The intestinal immune system plays an essential role in maintaining the delicate balance between mounting protective responses against invading pathogens and sustaining tolerance towards self-antigens and the endogenous microbiota. Disturbing this balance leads to intestinal inflammation, such as is seen in inflammatory bowel disease (IBD). IBD is characterised by alterations in the mucosa-associated microbiota, such as the increase in adherent and invasive Escherichia coli (AIEC) species in Crohn’s disease (CD) patients. Concomitantly, the inflamed mucosa exhibits an elevated rate of apoptosis in IBD, a phenomenon that also accompanies infection with a range of enteric bacterial pathogens. While phagocytosis of apoptotic cells by dendritic cells (DC) is required for self-tolerance in the healthy intestine, there is evidence to suggest that apoptotic cell uptake during infection activates protective T cell responses. In order to investigate the link between the recognition of apoptotic cells and intestinal inflammation, we used a range of different in vivo enteric bacterial infection models. Previously published work had implicated the AIEC strain NRG857c in the induction of chronic intestinal inflammation in vivo. We did not, however, find that NRG857c caused any signs of chronic colitis in mice, either by histological examination, or in-depth analysis of both innate and adaptive immune responses in the lamina propria and mesenteric lymph nodes (MLN). Due to the important role of DC in acquiring apoptotic cell antigen and priming protective T cell responses, we next characterised the expression of apoptotic cell receptors, specifically TIM4, on DC populations in steady state mucosal tissues. We demonstrated that TIM4 expression was enriched on CD11b- CD103+ DC, which have previously been shown to cross-present apoptotic cell-derived antigen. However, upon migration in mesenteric lymph, all intestinal DC populations upregulated TIM4, and migratory CD11b+ CD103+ had the highest frequency of TIM4+ cells in the MLN. However, blocking TIM4 did not affect DC migration in vivo. We also found that infection with C. rodentium elevated the percentage of TIM4+ DC in a population-specific manner, but that TIM4 was not essential for the induction of protective T cell responses during infection with either C. rodentium or S. Typhimurium. We therefore provide a detailed analysis of the intestinal immune response to bacterial infection, focussing specifically on the role of the apoptotic cell receptor TIM4 on intestinal DC populations.

No description available.

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.

This work explores the long-standing question of which forces drive the maintenance of MHC diversity in vertebrates. More precisely, it investigates whether a special form of heterozygote advantage can explain the characteristic features of MHC genes, as the large number of alleles, the characteristic distribution of allele frequencies, and the trans-species polymorphism. This special overdominance variant is based on the divergent allele advantage hypothesis (Wakeland et al., 1990), and therefore the corresponding model is called the divergent allele advantage model. The novel tool of diversity profiles (qDZ) will be used to characterise and compare allelic diversity of different overdominance models, and to contrast simulation results with observed data. Chapters 2 and 3 perform a stability analysis of a n-allele system at equilibrium. Different overdominance models are compared in a common framework, and a novel model, based on the divergent allele advantage hypothesis, is introduced while its properties are analysed. Chapter 4 explores a more realistic scenario that includes mutation and drift and compares a modified divergent allele advantage model to traditional models of heterozygote advantage. Implications of the results of the previous chapters are discussed in chapter 5. Finally, chapter 6 extends the analysis of the divergent allele advantage model to a structured population, thus additionally including migration, and explores whether the findings of the previous chapters also hold in a more realistic scenario of a metapopulation of interacting subpopulations. The results of this work demonstrate that the novel model based on the divergent allele advantage hypothesis can explain the main features of the diversity of the Major Histocompatibility Complex genetic region, but it predicts the existence of many genotypes that increase susceptibility to disease.

