overall DEG between the HIV-infected mature monocytes treated with or without meth. Each dot represents a gene that was either statistically significantly different(green) or unchanged (black). The y-axis represents -log false discovery rate (FDR), and the x-axis represents log 2-fold change. (D) A heat map representing normalized gene expression levels of the top 20 DEG between HIV-infected mature monocytes treated with meth or untreated. Each column represents an individual sample, and each row represents a gene. The color scale represents lower (blue) to higher (red) gene expression levels.

Figure 2

HIV-infected mature monocytes treated with meth compared to untreated HIV-infected cells have a predicted increase in viral infection and cytoskeletal rearrangement and decrease in cell activation. (A) Top functions predicted to be increased (red) or decreased (blue) in the meth treated compared to untreated HIV-infected cells. The y-axis represents the list of functions, and the numbers in the parenthesis represent the quantity of molecules that were differentially expressed after meth treatment. The x-axis represents activation z-score. (B–E) Heat maps representing normalized expression levels of some genes involved in (B) activation of leukocytes, (C) viral infection, (D) cell adhesion and matrix degradation, and (E) organization of cytoskeleton in HIV-infected mature monocytes treated with and without meth. Each column represents an individual sample, and each row represents a gene. The color scale represents lower (blue) to higher (red) gene expression levels. The dendrograms show unsupervised clustering of samples.

Figure 3

HIV-infected mature monocytes treated with meth have decreased expression of genes related to antigen presentation, pathogen recognition, and oxidative phosphorylation, and upregulation of genes related to cellular RHO-GD1 signaling pathways. (A) Top cellular pathways predicted to be increased (red) or decreased (blue) in meth treated compared to untreated HIV-infected cells. The y-axis represents the list of pathways, and the x-axis represents activation z-score. (B–E) Heat maps representing normalized expression levels of some genes involved in (B) neuroinflammation signaling pathways, (C) T-cell receptor signaling, (D) oxidative phosphorylation, and (E) RHO-GDI signaling in HIV-infected mature monocytes treated, or not, with meth. Each column represents an individual sample, and each row represents a gene. The color scale represents lower (blue) to higher (red) gene expression levels. The dendrograms show unsupervised clustering of samples.

Figure 4

MMP-9 protein is increased in HIV-infected mature monocytes treated with meth. (A) A representative western blot with corresponding total protein stain of HIV-infected mature monocytes either untreated or treated for 6 h with meth. (B) Fold change of normalized MMP-9 protein from HIV-infected mature monocytes treated with meth over untreated HIV-infected mature monocytes. Each colored dot represents an individual donor. The columns and error bars depict mean and standard deviation (SD), respectively. n=7–14. (C) Fold change of normalized MMP-9 protein in supernatants of HIV-infected mature monocytes treated with meth over untreated HIV-infected cells. Each colored dot represents an individual donor. The columns and error bars depict mean and standard error of the mean (SEM), respectively; n=7-10; **, p<0.005 by the one sample t-test.

Figure 5

Gelsolin protein is increased in HIV-infected mature monocytes treated with meth. (A) A representative gelsolin western blot with corresponding total protein stain of HIV-infected mature monocytes either untreated or treated for 6 h or 24 with meth. (B) Fold change of gelsolin of HIV-infected mature monocytes treated with meth over untreated HIV-infected cells. Each colored dot represents an individual donor. The column and error bars depict mean and standard deviation (SD), respectively. n = 5-9.

Figure 6

Meth-mediated effects on HIV-infected mature monocytes as identified by RNA sequencing and western blot analyses. Figure created with BioRender.com.