Microbial biofilm composition influences the host immune response

Periodontal disease (PD) is a multifactorial disease of the oral cavity affecting the majority of the population. Although not a direct cause of mortality, PD is a health concern because it affects the majority of the population and has a negative impact on oral health, ability to chew, appearance, quality of life, dental care costs and can lead to tooth loss. Dental plaque is a microbial biofilm, which is necessary but not sufficient for the development of periodontitis. The interactions between the biofilm and the host cells, both local tissue and immune cells, can lead to tissue destruction and ultimately tooth loss. Clinical management of periodontitis involves mechanical removal of plaque from the tooth surface. Treatment is time consuming, in some patients only partially successful and recurrence is common. Therefore, understanding how the host interacts with microbial biofilms in both health and PD will help improve treatments and identify novel targets for therapeutic and preventative strategies. The hypothesis of this thesis is that the bacterial composition of oral biofilms may modulate host cell responses which contribute to the pathogenicity of PD. The overarching aim of this research was to develop an in vitro co-culture model system to study how biofilm composition can influence the host immune response. The studies document the development of health-associated, intermediate and disease-associated biofilms with host tissue and immune cells, and the use of these models to test antimicrobial and anti-inflammatory compounds as potential treatments for PD. The biofilms developed were assessed for survival in cell culture conditions and batch reproducibility by PCR and morphology visualised using SEM. The health-associated biofilm included Streptococcus mitis, S. intermedius and S. oralis (3-species); the intermediate biofilm additionally included Veillonella dispar, Actinomyces naeslundii, Fusobacterium nucleatum and F. nucleatum spp. Vincentii (7-species); and the disease-associated biofilm included further addition of Porphyromonas gingivalis, Prevotella intermedia, and Aggregatibacter actinomycetemcomitans (10-species). These biofilms were co-cultured with an oral epithelial cell line and primary gingival epithelial cells, as well as neutrophils and a myeloid cell line. Host cell viability was assessed by AlamarBlue®/LDH and changes in mRNA and protein expression of chemokines and cytokines were assessed by quantitative PCR and ELISA/Luminex®, respectively. Cellular responses were further evaluated by microscopy and flow cytometry. Generally, co-culture of health associated biofilms with host cells resulted in minimal impact on cell viability and generally low inflammatory gene expression and protein release, with some genes including CXCL5 and CCL1 being downregulated compared to the cells only control. Intermediate biofilms caused some cell death and a marked upregulation of inflammatory genes and protein release, including a 302.7 fold increase of epithelial cell IL-8 gene expression compared to the cells only control. These intermediate biofilms elicited significant upregulation of CD40 and CD69 expression on the monocyte cell line compared with untreated controls. Co-culture of the 10 species disease associated biofilms with host cells resulted in significant host cell death of both epithelial cells and monocytes. The 10 species biofilm caused significantly increased pro-inflammatory gene expression, but only low levels of protein could be detected in the supernatants. Similar trends in upregulation of inflammatory gene expression but low levels of protein release was observed in co-culture with differentiated pro-monocytes, whereas upregulation of inflammatory gene expression and protein release in neutrophil co-cultures was observed. The effect of antimicrobial and anti-inflammatory compounds, resveratrol and chlorhexidine, was evaluated using this model system. Prior treatment of epithelial cells with resveratrol and biofilm with chlorhexidine significantly reduced IL-8 release from epithelial cells in co-culture with biofilms for 4 and 24 hours. In conclusion, this research has developed and validated 3 complex multi-species biofilms to study host: biofilm interactions in vitro. Furthermore, using these models in co-culture with multiple host cell types, clear differences in the host response to different biofilms were observed. The variations in inflammatory response of host cells and oral biofilms observed in this study help further understanding of the complex host: biofilm interactions within the oral cavity which contribute to PD. This model demonstrated its potential as a platform to test novel actives, highlighting its use a tool to study how actives can influence host: biofilm interactions within the oral cavity. Future use of this model will aid in greater understanding of host: biofilm interactions. Such findings are applicable to oral health and beyond, and may help to identify novel therapeutic targets for the treatment of PD and other biofilm associated diseases.