Gut microbiome-mediated bile acid metabolism regulates liver cancer via NKT cells

Abstract

Primary liver tumors and liver metastasis currently represent the leading cause of cancer-related death. Commensal bacteria are important regulators of antitumor immunity, and although the liver is exposed to gut bacteria, their role in antitumor surveillance of liver tumors is poorly understood. We found that altering commensal gut bacteria in mice induced a liver-selective antitumor effect, with an increase of hepatic CXCR6⁺ natural killer T (NKT) cells and heightened interferon-γ production upon antigen stimulation. In vivo functional studies showed that NKT cells mediated liver-selective tumor inhibition. NKT cell accumulation was regulated by CXCL16 expression of liver sinusoidal endothelial cells, which was controlled by gut microbiome-mediated primary-to-secondary bile acid conversion. Our study suggests a link between gut bacteria-controlled bile acid metabolism and liver antitumor immunosurveillance.

Fig. 1.. Depleting gut commensal bacteria inhibits liver tumor growth in multiple mouse models.

(A) MYC transgene was turned on at the age of 4 weeks. MYC-ON mice were fed with ABX or regular water. Ten weeks later, mice were killed and liver surface tumor nodules were counted. Representative liver images are shown. Data represent mean ± SEM of two pooled experiments. n = 5 for H₂O, 6 for ABX. P < 0.05, Student’s t test. (B) C57BL/6 mice were treated with ABX or H₂O for 3 weeks before receiving subcutaneous EL4 tumor cell injection. Four weeks later, liver metastases were determined. Representative images of five pooled experiments are shown. n = 17 for H₂O, 12 for ABX. P < 0.05, Chi-square test. (C) ABX- or H₂O-pretreated C57BL/6 mice were given intrasplenic B16 tumor cell injection. One and a half weeks later, liver metastases were measured. Representative images are shown. Data represent mean ± SEM of five pooled experiments n = 18 for H₂O, 15 for ABX. P < 0.05, Student’s t test. (D) BALB/c mice were treated with ABX or H₂O for 3 weeks. Then mice received A20 tumor cell tail vein injection. Three weeks later, liver metastases were counted. Representative images are shown. Data represent mean ± SEM of two pooled experiments. n = 8, P < 0.05, Student’s t test.

Fig. 2.. Hepatic NKT cells increase after depleting gut commensal bacteria.

(A) ABX- or H₂O-pretreated C57BL/6 mice were given subcutaneous EL4 tumor injection. Two and a half weeks later, liver infiltrating immune cells were measured. Data represent mean ± SEM of three pooled experiments. n = 15 for EL4-H₂O, 20 for EL4-ABX. P < 0.05, two-way analysis of variance (ANOVA). (B) Hepatic NKT, CD4, and CD8 T cell levels of MYC mice described in Fig. 1A. Data represent mean ± SEM of two pooled experiments. P < 0.05, two-way ANOVA. (C and D) Hepatic NKT cells in tumor-free C57BL/6 mice were fed with ABX or H₂O. Data represent mean ± SEM of two pooled experiments. n = 9 for H₂0,10 for ABX. P < 0.05, Student’s t test. (E) Hepatic NKT cells in tumor-free BALB/c mice fed with ABX or H₂O. Data represent mean ± SEM of two pooled experiments. n = 5, P < 0.05, Student’s t test. (F) Representative CXCR6 staining in hepatic NKT cells from three independent experiments. (G) Representative NKT cell staining in CXCR6⁺ liver infiltrating mononuclear cells from three independent experiments. (H) Composition of CXCR6⁺ liver infiltrating mononuclear cells in tumor-free C57BL/6 mice fed with ABX or H₂O. DN T: double negative T cells. Data represent pooled results from three experiments. (I) Levels of CXCR6⁺ liver-infiltrating cells in tumor-free C57BL/6 mice fed with ABX or H₂O. Data represent mean ± SEM of two pooled experiments. n = 9 for H₂0,10 for ABX. P < 0.05, Student’s t test. (J and K) IFN-γ and CD69 levels of hepatic NKT cells after in vivo stimulation by injecting aGalCer-loaded A20 tumor cells (Gal/A20) into vancomycin (Vanco) or H₂O-fed BABL/c mice. Data represent mean ± SEM of two pooled experiments. n = 9, P < 0.05, Student’s t test.

Fig. 3.. Hepatic NKT cells mediate the inhibition of liver metastasis in mice.

(A to D) B16 tumor cells were intrasplenically injected into mice pretreated with ABX or H₂O. One day before tumor injection, mice were given intraperitoneal injection of a combination of antibodies to CD4 (anti-CD4) (500 μg per mouse) and anti-CD8 (200 mg per mouse) or anti-CD8 alone (200 μg per mouse). Liver surface metastatic nodules were counted. Representative images are shown. Data represent mean ± SEM of two pooled experiments. n = 16 for H₂O, 14 for ABX, 13 for anti-CD4 + anti-CD8, 5 for anti-CD8. P < 0.05, one-way ANOVA. (E and F) Loss of hepatic NKT abrogated the inhibition of liver metastasis caused by ABX. CXCR6^−/−, CD1d^−/−, or wild-type mice pretreated with ABX or H₂O were given EL4 tumor cell tail vein injections. Liver surface metastatic nodules were counted. Data represent mean ± SEM of two pooled experiments. n = 10 for Wt-H₂O, 7 for Wt-ABX, 4 for CXCR6^−/−-H₂O, 5 for CXCR6^−/−-ABX, 5 for CD1d^−/−-H₂O, 4 for CD1d^−/−-ABX. P < 0.05, two-way ANOVA.

