The synovial environment in rheumatoid arthritis (RA) is a milieu of Damage Associated Molecular Patterns (DAMPs), cytokines and immune complexes, which can modulate the activation or polarisation of myeloid cells. GM-CSF, which is a pivotal myeloid cell growth factor, is also a pro-inflammatory cytokine that drives aspects of RA immunopathogenesis. Inhibition of GM-CSF signalling has been successful in both mouse models and in clinical trials for RA, however, the specific effect of GM-CSF on myeloid cells in a synovial setting is not well understood. The aim of this thesis was to investigate the impact of the synovial environment and GM-CSF on myeloid cells in RA. GM-CSF stimulation induced monocytes to secrete substantial amounts of the chemokine CCL17. However, this induction of CCL17 was significantly inhibitedupon co-stimulation with RA synovial fluid, but not osteoarthritis (OA) synovial fluid, whilst the expression of other chemokines was unaffected. TLR ligands also inhibited GM-CSF driven CCL17, however, through the use of MyD88/TRIF knockout mouse monocytes, we found RA synovial fluid inhibition of CCL17 was TLR-independent. Small Immune Complexes and IFNα also had the capacity to inhibit GM-CSF induction of CCL17, suggesting multiple mechanisms within the RA synovial fluid to prevent this induction. Despite the consistency of RA synovial fluid causing inhibition of the GM-CSF signalling pathway in comparison to OA synovial fluid, there were no distinct effects on macrophage polarisation. The RA synovial environment has more of an impact on monocyte activation in comparison to macrophage polarisation, as synovial fluid from other arthropathies had the comparable effects on macrophage phenotypes. This thesis concludes that RA synovial fluid contains several factors that inhibit GM-CSF induction of CCL17. This suggests a regulatory mechanism, preventing the excessive secretion of CCL17 by monocytes, thereby preventing exacerbation of immunopathogenesis.

Pertussis toxin (PT) is a major virulence factor produced by Bordetella pertussis. In its detoxified form (PTd), it is an important component of acellular pertussis vaccines although some residual FT activity may be present because of the limitations of the detoxification processes used. The in vivo histamine sensitisation test (HIST) in mice is currently used for the safety testing of these vaccines to determine the level of their residual FT activity. However, an alternative test is needed because of large assay variability and ethical concerns with regard to animal usage. The main objective of this study was to search for an alternative test to the HIST. The ADP-ribosylation enzyme activity of FT is thought to be the major factor responsible for the histamine-sensitising activity detected in vivo. In the present study, the enzymatic activities in different acellular pertussis-based combination vaccine formulations were measured by a recently-developed ADF-ribosylation assay and compared with their reactivities in the HIST. The results indicated that different products showed differences in ADP-ribosylation activity and, these did not correlate with their reactivity in the HIST. FT has two functionally-distinct domains: the enzymatic A-protomer and the B- oligomer that facilitates host-cell binding and entry of FT into the cell. This dual biological function could explain why the residual enzyme activity of FT in vaccines did not fully reflect the in vivo reactivity observed by the HIST. Thus, refinement of the in vitro test to include a step which monitored the B-subunit activity of FT was attempted. A quantitative FT carbohydrate-binding assay using glycoproteins or defined oligosaccharides was developed. PT was found to bind preferentially to multiantennary N-glycans, with the highest binding towards the fully sialylated structures. In contrast, PTd lost the ability to bind to sialylated multiantennary structures but retained some capacity to bind to neutral multiantennary structures. Different vaccine preparations had different levels of PT binding activity as well as enzymatic activity. It was concluded that, although the enzymatic activity of PT plays a more important role in the death of mice in the HIST, a high binding activity of the B- subunit could increase the in vivo toxic effect by aiding the accessibility of the A- subunit to its cellular targets. A mathematical equation was devised to establish a preliminary relationship between the enzymatic, carbohydrate-binding and HIST assays in a product-dependent manner. Further studies with a larger number of vaccines are required for a more meaningful statistical analysis. However the methods form a sound basis for the future development of an alternative assay to the histamine challenge test. The in vitro assays could also be useful for investigating the mechanisms of PT detoxification. Comparisons of A- and B-subunit activities of purified PT and vaccine preparations of PTd indicated that both subunits are modified after chemical detoxification. Different vaccine products had different levels of enzymatic and binding activities and it was concluded that different detoxification procedures, as well as formulation factors, could contribute to this variation. A CHO cell clustering assay is used as an alternative in vitro test to the HIST for assessing residual PT activity at the bulk stage of vaccine production. In a parallel study to the above, comparative proteomics was used to gain insights into the mechanism of PT-induced CHO cell clustering with a view to developing a mechanistic-based alternative assay for the safety testing of pertussis-based combination vaccines. A proteomic map of CHO cells was established and PT-induced CHO cell clustering appeared to be a complex process involving subtle changes in various cellular functions, mainly related to intracellular transport, cell stress and the cell cycle. The information obtained will be useful for future studies into the possible mechanisms of the effect of PT on CHO cells.

