Background: In rheumatoid arthritis (RA), chronic inflammation leads to progressive bone erosion, resulting in the release of calcium and phosphate into the local microenvironment. Elevated concentrations of these ions promote the formation of calciprotein particles (CPPs), which are colloidal complexes of calcium, phosphate, and serum proteins such as fetuin-A. CPPs are taken up by monocytes and macrophages, which can trigger inflammatory responses [1], although the precise mechanism remain incompletely understood. Extracellular vesicles (EVs) play a key role in intercellular communication in many diseases, suggesting that monocytes and macrophages may use EVs to interact with other immune cells, potentially amplifying inflammation in RA.

Objectives: This study aimed to determine whether calcium- and phosphate-associated extracellular vesicles (CaP-EVs) are present and detectable in patients with rheumatoid arthritis and healthy controls. Subsequently, CaP-EVs were quantified, and the effect of TNF inhibition on their serum concentration in RA patients was assessed.

Methods: Blood samples were collected from patients with rheumatoid arthritis (RA, n=67) and healthy controls (n=48) in serum gel monovettes. After complete clotting for 1 h at room temperature, samples were centrifuged, aliquoted, and serum was stored at -80 °C. For CaP-EV detection, serum samples were thawed at 37 °C for 2 min and diluted 1:10 in DMEM/10% HEPES (pH 8.0). Samples were stained for 1 h with 0.45 µM OsteoSense, 16 nM lactadherin-FITC, and 0.4 µM CellTrace Yellow, followed by removal of unbound dye by gel filtration. Samples were further diluted 1:10 in DMEM/10% HEPES (pH 8.0) and acquired on ImageStreamX Mark II in high-gain mode using a 60× objective and 488 nm, 561 nm, 642 nm and 785 nm lasers.

Results: Using a triple-staining approach—OsteoSense for calcium-phosphate complexes, lactadherin for phosphatidylserine, and CellTrace Yellow for enclosed membranes—we were able to detect and quantify intact CaP-EVs exposing phosphatidylserine on their surface. Phosphatidylserine is a lipid commonly exposed on extracellular vesicles that facilitates recognition and uptake by immune cells and its detection was used to confirm their vesicular nature and potential biological activity. RA patients displayed markedly elevated levels of CaP-EVs in serum compared to healthy controls, with concentrations even further enriched in synovial fluid. Notably, CaP-EV levels showed a strong correlation with DAS28 scores, implicating their close association with disease activity. Treatment with anti-TNF led to a substantial reduction in both CaP-EV concentrations and disease activity, highlighting the dynamic relationship between these vesicles and chronic inflammation.

Conclusions: Our data demonstrate that patients with rheumatoid arthritis exhibit significantly elevated concentrations of CaP-EVs in both serum and synovial fluid. CaP-EV levels correlate with disease activity and efficacious therapy is associated with a reduction in CaP-EV concentrations, implicating that CaP-EVs are involved in RA pathogenesis and may represent a novel therapeutic target.

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

Acknowledgments: NIL.

Disclosure of Interests: None declared.