Scatter plot showing expression levels of the 84 genes encoding for enzymes related to epigenetic modifications.

<p>The diagram represents the relative expression levels calculated in log (2^-ΔΔCt) for each one of the 84 genes (blue dots). The data was generated using the RT² Profiler PCR Array Human Epigenetic Chromatin Modification Enzymes (PAHS-085A, SA Biosciences). The analysis was performed comparing the data from uninfected cells at 6, 12, 24 and 36h (x-axis) versus infected cells (y-axis) at 6, 12, 24 and 36h after infection. Pink lines delimit the zone of change in gene expression between −3 and +3 fold. The central axis (dark blue line) represents the mean normalized endogenous control expression. Dots above the pink lines correspond to up-regulated genes and bellow the line to down-regulated genes. Genes showing up-regulation ≥ +3 and down-regulation ≤ −3 are indicated in the plot.</p

Modulation in the functional markers of activated CD4+ T cells after HIV-1 infection.

<p><b>(A)</b> Flow cytometry of purified CD4⁺ T cells at 36h post HIV-1 infection—dot plots of cell populations (gated on CD4⁺CD3⁺ cells) analyzed for the T cell early activation markers CD25, CD69 (percentages are shown in each quadrant) and graphical representation of the percentages of CD25⁺CD69⁺ cells (gated on CD4⁺CD3⁺ cells). <b>(B)</b> IL2 and IFN- γ mRNA relative expression in CD4+ T cells at 24h after HIV-1 infection. <b>(C)</b> GLUT-1 and ENO-1 mRNA relative expression in CD4+ T cells at 24h after HIV-1 infection. GAPDH was used as a normalizer of all reactions to calculate the relative expression by 2^-ΔΔCt method. Data are shown as mean ± SD of triplicates and are representative of three independent experiments using cells of three different healthy donors. Two-tailed Student’s t-test: *, p < 0.05. (<b>NI</b>) non-infected cells, (<b>I</b>) HIV-1 infected cells.</p

Activation status of the PBMCs prior to HIV-1 infection.

<p>PBMCs were gated on CD3⁺CD4⁺ and stained for the activation markers CD25 <b>(upper panel—right)</b>, CD69 <b>(upper panel—left)</b> and HLA-DR <b>(lower panel)</b>. Activated cells (green lines), non-activated cells (red lines) and unstained control (gray peaks). This histogram is representative of six independent experiments.</p

Classical markers of epigenetic transcriptional silencing and activation in activated PBMCs after HIV-1 infection.

<p><b>(A)</b> Graphical representation of protein ratios of epigenetic transcriptional silencing marker H3K27me3 over the total H3. <b>(B)</b> Graphical representation of protein ratios of epigenetic transcriptional silencing marker and H3K9m3 over the total H3. <b>(C)</b> Graphical representation of protein ratios of epigenetic transcriptional activation marker H3K4m3 over the total H3. Protein levels of each marker were calculated by the ratio of band intensities between specific markers (H3K27me3, H3K9me3 or H3K4me3) over the total H3 (normalizer) using the software ImageJ v. 1.45s (Public domain, NIH, USA). Dark bars—HIV-1 infected cells; White bars—non-infected cells (control group). <b>(D)</b> Representative Western blot image for each epigenetic marker (H3K27me3—upper panel, H3K9me3—middle panel, H3K4me3—lower panel and the total H3 as normalizer. The data represent the mean of three different measurements of the same experiment and the error bars indicate the differences between two independent experiments. 2way ANOVA: *** p< 0.001, ** p < 0.01 and *, p < 0.05. (<b>NI</b>) non-infected cells, (<b>I</b>) HIV-1 infected cells.</p

Classical markers of epigenetic transcriptional silencing and activation of non-activated PBMCs at 24h after HIV-1 infection.

