Robert A. Roth, PhD, Ashley R. Maiuri, PhD, Patricia E ...jpet.aspetjournals.org/content/jpet/early/2016/11/15/jpet.116... · TNFR: TNF receptor TVX: trovafloxacin This article has

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Title Page

Idiosyncratic, Drug-induced Liver Injury: Is Drug-Cytokine Interaction the

Linchpin?

Robert A. Roth, PhD, Ashley R. Maiuri, PhD, Patricia E. Ganey, PhD

Department of Pharmacology and Toxicology, Michigan State University, East Lansing,

MI, USA

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on November 15, 2016 as DOI: 10.1124/jpet.116.237578

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Running Title Page

Drug/Cytokine-Synergy in Idiosyncratic Liver Injury

Corresponding Author:

Robert Roth

Food Safety and Toxicology Bldg., Room 221

1129 Farm Lane, East Lansing, MI, 48824

517-353-9841

[email protected]

Number of text pages: 23

Number of figures: 3

Number of references: 117

Number of words in the abstract: 196

Number of words in the body of the manuscript: 6,718

Recommended section assignment: Toxicology

Non-standard abbreviations:

Apaf: apoptosis protease-associated factor

APAP: acetaminophen

Bid: Bcl interacting protein

CHOP: CCAAT-enhancer-binding protein homologous protein

ConA: concanavalin A

DOX: doxorubicin

ER: endoplasmic reticulum


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ERK: extracellular signal-regulated kinase

GM-CSF: granulocyte macrophage-colony stimulating factor

HLA: human leukocyte antigen

IDILI: idiosyncratic drug-induced liver injury

iNOS: inducible nitric oxide synthase

IFNγ: interferon-gamma

IRF: interferon regulatory factor

JAK: janus kinase

JNK: c-Jun N-terminal kinase

LPS: lipopolysaccharide

LVX: levofloxacin

MAPK: mitogen-activated protein kinase

MHC: major histocompatibility complex

MPT: mitochondrial permeability transition

NFκB: nuclear factor kappa B

NK: natural killer

NSAID: non-steroidal anti-inflammatory drug

PERK: protein kinase RNA-like ER kinase

ROS: reactive oxygen species

SNP: single nucleotide polymorphism

STAT: signal transducer and activator of transcription

TCDD: 2,3,7,8-tetrachoro-dibenzo-p-dioxin

TLR: toll-like receptor

TNFα: tumor necrosis factor-alpha


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TNFR: TNF receptor

TVX: trovafloxacin


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Abstract

Idiosyncratic, drug-induced liver injury continues to be a human health problem in part because

drugs that cause these reactions are not identified in current preclinical testing and because

progress in prevention is hampered by incomplete knowledge of mechanisms that underlie

these adverse responses. Several hypotheses involving adaptive immune responses,

inflammatory stress, inability to adapt to stress, and multiple, concurrent factors have been

proposed. Still, much remains unknown about how drugs interact with the liver to effect death of

hepatocytes. Evidence supporting hypotheses implicating adaptive or innate immune responses

in afflicted patients has begun to emerge and is bolstered by results obtained in experimental

animal models and in vitro systems. A commonality in adaptive and innate immunity is the

production of cytokines, including interferon-γ (IFNγ). IFNγ initiates cell signaling pathways that

culminate in cell death or inhibition of proliferative repair. Tumor necrosis factor-α (TNFα),

another cytokine prominent in immune responses, can also promote cell death. Furthermore,

TNFα interacts with IFNγ, leading to enhanced cellular responses to each cytokine. In this short

review we propose that the interaction of drugs with these cytokines contributes to idiosyncratic,

drug-induced liver injury, and mechanisms by which this could occur are discussed.


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Introduction

Idiosyncratic adverse drug responses occur in a minority of patients during drug therapy. The

liver is a frequent target of such reactions (Gunawan and Kaplowitz, 2007). For example,

trovafloxacin (TVX) is a broad-spectrum antibiotic that was introduced to the US market in 1998.

About a year later, several patients who consumed the drug suffered serious liver injury, leading

to curtailing of its use (Ball et al., 1999). Similarly, the nonsteroidal, anti-inflammatory drug

(NSAID) diclofenac has been associated with rare occurrence of liver injury in patients

(Boelsterli, 2003). Halothane was once a widely used, volatile anesthetic that caused severe

liver injury (“halothane hepatitis”) in approximately 1 in 30,000 patients who were anesthetized

with the drug; it has largely been replaced by other halogenated anesthetics that do not share

its IDILI liability (Ray and Drummond, 1991). These are but a few examples of drugs that cause

idiosyncratic, drug-induced liver injury (IDILI) reactions that remain a public health concern,

pose major challenges in drug development, and have led to the market withdrawal of otherwise

therapeutically effective drugs. Unlike typical (“intrinsic”) toxic responses to xenobiotic agents,

IDILI reactions happen at therapeutic dosing regimens and often occur with inconsistent

temporal patterns in relation to drug exposure (Zimmerman, 2000).

The infrequency with which IDILI reactions occur in humans and animals has rendered them

difficult to study. Importantly, these reactions are not predicted from tests used currently in

preclinical safety evaluation and often are not discovered in clinical trials, since the numbers of

volunteers in clinical trials are too few to reveal rare adverse reactions. The basis for the

reactions is incompletely understood, and consequently several hypotheses to explain them

have emerged. The most longstanding is that drugs with IDILI liability precipitate damaging

adaptive immune responses. Within the last several years, other hypotheses have been


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proposed, among which are the multiple determinant hypothesis, the inflammatory stress

hypothesis and the failure-to-adapt hypothesis. What is known is that IDILI responses are

driven by both sensitivity of the individual patient and characteristics of the drug. Determinants

of individual sensitivity include genetic differences and environmental stressors. Much effort

has been devoted in recent years to identifying factors that contribute to individual sensitivity.

Less is known about the specific events that drive hepatocellular injury during IDILI reactions.

This article offers a short review of the supporting evidence and a perspective about how

immune mediators interact with drug exposure to effect killing of hepatocytes, with a particular

emphasis on the role of the cytokines, tumor necrosis factor-alpha (TNFα) and interferon-

gamma (IFNγ).

