Damage‐associated molecular patterns: Their impact on the liver and beyond during acetaminophen overdose


Damage-Associated Molecular Patterns: Their
Impact on the Liver and Beyond During

Acetaminophen Overdose

See Article on Page 1971

A
cetaminophen (APAP) is one of the most
commonly used drugs for treating pain and
fever. Although it is safe at therapeutic dosage

levels, overdose of APAP causes severe liver injury (APAP-
induced liver injury; AILI), with the potential to progress
to liver failure. Up to 40% of patients who suffer from
liver failure will die (or undergo liver transplant).1 Data
combined from 22 specialty medical centers in the
United States revealed that AILI accounts for approxi-
mately half of acute liver failure cases and results in more
than 56,000 emergency room visits, 2,600 hospitaliza-
tions, and an estimated 458 deaths each year.1

APAP hepatotoxicity is initiated by the generation
of a chemically reactive metabolite (N-acetyl-p-benzo-
quinone imine; NAPQI). NAPQI depletes liver gluta-
thione and covalently binds to cellular proteins,
thereby causing mitochondrial dysfunction.2-6 Studies
using rodent models in the last four decades have
demonstrated that mitochondrial disruption is the key
underlying mechanism of AILI (Fig. 1FIG1)). Cova-
lent binding to mitrochondrial proteins by NAPQI
causes oxidative stress and mitochondrial membrane
permeability transition pore opening, which triggers
the collapse of membrane potential, cessation of aden-
osine triphosphate (ATP) production, and the release
of apoptosis-inducing factor and endonuclease G.7-11

Together, the mitochondrial dysfunction, energy crisis,
and nuclear DNA damage result in hepatocyte
necrosis.
In recent years, there has been a growing interest to

investigate whether downstream events of early hepato-
cyte necrosis contribute to the aggravation and pro-

gression of AILI. Necrotic cells release a number of
damage-associated molecular pattern (DAMP) mole-
cules, such as high-mobility group box-1, heat-shock
proteins, hyaluronan, fibronectin, cardiolipin, and
DNA fragments. Upon activation by DAMP mole-
cules, innate immune cells infiltrate the damaged area
and release cytokines and cheomokines, thereby caus-
ing tissue sterile inflammation.12-22 The soluble prod-
ucts of innate immune cells can exacerbate tissue dam-
age, as well as promote wound healing (Fig. 1).
Perhaps as a result of this dichotomy, the overall con-
tribution of innate immune cells, such as neutrophils,
to AILI remains unclear.
In this issue of HEPATOLOGY, Marques et al.23 report

on a study demonstrating that DAMP molecules
released from necrotic hepatocytes recruit and activate
neutrophils in the liver, which, in turn, amplify AILI.
Three experimental approaches were employed to elu-
cidate the pathological role of neutrophils in AILI.
Consistent with published reports,24,25 the present
study shows that neutrophil depletion by an anti-Gr-1
antibody significantly attenuates AILI. Furthermore,
the combined use of a CXC chemokine receptor 2 an-
tagonist and a formyl peptide receptor 1 (FPR1) an-
tagonist also blocks hepatic recruitment of neutrophils
and mitigates AILI. This approach is based on the
investigators’ previous finding that an intravascular gra-
dient of chemokines and mitochondria-derived formyl
peptides collaboratively guide neutrophils to sites of
liver necrosis.26 These two separate in vivo studies
demonstrate that liver injury initiated by APAP chal-
lenge is amplified by infiltrating neutrophils. The
investigators further examined the cytotoxic potential
of neutrophils against hepatocytes. Neutrophils isolated
from healthy individuals were cocultured with APAP-
treated HepG2 cells. Data show that the cytotoxicity
of HepG2 cells is enhanced by neutrophils in a cell-
contact–dependent manner, and that necrotic HepG2
cells significantly increase reactive oxygen species pro-
duction by netrophils. Collectively, these findings pro-
vide evidence to support a pathological role of neutro-
phils during AILI.
Traumatic injury is known to cause ‘‘septic-like’’ sys-

temic inflammatory response in the absence of infec-
tion.27 The underlying mechanism is recently

