Hindawi Publishing Corporation
Gastroenterology Research and Practice
Volume 2010, Article ID 710381, 12 pages
Toll-LikeReceptorsinthePathogenesis of AlcoholicLiverDisease
Department of Medicine, University of Massachusetts Medical School, LRB215, 364 Plantation Street, Worcester, MA 01605, USA
Correspondence should be addressed to Gyongyi Szabo, firstname.lastname@example.org
Received 20 May 2010; Accepted 20 July 2010
Academic Editor: Keigo Machida
Copyright © 2010 Jan Petrasek et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
In the multifactorial pathophysiology of alcoholic liver disease (ALD), inflammatory cascade activation plays a central role. Recent
studies demonstrated that Toll-like Receptors, the sensors of microbial and endogenous danger signals, are expressed and activated
in innate immune cells as well as in parenchymal cells in the liver and thereby contribute to ALD. In this paper, we discuss
the importance of gut-derived endotoxin and its recognition by TLR4. The significance of TLR-induced intracellular signaling
pathways and cytokine production as well as the contribution of reactive oxygen radicals is evaluated. The contribution of TLR
signaling to induction of liver fibrosis and hepatocellular cancer is reviewed in the context of alcohol-induced liver disease.
Alcohol abuse is a leading cause of morbidity and mortality
worldwide  and alcoholic liver disease (ALD), ranging
over 10 million Americans . Liver injury mediated by
alcohol involves both liver parenchymal and nonparenchy-
mal cells, including resident and recruited immune cells
that contribute to liver damage and inflammation . The
component of alcohol-induced liver disease dates back to
the observations that patients with ALD have increased
antibodies against Escherichia coli in plasma  and that
chronic alcohol administration increases gut-derived endo-
toxin in the portal circulation, activating resident liver
macrophages to produce several proinflammatory cytokines
[5, 6]. Recognition of Toll-like receptors (TLR) as the key
components involved in activation of the innate immune
mechanisms mediating alcohol-induced liver injury.
Criticalinthe Pathogenesis of ALD
blood from the intestine, exposing hepatocytes and cells in
the liver sinusoids not only to nutrients but also to gut-
derived microbial products. The gut mucosal epithelium
serves as an interface between the vast microbiota and
internal host tissues . Under normal circumstances, a
normal balance of gut barrier function, gut permeability,
and equilibrium of commensal and pathogenic microorgan-
isms in the gut lumen is maintained and mostly prevents
microbial translocation from the gut . Lipopolysaccharide
(LPS, endotoxin), a component of Gram-negative bacterial
wall, and other components derived from bacteria in the
intestinal microflora normally penetrate the mucosa only
in trace amounts, enter the portal circulation, and are
cleared by 80%–90% in the liver through uptake by Kupffer
cells (resident liver macrophages) and hepatocytes in a
manner that prevents cell damage or inflammation [9, 10].
These physiological uptake and detoxification are important
for preventing systemic reactions to gut-derived bacterial
Multiple lines of evidence support the hypothesis that
gut-derived endotoxin is involved in alcoholic liver injury
Figure 1(a). First, it has been shown that excessive intake
of alcohol increases gut permeability of normally nonab-
sorbable substances . Second, intestinal Gram-negative
bacteria, as well as blood endotoxin, are increased in acute
[12, 13] and chronic [12, 14, 15] alcohol feeding models.
Patients with alcoholic fatty liver, alcoholic hepatitis, and
alcoholic cirrhosis have 5- to 20-fold increased plasma
2 Gastroenterology Research and Practice
endotoxin compared to normal subjects [8, 16] although it
is unclear whether endotoxemia correlates with the extent
of liver dysfunction [17, 18]. Third, intestinal sterilization
with antibiotics  and displacement of Gram-negative
bacteria with Lactobacillus treatment  prevented alcohol-
induced liver injury. The mechanism underlying the disrup-
tion of the intestinal barrier appears to be multifactorial
. Disruption of tight junctions has been attributed to
acetaldehyde  and liver-derived inflammatory cytokines,
particularly TNF-α, that enter the systemic circulation and
further disrupt tight junctions, thus perpetuating intestinal
barrier dysfunction . Gut permeability may be also
increased by ethanol-induction of miR212, a microRNA that
downregulates proteins of the zona occludens in intestinal
cell culture and that was increased in colonic biopsy samples
in patients with ALD .
