Background:Dermal fibroblasts are responsible for the excessive extracellular matrix (ECM) formation observed in the skin of systemic sclerosis (SSc) patients and fibroblasts are therefore an obvious target for anti-fibrotic treatments. TGFβ, PDGF and IL-6 are known to be central cytokines in systemic sclerosis. Nintedanib, a tyrosine-kinase inhibitor approved for treatment of idiopathic pulmonary fibrosis, did not show effect on dermal fibrosis only on pulmonary fibrosis in SSc patients with interstitial lung disease (ILD). Tofacitinib, as Pan JAK inhibitor, has shown to inhibit dermal fibrosis in mouse models and shown positive indications in patients.Objectives:We investigated the direct effect of Nintedanib and Tofacitinib on ECM production from human dermal fibroblast using translational biomarkers of type I, III and VI collagens and fibronectin.Methods:Primary healthy human dermal fibroblasts were grown in DMEM media containing 0.4% fetal calf serum, Ficoll (to produce a crowded environment) and ascorbic acid for up to 17 days. The cells were stimulated with PDGF [3 nM] and/or TGFβ [1 nM] in combination with Nintedanib [1 nM-10 μM] treatment initiated at day 0 or 7 or Tofacitinib [3-100 nM] treatment initiated at culture start together. Media and treatments were changed twice a week. Non-activated cells (w/o) were used as control. Type I, III and VI collagen formation (PRO-C1, PRO-C3 and PRO-C6, respectively) and fibronectin (FBN-C) were evaluated by validated ELISAs (Nordic Bioscience). Statistical analysis included 1-way and 2-way ANOVA, AUC and Mann-Whitney U-test.Results:PDGF significantly increased collagen type III and VI formation and collagen type I formation minimally. PDGF did not induce changes in fibronectin levels. TGFβ increased collagen type I and VI formation but did not induce formation of collagen type III. TGFβ increased fibronectin levels, where PDGF did not.Nintedanib (≥100 nM) added either from day 0 or 7 reduced PDGF induced collagen type III and VI formation to the levels of w/o throughout the remainder of the study. In TGFβ treated fibroblasts, Nintedanib added either from day 0 or 7 reduced collagen type I and VI formation. The fibronectin levels were dose-dependently reduced by Nintedanib. The biomarker levels were at study end at the level of w/o. Nintedanib at a concentration of 1 uM and higher significantly decreased the biomarker levels. Nintedanib (≥100 nM) in fibroblasts stimulated with both TGFβ and PDGF significantly reduced collagen type I, III and VI collagen and fibronectin.A Tofacitinib concentration of 100 nM was toxic to the dermal fibroblasts as the cell viability was minimal at culture end. However, the viability of Tofacitinib (100 nM) in combination with TGFβ was decreased at study end, but only to half the viability of untreated cells. Tofacitinib dose-dependently decreased the TGFβ induced type I and III collagen formation and fibronectin in the dermal fibroblasts. Tofacitinib (100 nM) decreased the level of collagen type I and III formation to the level of w/o, where as the level of fibronectin was lowered by 80 % of TGFβ. Tofacitinib as low as 12.5 nM significantly lowered the collagen type I formation and fibronectin (both p<0.05) and Tofacitinib of 25 nM decreased collagen type III formation significantly (p<0.0001).Conclusion:Tofacitinib decreased the formation of the collagens and fibronectin. Nintedanib inhibited ECM production differently in PDGF and TGFβ induced dermal fibroblast, but in the combination of TGFβ and PDGF Nintedanib significantly decreased the ongoing fibrosis. In PDGF induced fibrosis, Nintedanib acted as an on-off switch, whereas the inhibition was dose-dependent in TGFβ induced fibrosis. This cell study indicates that Nintedanib and Tofacitinib inhibits collagen production in dermal fibroblasts.Figure:Disclosure of Interests:Anne Sofie Siebuhr Employee of: Nordic Bioscience, Morten Karsdal Shareholder of: Nordic Bioscience A/S., Employee of: Full time employee at Nordic Bioscience A/S., Pernille Juhl Employee of: Nordic Bioscience, Anne-Christine Bay-Jensen Shareholder of: Nordic Bioscience A/S, Employee of: Full time employee at Nordic Bioscience A/S.
Adenosine A2A
receptor promotes collagen type III synthesis via β-catenin activation in human dermal fibroblasts