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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Increasing evidence suggests that the presence of endotoxemia is of substantial clinical relevance to patients with cirrhosis, but it is unclear whether and how gut-derived LPS amplifies the tumorigenic response of the liver. We found that the circulating levels of LPS were elevated in animal models of carcinogen-induced hepatocarcinogenesis. Reduction of LPS using antibiotics regimen in rats or genetic ablation of its receptor Toll-like receptor 4 (TLR4) in mice prevented excessive tumor growth and multiplicity. Additional investigation revealed that TLR4 ablation sensitizes the liver to carcinogen-induced toxicity via blocking NF-κB activation and sensitizing the liver to reactive oxygen species (ROS)-induced toxicity, 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 LPS in 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 hepatocellular carcinoma (HCC) and manipulation of the gut flora to prevent pathogenic bacterial translocation and endotoxin absorption may favorably influence liver function in patients with cirrhosis who are at risk of developing HCC. (Hepatology 2010.)

Hepatocellular carcinoma (HCC) is closely associated with chronic inflammatory liver diseases, such as those caused by viral infection, alcohol consumption, or hepatic metabolic disorders. Chronic liver disease leads to continual injury and a wound-healing 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 common finding in the portal and systemic circulation of patients with cirrhosis.2 This accumulation is likely due to changes in the intestinal mucosal permeability and increased bacterial translocation, coupled with deficient clarification of the hepatic reticuloendothelial system.3 High levels of endotoxin may be responsible for the pathogenesis of the chronic inflammatory alterations that characterize cirrhosis and, hence, the major complications in this disease.4

TLR4 is a pattern recognition receptor that recognizes endotoxin and signals through adaptor molecules myeloid differentiation primary response gene (88) (MyD88) and TIR-domain-containing adapter-inducing interferon-β (TRIF) to activate transcription factors that initiate innate immunity.5 The liver is well equipped to respond to endotoxin because TLR4 is present on both parenchymal cells (hepatocytes) and nonparenchymal cells such as Kupffer cells. Both cell populations possess intact TLR4 signaling pathways.6,7 Kupffer cells are the best-characterized 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)-α and interleukin (IL)-6) and affect hepatic sinusoids to increase vascular permeability.9 Although hepatocytes also express low levels of the TLR4 receptor, they are only weakly responsive to LPS and may serve to uptake and remove endotoxin from the portal and systemic circulation.10 The effects of endotoxin in vivo on hepatic function and tumorigenesis are not well defined.

Robust clinical and epidemiologic data support the role of inflammation as a key player in HCC development.11 However, the exact molecular mechanisms and gatekeepers accounting for cellular transformation remain elusive. Given the important role of NF-κB signaling in mediating inflammatory signals, attention has been focused on its role in mediating the link between inflammation and the development of liver tumors.12 Inhibiting NF-κB obstructs later stages of tumor progression in multi-drug resistant (Mdr) 2-deficient mice, which develop HCC in the context of chronic bile duct inflammation.13 By contrast, mice lacking the I-kappa-B kinase-beta (IKKβ) specifically in hepatocytes exhibit a marked increase in chemically induced hepatocarcinogenesis, suggesting that NF-κB has a protective function against HCC development. Interestingly, compared with the deletion of IKKβ only in hepatocytes, the additional deletion in Kupffer cells results in a remarkable decrease in tumor load.14 These apparently contradictory conclusions may reflect the distinct roles for inflammatory signals in epithelial cells and inflammatory cells during HCC formation.

Here, we show that endogenous endotoxin accumulation regulates the survival and proliferation of hepatocytes and their preneoplastic derivatives during chemically induced hepatocarcinogenesis. The cytoprotective and protumorigenic effects of endotoxin are mainly due to elevated NF-κB activity in premalignant epithelial cells, which suppresses apoptosis, thus promoting the cells' survival and subsequent capacity to form tumors. Meanwhile, LPS engagement of the same pathway in tumor-associated inflammatory cells initiates transcription of proinflammatory cytokines, which in turn stimulate tumor proliferation. These results suggest that preventing plasma endotoxin accumulation could have a beneficial impact on liver function for patients with cirrhosis with the potential to progress to hepatoma.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

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 the Shanghai Experimental Center, Chinese Science Academy, Shanghai, and maintained at an animal facility under pathogen-free conditions. Male wild type (wt; C57BL/10SnJ), and TLR4-deficient (TLR4−/−; C57BL/10ScNJ) mice were obtained from the Model Animal Research Center of Nanjing University, Nan Jing, China. All animals received humane care according to the criteria outlined in the “Guide for the Care and Use of Laboratory Animals” prepared by the National Academy of Sciences and published by the NIH (publication 86-23 revised 1985). For detailed information related to animal experiments, see the Supporting Experimental Procedures.

