The following Abs were used in this study: anti-I-Ab FITC, anti-H-2Kb FITC, anti-CD11c PE, anti-CD8a
PE, anti-CD19 PE, anti-CD1d PE, anti-CD4 PerCP/Cy5.5, anti-CD80 PerCP/Cy5.5, anti-CD11c PerCP/Cy5.5, (Biolegend, San Diego, CA), anti-CD3 FITC, anti-CD86 PE, anti-CD40 PE, Selleckchem Liproxstatin 1 anti-NK1.1 APC (eBioscience, San Diego, CA). For intracellular staining, liver mononuclear cells were incubated with brefeldin A (10 μg/mL) (BD Biosciences, San Diego, CA) at 37°C for 1 hour, then incubated with anti-CD16/32 Abs, followed by staining with PerCP/Cy5.5-conjugated CD3 and PE-conjugated PBS57 loaded CD1d tetramer (originally produced by the NIH tetramer facility, and supplied through Dr. David Serreze), permeabilized with Cytofix/Cytoperm reagent (BD Biosciences), and stained with Alexa Fluor 488-conjugated anti-IFN-γ (clone XMG1.2), Alexa Fluor 488-conjugated anti-IL-4 (clone 11B11), or rat IgG1 isotype control (clone R3-34) (BD Biosciences). Stained cells were assessed on a FACSCalibur (BD Biosciences) using FlowJo softwares (Tree Star, Ashland, OR). Portions of the liver were excised and immediately fixed with 10% buffered formalin solution for 2 days at room temperature. Paraffin-embedded tissue sections
were then cut into 4-μm slices for routine hematoxylin and eosin (H&E), silver, and Azan staining. Scoring of liver inflammation was performed on coded H&E-, silver-, or Azan-stained sections of liver using a set of indices by a “blinded” pathologist (K.T.); these indices selleck chemicals quantitated the degree of portal inflammation, parenchymal inflammation, bile duct damage, granulomas, and fibrosis. Each section was scored as either 0 = no significant change, 1 = minimal, 2 = mild, 3 = moderate, PAK6 and 4 = severe pathology. Details of this scoring system have been described.21 Finally, to detect the presence of alpha-smooth muscle actin (α-SMA)-positive cells, an immunochemical analysis was performed with
a well-characterized monoclonal antibody (mAb) for α-SMA.22 Results are expressed as the mean ± standard error of the mean (SEM). All graphing and statistical analyses were performed using the Prism graphing program (GraphPad Software, San Diego, CA). P-values were calculated using a two-tailed unpaired Mann-Whitney test except in Table 1; the frequency of liver damage in Table 1 was evaluated using Fisher’s exact test. Significance levels were set at P = 0.05. First, to confirm the activity of α-GalCer, a nested substudy was performed in which we intravenously injected α-GalCer to naive mice and analyzed IFN-γ and IL-4 production in serum and in iNKT cells of mice. As shown in Fig. 1A, both IFN-γ and IL-4 were increased in mice injected with α-GalCer. Serum IFN-γ was detectable at 2 hours, peaked at approximately 6 hours, and was maintained until 24 hours after α-GalCer injection, whereas IL-4 peaked at 2 hours and became undetectable after 6 hours (Fig. 1A).