LPS Blocheaza Apoptoza in Cel Tumorale Hepatice

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Endotoxin Accumulation Prevents Carcinogen-Induced

Apoptosis and Promotes Liver Tumorigenesis in

Rodents

Le-Xing Yu,1* He-Xin Yan,1* Qiong Liu,1 Wen Yang,1 Hong-Ping Wu,1 Wei Dong,1 Liang Tang,1 Yan Lin,1

Ya-Qin He,1 Shan-Shan Zou,1 Chao Wang,1 Hui-Lu Zhang,1 Guang-Wen Cao,1,3 Meng-Chao Wu,1

and Hong-Yang Wang1,2

Increasing evidence suggests that the presence of endotoxemia is of substantial clinical rele- vance to patients with cirrhosis, but it is unclear whether and how gut-derived LPS ampli-fies the tumorigenic response of the liver. We found that the circulating levels of LPS wereelevated in animal models of carcinogen-induced hepatocarcinogenesis. Reduction of LPSusing antibiotics regimen in rats or genetic ablation of its receptor Toll-like receptor 4(TLR4) in mice prevented excessive tumor growth and multiplicity. Additional investigationrevealed that TLR4 ablation sensitizes the liver to carcinogen-induced toxicity via blocking NF-jB activation and sensitizing the liver to reactive oxygen species (ROS)-induced toxic-ity, but lessens inflammation-mediated compensatory proliferation. Reconstitution of TLR4 -expressing myeloid cells in TLR4 -deficient mice restored diethylnitrosamine (DEN)-induced hepatic inflammation and proliferation, indicating a paracrine mechanism of LPSin tumor promotion. Meanwhile, deletion of gut-derived endotoxin suppressed DEN-induced cytokine production and compensatory proliferation, whereas in vivo LPS pre-challenge promotes hepatocyte proliferation. Conclusion: Our data indicate that sustained LPS accumulation represents a pathological mediator of inflammation-associated hepato-cellular carcinoma (HCC) and manipulation of the gut flora to prevent pathogenic bacterialtranslocation and endotoxin absorption may favorably influence liver function in patients

with cirrhosis who are at risk of developing HCC. (HEPATOLOGY 2010;52:1322-1333)

Hepatocellular carcinoma (HCC) is closely associ-ated with chronic inflammatory liver diseases,such as those caused by viral infection, alcohol

consumption, or hepatic metabolic disorders. Chronicliver disease leads to continual injury and a wound-heal-ing response that causes a torrent of problems, including advanced hepatic fibrosis or cirrhosis.1 A high level of plasmatic endotoxin (lipopolysaccharide [LPS]), a cell-

wall component of gram-negative bacteria, is a commonfinding in the portal and systemic circulation of patients

with cirrhosis.2

This accumulation is likely due tochanges in the intestinal mucosal permeability andincreased bacterial translocation, coupled with deficient

clarification of the hepatic reticuloendothelial system.3

High levels of endotoxin may be responsible for thepathogenesis of the chronic inflammatory alterations thatcharacterize cirrhosis and, hence, the major complicationsin this disease.4

TLR4 is a pattern recognition receptor that recognizesendotoxin and signals through adaptor molecules mye-loid differentiation primary response gene (88) (MyD88)and TIR-domain-containing adapter-inducing interferon-b (TRIF) to activate transcription factors that initiate

innate immunity.5

The liver is well equipped to respondto endotoxin because TLR4 is present on both parenchy-mal cells (hepatocytes) and nonparenchymal cells such as

Abbreviations: TLR4, Toll-like receptor 4; DEN, diethylnitrosamine; HCC, hepatocellular carcinoma; LPS, lipopolysaccharide; EdU, 5-ethynyl-2 0-deoxyuridine;H&E, hematoxylin and eosin; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; ALT, alanine transarninase TNF a, tumor necrosis factor a; IL-6, Interleukin-6; SOD, superoxide dismutase; GSH, glutathione; MDA, malondialdehyde; BHA, butylated hydroxyanisole; A20, TNF a-induced-protein 3; ChIP, chromatin immunoprecipitation.

From the 1International Cooperation Laboratory on Signal Transduction, Liver Centre of SMMU, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai, China; 2 National Laboratory for Oncogene and Related Genes, Cancer Institute of Shanghai Jiao Tong University, Shanghai, China; and 3Department of Epidemiology, Second Military Medical University, Shanghai, China.

*These authors contributed equally to this work.Received March 9, 2010; accepted June 5, 2010.

1322



Kupffer cells. Both cell populations possess intact TLR4signaling pathways.6,7 Kupffer cells are the best-character-ized target of endotoxin in the liver,8 where they have a crucial role in causing hepatic damage by producing proinflammatory cytokines (e.g., tumor necrosis factor(TNF)-a and interleukin (IL)-6) and affect hepatic sinu-soids to increase vascular permeability.9 Although hepato-cytes also express low levels of the TLR4 receptor, they are only weakly responsive to LPS and may serve touptake and remove endotoxin from the portal and sys-temic circulation.10 The effects of endotoxin in vivo onhepatic function and tumorigenesis are not well defined.