Activation of Kupffer cells has been identified as one
of the key elements in the pathogenesis of alcohol-induced
liver damage. Kupffer cells are the largest population of
tissue macrophages, predominantly distributed in the lumen
of hepatic sinusoids, and exhibit endocytic activity against
bloodborne materials entering the liver [10, 24]. Triggering
of Toll-like receptor signaling drives Kupffer cells to produce
inflammatory cytokines and chemokines and to initiate the
inflammatory cascade . Indeed, the essential role of
Kupffer cells as a central component of the pathomechanism
of ALD has been demonstrated in studies in mice and rats
that show that inactivation of Kupffer cells with gadolinium
chloride or liposomal clodronate can almost fully ameliorate
alcohol-induced liver disease [26, 27].
3.Toll-Like Receptors InvolvedinALD
The innate immune system recognizes conserved pathogen-
associated molecular patterns, which are released during
bacterial multiplication or when bacteria die or lyse ,
through pattern recognition receptors, including Toll-like
receptors (TLRs) . TLR4 recognizes endotoxin from
Gram-negative bacteria, and TLR2 is essential for recog-
nition of microbial lipopeptides, while TLR1 and 6 com-
bined with TLR2 distinguish between triacyl- and diacyl-
lipopeptides . TLR3 recognizes viral double-stranded
RNA, and TLR5 recognizes bacterial flagellin [31, 32]. TLR7
and TLR8 bind viral single-stranded RNA , and TLR9
recognizes prokaryotic CpG-rich DNA . Kupffer cells
express TLR4, TLR2, TLR3, and TLR9 [35–37], and hepatic
stellate cells express TLR2, TLR4, and TLR9 [38, 39]. Liver
sinusoidal endothelial cells express TLR4 [40, 41], and
primary cultured hepatocytes express mRNA for all Toll-
like receptors although they express very low levels of TLR2,
TLR3, TLR4, and TLR5 and show weak responses in vivo
3.1. Role of TLRs in the Pathogenesis of Alcohol-Induced
Liver Injury. Activation of Kupffer cells via TLR4-dependent
mechanism plays a crucial role in the pathogenesis of
alcohol-induced liver injury [6, 19, 44, 45]. LPS, a compo-
immune responses through its binding to the TLR4 complex
and comprises three distinct parts: a carbohydrate (O-
antigen), the oligosaccharide core region, and a lipid portion
(Lipid A). Only the lipid A portion is immunogenic .
While TLR4 cannot directly bind LPS, the coreceptors CD14
and MD-2 bind LPS and upon LPS binding activate TLR4.
CD14 is a GPI-anchored protein, which also exists in soluble
form, and facilitates the transfer of LPS to the TLR4/MD-
2 receptor complex that modulates LPS recognition .
MD-2 is a soluble protein that noncovalently associates with
TLR4 and binds LPS directly to form a complex with LPS
in the absence of TLRs . The association between LPS
and CD14 is facilitated by LPS-binding protein (LBP), which
is a soluble shuttle protein . TLR4, CD14, and LBP
are critical in alcohol-induced liver injury. Alcoholic liver
injury was prevented in C3H/HeJ mice , which have
functional mutation in the TLR4 gene and have defective
response to bacterial endotoxin . Prevention of alcohol-
induced liver inflammation and injury in C3H/HeJ mice
was associated with decreased TNF-α expression, compared
to wild-type mice. Similar protection from alcohol-induced
liver injury was observed in mice deficient for LBP  and
CD14  whereas mice transgenic for human CD14 were
hypersensitive to LPS .
