Pharmacologic activation of HNF4α/γ Restores Epithelial Barrier Function in Crohn's disease.

Background and aims: Intestinal permeability barrier dysfunction is a hallmark of Crohn's disease (CD) that occurs before inflammation, persists during remission, and predicts relapse. Despite its clinical importance, no validated biomarkers or therapies directly target epithelial barrier restoration in CD. We investigated whether hepatocyte nuclear factors HNF4α and HNF4γ regulate tight junction (TJ) organization and barrier integrity in CD, and whether pharmacologic HNF4 activation can restore epithelial barrier function. Methods: To investigate the role of HNF4α/γ in TJ regulation and CD pathogenesis, we integrated transcriptomic data from the small intestinal epithelium of Hnf4α/γ double knockout (DKO) mice, with functional analyses of genetic ( Hnf4α/γ double knockout) and inflammatory ( Tnf ΔARE/+ ) mouse models, human CD tissues, and patient derived organoids (PDOs). Barrier function was assessed by FITC-dextran flux and transepithelial electrical resistance (TEER). Two plant-derived, non-toxic HNF4 agonists, N-trans-caffeoyltyramine (NCT) and N-trans-feruloyltyramine (NFT), were tested for therapeutic rescue of barrier function in CD. Results: Combined loss of Hnf4α/γ impaired epithelial barrier function by activating both pore and leak pathway, resulting in selective permeability to 4-10 kDa tracers. Integration with our transcriptomic data from the Hnf4α/γ double knockout (DKO) mice revealed broad repression of TJ-associated genes, including Tjp2 (encoding ZO-2), F11r (encoding JAM-A), MarvelD3 , Cldn4 , Cldn7 , and Cldn15 , together with induction of the pore-forming gene Cldn2 . HNFα/γ proteins were markedly downregulated in CD intestinal epithelium and in PDOs established from non-inflamed mucosa, establishing that the barrier defect is epithelial-intrinsic and independent of inflammation. In contrast, chronic TNF overexpression phenocopied this suppression through a normally transient epithelial injury response that became pathologically stabilized under sustained cytokine signaling in Tnf ΔARE/+ mice. Treatment of both CD PDOs and Tnf ΔARE/+ mouse organoids with NCT or NFT reactivated HNF4α/γ expression, normalized TJ gene profile, and restored TEER to near-control levels. Conclusion: HNF4α/γ are primary transcriptional regulators of epithelial TJ integrity whose loss defines a primary, inflammation-independent barrier defect in CD. Chronic inflammation can mimic but does not initiate this intrinsic epithelial dysfunction. Pharmacologic activation of HNF4α/γ with plant-derived, non-toxic, translationally ready agonists (NCT and NFT) restore barrier function, establishing the first barrier-healing therapy for CD.