Rheumatoid Arthritis (RA) is a chronic systemic inflammatory disease, which targets the synovial membrane and joints of patients, resulting in irreversible bone damage, and disability. The development of new treatments for RA is limited by our incomplete understanding of disease pathogenesis. Macrophages are critically important in the pathogenesis of RA due to their ability to initiate and amplify both systemic inflammation and local tissue damage through the production of cytokines such as Tumour necrosis factor (TNF) α and Interleukin (IL)-6. Our understanding of the molecular mechanisms underlying monocyte and macrophage activation in arthritis remains unclear. MicroRNAs (miRs) are a novel class of post-transcriptional regulators. They are short, non-coding RNA molecules that bind to complementary sequences, typically in the 3’untranslated region (UTR) of target genes, resulting in mRNA degradation or sequestration. miRs have been shown to impact various aspects of mammalian biology including cell proliferation, differentiation and the immune response. We identified a miR profile of SF CD14+ cells in RA. The majority of this study concentrated on miR-125a which was shown to be upregulated in SF CD14+ cells and PB CD14+ cells of patients who responded well to cDMARD therapy when compared to healthy controls. Using miR sponge technology we generated macrophage like, miR-125a null cells, and demonstrated that these cells display a hyper inflammatory response to LPS by producing significantly higher levels of TNFα, IL-6, CCL4 and CCL5 when compared to a reporter control. This phenotype was confirmed in primary human macrophages in which miR-125a was inhibited. These cells produced significantly higher levels of TNFα, IL-6, IL-12, CCL4 and lower levels of anti-inflammatory IL-10. Prediction algorithms identified members of the Toll Like Receptor 4 (TLR4) signalling pathway and inflammatory cytokines as potential miR-125a targets. Interestingly, overexpression of miR-125a in primary human macrophages resulted in increased IL-10 production, but also increased TNFα production, highlighting the complicated nature of miR regulation. This study has identified miR-125a as a potential negative regulator of macrophage activation, which may be impaired at sites of chronic inflammation. Future work will therefore aim to characterise the pathways regulated by this miR in macrophages, and the relationship to their activation within the RA joint.

The bacteria Ureaplasma has long been associated with a wide range of adverse health implications, including preterm birth, preterm premature rupture of the membrane and lung disorders, such as bronchopulmonary dysplasia in neonatal infants, but still, little is known about the pathogenic properties of Ureaplasma and possible direct association with adverse health complications. Estimated prevalence of Ureaplasma colonisation in sexually active adults is between 40 – 80%, therefore further understanding of its pathogenic properties and its ability to initiate an immune response is crucial. Specifically selected human cell-lines were examined in vitro to determine whether an innate immune response could be activated by Ureaplasma infection. If inflammatory immune responses were detected in human cell-lines, pathogenic properties of Ureaplasma would be confirmed, and its role in pregnancy and neonatal complications could be supported. Using a range of techniques, activation of immune response pathways were examined, as too were the production of detrimental pro-inflammatory cytokines that would strengthen the suggested associations of Ureaplasma infection with the above-mentioned complications. Myeloid-derived leukocytic monocytes, human bronchial epithelial cells and human amniotic epithelial cells were examined, as these would be the most relevant cell lines to determine if Ureaplasma could induce preterm birth, preterm premature rupture of the membrane and bronchopulmonary dysplasia. All cell lines studied showed immune response and inflammatory cytokine production after stimulation with Ureaplasma. This supports that Ureaplasma is capable of causing tissue damage in neonatal respiratory tracts that may lead to bronchopulmonary dysplasia and damage to the amniotic and chorion membranes that may lead to preterm premature rupture of the membrane. Ureaplasma was detected at the cell surface of human amniotic epithelial cells (HAECs) by TLR2 and TLR2/6 heterodimers. Results suggest that Ureaplamsma multiple banded antigen (MBA) is the strong ligand for TLR2 and TLR6 and stimulation of HAECs with MBA alone caused an immune response. TLR9 was responsible for the detection of internalised Ureaplasma, which is also able to initiate an immune response and inflammatory cytokine production. v Ureaplasma stimulation results in the production of the inflammatory cytokines TNF-α, IL-8 IL-6 via the NF-κB signaling pathway. Production of the potent inflammatory cytokine IL-1β was also observed, which would suggest the formation of inflammasome complexes. NLRs were investigated to find which NLR inflammasome were activated. It was shown that genetically knocking down NLRP7 significantly reduced the amount of IL-1β that was produced after Ureaplasma stimulation, suggesting that NLRP7 inflammsones are activated by Ureaplasma. Reduction in IL-1β was also observed, but to a lesser extent, when NLRP3 was knocked down. We decided to investigate the role of NLRP7 further and found a novel immune pathway, where NH3 causes activation and formation of the NRLP7 inflammasone. NH3 is produced as a bi-product of urease activity, which an essential process for Ureaplasma. The addition of a potent urease inhibitor to HAECs being stimulated with Ureaplasma significantly reduced the production of IL-1β, strongly supporting that NH3 plays a significant role in the detection of Ureaplasma infection and is responsible for causing the tissue damage that contributes to preterm premature rupture of the membrane leading to preterm birth. This investigation strongly supports that Ureaplasma is responsible for causing preterm birth and health complications in neonates, and that more robust treatment and monitoring of Ureaplasma is required, especially in pregnant women. These undertakings will hopefully reduce the rates of preterm birth and the associated health implications, in addition to reducing rates of bronchopulmonary dysplasia in neonates.

