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Role of innate and adaptive immunity during drug-induced liver injury
C. David Williams and Hartmut Jaeschke*
Received 23rd May 2012, Accepted 24th August 2012
DOI: 10.1039/c2tx20032e

Drug-induced liver injury (DILI) is a major human health concern and is the most frequent cause of FDA
boxed warnings and the removal of drugs from the market. Idiosyncratic DILI (IDILI) is highly variable
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in its time to onset and no one clear hypothesis exists to explain the mechanism. The general belief is
most cases of IDILI involve some immune mediated component; however no animal models can
recapitulate human IDILI. Despite numerous drugs that have IDILI potential, the most common cause of
DILI is acetaminophen (APAP) overdose. APAP in animal models and in patients is a dose-dependent
hepatotoxicant resulting in severe centrilobular necrosis and a robust inflammatory response. In this
review we will compare the existing hypotheses for potential causes of IDILI and discuss the potential
roles of immune involvement in DILI. Additionally we will focus on what we have learned from the
mechanisms of APAP toxicity ( protein adduction, mitochondrial dysfunction, oxidant stress, DNA
damage, release of damage associated molecular patterns (DAMPs)) and useful interventions to alleviate
APAP-toxicity (reduced protein binding, scavenging of reactive oxygen, induction of autophagy).
Mechanistically APAP-induced liver injury appears to be fundamentally different from IDILI, however,
there are potential critical events shared between APAP-induced liver injury and IDILI. The strategies and
methods currently being used to study APAP-induced liver injury are described in this review. This
improved insight into mechanisms of APAP-induced injury with initiation, propagation and inflammation
may also help to better understand IDILI.

Department of Pharmacology, Toxicology and Therapeutics, University
of Kansas Medical Center, Kansas City, KS, USA.
E-mail: hjaeschke@kumc.edu; Fax: +1 913 588 7501;
Tel: +1 913 588 7969
Introduction
The liver is generally considered a target of drug toxicity
because of its first-pass exposure to orally administered drugs
and its high capacity for xenobiotic metabolism. Phase I (oxi-
dation-reduction) and phase II (conjugation) reactions occur

Dr Clarence David Williams Dr Hartmut Jaeschke is cur-

received his doctorate degree in
toxicology from the University
of Kansas Medical Center and
currently works in the labora-
tory of Dr Jaeschke. Dave has
previously studied, in an indus-
try setting, the mechanisms of
immunogenicity to biologic
therapeutics. Currently his
work focuses on liver injury
and in particular the role of
inflammation in the progression
Clarence David Williams and resolution of injury. The
primary model that Dave focuses on is acetaminophen-induced
hepatotoxicity in both rodents and humans.
rently Professor and Chairman
of the Department of Pharma-
cology, Toxicology and Thera-
peutics at the University of
Kansas Medical Center in
Kansas City. He received a
MSc and PhD degree in bio-
chemistry and toxicology from
the University of Tübingen,
Germany. Since 1988 he held
faculty positions at Baylor
College of Medicine in Hoston,
Hartmut Jaeschke TX, The University of Arkansas
for Medical Sciences in Little Rock and The University of
Arizona in Tucson and was a scientist at the Upjohn Company.
Dr Jaeschke has published more than 270 original manuscripts
and invited reviews and book chapters on mechanisms of liver
pathophysiology and drug hepatotoxicity.

This journal is © The Royal Society of Chemistry 2012 Toxicol. Res., 2012, 1, 161–170 | 161

within hepatocytes to detoxify and facilitate the removal of xeno-
biotics, however in this process toxic metabolites can be gener-
ated and accumulate within hepatocytes. Additionally, many
phase III (transporter) reactions lead to the accumulation of
xenobiotics and their metabolites within the liver. These meta-
bolic capacities, in combination with the liver’s portal blood
supply, and unique immune system make it vulnerable to drug
toxicity.
Despite the relative infrequency of idiosyncratic drug-induced
liver injury (IDILI) it poses a very serious health concern with
potentially fatal consequences. In a retrospective study from
1998 to 2007 it was reported that of all causes of acute liver
failure, APAP was the predominant cause (46%) and DILI from
other drugs accounted for 11%.1 Additionally, the spontaneous
patient survival is quite different between etiologies; APAP
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showed a 65% spontaneous survival rate while the spontaneous
survival rate from other drugs was only 29%.1 In many cases,
IDILI cannot be detected in preclinical testing or clinical trials
because the occurrence is so infrequent that the study number is
insufficient to detect it (Table 1), however guidelines have been
implemented to minimize these risks. If a drug is suspected of
IDILI the decision making process if the drug should be discon-
tinued is generally determined by the frequency of patients that
fall into “Hy’s Law” (Table 2). If a patient meets the three cri-
teria there is a 10–50% chance of patient mortality.2
Drugs with the highest IDILI concern are antineoplastic
agents, NSAIDs, antivirals, antidepressants, and antimicrobials,3
and in the US, antimicrobials account for 46% of IDILI.4 In a
majority of cases (73%) a single prescription was implicated as
the cause of IDILI.5 Clinically, the only effective treatment for
DILI is to stop the administration of the drug and supportive care
(with the exception of N-acetyl-cysteine for APAP overdose and
potentially carnitine for valproic acid).6
There are several reasons to assume IDILI is immune
mediated. The most striking is the time to onset; depending on
the drug it could take months to over one year prior to initiation
Table 1 Frequencies of drug-induced altered liver functiona
Severity Clinical manifestation Frequency rate
Mild <3-fold ULN ALT 1/10 to 1/1000
Moderate Impaired hepatic function 1/100 to 1/10 000
(>3-fold ULN ALT)
Severe Acute liver failure or death 1/10 000 to 1/1 000 000
a
The frequency of IDILI is variable due to failures in reporting and
potential conflicting causes of liver injury (i.e. autoimmune hepatitis,
viral hepatitis and others), however the individual susceptibility for
altered liver function due to a drug is shown. Content adapted with
permission from Robert J. Fontana, MD, University of Michigan.