Since disruption of intestinal barrier by ethanol increases
permeability for macromolecular substances in general ,
it is likely that other bacterial components, in addition to
LPS, are translocated to the portal blood in alcoholics. In
particular, bacterial DNA was found in serum and ascites of
patients with advanced liver cirrhosis leading to increased
cytokine production in peritoneal macrophages [55–57].
Bacterial DNA, which is detected by TLR9, sensitizes the
liver to injury induced by LPS via upregulation of TLR4,
MD-2 and induction Th1-type immune response in the
liver [58, 59]. Hepatic expression of TLR9 was increased in
wild-type animals using the Lieber-DeCarli chronic alcohol
feeding model, and alcohol feeding sensitized to TLR9
ligand CpG to enhance TNF-α production . In patients
with alcoholic cirrhosis, purified B cells stimulated with
TLR9 ligand CpG ex vivo showed significant upregulation
of immunoglobulin A, compared to B cells from control
individuals , suggesting involvement of TLR pathways in
alcohol-induced hyperimmunoglobulinemia [61–63]. Also,
overexpression of TLR9, TLR4, and TLR2 was associated
In addition to TLR4 and TLR9, increased expression
of TLR1, 2, 6, 7, and 8 was observed in wild-type mice
using the Lieber-DeCarli chronic alcohol feeding model,
and feeding with alcohol resulted in sensitization to liver
inflammation and damage because administration of TLR1,
2, 4, 6, 7, 8, and 9 ligands increased expression of TNF-
α . Interestingly, expression of these TLRs in mice on
ethanol diet remained significantly increased in spite of
concurrent administration of antibiotics that ameliorated
liver injury . Other investigations found that deficiency
in TLR2 had no protective effect on alcohol-induced liver
injury in a mouse model of chronic ethanol feeding 
and that hepatic expression of TLR2 or TLR4 mRNA was
Gastroenterology Research and Practice3
+ other PAMPs
Figure 1: Pathophysiology of alcohol-induced liver injury. (a) Ethanol promotes translocation of LPS and other pathogen-associated
molecular patterns (PAMPs) from the gut to the portal vein and to the liver. In the liver, LPS induces activation and recruitment of
bone marrow-derived inflammatory cells. Activated bone marrow-derived cells synthesize inflammatory cytokines and reactive oxygen
species that induce liver injury. Chronic ethanol per se contributes to sensitization of monocytes/macrophages to LPS and to sensitization
of hepatocytes to the cytotoxic effect of inflammatory cytokines. The latter is brought about by accumulation of lipids, opening of
LPS in cooperation with its coreceptors, CD14 and MD-2. The signal is passed through MyD88-dependent or TRIF-dependent intracellular
pathways, which activate various transcription factors, including AP-1, NFκB, and IRF3, and induces proinflammatory cytokine and Type I
not changed by chronic alcohol feeding or by acute ethanol
administration , implying that the increased sensitivity
TLR2 or TLR4 genes and may be related to an imbalance of
proinflammatory/oxidative and cytoprotective mechanisms.
Taken together, it seems likely that sensitization to
TLR ligands in alcohol-induced liver damage is regulated
by multiple mechanisms, including those that are directly
dependent on gut-derived bacterial components and TLR
signaling, but also other mechanisms, such as lipid accumu-
lation in hepatocytes [66, 67], histone acetylation in ethanol-
exposed macrophages , or activation of Kupffer cells by
C3 and C5 components of the complement pathway .
