Gentamicin alleviates intestinal graft-versus-host disease by modulating butyrate-associated gut microbiota

Background Intestinal graft-versus-host disease (GVHD) is a common complication following allogeneic hematopoietic cell transplantation (allo-HSCT) and is commonly treated with antibiotics. Additionally, certain antibiotics used for gut-decontamination prophylaxis and neutropenic fever could affect GVHD-related mortality in human patients and mice. Objectives This study aimed to investigate the role of gentamicin and butyrate in mitigating intestinal GVHD and improving the prognosis post allo-HSCT. Study Design: An allo-HSCT mouse model was prepared to assess the effects of gentamicin and sodium butyrate supplementation. 16S rRNA sequencing was performed for microbiota analysis using fecal samples from mice. The effects of sodium butyrate on cell proliferation and apoptosis were analyzed using LS174T human goblet cells. Protein extracts from LS174T cells and mouse intestinal epithelial cells (IECs) were analyzed using western blotting. Samples from the small intestine and colon were evaluated using hematoxylin & eosin (H&E) and periodic acid-Schiff (PAS) staining. Finally, intestine slices were evaluated for Lgr5 and Muc2 expression. A Wilcoxon rank-sum test was used for microbiome analysis and survival was analyzed using Kaplan–Meier curves. The survival curves were compared using a log-rank test. Statistical significance was set at P < 0.05. Results We observed that the intestinal barrier was compromised in mice with GVHD. Gentamicin treatment after allo-HSCT significantly reduced the mortality and GVHD scores in recipient mice. Additionally, 16S rRNA gene sequencing showed that gentamicin altered the gut microbiota composition and decreased Clostridium levels. However, sodium butyrate supplementation in allo-HSCT mice after treatment with gentamicin significantly increased the mortality and intestinal GVHD severity, shortened the length of the colon, decreased colonic mucus layer thickness, and aggravated epithelial barrier damage in aGVHD mice. Further investigation revealed that sodium butyrate induced the apoptosis of goblet cells and inhibited the expression of Muc2 in vivo and in vitro . In addition, sodium butyrate inhibited the proliferation of intestinal stem cells. Interestingly, concurrent supplementation of gentamicin and sodium butyrate before transplantation significantly relieved GVHD. Conclusion Our results show that gentamicin alleviates GVHD by modulating butyrate associated gut microbiotas, while sodium butyrate weakens the benefit of gentamicin on GVHD by inducing goblet cell apoptosis, reducing Muc2 expression, and inhibiting intestinal stem cell proliferation. Thus, butyrate may have a double-edged effect on GVHD based on the exposure timing.