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Epithelial NFkB Activation Is Critical in TNF Mediated Increases in Intestinal Tight Junction Permeability
Christopher J. Chow, Zheng Zhang, Yueming Tang, David Ivancic, Terrence Barrett, Jonathan P. Fryer, Northwestern University, Chicago, IL
Increased intestinal epithelial permeability has been associated with SB inflammatory processes including Crohn¡¦s disease and SB allograft rejection. Proinflammatory cytokines such as TNF mediate changes in intestinal tight junction permeability. However, the underlying mechanisms have not been thoroughly evaluated. The purpose of this study was to determine whether activation of epithelial NFkB is involved in TNF mediated permeability changes in the SB. To evaluate intestinal barrier function in-vivo, we have adapted the in vivo perfusion system described by Meddings. Isolated jejunal loops continuously perfused with physiologic solution in a closed circuit system were evaluated for permeability to various sized molecular probes (Alexa 350 hydrazide, FITC-Dextran 3000, and Texas Red-Dextran 10,000). C57BL/6 mice were injected i.p. with recombinant mouse TNF (3ug in 200ul PBS) or PBS 1 hour prior to initiating the perfusion system. We found TNF administration significantly shifted transpepithelial water flux toward the bowel lumen and increased SB epithelial permeability to larger probes (Dextran 3000 and Dextran 10000, P<0.05 vs controls). Furthermore, RT-PCR data revealed alterations in mRNA expression for junctional proteins including claudins 4, occludin and p-MLC in TNF treated SB tissues measured between 3 to 24 hours after injection. These data indicate that TNF altered intestinal barrier function, possibly by modification of epithelial TJP assembly of epithelial. To elucidate the role of epithelial NFkB activation in TNF mediated changes of intestinal permeability, we utilized double transgenic mice (DTg) that selectively express an inducible mutated IkB-alpha(mIkB-alpha, a super-repressor of NFkB) gene in their intestinal epithelial cells. DTg mice and wt controls were pretreated with Doxycline for 21 days prior to evaluation to induce the transgene expression. Our data revealed that, following TNF administration, SB permeability to Dextran 10000 was significantly reduced (~90% reduction) in DTg mice compared with Doxycline-treated or non-treated wt controls, indicating that inhibition of epithelial NFkB activation reverses TNF-mediated barrier dysfunction. In conclusion, these data suggest that epithelial NFkB activation is critical in TNF mediated intestinal tight junction dysfunction, possibly via regulating the expression or assembly of tight junction associated proteins.
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