685 - IL-23 and IL-12 Exposure Leads to Increased Intestinal Permeability In Vitro

Publication Number: 685.5375

Katrina N. Russell, University of Central Florida College of Medicine, Orlando, 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

Background: The gut barrier is critical in protection from pathogenic bacteria and inflammatory milieu. Increased permeability of the intestinal epithelium enables translocation of bacteria and their toxins, leading to immune activation and a pro-inflammatory state. We have previously shown that treatment of intestinal epithelial cells with two pro-inflammatory cytokines, interleukin-23 (IL-23) or interleukin-12 (IL-12), leads to altered localization of the tight junction protein, Zona Occludens-1 (ZO-1), on immunocytochemistry. However, the impact of altered ZO-1 localization on membrane architectural changes and physiologic barrier function in response to these cytokines is unknown.

Objective: To determine the impact of IL-12, IL-23 and high IL-23 milk on intestinal epithelial barrier permeability in vitro.

Design/Methods: Immature rat small intestinal epithelial cells (IEC18) were grown to confluence on transwell inserts in 12-well plates and treated with recombinant IL-23 or IL-12 at 10ng/mL or 100ng/mL. IL-23 content of expressed breast milk was measured by ELISA. Additional monolayers were treated with milk with equivalent amounts of IL-23. Media was added to the basal compartments. FITC-Dextran70 (FD70), a 70kDa fluorescently tagged glucan, was added to the apical compartment at time of treatment. Media was collected from the basal compartment every 30 minutes from time 0 through 2 hours and fluorescence measured.

Results: FD70 flux was elevated in cells treated with rIL-23 and high IL-23 milk compared to untreated controls, with maximum fluorescence measured at 60 minutes post-treatment (p < 0.01). Fluorescence readings fell past baseline at 90 minutes and 120 minutes, which may represent membrane recovery or, more likely, loss of FD70 fluorescence over time. rIL-12 treatment showed a peak at 60 minutes with a 1.5-fold increase over control. Statistical analysis utilized a student's t-test.

Conclusion(s): Accumulation of FD70 in the basal compartment of treated monolayers compared to untreated controls strongly suggests that exposure to IL-23 and IL-12 results in impairment of the epithelial barrier, allowing translocation of FD70. This further supports our previous findings that showed disruption of tight junction proteins, including ZO-1, after treatment with IL-23 or IL-12. These data add functional evidence to support that disruption of tight junction proteins due to cytokine exposure is significant. This is a plausible mechanism for translocation of inflammatory milieu from the luminal compartment through the intestinal barrier, which is a key factor in the development of necrotizing enterocolitis.