714 - Alteration in Barrier Function and Cytokine Expression in IEC-18

Publication Number: 714.3940

Nina Pagani, University of Central Florida College of Medicine, Windermere, FL, United States; Rajarajeshwari Venkataraman, Nemours Children's Hospital, Orlando, FL, United States; Peter E. Phelan, Nemours Children's Hospital, Orlando, FL, United States; Jennifer Liedel, Nemours Children's Hospital/University of Central Florida College of Medicine, ORLANDO, FL, United States; Katrina N. Russell, University of Central Florida College of Medicine, Orlando, FL, United States

Background: Multiple factors contribute to the development of necrotizing enterocolitis, including the inflammatory response that precedes intestinal barrier dysfunction. We have previously shown that two member of the IL-12 family, IL-12 and IL-23, disrupt Zona Occludens (ZO-1) predisposing the intestinal barrier to subsequent injury. While most of the IL-12 family members appear to be proinflammatory, IL-27, which is a heterodimer of EBI3 and p28, has been proposed to have protective effects. Binding to the gp130 receptor, IL-27 activates JAK/STAT3 pathway. Phosphorylated STAT3 translocates to the nucleus and binds to the promoter of ZO-1, initiating protein synthesis and its translocation to the membrane for recovery of intestinal barrier.

Objective: To assess the effect of IL-27 on barrier function in vitro and further elucidate its effect on ZO-1 and maintenance of the intestinal barrier.

Design/Methods: The immature rat small intestinal epithelial cell line, IEC-18 and murine macrophage-monocyte cells, RAW264.7 were used for all experiments. IEC-18 cells were treated with recombinant IL-27. RAW264.7 cells with were stimulated with 1ug/mL LPS for 30 minutes to 48 hours. Supernatant was collected and utilized to treat IEC-18 to evaluate the role of the inflammatory response on the neonatal intestinal epithelium. Cytokine mRNA expression of ZO-1, IL-12 family subunits (eg. p28, EBI3) and receptor subunits (eg. gp130,) STAT3 and NF- κB was measured using RT-qPCR in both RAW264.7 and IEC-18 cells. Immunocytochemistry was performed to determine localization of ZO-1 over time in IEC-18 cells as a measure of barrier dysfunction.

Results: Following treatment of RAW264.7 cells with LPS, p28 and EBI3 subunit mRNA expression increases. Staining for ZO-1 showed no alteration in membrane location when treated with hrIL-27. When treated with the supernatant, ZO-1 decreased at the membrane at 1 hour, followed by recovery at 4 hours. IEC-18 cells treated with the stimulated supernatants showed increase2 protein expression of gp130 at 4 and 6 hours and increase in STAT3 at 30 minutes.

Conclusion(s): We have previously shown that treatment with IL-12 or IL-23 causes barrier disruption in vitro. However, elevated expression of EBI3 and p28 in response to stimulated supernatant or rIL-27 without ZO-1 disruption suggests a protective role of IL-27. Furthermore, increases in protein expression of gp130 and STAT3 further supports receptor engagement and activation of JAK/STAT pathway. Further investigation into the protective role of IL-27 is warranted.