Table 2 “Hy’s Law”: Determining the potential for serious drug-
induced liver injury
Hepatocellular Injury 3-fold ULN for ALT or AST
Hepatic Impairment 2-fold ULN for total bilirubin
Exclusion of other No evidence of viral hepatitis, confounding
causes drugs, acute liver disease or cholestasis (should
not present elevated serum ALP)
162 | Toxicol. Res., 2012, 1, 161–170
View Article Online
of IDILI which would indicate an adaptive immune response.
Additionally elevated transaminase activities can be observed
with some drugs up to one month following discontinuation of
drug.7 Some IDILI also involves the generation of antidrug anti-
bodies or autoantibodies.8,9
In most cases IDILI is regarded as dose-independent, however
this is most likely an incorrect statement. IDILI is very rarely
seen at doses of <10 mg per day and more than three fourth of
IDILI cases occur when the drug is given at >50 mg per day.10
The notion that IDILI is dose independent probably arises from
the fact that the overwhelming majority of patients taking the
drug are non-responders in regard to toxicity.8 In this review we
will compare and contrast what is known about mechanisms of
APAP-induced liver injury and IDILI.
Why is the liver a target of drug toxicity?
Drug disposition alone cannot explain IDILI because there is no
association with the accumulation of excess drug or metabolite
that would result in concentrations that are toxic to the liver.11
This is in contrast to what is seen in APAP overdose. It is inter-
esting however that deletion of certain drug detoxification
enzymes cause an increased risk for IDILI. Patient with gluta-
thione S-transferase M1 and T1 null mutations were 2.7-fold
more likely to develop IDILI; these odds ratios increased to
3.5-fold for antibacterial and 5.6-fold for NSAIDs.12 Also of
interest in this study was the predominance of women with this
double null genotype as there is generally a female predomi-
nance of IDILI cases.12 IDILI is classified by its bizarre and, as
to date, unexplainable toxicity unlike APAP which is explainable
based on its chemical structure and generation of excess reactive
metabolite.
Liver toxicity is the leading cause of removing drugs from the
market and issuance of boxed warnings.11 There is no one single
genetic, environmental or other factor that can be the sole cause
of IDILI. This is illustrated in the case of flucloxacillin. A
genome wide association study showed patients with the
HLA-B*5701 genotype were at a much higher risk of developing
IDILI to flucloxacillin with an incredibly high odds ratio (OR) of
80.6.13 This genotype was in no means the sole determinant of
DILI, however. Despite this very strong association, only 1 out
of every 500 to 1000 patients treated with flucloxacillin who
have the HLA-B*5701 genotype develop IDILI. The
HLA-B*5701 genotype is also associated with abacavir hyper-
sensitivity.10 This is a clear illustration of the multifactorial
nature of IDILI and certain genotypes and environmental queues
are risk factors rather than causative. This study also demon-
strates a direct link to risk of IDILI with the adaptive immune
system as HLA-B is an MHC class I (MHC-I) molecule that
presents self-antigen to CD8+ T cells. For additional detail of
MHC-related risk factors for IDILI we recommend a current
critical review by Daly and Day.14
The liver is considered a site of immune tolerance, and
perhaps in IDILI this tolerance is lost. Tolerance in the liver has
been demonstrated in several ways.15 Systemic donor-specific T
cell tolerance can be achieved after orthotopic liver transplan-
tation. Introduction of antigen via portal vein can promote toler-
ance. Liver transplantations can be performed between patients
This journal is © The Royal Society of Chemistry 2012

with MHC mismatches. The liver can function as a site for
clonal deletion of T cells and has been referred to as a “sink” for
activated T cells.16,17
Perhaps this tolerance is due in part to altered leukocyte distri-
bution relative to other organs, blood and secondary lymphoid
tissue. In the human liver the ratio of CD4+/CD8+ T cells is
1 : 3.5 but 2 : 1 in peripheral blood.18 Additionally the liver has a
much higher number of cells that blur the line between innate
and adaptive immune response. This includes the ‘unconven-
tional’ γδ T cells with frequency in the liver five-times that
found in peripheral blood,18 natural killer (NK) T cells
(CD3+CD56+) which account from 5% to 25% of liver lympho-
cytes but are rarely seen in blood19 and NK cells which account
for approximately one third of human liver lymphocytes.20
Additionally, between species the frequencies of these cell types
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can differ dramatically with invariant NK T cells being relatively
rare in the human liver.21
In the liver the accumulation of leukocytes occurs in a manner
substantially different from other organs mediated at least in part
by the blood supply and architecture of the liver. The sinusoidal
vasculature is small in diameter, low pressure and fenestrated,
and leukocyte accumulation is not dependent on selectins or
integrins.22,23 It was reported that leukocyte accumulation into
the liver in the endotoxin model was dependent on CD44-hyal-
uronan interaction,24 however this does not appear to be the
mechanism of neutrophil accumulation during APAP overdose
(Williams and Jaeschke, unpublished data). Most leukocytes that
leave the vasculature and enter into the parenchyma do so via the
sinusoids rather than postcapillary venules.25,26
Liver immunity and its role during APAP overdose
The most common model to study drug-induced hepatotoxicity
is APAP. APAP overdose results in centrilobular necrosis that is
not exacerbated by the recruitment of inflammatory cells.27
Despite extensive evidence of the benign role of inflammation in
regard to injury several groups still report that inflammatory cells
can increase injury, and this will be discussed in more detail in
later sections and has previously been reviewed in detail.27 If
biopsy is performed in IDILI patients the mode of cell death
appears to necrosis with inflammatory infiltrates,11 however the
mechanisms of T cell mediated death is generally apoptosis.
