Abstract
BACKGROUND: Clostridioides difficile is a major cause of nosocomial postantibiotic infections, often resulting in severe inflammation and watery diarrhea. Previous studies have highlighted the role of C. difficile flagellin FliC in activating Toll-like receptor 5 and triggering nuclear factor-κB (NF-κB) cell signaling, leading to the release of proinflammatory cytokines. However, the microRNA (miRNA)-mediated regulatory mechanisms underlying the FliC-induced inflammatory response remain unclear. METHODS: miRNA expression levels were analyzed in Caco-2 intestinal epithelial cells following FliC stimulation and infection with the epidemic C. difficile R20291 strain or its unflagellated mutant by reverse transcription-quantitative polymerase chain reaction. Chemical inhibitors were used to block NF-κB signaling, and their impact on miR-27a-5p expression was assessed. Knockdown and overexpression experiments with miRNA inhibitor and mimic respectively were conducted to elucidate the functional role of miR-27a-5p in FliC-induced inflammatory responses. Additionally, a mouse model of C. difficile infection was treated with miR-27a-5p to evaluate its therapeutic potential in vivo. RESULTS: miR-27a-5p showed significant FliC-dependent overexpression in Caco-2 cells. Inhibition of NF-κB signaling suppressed miR-27a-5p overexpression. Knockdown of miR-27a-5p increased NF-κB activation and cytokine production (tumor necrosis factor α and interleukin 8), while its overexpression had the opposite effect. Moreover, miR-27a-5p was overexpressed in the ceca of C. difficile-infected mice, correlating with intestinal interleukin 8 levels. Treatment of infected mice with the miR-27a-5p mimic reduced disease severity and intestinal inflammation. CONCLUSIONS: miR-27a-5p plays a crucial role in regulating C. difficile-induced inflammation, suggesting its potential as a therapeutic target for controlling severe infection. These findings offer valuable insights into potential therapeutic strategies for managing C. difficile infection and associated inflammatory complications.