Plasmodium chabaudi has been shown to undergo antigenic variation during the course of infection in mice. The importance of this model is the similarity and applicability of its features to infection of humans in P. falciparum. This thesis presents work performed using P. chabaudi to study various aspects of antigenic variation in asexual erythrocytic malaria parasites. The course of infection of P. chabaudi in N1H mice shows an initial acute parasitaemia which clears to subpatency. This is usually followed, after a period of days, by a second, and occasionally a third, recrudescent parasitaemia of lesser magnitude and duration. A cloned parent parasite population and cloned parasite populations derived from a recrudescence of the parent were tested in an indirect fluorescent antibody test on live, schizont-infected RBC (live IFAT) using a panel of hyperimmune sera raised against these populations and against one of the recrudescent clones after mosquito transmission. This test can detect antigens on the surface of parasitised RBC. The results of this analysis indicated that all the recrudescent clones were antigenically different from the parent and some were different from each other. In total, including the parent, six variant antigen types (VATs) were identified. Some of these also appeared to vary in immunogenicity. The effects of mosquito transmission on expression of variant antibodies was also examined using the panel of hyperimmune sera in the live IFAT. Mosquito transmission of two antigenically distinct recrudescent clone populations resulted in a change in antigenicity of both types to an apparently similar VAT, which had the same apparent identity as that of the original, post mosquito transmission but pre-cloning, parent population.

The increasing spread of antimicrobial resistant bacteria and the decline in the development of novel antibiotics have incited exploration of other avenues for antimicrobial therapy, such as the use of antibiotics that enhance the host’s defenses to infection. This study explores the influence of antibiotics on the innate immune responses to bacteria. The aims were to investigate antibiotic effects on bacterial viability, innate immune cells in response to bacteria and interactions between bacteria and the host. Five exemplar antibiotics at maximum serum concentration (C\(_m\)\(_a\)\(_x\)) and minimum inhibitory concentrations (MIC) were tested. \(Salmonella\) Typhimurium SL1344 was chosen as the model pathogen. Following incubation of SL1344 with C\(_m\)\(_a\)\(_x\) concentrations of ceftriaxone bacterial viability was undetectable. When SL1344 was incubated with ceftriaxone and ciprofloxacin treated neutrophils, there was reduced bacterial viability. Ciprofloxacin pre-treated neutrophils had reduced ability to phagocytose bacteria, while oxidative burst was increased following exposure to ceftriaxone. Adhesion of SL1344 to J774 macrophages pre-exposed to both concentrations of ciprofloxacin and ceftriaxone was increased, but only C\(_m\)\(_a\)\(_x\) of azithromycin and streptomycin. Expression of IL-1β and TNFα mRNA was greater in SL1344 infected macrophages pre-exposed to ciprofloxacin or ceftriaxone, than in macrophages exposed to antibiotics alone or SL1344 alone. In conclusion, it was found that clinical relevant concentrations of certain antibiotics enhance the response of immune cells and their interaction with bacteria, by increasing phagocytosis and killing in neutrophils, increasing bacterial adhesion to macrophages and increased cytokine production. These immunomodulatory potentials of antibiotics can be harnessed and exploited for broader therapeutic use.

Thiomarinol is a novel hybrid antibiotic produced by \(Pseudoalteromonas\) \(rava\) sp. nov. SANK 73390. It is structurally similar to a clinically significant antibiotic mupirocin, but includes an additional pyrrothine moiety joined to the mupirocin-like marinolic acid via an amide bond. Thiomarinol has been shown to be more potent against a wider range of microorganisms. This potency was hypothesised to be due to either an increase in inhibition of its target enzyme, isoleucyl-tRNA synthetase (IleS) or an increase in antibiotic uptake and/or inefficient efflux by bacterial cells or a combination of both. This thesis describes experiments that investigate the basis for this increase in potency.

Mammalian cell entry (MCE) domains are so called due to the reported ability of an Escherichia coli strain harbouring the mce1 gene from Mycobacterium tuberculosis to invade mammalian cells. Bioinformatic analyses presented here demonstrated that proteins containing a single MCE domain are widespread in bacteria and that proteins containing multiple MCE domains are specific to and have evolved within Proteobacteria. Gene neighbourhood analyses revealed that MCE domain containing proteins are components of transporters and that multi MCE domain containing proteins constitute a novel type of transporter. E. coli was shown to harbour three MCE proteins: the single MCE domain protein MlaD and two multi-domain proteins PqiB and YebT. All three proteins were shown to locate to the inner membrane and bind phospholipids. Phenotypic studies revealed that their functions overlap but are distinct. Infection studies with Salmonella showed that the proteins are important for systemic infection but are not required for mammalian cell entry. Phospholipid growth experiments with Salmonella demonstrated that they are important for phospholipid uptake. These findings suggest that MCE domain containing proteins in Proteobacteria are not directly involved in mammalian cell entry and instead play a role in other aspects of mammalian infection related to phospholipid trafficking.

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.

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.

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.

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.

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.