Interferon-gamma and its interaction with tumor necrosis factor-alpha cause diverse

cellular effects, including cell death.

Interferon-gamma (IFNγ). IFNγ is a cytokine that exists as a soluble dimer encoded by the

IFNG gene. It is not produced in substantial amounts by hepatic parenchymal cells but is

expressed and secreted by several immune cell types, including CD4+ T-helper cells (ie, Th1

cells), CD8+ cytotoxic T-cells, natural killer (NK) T-cells, NK cells and eosinophils. These cells

can be activated to express and release IFNγ by cytokines such as interleukin (IL)-12, IL-18 and

IL-27 (Trinchieri and Scott, 1995; Okamura et al., 1998; Batten and Ghilardi, 2007). Moreover,

interaction among different types of leukocytes occurs; for example, using CD1 to present

glycolipid stimuli, NKT-cells can activate NK cells to produce IFNγ (Carnaud et al., 1999;

Hayakawa et al., 2001). This stimulation involves IFNγ itself.


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The biologic activities of IFNγ result mostly from binding to two transmembrane receptors that

reside on hepatocytes and on certain nonparenchymal cells, including Kupffer cells. Ligation of

IFNγ receptors activates an intracellular signaling pathway involving janus kinase (JAK) and

signal transducer and activator of transcription (STAT). This activation results in the

transcription of dozens of genes, the protein products of which produce a variety of cellular

responses (Schroder et al., 2004). Many of these responses involve immune cells. For

example, IFNγ can increase the activity of antigen-presenting macrophages, promote leukocyte

adhesion required for migration into tissue and enhance NK cell activation. Kupffer cells

activated by IFNγ produce cytokines, including TNFα, which can modulate hepatocellular

function, promote an inflammatory response and participate in cell death (see below). Both

CD8+ and CD4+ T-cells have receptors for IFNγ (Whitmire et al., 2005). IFNγ promotes

differentiation of naïve CD4+ (Th0) cells into Th1 cells and inhibits their differentiation into Th2

cells. Since Th1 cells produce IFNγ, this differentiation can enhance IFNγ secretion and provide

positive feedback to increase Th1 differentiation.

In addition to these activities, IFNγ can inhibit proliferation of many types of cells. Proliferation

of hepatocytes occurs slowly in normal liver but can increase markedly when the liver is

stressed by partial hepatectomy or by exposure to pathogens or toxicants. Expression of IFNγ

receptors increases on hepatocytes when liver injury occurs, and receptor ligation by IFNγ

inhibits hepatocyte proliferation (Volpes et al., 1991; Dong et al., 2007). This occurs through

inhibition of cell cycle progression involving STAT-1 activation of IFNγ-responsive genes. One

result is the expression of IFN regulatory factor-1 (IRF-1), which in turn promotes expression of

p53 (Kano et al., 1999; Sun et al., 2006). STAT-1 and p53 activate the promoter for p21, the

expression of which leads to inhibition of S-phase progression and, consequently, to the

inhibition of cell proliferation (reviewed in Tura et al., 2001 and Horras et al., 2011).


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IFNγ can also induce cell death, including apoptosis of hepatocytes, by mechanisms

independent of p53 (Kano et al., 1997). Mechanisms by which this occurs are not well

understood (reviewed in Horras et al., 2011). According to one proposed mechanism, IFNγ acts

through STAT-1 activation and IRF-1 induction to express inducible nitric oxide synthase

(iNOS); this results in enhanced production of NO, which under conditions of redox stress can

initiate intracellular signaling that culminates in apoptosis. However, other cell death pathways,

some not involving STAT-1, can also be activated by IFNγ, depending on cell type. Finally,

IFNγ can cause expression of Fas ligand (FasL) on cells and thereby contribute to Fas-FasL-

induced cell killing (Boselli et al., 2007). Thus, IFNγ-mediated cell killing may occur by several

different mechanisms (Fig. 1).

Tumor Necrosis Factor-alpha (TNFα). IFNγ often acts through interaction with TNFα. TNFα is

a cytokine that can induce a variety of cellular responses and plays a critical role in liver

physiology. It is produced and released by a variety of immune cell types including, but not

limited to NK cells, macrophages, and T-cells (both CD4+ and CD8+). TNFα signaling can

initiate either hepatocyte proliferation or hepatocyte apoptosis, and an appropriate balance

between these two conditions is critical to preserving homeostasis in the liver (Wullaert et al.,

2007). It binds to and activates two distinct plasma membrane receptors, TNFα receptor 1

(TNFR1, p55) and TNFα receptor 2 (TNFR2, p75). TNFR1 is expressed constitutively in most

cell types, whereas the expression of TNFR2 is restricted mainly to immune cells. TNFR1 is

responsible for initiating most of the biological activities of TNFα (Chen and Goeddel, 2002).

Whether TNFα initiates intracellular signaling for cell survival and proliferation or for apoptosis

depends on the state of the cell (Wajant et al. 2003). To initiate cell survival, TNFα binding to

the extracellular domain of TNFR1 drives receptor trimerization followed by recruitment of a


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complex of adapter proteins, which can result in activation of NFκB. NFkB then translocates to

the nucleus, where it promotes transcription of many genes involved in cell survival and

proliferation and in inhibition of signaling for apoptosis (Wullaert et al. 2007).

When hepatocytes become stressed or damaged, TNFα can lead to activation of cell death

signaling pathways. Activation of TNFR1 can recruit and activate procaspase-8, resulting in

apoptosis via two possible routes. The first involves direct cleavage and activation of the

executioner caspases-3 and 7, which cleave a number of proteins leading to apoptosis. The

second, mitochondrial pathway can also be initiated by caspase-8 and entails cleavage and

activation of the proapoptotic protein, Bcl-interacting protein (Bid). The truncated form of Bid

(tBid) translocates to the outer mitochondrial membrane where it facilitates formation of the

mitochondrial permeability transition (MPT) pore. Formation of the MPT pore allows for release

of cytochrome c into the cytosol where it interacts with apoptosis protease-associated factor

(Apaf1) and procaspase-9, resulting in cleavage and activation of the latter. Activated caspase-9

subsequently cleaves and activates the executioner caspases 3 and 7, which effect apoptosis

(Green 1998, Wullaert et al. 2007).