Robust clinical and epidemiologic data support the roleof inflammation as a key player in HCC development.11

However, the exact molecular mechanisms and gatekeep-ers accounting for cellular transformation remain elusive.Given the important role of NF-jB signaling in media-

ting inflammatory signals, attention has been focused onits role in mediating the link between inflammation andthe development of liver tumors.12 Inhibiting NF-jBobstructs later stages of tumor progression in multi-drug resistant (Mdr) 2-deficient mice, which develop HCC inthe context of chronic bile duct inflammation.13 By con-trast, mice lacking the I-kappa-B kinase-beta (IKK b) spe-cifically in hepatocytes exhibit a marked increase inchemically induced hepatocarcinogenesis, suggesting thatNF-jB has a protective function against HCC develop-ment. Interestingly, compared with the deletion of IKK bonly in hepatocytes, the additional deletion in Kupffer

cells results in a remarkable decrease in tumor load.14

These apparently contradictory conclusions may reflectthe distinct roles for inflammatory signals in epithelialcells and inflammatory cells during HCC formation.

Here, we show that endogenous endotoxin accumula-tion regulates the survival and proliferation of hepato-cytes and their preneoplastic derivatives during chemi-cally induced hepatocarcinogenesis. The cytoprotectiveand protumorigenic effects of endotoxin are mainly dueto elevated NF-jB activity in premalignant epithelialcells, which suppresses apoptosis, thus promoting thecells’ survival and subsequent capacity to form tumors.

Meanwhile, LPS engagement of the same pathway in tu-mor-associated inflammatory cells initiates transcriptionof proinflammatory cytokines, which in turn stimulate

tumor proliferation. These results suggest that prevent-ing plasma endotoxin accumulation could have a benefi-cial impact on liver function for patients with cirrhosis

with the potential to progress to hepatoma.

Materials and Methods Animals. Pathogen-free male Sprague-Dawley rats

(weighing 160-180 g) and male C57BL/6 mice (6-8 weeks old, weighing 16-20 g) were obtained from theShanghai Experimental Center, Chinese Science Academy,Shanghai, and maintained at an animal facility underpathogen-free conditions. Male wild type (wt; C57BL/10SnJ), and TLR4 -deficient (TLR4 À/À; C57BL/10ScNJ)mice were obtained from the Model Animal ResearchCenter of Nanjing University, Nan Jing, China. All ani-mals received humane care according to the criteria out-

lined in the ‘‘Guide for the Care and Use of Laboratory Animals’’ prepared by the National Academy of Sciencesand published by the NIH (publication 86-23 revised1985). For detailed information related to animal experi-ments, see the Supporting Experimental Procedures.

Immunohistochemistry. All paraffin-embedded livertissues were stained with hematoxylin and eosin(H&E) for analysis of morphologic changes. The pri-mary antibodies were as follows: cyclin D1, phospho-c-Jun (Cell Signaling Technology) and F4/80 (Santa Cruz Biotechnology, Santa Cruz, CA). Apoptosis wasassessed by TUNEL staining of paraffin-embedded

slides (Calbiochem, La Jolla, CA). Proliferation wasassessed by immunostaining for 5-ethynyl-20-deoxyuri-dine (EdU; Ruibo Biotech, Guangzhou, China) or Ki-67 (Labvision, Fremont, CA) staining.

Bone Marrow Transplantation. Recipient mice werelethally irradiated with 9.0 Gy at a rate of 70 cGy/minuteusing a cobalt-source gamma-irradiator (the Irradiation Cen-ter of the Second Military Medical University). Irradiated re-cipient mice were i.v. injected with approximately 1 Â 107

bone marrow cells in 200 lL of PBS. They were subjectedto DEN treatment 5 weeks after transplantation. To demon-strate the success of BMT in TLR4 À/À and wt mice, the

blood and bone marrow of the chimeric mice were collected,and genomic DNA was extracted for detection of the Tlr4 gene by quantitative polymerase chain reaction (qPCR).

Supported by National Natural Science Foundation of China grant no. (30871293, 30921006). Chinese National Programs for High Technology Research and Development grant no. (2007AA02Z166). Chinese National Key Project grant no. (2008ZX10002). Shanghai Young Scholars grant Grant number: 07QA14070;Grant number 07SG45; Grant number 08400701000.

Address reprint requests to: Hong-Yang Wang, International Cooperation Laboratory on Signal Transduction, Eastern Hepatobiliary Surgery Hospital, Shanghai 200438, China. E-mail: [email protected]; fax: 86 21 6556 6851.

Copyright VC 2010 by the American Association for the Study of Liver Diseases.View this article online at wileyonlinelibrary.com.DOI 10.1002/hep.23845 Additional Supporting Information may be found in the online version of this article.