Fig. 4.. Altered bile acids mediate CXCL16 up-regulation in LSECs, NKT accumulation and liver tumor inhibition in mice.

(A) CXCL16 mRNA expression levels in liver tissues from ABX- or H₂O-treated tumor-free C57BL/6 mice. Data represent mean ± SEM of two pooled experiments. n = 9 for H₂O, 10 for ABX. P < 0.05, Student’s t test. (B) Primary LSECs were isolated from mice treated with ABX or H₂O. CXCL16 mRNA levels were measured by real-time polymerase chain reaction (PCR). Data represent mean ± SEM of three pooled experiments. n = 9, P < 0.05, two-way ANOVA. (C) Immunohisto-chemistry staining of CXCL16 and LYVE-1 in liver sections from ABX- or H₂O- treated mice. CXCL16⁺ (red)/LYVE⁺ (green) LSECs are highlighted by arrows and are shown in more detail in the insert. Scale bar, 50 μm. (D) Mice were fed with a 2% cholestyramine (CHOL) or control diet (Con). Hepatic NKT, CXCR6⁺, CD4 T, and CD8 T cells were measured. Data represent mean ± SEM of two pooled experiments. n = 5 for control, 9 for cholestyramine diet. P < 0.05, two-way ANOVA. (E) Liver bile acids profile of ABX- or H₂O-treated mice. Data represent mean ± SEM of two pooled experiments. n = 9, P < 0.05, Student’s t test. (F and G) Isolated LSECs were treated with different bile acids (F) or a combination of T-β-MCA with T-ω-MCA or ω-MCA (G). Data represent mean ± SEM of two pooled experiments. n = 5, P < 0.05, one-way ANOVA. (H) ABX-treated mice were given three times oral gavage of CDCA or ω-MCA three times at the dose of 6 mg per 15 g body weight. Hepatic NKT cell levels from different treatments were measured. Data represent mean ± SEM of two pooled experiments. n = 5, P < 0.05, two-way ANOVA. (I) A20 liver metastasis in vancomycin (Vanco) or H₂O-treated mice receiving LCA or CDCA. As indicated, one group of LCA-treated mice also received hydrodynamic injection to force CXCL16 expression in the liver. Data represent mean ± SEM of two pooled experiments. n = 15 for Vanco + LCA, others n = 10. P < 0.05, one-way ANOVA. (J) EL4 liver metastasis in ABX-treated CXCR6^−/− mice with or without ω-MCA feeding. Data represent mean ± SEM of two pooled experiments. n = 5.

Fig. 5.. Clostridium species influence bile acid-controlled NKT cell accumulation.

(A) Hepatic NKT cell levels from germ-free mice or matched SPF mice were measured. Data represent mean ± SEM of two pooled experiments. n = 5, P < 0.05, Student’s t-test. (B) cxcl16 mRNA expression in liver tissue from germ-free or SPF mice. Data represent mean ± SEM of two pooled experiments. n = 8 for SPF, 7 for GF. P < 0.05, Student’s t-test. (C) Naive C57BL/6 mice were fed with vancomycin (Vanco), neomycin (Neo) or cefoparazone (Cefo). Hepatic NKT levels were determined. Data represent mean ± SEM of three pooled experiments. n = 18 for H₂O, 14 for vancomycin, 14 for neomycin, 10 for cefoperazone. P < 0.05, one-way ANOVA. (D and E) Mice were treated with vancomycin for 1 week and then gavaged with C. scindens or vehicle (cessation). Twenty-four hours after C. scindens gavage, 16S rRNA sequencing analysis of stool samples was performed. The relative abundance of OTUs in the fecal bacterial are shown (D). Time-course study of hepatic NKT levels was performed (E). Data represent mean ± SEM of two pooled experiments. n = 10 for H₂O, D0, C. scindens D4, and Cessation D4; 5 for C. scindens D2, Cessation D2, C. scindens D7 and Cessation D7 P < 0.05, two-way ANOVA. (F and G) A20 liver tumors were induced in mice treated with vancomycin or H₂O. Mice were colonized with C. scindens or control C. innocuum as illustrated in (F). Cumulative liver tumor counts are shown in (G). Data represent mean ± SEM of two pooled experiments. n = 10 for H₂O and Vanco, n = 20 for C. scindens and C. innocuum. P < 0.05, one-way ANOVA. (H) SK-HEP1 cells were treated with different bile acids. CXCL16 mRNA levels were measured by real-time PCR. Data represent mean ± SEM of three pooled experiments. n > 10, P < 0.05, one-way ANOVA. (I) Correlation between primary bile acid CDCA and CXCL16 mRNA expression in nontumor liver tissues from hepatocellular carcinoma or cholangiocarcinoma patients of the TIGER cohort. Pearson correlation coefficient test was performed.