Dysregulation in miRNA expression profile in PBMC from PTSD patients and controls.

<p><b>A–F:</b> Total RNA from PBMC of patients with PTSD and normal controls were used in the analysis of miRNA expression by Affymetrix miRNA array hybridization. Comparison of miRNA expression profiles between individual controls and PTSD patients was shown by the heat map (<b>A</b>) and plot of the principal component analysis accounting for 60.1% variability (<b>B</b>). Seven up-regulated miRNAs in PBMC from PTSD patients were compared with those from controls (<b>C</b>). 64 down-regulated miRNA molecules (>2.5 fold change, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094075#pone-0094075-t001" target="_blank">Table 1</a>) in PBMC from PTSD patients were detected (<b>D</b>). Total RNA samples were also used in the confirmation of miRNA down-regulation (miR-125a and miR-181c) in PBMC from PTSD patients by real-time PCR (<b>E</b>). Wilcoxon rank sum test was used to compare the difference in miRNA expression in PBMC between controls and PTSD patients. <b>F–G:</b> Role of hsa-miR-125a in the regulation of IFN-γ. Complementary sequences between the seed sequence of miR-125a and 3′-untranslated region (3′UTR) of IFN-γ gene were compared (<b>F</b>). In silico studies were used to determine the complementary sequences between the seed sequence of miR-125a and 3′UTR of IFN-γ gene. The inhibitory effect of miR-125a on IFN-γ production in PBMC was determined (<b>G</b>). Hsa-miR-125a precursor (pre-miR125a) and pre-scramble control (scrambled premiR) were introduced into PBMC by electroporation. After PHA stimulation for two days, IFN-γ release from PBMC was determined in the culture supernatant by ELISA. Wilcoxon rank sum test was used to analyze the inhibitory effects of miR-125a on IFN-γ production in PBMC.</p

Comparison of cytokines IFN-γ, IL-17, PDGF-bb, RANTES and IL-4inplasma from controls and PTSD patients.

<p>Plasma samples were isolated from peripheral blood samples of patients with PTSD and normal controls by centrifugation. Then, Bio-Rad Bio-Plex method was used to determine the concentrations of multiple cytokines (including IL-1β, IL-1RA, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12(p70), IL-13, IL-15, IL-17, FGF-β, eotaxin, G-CSF, GM-CSF, IFN-γ, IP-10, MCP-1, MIP-1α, MIP-1β, PDGF-BB, RANTES, TNF-α and VEGF). Wilcoxon rank sum test was used to compare the difference of cytokines in the plasma between controls and PTSD patients. Plasma levels of IFN-γ (<b>A</b>), IL-17 (<b>B</b>), PDGF-bb (<b>C</b>), and RANTES (<b>D</b>) were found to be significantly different between controls and PTSD patients, whereas other cytokines such as IL-4 (<b>E</b>) did not have significant difference in plasma from controls and PTSD patients. The boxed data included 50% of measurements at medium level, the line in the box was the average value and the data beyond the upper line were the outliers. <i>p</i> values were presented.</p

Down-regulated miRNAs (>2.5 fold changes) detected by miRNA array hybridization assay in patients with post-traumatic stress disorder.

<p>Down-regulated miRNAs (>2.5 fold changes) detected by miRNA array hybridization assay in patients with post-traumatic stress disorder.</p

PBMC counts and correlation between clinical scores.

<p>The PTSD scores, anxiety scores and depression scores were used to determine the disease severity in PTSD patients. The higher score represented the more severe PTSD disease. PBMC were isolated from peripheral blood samples of patients with PTSD and healthy controls by Ficoll gradient centrifugation and viable cells were enumerated. Wilcoxon rank sum test was used to compare the difference of PBMC counts between controls and PTSD patients (<b>A</b>). Spearman rank correlation was used to analyze the correlation between anxiety scores and PBMC counts (<b>B</b>), between PTSD scores and anxiety scores (<b>C</b>), between PTSD scores and depression scores (<b>D</b>) and between anxiety scores and depression scores (<b>E</b>) in PTSD patients. Each dot or diamond symbol represents data from an individual patient or normal control. In panel <b>A</b>, the boxed data included 50% of measurements at medium level, the line in the box was the average value and the data beyond the upper line were the outliers. In panels <b>B–E</b>, both correlation coefficient (R) and <i>p</i> values were presented.</p

Comparison of T helper cell populations in PBMC between controls and PTSD patients and correlation between T helper cell populations and PTSD scores.