HEPATOLOGY, Vol. 52, No. 4, 2010 YU ET AL. 1323



Statistical Analysis. Data are expressed as means 6SE. Differences were analyzed by the Student t test,and P values < 0.05 were considered significant.

Results

Depletion of Host Microflora Suppresses Chemical

Hepatocarcinogenesis in Rats. Chronically exposing rats to diethylnitrosamine (DEN) provides a multistagehepatocarcinogenesis model for studying human HCC,

which allows one to distinguish tumor initiation frompromotion (Supporting Information Fig. 1A). Becausethe liver is directly downstream from the gut, wehypothesized that translocated microbes or endotoxincould promote chemically induced HCC. To address thisquestion, we first used the rat HCC model to assess theplasma levels of LPS at different stages of DEN-inducedhepatocarcinogenesis. Of interest, the plasma levels of

LPS were elevated during tumor progression (Fig. 1A),indicating that plasma endotoxin may be a critical cofac-tor in chemically induced hepatocarcinogenesis.

To address whether the circulating LPS in DEN-treated animals augmented tumor induction, we deter-

mined whether their removal with LPS antagonist poly-

myxin B (PMB),15

and neomycin that is bactericidalmainly for gram-negative organisms in gut, would affect

HCC development. Rats were treated with antibiotics

in their drinking water, from 4 days prior to DEN i.p.injection to 3 weeks after, and then analyzed for the

presence of plasma LPS. As expected, although the anti-

biotics alone caused no phenotypic manifestation in the

liver (Supporting Information Fig. 1B), antibiotic treat-ment significantly reduced the levels of LPS in their

plasma (Fig. 1A). Treating rats with antibiotics signifi-

cantly reduced the cytokines (TNFa and IL-6)

Fig. 1. Antibiotics treatment suppresses

chemical hepatocarcinogenesis in rats. (A) The plasma LPS concentration at the indi-

cated time after DEN injection in the four

groups (Antibiotics þ DEN, DEN alone, Antibi-

otics alone and untreated control, n ¼ 6-8).

**P < 0.01; *P < 0.05 versus antibiotics þ

DEN group. (B) DEN induced TNF a and IL-6

mRNA production in the livers of rats with or

without antibiotics treatment. (C) Gross and

histology appearance (H&E staining) of livers

from DEN-treated rats with or without antibiot-

ics treatment 21 weeks after DEN administra-

tion. (D) Numbers of tumors (!1 mm) and

maximum tumor sizes (diameters) in DEN and

AntibioticsþDEN groups 21 weeks after DEN

injection. (E) The ratio of liver/body weight change after DEN injection in the four groups

above. (F) Staining of Ki-67 and histological

quantification (lower panel) are also shown.

*P < 0.05. All scale bars ¼ 100 lm. HP,

high power field (400Â).

1324 YU ET AL. HEPATOLOGY, October 2010



production and liver fibrogenesis after DEN treatment

(Fig. 1B; Supporting Information Fig. 1C). Notably,DEN-induced HCC multiplicity was significantly decreased. (Fig. 1C). Although all the DEN-treated ratsdeveloped liver tumors 21 weeks after DEN injection,the number of detectable tumors (!1 mm), maximaldiameters of tumors and the relative liver weight weresignificantly decreased in antibioticsþDEN group 21

weeks after DEN injection (Fig. 1D,E). Consistently,antibiotic-treated rats have a significantly lower level of cell proliferation (Ki-67) in tumor mass (Fig. 1F), butthere was no significant difference in the apoptotic cells

between the two groups (Supporting Information Fig.

1D). These data confirmed the role of microbial LPS incontributing to tumor induction after DEN treatment.

Deletion of TLR4 Greatly Decreases Chemically Induced Hepatocarcinogenesis in Mice. A singleDEN injection to 15-day-old male mice also results inefficient HCC induction.16 Because LPS is thought toexert its effects primarily through its innate receptor,TLR4, we examined whether mice deficient in TLR4 mounted an altered susceptibility to develop HCC.Neither wild type (wt) nor TLR4 -deficient strainsexhibited spontaneous liver dysfunction or HCC, up to

Fig. 2. Loss of TLR4 decreases

DEN-induced tumor development in

mice. (A) Representative livers of 10-

month-old DEN-treated male wt and

TLR4À / À

mice. (B) Tumor incidence,

(C) numbers of tumors (!0.5 mm)

and (D) maximum tumor sizes (diame-

ters) in livers of male wt (n ¼ 23) and

TLR4À /À(n ¼ 28) mice 10 months af-

ter DEN injection. (E) Histological andimmunohistochemical analysis of wt

and TLR4À /À tumors. Histological

quantification of Ki-67, cyclin D1,

phospho-c-jun positive and apoptotic

cells (TUNEL staining) are shown in the

lower panels. (F) Expression of TNF aand IL-6 mRNA in wt and TLR4

À /À

tumors (n ¼ 5). HP, high-power field

(200Â). ***P < 0.001; **P < 0.01;

*P < 0.05. All scale bars ¼ 50 lm.