In addition to the pathways discussed above, TNFα signaling via TNFR1 can result in activation

of the mitogen-activated protein kinases (MAPKs), c-jun N-terminal kinase (JNK) and p38.

Importantly, activation of these MAPKs can promote signaling for either cell survival or

apoptosis depending on their subcellular localization, duration of activation, health state of the

cell and other factors (Cowan and Storey, 2003). For instance, when it is activated transiently,

JNK, in particular, activates transcription factors that promote cell survival, including AP-1 and

NFκB (Hasselblatt et al., 2007). However, when it is activated for a prolonged period of time,

JNK can lead to activation of substrates that promote cell death, including p53. Specifically,


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phosphorylation of p53 by JNK promotes its stabilization and resistance to proteasomal

degradation (Fuchs et al., 1998). Moreover, JNK can lead to activation of the transcription factor

c-MYC, which can give rise to apoptosis under certain conditions (Hoffman and Liebermann,

2008). Finally, persistent activation of JNK can result in a decrease in mitochondrial membrane

potential (by an unknown mechanism) leading to MPT, apoptosome formation and activation of

caspase-3 and thereby bring about apoptosis (Gross et al., 1999).

IFNγ -TNFα Interaction. Importantly, IFNγ and TNFα are sometimes incapable of causing the

responses described above on their own or are required in very large concentrations to do so;

however, a pronounced synergy between IFNγ and TNFα can lead to various responses at

relevant cytokine concentrations. For example, IFNγ and TNFα can synergize with each other

in causing DNA fragmentation and apoptosis in vitro in primary mouse hepatocytes (Morita et

al., 1995). Additionally, it has been suggested that IFNγ can synergize with TNFα and other

inflammatory mediators to induce expression of the iNOS gene; as noted above, in the

presence of redox stress, iNOS induction can lead to production of oxidizing species that

promote hepatocyte apoptosis (Vodovotz et al., 2004; Fig. 1).

Although interaction between IFNγ and TNFα seems to be important in some IDILI models

(addressed below), this interaction is incompletely understood at the molecular level. The IFNγ

-mediated binding of STAT-1 to gamma activation sequences (GASs) in DNA and the binding of

IRF-1 to IFN-activated response elements in DNA were enhanced in a human epithelial cell line

after cotreatment of the cells with IFNγ and TNFα, and this may be due, in part, to increased

expression of IFNγ receptor (IFNγ R) (Robinson et al., 2003). TNFα enhanced IFNγ-stimulated

JAK2 phosphorylation and activation in human sarcoma cells, and tyrosine phosphorylation of

IFNγ R chain 1 was elevated in cells cotreated with IFNγ and TNFα (Han et al., 1999). These


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results suggest that both the expression and activation of IFNγ R can be enhanced by TNFα

coexposure. Conversely, expression of TNFRs can be enhanced by IFNγ (Wang et al., 2006).

Moreover, in human monoblastic Mono-Mac-6 cells stimulated with lipopolysaccharide (LPS),

IFNγ prolonged TNFα expression (Lee and Sullivan, 2001). In microglial cells in vitro, IFNγ and

TNFα cooperated in enhancing expression of iNOS and other prooxidant enzymes (Mir et al.,

2009); the enhanced expression depended on MEK (MAPK kinase) and on extracellular signal-

regulated kinase (ERK) signaling that resulted in the release of TNFα (Mir et al., 2008). Also,

IFNγ activation of the JAK/STAT pathway potentiated TNFα-induced NFkB binding to DNA and

activated IRF-1 needed for iNOS expression. In the AML-12 hepatocyte cell line, cotreatment

with IFNγ and TNFα caused cell cycle arrest that was independent of apoptosis and mediated

by p53 and NO (Brooling et al., 2005). Together, these results suggest that the IFNγ -TNFα

interaction can involve (1) enhanced IFNγ R expression and activation by TNFα exposure; (2)

enhanced TNFR expression and prolonged TNFα expression by IFNγ exposure (Fig. 1); (3)

potentiation by IFNγ of TNFα-induced NFkB binding to DNA and (4) synergistic cell cycle arrest

that is mediated by NO. However, most of these studies were conducted in extrahepatic,

transformed cells so additional study is needed to understand the molecular mechanisms that

underlie IFNγ-TNFα interactions in liver parenchymal and nonparenchymal cells and whether

these interactions become significant only in stressed cells.

IFNγ and TNFα: uniting hypotheses regarding the etiology of IDILI

The Adaptive Immunity Hypothesis of IDILI. The adaptive immunity hypothesis has

remained for decades the most popular of the theories to explain IDILI. The classical thinking

has been that a reactive metabolite of a drug binds to a protein, and the resulting adducted


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protein acts as a hapten that is recognized by and sensitizes the adaptive immune system.

Drug rechallenge or continued drug exposure then precipitates an adaptive immune response

that injures the liver (Fig. 2, upper right). The “pharmacological interaction” hypothesis is a

more recent modification, proposing that a drug might bind directly and reversibly with antigen-

presenting molecules, stimulating a damaging immune response that does not require prior

sensitization to the drug (Wuillemin et al., 2013).

The observation that fever, skin rash and eosinophilia accompany some IDILI reactions has

been taken as evidence for an adaptive immune etiology. However, the most compelling

evidence for adaptive immune system involvement in IDILI has arisen from recent studies in

humans that revealed associations between HLA polymorphisms and cases of IDILI for several

drugs. For example, patients who suffered IDILI from amoxicillin/clavulanate had one or more

HLA single nucleotide polymorphisms (SNPs), suggesting involvement of the adaptive immune

system in the pathogenesis (Lucena et al., 2011). Most of the SNPs occurred in HLA Class II

genes. Importantly, although various HLA SNPs were associated with increased IDILI risk, the

predictive value of the SNPs was very small; this suggests that some other genetic or

environmental factor(s) is needed to precipitate IDILI, even in individuals with the associated

HLA SNPs. Interestingly, a SNP in the gene encoding TNFα was also found in the study by

Lucena et al. (2011) to be significantly associated with amoxicillin/clavulanate IDILI, suggesting

the possibility of a role for inflammatory cytokines. Indeed, the linkage of the TNFα gene to the

HLA-B locus raises the possibility that some IDILI reactions associated with various HLA-B

alleles could be due to linkage with variations in the TNFα gene (Inoko et al., 1987).