The nematode Caenorhabditis elegans has been a powerful experimental organism for almost half a century. Over the past ten years, researchers have begun to exploit the power of C. elegans to investigate the biology of a number of human pathogens. This work continues to uncover mechanisms of host immunity and pathogen virulence that are either analogous to those involved during pathogenesis in alternative animal hosts or mechanisms which are, thus far, unique to the worm. In this thesis, we present data that describes an immunological balance in C. elegans, whereby heightened tolerance to one pathogen, the enteric bacteria Salmonella Typhimurium, comes at the cost of increased susceptibility to another, the fatal fungal human pathogen Cryptococcus neoformans. We find that this susceptibility trade-off is mediated by the reciprocal activity of two immune genes: the lysozyme lys-7 and the tyrosine kinase abl-1. We suggest that ABL-1 controls two different DAF-16-dependent pathways to regulate this balance. Both pathways are necessary for wild type resistance to C. neoformans, whilst the activity of only one pathway is a requirement for the tolerance phenotype to S. Typhimurium. We infer from sequence data that LYS-7 has an atypical mode of action in C. elegans, which we hypothesise to be detrimental to the worm during S. Typhimurium pathogenesis and thus a contributing factor to the tolerance phenotype. Furthermore, we find that this tolerance has a Salmonella-dependency which we propose to be under the control of the alternative sigma factor, RpoS. Taken together, we describe an immunological balance in C. elegans for the first time, one that is mediated by both host and pathogen factors. We therefore suggest that the innate immune response of C. elegans has a higher level of immune complexity than previously believed, and that such trade-offs are evolutionarily ancient mechanisms.

The glycolipid α-galactosyl ceramide, α-GalCer, has been shown to stimulate the proliferation of murine spleen cells and activate the immune system. Stimulation occurs through binding of the glycolipid to the protein CD1d. Subsequent presentation of the CD1d−glycolipid complex to invariant Natural Killer T cells (iNKT cells) initiates the proliferation of a host of cytokines leading to an immune response The therapeutic potential of α-GalCer is currently being explored; however the induction of both Th1 and Th2 cytokines by this agent is likely to limit its therapeutic application. Significantly, analogues of α-GalCer have been shown to induce iNKT cell-derived cytokines more selectively through a skewed Th1-Th2 response. To date, very few alterations around the amide bond have been explored. To investigate its importance in iNKT cell stimulation, a range of α-GalCer and threitol ceramide (ThrCer) analogues has been synthesised in which the amide functionality in these two leads has been replaced with different carbonyl functional groups. These compounds have been tested for iNKT cell induction and in particular their Th1/Th2 response, which determined their therapeutic potential. Labelled derivatives of α-GalCer and ThrCer have also been designed and synthesised to find application in lipid trafficking studies.

Rheumatoid arthritis (RA) is a chronic disease characterised by inflammatory infiltration of the synovial membrane, with concomitant destruction of adjacent cartilage and bone. Elucidation of immunoregulatory networks within the synovium offers the potential for therapeutic intervention. Two such pathways were investigated in the present study. Interleukin-15 (IL-15) is a novel pleiotropic cytokine produced by macrophages and fibroblasts, which induces T cell migration and activation and B cell maturation and immunoglobulin production. IL-15 was identified in RA synovial fluids and synovial membrane cultures and, using immunohistochemistry, its expression was localised in the RA synovial membrane to the lining layer and T lymphocyte aggregates. Enhanced proliferation and cytokine production to IL-15 was observed in RA synovial fluid (SF) T cells in comparison to matched peripheral blood (PB) T lymphocytes, which in turn, were more sensitive to IL-15 induced proliferation than PBT cells from normal controls. Following IL-15 mediated activation, PBT cells were capable of inducing TNF production from a macrophage cell line, from syngeneic PB monocytes, and from synovial macrophage/synoviocyte co-cultures, through a cell-contact dependent mechanism, which required no T cell cytokine synthesis. RASFT cells exhibited similar properties, which were IL-15 dependent . IL-15 upregulated CD69 expression on CD45ROT cells and neutralisation studies determined that such CD69 expression, in combination with LFA-1 and ICAM-1, was partly responsible for cell-contact mediated macrophage activation by T cells. Finally, in a murine model, IL-15 injection induced significant local tissue T cell invasion, confirming previous observations of its chemotactic properties. IL-15 expression in RA synovial membrane therefore provides a mechanism whereby polyclonal T cell recruitment and activation can lead to macrophage activation and TNF production, without T cell cytokine synthesis.