Psoriasis is a common, debilitating systemic inflammatory disorder that is characterised by sharply demarcated, thick, erythematous scaly skin plaques. Such plaques commonly appear on skin that is subjected to repeated tensile trauma, such as elbows, knees and flexures. The mechanism by which these inflammatory lesions are spatially restricted is not known and yet knowledge of this could be of critical importance for our understanding of this disease. Chemokines are the principal regulators of leukocyte migration and play a critical role in the initiation and maintenance of inflammation. The atypical chemokine receptor ACKR2 (formerly D6) binds inflammatory CC-chemokines, but does not signal upon ligand binding; instead ACKR2 internalises and helps degrade such chemokines, after which it continues to cycle back to the cell surface. ACKR2 acts, through this mechanism, as a high-capacity scavenger of chemokines, and plays an important role in regulating inflammation. It is known that ACKR2 expression is high in unaffected skin in patients with psoriasis (remote from inflammatory plaques) and concurrently deficient in the plaques themselves. Additionally, human studies have shown that simple skin trauma in psoriasis patients causes a reduction in cutaneous ACKR2 expression at the site of trauma. However, the functional significance and the molecular mechanism by which it occurs are not understood. This thesis explored the role of ACKR2 in the spatial restriction of psoriasiform inflammation and the molecular mechanisms for its differential regulation. Through the use of disease relevant mouse models, primary human cell cultures and novel cell migration assays, the results presented here show that localised psoriasiform inflammation upregulates ACKR2 in remote tissues through the systemic release of cytokines. This remotely upregulated ACKR2 expression protects tissues from the further spread of inflammation. This protective effect is mediated by stromally expressed ACKR2 that acts to control inflammatory T-cell positioning within the skin. Tensile trauma of keratinocytes however, acted to reduce ACKR2 expression in the context of inflammation, which in turn provides a novel mechanism for the well-characterised phenomenon that occurs in psoriasis (and a range of skin condition) termed ‘koebnerisation’. Koebnerisation refers to the phenomenon by which relatively simple skin trauma induces the development of disease-specific skin lesions. Furthermore, this thesis defines novel disease-relevant regulators of ACKR2 expression. In silico analyses identified psoriasis-associated microRNAs that bound to the 3’-UTR of ACKR2, and reduced its expression at transcriptional and protein level. Importantly, trauma of keratinocytes induced ACKR2 downregulation concurrent with a substantial and significant increase in the expression of the identified ACKR2 targeting microRNAs. Together, this thesis defines a novel mechanism by which ACKR2-mediated regulation of chemokine function, cutaneous trauma, microRNAs and systemic cytokines, co-ordinately modulate the predisposition of remote tissue sites to the development of new lesions. Importantly, the results presented here have profound implications for how spatial restriction is imposed on inflammation. The data also highlight therapeutic ACKR2 induction as a plausible novel strategy for the limitation and treatment of psoriasiform- and potentially other forms of inflammation.