From these data it is very difficult to determine the mode of cell
death in IDILI because it is possible that at the time of biopsy
the injury has already degraded into secondary necrosis. Clearly
there are striking differences between IDILI and APAP toxicity.
APAP toxicity is dose dependent with a very rapid onset of
injury (within hours). Therefore the mechanisms of toxicity and
cell death are most likely different between idiosyncratic hepato-
toxic drugs and APAP, but understanding of intracellular events
of APAP toxicity (i.e. formation of reactive metabolite, covalent
binding of cellular proteins, mitochondrial dysfunction, release
of mitochondrial components, nuclear DNA damage) could lead
to more clues regarding susceptibility or outcome of IDILI.
The initiation of APAP toxicity is dependent on the metabolic
conversion of APAP to N-acetyl-p-benzoquinone imine
(NAPQI) in hepatocytes predominantly by CYP2E1 and to a
lesser extent by CYP1A2, CYP2D6 and potentially other P450
This journal is © The Royal Society of Chemistry 2012
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enzymes. The reactive metabolite then adducts cellular proteins;
of particular interest is the adduction of mitochondrial proteins.28
This mitochondrial binding propagates injury by initiating an
oxidant stress that leads to mitochondrial failure, the formation
of ROS and the mitochondrial release of endonucleases.29 These
endonucleases translocate to the nucleus and damage DNA.30
All of these events ultimately lead to cellular oncotic necrosis.31
NK and NK T cells
Despite the liver’s role in immune tolerance, it is also critical
during inflammation and capable of very quick and robust
inflammatory response. Of particular interest in this regard are
the liver NK and NKT cells that have the capacity to become
activated very quickly and produce large amounts of cytokines,
in particular IFN-γ. Examples of this massive inflammatory
response can be seen in hepatitis A and E infections where these
viral vectors are delivered to the liver via the portal vein.32 Inter-
estingly, depletion of Kupffer cells (KCs) results in decreased
NK cell numbers in the liver.21
NK and NK T cells were suspected to cause increased injury
during APAP overdose via IFN-γ production and enhanced neu-
trophil recruitment.33 It was later demonstrated that the DMSO
vehicle in these studies using C57BL/6 mice artificially recruited
and activated these cells types.34 It is known that even between
different strains of mice the surface marker expression of NK
cells is variable.21 It is therefore reasonable to conclude that
function or activation could be different between strains as well,
and it was shown that elimination of NK and NKT cells reduced
APAP-induced injury in IL-13 deficient mice.35 While these cell
types in C57BL/6 mice do not cause additional injury in APAP
overdose they are still suspected as potential participants in other
forms of DILI primarily because these cells function as innate
immune cells but perform these functions in ways similar to the
adaptive immune system. It was shown in a case report of two
patients with IDILI-induced hepatic failure that NK and NKT
cell quantity and expression of the costimulatory factor CD28
was different than control patients.36 Additionally, of potential
importance is the Fas/Fas ligand interaction that can be mediated
by these cells on hepatocytes.27 Generally, hepatocytes are resist-
ant to perforin/granzyme-induced cell death which means T cell
mediated killing could involve Fas/FasL interaction or TNF-α.37
Neutrophils
Neutrophils can occasionally be seen in normal, healthy liver,
however upon inflammatory insult these cells are rapidly
recruited into the liver and the number of total hepatic neutro-
phils can be increased several orders of magnitude.38 If proper
stimuli are present, these cells will extravasate from the vascula-
ture into the hepatic parenchyma and cause injury.39 Without
these signals the neutrophils will remain in the sinusoids,
undergo apoptosis and be cleared by KCs. Normally the half-life
of a neutrophil is 6–12 h but during active inflammation this can
be increased to 48 h.40
It has been extensively demonstrated that neutrophils do not
participate in APAP induced injury,27 however, some controversy
still exists. A neutropenia-inducing antibody results in protection
Toxicol. Res., 2012, 1, 161–170 | 163

against APAP toxicity but only if given 24 h prior to APAP,41–43
but not if given after APAP overdose despite functional inacti-
vation of neutrophils.44–46 Also, neutrophils show no enhanced
activation status during the APAP-induced injury phase,47
priming and activating neutrophils during APAP overdose by
IL-1β or endotoxin does not increase injury,47,48 CD18-47 and
ICAM-1-deficient mice44 are not protected, an anti-CD 18 anti-
body did not affect injury,49 and mice that have inhibited
NADPH oxidase or lack NADPH oxidase ( phagocytic respirat-
ory burst) have no difference in oxidant stress or APAP-induced
injury.44,50 Often times neutrophils are thought to be the cause of
enhanced injury because in genetic or pharmacologic interven-
tions mice with reduced injury will have lower hepatic neutrophil
counts or reduced myeloperoxidase staining,43,51 however, this is
most like due to the fact that mice with less injury, release less
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DAMPs, and therefore recruit fewer neutrophils. In simple
terms, blocking upstream events (injury) will reduce downstream
events (inflammation). A caveat of most immunological inter-
ventions is these studies often times do not assess drug meta-
bolism, protein binding or other intracellular events. Without
these control experiments results are difficult if not impossible to
interpret and a variety of off-target effects could occur. This
should be a particular concern when the data contradict many
published findings.
