Bacterial infection is a feature of long-term peritoneal dialysis (PD) and a cause of loss of peritoneal function and treatment failure. Understanding how local inflammation is initiated and peritoneal host defence mechanisms are activated in PD patients is key to reducing the detrimental consequences of peritonitis. The capacity of human peritoneal mesothelial cells (HPMC) to ingest bacteria has been described, and the ability of the Toll-like family of innate immune receptors (TLR) to trigger inflammatory responses to pathogens has been demonstrated. However, the pathogen recognition ability, the potential role of TLRs, the specific role in the early inflammatory response, and the regulation of HPMC’ putative ability for pathogen recognition have not been fully investigated. To address these issues, the present study aimed at characterising TLR expression and responses in HPMC and evaluating the capacity of modulators of pro-inflammatory responses, namely soluble TLR2 (sTLR2) and the IL-6/sIL-6R complex, to regulate TLR-mediated HPMC and peritoneal responses in vitro and in vivo. HPMC were found to respond to an array of bacterial pathogens via expression and function of a specific set of TLRs. HPMC responses were susceptible to modulation by sTLR2 and sIL-6R, resulting in inhibition of TLR2-driven HPMC responses. In vivo, sTLR2 and IL-6/sIL- 6R reduced neutrophil influx partly by inhibiting NF-κB activation in stromal cells of the peritoneum. IL-6 signalling counteracted TLR2-mediated responses by reducing peritoneal leukocyte recruitment and chemokine production. Notably, following the establishment of a mouse model of peritoneal bacterial infection, IL-6 signalling was confirmed to be beneficial to bacterial clearance. The results of this thesis confirm and extend the knowledge of the pivotal role that HPMC play in peritoneal responses to infection. The capacity of sTLR2 and IL-6/sIL-6R to modulate peritoneal responses demonstrated in this study may inform the design of new therapeutic strategies to reduce PD-associated peritonitis and thus improve treatment outcomes.

Investigations into Human Cytomegalovirus (HCMV) pathogenesis should be based on strains that closely reflect the causative agent of disease, however HCMV invariably mutates in vitro, generating phenotypically distinct laboratory-adapted strains. In particular, mutations are selected in the HCMV genome UL128 locus (UL128L) that encodes sub-units of a virion envelope pentameric complex that impedes virus propagation in fibroblasts (the cell type most commonly used in vitro), but is required for infection of several naturally targeted cell-types (e.g. epithelial, endothelial, and myeloid cells). Addressing this issue, the genome of wildtype HCMV strain Merlin was cloned as a stable bacterial artificial chromosome (BAC), however similarly to clinical isolates, viruses reconstituted from the Merlin-BAC grow poorly and are prone to de novo mutation in cell culture. Direct comparison to viruses from the Merlin-BAC revealed that viruses from the BACcloned versions of strains TR (TR-BAC), TB40E (TB40-BAC4) and VR1814 (FIX-BAC) could be propagated more efficiently in fibroblasts, despite containing intact UL128L ORFs. Unique nucleotide variations identified in TB40-BAC4 and FIX-BAC UL128L ORFs were transferred into the Merlin-BAC, generating variants that produced greater titres of cell-free virus following reconstitution. Virions from these novel Merlin variants displayed reduced pentameric complex content, but remained able to infect epithelial cells, albeit with slightly compromised efficiency. The greater fitness of viruses from these novel Merlin-BAC variants alleviated the selective pressures for the selection of de novo UL128L mutations in fibroblasts. The Merlin virion proteome was determined, with up to 30 novel components identified to provide a more comprehensive picture of wildtype HCMV virion composition. Comparison of virions from several Merlin variants demonstrated that varying pentameric complex content impacted the incorporation of other components, however virions from the novel Merlin variants produced in this work closely matched those from the parental Merlin variant containing wildtype UL128L ORFs. Thus, the novel Merlin-BAC variants produced in this work represent valuable reagents for future HCMV research.