Immunohistochemistry.

All paraffin-embedded liver tissues were stained with hematoxylin and eosin (H&E) for analysis of morphologic changes. The primary antibodies were as follows: cyclin D1, phospho-c-Jun (Cell Signaling Technology) and F4/80 (Santa Cruz Biotechnology, Santa Cruz, CA). Apoptosis was assessed by TUNEL staining of paraffin-embedded slides (Calbiochem, La Jolla, CA). Proliferation was assessed by immunostaining for 5-ethynyl-2′-deoxyuridine (EdU; Ruibo Biotech, Guangzhou, China) or Ki-67 (Labvision, Fremont, CA) staining.

Bone Marrow Transplantation.

Recipient mice were lethally irradiated with 9.0 Gy at a rate of 70 cGy/minute using a cobalt-source gamma-irradiator (the Irradiation Center of the Second Military Medical University). Irradiated recipient mice were i.v. injected with approximately 1 × 107 bone marrow cells in 200 μL of PBS. They were subjected to DEN treatment 5 weeks after transplantation. To demonstrate the success of BMT in TLR4−/− 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).

Statistical Analysis.

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

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Depletion of Host Microflora Suppresses Chemical Hepatocarcinogenesis in Rats.

Chronically exposing rats to diethylnitrosamine (DEN) provides a multistage hepatocarcinogenesis model for studying human HCC, which allows one to distinguish tumor initiation from promotion (Supporting Information Fig. 1A). Because the liver is directly downstream from the gut, we hypothesized that translocated microbes or endotoxin could promote chemically induced HCC. To address this question, we first used the rat HCC model to assess the plasma levels of LPS at different stages of DEN-induced hepatocarcinogenesis. Of interest, the plasma levels of LPS were elevated during tumor progression (Fig. 1A), indicating that plasma endotoxin may be a critical cofactor in chemically induced hepatocarcinogenesis.

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Figure 1. Antibiotics treatment suppresses chemical hepatocarcinogenesis in rats. (A) The plasma LPS concentration at the indicated time after DEN injection in the four groups (Antibiotics + DEN, DEN alone, Antibiotics alone and untreated control, n = 6-8). **P < 0.01; *P < 0.05 versus antibiotics + DEN group. (B) DEN induced TNFα 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 antibiotics treatment 21 weeks after DEN administration. (D) Numbers of tumors (≥1 mm) and maximum tumor sizes (diameters) in DEN and Antibiotics+DEN 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 = 100 μm. HP, high power field (400×).

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To address whether the circulating LPS in DEN-treated animals augmented tumor induction, we determined whether their removal with LPS antagonist polymyxin B (PMB),15 and neomycin that is bactericidal mainly 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 antibiotics alone caused no phenotypic manifestation in the liver (Supporting Information Fig. 1B), antibiotic treatment significantly reduced the levels of LPS in their plasma (Fig. 1A). Treating rats with antibiotics significantly reduced the cytokines (TNFα and IL-6) 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 rats developed liver tumors 21 weeks after DEN injection, the number of detectable tumors (≥1 mm), maximal diameters of tumors and the relative liver weight were significantly decreased in antibiotics+DEN 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), but there was no significant difference in the apoptotic cells between the two groups (Supporting Information Fig. 1D). These data confirmed the role of microbial LPS in contributing to tumor induction after DEN treatment.

Deletion of TLR4 Greatly Decreases Chemically Induced Hepatocarcinogenesis in Mice.

