Deciphering gene dysregulation across the immune system in ME/CFS with single-cell transcriptomics

项目概要-项目3 肌痛性脑脊髓炎，或慢性疲劳综合征（ME/CFS），是一种流行和严重的疾病， 症状包括注意力、记忆力和睡眠问题，以及肌肉骨骼疼痛。 ME/CFS没有确定的原因;既没有疫苗也没有有效的治疗方案。因此，我们认为， 迫切需要提高我们对这种疾病的基础科学的理解， 最终治愈的方法尽管我们对ME/CFS缺乏了解，但大量证据表明， 免疫失调是疾病的根本原因或主要后果。但 ME/CFS中涉及最多的特定失调白细胞（白色血细胞，WBC）的身份是 不清楚项目3，我们的ME/CFS合作研究中心应用程序的一部分，建议阐明 ME/CFS中的免疫失调，通过全面调查不同年龄组患者的基因调控缺陷， 所有类型的白细胞。在目的I中，我们建议使用单细胞RNA测序（scRNAseq）来询问 来自收集自ME/CFS患者和对照组群的外周血的白细胞的转录组。 这种新的方法提供了多种优势，超过所有以前的基因调控研究的疾病。第一、 scRNAseq对于所检查的特定细胞类型的身份本质上是无偏的。第二、 scRNAseq生成的数据具有显著的特异性和统计能力，例如，允许我们定义 单个免疫细胞的激活状态。第三，我们的scRNAseq方法将同时产生 血液中细胞类型谱的测量。因此，我们将能够发现坐标 不同细胞类型之间基因调控的变化。在Aim II中，我们将扩展scRNAseq的使用， 比较运动后患者和对照组的白细胞分布。这一目标的动机是需要 更好地了解ME/CFS患者运动引起的特定白细胞中的异常基因调控， 因为运动后不适是这种疾病的典型症状。最后，在目标III中，我们将研究可能的 在ME/CFS中，细胞外囊泡中发现的microRNA（ECV-miRNAs）的作用。细胞外囊泡是一种 新发现的一类由供体细胞分泌的颗粒，可将包括miRNA在内的货物运送到 受体细胞，从而改变受体细胞的状态。具体来说，我们将测试ECV的假设- 微RNA在ME/CFS中发生改变，检查运动激发前和运动激发后获得的样品， 这种改变是否导致患者免疫细胞的失调。因此，本研究的统一主题 目的是提供ME/CFS患者白细胞基因调控变化的明确数据，并研究 ECV-miRNAs是否参与了这种变化。因为检查的患者和对照组是 在U 54应用程序中很常见，我们将从白细胞中生成基因调控数据， 运动（项目3），以及神经学（项目1）和生理/分子数据（项目2）。我们 全面的数据将不仅是ME/CFS中心的关键资源，也是该领域所有研究人员的关键资源。

PROJECT SUMMARY- PROJECT 3 Myalgic encephalomyelitis, or chronic fatigue syndrome (ME/CFS), is a prevalent and severe disease with symptoms including problems with concentration, memory and sleep, together with musculoskeletal pain. There is no established cause for ME/CFS; neither vaccines nor effective treatment options exist. Therefore, there is an urgent need to advance our understanding of the basic science of this disease as an integral step towards an ultimate cure. Despite our lack of understanding of ME/CFS, substantial evidence implicates immune dysregulation either as an underlying cause or major consequence of the disease. Nevertheless, the identity of specific dysregulated leukocytes (white blood cells, WBC) that are most implicated in ME/CFS is unclear. Project 3, part of our ME/CFS Collaborative Researcher Center application, proposes to elucidate immune dysregulation in ME/CFS by comprehensively investigating gene regulation defects in patients across all types of leukocytes. In Aim I, we propose to use single-cell RNA sequencing (scRNAseq) to interrogate the transcriptomes of leukocytes from peripheral blood collected from a cohort of ME/CFS patients and controls. This novel approach offers multiple advantages over all previous gene regulatory studies of the disease. First, scRNAseq is inherently unbiased with respect to the identity of the specific cell-types examined. Second, scRNAseq generates data with remarkable specificity and statistical power, allowing us, for example, to define the activation state of individual immune cells. Third, our scRNAseq approach will generate simultaneous measurements across the spectrum of cell types in blood. Thus, we will be able to discover coordinate changes in gene regulation between different cell types. In Aim II, we will extend our use of scRNAseq to compare leukocyte profiles from patients and controls post-exercise. This Aim is motivated by the need to better understand aberrant gene regulation in specific leukocytes resulting from exercise in ME/CFS patients, as post-exertional malaise is a defining symptom of the disease. Finally, in Aim III, we will examine possible roles for microRNAs found in extracellular vesicles (ECV-miRNAs) in ME/CFS. Extracellular vesicles are a newly discovered class of particles secreted by donor cells, which deliver cargoes, including miRNAs, to recipient cells, thereby altering the state of the recipient cell. Specifically, we will test the hypothesis that ECV- miRNAs are altered in ME/CFS, examining samples obtained pre- and post- exercise challenge, and examine whether such alterations result in dysregulation of patient immune cells. Thus, the unifying theme of this study is to provide definitive data on gene regulatory changes in leukocytes from ME/CFS patients, and investigate whether ECV-miRNAs contribute to such changes. Because the patient and control cohorts examined are common across this U54 application, we will generate gene regulatory data from leukocytes before and after exercise (Project 3), together with neurological (Project 1) and physiological/molecular data (Project 2). Our comprehensive data will be a key resource not only for ME/CFS Centers, but for all researchers in the field.