Periodontal disease is associated with an inflammatory response to a pathogenic biofilm. The host response may cause gingival inflammation, which can progress to irreversible gingival recession, alveolar bone destruction and tooth loss. Enhanced understanding of the host-biofilm relationship may inform novel therapeutic approaches. A key molecule involved in inducing and mediating pro-inflammatory responses are the IL-17 cytokine family. An in vitro model system potentially provides a platform to investigate biofilm interaction with epithelial cells. The aim of this study was to develop in vitro mono-species and multi-species biofilms and investigate the survival of biofilms in cell culture conditions, and simultaneously assess the epithelial response to the bacterial biofilms and planktonic cells with respect to viability, apoptosis and inflammatory mediators. This study also looked to determine whether IL-17A is expressed within and released from periodontal tissues and to investigate its role in the regulation of epithelial cell cytokine and chemokine production. Mono- and multi-species biofilms of P. gingivalis, F. nucleatum, A. actinomycetemcomitans and S. mitis were developed, which were assessed for survival in cell culture conditions, recovery from biofilms and morphology. Gingival tissue from patients with chronic periodontitis or healthy controls were analysed for IL-17A gene expression by qPCR. Protein expression and cellular localization was determined by immunofluorescence. Single cell suspensions of gingival tissue were stimulated in vitro and IL-17A release assessed. Epithelial response after bacterial and IL-17A co-culture was assessed. The individual bacteria survived preferentially in multi-species biofilm compared with mono-species biofilm in cell culture conditions. The viability, apoptosis and inflammatory mediator response depended on the type (pathogen or commensal) and form (planktonic or biofilm) of bacteria. Diseased gingival tissues expressed significantly higher levels of IL-17A mRNA than healthy samples. IL-17A localised to mast cells in the inflamed gingival tissue, and was released in cell culture supernatants following stimulation. Stimulation of epithelial cells with IL-17A resulted in the transcriptional regulation and release of numerous cytokines and chemokines. The initial component of the entire investigation has provided a quantitative and qualitative assessment of both mono- and multi-species biofilms that can be used to investigate how oral biofilms interact with the host epithelium. The epithelial-biofilm co-culture model has demonstrated clear differences between (i) planktonic and biofilms, (ii) pathogens and commensals, and (iii) live and dead bacterial challenge. These observations and the utility of the model will provide a platform to investigate key questions relating to pathogen and host within the oral cavity and beyond. From this study, it appears that IL-17A plays an important role in the protective periodontal immune response to bacterial pathogens. The upregulation of acute inflammatory mediators (such as IL-8) will promote neutrophil recruitment and potentiate the removal of any invading microbial threat. Therefore it is important to understand the benefits of this cytokine, before systemic therapeutic agents are used to antagonise its actions. The hope for the future is to unravel the details of the mechanisms involved and thereby identify novel therapeutic targets for inflammatory and infectious disease.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:570011 |
| Date | January 2013 |
| Creators | Jose, Anto |
| Publisher | University of Glasgow |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://theses.gla.ac.uk/4162/ |
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