Deciphering Cell-types and Gene Signatures Associated with Disease Activity in Rheumatoid Arthritis using Single Cell RNA-sequencing

Objective: Single cell profiling of synovial tissue has previously identified gene signatures associated with rheumatoid arthritis (RA) pathophysiology, but synovial tissue is difficult to obtain. This study leverages single cell sequencing of peripheral blood mononuclear cells (PBMCs) from patients with RA and matched healthy controls to identify disease relevant cell subsets and cell type specific signatures of disease. Methods: Single-cell RNA sequencing (scRNAseq) was performed on peripheral blood mononuclear cells (PBMCs) from 18 RA patients and 18 matched controls, accounting for age, gender, race, and ethnicity). Samples were processed using standard CellRanger and Scanpy pipelines, pseudobulk differential gene expression analysis was performed using DESeq2, and cell-cell communication analysis using CellChat. Results: We identified 18 distinct PBMC subsets, including a novel IFITM3+ monocyte subset. CD4+ T effector memory cells were increased in patients with moderate to high disease activity (DAS28-CRP ≥ 3.2), while non-classical monocytes were decreased in patients with low disease activity or remission (DAS28-CRP < 3.2). Differential gene expression analysis identified RA-associated genes in IFITM3+ and non-classical monocyte subsets, and downregulation of pro-inflammatory genes in the Vδ subset. Additionally, we identified gene signatures associated with disease activity, characterized by upregulation of pro-inflammatory genes TNF, JUN, EGR1, IFIT2, MAFB, G0S2, and downregulation of HLA-DQB1, HLA-DRB5, TNFSF13B. Notably, cell-cell communication analysis revealed upregulation of immune-associated signaling pathways, including VISTA, in patients with RA. Conclusions: We provide a novel single-cell transcriptomics dataset of PBMCs from patients with RA, and identify insights into the systemic cellular and molecular mechanisms underlying RA disease activity. ### Competing Interest Statement MB research is funded by a grant from the French Society of Rheumatology, the Osteoarthritis Foundation grant and a doctoral fellowship from Sorbonne University. BYM is a paid consultant for SandboxAQ. CW research is funded by grants from the Lundbeck Foundation, Fabrikant Aage Lichtingers Foundation, Director Ib Henriksens stipend, Knud Hoejggaards Foundation, Jorcks Foundation and the Denmark-America Foundation. CW is a minor share holder in Ambu A/S, Bavarian Nordic A/S, Genmab A/S, H. Lundbeck A/S A and B and several other non-health related companies and mutual funds. U.K. is a paid consultant for Vevo Therapeutics. AJB is a co-founder and consultant to Personalis and NuMedii, consultant to Samsung, Mango Tree Corporation, and in the recent past, 10x Genomics, Helix, Pathway Genomics, and Verinata (Illumina), has served on paid advisory panels or boards for Geisinger Health, Regenstrief Institute, Gerson Lehman Group, AlphaSights, Covance, Novartis, Genentech, and Merck, and Roche, is a shareholder in Personalis and NuMedii, is a minor shareholder in Apple, Facebook, Alphabet (Google), Microsoft, Amazon, Snap, 10x Genomics, Illumina, CVS, Nuna Health, Assay Depot, Vet24seven, Regeneron, Sanofi, Royalty Pharma, AstraZeneca, Moderna, Biogen, Paraxel, and Sutro, and several other non-health related companies and mutual funds, and has received honoraria and travel reimbursement for invited talks from Johnson and Johnson, Roche, Genentech, Pfizer, Merck, Lilly, Takeda, Varian, Mars, Siemens, Optum, Abbott, Celgene, AstraZeneca, AbbVie, Westat, and many academic institutions, medical or disease specific foundations and associations, and health systems. Atul Butte receives royalty payments through Stanford University, for several patents and other disclosures licensed to NuMedii and Personalis. Atul Buttes research has been funded by NIH, Peraton (as the prime on an NIH contract), Genentech, Johnson and Johnson, FDA, Robert Wood Johnson Foundation, Leon Lowenstein Foundation, Intervalien Foundation, Priscilla Chan and Mark Zuckerberg, the Barbara and Gerson Bakar Foundation, and in the recent past, the March of Dimes, Juvenile Diabetes Research Foundation, California Governors Office of Planning and Research, California Institute for Regenerative Medicine, LOreal, and Progenity. None of these entities had any role in the design, execution, evaluation, or writing of this manuscript.