• Darab Ghadimi, Michael de Vrese, Knut J Heller, and Juergen Schrezenmeir.
• Department of Physiology and Biochemistry of Nutrition, Max Rubner-Institut, Kiel, Germany. ghadimi@bafm.de
• Inflamm. Bowel Dis. 2010 Mar 1;16(3):410-27.

Background And AimThe intestinal epithelium is constantly exposed to high levels of genetic material like bacterial DNA. Under normal physiological conditions, the intestinal epithelial monolayer as a formidable dynamic barrier with a high-polarity structure facilitates only a controlled and selective flux on components between the lumen and the underlining mucosa and even is able to facilitate structure-based macromolecules movement. The aim of this study was to test the effect of natural commensal-origin DNA on the TLR9 signaling cascade and the barrier integrity of polarized intestinal epithelial cells (IECs).Methods: Polarized HT-29 and T84 cells were treated with TNF-alpha in the presence or absence of DNA from Lactobacillus rhamnosus GG (LGG) and Bifidobacterium longum. TLR9 and interleukin-8 (IL-8) mRNA expression was assessed by semiquantitative and TaqMan real-time reverse-transcription polymerase chain reaction. Expression of TLR9 protein, degradation of inhibitor of kappa B alpha (IkappaBalpha), and p38 mitogen-activated protein kinase (p38 MAP) phosphorylation were assessed by Western blotting. To further reveal the role of TLR9 signaling, the TLR9 gene was silenced by siRNA. IL-8 secretion was measured by an enzyme-linked immunosorbent assay. Nuclear factor-kappa B (NF-kappaB) activity was assessed by the electrophoretic mobility shift assay (EMSA) and NF-kappaB-dependent luciferase reporter gene assays. As an indicator of tight junction formation and monolayer integrity of epithelial cell monolayers, transepithelial electrical resistance (TER) was repetitively monitored. Transmonolayer movement of natural commensal-origin DNA across monolayers was monitored using qRT-PCR and nested PCR based on bacterial 16S rRNA genes.ResultsIn response to apically applied natural commensal-origin DNA, polarized HT-29 and T84 cells enhanced expression of TLR9 in a specific manner, which was subsequently associated with attenuation of TNF-alpha-induced NF-kappaB activation and NF-kappaB-mediated IL-8 expression. TLR9 silencing abolished this inhibitory effect. Apically applied LGG DNA attenuated TNF-alpha-enhanced NF-kappaB activity by reducing IkappaBalpha degradation and p38 phosphorylation. LGG DNA did not decrease the TER but rather diminished the TNF-alpha-induced TER reduction. Translocation of natural commensal-origin DNA into basolateral compartments did not occur under tested conditions.ConclusionsOur study indicates that TLR9 signaling mediates, at least in part, the anti-inflammatory effects of natural commensal-origin DNA on the gut because TLR9 silencing abolished the inhibitory effect of natural commensal-origin DNA on TNF-alpha-induced IL-8 secretion in polarized IECs. The nature of the TLR9 agonist, the polarity of cells, and the tight junction integrity of IECs has to be taken into account in order to predict the outcome of TLR9 signaling. (Inflamm Bowel Dis 2010).

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