BUTYRATE PREVENTS THE TRANSLOCATION OF ESCHERICHIA COLI ACROSS EPITHELIA UNDER METABOLIC STRESS

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BUTYRATE PREVENTS THE TRANSLOCATION OF ESCHERICHIA COLI ACROSS EPITHELIA UNDER METABOLIC STRESS

K Lewis, D McKay
University of Calgary, Calgary, Alberta
Aims: Bacterial dysbiosis can occur in some patients with Crohn's disease, where reductions in butyrate-producing bacteria could result in local metabolic stress since butyrate is a major energy source for the epithelial lining of the colon. We have shown that monolayers of the human colon-derived T84 epithelial cell line when exposed to metabolic stress become leaky. Thus, we hypothesized that the degree of metabolic stress evoked by loss of butyrate-producing bacteria of the commensal flora could impact the integrity of the epithelial barrier and allow increased passage of bacteria from the lumen into the mucosa. We wish to test the ability of butyrate to block the increase in epithelial permeability evoked by co-treatment with the chemical stressor, dinitrophenol (DNP) and commensal non-pathogenic, non-invasive E. coli.
Methods: T84 cells were grown to confluence on transwell filters and were exposed to DNP (0.1mM) ± E. coli (strains HB101 and F18; 10⁶ cfu) ± sodium-butyrate (3mM). Transepithelial electrical resistance (TER; an indication of the integrity of the tight junctions) was assessed, as was bacterial translocation by the culture of 10 µL samples retrieved from the basolateral compartment of the culture well. The cells were examined by transition electron microscopy and NFkappaB activation was assessed by immunoblotting and ELISA for phosphorylated IkappaB.
Results: A single dose of butyrate significantly reduced bacterial internalization and translocation across metabolically stressed T84 cell monolayers at 4h post-infection (65% of butyrate + DNP + E. coli HB101 treated monolayers had no translocation whereas every T84 preparation exposed to DNP + E. coli HB101 displayed evidence of bacterial translocation). Repeated butyrate treatments increased this protective effect up to 24h post-infection. In contrast, butyrate did not prevent the drop in TER evoked by DNP + E. coli treatment. Electron microscopy revealed that butyrate prevented the cell membrane ruffling evoked by DNP + E. coli treatment and may induce the genesis of mitochondria in T84 cells. We have shown that NFkappaB activation mediates, at least in part, increased bacterial translocation across DNP + E. coli treated epithelia. Addition of butyrate to DNP + E. coli treated cells resulted in less IkappaB phosphorylation and hence NFkappaB activation as assessed by immunoblotting (densitometry: 33.7±8.9 vs.13.6±3.6 arb. Units, n=4) and ELISA.
Conclusions: We speculate that butyrate production in situ regulates epithelial permeability by suppression of NFkappaB signaling and this may be of particular importance during periods of metabolic stress. Moreover, stimulation of bacterial synthesis of butyrate by increasing dietary fiber intake could be of therapeutic value in individuals with risk factors that may predispose them to IBD.
Funded by CIHR and AHFMR.

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