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46 SURFACE EXPRESSION OF TLR9 IS UPREGULATED IN RESPONSE TO PATHOGENIC BACTERIAL DNA JB Ewaschuk, JL Backer, KL Madsen BACKGROUND: DNA isolated from Salmonella dublin (Sd) has been shown to induce IL-8 secretion in intestinal epithelial cells. In contrast, DNA from probiotic bacterial strains (Bifidobacteria breve - Bb) does not elicit an inflammatory response, and can inhibit IL-8 release in the presence of Sd DNA. Bacterial DNA is recognized by toll-like receptor 9 (TLR9). TLR9 is localized on the endoplasmic reticulum in immune cells, but its location in intestinal epithelial cells is unknown.
Division of Gastroenterology, University of Alberta, Edmonton, Alberta
OBJECTIVE: The aim of this study was to investigate TLR9 expression and localization in intestinal epithelial cells under basal conditions and in response to stimulatory and inhibitory bacterial DNA.
METHODS: HT-29 epithelial cells were treated with 50 µg/mL of purified Bb (inhibitory) and Sd (stimulatory) DNA. A time course of TLR9 mRNA expression and IL-8 secretion was determined by RT-PCR and ELISA respectively. Localization of TLR9 was determined by flow cytometry and immunofluorescence. Chloroquine was used to block endosomal TLR9 interactions.
RESULTS: TLR9 mRNA increased over time in response to Sd DNA, as did IL-8 secretion. In contrast, Bb DNA reduced levels of TLR9 mRNA, which correlated with the Bb DNA-induced inhibition of IL-8 secretion in response to Sd DNA. Immunofluorescence and flow cytometric analyses of nonpermeablized cells demonstrated cell-surface expression of TLR9 under basal conditions. Application of Sd but not Bb DNA increased surface expression of TLR9. Interestingly, chloroquine did not block IL-8 secretion in response to Sd DNA, suggesting either the involvement of a TLR9-independent pathway or alternatively an endosomal-independent pathway in intestinal epithelial cells that responds to pathogenic bacteria DNA.
CONCLUSION: TLR9 is expressed on the surface of intestinal epithelial cells, and its expression is differentially regulated by bacterial DNA. Bacterial DNA appears to interact differently with intestinal epithelial cells than with immune cells.