[5, 12-14] These inconclusive

results are likely the result of α-Galcer inducing iNKT production of a wide variety of cytokines, the synergistic effects of which remain largely unknown for the control of hepatitis. These questions urgently need to be addressed prior to the application of α-Galcer in additional clinical trials for treating liver disease. Injection of α-Galcer into mice induces iNKT activation, with rapid production of IL-4 but delayed production of IFN-γ, which results in mild hepatitis and liver injury.[15, 16] In the current study, we found that after α-Galcer injection, iNKT cells rapidly produce IL-4, which promotes liver neutrophil accumulation and hepatitis by way of a STAT6-dependent mechanism, whereas the subsequent production of IFN-γ acts in a negative

feedback loop to control α-Galcer-induced hepatitis by inducing neutrophil STA-9090 apoptosis by way of a STAT1-dependent mechanism. Eight- to 10-week-old male mice were used in this study. C57BL/6J, IFN-γ−/−, IFNGR−/−, IL-4−/−, and STAT6−/− mice on a C57B/6J background were purchased from the Jackson Laboratory (Bar Harbor, ME). Balb/c and IL-4R−/− mice on a Balb/c background were also purchased from the Jackson Laboratory. STAT1−/− mice were originally purchased from Taconic (Hudson, NY) and backcrossed into a C57BL/6J background for at least 11 generations. IFN-γ−/− IL-4−/− double knockout (dKO) mice were generated as a result of several steps of cross-breeding between IFN-γ−/− and IL-4−/− mice. All animals were maintained in a specific pathogen-free facility and were check details cared for in accordance with National Institutes of Health guidelines; this study was approved by the National Institute on Alcohol Abuse and Alcoholism Animal Care and Use Committee. To induce murine iNKT cell-driven acute experimental hepatitis, a single intravenous injection of α-Galcer (3 μg in 300 μL vehicle) was administered to each mouse. Control mice received 300 μL of vehicle.

α-Galcer (KRN7000) was purchased from Alexis Biochemicals (San Diego, CA) and dissolved in 0.5% polysorbate-20 (Tween-20) and diluted in sterile phosphate-buffered saline (PBS). Data are expressed as the mean ± SEM for each group and were analyzed using GraphPad Prism software (v. 5.0a; GraphPad Software, La Jolla, CA). To compare values Casein kinase 1 obtained from two groups, Student t test was performed. To compare values obtained from three or more groups, single-factor analysis of variance (ANOVA) was used, followed by Tukey’s post-hoc test. Statistical significance was designated at the P < 0.05 level. Additional Materials and Methods are included in the Supporting Materials. Injection of α-Galcer rapidly increased the serum levels of IL-4, with a peak effect at 3 hours postinjection, whereas the elevation of serum IFN-γ was delayed, with a peak effect at 16 hours postinjection (Fig. 1A).