Fig 1.
Meth does not induce catecholamines in innate immune cells in culture.
(A) Dopamine, (B) Norepinephrine and (C) Epinephrine were measured in the supernatant of the cell cultures after Meth stimulation using liquid chromatography tandem mass-spectrometry (LC-MS/MS). The supernatant of control and Meth-stimulated THP1 cells was harvested at baseline, 2 hours, 4 and 24 hours, and the levels of catecholamines were plotted against background measurements obtained with culture media. The values of the supernatant of control or Meth-stimulated cells were below the detection threshold, suggesting that Meth does not induce catecholamine release from innate immune cells.
Fig 2.
Meth affects levels of transcription factors with binding sites in the human CCR5 promoter.
(A) Western blot was used to examine the enrichment of transcription factors such as NFkB, GATA3, cFos, and CREB, among others, normalized against b-actin, over time. The picture shows westerns performed in one representative experiment out of 3 independent tests. (B) Changes in the abundance of transcription factors were estimated by band area and intensity density. Values represent the average of at least three separated experiments. *p<0.05 compared to controls.
Fig 3.
Nuclear translocation of CCR5-relevant transcription factors by Meth, dependent on ROS, effects of HIV Tat and of DA.
Translocation of (A) cJun, (B) NFkB, (C) GATA3 and (D) CREB in THP1 macrophages treated with 60uM Meth, 5uM NAC, 10ng/ml HIV Tat or 1uM DA. Transcription factors were detected at different time points with immunocytochemistry, analyzed using ImageJ using confocal images. A nuclear translocation index was derived as percentage of the total transcription factor stained measurement values that is present within the nuclear area, and derived from the difference between total and nuclear staining (See S1 Fig for model). Nuclear translocation indexes from 5 experiments were compared using two-way ANOVA, followed by Bonferroni’s posthoc test.
Fig 4.
RNA Pol accumulation and transcription of the CCR5 gene.
The pattern of RNA Pol II accumulation is a measure of the earliest detectable impact on the CCR5 promoter towards the gene transcription. (A) RNA Pol II accumulation pattern mapped onto the CCR5 gene sequence detected by RNA Pol ChIP-qPCR in THP1 cells stimulated with Meth (60uM), Tat (10ng/ml), or NAC (1nM), or DA (10nM) for 15 minutes before cell fixation and cross linking. Graph shows an Integrated Genome Browser (IGB 8.5) view of RNA Pol peak sequencing, and the alignment with the CCR5 sequence in the Homo sapiens genome. (B) Geometric mean of the total RNA Pol accumulation in the CCR5 gene in different conditions. (C) The effect of Meth, Tat and DA, on CCR5 transcription 2 hours after stimulation. CCR5 transcription was examined using SyBrGreen qRT-PCR, and normalized against the expression of housekeeping GAPDH. *p<0.05 compared to control conditions, One way ANOVA followed by Bonferroni’s posthoc test.
Fig 5.
Histone modification patterns in CCR5 promoter regions, in THP1 cells treated with Meth, Tat and DA.
Regulatory and enhancer histone modifications were detected by ChIP-qPCR, 30 minutes after stimulation. Suppressor H3K27me3 and H3K9me3, and enhancer H3K4me3 and H3K27Ac, were detected with specific antibodies on cross-linked chromatin, followed by qPCR to amplify 6 non-overlapping 500bp segments that covered the whole CCR5 gene. (A) Controls, (B) Meth, (C) Tat, (D) Meth+Tat, (E) DA.
Fig 6.
Effect of DA neurons and Meth stimulation on innate immune CCR5 transcription.
CCR5 gene transcription was examined in THP1 macrophages, treated with Meth or DA (1 uM), or co-cultured with SH-Sy5Y DA neurons, with or without a 1 hour pre-stimulation with Meth (60uM). Results normalized to the expression of GAPDH, represent the average ± SD of 2 experiments performed in quadruplicate. *p<0.05 in Bonferroni’s post hoc test.