Most theories and models of IDILI do not have neutrophils
being a major contributor to injury with a few exceptions. If neu-
trophils are participating in IDILI then it would most likely
occur as a secondary effect to damage caused by an adaptive
immune response. One exception is the case of halothane.
Halothane was used clinically as an inhaled anesthetic, but
patients upon reexposure had an increased risk of potentially
fatal hepatotoxicity.9 Halothane is converted to the reactive
metabolite trifluoroacetic acid which adducts hepatic protein and
initiates liver injury.9 It was shown that depletion of neutrophils
in this model could attenuate injury.52 It was later demonstrated
that NK T cells were critical for the recruitment of neutrophils in
this model.53
Kupffer cells (KCs)
Administration of LPS or other inflammatory insults results in
massive cytokine formation and accumulation of leukocytes in
the liver. KCs are instrumental in the removal of gut-derived
endotoxin and the phagocytosis and killing of bacteria. Within
minutes of the inflammatory stimuli KCs produce numerous
cytokines, chemokines and subsequently stimulate hepatocytes
( predominantly through IL-6) to produce acute phase proteins
which include pentraxins, protease inhibitors, coagulation
factors, complement components and others.37 Interestingly,
inactivation of KCs using gadolinium chloride (GdCl3) prevents
portal vein tolerance.54 This was determined by the adminis-
tration of alloantigen (spleen cells from different mouse strains)
with or without GdCl3 treatment and measurement of delayed
hypersensitivity reaction by foot swelling.54 In another model of
T cell mediated delayed hypersensitivity it was shown that KCs
were involved in the induction of tolerance for protein adducts
or haptens.55 This was determined by sensitization of mice to
2,4-dinitrochlorobenzene (DCNB) with or without KC depletion
164 | Toxicol. Res., 2012, 1, 161–170
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via liposome/clodronate. Additionally this tolerance could be
induced upon adoptive transfer of KCs from tolerized mice.55
KCs are also important in attenuating this inflammatory response
by producing high levels of IL-10.37
During APAP overdose distinct macrophage populations can
be observed during the injury phase and injury resolution. These
different populations have unique phenotypes which can be
determined by surface markers. Kupffer cells are often referred
to as M1 (classically activated) macrophages are typically
thought of as pro-inflammatory and drive cells toward a Th1
phenotype through high IL-12 production; traditionally these
cells are activated by IFN-γ or LPS. M2 (alternatively activated)
are typically anti-inflammatory, activated by IL-4 or IL-13,
highly phagocytic and promote tissue repair.56 During injury
resolution monocytes are recruited into the liver and become
M2-biased. Depletion of KCs prior to APAP overdose actually
results in increased injury which is mediated by the loss of IL-10
production.57,58 Also of importance during APAP overdose is
IL-6 which is also hepatoprotective and generally considered
pro-regenerative.59 IDILI most likely involves the loss of
immune tolerance. Alteration of the cytokine production profile
could cause of a shift away from immune tolerance which would
most likely begin with the resident tissue macrophages of the
liver.
Dendritic cells
As stated previously, KCs produce fairly high levels of IL-10
which is capable of altering the function of the liver’s other
antigen presenting cell type, dendritic cells (DCs). The liver con-
tains both plasmacytoid and myeloid DCs which are most com-
monly found within portal tracts.32 High levels of IL-10 tend to
drive DCs away from effector pathways, and DCs of the liver are
generally immature and express low levels of co-stimulatory
molecules, which tend to produce regulatory T cells.37
It was reported that dendritic cell depletion results in enhanced
APAP-induced injury.60 DC depletion was induced in CD11c.
DTR mice via diphtheria toxin prior to APAP; this resulted in
enhanced inflammatory cytokine and chemokine profiles and
increased liver injury. Conversely, expansion of DC populations
via Fms-related tyrosine kinase 3 ligand (Flt3L) decreased
APAP-induced injury. Additionally, neutrophil or NK cell
depletion in addition to DC depletion did not modulate the
injury further confirming these innate immune cells do not con-
tribute to APAP-induced injury.60 The immature phenotype of
DCs in the liver almost certainly participates in the maintenance
of tolerance and loss of tolerance could induce IDILI. It is not
clear if loss of tolerance would be mediated by DCs directly or
modulation of the microenvironment (i.e. exposure to cytokines
and stimulatory factors).
Nalp3 inflammasome
The NACHT, LRR and PYD domains containing protein 3
(Nalp3) inflammasome is assembled after the initiation of sterile
inflammation and is responsible for the maturation of IL-1β and
IL-18 through activation of caspase-1.61 It was reported that
mice deficient for each component of the Nalp3 are protected
This journal is © The Royal Society of Chemistry 2012

from APAP-induced injury62 and ATP released during early
injury signals through the P2X7 purinergic receptor to activate
the Nalp3 inflammasome.63 These studies were repeated and no
protection was observed in mice deficient for each component of
the Nalp3 inflammasome.64 Additionally, inhibiting caspase-1
prevented IL-1β maturation without altering liver injury, and
pharmacologic addition of recombinant IL-1β during APAP
overdose did not modulate liver injury.48 In addition, the puriner-
gic receptor antagonist can directly act on hepatocytes and
inhibit protein adduct formation, oxidant stress and c-Jun N-
terminal kinase (JNK) activation (Williams and Jaeschke, unpub-
lished data). These data suggest that this compound alters the
mechanisms of intracellular cell death, which occur upstream
and independently of the Nalp3 inflammasome. From these data
it is highly unlikely that the Nalp3 inflammasome contributes to
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APAP-induced injury but its role in IDILI is yet to be deter-
mined. This is just one of many examples where data from
immunological or pharmacological interventions are misinter-
preted because effects of these agents on intracellular signaling
events are not considered.
