490 - IL-12 and IL-23 impair ZO-1 function via the IL-12 Receptor

Poster Number: 490
Publication Number: 490.424

Katrina Russell, Nemours Children's Hospital-- Florida, Orlando, 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: Tight junction function is imperative to maintenance of intestinal epithelial barrier. One underlying mechanism of Necrotizing Enterocolitis(NEC) is barrier injury and dysfunction, enabling bacteria and/or their toxins to translocate causing inflammation. We have shown that cytokine rich breast milk and recombinant Interleukin-23(rIL-23) treatment lead to barrier dysfunction in a rat NEC model. There was an alteration in both mRNA and protein expression of the tight junction protein Zona Occludens-1(ZO-1) in vitro following treatment with IL-23 rich milk.  IL-12 disrupts tight junctions in models of barrier function. Importantly, IL-23 and IL-12 share a subunit, allowing IL-23 to bind to the IL-12 receptor. Thus it is unclear if the effects seen are a result of IL-12 or IL-23 receptor signalling.

Objective: We hypothesized that proinflammatory cytokines IL-23 and IL-12 impact the function of ZO-1 by acting through the IL-12 receptor, disrupting the intestinal epithelial barrier, causing inflammation that leads to NEC.

Design/Methods: Utilizing the IEC-18 cell line, localization of ZO-1 was assessed with fluorescent immunohistochemistry. Protein expression of ZO-1, IL-12 receptor and IL-23 receptor was assessed with Western blot in response to recombinant IL-12(rIL-12) or rIL-23. mRNA expression of ZO-1 was quantified. Cells were treated with increasing doses of rIL-23 and rIL-12 at intervals between 15 minutes and 4 hours. After treatment cells were fixed, permeabilized and stained or harvested for Western or real time PCR analysis. Stained cells were screened under fluorescent microscopy to assess the location of ZO-1. Statistical analysis utilized t test.

Results: Following treatment, less ZO-1 was present at the cell membrane. However, cells treated with rIL-12, not rIL-23, had more disruption of ZO-1 as demonstrated on fluorescent imaging. On Western blot, IL-12 receptor expression was increased in response to rIL-23 and rIL-12 treatment. Treated cells had increased IL-12 and IL-23 mRNA expression of ZO-1. This did not reach statistical significance.Conclusion(s): Treatment with rIL-12 and rIL-23 altered ZO-1 localization at the membrane.  Moreover, treatment with rIL-12 and rIL-23 increased IL-12 receptor protein expression. Exposure to both cytokines increased ZO-1 mRNA expression but this was not significant. The results strongly suggest that IL-23 and IL-12 impair barrier function through alterations in ZO-1 via the IL-12 signalling pathway. Further studies to elucidate the mechanism by which IL-12 and IL-23 impact ZO-1 leading to intestinal epithelial barrier dysfunction and NEC are warranted.