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

Abstract

Background & AimsCurrently 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).MethodsHuman 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.ResultsWe 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.ConclusionHere, 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.

Mechanistic evaluation of AMPK/SIRT1/FXR signaling axis, inflammation, and redox status in thioacetamide‐induced liver cirrhosis: The role ofCichorium intybuslinn (chicory)‐supplemented diet

“…NAD(P)H:quinone oxidoreductase‐1 (NQO1) enzyme catalyzes two‐electron reduction and detoxification of quinones and its derivatives, so protecting cells from oxidative stress and their sequel (Shoeib et al, ). TAA intoxication increased hepatic MDA, IL6 level; it significantly decreased NQO1 activity compared to controls as reported previously by Prestigiacomo et al (); Zhou et al (); Shoeib et al (). Meanwhile, C. intybus linn supplementation decreased hepatic MDA, IL6 level and increased NQO1 enzyme activity in relation to TAA‐intoxicated group.…”

“…TAA intoxication increased hepatic MDA, IL6 level; it significantly decreased NQO1 activity compared to controls as reported previously byPrestigiacomo et al (2017);Zhou et al (2017);Shoeib et al (2018). Meanwhile, C. intybus linn supplementation decreased hepatic MDA, IL6 level and increased NQO1 enzyme activity in relation to TAA-intoxicated group Ahmed (2009).…”

Human Liver Spheroids as a Model to Study Aetiology and Treatment of Hepatic Fibrosis

“…The cellular mechanisms involved in the initiation and progression of fibrosis are orchestrated by oxidative stress, which drives hepatocyte damage and non-parenchymal cell (NPC) activation, with transforming growth factor-β (TGF-β) being a central regulator in these processes [11]. Hepatic stellate cells (HSCs) undergo differentiation to a pro-fibrotic phenotype which is associated with liver fibrosis [12][13][14], making these NPCs integral to evaluating pharmaceutical interventions. Advances in tissue engineering support increased cellular longevity in vitro, which is achieved by a three-dimensional (3D) spatial arrangement of cells, and where applied to liver tissue, demonstrate improved hepatocyte viability and functionality [15][16][17].…”

Modeling Liver Biology and the Tissue Response to Injury in Bioprinted Human Liver Tissues

“…Aspects of fibrogenic liver injury have been reported in other in vitro model systems, including TGFb-treated spheroids composed of HepG2 cells, human telomerase reverse transcriptase (hTERT)-immortalized human HSC, and monocytederived macrophages, wherein fibrosis-associated genes were upregulated and collagen 1 was detectable by immunohistochemistry. 51 Additional studies demonstrated similar outcomes in allyl alcohol-or MTX-treated spheroids generated from HepG2 cells and primary human HSC. 52 Culture-or TGFb-activated rodent or human HSC are also used to test the ability of a compound to exacerbate or inhibit activationassociated endpoints, such as profibrotic gene expression or collagen secretion, but this strategy detects HSC effects only outside of the context of other cell types or tissue architecture.…”