Background: Ulcerative colitis (UC) may be exacerbated by Fusobacterium nucleatum (Fn) infection. However, the mechanism underlying Fn-mediated progression of UC has yet to be established. Here, we aimed to establish whether and how Fn-derived extracellular vesicles (Fn-EVs) participate in the development of experimental colitis through microRNAs (miRNAs).

Methods: EVs were isolated and purified by ultracentrifugation from Fn and Escherichia coli culture supernatants. Differentially expressed miRNAs in control intestinal epithelial cells (IECs) and Fn-EV-treated IECs were identified by miRNA sequencing. EVs were cocultured with IECs or administered to CARD3wt/CARD3-/- mice by gavage to assess inflammatory responses to and the mechanism of action of Fn-EVs.

Results: Fn-EVs promoted upregulation of proinflammatory cytokines (interleukin [IL]-1β, IL-6, tumor necrosis factor α), downregulation of anti-inflammatory IL-10 and intercellular tight junction proteins ZO-1 and occludin, and epithelial barrier dysfunction in IECs. Fn-EVs significantly aggravated experimental colitis in mice associated with Fn-EV-mediated downregulation of miR-574-5p expression and autophagy activation. Blockade of autophagy using chloroquine alleviates barrier damage exacerbated by Fn-EVs in vitro and in vivo. Inhibition of the miR-574-5p/CARD3 axis reduced the severity of colitis, epithelial barrier damage, and autophagy activation induced by Fn-EVs.

Conclusions: Here, we describe a new mechanism by which Fn-EVs mediate experimental colitis severity through miR-574-5p/CARD3-dependent autophagy activation, providing a novel target for UC monitoring and targeted therapy.