Background: Rheumatoid arthritis (RA) is a chronic inflammatory arthritis that causes significant morbidity and mortality and has no cure. Although early treatment strategies and biologic therapies such as TNFα blocking antibodies have revolutionised treatment, there still remains considerable unmet need. JAK kinase inhibitors, which target multiple inflammatory cytokines, have shown efficacy in treating RA although their exact mechanism of action remains to be determined. Stratified medicine promises to deliver the right drug to the right patient at the right time by using predictive ‘omic biomarkers discovered using bioinformatic and “Big Data” techniques. Therefore, knowledge across the realms of clinical rheumatology, applied immunology, bioinformatics and data science is required to realise this goal. Aim: To use bioinformatic tools to analyse the transcriptome of CD14 macrophages derived from patients with inflammatory arthritis and define a JAK/STAT signature. Thereafter to investigate the role of JAK inhibition on inflammatory cytokine production in a macrophage cell contact activation assay. Finally, to investigate JAK inhibition, following RA synovial fluid stimulation of monocytes. Methods and Results: Using bioinformatic software such as limma from the Bioconductor repository, I determined that there was a JAK/STAT signature in synovial CD14 macrophages from patients with RA and this differed from psoriatic arthritis samples. JAK inhibition using a JAK1/3 inhibitor tofacitinib reduced TNFα production when macrophages were cell contact activated by cytokine stimulated CD4 T-cells. Other pro-inflammatory cytokines such as IL-6 and chemokines such as IP-10 were also reduced. RA synovial fluid failed to stimulate monocytes to phosphorylate STAT1, 3 or 6 but CD4 T-cells activated STAT3 with this stimulus. RNA sequencing of synovial fluid stimulated CD4 T-cells showed an upregulation of SOCS3, BCL6 and SBNO2, a gene associated with RA but with unknown function and tofacitinib reversed this. Conclusion: These studies demonstrate that tofacitinib is effective at reducing inflammatory mediator production in a macrophage cell contact assay and also affects soluble factor mediated stimulation of CD4 T-cells. This suggests that the effectiveness of JAK inhibition is due to inhibition of multiple cytokine pathways such as IL-6, IL-15 and interferon. RNA sequencing is a useful tool to identify non-coding RNA transcripts that are associated with synovial fluid stimulation and JAK inhibition but these require further validation. SBNO2, a gene that is associated with RA, may be biomarker of tofacitinib treatment but requires further investigation and validation in wider disease cohorts.

Candida albicans and related Candida species are common members of the normal flora in humans; however, they are opportunistic pathogens and may cause superficial or systemic disease. Candidaemia is the most extensively studied nosocomial invasive fungal infection, and recent data show that Candida species are the fourth most commonly recovered blood culture isolates. It is now clear that this kind of infection is often due to the formation of Candida biofilms on catheters and other indwelling medical devices. This project investigates the role of quorum sensing in Candida biofilm formation, and the effect of prostaglandins, COX inhibitors and the combined effect of COX inhibitors with antifungal agents on biofilm development. Additional investigations monitor prostaglandin production by planktonic cells and biofilms of Candida albicans. Quorum sensing is known to be involved in bacterial biofilm formation, and in Candida, two signal molecules (farnesol and tyrosol) were recently identified. In this study the effects of farnesol on germ-tube and biofilm formation were investigated. Farnesol totally prevented germ-tube formation and inhibited biofilm formation by 30 % when 1mM farnesol was added at an early stage. Scanning electron microscopy revealed that only yeast cells were formed in biofilms grown in the presence of farnesol. Supernatants from biofilms formed after 24h and 48h inhibited germ-tube formation by 10 % and 29 %, respectively, indicating that they contained farnesol or farnesol-like activity. It has been reported that planktonic cultures of C. albicans produce tyrosol, another quorum-sensing molecule. In this study, HPLC confirmed that tyrosol was produced by both planktonic cells and biofilms of C. albicans, and biofilms produced significantly higher levels of tyrosol than planktonic cells. Overall, the results demonstrated that biofilm development in C. albicans is under the control of at least two quorum-sensing molecules. Farnesol acts as a negative signal and inhibits hyphal production. Tyrosol acts as a positive signal and promotes hyphal production. Prostaglandins are now