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In recent years, genome-wide association studies have identified polymorphisms in several genes which implicate the role of inflammation in the development and progression of Alzheimer's disease (AD). This is of particular relevance in rheumatoid arthritis (RA), a chronic inflammatory autoimmune disease, as these patients have higher levels of systemic inflammation than the general population. If AD is driven by inflammation, then RA may be the ideal model in which to observe the accelerated effects of systemic inflammation on cognitive decline for future interventional trials. Moreover, there have been several inflammatory biomarkers which have been found to be peripherally elevated in both diseases including tumour necrosis factor-alpha (TNFa), a proinflammatory cytokine which has been associated with an increased rate of neurodegeneration and risk of conversion from mild cognitive impairment (MCI) to AD dementia. This led to the development of a hypothesis which suggested that reduction in peripheral TNFa via TNF inhibitors (TNFi) may slow or prevent cognitive decline associated with AD. This thesis aimed to test this hypothesis by using preliminary data from the rheumatoid arthritis medication and memory study (RESIST), an 18-month observational study which aims to compare the rate of cognitive decline between TNFi and conventional synthetic anti-rheumatic drugs (csDMARDs) in patients with both RA and MCI. While analysis of screening data was able to demonstrate high prevalence of cognitive impairment in this RA population, analysis of preliminary longitudinal data found little evidence of a relationship between TNFi and better cognitive performance after 6/12 months. This analysis will be repeated using 18-month data when available which will more fully describe the relationship between TNFi and cognitive outcomes. Nevertheless, this preliminary research is a valuable contribution in a topic that is largely under researched and will help to inform future longitudinal studies of TNFi as a preventative treatment for AD.

The interaction between epithelial cells and macrophages is integral to mucosal immune fate: determining the decision between tolerance and immune activation/inflammation. Endotoxin tolerisation (ET) is a circumstance where cells go through a hypo-responsive state, unable to respond to further endotoxin-LPS challenge. Mucosal macrophages (MΦs) have a dual functionality that determines tolerance to commensal organisms or immune response to entropathogens such as E. coli. In the case of mucosal inflammatory pathologies, such as Crohn’s disease, this state of tolerance is broken, resulting in destruction of gut mucosal tissue where the macrophage phenotype has been altered from a regulatory M2-like subset phenotype to an inflammatory M1-like subset phenotype, responding to both pathogenic and commensal bacteria. Chronic inflammation by bacterial pathogen related molecular patterns (PAMPs), such as LPS, is well established to induce tolerisation. The aims of this project were firstly, to characterise the control of macrophage differentiation in a mucosal setting by investigating the immunomodulatory effects of PAMPs, such as LPS in presence or absence of TNFα and to investigate ET mechanisms associated with MΦ subsets responding to the entropathogen E. coli K12-LPS. Secondly, to investigate the effect of epithelial cells on macrophage subsets behaviour upon inflammation and ET. M1- and M2-like MΦs were generated in vitro from the THP-1 monocyte cell line by differentiation with PMA and vitamin D3, respectively, whereas differentiated epithelial cells (Caco-2) were obtained by long term culturing for 21 days. A transwell co-culture system of Caco2 cells and MΦ subsets was developed to mimic the cell-to-cell cross-talk between epithelial cells and immune cells. Mono- and co-culture models were pre-treated with either LPS, TNFα or IL-1β prior to stimulation by PAMPs. TNFα, IL-1β, IL-18, IL-6 and IL-10 were qualified by ELISA. Cytokines, PRRs and endogenous negative regulatory molecules were detected by RT-PCR and WB and epithelial barrier function was measured by trans epithelial electrical resistance (TEER). ET induced by K12-LPS failed to demonstrate a differential subset-specific response in MΦ mono-culture system whereas, LPS differentially suppress LPS induced cytokine expression in MΦ co-culture system. Tolerised M1- and M2-like MΦs exhibited a significant reduction in expression and secretion of pro-inflammatory cytokines and comparable levels of anti-inflammatory cytokine, IL-10. The suppression of pro-inflammatory cytokine in these MΦs appeared to be linked to the differential TLR4 expression and up-regulation of negative regulators, such as IRAK-M and Tollip. In addition, MΦ subsets differentially responded to inflammation induced by pro-inflammatory cytokines, TNFα and IL-1β in mono- and co-culture models. In conclusion, tolerisation induced in MΦs is presented by the suppression of pro-inflammatory cytokine, which is associated with corresponding up-regulation of IL-10, TLR4 receptor and the negative regulators, in a subset-independent manner. In the case of cross-talk between epithelial cells and macrophages however, a differential sensitivities to ET was displayed. These findings allow more understanding of MΦ subsets functions and ET mechanisms, which may be beneficial for the development of in-vitro models of MΦ subsets and therapeutics targeting Crohn’s diseases.

