Nintedanib downregulates the transition of cultured systemic sclerosis fibrocytes into myofibroblasts and their pro-fibrotic activity

Background Circulating fibrocytes are an important source of fibroblasts and myofibroblasts, which are involved in fibrotic processes, including systemic sclerosis (SSc). The study aimed to investigate the effect of nintedanib (a tyrosine kinase inhibitor) in inhibiting the in vitro transition of circulating SSc fibrocytes into myofibroblasts and their pro-fibrotic activity. Methods Circulating fibrocytes were obtained from 18 SSc patients and 5 healthy subjects (HSs). Cultured SSc fibrocytes were maintained in growth medium (untreated cells) or treated with nintedanib 0.1 and 1 mu M for 3 and 24 h. Fibroblast-specific protein-1 (S100A4) and alpha-smooth muscle actin (alpha SMA), as markers of fibroblast/myofibroblast phenotype, together with type I collagen (COL1) and fibronectin (FN), were investigated by qRT-PCR and Western blotting. Non-parametric tests were used for statistical analysis. Results Significantly elevated gene and protein expressions of alpha SMA, S100A4, COL1, and FN were observed in SSc fibrocytes compared to HS fibrocytes (gene: alpha SMA p < 0.001; others p < 0.0001; protein: all p < 0.05). Interestingly, an increased gene and protein expression of alpha SMA and S100A4 was found in fibrocytes from SSc patients positive for anti-Scl70 and with interstitial lung disease (ILD) (Scl70(+)ILD(+)) compared to Scl70(-)ILD(-) patients (S100A4: gene: p < 0.01; protein: p < 0.05), whereas no differences were observed for COL1 and FN. Nintedanib reduced gene and protein expression of alpha SMA, S100A4, COL1, and FN in SSc fibrocytes compared to untreated ones with different statistical significance. Noteworthy, nintedanib significantly downregulated gene and protein expression of alpha SMA, S100A4, COL1, and FN in Scl70(+)ILD(+) fibrocytes (all p < 0.05), whereas only that of S100A4 and FN was significantly downregulated (p < 0.05) in Scl70(-)ILD(-) fibrocytes compared to the related untreated cells. Conclusions Nintedanib seems to downregulate in vitro the transition of fibrocytes into myofibroblasts and their pro-fibrotic activity, particularly in cells isolated from Scl70(+)ILD(+) SSc patients