known to be produced by C. albicans and may play an important role in fungal colonization. Their synthesis in mammalian cells is decreased by inhibitors of the cyclooxygenase isoenzymes required for prostaglandin formation, hi this study, the effects of nonsteroidal anti-inflammatory drugs (all cyclooxygenase inhibitors) on biofilm formation by three strains of C. albicans were investigated. Seven out of nine drugs tested at a concentration of 1 mM inhibited biofilm formation. Aspirin, etodolac and diclofenac produced the greatest effects, with aspirin causing up to 95% inhibition. Surviving cells had a wrinkled appearance, as judged by scanning electron microscopy, and consisted of both yeasts and hyphae. The effect of aspirin on viability of both biofilm and planktonic cells was pH dependent, with the greatest effect at pH 3 or in unbuffered yeast nitrogen base medium. The effect of aspirin on metabolic activity and viability of mature C. albicans biofilms was similarly pH dependent. At pH 3, aspirin inhibited metabolic activity of mature C. albicans biofilms more than fluconazole or a mixture of fluconazole and aspirin. This study also demonstrated that both biofilms and planktonic cells of C. albicans synthesize extracellular prostaglandin(s) during growth at 37°C, but biofilm cells secrete significantly more when production is determined on the basis of cell dry weight. Prostaglandin synthesis by both cell types was sensitive to the cyclooxygenase inhibitors aspirin, diclofenac and etodolac. A morphological mutant blocked in two signalling pathways (cph1/cph1 efg1/efg1) produced prostaglandin levels similar to those of the parent strain but formed yeast-only biofilms. Unicellular organisms such as fungi have been reported to interact with hormones, such as fungal sex hormones and mammalian hormones, hi this study the effect of several steroids including progesterone, corticosterone, dexamethasone, prednisolone, hydrocortisone and estradiol on C. albicans biofilm development was investigated. The results showed that biofilm formation was not affected by any of these compounds. However, at 1μM some steroids such as progesterone and dexamethasone inhibited germ-tube formation by more than 25%. At 100 to 1000 μM, all steroids inhibited germ-tube formation.

The Gram-negative bacterial pathogen Pasteurella multocida causes economically significant infections of domesticated animals. Very little is known about the roles of P. multocida outer membrane proteins (OMPs) in host-specificity and virulence. This study aimed to compare the outer membrane proteomes of eight representative P. multocida isolates associated with diseased cattle (two), sheep (two), pigs (two) and poultry (two).

Background: ST2 has been identified in playing an important role in Th2-mediated inflammation and asthma. IL-33 acts as the ligand for ST2; it is a novel cytokine that induces innate Th2/type-2 responses when delivered to the lung. The hierarchy of IL-33 and type-2 cytokines and chemokines in Th2 inflammation in the lung has not been fully elucidated. Furthermore, the role of IL-33 in the adaptive response in allergic mediated airways disease is unclear. Epithelial cells (ECs) are increasingly recognised as having an immunological role in airway inflammation and asthma, in particular releasing cytokines such as IL-33. Little is known about whether ST2 is expressed on these cells and what function IL-33 responsive ECs may have in Th2 diseases. Soluble ST2 (sST2) has emerged as a biomarker correlating with disease activity in cardiovascular disease. It is not known if there is a clear association between sST2 and asthma, nor whether measurable IL-33 concentrations are present and if so, their association with disease severity. The influence of smoking and corticosteroid treatment on these parameters has also not been determined. Aim: To ascertain the levels of systemic sST2 and IL-33 in asthmatic patients. To determine cytokine, chemokine and airway dynamics of IL-33-driven innate airway inflammation. To determine the role of epithelial cells in IL-33-driven innate airway inflammation. To investigate the function of ST2/IL-33 axis in the innate and adaptive responses in allergic airways inflammation and asthma. Methods and Results: sST2 and IL-33 levels in plasma of never smokers, ex-smokers and smokers were determined by immunoassay before and after a corticosteroid trial. Corticosteroid treatment resulted in increased sST2 levels in all smoking status patient groups; there was no effect attributable to smoking. Time course and dosage interval experiments were performed in mice treated with intranasal IL-33. IL-5, IL-13, eotaxin/CCL11 and eotaxin2/CCL24 mediated eosinophilic airway inflammation (AI). Treatment of mice with both anti- CCL11 and -CCL24 partially ameliorated the AI. IL-4 gene deficient