Tight Junction Proteins Are Deficient in the Ganglionic and Aganglionic Colon in Children with Hirschsprung Disease
Lorena Rincon-Cruz1, Shabnam Abhati2, Jerrold Turner2, Prathima Nandivada1
1Boston Children's Hospital, Boston, MA; 2Brigham and Women's Hospital, Boston, MA
Background: Despite successful pull-through procedures, children with Hirschsprung disease (HSCR) may continue suffering from Hirschsprung-associated enterocolitis (HAEC) and constipation. To test for dysregulation of intestinal epithelial barrier function indirectly, we assessed tight junction protein and regulatory enzyme expression in HSCR resection specimens and controls.
Methods: Tissue microarrays were created from colonic resection specimens from 28 children with HSCR disease and 13 age-matched controls undergoing colorectal biopsies. We assessed expression of tight junction proteins claudin-2, claudin-4, claudin-15, zonula occludens-1 (ZO-1), occludin, myosin light chain kinase (MLCK) expression, and myosin II regulatory light chain (MLC) phosphorylation using immunohistochemistry and quantitative morphometry with CellProfiler. Median fluorescence intensity was normalized to ZO-1 to control for variations in tissue handling.
Results: Expression of claudin-2, claudin-15, and occludin (Fig. 1a, p<0.0001; Fig. 1b, p=0.0013; Fig. 1c, p < 0.0001, respectively) were all reduced in ganglionic HSCR segments relative to controls. Similarly, when compared to age-matched controls, claudin-2, claudin-15, and occludin (Fig. 1a, p=0.0013; Fig. 1b, p=0.001, Fig. 1c, p < 0.0001, respectively) expression in aganglionic segments was reduced. No differences were detected in levels of claudin-4, MLCK expression or MLC phosphorylation. Expression of all proteins was similar between ganglionic and aganglionic segments of each patient.
Conclusion: Expression of claudin-2, claudin-15, and occludin were decreased in ganglionic and aganglionic HSCR colonic segments relative to controls. These data may explain how epithelial barrier dysregulation can impact HSCR.
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