Effective immunity is fundamental to life on a dirty planet. Appropriate immune responses control infections and protect against cancer. Inappropriate immune responses lead to autoimmunity and allergy. A fine balance between aggression and tolerance is therefore central to effective immune function at the system level. This is a particular problem for T cells, which recognize peptide antigens bound to host major histocompatibility complex (MHC) molecules. Faced with a composite antigenic structure, the distinction between “foreign and dangerous” and “self and harmless” becomes both difficult and imperative, especially when the antigen persists. In this thesis, antigen-specific T cell responses were investigated under conditions of chronic antigenic stimulation to inform our understanding of this process. In T cell receptor transgenic mice, continuous antigenic stimulation without adjuvant lead to increased in vivo turnover of antigen-specific CD4+ T cells but “aborted” immune activation, characterized by depletion of these cells from the circulation and spleen. Full immune activation and expansion of antigen-specific memory/effector CD4+ T cells required the presence of adjuvant, in this case IL-1β, which induces an inflammatory environment. Further isotope labelling studies in human immunodeficiency virus-infected subjects suggested that the surface marker CD57 demarcates a “steady state” within the CD8+ T cell memory compartment, whereby CD57+ cells have lower in vivo turnover rates compared to their CD57- counterparts. These observations provide a potential mechanistic explanation for the preferential accumulation of CD57+CD8+ cells under conditions of chronic antigenic stimulation. Another persistent pathogen, cytomegalovirus (CMV), expresses a viral interleukin (IL)-10 homologue. Memory T cell inflation and antiviral cytokine production in murine CMV(MCMV)-infected mice were suppressed by IL-10. Conversely, IL-10 blockade or deficiency lead to the inflation of certain antigenspecific T cell populations and reduced viral load, most likely as a consequence of the enhanced immune response. Reactivation of human CMV was also apparent in subjects with dasatinib-associated large granular lymphocyte expansions. Consistent with a causative association, the expanded T cell and NK cell populations in these subjects were oligoclonal and exhibited a late differentiated (CD27-CD57+) phenotype, indicative of chronic antigenic stimulation. In addition, CD8high and CD8low T cells were observed within both the total and CMV-specific CD8+ T cell compartments, consistent with CMVdriven activation. In summary, these data show that antigen alone is not sufficient to induce full immune activation, even under conditions of chronic stimulation. Additional signals, such as those provided by an inflammatory environment, are required to trigger full T cell activation and expansion. Persistent viruses attempt to undermine this process, for example by the expression of homologues that mimic host immune regulators. Even in the presence of viral reactivation and immune system perturbations, however, the T cell compartment can demonstrate remarkable resilience in its ability to generate fully differentiated and functional expansions. The persistence of certain memory T cell subsets under such conditions appears to play an important role in the immune response to chronic “dangerous” antigens.

The interaction between host genetics and epidemiological processes in ovine footrot was investigated using a combination of data analysis and simulation modelling. The study’s aims were to determine the potential for genetic selection to be used to reduce the prevalence of footrot in the UK and to assess different strategies for use of conventional epidemiological interventions. A stochastic simulation model was developed, incorporating host genetics for traits controlling footrot resistance, bacterial population dynamics, sheep population dynamics and epidemiological processes. Sensitivity analysis of the model showed survival time of Dichelobacter nodosus in the environment and infection rate were the key determinants of disease outcomes. Antibiotics were predicted to be the most effective conventional control method, reducing prevalence of footrot to 1-2% when administered promptly. Pasture rotation, selective culling and vaccination were all predicted to reduce prevalence but to a lower extent. Analysis of field data confirmed the likely role for some degree of host genetic control of footrot resistance, i.e. resistance appears to be lowly to moderately heritable. Using the simulation model it was then shown that genetic selection could be effective at reducing footrot prevalence. In combination with antibiotic treatment or pasture rotation elimination of footrot from an individual flock could be achieved. Genetic selection was predicted to be effective at reducing prevalence and improving resistance but the choice of selection criteria impacts the results seen. It is likely that progress would be slower in field situations because footrot traits would be diluted by simultaneous selection for other traits affecting profitability. Field studies are required to determine optimal combinations of interventions and genetic selection and to validate modelling outcomes. Combined data from longitudinal disease observations, genetic information and bacterial samples are necessary to address current knowledge gaps and to further advance understanding of host and disease processes in ovine footrot.

Infection of bovine leukocytes by the intracellular protozoan parasites Theileria annulata and Theileria parva represents a unique model of reversible host cell transformation; characterised by uncontrolled proliferation and protection against apoptosis. Host cell transformation is known to involve parasite dependent manipulation of a number of signalling pathways that result in constitutive activation of pro-inflammatory transcription factors. Activation of these factors has been shown to confer protection against apoptosis or influence the metastatic potential of the infected cell, respectively. However, transcription factors have the potential to be detrimental as well as beneficial to the infected cell and the infected animal. This study investigated the potential of the parasite to manipulate the outcome of cellular activation by comparing the outcome of stimulating Theileria-infected and uninfected bovine cells with LPS. The results clearly indicate that prolonged stimulation of uninfected cells generated a phenotype associated with loss of proliferation, cell death and activation of NF-B. In contrast, infected TBL20 cells were refractory to stimulation, despite displaying high levels of constitutively activated NF-B.

