Increased colonization of lactococcus lactis could prevent development of non-alcoholic fatty liver disease: evidence from a human microbiota-associated rodent model

Abstract

Background: Gut microbiota dysbiosis is increasingly linked to the development of non-alcoholic fatty liver disease (NAFLD). Yet, its causal relationship has not been well established. We aimed to investigate gut microbiota species related to NAFLD development in human microbiota-associated rodents using fecal microbiota transplantation (FMT). Methods: We recruited Asian human FMT donors categorized as three groups (n=8 each): obese NAFLD donors; non-obese NAFLD; and non-obese, non-diabetic healthy controls, with hepatic steatosis quantified using controlled attenuation parameter measurements via transient elastography (Fibroscan, Echosens). After antibiotic administration, collected human fecal samples were transplanted individually into C57BL/6J mice, categorized as FMT-obese, FMT-lean and FMT-healthy. We collected liver, adipose tissue and blood from mice after a 12-week high-fat diet for assessment of histology, lipid metabolism, hepatic inflammation and intestinal barrier function. Shotgun metagenomics sequencing was used to profile gut microbiome. Results: Obese NAFLD donors had a significantly higher controlled attenuation parameter measurement and BMI than non-obese NAFLD and healthy controls. Non-obese NAFLD donors had a distinct gut microbiota compared with healthy controls (p=0.03) and obese NAFLD donors (p=0.0479). Compared to FMT-healthy mice, FMT-obese had significantly higher plasma triglyceride levels and intrahepatic triglyceride content (p<0.01); and increased hepatic lipid accumulation and adipocyte size (p<0.05); but lower expression of tight junction protein zonula occludens-1, an intestinal barrier marker (p=0.03). No difference was observed between FMT-healthy vs. FMT-lean mice. Gut microbiota composition of FMT-obese mice was significantly distinct from FMT-healthy (p=0.03) and FMT-lean (p=0.038). Biomarker analysis confirmed a lower abundance of Lactococcus lactis in FMT-obese mice than FMT-healthy mice (43.50% vs. 24.82%, FDR-p=0.04). As low levels of L. lactis were not well-detected in human fecal metagenomes, we confirmed their colonization by targeted qPCR, which indicated a consistent trend of decreased colonization in obese NAFLD donors. L. lactis correlated positively with tricarboxylic acid cycle pathway, a major energy-producing pathway involved in triglyceride breakdown (rho=0.59, p=0.002), but correlated negatively with plasma triglyceride levels (rho=-0.45, p=0.03). Conclusion: In this study, we proposed the potential causative role of L. lactis in preventing NAFLD development. Future animal and human interventional studies are warranted to determine its therapeutic potential in NAFLD.