1227: The Composition of Circulating Immune Cells is Associated with Nociplastic Pain in Patients with Rheumatoid Arthritis

Background/Purpose: Over half of patients with RA report clinically meaningful pain, despite treatment with disease-modifying antirheumatic drugs (DMARDs). While joint inflammation is a known cause of pain in patients with rheumatic diseases, emerging data indicate that many patients also suffer from centralized or nociplastic pain. In our prior studies, we have used quantitative sensory testing (QST) to demonstrate that patients with RA have altered CNS pain processing compared to healthy controls and these alterations are associated with clinically meaningful outcomes. 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.

Methods: In this study, we aimed to recruit 50 RA patients with minimal joint inflammation but varying levels of systemic inflammation and pain. All patients consented under the IRB protocol underwent QST to assess centralized pain and provided blood samples for immune profiling using state-of-the-art multi-parameter flow cytometry with a custom panel designed to identify key immune cell types and activation states. Frozen peripheral blood mononuclear cells (PBMCs) from patients in the study with the highest and lowest levels of nociplastic pain were additionally used to generate single-cell RNA-sequencing (scRNA-seq) libraries. These samples were individually labelled using hash tag oligomers (HTO) and pooled for library prep using 10X Genomics technology to reduce batch effect. The resulting sequencing data was processed with cellranger pipeline and analyzed using the Seurat package in R.

Results: We were able to collect 44 usable samples from patients who successfully consented and completed all study requirements. Of these, ~43% reported mild or moderate pain at the time of sample collection as measured by the numeric rating scale. Based on preliminary analyses of scRNA-seq from 6 patients among the highest and lowest pain levels, we annotated 3 subpopulations of T cells (Naïve CD4, Memory CD4, and CD8), NK cells, B cells, Plasma cells, Dendritic cells (DCs), and 2 subpopulations of Monocytes (CD14+ and CD16+/FCGR3A+). The high pain group exhibited decreased proportions of T cells and increased monocytes and DCs compared to the low pain group.

Conclusion: Our preliminary results indicate that the composition of circulating immune cells is associated with nociplastic pain in RA patients. With further analysis, we aim to validate these results in the larger dataset and determine whether there are robust transcriptional changes in specific cell types. Moreover, we are currently in the process of analyzing immunophenotyping data to assess composition and activation across pain levels. These studies provide novel insights into the role of circulating immune cells in altered CNS pain regulation in RA patients.