Investigating immunophenotypic and transcriptional heterogeneity as biomarkers of pain centralization in rheumatoid arthritis

超过一半的类风湿性关节炎（RA）患者报告疼痛，尽管治疗具有很强的疾病修饰， 抗风湿药（DMARDs）。我们的研究小组是最早证明这种疼痛与 远离发炎关节的区域疼痛敏感性增加，暗示大脑 和脊髓调节疼痛与此过程相关的疼痛被称为“集中性疼痛”，并且经常被称为“中枢性疼痛”。 用阿片类镇痛药治疗然而，阿片类药物的效果最低，而且经常被滥用。发展 非阿片类镇痛药的研究受到我们对中枢性疼痛如何影响的有限理解的阻碍。 表型与细胞水平的变化有关。在研究环境中，集中性疼痛通常被评估为 定量感官测试（QST）。然而，QST是耗时的，需要大量的评估人员， 训练，并且容易产生测量误差。在制定可量化的衡量标准方面， 改变的细胞状态来区分集中性疼痛的患者。必须满足这一需要， 实现我们的长期目标，为患有以下疾病的患者开发安全有效的非阿片类镇痛药 风湿性疾病这个高风险、高回报的提案的目的是产生前沿的见解， RA患者外周血单个核细胞（PBMC）与集中性疼痛的关系 我们的建议强调了一个公正的，高通量的方法，使用多参数流式细胞术， 单细胞RNA测序（scRNA-seq），以鉴定PBMC的特征， DMARD治疗后的集中性疼痛。我们的中心假设是， 循环免疫细胞，特别是单核细胞的表达将使RA患者具有集中的免疫功能。 疼痛表型与没有集中性疼痛表型的那些。在目标1中，我们将确定关联 PBMC的免疫表型特征与集中性疼痛之间的关系。我们将招募50名RA患者， 最小的关节炎症，但不同程度的疼痛后DMARD治疗。这些患者将接受QST 为了测试集中性疼痛（通过肌肉的压力疼痛阈值评估）是否与 其中：a）循环中每种细胞类型的比例，和B）每种细胞类型的激活状态的连续测量 细胞类型。在目标2中，我们将确定循环PBMC转录异质性的差异， 具有集中性疼痛表型的患者与不具有集中性疼痛表型的患者之间的差异。我们 将对目标1中具有最高（N = 10）和最低（N = 10）水平的患者子集进行scRNA-seq 集中的疼痛。我们将研究转录亚群和细胞类型特异性基因 两组之间的表情。我们的提案具有很高的影响潜力，因为如果成功， 新的见解循环PBMC的作用改变疼痛调节类风湿关节炎患者，典型的 全身炎症状况。本建议书中的数据将用于通知R01应用程序， RA患者集中性疼痛的可重复外周血生物标志物

Over half of patients with rheumatoid arthritis (RA) report pain despite treatment with strong disease-modifying antirheumatic drugs (DMARDs). Our research group was one of the first to show that this pain is associated with increased pain sensitivity in areas distant from inflamed joints, suggestive of abnormalities in the way the brain and spinal cord regulate pain. The pain associated with this process is termed “centralized pain” and is frequently treated with opioid analgesics. However, opioids are minimally effective and often misused. The development of non-opioid analgesics has been hindered by our limited understanding of how the centralized pain phenotype relates to changes at the cellular level. In the research setting, centralized pain is often assessed using quantitative sensory testing (QST). However, QST is time-consuming, requires significant assessor training, and is prone to measurement error. There is a critical unmet need to develop quantifiable measurements of the altered cellular state that distinguish patients with centralized pain. It is imperative to address this need to achieve our long-term goal of developing safe and efficacious non-opioid pain analgesics for patients with rheumatic diseases. The objective of this high-risk, high-reward proposal is to generate cutting-edge insights into the relationship between peripheral blood mononuclear cells (PBMCs) and centralized pain in patients with RA. Our proposal emphasizes an unbiased, high-throughput approach, using multi-parameter flow cytometry and single-cell RNA sequencing (scRNA-seq) to identify characteristics of PBMCs that are associated with centralized pain after DMARD therapy. Our central hypothesis is that differences in the composition and gene expression of circulating immune cells, particularly monocytes, will characterize RA patients with a centralized pain phenotype vs. those without a centralized pain phenotype. In Aim 1, we will determine the association between the immunophenotypic profile of PBMCs and centralized pain. We will enroll 50 RA patients with minimal joint inflammation but varying levels of pain after DMARD treatment. These patients will undergo QST to test whether centralized pain, assessed by pressure pain thresholds at the trapezius muscle, is associated with: a) the proportion of each cell type in circulation, and b) continuous measures of activation status for each cell type. In Aim 2, we will identify differences in the transcriptional heterogeneity of circulating PBMCs between patients with a centralized pain phenotype vs. those without a centralized pain phenotype. We will perform scRNA-seq on a subset of patients from Aim 1 with the highest (N = 10) and lowest (N = 10) levels of centralized pain. We will investigate differences in transcriptional subpopulations and cell-type-specific gene expression between the two groups. Our proposal has high impact potential because, if successful, it will deliver novel insights into the role of circulating PBMCs in altered pain regulation in patients with RA, the prototypical systemic inflammatory condition. Data from this proposal will be used to inform an R01 application to identify a reproducible peripheral blood biomarker for centralized pain in patients with RA.