The NF-κB transcription factor is expressed in the majority of mammalian cells and regulates a large number of genes with important functions in a variety of cellular processes including cell growth, division, apoptosis and inflammatory responses. Perturbation of NF-κB response has been implicated in a variety of diseases such as asthma and inflammatory bowel disease, in addition to various forms of cancer. Through experiments at the single cell level it has been shown that NF-κB displays complex temporal activation, notably including nucleo-cytoplasmic oscillations. It has been observed that these oscillations occur in a heterogeneous manner; as such they are masked when measured at the population level. In contrast, pulsed TNFα treatment at 100 min intervals produces regular and synchronous nuclear peaks of NF-κB. Such pulsatile stimulation may reflect more accurately physiological conditions. The work in this project uses a Systems Biology approach consisting of bioinformatic, mathematical, and experimental methodologies to investigate how NF-κB can regulate such a diverse set of gene responses. Previously published studies have proposed that target gene expression levels following NF-κB activation (continuous TNFα) can be explained by a combination of key parameters, including transcript degradation rate, transcript structure, and transcription initiation rate. Initial work in this project highlighted that these explanatory factors are not sufficient to describe the observed temporal order of gene transcription. The roles of miRNAs and NF-κB subunit phosphorylation in regulation were additionally explored. A large set of genes was identified that are activated more strongly by pulsed TNFα than by continuous TNFα treatment. This suggests a new unreported mechanism of gene regulation, the possible causes of which are examined in this thesis. The gene list was refined by altering pulse frequency, which revealed an enrichment of NF-κB targets correlated with the regularity of these pulses. Temperature shift and anti-inflammatory drug treatment (Diclofenac) were shown to have a profound effect on NF-κB oscillation frequency. These perturbations provide an alternative method to study the effects of NF-κB oscillation frequency on specific target genes, independent of a pulse regime. Integration and analysis of these datasets suggested that a core, frequency-encoded set of genes regulated by NF-κB might exist. It is proposed that such genes may respond optimally to specific frequencies of NF-κB activation, implying a potential frequency threshold. The presence of such genes may explain the need for the complex systems that control NF-κB timing. It was noted that there was an enrichment of genes encoding transcription factors within the frequency encoding set, in addition to proteins which are known to be involved in the control of inflammation.

Inflammatory bowel disease (IBD) continues to affect millions worldwide, with an increasing prevalence that highlights the urgent need for deeper understanding of its underlying immune mechanisms. The cytokine interactions, especially those mediated by cells from the TH1 and TH17 lymphocyte subsets, are crucial in orchestrating the immune landscape of IBD. TH1 cells are well known for producing TNF-α and IFNγ, which have been extensively studied for their roles in conjunction with each other within the context of IBD (Fish, 1999). TH17 cells secrete IL-22 and IL-17, with existing studies primarily focusing on IL-22’s interaction with IL-17 rather than its interplay with other cytokines such as IFNγ. Our study focuses on the co-stimulatory effects of IL-22 and IFNγ using organoids derived from mouse small intestines to model epithelial interactions. We found that IFNγ interferes with the capacity of IL-22 to up-regulate antimicrobial peptides, which is essential in mucosal defense. Additionally, higher concentrations of IL-22 enhance IFNγ's ability to stimulate TNF-α gene expression and CXCL10 protein production, indicating a dose-dependent relationship. This co-stimulation also led to an increased rate of cell death, influenced partly by TNF-α. These findings suggest that IL-22, typically seen as an anti-inflammatory agent, can assume a pro-inflammatory role when combined with IFNγ, complicating its effects on epithelial cells. This study highlights the need to consider specific cytokine interactions in developing more effective IBD treatments.

