Suter-Dick, Laura
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Methotrexate-induced liver injury is associated with oxidative stress, impaired mitochondrial respiration, and endoplasmic reticulum stress in vitro
Low-dose methotrexate (MTX) is a standard therapy for rheumatoid arthritis due to its low cost and efficacy. Despite these benefits, MTX has been reported to cause chronic drug-induced liver injury, namely liver fibrosis. The hallmark of liver fibrosis is excessive scarring of liver tissue, triggered by hepatocellular injury and subsequent activation of hepatic stellate cells (HSCs). However, little is known about the precise mechanisms through which MTX causes hepatocellular damage and activates HSCs. Here, we investigated the mechanisms leading to hepatocyte injury in HepaRG and used immortalized stellate cells (hTERT-HSC) to elucidate the mechanisms leading to HSC activation by exposing mono- and co-cultures of HepaRG and hTERT-HSC to MTX. The results showed that at least two mechanisms are involved in MTX-induced toxicity in HepaRG: (i) oxidative stress through depletion of glutathione (GSH) and (ii) impairment of cellular respiration in a GSH-independent manner. Furthermore, we measured increased levels of endoplasmic reticulum (ER) stress in activated HSC following MTX treatment. In conclusion, we established a human-relevant in vitro model to gain mechanistical insights into MTX-induced hepatotoxicity, linked oxidative stress in HepaRG to a GSH-dependent and -independent pathway, and hypothesize that not only oxidative stress in hepatocytes but also ER stress in HSCs contribute to MTX-induced activation of HSCs.
Single Cell Gene Expression analysis in a 3D microtissue liver model reveals cell type-specific responses to pro-fibrotic TGF-β1 stimulation
3D cell culture systems are widely used to study disease mechanisms and therapeutic interventions. Multicellular liver microtissues (MTs) comprising HepaRG, hTERT-HSC and THP-1 maintain multicellular interactions and physiological properties required to mimic liver fibrosis. However, the inherent complexity of multicellular 3D-systems often hinders the discrimination of cell type specific responses. Here, we aimed at applying single cell sequencing (scRNA-seq) to discern the molecular responses of cells involved in the development of fibrosis elicited by TGF-β1. To obtain single cell suspensions from the MTs, an enzymatic dissociation method was optimized. Isolated cells showed good viability, could be re-plated and cultured in 2D, and expressed specific markers determined by scRNA-seq, qRT-PCR, ELISA and immunostaining. The three cell populations were successfully clustered using supervised and unsupervised methods based on scRNA-seq data. TGF-β1 led to a fibrotic phenotype in the MTs, detected as decreased albumin and increased αSMA expression. Cell-type specific responses to the treatment were identified for each of the three cell types. They included HepaRG damage characterized by a decrease in cellular metabolism, prototypical inflammatory responses in THP-1s and extracellular matrix remodeling in hTERT-HSCs. Furthermore, we identified novel cell-specific putative fibrosis markers in hTERT-HSC (COL15A1), and THP-1 (ALOX5AP and LAPTM5).
Exosomal microRNAs Release as a Sensitive Marker for Drug-Induced Liver Injury In Vitro
How to Foster ‘New Approach Methodology’ Toxicologists
The need to reduce, refine and replace animal experimentation has led to a boom in the establishment of new approach methodologies (NAMs). This promising trend brings the hope that the replacement of animals by using NAMs will become increasingly accepted by regulators, included in legislation, and consequently more-often implemented by industry. The majority of NAMs, however, are still not very well understood, either due to the complexity of the applied approach or the data analysis workflow. A potential solution to this problem is the provision of better educational resources to scientists new to the area — showcasing the added value of NAMs and outlining various ways of overcoming issues associated with knowledge gaps. In this paper, the educational exchange between four institutions — namely, two universities and two SMEs — via a series of video training sessions, is described. The goal of this exchange was to showcase an exemplary event to help introduce scientists to non-animal approaches, and to actively support the development of resources enabling the use of alternatives to laboratory animals.