A single DEN injection to 15-day-old male mice also results in efficient HCC induction.16 Because LPS is thought to exert 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 strains exhibited spontaneous liver dysfunction or HCC, up to 1 year of age (data not shown). Upon DEN injection on postnatal day 15, all wt males developed typical HCCs within 10 months, but tumor incidence was 25% lower in TLR4−/− mice (Fig. 2A,B). Furthermore, a dramatic decrease in the number of detectable HCCs was observed in TLR4−/− mice relative to wt controls (Fig. 2C). The maximal tumor diameters were also significantly smaller in TLR4−/− mice compared to wt controls (Fig. 2D). Consistantly, malignant liver tumors in TLR4−/− mice displayed strongly decreased proliferation (Ki-67) and lower levels of phospho-c-Jun and cyclin D1, which are needed for cell proliferation, compared to in wt mice. Furthermore, apoptotic tumor cells were more frequently observed in tumors from TLR4−/− mice than in tumors from wt mice (Fig. 2E). Moreover, the serum ALT was modestly reduced in tumor-bearing TLR4−/− mice compared with tumor-bearing wt mice, indicating a lower tumor load indirectly (Supporting Information Fig. 2A).

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Figure 2. Loss of TLR4 decreases DEN-induced tumor development in mice. (A) Representative livers of 10-month-old DEN-treated male wt and TLR4−/− mice. (B) Tumor incidence, (C) numbers of tumors (≥0.5 mm) and (D) maximum tumor sizes (diameters) in livers of male wt (n = 23) and TLR4−/−(n = 28) mice 10 months after DEN injection. (E) Histological and immunohistochemical analysis of wt and TLR4−/− 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 TNFα and IL-6 mRNA in wt and TLR4−/− tumors (n = 5). HP, high-power field (200×). ***P < 0.001; **P < 0.01; *P < 0.05. All scale bars = 50 μm.

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Because TLR4 activation of innate immune cells resulted in the production of several inflammatory cytokines that stimulated tumor growth, we thus assessed whether the absence of TLR4 influences cancer-linked inflammatory responses. Indeed, in addition to the smaller number and size of tumors in TLR4−/− mice, these lesions were consistently associated with reduced infiltration of macrophages (F4/80 staining) compared to wt mice (Supporting Information Fig. 2B). Concordantly, the expression levels of hepatomitogens (TNFα and IL-6) were evidently reduced in TLR4−/− HCCs relative to controls (Fig. 2F). However, unlike the DEN-induced rat HCC model, no evident liver fibrosis was found in this model (Supporting Information Fig. 2C). Thus, the loss of TLR4 protects the liver from chemically induced carcinogenesis, possibly because of less pronounced inflammation, reduced proliferation, and enhanced apoptosis in tumor cells.

Loss of TLR4 Exacerbates DEN-Induced Liver Injury but Lessens Compensatory Proliferation.

The finding that loss of TLR4 reduced the susceptibility of mice to chemical hepatocarcinogenesis prompted us to examine the early effects of DEN on cell behavior and signal transduction. At 24 or 48 hours after DEN injection, TLR4−/− males displayed a considerable elevation of ALT in serum and an increased number of TUNEL-positive cells in liver, indicating the presence of exacerbated hepatocyte damage (Fig. 3A,B,D). The histological evidence of damage was likewise increased in TLR4−/− mice compared to wt mice (Supporting Information Fig. 3). DEN administration led to a rapid increase in expression of the p53 target genes p21 and Mdm2, but the response was similar in wt and TLR4−/− mice, excluding the possibility that TLR4 affects DEN metabolism (Supporting Information Fig. 4). These data suggest that deletion of TLR4 may result in more DEN-induced cell death.

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Figure 3. TLR4 deficiency exacerbates liver injury but attenuates compensatory hepatocyte proliferation after DEN treatment. (A) ALT levels in mice of different genotypes were determined at the indicated times after DEN injection. *P < 0.05. (B) TUNEL assay of liver sections from wt and TLR4−/− mice 48 hours after DEN administration. Scale bars = 50 μm. (C) Representative pictures of immunofluorescence staining for incorporating EdU in the livers of DEN-treated wt and TLR4−/− mice at 72 hours. Yellow arrows showed EdU-positive cells, and slides were counterstained with Hoechst 33342. Scale bars = 100 μm. (D) Histological quantification of apoptotic cells at 48 hours (left panel, HP: 200×) and EdU-positive proliferating cells at 72 hours and 96 hours (right panel, HP: 400×) after DEN injection, respectively. *P < 0.05. (E) wt and TLR4−/− mice were treated with DEN. At the indicated times the livers were removed and lysed to assess Jnk and Erk activation by western blot with antibodies against the indicated phosphoproteins. The densitometric analyses of the bands were shown in the right.