Replication-deficient adenovirus (Ad) and modified vaccinia virus Ankara (MVA) vectors expressing single Plasmodium falciparum antigens can induce potent T cell and antibody responses and have entered clinical testing using a heterologous prime-boost immunisation approach (Ad_MVA). This thesis describes a number of pre-clinical approaches aimed at enhancing the efficacy of these viral vectored vaccines targeting the blood-stage of malaria. First, the development of a highly efficacious malaria vaccine is likely to require a multi-antigen and/or multi-stage subunit vaccine. The utility of an Ad_MVA immunisation regime combining vaccines expressing the 42kDa C-terminus of the blood- stage antigen merozoite surface protein 1 (MSP142) and the pre-erythrocytic antigen circumsporozoite protein (CSP) in the P. yoelii mouse model was investigated. It was found that vaccine co- administration leads to maintained antibody responses and efficacy against blood-stage infection, but reduced secondary CD8+ T cell responses and efficacy against liver-stage infection. CD8+ T cell interference can be minimised by co-administering the MVA vaccines at separate sites, resulting in enhanced liver-stage efficacy. The mechanisms of CD8+ T cell interference were explored. Second, Ad_MVA regimes expressing blood-stage antigens that can protect against P. chabaudi and P. yoelii blood-stage infection were tested against P. berghei, but did not confer protection. Similarly, IgG from rabbits immunised against P. falciparum MSP1 (PfMSP1) could not protect mice from a chimeric P. berghei parasite expressing PfMSP1. Third, two molecular adjuvants, the C4bp α-chain oligomerisation domain (IMX108/313) and the Fc fragment of murine IgG2a, were tested for their ability to enhance immunogenicity of recombinant adenoviruses when fused at the C-terminus of a blood-stage antigen. IMX108/313 was found to adjuvant T cell responses of small (< 80kDa) antigens and this was associated with antigen oligomerisation. However, the Fc fragment did not adjuvant responses. Finally, it was found that using a strong early promoter to drive antigen expression enhances the immunogenicity of single administration MVA vaccines, but that this did not enhance post-boost immunogenicity in an Ad_MVA regime.

The purpose of this study was to investigate lipocalin-2 (Lcn2) as a potential reactive astrocyte marker in the glaucomatous optic nerve. Glaucoma is a disease of the optic nerve that results in irreversible vision loss secondary to retinal ganglion cell damage and astrocyte reactivity in response to glaucomatous injury. Due to the role of Lcn2 in reactive astrocytes of glaucomatous optic nerves being poorly elucidated, we studied Lcn2 in the setting of optic nerve crush (ONC), DBA/2J, and microbead injection (ocular hypertension) models. In situ hybridization and immunohistochemistry (IHC) were used to detect and localize Lcn2 in tissue samples. Quantification was performed using qPCR. We found that Lcn2 expression is upregulated in the ONC and DBA/2J models, but not in the microbead injection model. IHC revealed Lcn2+ cells starting 4 h following ONC. However, in contrast to what was expected, these cells were not astrocytes or microglia. Instead, the co-labeling of Lcn2+ cells with CD3 indicates a T-cell population. qPCR screening of ONH astrocytes cultured in Lcn2 revealed that neither the neurotoxic A1 nor neuroprotective A2 reactive astrocyte phenotype was acquired. Instead, Lcn2 differentially upregulated the inflammatory marker TNF-α in astrocytes, thus demonstrating that Lcn2 is not a marker for reactive astrocytes. These findings propose a link between Lcn2 and TNF-α that may be key in decreasing ON inflammatory degeneration.