1 year of age (data not shown). Upon DEN injectionon postnatal day 15, all wt males developed typicalHCCs within 10 months, but tumor incidence was25% lower in TLR4 À/À mice (Fig. 2A,B). Furthermore,a dramatic decrease in the number of detectable HCCs

was observed in TLR4

À/À

mice relative to wt controls(Fig. 2C). The maximal tumor diameters were also sig-nificantly smaller in TLR4 À/À mice compared to wtcontrols (Fig. 2D). Consistantly, malignant liver tumorsin TLR4 À/À mice displayed strongly decreased prolifer-ation (Ki-67) and lower levels of phospho-c-Jun andcyclin D1, which are needed for cell proliferation, com-pared to in wt mice. Furthermore, apoptotic tumorcells were more frequently observed in tumors fromTLR4 À/À mice than in tumors from wt mice (Fig. 2E).Moreover, the serum ALT was modestly reduced in tu-mor-bearing TLR4 À / À mice compared with tumor-

bearing wt mice, indicating a lower tumor load indi-rectly (Supporting Information Fig. 2A).

Because TLR4 activation of innate immune cellsresulted in the production of several inflammatory cytokines that stimulated tumor growth, we thusassessed whether the absence of TLR4 influences can-cer-linked inflammatory responses. Indeed, in additionto the smaller number and size of tumors in TLR4 À/À

mice, these lesions were consistently associated withreduced infiltration of macrophages (F4/80 staining)compared to wt mice (Supporting Information Fig.2B). Concordantly, the expression levels of hepatomito-

gens (TNFa and IL-6) were evidently reduced inTLR4 À/À HCCs relative to controls (Fig. 2F). How-ever, unlike the DEN-induced rat HCC model, no evi-dent liver fibrosis was found in this model (Supporting Information Fig. 2C). Thus, the loss of TLR4 protectsthe liver from chemically induced carcinogenesis, possi-bly because of less pronounced inflammation, reducedproliferation, and enhanced apoptosis in tumor cells.

Loss of TLR4 Exacerbates DEN-Induced Liver Injury but Lessens Compensatory Proliferation. Thefinding that loss of TLR4 reduced the susceptibility of mice to chemical hepatocarcinogenesis prompted us to

examine the early effects of DEN on cell behavior andsignal transduction. At 24 or 48 hours after DENinjection, TLR4 À/À males displayed a considerable ele-vation of ALT in serum and an increased number of TUNEL-positive cells in liver, indicating the presenceof exacerbated hepatocyte damage (Fig. 3A,B,D). Thehistological evidence of damage was likewise increasedin TLR4 À/À mice compared to wt mice (Supporting Information Fig. 3). DEN administration led to a rapid increase in expression of the p53 target genesp21 and Mdm2, but the response was similar in wt

and TLR4 À/À mice, excluding the possibility thatTLR4 affects DEN metabolism (Supporting Informa-tion Fig. 4). These data suggest that deletion of TLR4may result in more DEN-induced cell death.

The mammalian liver possesses an extraordinary capacity for compensatory growth and thereby main-tains liver mass after liver loss or injury.17 We analyzed5-ethynyl-20-deoxyuridine (EdU) incorporation 72 and96 hours after DEN administration.18 As compared

with wt mice, loss of TLR4 resulted in a substantialdecrease in proliferating hepatocytes (Fig. 3C,D). De-letion of TLR4 significantly reduced the magnitudeand duration of Jnk and Erk mitogenic signals afterDEN exposure compared to wt mice (Fig. 3E). There-fore, both the enhanced cell apoptosis and reducedproliferative response likely account for the observedlower susceptibility of TLR4 À/À mice to chemical

hepatocarcinogenesis.Despite the worsened tissue damage in TLR4 À/À

mice, these lesions were consistently associated withlower production of proinflammatory cytokines (TNFaand IL-6) than that observed in TLR4 -proficient mice(Fig. 4A). It is yet to be determined whether thereduced inflammatory response in TLR4 À/À mice wasresponsible for retardation of compensatory prolifera-tion. Therefore, we generated TLR4 -chimeric miceusing irradiation and bone-marrow transplantation(BMT). Successful BMT in all mice was confirmed by assessing expression of TLR4 using genomic DNA

from tail, blood, bone marrow (Fig. 4B). Expression of TLR4 on Kupffer cells (CD11bþ) isolated from thechimeric mice was demonstrated by flow cytometry (Supporting Information Fig. 5). As expected, chimericmice containing TLR4 À/À bone marrow showed a sig-nificant reduction in TNFa and IL-6 production in thelivers in response to DEN compared to mice trans-planted with wt bone marrow (Fig. 4C), and the levelsof circulating TNFa and IL-6 were also lower in chi-meric mice containing TLR4 À/À bone marrow (Fig.4D). In contrast, chimeric mice containing TLR4 -wtbone marrow, but TLR4 À/À resident liver cells (wt/

TLR4 À/À), had markedly elevated inflammatory responses relative to TLR4 À/À/TLR4 À/À mice. The res-toration of inflammatory activation in TLR4 À/À micecoincided with the presence of extended areas of epi-thelial proliferation, as visualized by immunohisto-chemical staining for Ki-67 (Fig. 4E). Kupffer cells arethe main targets of LPS in the liver, and they have a pivotal role in the induction of TNFa and IL-6. Asinactivation of Kupffer cells has been shown to cause a significant reduction in cytokine production and com-plementary proliferation in response to DEN,14 these




data clearly indicate that TLR4 in Kupffer cells wasgenerally required for inflammatory cytokine produc-

tion and compensatory proliferation in response toDEN exposure.

