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Chronic inflammation is considered a key contributor to individual differences in brain ageing. However, there remains conflicting evidence about the exact brain structural and functional consequences of chronic inflammation. Part of this ambiguity comes from the lack of robust biomarkers used to characterise chronic inflammation, with most studies conflating acute vs chronic inflammation and relying on single time point sampling of highly phasic inflammatory markers from blood. Recent research indicates the potential of epigenetics to circumvent this issue, in particular the use of DNA methylation (DNAm) to provide a biological archive of inflammatory burden. DNAm is an epigenetic mechanism that regulates gene expression and acts as an interface by which lifestyle and environment can influence phenotype; alterations in DNAm are increasingly investigated as proxies for certain exposures, traits, and conditions. This thesis explores chronic inflammation and its relation to brain health using a multi-omic approach, examining associations of proteomic and DNA methylation signatures of inflammation with brain measures from structural and diffusion MRI and cognitive ability at different stages of the lifecourse. Following introductory chapters presenting overviews of epigenetics, inflammation and neuroimaging and cognitive metrics, the empirical work of this thesis examines these interrelationships in three population cohorts covering the human lifespan: from infancy in Theirworld Edinburgh Birth cohort (TEBC; age < 1 year), to mid to late adulthood in Stratifying Longitudinal Resilience & Depression Longitudinally cohort (STRADL; age ~ 60 years, range 28 - 81 years), to older-age in the Lothian Birth Cohort 1936 (LBC1936; age ~ 73 years). First, neuroimaging and cognitive associations of chronic inflammation in older age are examined. In 521 individuals from the LBC 1936 cohort, associations between a DNA methylation predictor of C-Reactive Protein (DNAm CRP) and brain structure and cognition are shown to be consistently stronger (6.4-fold greater on average) than those with traditional serum CRP measures: with higher DNAm CRP levels significantly associated with global and regional brain atrophy (β range |0.200| to |0.150|), differences in white matter microstructure and white matter hyperintensity burden (β range |0.099| to |0.162|), and poorer global and domain- specific cognitive functioning (β range |0.095| to |0.158|). This paper also demonstrates that the association between inflammation and cognitive ability is partially mediated by brain structure (up to 29.7%), dependent on lifestyle and health factors. The second empirical chapter replicates this association of an epigenetic inflammatory signature with brain and postnatal health outcomes in early life. In a neonatal cohort of 258 infants, the relationship between DNAm CRP with perinatal health and neuroimaging outcomes is investigated. The results support the theory that DNAm may be leveraged to capture a more cumulative impact of inflammatory burden, with DNAm CRP being higher in preterms compared to term infants and higher DNAm CRP levels associating with perinatal inflammatory-related morbidities (such as late onset sepsis and histologic chorioamnionitis) both individually and in aggregate (OR range |2.00 | to |4.71|). The main finding - that elevated DNAm CRP associates with poorer measures of white matter, both globally and regionally (β range |0.206| to |0.371|) - is considered in the context of how early birth associates with an increase in immune-related risks, which coincide with windows of neurodevelopmental plasticity, highlighting the vulnerability of developing white matter to inflammatory insults. Finally, the brain health associations of signatures of inflammation in midlife are considered. In 709 participants from the STRADL cohort, DNAm signatures for CRP alongside a range of other inflammatory-immune mediators are constructed and compared against protein levels in relation to neuroimaging and cognitive metrics. 73 unique DNAm signatures associated with numerous aspects of global brain structure and cognitive ability (β range |0.097| to |0.200|), alongside regional atrophy across the brain's cortex (β range |0.087| to |0.260|) and focal vulnerability of specific white matter tract microstructure (β range |0.103| to |0.185|). Many of these DNAm-brain associations were larger than the analogous proteome-brain associations, broadly independent of immune-cell proportions, clinical risk factors, and had previously been linked to various age-related diseases, reinforcing the central role that inflammation plays in health trajectories. The results of this chapter suggest that associations with chronic inflammation and brain structure are apparent in mid to late adulthood and may precipitate and underlie changes in cognitive functioning seen in older-age. This PhD characterises the global and regional associations of chronic inflammation on brain structure at different stages of life, elucidating new perspectives on how these mechanisms may be contributing to individual differences in cognitive ability. I highlight the potential of the developing field of epigenetics to offer a solution to the traditional limitations of assessing inflammatory burden in human cohorts, and how this may be used to gain a better understanding of how inflammation relates to aberrant neurodevelopment and cognitive decline. These findings provide new insights about the extent of inflammation's impact on brain health, as well as highlighting the utility of DNAm for risk prediction and stratification in relation to brain health outcomes.