3.1.1. MyD88-Independent and Dependent TLR Pathways in
ALD. TLR4 is unique among TLRs in its ability to activate
two distinct signaling pathways Figure 1(b). One pathway
is activated by the adaptors TIRAP (Toll/interleukin-1-
receptor- (TIR-) domain-containing adaptor protein) and
MyD88, which leads to activation of NF-κB and to the
induction of proinflammatory cytokines. The second path-
way (MyD88-independent) is activated by the adaptors TRIF
the TBK/IKKε kinase and interferon regulatory factor 3
(IRF3) to induce Type I IFNs as well as NF-κB activation
[70, 71]. The two TLR4-dependent signaling pathways are
induced sequentially, and the TRAM-TRIF pathway is only
operational from early endosomes following endocytosis of
Recent evidence suggests that TLR4 downstream signal-
ing in ALD is mediated predominantly through MyD88-
independent pathway, rather than through the MyD88-
dependent mechanism. Alcohol feeding with the Lieber-
DeCarli diet [73, 74] resulted in significant steatosis and
liver damage in MyD88-deficient mice compared to mice
on pair-fed diet, and the extent of alcohol-induced changes
was comparable in alcohol-fed MyD88-deficient and wild-
type mice . The involvement of the MyD88-independent
TLR4 signaling pathway was indicated by upregulation
of IRF7, an IRF3-inducible gene, in Kupffer cells .
In a different study it was reported that mice deficient
in TRIF, which is a key TLR4 downstream adaptor in
the MyD88-independent pathway, were protected against
alcohol-induced liver disease, and it is likely that IRF3, a
transcription factor downstream to TLR4/TRIF, binds to the
findings, together with the observation that mice deficient
in IRF3 show protection against alcohol-induced liver injury
signaling via MyD88-independent pathways is critical in
induction of alcoholic liver disease.
3.1.2. Endotoxin Sensing and Loss of TLR Tolerance in
ALD. Lipopolysaccharide is the most potent inducer of
inflammatory cytokines, particularly TNF-α, in monocytes
and macrophages. However, when LPS challenge is provided
following an initial insult with LPS, induction of TNF-α is
severely attenuated, a phenomenon called “LPS tolerance.”
4 Gastroenterology Research and Practice
Recent studies demonstrated that upregulation of negative
regulators of TLR signaling play a central role in TLR
tolerance [77–79]. Experimental evidence suggests, however,
that TLR tolerance can be broken by multiple sequential
LPS administration in vivo and in vitro . When mice
were injected with a single dose of LPS, a second LPS
compared to the initial dose demonstrating TLR4 tolerance
. However, when LPS was given in 3-day intervals for 5
repeated times, the TLR tolerance was lost and serum TNF-
α levels induced by the last dose of LPS were comparable
to TNF-α induced by a single LPS administration . The
bimodal effects of LPS on TNFα production are reminiscent
to the opposite modulation of inflammation by acute and
prolonged alcohol use. At the cellular and molecular level,
acute alcohol administration inhibited while chronic alcohol
ticularly when LPS was used as an inflammatory insult .
Increased TNF-α production and NF-κB activation were
In vitro studies showed that prolonged alcohol exposure
of monocytes for 4 days or longer in vitro augmented LPS-
induced TNF-α production compared to alcohol-na¨ ıve cells
. The involvement of the TLR4 signaling pathway was
suggested by increased IRAK-1 phosphorylation, increased
IKK kinase activity, increased NF-κB nuclear translocation
and DNA transactivation in human monocytes . This
upregulation of TLR4 signaling in the presence of dimin-
ished expression of IRAK-M in monocytes after prolonged
alcohol treatment. Overexpression of IRAK-M prevented
the increased LPS-induced TNF-α production in chronic
alcohol-treated cells suggesting that loss of IRAK-M is likely
to contribute to the loss of TLR tolerance in monocytes after
prolonged alcohol exposure .
