Background: Rheumatoid arthritis (RA) is a complex chronic inflammatory disease with high heterogeneity. Characterizing the molecular mechanisms linked to different phenotypes might open new opportunities for the development of personalized therapeutic approaches.

Objectives: 1-To characterize the circulating inflammatory proteome in both early and established RA patients. 2- To identify common and unique inflammatory signatures associated with disease activity in both RA groups.

Methods: A cohort of 125 RA patients was included in the study, comprising 72 with early RA (diagnosed <2 years) and 54 with established RA (diagnosed >10 years). Serum samples were collected from all subjects for the analysis of the inflammatory proteome using the novel high-throughput proteomic technology ‘Proximity Extension Assay’ (PEA-Olink, Cobiomic). This specific and sensitive technology allows for the unique characterization of inflammatory proteins through qPCR, using antibodies linked to DNA oligonucleotides. Correlation and association studies were conducted to identify shared and distinctive signatures between both patient groups. Finally, functional enrichment analysis was performed using the STRING platform to gain insights into the biological meaning of the identified signatures.

Results: Clinically, early and established RA patients were treated with csDMARDs, excluding ts/bDMARDs, and did not differ in disease activity (mean DAS28 4.3 vs. 4.4), age (53 vs. 55 years), and sex (75% vs. 77% females). The analysis of the circulating inflammatory proteome showed that established RA patients displayed significantly higher levels of several inflammatory mediators compared with early RA patients, including CSF-1, IFN-g, FGF-23, TNF, Flt3L, EN-RAGE, OPG, DNER, IL-10RB, Beta-NGF, IL-18R1, AXIN1, CD40, CXCL5, STAMBP, SIRT2. Correlation studies identified common and distinctive associations among disease activity and inflammatory molecules in each group of patients. Thus, in early RA patients, 20 inflammatory markers positively correlated with DAS28, while in established RA patients, almost twice the number of inflammatory proteins (36) were also positively correlated. Functional enrichment of these inflammatory signatures identifies that proteins correlated to disease activity in early RA were mainly associated with pathways such as myeloid migration and macrophages and neutrophils chemotaxis, while proteins correlated with DAS28 in established RA were also associated with complementary pathways such as plasma cell differentiation and regulatory T-cell cytokine production. This data might suggest that disease activity in RA patients is orchestrated differently at the beginning of the disease compared to the progression of the pathology, where a more complex molecular network seems to drive the disease. Interestingly, a common signature of inflammatory mediators was positively correlated in both groups of patients, including CASP-8, CSF-1, CXCL1, CXCL10, CXCL9, EN-RAGE, GDNF, HGF, IL-15RA, IL6, LAP TGF-beta-1, MCP-3, TNFRSF9, VEGFA, highlighting their key role in the pathophysiology of RA.

Conclusion: Established and early RA patients exhibit distinctive circulating inflammatory signatures associated with disease activity, with early RA patients being linked to myeloid activity, and established RA patients displaying a more complex profile, also associated with adaptive immunity pathways. These data might have therapeutic implications, as they could drive the development of personalized clinical strategies for RA patients.

REFERENCES: NIL.

Acknowledgements: EU/EFPIA Innovative Medicines Initiative Joint Undertaking 3TR, Projects no. PI21/0591 & CD21/00187 funded by Instituto de Salud Carlos III (ISCIII) and co-funded by the European Union. Project no. RD21/0002/0033 funded by ISCIII and funded by the European Union-NextGeneration EU, via Plan de Recuperación, Transformacion y Resiliencia (PRTR) and MINECO (RYC2021-033828-I, and PID2022-141500OA-I00).

Disclosure of Interests: None declared.