THU0347 TESTING THE IN VITRO EFFECTS OF NINTEDANIB ON CIRCULATING FIBROCYTES AND RESIDENT SKIN FIBROBLASTS FROM THE SAME SYSTEMIC SCLEROSIS PATIENTS: PRELIMINARY RESULTS
Authors: Maurizio Cutolo, Paola Contini, Giulia Martinelli, Paola Montagna, Samuele Tardito, Sabrina Paolino, Massimo Patanè, Carmen Pizzorni, Elisa Alessandri, Veronica Tomatis, Vanessa Smith, Stefano Soldano
Journal: Annals of the Rheumatic Diseases (2019)

<h3>Background:</h3> The fibrosis in systemic sclerosis (SSc) progresses from microvascular alterations, immune system activation, and increased extracellular matrix protein synthesis into the skin and internal organs, primarily mediated by myofibroblasts.<sup>1</sup> Myofibroblasts are characterized by a higher expression of α-smooth muscle actin (αSMA) and by the overproduction of type I collagen (COL1) and fibronectin (FN).<sup>2</sup> Although myofibroblasts primarily derive from fibroblasts differentiation and transition, circulating fibrocytes represent a further important source.<sup>3</sup> Nintedanib is a tyrosine kinase inhibitor that interacts with several inflammatory and profibrotic pathways implicated in the pathogenesis of fibrosis, including those of platelet derived growth factor receptors, vascular endothelial growth factor receptors and fibroblast growth factor receptors.<sup>4</sup> <h3>Objectives:</h3> To investigate, in primary cultures, the effects of nintedanib on the differentiation of circulating fibrocytes and on the profibrotic activity of skin fibroblasts isolated from the same SSc patients. <h3>Methods:</h3> Circulating fibrocytes and fibroblasts were obtained from peripheral blood and skin biopsies, respectively, from three untreated SSc patients with diffuse skin involvement (mean age 55±6 yrs). To investigate the complete differentiation, fibrocytes were maintained in DMEM at 20% of foetal bovine serum, either with or without treatment with nintedanib at the concentrations of 0.1μM and 1μM for 8 days.<sup>5</sup> Fibrocytes were characterized as CD45<sup>+</sup>CXCR4<sup>+</sup>COL1<sup>+</sup>cells, and their percentage was detected by Flow Cytometry analysis.<sup>6</sup> Fibroblasts were grown until the 3<sup>dr</sup> culture passage and then maintained in normal growth medium with or without nintedanib treatment for 4, 24 and 48 hours. The gene expressions of αSMA, COL1 and FN were evaluated by qRT-PCR. <h3>Results:</h3> Nintedanib reduced the percentage of differentiated SSc fibrocytes (CD45<sup>+</sup>CXCR4<sup>+</sup>COL1<sup>+</sup>cells), already at the concentration of 0.1μM compared with untreated fibrocytes. In cultured SSc skin fibroblasts, nintedanib 1μM downregulated αSMA, COL1 and FN expressions already after 4 hours of treatment, and this effect was also maintained after 24- and 48-hours' treatment. Nintedanib 0.1μM downregulated the αSMA expression at all timepoints, whereas the downregulation of COL1 and FN expressions was observed at 4 and 24 hours, with no more effect at 48 hours of treatment compared with untreated cells. <h3>Conclusion:</h3> Initial experiments seem to indicate an evident <i>in vitro</i> concentration-dependent antifibrotic activity of nintedanib on SSc fibrocytes and skin fibroblasts from the same SSc patients; in particular by reducing the fibrocyte differentiation as potential source of myofibroblasts and contrasting the profibrotic activity of already activated fibroblasts/myofibroblasts. These results have translational implications in clinical trials using nintedanib in SSc patients. <h3>References:</h3> [1] Furue M, et al. Immunol Res 2017;65:790−7. [2] Kendall RT, et al. Front Pharmacol 2014;5:123. [3] Strieter RB, et al. J Leukocyte Biol 2009;88:111−8. [4] Huang J, et al. Ann Rheum Dis 2017;76:1941–8. [5] Cutolo M, et al. Arthritis Res Ther 2018;20:157. <h3>Disclosure of Interests:</h3> None declared