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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.

The interaction between the tyrosine kinase receptor Flt3 and its ligand leads to signalling during the commitment of haematopoietic stem cells (HSCs). Constitutive activation of the Flt3/FL pathway is a key factor in enhanced survival and expansion in acute myeloid leukaemia (AML). Although there is extensive knowledge regarding mutations leading to the constitutive activation of Flt3 receptor activity, the molecular mechanisms underlying the regulation of the \(flt3\) gene in HSCs, and how such mechanisms might be altered in leukaemia, are still poorly understood. Here, by using HSC and leukaemic cell lines, I locate several regulatory elements in the \(flt3\) locus by DNaseI mapping and have characterized their epigenetic environment. Analysis of the methylation and acetylation status of histones H3 and H4 around \(flt3 cis\)-regulatory regions highlights a distinct combination of epigenetic modifications specific to AML cells in a region that distinguishes the Flt3\(^-\) and Flt3\(^+\) stages of HSC differentiation. Moreover, I show the link between the \(in vivo\) binding of C/EBP and c-Myb on regulatory elements and epigenetic remodelling in the differential regulation of \(flt3\) in leukaemic cells. Finally, I identify the histone modifiers TIP60 and CBP as potential mediators of the epigenetic regulation of \(flt3\) in AML cells.

This study examined IgG subclass in ANCA associated vasculitis and glomerular endothelial cell (GEC) phenotype predisposes to injury. Using the flow model, interaction of neutrophils with normal immunoglobulin subclasses was compared to interaction with subclasses of ANCA IgG. Neutrophils were captured by normal IgG3>IgG1>IgG2/IgG4. Blockade of CD32 affected IgG3, CD16, IgG1/2. Neutrophils exposed to soluble ANCA IgG1/3 adhered to cytokine-activated endothelial cells, as did IgG4, not previously thought to bind constitutively expressed CD16/CD32. Fc blockade reduced binding. GEC were compared with human umbilical vein endothelial cells. Surface VCAM-1 was reduced on GEC and GEC demonstrated reduced leukocyte capture. RNA array analysis demonstrated a reduction in the GEC gene responsible for post translational modification of VCAM-1 to a sialoglycoprotein. VCAM-1 expression by GEC may be a protective mechanism to reduce inflammatory responses, potentially disrupted in disease. ANCA subclass and endothelial phenotype are important vasculitis pathogenesis: this may be useful in designing targeted therapy reducing overall immunsuppressive load. Additionally modification of specific adhesion molecule profiles on endothelial cells may enable alteration of conditions of one vascular bed whilst reducing impact on unaffected sites.

The neutrophil is central to the development of COPD. To enter lung, neutrophils must migrate accurately from the circulation to inflamed tissue. It is unclear which migratory stimuli are important and whether COPD neutrophils vary in their migratory behaviour, either to controls or patients with similar lung disease. COPD sputum and plasma samples were collected on 11 occasions over one month. Significant correlations were demonstrated between the inflammatory biomarkers and between inflammatory biomarkers and markers of disease. IL-8 correlated most strongly both with other inflammatory mediators, neutrophil counts and indices of disease. Neutrophils from healthy older subjects migrated with maintained speed but reduced accuracy to IL-8. Differences could not be accounted for by surface receptor expression or shedding, but inhibition of CXCR2 gave young neutrophils and old migratory phenotype, suggesting altered downstream signalling. COPD neutrophils migrated with increased speed and reduced accuracy compared with control groups. They formed less pseudopodia when migrating, and had reduced surface expression of CXCR1 and CXCR2. Inhibitory studies suggested that CXCR2 was the predominant receptor in migration to biological samples. Treating COPD cells with a PI3 Kinase inhibitor differentially altered their migration, reducing speed but increasing accuracy, so that cells now resembled those from controls.