In these experiments, SIM-A9 microglia were exposed to Staphylococcus aureus cell wall components lipoteichoic acid (LTA) and peptidoglycan (PGN) at a concentration of 5 ug/mL for six, twelve, eighteen, and twenty-four hours and the cytokine expression, cytoskeleton organization, and cell viability of the cells were observed. Following LTA and PGN exposure, TNF-a; secretion increased at each time interval and was highest observed at 24 hours. No significant IL-10 secretion was detected. Over the 24 hour period, cell viability and cytotoxicity of LTA and PGN treatment groups were not significantly different from the control, indicating the observed inflammatory cytokine response was not due to cell death. These data suggest that LTA and PGN play a predominantly inflammatory role in the first twenty-four hours of S. aureus CNS infection.

Mastitis, an inflammation in the mammary gland, is one of three major diseases in the dairy industry. One in three cows will encounter the disease which is also a problem in humans and other species. While E. coli bacterial infections lead to acute mastitis, S. aureus lead to chronic mastitis. Dynamics of the regulation and identification of differentially expressed genes between two bacterial infections are important factors for understanding mastitis and assist in the development of pharmaceutical and breeding targets. Previous studies have identified differentially expressed genes. However, they have not compared expression between two bacterial infections over time. Neither have the dynamics of the signalling and gene network regulation that leads to the differential expressions been investigated. This thesis aims to provide new insight into immune defence in mastitis by analysing dynamics of the signalling and gene regulation in mammary epithelial cells. The main focus is to develop a mathematical model of the signalling and gene network regulation in mastitis. First, the genes differentially regulated between the two clinical presentations of the disease are identified. Time series microarray experiments of E. coli and S. aureus challenged mammary epithelial cells are analysed, and confirm that each type of mastitis has a significantly different gene expression time profile from healthy cells. The differentially expressed gene time profiles are then compared between the bacterial challenges. RANTES is identified as the key cytokine which is responsible for two distinctly different time profiles between the bacterial challenges. In this second part the mathematical model is developed and a systems biology approach applied to investigate the complex dynamics of signalling proteins and gene network regulation of three different cytokines (RANTES, IL8 and TNFα) in mastitis. A modification to a conversion method allows us to use relative microarray expression data in the model. The method opens up a large amount of datasets for use in future modelling. The model explains signalling and gene network regulation of three cytokines in acute mastitis. No fit could be found for the S. aureus experimental data indicating that there is a difference in the regulatory mechanisms between the two types of mastitis. In the third part sensitivity analysis is used to investigate the role of parameters on the model output. The analysis reveals that each cytokine is sensitive to specific parameter changes. This indicates different dynamics in the regulatory mechanism. As a result, pharmaceutical and breeding targets need to be evaluated in the context of all cytokines to prevent undesirable side effects. The importance of modelling prior to experimental design is also revealed; each cytokine has a specific time frame for the most informative experimental measurement. In the fourth part robustness analysis is used to investigate the role of the bacterial load on the model output. Robustness analyses indicate that robustness does not originate in the nuclear NFκB time profile and is specific for each cytokine. Finally, future directions of the model and biological experiments are discussed.

