Methods: Peripheral blood mononuclear cells (PBMCs) and neutrophils from 48 treatment-naïve RA patients were cultured for 24 and 12 hours, respectively, with autologous serum and Baricitinib (JAKinib) at a concentration of 10 micromolar. Changes in PBMC and neutrophil proliferation, adhesion, and NETosis-derived products were quantified using specialized kits. Changes in the secreted inflammatory proteins by PBMCs and neutrophils were assessed using proximity extension assay (PEA) technology (Olink), analyzing a 92-protein inflammation panel.The proteomic signature identified as deregulated by ex vivo JAKinibs treatment was subsequently analysed in the serum of a second cohort of 66 RA patients initiating JAKinibs treatment to evaluate its association with clinical response. Additionally, the signature was examined in the serum of a third cohort of 82 RA patients starting TNFi therapy to assess its correlation with clinical response and its specificity for JAKinibs.

Results: Ex vivo treatment with Baricitinib demonstrated its ability to reduce PBMC and neutrophil proliferation and adhesion and prevent NETosis formation in 70% of treatment-naïve RA patients. Autologous serum enhanced inflammatory protein secretion in both PBMCs and neutrophils, affecting cytokines, chemokines, and growth factors. Baricitinib reversed this inflammatory protein secretion, highlighting its key role in pathways associated with RA pathophysiology. Using serum samples from a second cohort of 66 active RA patients starting JAKinibs treatment, an unsupervised analysis of the specific inflammatory signature modulated ex vivo by JAKinibs was performed. This analysis before therapy stratified patients into two distinct subgroups: one with low protein expression and another with high expression levels of this signature. Clinically, elevated levels of the Baricitinib-modulated proteins were observed in patients with active disease (DAS28-CRP), higher acute phase reactants, and elevated RF titres before therapy. The cluster with high expression at baseline experienced a significant reduction in disease activity, as measured by ΔDAS28-CRP after 3, 6, and 12 months of JAKinib treatment. Furthermore, this subgroup showed a significantly higher rate of clinical remission at 3 months (DAS28-CRP < 3.2) compared to the low expression subgroup. Machine-learning algorithms identified both, clinical and molecular signatures as potential predictors of response to JAKinibs treatment with high accuracy, which was further increased when both features were integrated in a mixed model (AUC: 0.8). An unsupervised analysis of the serum from a third cohort of 82 RA patients starting TNFi treatment revealed two subgroups based on the same Baricitinib-modulated protein signature. However, patients with high baseline levels of this protein signature showed no changes in DAS28-CRP. Moreover, elevated levels of this protein signature predicted a low rate of disease remission at 3 months of TNFi treatment, suggesting the specificity of this molecular signature as predictor of beneficial therapeutic effects of JAKinibs.

Conclusion: The molecular analysis of changes induced by JAK inhibitors through ex vivo assays revealed a specific immune-proteomic signature. This signature stratifies RA patients into subgroups with differential clinical responses, demonstrating its potential as a robust predictor of therapeutic response to JAKinibs. Importantly, the signature appears to be specific to JAKinibs and not applicable to TNFi therapies, highlighting its relevance for precision medicine approaches in RA management.

REFERENCES: NIL.

Acknowledgements: Supported by the EU/EFPIA Innovative Medicines Initiative Joint Undertaking 3TR, ISCIII (PI24/00959, CD21/00187 and RICOR-21/0002/0033), and RYC2021-033828-I; co-financed by European Union.

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.