Fig 7.
Modulation of the surface expression of CCR5 in THP1 cells by Meth, Tat and DA.
FACS analysis was performed on THP1 cells stimulated with 1 or 10 uM of DA for 2 hrs, alone or in combination with Meth (60uM) and HIV Tat (10ng/ml). A) Representative scatter plot showing the expression of CCR5 and of the myeloid marker CD11b in cells that were not-treated (NT) or treated with 1uM DA. B) Overlapping representative histograms showing the CCR5 fluorescence intensity of NT, DA-treated cells (1uM) and DA+ Meth-treated cells. C) Overlapping representative histograms showing CCR5 fluorescence intensity in NT, Tat and DA (1uM) + Tat-treated cells. D) Overlapping representative histograms showing CCR5 fluorescence intensity in NT, and treatments with Meth + Tat, or Meth + Tat + DA (1uM) simultaneously in THP1 cells. *p<0.05 in comparison to NT. E) CCR5 density per cell was obtained by normalizing the geometric mean fluorescence to the number of acquired cell counts. Results represent the average ± SEM of 3 experiments performed in triplicate.
Fig 8.
Expression of DRD receptors by THP1 cells and their role in the modulation of CCR5 transcription.
(A) Transcription of all 5 DRDs on THP1 macrophages, in the presence or absence of Meth and/or HIV Tat peptide. THP1 cells were incubated with vehicle, Meth, DA or Meth and DA for 2 hrs, in the presence or absence of specific DRD receptor agonists and antagonists. Using qRT-PCR, CCR5 expression was tested upon a 30 min pre-treatment with (B) DRD1-like agonist SKF38393 or antagonist SCH 23390 HCl; or (C) DRD2 agonist Ropinirole or antagonist Haloperidol; or (D) DRD3 agonist Pramipexole or antagonist Pnu 177864; or (E) DRD4 agonist PD168077 or antagonist L745,870. Results represent the average ± SD of 3 experiments performed in duplicate. *p<0.05 compared to Control (Ctr) conditions within each treatment, ANOVA followed by Bonferroni’s test.
Fig 9.
Surface expression of CCR5 in the presence of selective DRD isoform agonists.
Control (A, B, C, D, I, J, K, L) and Meth-treated (E, F, G, H, M, N, O, P) THP1 cells were exposed for 1 hour to DRD1–5 agonist SKF 38393 (A, B, E, F), DRD2 agonist (C, D, G, H), DRD3 agonist Pramipezole (I,J, M, N) and to DRD4 agonist PD 168077 (K, L, O, P). The cells were stained with PE- labeled CD11b and APC-labeled CCR5. Scatter plots show the superposition of CCR5 and CD11b subsets in non-treated cells (NT), and cells treated with antagonists in each panel’s legend, with or without simultaneous Meth (60uM). (Q) CCR5 levels measured by the geometric mean fluorescence of cells that received the different DRD agonists, in the presence or absence of Meth. The results represent the average ± SD of 3 independent experiments performed in duplicate. *p<0.05 in Bonferroni’s test, and in comparison to respective controls, or white bars are compared to not treated (NT) without Meth, and black bars are compared to Meth alone NT controls.
Fig 10.
Infection of THP1 cells with HIV is enhanced by DA in a CCR5-dependent fashion.
THP1 cells were exposed to HIV-pR7-GFP, and the internalization of viral particles was measured using flow cytometry. (A) Mean fluorescence histogram of non-treated, uninfected cells (black), HIV-infected (red), and HIV-infected in the presence of 1uM of DA. (B) Geometric mean fluorescence of intracellular HIV-pR7-GFP in control cells and cells exposed to DA, in the presence or absence of a CCR5-blocking compound TAK799. Results represent the average and standard deviation of 5 independent experiments performed in duplicate. *p<0.05 in Bonferroni’s post hoc test, where comparisons were assigned to respective controls.