NAC Inhibits Va14iNKT Cell Activation retained near normal architecture. (c & e) Western blot analysis of hepatic active caspase 3, T-bet, pSTAT-1 expression levels and nitrotyrosine formation in WT and IFN-c2/2 mice after PBS or NAC treatment during Fas mAb-induced FLF. (d) TUNEL staining of liver sections from WT and IFN- c2/2 mice treated with PBS during Fas mAb-induced FLF showed intense TUNEL staining characteristic of apoptosis whereas WT and IFN-c2/2 mice treated with NAC mice showed minimal TUNEL staining. Figure S1 in a are presented as mean 6 s.e.m with n = 3–6 mice/group. *P,0.05, ?P,0.05 by one-way analysis of variance followed by Newman-Kuels post hoc test. All experiments were performed twice. doi:10.1371/journal.pone.0038051.g003 nitrosative stress (i.e. nitrotyrosine formation) were suppressed by the deficiency in Va14iNKT cells during Fas mAb-dependent FLF. Therefore, we propose that Va14iNKT cells positively regulates the expression of Th1 differentiating signaling media- tors, IFN-c, STAT-1 and T-bet, in the liver as well as liver apoptosis and nitrosative stress during Fas mAb-dependent FLF. To provide proof-of-principle that the pro-inflammatory/ pathological effects of intrahepatic Va14iNKT cells could be directly mediated by IFN-c, we examined the effects of IFN-c deficiency on the development of Fas mAb-dependent FLF. Astonishingly, IFN-c mutant mice were similarly susceptible to Fas mAb-induced FLF as WT mice. In correlation, a previous study demonstrates that IFN-c can exert liver inflammation/injury independent of Fas [49]. Furthermore, hepatic apoptosis was not alleviated by IFN-c deficiency since expression of active caspase 3 and TUNEL positive cells in the liver of IFN-c mutant mice was comparable to WT mice. Although STAT-1 and T-bet are usually critical to IFN-c signaling, we found that IFN-c is dispensable for hepatic induction of pSTAT-1 and T-bet following Fas mAb treatment since their expression was not inhibited by IFN-c deficiency. In view of the fact that pSTAT-1 is an upstream transcription factor known to induce caspase 3-dependent apoptosis [46,47] and T-bet activation [46,48], we speculated that endogenous factor(s) upstream of IFN-c may be early activator(s) of STAT-1 and T-bet. With this in mind, our subsequent experiments were designed to establish if treating mice with NAC to block ROS, an endogenous mediator produced in the liver (i.e. hepatocytes) during Fas mAb-induced FLF [50,51,52], may suppress IFN-c signaling. Alternatively, NAC may directly inhibit pSTAT-1 and T-bet activation independent of IFN-c. Figure 4. Effect of NAC treatment on intrahepatic Va14iNKT cell accumulation during Fas mAb-induced FLF. (a) Representative FACS dot plot of Va14iNKT cells levels in the liver after PBS or NAC treatment during Fas mAb-induced FLF. (b) FACS analysis of Va14iNKT cells level in the liver after PBS or NAC treatment in response to Fas mAb-induced FLF. (c) FACS analysis of Va14iNKT cells level in the liver after PBS or NAC treatment only (i.e. in the absence of agonistic Fas mAb). Results in b and c are shown as mean 6 s.e.m with n = 4–6 mice/group with *P,0.05 by Student’s unpaired t test. All experiments were conducted twice. Broken lines denote levels in untreated mice. doi:10.1371/journal.pone.0038051.g004 PLoS ONE | www.plosone.org 8 June 2012 | Volume 7 | Issue 6 | e38051