The adaptive immune response is complex and interacts with the innate immune system to

effect killing of pathogens and damage to host tissue. Haptens comprising drug metabolite-


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protein adducts are endocytosed and degraded into peptides by antigen-presenting cells, which

can then present specific peptides bound to HLA II molecules to CD4+ T-helper cells (Th cells)

or to CD8+ T-cells that express specific receptors for the peptide-HLA complex. This binding

activates the T-cells, and upon a second, costimulatory signal (provided by CD28, CD80, CD86)

and subsequent autocrine signals, the cells proliferate and differentiate. One antigen-

presenting pathway involving CD8+ T-cells results in differentiation into cytotoxic T-cells, which

can kill pathogen-infected cells by expressing cytotoxic proteins (see below). Another pathway

results in differentiation of Th0 cells into either Th1 cells or Th2 cells. Differentiated Th1 cells

produce several cytokines, a major one of which is IFNγ. Th2 cell differentiation results in a

population of B-cells that produce antibodies. Th2 cells also produce factors such as IL-3, IL-5

and GM-CSF (granulocyte macrophage-colony stimulating factor) that stimulate differentiation of

myeloid precursor cells into eosinophils; these cells produce large amounts of IFNγ in response

to TNFα, IL-12 or IL-4 (Spencer et al., 2009).

Killing of host cells by activated T-cells can occur through several mechanisms. Perforin

expressed by cytotoxic CD8+ T-cells can incorporate into the plasma membranes of stressed

host cells, forming a pore and allowing the passage of granzyme B into the cells, which initiates

caspase-dependent cell death signaling (Fig. 1). Cytotoxic CD8+ T-cells can also express FasL

on their surfaces, which can bind to Fas on target cells to initiate killing through caspase-

dependent apoptotic pathways. Cytotoxic CD8+ T-cells also express IFNγ receptors, the

activation of which promotes CD8+ T-cell expansion as well as cell killing by these mechanisms

(Whitmire et al., 2005). Alternatively, IFNγ produced by CD4+ Th1 or other cells can initiate cell

death signaling by binding to an IFNγ R on the surface of host cells (Fig. 1). Such binding

initiates intracellular signaling involving STAT-1 activation of IFNγ -responsive genes, including

the gene expressing IRF-1, which in turn induces iNOS and other genes that could be involved

in initiation of cell death. IFNγ can also act indirectly by stimulating macrophages to release


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cytotoxic factors such as NO, reactive oxygen species (ROS) and TNFα (Fig. 1); as noted

above, TNFα acts synergistically with IFNγ in hepatocellular killing.

In addition to its ability to stimulate T-cell-mediated killing, IFNγ might influence liver

regeneration by inhibiting hepatocyte proliferation. In a study in partially hepatectomized rats,

IFNγ stimulated MHC II antigen expression on Kupffer cells; the authors speculated that these

cells then present hepatocytes as antigen to Th cells and cytotoxic T-cells, which suppresses

reparative hepatocyte proliferation (Sato et al., 1993).

Despite the longstanding popularity of the adaptive immunity hypothesis, no animal models

have emerged in which substantial liver injury occurs from an adaptive immune response after

sensitization with a drug that has caused IDILI in people (Ng et al., 2012; Metushi et al., 2015b).

Recent animal models have been developed which implicate a role for adaptive immunity in

IDILI responses to the drugs amodiaquine and halothane; however, it is important to note that

the liver injury produced in these models is mild and does not reflect the severity of liver injury

that occurs in human IDILI responses to these drugs. Nevertheless, these models provide some

insight concerning the mechanisms underlying adaptive immune-mediated IDILI responses.

Metushi et al. (2015a) found in mice that depletion of NK cells attenuated the mild liver injury

induced by amodiaquine exposure. When activated, NK cells release IFNγ which is known to

activate signaling pathways that lead to cell death. Additionally, Chakraborty et al. (2015) found

that depletion of CD4+ T cells, which also release IFNγ, protected mice from the delayed onset

of halothane hepatitis. Accordingly, it is possible that IFNγ by itself or in the presence of other

cytokines promotes hepatocellular killing in cases of human IDILI induced by amodiaquine or

halothane.


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Insight into which pathways predominate in T-cell-mediated killing of hepatocytes has been

provided by studies by Dr. G. Tiegs and colleagues in an animal model employing concanavalin

A (con A). This plant lectin is a T-cell mitogen that causes T-cell-dependent liver injury in mice.

Antibody-mediated depletion of CD4+ T-cells protected completely against liver injury from con

A, whereas depletion of CD8+ T-cells did not prevent liver injury, suggesting the importance of

Th1 cells (Tiegs et al., 1992; Tiegs, 1997; Cao et al., 1998). NKT cells also appear to contribute

(Erhardt and Tiegs, 2010; Zhang et al., 2013). Con A treatment was associated with

appearance of IFNγ and TNFα in plasma, and inactivation of macrophages or neutralizing either

one of these cytokines prevented con A-induced liver injury. Moreover, liver injury development

correlated with IFNγ activation of STAT-1 and IRF-1 and with TNFα activation of the JNK

pathway (Streetz et al., 2001; Hong et al., 2002). Findings in IL-27 conditional knockout mice

supported the importance of dysregulated production of IFNγ by CD4+ T-cells in con A-induced

liver injury in mice (Zhang et al., 2012). Interestingly, IL-5 derived from NKT cells led to

maturation of eosinophils, which contributed to the liver injury in this model (Louis et al., 2002).

These results suggest that IFNγ is critically important in mediating liver injury from Th1 cell

activation and that it acts synergistically with TNFα produced by Kupffer cells.

Recent studies with 2,3,7,8-tetrachoro-dibenzo-p-dioxin (TCDD) showed that this environmental

contaminant enhances con A-induced liver injury in mice and that both IFNγ and NK cells, which

produce IFNγ, are involved (Fullerton et al., 2013). This result indicates that xenobiotic agents

can interact with Th1 cell-dependent pathways to cause hepatocellular killing by mechanisms

involving IFNγ and IFNγ-producing cells and suggests the possibility that some drugs

associated with IDILI might evoke similar responses.

