Post-Translational Modifications of Neutrophil Proteases Shape Fibroblast-Like Synoviocyte Responses in Rheumatoid Arthritis

Objectives Rheumatoid arthritis (RA) is driven by persistent inflammation within the synovium, where activated fibroblast-like synoviocytes (FLS) perpetuate tissue damage and immune activation.[1] Neutrophils, abundant in early synovial infiltrates, release serine proteases such as Proteinase-3 (PR3), Cathepsin-G (Cat-G), and Neutrophil Elastase (NE), which regulate immune responses, but when dysregulated, promote inflammatory pathology.[2] During pre-clinical RA, genetic and environmental factors trigger post-translational modifications (PTMs) such as citrullination and carbamylation. These PTMs can alter protease activity, immune recognition, and inflammatory potential.[3] The objective of this study was to determine how PTM-modified neutrophil proteases modulate FLS inflammatory signaling, cytokine secretion, and their role in driving autoimmunity and chronic inflammation in RA. Methods Primary human FLS were cultured and stimulated with native or PTM-modified neutrophil proteases. Cytokine and chemokine secretion (IL-6, IL-1β, TNF-α) was quantified by ELISA and Olink multiplex analysis. Protease-activated receptor (PAR) signaling was assessed using PAR2 inhibitors and phospho-ERK immunoblotting. Serum autoantibody reactivity to citrullinated PR3 was evaluated in RA patients and at-risk relatives. Results FLS exposed to PTM-modified neutrophil proteases showed markedly elevated secretion of IL-6, IL-1β, and TNF-α compared with unmodified controls (Figure 1A). This activation was significantly reduced by PAR2 inhibition, implicating a PAR2–ERK signaling axis in FLS inflammatory activation (Figure 1B). Flow cytometric analysis demonstrated rapid ERK phosphorylation following cit-PR3 stimulation, peaking at 15 minutes—much stronger than responses to PR3 (Figure 1C). Western blot confirmed prolonged pERK1/2 activation after cit-PR3 treatment, which was reduced by PAR2 blockade (Figure 1D). Olink proteomic profiling revealed that cit-PR3 shifted the FLS secretome toward a pro-inflammatory, immune-recruiting phenotype, with upregulation of CXCL1, CXCL5, CCL2, CCL3, and enrichment of granulocyte chemotaxis and cytokine-mediated response pathways. PCA distinguished cit-PR3–treated FLS from PR3, reflecting distinct secretome remodeling (Figure 1E-H). Time-course analysis showed persistent IL-6 elevation up to 24 h, consistent with chronic synovial activation (Figure 1I). Autoantibody profiling further revealed stepwise increases in anti-cit-PR3 IgG from ACPA− relatives to ACPA+ relatives to RA patients, linking PR3 modification to early autoimmune responses. Together, these findings identify cit-PR3 as a potent driver of FLS inflammatory signaling and a potential early biomarker for RA. Figure 1: (A) Cultured fibroblast-like synoviocytes (FLS) stimulated with native or PAD2-citrullinated PR3 show enhanced IL-6 and TNF-α secretion upon cit-PR3 exposure. (B) PAR2 inhibition markedly reduces IL-6 release, implicating PAR2 in protease-mediated inflammatory signaling. (C) Flow cytometry histograms showing time-dependent ERK phosphorylation in FLS, with cit-PR3 inducing stronger and more sustained pERK activation than PR3, LPS, or untreated controls. (D) Western blot demonstrates increased and sustained ERK phosphorylation following cit-PR3 treatment, blunted by PAR2 blockade. (E) Principal component analysis (PCA) demonstrates clear segregation of cit-PR3–treated FLS from PR3 and LPS controls, highlighting distinct secretome remodeling. (F) Volcano plot illustrating significantly upregulated proteins (e.g., CXCL1, CXCL5, CCL2, CCL3, CSF1, TSLP) following cit-PR3 treatment compared with PR3. (G) Heatmap of differentially expressed proteins highlights a distinct pro-inflammatory secretome induced by cit-PR3. (H) Pathway enrichment analysis showing activation of granulocyte chemotaxis and cytokine-mediated signaling pathways. (I) Time-course analysis shows prolonged IL-6 elevation up to 24 h after cit-PR3 stimulation, indicating persistent activation. (J ) Anti-cit-PR3 IgG levels increase progressively from ACPA− relatives to ACPA+ relatives and RA patients, supporting its potential as on early autoimmune biomarker. Conclusion Our findings establish PTM-modified neutrophil proteases as potent modulators of FLS behavior and contributing amplification of inflammation and autoimmunity in RA. These translational findings will be critical in developing targeted therapies that modulate dysregulated neutrophil function and pathogenic PTMs—broadening the therapeutic arsenal, preventing early disease onset, and ultimately improving patient quality of life. References [1.] Carmona-Rivera C. Curr Osteoporosis Rep 2024;22:280-9. [2.] Pham C. Int J Biochem Cell Biol 2008;40:1317-33. [3.] Suskiewicz MJ. BioEssays 2024;46:202300178.