Background <p>Defensins, small cationic peptides with strong antimicrobial activity, are key effectors of innate immunity. α-defensins, human neutrophil peptides, are produced primarily by neutrophils and serve as an essential component of the airway defense system against invading pathogens. However, accumulating evidence indicates that α-defensins released from human neutrophils are markedly elevated in various lung diseases, where excessive α-defensins exert cytotoxic effects on epithelial and immune cells.</p> Objective <p>We investigated how α-defensins influence macrophage inflammatory responses and aimed to elucidate the molecular mechanisms underlying α-defensin-induced macrophage activation.</p> Methods <p>Through RNA-seq analysis, we identified key molecules potentially involved in α-defensin-induced inflammatory signaling and validated these candidates using qRT-PCR and western blotting.</p> Results <p>Our results show that α-defensins significantly upregulate both the gene expression and protein levels of RNF31 in macrophages, leading to enhanced phosphorylation of NF-κB p65 and increased production of pro-inflammatory cytokines. Furthermore, when co-cultured with lung epithelial cells, α-defensin-stimulated macrophages induced NLRP3 expression in epithelial cells, suggesting that macrophage-epithelial crosstalk contributes to α-defensin-driven airway inflammation.</p> Conclusion <p>Together, our results reveal that α-defensins promote macrophage-driven inflammation through RNF31-dependent NF-κB activation and subsequent macrophage-epithelial communication, providing new insight into the inflammatory mechanisms of lung injury. These findings uncover a previously unrecognized α-defensin-RNF31 signaling pathway that amplifies macrophage-mediated airway inflammation, highlighting RNF31 as a potential therapeutic target for inflammatory lung diseases.</p>