Intrahepatic Cholestasis of Pregnancy Increases Inflammatory Susceptibility in Neonatal Offspring by Modulating Gut Microbiota

Biological sequence data is accumulating rapidly, motivating the development of improved high-throughput methods for sequence classification. UBLAST and USEARCH are new algorithms enabling sensitive local and global search of large sequence databases at exceptionally high speeds. They are often orders of magnitude faster than BLAST in practical applications, though sensitivity to distant protein relationships is lower. UCLUST is a new clustering method that exploits USEARCH to assign sequences to clusters. UCLUST offers several advantages over the widely used program CD-HIT, including higher speed, lower memory use, improved sensitivity, clustering at lower identities and classification of much larger datasets. Binaries are available at no charge for non-commercial use at http://www.drive5.com/usearch.

The gut microbiota is considered a metabolic "organ" that not only facilitates harvesting of nutrients and energy from the ingested food but also produces numerous metabolites that signal through their cognate receptors to regulate host metabolism. One such class of metabolites, bile acids, is produced in the liver from cholesterol and metabolized in the intestine by the gut microbiota. These bioconversions modulate the signaling properties of bile acids via the nuclear farnesoid X receptor and the G protein-coupled membrane receptor 5, which regulate numerous metabolic pathways in the host. Conversely, bile acids can modulate gut microbial composition both directly and indirectly through activation of innate immune genes in the small intestine. Thus, host metabolism can be affected through microbial modifications of bile acids, which lead to altered signaling via bile acid receptors, but also by altered microbiota composition.

INTRODUCTION: Primary liver tumors and liver metastasis currently represent the leading cause of cancer-related deaths. The liver intimately cross-talks with the gut and performs many essential functions related to digestion, metabolism of nutrients, and clearance of bacterial metabolites. Diseased livers are often associated with altered gut bacterial composition, or dysbiosis, and it has been suggested that gut bacterial products contribute to malignant transformation of hepatocytes. The liver is exposed to the gut microbiome through the portal vein and is an immunological organ that is heavily populated by immune cells. Emerging studies have shown that gut commensal bacteria are important regulators of antitumor immunity. Although it has been established that the gut microbiome influences the efficacy of cancer immunotherapy, the role of gut bacteria in antitumor surveillance in the liver is poorly understood. RATIONALE: The liver is exposed to gut bacterial metabolites and products by way of blood from the intestine, which comprises 70% of the whole liver blood supply. Changes in the gut microbiome may affect immune cell function in the liver, and commensal bacteria can mediate the metabolism of primary into secondary bile acids, which recirculate back into the liver through the enterohepatic circulation. Given that bile acids are known to be involved in liver cancer development, we focused on the role of bile acids in immunosurveillance of tumors growing in the liver. We altered gut bacteria and examined changes of hepatic immune cells and antitumor immunity directed against liver tumors. Uncovering how the gut microbiome uses bile acids to shape immunity to liver cancer may have future therapeutic applications. RESULTS: Using one primary liver model and three liver metastasis models, we found that altering commensal gut bacteria induced a liver-selective antitumor effect. A selective increase of hepatic CXCR6 + natural killer T (NKT) cells was observed, independent of mouse strain, gender, or presence of liver tumors. The accumulated hepatic NKT cells showed an activated phenotype and produced more interferon-γ upon antigen stimulation. In vivo studies using both antibody-mediated cell depletion and NKT-deficient mice confirmed that NKT cells mediated the inhibition of tumor growth in the liver. Further investigation showed that NKT cell accumulation was regulated by the expression of CXCL16, the solo ligand for CXCR6, on liver sinusoidal endothelial cells, which form the lining of liver capillaries and the first barrier for the blood coming from the gut entering the liver. Primary bile acids increased CXCL16 expression, whereas secondary bile acids showed the opposite effect. Removing gram-positive bacteria by antibiotic treatment with vancomycin, which contains the bacteria mediating primary-to-secondary bile acid conversion, was sufficient to induce hepatic NKT cell accumulation and decrease liver tumor growth. Feeding secondary bile acids or colonization of bile acid-metabolizing bacteria, reversed both NKT cell accumulation and inhibition of liver tumor growth in mice with altered gut commensal bacteria. In nontumor liver tissue from human patients with primary liver cancer, primary bile acid cheno-deoxycholic acid (CDCA) levels correlated with CXCL16 expression, whereas an inverse correlation was observed with secondary bile acid glycolithocholate (GLCA), suggesting that the finding may apply to humans. CONCLUSION: We describe a mechanism by which the gut microbiome uses bile acids as messengers to control a chemokine-dependent accumulation of hepatic NKT cells and antitumor immunity in the liver, against both primary and metastatic liver tumors. These findings not only have possible implications for future cancer therapeutic studies but also provide a link between the gut microbiome, its metabolites, and immune responses in the liver. 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. Gut microbiome modulates liver cancer through bile acid-regulated NKT cells. Gut microbiome uses bile acids as a messenger to regulate chemokine CXCL16 level on liver sinusoidal endothelial cells (LSEC) and thus controls the accumulation of CXCR6 + hepatic NKT cells. The accumulated NKT cells have an activated phenotype and inhibit liver tumor growth.