P89 Osteopontin promotes lymphocyte recruitment in steatohepatitis

IntroductionSteatohepatitis is the critical step in the progression to fibrosis, and is characterised by increased inflammatory cell recruitment from the circulation. The cytokine Osteopontin (OPN) is intricately involved in cell-recruitment and tissue-repair, and we reported that OPN is significantly upregulated during non-alcoholic steatohepatitis (NASH).AimThus, we hypothesised that OPN promotes steatohepatitis by supporting leucocyte migration across hepatic sinusoidal endothelium.MethodWild-type mice were fed chow or methionine-choline deficient (MCD) diet to induce NASH. After 4 weeks, mice were sacrificed; severity of disease assessed by serum aminotransferase (AST), hepatic OPN quantified by QRTPCR and immunohistochemistry, serum OPN measured by ELISA. In separate experiments, MCD-fed mice were treated with anti-OPN or IgG, and flow cytometry used to quantify numbers of liver infiltrating lymphocytes (LIL). Primary human hepatic sinusoidal endothelial cells (HSEC) were stimulated with recombinant (r) OPN (0–1000 ng/ml), and expression of adhesion molecules (ICAM-1, VCAM-1, CD31) quantified by western blot. To assess lymphocyte transendothelial migration, lymphocytes were perfused over rOPN- or vehicle-treated-HSEC, with or without TNFa (20 ng/ml) + IFNa (100 ng/ml). In separate experiments, TNFa+IFNa stimulated-HSEC were treated with sham or OPN-aptamers and total lymphocyte adhesion recorded. Human livers with NASH were immunostained for OPN, and FACS used to quantify LIL isolated from control or NASH-cirrhotic patients.ResultsIn mice, diet-induced NASH upregulated expression of hepatic OPN by threefold (p