The Fecal Mycobiome in Non-alcoholic Fatty Liver Disease

Background & Aims: Studies investigating the gut-liver axis have largely focused on bacteria, whereas little is known about commensal fungi. We characterized fecal fungi in patients with non-alcoholic fatty liver disease (NAFLD) and investigated their role in a fecal microbiome-humanized mouse model of Western diet-induced steatohepatitis.

Methods: We performed fungal internal transcribed spacer 2 sequencing using fecal samples from 78 patients with NAFLD, 16 controls and 73 patients with alcohol use disorder. Anti-Candida albicans (C. albicans) IgG was measured in blood samples from 17 controls and 79 patients with NAFLD. Songbird, a novel multinominal regression tool, was used to investigate mycobiome changes. Germ-free mice were colonized with feces from patients with non-alcoholic steatohepatitis (NASH), fed a Western diet for 20 weeks and treated with the antifungal amphotericin B.

Results: The presence of non-obese NASH or F2-F4 fibrosis was associated with a distinct fecal mycobiome signature. Changes were characterized by an increased log-ratio for Mucor sp./Saccharomyces cerevisiae (S. cerevisiae) in patients with NASH and F2-F4 fibrosis. The C. albicans/S. cerevisiae log-ratio was significantly higher in non-obese patients with NASH when compared with non-obese patients with NAFL or controls. We observed a different fecal mycobiome composition in patients with NAFLD and advanced fibrosis compared to those with alcohol use disorder and advanced fibrosis. Plasma anti-C. albicans IgG was increased in patients with NAFLD and advanced fibrosis. Gnotobiotic mice, colonized with human NASH feces and treated with amphotericin B were protected from Western diet-induced steatohepatitis.

Conclusions: Non-obese patients with NAFLD and more advanced disease have a different fecal mycobiome composition to those with mild disease. Antifungal treatment ameliorates diet-induced steatohepatitis in mice. Intestinal fungi could be an attractive target to attenuate NASH.

Lay Summary: Non-alcoholic fatty liver disease is one of the most common chronic liver diseases and is associated with changes in the fecal bacterial microbiome. We show that patients with non-alcoholic fatty liver disease and more severe disease stages have a specific composition of fecal fungi and an increased systemic immune response to Candida albicans. In a fecal microbiome-humanized mouse model of Western diet-induced steatohepatitis, we show that treatment with antifungals reduces liver damage.