Conditional Macrophage Wls Deletion in Chronic Liver Injury

All animal experiments were performed with approval from the University of Queensland Animal Ethics Committee (MED/PAH/156/13/PAHRF/NHMRC, UQDI/571/12/NHMRC/AIDRCC). To conditionally delete Wls from macrophages, homozygous Wls^flox/flox mice [40 (link)] were crossed with LysM-Cre transgenic mice [41 (link)] for 4–10 generations. Offspring with the genotype Wls^flox/flox; LysM-Cre-positive represents Wls macrophage knockouts, whilst Wls^flox/flox; LysM-Cre-negative was used as controls. Six to 9-week-old mice were administered with 30 mg/L thioacetamide (TAA, Sigma) in drinking water for 12 weeks [71 (link)] or a modified choline-deficient ethionine-supplemented (CDE) diet for 6 weeks [48 (link)], to induce chronic liver injury and fibrosis. The modified CDE diet was optimised by Professor George Yeoh [48 (link)] (UWA, Australia) and was custom made by MP Biosciences (Santa Ana, USA). The diet consisted of 70 % choline-deficient diet (Cat#0296021410) and 30 % control (choline-sufficient) diet (Cat#0296041210). At the end of the treatment period, mice were euthanised and a blood sample taken for serum isolation. Livers were perfused with 10 ml of PBS via the portal vein in situ prior to tissue harvest to minimise blood contamination. Portions of the liver were fixed in 10 % neutral buffered formalin and embedded in paraffin or homogenised in TRI reagent and stored at −80 °C for RNA isolation. The remainder of the liver was used to isolate non-parenchymal cells. Serum ALT levels were measured using the MaxDiscovery Alanine Transaminase Color Endpoint Assay (Bioo Scientific, Austin, TX, USA).