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，尽管接受了强烈的疾病修饰治疗 抗风湿药物(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度增加，提示大脑方式异常 而脊髓可以调节疼痛。与这一过程相关的疼痛称为“集中性疼痛”，通常是 用阿片类镇痛剂治疗。然而，阿片类药物的效果微乎其微，而且经常被滥用。最新进展 非阿片类止痛药的研究一直受到我们对集中性疼痛的有限了解的阻碍 表型与细胞水平的变化有关。在研究环境中，集中疼痛通常被评估 使用定量感觉测试(QST)。然而，QST是耗时的，需要大量的评估人员 训练，容易产生测量误差。有一个严重的未得到满足的需求，即开发可量化的衡量标准 细胞状态的改变来区分集中性疼痛的患者。解决这一需求势在必行 实现我们开发安全有效的非阿片类止痛药的长期目标 风湿病。这项高风险、高回报的提案的目标是产生对 类风湿关节炎患者外周血单个核细胞与集中性疼痛的关系 我们的建议强调一种公正、高通量的方法，使用多参数流式细胞术和 单细胞RNA测序(scRNA-seq)以确定与以下相关的PBMC的特征 DMARD治疗后的集中性疼痛。我们的中心假设是组成和基因上的差异 循环免疫细胞的表达，特别是单核细胞，将是集中的RA患者的特征 疼痛表型与那些没有集中疼痛表型的患者相比。在目标1中，我们将确定关联 外周血单核细胞的免疫表型与集中性疼痛之间的关系。我们将招募50名患有RA的患者 DMARD治疗后关节炎症轻微，但疼痛程度不同。这些患者将接受QST 通过斜方肌压痛阈值来评估集中性疼痛是否与 使用：a)每种细胞类型在循环中的比例，以及b)每种细胞的激活状态的连续测量 单元类型。在目标2中，我们将确定循环中PBMCs转录异质性的差异 有集中疼痛表型的患者与没有集中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)水平的患者的子集进行scRNA-seq 集中的痛苦。我们将研究转录亚群和细胞类型特定基因的差异。 两组之间的表达。我们的提议具有很大的影响潜力，因为如果成功，它将提供 循环PBMCs在类风湿关节炎患者疼痛调节改变中作用的新见解 全身炎症状态。此提案中的数据将用于通知R01应用程序以识别 RA患者集中性疼痛的可重复性外周血液生物标记物。

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.