NAC Inhibits Va14iNKT Cell Activation Figure 1. Th1 differentiating signaling in the liver is dysregulated by Va14iNKT cells deficiency during Fas mAb-induced FLF. (a) Serum ALT levels of na¨ıve WT mice, Fas mAb-treated WT and Ja182/2 mice at 4.5 h. (b–d) H & E staining of liver sections from na¨ıve WT mice, Fas mAb-treated WT and Ja182/2 mice at 4.5 h. Livers from Fas mAb-treated WT mice (c) showed extensive damage with destruction of hepatocytes and distortion of normal liver architecture. The hepatocytes show hemorrhagic necrosis (white arrows) and characteristic signs of apoptosis (black arrows) including chromatin condensation and cellular shrinkage. In comparison, livers from Fas mAb-treated Ja182/2 mice showed only minimal damage and retained the normal architecture (d). Liver from a na¨ıve WT mouse is illustrated in (b) for comparison. (e) Western blot analysis of active caspase 3, T-bet, pSTAT-1, nitrotyrosine and GAPDH expression in the liver of PBS-treated WT mice and agonistic Fas mAb-treated WT and Ja182/2 mice at 4.5 h. (f) TUNEL staining of liver sections from WT and Ja182/2 mice at 4.5 h after Fas mAb injection in which WT mice showed intense TUNEL staining characteristic of apoptosis whereas Ja182/2 mice showed less/reduced TUNEL staining. (g) HPLC measurement of hepatic GSH levels in PBS- treated WT mice and Fas mAb-treated WT and Ja182/2 mice at 4.5 h. Figure S1 in a and g are presented as mean 6 s.e.m with n = 5 mice/group; *P,0.05 by one-way analysis of variance followed by Newman-Kuels post hoc test. All experiments were conducted twice. doi:10.1371/journal.pone.0038051.g001 ciences). For measurement of intracellular IFN-c, TNF-a and active caspase 3, tetramer positive Va14iNKT cells were first permeabilized using the Cytoperm/fix kit (BD Pharmingen) and then stained with either fluorochrome-labeled murine IFN-c mAb (clone XMG1.2; BD Pharmingen) [5,35], fluorochrome-labeled murine TNF-a mAb (clone MP6-XT22; eBioscience) [20] or fluorochrome-labeled active caspase 3 mAb (clone C92-605; BD Pharmingen) [5,16,20]. In all experiments, cells were analyzed directly ex vivo without cell culture treatment with brefeldin A or monensin. Corresponding isotype antibody/tetramer was used to set analysis gates. In addition, viable lymphocyte populations were gated using forward and side scatter characteristics and data analyzed using the FACS Calibur and FACS Scan Diva software (BD Pharmingen). Statistical Analysis All data are shown as mean 6 SEM. For comparisons of means between 2 experimental groups, a Student unpaired t test was used. Comparison among three or more experimental groups was performed using a one-way ANOVA, followed by Newman-Kuels post hoc test. A value of p,0.05 was considered significant. Results Resistance of Va14iNKT Cells Deficient Mice to FLF is Associated with Decreased Th1 Differentiating Signaling in Liver We first confirmed our recent observation [5] that the presence of hepatic Va14iNKT cells promote acute FLF in response to agonistic Fas mAb treatment. Specifically, we found that Fas mAb administration into WT mice caused a significant increase in serum ALT level whereas Ja182/2 mice were highly resistant to acute FLF as reflected by almost complete suppression (.90% reduction) of serum ALT (Figure 1A). In parallel, liver sections from WT mice exhibited extensive hepatocyte apoptosis and necrotic damage following Fas mAb treatment relative to livers from Ja182/2 mice which displayed mild hepatocyte damage (Figure 1C and D). Specifically, the degree of hepatic inflamma- tion and hepatocyte damage in WT mice after Fas mAb treatment was graded as severe (.50%) relative to mild (,25%) in Ja182/2 mice. As expected, normal serum ALT levels was observed in both naive WT and J Ja182/2 mice (Figure 1A). In the present study, we provide new data demonstrating that resistance of Ja182/2 mice to FLF was associated with a dramatic decrease in hepatic apoptosis as revealed by reduced expression of active caspase 3 and TUNEL staining in the liver (Figure 1E and 1F). The finding that active caspase 3 expression was not completely suppressed in Ja182/2 mice after Fas mAb treatment suggests that other hepatic cells apart from intrahepatic Va14iNKT cells may also contribute to apoptosis. It is notable that reduced susceptibility of Ja182/2 mice to FLF was also accompanied by striking reductions in hepatic expression of Th1 differentiating signaling molecules, pSTAT-1 and T-bet (Figure 1E). To determine whether oxidative and nitrosative stress may also contribute to the development of FLF, we measured nitrotyrosine formation (a product of nitrosative stress) and the ROS scavenger, GSH. We observed a striking increase in nitrotyrosine formation in the liver of WT mice but not Ja182/2 mice after Fas mAb administration (Figure 1E). Remarkably, we also found that Fas mAb-mediated FLF in WT mice caused a significant decrease in hepatic GSH (relative to PBS-treated WT mice), but GSH levels were restored in the absence of Va14iNKT cells (i.e. in Ja182/2 mice) during mild FLF to levels seen in PBS-treated WT mice (Figure 1G). Agonistic Fas mAb Promotes Intrahepatic Va14iNKT Cell Activation We next verified by flow cytometry that hepatic Va14iNKT cells were activated following agonistic Fas mAb administration in WT mice as denoted by upregulation of the activation marker, CD25, on cell surface (Figure 2A and B) and by increased intracellular IFN-c expression by hepatic Va14iNKT cells (Figure 2C and D). In addition, we established that the ROS scavenger, NAC, effectively suppressed hepatic Va14iNKT cells CD25 and IFN-c expression in WT mice during Fas mAb- mediated FLF (Figure 2A, B, C, D). Although CD25 expression by hepatic Va14iNKT cells in NAC-treated WT mice during Fas mAb-mediated FLF was 2-fold higher than PBS control, it was not significant (Figure 2B). In contrast, hepatic Va14iNKT cells IFN-c expression in NAC-treated WT mice during Fas mAb-mediated FLF was significantly higher (i.e. 3-fold) than PBS control (Figure 2C). Moreover, the number of CD25-positive cells but not IFN-c positive cells in the liver of WT mice after NAC/Fas mAb treatment was significantly higher than PBS control (Figure S1). It is noteworthy that Va14iNKT cells from the liver of Fas mAb-treated WT mice lack intracellular TNF-a (Figure 2E) and active caspase 3 (Figure 2F). Pathophysiological Role of IFN-c During Fas mAb- dependent FLF In view of our preceding findings, we next treated WT and IFN-c2/2 mice with agonistic Fas mAb to evaluate whether IFN-c is an essential and direct participant in FLF. As shown in Figure 3A, both WT and IFN-c2/2 mice were similarly susceptible to acute FLF since serum ALT levels in both mice strains were comparable. In correlation, histological evaluation of liver sections showed that the degree of hepatic inflammation and hepatocyte damage in both strains of mice was severe (.50%; Figure 3B, top panels). Histological scoring criteria was based on the severity/magnitude of inflammation, and the degree of hepatocyte degenerative changes including hepatocyte necrosis, hemorrhage and frequency of acidophilic bodies [5,35]. Likewise, active caspase 3 expression and tunnel staining in the liver during IFN-c deficiency was comparable to levels in WT mice during Fas mAb-mediated acute FLF (Figure 3C and D). It PLoS ONE | www.plosone.org 4 June 2012 | Volume 7 | Issue 6 | e38051