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ABSTRACT FROM 12th INTERNATIONAL CONGRESS ON IMMUNOLOGY -- EIF5A is Required for Interferon Gamma Signaling in HT-29 Cells

C. A. Taylor1, L. Reznikov2, A. N. Boone1, C. A. Dinarello2, and J. E. Thompson1

1Department of Biology, University of Waterloo, Waterloo, ON, Canada, N2L 3G1

2Department of Medicine, University of Colorado, Denver, CO 80262

RATIONALE: Pro-inflammatory cytokines and chemokines released by intestinal epithelial cells play an important role in the inflammatory response that leads to tissue damage during inflammatory bowel disease. Eukaryotic initiation factor 5A (eIF5A) is a nucleocytoplasmic shuttle protein that is believed to shuttle specific classes of mRNA from the nucleus to the cytoplasm for translation. While traditionally thought to be important during cell proliferation, eIF5A has recently been found to function during apoptosis and siRNAs targeting eIF5A have been found to be protective against cytokine-induced cytotoxicity. In order to further understand the role of eIF5A in cytokine signalling we have examined the ability of an siRNA against eIF5A to block interferon gamma (IFNgamma) signalling.

METHODS: HT-29 cells were used as a model system. siRNA was synthesized by Dharmacon. Secreted TNF-alpha and IL-8 were measured by ELISA and liquid-phase electrochemiluminescence (ECL), respectively. Analysis of eIF5A, TLR4, TNFR1, and iNOS protein was performed by Western blot analysis and densitometry. RESULTS: eIF5A siRNA was able to effectively inhibit TNF-alpha production by HT-29 cells primed with IFNgamma and challenged with LPS. A reduction in IL-8 production upon IFNgamma treatment or combined TNF-alpha and IFNgamma treatment was also observed following siRNA transfection. Western blot analysis revealed that the eIF5A siRNA was able to reduce IFNgamma-mediated upregulation of TLR4, TNFR1, and iNOS.

CONCLUSIONS: Inhibition of eIF5A by siRNA interferes with IFNgamma signalling and reduces the ability of intestinal epithelial cells to respond to LPS and TNF-alpha via TLR4 and TNFR1, respectively. Inhibition of eIF5A appears to have a direct immunoregulatory effect on intestinal epithelial cells and may be a therapeutic target for inflammatory bowel disease.