Cardiovascular disease (CVD) is the leading cause of death in the UK, accounting for more than a third of all deaths, with atherosclerotic coronary artery disease (CAD) being the commonest type of CVD. Recently, it has become recognised that in addition to traditional cardiovascular (CV) risk factors such as dyslipidaemia, hypertension, smoking, and diabetes, inflammation plays an important role in the development and progression of atherosclerosis. A concept has emerged that atherosclerosis to some extent can be viewed as a chronic inflammatory autoimmune disease in which the adaptive immune system is targeted against vascular self-antigens modified by hypercholesterolaemia, involving both the innate and adaptive immune response. Much work has been done in determining how the immune system is involved, however relatively little is known about natural killer (NK) cells – an important component of the innate immune system which acts against virally infected cells and neoplastic transformation. In addition NK cells possess cytolytic ability and provide an early source of immunoregulatory cytokines. Recently, there has been increasing evidence to support a role for NK cells in the development of atherosclerosis. The work in this thesis examines NK cell function with the aim of determining whether any changes in the function of this immune cell could have a role in the development of CVD. In order to do this, we chose two patient populations at high CV risk and compared NK cell subsets and function to healthy controls. Firstly, 66 patients with dyslipidaemia on a variety of lipid lowering treatments attending a lipid clinic, and secondly 143 patients with chronic kidney disease (CKD) including 11 with end-stage renal disease (ESRD) on hospital haemodialysis (HD). It is known that CVD is the leading cause of death in patients with CKD, and in ESRD patients have a 20-100 fold risk of premature CV death compared to age matched controls from the general population. The increased CV risk results from additional risk factors that are unique to this patient population, but in particular, these patients have an immune dysfunction that is not completely understood and a resultant inflammatory state. We determined T-cell, NKT-cell, and NK cell subsets from peripheral blood mononuclear cells (PBMCs) by flow cytometry. We then isolated NK cells from PBMCs and assessed NK cell function using a 51-Chromium release assay. These results were then correlated with clinical and laboratory results. In the patients with dyslipidaemia, we did not find any correlations between lipid levels and NK cell numbers, subsets, or cytoxicity. The presence of statin therapy or any other lipid lowering treatment did not result in a reduction in NK cell cytotoxicity. In the CKD patient group, we found a correlation between NK cell cytotoxicity and creatinine, although this did not retain significance after multivariate analysis. Interestingly, we also found a correlation between NK cell cytotoxicity and serum phosphate level, which did remain significant after multivariate regression. We are the first to report a relationship between phosphate and NK cytotoxicity. This is an interesting finding as there is increasing evidence supporting a role for hyperphosphataemia in CVD and increased mortality in both the general population and particularly in patients with ESRD. Phosphate has been shown in some studies to be an independent predictor of inflammation, and may provide the link between the high risk of CVD and CKD. The next part of this thesis was an in vitro study of membrane cholesterol in NK cells. The cell membrane supports cholesterol-rich microdomains termed “lipid rafts”, which concentrate receptors and signal transduction molecules to facilitate high efficiency signal transduction. Statins have a number of pleiotropic effects which have been explained by reduced production of isoprenoid intermediates, and depletion of cell membrane rafts. This study aimed to investigate the effects of membrane cholesterol manipulation on NK cell function, and specifically, whether the actions of statins on NK cells are due to depletion of membrane cholesterol or inhibition of isoprenylation. The NK92MI cell line was used. Cells were either cholesterol loaded, or cholesterol depleted using statins, and NK cell function assessed using a 51-Chromium release assay. Cholesterol was successfully incorporated into the membrane and rafts and was concentration dependent. The addition of cholesterol to statin treated cells restored the cholesterol content in the cell membrane and in rafts. NK cell cytotoxicity decreased with statin treatment in correspondence with raft levels, however in contrast with the increased raft levels of cells which were cholesterol loaded, NK cytotoxicity was also decreased. Measurement of active Ras (a small G-protein that is localised by isoprenylation in membrane rafts when activated), showed that statin treatment reduced Ras within the raft which was not rescued by the addition of cholesterol, suggesting that statins deplete membrane cholesterol and rafts as well as inhibiting isoprenylation. Replenishment of membrane cholesterol restores non-isoprenylated, raft-associated proteins, but does not correct the functional effects of statins. The final part of this thesis aimed to evaluate the relationship between NK cells and potential CV risk biomarkers in these two patient populations at high risk of CVD. High sensitivity C-reactive protein (hsCRP), interleukin-6 (IL-6), pentraxin-3 (PTX-3), adiponectin, and soluble ST2 (sST2) levels were determined by ELISA, and correlated with NK cell numbers, phenotype, and function, as well as other routine biochemical and haematological parameters. We were not able to determine any definite relationships between the biomarkers studied and NK cell function, although there was an association between PTX-3 and NK cytotoxicity that was only found in inflamed (hsCRP>2mg/L) patients. In conclusion, these studies have provided further insight into the role of NK cells in a group of patients that have not previously been studied: patients with a range of CKD, in addition to patients with dyslipidaemia. This study is the first to associate NK cell cytotoxicity with serum phosphate levels which may have clinical implications. Further studies are needed to clarify whether other immune abnormalities occur in the context of hyperphosphataemia, and the causes and consequences of this. We have also demonstrated that statins deplete membrane lipid rafts as well as inhibit isoprenylation, suggesting a novel dual mechanism of action which merits further investigation.

