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Abstract Background/Aims Calcinosis may result from localised trans-differentiation of tissue resident stem cells in the subcutaneous layer of affected skin in systemic sclerosis (SSc), as a severe disabling manifestation linked to ischaemia and local trauma. Polarised macrophages synergistically promote osteogenesis: M1 macrophages recruit mesenchymal stem cells leading to osteogenic differentiation, while M2 are responsible for osteoblast formation. Previous studies have implicated two pathways-Activin-A signalling and CD206 expression by macrophages - in heterotopic osteogenesis. We investigated the pathogenesis of calcinosis through macrophages, using a novel tissue culture model created by stimulation of adipose derived mesenchymal stem cells (ASCs) with SSc macrophages and explored the relevance of the Activin-A pathway. Methods Activin-A was measured by ELISA in diffuse cutaneous systemic sclerosis (dcSSc) patients' (n = 72), and healthy control (HC) plasma samples (n = 42). Macrophages were derived from peripheral blood monocytes of dcSSc patients (n = 4) and cultured under basal and BzATP-stimulated conditions. dcSSc macrophages were maintained in co-culture with ASCs in osteogenic media ±anti-activin-A neutralising antibody (anti-AA). Osteogenesis was assessed using Alizarin Red. Results Activin-A was increased in dcSSc patients’ plasma when compared to HC, most notably in anti-centromere antibody (ACA) subgroup (plasma activin-A 521±199pg/ml vs 255 ± 170pg/ml p = 0.000065) (Figure 1A). Moreover, there was synthesis of Activin A by SSc macrophages, enhanced by stimulation with BzATP (0.1µM) (basal Activin A 3.3 (0-14.5), BzATP stimulated 21.7 (7.7-418) pg/ml p NS) (Figure 1B). In co-cultures, the addition of SSc macrophages to ASCs led to significantly enhanced synthesis of Activin A, which was reduced by the addition of anti-AA (ASC monoculture 987+/-87, SSc macrophages+AScs 1599+/- 122, p 0.0065, SSc macrophages+ASCs+anti-AA 1034+/-108 pg/ml, p 0.013) (Figure 1C). Moreover, adding SSc macrophages to ASC cultures significantly enhanced calcinosis, as measured by Alizarin red stain, but anti-AA did not fully supress the Alizarin staining (ASC: 0.5, 0.4-0.7; ASC+M: 13.0, 5.8-17.2 p = 0.0022; ASC+M+anti-AA p NS median, IQR, red pixels per field) (Figure1D). Conclusion Elevated plasma Activin-A, most notable in ACA subgroup, is consistent with systemic upregulation of the Activin-A pathway in SSc. The interaction of activated macrophages with ASCs represents a potential model for calcinosis. Targeting Activin-A might benefit in this severe complication. Disclosure K. Liang: None. S. Lopez Garces: None. T. Searle: None. H. Lopez: Corporate appointments; CEO of Murigenics. G. Martin: Corporate appointments; Senior Scientific Officer Riptide. C. Denton: None. D. Abraham: None. R.J. Stratton: None.
Liang et al. (Mon,) studied this question.