Introduction: Metabolic dysfunction-associated steatotic liver disease (MASLD) affects over a third of the world population and is increasing at an alarming rate. No FDA-approved pharmacological agents exist to treat MASLD or Metabolic dysfunction-associated steatohepatitis (MASH, advanced MASLD). Fibrosing MASH will soon be the leading indication for liver transplant. Dietary cholesterol associates with MASLD in humans and drives fibrosing MASH in specific pathogen-free (SPF) mice harboring microbial communities, but germ-free (GF) mice devoid of microbes remain protected. Microbial translocation from gut to liver correlates with MASH in humans and animal models, yet mechanisms responsible are unknown. We hypothesized cholesterol-induced gut dysbiosis enhances microbial translocation to the liver, driving hepatic immune dysregulation, promoting fibrosing MASH. Methods/Results: Male 16-week-old GF and SPF mice were provided fructose+glucose drinking water and fed high-fat (HF) or HF+2% cholesterol (HFHC) diets for up to 26 weeks. HFHC rapidly increased plasma Alanine transaminase (indicator of liver damage) in SPF mice relative to all other groups at week 4 (P<0.01). NAFLD Activity Score of liver cross-sections was increased in HFHC-fed SPF mice at week 8 (P<0.05). Picrosirius red staining showed HFHC increased liver fibrosis in SPF mice at week 24 relative to all other groups (P<0.01). At week 26, single-cell RNA sequencing of hepatic non-parenchymal cells (n=2-3/treatment) showed an increased proportion of activated Ccr7^low,Ccr5^high Cd8⁺ T-cells in HFHC-fed SPF and GF mice, suggesting cholesterol level, but not microbes, impact Cd8⁺ T-cell activation. However, trajectory analysis showed exhausted Pd-1^high,Gzmk^high Cd8⁺ T-cells were most abundant in HFHC-fed SPF mice. Further, pathogen-specific Cd4⁺ T effector cells (PSC) that express Ccl4 and recruit Ccr5⁺ Cd8⁺ T-cells were enriched ~7-fold in HFHC-fed SPF mice relative to all other groups. Bray-Curtis β-diversity analysis of 16S rRNA amplicon sequencing performed via Illumina MiSeq showed HFHC shifted cecal microbial community membership at week 8 (PERMANOVA P=0.003), while liver microbial communities were not different. Yet, at 24 weeks, HFHC shifted both cecum and liver microbial community membership (PERMANOVA P<0.01) suggesting HFHC-induced shifts in gut microbes occur early, proceeding shifts in liver microbes. Conclusion: These data show high dietary cholesterol intake alters the gut-liver axis, leading to altered hepatic microbiota late in disease, which may induce antigen-specific PSCs that recruit and drive exhaustion of Ccr5⁺ Cd8⁺ T-cells. Future studies are required to decipher further mechanistic connections, which may help to identify novel microbial-based interventions to prevent fibrosing MASH.

Yongjun Liu

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