Abbreviations: AILI, APAP-induced liver injury; APAP, acetaminophen;
ATP, adenosine triphosphate; DAMP, damage-associated molecular pattern;
FPR1, formyl peptide receptor 1; IL, interleukin; mtDNA, mitrochondrial
DNA; NAPQI, N-acetyl-p-benzoquinone imine; TLR-9, Toll-like receptor 9.
Address reprint requests to: Cynthia Ju, Ph.D., School of Pharmacy,

University of Colorado Health Sciences Center, 4200 East 9th Avenue, C238,
Denver, CO 80246. E-mail: Cynthia.ju@uchsc.edu; fax: 303-724-7266.
Copyright VC 2012 by the American Association for the Study of Liver Diseases.
View this article online at wileyonlinelibrary.com.
DOI 10.1002/hep.25920

Potential conflict of interest: Nothing to report.

1599



elucidated by the detection of mitrochondrial DNA
(mtDNA) and formyl peptides released in the serum
of trauma patients.28 Circulating mitochondrial
DAMPs activate neutrophils through Toll-like receptor
9 (TLR-9) and FPR1, respectively, thereby eliciting
neutrophil-mediated organ injury.28 Because APAP
causes mitochrondria damage, it is likely that mito-
chondrial contents are released into the circulation.
Evidence supporting this hypothesis is provided by a
recent study detecting circulating mitochondrial bio-
markers, including mtDNA and glutamate dehydro-
genase, in the serum of patients with AILI.29 Similarly,
the present study shows a significant increase in serum
mtDNA levels in acute liver failure patients, compared
to healthy volunteers. The elevation of circulating
mtDNA is also observed in APAP-treated mice. The
effect of mtDNA release on AILI is revealed by a sig-
nificant decrease of liver injury in TLR-9�/� mice,
compared to wild-type mice. This finding is consistent
with a published report of protection against AILI by
TLR-9 antagonists and in TLR-9�/� mice.30

APAP-induced liver failure is accompanied by other
tissue complications, such as encephalopathy, coagul-

opathy, renal failure, metabolic derangements, cardio-
vascular compromises, and severe lung injury.31,32

Toxic effects on remote tissues may be the result of the
original insult of APAP, but, in many cases, are conse-
quences of severe liver injury. A novel finding of the
present study is the observation of marked lung injury
in mice treated with APAP. Data suggest that lung
injury is the result of the systemic release of mitochon-
drial DAMPs, because blockade of FPR1 or deletion
of TLR-9 significantly reduced lung injury. From their
results, the investigators summarize a mechanistic
model for APAP-induced liver damage and lung
inflammation. APAP-initiated hepatocyte necrosis
causes the release of mitochondrial DAMPs and che-
mokines, which lead to hepatic recruitment of neutro-
phils that amplify liver damage. The release of mito-
chondrial DAMPs also triggers systemic inflammation
and causes organ injury at remote sites.
Many studies clearly support that neutrophils are

recruited into the liver after cellular damage initiated
by APAP challenge. However, the key unresolved ques-
tion is whether or not the infiltrated neutrophils are
activated and aggravate AILI. Data from some studies,
including the present one, provide evidence for a path-
ological role of neutrophils in AILI. However, other
studies have demonstrated that (1) neutrophils recruited
into the liver are not activated,33 (2) blocking neutro-
phil recruitment does not affect AILI,33,34 and (3) even
activating neutrophils by endotoxin or interleukin (IL)-
1b does not worsen AILI.33,35 Aside from the dichot-
omy of tissue-damaging and -repair functions of neu-
trophils, these discrepancies can be, at least in part,
explained by different experimental protocols employed
by various research groups. For example, two critical
experimental conditions that can significantly affect the
severity and kinetics of AILI include the mouse strains,
as well as the dose and route of administration of
APAP. Therefore, direct comparisons can only be made
when the experimental approaches are unified.

CYNTHIA JU, PH.D.
School of Pharmacy
University of Colorado Health Sciences Center
Denver, CO

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