mice were protected from IL-33-induced inflammation. BALB/c mice displayed airways hyperreactivity following IL-33 treatment. Murine, human cell line and primary human ECs were assessed for ST2 expression by immunohistochemistry and fluorescence activated cell sorting (FACS). ST2 expression was clearly demonstrated in the ECs. Subsequently ECs were treated with IL-33 in an in vitro setting including in a pseudostratifed epithelium model. ECs produced a range of inflammatory and angiogenic mediators in response to IL-33. In particular IL-33 driven EC-derived VEGF promoted angiogenesis in vitro. Intranasal IL-33 induced increased endothelial cells and vascular remodelling in vivo. Experimental allergic airways inflammation (AAI) was generated in BALB/c mice which were co-treated with IL-33 or PBS at allergen sensitisation. IL-33 induced the polarisation of IL-5+IL-4- T cells in the draining lymph nodes and these mice developed more severe inflammation. AAI was induced in WT, ST2-deficient, IL-4-deficient and ST2/IL-4 deficient mice. These experiments showed IL-4 was necessary for generation of AAI which could not be overcome by ST2-pathway stimulation in an adjuvant-free model. Conclusions: The data presented further extends the current understanding of the ST2/IL-33 axis in the innate and adaptive aspects of Th2 inflammation in AAI and asthma. In particular the hierarchy of mediators and cells involved in Th2 inflammation, including at the sensitisation phase, have been explored. This identifies ST2/IL-33 as a potential target in the development of biological therapies for asthma.

Chemokines are a highly conserved family of chemoattractant cytokines that are key to the movement of cells around the body under both inflammatory and homeostatic conditions. Chemokines bind to seven transmembrane G protein coupled receptors that signal and induce cell movement upon ligand binding. As well as the ‘classical’ chemokine receptors, there also exists a family of atypical chemokine receptors that do not induce a canonical signalling response upon ligand binding. These atypical chemokine receptors (ACKR) have been shown to modify the chemokine response through processes such as the scavenging of inflammatory chemokines. One such receptor with this scavenging function is ACKR2 which has been shown to bind and internalise all of the inflammatory CC chemokines. The functional repertoire of ACKR2 continues to be expanded and it is now thought to have a role in inflammation, lymphatic drainage and lymphatic vessel development. It has been shown that the absence of this receptor results in impaired resolution of inflammation and, as a result, increased inflammatory pathologies in vivo. In models of skin inflammation a lack of ACKR2 has been shown to result in increased pathology and impaired inflammatory resolution. Multiple models of cutaneous inflammation, including excisional wound healing and chemically induced damage, were used to further investigate the role of ACKR2 in this context. Work on wound healing suggested that although ACKR2 appears to play no role in wound closure it does have a role in the formation of scar tissue in an excisional wound. Our data suggest that ACKR2 has a role in collagen deposition in developing and maturing scars. We also found that ACKR2 had a protective role in chemically-induced models of skin inflammation. We then looked at the role of ACKR2 in ocular inflammation. The main work performed in this section involved the use of the experimental autoimmune uveitis (EAU) model. Here we found that ACKR2 had a protective effective resulting in reduced pathology and infiltration of inflammatory leukocytes. This work also suggested, using in vitro analysis, that a human retinal pigmented epithelial cell line expresses functional ACKR2 protein and that our findings may be relevant to human disease. Finally we looked at the role of ACKR2 in the inflammatory autoimmune disease rheumatoid arthritis (RA). By taking samples of peripheral blood from RA patients we assessed the transcript levels of Ackr2 and correlated them with clinical measurements. Our findings suggested that, in patients with ‘well-controlled’ RA, there was an increase in the transcription of Ackr2 in peripheral blood leukocytes. Additional work using in vitro methods suggest that the hypoxic nature of the rheumatoid joint, and some of the drugs used to treat the disease, may increase the transcription of Ackr2. Overall the findings in this work suggest novel roles for ACKR2 in the skin and the eye. They also shed light on further environmental factors that may alter the local expression of ACKR2 in the rheumatoid joint. Taken together this work suggests that ACKR2 may have great therapeutic potential and, furthermore, this potential may be relevant to a wider range of tissues than previously thought.

