diminish liver injury in concanavalin A or Fas ligand-induced hepatitis [25,26], whereas DC-derived IL-15 enhanced endotoxin shock injury through the liver [27]. Moreover, IL-15 mediates the crosstalk between conventional and plasmacytoid DCs for immune activation [28]. Interestingly, IL-15 promoted hepatocyte mitosis and liver proliferation in healthy mice and those with hepatecto- my, respectively [29]. In this study, we aimed to study the role of IL-15 in a sterile APAP-induced fulminant hepatitis model in IL-15-knockout (Il152/2) and wild-type (WT) mice. By treatment of mice with a hepatotoxic dose of APAP and evaluation of animal survival, liver damage, APAP metabolism in livers and the inflammatory response, we were able to elucidate whether IL-15 was involved in APAP hepatitis. Materials and Methods Animals and materials Male C57BL/6J mice were purchased from National Labora- tory Animal Center, Taipei, Taiwan. C57BL/6NTac/Il152/2 mice, obtained from Taconic Farms (Terrytown, NY), were backcrossed to C57BL/6J background for 4 generations. This substrain, as C57BL/6J/Il152/2 (presented as Il152/2 in our study) mouse, was used in our later study. All mice (9–12 weeks old) were kept in a pathogen-free condition in compliance with institutional animal care and use committee guidelines (project I.D., IACUC NO. 10-043). All chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA) unless otherwise stated. Recombinant murine IL-15, with specific activity of $26105 units/mg, was purchased from Perprotech (Rocky Hill, NJ, USA). Rat anti-IL-15 neutralizing antibody was obtained from eBiscience (San Diego, CA, USA). Recombinant IL-15 was intraperitoneally or subcutaneously injected 20 minutes before APAP challenge. IL-15 neutralizing antibody was intraperitoneally injected 30 minutes prior to APAP administration. APAP was dissolved in warm saline and intraperitoneally injected into overnight-fasted mice to induce hepatitis, and 350 mg/kg of APAP (sub-lethal dose) were generally used in this study unless otherwise mentioned. Animal survival was monitored for 5 days. 1.5 mg/kg of vinblastine (vin) or 30 mg/kg of gadolinium chloride (GdCl3) was intravenously given to animals for neutrophil or KC elimination, respectively. Evaluation of liver injury Serum alanine and aspartate aminotransferase (ALT/AST) levels were determined by the test kit DRY-CHEM SLIDE ALT/ AST (Fujifilm, Tokyo, Japan). The liver was sectioned and fixed overnight in 10% formalin solution, dehydrated, paraffin-embed- ded, cut at 4-mm thickness and stained with haematoxylin and eosin (H&E) for histological examination. The liver necrotic area was quantified by 2 individual pathologists with Image-Pro Plus (Media Cybernetics, Bethesda, MD) and the necrotic area (%) was expressed as necrosis area/total area of liver section. Hepatocyte isolation and culture, and KC-enriched fractionation Hepatocytes were isolated from mice by a 2-step collagenase perfusion protocol. The hepatic portal vein was ligated and perfused with Ca2+/Mg2+-free Hank’s balanced salts solution (HBSS; Gibco, Invitrogen, Carlsbad, CA) with 5 mM EGTA, than 0.025% type IV collagenase containing HBSS. After perfusion, liver was excised and hepatocytes were suspended in serum free L-15 medium and pass through 100 mm strainer. The filtrate was centrifuged at 45 g for 2 min at 4uC. The supernatant Effect of IL-15 on APAP Hepatitis as the non-parenchymal cell (NPC) fraction was centrifuged at 350 g for 5 min at 4uC and, used to analyze mononuclear cell population or KC enrichment. After perfusion, hepatocyte viability (more than 85%) was determined by trypan blue exclusion. Hepatocytes were suspended in 10% fetal bovine serum containing William’s Essential Medium (WEM) and cultured in 0.02% collagen-coated plate; after 4 hrs, the indicated concentration of APAP was added to analyze cytotoxicity to hepatocytes. The APAP toxicity was quantified by use of the Cell Counting Kit-8 (CCK-8). NPCs underwent a 25%/50% two-step Percoll gradient to isolated KC-enriched cells. Hepatic glutathione (GSH) measurement Hepatic total GSH was measured by 5,59-dithiobis(2-nitroben- zoic acid) (DTNB) assay. Frozen liver samples were homogenized with 4 volumes (v/w) of 5% trichloroacetic acid, and supernatant was incubated with DTNB for 15 min. Hepatic GSH concentra- tion was measured by colorimetry at optical density 412 nm. Determination of cytokines by ELISA Murine IL-15, IL-1b, IFNc and TNFa levels in serums or livers were measured by ELISA kits (R&D research). Serum samples were collected by heart puncture and liver proteins were collected from frozen liver samples homogenized in protein lysis buffer. Immunohistochemical staining of liver samples The levels of hepatic inducible NO synthase (iNOS) and nitrotyrosine were evaluated in deparaffinized and antigen- retrieved liver sections by LSAB2 system-HRP kits (DakoCytoma- tion, Produktionsvej, Denmark). Liver sections were incubated with primary antibodies against iNOS or nitrotyrosine (Upstate Biotechnology, Lake Placid, NY) as the manufacture’s instructions. Immunohistochemical staining was analyzed by use of Image-Pro Plus (Media Cybernetics, Bethesda, MD), with positive area (%) = positive signal area/total area of liver section. Western blot analysis The snap-frozen liver samples were thawed on ice, then homogenization with 9 volumes (v/w) of protein lysis buffer [25 mM HEPES (pH 7.3) containing 5 mM EDTA, 5 mM DTT, 0.1% CHAPS and protease inhibitor cocktail (Roche, Penzberg, Germany)]. Protein underwent SDS-PAGE, then were transferred to PVDF membranes (Millipore, Billerica, MA), which were incubated with antibodies against HO-1 (Epitomics, Inc., Burlingame, CA), CYP2E1 (Abcam, Cambridge, UK), CYP1A2, glutamate-cysteine ligase, catalytic subunit (GCLC), and heat shock protein 70 (Hsp70/HSC70; Santa Cruz Biotechnology, Santa Cruz, CA). Target signals were detected by use of enhanced chemiluminescence (Millipore, Billerica, MA) and LAS-3000 (Fujifilm, Tokyo, Japan). The relative protein expression was quantified by densitometry with Image J (NIH). Real-time PCR Hepatic RNA was isolated by Trizol reagent (Gibco, Invitrogen, Carlsbad, CA) from ,50 mg of liver sample. cDNA was reversely transcribed by the H minus reverse transcription kit (Fermentas, Thermo Fisher Scientific, Waltham, MA). Total 0.05 mg RNA- transcripted cDNA was processed by the SYBR green method with ABI-7500 (Applied Biosystems, Inc., Foster city, CA). The primer sequences for target genes are listed in Table S1. The expression of target genes was normalized to that of glyceralde- PLOS ONE | www.plosone.org 2 September 2012 | Volume 7 | Issue 9 | e44880