The paucity of animal models employing IDILI-associated drugs has limited the progress into

understanding the contribution of the adaptive immune system to IDILI and the factors that


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might govern such reactions. Nevirapine used in the treatment of HIV infections has caused

skin and liver reactions in patients. Uetrecht and colleagues developed a model of nevirapine-

induced skin injury in Brown Norway rats that is clearly adaptive immune-mediated (Popovic et

al., 2010). The skin rash that developed depended on CD4+ T-cells, and cells isolated from

rechallenged rats released IFNγ and other cytokines. No liver injury developed in this animal

model; however, if nevirapine-induced liver injury in humans arises from the same mechanism

as the skin rash in rats, then IFNγ could be a player in the IDILI pathogenesis from this drug.

Clearly, more animal models in which liver injury occurs from IDILI-associated drugs by an

adaptive immune-mediated mechanism are needed to understand the importance of IFNγ,

TNFα and other mediators in such reactions.

The Multiple Determinant Hypothesis of IDILI. It has been theorized that some IDILI

reactions result from the intersection of several factors or events (ie, “determinants”) that

together precipitate hepatocellular necrosis (Li, 2002; Ulrich, 2007). Inasmuch as the probability

of a reaction would be the product of the probabilities of each factor/event, and since the

product would usually be very small, this hypothesis could explain why IDILI responses are

typically rare. Important factors are proposed to relate to chemical properties of the drug, drug

exposure and metabolism/bioactivation, and genetic and/or environmental factors that

determine individual susceptibility to injury. Examples of likely genetic factors could include

polymorphisms in drug metabolizing enzymes or transporters that could lead to enhanced

production of a toxic metabolite or altered hepatic accumulation of a drug or its metabolites,

respectively. Other factors could include race/ethnicity, nutrition, pre-existing chronic liver

diseases and differences in intestinal microbiome. For example, recent studies indicate that

alterations in the intestinal microbiome determine sensitivity of animals to liver injury from

numerous hepatotoxiciants (Lv et al., 2014; Chiu et al, 2015; Shen et al., 2015; Dubey et al.,

2015; Tian et al., 2015).


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A murine model of halothane-induced liver injury has been developed based on this hypothesis.

Halothane is metabolized to a reactive metabolite that binds covalently to cellular

macromolecules, and the formation of halothane-protein adducts is thought to be required for

liver injury. Among the known human risk factors for halothane hepatitis are female sex, middle

age and genetic predisposition (Inman and Mushin, 1974; Cousins et al., 1989). Fasting may be

an additional risk factor, since all patients are fasted prior to surgery that requires general

anesthesia. Halothane given to fasted, mature, female Balb/c mice caused pronounced liver

injury (Dugan et al., 2010). Fed mice were less sensitive to the liver injury, male Balb/c mice

and female C57Bl/6 mice were markedly resistant, and immature female Balb/c mice were less

sensitive too. Moreover, isoflurane, which is not extensively metabolized to a reactive

intermediate and does not share the human IDILI liability of halothane, failed to cause liver

injury in the mouse model, indicating that chemical structure of the anesthetic is a determinant

of the hepatotoxic response. These results are consistent with the multiple determinant

hypothesis, inasmuch as the confluence of factors known to increase risk in humans

(femaleness, mature age, halothane structure, genetics) was required to produce halothane

hepatitis in mice.

In this murine model of halothane hepatitis, plasma IFNγ concentration was elevated 10-fold in

halothane-treated females compared with similarly treated male mice or ovariectomized female

mice, which were insensitive to injury (Dugan et al., 2011). IFNγ knockout mice were resistant to

halothane-induced liver injury, indicating the importance of IFNγ in the pathogenesis. Halothane

treatment increased the activation (CD69 expression) of NK cells, and inactivation of NK cells

attenuated both the rise in plasma IFNγ and the liver injury. A recent study also implicated

eosinophils in this model (Proctor et al., 2013); these cells are another potential source of IFNγ.

Interestingly, TNFα concentration in plasma also rose as a result of halothane exposure. These


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results suggested that IFNγ released from NK cells and perhaps eosinophils plays an essential

role in the development of severe halothane-induced hepatotoxicity in mice and raised the

possibility of synergistic interaction between IFNγ and TNFα in the pathogenesis.

The Inflammatory Stress Hypothesis of IDILI. The erratic temporal and dose-response

relationships that characterize idiosyncratic reactions suggest that some event occurring during

the course of therapy precipitates IDILI. If this is true, then the precipitating event must happen

occasionally and irregularly to account for the infrequent and erratic occurrence of these

reactions. Inflammatory cell infiltrates often characterize liver lesions in patients who suffer

IDILI (e.g., see Khouri et al., 1987; Fukano et al., 2000; Murphy et al., 2000). This raised the

possibility that some IDILI reactions might be explained by an episode of modest inflammation

occurring during the course of drug therapy that interacts with some action of the drug to initiate

liver injury. Such inflammatory episodes occur commonly in people and are associated with

various diseases, infections and intestinal translocation of inflammagenic bacterial products

such as endotoxin (ie, lipopolysaccharide, LPS). Indeed, episodes of mild, subclinical

endotoxemia appear to be a normal occurrence in people (reviewed in Roth et al., 1997; Ganey

and Roth, 2001; Ganey et al., 2004).

LPS and other inflammagens bind to “pattern recognition receptors” such as toll-like receptors

(TLRs) on cells of the innate immune system (Fig. 2, upper left). This initiates intracellular

signaling that leads to activation of transcription factors and expression of inflammatory

mediators such as TNFα and IFNγ (Arbour et al., 2000; Beutler, 2000). These mediators are

essential in defense against pathogens, but as noted above they are also capable of altering

homeostasis of host cells. It is well known that modest activation of the innate immune system

by inflammagens such as small doses of LPS can markedly augment hepatotoxic responses to

numerous chemicals, including some drugs (Ganey et al., 2004; Roth and Ganey, 2010).


