Fermentation-derived microbial metabolite DHNA modulates intestinal innate immune responses through AhR-associated pathways

1,4-Dihydroxy-2-naphthoic acid (DHNA), a microbial metabolite produced during fermentation by Propionibacterium freudenreichii, is known as a bifidogenic growth stimulator used in functional foods. Although DHNA has been reported to exert anti-inflammatory effects, its role in intestinal immune regulation remains incompletely understood. In this study, we investigated whether DHNA modulates innate immune responses in intestinal epithelial and immune cell models in association with aryl hydrocarbon receptor (AhR) signaling. DHNA induced AhR-associated nuclear signaling in intestinal epithelial cells without detectable cytotoxicity. In THP-1-derived immune cell models, DHNA enhanced macrophage-associated bacterial clearance and induced production of the antimicrobial peptide human β-defensin-2 (hBD-2). In a Caco-2/THP-1 Transwell co-culture system, DHNA increased IFN-γ mRNA expression, whereas in THP-1-derived macrophage-like cells, DHNA increased IL-12p35 and IFN-γ protein levels. Pharmacological inhibition and AhR knockdown experiments indicated that several of these responses were associated, at least in part, with AhR signaling. These findings suggest that DHNA modulates antimicrobial and cytokine responses through AhR-associated pathways, supporting a potential immunomodulatory role for this fermentation-derived metabolite in the intestinal environment.