This thesis was Concerned with the study of gut immunity to an intestinal trematode. Me parasite used was the strigeid Diplostomum phoxini. Adult D, phoxini normally parasitise the intestine of fish eating birds, but will readily establish and reach sexual maturity in laboratory infected mice. After reaching sexual maturity in about 4 dayst the'worms are lost shortly afterwards, and there is strong evidence that this loss is immunologically mediated by the host. The aims of this thesis were to establish the kinetics of the infection in mice and to investigate aspects of the immune response of the host to the parasite. The first chapter was concerned with the kinetics of establishment and rejection of both a Drimary and secondary infection of D. Rhoxini. The kinetics of heavy (200 metacercariae) and light (20 metacercariae) infections were com-Dared in an attempt to economise on the number of parasites required, especially with regards to immunization schedules. Results showed that in NIH mice, at least 80% of the administered worms would establish in the intestine. This wom T)opulation remained stable until rejectiong the onset of which was, related to the level of infection, i. e. heavy worm burdens were expelled earlier than light infections. The most important experiment with regards to immunity was the demonstration that a secondary infection was ex-oelled earlier than a primary infection, showing that loss was Drobably immunologically mediated and that 'memory cells" were established as a result of a primary infection in a mouse.This thesis was Concerned with the study of gut immunity to an intestinal trematode. Me parasite used was the strigeid Diplostomum phoxini. Adult D, phoxini normally parasitise the intestine of fish eating birds, but will readily establish and reach sexual maturity in laboratory infected mice. After reaching sexual maturity in about 4 dayst the'worms are lost shortly afterwards, and there is strong evidence that this loss is immunologically mediated by the host. The aims of this thesis were to establish the kinetics of the infection in mice and to investigate aspects of the immune response of the host to the parasite. The first chapter was concerned with the kinetics of establishment and rejection of both a Drimary and secondary infection of D. Rhoxini. The kinetics of heavy (200 metacercariae) and light (20 metacercariae) infections were com-Dared in an attempt to economise on the number of parasites required, especially with regards to immunization schedules. Results showed that in NIH mice, at least 80% of the administered worms would establish in the intestine. This wom T)opulation remained stable until rejectiong the onset of which was, related to the level of infection, i. e. heavy worm burdens were expelled earlier than light infections. The most important experiment with regards to immunity was the demonstration that a secondary infection was ex-oelled earlier than a primary infection, showing that loss was Drobably immunologically mediated and that 'memory cells" were established as a result of a primary infection in a mouse.IgM was specific anti worm antibody. The final cha-nter was-concerned with the non-specific responses of the mouse. The intra epithelial globule leukocyte, lamina T)roDoria mast cell and goblet cell resnonse to infection was investigated. Both the intra epithelial globule leukocytes and lamina Droporia mast cells increased in numbers in response to infection, with the greatest resnonse observed in the intra epithelial globule leukocyte DoDulation. Wo increase in goblet cell numbers was observed. The transfer of immune mesenteric lymph node cells in the presence of D. phoxini also accelerated the increase in the intra eDithelial globule leukocyte and lamina Droporia mast cell DoDulations which suggests that these cells may be under lymphocyte control. Finally the results of the e"eriments on Do Rhoxini were discussed in relation to the more well known, but still far from well understood, nematode and cestode models.

D6 is a novel chemokine receptor, homologous to other members of the CC-chemokine receptor family, which recognises a number of inflammatory CC-chemokines with high affinity. The aims of this thesis were to further our understanding of the biology of D6, chiefly through characterisation of immune responses in D6-deficient animals. Firstly, as described in Chapter 3, I analysed the cellular composition of lymphoid tissues of D6 KO mice. These studies revealed higher proportions of CD11c+ and F4/80+ cells in the D6 KO spleen compared with WT controls, suggesting that increased accumulation of myeloid lineage cells was occurring at this site. In Chapter 4, I examined the role of D6 in myeloid cell responses, by comparing monocyte recruitment to the inflamed peritoneum and dendritic cell development from bone-marrow (BM) cultures. I found that while the accumulation of inflammatory monocytes/macrophages appeared quantitatively similar in WT and D6 KO animals, D6 KO cells expressed greater levels of CD11c, suggesting preferential accumulation of DC-like cells in the inflamed peritoneum. D6 may influence the development and function of myeloid lineage cells. As D6 is expressed at high levels in the small and large intestine, I next investigated both tolerogenic and inflammatory intestinal responses in D6 KO animals. As detailed in Chapter 5 of this thesis, the induction of oral tolerance in response to a high dose feeding protocol was normal in D6 KO mice. However, D6 KO mice showed increased resistance to experimental colitis. As described in Chapter 6, various D6 KO populations displayed differential chemokine receptor profiles compared with their WT counterparts. The results suggest a role for D6 in the normal development of leukocytes populations, with absence of this atypical receptor leading to the dysregulated expression of other chemokine receptors. Taken together, my data suggest that the biological functions of D6 may be more complicated than previously appreciated. Indeed, I found no evidence for a decoy role of D6 in vivo, but D6-deficient animals were characterised by altered leukocyte development, aberrant chemokine receptor expression and increased resistance to experimental colitis induction.