TLR4 Ablation Blocks NF-jB Activation and Sen-sitizes the Liver to ROS-Induced Toxicity. NF-jB isinvolved in signal transduction of various extracellularstress stimuli including DEN treatment14 and regulatesboth proinflammatory and protective responses in theliver.19,20 We detected a marked decrease in nuclearstaining of NF-jB, which is predominantly adjacentto the centrilobular area, in livers of DEN-treated-TLR4 À/À mice compared to DEN-treated wt mice (Fig.

5A). ChIP assay revealed reduced binding of NF-jB tothe promoter regions of its downstream genes including

Bcl-xl, A20 and MnSOD (Supporting Information Fig.6A) in TLR4 À / À mice than wt mice. Consistently, quan-titative PCR analysis revealed evidently decreased expres-sion of A20 and Bcl-xl (Fig. 5B). Previous results suggestthat ROS production contributes to DEN-induced cellapoptosis, whereas NF-jB inhibits oxidative stressthrough controlling expression of Mn-superoxide dis-mutase (MnSOD), a mitochondrial enzyme that detoxi-fies superoxide anions.21 Indeed, TLR4 À/À mice exhib-ited decreased expression MnSOD but notCuZnSOD (Fig. 5C). The levels of reduced glutathione

Fig. 3. TLR4 deficiency exacerbates liver

injury but attenuates compensatory hepa-

tocyte proliferation after DEN treatment.(A) ALT levels in mice of different geno-

types were determined at the indicated

times after DEN injection. *P < 0.05. (B)

TUNEL assay of liver sections from wt and

TLR4À /À

mice 48 hours after DEN admin-

istration. Scale bars ¼ 50 lm. (C) Repre-

sentative pictures of immunofluorescence

staining for incorporating EdU in the livers

of DEN-treated wt and TLR4À /À

mice at

72 hours. Yellow arrows showed EdU-posi-

tive cells, and slides were counterstained

with Hoechst 33342. Scale bars ¼ 100

lm. (D) Histological quantification of apo-

ptotic cells at 48 hours (left panel, HP:

200Â) and EdU-positive proliferating cells

at 72 hours and 96 hours (right panel,

HP: 400Â) after DEN injection, respec-

tively. *P < 0.05. (E) wt and TLR4À /À

mice were treated with DEN. At the indi-

cated times the livers were removed and

lysed to assess Jnk and Erk activation by

western blot with antibodies against the

indicated phosphoproteins. The densito-

metric analyses of the bands were shown

in the right.




(GSH), a major cellular antioxidant, were much lower inthe livers of DEN-treated TLR4 À/À mice than in simi-

larly treated wt mice (Fig. 5D). Conversely, DEN-induced lipid peroxidation, assessed by malondialdehyde(MDA) content,22 was substantially higher in TLR4 À/À

mice than in wt mice after DEN treatment (Fig. 5E).To further evaluate the contribution of oxidative

stress to the pathogenesis of liver disease in TLR4 À/À

mice, we placed a group of TLR4 À/À mice on a dietsupplemented with the antioxidant butylated hydrox-yanisole (BHA) for 2 days and then injected withDEN. TLR4 À/À mice on the BHA diet showed a strik-ing improvement of liver damage upon DEN expo-

sure, as shown by a drop of serum ALT to almost nor-mal levels and a strong reduction of hepatocyte

apoptosis (Fig. 5F; Supporting Information Fig. 6B).These findings indicate that loss of TLR4 enhancedDEN-induced liver damage through a mechanismlikely to depend on oxidative stress accumulation,

which is possibly due to the lack of NF-jB activation.TLR4 Protects Hepatocytes Against Carcinogen-

Induced Apoptosis. To determine the role of TLR4 inprotecting hepatocyte from apoptosis, we used TLR4-chimeric mice to assess whether the DEN-inducedinjury required TLR4 expression on liver parenchymalcells. Interestingly, a significant increase in serum ALT

Fig. 4. TLR4 expressed on bone marrow–

derived cells promotes inflammatory cytokine

production and subsequent compensatory he-

patocyte proliferation. (A) Mice of the indi-

cated genotypes were injected with DEN (n ¼

3 mice per time point), and liver RNA was

extracted at the indicated times. The levels of

TNF-a and IL-6 mRNAs were determined by

qPCR. (B)TLR4 genotyping using genomic DNA

from tail, blood, and bone marrow; the PTPN6

gene was used as an internal control. (C) Liv-

ers of chimeric mice were removed at 24hours after DEN injection for RNA extraction.

