IL-33 and IL-17 family cytokines (IL-17A – IL-17F) have been shown to play roles in the pathogenesis of chronic inflammatory diseases such as rheumatoid arthritis and inflammatory bowel disease. However knowledge of their role in periodontal disease pathogenesis is limited. The aim of this study was therefore to determine clinical associations between IL-33 and IL-17 family cytokines and chronic periodontitis. In addition, to begin to investigate the biological significance of these associations using in vitro model systems. 97 patients with chronic periodontitis and 77 healthy volunteers were recruited in Glasgow and Newcastle. Serum, gingival crevicular fluid (GCF) and saliva were analysed for levels of IL-33 and IL-17 family cytokines by ELISA. Periodontal tissues from 17 chronic periodontitis patients and 10 healthy subjects from Glasgow were also investigated for IL-33 and IL-17 family cytokines mRNA expression by real time PCR. Immunohistochemical analysis was also performed on tissue to investigate expression of IL-33 and IL-17E at the protein level. In vitro experiments were performed using the OKF6/TERT-2 oral keratinocyte cell line and primary human gingival epithelial (PHGE) cells. The cells were stimulated with either a live Porphyromonas gingivalis monospecies biofilm or recombinant cytokines and changes in expression of cytokines, chemokines and their receptors evaluated by real-time PCR, immunocytochemical analysis or ELISA. In addition, transcriptional activity was monitored by analysis of changes in the phosphorylation (activation) of the NF-κB p65 subunit transcription factor using serum, GCF and saliva. IL-17A and IL-17A/F levels were higher in chronic periodontitis patients, but serum IL-17E was lower. IL-17A, IL-17A/F and the serum IL-17A:IL-17E ratio correlated positively with clinical parameters. IL-33, and IL-17 family cytokine (except IL-17B) gene transcripts were higher in tissue of chronic periodontitis patients. In addition, IL-33, ST2, IL-17E and IL-17RB proteins are expressed in periodontal tissues. Furthermore, IL-33 protein expression is upregulated in tissue of chronic periodontitis patients. In vitro models showed that IL-33 and its receptors (ST2 and ST2L) are expressed by oral keratinocytes (OKF6/TERT-2 cells and PHGE cells) and IL-33 expression up-regulated in response to P. gingivalis. However, IL-33 failed to induce expression of a range of inflammatory mediators and receptors in OKF6/TERT-2 cells. In vitro, IL-17E inhibited P. gingivalis monospecies biofilm and IL-17A induced expression of chemokines (IL-8 and/or CXCL5) by OKF6/TERT-2 cells at the transcriptional level by blocking the phosphorylation (activation) of the NF-κB p65 subunit. This study demonstrates clinical associations between IL-33 and IL-17 family cytokines and chronic periodontitis. The expression of IL-33 by oral keratinocytes and its up regulation upon exposure to P. gingivalis suggest it plays a role in the innate immune response to pathogens within the periodontium. However, the role of IL-33 in the periodontal inflammatory response remains to be elucidated. The negative correlations between serum levels of IL-17A and IL-17E and correlations with disease parameters, combined with their differing effects on the induction of expression of key neutrophil chemoattractants (CXCL5 and CXCL8), suggest opposing roles in periodontal immunity. Indeed, it can be hypothesised that the differential regulation of chemokine expression is due to IL-17A having pro- and IL-17E having anti-inflammatory properties. Indeed, as neutrophils play a key role in the early events associated with periodontal disease progression, the data suggests IL-17E is a rational target for therapeutic intervention.

