Sepsis kills more people than car accidents, breast cancer, and bowel cancer combined. The key areas integral for ensuring improvements in the care of sepsis patients include improved risk stratification, better microorganism identification techniques, and a reduction in the burden of second-hit nosocomial infections. This work addresses each of these key areas in turn, with the ultimate aim of improving patient care through applied translation research. Firstly, this work will combine small-scale yet complex immunological data with new statistical modelling techniques to form a new approach to microbe identification based on “immune fingerprints”. This new approach will allow discrimination between Grampositive and Gram-negative infecting organisms using a small set of immune markers suitable for development into point-of-care technology. These immune fingerprints will also be used to improve the diagnosis of sepsis and provide risk stratification models. Secondly, this thesis will offer new insights into immunosuppression that may impact upon current and future clinical trials. Specifically, it will suggest that aminobisphosphonates may help in the treatment of sepsis related-immunosuppression and that sepsis neutrophils gain the ability to act as antigen presenting cells.

Innate lymphoid cells (ILCs) are an emerging family of cells which have been well-characterised within the gut and peripheral tissues. Despite being implicated in shaping adaptive immune responses, relatively little is known about their function within lymph nodes (LNs), important sites for the generation of this type of response. The aim of this investigation was to characterise ILC populations within a range of different LNs, both at steady state and using a draining LN model to understand their role in an immune response. Mice in which a subset of ILC, or key functional molecules, are deficient will be used to better understand their function within LNs, with a focus on adaptive immune responses. My results reveal that ILCs are present in all LNs analysed, however, differences in the ILC composition of mucosal tissue and peripheral tissue-draining LNs indicate site-specific requirements for these cells. Differing dependencies on CCR7 for ILC entry into LNs were observed, consistent with migration of these cells into these secondary lymphoid tissues. Within LNs, group 3 ILCs were found to express major-histocompatibility complex class-II and specifically locate to sites where adaptive immune responses are initiated and maintained. Notably, ILCs accumulated in draining LNs following immunisation and whilst their roles here remain unclear they are unlikely to be involved in the priming of naïve CD4+ T cells. In summary, I show that subsets of ILC3 are enriched in LNs which drain mucosal sites and can be found to locate to crucial sites within LNs following their entry by a CCR7-dependent mechanism. These data support a role for ILC3 in adaptive immune responses.

Tuberculosis is a global problem, with little change in antibiotic therapy over the last fifty years, but with the emergence of both multi-drug resistant disease and extensive drug resistant disease further treatments are needed to ensure successful management of the disease. Severe vitamin D deficiency is prevalent in patients with tuberculosis and the immunomodulatory mechanisms of elements of the vitamin D axis, including vitamin D binding protein (DBP) and vitamin D receptor (VDR) have been explored in part. Aims This thesis will ascertain the role of polymorphisms in vitamin D axis genes in determining response to vitamin D in tuberculosis patients. Additionally it will aim to determine whether the interaction between underlying genotype, baseline vitamin D level and other elements of the vitamin D axis has the potential to influence clinical outcome and further investigate in vitro effects of vitamin D and elements of the vitamin D axis in influencing the frequency and functionality of monocytes and T cells, both of which are key elements in the immune response to tuberculosis infection. Results Associations between vitamin D baseline and response to supplementation appear to have a genetic association with DBP, VDR and DHCR7 genotypes and varying DBP haplotypes appear to determine the level of DBP at baseline measurement. The effect of vitamin D has an immunomodulatory role in both monocyte response and T regulatory activity, with a clear effect of vitamin D on cytokine response. Conclusion There appears to be a role for vitamin D in the treatment of tuberculosis but further questions are raised regarding the benefits and risks of immune response modulation in an inflammatory/ cytopathogenic condition such as tuberculosis.

Invasive non-typhoidal Salmonellosis is a major cause of bloodstream infections in Sub-Saharan Africa, mainly caused by Salmonella enterica serovars Typhimurium and Enteritidis. Naturally shed outer membrane vesicles from Gram-negative bacteria are being explored to generate cost-effective vaccines against many infections, since antigens within vesicles maintain their natural conformation and orientation. Shedding can be enhanced through genetic modification and the resulting vesicles, Generalized Modules for Membrane Antigens (GMMA) offer potential as vaccines. We explored the potential of expressing a known immunogenic antigen of S. Typhimurium, OmpD, in GMMA derived from E. coli as a vaccine. Further, we showed that immunization with GMMA derived from S. Typhimurium (STm-GMMA) induced protection in mice. The response to STm-GMMA immunization included the generation of antibodies to two immunodominant antigens, lipopolysaccharide and porins. Strikingly, the IgG response towards these two antigens was induced with different rates during first week, with a dramatic induction of IgG targeting porins, and not LPS, in a T-cell independent manner. Nevertheless, the antibody response against both antigens persisted for over 200 days in sites including the bone marrow. Results from this thesis shows that STm-GMMA is both attractive as a vaccine and as a tool to facilitate investigations of B-cell responses.

Type 1 diabetes (T1D) results from autoimmune destruction of pancreatic beta cells. Preservation of beta cell function reduces the risk of the complications of T1D. Regular exercise preserves beta cell function rat models of T1D and patients with other forms of diabetes. We wished to examine whether exercise could preserve beta cell function in T1D and whether this was influenced by adipokine receptor expression and function. We undertook a qualitative study to explore barriers to exercise in patients newly diagnosed with T1D. This showed that patients lacked confidence managing diabetes for exercise, and were poorly supported by healthcare professionals. Using these results, we then undertook a pilot clinical trial aiming to determine recruitment and retention, adherence to exercise, and exploring whether exercise preserves beta cell function in patients newly diagnosed with T1D. We show successful recruitment to an unsupervised exercise intervention study. We did not detect a beneficial effect of exercise on beta cell function in this pilot trial, but identified several areas that will need to be addressed in designing a larger scale study. Finally, we demonstrate improved adiponectin receptor expression and adiponectin mediated suppression of T cell endothelial migration in the months after diagnosis with T1D.