Major theories of IDILI
There are a number of hypotheses regarding the cause of IDILI.
Most likely no one hypothesis alone can explain the mechanism
of all cases of IDILI and several theories might occur sequen-
tially or concurrently to explain the mechanism. As described
previously IDILI most likely involves an adaptive immune
response which is fundamentally different from what is observed
in APAP-induced injury. The accepted dogma of adaptive
immune activation requires presentation of antigen loaded on an
MHC molecule to the T cell receptor (TCR) of a T cell.
Additionally, a second costimulatory signal is required which
could be a B7 molecule (CD80 or CD86) on antigen presenting
cell (APC) interacting with CD28 on the T cell. When both of
these signals occur an immune response is mounted.65 Without
this second costimulatory signal the T cell becomes anergic for
the maintenance of immune tolerance. This central dogma of
immunology gives rise to the “danger hypothesis”.65 APCs
present antigen to CD4+ T cells via MHC class II (MHC-II); all
other cells present antigen to CD8+ T cells via MHC class I
(MHC-I). Classically this means exogenous antigens are pres-
ented via the MHC-II and endogenous antigens are presented via
MHC-I. There are exceptions to this rule however with the most
predominant being that of “cross-presentation”.66 In this process
antigens from the extracellular environment can be processed,
loaded into MHC-I and presented to CD8+ T cells.
“Danger hypothesis in IDILI”
Due to the environment and diet we are constantly exposed to
countless exogenous substances through multiple routes of deli-
very yet mount immune response to very few of these things.
For this reason the “danger hypothesis” was proposed.8,9 It is
believed that the immune system responds to “danger” (i.e. cell
injury) rather than specifically to “non-self”.64 In this theory
necrotic cell death is referred to as “bad death” and only bad
death triggers immune response mediated by the co-stimulatory
This journal is © The Royal Society of Chemistry 2012
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factors described previously. Additionally, this theory proposes
that only professional APCs (i.e. DCs) can prime naïve T cells
where subsequent activation can be performed by other APCs
(macrophages and B cells). Effector T cells that are presented
antigen without the required co-stimulatory factors will become
senescent. Initially, it is thought that cells dying by apoptosis are
non-inflammatory and therefore tolerogenic and necrotic cells
are pro-inflammatory and therefore immunogenic, however it has
been shown that this is not always the case. Antigens from apop-
totic cells can be cross-presented to CD8+ T cells and prime an
immune response, and cells that die at the peak of an immune
response can be immunogenic but cells dying as inflammation
wanes can induce tolerance.67 It was also shown that DCs that
phagocytosed necrotic cells debris presented antigen to both
CD4+ and CD8+ T cells, but DCs that phagocytosed apoptotic
cells only presented antigen to CD8+ T cells.68
The first step of the danger hypothesis involves some initial
cell injury or at the very least some cell stress to cause the initial
danger signal. Potentially these danger signals could be cyto-
kines released by innate immune cells or DAMPs released by
the injured cells. High mobility group box-1 (HMGB-1) protein
currently is one of the most commonly studied and measured
DAMPs. APAP causes the release of HMGB-1.64,69 In agree-
ment with one of Matzinger’s original hypotheses, oxidation of
HMGB-1 which occurs in apoptotic but not necrotic cells was
critical to neutralizing its stimulatory activity and blocking oxi-
dation sites prevented tolerance.70 Potentially the oxidation state
of HMGB-1 could be of prognostic value during drug-induced
liver injury.71
“Hapten hypothesis”
A hapten is a small molecule (typically incapable of initiating an
immune response) that covalently binds to a larger molecule
(cellular protein) and becomes immunogenic. This theory of
IDILI involves the concept of a drug or more-likely the drug’s
reactive metabolite binding to a cellular protein and triggering
the initiation of an immune response. This is a rational theory
because of the high expression of CYP450’s (drug metabolizing
enzymes) and drug transporters found in the liver, therefore most
of these reactive metabolites are present within the liver as is the
case with the conversion of APAP to the reactive metabolite
NAPQI. Adduction of cellular proteins by the reactive meta-
bolite then can initiate an adaptive immune response following
antigen processing and MHC presentation; this new immuno-
logical entity is referred to as a hapten. The haptenized protein
can elicit a T cell response to the drug, the protein itself or a
mixed response can occur depending on the antigen presen-
tation.9,72 If the drug metabolite adducts a cellular protein and
results in cell death then these aberrant antigens during an
inflammatory response (increase costimulatory factors) could be
presented by APCs and trigger a CD4+ response, so this theory
generally involves the danger hypothesis to propagate the injury.
Evidence for the pathogenic role of haptens can be seen with
drugs like tienilic acid, dihydralazine, halothane, phenytoin, car-
bamazepine and others.9 In these cases, common targets of
haptenation are the enzymes responsible for metabolism of the
drug as well as other liver-specific proteins.73 It has been
Toxicol. Res., 2012, 1, 161–170 | 165

demonstrated that these antibodies or autoantibodies can react
with P450 enzymes (CYP2D6, CYP2C9, CYP1A2, and others)
and UDP glucuronyltransferases.74,75 It is unclear if these anti-
bodies exert a direct toxicity. However, it does clearly demon-
strate that immune cell activation has occurred, and in the case
of autoantibodies immune tolerance has been compromised.8,9
An argument against the hapten hypothesis is some IDILI
drugs do not form reactive metabolites to covalently bind pro-
teins.8,76 Additionally, it should be noted, that covalent binding
should be regarded as a bioactivation event and not one of overt
toxicity, and conversely not all drugs must be metabolized to
trigger an adverse drug reaction.77 Additionally, the efficacy of
some drugs is dependent on covalent binding like aspirin and
proton-pump inhibitors.78
Not all drugs have to form a reactive metabolite to cause
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IDILI and generation of reactive metabolite does not mean an
immune response will be generated even in the presence of sub-
stantial injury and inflammation which is the case with APAP. It
was hypothesized that APAP toxicity creates a tolerance to
APAP protein adducts.79 In a mouse model of APAP overdose
lymphocyte apoptosis could be seen in the spleen, thymus and
liver-draining lymph nodes, and APAP overdose also suppressed
the hypersensitivity response of DNCB.79 This theory might be
a mechanism of how progression of IDILI is avoided in most
people with minor or subclinical drug toxicity.
