Josefine Hirschfeld1, Henrik Dommisch2, Philipp Skora3, Gabor Horvath4, Eicke Latz4, Achim Hoerauf5, Tobias Waller3, Toshihisa Kawai6, Søren Jepsen3, James Deschner7, Isabelle Bekeredjian-Ding8. 1. 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. 2. 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. 3. Center for Dental and Oral Medicine, Department of Periodontology, Operative and Preventive Dentistry, University Hospital Bonn, Welschnonnenstraße 17, D-53111 Bonn, Germany. 4. Institute of Innate Immunity, Biomedical Centre, University Hospital Bonn, Sigmund-Freud-Str. 25, D-53127 Bonn, Germany. 5. Institute of Medical Microbiology, Immunology and Parasitology, University Hospital Bonn, Sigmund-Freud-Str. 25, D-53127 Bonn, Germany. 6. The Forsyth Institute, Department of Immunology and Infectious Diseases, 245 First Street, Cambridge, MA 02142, USA. 7. Center for Dental and Oral Medicine, Department of Experimental Dento-Maxillo-Facial Medicine, University Hospital Bonn, Welschnonnenstraße 17, D-53111 Bonn, Germany. 8. 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.
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.
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.
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