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Accordingly, when an inflammatory episode of sufficient magnitude occurs during drug therapy,

it could interact with a drug to render an individual susceptible to a hepatotoxic reaction that

would not otherwise occur (i.e., an IDILI response). The episodic and variable nature of

exposure to LPS and other inflammagens and genetic variations (e.g., in TLRs or genes

encoding cytokines) that influence individual responses to inflammagens could explain

individual susceptibility, the infrequency of idiosyncratic reactions and their erratic temporal

relationship to drug exposure.

The inflammatory stress hypothesis led to attempts to determine if liver injury could be produced

in animals by concurrent exposure to an IDILI-associated drug and an otherwise noninjurious

inflammatory episode. Indeed, several drugs that cause human IDILI also caused liver injury in

animals upon cotreatment with a small, nontoxic dose of LPS, whereas drugs without human

IDILI liability failed to synergize with LPS to cause hepatotoxicity (summarized in Deng et al.,

2009 and Shaw et al., 2010). It is of interest that drugs frequently associated with human IDILI

are nonsteroidal anti-inflammatory drugs and antibiotics, ie, drugs used in conditions associated

with inflammation. Drug-LPS interaction models in rodents have now been developed with

chlorpromazine, halothane, ranitidine, diclofenac, sulindac, amiodarone, doxorubicin (DOX) and

TVX (see Deng et al., 2009 and Shaw et al., 2010). In each of these models, liver injury occurs

from drug-LPS cotreatment but not from exposure to either agent alone. Thus, the inflammatory

stress hypothesis has provided the first animal models in which pronounced liver injury occurs

from numerous drugs associated with human IDILI.

An example is the TVX-LPS model of drug-inflammation interaction in mice. A robust

hepatotoxic response occurred when a small (ie, nonhepatotoxic but modestly inflammatory)

dose of LPS was given to mice 3 hr after doses of TVX that were nontoxic by themselves (Shaw

et al., 2007). Interestingly, levofloxacin (LVX), a drug in the same pharmacological class that


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does not share the same propensity for causing IDILI, showed no synergistic interaction with

LPS. Of interest, both IFNγ and TNFα were elevated in the plasma of TVX-LPS cotreated

mice, and either (1) neutralization of TNFα with etanercept or knockout of either TNFR1 or

TNFR2 or (2) knockout of the gene encoding IFNγ provided protection from hepatocellular

necrosis (Shaw et al., 2008; Shaw et al., 2009a; Shaw et al., 2009b). Interestingly, TVX

enhanced LPS-stimulated TNFα production in the RAW 264.7 murine macrophage cell line in

vitro (Poulsen et al., 2014a and Poulsen et al., 2014b), suggesting that TVX can directly

enhance macrophage activation and TNFα release.

Together, these results indicated that both IFNγ and TNFα are critical for the

hepatopathogenesis of LPS-TVX interaction in vivo. Moreover, the liver injury in TVX-LPS

cotreated mice depended on a modest prolongation of TNFα appearance in the plasma by TVX

above that caused by LPS alone (Shaw et al., 2009a). IFNγ knockout reduced plasma TNFα,

and conversely neutralization of TNFα markedly reduced the appearance of IFNγ in plasma,

suggesting that each cytokine amplified the production of the other in a dysregulated cycle

(Shaw et al., 2008).

Replacing LPS administration with TNFα administration in this murine model also led to

hepatotoxic interaction with TVX (Shaw et al., 2009a). Interestingly, TNFα administration alone

caused appearance of IFNγ in plasma, and coadministration of TVX enhanced this response.

These findings emphasize the importance of TNFα in initiating IFNγ production and are

consistent with studies in vitro, since TNFα, by acting through macrophages or other cell types,

can stimulate IFNγ production by cultured NK cells and conversely IFNγ can enhance TNFα

production by macrophages (Berner et al., 2005; Makarenkova et al., 2005; Vila-del Sol et al.,

2008).


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Transcriptomic analysis of the livers of TVX-LPS-cotreated mice revealed selective

enhancement of expression of several genes involved in IFNγ signaling (Shaw et al., 2008).

These genes included IRF-1, which is involved in IFNγ -mediated apoptosis and inhibition of cell

proliferation. In the human HepG2 hepatocyte cell line, TVX enhanced cell killing from exposure

to a combination of IFNγ and TNFα, indicating that the IDILI-associated antibiotic increased the

sensitivity of hepatocytes to killing from this cytokine combination (Maiuri et al., 2016b).

Studies in other inflammatory stress models also point to importance of IFNγ. One study

revealed that DOX-LPS cotreatment synergistically enhanced liver injury in rodents, and this

enhancement depended on IFNγ (Hassan et al., 2008). Ju and colleagues showed that the viral

RNA mimetic and TLR3 agonist, polyinosinic-polycytidylic acid (polyI:C), markedly enhanced

halothane-induced liver injury in mice. This was accompanied by activation of Kupffer cells and

NK cells and upregulation of TNFα expression (Cheng et al., 2009). Although IFNγ was not

evaluated in that study, it is produced by NK cells activated by polyI:C (Zhang et al., 2009), so

that it seems likely that IFNγ interaction with TNFα contributes to injury in that model. Recently,

we have shown that IFNγ synergizes with TNFα in sensitizing hepatocytes in vitro to killing by a

number of drugs that cause IDILI in people (Maiuri et al., 2015 and Maiuri et al., 2016b).

Together, these results suggest that IFNγ -TNFα interactions could be critical to IDILI

pathogenesis from a number of IDILI-associated drugs.

The Failure-to-adapt Hypothesis of IDILI. A majority of human volunteers given a therapeutic

dose of acetaminophen (APAP) daily for two weeks experienced early, modest increases in

serum ALT activity which subsequently subsided toward normal despite continued drug

treatment (Watkins et al., 2006). An interpretation of this result was that APAP causes minor

hepatotoxicity, to which the liver adapts over time, thereby returning to normal. It is possible

that this injury-adaptation phenomenon occurs commonly with many drugs and that people who


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develop IDILI responses to a drug are those whose livers fail to adapt, permitting progression to

fulminant injury (Watkins, 2005). Such adaptation to minor injury has been demonstrated in

mice treated with amodiaquine, a drug that has caused IDILI in humans (Metushi et al., 2015b).

Stimulation of immune cells by TNFα or IFNγ leads to IL-10 expression, which in turn

downregulates TNFα and IFNγ production, thereby preventing their organ-damaging actions.

