Promotion of hepatocellular carcinoma by the intestinal microbiota and TLR4

Abstract

Increased translocation of intestinal bacteria is a hallmark of chronic liver disease and contributes to hepatic inflammation and fibrosis. Here we tested the hypothesis that the intestinal microbiota and Toll-like receptors (TLRs) promote hepatocellular carcinoma (HCC), a long-term consequence of chronic liver injury, inflammation, and fibrosis. Hepatocarcinogenesis in chronically injured livers depended on the intestinal microbiota and TLR4 activation in non-bone-marrow-derived resident liver cells. TLR4 and the intestinal microbiota were not required for HCC initiation but for HCC promotion, mediating increased proliferation, expression of the hepatomitogen epiregulin, and prevention of apoptosis. Gut sterilization restricted to late stages of hepatocarcinogenesis reduced HCC, suggesting that the intestinal microbiota and TLR4 represent therapeutic targets for HCC prevention in advanced liver disease.

Figure 1. TLR4 contributes to hepatocarcinogenesis

A. Tlr4^WT mice (n=10) and Tlr4^mut (n=9) were injected with DEN (100 mg/kg i.p.) at the age of 12 weeks followed by 6 injections of CCl₄ (0.5 ml/kg i.p.) and sacrificed 10.5 months after DEN. Shown are tumor number, largest tumor size, liver-body weight ratio, H&E sections and representative images. B. Tlr4^WT (n=14) and Tlr4^mut mice (n=15) were treated with DEN followed by 2 injections of CCl₄. Gene expression and ALT and AST levels were determined 48h after the second CCl₄ injection. Fold induction in comparison to untreated livers. Data are represented as means ± SD. * p<0.05, ** p<0.01. See also Figure S1.

Figure 2. The intestinal microbiota promotes hepatocarcinogenesis

A. WT mice (C3H/HeOuJ) were treated with DEN plus 6 injections of CCl₄ and either did (n=11) or did not (n=7) receive antibiotics (ampicillin, neomycin, metronidazole, vancomycin) in their drinking water starting 2 weeks before and throughout the entire period. Shown are representative images of ceca, tumors and H&E sections. B. WT mice were treated with DEN plus 2 injections of CCl₄, and either did (“Abx”, n=14) or did not (“Ctrl”, n=9) receive the above-described antibiotics cocktail 2 weeks before DEN injection until they were sacrificed 48h after the last CCl₄ injection. Gene expression is expressed as fold induction in comparison to untreated liver. Liver injury was assessed by ALT and AST measurements C. Tlr4^WT, Tlr4^mut and Tlr4^WT mice treated with antibiotics were injected with DEN and two subsequent injections of CCl₄ and sacrificed 48h later. RNA of livers from untreated Tlr4^WT mice (n=3), DEN plus CCl₄-treated Tlr4^WT (n=4), DEN plus CCl₄-treated Tlr4^mut mice (n=4) and antibiotics plus DEN plus CCl₄-treated Tlr4^WT mice (n=4) were used for microarray analysis. The left heatmap shows changes of 1752 genes in the Tlr4^mut and antibiotics-treated mice that were (i) at least 1.8-fold up- or downregulated by DEN plus CCl₄ treatment in Tlr4^WT mice in comparison to untreated mice, and (ii) 1.8-fold up- or downregulated in DEN plus CCl₄-treated Tlr4^mut mice in comparison to DEN plus CCl₄-treated Tlr4^WT mice. The right heatmap shows genes fulfilling the same conditions as described above restricted to the GO categories 0007049, 0009611 and 0005578. Data are represented as means ± SD. * p<0.05, ** p<0.01. See also Figure S2.

