K. P. Kuruvilla1,3, J. F. Pierre2, A. Gosain1,3 1University of Tennessee Health Science Center,Department Of Surgery,Memphis, TN, USA 2University of Tennessee Health Science Center,Department Of Pediatrics,Memphis, TN, USA 3Children’s Foundation Research Institute, Le Bonheur Children’s Hospital,Division Of Pediatric Surgery,Memphis, TN, USA
Introduction: Hirschsprung disease (HSCR) results from incomplete development of the enteric nervous system (ENS). Hirschsprung-Associated Enterocolitis (HAEC) is the most significant and life-threatening complication of HSCR and the occurrence of HAEC even after surgical resection of bowel lacking an ENS suggests that pathophysiology of HAEC is multifactorial. We have previously shown that EdnrBNCC-/- mouse model of HSCR exhibit colonic dysbiosis prior to development of HAEC. We have also observed that tight junctions (TJ), which are critical determinants of intestinal epithelial barrier integrity, mature earlier in EdnrBNCC-/- mice (vs. wildtype) but then are downregulated just prior to HAEC onset. In this study, we sought to determine the impact of the dysbiotic EdnrBNCC-/- microbiome in regulating the expression of TJ using pseudo germ-free (PGF) mice. We hypothesized that the dysbiotic EdnrBNCC-/- microbiome would trigger downregulation of TJs and promote HAEC.
Methods: Wild-type PGF pups were generated by treatment of pregnant dams with broad spectrum antibiotics and antifungals and maintained PGF until weaning. At postnatal day P21, PGF animals received oral gavage of either EdnrBNCC-/- microbiota for fecal microbiota transplant (FMT) or sterile phosphate buffered saline (PBS) as control. Mice were separately housed and followed for 1 week (P28). Prior studies have shown that P21 coincides with the onset of HAEC in EdnrBNCC-/- mice and P28 is the mean time of death from HAEC.
Results: 16S pyrosequencing and total copy number confirmed the PGF status of neonatal pups. PGF pups showed reduction in body weight gain as compared to non-PGF WT pups beginning about P10. However, upon weaning and gavage with either FMT or PBS, both groups started gaining body weight. In the seven days following gavage, neither FMT nor PBS-treated mice showed signs of HAEC based on clinical severity scoring (hunched posture, ruffled hair coat, lethargy and weight loss). Immunohistochemistry revealed increased expression of the TJ proteins occludin (Ocln) and zona occludens-1 (ZO-1) in the ileum and colon of the FMT group as compared to PBS. There were no differences in the expression of claudin-3 (Cldn3) or E-cadherin (adherens junction protein).
Conclusions: Increased expression of TJ proteins (Ocln and ZO-1) in FMT mice indicates that the dysbiotic EdnrBNCC-/- microbiome aids in strengthening the epithelial barrier of these intestinal tissues. This result is consistent with our prior, in vivo, observations in EdnrBNCC-/- mice prior to HAEC and suggests that EdnrBNCC-/- microbiome alone is not sufficient to cause HAEC. The specific components of the EdnrBNCC-/- microbiome contributing to TJ maturation and the mechanistic pathways are under active investigation.