Expression of the indicated genes were deter-

mined by qPCR. (D) The serum TNF a and IL-6

were determined by ELISA. (E) Chimeric mice

were treated with DEN, and 48 hours later, liv-

ers were removed for hepatic proliferation

determination by Ki-67 staining. The average

number of Ki-67 positive cells per high power

field (400Â) was counted (right panel). *P <

0.05.




levels were present in TLR4 À/À/TLR4 À/À(TLR4 À/À

bone marrow TLR4 À/À mice), whereas minimalalteration was noted in samples derived from wt/wt,

wt/TLR4 À/À(wt bone marrow TLR4 À/À mice) mice,and, notably, TLR4 À/À/wt chimeric mice (Fig. 6A).

The apoptotic cells was consistent with the serum ALTestimation of liver damage (Supporting InformationFig. 7A). Thus, intact TLR4 expression on parenchy-mal and nonparenchymal cells seems to be both neces-sary for prevention of DEN-induced cell apoptosis.

We next investigated whether plasma LPS isrequired for the protective effect of TLR4 on DEN-induced apoptosis. Indeed, plasma LPS levels wereconsiderably elevated at 24 and 48 hours after DENinjection (Fig. 6B). However, compared to the controlgroup, administration of LPS simultaneously with or

12 hours prior to DEN resulted in a significantincrease in serum ALT at 24 hours after DEN treat-ment (Fig. 6C,E), indicating the presence of exacer-bated hepatocyte damage. Intriguingly, the serum levelsof ALT were drastically decreased in the LPS pre-con-

ditioning group at 48 hours post-DEN treatment, whereas the control mice displayed exaggerated liverdamage. The apoptotic liver cells (TUNEL positive) inLPS-treated mice were also decreased dramatically 48hours after DEN administration (Fig. 6D,F). Thesedata indicate that plasma LPS accumulation inducestransient liver inflammation and injury and also trig-gers a cascade of cellular events that prevent DEN-induced apoptosis. Interestingly, DEN induced a tran-sient increase in TLR4 expression in wt mice (Support-ing Information Fig. 7B), suggesting that TLR4 up-

Fig. 5. Loss of TLR4 suppresses NF-jBactivation and sensitizes the liver to ROS-

induced toxicity. (A) Immunostaining for NF-

jB (p65) in liver specimens prepared 4 hours

after DEN injection. Arrowheads indicate posi-

tive nuclear staining. Average number of p65-

positive nuclei per high-power field is shown

(right panel). Scale bars ¼ 50 lm. (B,C)

Mice were treated with DEN. The levels of

A20, Bcl-xl(B), CuZnSOD and MnSOD(C)

mRNA in the livers at indicated times were

determined by qPCR (n ¼ 3 per time point).

(D) Liver lysates prepared at the indicated

times after DEN injection were analyzed for

GSH content. (E) Lipid peroxidation was

examined at the indicated times after DEN

injection by measuring MDA in liver homoge-

nates. (F) The serum ALT level at the indi-

cated times in TLR4À /À

mice pretreated with

or without BHA after DEN administration. **P

< 0.01; *P < 0.05 versus control mice.




regulation might contribute to the repertoire of

defense mechanisms used by the hepatocyte againstcarcinogen-induced damage.

Gut-Derived LPS Promotes DEN-Induced Com- pensatory Proliferation. We next investigated whethergut-derived LPS is required for the DEN-induced he-patocytes compensatory proliferation. We treated mice

with a cocktail of nonabsorbable broad-spectrum anti-biotics23 and injected with DEN. This antibiotic cock-tail efficiently suppressed the increase in plasma LPSafter DEN injection (Fig. 7A). As shown in the Fig.7B and S8A, mice receiving this cocktail showed a sig-nificantly decrease in serum ALT and cell apoptosis,

indicating the presence of reduced hepatocyte damage.Moreover, the production of TNFa and IL-6 was sup-pressed (Fig. 7C), and the proliferating hepatocytes

were also significantly decreased (Fig. 7D) in the anti-biotics treated mice. Meanwhile, this cocktail treat-ment did not change the DEN metabolic enzymes(Supporting Information Fig. 8B), thus excluding any effects of antibiotics on DEN metabolism. In contrast,compared to control groups, administration of LPS 12hours prior to DEN resulted in a significant increasein proliferating hepatocytes (Fig. 7E). These data

clearly show that LPS can activate TLR4 signaling and

promote DEN induced hepatcytes proliferation.

