Coordinated HNF4α and HNF4γ Suppression Defines a Reversible Ileal Epithelial Barrier Defect in Crohn,s Disease

Background & Aims: Ileal barrier dysfunction in Crohn,s disease (CD) can occur in macroscopically non-inflamed mucosa, persist despite clinical and endoscopic remission, and predict adverse outcomes. The epithelial mechanisms underlying this defect remain unclear. We investigated whether coordinated reduction of hepatocyte nuclear factor 4, (HNF4) and HNF4{gamma} defines an epithelial-intrinsic, barrier-defective state in ileal CD. Methods: Barrier-associated transcriptional programs were compared in small-intestinal and colonic epithelium from wild-type mice and mice with intestinal epithelial deletion of Hnf4, Hnf4{gamma}, or both paralogs. Paracellular permeability was assessed in wild-type and Hnf4/{gamma} double-knockout (Hnf4{gamma}DKO) small-intestinal organoids. HNF4, HNF4{gamma}, and the direct HNF4 transcriptional target claudin-15 (CLDN15) were evaluated in human ileal CD tissue, patient-derived organoids (PDOs) generated from macroscopically non-inflamed ileal mucosa, and ileal tissue and organoids from Tnf{delta}ARE/+ mice. The effects of HNF4 agonists N-trans-caffeoyltyramine (NCT) and N-trans-feruloyltyramine (NFT) were assessed in CD PDOs and Tnf{delta}ARE/+ organoids. Results: Combined, but not individual, loss of HNF4-paralog broadly disrupted small-intestinal transcriptional programs governing tight junction (TJ) organization, epithelial polarity, junctional-cytoskeletal coupling, and TJ repair, with limited effects in the colon. Hnf4{gamma}DKO organoids exhibited size-selective paracellular permeability in the absence of inflammatory and stromal cells, demonstrating that combined Hnf4{gamma} loss is sufficient to produce an epithelial-intrinsic barrier defect. HNF4, HNF4{gamma}, and CLDN15 were coordinately reduced in human ileal CD epithelium and this HNF4-low phenotype was retained in PDOs from macroscopically non-inflamed ileal mucosa. Chronic TNF-driven ileitis reproduced this HNF4-low state, which was retained ex vivo in organoids. HNF4 agonists restored HNF4, HNF4{gamma}, and CLDN15 expression and improved transepithelial electrical resistance (TEER) in CD PDOs. They also increased HNF4 and CLDN15 expression in Tnf{delta}ARE/+ organoids. Conclusions: Coordinated HNF4/{gamma} suppression defines an epithelial-intrinsic, barrier-defective state in ileal CD that is present in PDOs derived from macroscopically non-inflamed ileal mucosa and retained ex vivo without continued exposure to the inflammatory tissue environment. Its pharmacologic reversibility identifies incomplete restoration of the HNF4-dependent epithelial program as a potential mechanism of persistent ileal barrier dysfunction and supports further evaluation of HNF4-directed barrier-restorative strategies.