Search bioRxiv⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2025.10.27.684895

Pharmacologic activation of HNF4α/γ restores epithelial barrier function in Crohn's disease.

Halder, D.·Ghazi, A.·Wang, Y.·Hou, J. K.·Verzi, M.·Khurana, S.
View original source ↗Export metadata ↓

Abstract

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.

Explore related subjects

pathologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Halder, D., Ghazi, A., Wang, Y., Hou, J. K., Verzi, M., Khurana, S.. 2025-10-27. Pharmacologic activation of HNF4α/γ restores epithelial barrier function in Crohn's disease.. https://doi.org/10.1101/2025.10.27.684895

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Autophagic flux is increased in peripheral blood mononuclear cells in atherosclerotic vascular disease and associates inversely with adverse cardiovascular events

Background: Autophagy is a homeostatic pathway supporting stress adaptation and is dysregulated in atherosclerosis. Its potential as a biomarker or therapeutic target in atherosclerotic vascular disease (ASVD) remains incompletely defined. We measured autophagic flux in peripheral blood mononuclear cells (PBMCs) from patients with peripheral arterial disease (PAD) or carotid stenosis (CS), compared with healthy controls, and explored clinical outcome associations. Methods: Ninety-four patients with PAD or CS and 19 healthy controls were studied. Autophagic flux was quantified from fresh blood using a validated ex vivo chloroquine inhibition ELISA measuring LC3BII accumulation. Major adverse cardiovascular events (MACE) and major adverse limb events (MALE) were ascertained over a median follow up of 828 days. Results: The ASVD cohort comprised claudication (n = 16), chronic limb threatening ischemia (CLTI; n = 49), and CS (n = 29). Autophagic flux was higher in ASVD than controls (mean 281.4 vs. 182.3 ng LC3BII/mg protein/h; p < 0.0001) and remained independently associated after multivariable adjustment. Within CLTI, concurrent infection was associated with lower flux (p = 0.001), approaching control levels (p = 0.327). In CLTI, higher flux quartiles were associated with lower MACE risk, most strongly for quartile 3 (hazard ratio 0.07 vs. quartile 1, 95% CI 0.01 to 0.50; p = 0.009). Conclusion: Autophagic flux is elevated in PBMCs from ASVD patients, independent of age and sex. Attenuated flux in CLTI with concurrent infection may indicate autophagic exhaustion in advanced disease. The association between higher flux and lower MACE in CLTI suggests prognostic utility, warranting evaluation in larger prospective studies.

pathology↗
bioRxiv2026

Quantitative Model of the Ocular Immune Response during Seasonal Allergic Conjunctivitis

Allergic conjunctivitis is an inflammation of the conjunctiva caused by allergen; it is common disorder affecting up to 40% of the population. In this work, we study seasonal allergic conjunctivitis (SAC), also called "hay fever eyes", which is caused by exposure to airborne pollens. We develop a mathematical model quantifying the ocular immune system response to the allergens. First, we present a simplified qualitative description of the immunopathogenesis of SAC. Then, we express each chosen immunopathological mechanism mathematically to construct a system of thirty-one ordinary differential equations. We compare summary statistics of the predicted observable immune signals to experimental measurements and find our model captures key qualitative features of SAC progression. We then compare our predicted time series of histamine concentration to symptom scores and find a strong correlation suggesting the model predicts relevant clinically trends. Next, we calibrate the model through multi-step process. We find the most influential parameters are the production and depletion rates of IL-4, and the production rates of IL-5 and IL-8. These cytokines are targeted in treatments for asthma, atopic dermatitis, and severe eosinophilic associated disorder and suggest potential therapeutic targets for SAC. Our calibrated model mimics most of the summary statistics of the experimentally observable immune signals with discrepancies for IL-5 and IL-13 indicating that additional immunopathological mechanisms could be important.

pathology↗
bioRxiv2026

Dysregulated Platelet GPIb alpha - VWF Signalling in Abdominal Aortic Aneurysm formation and Progression

Background: Platelets are critical drivers of thrombo-inflammatory responses in different cardiovascular diseases. Abdominal aortic aneurysm (AAA) is a progressive, life-threatening vascular disorder mainly characterised by chronic inflammation, extracellular matrix degradation, and the formation of a platelet-rich intraluminal thrombus (ILT). Experimental and clinical evidence identified platelets as main players in AAA pathology as evidenced by elevated platelet activation and procoagulant activity that critically contribute to AAA progression. Methods: The present study investigated the contribution of glycoprotein (GP)Ib alpha, the von Willebrand factor (VWF)-binding subunit of the platelet GPIb-IX-V complex, to AAA initiation and progression in experimental AAA using the ePPE mouse model and in patients. Results: Genetic ablation of platelet GPIb alpha significantly attenuated early aneurysm expansion in experimental AAA, indicating a critical role for GPIb alpha during the initial stages of aneurysm development. This initial effect was compensated at later time points showing no differences in aneurysm progression between groups. Notably, genetic deletion of GPIb alpha induced a constitutively hyperactive platelet phenotype already in naive mice that was further amplified during experimental AAA. This elevated platelet hyperactivity was mainly due to increased GPVI activation of platelets 28 days post-surgery. To assess the clinical relevance, spatial profiles of human ILT specimens from patients with AAA were analysed. In the ILT, we detected a highly compartmentalised distribution of GPIb alpha and VWF with pronounced enrichment within the luminal layer. In parallel, circulating VWF activity as well as platelet surface expression of GPIb alpha were significantly increased in patients with AAA. Conclusion: Collectively, these findings identify a dysregulated GPIb alpha-VWF axis in human AAA pathology, mainly characterised by enhanced platelet GPIb alpha surface expression and increased activity of circulating VWF.

pathology↗