Introduction: While many studies on rheumatoid arthritis have focused on the active phase of the disease, the events that lead to the development of autoimmunity remain poorly defined. We have developed a model of breach of self tolerance, where a Th1 response to irrelevant antigen (OVA) results in arthropathy associated with spontaneous induction of autoreactive T and B cell responses, which allows the investigation of the immnologival events that lead to the development of autoreactivity. Employing this model the role of Th17 cells, a a subset of IL-17 producing CD4+ T important in autoimmunity, was investigated in the development of autoimmunity. In addition, the relative ability of Th1 and Th17 polarised populations in supporting B cell responses was analysed. Finally, in this thesis the role of sterile damage regulation in the development of autoimmunity was assesed, by investigating the role of Siglec-G, a molecule involved in DAMP-signalling regulation, in this process. Results: Transfer of OVA specific Th17 cells induced similar levels of inflammation as Th1 cells, and could induce a breach of self tolerance as demonstrated by CII specific T and B cell responses. While the CII specific T cells in the Th1 recipients produced IFNγ and not IL-17, surprisingly the CII T cell responses in the Th17 recipients were predominantly IFNγ producers. Whereas the transferred OVA specific Th1 population retained its phenotype, the transferred Th17 population displayed significantly reduced IL-17 production. However, cells polarised under Th17 conditions expanded in a higher degree and persisted for longer time in response to immunisation. This resulted in a higher ability of Th17 polarised population in supporting B cell responses. Finally in this thesis, preliminaty data for a role of Siglec-G in the development of autoimmunity were presented, as Siglec-G deficient mice were protected from the development of autoreactive B cell responses. Conclusion: The results of this thesis suggest that the developing autoimmuniy in both Th1 and Th17 models is mediated by Th1 cells. These studies highlight the plasticity of transferred cell populations in vivo, and support the use of blocking and fate-mapping studies to definitively address how auto-reactive responses develop.

Avian influenza viruses are considered to be key contributors to the emergence of human influenza pandemics. While aquatic birds and ducks are the major reservoir for influenza viruses, they are typically resistant to the effects of viral infection, in contrast to the frequently severe disease observed in chickens. In order to understand whether differences in receptors might contribute to this observation, anatomical distribution of influenza virus receptors (sialic acid SAα2,3-Gal and SA α2,6-Gal) in key organs of both species was studied using lectin histochemistry with linkage specific lectins, and receptor binding assays with swine H1N1 (classical A/sw/Iowa/15/30) and avian H2N3 (A/mallard duck/England/7277/06) influenza viruses. Widespread presence of both SAα 2,6-Gal and SAα2,3-Gal receptors were found in all major organs examined in both chickens and ducks. Interestingly, the predominant receptor type in chicken tracheal epithelium (TE) was SAα2,6-Gal whereas SAα2,3-Gal receptors were most abundant in duck TE. Paradoxically, infection of primary cell cultures (duck and chicken lung cells and embryo fibroblasts) with the swine H1N1, the low pathogenicity avian H2N3, and a highly pathogenic H5N1 (A/turkey/England/50-92/91) virus resulted in more extensive and rapid cell death in duck cells than in chicken cells. Infected duck cells displayed morphological features of apoptosis, increased DNA fragmentation and activation of caspase-3/7. Infected duck cells produced comparable levels of viral RNA but less infectious virus than infected chicken cells. Notably, such rapid cell death was not observed in duck cells infected with a contemporary Eurasian lineage H5N1 virus (A/turkey/Turkey/1/05) which has been shown to be fatal to ducks. Gene expression profiling of infected chicken and duck cells, 24hrs post-infection, with a chicken Affymetrix microarray platform revealed differential transcription of many genes between the two avian species. In particular, the array results suggested a possible role of BCoR, HSPA-9, STAT-3, AVEN, BCLAF1, IL-18, IFN-α, and TNF-α genes in mediating the contrasting species phenotypic response to influenza infections. In summary, rapid cell death in duck cells, mediated at least in part by apoptosis, results in reduced infective virus production and may well be an important protective host response of resistant ducks. By contrast, longer surviving infected chicken cells produce much higher infective virus load along with high levels of pro-inflammatory cytokines which could account for the susceptibility of chickens to influenza infections.

