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BACKGROUND AND AIMS The molecular mechanisms underlying successful fecal microbiota transplantation (FMT) for recurrent Clostridioides difficile infection (rCDI) remain poorly understood. The primary objective of this study was to characterize alterations in microRNAs (miRs) following FMT for rCDI. METHODS Sera from two prospective multicentre randomized controlled trials were analyzed for miRNA levels using the Nanostring nCounter platform and quantitative RT-PCR. Additionally, rCDI-FMT and toxin-treated animals and ex vivo human colonoids were employed to compare intestinal tissue and circulating miRNAs. miRNA inflammatory gene targets in colonic epithelial and peripheral blood mononuclear cells were evaluated by qPCR and 3'UTR reporter assays. Colonic epithelial cells were employed for mechanistic, cytoskeleton, cell growth and apoptosis studies. RESULTS miRNA profiling revealed upregulation of 64 circulating miRNAs 4- and 12-weeks following FMT compared to screening, of which the top 6 were validated in the discovery cohort by RT-qPCR. In a murine model of relapsing-CDI, RT-qPCR analyses of sera and cecal RNA extracts demonstrated suppression of these miRNAs, an effect reversed by FMT. In mouse colon and human colonoids, TcdB mediated the suppressive effects of CDI on miRNAs. CDI dysregulated Drosha, an effect reversed by FMT. Correlation analyses, qPCR and 3'UTR reporter assays revealed that miR-23a, miR-150, miR-26b, miR-28 target directly the 3'UTR of IL12B, IL18, FGF21 and TNFRSF9, respectively. miR-23a and miR-150 demonstrated cytoprotective effects against TcdB. CONCLUSION These results provide novel and provocative evidence that modulation of the gut microbiome via FMT induces alterations in circulating and intestinal tissue miRNAs. These findings contribute to a greater understanding of the molecular mechanisms underlying FMT and identify new potential targets for therapeutic intervention in rCDI.