MASH-in-a-dish reveals fatty acid-driven itaconate crosstalk between Kupffer cells and hepatocytes

Metabolic dysfunction-associated steatohepatitis (MASH) is characterized by lipid-driven sterile inflammation and fibrosis that leads to liver failure and liver cancer. Metabolites are increasingly recognized as signaling molecules and regulate disease progression through cell-cell crosstalk. To overcome the challenges of studying metabolite crosstalk between different cell types in MASH livers, we developed a novel in vitro MASH model, "MASH-in-a-dish", a Transwell co-culture system of mouse hepatocyte-derived organoids (HepOrgs), Kupffer cells (KCs), and hepatic stellate cells (HSCs) in a chemically defined liver co-culture medium. MASH-in-a-dish models key clinical features of clinical MASH, including steatosis, hepatocyte ballooning, inflammation and fibrosis. MASH-in-a-dish allowed for rapid and specific separation of co-cultured HepOrgs, KCs and HSCs, enabling cell type-specific metabolomics and stable-isotope fluxomics analysis. Using MASH-in-a-dish, we identified 19 MASH-associated, cell interaction-responsive metabolites and demonstrated that fatty acids, via the carnitine shuttle, drive KC itaconate production, which aggravated oxidative stress, steatosis, and cell damage in lipid-stressed HepOrgs. Inhibiting itaconate production by KC-specific Acod1 knockout in the MASH-in-a-dish system mitigated hepatocyte damage. Our results show that MASH-in-a-dish is a powerful in vitro platform to study cell-cell interactions in MASH pathogenesis and identify itaconate crosstalk between Kupffer cells and hepatocytes as a novel pathogenic mechanism and a potential therapeutic target in MASH.