NAC Inhibits Va14iNKT Cell Activation Figure 5. A proposed model depicting the sequential molecular and cellular events of NAC therapy regulation of Va14iNKT cells signaling in the liver during Fas mAb-induced FLF. An endogenous mediator inhibited by NAC (possibly ROS) mediates Fas mAb-dependent FLF by promoting intrahepatic Va14iNKT cells signaling, upregulation of pSTAT-1 and pSTAT-1-regulated genes, caspase 3 and T-bet, induction of hepatocyte damage and fatal/lethal immunopathological events in the liver that ultimately leads to FLF. doi:10.1371/journal.pone.0038051.g005 There is growing evidence that Fas mAb-induced acute FLF is a result of complex pathophysiological events involving injurious factors such as ROS. For example, studies highlighting the deleterious consequences of GSH depletion in Fas mAb-mediated FLF underscore the importance of this anti-oxidant [30,36]. Likewise, administration of MnTBAP (a nonpeptidyl mimic of superoxide dismutase) [51] prevents Fas mAb-induced FLF. Further, GSH enrichment alleviates Fas mAb-induced acute FLF [30]. For this reason, we next determined the effect of NAC treatment on IFN-c signaling during Fas mAb-induced FLF by treating IFN-c2/2 mice with NAC. We demonstrate for the first time that NAC therapy diminished hepatic injury in IFN-c2/2 mice during Fas mAb-dependent FLF probably via suppression of hepatic Th1 signaling molecules (i.e. pSTAT-1 and T-bet), apoptosis and nitrotyrosine formation. However, our observation that pSTAT-1 and T-bet expression in both vehicle and NAC treatment groups in IFN-c2/2 mice was higher than in WT mice suggests a possible inhibitory role of IFN-c2 that may not involve caspase 3. This observation warrants further investigation. Taken together, these studies provide important evidence that NAC therapy regulates IFN-c signaling and effector functions during Fas mAb-induced FLF. An important question our study also addressed is why Ja182/2 mice are resistant to development of FLF and hepatic apoptosis following Fas mAb administration. It is notable that Fas stimulation induces hepatic GSH depletion by triggering a cellular efflux of reduced GSH [53,54]. Conversely, preventing Fas induced GSH depletion attenuates apoptosis [30]. In agreement, we observed that GSH levels in the liver of WT mice was significantly depleted in response to agonistic Fas mAb treatment, but completely restored by V Va14iNKT cell de- ficiency to levels seen in PBS-treated WT mice. As mentioned previously, hepatic apoptosis (as denoted by active caspase 3 and TUNEL staining), pSTAT-1 and T-bet levels in the liver were markedly diminished by Va14iNKT cell deficiency during Fas mAb-induced FLF. On the basis of these findings, we surmised that fortification of hepatic GSH due to absence of Va14iNKT cells generates anti-inflammatory responses that suppresses IFN-c signaling and effector functions in the liver to prevent/limit the development of FLF and hepatic apoptosis in Ja182/2 mice. We previously discussed that intrahepatic Va14iNKT cells are activated during agonistic Fas mAb-induced FLF to express extracellular CD25 and intracellular IFN-c. However, it is not PLoS ONE | www.plosone.org 9 June 2012 | Volume 7 | Issue 6 | e38051