Previous studies have demonstrated that chronic alcohol
use results in increased levels of LPS in the portal and
systemic circulation that may mediate or amplify the loss of
TLR4 tolerance after chronic alcohol treatment. The role of
TLR tolerance and of the loss thereof may deserve further
The importance of molecular mechanisms culminating in
nuclear events leading to activation of a wide array of tran-
scription factors in various liver cell types is widely studied
in progression of alcoholic liver injury. These transcription
factors bind to the promoter regions in target genes resulting
in induction of cytokines, chemokines, and various other
Studies using rodent models of alcoholic liver injury show
that exposure to chronic alcohol increases expression of
genes related to fatty acid synthesis and decreases fatty acid
proliferator factor α (PPARα) play a pivotal role in fatty
acid metabolism and are altered during chronic alcohol
consumption. Liver-specific overexpression of SREBP-1α
and SREBP-1c is observed in alcoholic liver injury leading
to increased hepatic triglyceride content . On the other
hand PPARα expression and DNA binding activity were
decreased in alcoholic livers resulting in decreased fatty
acid oxidation . Similar to alterations in transcription
factors related to fatty acid metabolism, chronic alcohol-
induced inflammatory mediators are also modulated by key
transcription factors. The most studied transcription factor
is NFκB, and alteration in its DNA binding activity has been
observed in livers following chronic alcohol consumption
 as well as in isolated monocytes/macrophages .
Chronic alcohol increased NFκB activity in monocytes and
macrophages leading to an upregulation in various inflam-
matory cytokine and chemokine genes [89, 90]. Another
transcription factor modulated by chronic alcohol exposure
is AP-1 wherein increased expression and activity were
of PPARγ, another transcription factor, was beneficial and
prevented chronic alcohol-induced liver injury in mice .
While PPARγ is thought to be involved in anti-inflammatory
cytokine production, its exact mechanism in alcoholic livers
is not known. Furthermore Egr-1, another zinc finger tran-
scription factor, is up-regulated in LPS-stimulated isolated
to alcoholic liver injury, indicating a role for the Egr-1-
ERK pathway in the pathogenesis of alcoholic liver injury
. Thus, studies so far suggest that transcription factors
play an important role in alcoholic liver injury, and future
investigations are necessary to determine the complexity of
regulation of the target genes in various liver types during
alcoholic liver disease.
Alcoholic steatohepatitis is characterized by infiltration of
various inflammatory cells in the liver, including mono-
cytes, macrophages, neutrophils, and lymphocytes, which
occurs as a consequence of activation of inflammatory
mediators induced by TLR signaling [95, 96]. In humans
with alcoholic steatohepatitis, serum TNF-α, IL-6, and IL-8
levels are increased, and their levels correlate with markers
of the acute-phase response, liver function, and clinical
outcome . There is also evidence for activation of
circulating monocytes in individuals with ALD, based on
increased TNF-α production and increased NFκB activation
Induction of TNF-α by TLR4 signaling and by reactive
oxygen species in Kupffer cells has been identified as a major
component in ALD [85, 101, 102]. The effect of TNF-α in
hepatic inflammation and hepatocyte apoptosis is mediated
through TNF receptor TNF-R1 . Binding of TNF-α to
resulting in the activation transcription factors including
NFκB and c-Jun-N-terminal kinase  and in activation
of proapoptotic Fas-associated death domain .
Circulating levels of TNF-α and TNF-R1 are higher in
patients with alcoholic steatohepatitis than in heavy drinkers
with inactive cirrhosis, heavy drinkers who do not have
Gastroenterology Research and Practice5
liver disease, and individuals with neither alcoholism nor
liver disease [45, 83, 106]. High serum levels of TNF-
α and TNF-R1 correlated with mortality in patients with
acute alcoholic hepatitis [106–108]. Hepatic expression of
TNF-R1 is enhanced in chronic ethanol consumption ,
and liver injury is substantially reduced when alcohol diet
is administered in TNF receptor 1 (TNF-R1)—knockout
mice or in rats that have been pretreated with anti-TNF-
α antibodies or thalidomide, which reduces production of
TNF-α [110, 111].
Under normal circumstances, hepatocytes are resistant
to the proapoptotic effect of TNF-α; however, several
conditions prime hepatocytes to TNF-α-mediated cell death
in the setting of chronic alcohol consumption [112–115].
