Crosstalk through cholinergic and aryl hydrocarbon receptor pathways in the anti-inflammatory actions of microbial...

The intestinal epithelium maintains immune and metabolic homeostasis at the host-microbiota interface, and its disruption contributes to inflammatory bowel disease (IBD). Microbial metabolites such as short-chain fatty acids (SCFAs) and indole derivatives are known to regulate epithelial and immune functions, but their roles in cholinergic signaling and aryl hydrocarbon receptor (AhR) pathways remain incompletely understood. Caco-2 intestinal epithelial cells were stimulated with lipopolysaccharide (LPS; 10 µg/mL) and co-treated with SCFAs (acetate, propionate, butyrate) or indole derivatives (IAA, IPA, IBA; 1000 µM). The AhR antagonist CH223191 was used to assess receptor involvement. Inflammatory markers (TNF-α, IL-6, COX-2), barrier integrity (FITC-dextran permeability, ZO-1 and occludin), cholinergic components (CHAT, ACHE, BCHE, α7nAChR, mAChR1, mAChR3), and AhR activation (nuclear localization, CYP1A1 expression) were analyzed using qPCR, ELISA, immunostaining, and enzymatic assays. Molecular docking was performed to predict metabolite-protein interactions. LPS induced inflammatory cytokine expression and barrier disruption, whereas SCFAs and indole derivatives significantly reduced inflammation, restored tight-junction organization, and decreased permeability. These metabolites enhanced CHAT and muscarinic receptor expression while suppressing ACHE upregulation, indicating restoration of cholinergic balance. Both groups reactivated AhR signaling, and AhR inhibition disrupted cholinergic regulation, supporting an AhR-dependent mechanism. Butyrate and indole-3-butyric acid exhibited the strongest protective effects. These findings identify a novel AhR-cholinergic axis in intestinal epithelial regulation and highlight microbial metabolites as promising therapeutic candidates for IBD.