Failure of this regulation by IL-10 in susceptible patients could result in unrestricted and

damaging cytotoxic actions of cytokines such as TNFα and IFNγ. In this regard, it is of interest

that one study found worse outcomes from IDILI in patients who had IL-10 polymorphisms that

resulted in lower plasma IL-10 concentrations (Pachkoria et al., 2008) and another found an

association between polymorphisms resulting in low plasma IL-10 and diclofenac-induced liver

injury (Aithal et al., 2004). Accordingly, it is possible that in susceptible patients an inability of

IL-10 to control production of cytokines such as TNFα and IFNγ could lead to hepatocellular

death from these cytokines, which would present clinically as “failure to adapt” to their damaging

effects.

Recovery from liver injury typically entails proliferation of hepatocytes. For example, after loss

of liver tissue from partial hepatectomy the organ responds with cell proliferation that restores

liver mass and function. In a phenomenon that has been termed “autoprotection,” repeated,

small doses of APAP given to rodents reduced liver injury when a larger, hepatotoxic dose was

subsequently administered, and cell proliferation stimulated by the APAP pretreatments was

thought to play a major role in the reduced hepatic sensitivity (Dalhoff et al., 2001). Indeed,

tissue repair via cell proliferation has been shown to be critical to halting the progression of

injury for numerous hepatotoxicants, including APAP, and inhibiting cell proliferation results in

injury progression (Chanda et al., 1995; Mehendale, 2005). These observations suggest the

possibility that failure to adapt to modest injury caused by drugs might be due at least in part to


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reduced hepatocellular proliferative ability in susceptible individuals, leading to injury

progression and IDILI rather than adaptation (Fig. 2, bottom right).

As noted above, IFNγ is known to cause cell cycle arrest in hepatocytes in vitro and in vivo and

might therefore play a role in inhibiting cell proliferation during treatment with certain drugs

(Kano et al., 1999; Tura et al., 2001; Brooling et al., 2005; Sun et al., 2006; Dong et al., 2007).

Moreover, numerous drugs that cause human IDILI have been shown to inhibit cell proliferation

in vitro. Examples include diclofenac (Rajabalian et al., 2009), sulindac (Chennamaneni et al.,

2012), TVX (Beggs et al., 2015), halothane (Waxler et al., 1994), DOX (Supino et al., 1997) and

chlorpromazine (Basta-Kaim et al., 2006). Additionally, a series of NSAIDs associated with IDILI

inhibited HepG2 cell proliferation in vitro, whereas an NSAID not associated with IDILI, aspirin,

did not have this effect (Maiuri et al. 2014). This raises the possibility that a drug’s ability to

inhibit proliferative repair could contribute to IDILI. Although speculative, of interest is the

possibility that IFNγ produced during drug exposure might interact synergistically with direct,

antiproliferative effects of a drug to inhibit hepatocellular regeneration, thereby prompting failure

to adapt.

Although these cytokines might prompt adaptation failure by inhibiting hepatocyte proliferation,

an effect on immune cells is also possible. There exists some evidence that hepatic failure to

adapt might be due to a failure of immune tolerance in the liver (reviewed in Dara et al., 2016).

Accordingly, cytokine-induced inhibition of proliferation of lymphocytes that are needed for

immune tolerance could contribute to the initiation of severe IDILI.

Drug Interaction with Cytokine-mediated Cell Death Signaling


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Drugs associated with IDILI synergize with cytokines including IFNγ and TNFα in vitro to kill

hepatocytes (Cosgrove et al., 2009; Zou et al. 2009; Gandhi et al. 2010; Fredriksson et al. 2011;

Lu et al. 2013; Beggs et al. 2014; Maiuri et al. 2015). TNFα augmented the cytotoxicity of

sulindac sulfide in primary rat hepatocytes and in HepG2 cells (Zou et al., 2009). The interaction

between sulindac sulfide and TNFα depended on caspase 3/7 activity and also involved

sulindac sulfide-induced oxidative stress (Zou et al., 2010). TNFα potentiated cytotoxicity of

chlorpromazine in primary mouse hepatocytes via activation of JNK (Gandhi et al., 2010). TVX

synergized with TNFα to cause cell death in HepG2 cells and primary mouse hepatocytes

(Beggs et al., 2015). The TVX/TNFα-induced cytotoxic interaction depended on the MAPKs JNK

and ERK and also on ataxia telangiectasia Rad3-related (ATR), which is activated in response

to replication stress and DNA damage (Beggs et al., 2014, Beggs et al., 2015). Several drugs

associated with IDILI synergized with an inflammagen mixture containing TNFα, IFNγ, IL-1α,

and LPS, causing cytotoxicity in HepG2 cells and primary human hepatocytes (Cosgrove et al.,

2009). Another study demonstrated that diclofenac synergized with TNFα to kill HepG2 cells,

and this depended on caspase activation and JNK activation (Fredriksson et al., 2011).

Additionally, diclofenac dysregulated NFkB signaling, which likely promoted apoptosis by

interfering with its ability to dampen the apoptotic pathway. In a subsequent study, endoplasmic

reticulum (ER) stress sensors protein kinase RNA-like ER kinase (PERK) and CCAAT-

enhancer-binding protein homologous protein (CHOP) were also involved in the cytotoxic

interaction mediated by TNFα in combination with either diclofenac or carbamazepine

(Fredriksson et al., 2014). Maiuri et al. (2015) found that NSAIDs associated with IDILI

synergize with TNFα to kill HepG2 cells, and IFNγ enhanced this interaction. Interestingly, an

NSAID not associated with IDILI, aspirin, did not synergize with cytokines to kill cells. The

cytotoxic interaction between NSAIDs associated with IDILI and the cytokines TNFα and IFNγ

depended on activation of caspases and MAPKs. An interesting observation was that certain


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NSAIDs were more likely to synergize with IFNγ than others, and this propensity was associated

with degree of IDILI liability and the chemical structure of the NSAID (Maiuri et al., 2015).

As noted above, many of the actions of IFNγ are mediated by intracellular activation of STAT-1.

