Development of a diagnostic platform for PTSD blood biomarkers

项目摘要 创伤后应激障碍（PTSD）是一种应激反应性障碍，临床表现为 当患者在暴露于创伤性事件后被触发时是明显的。敏感性受损 糖皮质激素（GC）和外周血单核细胞（PBMC）基因表达谱的改变， 包括与GC信号传导和炎症细胞因子产生有关的基因，正处于 外周和中枢易感性通路，代表有前途的PTSD生物标志物。然而，他们的小 病例-对照效应量一直是发展可行的诊断方法的一个重要障碍。 疾病的生物标志物。值得注意的是，这些研究是在创伤后的基线条件下进行的 并且不能完全概括对不利的病理生理学触发因素（例如暴露）的分子反应 来刺激荷尔蒙我们开发了一种新的体外模型，使PBMC对合成GC敏感， 地塞米松（DEX）。我们的模型捕捉了个体对GC敏感性的差异， 显著放大PTSD基因表达效应大小到一个范围内，可能有助于发展 可行的生物标志物。尽管如此，GC敏感性的细胞类型特异性差异尚未被完全理解， 创伤后应激障碍和代表了发展可翻译的生物标志物的重要一步， 治疗学在这里，我们建议将我们的体外模型与单细胞细胞索引相结合， 通过测序（CITE-seq）和染色质免疫沉淀（ChIP）-seq， 研究PTSD中GC激活的单细胞转录反应。具体来说，我们将调查 目的1）使用CITE-seq方法对媒介物和DEX处理的细胞类型特异性转录差异 PBMC来源于创伤暴露的患有PTSD（+; n=20）和不患有PTSD（-; n=20）的退伍军人， 以及健康无创伤参与者（HC; n=20）。在此过程中，我们将研究约600，000个单细胞（约5，000个 每个个体的每个条件的细胞数）。流式细胞术将定量GR的密度和比例 在五种主要免疫细胞类型上的表达。由于GR具有位点特异性DNA结合活性， 在细胞和条件之间，我们将目标2）在五个主要免疫细胞亚群上进行抗GR ChIP-seq数据 以鉴定全基因组范围的细胞类型特异性GR结合位点。整合CITE-seq、GR表达和ChIP- 在DEX暴露的免疫细胞亚群中的seq将提供对免疫细胞亚群的更细致入微和准确的见解。 与PTSD中GC敏感性改变相关的细胞和分子扰动。我们已经 在用DEX处理的大量PBMC中测量全基因组RNA-seq， PTSD+和PTSD-的区别成功完成这项提案将揭示细胞类型- 特异性转录反应，将作为生物标志物的发展的明确指导， PTSD压力反应，脆弱性和弹性。

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.