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Figure 4. TLR4 expressed on bone marrow–derived cells promotes inflammatory cytokine production and subsequent compensatory hepatocyte proliferation. (A) Mice of the indicated genotypes were injected with DEN (n = 3 mice per time point), and liver RNA was extracted at the indicated times. The levels of TNF-α 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) Livers of chimeric mice were removed at 24 hours after DEN injection for RNA extraction. Expression of the indicated genes were determined by qPCR. (D) The serum TNFα and IL-6 were determined by ELISA. (E) Chimeric mice were treated with DEN, and 48 hours later, livers were removed for hepatic proliferation determination by Ki-67 staining. The average number of Ki-67 positive cells per high power field (400×) was counted (right panel). *P < 0.05.

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The mammalian liver possesses an extraordinary capacity for compensatory growth and thereby maintains liver mass after liver loss or injury.17 We analyzed 5-ethynyl-2′-deoxyuridine (EdU) incorporation 72 and 96 hours after DEN administration.18 As compared with wt mice, loss of TLR4 resulted in a substantial decrease in proliferating hepatocytes (Fig. 3C,D). Deletion of TLR4 significantly reduced the magnitude and duration of Jnk and Erk mitogenic signals after DEN exposure compared to wt mice (Fig. 3E). Therefore, both the enhanced cell apoptosis and reduced proliferative response likely account for the observed lower susceptibility of TLR4−/− mice to chemical hepatocarcinogenesis.

Despite the worsened tissue damage in TLR4−/− mice, these lesions were consistently associated with lower production of proinflammatory cytokines (TNFα and IL-6) than that observed in TLR4-proficient mice (Fig. 4A). It is yet to be determined whether the reduced inflammatory response in TLR4−/− mice was responsible for retardation of compensatory proliferation. Therefore, we generated TLR4-chimeric mice using 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 (CD11b+) isolated from the chimeric mice was demonstrated by flow cytometry (Supporting Information Fig. 5). As expected, chimeric mice containing TLR4−/− bone marrow showed a significant reduction in TNFα and IL-6 production in the livers in response to DEN compared to mice transplanted with wt bone marrow (Fig. 4C), and the levels of circulating TNFα and IL-6 were also lower in chimeric mice containing TLR4−/− bone marrow (Fig. 4D). In contrast, chimeric mice containing TLR4-wt bone marrow, but TLR4−/− resident liver cells (wt/TLR4−/−), had markedly elevated inflammatory responses relative to TLR4−/−/TLR4−/− mice. The restoration of inflammatory activation in TLR4−/− mice coincided with the presence of extended areas of epithelial proliferation, as visualized by immunohistochemical staining for Ki-67 (Fig. 4E). Kupffer cells are the main targets of LPS in the liver, and they have a pivotal role in the induction of TNFα and IL-6. As inactivation of Kupffer cells has been shown to cause a significant reduction in cytokine production and complementary proliferation in response to DEN,14 these data clearly indicate that TLR4 in Kupffer cells was generally required for inflammatory cytokine production and compensatory proliferation in response to DEN exposure.

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Figure 5. Loss of TLR4 suppresses NF-κB activation and sensitizes the liver to ROS-induced toxicity. (A) Immunostaining for NF-κB (p65) in liver specimens prepared 4 hours after DEN injection. Arrowheads indicate positive nuclear staining. Average number of p65-positive nuclei per high-power field is shown (right panel). Scale bars = 50 μm. (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 = 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 homogenates. (F) The serum ALT level at the indicated times in TLR4−/− mice pretreated with or without BHA after DEN administration. **P < 0.01; *P < 0.05 versus control mice.

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TLR4 Ablation Blocks NF-κB Activation and Sensitizes the Liver to ROS-Induced Toxicity.