Inflammation is a complex, dynamic process, which despite extensive research, is still far from fully understood. The use of model organisms provides unique insights into the mechanisms underlying inflammatory cell recruitment. Previous work using the fruit fly Drosophila melanogaster has demonstrated that following laser-induced epithelial wounding, hydrogen peroxide is rapidly produced, and it is this early damage signal that leads to an inflammatory response, via direct activation of the redox sensitive tyrosine kinase Src42a within responding macrophages. Src42a phosphorylates Draper, the key damage receptor in fly macrophages, which in turn recruits a downstream kinase, Shark, resulting in macrophage recruitment to wounds. More recently, the phosphatase Pez has been demonstrated to interact with both Src42a and Draper and is required for efficient inflammatory macrophage recruitment to sites of damage in vivo. Importantly, macrophages are insensitive to tissue damage until they become 'primed', by engulfment of an apoptotic corpse, leading to an increase in Draper expression. Given macrophage insensitivity to tissue damage prior to priming, we utilised the tractable genetics of Drosophila and undertook RNA sequencing of both primed and un-primed cells in vivo, identifying many differentially expressed genes. Selected candidates were used in macrophage specific, RNAi knock down wounding studies, revealing novel players in the regulation of macrophage recruitment to wounds. The mechanism underlying Pez signalling within the macrophage is poorly understood; we utilised live imaging in Drosophila embryos to investigate Pez intracellular dynamics during inflammatory cell migration. Microtubule mediated transport and clathrin mediated endocytosis have been excluded, however, we show that dynamic Pez puncta correlate with retrograde actin flow providing a potential mechanism for the regulation of Pez trafficking in macrophages in vivo. Evolutionary conservation of Pez and Draper signalling in modulating wound induced, inflammatory cell migration has previously been demonstrated using zebrafish larvae in a tail fin transection model. Here, we show that the Pez homologue in mouse, PTPN21, and the murine Draper homologues, MEGF11 and PEAR1, play a role in blood monocyte and tissue resident macrophage population dynamics through the use of congenic CD45 bone marrow chimeric mice. Specifically, PEAR1 knock out leads to a decrease in circulating classical monocytes, whilst PTPN21, MEGF11 and PEAR1 knock out leads to a decrease in alveolar macrophage, but not peritoneal macrophage, numbers. The scope for inflammation research is almost unlimited and in order to further dissect the cellular processes that underlie the inflammatory response in vivo, model organisms must be utilised. Here, we have demonstrated that different model organisms can be exploited in a synergistic manner, facilitating experiments and answering questions that would be impossible to undertake using a single system.

Allergic asthma caused by house dust mites (HDM) is highly prevalent and has links to the onset and exacerbation of asthma symptoms and the induction of an IgE-mediated immune response. HDM allergens contain proteolytic activities, including serine proteases such as trypsin-like proteases (TLPs) and chymotrypsin-like proteases (CTLPs), as well as cysteine proteases, which can interfere with various cellular functions. The aim of this study is to explore the impact of house dust mites (HDM) and its protease components on asthma epithelium, specifically focusing on their effect on calcium signalling and pro-inflammatory responses in asthmatic human primary bronchial epithelial cells (hPBECs). We discovered that HDM proteases, particularly serine proteases, activate protease-activated receptors (PARs) associated calcium signalling and lead to increased expression of pro-inflammatory alarmins, such as Interleukin 33 (IL-33) and thymic stromal lymphopoietin (TSLP). Furthermore, in a differentiated culture model of asthma hPBECs, we observed HDM-induced calcium mobilization involved PAR-2 and PAR-4 activation, as well as plasma membrane calcium channels from transient receptor potential vanilloid family TRPV1 and TRPV2. This study also explored the impact of HDM and HDM proteases on macrophage-like THP-1 cells, demonstrating increased secretion of pro-inflammatory cytokines and chemokines including IL-10, IL-6, Cxcl1 chemokine (CXC motif) ligand 1(CXCL1), tumour necrosis factor alpha (TNF-α), IL-8, along with elevated CTLPs activities and matrix metalloproteinase-9 (MMP-9), active-matrix metalloproteinase-2 (MMP-2) activities in cell condition media of macrophage-like THP-1 cells. Taken together, this study enhances our knowledge of the role of serine proteases in HDM-induced inflammation and regulation of ion channel function in asthma.

