Breaches in the intestinal mucosal barrier lead to sepsis and death if epithelial coverage is not rapidly restored, particularly in neonates. The reason for higher mortality in infants as compared to more mature individuals with intestinal injury has not been explained. High infant mortality results from diseases associated with ischemia/ reperfusion (I/R) injury, including necrotizing enterocolitis and volvulus. In juvenile pigs (6-8-weeks of age), we have extensively studied the remarkably rapid intestinal repair marked by epithelial cell migration (restitution). However, in a recently published study featured in the revised version of this proposal, we have shown an intriguing age-dependent deficiency in the recovery of mucosal repair following ischemic injury with a near-total lack of restitution in neonatal piglets. Interestingly, this defect in repair can be rescued by a homogenate of ischemic injured juvenile homogenate, suggesting more mature cell populations stimulate restitution. One cell population of particular interest is the enteric glial cell (EGC) network, which we have shown matures postnatally in pigs. In the revised proposal, we have now shown that porcine EGC-conditioned media stimulates cell migration (restitution) in wounded neonatal porcine IPEC-J2 cells. However, a gap remains in our knowledge regarding the signaling mechanisms between epithelium and underlying EGC that results in restitution. To address this gap, we have performed RNAseq analyses of ischemic-injured neonatal and juvenile mucosa, and created an unbiased discovery pipeline approach to focus on significantly deficient signaling pathways in the neonate. One such pathway includes EGF, which is secreted by the EGC as proEGF, and epithelial annexin A2, which initiates cell migration by signaling internalization of ?1-integrin. Our central hypothesis is that repair of ischemic-injured mucosa is reliant on postnatal migration of EGC into the lamina propria in order to signal wounded epithelium to efficiently restitute. We will test this hypothesis with two specific aims: 1) Determine age-dependent defects in migration signaling mechanisms in wound-adjacent intestinal epithelial cells. Our working hypothesis is that there is an age-dependent development of epithelial restitution in response to injury at least in part via annexin A2 signaling. 2) Determine perinatal changes in mucosal EGC network structure, density and secretome. Our working hypothesis is that there are insufficient EGCs secreting barrier-promoting factors, including proEGF, in proximity to the epithelium to stimulate restitution in neonates. To examine these specific aims, we will use our unique age-dependent porcine model of mucosal repair, a wounded porcine neonatal IPEC-J2 cell model, primary culture of porcine EGC, EGC secretome analyses via mass spectrometry, and state of the art imaging techniques for glial cells, including immunolabeling-enabled three-dimensional imaging of solvent-cleared organs (iDISCO). Using an unbiased discovery pipeline approach, we expect to be able to define signaling mechanisms that are deficient in neonatal injured intestine that can ultimately be targeted to enhance survival of patients with intestinal failure
/PUBLIC HEALTH RELEVANCE STATEMENT The pig has anatomical and physiological similarities to humans, providing a highly relevant translational model of digestive disease. We will focus on the mechanisms of restitution that result in poor intestinal recovery in infants and piglets with severe mucosal injury as compared to more mature individuals. We expect to identify age-dependent novel mechanisms whereby the enteric glial cells (EGC) signal wounded epithelial cells to restitute in order to repair wounded intestinal mucosa, thereby ultimately improving survival of neonates with intestinal disease.
