The Role of Nuclear Factor-kappa B in IBD

A vast array of recent data has solidified the important role of the transcription factor nuclear factor-kappa B (NF-κB) in the pathogenesis of Inflammatory Bowel Disease (IBD). NF-κB is believed to play a pivotal role in both animal models of IBD, as well as in human IBD. Moreover, genes that are regulated by NF-κB (e.g., IL1-β TNF-α IL-8, IL-12) are believed to be involved in the pathogenesis of IBD. Elevated amounts of NOD2 protein have been found in colonic epithelial cells from patients with IBD, and NOD2 expression in colonic epithelial cells appears to be regulated by NF-κB. Also, the NOD2 protein is involved in NF-κB activation following intacellular stimulation with the bacterial product, peptidoglycan. Interestingly, mutations in the NOD2 gene have recently been identified in patients with Crohn's Disease. From a therapeutic standpoint, inhibitors of the NF-κB signaling cascade are effective, for reducing the severity of colitis in animal models of IBD. Also, initial data from an ongoing clinical trial suggests that a specific anti-sense inhibitor of NF-κB may be effective in the treatment of IBD.

My primary research interest over the past five years has been investigating the role of NF-κB in IBD. This research utilized both relevant cell lines and animal models of IBD. Specifically, we investigated NF-κB activation in both macrophage and colonic epithelial cell lines. Since this transcription factor also plays a role in the regulation of apoptosis, we aslo investigated DNA fragmentation in these cell lines. Relatively specific small molecular weight inhibitors of NF-κB (i.e., gliotoxin and caffeic acid phenetyl ester [CAPE]) were used in these studies, in order to evaluate the effects of NF-κB inhibition on both cytokine production and DNA fragmentation.

Both of these NF-κB inhibitors were shown to dose dependently reduce bacterial derived LPS and PG-PS induced TNF-α production in a rat macrophage cell line. Moreover, these inhibitors also reduced TNF-α induced IL-8 production in the SW620 colonic epithelial cell line. In the same cell lines, gliotoxin and CAPE dose dependently attenuated the nuclear binding of NF-κB. Also, these inhibitors were found to induce apoptosis of rat macrophages, following 24 hours of exposure. This is consistent with an anti-apoptotic role of NF-κB in macrophages. However, when SW620 colonic epithelial cells were stimulated with Fas Ligand, TNF-α and Interferon-γ to induce apoptosis, the NF-κB inhibitors significantly attenuated the induction of apoptosis by these relevant pro-inflammatory agents. These results suggested that NF-κB inhibitors can have varying effects on apoptosis, which is dependent on the both the cell type and stimulus.

The 20S proteasome plays an important role in the NF-κB signaling pathway. Other in vitro testing from my previous laboratory suggested that CAPE and gliotoxin inhibited the proteolytic activity of the 20S proteasome to varying degrees. Particularly for CAPE, such inhibition may have been responsible for the downstream inhibition of NF-κB binding, which was observed in relevant cell lines.

In order to more critically evaluate the role of NF-κB in colonic inflammation, we established two rat models of IBD. Specifically, the administration of dextran sodium sulfate (DSS), or bacterial peptidoglycan polysaccharide (PG-PS) resulted in colonic inflammation, which mimics to some degree human IBD. In both IBD models, we observed up-regulation of colonic NF-κB. In the DSS model, expression of NF-κB was observed primarily in mucosal lamina propria inflammatory cells (e.g., macrophages), but also in colonic epithelial cells. In contrast, with PG-PS induced colitis, NF-κB expression was noted most prominently within inflammatory cells of the sumucosa, and particularly in areas of granuloma formation. Using these two colitis models, we also demonstrated that gliotoxin and CAPE could attenuate various clinical, macroscopic, histologic, and functional indices of colonic inflammation. As a whole, this data sugested that NF-κB played a key role in the pathogenesis of colitis, for these two rat models of IBD.

Within the Department of Surgery, we are continuing to investigate the role of the NF-κB signaling pathway, as related to IBD. This work includes the use of cell lines, as well as murine models of IBD. Using these general research approaches, we are also interested in exploring possible interactions between the NF-κB and STAT6 signaling pathways. Specifically, we are using EBV-transformed B lymphocyte cell lines from IBD patients, in order to further investigate these transcription factors.