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Introduction: Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by synovial inflammation and bone erosion. Bone erosion increases local concentrations of calcium and phosphate, promoting the formation of calcium–phosphate complexes such as calciprotein particles (CPPs). CPPs can be taken up by monocytes and macrophages and trigger inflammatory responses in the joint. Notably, monocytes from RA patients take up CPPs more efficiently and produce higher levels of IL-1β than monocytes from healthy controls [1]. Identifying biomarkers that reflect such pathogenic processes remains an important goal in RA research, and extracellular vesicles (EVs) have emerged as promising candidates and regulators of inflammatory signaling. Here, we investigated whether calcified EVs are present in RA and explored their cellular origin and inflammatory potential.

Methods: Serum (n=67) and synovial fluid (n=13) samples from RA patients and healthy controls (n = 48) were analyzed for calcified EVs using imaging flow cytometry and a triple-staining strategy: OsteoSense for CPPs, lactadherin for phosphatidylserine exposure, and CellTrace Yellow for enclosed membranes. To investigate their origin, RA monocytes were stimulated with CPPs in vitro, and EV release into culture supernatants was quantified. Functional effects were assessed by transferring conditioned supernatants to naïve monocytes and measuring IL-1β production.

Results: RA patients exhibited significantly elevated levels of calcified EVs in serum (mean: 2.5×106 EVs/ml) compared with healthy controls (mean: 1.3×106 EVs/ml; p=0.0008), with even higher concentrations detected in synovial fluid (mean: 5.4×106 EVs/ml; p=0.0475). Importantly, calcified EVs correlated significantly with DAS28 (p<0.0001) and with the number of swollen joints (p<0.0001). In vitro, stimulation of monocytes with CPPs induced the release of calcified EVs (mean: 2.1×106 EVs/ml), whereas CPP formation in cell-free conditions did not generate EVs (mean:9.9×104 EVs/ml, p=0.0312). Transfer of supernatants from CPP-stimulated monocytes to naïve monocytes induced approximately two-fold higher IL-1β production compared with direct CPP-stimulation (p=0.0228), indicating that calcified EVs amplify inflammatory signaling.

Conclusion: Calcified EVs are elevated in the circulation and synovial fluid of RA patients and can be generated by monocytes in response to CPPs. These findings suggest that calcified EVs link mineral imbalance to inflammatory amplification in rheumatoid arthritis and may serve as biomarkers of disease activity.

Literatur

[1] Jäger E, Murthy S, Schmidt C, Hahn M, Strobel S, Peters A, Stäubert C, Sungur P, Venus T, Geisler M, Radusheva V, Raps S, Rothe K, Scholz R, Jung S, Wagner S, Pierer M, Seifert O, Chang W, Estrela-Lopis I, Raulien N, Krohn K, Sträter N, Hoeppener S, Schöneberg T, Rossol M, Wagner U. Calcium-sensing receptor-mediated NLRP3 inflammasome response to calciprotein particles drives inflammation in rheumatoid arthritis. Nat Commun. 2020 Aug 25;11(1):4243. DOI: 10.1038/s41467-020-17749-6