NF-κB is involved in signal transduction of various extracellular stress stimuli including DEN treatment14 and regulates both proinflammatory and protective responses in the liver.19,20 We detected a marked decrease in nuclear staining of NF-κB, which is predominantly adjacent to the centrilobular area, in livers of DEN-treated-TLR4−/− mice compared to DEN-treated wt mice (Fig. 5A). ChIP assay revealed reduced binding of NF-κB to the promoter regions of its downstream genes including Bcl-xl, A20 and MnSOD(Supporting Information Fig. 6A) in TLR4−/− mice than wt mice. Consistently, quantitative PCR analysis revealed evidently decreased expression of A20 and Bcl-xl(Fig. 5B). Previous results suggest that ROS production contributes to DEN-induced cell apoptosis, whereas NF-κB inhibits oxidative stress through controlling expression of Mn-superoxide dismutase (MnSOD), a mitochondrial enzyme that detoxifies superoxide anions.21 Indeed, TLR4−/− mice exhibited decreased expression MnSOD but not CuZnSOD(Fig. 5C). The levels of reduced glutathione (GSH), a major cellular antioxidant, were much lower in the livers of DEN-treated TLR4−/− mice than in similarly treated wt mice (Fig. 5D). Conversely, DEN-induced lipid peroxidation, assessed by malondialdehyde (MDA) content,22 was substantially higher in TLR4−/− mice than in wt mice after DEN treatment (Fig. 5E).

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Figure 6. LPS/TLR4 protects against DEN-induced apoptosis. (A) Bone marrow-transplanted chimeric mice were treated with DEN, and the serum ALT was measured at the indicated times. *P < 0.05, versus other mice. (B) The concentration of plasma LPS was determined after DEN injection in both genotypes 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 number 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 (400×) was counted. **P < 0.01; *P < 0.05, versus mice treated with DEN alone.

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To further evaluate the contribution of oxidative stress to the pathogenesis of liver disease in TLR4−/− mice, we placed a group of TLR4−/− mice on a diet supplemented with the antioxidant butylated hydroxyanisole (BHA) for 2 days and then injected with DEN. TLR4−/− mice on the BHA diet showed a striking improvement of liver damage upon DEN exposure, as shown by a drop of serum ALT to almost normal levels and a strong reduction of hepatocyte apoptosis (Fig. 5F; Supporting Information Fig. 6B). These findings indicate that loss of TLR4 enhanced DEN-induced liver damage through a mechanism likely to depend on oxidative stress accumulation, which is possibly due to the lack of NF-κB activation.

TLR4 Protects Hepatocytes Against Carcinogen-Induced Apoptosis.

To determine the role of TLR4 in protecting hepatocyte from apoptosis, we used TLR4-chimeric mice to assess whether the DEN-induced injury required TLR4 expression on liver parenchymal cells. Interestingly, a significant increase in serum ALT levels were present in TLR4−/−/TLR4−/−(TLR4−/− bone marrow [RIGHTWARDS ARROW]TLR4−/− mice), whereas minimal alteration was noted in samples derived from wt/wt, wt/TLR4−/−(wt bone marrow [RIGHTWARDS ARROW]TLR4−/− mice) mice, and, notably, TLR4−/−/wt chimeric mice (Fig. 6A). The apoptotic cells was consistent with the serum ALT estimation of liver damage (Supporting Information Fig. 7A). Thus, intact TLR4 expression on parenchymal and nonparenchymal cells seems to be both necessary for prevention of DEN-induced cell apoptosis.

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Figure 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 plasma LPS 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-α and IL-6 mRNAs in the livers were determined by qPCR. (D) The average 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, 400×; *P < 0.05; **P < 0.01.

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We next investigated whether plasma LPS is required for the protective effect of TLR4 on DEN-induced apoptosis. Indeed, plasma LPS levels were considerably elevated at 24 and 48 hours after DEN injection (Fig. 6B). However, compared to the control group, administration of LPS simultaneously with or 12 hours prior to DEN resulted in a significant increase in serum ALT at 24 hours after DEN treatment (Fig. 6C,E), indicating the presence of exacerbated hepatocyte damage. Intriguingly, the serum levels of ALT were drastically decreased in the LPS pre-conditioning group at 48 hours post-DEN treatment, whereas the control mice displayed exaggerated liver damage. The apoptotic liver cells (TUNEL positive) in LPS-treated mice were also decreased dramatically 48 hours after DEN administration (Fig. 6D,F). These data indicate that plasma LPS accumulation induces transient liver inflammation and injury and also triggers a cascade of cellular events that prevent DEN-induced apoptosis. Interestingly, DEN induced a transient increase in TLR4 expression in wt mice (Supporting Information Fig. 7B), suggesting that TLR4 up-regulation might contribute to the repertoire of defense mechanisms used by the hepatocyte against carcinogen-induced damage.

Gut-Derived LPS Promotes DEN-Induced Compensatory Proliferation.

