The role of gut microbiome in the development of non-alcoholic fatty liver disease

Abstract

The global rise in the prevalence of non-alcoholic fatty liver disease (NAFLD) poses a significant threat and burden to health care systems worldwide. The gut microbiome plays a pivotal role in maintaining host homeostasis. Microbial dysbiosis has been increasingly associated with the development of NAFLD, however whether or not this relationship is causal in nature has yet to be established. This thesis can be divided into two major parts: 1) Paneth cells modulate the gut microbiome via secretion of antimicrobial peptides with homeostatic functions, but its role in the pathogenesis of NAFLD remains unclear. Whether Paneth cell associated microbial alterations are involved in the development of NAFLD was determined by injection of dithizone which can disrupt cell granulates. 2) A human microbiota-associated murine model was established by fecal microbiota transplantation (FMT) using human feces from healthy controls, lean NAFLD and obese NAFLD donors to determine the causal effects of the gut microbiome, and to identify fecal biomarkers for NAFLD. Controlled attenuation parameter measurements by vibration-controlled transient elastography were adopted to quantify the donor's liver fat non- invasively. This thesis aims to investigate potential causality between the gut microbiome and NAFLD, and to identify altered bacterial species or biomarkers that may play a role in NAFLD development. The results show that 1) dithizone-treated HFD mice had significant reductions in liver weight gain, hepatic triglyceride content, and plasma antimicrobial peptide levels when compared to non-treated HFD mice. Bacterial taxonomic profiling found that dithizone-treated HFD mice had a lower Firmicutes/Bacteroidetes ratio and a higher abundance of Bacteroides ASV21 and ASV42 than non-treated HFD mice. Bacteroides correlated negatively with antimicrobial peptide expression and positively with L-methionine and tetrahydrofolate biosynthesis. 2) FMT-Obese mice had significantly higher plasma and intrahepatic triglyceride content, adipocyte size, macrophage infiltration, and production of reactive oxygen species; but a lower ileal expression of zonula occludens-1 than FMT-Healthy mice. Microbiome analysis confirmed a significantly higher abundance of Lactococcus lactis in FMT-Healthy mice than FMT-Obese mice. Five Lactococcus lactis and two Lactococcus lactis subsp. lactis strains were annotated. Lactococcus lactis correlated positively with biotin biosynthesis pathways, but negatively with plasma triglyceride levels. An altered intrahepatic bacterial DNA profile was associated with riboflavin production, which plays a beneficial role in preventing NAFLD development, in FMT-Lean mice when compared to FMT-Obese mice. The metabolomic profiles of fecal samples in FMT-Healthy mice demonstrated an increase in beneficial microbial fermentation products. Notably, fumaric acid was only detected in feces of FMT- Healthy mice and was undetectable in both FMT-Lean and FMT-Obese mice, signifying its potential use as a fecal biomarker. Collectively, these findings suggest that Paneth cell alterations protect against high-fat diet induced liver steatosis, possibly through the gut microbiome-related modulation involving Bacteroides. Stool microbes from healthy human donors ameliorate the severity of NAFLD in mice, signifying a potential causal relationship. The presence of fumaric acid and Lactococcus lactis in feces was linked to reduced NAFLD risk. These results highlight that future microbiome-specific therapies targeting Bacteroides, Lactococcus lactis, or fumaric acid may have a potential therapeutic function in NAFLD.