Medical Student University of Central Florida College of Medicine Orlando, Florida, United States
Background: Intestinal barrier dysfunction contributes to the development of necrotizing enterocolitis (NEC). Impaired barrier function may allow bacteria and their toxins to translocate and cause inflammation. Zona occludens proteins are a group of proteins that are critical for maintenance of this barrier. We have previously shown that treatment with recombinant interleukin-23 (rIL-23), recombinant interleukin-12 (rIL-12) and cytokine-rich breast milk disrupted zona occludens-1 (ZO-1) at the membrane. Although it remains unclear by which receptor signaling occurs, we have previously shown that the IL-12 receptor subunits have been upregulated. Objective: Assess the effect of exposure to lipopolysaccharide (LPS) endotoxin on intestinal barrier integrity in vitro in the presence and absence of activated macrophages. Design/Methods: ZO-1 localization was assessed with fluorescent immunohistochemistry (IHC) in the IEC18 cells, a rat immature small intestinal cell line. We also assessed expression of gp130, a receptor in the IL-12 family. IEC18 cells were treated with 1 ug/mL of E. coli-derived LPS or cytokine-rich breast milk for 30 minutes, 1 hour, 2 hours or 4 hours. IEC18s were also co-cultured with a mouse macrophage cell line, RAW264.7. The co-cultured cells were treated with 1 ug/mL LPS over the same time course and assessed by fluorescent IHC. Proteins were extracted from treated cells and protein expression of ZO-1 and gp130 were assessed by Western Blot. Results: Following treatment of IEC-18s with LPS, ZO-1 fluorescence was reduced at all timepoints. However, when IEC-18s were co-cultured with RAW264.7 cells, recovery of ZO-1 localized to the membrane was observed. Western blot showed increased expression of gp130 over time in response to IEC18 treatment with LPS. IHC showed maximal expression of gp130 at 30 minutes following treatment, although staining at 1 and 3 hours was still higher than baseline.
Conclusion(s): The intestinal barrier was transiently disrupted following treatment with cytokine-rich milk. Barrier disruption was sustained following exposure to LPS which could predispose to NEC. However, in the presence of activated macrophages, the barrier function was rescued. Increased expression of gp130 with limitation in ZO-1 disruption suggests signaling through this membrane receptor following membrane insult is protective. Further studies are needed to elucidate the mechanism by which gp130 maintains the barrier and prevents injury leading to NEC.
