Dissecting itaconate signalling in MASLD: a lentiviral approach

Abstract

Metabolic dysfunction-associated steatotic liver disease (MASLD) is estimated to affect 40% of the world population, with 20% of these patients advancing to the more advanced metabolic dysfunction- associated steatohepatitis (MASH, formerly NASH) every year. To study this disease, the NASH-in-a-dish system was developed to examine cell-cell crosstalk between murine hepatocyte organoids, hepatic stellate cells (HSCs) and Kupffer cells (KCs). This model was used to identify several metabolites that were differently regulated in NASH specifically in these crosstalk conditions. We focused on one metabolite here, itaconate, and devised a method to study the effects of itaconate by knockdown or knockout and overexpression of the enzymes that break itaconate down into pyruvate. Here, we used lentivirus to transduce each of the cells involved in the NASH-in-a-dish system. Among these cells, we transduced HSCs, the cells primarily responsible for fibrosis and fibrogenesis in the liver, in order to determine the effects of itaconate on these processes in the liver. In order to transduce murine hepatocyte organoids, we overcame the lowered expression of low- density lipoprotein receptor (LDLR) of murine hepatocytes, their innate immune response to lentiviral transduction and finally the virus-inactivating effects of Matrigel through a combination of spinoculation and lentivirus concentration. We found that concentrated lentivirus in combination with spinoculation is successful in transducing murine hepatocyte organoids. We also found that itaconate may have a protective role to play in fibrosis and fibrogenesis in the NASH liver.