PP401 - PARENTERAL NUTRITION ASSOCIATED LPS/TLR4 SIGNALING DISRUPTS WNT/Β-CATENIN–DEPENDENT EPITHELIAL HOMEOSTASIS AND REVERSED BY GSK3Β INHIBITION IN ENTEROIDS

PP401

PARENTERAL NUTRITION ASSOCIATED LPS/TLR4 SIGNALING DISRUPTS WNT/Β-CATENIN–DEPENDENT EPITHELIAL HOMEOSTASIS AND REVERSED BY GSK3Β INHIBITION IN ENTEROIDS

A. Bagwe¹, C. Manithody¹, S. Mehta¹, S. Kolli¹, T. Ohkura¹, K. Desanti¹, M. Johnson¹, J. Arnaut¹, M. Deen ¹, F. Medina¹, S. Onishi¹, U. Ezekiel ², A. K. Jain^1,*

¹Department of Pediatrics, ²Clinical Health Sciences , Saint Louis University School of Medicine, Saint Louis, United States

Rationale: Total parenteral nutrition (TPN) and lipopolysaccharide (LPS) disrupt intestinal epithelial homeostasis via TLR4 activation, weakening barrier integrity and suppressing Wnt/β-catenin signaling. We used neonatal porcine 3D enteroids to define epithelial inflammatory mechanisms and test whether GSK3β inhibition with CHIR99021 rescues LPS-induced injury. 

Methods: Enteroids from neonatal Yorkshire pig small bowel were cultured in Matrigel for 7 days, then exposed to 1, 2, or 5 µg/mL LPS for 24 h (dose-response) or to control, LPS, CHIR99021, or LPS+CHIR99021 (rescue studies). qPCR assessed TLR4, Wnt signaling, epithelial barrier, mucus, and cytokine genes. Serum cytokines from EN and TPN animals served as in vivo references.

Results: LPS activated innate signaling, increasing TLR4 (peak at 2 µg/mL) and IL-8/IL-10. At 5 µg/mL, ZO-1 was reduced (3.6-fold), indicating barrier loss. LPS suppressed Wnt/β-catenin signaling (β-catenin 0.29-fold, Cyclin D1 0.37-fold) and decreased epithelial markers (E-cadherin 0.43-fold, Occludin 0.62-fold, Claudin-3 0.69-fold, Muc2 0.42-fold), with increased IL-1β/IL-8 and reduced IL-10. CHIR99021 restored Wnt signaling (β-catenin 1.87-fold, Cyclin D1 1.20-fold), improved barrier markers (E-cadherin 1.13-fold, Occludin 1.18-fold, Claudin-3 0.83-fold, Muc2 1.10-fold), reduced IL-1β (0.69-fold) and IL-8 (0.59-fold), and increased IL-10 (1.48-fold). CHIR alone enhanced epithelial programs (β-catenin 6.20-fold, Claudin-1 2.99-fold, Occludin 2.00-fold, Muc2 3.60-fold). In vivo, TPN elevated IFN-γ vs EN (p=0.009) and FMT (p=0.001), and IL-8 and LPS vs EN (p=0.011, p<0.0001), while IL-10 was unchanged.

Conclusion: LPS drives TLR4-dependent inflammatory injury with Wnt suppression, barrier disruption, and impaired mucus production. GSK3β inhibition restores Wnt activity, cytokine balance, and epithelial resilience.

Disclosure of Interest: None declared