Figure 3. LPS promotes hepatocarcinogenesis but does not change the intestinal bacterial microbiota

A. Tlr4^WT (C3H/HeOuJ) mice were treated with DEN plus 6 injections of CCl₄ and either received LPS (300 µg/kg/d, n=9) or PBS (n=10) via subcutaneous pumps starting one week before the first CCl₄ injection for 12 weeks. Mice were sacrificed 8.5 months after DEN injection to determine tumor number, size and liver-body weight ratio. * p<0.05, ** p<0.01. B. Unifrac analysis of 16s sequencing results of the cecal microbiome in Tlr4^WT (n=3) and Tlr4^mut (n=4) mice, Tlr4^WT mice treated with subcutaneous PBS (n=4) or LPS (n=4) pumps for 2 weeks, or Tlr4^WT mice receiving antibiotics (n=4) for 2 weeks. C. Taxonomic distribution of the cecal microbiome in Tlr4^WT and Tlr4^mut mice, Tlr4^WT mice treated with subcutaneous PBS or LPS pumps for 2 weeks, or mice receiving antibiotics for 2 weeks. The chart contains a total of 118 taxa at the genus level and the top 20 taxa are annotated on the left with most abundant taxa listed on top. Data are represented as means ± SD. * p<0.05, ** p<0.01. See also Figure S3.

Figure 4. Germ-free status decreases hepatocarcinogenesis

Germ-free (GF) C57Bl/6 mice were subjected to DEN (25 mg/kg i.p.) injection, split into specific-pathogen free (SPF) and GF groups, and then received 10 CCl₄ (0.5 ml/kg i.p.) injections. A. Ceca of GF and SPF mice are shown on the far left and representative livers with tumors are shown on the right. B. Tumor number, size and liver-body weight ratio was determined in SPF mice (n=9) and GF mice (n=10). Data are represented as means ± SD. * p<0.05, ** p<0.01.

Figure 5. Resident liver cells mediate TLR4-dependent tumor promotion

A. Tlr4^WT and Tlr4^mut mice were subjected to BMT (n≥10 per group) at 8 weeks followed by treatment with DEN plus 8 injections of CCl₄. Mice were sacrificed 10.5 months after DEN. Shown are representative images of tumors and H&E sections, as well as tumor number, size and liver body weight ratio. B. Tlr4^WT (n=7) and Tlr4^mut mice (n=6) were transplanted with wild-type bone marrow and treated with DEN plus 2 injections of CCl₄ at the age of 8 weeks. Proliferation markers were evaluated by qPCR and shown as fold induction in comparison to untreated mice. C. TLR4 chimeric mice with Tlr4^WT resident cells and Tlr4^mut BM (n=3) were treated with DEN plus CCl₄ as described above. 10.5 months after DEN, LPS (10 mg/kg i.v.) was injected and NF-κB p65 nuclear translocation was determined by immunohistochemistry and confocal microscopy in non-tumor liver areas. Hepatic stellate cells were visualized by desmin staining and nuclei were visualized by Hoechst staining. Green and white arrows indicate NF-κB translocation in hepatic stellate cells and hepatocytes respectively. The percentage of hepatic stellate cells (HSC) and hepatocytes (Hep) with positive nuclear p65 was determined. Data are represented as means ± SD. * p<0.05, ** p<0.01. See also Figure S4.