Campylobacter is a bacterial pathogen commonly responsible for foodborne gastroenteritis worldwide. Due to the rapid development of resistance to antibiotics, many alternative interventions have been studied and bacteriophage therapy is one of them. To maximise the impact of the intervention and to ensure it remains sustainable it important to study the ecology and coevolution of Campylobacter and the bacteriophage that infect the pathogen. Coevolution interactions between Campylobacter and bacteriophage drive rapid molecular change and contribute to a higher mutation rates for both parties. In this study genetic modifications were examined in the bacterial host and phage arising either in vivo or in vitro. We observed the impact of C. jejuni containing Mu-like pro-phage on campylobacters populating the caeca of commercial broiler flocks. The Mu-containing campylobacters initially colonised and became the dominant strain, only to be out-competed before depopulation of the broiler house. The presence of the transposable Mu-like prophage ultimately proved to be a limitation in the fitness of the host. Campylobacter-specific phage CP30 is a T4-like phage of the Eucampyvirinae that was isolated from a farm with several other phage showing differences in host range of contemporary farm isolates. After serial passage the phage population acquired sequence variants. One newly characterised sequence type, CP30C, was defective in a tail fibre protein and revealed reduced adsorption ability and sensitivity against C. jejuni host strains. Whole genome sequencing identified host mutation in C. jejuni carrier state strains that maintain viability despite the continual production of virulent phage. A point mutation in the flhF gene (flhF(T368A)) was hypothesised to contribute to the non-motile phenotype of carrier state strains. Expression of flhF(T368A) in a flhF knockout background provedto have impaired motility, exhibit structural defects in flagella synthesis, were less susceptible to phage infection and show down regulation of σ28 dependent and σ54 dependent flagellar associated genes. Recombinant protein expression of FlhF demonstrated the protein to have GTPase activity and the FlhF(T368A) to have reduced enzyme activity, greater thermal sensitivity and to be impaired in protein folding compared to wild type FlhF.

Typhoid fever remains a major public health concern in low-income countries affecting millions of people each year. Research on effective vaccines against Salmonella Typhi has been directed toward the development of glycoconjugates. Several conjugation parameters affect the magnitude, quality and persistence of immune response. A systematic investigation of the effect of each variable on immunogenicity was conducted, synthesizing and testing in mice a panel of Vi-based conjugates differing for saccharide size, carrier protein, saccharide to protein ration and conjugation chemistry. Saccharide size and carrier protein were the parameters mainly impacting immunogenicity. Differently from full-length Vi (165 kDa) conjugates, fragmented Vi (< 82 kDa) –CRM₁₉₇ showed anamnestic response and induced minimal antibody responses in T-cell knockout mice. Unexpectedly, fragmented Vi conjugates induced lower persistent antibody levels compared to full-length Vi conjugates. Fragmented Vi conjugates offer benefits in terms of manufacture, and random chemistry is preferable because of higher conjugation yields obtained compared to selective chemistry. In view of Vi conjugate vaccine introduction into vaccination schedules, CRM₁₉₇ may have advantages as an optimal carrier protein to avoid any negative effect of pre-existing anti-carrier immunity. Therefore, this systematic investigation of conjugation parameters represents a model for rational design of glycoconjugates.