Gut microbiota modulates the effects of host-derived fecal microRNAs on cultured gut microbiota in mice

We previously reported that murine fecal microRNAs (miRNAs) alter the composition of cultured gut microbiota and selectively increase Enterococcus. Since host miRNA expression is influenced by microbial colonization, this study investigated whether fecal miRNAs derived from germ-free (GF) and specific pathogen-free (SPF) mice differentially affect the composition and metabolic activity of cultured gut microbiota. Fecal miRNAs isolated from GF and SPF mice were added to cultures of murine gut microbiota. 16S rRNA gene sequencing showed that GF mice- and SPF mice-derived miRNAs altered microbial community structures in distinct ways. ANCOM identified Enterococcus as the only taxon significantly increased by miRNA treatment. GF mice-derived miRNAs significantly enhanced fermentation, as evidenced by a lower culture pH and higher concentrations of acetate, propionate, and lactate compared with SPF mice-derived miRNAs or vehicle controls. Correlation analysis demonstrated positive associations between organic acid levels and secondary fermenters, including Alloprevotella, Muribaculum, Lachnoclostridium, and Peptococcaceae. In contrast, typical saccharolytic butyrate producers such as Blautia, Lachnospiraceae, and Oscillospiraceae showed negative correlations. Microarray analysis revealed differences in the fecal miRNA profiles of GF and SPF mice, supporting the hypothesis that microbial exposure modulates fecal miRNA composition. Fecal miRNAs derived from antibiotic-treated mice did not reproduce the broad fermentation-promoting effects observed with GF mice-derived miRNAs, although lactate concentration increased. These observations suggest that fecal miRNAs promote fermentation by stimulating Enterococcus and downstream cross-feeding networks, and that prior microbial exposure attenuates this effect. Consequently, fecal miRNAs appear to represent a coevolved host mechanism that modulates microbial fermentation to maintain intestinal homeostasis.