Effect of IL-15 on APAP Hepatitis Figure 8. Injection of GalN reduces the enhanced susceptibility to AILI in Il152/2 and WT mice. Mice were pre-treated with or without 700 mg/kg of GalN 20 minutes before APAP challenge. (A) ALT levels in serum and (B) liver histopathological changes at 6 hr after APAP (H&E staining; magnification 64). (C) The quantification results of hepatic necrotic area by software Image-Pro Plus analysis. (D) Hepatic GSH levels at 0.5 hr with or without APAP injection. *P,0.05; **P,0.01; n.s., not significant. Data are mean 6 SEM from 4,6 mice per group. doi:10.1371/journal.pone.0044880.g008 (Figure 5), indicating their important roles in the increased APAP hepatotoxicity of Il152/2 mice. Induction of iNOS, by pro-inflammatory cytokines such as IL- 1b, IFNc and TNFa in hepatocytes results in RNS formation [37], which further potentiates the APAP-induced hepatotoxicity [35]. It had been reported anti-oxidative proteins, Hsp70 and HO-1, protected mice against APAP hepatotoxicity [5,36]. In a rat heat- shock model, Hoetzel et al. demonstrated that an NO donor could diminish HO-1 or Hsp70 induction by suppressing activator protein-1 (AP-1) DNA binding activity or modulating protein translation, respectively [44]. Our results in Il152/2 mice (Figure 6) suggested that excess hepatic iNOS and RNS production might potentiate APAP toxicity by suppressing HO-1 and Hsp70 induction (Figure 7,S3). Through transcriptional arrest of hepatocytes, GalN with endotoxin or TNFa had been reported to induce acute apoptotic hepatitis in mice [45], whereas GalN pre-treatment markedly diminished the increased APAP hepatitis in Il152/2 mice versus a minor advantage in WT controls (Figure 8A–C). However, the apoptotic pathway was interrupted in our AILI model through mitochondria damage [46] and, moreover, GalN might block the secondary hepatic transcriptional activation from inflammatory mediators. In addition, the divergent responses between Il152/2 and WT mice to AILI might be partly attributed to the excess inflammation in the former. The detailed mechanism of GalN protection against APAP hepatitis needed further investigation. But, the protective effect of GalN was, at least, not through alteration of hepatic GSH contents in mice at early stage of AILI (Figure 8D). Exogenous IL-15 administration into Il152/2 mice or IL-15 neutralizing antibody injection into WT counterparts could not alter the APAP hepatotoxicity (Figure 9), implicating the extracellular IL-15 was insufficient to affect the present fulminant AILI model. Moreover, increased serum IL-15 levels during AILI might be considered as the compensatory effect of hepatic inflammation and the activation of KCs. Although exogenous IL-15 did not affect APAP hepatitis, endogenous IL-15 seemed to play an interesting and pivotal role in AILI of our mice. Prolonged innate inflammatory response induced by APAP- damaged hepatocyte determines the prognosis of liver injury [6]. APAP-induced inflammation was modulated by innate immune cells such as neutrophils [8], KCs [11] and DCs [9]. Neutrophil elimination did not protect against APAP hepatotoxicity in WT and Il152/2 mice (Figure 10A), which indicates a minor role of neutrophils in AILI [39]. Previously, KC produced hepato- protective factors (e.g., IL-10 and cyclooxygenase derived medi- ators) and depletion of KCs enhanced APAP hepatitis [11]. In ischemia-reperfusion (IR) liver injury, GdCl3 (30 mg/kg) protected mice via reduction of TNFa and IFNc levels in liver effluents of balb/c mice [47], indicating the inflammatory contribution of KCs. However, Jaeschke et. al. [39] suggested the protective role of KCs, but not inflammatory effecter, during AILI, and hepatic inflammation might be enhanced by other NPCs, such as endothelial cells [19,39]. During AILI, cyclooxygenase products from KCs not only suppressed inflammation but also up-regulated PLOS ONE | www.plosone.org 10 September 2012 | Volume 7 | Issue 9 | e44880