<p><b>A–D:</b> Comparison of T helper cell populations in PBMC between controls and PTSD patients. PBMC from PTSD and normal controls were stimulated with 50 µg/mL of PHA for 3 days. Cells were stained with FITC-anti-IFN-γ, PE-anti-IL-4 and APC-anti-CD4 antibodies, and Th1 and Th2 cell populations were analyzed by flow cytometry. After staining with FITC-anti-IL-17A, PE-anti-FoxP3 and APC-anti-CD4 antibodies, Th17 and regulatory T cell (Treg) populations were analyzed by flow cytometry. Wilcoxon rank sum test was used to compare the difference of Th1 (<b>A</b>), Th17 (<b>B</b>), Treg (<b>C</b>) and Th2 (<b>D</b>) cell populations in controls and PTSD patients. The boxed data included 50% of measurements at medium level, the line in the box was the average value and the data beyond the upper line were the outliers. <i>p</i> values were also presented. <b>E–I:</b> Correlation between T helper cell populations and clinical scores in PTSD patients. PBMC from PTSD and normal controls were stimulated with 50 µg/mL of PHA for 3 days and stained for Th1, Th2, Th17 and Tregs as described above. Spearman rank correlation was used to analyze the correlation between PTSD scores and percentages of T helper cell populations from PTSD patients. The correlation between PTSD scores and T helper/Treg populations in PTSD patients was shown by the heat map (<b>E</b>), and the correlation between PTSD scores and Th1 (<b>F</b>), Th17 (<b>G</b>), Treg (<b>H</b>) and Th2 (<b>I</b>) cell populations in PTSD patients was presented. In panels <b>F–I</b>, correlation coefficient (R) and <i>p</i> values were presented.</p

Pathway Analysis depicting a network of dysregulated miRNAs and target genes and their relationship in regulation of immune functions and pathways.

<p><b>A.</b> Dysregulated miRNAs from PTSD patients were found to be involved in immunological pathways when Ingenuity Pathway Analyses were performed. The green tags represent down-regulated miRNAs and red tags up-regulated miRNAs in PBMC from PTSD patients when compared to controls. Circles represent genes, ovals are transcriptional regulators, diamonds are enzymes and triangles are kinases. Solid arrows represent direct action and dashed arrows indirect action. <b>B.</b> Depicts the relationship between miRNAs from PTSD patients with their target genes. First, both upregulated and downregulated miRNAs of PTSD patients were selected following Ingenuity Pathway analysis and then the relationship between miRNAs and genes were analyzed using Cytoscape software. Cytoscape analysis showed relationship between miRNAs and immunological pathways, especially between miRNAs and cytokine-associated genes (IL-1RA, IL-2, IL-2RB, IFN-γ, IL-12, IL-10, IL-17, IL-17R, TGF-β, IL-23A, etc.) playing a role in T cell development and immune functions. The larger circles represent miRNAs that are involved in regulation of a large number of genes, when compared to miRNAs represented by smaller circles. <b>C.</b> Shows the role of dysregulated miRNAs from PTSD patients in various immunological mechanisms and pathways. Upregulated and down-regulated miRNAs from PTSD patients were analyzed using Cytoscape (ClueGo) software. miRNAs from PTSD patients were found to be involved in several immunological and biological pathways.</p

Comparison of cell populations in PBMC between controls and PTSD patients.

<p>PBMC were isolated from peripheral blood samples of patients with PTSD and normal controls as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094075#pone-0094075-g001" target="_blank">Fig. 1</a>. After staining with FITC-anti-CD3, PE-anti-CD8 and APC-anti-CD4 antibodies, CD4 and CD8 T cells were analyzed by flow cytometry. After staining with FITC-anti-CD19 and APC-anti-CD3 antibodies, B cells (CD19+) were analyzed by flow cytometry. After staining with PE-anti-CD56 and APC-anti-CD3 antibodies, NK (CD56+) and NKT cells (CD56+CD3+) were analyzed by flow cytometry. The difference in the percentages of various lymphocyte subsets in PBMC between normal controls and PTSD patients was shown (<b>A</b>). The differences in absolute numbers of CD4 T cells (<b>B</b>), CD8 T cells (<b>C</b>), B cells (<b>D</b>), NK cells (<b>E</b>) and NKT cells (<b>F</b>) in PBMC between individual controls and PTSD patients were also presented. Wilcoxon rank sum test was used to compare the difference between controls and PTSD patients. In panels <b>B–F</b>, the boxed data included 50% of measurements at medium level, the line in the box was the average value and the data beyond the upper line were the outliers. <i>p</i> values were also presented.</p