Discussion

Although the liver is constantly exposed to micro-bial products from the enteric microflora, no obviousinflammation occurs in the healthy liver. This lack of response is to some extent explained by very specificimmunologic properties of the liver,24 namely ‘‘livertolerance’’. A breakdown of tolerance may induce aninappropriate immune response, resulting in acute andchronic inflammatory liver diseases. Activation of

innate immunity, specifically TLR4 signaling, hasemerged as a central component of the liver’s inflam-matory response to gut bacteria under pathologic con-ditions.8,25 Abundant data demonstrate that TLR4ligand endotoxin is elevated in experimental models of hepatic fibrosis 2 and patients with cirrhosis,26,27 but ithas been unclear whether and how the LPS/TLR4pathway amplifies the tumorigenic response of theliver. We have now observed increased endotoxin levelsin experimental liver cancer models upon administra-tion of the chemical carcinogen DEN. The attenuation

Fig. 6. LPS/TLR4 protects against DEN-

induced apoptosis. (A) Bone marrow-trans-

planted chimeric mice were treated with DEN,

and the serum ALT was measured at the indi-

cated times. *P < 0.05, versus other mice.

(B) The concentration of plasma LPS was

determined after DEN injection in both geno-

types of mice. *P < 0.05, versus untreated

mice. (C,D) wt mice were coinjected i.p. with

DEN and LPS. The serum ALT was measured

at the indicated points (C), the average num-

ber of TUNEL positive cells per high-power

field was counted (D). **P < 0.01, versus

mice treated with DEN alone. (E,F) wt mice

were injected with LPS 12 hours before DEN

administration. The serum ALT was measured

at the indicated points, the average number

of TUNEL positive cells per high-power field

(400Â) was counted. **P < 0.01; *P <

0.05, versus mice treated with DEN alone.




of DEN-induced endotoxemia and liver damage by antibiotics indicates that enhanced intestinal perme-ability to endotoxins appears to be the primary causeof chemically induced endotoxemia. Gut barrier dys-function leading to elevated intestinal permeability isalso considered the main cause of endotoxemia in alco-holic liver disease.28 Reduction of the levels of LPSresulted in suppression of inflammatory response, andthis may be the primary cause for the reduced liver fi-brosis and tumor development in the antibio-ticsþDEN treated rats and lower tumor load inTLR4 À / À mice.

Systemic infusion of endotoxin in healthy subjectscauses the release of proinflammatory mediators likeTNFa, IL-6 and inflammatory infiltration within theliver parenchyma and portal tracts.29 This capacity of proinflammatory immune system activation seems toplay a key role in the pathogenic effects of endotoxinand its receptor, TLR4, in liver diseases.8 During alco-hol-induced liver injury, proinflammatory mediatorsand liver injury are significantly reduced in TLR4-mutated mice despite elevated endotoxin levels.30

TLR4-mutant mice also strongly displayed less liver fi-

brosis upon bile duct ligation, indicating that the LPS-TLR4 pathway plays an important role in hepaticfibrogenesis25 Similarly, we found ablation of TLR4reduced the generation of inflammatory cytokines inDEN-induced liver early damage and cancer formationlater on. Production of these cytokines depends onLPS/TLR4 in hematopoietic-derived Kupffer cells, asdepletion of Kupffer cells14 or antibiotics treatment toreduce LPS levels prior to DEN treatment inhibitedthe induction of the inflammatory mediators. In agree-ment, inhibition of TLR4 activation in myeloid cells,exerted through transplantation of TLR4 À / À bone mar-

row, inhibited inflammatory responses following DEN-induced hepatic insult.

Because mature livers have extremely low rates of cellturnover, DEN-exposed hepatocytes do not yield geneti-cally transformed progeny in the absence of hepatomi-togens. TNFa and IL-6 were identified as the majorKupffer cell-produced factors that enhance the growthof surviving DEN-initiated hepatocytes.14 In light of the compensatory proliferation that promotes chemicalhepatocarcinogenesis was significantly reduced inKupffer cell-depleted mice,14 in chimeric mice

Fig. 7. Gut-derived LPS promotes DEN-

induced compensatory proliferation. Wt mice

were treated with the cocktail of antibiotics

and then injected with DEN. (A) The plasmaLPS levels were measured 48 hours after

DEN treatment. (B) ALT levels were measured

at the indicated times after DEN injection. (C)

The levels of TNF-a and IL-6 mRNAs in the

livers were determined by qPCR. (D) The aver-

age number of Ki-67–positive cells per high-

power field in the livers were shown. (E) The

average number of ki-67–positive cells per

high-power field in the livers from DEN-

treated mice pretreated with LPS and controls

at the indicated times. HP, 400Â; *P <

0.05; **P < 0.01.




containing TLR4 À / À bone marrow and in antibioticstreated mice, it is reasonable that activation of TLR4signaling by LPS in Kupffer cells is essential for driving expression of these proliferation-stimulating cytokines.Consistently, ablation of Myd88 led to a reduced inci-dence of HCC in response to treatment with DEN.31

Therefore, we concluded that LPS engagement of TLR4 in myeloid cells, specifically Kupffer cells, in theliver of mice subjected to DEN treatment producesparacrine-acting, tumor-promoting cytokines that notonly cause inflammation but also stimulate the prolifer-ation of adjacent premalignant hepatocytes.