We next investigated whether gut-derived LPS is required for the DEN-induced hepatocytes compensatory proliferation. We treated mice with a cocktail of nonabsorbable broad-spectrum antibiotics23 and injected with DEN. This antibiotic cocktail efficiently suppressed the increase in plasma LPS after DEN injection (Fig. 7A). As shown in the Fig. 7B and S8A, mice receiving this cocktail showed a significantly decrease in serum ALT and cell apoptosis, indicating the presence of reduced hepatocyte damage. Moreover, the production of TNFα and IL-6 was suppressed (Fig. 7C), and the proliferating hepatocytes were also significantly decreased (Fig. 7D) in the antibiotics treated mice. Meanwhile, this cocktail treatment 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 12 hours prior to DEN resulted in a significant increase in proliferating hepatocytes (Fig. 7E). These data clearly show that LPS can activate TLR4 signaling and promote DEN induced hepatcytes proliferation.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Although the liver is constantly exposed to microbial products from the enteric microflora, no obvious inflammation occurs in the healthy liver. This lack of response is to some extent explained by very specific immunologic properties of the liver,24 namely “liver tolerance”. A breakdown of tolerance may induce an inappropriate immune response, resulting in acute and chronic inflammatory liver diseases. Activation of innate immunity, specifically TLR4 signaling, has emerged as a central component of the liver's inflammatory response to gut bacteria under pathologic conditions.8,25 Abundant data demonstrate that TLR4 ligand endotoxin is elevated in experimental models of hepatic fibrosis2 and patients with cirrhosis,26,27 but it has been unclear whether and how the LPS/TLR4 pathway amplifies the tumorigenic response of the liver. We have now observed increased endotoxin levels in experimental liver cancer models upon administration of the chemical carcinogen DEN. The attenuation of DEN-induced endotoxemia and liver damage by antibiotics indicates that enhanced intestinal permeability to endotoxins appears to be the primary cause of chemically induced endotoxemia. Gut barrier dysfunction leading to elevated intestinal permeability is also considered the main cause of endotoxemia in alcoholic liver disease.28 Reduction of the levels of LPS resulted in suppression of inflammatory response, and this may be the primary cause for the reduced liver fibrosis and tumor development in the antibiotics+DEN treated rats and lower tumor load in TLR4−/− mice.

Systemic infusion of endotoxin in healthy subjects causes the release of proinflammatory mediators like TNFα, IL-6 and inflammatory infiltration within the liver parenchyma and portal tracts.29 This capacity of proinflammatory immune system activation seems to play a key role in the pathogenic effects of endotoxin and its receptor, TLR4, in liver diseases.8 During alcohol-induced liver injury, proinflammatory mediators and liver injury are significantly reduced in TLR4-mutated mice despite elevated endotoxin levels.30TLR4-mutant mice also strongly displayed less liver fibrosis upon bile duct ligation, indicating that the LPS-TLR4 pathway plays an important role in hepatic fibrogenesis25 Similarly, we found ablation of TLR4 reduced the generation of inflammatory cytokines in DEN-induced liver early damage and cancer formation later on. Production of these cytokines depends on LPS/TLR4 in hematopoietic-derived Kupffer cells, as depletion of Kupffer cells14 or antibiotics treatment to reduce LPS levels prior to DEN treatment inhibited the induction of the inflammatory mediators. In agreement, inhibition of TLR4 activation in myeloid cells, exerted through transplantation of TLR4−/− bone marrow, inhibited inflammatory responses following DEN-induced hepatic insult.

Because mature livers have extremely low rates of cell turnover, DEN-exposed hepatocytes do not yield genetically transformed progeny in the absence of hepatomitogens. TNFα and IL-6 were identified as the major Kupffer cell-produced factors that enhance the growth of surviving DEN-initiated hepatocytes.14 In light of the compensatory proliferation that promotes chemical hepatocarcinogenesis was significantly reduced in Kupffer cell-depleted mice,14 in chimeric mice containing TLR4−/− bone marrow and in antibiotics treated mice, it is reasonable that activation of TLR4 signaling by LPS in Kupffer cells is essential for driving expression of these proliferation-stimulating cytokines. Consistently, ablation of Myd88 led to a reduced incidence of HCC in response to treatment with DEN.31 Therefore, we concluded that LPS engagement of TLR4 in myeloid cells, specifically Kupffer cells, in the liver of mice subjected to DEN treatment produces paracrine-acting, tumor-promoting cytokines that not only cause inflammation but also stimulate the proliferation of adjacent premalignant hepatocytes.

