The ROS Scavenger, NAC, Regulates Hepatic Va14i NKT Cells Signaling during Fas mAb-Dependent Fulminant Liver Failure Isaac Downs1, Jianfeng Liu1, Tak Yee Aw1, Patrick A. Adegboyega2, Maureen N. Ajuebor1* 1 Department of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center-Shreveport, Shreveport, Louisiana, United States of America, 2 Department of Pathology, Louisiana State University Health Sciences Center-Shreveport, Shreveport, Louisiana, United States of America Abstract Uncontrolled systemic activation of the immune system is an early initiating event that leads to development of acute fulminant liver failure (FLF) in mice after treatment with agonistic Fas mAb. In this study, we demonstrate that treatment of mice with N-acetylcysteine (NAC), an ROS scavenger and glutathione (GSH) precursor, almost completely abolished Fas mAb-induced FLF through suppression of Va14iNKT cell activation, IFN-c signaling, apoptosis and nitrotyrosine formation in liver. In addition, enrichment of the liver with GSH due to Va14iNKT cells deficiency, induced an anti-inflammatory response in the liver of Ja182/2 mice that inhibited apoptosis, nitrotyrosine formation, IFN-c signaling and effector functions. In summary, we propose a novel and previously unrecognized pro-inflammatory and pro-apoptotic role for endogenous ROS in stimulating Th1 signaling in Va14iNKT cells to promote the development of FLF. Therefore, our study provides critical new insights into how NAC, a ROS scavenger, regulates Th1 signaling in intrahepatic Va14iNKT cells to impact inflammatory and pathological responses. Citation: Downs I, Liu J, Aw TY, Adegboyega PA, Ajuebor MN (2012) The ROS Scavenger, NAC, Regulates Hepatic Va14iNKT Cells Signaling during Fas mAb- Dependent Fulminant Liver Failure. PLoS ONE 7(6): e38051. doi:10.1371/journal.pone.0038051 Editor: Nai Sum Wong, University of Hong Kong, Hong Kong Received June 7, 2011; Accepted May 2, 2012; Published June 6, 2012 Copyright: 2012 Downs et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by NIH grants to MNA (NIAID R56AI085150) and TYA (NIDDK DK44510).The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: majueb@lsuhsc.edu Introduction Fas (CD95), a 45-kDa type I membrane protein, is expressed on numerous cell types including lymphoid cells (NK cells, T cells, Va14iNKT cells) [1,2,3,4,5] and non-lymphoid cells such as hepatocytes [1,2,3]. Fas, a member of the TNF receptor superfamily, plays a vital role in regulating apoptosis in many cell types and is typically stimulated by FasL or agonistic Fas mAb [1,2]. Upon ligand binding, Fas-associated protein with death domain and procaspase 8 are recruited to initiate caspase 8 proteolytic autocleavage, leading to activation of the effector caspase, caspase 3, and ultimately cell death [1,2]. Fas activation is often observed in diseases affecting many organ systems including heart, lung and kidney. Of significant relevance, Fas activation is a primary trigger for apoptotic death of hepatocytes [1,2]. The fundamental concept that the liver is highly sensitive to Fas- mediated apoptosis was first demonstrated in 1993 by Ogasawara and colleagues [1] where systemic administration of agonistic Fas mAb (Jo2) caused acute FLF, and ultimately mice mortality within a few hours due to diffuse hemorrhage and massive apoptosis of hepatocytes [1]. Although Fas activation is widely associated with caspase-mediated cell death, growing evidence have increasingly highlighted an important pro-inflammatory role for Fas in promoting NF-kB/AP-1 activation [6,7,8], chemokine/cytokine production [6,7,9] and leukocyte infiltration [6,7,9] in tissue sites. Va14iNKT cells are thymic-derived innate T lymphocytes that express a highly restricted TCR characterized by a Va14-Ja18 rearrangement [10]. Distinct from conventional T cells, Va14iNKT cells respond to glycolipid antigens presented by CD1d bearing antigen presenting cells [11]. Since the identity of the endogenous glycolipid ligand that is responsible for Va14iNKT cell selection and development in the thymus remains elusive [12,13,14], characterization of several exogenous glycoli- pids that Va14iNKT cells respond to, a-galactosylceramide (GalCer) and its derivatives, has greatly facilitated the research into the functional role of Va14iNKT cells in health and diseases [13,14]. Notably, CD1d tetramers loaded with the prototypical synthetic glycolipid antigen, a-GalCer, has been used to reveal that murine liver has the highest frequency of resident Va14iNKT cells [15,16]. Va14iNKT cells are activated in a TCR-dependent manner by lipids presented by CD1d [17] or by TCR independent mechanisms involving toll like receptors [17,18,19,20]. Following activation, Va14iNKT cells may display cytotoxicity via Fas-FasL and TRAIL-dependent death pathways [17], much like NK cells [21]. However, their major function is thought to be rapid release of copious amounts of immunopolarizing cytokines (including IFN-c, IL-4 and TNF-a) and chemokines leading to stimulation or suppression of immune responses [17]. Through these mediators, activated Va14iNKT cells can ‘‘bridge’’ the innate and adaptive immune systems by interacting with and transactivating immune cells [22,23,24]. This ability to respond rapidly at the onset of the immune response underscores the role of Va14iNKT cells in immune response regulation. Consequently, Va14iNKT cells have been demonstrated to play a critical role in several immune PLoS ONE | www.plosone.org 1 June 2012 | Volume 7 | Issue 6 | e38051