Elsevier

Journal of Hepatology

Volume 64, Issue 3, March 2016, Pages 641-650
Journal of Hepatology

Research Article
Functional analysis of the relationship between intestinal microbiota and the expression of hepatic genes and pathways during the course of liver regeneration

https://doi.org/10.1016/j.jhep.2015.09.022Get rights and content

Background & Aims

The pathways regulating liver regeneration have been extensively studied within the liver. However, the signaling contribution derived from the gut microbiota to liver regeneration is poorly understood.

Methods

Microbiota and expression of hepatic genes in regenerating livers obtained from mice at 0 h to 9 days post 2/3 partial hepatectomy were temporally profiled to establish their interactive relationships.

Results

Partial hepatectomy led to rapid changes in gut microbiota that was reflected in an increased abundance of Bacteroidetes S24-7 and Rikenellaceae and decreased abundance of Firmicutes Clostridiales, Lachnospiraceae, and Ruminococcaceae. Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) was used to infer biological functional changes of the shifted microbiota. RNA-sequencing data revealed 6125 genes with more than a 2-fold difference in their expression levels during regeneration. By analyzing their expression pattern, six uniquely expressed patterns were observed. In addition, there were significant correlations between hepatic gene expression profiles and shifted bacterial populations during regeneration. Moreover, hepatic metabolism and immune function were closely associated with the abundance of Ruminococcacea, Lachnospiraceae, and S24-7. Bile acid profile was analyzed because bacterial enzymes produce bile acids that significantly impact hepatocyte proliferation. The data revealed that specific bacteria were closely associated with the concentration of certain bile acids and expression of hepatic genes.

Conclusions

The presented data established, for the first time, an intimate relationship between intestinal microbiota and the expression of hepatic genes in regenerating livers.

Introduction

Commensal bacteria are implicated in digestive tract health and disease. It is known that intestinal microbiota plays a role in regulating host cell proliferation and tissue repair [1], [2]. For example, germ-free mice have reduced intestinal epithelial cell turnover due to reduced proliferation, apoptosis, and crypt-to-tip cellular migration [3]. Germ-free mice also exhibit increased cancer incidence compared to conventional mice [4]. In addition, increased bacterial load and dysbiosis are found in colonic biopsies of patients with colorectal adenoma or cancer [5]. Moreover, Gram-negative bacteria-generated lipopolysaccharide (LPS) stimulates liver regeneration and tissue repair through Toll-like receptor 4 (TLR4) signaling [6]. Gut microbiota also affects metabolic phenotype of the mammalian host and participates in microbial-host co-metabolism [7]. Alterations in gut bacterial communities are associated with metabolic disorders [8], metabolic syndrome [9], obesity [10], [11], [12], and non-alcoholic steatohepatitis [13]. There is an intrinsic link between proliferation and metabolism. Cell proliferation elevates metabolic demands to generate the energy and precursors for biosynthesis of macromolecules, and yet metabolic disorder dampens proliferation. Thus, through the gut-liver axis, intestinal microbiota, which is implicated in both proliferation and metabolism, may significantly regulate liver regeneration.

The liver is a major organ for host metabolism that can remarkably regenerate itself in response to partial resection or injury [14]. Liver regeneration requires activation of an array of genes and networks of signal transducers. Bile acids (BAs) have been identified as key metabolic signals during liver regeneration, and BA levels are tightly regulated by both host and microbiota [15]. There exists a “gut-liver axis” that facilitates bidirectional communication between intestinal microbes and BAs [1]. In one direction, the gut microbiota plays a pivotal role in regulating BA homeostasis. On the other end, BAs influence the gut microbiota profile. Although the bidirectional relationship of BAs and microbiota in the gut-liver axis has been investigated in humans and mice, whether it is linked to the regenerative process after liver resection remains largely unclear [16].

Previous studies have demonstrated the significance of BAs and its receptor farnesoid X receptor (FXR) in regulating liver regeneration [15]. However, the interplay between BAs, gut microbiota, and hepatic gene profiles during liver regeneration has not been defined. This is the first study to demonstrate the dynamic shift of hepatic transcripts and pathways in relation to gut microbiota as well as BA profiles in partial hepatectomy (PHx)-induced liver regeneration.

Section snippets

Animal experiments and sample collection

See Supplementary material and methods for sources of materials and methodological details.

Statistical analysis

Data are given as mean ± SD. Statistical analysis was performed using Student’s t test or one-way analysis of variance. Significance was defined by p <0.05.

PHx-induced liver regeneration

After 2/3 liver resection, liver mass was restored to its original size at 7 to 9 days, consistent with previously reported findings (Supplementary Fig. 1A) [17], [18], [19]. Ki67 immunostaining of liver sections revealed that cell proliferation started 1 day after PHx, peaked on day 2, and ceased on day 9 (Supplementary Fig.1B, C).

Alteration in microbial communities during liver regeneration

To characterize changes in the intestinal microbiota associated with regeneration, we constructed and sequenced 16S rRNA amplicon libraries from cecal contents. Mice

Discussion

The presented data, for the first time, analyzed changes in intestinal commensal microbiota occurring in mice whose livers are undergoing regeneration. Functional analysis demonstrated specific and unique functions of gut microbiota at each stage of liver regeneration. Accordingly, hepatic gene profiling also revealed unique expression patterns that can be associated with specific biological pathways involved in the regenerative process. Moreover, based on these unique functions of microbiota

Financial support

This study is supported by grants funded by National Institutes of Health CA53596, DK092100, and U01CA179582.

Conflict of interest

The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.

Acknowledgement

The authors thank Thinh Chau and Lisa Teixeira for editing the manuscript. This study is supported by grants funded by National Institutes of Health CA53596, DK092100, and U01CA179582.

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