7E). Last, starvation caused greater elevations of serum BOH levels in Jα18−/− mice, compared to WT mice (Fig. 7F). These data confirm that it was the deletion of NKT cells that rendered mice more susceptible to AILI. Our data demonstrate that NKT cell-deficient mice are more susceptible to AILI than WT mice. This is the result, in part, to starvation-induced up-regulation

of CYP2E1 protein phosphatase inhibitor library expression and activity, which is associated with marked increases in hepatic APAP protein adduct formation. Starvation also caused greater elevations of ketone bodies in NKT cell-deficient mice, which may account for the increase in CYP2E1 protein levels. Upon activation, NKT cells rapidly produce cytokines, such as interleukin (IL)-4 and IFN-γ.9 Many studies have shown both protective and pathological functions of these 3-MA order cytokines in liver disease models.22, 23 Based on these findings, we examined whether differential production of these cytokines between WT and CD1d−/− mice may explain the increased susceptibility of NKT cell-deficient mice to AILI. However, message levels of a number of cytokines were similar in liver tissues and isolated liver mononuclear cells (in which NKT cells are enriched) from APAP-treated WT and CD1d−/− mice (data not shown). These results suggest that APAP treatment does not trigger NKT cells to produce protective cytokines. It is established that

APAP mafosfamide metabolism to NAPQI and its covalent modification of liver proteins are essential in triggering hepatocyte damage.16 Because a series of downstream events, such as mitochondrial dysfunction, ATP depletion, and DNA damage, take place before

ALT release, there is a delay between NAPQI generation and increase of serum ALT levels. Compared to WT mice, CD1d−/− mice had significantly higher levels of APAP protein adducts as early as 2 hours post-APAP (Fig. 2); however, depending on the dose of APAP, a significantly higher ALT level was not observed until 8 (Supporting Fig. 2) or 24 hours after APAP challenge (Fig. 1). GSH plays a pivotal role in AILI through scavenging of NAPQI. It has been demonstrated that mice deficient in both IL-10 and IL-4 (IL-10/4−/− mice) are more susceptible to AILI, compared to WT mice. This appears to be the result of lower GSH levels in IL-I0/4−/− mice before, and more dramatically after, APAP challenge.24 We observed no differences in total GSH levels in livers of naïve or starved WT and CD1d−/− mice (Fig. 4). Although there appears to be a slight delay in GSH rebound in the CD1d−/− mice at 8 hours, GSH levels were similar in WT and CD1d−/− mice at 19 hours after APAP treatment. Furthermore, we did not observe significant differences in expression and holoenzyme formation of glutamate cysteine ligase, the rate-limiting enzyme for GSH synthesis (data not shown).