hyde 3-phosphate dehydrogenase (GAPDH) and calculated relative to the control. Statistical analysis Data are presented as mean 6 SEM. Statistical significance was determined by the Student’s t test and the log-rank test by use of GraphPad Prism 5 (GraphPad Software, La Jolla, CA). P,0.05 was considered statistically significant. (See Additional Supporting Methods S1.) Results Deficiency of IL-15 enhanced susceptibility to APAP- induced fulminant hepatitis in mice Although IL-15 was previously shown to attenuate Jo-2 and concanavalin A-induced liver injuries [25,26], its role in drug- induced liver injury remained elusive. In our model of hepatitis induced by 400 mg/kg (lethal dose) of APAP in mice, the 5-day mortality was higher for Il152/2 than WT mice [100% (8/8) vs. 60% (6/10)] (Figure 1A). To confirm this result, we examined the serum activities of the liver enzymes ALT and AST in Il152/2 and WT mice. Four hours after APAP injection, the enzyme levels significantly increased and peaked between 8 and 12 hours post- APAP injection in Il152/2 mice, as compared to WT counterparts (Figure 1B,C). Another non-lethal dose (200 mg/kg) of APAP was further given to demonstrate this increased APAP sensitivity in Il152/2 mice. Compared with WT controls, Il152/2 mice showed higher serum ALT levels at 8 hr post-APAP injection (Figure 1D), which were further confirmed by histopathological examination of damaged livers, with more extensive centrilobular necrosis in Il152/2 mice (Figure 1E). Thus, deficiency of IL-15 increased the susceptibility to AILI in mice. IL-15 was induced in the KC-enriched fraction in APAP- induced hepatic injury Because of enhanced APAP hepatotoxicity in Il152/2 mice, we further examined IL-15 serum levels and hepatic expression in mice. The serum IL-15 levels significantly increased at 4 hr and reached to peak at 8 hr post-APAP in WT mice (Figure 2A), which were in parallel to liver damages by ALT levels. In contrast, expression of hepatic IL-15 was reduced at 8 but not 2 hr post- APAP in WT mice (Figure 2B), indicating IL-15 was not up- regulated in hepatic parenchymal cells during AILI. Monocytes (e.g., KCs) were previously reported to be the dominant IL-15- producing cells [30]. Hence, we examined the expression of IL-15 in the KC-enriched cell fraction. WT mice showed 7- to 8-fold increased mRNA expressions of IL-15 at 8 hr after APAP injection (Figure 2C). Thus, IL-15 was predominantly up-regulated in KCs but not in hepatocytes during AILI. Enhanced susceptibility to AILI in Il152/2 mice is not mediated by altered metabolism and ROS detoxification of APAP in hepatocytes To exclude that APAP metabolites participate in the heightened APAP hepatotoxicity in Il152/2 mice, we examined the expres- sions of CYP2E1 and CYP1A2, the main enzymes to oxidize excess APAP to toxic NAPQI [1] in liver. Hepatic CYP2E1 and CYP1A2 expressions were similar in WT and Il152/2 mice at 0 hr (Figure 3A,B). Additionally, GSH depletion in liver indirectly reflects the elimination of APAP metabolites. Both groups of mice showed similar GSH levels at 0.5 and 2 hr, with the lowest GSH level at 2 hr post-APAP (Figure 3C). Interestingly, delayed GSH recovery was observed at 8 and 12 hr post-APAP in Il152/2 mice Effect of IL-15 on APAP Hepatitis but not in WT mice. We further analyzed in vitro APAP sensitivity of isolated hepatocytes from individual mice. The hepatocyte viability after 4, 6 and 8 hrs of APAP treatment did not differ between WT and Il152/2 mice (Figure 3D). To explore the causes of diminished GSH recovery in Il152/2 mice, we determined synthetic efficiency of GSH by analyzing GCLC expression [31]. However, GCLC levels did not differ in both groups of mice at 0 and 8 hr post-APAP (Figure 4A,B). This finding indicated the delayed GSH recovery in Il152/2 mice was probably attributed to the less residual functional hepatocytes after APAP injection. Nuclear factor erythroid 2-related factor 2 (Nrf2)- related and ROS detoxification genes also mediate the sensitivity of APAP hepatotoxicity [32,33]. Therefore, we further examined the activation of Nrf2-related genes, such as NAD(P)H:quinone oxidoreductase 1 (NQO-1), glutathione S-transferase pi 1 (GstPi- 1), multidrug resistance protein 2 (MRP-2), MRP-3, etc. and ROS detoxification genes, such as superoxide dismutase-1 (SOD-1), superoxide dismutase-2 (SOD-2), catalase, etc. Except for the minimal suppression of SOD-1 in Il152/2 mice, both groups did not differ in mRNA expression levels of these genes (Figure 4C,D). Thus, metabolism of APAP and induction of ROS detoxification genes in hepatocytes do not contribute to the heightened APAP sensitivity in Il152/2 mice. APAP treatment induced a stronger inflammatory response in Il152/2 mice than WT mice Because inflammation caused by necrotic hepatocytes partici- pated in AILI [6], we further examined the APAP-induced inflammatory response in mice. The mRNA levels of hepatic pro- inflammatory cytokines IL-1b, TNFa and IL-6 were higher at 8 hr post-APAP in Il152/2 mice than WT counterparts, with a higher IL-1b level at 2 hr (Figure 5A). Moreover, the serum levels of IL- 1b, TNFa and IFNc and liver levels of IL-1b and IFNc were also higher in Il152/2 mice than WT controls (Figure S1). Il152/2 mice showed stronger induction of the adhesion molecules, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion protein-1 (VCAM-1) as well as neutrophilic chemokines, such as macrophage inflammatory protein-1 alpha (MIP-1a), KC/ GRO and MIP-2a at 8 hr post-APAP (Figure 5B,C). Because of the enhanced production of hepatic neutrophilic chemokines in Il152/2 mice, we investigated the profile of infiltrated monocytes in mouse livers. NPCs with CD11bhighF4/ 80high expression were referred to KCs [34], and the population of CD11bhighF4/80low cells were confirmed to be neutrophils by anti- Gr-1 antibody (data not shown) [34]. In the resting state, .90% and ,4% of hepatic monocytes were KCs and neutrophils, respectively. APAP-induced neutrophil infiltration of liver was greater in Il152/2 than WT mice at 8 hr after challenge, while the infiltration of KCs was relatively lesser (Figure S2). Therefore, APAP treatment increased inflammatory cytokine production and the extent of inflammatory cell infiltration in livers of Il152/2 mice. APAP-injected Il152/2 mice showed discordant induction of hepatic iNOS and HO-1 It has been reported that, NO, a product of iNOS, contributes to the APAP-induced hepatotoxicity [35]. We therefore tested hepatic iNOS induction by immunostaining in APAP-treated mice. The Il152/2 mouse livers showed stronger iNOS inductions than those of WT livers at 4 and 8 hr post-APAP injection (Figure 6A,C), with a similar trend of nitrotyrosine accumulation in both groups of mice (Figure 6B,C). Collectively, APAP-induced PLOS ONE | www.plosone.org 3 September 2012 | Volume 7 | Issue 9 | e44880