PurposeTo investigate whether Leukotriene B4 receptor 2 (BLT-2), an upstream regulator of tight junction protein (TJP) Claudin-4, and TJPs could be etiologic factors in Hirschsprung-associated enterocolitis (HAEC) after pull-through (PT) for Hirschsprung disease (HD). MethodsNormoganglionic colon (HD-N) and aganglionic rectum (HD-A) specimens from rectal/rectosigmoid (R/RS) or descending/transverse (D/T) HD were assessed using quantitative polymerase chain reaction (qPCR) for Occludin, TJP-1, TJP-2, Junctional adhesion molecule (JAM)-1, JAM-2, Claudin-1, Claudin-3, Claudin-4, and BLT-2 and immunoblotting for Claudin-4 using fresh specimens obtained intraoperatively (2021-2024; n=17; R/RS=15 and D/T=2). Claudin-4 immunohistochemistry was also evaluated quantitatively using preserved (n=29; R/RS=20 and D/T=9; 2009-2021) and fresh HD specimens for comparison with anorectal malformation patients having colostomy closure as controls (n=42) and between HD-A versus HD-N, R/RS versus D/T, and HAEC(+) versus HAEC(-). Technically inadequate or transitional zone PT were excluded. ResultsSubjects were 123 PT cases. Mean ages at PT/colostomy closure (years) were R/RS: 2.7±2.9, D/T: 1.6±2.2, and controls: 1.4±0.7. Postoperative HAEC occurred 18 times in 14 PT cases (grade I=5, grade II=13). Post-PT HAEC was significantly more frequent in D/T (50.0% versus 6.4%; p<0.001); Claudin-4 was significantly lower in HD-N from post-PT HAEC cases, especially D/T (p<0.05) on immunohistochemistry. Claudin-4 was significantly lower in HD-N/HD-A compared with controls on immunoblotting (p<0.05) and immunohistochemistry (p<0.001). qPCR showed TJP-1, TJP-2, JAM-1, JAM-2, Claudin-4, and BLT-2 were significantly lower in HD-N/HD-A compared with controls. ConclusionsLower Claudin-4 and BLT2 in post-PT HAEC HD-N (especially D/T) suggests generalized epithelial barrier derangement with possible etiologic implications for HAEC. Level of EvidenceⅡ