Background: Around 30% of the patients with rheumatoid arthritis (RA) are seronegative for both rheumatoid factor (RF) and anti-citrullinated protein antibodies (ACPA). In this subset of patients, the lack of biological markers complicates diagnosis and could delay treatment initiation. Seronegative RA presents with a weaker HLA-DRB1 association [1, 2] and a lower infiltration of CXCL13 ⁺ CD4 ⁺ T cells in the synovium compared to ACPA+ RA [3, 4], suggesting that the adaptive immune system plays a less prominent role in the pathogenesis of seronegative RA.

Objectives: We hypothesized that innate immune mechanisms may play a more prominent role in ACPA- RA. Our overall objective was to uncover molecular pathways involved in the pathogenesis of ACPA-RA by identifying unique transcriptomic signatures in synovial mononuclear myeloid cells.

Methods: We performed 10X single cell RNA sequencing on immune cells from paired synovial fluid and peripheral blood (PB) from patients with ACPA+ and ACPA- RA (n=4 in each group). We confirmed our finding in a published single cell dataset comprising synovial biopsies from patients with ACPA- (n=6) and ACPA+ RA (n=21), naïve to treatment. We performed full spectrum flow cytometry on synovial fluid mononuclear cells from patients with ACPA- RA, ACPA+ RA and psoriatic arthritis (PsA) as a disease control group (n=8 to 9 in each group). Here, we assessed the frequency of dendritic cell populations and their capacity to produce pro-inflammatory cytokines upon Toll-Like Receptor stimulation. Finally, we measured interferon (IFN) cytokines in synovial fluid and sera of patients with ACPA- RA, ACPA+ RA and PsA (n=9 to 12 in each group).

Results: Our analysis revealed an elevated expression of type I IFN-stimulated genes (ISG) and a higher IFN score in SF of ACPA- RA as compared to ACPA+ RA. This IFN signature was derived from synovial dendritic cells, macrophages and T cells and was not detected in PB of ACPA- RA. We also identified a type I IFN signature in synovial tissues of two patients with ACPA-RA in an independent dataset. A subset of mature conventional dendritic cell 2 (cDC2) was enriched in SF of ACPA- compared to ACPA+ RA patients. The levels of IFNs were increased in synovial fluid as compared to blood in all disease groups but were not different between ACPA- and ACPA+ RA.

Conclusion: In this study, we demonstrate a distinct type I IFN gene response signature in immune cells within the synovial joint, but not in peripheral blood, of patients with ACPA- RA. The lack of difference in the levels of IFNs suggest that IFN priming might occur either in a local niche in the synovial joint or in the lymph nodes, or even at a different disease stage. These findings provide a foundation for future research to explore the initiation of type I IFN responses and their role in the pathogenesis of ACPA- RA.

REFERENCES: [1] Huizinga TW, Amos CI, van der Helm-van Mil AH, Chen W, van Gaalen FA, Jawaheer D, et al. Refining the complex rheumatoid arthritis phenotype based on specificity of the HLA-DRB1 shared epitope for antibodies to citrullinated proteins. Arthritis Rheum. 2005;52(11):3433-8.

[2] Ding B, Padyukov L, Lundström E, Seielstad M, Plenge RM, Oksenberg JR, et al. Different patterns of associations with anti-citrullinated protein antibody-positive and anti-citrullinated protein antibody-negative rheumatoid arthritis in the extended major histocompatibility complex region. Arthritis Rheum. 2009;60(1):30-8.

[3] Rao DA, Gurish MF, Marshall JL, Slowikowski K, Fonseka CY, Liu Y, et al. Pathologically expanded peripheral T helper cell subset drives B cells in rheumatoid arthritis. Nature. 2017;542(7639):110-4.

[4] Argyriou A, Wadsworth MH, 2nd, Lendvai A, Christensen SM, Hensvold AH, Gerstner C, et al. Single cell sequencing identifies clonally expanded synovial CD4(+) T(PH) cells expressing GPR56 in rheumatoid arthritis. Nat Commun. 2022;13(1):4046.

Acknowledgements: NIL.

Disclosure of Interests: Alexandra Argyriou: None declared, Marc H Wadsworth II Pfizer Inc., Chirag Krishna Pfizer Inc., Christina Gerstner: None declared, Begum Horuluoglu: None declared, Merel Sijbranda: None declared, Lars Ronnblom AstraZeneca, Maija-leena Eloranta: None declared, Marie Wahren-Herlenius: None declared, Aase Hensvold: None declared, Aaron Winkler Pfizer Inc., Vivianne Malmström: None declared, Karine Chemin: 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.