C57BL/6 wild-type (WT) mice were purchased from Japan CLEA (Tokyo, Japan), and TNFRsf1a−/− mice (C57BL/6 background) from Jackson Laboratory
(Bar Harbor, ME). CCR9−/− mice (C57BL/6 background) were previously described.24 Mice were maintained under specific pathogen-free conditions in the Animal Care Facility of Keio University School of Medicine. Experiments were performed with age- and sex-matched mice at 8-12 weeks of age. All experiments were approved by the regional Animal Study Committees and were performed according DNA-PK inhibitor to institutional guidelines. To induce acute liver injury, mice received an intraperitoneal (IP) injection of 0.6 mL/kg body weight of carbon APO866 purchase tetrachloride (CCl4, Sigma Aldrich, St. Louis, MO) mixed with olive oil, and were sacrificed at 24 hours after IP. To induce liver fibrosis, 0.2 mL/kg CCl4 was injected three times weekly for 6 weeks. As a second model, 200 mg/kg thioacetamide (TAA; Sigma Aldrich) plus 1 mg/kg leptin (R&D Systems, Minneapolis, MN)
was injected Tyrosine-protein kinase BLK three times weekly for 4 weeks as previously described.25 Mice were sacrificed 24 hours after the last administration. Livers were removed, fixed in 10% formalin, and embedded in paraffin. Sections were stained with hematoxylin-eosin (H&E), or with silver (Ag) for reticular fibers. Serum alanine aminotransferase (ALT) levels were measured using a lactate dehydrogenase
(LDH)-UV kinetic method (SRL, Tokyo, Japan). Hepatic collagen contents were evaluated by Sirius red staining of paraffin-embedded sections. Sirius red-positive areas were quantified in five nonoverlapping random fields (magnification 100×) on each slide using the imaging software ImageJ (NIH, Bethesda, MD). Liver nonparenchymal mononuclear cells were isolated from the liver as previously described.26 Details are described in the Supporting Methods. After blocking with anti-FcR (CD16/32, BD Pharmingen, Franklin Lakes, NJ) for 20 minutes, cells were incubated with specific fluorescence-labeled monoclonal antibodies (mAbs) at 4°C for 30 minutes.27 Antimouse mAbs used are listed in Supporting Table 1.