Remarkably, TLR4 stimulates both liver cell proliferation and survival, which explains the profound tumor-suppressive phenotype observed in TLR4−/− mice. Although TLR4 ablation did not result in spontaneous chronic liver pathology, these animals had increased sensitivity to disease in a model of DEN-induced liver injury. By contrast, mice pretreated with LPS were protected against DEN-induced acute liver injury. Evading apoptosis is generally considered as a classic cellular mechanism contributing to cancer.32 Our results demonstrate that TLR4 activation is a survival signal allowing tumor cells to escape apoptosis; thus, inhibition of endotoxin accumulation has anti-oncogenic effects. Therefore, the increased epithelial apoptosis during tumor promotion and the decreased inflammatory compensatory proliferation may eventually halt liver tumor progression in TLR4−/− mice. Because NF-κB is a major downstream signaling component of TLR4 signaling, similar observations were also made in mice with deletion of IKKβ in hepatocytes. These mice exhibited a marked increase in hepatic injury, compensatory proliferation, and development of HCC, whereas an additional deletion of IKKβ in liver myeloid cells prevented tumor development by depriving the transformed hepatocytes of essential growth factors.14 Toxin-mediated liver injury occurs largely through the generation 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-κB activation is cytoprotective in a broad spectrum of liver injuries mediated by death-receptor ligands and liver toxins. It is of note that the injection of exogenous LPS transiently exaggerated DEN-induced liver damage. This enhanced damage is most 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 aminotransferase (ALT) level in these treated mice suggests that the activation of TLR4 signaling in hepatocytes tilts the balance toward liver protection upon DEN exposure.

Previous studies have shown that there is a positive correlation between cell death and tumor load.14,31,35 It has been suggested that the response of stromal cells such as Kupffer cells to the death of hepatocytes is crucial to the proliferation and expansion of pre-cancerous cells and tumor promotion.14 However, in the TLR4−/− mice aggravated liver injury did not lead to an increased tumor load. Our data showed that DEN-induced liver injury was accompanied by elevation of plasma LPS level. LPS can promote the cytokine production and hepatocytes compensatory proliferation by activating TLR4 expressed on myeloid cells, also it may have a protective role on the initiated cells by activating TLR4 on hepatocytes. TLR4 deficiency ablated the effects of the LPS, so the TLR4−/− mice displayed more severe liver damage, lower cytokine production and hepatocytes proliferation. Accordingly, the chimeric mice with wild type bone marrow had higher levels of cytokines (TNFα and IL-6) and more proliferating hepatocytes than mice with TLR4−/− bone marrow. In addition to LPS, TLR4 has many endogenous ligands, such as high mobility group box (HMGB) 1, Heat Shock Proteins (HSPs), most of which were released by necrotic cells36 In conclusion, our data showed that LPS/TLR4 played an important role in the DEN-induced hepatocarcinogenesis.

Recognition of commensal bacteria by TLR4 is crucial in the control of intestinal epithelial homeostasis and protection from direct injury, the disturbance of which can result in severe chronic inflammatory bowel disease (IBD)23 Here, we show that TLR4 activation also affected tissue homeostasis in the adult liver following injury. A pathological increase in plasma LPS, because of abnormal entero-hepatic circulation as well as delayed endotoxin clearance by liver, resulted in promotion of chemical hepatocarcinogenesis. Our findings emphasize the existence of a crucial balance between gut and liver homeostasis, which is closely linked by ligands derived from indigenous microflora. Deregulated liver homeostasis may promote intestinal bacterial overgrowth and structural changes in intestinal mucosa, which in turn cause plasma endotoxin accumulation and induce the protective and growth-promoting effects of TLR4 activation in transformed liver cells. Our findings suggest that reducing gut injury, improving blood flow to the gastrointestinal tract, and lessening the gut translocation of endotoxin may improve liver function in patients with cirrhosis with potential to progress into HCC. More importantly, it would be interesting to determine whether the manipulation of gut-flora with anti-endotoxin effects will prove beneficial in preventing or delaying HCC development.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

We thank Dong-Ping Hu, Dan-Dan Huang, Shan-Hua Tang, Lin-Na Guo, and Dan Cao for their technical assistances. We also thank Professor Gen-Sheng Feng for reviewing this manuscript.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

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