“Failure to adapt hypothesis”
Another concept with IDILI is the notion of a lack of adaptation
to a toxic insult in which injury caused by the drug or metabolite
triggers a response or repair mechanism. This injury could be
initiated in the mitochondria (as discussed later), in the endoplas-
mic reticulum as unfolded protein response or other cellular
stressors.
Occasionally, patients on a drug with known IDILI potential
will present with serum ALT >3 times upper limit of normal
(ULN) but return to baseline levels even when continued on the
drug.9 This concept is known as adaption. Why will some
patients return to normal and other progress to severe liver
injury? The response mechanisms could involve phase I, II or III
metabolic processes; the induction of anti-oxidant responses,
enhanced autophagy, enhanced cell proliferation or other protec-
tive measures. On the other hand, this could be indicative of the
development of immune tolerance. It is possible the loss of toler-
ance could be mediated by KCs. This could occur by generation
of reactive metabolite within the KCs9 or cross-presentation of
antigen. The mode of this initial cell death could also be impor-
tant. It has been shown that caspases or other cellular proteases
upon activation may create new antigenic epitopes within the
cell.67
“Pharmacologic interaction (P-I) hypothesis”
This theory involves the direct binding of drug to TCR and T
cell activation upon MHC interaction with an APC, or the drug
non-covalently interacts with MHC and then presents it to a
responsive T cell.80 This concept can explain why drugs that do
not form reactive metabolites can initiate IDILI, however this
hypothesis is fundamentally different from the danger hypothesis
166 | Toxicol. Res., 2012, 1, 161–170
View Article Online
because T cell activation in this hypothesis does not require co-
stimulatory factors. This theory was first used to describe the
IDILI potential of sulfamethoxazole. In this study T cell clones
could be activated by drug even after the fixation of APCs indi-
cating antigen processing was not required.81 While this theory
appears to be plausible for some drugs it does not apply for all
IDILI drugs. It has been shown that the P-I hypothesis can be
applied to other drugs like carbamazepine, lamotrigine, lidocaine
and others;80 however, these drugs also can produce reactive
metabolites, so perhaps P-I hypothesis occurs in concert with
another model of IDILI.
“Mitochondria hypothesis” (SOD2+/− model)
This hypothesis involves a subclinical mitochondrial dysfunction
that gradually accumulates until a critical threshold is met at
which time liver injury rapidly occurs.82 Mitochondria lack
DNA repair mechanisms, and cumulative damage over time
might induce toxicity. Typically speaking DNA damage is
associated with hard electrophiles which have a high positive
charge density; these electrophiles are also more likely to react
with amino acid residues like lysine. Soft electrophiles (i.e.
NAPQI) generally do not attack nucleic acids and interact with
amino acids like cysteine and are predominantly detoxified by
GSH.77,83 However during mitochondrial dysfunction ROS gen-
erated within the mitochondria is the most likely cause of mito-
chondrial failure. Mitochondrial DNA damage is a late event
(after drug metabolism) which can be prevented by scavenging
ROS in the mitochondria.84
The inner membrane of mitochondria contains high levels of
cardiolipin which has many unsaturated bonds potentially
making in vulnerable to lipid peroxidation.82 This would be an
organelle-specific toxicity potentially leading to impaired mito-
chondrial respiration and damage as previously described. It is
unknown if lipid peroxidation (on a cellular level) is a relevant
mechanism of cell death in IDILI, but it is not a relevant general
mechanism during APAP overdose.85,86
Sod2-heterozyous mice are more susceptible to mitochondrial
injury, and SOD is critical for the detoxification of superoxide to
prevent peroxynitrite formation.87–89 It was shown that in
Sod2+/− mice troglitazone caused delayed hepatic injury.90 This
injury is consistent with a delay in toxicity seen in patients,
however these findings could not be repeated in a subsequent
study using a very similar model.87 Additionally, long-term
administration of flutamide resulted in oxidant stress, mitochon-
drial dysfunction and hepatic injury in Sod2+/− mice.91 Clini-
cally it has been shown that patients with SOD2 Val16Ala
mutation are prone to mixed cholestatic injury (OR = 2.3) and
patients homozygous for this mutation with this type of injury
are more susceptible to drugs with known mitochondrial hazards
(OR = 3.6).92
If a mitochondrial threshold effect does occur and causes toxi-
city this will not explain why patients with idiosyncratic toxici-
ties are susceptible to toxicity upon re-challenge. Most likely the
mitochondria would be an initiating event in IDILI that would
then proceed to an immune response to propagate the injury.
Thus, the Sod2+/− model could be useful to evaluate the poten-
tial of drugs to cause a mitochondrial stress but does not mimic
the entire pathophysiology of IDILI.