IFNγ phosphorylates STAT-1 at the tyrosine701 site, but maximal activation of STAT-1 as a

transcription factor necessitates additional phosphorylation at serine727. Studies in HepG2

cells revealed that diclofenac stimulates phosphorylation of IFNγ at serine727. This

phosphorylation is mediated in part via JNK and ERK and explains how IFNγ initiates

enhancement of cytotoxicity from diclofenac-TNFα interaction (Maiuri et al., 2015). In contrast,

the propionic acid derivative ibuprofen, which causes IDILI that is of less clinical concern,

ablated STAT-1 phosphorylation in response to IFNγ. This finding could explain the differential

propensities of certain NSAIDs to synergize with IFNγ to enhance cell death. As mentioned

earlier, diclofenac causes ER stress (Fredriksson et al., 2014). The cytotoxic interaction

between diclofenac and TNFα/IFNγ required availability of intracellular Ca++ which is known to

be released from the ER during ER stress (Maiuri et al., 2016).

These in vitro studies demonstrate that a number of drugs associated with IDILI activate various

stress responses in the cell which ultimately lead to a cytotoxic interaction with TNFα and IFNγ.

For instance, TVX caused DNA damage and induced replication stress leading to cytotoxic

synergy with TNFα (Beggs et al., 2015). Sulindac sulfide caused oxidative stress which led to

synergy with TNFα, and diclofenac caused ER stress resulting in synergy with both TNFα and

IFNγ. Moreover, many of these drugs, irrespective of pharmacological class, led to activation of

the MAPKs JNK and ERK, and this MAPK activation was required for synergy with these two

cytokines. Importantly, although drugs associated with IDILI induce cellular stress via different

mechanisms, in the presence of cytokines these diverse stress responses ultimately culminate


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in persistent MAPK activation which is crucial to the drug/cytokine cytotoxic interaction in

hepatocytes (Fig. 3).

Conclusion

IFNγ and TNFα are produced during innate and adaptive immune responses (Fig. 2); each can

enhance production of the other. Enhanced IFNγ production and interaction with TNFα occurs

and plays a critical role in liver injury that develops in animal models based on different

hypotheses about IDILI etiology. In addition, recent studies in vitro are revealing interaction

between IDILI-associated drugs and TNFα/IFNγ that lead to hepatocyte killing (Fig. 3). These

observations suggest that TNFα and IFNγ, perhaps acting synergistically with each other, could

be critical to the pathogenesis of IDILI, irrespective of the mechanism by which a drug increases

their production. IFNγ probably contributes to IDILI pathogenesis by (1) enhancing the

production of cytokines such as TNFα, (2) promoting death of hepatocytes and/or (3) inhibiting

proliferative repair of liver. It seems likely that one or more biological activities and TNFα act in

concert with direct actions of a drug to precipitate IDILI, but more study is needed to understand

how cytokines interplay with drug exposure in producing adverse outcomes.


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Authorship Contributions

Wrote or contributed to the writing of the manuscript: Roth R.A., Maiuri A.R., Ganey P.E.


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Figure 1. IFNγ-mediated hepatocyte killing by T cells. Both CD8+ T-cells and CD4+ T-cells

as well as NK and other cells produce and release interferon-gamma (IFNγ), which can kill cells

through activation of cell death pathways involving STAT1 and IRF-1 activation (right). This

killing can be enhanced by TNFα and/or by ROS/NO released from inflammatory cells, including

macrophages such as Kupffer cells (Mphage). IFNγ can also kill hepatocytes via autocrine or

paracrine activation of CD8+ T-cells involving pathways initiated by Fas-Fas ligand (FasL)

interaction or by formation of perforin pores that allow granzyme B translocation (left). During

adaptive immune responses, CD8+ T-cells recognize altered hepatocytes via receptors (TCRs),

prompting release of IFNγ. See text for references.

Figure 2. Hypotheses regarding drug-cytokine interactions in IDILI pathogenesis.

Evidence in humans and animals suggests that drugs can be bioactivated to reactive

metabolites that can initiate an adaptive immune response (top right). Alternatively, initiation of

an innate immune response through Toll-like receptor activation (top left) can prompt an

hepatotoxic interaction with drugs. Both of these immune responses result in the generation of

mediators such TNFα and IFNγ. Some drugs also directly increase production of these

cytokines by activated immune cells. At the level of the hepatocyte, IDILI-associated drugs can

enhance hepatocellular sensitivity to the cytotoxic actions of TNFα and IFNγ (bottom center). In

addition, IFNγ and at least some IDILI-associated drugs inhibit hepatocyte proliferation, thereby

impairing the ability of the liver to adapt to drug-induced stress (bottom right).

DAMPs, damage associated molecular pattern molecules. Other abbreviations are

defined in the text.

Figure 3. Cell death signaling in IDILI drug-cytokine interaction. Hypothesized pathway of

IDILI drug/cytokine synergy based on studies in vitro (see text for references). TNFα exposure


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causes early and transient activation of JNK that is innocuous by itself, probably in part due to

activation of the NFkB survival pathway. MAPKs, in particular JNK and ERK, play key roles in

IDILI drug/cytokine-mediated cell death. Some drugs including diclofenac and carbamazepine

induce increased intracellular calcium and ER stress that leads to activation of MAPKs and

synergy with cytokines to cause cell death whereas others such as TVX induce the DNA

damage response pathway which also leads to synergy with TNFα. In the context of early

activation of JNK by TNFα, JNK activation is prolonged by drug exposure, leading to activation

of cell death signaling. Simultaneous disruption by diclofenac of the NFkB-mediated cell

survival pathway amplifies diclofenac/TNFα-mediated cell death signaling. Activation of JNK

also leads to ERK activation, which results in phosphorylation of cytosolic STAT1 at serine727.

Full activation of STAT1 requires additional phosphorylation at tyrosine701, which is effected by

IFNγ exposure. By as yet unknown mechanisms, this amplifies cell killing initiated by

drug/TNFα interaction. Not shown is ER stress-mediated activation of an additional apoptotic

pathway that involves expression of CHOP.


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Robert A. Roth, PhD, Ashley R. Maiuri, PhD, Patricia E ...jpet.aspetjournals.org/content/jpet/early/2016/11/15/jpet.116... · TNFR: TNF receptor TVX: trovafloxacin This article has