Neonatology and Critical Care Nemours Children's Hospital Orlando, Florida, United States
Background: Multiple factors contribute to inflammation underlying intestinal barrier dysfunction and NEC. We have demonstrated that the IL-12 family members, IL-23 and IL-12, cause barrier dysfunction and NEC like injury in cellular and animal models. It is unclear if injury is mediated via the IL-23R or 12R,. There is alteration of ZO-1 distribution, disrupting tight junctions between intestinal epithelial cells. Additional members of the IL-12 family were recently identified. They share subunits which dimerize to create 5 individual cytokines and receptors (Table 1.) Objective: The two objectives were: 1.) characterize expression of IL-12 family cytokines in IEC-18 and RAW 246.7 cells, and 2.) elucidate changes in LPS induced expression of these cytokines and downstream mediators. Design/Methods: Baseline mRNA expression of cytokine and receptor subunits and downstream mediators, IL-10 and TNFa, was quantified with real time PCR in both cell types. These were compared to changes in expression following exposure to LPS alone and with IFNg. Supernatant from RAW cells was added to IEC-18 cells to replicate the role of secreted cytokines in vivo. Changes in protein expression were determined by Western blot and compared by densitometry. Statistical analysis utilized Students t test. Results: Baseline expression of cytokine subunits was minimal in IEC-18 cells. Following LPS exposure, IEC-18 cells had increased expression of EBI3 at 2 and 4hrs, paralleled by higher IL-12R expression(p < 0.01.) Expression of gp130 in IEC-18s did not change. Further increases in EBI3 and IL-12R followed IFNg treatment when compared to LPS alone. Treatment of RAW cells with LPS and IFNg induced p40 expression significantly at all timepoints with lesser increase in p19. p35 expression was minimal. IEC-18 monolayers exposed to supernatant from treated RAW cells increased gp130 at 1hr, EBI3 at 2 hours and IL-12 and 23R at 4hrs. Change in gp130 expression temporally correlated with changes in ZO-1. Western blot of downstream cytokines showed increase in TNFa and then IL-10 (p < 0.01.)
Conclusion(s): We previously showed that treatment of IEC-18 cells with IL-12 or 23 reduces ZO-1, allowing translocation of LPS. LPS and IFNg treatment increased EBI3 and gp130 in IECs. IL-12R expression increased but not IL-23, suggesting that observed ZO-1 barrier disruption is mediated via IL-12R not IL-23R. Increase in p40 and EBI3 without an increase in p35 also suggests IL-23 and IL-39 predominate. However, further evaluation of EBI3 and p28 are needed to further elucidate mechanisms by which IL-12 family members facilate injury leading to NEC. Slide1.jpeg