No description available.

The aetiology of Common Variable Immunodeficiency (CVID) has fascinated immunologists since Dr. Janeway reported the first case in 1953. While the advances in molecular biology have enlightened us on the aetiology in some patients, the majority is not caused by inherited genetic disorders. A convincing mechanism accounting for the intrinsic variable and partial nature of the condition has yet been proposed. CVID separates itself from other primary antibody deficiencies by the procurement of an abnormal T-cell compartment. Data from this study support that both T-cells and B-cells are subjected to similar deficiency. Investigation of the T-cell receptor repertoire by next-generation sequencing and multi-parametric flow cytometry suggests a severe reduction in naïve T-cell output from the thymus. Similarly, the study of long-lived plasma cell generation and survival highlighted the greatest functional deficits in the naïve B-cell pool, altogether supporting an acquired arrest in lymphogenesis. Using DNA methylation as a surrogate marker for pre-VDJ clonality, this study further shows that some CVID patients exhibited clonal haematopoiesis, adjoining CVID to other clonal haematopoiesis related acquired haematological disorders. Further work is being focused on using high resolution techniques to confirm this association and mechanistically define the development of antibody deficiency in adulthood.

Macrophages are key players in both regulatory and inflammatory immune responses. They are implicated in the pathogenesis of rheumatoid arthritis (RA) where they accumulate in the synovium and produce pro-inflammatory cytokines including TNFα and IL-6. The rheumatoid synovium is metabolically distinctive, with low oxygen perfusion and high concentrations of lactate and reactive oxygen species (ROS). Macrophages are known to respond to metabolic signals, therefore we wanted to explore whether metabolic phenotypes of differentiated macrophages could play a role in the persistence of RA. We used an in vitro model of pro-inflammatory “classically activated” and “alternatively activated” macrophages to study macrophage behaviour using metabolomic and transcriptomic techniques. Differentiation with GMCSF and M-CSF produced macrophages with distinctive profiles. GM-CSF macrophages were metabolically active, metabolising glucose, glutamine and fatty acids, while M-CSF macrophages utilised fatty acid β-oxidation alone. Activation of macrophages with LPS, LPS+IFNγ or IL-4 produced metabolic changes, however, differences between MCSF groups were modest. LPS activation of GM-CSF macrophages drove both depletion of intracellular metabolites and transcriptional downregulation. In contrast, IL-4 activation of M-CSF macrophages was metabolically activating. We propose that the metabolic adaptability of GM-CSF macrophages may put them at an energetic advantage in the hypoxic, ROS-enriched rheumatoid synovium.

CD4\(^+\) T cells modulate an immune response through the production of effector cytokines. In some circumstances the effector function of CD4\(^+\) T cells is diminished, which may have beneficial (peripheral tolerance) or detrimental (exhaustion, senescence) consequences. Here I characterise a population of CD4\(^+\) cells in human peripheral blood which exhibit complete hyporesponsiveness to in vitro stimulation, as indicated by an absence of CD69 upregulation and the failure to secrete any of thirteen candidate cytokines. These T cells had an effector memory phenotype (CD45RA\(^-\)CCR7\(^-\)\(^/\)\(^+\)CD62L\(^{lo}\)CD27\(^{lo}\)), but their intermediate expression of PD-1 did not suggest a state of exhaustion. Although regulatory T cells (CD25\(^{hi}\)CD127\(^{lo}\)) contributed to the hyporesponsive population it was not predominated by this phenotype. However, the possibility that these hyporesponsive cells represent a non-classical regulatory subset could not be excluded. CD4\(^+\) T cells can enter the central nervous system (CNS) via the blood cerebrospinal fluid barrier, but their biological activity and recruitment pathways are under-defined. Preliminary studies had suggested that hyporesponsive CD4\(^+\) T cells were enriched in uninflamed human cerebrospinal fluid (CSF). However, this investigation found that CSF and brain-derived CD4\(^+\) T cells readily upregulate CD69 upon activation (mouse, human) and have robust IFNγ responses (rat). This evidence supports a role for CD4\(^+\) T cells in CNS immune surveillance and immunity. This investigation also showed that the proportion of CCR7\(^+\) and CCR7\(^-\) memory CD4\(^+\) T cells in the CSF was a direct reflection of the distribution in the peripheral blood in both mouse and man. This suggests that CSF recruitment is not CCR7-dependent as is previously described, and shows effector memory cells enter the CSF space in the absence of neuropathology. Such findings have implications for the understanding of normal immune function in the CNS, and the protective or pathogenic contribution of CD4\(^+\) T cells to neuroinflammatory disorders such as multiple sclerosis.

