Abstract
Nonalcoholic steatohepatitis (NASH) is the most severe form of nonalcoholic fatty liver disease (NAFLD), which to date has no approved drug treatments. There is an urgent need for better understanding of the genetic and molecular pathways that underlie NAFLD/NASH, and currently available preclinical models, be they in vivo or in vitro, do not fully represent key aspects of the human disease state. We have developed a human in vitro co‐culture NASH model using primary human hepatocytes, Kupffer cells and hepatic stellate cells, which are cultured together as microtissues in a perfused three‐dimensional microphysiological system (MPS). The microtissues were cultured in medium containing free fatty acids for at least 2 weeks, to induce a NASH‐like phenotype. The co‐culture microtissues within the MPS display a NASH‐like phenotype, showing key features of the disease including hepatic fat accumulation, the production of an inflammatory milieu, and the expression of profibrotic markers. Addition of lipopolysaccharide resulted in a more pro‐inflammatory milieu. In the model, obeticholic acid ameliorated the NASH phenotype. Microtissues were formed from both wild‐type and patatin‐like phospholipase domain containing 3 (PNPLA3) I148M mutant hepatic stellate cells. Stellate cells carrying the mutation enhanced the overall disease state of the model and in particular produced a more pro‐inflammatory milieu. Conclusion: The MPS model displays a phenotype akin to advanced NAFLD or NASH and has utility as a tool for exploring mechanisms underlying the disease. Furthermore, we demonstrate that in co‐culture the PNPLA3 I148M mutation alone can cause hepatic stellate cells to enhance the overall NASH disease phenotype.
Highlights
Nonalcoholic steatohepatitis (NASH) is the most severe form of nonalcoholic fatty liver disease (NAFLD), which to date has no approved drug treatments
We previously developed a model of hepatic steatosis using a 3D perfused microphysiological system (MPS), which enabled primary human hepatocyte (PHH) to be cultured for 2 weeks in the presence of free fatty acid (FFA), allowing the chronic effects of triglyceride accumulation to be analyzed.[21]. The perfused MPS maintains highly metabolically active PHHs for extended periods[22,23,24] and has been demonstrated to support PHHs and human Kupffer cell (HK) co-cultures to study the effects of liver inflammation on drug metabolism, drug–drug interactions, and liver toxicity.[22,23,24]
Both models were compared for a range of biomarkers expressed in NAFLD/NASH,(28) and the NASH model was found to display a pro-inflammatory phenotype with the detection of IL-6 secretion (Fig. 1A), tumor necrosis factor α (TNF-α) secretion (Fig. 1B), and the expression of a range of pro-inflammatory genes (Fig. 1C)
Summary
Nonalcoholic steatohepatitis (NASH) is the most severe form of nonalcoholic fatty liver disease (NAFLD), which to date has no approved drug treatments. The co-culture microtissues within the MPS display a NASH-like phenotype, showing key features of the disease including hepatic fat accumulation, the production of an inflammatory milieu, and the expression of profibrotic markers. Microtissues were formed from both wild-type and patatin-like phospholipase domain containing 3 (PNPLA3) I148M mutant hepatic stellate cells. Stellate cells carrying the mutation enhanced the overall disease state of the model and in particular produced a more pro-inflammatory milieu. We demonstrate that in co-culture the PNPLA3 I148M mutation alone can cause hepatic stellate cells to enhance the overall NASH disease phenotype. NASH is a serious condition, defined as a combination of hepatic steatosis, inflammation, hepatic damage, and pericellular liver fibrosis.[2] The genetic basis of NAFLD has started to be explored, and the I148M mutation in the patatin-like phospholipase domain containing. Significant numbers of compounds are entering clinical development aimed at treating NAFLD/ NASH, using a wide range of therapeutic approaches and targeting a diverse set of molecular pathways
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