Modeling idiosyncrasy: A novel animal model of drug‐induced liver injury
Authors: Gwilym J. Webb, David H. Adams
Journal: Hepatology (2014)

Potential conflict of interest: Nothing to report. See Article on Page 1332 Drug‐induced liver injury (DILI) may be divided into the predictable, such as that seen in acetaminophen overdose, and the idiosyncratic, when the adverse hepatic reaction is unexpected on the basis of the pharmacological action of the drug administered.1 A division of idiosyncratic DILI (iDILI) into metabolic and immune‐mediated reactions has been proposed. However, a wide variety of contributing, and probably interacting, factors have been identified and discriminating the proposed subentities remains challenging (Fig. 1).1Figure 1: Summary diagram of factors proposed to contribute to the development of drug‐induced liver injury. See references Aithal et al.,1 Roth and Ganey,2 Fontana,3 and Russmann et al.4 and text. Abbreviations: BSEP, bile salt export pump; HIV, human immunodeficiency virus; HLA, human leukocyte antigen; PXR, pregnane X receptor.Idiosyncratic DILI is a major health‐care problem: it accounts for some 11% of those developing acute liver failure in the United States and carries a particularly poor prognosis.5 It is a leading cause of product withdrawal from the pharmaceuticals market, and fear of hepatic toxicity results in potentially unnecessary monitoring and physician‐initiated drug discontinuation.1 With ever‐increasing numbers of drugs receiving regulatory approval and rising rates of polypharmacy, iDILI is likely to be a growing problem. The novel animal model of DILI presented by Metushi et al. in this issue of Hepatology is thus of great interest.7 Despite the condition's significance, reliable data on the incidence of iDILI have been challenging to collate because of the condition's heterogeneity, variability in its reporting and classification, and incomplete data on numbers at risk. A recent population‐wide study from Iceland has recorded an annual incidence of just under 20 per 100,000 population, with only a minority progressing to liver failure; and this represents the best estimate to date.8 Amodiaquine, the drug used in Metushi et al.'s study, has been considered for withdrawal because of adverse reactions including iDILI despite its major utility as an antimalarial.9 Although many associations with iDILI have been identified, none is sufficiently understood to explain the idiosyncrasy that is its characteristic. Reliable methods of modeling DILI are needed, both to aid early prediction of adverse reactions and to elucidate the underlying pathophysiology.2 In this issue of Hepatology, Metushi et al. introduce a novel model of amodiaquine‐induced liver injury in a mouse rendered susceptible by the inhibition of normally tolerogenic immune pathways. In so doing, they have taken a step forward in modeling idiosyncrasy and provide further evidence of the clinical consequences of abnormalities in immune regulation. In previous work the Uetrecht group identified delayed increases in transaminases with subsequent resolution over weeks following amodiaquine administration to immunologically intact mice.10 Importantly, this dose resulted in similar bioavailability to that seen in humans, and the resolution in inflammation despite continued exposure is analogous to the clinical response that is termed "adaptation." On analyzing the immune cell profile of mice that appeared to have undergone adaptation, there was an induction of the inhibitory molecule programmed death receptor 1 (PD‐1) associated with liver infiltration by regulatory T cells (Treg), implicating recognized tolerogenic pathways. In support of the role of Tregs, the amodiaquine‐induced elevation of transaminases was increased when the experiment was repeated in recombination activating gene–deficient mice, which lack B and T cells. The healthy immune system is in balanced homeostasis as a consequence of active regulatory pathways that constantly suppress immune activation in response to harmless antigens. The molecular mechanisms that underlie this regulation are complex and involve several molecular pathways including the molecules PD‐1, cytotoxic T‐lymphocyte‐associated protein 4 (CTLA‐4), and the Src homology 3 binding protein Cbl‐b. The molecule PD‐1 is expressed by hematopoietic cells, T cells, B cells, and natural killer T cells. It reduces immune activity through several mechanisms including promotion of Treg development and function and inhibition of effector functions through interaction with its ligand PDL‐1;11 Cbl‐b is an E3 ubiquitin ligase that down‐regulates effector T‐cell function. Mice deficient in Cbl‐b develop generalized autoimmunity in later adult life, and