Trypanosoma brucei is the causative agent of both sleeping sickness in humans and the related veterinary disease, Nagana. Both diseases have a wide distribution across sub-Saharan Africa and affect some of the poorest areas of the world. T. brucei can be segregated into three morphologically identical sub-species based on host, geography and pathology. T. b. brucei is limited to domestic and wild animals throughout sub-Saharan Africa and is non-infective to humans due to trypanosome lytic factors found in human serum. T. b. gambiense and T. b. rhodesiense are human infective sub-species, named due to their relative geographic locations. T. b. gambiense is the dominant form of the disease, causing over 90% of reported cases. Study of T. b. gambiense is complicated in that there are two distinct groups. Group 1 is invariably resistant to lysis and by far the more prevalent group. Group 2 T. b. gambiense exhibit a variable resistance phenotype and are only found at a small number of Côte d’Ivoire disease foci. There are two trypanosome lytic factors in human serum (TLF-1 & 2), both containing the proteins Apolipoprotein L1 (ApoL1) and Haptogoblin-related protein (Hpr). It has been conclusively demonstrated that the lytic component of TLF is ApoL1, although Hpr is required for maximal lysis by facilitating uptake of TLF particles via the HpHbR cell surface receptor. This thesis has exposed several features of the human infectivity phenotype in both groups of T. b. gambiense, an area of research for which data has been lacking due to the difficulty of working with the organism. Fluorescence microscopy indicated that group 1 T. b. gambiense exhibit avoidance of TLF-1 particles by down-regulating HpHbR receptor expression and function. However, they are also able to resist the effects of recombinant ApoL1, suggesting an additional neutralisation or compensatory mechanism. Due to group 1 T. b. gambiense avoidance of TLF-1, TLF-2 is the more important lytic particle for this sub-species group and future research must take this into consideration. Unlike group 1, group 2 T. b. gambiense displays a variable human serum resistance phenotype that involves a neutralisation or compensatory mechanism for ApoL1, with no significant avoidance of lytic particles. Despite the high variability of the phenotype of group 2 T. b. gambiense, Quantitative Trait Analysis (QTL) using twenty-five F1 progeny from a T. b. brucei / group 2 T. b. gambiense cross indicated a strong heritable component to human serum resistance largely determined by a 30 gene locus on chromosome 8. Finally, a six multi-locus genotype population analysis of a Côte d’Ivoire T. b. gambiense focus was conducted, revealing little relationship between the two groups of T. b. gambiense in the field. The differences in the human serum resistance phenotypes and population genetics of both groups of T. b. gambiense revealed both prior and during this study make it appear likely that the two groups have evolved distinct human serum resistance strategies.

Throughout our life cycle, the human body is exposed to harmful microorganisms. The innate immune system is a fundamental factor in the human body, which helps eliminate foreign organisms through specific signalling pathways with the involvement of immune receptors and signalling molecules. Viruses have evolved to infect the host and bypass the host immune responses,however a plethora of Pattern recognition receptors exist in the cell that are capable of detecting viral pathogens and mounting an innate immune response. Herpes simplex Virus Type 2 and Cytomegalovirus are common human pathogens that cause genital ulcerations, organ failure and mental health problems like encephalitis. In this study, we have aimed to identify the host’s innate immune response to HSV2 and HCMV infection in primary vaginal cells as well as Hela cells. Our data have shown that these viruses are recognized by TLR2 on the cell surface followed by intracellular PRRs such as TLR9, DAI, and IFI16, which trigger cytokine activation and release. Confocal imaging has revealed that these PRRs are located in different cell compartments and during viral cell entry and replication they can identify viral presence at specific parts of the cell. Therefore it seems that different PRRs are strategically placed in different cell locations to detect virus invasion and replication in order to activate cytokine secretion and protect the host. When different agonists for PRRs were used it was revealed that they were effective against Herpes virus infection thus indicating that a combination of PRRs agonists especially ones that trigger different cytokines could provide a wider spectrum prophylaxis to the host and they can be used to generate efficient treatment against HSV2 and HCMV infection.