Mucin 1 (MUC1) is a plasma membrane-bound glycoprotein that plays a protective role in corneal epithelial cells. Two full-length splice variants of MUCl: MUCl/B and MUCl/A, that differ by the inclusion of 27 bp from intron 1 and a SNP in MUCl/A, but have identical C-terminal cytoplasmic domain (CD) sequences, were identified in human conjunctival tissue. I tested the hypothesis that MUC1 splice variants are key immunoregulators that act on the ocular surface to protect the ocular surface from inflammation and that their expression correlates with dry eye status. The expression of MUC1/A and MUCl/B splice variant was examined in non-Sjogren's aqueous deficient and evaporative dry eye patients and compared to normal controls. The frequency of MUC1/A gene expression was lower in patients with non-Sjogren's aqueous deficient dry eye compared to controls, indicating a link between MUC1/A and the symptoms associated with dry eye disease. Overexpression of MUC1/A and MUC1/B in COS-7 cells resulted in equal protein expression and plasma membrane localization. MUCl/B and MUCl/A splice variants differed in their ability to modulate the TNFa-induced inflammatory responses. The MUCl/B splice variant, and not MUCl/A, inhibited the induction of IL-1ß and IL-8 expression by TNFa at 4 h. In contrast, MUC1/A stimulated TNFa-driven IL-8 mRNA and protein expression after 24 h of treatment. MUC1/B, but not MUC1/A, inhibited TNFa-induced luciferase activity from an NF-KB reporter. Both MUC1/B and MUCl/A blocked the induction of miR-21 by TNFa. In addition, MUC1/A, but not MUCl/B, increased basal expression of TGFß. These data demonstrate for the first time that MUCl/A and MUC1/B are genetic susceptibility factors with different anti-inflammatory activities.

The present study examined adverse effects of sunlight exposure (mimicked by intradermal cis-urocanic acid, cUCA) on local and systemic immune responses, with or without co-exposure to the immunotoxic insecticide permethrin. A single exposure to cUCA caused diminished splenic macrophage phagocytosis that was persistent up to 30 days post-exposure. Five-day exposure to cUCA subtly increased splenocyte proliferation in response to the T cell mitogen Concanavalin A. Four-week exposure to cUCA caused increased splenic lymphocyte cellularity, thymic hypocellularity, and enhanced hydrogen peroxide production by splenic leukocytes. Single exposure to topical permethrin resulted in decreased thymic and splenic weight and cellularity, and inhibited antibody production by splenic B cells. cUCA worsened the negative effect of permethrin on both thymic weight and cellularity, and depressed splenocyte blastogenesis, hydrogen peroxide production, and antibody production. Five-day exposure to either cUCA or permethrin also caused persistent decreased contact hypersensitivity responses, an effect that became more than additive when the chemicals were administered concurrently. Defects in antigen processing and presentation by cutaneous Langerhans cells were evaluated as possible contributing mechanisms to the cutaneous immunosuppression, using mice with deleted genes. Vehicle-exposed IFNg knockout mice displayed approximately a 22.1% depression in the ear swelling response as compared to control C57BL/6N mice, suggesting that this cytokine may be required for mounting a control-level hypersensitivity response. Ear swelling in cUCA-exposed IFNg knockout mice displayed a 21.4% depressed response as compared to cUCA-exposed wild-type C57BL/6N mice, again suggesting that IFNg is an important cytokine in the contact hypersensitivity (CH) response. TNFaR knockout mice exposed to cUCA displayed 33.9% greater ear swelling than cUCA-exposed wild-type C57BL/6N mice, suggesting that increased TNFa may be involved in inhibited CH by cUCA. TNFaR knockout mice exposed to permethrin displayed 33.9% greater ear swelling than permethrin-exposed C57BL/6N mice, suggesting that increased TNFa may also be involved in inhibited CH by permethrin. C57BL/6N mice exposed to cUCA + permethrin displayed severe reduction of the CH response to 8.7% of the control level. IFNg knockout mice exposed to permethrin + cUCA showed essentially identical depression of the CH response as IFNg knockout mice exposed to either permethrin or cUCA alone. These results suggest that IFNg is required for the greater than additive immunotoxic effect that occurred when these two agents were co-administered. TNFaR knockout mice exposed to cUCA + permethrin displayed 8.7 fold greater ear swelling than similarly exposed C57BL/6N mice, again suggesting that increased TNFa is involved in inhibited CH by both cUCA and permethrin.