title: Toll-like receptor (TLR) 2 and TLR5, but not TLR4, are required for Helicobacter pylori-induced NF-kappa B activation and chemokine expression by epithelial cells.
abstract: Infection with Helicobacter pylori, a Gram-negative, microaerophilic, flagellated bacteria that adheres to human gastric mucosa, is strongly associated with gastric ulcers and adenocarcinoma. The mechanisms through which gastric epithelial cells recognize this organism are unclear. In this study we evaluated the interactions between the Toll-like receptors (TLRs) and H. pylori-mediated NF-kappa B activation and the induction of chemokine mRNA expression. By reverse transcriptase-PCR we determined that MKN45 gastric epithelial cells express low but detectable amounts of TLR2, -4, and -5 but no MD-2. To determine which, if any, TLRs may play a role in the response of epithelial cells to H. pylori, HEK293 cells were cotransfected with the NF-kappa B-Luc reporter, CD14 and MD2 expression plasmids, and expression plasmids for TLR2, TLR4, or TLR5. Infection of the cultures with H. pylori (strain 26695) induced NF-kappa B activity in cells transfected with TLR2 and TLR5, but not TLR4. Consistent with the HEK293 experiments, H. pylori-induced NF-kappa B activation was decreased in MKN45 gastric epithelial cells by transfection of dominant-negative versions of TLR2 and TLR5 but not TLR4. Highly purified lipopolysaccharide from H. pylori strain 26695 activated NF-kappa B in HEK293 via TLR2 but not TLR4. Partially purified flagellin from H. pylori was also capable of inducing NF-kappa B activation in HEK cells transfected with TLR5. Additionally, chemokine gene expression was induced by H. pylori in HEK293 cells following stable transfection with TLR2 or TLR5 expression plasmids. These studies demonstrate that gastric epithelial cells recognize and respond to H. pylori infection at least in part via TLR2 and TLR5. Furthermore, the unique lipopolysaccharide of H. pylori is a TLR2, not a TLR4 agonist.
authors.affiliation: University of Virginia Health System, Digestive Health Center of Excellence and Department of Microbiology, Charlottesville, Virginia 22908-0708, USA. mfs3k@virginia.edu
authors.forename: Michael F
authors.initials: MF
authors.lastname: Smith
authors.forename: Anastasia
authors.initials: A
authors.lastname: Mitchell
authors.forename: Guolian
authors.initials: G
authors.lastname: Li
authors.forename: Song
authors.initials: S
authors.lastname: Ding
authors.forename: Ann Marie
authors.initials: AM
authors.lastname: Fitzmaurice
authors.forename: Kieran
authors.initials: K
authors.lastname: Ryan
authors.forename: Sheila
authors.initials: S
authors.lastname: Crowe
authors.forename: Joanna B
authors.initials: JB
authors.lastname: Goldberg
mesh_terms: D002460:Cell Line; D018925:Chemokines / Q000378:metabolism*; D018076:DNA, Complementary / Q000378:metabolism; D004789:Enzyme Activation; D004847:Epithelial Cells / Q000378:metabolism* / Q000382:microbiology; D005408:Flagellin / Q000378:metabolism; D005799:Genes, Dominant; D017930:Genes, Reporter; D016480:Helicobacter pylori / Q000378:metabolism*; D006801:Humans; D018950:Lipopolysaccharide Receptors / Q000096:biosynthesis; D008070:Lipopolysaccharides / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000235:genetics / Q000502:physiology*; D016328:NF-kappa B / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D012333:RNA, Messenger / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000235:genetics / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D013270:Stomach / Q000382:microbiology; D013997:Time Factors; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4; D051198:Toll-Like Receptor 5; D051193:Toll-Like Receptors; D014162:Transfection
chemical_list: D018925:Chemokines; D018076:DNA, Complementary; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D016328:NF-kappa B; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; C495342:TLR2 protein, human; C495345:TLR4 protein, human; C495346:TLR5 protein, human; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4; D051198:Toll-Like Receptor 5; D051193:Toll-Like Receptors; D005408:Flagellin