Breaking the Barrier: Pro-inflammatory Stool from Infants with CHD Triggers Barrier Dysfunction within Intestinal Organoids

Background Infants with congenital heart disease (CHD) undergoing cardiopulmonary bypass (CPB) frequently develop gut injury and barrier dysfunction. The effects of the post-operative gut milieu on the intestinal epithelium remain poorly defined. Given ethical challenges in obtaining intestinal tissue from these patients, organoids offer a solution to study post-CPB intestinal changes. Methods Porcine intestinal organoids were converted from basal-out to apical-out polarity to enable luminal exposure. Organoids were treated with pre-operative and post-operative fecal supernatants derived from a neonate with CHD undergoing CPB and a non-CHD surgical control. Microbial composition and metabolite profiles were analyzed, and epithelial responses were assessed following 72-hour exposure. Results Apical-out organoids enabled direct luminal interrogation. Post-CPB stool exhibited enriched pro-inflammatory organisms and reduced microbial diversity, increased pro-inflammatory eicosanoids, and depletion of SCFA versus control. Post-CPB fecal supernatant induced epithelial barrier dysfunction and injury with redistribution of Claudin-2 and Claudin-3, and reduced fatty acid binding protein 2. There was altered PGE2 signaling including upregulation of PGE2 synthase, downregulation of prostaglandin dehydrogenase, and altered EP2 receptor localization. Conclusion Post-operative stool following CPB contributes to epithelial injury and maladaptive responses characterized by PGE2-axis remodeling, tight junction reorganization, and loss of mature enterocyte features. This model of CHD stool exposure in organoids provides a robust translational platform for mechanistic studies and therapeutic targeting of gut injury following CPB.