Dermal fibroblasts have different extracellular matrix profiles induced by TGF-\u03b2, PDGF and IL-6 in a model for skin fibrosis

Pernille Juhl,Anne-Christine Bay-Jensen,Morten Asser Karsdal,Anne Sofie Siebuhr,Sandie Bondesen,Clare Louise Hawkins,Michael Jonathan Davies

doi:10.1038/s41598-020-74179-6

Pernille Juhl, Anne-Christine Bay-Jensen + Show 5 more

Open Access

https://doi.org/10.1038/s41598-020-74179-6

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Abstract

Different stimulants might induce different extracellular matrix profiles. It is essential to gain an understanding and quantification of these changes to allow for focused anti-fibrotic drug development. This study investigated the expression of extracellular matrix by dermal fibroblast mimicking fibrotic skin diseases as SSc using clinically validated biomarkers. Primary healthy human dermal fibroblasts were grown in media containing FICOLL. The cells were stimulated with PDGF-AB, TGF-β1, or IL-6. Anti-fibrotic compounds (iALK-5, Nintedanib) were added together with growth factors. Biomarkers of collagen formation and degradation together with fibronectin were evaluated by ELISAs in the collected supernatant. Immunohistochemical staining was performed to visualize fibroblasts and proteins, while selected gene expression levels were examined through qPCR. TGF-β and PDGF, and to a lesser extent IL-6, increased the metabolic activity of the fibroblasts. TGF-β primarily increased type I collagen and fibronectin protein and gene expression together with αSMA. PDGF stimulation resulted in increased type III and VI collagen formation and gene expression. IL-6 decreased fibronectin levels. iALK5 could inhibit TGF-β induced fibrosis while nintedanib could halt fibrosis induced by TGF-β or PDGF. Tocilizumab could not inhibit fibrosis induced in this model. The extent and nature of fibrosis are dependent on the stimulant. The model has potential as a pre-clinical model as the fibroblasts fibrotic phenotype could be reversed by an ALK5 inhibitor and Nintedanib.

Highlights

Different stimulants might induce different extracellular matrix profiles
An increase in the metabolic activity of the fibroblasts was observed in each case, which was significant in experiments with TGF-β (P = 0.003) and platelet-derived growth factor (PDGF) (PDGF: P < 0.0001) (Fig. 1A)
Fibroblasts are the primary effector cell in fibrosis and dermal fibroblasts are responsible for the excessive skin fibrosis found in diseases as systemic sclerosis (SSc)

Summary

Introduction

Different stimulants might induce different extracellular matrix profiles. It is essential to gain an understanding and quantification of these changes to allow for focused anti-fibrotic drug development. Continuous fibroblast activation is facilitated by cytokines and growth factors such as interleukin 6 (IL-6), platelet-derived growth factor (PDGF), and transforming growth factor-beta (TGF-β), which are released by the immune and vascular systems. This results in excessive ECM production, especially different collagens, manifested as skin and internal organ fibrosis. It has been shown to induce differentiation to myofibroblasts and to stimulate ECM production directly[3,6] Activated immune cells, such as macrophages, together with fibroblasts are the main sources of TGF-β production, with these cell types known to be upregulated in S Sc2. PDGF has been shown to be upregulated in SSc and is responsible for inducing fibroblast p roliferation[9]

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