022

TUMOUR NECROSIS FACTOR-alpha (TNFalpha) ENHANCES THE BARRIER DEFECT EVOKED IN EPITHELIAL CELL MONOLAYERS BY CONCOMITANT EXPOSURE TO METABOLIC STRESS AND NON-INVASIVE, NON-PATHOGENIC ESCHERICHIA COLI: INVOLVEMENT OF NFkappabeta

K Lewis², J Caldwell¹, C Reardon¹, MH Perdue², SD Söderholm⁴, PM Sherman³, DM McKay
¹University of Calgary, Calgary, Alberta; ²McMaster University, Hamilton, Ontario; ³University of Toronto, Toronto, Ontario; ⁴University of Linköping, Sweden

Many insults, including ischemia, inflammation and perturbations in the microbiota can result in metabolic stress in the gut epithelium as defined by abnormal mitochondrial structure and reduced ATP. We have shown that monolayers of the human colonic T84 epithelial cell line exposed to metabolic stress (induced by the uncoupler of oxidative phosphorylation, dinitrophenol (DNP)) and E. coli (strain HB101) display decreases in barrier function. Given the prominent role that TNFalpha can play in IBD, we assessed paracellular (ie, transepithelial resistance [TER]) and transcellular permeability (bacterial transcytosis graded on a 0-5 point logarithmic scale) in T84 cell monolayers treated with DNP (0.1mM)+E. coli (10⁶ cfu)±TNFalpha (10ng/ml). The NFkappabeta inhibitor, PDTC (50µM) was employed in additional experiments. Consistent with earlier data, DNP and E. coli individually had negligible effects on TER, while TER was reduced to ~60% of pre-treatment values 24 hours after DNP+E. coli exposure: this paracellular permeability defect was enhanced by the inclusion of TNFalpha (TER dropped to 38±10% or pretreatment values; n=10). This effect likely was not due to the induction of apoptosis, since we failed to observe increased caspase-3 cleavage by immunoblotting. Bacterial translocation occurred in 8% of E. coli only treated epithelia, whereas TNFalpha treatment resulted in transcytosis in 43% of monolayers. Similarly, the enhanced E. coli transcytosis observed in DNP+E. coli treated monolayers was exaggerated by TNFalpha co-treatment: 2 of 20 (10%) and 12 of 36 (33%) monolayers were scored grade =>3 (i.e. =>100 cfu), respectively. Use of PDTC, to block NFkappabeta mobilization by TNFalpha, failed to alleviate the TER drop but significantly ablated the increased bacterial transcytosis observed following E. coli+TNFalpha or E. coli+DNP+TNFalpha treatment (at the doses used neither TNFalpha nor PDTC exhibited anti-bacterial effects). These data indicate differential regulation of epithelial paracellular and transcellular permeability pathways, and identify epithelial transcytosis as a significant route by which non-invasive bacteria can enter the gut mucosa. Thus, release of TNFalpha due to inflammation or bacterial penetration into the intestinal mucosa can add to the epithelial barrier dysfunction caused by metabolic stress, which could result in pathophysiological reactions initiating, exaggerating and triggering relapses in IBD.
Funded by CCFC and CIHR

PREVIOUS NEXT