PMO-121Osteopontin promotes natural killer T (NKT) cell accumulation in nonalcoholic steatohepatitis (NASH)

IntroductionProgressive steatohepatitis is characterised by increased inflammatory cell infiltration. Osteopontin (OPN) is a cytokine intricately associated with immune-cell accumulation, and we reported that NKT cells accumulate to promote hepatic injury in NASH. We hypothesise that OPN promotes NASH progression by supporting NKT migration across hepatic sinusoidal endothelium.MethodsMice were fed chow or methionine-choline deficient (MCD) diet to induce NASH. After 4weeks, mice were sacrificed; severity of disease assessed by serum aminotransferase (AST), liver OPN quantified by QRTPCR and immunohistochemistry, blood OPN measured by ELISA. In separate studies, MCD-fed mice were treated with sham or OPN aptamers, and FACS used to quantify liver NKTs. Primary human hepatic sinusoidal endothelial cells (HSEC) were stimulated with recombinant (r)OPN (0-1000ng/ml), with or without TNFa (20ng/ml)+IFNg (100ng/ml), and expression of adhesion molecules (ICAM1, VCAM1) and chemokines (CXCL9, 10, 11, 16) assessed. To assess lymphocyte migration, lymphocytes were perfused over rOPN-or vehicle-treated-HSEC, with or without TNFa+IFNg. In separate experiments, TNFa+IFNg stimulated-HSEC were treated with OPN aptamers or blocking antibodies, and total lymphocyte adhesion recorded. Human NASH livers were immunostained for OPN, plasma measured for OPN, and liver NKT numbers from normal or NASH-cirrhotic patients quantified by FACS.ResultsIn mice, MCD-induced NASH upregulated expression of liver OPN by threefolds (p