Identification of miR-199a-5p, miR-214-3p and miR-99b-5p as fibrosis-specific extracellular biomarkers and promoters of HSC activation
Liver fibrosis is characterized by the accumulation of extracellular matrix (ECM) resulting in the formation of fibrous scars. In the clinic, liver biopsies are the standard diagnostic method despite the potential for clinical complications. miRNAs are single-stranded, non-coding RNAs that can be detected in tissues, body fluids and cultured cells. The regulation of many miRNAs has been linked to tissue damage, including liver fibrosis in patients, resulting in aberrant miRNA expression/release. Experimental evidence also suggests that miRNAs are regulated in a similar manner in vitro and could thus serve as translational in vitro–in vivo biomarkers. In this work, we set out to identify and characterize biomarkers for liver fibrosis that could be used in vitro and clinically for research and diagnostic purposes. We focused on miRNAs released from hepatic 3D cultures exposed to methotrexate (MTX), which causes fibrosis, and acetaminophen (APAP), an acute hepatotoxicant with no clinically relevant association to liver fibrosis. Using a 3D in vitro model, we corroborated compound-specific responses as we show MTX induced a fibrotic response, and APAP did not. Performing miRNA-seq of cell culture supernatants, we identified potential miRNA biomarkers (miR-199a-5p, miR-214-3p, niRNA-125a-5p and miR-99b-5p) that were associated with a fibrotic phenotype and not with hepatocellular damage alone. Moreover, transfection of HSC with miR-199a-5p led to decreased expression of caveolin-1 and increased α-SMA expression, suggesting its role in HSC activation. In conclusion, we propose that extracellular miR-214-3p, miR-99b-5p, miR-125a-5p and specifically miR-199a-5p could contribute towards a panel of miRNAs for identifying liver fibrosis and that miR-199a-5p, miR-214-3p and miR-99b-5p are promoters of HSC activation.
The role of bile salts in cholestatic injury and fibrosis using a human 3D in vitro model
Evaluation of dioxin induced transcriptomic responses in a 3D human liver microtissue model
Three-dimensional human liver microtissue model provides a promising method for predicting the human hepatotoxicity of environmental chemicals. However, the dynamics of transcriptional responses of 3D human liver microtissue model to dioxins exposure remain unclear. Herein, time-series transcriptomic analysis was used to characterize modulation of gene expression over 14 days in 3D human liver microtissues exposed to 2,3,7,8-tetra-chlorodibenzo-p-dioxin (TCDD, 31 nM, 10 ng/ml). Changes in gene expression and modulation of biological pathways were evaluated at several time points. The results showed that microtissues stably expressed genes related to toxicological pathways (e.g. highly of genes involved in external stimuli and maintenance of cell homeostasis pathways) during the 14-day culture period. Furthermore, a weekly phenomenon pattern was observed for the number of the differentially expressed genes in microtissues exposed to TCDD at each time point. TCDD led to an induction of genes involved in cell cycle regulation at day three. Metabolic pathways were the main significantly induced pathways during the subsequent days, with the immune/inflammatory response enriched on the fifth day, and the cellular response to DNA damage was identified at the end of the exposure. Finally, relevant transcription patterns identified in microtissues were compared with published data on rodent and human cell-line studies to elucidate potential species-specific responses to TCDD over time. Cell development and cytochrome P450 pathway were mainly affected after a 3-day exposure, with the DNA damage response identified at the end of exposure in the human microtissue system but not in mouse/rat primary hepatocytes models. Overall, the 3D human liver microtissue model is a valuable tool to predict the toxic effects of environmental chemicals with a relatively long exposure.
Assessment of fibrotic pathways induced by environmental chemicals using 3D-human liver microtissue model
Exposure to environmental chemicals, particularly those with persistent and bioaccumulative properties have been linked to liver diseases. Induction of fibrotic pathways is considered as a pre-requirement of chemical induced liver fibrosis. Here, we applied 3D in vitro human liver microtissues (MTs) composed of HepaRG, THP-1 and hTERT-HSC that express relevant hepatic pathways (bile acid, sterol, and xenobiotic metabolism) and can recapitulate key events of liver fibrosis (e.g. extracellular matrix-deposition). The liver MTs were exposed to a known profibrotic chemical, thioacetamide (TAA) and three representative environmental chemicals (TCDD, benzo [a] pyrene (BaP) and PCB126). Both TAA and BaP triggered fibrotic pathway related events such as he-patocellular damage (cytotoxicity and decreased albumin release), hepatic stellate cell activation (transcriptional upregulation of α-SMA and Col1α1) and extracellular matrix remodelling. TCDD or PCB126 at measured con-centrations did not elicit these responses in the 3D liver MTs system, though they caused cytotoxicity in HepaRG monoculture at high concentrations. Reduced human transcriptome (RHT) analysis captured molecular re-sponses involved in liver fibrosis when MTs were treated with TAA and BaP. The results suggest that 3D, multicellular, human liver microtissues represent an alternative, human-relevant, in vitro liver model for assessing fibrotic pathways induced by environmental chemicals.