This journal is © The Royal Society of Chemistry 2012

“Inflammagen model”
This model uses drugs with IDILI potential and co-treats
animals with LPS. The assumption behind this approach is that
the hepatotoxicity of idiosyncratic drugs is not detectable
because of interfering events (death of animals). In addition an
inflammagen like LPS causes a leftward shift of the dose-
response curve and thus makes the toxicity detectable. The goal
of this model is to induce hepatotoxicity in a large percentage of
treated animals which will allow for better statistical analysis and
provide an economical way to test for IDILI.93 The inflammagen
model has been used for drugs like amiodarone, chlorpromazine,
diclofenac, trovafloxacin, sulindac and others.93 In these models
the dosing of drug and LPS was highly variable; occasionally
LPS was given prior to drug, sometimes post-drug, sometimes
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drug was given multiple times.93 In these studies the liver injury
ranged from moderate to severe liver necrosis with a relatively
rapid onset.
However, there are concerns regarding the relevance of this
model. First, the generally used dose of endotoxin (44 × 106 EU
kg−1 or ∼4 mg kg−1) results in plasma levels approximately five
orders of magnitude higher than the maximum serum concen-
tration measured in septic patients with confirmed Gram-negative
infection.94 And, sepsis has not been identified to precede or
accompany the onset of IDILI. Second, the mechanism of liver
injury in the inflammagen model appears to always involve
TNF-α, neutrophils and the coagulation cascade93 independent
of the drug. Thus it is likely that the model mimics LPS-induced
liver injury where a subtoxic dose of LPS causes neutrophil-
mediated liver injury when additional stress (exposure to drug) is
applied.
Support of these concerns comes from the application of the
inflammagen model to APAP toxicity. Pretreatment with a high
dose of LPS before a subtoxic dose of APAP caused severe but
delayed liver injury.95 The interpretation was that LPS caused a
leftward shift of toxicity implying the same mechanism of
toxicity just with a lower dose of APAP in the presence of LPS
pretreatment.95 However the time course of toxicity with LPS
suggests a different mechanism. Pretreatment with LPS will
cause the recruitment of primed neutrophils into the liver vascu-
lature.25 The subtoxic dose of APAP still causes GSH depletion
and protein adduct formation (McGill and Jaeschke, unpublished
data), which in the centrilobular area is likely a sufficient stress
signal for the neutrophils to extravaste and attack39 resulting in a
neutrophil-mediated liver injury. Thus, a high dose of APAP and
low dose of APAP with LPS pretreatment have fundamentally
different mechanisms of liver injury.
Role of autophagy in DILI
As mentioned previously autophagy could play a major role in
the adaptation process to prevent the progression of IDILI. It is
becoming more apparent that autophagy is critical for various
functions in both the innate and adaptive immune response.
Current investigations involve the modulation of autophagy in
vaccine efficacy and chronic inflammatory disease. Dysfunction
or altered regulation of autophagy can lead to loss of immune
tolerance, which is most likely a critical component to the induc-
tion and progression of IDILI. Autophagy has a multitude of
This journal is © The Royal Society of Chemistry 2012
View Article Online
immune functions.96,97 These functions include digestion and
removal of intracellular pathogens, promotion of Th1-Th2 polar-
ization during intracellular infection, modulation of TLR
response and inflammation via pathogen associated molecular
pattern (PAMP) delivery, presentation of endogenous peptides to
MHC-II molecules, regulation of T cell homeostasis, and
immune tolerance. Autophagy can result in the “non-classical”
MHC class II presentation of autophagosomal peptides98 and
self-antigen presentation on MHC-II by DCs might be respon-
sible for CD4+ mediated peripheral tolerance and immature den-
dritic cells (like those observed in the liver) have a high rate of
autophagy and are considered pro-tolerogenic.96 Following
pattern recognition receptor signaling, autophagy is enhanced
thereby promoting non-classical MHC-II presentation.97 Interest-
ingly autophagy has been shown to be critical for type I inter-
feron production, however, loss of autophagy can also up-
regulate several pro-inflammatory cytokines and generation of
ROS in macrophages.99 Autophagy is also critical in T cell
homeostasis and activation. It has been shown that autophagy is
induced during T-cell activation and it has been reported that it
is required for clonal expansion.100,101 In this regard, autophagy
promotes T cell expansion. Conversely, if autophagy is blocked
the immune synapse (MHC-TCR interaction) can become hyper-
stablized. Ultimately this leads to enhanced T cell activation.102
In addition to the potential implications of autophagy within
the immune system autophagy within the liver could be, equally,
if not more important. Autophagy of damaged mitochondria or
other cellular organelles could be critical to limit oxidant stress
or ER stress as was previously described in the “failure to adapt”
section. The role of autophagy and DILI has only very recently
been evaluated.
Just this year several papers have shown the role of autophagy
during APAP overdose however caution must be used when
using these models as we will discuss in more detail. The
primary hypothesis regarding the role of autophagy after APAP
is that of hepatoprotection which is not surprising since auto-
phagy is normally considered a cell survival pathway. Following
overdose, APAP adducts cellular proteins potentially making
some of them non-functional; of particular interest is the adduc-
tion of mitochondrial proteins ultimately resulting in mitochon-
drial dysfunction. As some mitochondria begin to fail an oxidant
stress is generated, mitochondrial membrane potential is lost,
and mitochondria swell then rupture releasing endonucleases;
the combination of these events ultimately lead to cell death by
oncotic necrosis.29,86,89 During this process there must be some
critical threshold for mitochondrial loss that determines cell fate;
the most centrilobular hepatocytes which have the highest P450
enzyme activities and lowest GSH levels presumably have the
highest reactive metabolite burden and therefore are the most
susceptible. Depending on the dose, the centrilobular injury can
expand and potentially invade midzonal or even periportal areas,
however the injury always begins with the most centrilobular
hepatocytes. The hepatocytes at the threshold between necrotic
and healthy tissue are the cells where autophagy plays a role.
