Abstract
BACKGROUND & AIMS: Hepatic recruitment of monocyte-derived macrophages (MoMFs) contributes to the inflammatory response in non-alcoholic steatohepatitis (NASH). However, how hepatocyte lipotoxicity promotes MoMF inflammation is unclear. Here we demonstrate that lipotoxic hepatocyte-derived extracellular vesicles (LPC-EVs) are enriched with active integrin β(1) (ITGβ(1)), which promotes monocyte adhesion and liver inflammation in murine NASH. METHODS: Hepatocytes were treated with either vehicle or the toxic lipid mediator lysophosphatidylcholine (LPC); EVs were isolated from the conditioned media and subjected to proteomic analysis. C57BL/6J mice were fed a diet rich in fat, fructose, and cholesterol (FFC) to induce NASH. Mice were treated with anti-ITGβ(1) neutralizing antibody (ITGβ(1)Ab) or control IgG isotype. RESULTS: Ingenuity® Pathway Analysis of the LPC-EV proteome indicated that ITG signaling is an overrepresented canonical pathway. Immunogold electron microscopy and nanoscale flow cytometry confirmed that LPC-EVs were enriched with activated ITGβ(1). Furthermore, we showed that LPC treatment in hepatocytes activates ITGβ(1) and mediates its endocytic trafficking and sorting into EVs. LPC-EVs enhanced monocyte adhesion to liver sinusoidal cells, as observed by shear stress adhesion assay. This adhesion was attenuated in the presence of ITGβ(1)Ab. FFC-fed, ITGβ(1)Ab-treated mice displayed reduced inflammation, defined by decreased hepatic infiltration and activation of proinflammatory MoMFs, as assessed by immunohistochemistry, mRNA expression, and flow cytometry. Likewise, mass cytometry by time-of-flight on intrahepatic leukocytes showed that ITGβ(1)Ab reduced levels of infiltrating proinflammatory monocytes. Furthermore, ITGβ(1)Ab treatment significantly ameliorated liver injury and fibrosis. CONCLUSIONS: Lipotoxic EVs mediate monocyte adhesion to LSECs mainly through an ITGβ(1)-dependent mechanism. ITGβ(1)Ab ameliorates diet-induced NASH in mice by reducing MoMF-driven inflammation, suggesting that blocking ITGβ(1) is a potential anti-inflammatory therapeutic strategy in human NASH. LAY SUMMARY: Herein, we report that a cell adhesion molecule termed integrin β(1) (ITGβ(1)) plays a key role in the progression of non-alcoholic steatohepatitis (NASH). ITGβ(1) is released from hepatocytes under lipotoxic stress as a cargo of extracellular vesicles, and mediates monocyte adhesion to liver sinusoidal endothelial cells, which is an essential step in hepatic inflammation. In a mouse model of NASH, blocking ITGβ(1) reduces liver inflammation, injury and fibrosis. Hence, ITGβ(1) inhibition may serve as a new therapeutic strategy for NASH.