title: Neutrophil extracellular trap formation in supragingival biofilms.
abstract: BACKGROUND
  Oral biofilms are the causative agents of the highly prevalent oral diseases periodontitis and caries. Additionally, the host immune response is thought to play a critical role in disease onset. Neutrophils are known to be a key host response factor to bacterial challenge on host surfaces. Release of neutrophil extracellular traps (NETs) as a novel antimicrobial defense strategy has gained increasing attention in the past years. Here, we investigated the influx of neutrophils into the dental plaque and the ability of oral bacteria to trigger intra-biofilm release of NETs and intracellular proteins.
  
  
  METHODS
  Supragingival biofilms and whole saliva were sampled from systemically healthy subjects participating in an experimental gingivitis study. Biofilms were analysed by immunofluorescence followed by confocal and fluorescence microscopy. Moreover, concentrations of cytokines and immune-associated proteins in biofilm suspensions and saliva were assessed by ELISA. Neutrophils obtained from blood were stimulated with twelve bacterial species isolated from cultured biofilms or with lipopolysaccharide to monitor NET formation.
  
  
  RESULTS
  Neutrophils, NETs, neutrophil-associated proteins (myeloperoxidase, elastase-2, cathepsin G, cathelicidin LL-37), interleukin-8, interleukin-1β and tumor necrosis factor were detected within plaque samples and saliva. All tested bacterial species as well as the polymicrobial samples isolated from the plaque of each donor induced release of NETs and interleukin-8. The degree of NET formation varied among different subjects and did not correlate with plaque scores or clinical signs of local inflammation.
  
  
  CONCLUSIONS
  Our findings indicate that neutrophils are attracted towards dental biofilms, in which they become incorporated and where they are stimulated by microbes to release NETs and immunostimulatory proteins. Thus, neutrophils and NETs may be involved in host biofilm control, although their specific role needs to be further elucidated. Moreover, inter-patient variability suggests NET formation as a potential factor influencing the individual course of disease.
authors.affiliation: Center for Dental and Oral Medicine, Department of Periodontology, Operative and Preventive Dentistry, University Hospital Bonn, Welschnonnenstraße 17, D-53111 Bonn, Germany. Electronic address: josefine.hirschfeld@ukb.uni-bonn.de.
authors.forename: Josefine
authors.initials: J
authors.lastname: Hirschfeld
authors.affiliation: Institute for Dental and Oral Medicine, Department of Periodontology and Synoptic Dentistry, Charité Berlin, Aßmannhauser Straße 4-6, D-14197 Berlin, Germany; Department of Oral Health Sciences, University of Washington, Seattle, WA 98195, USA.
authors.forename: Henrik
authors.initials: H
authors.lastname: Dommisch
authors.affiliation: Center for Dental and Oral Medicine, Department of Periodontology, Operative and Preventive Dentistry, University Hospital Bonn, Welschnonnenstraße 17, D-53111 Bonn, Germany.
authors.forename: Philipp
authors.initials: P
authors.lastname: Skora
authors.affiliation: Institute of Innate Immunity, Biomedical Centre, University Hospital Bonn, Sigmund-Freud-Str. 25, D-53127 Bonn, Germany.
authors.forename: Gabor
authors.initials: G
authors.lastname: Horvath
authors.affiliation: Institute of Innate Immunity, Biomedical Centre, University Hospital Bonn, Sigmund-Freud-Str. 25, D-53127 Bonn, Germany.
authors.forename: Eicke
authors.initials: E
authors.lastname: Latz
authors.affiliation: Institute of Medical Microbiology, Immunology and Parasitology, University Hospital Bonn, Sigmund-Freud-Str. 25, D-53127 Bonn, Germany.
authors.forename: Achim
authors.initials: A
authors.lastname: Hoerauf
authors.affiliation: Center for Dental and Oral Medicine, Department of Periodontology, Operative and Preventive Dentistry, University Hospital Bonn, Welschnonnenstraße 17, D-53111 Bonn, Germany.
authors.forename: Tobias
authors.initials: T
authors.lastname: Waller
authors.affiliation: The Forsyth Institute, Department of Immunology and Infectious Diseases, 245 First Street, Cambridge, MA 02142, USA.
authors.forename: Toshihisa
authors.initials: T
authors.lastname: Kawai
authors.affiliation: Center for Dental and Oral Medicine, Department of Periodontology, Operative and Preventive Dentistry, University Hospital Bonn, Welschnonnenstraße 17, D-53111 Bonn, Germany.
authors.forename: Søren
authors.initials: S
authors.lastname: Jepsen
authors.affiliation: Center for Dental and Oral Medicine, Department of Experimental Dento-Maxillo-Facial Medicine, University Hospital Bonn, Welschnonnenstraße 17, D-53111 Bonn, Germany.
authors.forename: James
authors.initials: J
authors.lastname: Deschner
authors.affiliation: Institute of Medical Microbiology, Immunology and Parasitology, University Hospital Bonn, Sigmund-Freud-Str. 25, D-53127 Bonn, Germany; Division of EU Co-operation/Microbiology, Paul-Ehrlich Institut, Paul-Ehrlich-Str. 51-59, D-63225 Langen, Germany.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Bekeredjian-Ding
mesh_terms: D000328:Adult; D001419:Bacteria / Q000276:immunology*; D018407:Bacterial Physiological Phenomena; D018441:Biofilms / Q000254:growth & development*; D065206:Extracellular Traps / Q000378:metabolism*; D005260:Female; D005881:Gingiva / Q000276:immunology* / Q000382:microbiology*; D064368:Healthy Volunteers; D006801:Humans; D008297:Male; D008875:Middle Aged; D010865:Pilot Projects; D055815:Young Adult
keywords: Bacteria; Interleukin-8; NET; Neutrophils; Oral biofilm