<p><b>(A)</b> Representative Western blot image for each epigenetic marker (H3K27me3—upper panel and H3K4me3—lower panel) and the total H3 as normalizer. <b>(B)</b> Graphical representation of protein ratios of epigenetic transcriptional silencing marker and H3K27m3 over the total H3. <b>(C)</b> Graphical representation of protein ratios of epigenetic transcriptional activation marker H3K4m3 over the total H3. Protein levels of each marker were calculated by the ratio of band intensities between specific markers (H3K27me3, H3K9me3 or H3K4me3) over the total H3 (normalizer) using the software ImageJ v. 1.45s (Public domain, NIH, USA). Dark bars—HIV-1 infected cells. <b>(D)</b> Percentage of 5’-methylcytosine content in genomic DNA. Data represent the mean of three different measurements of the same experiment and the error bars indicate the differences between two independent experiments. 2way ANOVA: *** p< 0.001, ** p < 0.01 and *, p < 0.05. <b>(E)</b> Flow cytometry of non-activated purified CD4⁺ T cells 36h post HIV-1 infection—dot plots of cell populations (gated on CD4⁺CD3⁺ cells) analyzed for the T cell early activation markers CD25, CD69 (percentages are shown in each quadrant) and graphical representation of the percentages of CD25⁺CD69⁺ cells (gated on CD4⁺CD3⁺ cells). Data are shown as mean ± SD of triplicates and are representative of three independent experiments using cells of three different healthy donors. Two-tailed Student’s t-test: *, p < 0.05. Dark bars—HIV-1 infected cells, White bars—non-infected cells, NA—non-activated cells. (<b>NI</b>) non-infected cells, (<b>I</b>) HIV-1 infected cells.</p

Experimental design and global DNA Methylation levels in HIV-1 infected activated PBMCs.

<p><b>(A)</b> Schematic representation of the experimental design and conditions: PBMC from healthy donors were separated by Ficoll gradient (in some experiments CD4⁺ cells were purified by positive selection). Cell viability was accessed by Trypan Blue exclusion. Cells were cultivated with a poor medium (RPMI+0.1% Bovine Albumin) for 24h in order to minimize any undesired previous activation. Next, cells were washed and resuspended in RPMI (supplemented)+10%FBS and activation stimulus (PHA+IL2) was added (activated group) or left without activation stimulus (non-activated group). After 36 hours of stimulation, the purity and activation status of cells were checked by flow cytometry using common human CD4⁺ T cell activation markers (CD25, HLA-DR and CD69). Next, 2x10⁷ cells were infected with HIV at a MOI (multiplicity of infection) of 0.05. The infections were carried out during the indicated time intervals (6h, 12h, 24h or 36h). After infection periods, cells were harvested, washed with PBS and cellular pellets were submitted to genomic DNA and RNA and protein extraction. <b>(B) and (C)</b> Experimental duplicates of 36h time-point agarose gel electrophoresis of genomic DNA from PBMCs after digestion with restriction enzymes HpaII and MspI. Gels were stained with SybrSafe dye (1:10.000—Invitrogen). HPA II—genomic DNA digested with Hpa II; MSP I genomic DNA digested with Msp I; C- Non-digested genomic DNA (Control). (<b>NI</b>) DNA from non-infected cells, (<b>I</b>) DNA from HIV-1 infected cells. The images are a representative of an experimental duplicate in which the cells were collected at 36h after HIV-1 infection. The experiments were performed in biological duplicates. <b>(D)</b> Percentage of 5’-methylcytosine content in genomic DNA at different time points. Dark bars—HIV-1 infected cells; White bars—non-infected cells. The data represent the mean of three different measurements and the error bars indicate the differences between three independent experiments. 2way ANOVA: *** p< 0.001, ** p < 0.01 and *, p < 0.05.</p

2015206 Raw data.xlsx

<div>Dataset associated with Nature Medicine (2016), doi:10.1038/nm.4106</div><div><br></div><div>Type I interferons and microbial metabolites of tryptophan modulate astrocyte activity and central nervous system inflammation via the aryl hydrocarbon receptor</div><div><br></div><div>1. RNA-Sequencing data of astrocytes during late stage Experimental autoimmune encephalomyelitis (EAE experiments) as compared to naive mice</div><div><br></div><div>2. Nanostring data of astrocytes and microglia during EAE</div><div><br></div><div><br></div