Dental caries remains one of the most common chronic infectious childhood diseases and individuals remain susceptible to the disease throughout their lifetime. The disease continues to inflict a substantial economic burden. Moreover, dental caries demonstrates considerable socioeconomic disparities with the lowest socioeconomic groups suffering the greatest burden of disease. There is an unmet need to improve prevention and therapeutics and yet there remain fundamental gaps in the knowledge of the interrelationships between caries-associated risk factors, in particular how the immune system interacts with the evolving cariogenic biofilm in young children. This thesis sought to investigate the immune response to cariogenic biofilms. Three different approaches were used to achieve this. Firstly, the salivary immune response and development of the oral biofilm in very young children were investigated prior to the onset of caries, as part of a pilot longitudinal clinical study, using a dental public health program as a platform. Secondly, the initiation of adaptive immune responses to S. mutans exposure were investigated using a series of In vitro and In vivo studies. Thirdly, a novel S. mutans In vitro biofilm model was developed and optimised. Childsmile is a dental health improvement programme for children in Scotland and provides children with specific dental health interventions depending on need, from birth and up to 16-years of age. To achieve the first and primary aim of this thesis, plaque and saliva samples were collected from children aged one-year and again at age three-years. At follow-up, dental disease scores were also measured. Additionally, the biological mechanisms underlying the socioeconomic disparities in the dental health of young children were investigated, including the measurement of salivary cortisol as a surrogate measure of stress. Sixty-three Childsmile participants aged one-year were recruited to the study at baseline. Twenty-three children aged three-years were successfully recalled at follow-up. This work demonstrated that variables hypothesised to influence the development of carious disease can be collected and successfully quantified in children aged one- to three-years. Nonetheless, it was extremely challenging to recruit children of this age and the data were compromised by the small sample sizes. During the study period both the intensity and incidence of S. mutans colonisation increased in the dental plaque of children aged one- to three-years. Coincidentally, concentrations of salivary antimicrobial proteins increased, including lactoferrin, LL37, calprotectin, the HNPs 1-3 and sIgA antibody titres specific for oral streptococci. It could not be determined from these studies whether the increased colonisation with S. mutans or the concentrations of salivary antimicrobial proteins influenced the prevalence of dental caries. The major limitation of this study was the low recruitment rates which resulted in low power to detect statistically significant differences. As a consequence there was insufficient evidence to identify the potential biological pathways that may underlie the socioeconomic disparities of dental caries. From this pilot study a number of valuable lessons were learned regarding the recruitment of children of this age and recommendations for future clinical studies conducted within Childsmile are made. In children with high risk of developing dental caries effective salivary antibody responses are required to provide protection. The mechanisms leading to effective antibody responses remain unclear. Thus, the second aim of this thesis was to investigate the initiation of an adaptive immune response to S. mutans, in an attempt to elucidate the mechanisms that lead to effective antibody production. Using a novel system, In vitro evidence indicated that S. mutans does not elicit a robust inflammatory immune response upon colonisation of the host. Dendritic cells exposed to S. mutans were not functionally mature and failed to induce antigen-specific T cell proliferation. Furthermore, In vivo, dendritic cells failed to become activated in response to oral exposure to S. mutans. An In vitro S. mutans sucrose-dependent biofilm model was developed and optimised. Using this model an antibody fragment known as a minibody, denoted ‘SS2’ was demonstrated to inhibit S. mutans biofilm formation. This biofilm model represents an important first step for examining the potential of therapeutic molecules to inhibit S. mutans biofilm formation, prior to their application in In vivo models of dental caries and possible subsequent use in human clinical trials. Data described here indicate that S. mutans colonises the oral cavity at a time when children are immunologically immature. Increased colonisation by S. mutans coincides with the maturation of salivary immune responses. Moreover, In vitro and In vivo evidence suggest that S. mutans does not elicit a robust immune response upon colonisation of the host. Thus, early acquisition of S. mutans in a relatively immunologically immature host together with the absence of an inflammatory immune response likely aids the colonisation of S. mutans and its persistence within the oral biofilm and subsequent contribution to dental caries.