Gut microbiota-derived tryptamine activates AhR-NRF2 signaling to modulate airway epithelial barrier function in allergic asthma

BACKGROUND: Allergic asthma is a chronic inflammatory airway disease characterized by epithelial barrier dysfunction and dysregulated immune responses. Emerging evidence indicates that gut microbiota dysbiosis contributes to asthma pathogenesis through the gut-lung axis, with microbial metabolites such as tryptamine (TRP) playing critical immunomodulatory roles. However, the precise mechanisms linking gut microbiota-derived tryptophan metabolites to airway epithelial barrier integrity remain incompletely understood. OBJECTIVE: This study aims to investigate the role of gut microbiota-dependent tryptamine-aryl hydrocarbon receptor (AhR)-nuclear factor erythroid 2-related factor 2 (NRF2) signaling in regulating airway epithelial barrier function in house dust mite (HDM)-induced allergic asthma. METHODS: HDM-induced allergic airway inflammation was established in C57BL/6 mice through intratracheal sensitization followed by intranasal challenge. Airway hyperresponsiveness (AHR) was measured using an Animal Lung Function System with methacholine challenge. Gut microbiota composition was analyzed by 16S rRNA gene sequencing, and functional prediction was performed using PICRUSt2. Fecal tryptophan metabolites were quantified by targeted metabolomics. The effects of exogenous TRP administration (10 mg/kg/day) on airway inflammation, AhR-NRF2 signaling, and tight junction protein expression were evaluated in vivo and in vitro using human bronchial epithelial cells (16HBE). NRF2-specific siRNA was employed to validate the necessity of NRF2 in the AhR-NRF2 axis. RESULTS: HDM-challenged mice exhibited significant gut microbiota dysbiosis, characterized by reduced abundance of beneficial bacteria (Lactobacillus, Bifidobacterium) and decreased fecal TRP levels. HDM-challenged mice also showed significantly enhanced AHR compared to controls. TRP administration attenuated HDM-induced airway inflammation and AHR, promoted AhR nuclear translocation, upregulated NRF2 expression, and enhanced tight junction protein (Claudin-1, Occludin, E-cadherin) expression. In vitro studies confirmed that TRP activated AhR-NRF2 signaling and restored barrier function in HDM-stimulated 16HBE cells, effects that were blocked by the AhR antagonist CH-223191. Importantly, NRF2 knockdown by siRNA abolished the protective effects of AhR overexpression on tight junction protein expression, demonstrating that NRF2 is an essential downstream mediator of the AhR-NRF2 axis. CONCLUSIONS: These findings demonstrate that HDM-induced allergic airway inflammation is associated with gut microbiota dysbiosis and impaired tryptophan metabolism. TRP, through activation of the AhR-NRF2 signaling pathway, enhances tight junction protein expression and restores airway epithelial barrier integrity, highlighting the therapeutic potential of targeting the gut microbiota-tryptophan-AhR-NRF2 axis in asthma.