Filarial nematode infections remain a significant threat of morbidity in the Tropics. Collectively known as filariasis, these helminth infections can result in gross inflammatory pathology of the infected host. However, more commonly, the host remains relatively asymptomatic, exhibiting a somewhat suppressed, non-inflammatory immune response to the parasite, permitting the longevity of infection commonly seen with many helminth infections. In addition to promoting parasite survival, such immunomodulatory actions appear conducive to host health by limiting development of pathological lesions resulting from aggressive, pro-inflammatory responses. This can prove to be advantageous for humans, as although nematode infection can result in severe pathology, the majority of infected people exhibit little evidence of an inflammatory response or overt tissue destruction/disruption commonly associated with autoimmune disorders. Consistent with this, there is increasing evidence supporting an inverse relationship between worm infection and Th1/17-based inflammatory disorders such as rheumatoid arthritis, inflammatory bowel disease, type-1 diabetes and multiple sclerosis. Moreover, in the developed world, there has been an alarming increase in these inflammatory diseases, coincident with recent improvements in hygiene; a trend not observed in parasite-endemic countries. Therefore, the ”hygiene hypothesis” proposes that these trends are directly associated, by predicting that in the absence of helminth infection, there is a lack of parasite-induced immunoregulation that can result in an over-active immune response in susceptible individuals, resulting in the development of autoimmunity. Filarial nematodes are known to secrete immunomodulatory excretory-secretory products during infection, which act to modulate inflammatory host immune responses and thus, protect the parasite from elimination. ES-62, a phosphorylcholine-containing glycoprotein secreted by the rodent filarial nematode, Acanthocheilonema viteae, has previously been shown to modulate the responses of several innate cells to promote anti-inflammatory immune responses in vitro and in vivo. Indeed, this laboratory demonstrated that ES-62 exhibited anti-inflammatory properties that extended to potential therapeutic action in autoimmune inflammatory diseases such as Rheumatoid Arthritis (RA). This was evidenced by studies demonstrating that ES-62 not only reduced severity of disease in the murine model of collagen induced arthritis (CIA), but also acted to reduce pro-inflammatory cytokine and auto-antibody production from blood and synovial cultures derived from human RA patients. Perhaps rather surprisingly therefore, ES-62 was subsequently found not to offer any protection in some other models of inflammation, including the NOD mouse model of Type 1 Diabetes, the Plasmodium chabaudi model of malaria and the Toxoplasma gondii model of toxoplasma, all of which have been linked with Th1-like pathology. However, given the recent reassessment of CIA as a Th17-, rather than Th1-, mediated disease, it has been hypothesised that ES-62 exhibits therapeutic potential in models, such as CIA, which reflect Th17- rather than Th1 mediated pathology. As the previous CIA/RA studies did not address the role of the Th17 family of pro-inflammatory cytokine mediators, which have been implicated as orchestrating much of the pathology seen in CIA, the core aim of this thesis was to define whether ES-62 was mediating its therapeutic action in autoimmune inflammatory disorders by targeting this Th17 population. Indeed, in chapter 3 of this thesis, it was found that the significant inhibition of disease scores in ES-62-treated CIA mice was accompanied by a significant reduction in levels of IL-17-producing cells, as detected by flow cytometry. Moreover, such analysis revealed that ES-62 reduced IL-17 production from both CD4+ (Th17) cells and γδ cells, which were the two major IL-17-producing populations in the LN of CIA mice. By contrast, ES-62 was not found to modulate the levels of IFNγ-producing cells, either in terms of CD4 or CD8 T cells, which constitute the major cellular compartments generating this cytokine in CIA, supporting the hypothesis that ES-62 targets Th17/IL-17-, rather than Th1-associated inflammation. In addition to suppressing CIA, pilot studies had shown that prophylactic treatment with ES-62 significantly inhibited the development of glomerulonephritis and articular inflammation in MRL/lpr mice, two pathologies commonly associated with SLE in humans. The anti-inflammatory actions of ES-62 were evidenced by a reduction of