Hepatocytes from rats chronically fed alcohol have increased
TNF-α induced cytotoxicity associated with mitochondrial
permeability transition pore opening  and with a pro-
found effect of alcohol on mitochondrial functional integrity
[115, 116]. Also, decreased mitochondrial glutathione in
alcohol-fed rats  or inhibition of hepatic transmethy-
lation reactions by S-adenosylhomocysteine  has been
shown to sensitize hepatocytes to TNF-α mediated cyto-
toxicity. Moreover, animal models of alcohol-induced liver
injury show impaired function of proteasomes that increases
hepatocyte sensitivity to TNF-α-mediated apoptosis .
Interestingly, although upregulation of TNF-R1 is observed
in the livers of patients with alcoholic steatohepatitis , a
recent in vitro study showed that free fatty acids sensitized
HepG2 cells to TRAIL-mediated apoptosis, but not to
cytotoxicity mediated by TNF-α .
In addition to the metabolic changes involved in sensi-
tization to TNF-α cytotoxicity, the net effect of TNF-α on
hepatocytes is influenced by other cytokines. For example,
in mice that are deficient in IL-6, increased production
of TNF-α induced by partial hepatectomy promotes death
of hepatocytes instead of stimulating their proliferation
. Similarly, deficiency of IL-10, an anti-inflammatory
cytokine inducible by adiponectin , exacerbates TNF-
α-mediated liver injury in mice by alcohol . Conversely,
mice that are deficient in interleukin-12 , interferon-
γ , or interleukin-18  are protected against
TNF-α-induced liver damage. The subtle balance between
hepatocyte proliferation and apoptosis is also regulated by
an autocrine cascade involving the pro-proliferative TGF-
α and IL-1 receptor antagonist, and the antiproliferative
6.Toll-Like Receptors andOxidative
Cellular responses induced by oxidative stress play an
important role in innate immune cell activation. Kupffer
cells produce reactive oxygen species (ROS) in response to
chronic alcohol exposure as well as endotoxin . Inter-
action of NADPH with TLR4 is involved in LPS-mediated
ROS generation and NFκB activation and production of
inflammatory cytokines in neutrophils  and in human
monocytes . Pretreatment of chronic alcohol fed rats
with inhibitor of NADPH oxidase diphenyleneiodonium
(DPI) normalized ROS production, decreased LPS-induced
ERK1/2 phosphorylation, and inhibited increased TNF-α
production in Kupffer cells . Inhibition of NADPH
oxidase prevented steatosis, upregulation of TLR2, 4, 6, and
9 mRNA, and sensitization to respective ligand-induced liver
injury , indicating a crosstalk between oxidative stress
and TLR pathways in ALD. Protection from alcohol-induced
liver injury was observed in p47 phox−/− mice, deficient in
the main cytosolic component of NADPH oxidase, further
induced inflammatory response and liver injury .
7.TLR Signalingas Target for Therapy of ALD
Recently, a number of different approaches that modulate
TLR signaling have been developed. These approaches
include modulation of TLR ligand release from the intestine
by probiotics [129, 130], activation of TLR signaling by syn-
thetic TLR ligands [131–133], inhibition of TLR activation
by small molecule inhibitors [134–136], and interference
with cytokines induced by TLR signaling [137–139]. So
far, probiotics and anticytokine therapeutic approaches
have progressed into clinical trials in patients with ALD
[129, 139, 140].
Modulation of intestinal microbiota using probiotics has
been shown to reduce bacterial translocation [141, 142],
circulating endotoxin levels in animal models , and
bacterial infection, a marker for bacterial translocation, in
patients with liver cirrhosis [144, 145]. Beneficial effects
of probiotics have been reported in an animal model of
alcohol-induced liver injury  and of LPS-induced liver
injury [142, 146]. Patients with alcoholic liver cirrhosis
treated with Lactobacillus casei Shirota three times daily
for 4 weeks showed restoration of deranged neutrophil
phagocytic capacity, compared to controls . A recent
open-label pilot trial showed that a 5-day administration
of Bifidobacterium bifidum and Lactobacillus plantarum in
alcohol-addicted psychiatric patients with mild alcoholic
hepatitis ameliorated serum markers of liver injury to
a significantly higher extent compared to control group
treated with abstinence only . These data suggest that
modulation of the bowel flora may play a role in the
pathogenesis and treatment of ALD and indicate a need for
larger and rigorously designed clinical trials to support the
use of probiotics in ALD.