Feline chronic gingivostomatitis (FCGS) is an inflammatory disease of the oral cavity that causes severe pain and distress. No specific treatment methods are available and little is known about its aetiology. The aims of this study were:- 1) to identify the bacterial flora, including uncultivable and potentially novel species, in healthy cats and those with FCGS, using 16S rRNA gene sequencing in combination with conventional culture methods; 2) to investigate the viral status of cats with and without FCGS; 3) to assess the immune response by investigating the expression of cytokine and Toll-like receptor (TLR) genes in tissue biopsies from normal cats and those with FCGS; 4) to investigate the histopathological changes in tissue biopsies from normal cats and those with FCGS, 5) to assess putative risk factors for FCGS by the use of a questionnaire-based study. Oral swabs, mucosal biopsies and blood were collected and the location of the oral lesions was recorded. A total of 32 cats with FCGS and 16 normal cats were included in the study. Bacteria were identified from swabs by use of 16S rRNA gene sequencing and by conventional culture methods. Blood samples and swabs were used for diagnosis of infection with feline leukaemia virus (FeLV), feline immunodeficiency virus (FIV), feline herpes virus 1 (FHV-1), feline calicivirus (FCV) and for blood biochemistry and haematology. Gene expression levels for TLR2, TLR3, TLR4, TLR7 and TLR9, and cytokines IL-1β, IL-4, IL-6, IL-10, TNF-α and IFN-γ mRNA were determined using quantitative PCR in biopsy samples from healthy cats and cats with FCGS. Histopathological examination of the tissue biopsies was done using hematoxylin and eosin (H&E) staining. In the healthy group, 16S rRNA gene sequencing demonstrated that the most prevalent bacteria were part of the Proteobacteria and Bacteroidetes phyla, plus a group of uncultured bacteria. The most prevalent species in the healthy group were Xanthomonadaceae bacterium (6.2 % of clones analysed), Capnocytophaga canimorsus (5.4%), Capnocytophaga cynodegmi (4.8%), Bergeyella species (4.5%) and Pasteurella multocida subspecies septica (4.4%). Uncultured bacteria accounted for 29% of the clones analysed. In the FCGS group most of the identified species were part of the phylum Proteobacteria. The most prevalent species in the FCGS group were P. multocida subsp. multocida (14.1%) P. multocida subsp. septica (11.5%), Pseudomonas sp. (7.3%), Tannerella forsythia (6.6%) and Porphyromonas circumdentaria (5.6%). A variety of uncultured bacteria represented 7.7% of all analysed FCGS clones. The culture data showed the most prevalent bacteria in the healthy group were P. multocida subsp. septica (9.9%), and uncultured bacteria (30.5%). In the FCGS group the most prevalent isolates were P. multocida subsp septica and P. multocida subsp. multocida (both 9.9%). Uncultured bacteria accounted for 21.7% of all isolates. FCV was detected in 71% of cats with FCGS and in 13.3% of normal cats. FeLV antigen was detected in 33.3% of normal cats but not in any cats with FCGS. FIV antibodies were detected in 3.4% of cats with FCGS and in 33.3% of normal cats. FHV-1 was detected in 6.9% of cats with FCGS, but was not detected any of the normal cats. In the FCGS group a significant increase was seen in the expression of TLR2 and TLR7 genes as well as TNF-α, IFN-γ, IL-1β and IL-6 cytokine genes. The healthy cats and cats with FCGS in the study that were found to harbour T. forsythia and P. circumdentaria showed an increase in the expression of several TLR and cytokine genes when compared to the group of cats in which these bacterial species were absent. The most severely inflamed sites in the oral cavity of cats with FCGS included the tissue lateral to the palatoglossal folds and the maxillary attached gingiva. Histopathological analysis of the tissue from the palatoglossal folds showed two types of infiltrates:- 1) a combination of lymphocytes and plasma cells, most often seen in the milder inflamed tissue samples; 2) a predominantly plasmacytic infiltration, most often seen in the severely inflamed tissue samples. Preliminary data from a questionnaire-based epidemiological study showed that the presence of potential environmental stress factors such as no ability to roam outdoors and the presence of more than one cat in the household is significantly higher in cats with FCGS when compared to normal cats. This study highlights the possibility of a multifactorial aetiology for FCGS in which FCV, specific bacteria and stress factors may play an important role. Although species from the Bacteroidetes phylum appeared to be capable of eliciting an immune response, these were not the most prevalent species in the FCGS group. A shift could be seen in the composition of the bacterial flora when healthy cats and those with FCGS were compared.

Implanted titanium (Ti) devices such as dental implants have been shown to produce metallic species within adjacent tissues. The effect of the presence of these species within oral epithelial tissues is currently not well characterised or known. This thesis investigates the effects of TiO\(_2\) nanoparticles (TiO\(_2\) NPs) at a range of concentrations on oral epithelial cells in the context of cell viability, cellular functions and interactions via a variety of methods. A co-culture model was established, and the difficulties of using a nano-scale insoluble stimulus were explored, and high content screening techniques were shown to be potentially more appropriate methods than conventional assays in this context. Interactions between TiO2 NPs and oral epithelial cells were imaged and investigated using a variety of imaging techniques. Oral epithelial cells were shown to take up TiO\(_2\) NPs within vacuole type structures. Cell viability appeared to not be affected at lower concentrations. Gene expression changes of oral epithelial cells in response to TiO\(_2\) NPs in the presence and absence of pathogenic bacteria were investigated. Cytokines important in cell-cell signalling were shown to bind TiO\(_2\) NPs, therefore creating potential for TiO\(_2\) NPs within tissues to modify immune responses within tissues adjacent to implanted Ti devices.

Oral keratinocyte behaviour were analysed in two and three dimensional cultures of an immortalised human H400 cellline and primary rat keratinocytes (PRKs) using a novel method of quantitative microscopy, RT-PCR data and immunohistochemistry profiles. Monolayer cultures were established in high and low calcium media at different cell densities and analysed prior to generating 3D organotypic cultures (OCs) onde-epidermalised dermis (DED), polyethylene terephthalate porous membrane (PET) and collagen gels for up to 14 days.H400 and PRKs proliferation in monolayer cultures was greater in low calcium medium compared with high calcium medium.Gene expression analysis indicated that adhesion and structural molecules including E-cadherin, plakophilin, desmocollin-3, desmogleins-3 and cytokeratins-1, -5, -6, -10, -13 were up-regulated by days 6 and 8 compared with day 4in high calcium medium. Immunohistochemical profiles and gene expression data of OCs on DED recapitulated those of normal oral epithelium. The final thickness of OCs as well as the degree of maturation/stratification was significantly greater on DED compared with other scaffolds used. Quantitative microscopy approaches enabled unbiased architectural characterisation of OCs and the ability to relate stratified organotypic epithelial structures to the normal oral mucosa. H400 and PRK OCs on DED at the air liquid interface demonstrated similar characteristics in terms of gene expression and protein distribution to the normal tissue architecture.