Modeling dysbiosis of human NASH in mice: Loss of gut microbiome diversity and overgrowth of Erysipelotrichales - PubMed (original) (raw)

Modeling dysbiosis of human NASH in mice: Loss of gut microbiome diversity and overgrowth of Erysipelotrichales

James K Carter et al. PLoS One. 2021.

Abstract

Background & aim: Non-alcoholic steatohepatitis (NASH) is a severe form of non-alcoholic fatty liver disease (NAFLD) that is responsible for a growing fraction of cirrhosis and liver cancer cases worldwide. Changes in the gut microbiome have been implicated in NASH pathogenesis, but the lack of suitable murine models has been a barrier to progress. We have therefore characterized the microbiome in a well-validated murine NASH model to establish its value in modeling human disease.

Methods: The composition of intestinal microbiota was monitored in mice on a 12- or 24-week NASH protocol consisting of high fat, high sugar Western Diet (WD) plus once weekly i.p injection of low-dose CCl4. Additional mice were subjected to WD-only or CCl4-only conditions to assess the independent effect of these variables on the microbiome.

Results: There was substantial remodeling of the intestinal microbiome in NASH mice, characterized by declines in both species diversity and bacterial abundance. Based on changes to beta diversity, microbiota from NASH mice clustered separately from controls in principal coordinate analyses. A comparison between WD-only and CCl4-only controls with the NASH model identified WD as the primary driver of early changes to the microbiome, resulting in loss of diversity within the 1st week. A NASH signature emerged progressively at weeks 6 and 12, including, most notably, a reproducible bloom of the Firmicute order Erysipelotrichales.

Conclusions: We have established a valuable model to study the role of gut microbes in NASH, enabling us to identify a new NASH gut microbiome signature.

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Conflict of interest statement

The authors have read the journal's policy and have the following competing interests: JJF is on the scientific advisory board of Vedanta Biosciences. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1

Fig 1. Hepatocyte injury, fibrosis, and cancer in NASH mice.

Six to eight week old mice were fed regular chow or WD for 24 weeks. NASH mice were injected once weekly with low dose CCl4. Representative liver histology demonstrated by A) H&E stain and B) picosirius red stain for fibrillar collagen to label areas of fibrosis. C) NAFLD activity score (NAS) was evaluated by a blinded pathologist. NASH mice have significantly elevated NAS, indicative of robust disease. D) Liver enzymes were measured in serum collected at the time of sacrifice. E) Expression of selected markers of hepatic stellate cell activation measured by quantitative real-time PCR (qRT-PCR). F) Liver tumors counted on the liver surface at 24 weeks. G) Endotoxin measured in serum collected at 12 or 24 weeks. Image magnification is 200x for H&E (A) and 50x for Sirius Red (B). For (A-F) n = 7 control and n = 3 NASH mice were included. (G) n = 3 mice per group. P values are encoded as * = p < 0.05, ** = p < 0.01, and *** = p < 0.001.

Fig 2

Fig 2. Loss of abundance and altered composition of microbiome in NASH mice.

Feces were collected from chow-fed controls or NASH mice at weeks 0, 12, and 24. Composition of the microbiome was assessed 16S rRNA amplicon sequencing. A) Microbiota density in feces. NASH mice had significantly lower bacterial density than controls. B) Faith’s phylogenetic diversity, calculated to assess species diversity. NASH mice displayed reduced alpha diversity. C) Principal Coordinate Analysis plotting weighted UniFrac distances. NASH mice at 12 and 24 weeks cluster separately from week 0 baseline or chow-fed controls. (A-C) fecal samples from 3–7 mice per condition, per timepoint. P values are encoded as * = p < 0.05, ** = p < 0.01, and *** = p < 0.001.

Fig 3

Fig 3. NASH shifts abundance of Bacteroidales and Erysipelotrichales.

A) Relative abundance of specific taxa at baseline or after 12, and 24 weeks of NASH protocol. B) Bacteroidales abundance is reduced in NASH while C) Erysipelotrichales increases dramatically. Fecal samples were sequenced from 3–7 mice per condition, per timepoint. P values are encoded as * = p < 0.05, ** = p < 0.01, and *** = p < 0.001.

Fig 4

Fig 4. Separate effects of NASH and WD on the microbiome.

Mice were randomized to control, CCl4-only, WD-only, or complete NASH protocol and followed for 12 weeks. A) Microbiota density decreased in both WD-only and NASH mice, as did B) Faith’s Phylogenetic Diversity. C) Principal Coordinate Analysis of Bray-Curtis dissimilarity distances shows changes to beta diversity. WD-only and NASH have separate impacts on population structure. Fecal samples were sequenced from 3–10 mice per condition (mean = 5), except NASH week 12 where n = 2. P values are encoded as * = p < 0.05, ** = p < 0.01, and *** = p < 0.001.

Fig 5

Fig 5. Erysipelotrichales expansion is specific to NASH.

A) Relative abundance of specific taxa over 12 weeks. B) Erysipelotrichales bacteria expand in NASH but not other conditions whereas C & D) decreased abundance of Verrucomicrobiales and Bacteroidales is observed in both WD-only and NASH. Fecal samples sequenced from 3–10 mice per condition (mean = 5), except NASH week 12 where n = 2. P values are encoded as * = p < 0.05, ** = p < 0.01, and *** = p < 0.001.

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