While the role of TNF-α in the development of ALD has
been well characterized , clinical investigations of the
therapeutic efficacy of antibodies to TNF-α (e.g., infliximab)
to treat patients with acute alcoholic hepatitis have generated
variable results [139, 148]. There is particular concern
about off-target effects of completely inhibiting TNF-α
function. For example, since TNF-α is a critical component
of immunity, infectious disease is a primary concern during
TNF-α therapy [139, 149]. Moreover, TNF-α is required
for normal liver regeneration as hepatocyte proliferation
in response to injury is impaired in mice lacking TNF-α
receptors . Etanercept, a TNF-α neutralizing antibody,
6 Gastroenterology Research and Practice
appeared to increase short-term survival of patients with
alcoholic hepatitis in a small pilot study  although a
subsequent randomized, placebo-controlled trial conducted
by the same investigators showed a worse 6-month survival
rate in the group treated with etanercept than in the placebo
8.Alcohol: TLR Signalingand LiverFibrosis
Alcohol-induced liver fibrosis is characterized by exces-
sive deposition of extracellular matrix components due to
increased matrix production and decreased matrix degra-
dation . Ethanol contributes to liver fibrosis in several
aspects, including the upregulation of collagen transcription
in hepatic stellate cells by acetaldehyde or reactive oxygen
species from ethanol-exposed hepatocytes [154–157]. Also,
activates hepatic stellate cells and Kupffer cells .
In addition, cytokines secreted by Kupffer cells activated
by alcohol/LPS are of key importance in activation and
transformation of hepatic stellate cells and induction of
alcoholic liver fibrosis [153, 159, 160]. Recently, it has
been shown that the crosstalk between Kupffer cells and
hepatic stellate cells involves TLRs on both cell types .
Activated hepatic stellate cells express TLR4, CD14, and
MD2. Stimulation of activated hepatic stellate cells with LPS
resulted in a rapid activation of NF-κB, c-Jun N-terminal
kinase and in upregulation of chemokines and adhesion
Interestingly, stimulation of hepatic stellate cells with
LPS alone is not sufficient for their transformation into
myofibroblasts. However, pretreatment with LPS strongly
enhances response of hepatic stellate cells to TGF-β, which
is a major profibrogenic cytokine derived predominantly
from activated Kupffer cells . The increased sensitivity
of LPS-pretreated hepatic stellate cells to TGF-β has been
linked to a TLR4-dependent downregulation of the TGF-
β pseudoreceptor Bambi in HSCs, which is a negative
regulator of TGF-β signaling . Taken together, these
findings suggest that LPS influences hepatic fibrosis via
TLR4-dependent modification of TGF-β signaling in hepatic
stellate cells and that hepatic stellate cells represent the
primary liver cell compartment integrating inflammatory
and fibrogenic pathways .
Additional components of the TLR system have been
investigated as possible modulators of the fibrogenic process.
Upon hepatocyte apoptosis, which is significantly increased
in alcoholic liver disease, degradation of nuclear DNA
activates immune cells via TLR9 . Activation of TLR9
has been shown to modulate the biology of hepatic stellate
cells, including inhibition of cell migration and upregulation
of collagen production .
9.Alcohol: TLR Signalingand
Alcoholic liver cirrhosis is a premalignant condition with
approximately fourfold increase in the risk of hepatocellular
carcinoma (HCC) . The five-year cumulative incidence
of HCC reaches 8% . In addition, alcohol shows
synergy with chronic hepatitis infection . For example,
the relative risk of developing HCC was 50-fold higher in
heavy drinkers with chronic hepatitis C (HCV) whereas
nondrinking HCV patients showed 15-fold increased risk,
compared to abstaining controls without HCV .
