Effect of IL-15 on APAP Hepatitis Figure 3. Hepatic metabolism of APAP does not contribute to the enhanced susceptibility to AILI in Il152/2 mice. The changes in APAP metabolic enzyme levels by analysis of hepatic mRNA and protein levels of CYP2E1 and CYP1A2 by (A) quantitative PCR and (B) western blot analysis and quantification. GAPDH served as the loading control. (C) Hepatic GSH levels at 0, 0.5, 2, 8 and 12 hr after APAP treatment. (D) Viability of isolated primary hepatocytes after treatment with 0 to 50 mM of APAP for 4 to 8 hr. *P,0.05; n.s., not significant. Data are mean 6 SEM from 7,8 mice per group. doi:10.1371/journal.pone.0044880.g003 reactive nitrogen species (RNS) formation was more pronounced in Il152/2 than WT mice. HO-1, also known as Hsp32, had been reported to protect against AILI [5]. After APAP treatment, hepatic HO-1 mRNA was induced within 2 hr and peaked at 4 hr, then returned to normal after 24 hr post-APAP in WT mice (Figure 7A). Despite similar hepatic HO-1 mRNA levels at 2 hr post-APAP in WT and Il152/2 mice, hepatic HO-1 levels were suppressed at 8 hr post- APAP in Il152/2 mice (Figure 7B). This finding was further confirmed by decreased HO-1 protein expression in livers of Il152/2 mice at the same time (Figure 7C). Previously, HO-1 was predominantly induced in parenchymal cells after APAP injection in rat [5]. Compared with WT counterparts, Il152/2 mice showed suppressed induction of HO-1 in hepatocytes (Figure 7D,E). Likewise, Hsp70 induction protected against APAP hepatitis in mice [36]. In our study, both mRNA and protein levels of hepatic Hsp70 were lesser in Il152/2 than WT mice after APAP injection (Figure S3). Increased susceptibility to AILI is abrogated by liver- specific transcriptional inhibitor in mice Pro-inflammatory cytokines such as IL1-b, TNFa and IFNc induce iNOS expressions in hepatocytes [37]. To further clarify the effecter cells in heightened APAP hepatitis of Il152/2 mice, galactosamine (GalN) was given to mice for specific inhibition of hepatocyte transcription [38]. Unexpectedly, pre-treatment with GalN abrogated the increased hepatotoxicity in Il152/2 mice in terms of serum ALT level at 6 hr post-APAP, with a minor effect on WT counterparts (Figure 8A, B & C). The reduced APAP hepatitis in Il152/2 mice, by GalN pre-treatment, was further confirmed by the serum ALT levels at 4 hr (APAP+GalN: 42506580 U/l, APAP+saline 1125062038 U/l, P,0.05) and 8 hr (APAP+GalN: 1701061493 U/l, APAP+saline: 2986064273 U/l, P,0.05), respectively. However, the GalN reduction effect on APAP hepatitis in WT mice was not as much as that in Il152/2 counterparts. With the histopathological examination and liver necrotic quantization, GalN pre-treatment reduced the APAP-related extensive cell-content release of the injured hepatocytes in Il152/2 mice (Figure 8B,C). Similar GSH depletions at 0.5 hr post-APAP were observed in WT and Il152/2 mice, with or without GalN pre-treatment (Figure 8D), which further indicated APAP metabolism, was not affected by GalN pre-treatment. Moreover, without APAP challenge, GalN pre- treatment did not affect hepatic GSH levels in WT and Il152/2 mice. Thus, heightened susceptibility to AILI in Il152/2 mice can be ameliorated by GalN pre-treatment and this effect seems to be not through alteration of hepatic GSH contents at early stage of APAP hepatitis. PLOS ONE | www.plosone.org 5 September 2012 | Volume 7 | Issue 9 | e44880