Alongside tumour resection, cancer radiotherapy is a gold standard intervention to manage cancer progression and is given to 27% of cancer patients in a curative or palliative capacity as part of their treatment in England each year (NCRAS, 2017). Yet, by virtue of killing cancer cells, radiotherapy causes acute tissue damage and inflammation at the cancer site, and there is growing evidence to suggest that this inflammation may contribute to the re-population of surviving cancer cells. Despite its routine use within the clinic, it is poorly defined how γ-irradiation causes cancer cell death, and less still is known of the ensuing inflammatory response to surviving cancer cells. I utilise the translucent zebrafish model to visualise how radiotherapy affects cancer progression in vivo and how, if at all, it alters the inflammatory response to cancer cells. I show that radiotherapy stalls cancer expansion by inducing significant dsDNA damage to pre-neoplastic cells which leads to reduced levels of mitosis and increased levels of cancer apoptosis. Furthermore, radiotherapy alters the inflammatory response to pre-neoplastic cells and increases the frequency of neutrophil/pre-neoplastic cell interactions when compared to unirradiated control larvae. These data indicate that as a consequence of tissue damage caused by radiotherapy, the inflammatory response to pre-neoplastic cells is enhanced. The use of anti-inflammatory drugs to suppress this newly identified radiotherapy-induced inflammatory response to cancer may reduce the growth of cancer cells which survive irradiation and improve rates of progression-free survival following cancer radiotherapy. Pre-neoplastic clones release a damage signal, hydrogen peroxide (H2O2), which recruits inflammatory cells, and these interactions are known to promote clonal expansion. However, immune cells are still recruited to clones that cannot produce H2O2, suggesting that other damage signals may also trigger inflammation and expedite cancer development. High-mobility group box protein 1 (Hmgb1) is a nuclear protein and a damage-associated molecular pattern (DAMP) released at wounds. Hmgb1 overexpression is associated with inflammation and tumour progression, but its role during early clone development is poorly understood. I show that in zebrafish larvae, tissue levels of Hmgb1a are enriched at wounds and in the immediate vicinity of growing RasV12.GFP clones. The subsequent knockout of hmgb1a showed reduced neutrophil recruitment to both wounds and pre-neoplastic clones, while the response of macrophages remained unaffected. Additionally, live imaging showed delayed recruitment of neutrophils to wounds, and shorter lesion interactions upon arrival. Similarly, neutrophil interactions with pre-neoplastic clones were less frequent in hmgb1a-null mutants and lasted for less time when compared to interactions in wild type larvae. Importantly, hmgb1a knockout larvae displayed fewer and smaller pre-neoplastic clones than their wild type siblings. These results suggest that enhanced levels of Hmgb1 around pre-neoplastic clones mimic a wound and enhance the recruitment of neutrophils to the neoplasm, which, in turn, promotes cancer progression. These data indicate that therapies targeting Hmgb1 might be useful in dampening the inflammatory response to cancer to inhibit its growth.

