Pharmacologic activation of HNF4α/γ Restores Epithelial Barrier Function in Crohn’s disease
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Background and aims
Intestinal permeability barrier dysfunction is a defining feature of Crohn’s disease (CD) that precedes inflammation, persists during remission, and predicts relapse. Despite its clinical importance, no validated biomarkers or therapies directly target epithelial barrier restoration. 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 function.
Methods
We integrated our previously defined HNF4α/γ-regulated epithelial transcriptome with 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, with 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 inflammation independent. In contrast, chronic TNF overexpression in the Tnf ΔARE/+ mice phenocopied this suppression through a normally transient epithelial injury response that became pathologically stabilized under sustained cytokine signaling. 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 master transcriptional regulators of epithelial TJ integrity whose loss defines a primary barrier defect in CD. Chronic inflammation can mimic but does not initiate this intrinsic defect. 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.
