O1090: Tight Junction Proteins Are Deficient in the Ganglionic and Aganglionic Colon in Children with Hirschsprung Disease

Background: Despite successful pull-through procedures, children with Hirschsprung disease (HSCR) may continue to suffer from Hirschsprung-associated enterocolitis (HAEC) and constipation. Although dysregulation of intestinal epithelial barrier function has been hypothesized to be a contributor to development of HAEC and constipation, but the veracity of this hypothesis remains to be tested.
Permeability of the intestinal epithelial barrier is, primarily, a function of tight junction conductance. In order to assess tight junction function indirectly, we assessed tight junction protein expression and regulatory enzyme expression and activity in HSCR resection specimens and controls.

Methods: Tissue microarrays were created from paraffin-embedded colonic resection specimens from 28 children with HSCR disease and 13 age-matched controls undergoing colorectal biopsies for constipation. Multiplex immunohistochemistry was used to assess expression of tight junction proteins claudin-2, claudin-4, claudin-15, zonula occludens-1 (ZO-1), and occludin as well as myosin light chain kinase (MLCK) expression and myosin II regulatory light chain (MLC) phosphorylation. Semi-automated quantitative morphometry was performed using CellProfiler. Median fluorescence intensity of each protein or phosphorylated MLC was normalized to ZO-1 to control for variations in tissue handling, including section thickness. Labeling of ganglionic and aganglionic specimens from the same patient were compared used Wilcoxon signed-rank test. Comparisons between HSCR patient and controls used the Mann-Whitney test. Statistical significance was taken at p< 0.05.

Results: Expression of claudin-2 (Fig. 1a, 0.78 vs. 0.98, p < 0.0001), claudin-15 (Fig. 1b, 1.54 vs. 2.20, p =0.0013), and occludin (Fig. 1c, 0.40 vs. 0.82, p < 0.0001) were all reduced in ganglionic HSCR segments relative to controls. Neither claudin-4 nor MLCK expression or activity, assessed as MLC phosphorylation, differed between ganglionic segments and age-matched controls. Similarly, when compared to age-matched controls, claudin-2 (Fig. 1a, 0.67 vs. 0.98, p =0.0013), claudin-15 (Fig. 1b, 1.23 vs. 2.20, p =0.0001), and occludin (Fig. 1c, 0.38 vs. 0.82, p < 0.0001) expression in aganglionic segments was reduced but there were no differences in claudn-4 or MLCK expression or MLC phosphorylation. Expression of all proteins was similar within ganglionic and aganglionic segments of each patient.

Conclusion: Expression of tight junction proteins claudin-2, claudin-15, and occludin are decreased in ganglionic and aganglionic HSCR colonic segments relative to controls. Notably, claudins 2 and 15 both form paracellular cation (Na+) and water channels whose expression contributes to luminal hydration. Reduced expression of these proteins may, therefore, contribute to constipation. Occludin loss has been shown to enhance epithelial survival and could, therefore, represent an adaptive response. These data may explain how epithelial barrier dysregulation can impact HSCR.