Neovascular Age-Related Macular Degeneration (nAMD) can display, in the late stage, the development of a fibrotic plaque in the subretinal space which we refer to as macular/subretinal fibrosis. Subretinal fibrosis is a major cause of anti-Vascular Endothelial Growth Factor (VEGF) drug resistance in nAMD. Unfortunately, it remains unpreventable and untreatable due to the incomplete understanding of the disease aetiology. Over the years, evidence regarding the critical role of C-C Chemokine Receptor type 2 (CCR2) and CX3C Chemokine Receptor 1 (CX3CR1) subsets of macrophages in the progression of AMD have emerged. However, their role in the development of subretinal fibrosis remains unknown. This study aimed to understand how subretinal fibrosis develops secondary to choroidal neovascularisation (CNV) by investigating the role of CCR2+ infiltrating macrophages and CX3CR1+ macrophages/microglia. In Chapter 3, we demonstrated that CX3CR1+ microglial cells were importantly involved in the development of CNV in the laser-induced CNV model. This was also confirmed in aged LysMCre-Socs3fl/flCx3cr1gfp/gfp (DKO) mice, which display microglial dysfunction and micro-retinopathy with age, indicating a potential role of microglia in retinal angiogenesis. In Chapter 4, we showed, using a mouse model of two-stage laser-induced subretinal fibrosis (TSLSF), that CX3CR1+ microglia depletion did not affect subretinal fibrosis progression. However, aged DKO mice displayed larger fibrovascular lesions suggesting that subretinal fibrosis may be supported by uncontrolled microglial activation. Finally in Chapter 5, we demonstrated, using a mouse model of TSLSF, that both phagocyte subsets may play distinct roles in the progression of subretinal fibrosis as our results indicated that CCR2+ monocytes play a critical role in the development of the fibrovascular phenotype during the chronic stage of inflammation. Overall, this study gives new insights in the understanding of the phagocyte infiltration dynamic in the subretinal space following injury and their implication towards the progression of subretinal fibrosis secondary to CNV.

During development, there are windows in which the brain is particularly susceptible to the impact of challenges - these include the pre-natal, early post-natal, and peri-pubertal periods. As such, adverse childhood experiences (ACEs), such as stress exposure, confer greatly increased risk of developing anxiety and depression in adult life. Previous work has found evidence for dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis, alterations in brain structure, and elevated inflammation markers in adults exposed to adverse and traumatic experiences during childhood. Recently, chronic, low-level inflammation has also been associated with depression and anxiety risk, making stress and inflammation potentially cumulative risk factors for the development of psychiatric illness. A rat model of an early life stress paradigm combined with an adolescent immune challenge was designed to evaluate the contributions of these insults to brain development and future behaviour. The early life stress model chosen was the limited bedding and nesting material (LBN) paradigm: a naturalistic model of maternal deprivation previously validated to produce aberrations in maternal behaviour, and anxiety-like traits as well as HPA axis dysregulation in the offspring. Assessment of maternal behaviour during the LBN period revealed disruption to maternal behaviour, including evidence for fragmentation of maternal care, which has been strongly linked to poor outcomes in offspring. Offspring exposed to LBN or control conditions during postnatal days 2-10 were then given an immune challenge via administration of a low-dose of the endotoxin lipopolysaccharide (LPS) or vehicle control injections over five consecutive days during puberty onset. Weight data, and plasma interleukin-6 and corticosterone concentrations from blood samples taken on the first and last days of this inflammatory challenge revealed evidence of a mild inflammatory response. In order to study the influence of these early adverse experiences on long-term behavioural outcomes representative of depression and anxiety, rats were subjected to a battery of behavioural tests once they reached adulthood (>8 weeks). The results revealed a moderate impact of early life stress on anxiety-like behaviour, although the direction of this effect varied across two experimental cohorts. Gene expression was determined in brain regions-of-interest using quantitative polymerase chain reaction (qPCR). This revealed reduced expression of the mineralocorticoid receptor in the hippocampus, and of the glucocorticoid receptor in the amygdala of male LBN condition animals. In addition, both male and female LPS condition animals showed reduced glucocorticoid receptor expression in the hypothalamus, with an interacting effect of stress condition in the males. LPS treatment also significantly reduced Iba1 expression - a marker of microglial abundance - in hippocampus and hypothalamus samples from the adult rat brain. These results demonstrate a long-lasting impact of both elements of the 'double-hit' animal model on markers of stress responsivity and on the neuro-immune system. We do see some evidence for an interaction between the two challenges on the expression of the glucocorticoid receptor, but otherwise the two 'hits' chiefly appear to impact outcomes separately. In order to study the impact of the double-hit model on brain