processes, from prevention of inflammation and autoimmunity to protection against various pathogens, including bacteria and viruses [25,26]. We recently demonstrated that Ja182/2 mice, which are specifically deficient in Va14iNKT cells, are highly resistant to agonistic Fas mAb-induced acute FLF [5]. But the endogenous mechanism(s) regulating the pathophysiological activities of hepatic Va14iNKT cells are not known. In the present study, we hypothesized that activation of the Fas receptor on liver parenchymal cells, hepatocytes, by agonistic Fas mAb, initiates an inflammatory response that induces an endogenous mediator, possibly ROS, to regulate the pathophysiological effects of intrahepatic Va14iNKT cell signaling during acute FLF. Materials and Methods Mice Male C57BL/6 mice and IFN-c2/2 mice (on C57BL/6 background) were purchased from the Jackson Laboratory (Bar Harbor, ME). Breeding pairs of Ja182/2 mice (on C57BL/6 background) were kindly provided by Dr. M. Taniguchi (RIKEN Research Center for Allergy & Immunology, Yokohoma, Japan) [27] and bred in a pathogen-free breeding facility at LSUHSC- Shreveport [5,20]. All mice were fed a standard chow pellet diet, had free access to water and were maintained on a 12 h light/dark cycle in a pathogen-free facility. All experiments were conducted in accordance with National Institutes of Health and LSUHSC- Shreveport guidelines for animal care. All experiments were approved by LSUHSC-Shreveport Animal Care and User Committee (Proposal #: P11-043). Agonistic Fas (CD95) mAb-mediated FLF Agonistic Fas mAb (clone Jo2; 0.5 mg/g of body weight; BD Pharmingen; San Diego, CA) was administered intraperitoneally to mice for 4.5 h to induce liver injury as we recently described [5]. This dose of Fas mAb does not cause mice mortality. Control mice received an equivalent volume of sterile PBS [28,29,30]. At indicated time-point, mice were anesthetized with a mixture of xylazine and ketamine hydrochloride and blood serum collected. All livers were then perfused with ice-cold sterile PBS (to remove blood elements) and harvested for experimental assays described below. In some experiments, mice were treated with a single dose of freshly prepared ROS scavenger, N-acetylcysteine (NAC; 300 mg/kg, i.p.; Sigma) [20,31] immediately after Fas mAb treatment. Glutathione (GSH) Measurement Perfused livers were snap-frozen in liquid nitrogen immediately after excision from mice. Total GSH in liver was determined in trichloroacetic acid supernatants by high-performance liquid chromatography (HPLC) using a modified protocol of Reed et al. [32] as we previously described [33,34]. Briefly, experimental samples were derivatized with 6 mM iodoacetic acid and 1% 2,4-dinitrofluorobenzene to yield the S-carboxymethyl and 2,4- dinitrophenyl derivatives, respectively. Separation of GSH de- rivative was performed on a 25064.6-mm Alltech Lichrosorb NH2 10-mm column using a Shimadzu HPLC system. Proteins in the acid pellet were solubilized in 0.1 M NaOH, and protein was determined using the Bio-Rad Protein Assay kit (Bio-Rad, Hercules, CA). GSH concentration was determined by compar- ison with purified GSH standards derivatized in the same manner. NAC Inhibits Va14iNKT Cell Activation Western Blot Analysis Perfused mice livers were homogenized in RIPA buffer (50 mM Tris-HCl pH 7.4, 1% Nonidet-P40, 0.25% Sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM Dithiothreitol and protease inhibitors). Equal volumes of 2x sample buffer were added to liver protein extract. Next, liver protein samples (50 mg/lane) were fractionated by SDS-PAGE and then transfered onto PVDF membrane (Thermo Scientific; Rockford, IL). After which, membranes were blocked with 5% fat-free milk for 1 h at room temperature followed by overnight incubation with the following primary antibodies at depicted dilutions/concentrations: active caspase 3 (1:1000); pSTAT-1 (1:500); T-bet (1:1000), GAPDH (1:1000) and Nitrotyrosine (1:1000). Membranes were then counterstained with corresponding horseradish peroxidase-conju- gated secondary antibodies for visualization by Pierce ECL western blotting reagent (Thermo Scientific). Each membrane was stripped in buffer (0.5 mM Tris-HCl pH 6.8, 10% SDS, 0.08% Mercaptoethanol) and probed for GAPDH to verify equal protein loading in samples. Active caspase 3 Ab (clone 269518) was obtained from R & D systems (Minneapolis, MN) whereas pSTAT-1 (Tyr701) Ab was supplied by BD Pharmingen. Antibodies for T-bet (4B10) and GAPDH were all purchased from Santa-Cruz Biotech (Santa-Cruz, CA) and Nitrotyrosine mAb (clone HM11) was supplied by Invitrogen (Camarillo, CA). Biochemical and Histological Liver Injury Acute liver injury was evaluated by biochemical and histological means. Biochemical assessment of liver damage was determined by serum levels of the liver enzyme, alanine aminotransferase (ALT) using a commercial kit (Thermo Electron, Waltham) [5,35]. For histological evaluation, paraffin embedded liver sections (5 mm thick) were deparaffinized, stained with H & E according to standard protocols and then analyzed by light microscopy in a blinded fashion by a pathologist (PAA). The degree of inflammation in the liver and hepatocyte damage was graded as none (0), mild (,25%), moderate (25%–50%) and severe (.50%) using a combination of indices: severity of the inflammation and degree of hepatocyte degenerative changes including hepatocyte necrosis, hemorrhage and frequency of acidophilic bodies [5,35]. In Situ Analysis of Liver Apoptosis Using TUNEL Paraffin-embedded liver sections were dewaxed in xylene and rehydrated by passage through a graded series of ethanol solutions, and then PBS. Sections were permeabilized with proteinase K (20 mg/ml in 10 mM Tris-HCl, pH 7.4–8.0) at 37uC for 15 min, washed and then stained with fluorescein nucleotide mixture with terminal deoxynucleotidyl transferase (TdT) from In Situ Cell Death Detection kit (Roche Applied Science; Indianapolis, IN). Sections were viewed and photographed using standard fluores- cent microscopic techniques. Hepatic Lymphocytes Isolation and Flow Cytometry Hepatic lymphocytes were isolated using our published proto- cols [16,20,35]. To specifically identify Va14iNKT cells by flow cytometry, isolated hepatic lymphocytes were preincubated with anti-mouse CD16/32 mAb (clone 2.4G2; BD Pharmingen) to block FccRs and then incubated simultaneously with fluoro- chrome-labeled TCRb mAb (clone H57-597; eBiosciences, San Diego, CA) and fluorochrome-labeled Va14iNKT cell tetramer (CD1d-PBS57; NIH Tetramer Core Facility, Atlanta) [5,20,35]. CD25 expression on the surface of tetramer positive hepatic Va14iNKT cells was determined by FACS after staining with fluorochrome-labeled murine CD25 mAb (clone PC61.5; eBios- PLoS ONE | www.plosone.org 2 June 2012 | Volume 7 | Issue 6 | e38051