their effector T cells appear less susceptible to regulation by Treg.12 The CTLA‐4 protein is constitutively expressed on Treg and binds avidly for CD80 and CD86, the two ligands of the major costimulatory molecule CD28, thereby reducing T‐cell activation.13 Mice entirely deficient in CTLA4 die at a few weeks of age from catastrophic multisystem autoimmunity.14 When mice deficient in either PD‐1 or Cbl‐b or treated with CTLA‐4 blocking antibody were given amodiaquine, there was a small increment in hepatic inflammation. However, using a combination of genetic knockout of PD‐1 and antibody blockade of CTLA‐4, Metushi et al. prevented adaptation (Fig. 2), resulting in increased inflammation and necrosis. As the authors note, there is an important analogy to cancer therapy where immune "checkpoint blockade" is used to uncover potent antitumor immune responses that are suppressed in cancer: the same combination of CTLA‐4 and PD1 blockade increases survival in patients with melanoma.15Figure 2: Simplified schematic representation of changes in liver inflammation in response to drug. *Point of adaptation; A, rapid onset of hepatic inflammation, perhaps due to metabolic interaction and which may or may not resolve (e.g., acetaminophen overdose or glutathione depletion and amodiaquine; see text); B, failure of adaptation (dashed line; e.g., amodiaquine in PD‐1‐deficient, anti‐CTLA‐4‐treated mice); C, successful adaptation (e.g., amodiaquine in intact mice); D, no clinically detectable response.Humans with heterozygous CTLA‐4 deficiency have recently been reported and demonstrate variable multiorgan autoimmunity, which involves the liver but to a lesser extent than other systems.16 Furthermore, administration of the humanized CTLA‐4‐blocking monoclonal antibody ipilimumab is associated with multiple immune‐mediated phenomena including skin and gastrointestinal inflammation and, less frequently, immune‐mediated hepatitis; hepatitis is also reported in anti‐PD‐1‐ and anti‐CTLA4‐treated cancer patients.15 There is more than one way to render amodiaquine hepatotoxic. In a different article, mice given amodiaquine after depletion of hepatic glutathione reserves developed more severe hepatitis.18 In separate work, the combination of lipopolysaccharide and amodiaquine was significantly more potent in generating liver injury than either agent on its own, with the ability of lipopolysaccharide to activate the innate immune system potentially contributing to a break in immune tolerance.19 As always with the best new science, Metushi and coworkers' study raises many avenues for onward experimental exploration: • Does dual immune blockade render mice susceptible to drug stimuli that are usually innocuous? An example would be chloroquine, which is related to amodiaquine but much less associated with liver injury. Were the model, or a derivative, able to discriminate effectively, this would be hugely valuable. • The DILI responses have been associated with specific major histocompatibility complex class I polymorphisms,20 and the autoimmune manifestations of PD‐1 deficiency in mice appear partly dependent on major histocompatibility complex class I type too:11 how transferable are these observations between mouse strains and MHC types? • Will mice treated with blocking antibodies to PD‐1 behave similarly to those that are constitutively deficient? If so, this would simplify the use of this model and avoid false‐positives resulting from the multisystem autoimmunity seen in older PD‐1‐deficient mice. • Metushi et al. demonstrated marked sex differences in the hepatic immunological response to amodiaquine. These contrast with the modest female predominance reported in DILI in humans and are worthy of further assessment.8 • The increase in Tregs in the inflamed livers of the mice that received immune blockade suggests normal recruitment but defective regulatory function. This is similar to the situation in autoimmune liver disease, and if the defects could be mapped, they could reveal therapeutic targets.21 • Perhaps most intriguingly, if certain immune interventions can trigger or unmask DILI, can an opposite intervention alter the disturbed immune balance toward tolerance and stop the inflammation? The ever‐increasing armory of targeted immunomodulatory agents makes this an exciting route to explore. Fundamentally, the difficulty with modeling iDILI remains that the human disease is unpredictable but that any useful animal model must be predictable and reproducible. Nevertheless, work like that discussed here has the potential to advance our understanding of iDILI immunopathology and to provide models to guide drug development and toxicity testing. Author names in bold designate shared co‐first authorship.