Background: Inflammatory microenvironment in RA synovium is highly complex, characterized by the expansion of fibroblast-like synoviocytes (FLS) and the infiltration of immune cells such as T cells, B cells, and macrophages. Recent advancements in single-cell RNA sequencing and functional studies have identified several pathogenic cell subsets within the RA synovium. However, these studies primarily focus on individual cells and their function or frequencies, and less is known about the spatial organization and cellular neighborhoods (CNs) that shape the inflammatory microenvironment in RA. Thus, understanding the CNs within the inflammatory microenvironment and signals that drive interactions between cells in the RA synovium are critical for uncovering the underlying mechanisms of disease pathogenesis and further benefiting to develop new strategies to tackle RA.

Objectives: This study aims to dissect CNs within RA synovium to identify key interacting cell types and potential therapeutic targets.

Methods: Synovial tissues from 10 RA patients and 4 osteoarthritis (OA) patients were collected for imaging mass cytometry (IMC). A combined analysis of IMC and single-cell RNA sequencing data was performed to elucidate cell interactions based on spatial localization and receptor-ligand relationships. Co-culture experiments were conducted to validate the effects and mechanism of fibroblast-like synoviocyte (FLS)-macrophage interaction. The impact of interrupting cell interaction signals on collagen-induced arthritis (CIA) mice was further evaluated.

Results: We identified 15 metaclusters of cell populations and observed close interaction between fibroblast-like synoviocytes (FLS) and macrophages in RA synovium. Further investigation revealed 20 distinct CNs, in which CN15, predominantly composed of FLS and macrophages, exhibited specific spatial distribution patterns and presented significantly more frequently in RA compared to OA. Single-cell RNA sequencing analysis showed that FLS specifically upregulated complement C3 while macrophages displayed high level of C3 receptor, C3aR1. Interestingly, C3a derived from FLS enhanced type I interferon response in macrophages, and blockade of the C3a-C3aR1 signaling reduced secretion of IFN-β in macrophages, thereby affecting FLS activation. Further, we demonstrated that inhibition of C3aR1 signaling attenuated the severity of CIA mice with decreased immune cell infiltration, FLS activation, and less bone destruction.

Conclusion: Our study suggests that FLS promote synovial inflammation via interaction with macrophages through a C3a-C3aR1 signaling-mediated positive feedback regulation. Thus, targeting C3a-C3aR1 signaling might provide a new therapeutic strategy for the treatment of RA.

REFERENCES: [1] Rauber S, Mohammadian H, Schmidkonz C, Atzinger A, Soare A, Treutlein C, Kemble S, Mahony CB, Geisthoff M, Angeli MR et al : CD200(+) fibroblasts form a pro-resolving mesenchymal network in arthritis. Nat Immunol 2024, 25 (4):682-692.

[2] Zhang F, Jonsson AH, Nathan A, Millard N, Curtis M, Xiao Q, Gutierrez-Arcelus M, Apruzzese W, Watts GFM, Weisenfeld D et al : Deconstruction of rheumatoid arthritis synovium defines inflammatory subtypes. Nature 2023, 623 (7987):616-624.

Acknowledgements: NIL.

Disclosure of Interests: None declared.

© The Authors 2025. This abstract is an open access article published in Annals of Rheumatic Diseases under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ ). Neither EULAR nor the publisher make any representation as to the accuracy of the content. The authors are solely responsible for the content in their abstract including accuracy of the facts, statements, results, conclusion, citing resources etc.