Gene expression of the Toll-like receptor 9 and related intracellular signaling cascade in response to DNA from commensal bacteria and immunostimulatory DNA sequence by polarized intestinal epithelial cells using gene silencing technology

Rationale: The aim of this study was to explore the effect of probiotic DNA on gene expression of toll-like receptor 9 (TLR9) and TLR9-related intracellular signalling cascade by the polarized intestinal epithelial cells (IECs). Methods: Polarized HT-29 cells were stimulated with TNFα in the presence or absence of DNA from Lactobacillus rhamnosus GG and Bifidobacterium longum, calf thymus DNA, CpG-B ODN and ODN backbone. TLR9 mRNA was assessed by TaqMan RT-PCR. Expression of TLR9 protein, degradation of inhibitor of kappa B alpha (IkBα) and p38 mitogen-activated protein (MAP) kinase phosphorylation were studied by Western blotting. The effect of LGG DNA on NF-kappa B (NF-kB) activity was assessed by EMSA. To further reveal the role of TLR9 signaling, siRNA gene silencing technology was used to target the TLR9 gene and TLR9 gene was knocked down by TLR9 siRNAs. IL-8 secretion was measured by ELISA. As a number of TNFα-induced genes are under the control of NF-kB transcription complex, we also aimed to find whether LGG DNA acted directly on TNFα-induced NF-kB transcriptional activity. For this purpose, HT-29 cells were transiently transfected with a NF-κB-responsive luciferase reporter gene, pre-treated with LGG DNA (25, 50 and 100µg/ml) for 2h and stimulated with TNFα (10ng/ml) for 12h. NF-kB-dependent luciferase activity was then measured. Results: Probiotic DNA and CpG-B ODN but not calf thymus DNA and ODN-backbone up-regulated TLR9 mRNA and protein expression. In response to TNFα, HT-29 cells secreted significantly more IL-8 in comparison to a control. Apically applied LGG DNA reduced TNFα-induced IL-8 secretion. TLR9 silencing using TLR9 siRNA abolished this inhibitory effect. LGG DNA also reduced NF-kB-DNA binding in TNFα-stimulated HT-29 cells by reducing IκBα-degradation and p38-phosphorylation, two key factors for the activation of NF-kB and subsequent translocation of NF-kB into the nucleus, where it regulates various genes. Pre-treatment with LGG DNA detracted TNFα-induced NF-kB-driven luciferase activity, however, not significantly. TNF-α alone significantly decreased transepithelial electrical resistance (TER). LGG DNA reinforced the TNF-α-reduced TER, albeit not significantly. Conclusion: Polarized HT-29 cells recognize probiotic DNA, synthetic ISS-ODNs, and to only negligible extent eukaryotic DNA and ODN backbone via TLR9. Apically applied probiotic DNA induce TLR9 expression in polarized HT-29 cells resulting in an attenuation of TNFα-induced NF-κB activation and NF-kB-mediated IL-8 secretion. This implies that TLR9 may mediate, at least in part, the anti-inflammatory effects of probiotic DNA on the gut, since TLR9 silencing abolished the inhibitory effect of probiotic DNA on IL-8 secretion. These results suggest that nature of DNA, polarity of cells, surface-specific expression of TLR9, and tight junction (TJ) integrity of intestinal epithelial cells, have to be taken into account to predict the uptake of DNA IECs and to predict the outcome of TLR9 and TLR9-related intercellular pathways. Additional studies are required to understand the pleiotropic actions of TLR9 through genomic and non genomic pathways.