993 WASp Deficiency in Innate Immune Cells Leads to Severe Colitis Associated With Impairment in IL-10 Production

Background: Defective intestinal epithelial tight junction (TJ) barrier, leading to increased antigenic penetration, has been recognized as an important pathogenic mechanism contributing to the development of intestinal inflammation in Crohn's disease.Interleukin-6 (IL-6) is a pro-inflammatory cytokine which is elevated in various inflammatory diseases including Crohn's disease.Previous studies have shown that IL-6 induced increase in intestinal epithelial TJ permeability is mediated in part by an increase in claudin-2 expression.However, the cellular and molecular mechanisms that regulate the claudin-2 gene activity and TJ permeability remains unclear.Aim: The aim of this study was to delineate the molecular mechanisms involved in IL-6 induced increase in intestinal epithelial TJ permeability.The major focus of these studies was to elucidate the AP-1 regulation of claudin-2 expression and TJ barrier.Methods: Filter-grown Caco-2 cells were used for TJ permeability studies.Various molecular and biochemical methods were used to determine claudin-2 gene activity and expression.Results: 1) IL-6 caused a time-dependent drop in Caco-2 transepithelial resistance and a size-selective increase in paracellular flux to small permeability markers (< 4 Å in radius).IL-6 did not affect TJ permeability to molecules > 4 Å in radius.2) IL-6 caused a timedependent increase in claudin-2 expression, and did not affect other TJ protein expression including occludin and claudin-1, 3, 4, or 8. 3) IL-6 up-regulation of claudin-2 expression was preceded by the activation of JNK pathway.Inhibition of JNK activation by inhibitor (SP600125) or siRNA prevented the IL-6 induced increase in claudin-2 expression and TJ permeability.4) IL-6 induced activation of JNK pathway resulted in AP-1 activation.5) A 3 kb of claudin-2 promoter region was identified by 5' RACE, and cloned into pGL3 basic vector.6) Minimal promoter region was determined by progressively 5' deletion.Four AP-1 binding sites were identified within this region.7) IL-6 induced an increase in claudin-2 promoter activity, mRNA and protein expression.8) SiRNA of AP-1 inhibited the IL-6 induced increase in claudin-2 promoter activity, expression, and increase in TJ permeability.Conclusion: IL-6 induced increase in Caco-2 TJ permeability was mediated by JNK pathway regulated activation of claudin-2 gene.The JNK pathway induced activation of AP-1 led to a step-wise binding of AP-1 to claudin-2 promoter and activation of promoter activity, increase in claudin-2 gene and protein expression, and claudin-2 dependent increase in TJ permeability.Thus, these studies demonstrate a novel mechanism of intestinal TJ barrier regulation.