In rheumatoid arthritis (RA), chronic inflammation and destruction of the joint is driven by local production of cytokines. My aims were to characterise cytokine mRNA expression in multiple synovial fluid cell populations in early and established RA and to study these, in addition to whole synovial tissue, in early synovitis patients in relation to disease outcome. I established a novel method to determine cytokine mRNA expression in synovial fluid CD4 T cells, CD8 T cells, B cells, macrophages and neutrophils directly ex vivo. I made several novel observations, for example RA synovial fluid B cells expressed high levels of RANKL. As RANKL drives bone resorption, this suggests a potential role for B cells in bone erosion in RA. RANKL protein was expressed by B cells in synovial tissue, and mainly by memory B cells in synovial fluid. Furthermore, synovial RANKL levels were reduced after treatment with the B cell depleting therapy, rituximab. Overall, both in whole synovial tissue and in sorted cells, the cytokine mRNA expression profile was very similar in early synovitis patients who subsequently developed RA or had resolving synovitis, and in patients with early or established RA. In comparison, cytokines and chemokines were upregulated in early and established arthritis patients compared to uninflamed controls. The finding that cytokine mRNA expression is largely similar in early synovitis patients who develop RA or have resolving disease, and in the early and established phases of RA, suggests that cytokine expression is reflective of general synovial inflammation, rather than being specific for early synovitis outcome or stage of RA.

This study examined the relationship between Mycobacterium tuberculosis global clades and patient origin. In the UK, the rate of tuberculosis is higher amongst patients who originated from the Indian subcontinent (ISC), where two dominant lineages are present, the Central Asian Strain (CAS) and the East African Indian (EAI) lineage. Mycobacterial interspersed units containing variable number of tandem repeats DNA fingerprinting of M. tuberculosis strains isolated from UK patients who originated from the ISC, as defined by novel software, identified that CAS was the most prevalent clade (39%) and EAI was the third most prevalent clade (15%). To further elucidate the relationship between host origin and strain lineage, two rigorous new models of infection were developed which assessed mycobacterial growth and host cell response. The monocyte-derived macrophage model was more appropriate for measuring cytototoxicity than the THP-1 cell model as in the absence of infection, 50% of THP-1 cells died compared to 2% of macrophages. CAS strains caused 1.5 fold more cell necrosis and their growth was four fold higher than EAI strains in the monocyte-derived macrophage model. Finally, the response of polarised monocyte-derived macrophages from Asian and Caucasian donors to different M. tuberculosis lineages was compared. CAS strains grew preferentially better in M2 macrophages from Asian donors. The prevalence of CAS in the Midlands is likely to be due to a combination of specific strain importation and increased ability of this strain to transmit to the population present in the Midlands.

Chronic systemic immunosuppression in pancreatic islet transplantation restricts its clinical application. This study aims to explore the potential of cell-mediated immune-modulation as an alternative to conventional immunosuppressive regimens; specifically investigating the innate immunosuppressive properties of human amniotic epithelial cells (AEC). Cell constructs composed of human islets and AEC (islet:AEC) were bio-engineered in rotational culture. Insulin secretory capacity and immuno-modulatory potential were characterised using appropriate in vitro assays. Fluorescence immunocytochemistry and multiplex arrays was used to identify putative mediators of the immunosuppressive response in isolated AEC monocultures. Islets and islet:AEC constructs demonstrated sustained, physiologically-appropriate insulin secretion. Resting peripheral blood mononuclear cells (PBMC) were activated on exposure to human islets but this response was significantly (p<0.05) attenuated in islet:AEC constructs. Phytohaemagglutinin (5\( \mu \)g/ml)-induced PBMC proliferation was sustained on contact with unmodified islets but abrogated in AEC and islet:AEC constructs. CD4+ and CD8+ T-cell proliferation was responsive to AEC; their in vitro expansion both in response to CD3/CD28 activation and contact with human islets being suppressed by the presence of AEC. Transplanted islets may thus benefit from an immune-privilege status conferred on them as a consequence of their close proximity to human AEC. Such an approach may diminish the requirement for generalised systemic immunosuppression in islet transplantation.

Rheumatoid arthritis is an autoimmune disease that affects approximately 1% of the population, where chronic inflammation of the synovial joints can lead to active destruction of cartilage and bone. New therapeutic targets are discovered by investigating genes or processes that exacerbate or ameliorate disease progression. Mouse models of inflammatory arthritis are commonly employed for this purpose, in conjunction with biomedical imaging techniques and suitable measures of disease severity. This thesis investigated the hypothesis that a statistical model of non-pathological bone shape variation could be used to quantify bone destruction present in micro-CT images. A framework for constructing statistical shape models of the hind paw was developed, based on articulated registration of a manually segmented reference image. Successful registration of the reference towards ten healthy hind paw samples was followed by statistical shape analysis. Mouse models of inflammatory arthritis were then investigated and compared by identifying bone abnormalities as deviations from the model statistics. Validation of the model against digital phantoms and clinical scores indicates that the method is largely successful in this effort. Application of the method in a novel study of macrophage-mediated inflammation shows promising results that are supportive of previous findings.