Figure 6. Epiregulin is an LPS-inducible promoter of hepatocarcinogenesis

A. Ereg and Hgf mRNA were determined in Tlr4^mut mice (n=14), Tlr4^mut (n=15) and antibiotics-treated Tlr4^WT (n=9) after DEN and 2×CCl₄ injections. Fold induction vs. untreated livers. B. Ereg and Hgf mRNA were determined in TLR4-chimeric mice with Tlr4^WT resident cells and Tlr4^WT BM (n=6) or Tlr4^mut resident cells and Tlr4^WT BM (n=6) after treatment with DEN and 2 CCl₄ injections. Fold induction vs. untreated livers. C. Western blot of HGF from Tlr4^WT mice, Tlr4^mut mice and antibiotics-treated mice after DEN and 2×CCl₄ injections. D. Hepatocytes (Hep), Kupffer cells (KC) and hepatic stellate cells (HSC) were isolated from liver treated with DEN and 2 injections of CCl₄ followed by analysis of Ereg mRNA expression by qPCR (left panel). Shown is a fold-induction vs. whole liver. Ereg mRNA was isolated in liver extracts from CCl₄-treated mice injected with LPS (10 mg/kg i.v.) or PBS 4 days after the last CCl₄ injection (second panel from left). Activated hepatic stellate cells were isolated from CCl₄-treated and bile duct-ligated livers. Epiregulin was determined by ELISA 24hr after plating. E. Hepatic stellate cells from DEN plus 2×CCl₄ treated mice were stimulated with LPS (100 ng/ml) in the presence of either an adenoviral IκB superrepressor (AdIκBsr) or empty shuttle virus (AdSh) by qPCR (left panel) or ELISA (right panel) F. EREG expression was determined in normal liver (“NL”, n=4) and livers of patients with alcoholic hepatitis (“AH”, n=10, left panel) and in activated human hepatic stellate cells stimulated with LPS (100 ng/ml, right panel). G. WT (n=11) and Ereg^KO mice (n=14) were treated with DEN plus 22 injections of CCl₄. Mice were sacrificed 6.5 months after DEN. Shown are representative images of tumors as well as tumor number, size of the largest tumor, and liver body weight ratio. H. Ereg expression was analyzed in non-tumorous regions of DEN plus CCl₄-treated Tlr4^WT (n=12), Tlr4^mut mice (n=9) and antibiotics-treated Tlr4^WT mice (n=6) 10.5 months after DEN. Fold induction vs. untreated liver. Data are represented as means ± SD. * p<0.05, ** p<0.01. nd, non-detectable

Figure 7. The intestinal microbiota promotes HCC in late stages of hepatocarcinogenesis

A. WT mice (C3H/HeOuJ) were treated with DEN (100 mg/kg i.p. at the age of 6 weeks) and 12 CCl₄ (0.5 ml/kg i.p.) injections and were either not gut-sterilized (“N”, n=7), gut-sterilized starting 2 weeks before DEN injection until 2 weeks after the last CCl₄ injection (“E”, n=10), or 2 weeks after the last CCl₄ injection until they were sacrificed (“L”, n=8) followed by determination of tumor number, size and liver-body weight ratio. B. WT mice treated as described above were sacrificed at the time point when the gut-sterilization treatment was started to determine whether macroscopic or microscopic tumors were present. C. WT mice were treated with DEN (25 mg/kg i.p.) at day 15 postpartum followed by 14 weekly injections of CCl₄. After confirming the presence of tumor by ultrasound (data not shown), mice were treated with (“Abx”, n=7) or without (“Ctrl”, n=6) antibiotics (ampicillin, vancomycin, neomycin and metronidazole) starting 2 weeks after the last CCl₄ injection. After 6 weeks of antibiotics, mice were sacrificed and tumor number, size and liver-body weight ratio was determined. Data are represented as means ± SD. * p<0.05, ** p<0.01. ns, non-significant. See also Figure S5.

Figure 8. TLR4 and the intestinal microbiota protect from apoptosis

A–B. Livers from Tlr4^WT (n=11), Tlr4^mut (n=9) or antibiotics-treated Tlr4^mut mice (n=7) were stained for Ki-67 (A.) or cleaved-caspase 3 (B.) followed by quantification. Correlation between the number of cleaved-caspase 3 positive cells and tumor number, size and liver-body weight ratio was determined. C–D. Tlr4^WT were either not gut-sterilized (n=7), or gut-sterilized 2 weeks after the last CCl₄ injection until they were sacrificed (n=8). Livers were stained for Ki-67 and cleaved-caspase 3 followed by quantification (B). Correlation between the number of cleaved-caspase 3 positive cells and tumor number, size and liver-body weight ratio was determined (C). E. Nos2, Birc3 and Birc5 mRNA levels were compared between livers from Tlr4^WT (n=12) and Tlr4^mut (n=9), and between untreated (n=9) and antibiotics treated (n=6) Tlr4^WT. Data are represented as means ± SD. * p<0.05. See also Figure S6.