These cells remove damaged mitochondria and adducted pro-
teins thereby preventing lethal oxidant stress and the release of
endonucleases allowing cell survival.
The first paper investigating the role of autophagy during
APAP overdose demonstrated that APAP does in fact activate
Toxicol. Res., 2012, 1, 161–170 | 167

autophagy and this activation is protective.103 This was deter-
mined by using both inhibitors (3-methyladenine or chloroquine)
and activators (rapamycin) of autophagy in vivo and in primary
cultured mouse hepatocytes. The predominant mechanism of
protection appears to be mitophagy (autophagy of mitochondria)
which is then capable of reducing APAP-induced injury.
Most recently it was shown that mice deficient for Atg5 in the
liver were actually resistant to APAP toxicity.104 This finding is
contradictory to the dogma that autophagy is protective, but it
was determined that the protection from APAP toxicity was due
to the aberrant phenotype seen in these mice. Atg5 was deleted
from hepatocytes under the albumin-Cre promoter. The Atg5
liver knockout mice develop mild liver injury resulting in
increased apoptosis and compensatory proliferation. Additionally
the liver becomes preconditioned and Nrf2 activation increases
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the expression of target genes which increases basal GSH levels,
and therefore these mice are protected by compensatory effects
rather than by loss of Atg5. Clinically the induction of Nrf-2
could be indicative of a “danger signal” because it is activated
by oxidant stress or the induction of autophagy.
Recently, another study has demonstrated that loss of auto-
phagy can promote APAP toxicity. In this study Atg7 conditional
knockout animals were generated under Mx1-Cre by treating
mice with poly I:C.105 The poly I:C treatment is capable of mo-
dulating the metabolic activation of APAP, and this study shows
a mechanism of cell death in the Atg7-deficient mice quite
different from control mice, however. The Atg7-deficient mice
have caspase processing and apoptosis. It has previously been
reported that the poly I:C treatment in Atg7-Mx1-Cre mice
results in hepatomegaly and liver injury.106 The abnormal pheno-
type of these mice is a major concern, therefore, care must be
used when interpreting data from these types of models.107
The role of autophagy and DILI is now beginning to be esta-
blished. Potentially, autophagy might be critical in the clearance
of adducted proteins, removal of damaged organelles or modu-
lation of immune tolerance (Fig. 1).
Conclusions
During APAP overdose there are several critical stages which
can be defined as initiation and propagation. The initiating event
is reactive metabolite formation resulting in protein adduction.
The injury is then propagated by mitochondrial dysfunction and
ROS production. In APAP overdose, both of these events occur
independently of immune cell infiltration27 and depletion of resi-
dent immune cells (KCs and DCs) actually causes increased
injury.57,60,108 A different situation most likely occurs in IDILI,
but this toxicity probably involves initiation and propagation
stages as well (Fig. 1). The initiation stage likely involves the
generation of reactive metabolites or the parent drug impairs
some cell function (mitochondrial respiration or autophagy); this
could lead to formation of a hapten or trigger mild liver injury.
The propagation stage most likely involves an immune com-
ponent which then greatly enhances the liver injury. In theory the
generation of reactive metabolites or location of parent drug will
determine the type of immune response by the antigenic presen-
tation via MHC-I or MHC-II. If the reactive metabolite is gener-
ated within the cell and then presented via MHC-I this will lead to
168 | Toxicol. Res., 2012, 1, 161–170
View Article Online
Fig. 1 There are multiple hypotheses regarding the initiation and
propagation of IDILI. This illustration depicts how several of these
events could occur and potential key events that could involve auto-
phagy. Autophagy could clear adducted or nonfunctional proteins or
could remove damaged organelles. If these processes are insufficient
drug or metabolite could cause cell stress or death within the hepatocyte.
This could then trigger an immune response and activation. This acti-
vation could then trigger various downstream effector functions in
additional immune cells. Propagation of injury can then occur through
these adaptive or innate immune responses. Autophagy could potentially
attenuate these responses through the modulation of TLR/ligand inter-
action or destabilization of the immune synapse. These roles of auto-
phagy during IDILI are speculative however convincing evidence of the
participation of autophagy during APAP overdose has been shown. The
figure is a modification of the concepts previously described.72
CD8+ T cell recognition; however in absence of co-stimulatory
factors this should result in immune tolerance. If the reactive
metabolite is released and then phagocytosed by an APC, these
antigens will be presented to CD4+ T cells via MHC-II. These
helper T cells can then lead to B cell or CTL activation.72,109
Clearly, there will be no easy answer to explain the cause of
IDILI. As evidenced by genetic polymorphisms there are clearly
risk factors involved with the development of IDILI but genetics
alone do not explain the cause. No better example illustrates this
point than the HLA-B*5701 genotype with flucloxacillin, which
greatly increases the risk but is by no means the determinant of
DILI.14 There are multiple factors involved like dose of drug,
gender, age, alcohol or concomitant drug use, underlying disease
state, environmental exposures, and potentially other as yet un-
identified risk factors.110,111 Additional risk could be attributed
to enhanced production or impaired detoxification of oxidant
stress, or altered mitochondrial respiration due to inherited or
acquired mitochondrial dysfunction.112 Because of these com-
plexities most likely not all IDILI will share the same mechan-
ism of toxicity. Therefore, uncovering new risk factors for
patients and trying to develop animal models of IDILI is critical
to reduce this human health concern.
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