title: Induction of TLR-2 and TLR-5 expression by Helicobacter pylori switches cagPAI-dependent signalling leading to the secretion of IL-8 and TNF-α.
abstract: Helicobacter pylori is the causative agent for developing gastritis, gastric ulcer, and even gastric cancer. Virulent strains carry the cag pathogenicity island (cagPAI) encoding a type-IV secretion system (T4SS) for injecting the CagA protein. However, mechanisms of sensing this pathogen through Toll-like receptors (TLRs) and downstream signalling pathways in the development of different pathologies are widely unclear. Here, we explored the involvement of TLR-2 and TLR-5 in THP-1 cells and HEK293 cell lines (stably transfected with TLR-2 or TLR-5) during infection with wild-type H. pylori and isogenic cagPAI mutants. H. pylori triggered enhanced TLR-2 and TLR-5 expression in THP-1, HEK293-TLR2 and HEK293-TLR5 cells, but not in the HEK293 control. In addition, IL-8 and TNF-α cytokine secretion in THP-1 cells was induced in a cagPAI-dependent manner. Furthermore, we show that HEK293 cells are not competent for the uptake of T4SS-delivered CagA, and are therefore ideally suited for studying TLR signalling in the absence of T4SS functions. HEK293 control cells, which do not induce TLR-2 and TLR-5 expression during infection, only secreted cytokines in small amounts, in agreement with T4SS functions being absent. In contrast, HEK293-TLR2 and HEK293-TLR5 cells were highly competent for inducing the secretion of IL-8 and TNF-α cytokines in a cagPAI-independent manner, suggesting that the expression of TLR-2 or TLR-5 has profoundly changed the capability to trigger pro-inflammatory signalling upon infection. Using phospho-specific antibodies and luciferase reporter assays, we further demonstrate that H. pylori induces IRAK-1 and IκB phosphorylation in a TLR-dependent manner, and this was required for activation of transcription factor NF-κB. Finally, NF-κB activation in HEK293-TLR2 and HEK293-TLR5 cells was confirmed by expressing p65-GFP which was translocated from the cytoplasm into the nucleus. These data indicate that H. pylori-induced expression of TLR-2 and TLR-5 can qualitatively shift cagPAI-dependent to cagPAI-independent pro-inflammatory signalling pathways with possible impact on the outcome of H. pylori-associated diseases.
authors.affiliation: Institute for Medical Microbiology, Otto von Guericke University, Magdeburg, Germany.
authors.forename: Suneesh
authors.initials: S
authors.lastname: Kumar Pachathundikandi
authors.forename: Sabine
authors.initials: S
authors.lastname: Brandt
authors.forename: Joseph
authors.initials: J
authors.lastname: Madassery
authors.forename: Steffen
authors.initials: S
authors.lastname: Backert
mesh_terms: D000942:Antigens, Bacterial / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D002460:Cell Line; D044404:Genomic Islands / Q000235:genetics*; D057809:HEK293 Cells; D016481:Helicobacter Infections; D016480:Helicobacter pylori / Q000502:physiology*; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism; D018836:Inflammation Mediators / Q000378:metabolism; D053592:Interleukin-1 Receptor-Associated Kinases / Q000378:metabolism; D016209:Interleukin-8 / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D051195:Toll-Like Receptor 2 / Q000235:genetics* / Q000378:metabolism; D051198:Toll-Like Receptor 5 / Q000235:genetics* / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000378:metabolism; D015533:Transcriptional Activation; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*; D015854:Up-Regulation / Q000235:genetics
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; C508606:CXCL8 protein, human; D020672:I-kappa B Proteins; D018836:Inflammation Mediators; D016209:Interleukin-8; D016328:NF-kappa B; D012333:RNA, Messenger; C495342:TLR2 protein, human; C495346:TLR5 protein, human; D051195:Toll-Like Receptor 2; D051198:Toll-Like Receptor 5; D018808:Transcription Factor AP-1; D014409:Tumor Necrosis Factor-alpha; C080866:cagA protein, Helicobacter pylori; D010768:Phosphoserine; C507768:IRAK1 protein, human; D053592:Interleukin-1 Receptor-Associated Kinases