Although clinical evidence suggests that many cases of DILD are mediated by hepatic protein adducts of drugs and the adaptive and innate immune systems, detailed experimental proof for this mechanism of toxicity has remained elusive due to the lack of animal models. We have hypothesized that DILD is as rare in animals as it is in humans due at least in part to the tolerogenic nature of the liver, which consists of multiple negative regulators of the adaptive immune system. This idea has now been tested in an established murine model of halothane-induced liver injury where the toxicity is initiated by the metabolism of halothane to form trifluoroacetylated liver proteins and enhanced by the innate immune system. Twenty-four hours after female Balb/cJ mice were treated with halothane, analysis of the liver revealed perivenous necrosis and an infiltration of CD11b+ Gr-1High neutrophils, as reported by other researchers. Further study revealed that the neutrophils contained a subpopulation of myeloid-derived suppressor cells (MDSC) that inhibited the proliferation of both CD4+ and CD8+ T cells isolated from naive mice. When MDSC were depleted from the liver with Gr-1 antibodies prior to two treatments with halothane, enhanced liver injury was observed nine days after the second exposure of halothane as compared to mice that were pretreated with isotype control antibodies before halothane treatments. Moreover, the liver injury was associated with elevated levels of hepatic eosinophils and hepatic T cells and serum antibodies that both reacted with trifluoroacetylated liver proteins isolated from halothane treated mice. Liver eosinophilia has been associated with incidences of drug-induced liver disease (DILD) for more than 50 years, though its role in the etiology of this disease has remained unclear. We reported for the first time a pathogenic role of eosinophils in DILD using a murine model of halothane-induced liver injury (HILI). When female Balb/cJ mice were administered halothane, eosinophils were detected in the liver within 12 hours and increased thereafter proportionally to liver damage. Eosinophil associated chemokines, eotaxins, and the activator/growth factor interleukin-5 (IL-5) increased in response to halothane-treatment. Immunohistochemical staining for major basic protein (MBP), a cytotoxic eosinophil granule protein, revealed that eosinophils accumulated exclusively around areas of hepatocellular necrosis and appeared to show signs of degranulation as MBP staining was more diffuse than in the livers from vehicle controls. The severity of HILI was decreased significantly when the study was repeated in wild-type mice partially depleted of eosinophils and in the eosinophil knockout mice. Conversely, animals with selective depletion of neutrophils, which have been previously reported to play a pathogenic role in this model, failed to reduce the extent of HILI when levels of eosinophils remained unchanged. These findings indicate that eosinophils, not neutrophils, have a pathologic role in HILI in mice. More recently, we have shown that the epithelial derived cytokine, thymic stromal lymphopoetin (TSLP), and its corresponding receptor TSLPR also play a pathogenic role in HILI in mice that is mediated by type 2 immunity. In this regard,the severity of HILI was reduced in both TSLPR and IL-4 knockout mice and was accompanied by decreases in serum levels of IL-5 and eotaxins and hepatic eosinophilia. In addition, we found that murine and human hepatocytes treated with IL-4 secreted TSLP and eotaxins. Conclusion:Collectively, this data provides a rational approach for developing animal models of DILD that are mediated by the adaptive and innate immune system and suggests that deficiencies in liver tolerance may predispose patients to DILD. These findings also establish a pathologic role for type 2 immunity in HILI in mice and suggest that similar signaling pathways may be involved in DILD caused by a variety of drugs in humans.

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National Heart, Lung, and Blood Institute
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Proctor, William R; Chakraborty, Mala; Fullerton, Aaron M et al. (2014) Thymic stromal lymphopoietin and interleukin-4 mediate the pathogenesis of halothane-induced liver injury in mice. Hepatology 60:1741-52
Proctor, William R; Chakraborty, Mala; Chea, Lynette S et al. (2013) Eosinophils mediate the pathogenesis of halothane-induced liver injury in mice. Hepatology 57:2026-36
Masson, Mary Jane; Collins, Lindsay A; Pohl, Lance R (2010) The role of cytokines in the mechanism of adverse drug reactions. Handb Exp Pharmacol :195-231