Remarkably, TLR4 stimulates both liver cell prolifera-tion and survival, which explains the profound tumor-suppressive phenotype observed in TLR4 À/À mice.

Although TLR4 ablation did not result in spontaneouschronic liver pathology, these animals had increased sen-

sitivity to disease in a model of DEN-induced liverinjury. By contrast, mice pretreated with LPS were pro-tected against DEN-induced acute liver injury. Evading apoptosis is generally considered as a classic cellularmechanism contributing to cancer.32 Our results dem-onstrate that TLR4 activation is a survival signal allow-ing tumor cells to escape apoptosis; thus, inhibition of endotoxin accumulation has anti-oncogenic effects.Therefore, the increased epithelial apoptosis during tu-mor promotion and the decreased inflammatory com-pensatory proliferation may eventually halt liver tumorprogression in TLR4 À/À mice. Because NF-jB i s a

major downstream signaling component of TLR4 sig-naling, similar observations were also made in mice

with deletion of IKK b in hepatocytes. These miceexhibited a marked increase in hepatic injury, compen-satory proliferation, and development of HCC, whereasan additional deletion of IKK b in liver myeloid cellsprevented tumor development by depriving the trans-formed hepatocytes of essential growth factors.14 Toxin-mediated liver injury occurs largely through the genera-tion of ROS and direct mitochondrial damage, leading to hepatic necrosis with a lesser degree of apoptosis.33

By inducing both the antioxidant superoxide dismutase

(SOD) and antiapoptotic regulators (e.g., A20 and Bcl- xl ), LPS-induced NF-jB activation is cytoprotective ina broad spectrum of liver injuries mediated by death-re-ceptor ligands and liver toxins. It is of note that theinjection of exogenous LPS transiently exaggeratedDEN-induced liver damage. This enhanced damage ismost likely due to the synergistic effects of DEN-induced hepatic insult and cytokine-induced toxicity following the activation of Kupffer cells by LPS.34

However, the rapid decrease in serum alanine amino-transferase (ALT) level in these treated mice suggests

that the activation of TLR4 signaling in hepatocytestilts the balance toward liver protection upon DENexposure.

Previous studies have shown that there is a positivecorrelation between cell death and tumor load.14,31,35 Ithas been suggested that the response of stromal cells suchas Kupffer cells to the death of hepatocytes is crucial tothe proliferation and expansion of pre-cancerous cellsand tumor promotion.14 However, in the TLR4 À/À miceaggravated liver injury did not lead to an increased tumorload. Our data showed that DEN-induced liver injury

was accompanied by elevation of plasma LPS level. LPScan promote the cytokine production and hepatocytescompensatory proliferation by activating TLR4 expressedon myeloid cells, also it may have a protective role on theinitiated cells by activating TLR4 on hepatocytes. TLR4deficiency ablated the effects of the LPS, so the TLR4 À/À

mice displayed more severe liver damage, lower cytokineproduction and hepatocytes proliferation. Accordingly,the chimeric mice with wild type bone marrow hadhigher levels of cytokines (TNFa and IL-6) and moreproliferating hepatocytes than mice with TLR4 À/À bonemarrow. In addition to LPS, TLR4 has many endoge-nous ligands, such as high mobility group box (HMGB)1, Heat Shock Proteins (HSPs), most of which werereleased by necrotic cells36 In conclusion, our data showed that LPS/TLR4 played an important role in theDEN-induced hepatocarcinogenesis.

Recognition of commensal bacteria by TLR4 is cru-

cial in the control of intestinal epithelial homeostasisand protection from direct injury, the disturbance of

which can result in severe chronic inflammatory boweldisease (IBD)23 Here, we show that TLR4 activationalso affected tissue homeostasis in the adult liver fol-lowing injury. A pathological increase in plasma LPS,because of abnormal entero-hepatic circulation as wellas delayed endotoxin clearance by liver, resulted inpromotion of chemical hepatocarcinogenesis. Our find-ings emphasize the existence of a crucial balancebetween gut and liver homeostasis, which is closely linked by ligands derived from indigenous microflora.

Deregulated liver homeostasis may promote intestinalbacterial overgrowth and structural changes in intesti-nal mucosa, which in turn cause plasma endotoxinaccumulation and induce the protective and growth-promoting effects of TLR4 activation in transformedliver cells. Our findings suggest that reducing gutinjury, improving blood flow to the gastrointestinaltract, and lessening the gut translocation of endotoxinmay improve liver function in patients with cirrhosis

with potential to progress into HCC. More impor-tantly, it would be interesting to determine whether




the manipulation of gut-flora with anti-endotoxineffects will prove beneficial in preventing or delaying HCC development.

Acknowledgment: We thank Dong-Ping Hu, Dan-Dan Huang, Shan-Hua Tang, Lin-Na Guo, and Dan

Cao for their technical assistances. We also thank Pro-fessor Gen-Sheng Feng for reviewing this manuscript.

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LPS Blocheaza Apoptoza in Cel Tumorale Hepatice

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