Coinfection with bacteria and viruses is an understudied area of microbiology, despite its potential to modulate pathogen abundance and host survival. We investigated the effect of bacteria on virus infection and developed an in vitro system to study the first step: viral internalization. Our studies show that multiple bacterial species promote the entry of a diverse panel of viruses into lung and gut epithelial cells. Bacteria expressing the toll-like receptor (TLR)5 agonist, flagellin, are most efficient at inducing viral uptake and studies using recombinant flagellin or aflagellate bacterial strains confirm that flagellin has pro-viral activity. Flagellin promotes epithelial cells to support virus entry via TLR5-dependent activation of NF-KB. To extend these observations and study the role of flagellin in the complete viral replicative lifecycle, we studied human immunodeficiency virus (HIV)-1 replication in T cells. Flagellin augments HIV-1 entry and promoter activity and increases the production of extracellular virus. The data presented in this thesis highlight a new role for bacterial flagellin to promote diverse virus infection of epithelial barriers and enhance the spread of HIV-1. This has significant implications for understanding how exposure to multiple pathogens can alter susceptibility to infection and its associated pathogenesis.

Ageing is accompanied by impairments in immune responses. In this thesis, an age associated numerical and functional deficit in a novel subset of immunosuppressive CD19\(^+\)\(^v\)\(^e\) CD24\(^h\)\(^i\) CD38\(^h\)\(^i\) B cells is reported which might be an additional factor contributing towards increased risk of systemic autoimmunity with advancing age. The immune system is profoundly affected by environmental factors such as stress. This thesis also examined the synergistic effect of the physical stress of a hip fracture and the psychological stress of depressive symptoms on immune function of older adults. It was observed that 37% of our hip fracture patients had developed depressive symptoms 6 weeks post-surgery. A significant decline in neutrophil, monocyte and NK cell functioning was reported in hip fracture patients with depressive symptoms, but not in those with hip fracture alone. Additionally, an increased cortisol: DHEAS ratio was also only found in hip fracture patients with depressive symptoms. An elevated level of pro-inflammatory (IL6, TNFα) and anti-inflammatory (IL10) cytokines were observed in hip fracture patients with depressive symptoms. In the adaptive immune system, a reduction in circulating T cells and an accumulation of senescent and activated T cells was also found in the hip fracture patients who developed depressive symptoms. Finally, the age associated numerical and functional deficit in IL10 production by CD19\(^+\)\(^v\)\(^e\) CD24\(^h\)\(^i\) CD38\(^h\)\(^i\) B cells was further suppressed on exposure to chronic stress in hip fracture patients with depressive symptoms.

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.

The thymus is organised into functionally distinct microenvironments that facilitates development of a diverse and self-tolerant T cell repertoire. Following positive selection, thymocytes undergo chemotactic migration from the cortex to the medulla, a site that mediates negative selection of potentially autoreactive CD4+ and CD8+ single positive (SP) thymocytes. Importantly, current models suggest that the medulla also fosters the continued maturation of SP thymocytes, post-selection. However, the mechanisms of thymic medulla function remain unclear. Using a novel approach based on chemokine receptor expression, we have mapped stages in the positive selection process, and developed a model to study αβT cell development in the absence of medullary thymic epithelial cells (mTEC), but in the presence of an otherwise intact immune system. We show that mTEC are dispensible for the continued development of newly selected CD4+CD69+ SP thymocytes, yet are essential for the generation of FoxP3+ regulatory T cells and their FoxP3-CD25+ progenitors. In addition, although CCR4 represents a marker of early stage CD4+ SP positive selection, it is dispensable for SP medullary accumulation and intrathymic development. Collectively these findings highlight differences in the developmental requirements of conventional and regulatory CD4+ T cells, and rule out a role for CCR4 in cortex to medulla migration.