Inhibition of Proinflammatory Molecule Production by Adenovirus-Mediated Expression of a Nuclear Factor κB Super-Repressor in Human Intestinal Epithelial Cells

Abstract

NF-κB plays a major role in the transcriptional regulation of many proinflammatory genes in multiple cell lineages, including intestinal epithelial cells (IEC). Activation of NF-κB requires both phosphorylation and degradation of its natural cytoplasmic inhibitor, IκB. We tested whether a super-repressor of NF-κB activity, which is a mutated nondegradable IκBα resistant to phosphorylation and degradation, could be delivered into IEC using an adenoviral vector (Ad5IκB) and determined the anti-inflammatory potential of this inhibitor following different stimuli. We showed for the first time that recombinant adenovirus efficiently infected (>80%) transformed as well as primary IEC. Cytoplasmic levels of the NF-κB super-repressor protein were more than 50-fold higher than those of endogenous IκB, and this mutated IκB was resistant to IL-1β-induced degradation. Immunofluorescent RelA nuclear staining was strongly inhibited in Ad5IκB-infected IEC compared with control Ad5LacZ, and NF-κB, but not AP-1 binding activity, was reduced by more than 70% as measured by electrophoretic mobility shift assay (EMSA). Induction of inducible nitric-oxide synthase (iNOS), IL-1β, and IL-8 genes by IL-1β, TNF-α, or PMA was blocked in Ad5IκB-infected cells but not in Ad5LacZ controls as assayed by RT-PCR and ELISA. In addition, IL-1β-induced IL-8 secretion was totally inhibited by Ad5IκB in primary colonic IEC. We conclude that an adenoviral vector efficiently transfers a nondegradable IκB in both transformed and native IEC. The strong inhibition of NF-κB activity and the resulting down-regulation of multiple proinflammatory molecules by Ad5IκB suggests an exciting approach for in vivo intestinal gene therapy and illustrates the key role of NF-κB in transcriptional regulation of the inflammatory phenotype of IEC.