20 - Alteration in ZO-1 and TNFα Expression in IEC18 Cells in Response to Milk or IL-12

Poster Number: 20
Publication Number: 20.201

Mackenzie Simon-Collins, Johns Hopkins University, Jacksonville, FL, United States; Katrina Russell, Nemours Children's Hospital, Sanford, FL, United States; Rajarajeshwari Venkataraman, Nemours Children's Hospital, Orlando, FL, United States; Peter Phelan, Nemours Children's Hospital, Orlando, FL, United States; Jennifer L. Liedel, Nemours Children's Hospital, Orlando, FL, United States

Background: Necrotizing enterocolitis(NEC) is a condition that affects many premature infants. No single identifiable factor causes NEC to develop, rather it results from multiple factors intrinsic to the infant which are modulated by environmental stressors. We have shown that cytokine rich breast milk and recombinant Interleukin-23(rIL-23) treatment lead to intestinal epithelial barrier dysfunction in a rat NEC model and changes in Zona Occludens-1(ZO-1) in vitro. Interleukin-12(IL-12) has also been shown to impact the intestinal barrier.  IL-23 and IL-12 are both able to act via the IL-12 receptor. It is unclear if the effects seen are a result of IL-12 or IL-23 receptor signaling.

Objective: To assess the effects of IL-12, IL-23 and expressed breast milk(EBM) on inflammation and ZO-1 localization in an in vitro model.

Design/Methods: IEC-18 cells were treated with rIL-23, rIL-12 or EBM. After treatment cells were fixed, permeabilized and stained or harvested for Western or real time PCR analysis.  Localization of ZO-1 was assessed with fluorescent immunohistochemistry. Protein expression of ZO-1, IL-12 receptor (IL-12R) and IL-23R was assessed with Western blot in response to recombinant IL-12(rIL-12), rIL-23 and 2 EBM samples. mRNA expression of Tumor Necrosis Factor α(TNFα) and ZO-1 was quantified. Statistical analysis utilized t test.

Results: All cells treated with cytokines had evidence of altered ZO-1 localization when compared to control. This was more evident in cells exposed to EBM sample 2 or rIL-12.  Normalization of ZO-1 was seen at 2 hours.  In rIL-12 treated cells, there was a significant increase in TNFα expression as measured by PCR.  Similar increased expression was seen in cells treated with EBM sample 2 (p<0.02.)  A reduction in ZO-1 expression was detected after exposure to this sample.  Protein expression of IL-23, IL-12 and their respective receptors was unchanged.

Conclusion(s):

Less cell surface ZO-1 was seen in those cells treated with EBM sample 2 and rIL-12. All treated cells showed some evidence of recovery at 2 hrs. Significantly elevated mRNA expression of TNFα in EBM and IL-12 treated cells, with a concomitant decrease in ZO-1 expression, was seen.  IL-12 and IL-23 were not detected in the EBM whey fraction, but were expressed in treated cells. Exposure to EBM and IL-12 led to increased TNFα expression. A reduction in cell surface ZO-1 suggests that factors in milk as well as the cytokines, IL-12 and IL-23, are capable of disrupting the gut barrier. Further study is needed to understand the mechanism by which IL-12, IL-23 and EBM contribute to barrier dysfunction and inflammation.