Dysregulated TP53 Among PTSD Patients Leads to Downregulation of miRNA let-7a and Promotes an Inflammatory Th17 Phenotype

Abstract

Post-traumatic stress disorder (PTSD) is a psychiatric disorder and patients diagnosed with PTSD often express other comorbid health issues, particularly autoimmune and inflammatory disorders. Our previous reports investigating peripheral blood mononuclear cells (PBMCs) from PTSD patients showed that these patients exhibit an increased inflammatory T helper (Th) cell phenotype and widespread downregulation of microRNAs (miRNAs), key molecules involved in post-transcriptional gene regulation. A combination of analyzing prior datasets on gene and miRNA expression of PBMCs from PTSD and Control samples, as well as experiments using primary PBMCs collected from human PTSD and Controls blood, was used to evaluate TP53 expression, DNA methylation, and miRNA modulation on Th17 development. In the current report, we note several downregulated miRNAs were linked to tumor protein 53 (TP53), also known as p53. Expression data from PBMCs revealed that compared to Controls, PTSD patients exhibited decreased TP53 which correlated with an increased inflammatory Th17 phenotype. Decreased expression of TP53 in the PTSD population was shown to be associated with an increase in DNA methylation in the TP53 promotor region. Lastly, the most significantly downregulated TP53-associated miRNA, let-7a, was shown to negatively regulate Th17 T cells. Let-7a modulation in activated CD4+ T cells was shown to influence Th17 development and function, via alterations in IL-6 and IL-17 production, respectively. Collectively, these studies reveal that PTSD patients could be susceptible to inflammation by epigenetic dysregulation of TP53, which alters the miRNA profile to favor a proinflammatory Th17 phenotype.

Keywords: T helper 17 cells; inflammation; microRNA; post-traumatic stress disorder; tumor protein 53.

Figure 1

Dysregulated miRNA in PBMCs from PTSD patients are associated with TP53. miRNA array data from PBMCs from Control (n=4) and PTSD subjects (n=8) was analyzed using IPA analysis software. (A) Volcano plot depicting miRNA fold change values (x-axis) of PTSD vs. Control plotted against p-values (y-axis). Yellow dots represent TP53-associated miRNAs. (B) Heat Map depicting 21 TP53-associated miRNA that were significantly downregulated in PTSD samples compared to control. (C–G) Box and whisker plots depicting top 5 downregulated TP53-associated miRNA between Control and PTSD samples to include (C) let-7a, (D) miR-145, (E) miR-16, (F) miR-122, (G) miR-321b. Box and whisker bars depict maximum and minimum values, with middle bar depicted at the median. (H, I) Top ranked networks (as determined by IPA-derived –log p-values and # of molecules) for 21 total dysregulated miRNA associated with TP53 to include (H) Disease and Functions, as well as I) Molecular and Cellular Functions. Significance in linear fold change was determined as any value ≥1.5 or ≤ -1.5 fold, and p-value was determined significant if this value ≤ 0.05 using an unpaired standard t test. P-values: ≤ 0.005 (***), ≤ 0.001 (****).

Figure 2

Network of TP53 interactions with dysregulated miRNA from PTSD patients. IPA-derived network depicting dysregulated miRNA associated with TP53 from mean fold changes of Control (n=4) vs. PTSD (n=8) PMBCs. miRNA are depicted in green to denote downregulation (darker green denotes more downregulation) or red to denote decreased expression. Genes depicted in the network are predicted to be activated (orange) or inhibited (blue) based on interactions present, as noted in the Prediction Legend. A separate legend (lower right corner) is provided to identify components depicted in the network. Solid lines indicate direct interactions, and doted lines indicate either indirect or highly-predicted interactions.

Figure 3

Expression of TP53 in PBMCs is downregulated in PTSD samples compared to Control. (A–C) Normalized expression data was analyzed from public database (ID. GSE860) between PTSD (n=17) and Controls (n=15), with two time points during initial emergency room visit for trauma (ER) and 4 months later (4MTHS). (A) Box and whisker plot depicting fold change expression of TP53. (B) Box and whisker plot depicting fold change expression of IL-17A. (C) Correlation plot between TP53 and IL-17A fold change expression. (D–I) PBMC gene expression data was collected from VMC PTSD (n=19) and Control volunteers (n=6). (D) Box and whisker plot depicting TP53 expression by PCR. (E) Box and whisker plot depicting Mdm2 expression by PCR. Correlation plots depicting (F) TP53 vs. PTSD score, (G) TP53 vs age of cohort, (H) TP53 vs. Depression score, and (I) TP53 vs. Anxiety score. Box and whisker bars depict maximum and minimum values, with middle bar depicted at the median. For box and whisker plots, significance was determined using an unpaired standard t test. For correlation plots, Pearson correlation test was used to determine significance. Significance was defined as having p-value ≤ 0.05 (*).

Figure 4

PTSD PBMCs had increased DNA methylation in TP53 promoter region. MeDIP-seq was performed on randomly selected PTSD (n=5) and Control (n=5) PBMC samples. Visualization of the methylated genome was acquired in the IGB genome browser (www.bioviz.org) for (A) individual samples, and (B) combined samples within respective groups. Blue sections indicate PTSD samples, and yellow sections represent Controls. Green section indicates the TP53 gene, while the red section represents one of the known TP53 promoter regions (~5kb) provided in the Ensembl genome browser (www.ensembl.org). For combined data, a line at 1.5 value was drawn to better visualize methylation intensity between the two groups. Differences observed are qualitative measures in which increased signal intensity (e.g. peak height and width) signify more DNA methylation. Signal intensity in the PTSD group was greater overall in the TP53 promoter compared to Control samples.

Figure 5

Let-7a modulation affects Th17 development and function. (A) CD4+ cells were purified using magnetic bead separation from PBMCs provided by healthy control donor, which was validated using flow cytometry to ensure purity (>95%). (B) Representative flow cytometry plots depicting Th17 cells (CD4+IL-17A+) in unactivated or CD3/CD28 activation cultures (24 hours) in the absence or presence of mock, let-7a mimic, or let-7a inhibitor. Red numbers indicating double positive TH17 percentage. (C) Violin plot depicting Th17 percentages as assessed by flow cytometry. (D) Violin plot depicting expression of let-7a as assessed by PCR. (E) Violin plot depicting IL-17 concentration as assessed by ELISA from cell culture supernatants. (F) Violin plot depicting IL-6 concentration as assessed by ELISA from cell culture supernatants. In vitro data are representative of at least 3 independent experiments, with 3 samples per culture condition. One way ANOVA was used to determine significance between groups. Significance was determined as p-value ≤ 0.05(*), ≤ 0.01(**), ≤ 0.001(****).