Nontyphoidal Salmonella (NTS) are a major cause of invasive disease in young children and HIV-infected adults in Sub-Saharan Africa. To develop a vaccine for NTS, an improved understanding of immunity to NTS is required. This thesis investigates the role of opsonic antibody in protection against NTS. First, we defined the optimal serum handling conditions to preserve complement function. We determined minimal titres of antibody and complement required for cell-mediated killing of Salmonella and found they are higher than for cell-free killing. We demonstrated impaired blood cell killing of NTS opsonised with sera from HIV-infected Africans. Developing a method to purify anti-LPS antibodies, we showed that high titres of anti-LPS antibodies in these sera inhibit cell-mediated killing while lower titres are opsonic and induce cell-mediated killing of NTS. For most children, antibody acquired during NTS bacteraemia effected cell-mediated killing of NTS. High antibody titres were not necessarily protective, but for some sera, dilution prior to opsonisation, induced killing. The sensitivity of Malawian NTS isolates to opsonic antibody varied, with resistance to cell-mediated and bactericidal killing correlating. Overall, this thesis emphasises the importance of opsonic antibody in protecting against NTS and supports the development of a vaccine which induces antibody to Salmonella.

Nonalcoholic fatty liver disease (NAFLD), and in particular its inflammatory component steatohepatitis (NASH), are associated with significant risk of liver/cardiovascular morbidity and death. My findings highlight that NAFLD is now the commonest cause of liver disease in primary care, yet significant numbers with advanced fibrosis remain undetected. Application of simple non-invasive scoring systems could aid with identifying those in greatest need of intervention. By adopting an integrative physiological approach with functional measures of lipid and carbohydrate flux, I demonstrated that patients with NASH (vs. healthy controls) have marked adipose tissue dysfunction (especially in abdominal subcutaneous adipose tissue), alongside increased hepatic and muscle insulin resistance (IR). Targeting adipose-derived lipotoxicity should be the mainstay of therapy in NASH. Glucagon-like peptide-1 (GLP-1) based therapy (liraglutide) appears to be safe and well tolerated in patients at risk of underlying NAFLD. My prospective randomised-controlled study highlighted that liraglutide reduces metabolic dysfunction, hepatic lipogenesis, hepatic/adipose IR and inflammation in patients with NASH. My in vitro studies in human hepatocytes indicate that the anti-steatotic effects are not solely reliant on improvements in weight and/or glycaemic control. Taken together, my findings highlight that GLP-1 based therapies have all the metabolic and clinical attributes to make them a promising therapeutic option in patients with NASH. However, the safety and histological efficacy of such awaits the completion of my 48-week Phase II ‘LEAN’ trial, which is integral as to whether larger clinical trials are warranted.

Short sleep duration and poor sleep continuity have been related to adverse health outcomes. Sleep disturbances are more frequent among older people, who also experience a reduction in immune function (immunosenescence). This thesis tested the hypothesis that sleep disruption in old age contributes to immunosenescence. 93 healthy older subjects had their sleep recorded by actigraphy and immunological parameters were assessed. The data indicated that sleep continuity and duration in older adults does not influence innate immune function but was associated with changes in blood cell numbers, in particular an increase in the granulocyte:lymphocyte ratio. Differences in serum IL-4 and adiponectin were associated with long sleep duration and poor sleep continuity was also associated with raised serum cortisol. However, preliminary data obtained from a small pilot study of partial sleep deprivation in young and old adults did not show similar changes therefore causality was not confirmed. In vitro experiments were performed to evaluate whether adiponectin, whose levels change with age, affected neutrophil apoptosis and phagocytosis. Adiponectin extended lifespan of neutrophils and inhibited bacterial phagocytosis. The findings suggest that sleep dysregulation does not contribute to immunosenescence and in vitro studies add weight to the literature showing immunomodulatory roles for adiponectin.

Nasal carriage of \(Staphylococcus\) \(aureus\) is a major risk factor for surgical infection. An observational longitudinal clinical study was carried out to determine the impact of antibiotic surgical prophylaxis (ASP) on the nasal microbial community, and carriage of staphylococci. Daily nasal samples were taken from 79 study patients, 63 patients received ASP regimens and 16 patients received no antibiotics. Samples were analysed using a culture dependent technique, and a novel culture independent technique using the \(tuf\) gene developed in this thesis. The composition of each individual patient’s own nasal microbial community influenced the observed effect of ASP administration. The overall effect of ASP was a reduction in total aerobic bacterial load and altered the nasal bacterial composition. Both culture and \(tuf\) gene analysis of staphylococcal carriage was comparable, showing an increase in CNS and reduction in \(S\). \(aureus\) after administration of ASP. Analysis of \(tuf\) gene revealed greater staphylococcal species diversity in the nose than by culture, and demonstrated that \(S\). \(aureus\) carriage was not eradicated by ASP. Administration of ASP increased nasal carriage of antibiotic resistant staphylococci. This thesis has demonstrated that ASP impacts on nasal carriage of staphylococci in surgical patients.