proteinuria levels and footpad swelling, respectively, but were not associated with modulation of lymphadenopathy, splenomegaly or hypergammaglobulinemia occurring in tandem with autoimmune pathology in such MRL/lpr mice. Studies in chapter 4 of this thesis characterised the cytokine profile of MRL/lpr lupus prone mice, in comparison to congenic MRL/MP control mice, throughout the course of disease induction and progression. These studies showed that IL-17 production was detected in the lymph nodes and serum of MRL/lpr mice, before the onset of lupus pathology. Furthermore, γδ T cells were the major IL-17 producing cell type examined in these mice. Prophylactic treatment with ES-62 resulted in reduced cytokine (IL-17 and IFNγ) release by lymph node cells and splenocytes in response to ex vivo stimulation with the mitogen, ConA, although this did not prove to be significant. As IL-17 was one of the cytokines targeted, this suggested that, as with the CIA model, ES-62 mediated its anti-inflammatory effects through modulation of this cytokine. Consistent with this, it was also found that exposure to ES-62 in vivo reduced the levels of IL-17-producing cells, particularly the γδ T cell population at the earliest time-point examined. By contrast, again as with the CIA model, there was no reduction in the levels of IFNγ-producing cells in ES-62 treated mice, confirming that ES-62 was not acting directly on these Th1 cells. One of the hallmarks of the MRL/lpr model of lupus is the accumulation of B220+CD3+ Double Negative (DN) T cells, which account for much of the lymphadenopathy observed. These cells, which would otherwise have been deleted during development, are known to survive and accumulate due to the lpr (lymphoproliferative) mutation of the Fas gene in these mice. Despite being the predominant LN cell population following onset of disease in MRL/lpr mice, DN T cells have not generally been considered to be pathogenic in this model, but rather proposed to exhibit an anergic-like phenotype. However, some studies have suggested that they can produce cytokines, such as IFNγ and more recently it has been shown that they are a source of IL-17 [1, 2]. Moreover, DN T cells producing IL-17 have been found in the kidneys of SLE patients, suggesting they might indeed play a pathogenic role in lupus-like inflammation [3]. The data presented here established that such DN T cells are capable of producing a range of cytokines, including IL-10, IL-17 and IFNγ and indeed, the most novel finding of these studies was that DN T cells are capable of producing IL-22, a cytokine generally associated with Th17 cells. While the function of this IL-22 production is unclear, it was seen that the percentage of DN T cells producing IL-22 were significantly inhibited by exposure to ES-62 in vivo and hence, whilst ES-62 did not appear to influence the expansion of these cells, it did modulate their effector function by suppressing their capacity to produce this cytokine, although the absolute numbers of cells was not significant reduced. In summary, the novel findings presented in this thesis support the theory that parasite-derived products such as ES-62 may protect against development of the autoimmune inflammatory diseases prevalent in developed society.

Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 is a formidable pathogen that uses a type-III secretion system to inject bacterial ‘effector’ proteins directly into host cells. Most effectors that are encoded within the locus of enterocyte effacement (LEE) have been studied extensively. This study aimed to characterise a selection of recently discovered non-LEE-encoded effectors using a variety of model systems. Firstly, a β-lactamase translocation assay was used to demonstrate translocation of novel effectors into host cells. The localisation of selected effectors was then investigated using mammalian cells and a yeast cell model. The effector EspM2 was shown to induce the formation of actin stress fibres in transfected HeLa cells and caused growth retardation when expressed in yeast. A number of NleG effectors also caused growth retardation and morphological changes when expressed in yeast. Growth retardation caused by the effector NleG8-2 was shown to be dependent on three conserved cysteine, aspartic acid and histidine residues. Transcriptomics and a high copy yeast gene suppression screen revealed that NleG8-2 may disrupt yeast physiology by affecting the secretory pathway. This study confirms that the effector repertoire of EHEC O157:H7 is much larger than previously imagined and provides insight into the function of selected novel effectors.