Development of a diagnostic platform for PTSD blood biomarkers

PROJECT SUMMARY Post-traumatic stress disorder (PTSD) is a disorder of stress reactivity with clinical manifestations that are evident when patients are triggered following exposure to a traumatic event. Impaired sensitivity to glucocorticoids (GCs) and alterations in peripheral blood mononuclear cell (PBMC) gene expression profiles, including genes implicated in GC signaling and inflammatory cytokine production, are at the crossroads of peripheral and central susceptibility pathways and represent promising PTSD biomarkers. However, their small case-control effect sizes have been a significant barrier to the development of actionable diagnostic biomarkers for the disorder. Notably, such studies have been performed post-trauma under baseline conditions and do not fully recapitulate the molecular response to adverse pathophysiological triggers, such as exposure to stress hormones. We developed a new in vitro model that sensitizes PBMCs to the synthetic GC, dexamethasone (DEX). Our model captures differences between individuals in sensitivity to GCs and significantly amplifies PTSD gene expression effect sizes into a range that may facilitate the development of actionable biomarkers. Still, cell type-specific differences in GC sensitivity have not been fully appreciated in PTSD and represent an important step forward for the development of translatable biomarkers and therapeutics. Here, we propose to combine our in vitro model with single-cell cellular indexing of transcriptomes and epitomes by sequencing (CITE-seq) and chromatin immunoprecipitation (ChIP)-seq to investigate the single-cell transcriptional responses to GC activation in PTSD. Specifically, we will investigate Aim 1) cell type-specific transcriptional differences using the CITE-seq approach on vehicle and DEX treated PBMCs derived from trauma-exposed combat veterans with PTSD(+; n=20) and without PTSD(-; n=20), as well as healthy trauma-free participants (HC; n=20). In doing so, we will study ~600,000 single cells (~5,000 cells per condition for each individual). Flow-cytometry will quantify the density and proportion of GR expression on five major immune cell types. Because the GR has site-specific DNA binding activity that differs between cells and conditions, we will Aim 2) perform anti-GR ChIP-seq data on five major immune cell subsets to identify genome-wide, cell type-specific GR binding sites. Integrating CITE-seq, GR expression and ChIP- seq across DEX exposed immune cell subsets will provide more nuanced and accurate insights into the cellular and molecular perturbations associated with altered GC sensitivity in PTSD. We have already measured genome-wide RNA-seq in bulk PBMCs treated with DEX and uncovered robust transcriptional differences distinguishing PTSD+ from PTSD-. Successful completion of this proposal will uncover cell type- specific transcriptional responses that will serve as definitive guides for the development of biomarkers for PTSD stress responsivity, vulnerability and resiliency.