675 - The Role of Localized Prostaglandin E2 in the Preterm Infant Gut

Publication Number: 1652.675

Melissa Olsen, University of South Florida, Tampa, FL, United States; Hannah Wapshott-Stehli, University of South Florida, Wesley Chapel, FL, United States; Madison R. Huszar, University of South Florida, Tampa, FL, United States; Thao Ho, University of South Florida, Tampa, FL, United States

Background: Preterm infant digestive tracts differ from full-term and adult ones because of their underdeveloped nature. The study of the immature preterm infant gut requires validation of pathways known to exist in mature intestinal cells. For this work, we are studying the localized role of prostaglandin E2 (PGE2). The localized, short-term activation PGE2 promotes intestinal wound healing in mouse and adult human intestinal enteroids. This mechanism involves PGE2 binding the EP4 receptor on intestinal cells and activating a signal cascade that leads to the expression of wound-associated epithelial (WAE) cells. These specialized cells cover the injured area, quickly giving enterocytes and crypts a chance to reform. In a case-control study of fecal metabolomics from our group, preterm infants with severe anemia had lower relative abundance of fecal prostaglandins compared to non-anemic infants.

Objective: Our objective was to determine if the pathway that involves PGE2 and the EP4 receptor is intact in immature human intestinal epithelial cells using human tissue-derived enteroids models.

Design/Methods: Enteroids were plated onto 12-well plates in Ultimatrix domes and grown for 72 hours in 85:15 media (85% Human Colonoid Media [HCM] and 15% Intesticult Organoid Growth Medium) containing 100µg/mL Primocin and 10µM Y-27632. The cells were then either exposed to methyl acetate (vehicle control), EP4 inhibitor L-161982, or 16, 16-Dimethyl PGE2 (stable form of PGE2) for 2, 5, 8, and 24 hours. RNA was extracted at each timepoint, and the relative expression of WAE cell marker cd55 was then analyzed using RT-qPCR.

Results: Initial exposure to PGE2 induced the expression of wound-associated epithelial cells in our immature intestinal enteroid cell line as shown by increase in relative cd55 gene expression (Figure 1). This increased expression was significant compared to time 0 vs 2, 5, and 8 hours after exposure (Figure 1) and between control and EP4 inhibitor cells vs PGE2 cells for hours 2, 5, and 8 after exposure (Figure 2).

Conclusion(s): Now that the pathway for PGE2 WAE cell induction has been demonstrated in immature enteroids, we will continue with monolayer and barrier function experiments to further elucidate the role of localized prostaglandins in preterm intestinal wound healing. In addition, the longitudinal quantitative measurements of fecal prostaglandin metabolites will be compared between very low birth weight preterm infants with and without severe anemia.

Enteroid cd55 expression after PGE2 exposure
PAS Figure 1.jpegFigure 1. Enteroid relative cd55 expression after PGE2 exposure. Enteroids were grown for 72hrs before the addition of 10µM PGE2. RNA was extracted and RT-qPCR was run to visualize the relative expression of the cd55 gene compared to the cell population before PGE2 exposure. Data points are a mean of five replicates, and error bars represent the standard deviation. Significance was determined using a Kruskal-Wallis nonparametric test with Dunn’s multiple comparisons test between the 0hr control and each timepoint. Created in https://BioRender.com.

Relative cd55 expression under different exposure conditions
PAS Figure 2.jpegFigure 2. Enteroid relative cd55 expression after different exposure conditions. Enteroids were grown for 72hrs before the addition of 10µM PGE2, 10µM L-161982, or methyl acetate (vehicle control). RNA was extracted at 2, 5, 8, and 24 hours, and RT-qPCR was run to determine the relative expression of cd55 under each condition. Data points are a mean of five replicates, and error bars represent the standard deviation. Significance between each group at the same hour after exposure was determined using a Two-way ANOVA with Turkey multiple comparisons test. Created in https://BioRender.com.