NAC Inhibits Va14iNKT Cell Activation caspase 3 (f; Adenovirus used as a positive control) expression levels by intrahepatic Va14iNKT cells at 4.5 h after PBS or agonistic Fas mAb treatment. All experiments were performed twice. Figure S1 in b and c are presented as mean 6 s.e.m with n = 4 mice/group; *P,0.05 by one-way analysis of variance followed by Newman-Kuels post hoc test. doi:10.1371/journal.pone.0038051.g002 is also notable that the strong expression levels of pSTAT-1 and T-bet in the liver of WT mice during FLF was not suppressed by IFN-c deficiency (Figure 3C). Likewise, nitrotyrosine formation in the liver of WT mice was comparable to IFN-c2/2 mice during Fas mAb-mediated acute FLF (Figure 3E). These data strongly suggests that IFN-c may not be a key and/or direct mediator of FLF in response to agonistic Fas mAb treatment. NAC Therapy Alleviates Fas mAb-dependent FLF During IFN-c Deficiency We demonstrated in Figure 1G that resistance of Ja182/2 mice to hepatic failure may be associated with elevated GSH levels. GSH has been reported to ameliorate Fas mAb-dependent FLF [30,36]. For this reason, we evaluated the effects of NAC, a GSH precursor and ROS scavenger, on the development of FLF in WT and IFN-c2/2 mice in response to agonistic Fas mAb. Impor- tantly, we observed that WT mice were strikingly resistant to Fas mAb-dependent FLF following NAC treatment as denoted by considerably lower ALT levels relative to Fas mAb-treated WT mice given PBS (Figure 3A). Equally significant, FLF in IFN-c2/2 mice was almost completely resolved by NAC therapy relative to Fas mAb-treated IFN-c2/2 mice administered PBS (Figure 3A). In parallel, histological injury in both strains of mice was found to be minimal (Figure 3B) after NAC therapy since the scale of hepatic inflammation and hepatocyte damage in both strains of mice were graded as very mild to none. Notably, NAC therapy in WT and IFN-c2/2 mice also markedly suppressed hepatic apoptosis as revealed by active caspase 3 (Figure 3C) and TUNEL staining (Figure 3D). Additionally, pSTAT-1 (Figure 3C), T-bet (Figure 3C) expression and nitrotyrosine formation (Figure 3E) in the liver of WT and IFN-c2/2 mice were all suppressed by NAC therapy. These findings suggest a fundamental role for endogenous ROS in regulating Th1 differentiating signaling and nitrosative stress in the liver during Fas mAb-dependent FLF. NAC Therapy Prevents Hepatic Va14iNKT Cell Accumulation During FLF Given the anti-inflammatory effects of NAC therapy on Fas mAb-dependent FLF and Th1 differentiating signaling, we next determined whether NAC may also inhibit intrahepatic Va14iNKT cell accumulation. Indeed, we found that NAC therapy effectively diminished the frequency of Va14iNKT cells in the liver of WT mice undergoing acute FLF (Figure 4A and B). It is also notable that the frequency of Va14iNKT cells in the liver of WT mice treated only with NAC (i.e. in the absence of agonistic Fas mAb) did not significantly differ from WT mice given only PBS (Figure 4C), suggesting that endogenous ROS produced in inflamed liver during agonistic Fas mAb-mediated FLF may be driving the effects seen on intrahepatic Va14iNKT cell accumu- lation. Effects of NAC Therapy on Va14iNKT TCR Downregulation These experiments were designed to determine whether the suppressive effects of NAC therapy on Va14iNKT cells accumu- lation in the liver during agonistic Fas mAb-mediated FLF could be due to down-modulation of surface TCR. Our results showed that surface TCRb on Va14iNKT cells was not downregulated by NAC therapy during Fas mAb-induced FLF since the geometric mean fluorescence intensity (MFI) of surface TCRb after NAC treatment was comparable to WT mice administered PBS (MFI: 15486354 in WT/NAC/Fas mAb relative to 15396343 in WT/ PBS/Fas mAb; n = 6 mice/group). Discussion Engagement of the Fas receptor typically leads to apoptosis [1,2,3]. The importance of the Fas/FasL system in hepatic apoptosis has been convincingly demonstrated in both experi- mental and clinical liver injury models including viral and autoimmune hepatitis, alcoholic liver disease and acute liver failure [1,2,3,5,37,38]. Therefore, strategies for downregulating the Fas/FasL system might have therapeutic value in the treatment of these human diseases. In addition to its role in caspase-mediated cell death, emerging studies have increasingly proposed an inflammatory role for agonistic Fas mAb in stimulating intracellular signaling pathways in target cells, such as hepatocytes, astrocytes and epithelial cells, leading to NF-kB and/or AP-1 activation [6,7,8], chemokine/cytokine production [6,7] and leukocyte infiltration [6,7,9] in tissue sites. Va14iNKT cells represent a critical link between the innate and adaptive immune systems and play an important immunoregulatory role in hepatic, cardiovascular, infectious and autoimmune diseases as well as in tumor immunity. We recently demonstrated that mice deficient in Va14iNKT cells (i.e. Ja182/2 mice) are highly resistant to acute FLF in response to Fas mAb treatment [5]. But, there are notable deficiencies in our knowledge regarding whether; (i) agonistic Fas mAb directly stimulates intrahepatic Va14iNKT cells to induce effector functions or (ii) inflammatory mediator(s) are produced in the liver in response to agonistic Fas mAb treatment to alter/regulate the biological/functional effects of intrahepatic Va14iNKT cells. The current study highlights a novel dual pro-inflammatory and pro-apoptotic role for endogenous ROS in mediating agonistic Fas mAb-dependent acute FLF by promoting intrahepatic Va14iNKT cell activation and effector functions. During inflammatory responses, Va14iNKT cells are rapidly activated by TCR-dependent and independent mechanisms [17,18,19,20] to produce significant amounts of immunopolariz- ing cytokines including the Th1 cytokine, IFN-c [16] and TNF- a [20]. For this reason, we initially ascertained the activation status of intrahepatic Va14iNKT cells in response to agonistic Fas mAb treatment. We observed by FACS analysis that hepatic Va14iNKT cells are activated to upregulate extracellular CD25 and intracellular IFN-c expression but not TNF-a. Our approach of using intracellular IFN-c production and/or extracellular CD25 expression to denote Va14iNKT cell activation is widely supported by multiple studies from our laboratory [5,20] and others [22,39,40,41,42,43,44,45]. Since many of the effects of IFN-c are STAT-1 and T-bet dependent [46,47,48], we also determined by western blotting if these Th1 differentiating signaling molecules are differentially regulated in the presence and absence of Va14iNKT cells following Fas mAb administration. Consistent with this notion, we found that pSTAT-1 and T-bet levels in the liver were markedly diminished in the absence of Va14iNKT cells. Additionally, markers of apoptosis (i.e. active caspase 3 and TUNEL staining) and PLoS ONE | www.plosone.org 6 June 2012 | Volume 7 | Issue 6 | e38051