IL-21 is a potent immune modulator crucial for the generation of protective anti-viral and humoral responses. Nonetheless, the detrimental effects of IL-21 are well documented in a variety of autoimmune diseases including type-1 diabetes. Although elevated IL-21 mRNA expression has been reported in mouse models of diabetes, it remained unclear which cells are responsible for its production and whether this cytokine was expressed by cells that infiltrate the pancreas. We addressed these questions by evaluating IL-21 production in the DO11xRIP-mOVA mouse model of type-1 diabetes. Our findings demonstrated that conventional CD4 T cells are the main source of IL-21 protein and T cell expression of this cytokine was markedly enriched within the pancreas, suggesting a potential role at the site of the autoimmune attack. Since both dendritic cells and B cells are abundant within the pancreatic lesion, we explored the capacity of these cells to contribute to T cell IL-21 production. Our investigation revealed that bone marrow-derived dendritic cells are constitutively able to support T cell IL-21 production, whereas B cell stimulated cultures required additional stimuli such as the provision of exogenous IL-6. Interestingly, our study identified a novel CD25+ innate lymphoid cell population in the pancreas, which appears to be a counterpart of the innate lymphoid cell populations recently described in the gut and lungs. Pancreas-derived CD4-CD25+ innate lymphoid cells could promote T cell IL-21 production in vitro, raising the possibility that this population contributes to T cell IL-21 production within the autoimmune lesion. Collectively, these data suggest that IL-21 production is a characteristic feature of pancreas-specific CD4 T cell responses. A better understanding of how this cytokine is elicited and how it contributes to autoimmune pathology is likely to be valuable for future therapeutic interventions.

The pattern recognition receptor Dscam is a key molecule mediating innate immunity and wiring of the nervous system in Drosophila. Intriguingly, massive molecular diversity is generated by alternative splicing in three exon clusters of Dscam. Upon pathogen exposure in Anopheles gambiae, the AgDscam splicing pattern changes to express isoforms that bind pathogens with higher affinity. In order to test the generality of Dscam splicing regulation in Drosophila, similar experiments involving microbial exposure were carried out, which also showed changes in Dscam splicing pattern. Mutants in RNA regulatory pathways and in the RNA binding protein ELAV were analyzed due to their similar mutant phenotypes in nervous system development as Dscam. In each of these mutants, alterations of Dscam alternative splicing in a cluster specific manner were observed, eluding a unique mechanism for any of the analyzed pathways. In ELAV mutants, one of the three clusters of alternatively spliced exons is dramatically mis-regulated. Since no ELAV binding site is present in this cluster, genes downstream of ELAV could mediate mis-regulation of alternative splicing. From the analysis of mutants in ELAV differentially regulated genes it was concluded that Dscam alternative splicing is most prominently affected by chromatin remodeling factors, along with RNA binding proteins, DNA binding proteins and small-RNA processing factors. A heterologous transgene for expression of Dscam pre-mRNA in Drosophila was also developed to characterize the role of the chromatin state in alternative splicing.

In alpha-1-antitrypsin deficiency (A1ATD) there is excess neutrophil elastase activity, resulting in proteolytic destruction of the lung parenchyma. I hypothesised that the peptide fragments of proteins present in the lung might be detectable in plasma by mass spectrometry and that they might be useful biomarkers of disease activity and treatment efficacy. Calcium ionophore, neutrophil elastase and proteinase 3 were added to plasma from patients with A1ATD to create an in vitro model of the destructive processes. MALDI-based peptide profiling of plasma from patients pre and post treatment with intravenous A1AT was undertaken and MS/MS performed to identify differences. Plasma was also depleted of abundant plasma proteins, labelled with isobaric tags and analysed by shotgun proteomics. The readily detectible components of the plasma proteome remained unchanged with intravenous A1AT. Addition of ionophore, elastase and proteinase 3 to patient blood generated predominantly fragments of fibrinogen. In patients treated with intravenous A1AT, fragments of A1AT increased significantly with treatment: - 2 of these were fragments of a short C-terminal segment of the A1AT protein and were also present in healthy subjects. The shotgun experiments did not identify any robust biomarkers and illustrate the challenging nature of plasma proteomics.

