Pro-fibrotic compounds induce stellate cell activation, ECM-remodelling and Nrf2 activation in a human 3D-multicellular model of liver fibrosis - PubMed (original) (raw)
Pro-fibrotic compounds induce stellate cell activation, ECM-remodelling and Nrf2 activation in a human 3D-multicellular model of liver fibrosis
Vincenzo Prestigiacomo et al. PLoS One. 2017.
Abstract
Background & aims: 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).
Methods: 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.
Results: 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.
Conclusion: 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.
Conflict of interest statement
Competing Interests: The authors declare that they have no conflict of interest that may have influenced the data generation or interpretation.
Figures
Fig 1. TNF-α and TGF-β1 promote αSMA production in monolayer culture of hTERT-HSC.
hTERT-HSC cells were treated for 2, 5 or 10 days with TNF-α (50ng/mL) or TGF-β1 (1ng/mL). After treatment, the cells were fixed and stained against αSMA (green) and nuclei (DAPI, blue). Pictures taken using fluorescence microscopy. Scale bar: 50μm.
Fig 2. LPS induces TNF-α production in PMA-treated THP-1 cells.
THP-1 were treated with 0, 5, 10 and 25 ng/mL PMA for 48h prior exposure to 1 μg/mL LPS. The concentration of TNF-α was measured from the 48h culture supernatants by ELISA. These findings indicate that PMA differentiated THP-1 cells are well differentiated and yet respond adequately to a subsequent low-concentration of LPS. Values are presented as duplicates.
Fig 3. Staining of formalin fixed paraffin embedded human microtissues generated with HepaRG-cells or HepaRGs/THP-1/hTERT-HSC.
Microtissues were stained with Hematoxylin & Eosin (H&E), mesenchymal NPC-marker vimentin and macrophages marker CD68. Microtissues were kept in culture during 14 days before performing histological staining. Co-culture systems showed positive staining for vimentin and CD68 indicating the presence of the three different cell types. Arrows indicate CD68-positive cells. Dendritic stellate cells are shown in the zoom of vimentin staining picture. Scale bar: 200μm (20X magnification) and 100μm (40X magnification).
Fig 4. Viability response of human liver microtissues treated with pro-fibrotic compounds.
(A) Effect of LPS, TNF-α and TGF-β1 on viability of human liver microtissues was assessed by MTT assay. Microtissues were incubated with 0.5mg/mL MTT solution for 4h after 14 days of exposure to the tested compounds. DMSO and Sörensen buffer were then added into the wells and absorbance was read at 550nm using FlexStation 3. Values are expressed as percentage of negative control. *; P ≤ 0.05, **; P ≤ 0.01, ***; P ≤ 0.001 vs vehicle control. (n = 6, mean ± SD). (B) Effect of MTX, TAA and TGF-β1 on the ATP production. ATP content was measured using CellTiter-Glo® Luminescent Cell Viability Assay 2.0 after 14 days of exposure to MTX, TAA and TGF-β1. Values are expressed as percentage of negative control. ***; P ≤ 0.001 vs vehicle control (n = 6, mean ± SD).
Fig 5. Gene expression of fibrotic markers and cytokines in human liver microtissues exposed to LPS, TNF-α and TGF-β1.
mRNA was extracted using TRIzol conventional procedure and fold induction were calculated as 2^(-ΔΔCT) for each sample and vehicle control and expressed as mean fold induction ± S.E.M of three replicates with six MTs each. Actin was used as reference gene for each sample. † pool of 16 microtissues analysed as duplicate; no statistical analysis were performed on these samples. ND: no-detected values. *; P ≤ 0.05, **; P ≤ 0.01, ***; P ≤ 0.001 vs vehicle control.
Fig 6. Albumin production and cell proliferation in liver microtissues.
Formalin fixed paraffin embedded slides of HepaRG/THP-1 macrophages/hTERT-HSC microtissues were stained with Albumin and Ki67 antibodies after 14 days of treatment with MTX, TAA and TGF-β1. Microtissues were fixed in 4% PFA and embedded in 2% agarose prior paraffinization. Microtissues showed decrease in albumin production after MTX, TAA and especially TGF-β1 exposure. Ki67 shows strong induction of cell proliferation in the microtissues after TGF-β1 exposure. Scale bar: 200μm. Graphics show Quantitative IHC Staining Value (QISV) as mean ± S.D. (N = 5). *; P ≤ 0.05, **; P ≤ 0.01, ***; P ≤ 0.001 vs vehicle control.
Fig 7. Immunostaining of formalin fixed paraffin embedded human microtissues after exposure to MTX, TAA and TGF-β1.
Formalin fixed paraffin embedded slides of HepaRG/THP-1 macrophages/hTERT-HSC microtissues were stained with Hematoxylin & Eosin (H&E), vimentin, α-smooth muscle actin (αSMA), collagen I and CD68 after 14 days of treatment with MTX, TAA and TGF-β1. Microtissues were fixed in 4% PFA and embedded in 2% agarose prior paraffinization. Microtissues showed increase in the vimentin, αSMA, collagen I and CD68 positive cells after MTX, TAA and TGF-β1 exposure. Vimentin and CD68 stainings show proliferation of stellate cells and THP-1 macrophages in the microtissues, suggesting the onset of inflammation process, while αSMA and collagen I indicate activation of stellate cells and deposition of collagen. Scale bar: 200μm. Graphics show Quantitative IHC Staining Value (QISV) as mean ± S.D. (N = 5). *; P ≤ 0.05, **; P ≤ 0.01, ***; P ≤ 0.001 vs vehicle control.
Fig 8. Effect of MTX and TAA on gene expression of fibrotic markers in human liver microtissues.
mRNA was extracted using TRIzol conventional procedure and fold induction were calculated as 2^(-ΔΔCT) for each sample and negative control and expressed as mean fold induction ± S.E.M. of three replicates with six MTs each. Actin was used as reference gene for each sample. *; P ≤ 0.05, **; P ≤ 0.01, ***; P ≤ 0.001 vs vehicle control.
Fig 9. Secretion of Collagen I and MMP2 in supernatant medium after exposure of microtissues to pro-fibrotic compounds.
Protein amount was assessed by ELISA in supernatant medium after 14 days of exposure to MTX, TAA and TGF-β1. *; P ≤ 0.05, **; P ≤ 0.01, ***; P ≤ 0.001 vs vehicle control (n = 4, mean ± SD).
Fig 10. Molecular signalling between hepatic stellate cells, Kupffer cells and hepatocytes after liver injuries.
The picture shows the potential complex interactions between matrix-producing hepatic stellate cells and liver-resident macrophages and hepatocytes after liver injuries such as pro-fibrotic compounds (MTX and TAA). Some of these interactions have been previously published, some are supported by our data and some are still speculative. In our liver microtissues THP1 macrophages (as surrogate of Kupffer cells) produced large amount of TNF-α, IL6 and TGF-β1 detected by increase in gene expression. HepaRG (hepatocyte-like cells) also produced cytokines (IL-6) after specific stimulation such as LPS and TNF-α and showed hepatocyte damage after exposure to TGF-β1 as demonstrated by decreased albumin staining. The cytokines were able to enhance stellate cell activation (increase in αSMA production) and collagen deposition. Furthermore increase in expression of oxidative related genes, such as Nrf2 and Keap1, was measured at high concentrations of MTX and TAA suggesting the involvement of this cellular defence. Arrows indicate measured parameters.
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