P140 Macrophage-mediated mechanisms and clinical predictors of calcinosis in systemic sclerosis

Abstract Background/Aims Calcinosis is a debilitating complication of systemic sclerosis (SSc), resulting from ectopic deposition of hydroxyapatite in skin and subcutaneous tissue, and frequently associated with digital ulcers, pain, and infection. The pathogenesis remains poorly understood but appears to involve aberrant osteogenic differentiation in subcutaneous adipose tissues. Activin A is a candidate pathway implicated in dysregulated osteogenesis. Methods We retrospectively analysed clinical records from a tertiary SSc cohort comprising 79 patients (28 with calcinosis, 51 without). All patients participating in this study fulfilled the 2013 ACR/EULAR classification for systemic sclerosis. Comprehensive clinical, serological, and organ involvement data were extracted (age, gender, subtype, duration, digital ulcers, interstitial lung disease, pulmonary arterial hypertension, gastrointestinal, renal, myositis, joint involvement, and autoantibody status). Clinical associations with calcinosis were explored using both simple univariate group comparisons and multivariable regression analysis. In parallel, plasma levels of Activin A and in vitro data on Activin A release and macrophage-mesenchymal stem cell (MSC) models of calcinosis were investigated using a co-culture system in which adipose-derived MSCs were differentiated towards osteocytes by SSc macrophages. Cultures were maintained in osteogenic medium, with macrophages introduced at day 14 and 17 of differentiation. Calcium deposition was assessed by Alizarin Red S staining at day 21, and Activin A release was measured in plasma and culture supernatants by ELISA (Biotechne, Quantikine Activin A, Cat. #353043). Pharmacological inhibition with a CD206 antagonist (10 μM) and an Activin A neutralising antibody (R 0.001). SSc-derived macrophages promoted greater osteogenic foci in MSC co-cultures compared with controls (1.33 ± 0.29 vs 0.83 ± 0.29, p 0.05). Alizarin Red staining confirmed increased hydroxyapatite deposition, which was attenuated by CD206 inhibition (0.33 ± 0.29, p 0.01) and by Activin A neutralisation (0.50 ± 0.29, p 0.05). Treatment with the CD206 inhibitor also significantly modulated macrophage activation marker expression (CD206 by qPCR p = 0.0006). Conclusion These findings confirm a strong association of calcinosis with digital ulceration, a significant and a severe complication in both limited and diffuse subsets. Activin A is elevated systemically in SSc and implicated in macrophage:MSC cross talk promoting osteogenic differentiation leading to calcinosis development. Disclosure K. Shetty: None. S. Lopez Garces: None. B. Ahmed Abdi: None. K. Liang: None. Z. Kannan: None. H. Lopez: None. C. Yates: None. V. Ong: None. C. Denton: None. D. Abraham: None. S.N. Ahmad: None. R.J. Stratton: None.