Methods: This longitudinal analysis spanned 103 early RA patients across three independent cohorts, all DMARD-naïve. Synovial explants obtained by ultrasound-guided biopsies from 13 patients were cultured in vitro for 24 hours. Paired PBMCs and neutrophils from their blood were cultured for 24 and 12 hours, respectively. Protein secretion in culture supernatants was quantified using proximity extension assay technology, profiling a 92-protein inflammation-related panel. Proteomic profiles were also evaluated in serum samples from the same subjects, and correlation analyses were performed to define shared and compartment-specific contributors to the circulating inflammatory proteome. The resulting compartment-derived secreted protein signature was validated in an independent cohort of 74 patients with early RA naïve to conventional and biologic DMARDs by evaluating its association with baseline disease activity and clinical response to MTX at 3 and 6 months. To assess MTX-related effects in vivo , longitudinal serum samples from a third cohort of 16 patients with early RA naïve to conventional and biologic DMARDs were analyzed at baseline and after 6 months of MTX therapy. Analyses were restricted to patients achieving clinical response to MTX. Proteins significantly modulated in vivo in these patients were mapped onto compartment-specific secreted protein signatures to identify the cellular sources driving treatment-related proteomic changes.

Results: In the discovery cohort, correlation analyses between serum proteins and cell culture supernatants from synovial tissue, PBMCs and neutrophils identified both shared and compartment-specific contributors to the circulating inflammatory proteome. A minimal core consisting of a single protein common to all three compartments (CXCL9) defined a convergent inflammatory axis detectable in serum. PBMCs and neutrophils shared six proteins tied to growth factor signaling, cell survival, and chemotaxis. Synovial tissue shared one protein with neutrophils but none exclusively with PBMCs. Synovial-specific proteins featured pro-inflammatory cytokines like IL-17A and IL-1α; PBMC-specific ones emphasized immune regulation, apoptosis, and chemotaxis; neutrophils dominated with exclusive proteins in extracellular matrix remodeling, proteolysis, TNF/TWEAK signaling, and myeloid activation/survival pathways (e.g., MMP-1, MMP-10, TWEAK, TRANCE, CSF-1, uPA). Unsupervised clustering of the compartment-derived secreted protein signature in a second independent cohort of 74 patients with early RA naïve to conventional and biologic DMARDs identified two patient groups with distinct baseline serum protein profiles. Patients with higher expression of the secreted signature exhibited higher baseline disease activity and a better clinical response to MTX at 3 and 6 months of treatment. Longitudinal in vivo serum analysis in a third cohort of 16 patients with early RA treated with MTX, all achieving clinical response, revealed significant modulation of multiple circulating proteins after 6 months of therapy. A substantial proportion of MTX-modulated proteins overlapped with the compartment-derived secreted protein signature. Integration of in vivo serum changes with compartment-specific secreted protein profiles showed that MTX-associated proteomic modulation was predominantly linked to neutrophils, followed by PBMCs and synovial tissue.

Conclusions: This study unveils a compartment-derived secreted protein signature that not only shapes the circulating inflammatory proteome in early RA but also powerfully stratifies patients into clinically distinct subgroups with divergent baseline disease activity and MTX responsiveness, paving the way for biomarker-driven patient selection. Neutrophils emerge as the dominant cellular driver of MTX-induced proteomic resolution in responders, revealing a previously unrecognized myeloid-centric mechanism underpinning the drug’s efficacy and highlighting novel therapeutic targets within neutrophil-mediated pathways like proteolysis, TNF/TWEAK signaling, and extracellular matrix remodeling. These actionable serum biomarkers hold transformative potential for precision medicine in RA, enabling prospective identification of MTX responders, optimization of first-line therapies, and acceleration of personalized treatment strategies.

REFERENCES: NIL.

Acknowledgments: NIL.

Disclosure of Interests: None declared.