POS0366 PRO-INFLAMMATORY EFFECTS OF HUMAN APATITE CRYSTALS EXTRACTED FROM PATIENTS SUFFERING FROM CALCIFIC TENDINOPATHY
Authors: J. Herman, B. Le Goff, J. De Lima, R. Brion, C. Chevalier, F. Blanchard, C. Darrieutort-Laffite
Journal: Annals of the Rheumatic Diseases (2021)

Background: Calcific tendonitis of the rotator cuff is due to carbonated apatite deposits in the shoulder tendons. During the evolution of the disease, an acute inflammatory episode may occur leading to the disappearance of the calcification. Although hydroxyapatite crystals-induced inflammation has been previously studied with synthetic crystals, no data are available with calcifications extracted from patients suffering from calcific tendinopathy. The objective of the study was to explore the inflammatory properties of human calcifications and the pathways involved. Objectives: The objective of the study was to explore the inflammatory properties of human calcifications and the pathways involved. Methods: Human calcifications were obtained from patients treated for their shoulder pain related to a calcific tendinopathy of the rotator cuff. Calcifications were extracted by ultrasound-guided lavage and aspiration as previously described [1]. Human calcifications and synthetic hydroxyapatite (sHA) were used in vitro to stimulate human monocytes and macrophages, the human myeloid cell line THP-1 and human tenocytes. The release of IL-1β, IL-6 and IL-8 by cells was quantified by ELISA. Gene expression of pro- and anti-inflammatory cytokines was evaluated by quantitative PCR. NF-kB activation and NLRP3 involvement was assessed in THP-1 cells using a NF-kB inhibitor and a Caspase 1 inhibitor. The inflammatory properties were then assessed in vivo using a mouse air pouch model. The membrane thickness and infiltrate were assessed 6 and 24 hours after the injection of human calcifications or synthetic hydroxyapatite using hematoxylin and eosin staining. Macrophages, neutrophils and lymphocytes infiltrates were assessed by immunohistochemistry. Total RNA was extracted from the membranes and expression of IL-1β, IL-6 and TNFβ was quantified by PCR. Results: Human calcifications were able to induce a significant release of IL-1β when incubated with monocytes, macrophages and THP-1 only if they were first primed with LPS (lipopolysaccharide) for monocytes and macrophages or PMA (Phorbol 12-myristate 13-acetate) for THP-1. No IL-1β was detected in tenocytes’ supernatants. Stimulation of THP-1 by human calcifications led to similar levels of IL-1β when compared to synthetic hydroxyapatite although these levels were significantly inferior in monocytes and macrophages. IL-6 and IL-8 levels were not increased in the supernatants after crystal stimulation. Patient’s crystals enhanced mRNA expression of pro-IL-1β, as well as IL-18, NF-kB and TGFβ when IL-6 and TNFα expression were not. IL-1β production was reduced by the inhibition NF-kB as well as Caspase 1 indicating the role of NLRP3 inflammasome. In vivo, injection of human calcifications or synthetic hydroxyapatite in air pouch led to significant increase in membrane thickness with an infiltrate mainly composed of macrophages. Significant overexpression of IL-1β was only observed in the synthetic hydroxyapatite group. Conclusion: As synthetic hydroxyapatite, human calcifications were able to induce an inflammatory response resulting in the production of IL-1β after NF-kB activation and through NLRP3 inflammasome. In some experiments, IL-1β induction was lower with human calcifications compared to synthetic apatite. Differences in size, shape and protein content may explain this observation. References: [1]Darrieutort-Laffite C, Arnolfo P, Garraud T, Adrait A, Couté Y, Louarn G, et al. Rotator Cuff Tenocytes Differentiate into Hypertrophic Chondrocyte-Like Cells to Produce Calcium Deposits in an Alkaline Phosphatase-Dependent Manner. J Clin Med. 2019 Sep 26;8(10):1544. doi: 10.3390/jcm8101544. Acknowledgements: Fondation Arthritis, Recherche et Rhumatismes and French Society for Rheumatology for their financial support Disclosure of Interests: None declared