Studies investigating the synergism between alcohol and
HCV focused at the structural HCV core [169–171] and
the nonstructural NS5A proteins [172, 173]. The HCV core
protein causes overproduction of reactive oxygen species
, induces insulin resistance , and inhibits very low
density lipoprotein secretion from hepatocytes, contributing
to steatosis . However, although HCV core-transgenic
mice fed with ethanol for 9 months have shown increased
incidence of HCC, the mechanism of synergism between the
HCV core protein and ethanol in hepatic carcinogenesis is
not known .
Recently, the role of TLR4 in the synergism between
alcohol and HCV nonstructural protein NS5A in hepatic
oncogenesis has been proposed . In a study with
NS5A transgenic mice (NS5A Tg), it was reported that
NS5A induces TLR4 expression in the liver. NS5A Tg mice
dose of LPS and showed aggravated alcoholic steatohepatitis
after 4-week intragastric ethanol feeding . Importantly,
the adjuvant effect of NS5A was blunted in NS5A Tg
mice who were deficient in TLR4 or who underwent gut
sterilization with antibiotics, indicating the importance of
endotoxin and TLR4 signaling in the synergism between
alcohol/LPS and NS5A.
Furthermore, one-fourth of NS5A Tg mice fed Lieber-
DeCarli ethanol diet for 12 months developed HCC, in
contrast to no tumors found in WT or TLR4−/−NS5A mice,
demonstrating that alcohol and NS5A synergistically induce
liver tumors through TLR4 signaling . Microarray
analysis showed that NS5A Tg mice fed ethanol have
increased liver expression of the stem/progenitor cell marker
Nanog, which is involved in the genesis of CD133+cancer
stem cells. Nanog induction was dependent on NS5A and
alcohol and was abrogated in TLR4−/−NS5A Tg mice fed
alcohol. Further experiments demonstrated that Nanog is a
novel downstream gene of TLR4 signaling.
Transplantation of p53-deficient hepatic progenitor cells
transduced with Nanog or TLR4 resulted in spontaneous
tumor development after 80 days or after repetitive LPS
injections for 25 weeks, respectively. The tumor incidence
by coexpression of short hairpin RNA against Nanog, indi-
cating that Nanog expression is involved in tumor formation
that Nanog-positive cancer stem cells did not upregulate
TGF-β signaling after TLR4 activation . Defective
TGF-β pathway leads to spontaneous development of
Taken together, the recent data [172, 173] suggest that
alcohol and HCV NS5A induce synergistic tumor develop-
ment via induction and activation of TLR4 in mice and that
this synergism involves the stem cell marker Nanog, which is
Gastroenterology Research and Practice7
a TLR4-downstream regulated gene. These findings indicate
that inhibition of TLR4 signaling may provide a therapeutic
option for HCV-associated liver tumors.
In conclusion, there is clear evidence that alcohol con-
sumption leads to increased intestinal permeability and
endotoxemia, which results in activation of innate immunity
via TLR4 signaling. Recent studies have contributed to
the dissection of molecular mechanisms of TLR4 signal-
ing in ALD, indicating the indispensable role of MyD88-
independent pathway in mediating the effects of gut-derived
endotoxin in ALD and suggesting the role of other TLRs
in modulation of alcohol-induced liver injury. Moreover,
novel data provide insight into the mechanisms of prolonged
alcohol exposure on TLR4-induced inflammation and loss
of LPS tolerance and the interplay between proinflammatory
and anti-inflammatory cytokines mediating TLR-induced
cytotoxicity. Further studies are needed to evaluate crosstalk
between liver parenchymal and nonparenchymal cells.
Understanding the cell-specific role of TLR signaling in ALD
will further provide new insights into the pathogenesis of
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