title: TLR9 mediates S. aureus killing inside osteoblasts via induction of oxidative stress.
abstract: BACKGROUND
  Staphylococcus aureus is the principle causative pathogen of osteomyelitis and implant-associated bone infections. It is able to invade and to proliferate inside osteoblasts thus avoiding antibiotic therapy and the host immune system. Therefore, development of alternative approaches to stimulate host innate immune responses could be beneficial in prophylaxis against S. aureus infection. TLR9 is the intracellular receptor which recognizes unmethylated bacterial CpG-DNA and activates immune cells. Synthetic CpG-motifs containing oligodeoxynucleotide (CpG-ODNs) mimics the stimulatory effect of bacterial DNA.
  
  
  RESULTS
  Osteoblast-like SAOS-2 cells were pretreated with CpG-ODN type-A 2216, type-B 2006, or negative CpG-ODN 2243 (negative control) 4 h before infection with S. aureus isolate EDCC 5055 (=DSM 28763). Intracellular bacteria were streaked on BHI plates 4 h and 20 h after infection. ODN2216 as well as ODN2006 but not ODN2243 were able to significantly inhibit the intracellular bacterial growth because about 31 % as well as 43 % of intracellular S. aureus could survive the pretreatment of SAOS-2 cells with ODN2216 or ODN2006 respectively 4 h and 20 h post-infection. RT-PCR analysis of cDNAs from SAOS-2 cells showed that pretreatment with ODN2216 or ODN2006 stimulated the expression of TLR9. Pretreatment of SAOS-2 cells with ODN2216 or ODN2006 but not ODN2243 managed to induce reactive oxygen species (ROS) production inside osteoblasts as measured by flow cytometry analysis. Moreover, treating SAOS-2 cells with the antioxidant Diphenyleneiodonium (DPI) obviously reduced S. aureus killing ability of TLR9 agonists mediated by oxidative stress.
  
  
  CONCLUSIONS
  In this work we demonstrated for the first time that CPG-ODNs have inhibitory effects on S. aureus survival inside SAOS-2 osteoblast-like cell line. This effect was attributed to stimulation of TLR9 and subsequent induction of oxidative stress. Pretreatment of infected SAOS-2 cells with ROS inhibitors resulted in the abolishment of the CPG-ODNs killing effects.
authors.affiliation: Laboratory of Experimental Trauma Surgery Giessen, Justus-Liebig-University Giessen, 35394, Giessen, Germany. walid.mohamed@mikrobio.med.uni-giessen.de.|Institute of Medical Microbiology, German Centre of Infection Research, Justus-Liebig-University Giessen, 35392, Giessen, Germany. walid.mohamed@mikrobio.med.uni-giessen.de.
authors.forename: Walid
authors.initials: W
authors.lastname: Mohamed
authors.affiliation: Institute of Medical Microbiology, German Centre of Infection Research, Justus-Liebig-University Giessen, 35392, Giessen, Germany.
authors.forename: Eugen
authors.initials: E
authors.lastname: Domann
authors.affiliation: Institute of Medical Microbiology, German Centre of Infection Research, Justus-Liebig-University Giessen, 35392, Giessen, Germany.
authors.forename: Trinad
authors.initials: T
authors.lastname: Chakraborty
authors.affiliation: Laboratory of Experimental Trauma Surgery Giessen, Justus-Liebig-University Giessen, 35394, Giessen, Germany.
authors.forename: Gopala
authors.initials: G
authors.lastname: Mannala
authors.affiliation: Laboratory of Experimental Trauma Surgery Giessen, Justus-Liebig-University Giessen, 35394, Giessen, Germany.
authors.forename: Katrin S
authors.initials: KS
authors.lastname: Lips
authors.affiliation: Laboratory of Experimental Trauma Surgery Giessen, Justus-Liebig-University Giessen, 35394, Giessen, Germany.|Department of Trauma Surgery Giessen, Justus-Liebig-University Giessen, 35385, Giessen, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Heiss
authors.affiliation: Laboratory of Experimental Trauma Surgery Giessen, Justus-Liebig-University Giessen, 35394, Giessen, Germany.|Department of Trauma Surgery Giessen, Justus-Liebig-University Giessen, 35385, Giessen, Germany.
authors.forename: Reinhard
authors.initials: R
authors.lastname: Schnettler
authors.affiliation: Laboratory of Experimental Trauma Surgery Giessen, Justus-Liebig-University Giessen, 35394, Giessen, Germany.|Department of Trauma Surgery Giessen, Justus-Liebig-University Giessen, 35385, Giessen, Germany.
authors.forename: Volker
authors.initials: V
authors.lastname: Alt
mesh_terms: D018441:Biofilms / Q000187:drug effects; D045744:Cell Line, Tumor; D004269:DNA, Bacterial / Q000276:immunology; D005434:Flow Cytometry; D006801:Humans; D007113:Immunity, Innate; D009838:Oligodeoxyribonucleotides / Q000276:immunology / Q000494:pharmacology*; D009861:Onium Compounds / Q000494:pharmacology; D010006:Osteoblasts / Q000276:immunology* / Q000382:microbiology*; D018384:Oxidative Stress / Q000187:drug effects / Q000276:immunology*; D013203:Staphylococcal Infections / Q000188:drug therapy / Q000276:immunology / Q000382:microbiology; D013211:Staphylococcus aureus / Q000187:drug effects / Q000276:immunology* / Q000502:physiology; D051217:Toll-Like Receptor 9 / Q000819:agonists / Q000096:biosynthesis / Q000276:immunology* / Q000378:metabolism
chemical_list: C408982:CPG-oligonucleotide; C513533:CpG ODN 2006; C526902:CpG ODN 2216; D004269:DNA, Bacterial; D009838:Oligodeoxyribonucleotides; D009861:Onium Compounds; C495371:TLR9 protein, human; D051217:Toll-Like Receptor 9; C007517:diphenyleneiodonium
keywords: CpG-ODN; Oxidative stress; S. aureus; TLR9