known whether agonistic Fas mAb is capable of directly stimulating intrahepatic Va14iNKT cells. Work by us [5,16] and others [4,40,55] in several animal models have previously demonstrated that intrahepatic Va14iNKT cell activation during inflammatory responses is associated with a significant decline in the frequency of hepatic Va14iNKT cells due to death by apoptosis, a process known as activation-induced cell death (AICD). In the present study, we provide evidence supporting the notion that agonistic Fas mAb is unlikely to directly stimulate intrahepatic Va14iNKT cells to undergo AICD. Specifically, Fas mAb treatment in WT mice did not cause a decline in intrahepatic Va14iNKT cells since the frequency of intrahepatic Va14iNKT cells in Fas mAb-treated WT mice was comparable to PBS-treated WT mice. Furthermore, intrahepatic Va14iNKT cells lack intracellular active caspase 3 upon Fas mAb treatment. Hence, we next assessed the effect of NAC therapy on intrahepatic Va14iNKT cell activation during Fas mAb-induced FLF. Recently, we showed that NAC therapy inhibits intrahepatic Va14iNKT cell activation during poly I:C-induced liver in- flammation [20]. To further explore the effect of NAC therapy on intrahepatic Va14iNKT cell activation in this study, Va14iNKT cells extracellular CD25 and intracellular IFN-c expression were determined. Significantly, we found that NAC therapy effectively curbed intrahepatic Va14iNKT cell activation (i.e. extracellular CD25 and intracellular IFN-c expression) in WT mice during Fas mAb-induced FLF. Equally important, liver pathology, hepatic apoptosis and IFN-c signaling in WT mice were all diminished by NAC treatment during FLF. It is also notable that the frequency of Va14iNKT cells in the liver of WT mice undergoing Fas mAb- induced FLF was also significantly decreased by NAC therapy. In contrast, NAC only treatment of WT mice (i.e. in the absence of agonistic Fas mAb) did not alter the frequency of Va14iNKT cells in the liver relative to WT mice treated only with vehicle (i.e. in the absence of agonistic Fas mAb). These results indicate that NAC effectively suppresses the endogenous mediator produced by inflamed liver to drive the effects seen in intrahepatic Va14iNKT cells during agonistic Fas mAb-mediated FLF. It is generally accepted that the decline/disappearance of Va14iNKT cells References 1. Ogasawara J, Watanabe-Fukunaga R, Adachi M, Matsuzawa A, Kasugai T, et al. (1993) Lethal effect of the anti-Fas antibody in mice. Nature 364: 806–809. 2. Song E, Lee SK, Wang J, Ince N, Ouyang N, et al. (2003) RNA interference targeting Fas protects mice from fulminant hepatitis. Nat Med 9: 347–351. 3. Ryo K, Kamogawa Y, Ikeda I, Yamauchi K, Yonehara S, et al. (2000) Significance of Fas antigen-mediated apoptosis in human fulminant hepatic failure. 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Rensing-Ehl A, Hess S, Ziegler-Heitbrock HW, Riethmuller G, Engelmann H (1995) Fas/Apo-1 activates nuclear factor kappa B and induces interleukin-6 production. J Inflamm 45: 161–174. 9. Saas P, Boucraut J, Quiquerez AL, Schnuriger V, Perrin G, et al. (1999) CD95 (Fas/Apo-1) as a receptor governing astrocyte apoptotic or inflammatory responses: a key role in brain inflammation? J Immunol 162: 2326–2333. 10. Godfrey DI, MacDonald HR, Kronenberg M, Smyth MJ, Van Kaer L (2004) NKT cells: what’s in a name? Nat Rev Immunol 4: 231–237. 11. Gumperz JE, Roy C, Makowska A, Lum D, Sugita M, et al. (2000) Murine CD1d-restricted T cell recognition of cellular lipids. Immunity 12: 211–221. NAC Inhibits Va14iNKT Cell Activation during inflammatory responses may result from TCR down- regulation and/or apoptosis [4,5,16,39,55,56,57]. In the current study, we observed that surface TCR on Va14iNKT cells was not down-regulated by NAC therapy during Fas mAb-induced FLF. Furthermore, NAC therapy did not promote apoptosis of intrahepatic Va14iNKT cells following agonistic Fas mAb administration. Although beyond the scope of the current study, it is conceivable that NAC therapy may suppress the production of chemoattractant(s) critical to Va14iNKT cells accumulation in the liver during FLF. This is an area that warrants further in- vestigation. In summary, the current study reveals new insights into how NAC therapy regulates IFN-c signaling in Va14iNKT cells to impact inflammatory and pathological responses in the liver (Figure 5) and possibly other tissue sites (such as heart, lung and kidney) where Fas activation is often observed. Supporting Information Figure S1 Effect of NAC treatment on intrahepatic Va14iNKT cell CD25 expression during agonistic Fas mAb-induced FLF. The number of CD25-expressing Va14iNKT cells in the liver after PBS or NAC treatment during Fas mAb-induced FLF at 4.5 h. All experiments were performed twice. Data is presented as mean 6 s.e.m with n = 4 mice/group (Figure S1); *P,0.05 vs. PBS group (no Fas mAb treatment); *P,0.05 vs. NAC/Fas mAb-treated group. #P,0.05 vs PBS group (no Fas mAb treatment); #P,0.05 vs. PBS/Fas mAb- treated group. Analysis performed by one-way analysis of variance followed by Newman-Kuels post hoc test. (TIF) Author Contributions Conceived and designed the experiments: MNA ID TA. Performed the experiments: MNA ID JL PAA. Analyzed the data: MNA ID JL PAA TA. Contributed reagents/materials/analysis tools: MNA ID JL PAA TA. Wrote the paper: MNA ID JL PAA TA. 12. Brutkiewicz RR (2006) CD1d ligands: the good, the bad, and the ugly. J Immunol 177: 769–775. 13. Porubsky S, Speak AO, Luckow B, Cerundolo V, Platt FM, et al. (2007) From the Cover: Normal development and function of invariant natural killer T cells in mice with isoglobotrihexosylceramide (iGb3) deficiency. Proc Natl Acad Sci U S A 104: 5977–5982. 14. 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J Immunol 181: 4452–4456. 19. Nagarajan NA, Kronenberg M (2007) Invariant NKT cells amplify the innate immune response to lipopolysaccharide. J Immunol 178: 2706–2713. 20. Gardner TR, Chen Q, Jin Y, Ajuebor MN (2010) Toll-Like Receptor 3 Ligand Dampens Liver Inflammation by Stimulating V{alpha}14 Invariant Natural Killer T Cells to Negatively Regulate {gamma}{delta}T Cells. Am J Pathol 176: 1779–1789. 21. Johansson S, Berg L, Hall H, Hoglund P (2005) NK cells: elusive players in autoimmunity. Trends Immunol 26: 613–618. PLoS ONE | www.plosone.org 10 June 2012 | Volume 7 | Issue 6 | e38051