Background: Tenosynovial inflammation is a hallmark of rheumatoid arthritis [1] (RA), even during the preclinical phase [2]. While synovial tissue has been extensively studied, leading to the identification of novel molecular players with putative prognostic and therapeutic values [3, 4], the tenosynovium remains unexplored.

Objectives: To characterize tenosynovial cellular and molecular features across RA trajectory and compare it with adjacent synovial tissue samples.

Methods: Tenosynovium was retrieved by minimally invasive ultrasound (US)-guided biopsy from 59 patients: n=16 with ACPA ^pos and/or RF ^pos Clinical Suspect Arthralgia [5] (CSA) and n=43 with RA (n=10 naïve, n=14 resistant to csDMARDS, n=13 resistant to bDMARDs, n=2 difficult to treat and n=4 in remission). RA patients with synovitis in an adjacent joint to the tendon of interest also underwent paired US-guided synovial tissue biopsy (n=14). Lining hyperplasia and the degree of inflammatory infiltrate were assessed using H&E staining and immunohistochemistry. Among them, tenosynovium of 2 CSA and 14 active RA patients (n=5 naïve, n=5 resistant to csDMARDS, n=2 resistant to bDMARDs, and n=2 difficult to treat) were further analysed using single-cell RNA sequencing (scRNAseq, 10xGenomics). As a comparison, these data were integrated with a scRNAseq dataset of active RA synovium of the wrist [3] (n=17). Data processing and integration were conducted using the Seurat and Harmony packages. Cell clusters were manually annotated based on differentially expressed genes identified with MAST (adjusted P<0.05 by Bonferroni correction). Differential abundance testing was performed with MiloR package.

Results: Lining hyperplasia and inflammatory infiltrate (CD68 ^pos , CD3 ^pos and CD20 ^pos cells) of tenosynovium reflected the disease activity, being significantly higher in naïve or resistant to cs/bDMARDs patients compared to CSA and remission RA (One-way ANOVA: p<0.0001 for all comparisons). Paired tenosynovium and synovium samples from adjacent anatomical locations in active RA showed comparable enrichment of CD68 ^pos , CD20 ^pos and CD3 ^pos cells (p>0.05). A total of 60455 cells were successfully sequenced, revealing a tenosynovial atlas composed of stromal (e.g. fibroblasts, endothelial cells, mural cells) and immune (e.g. myeloid cells, T cells, B cells, plasmacells) clusters. Within the myeloid compartment (10139 cells), 13 clusters were identified. CSA patients exhibit an enrichment with homeostatic clusters, including TREM2 ^pos macrophages, FOLR2 ^high LYVE1 ^pos macrophages and FOLR2 ^high SELENOP ^high APOE ^high macrophages, while pathogenic clusters, such as S100A12 ^pos macrophages, SPP1 ^pos macrophages, TNF ^pos macrophages and CLEC10A ^pos macrophages, were prominent in naïve to treatment patients and persisted in non-responders. Within the fibroblast compartment (10092 cells), 9 clusters were identified, showing distinct distribution with disease progression. Difficult-to-treat patients showed an enrichment of perivascular CD34 ^pos MFAP5 ^pos fibroblasts, CXCL14 ^pos MFAP5 ^pos fibroblasts and HSPH1 ^pos fibroblasts, compared to naïve patients. Comparative analysis of the myeloid and the fibroblast compartment between the tenosynovium and adjacent synovium in the wrist of active RA patients revealed a differential clusters distribution. In particular, the tenosynovium was significantly enriched with perivascular CD34 ^pos MFAP5 ^pos fibroblasts, and TNF ^pos macrophages, whereas the wrist synovium was enriched with FOLR2 ^high APOE ^pos PDE4C ^pos macrophages. Other clusters, such as PRG4 ^pos lining fibroblasts and CXCL12 ^pos HLA ^pos fibroblasts, showed similar enrichment between the two compartments but exhibited tissue-specific signatures, suggesting distinct, locally defined pathogenetic functions.

Conclusion: Tenosynovial tissue analysis revealed distinct cellular and molecular profiles across the RA trajectory, and tissue-specific clusters that distinguish it from synovial tissue. Further investigation could provide valuable insights into the mechanisms underlying RA onset and progression.

REFERENCES: [1] Rogier C et al. Ann Rheum Dis. 2020

[2] Niemantsverdriet et al. ClinExpRheumatol. 2018

[3] Alivernini S et al. Nat Med. 2020

[4] Zhang F et al. Nature 2023

[5] Van Steenbergen HW et al. Ann Rheum Dis. 2015

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.