AB0061 INVOLVEMENT OF CELL-FREE DNA IN THE DIFFERENTIATION OF OSTEOCLASTS – IMPLICATIONS FOR CLINICAL RESEARCH
Authors: P. Škubica, J. Horinkova, M. Gregová, K. Pavelka, M. Husakova, P. Dankova
Journal: Annals of the Rheumatic Diseases (2020)

Background: Cell-free DNA (cfDNA) is a molecule with undisputable immunogenic potential [1] . Its role is well established in etiopathogenesis of systemic lupus erythematosus and many reports suggest it may play an important role in other rheumatic diseases [2] . Patients suffering from rheumatic diseases are at higher risk of osteoporosis [3] , which may be a consequence of increased generation of osteoclasts (OC). Traditionally, increased formation of OC is attributed to changes in cytokine network [4] . It is not yet known whether cfDNA can also act on mononuclear precursors and influence their differentiation into OC. Objectives: The aim of this study was to assess role of cfDNA in differentiation of bone resorbing cells in rheumatic diseases. For this purpose, deoxyribonuclease I (DNAse) was employed to eliminate cfDNA from sera of patients and healthy controls (HC) used for cell culture. Methods: In order to evaluate effects of cfDNA on osteoclastogenesis, an in vitro experiment of peripheral blood monocytes (PBM) cultures was designed. Axial spondyloarthritis (AxSpA) was chosen as a model of rheumatic disease. Sera of AxSpA patients (total n=13) and healthy controls (total n=13) were collected and subsequently used in cell cultures. PBM of 13 healthy donors were cultured in presence of 25 ng/ml macrophage colony-stimulating factor (M-CSF), 0,5 ng/ml receptor-activator of nuclear factor kappa B ligand (RANKL) and 10 % of pooled AxSpA or HC sera, respectively. Serum counterpart treated with DNAse was used in parallel. After 14 days, cells were stained for tartrate-resistant acid phosphatase (TRAP) and counted. TRAP positive cells with 3 or more nuclei were recognized as OC. Results: On average, differentiation of OC in presence of AxSpA sera led to higher osteoclastogenesis compared to DNAse-treated counterpart (137 vs. 61; p &lt; 0.01). In a similar way, treatment of HC sera with DNAse greatly diminished osteoclastogenesis compared to untreated sera (17 vs. 111; p &lt; 0.05). Overall, the effect of cfDNA depletion in HC sera regarding OC differentiation was more than 2,5× higher than the effect of cfDNA depletion in AxSpA sera (p &lt; 0.001). Conclusion: Results of our pilot study indicate possible involvement of cfDNA in osteoclastogenesis. Decrease in number of OC after cfDNA depletion in both AxSpA sera and HC sera suggests that cfDNA effect in osteoclastogenesis is non-specific with respect to healthy condition and may represent universal link between innate immunity and bone metabolism. Lower effect of cfDNA depletion in AxSpA sera on osteoclastogenesis is very possibly owing to presence of other inflammatory molecules, which influence osteoclastogenesis. Future research might identify cfDNA as useful therapeutic target to decrease osteoporosis risk not only in patients with rheumatic and autoimmune diseases, where pathological bone resorption is imminent, but also in general aging population. References: [1]Pisetsky, D. S. (2012). The origin and properties of extracellular DNA: from PAMP to DAMP. Clinical Immunology, 144(1), 32-40. [2]Lood, C. J., &amp; Duvvuri, B. (2019). Cell-free DNA as a biomarker in autoimmune rheumatic diseases. Frontiers in immunology, 10, 502. [3]Maruotti, N., Corrado, A., &amp; Cantatore, F. P. (2014). Osteoporosis and rheumatic diseases. Reumatismo, 125-135. [4]Amarasekara, D. S., Yu, J., &amp; Rho, J. (2015). Bone loss triggered by the cytokine network in inflammatory autoimmune diseases. Journal of immunology research, 2015. Acknowledgments: Project MH CR 00023728 &amp; MEYS CR Progres Q43 Disclosure of Interests: Patrik Škubica: None declared, Jana Horinkova: None declared, Monika Gregová Consultant of: Novartis, Abbvie, Paid instructor for: Novartis, Speakers bureau: Novartis, Abbvie, MSD, Karel Pavelka Consultant of: Abbvie, MSD, BMS, Egis, Roche, UCB, Medac, Pfizer, Biogen, Speakers bureau: Abbvie, MSD, BMS, Egis, Roche, UCB, Medac, Pfizer, Biogen, Marketa Husakova Speakers bureau: Novartis, Pavlina Dankova: None declared