Involvement of natural killer T cells in halothane-induced liver injury in mice
Graphical abstract
Introduction
The liver is a primary target of adverse drug reactions because it is the predominant site for the biotransformation of drugs. The severity of drug-induced liver injury (DILI) can range from mild and transient increases in serum transaminase levels with no clinical consequences, to acute and chronic hepatitis, cholestatic disease, cirrhosis, fulminant hepatic failure (requiring liver transplantation) and even death. Compared with other types of liver disease, DILI has a greater risk of developing into fulminate hepatic failure. It is estimated that DILI accounts for over 50% of liver failure cases in the US [1]. DILI is also the most common reason for the withdrawal of FDA-approved drugs from the market [2]. In recent years, several drugs, most notably troglitazone and bromfenac have been withdrawn from the market during post-marketing surveillance due to severe hepatotoxicity. As such, it is imperative to gain a thorough understanding of the underlying mechanisms of DILI before strategies for developing diagnostic tests and designing enhanced clinical trials can be achieved. The enhanced usage and implementation of animal studies will provide insights into the complex molecular and cellular mechanisms of DILI.
We have previously reported the development of a murine model of liver injury caused by an inhalation anesthetic, halothane [3]. This model shares similar characteristics to those found in 20% of patients who develop acute halothane hepatotoxicity, which progresses into life-threatening halothane hepatitis in a small percentage of such patients. Based on our model, it was revealed that although the generation of a chemically reactive metabolite via halothane metabolism is necessary to initiate hepatocyte damage, various innate immune cells appear to play critical roles in the progression of tissue damage. The data showed that depletion of neutrophils markedly attenuated halothane-induced liver injury (HILI) in mice, indicating a pathological role for neutrophils. Natural killer T (NKT) cells, which express surface markers of both NK and T cells, represent another important type of hepatic innate immune cells. In mice, as a proportion of mature T cells, NKT cells are most frequent in the liver (30–50%) and bone marrow (20–30%), while they represent a smaller proportion of T cells in the spleen (3%) and lymph nodes (0.3%) [4]. Numerous reports have described the important roles of murine hepatic NKT cells in inhibiting hepatitis B viral replication [5], as well as suppressing cancer liver metastasis and the development of hepatocellular carcinoma [6].
In contrast to their protective roles, NKT cells have also been demonstrated to exhibit pathological effects in animal models of liver injury. CD1d−/− mice, which lack NKT cells, were shown to be highly resistant to concanavalin A-induced hepatitis, a mouse model of human autoimmune and viral hepatitis [7]. Mechanistic studies have further revealed the link between the indispensable pathological role of NKT cells and their production of interleukin (IL)-4, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and FasL, all of which are critical in the development of concanavalin A-induced hepatitis [7], [8], [9], [10]. In another murine model of hepatitis caused by the injection of the exogenous ligand of NKT cell receptor, alpha-galactosylceramide (α-GalCer), the production of TNF-α by NKT cells was found to be the key mediator of liver injury [11]. Furthermore, the prompt production of IFN-γ by NKT cells has been suggested to play a central role in endotoxin-induced liver injury [12], [13].
In spite of two studies investigating the role of NKT cells in a mouse model of acetaminophen overdose-induced liver injury [14], [15], the role of NKT cells in DILI remains largely unknown. The former of these studies demonstrated that depletion of both NK and NKT cells yielded a significant decrease in acetaminophen-induced liver injury and improved survival [14]. The data suggested that the secretion of IFN-γ by NK/NKT cells may induce inflammatory chemokines, enhance leukocyte recruitment and increase FasL expression, thereby contributing to liver injury. However, a more recent report revealed that the pathological role of NK/NKT cells in acetaminophen-induced liver injury was due to the presence of dimethyl sulfoxide used to aid in the dissolution of acetaminophen [15]. The latter study demonstrated that the low amounts of dimethyl sulfoxide caused a significant increase in both the number and activation of hepatic NK/NKT cells. These findings suggest that NKT cells, activated by either drug-dependent or independent stimuli, may play a critical role in the underlying mechanisms of DILI. Provided that NKT cells are readily activated by self-lipid antigens, bacterial and viral antigens, cytokines, as well as “danger signals” released from damaged cells, these factors may represent key risk factors that modulate an individual's susceptibility to DILI.
The aim of the present study was to investigate the role of NKT cells in HILI in mice. Susceptibility to HILI was compared between NKT cell-depleted CD1d−/− mice and their wild-type (WT) counterparts. Our data demonstrated that CD1d−/− mice developed minimal liver injury upon halothane challenge and exhibited a much lower number of infiltrating neutrophils in the liver compared with WT mice. These findings suggest that NKT cells are activated upon halothane exposure and that they subsequently play a critical role in the development and progression of HILI.
Section snippets
Materials
All chemicals and reagents were obtained from Sigma (St. Louis, MO), unless stated otherwise. All antibodies used for flow cytometric analysis were purchased from eBioscience (San Diego, CA), except for phycoerythrin (PE)-conjugated α-GalCer-loaded mouse CD1d-tetramers (CD1d-tetramer, National Institutes of Health Tetramer Core Facility). Rabbit polyclonal anti-trifloroacetylchloride (TFA) antisera (anti-TFA antisera) were a kind gift from Dr. Lance Pohl (National Institutes of Health,
NKT cell-deficient CD1d−/− mice are resistant to HILI
NKT cell depletion in vivo has often been achieved by administering either an anti-NK1.1 antibody or α-GalCer to mice. However, the anti-NK1.1 antibody depletes not only NKT cells, but also NK cells as well. α-GalCer-induced NKT cell depletion is preceded by activation of these cells, which may cause the further activation of additional cells, including NK cells. Together, these activated cells are capable of producing pro-inflammatory cytokines, such as IFN-γ, that can inhibit CYP450 activity
Discussion
DILI is a significant cause of patient morbidity and mortality. The idiosyncratic nature and poor diagnosis of such reactions represent a major safety issue in drug therapy and new drug development. Research aimed at understanding the pathogenesis of DILI has been hampered by the lack of animal models. Our previous development of a murine model of HILI demonstrated a strain-dependent susceptibility that was not due to variations in the metabolism of halothane to TFA but rather the extent of
Acknowledgments
The authors wish to thank Dr. Lance Pohl (NIH, Bethesda, MD) for the generous gift of anti-TFA antisera. This work was supported by U.S. National Institutes of Health grant R01ES012914 (to Cynthia Ju).
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