Pro-fibrotic compounds induce stellate cell activation, ECM-remodelling and Nrf2 activation in a human 3D-multicellular model of liver fibrosis

• Published in: PloS one 2017-06, Vol.12 (6), p.e0179995-e0179995
• Main Authors: Prestigiacomo, Vincenzo, Weston, Anna, Messner, Simon, Lampart, Franziska, Suter-Dick, Laura
• Format: Article
• Language: English
• Subjects: Alcohol; Bile; Biology and Life Sciences; Biomarkers; Biotechnology; Cell activation; Cell culture; Cell Line; Cell lines; Collagen; Cytokines; Deposition; Development and progression; Electrochemical machining; Extracellular matrix; Extracellular Matrix - metabolism; Fibronectin; Fibrosis; Gene expression; Genetic aspects; Genotype & phenotype; Hepatic Stellate Cells - drug effects; Hepatic Stellate Cells - metabolism; Hepatic Stellate Cells - pathology; Hepatocytes; Hepatology; Humans; In vitro methods and tests; Interleukin 6; Kupffer cells; Life sciences; Lipopolysaccharides; Lipopolysaccharides - pharmacology; Liver; Liver Cirrhosis - metabolism; Liver Cirrhosis - pathology; Liver diseases; Macrophages; Macrophages - drug effects; Macrophages - metabolism; Medicine and Health Sciences; Metabolism; Methotrexate; Methotrexate - pharmacology; NF-E2-Related Factor 2 - metabolism; Oxidative stress; Physiological aspects; Players; Proteins; Rodents; Stellate cells; Stimulation; Telomerase reverse transcriptase; Thioacetamide; Thioacetamide - pharmacology; Three dimensional models; Transforming Growth Factor beta1 - pharmacology; Transforming growth factor-b1; Transforming growth factors; Tumor Necrosis Factor-alpha - pharmacology
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0179995

Currently most liver fibrosis research is performed in vivo, since suitable alternative in vitro systems which are able to recapitulate the cellular events leading to liver fibrosis are lacking. Here we aimed at generating a system containing cells representing the three key players of liver fibrosis (hepatocyte, Kupffer cells and stellate cells) and assess their response to pro-fibrotic compounds such as TGF-β1, methotrexate (MTX) and thioacetamide (TAA). Human cell lines representing hepatocytes (HepaRG), Kupffer cell (THP-1 macrophages) and stellate cells (hTERT-HSC) were co-cultured using the InSphero hanging drop technology to generate scaffold-free 3D microtissues, that were treated with pro-fibrotic compounds (TGF-β1, MTX, TAA) for up to 14 days. The response of the microtissues was evaluated by determining the expression of cytokines (TNF-α, TGF-β1 and IL6), the deposition and secretion of ECM proteins and induction of gene expression of fibrosis biomarkers (e.g. αSMA). Induction of Nrf2 and Keap1, as key player of defence mechanism, was also evaluated. We could demonstrate that the multicellular 3D microtissue cultures could be maintained in a non-activated status, based on the low expression levels of activation markers. Macrophages were activated by stimulation with LPS and hTERT-HSC showed activation by TGF-β1. In addition, MTX and TAA elicited a fibrotic phenotype, as assessed by gene-expression and protein-deposition of ECM proteins such as collagens and fibronectin. An involvement of the antioxidant pathway upon stimulation with pro-fibrotic compounds was also observed. Here, for the first time, we demonstrate the in vitro recapitulation of key molecular and cellular events leading to liver fibrosis: hepatocellular injury, antioxidant defence response, activation of Kupffer cells and activation of HSC leading to deposition of ECM.