Common variable immunodeficiency (CVID) is a primary antibody deficiency of unknown aetiology, the diagnosis of which is made by the exclusion of known causes of hypogammaglobulinaemia. In addition to recurrent and severe infections patients demonstrate a variable phenotype which may include other features such as granuloma and autoimmunity. Given the heterogeneous nature of this condition it appears likely that the label CVID encompasses a number of different conditions. The ability to classify CVID subgroups would be advantageous both clinically and from the research point of view. Accurate subgroup classification would allow the targeting of monitoring and treatment to those patients most at risk of complications. Furthermore, current research into the pathogenesis of CVID is hindered by the grouping of clinically and biologically distinct conditions together. To date, attempts at classification, such as by flow cytometry, have failed to accurately demarcate subgroups. A total of 53 CVID patients were recruited to the study, and peripheral blood RNA was extracted, stored and analysed by gene expression microarray technology. The clinical and immunological data pertaining to these patients was gathered, analysed and used to allow bioinformatic analysis of the microarray data. The clinical data demonstrated a statistically significant tendency for some of the non-infective complications of CVID to cluster together, possibly suggesting a separate clinical subgroup. Flow cytometric analysis showed that in addition to previously described B and CD4+ T cell phenotyping, CD8+ phenotyping may be potentially useful and there was a correlation between decreased proportions of naïve CD8+ T cells and the presence of granulomatous disease. The analysis of the microarray data demonstrated a number of processes where there was differential gene expression between the clinical phenotypes, for example genes involved in the response to IL-1 in patients with granulomatous disease. Differential expression of genes involved in apoptosis was of particular interest and a consistent finding in the granuloma, autoimmunity and any complication subgroups.

Toll-like receptors (TLRs) and the complement system play a crucial role in the innate immune response by mediating the initial recognition of, and prompt response to a variety of microorganisms. The concerted activation of TLRs and complement ensures efficient clearance of infection. Previous studies have documented synergism between TLRs and the receptor for the pro-inflammatory peptide C5a (C5aR/CD88), and regulation of TLR-induced pro-inflammatory responses by C5aR, suggesting crosstalk between TLRs and C5aR. However, it is unclear whether and how TLRs modulate C5a-induced pro-inflammatory responses. This study tested the hypothesis that a genuine, bi-directional signalling crosstalk between TLRs and C5a receptors exists, involving not only modulation of TLR-mediated responses by C5a receptor activation, but also modulation by TLR activation of the extent and/or quality of cellular responses to C5a. The experiments described in this thesis confirmed this hypothesis by demonstrating a marked positive modulatory effect of TLR activation on cell sensitivity to C5a in vitro and ex vivo and identifying underlying mechanistic targets. Pre-exposure of peripheral blood mononuclear cells and whole blood to diverse TLR ligands or bacteria enhanced C5a-induced pro-inflammatory responses. This effect was not observed in TLR4-signalling-deficient mice. TLR-induced hypersensitivity to C5a did not result from C5aR up-regulation or modulation of C5a-induced calcium mobilization. Rather, TLRs targeted the second C5a receptor, C5L2 (acting as a negative modulator of C5aR) by reducing C5L2 expression and activity. TLR-induced hypersensitivity to C5a was mimicked by blocking C5L2 and was not observed in C5L2KO mice. Furthermore, TLR activation inhibited C5L2 expression upon C5a stimulation. Expression of the key adaptor molecule β-arrestin 1, which mediates the inhibitory effects of C5L2 on C5aR, was also found to be negatively regulated by TLR activation. These findings identify a novel pathway of crosstalk within the innate immune system that amplifies innate host defence at the TLR-complement interface. Unravelling the mutually regulated activities of TLRs and complement may reveal new therapeutic avenues to control inflammation.

Recent years have witnessed a rapid proliferation of gene editing in mammalian cells due to the increasing ease and reduced cost of targeted gene knockout. There has been much excitement about the prospect of engineering T-cells by gene editing in order to provide these cells with optimal attributes prior to adoptive cell therapy for cancer and autoimmune disease. I began by attempting to compare short hairpin RNA (shRNA) and zinc finger nuclease (ZFN) approaches using the CD8A gene as a target for proof of concept of gene editing in Molt3 cells. During the course of my studies the clustered regularly interspaced short palindromic repeats (CRISPR) mechanism for gene editing was discovered so I also included CRISPR/Cas9 in my studies. A direct comparison of the three gene editing tools indicated that the CRISPR/Cas9 system was superior in terms of ease, efficiency of knockout and cost. As the use of gene editing tools increases there are concerns about the inherent risks associated with the use of nuclease based gene editing tools prior to cellular therapy. Expression of nucleases can lead to off target mutagenesis and malignancy. To circumvent this problem I generated a non-nuclease based gene silencing system using the CD8A zinc finger (ZF) fused to a Krüppel associated box (KRAB) repressor domain. The ZF-KRAB fusion resulted in effective silencing of the CD8A gene in both the Molt3 cell line and in primary CD8+ T-cells. Importantly, unlike CRISPR/Cas9 based gene editing, the ZF-KRAB fusion was small enough to be transferred in a single lentiviral vector with a TCR allowing simultaneous redirection of patient T-cell specificity and alteration of T-cell function in a single construct. To improve the efficiency of gene editing with CRISPR/Cas9 I developed an ‘all in one’ CRISPR/Cas9 system which incorporated all elements of the CRISPR/Cas9 gene editing system in a single plasmid. The ‘all in one’ system was utilised to derive MHC-related protein 1 (MR1) deficient clones from the A549 lung carcinoma and THP-1 monocytic cell lines in order to study MR1 biology. Mucosal-associated invariant T-cell (MAIT) clones were not activated by MR1 deficient A549 or THP-1 clones infected with bacteria.