Prophylactic Lipoxin A ₄ Attenuates Clostridioides difficile Infection by Augmenting Epithelial Barrier and Resolving Inflammation

Clostridioides difficile infection (CDI) is a leading healthcare-associated diarrhea with high recurrence rates, partially due to antibiotic-induced dysbiosis and dysregulated host inflammation. Specialized pro-resolving mediators (SPMs), such as Lipoxin A ₄ (LXA ₄ ), offer promise in controlling excessive inflammation and promoting tissue repair, yet their role in CDI remains unexplored. Here, we developed a compact, gas-tight gut-on-a-chip (GOC) system that reconciles the anaerobic requirements of C. difficile with the oxic needs of human intestinal epithelium, enabling physiologically relevant co-culture within a standard incubator. A CDI in vitro model was established based on this GOC system. Using the model, we demonstrated that prophylactic administration of LXA ₄ significantly preserved epithelial barrier integrity, attenuated pro-inflammatory cytokine secretion (IL-8 and IFN-γ), and reduced bacterial colonization. Transcriptomic analysis revealed that LXA ₄ pretreatment upregulated genes involved in cell junction organization while downregulated immune activation pathways. These protective effects were validated in a murine CDI model, where LXA ₄ pretreatment reduced weight loss, pathological damage, and fecal bacterial burden. Furthermore, prophylactic administration of LXA ₄ synergized with vancomycin treatment further enhanced antibiotic efficacy while allowing a 50% dose reduction without compromising therapeutic outcomes. Our study establishes a robust approach for CDI research and highlights the prophylactic and adjuvant potential of inflammation-resolving strategies, offering a novel approach to mitigate CDI incidence and improve treatment outcomes.