structure, post-mortem structural magnetic resonance imaging (MRI) scans were conducted in the male rats. Volumetric analysis using a semi-automated protocol for cortical and subcortical parcellation of MRI scans revealed stress-induced reductions in global metrics of brain volume, as well as changes in the volumes of the subiculum (a sub-region of the hippocampus) and the neocortex. A significant interaction between the two challenges on the volume of the superior colliculus - a region involved in threat perception - was observed. These MRI findings indicate effects of early life stress on gross brain development, as well as some regionally-specific effects on brain volumes which may underlie functional outcomes of both early life stress and an adolescent immune challenge. In order to provide a translational context for the outcomes of this novel animal model, analysis of a large cohort neuroimaging study examining the neurostructural sequelae of childhood trauma in two independent, UK-based adult populations was undertaken. There was strong evidence for associations between reports of childhood trauma and the incidence of depression and other psychiatric symptomology in adulthood. Volumes of the whole brain, grey matter, and white matter were found to be lower in adults reporting traumatic childhood events. This indicates a long-lasting effect of childhood adversity on brain structure. In addition, reduced volumes of hippocampal and thalamic structures were negatively associated with self-reported severity of childhood trauma. These data are consistent with the finding of lower global volume in the brains of rats exposed to early life stress. The data presented indicate exposure to early life stress induces alterations in stress-related behaviour, central gene expression, and brain structure in animals. Additionally, long-lasting changes in the resident immune cells of the brain after a mild systemic inflammatory event in adolescence are demonstrated. There is limited evidence for cross-talk between the two hits in this model, although there is evidence of a combined influence on the volume of the superior colliculus in post-mortem MRI data. Data derived from human neuroimaging cohorts provides a translational link to the animal models, with evidence of global brain volume reductions in both humans and experimental animals.

RPE dysregulation is known to be involved in age-related macular degeneration (AMD) which is the leading cause of vision loss in the elderly of developed nations. There are two types of AMD which are known as neovascular AMD (nAMD) and geographic atrophy (GA). nAMD patients may be treated with anti-VEGF intravitreal injections however, 20-40% of nAMD patients do not adequately or fully respond to anti-VEGF treatment. Therefore, new treatment options are urgently required. Levels of the acute stress response protein, lipocalin-2 (LCN2), were significantly increased in the blood plasma of nAMD patients with secondary fibrosis compared to healthy controls. Immunohistochemical studies demonstrated that LCN2 is upregulated in the murine RPE but concluded that LCN2 does not appear to have a role to play in fibrosis. LCN2 production by RPE cells was almost undetectable under normal conditions but LCN2 levels were significantly upregulated in response to the proinflammatory conditions induced by the TLR1/2 ligand Pam3CSK4, the TLR4 ligand LPS, and the TLR6/2 ligand FSL-1. However, the ligands of the other tested TLRs did not have any significant effect on LCN2 production. IL-1α, IL-1β, and IL-33 all significantly upregulated LCN2 production from B6-RPE07 but IL-18 had no effect. LCN2 expression was upregulated in the RPE and retina in mice with EAU in comparison to wild type (WT) mice. Recombinant LCN2 (rmLCN2) did not play a role in wound healing of B6-RPE07 cells nor did rmLCN2 promote epithelial-mesenchymal transition (EMT) in primary RPE cells. However, rmLCN2 did significantly reduce IL-33 gene expression in primary RPE cells and rmLCN2 rescued B6-RPE07 cells from oxidative stress. These results suggest LCN2 may have an anti-inflammatory role to play within the RPE. Future studies to understand LCN2's function in the RPE under inflammatory conditions may help with the development of more effective AMD treatment strategies.

Nociception is a protective mechanism alerting an organism to noxious stimuli and potential harm. However, dysregulation of the nociceptive system can result in chronic pain, which has a prevalence of approximately 40 % in the adult population. Current therapeutics are often inefficacious, and the growing opioid crisis demonstrates a clear need to develop new analgesics and improved pain management strategies. A common feature of inflammation is tissue acidosis, and the raised extracellular proton concentration can activate acid-sensing ion channel 3 (ASIC3), which is most highly expressed in those sensory neurones tuned to detect noxious stimuli, i.e., nociceptors. To date, the most potent non-peptide ASIC3 inhibitors act at micromolar concentrations *in vitro* on rat-ASIC3 (rASIC3), but are non-selective, thus inhibiting other ASIC subunits. Intriguingly, certain non-steroidal anti-inflammatory drugs (NSAIDs) directly inhibit rASIC3 but are neither potent ligands nor is it understood precisely how they interact with ASIC3. Here, I aimed to use *in silico* modelling to not only predict the plausible binding mode of some known ASIC3 modulators to this channel, but to further identify new ligands that can modulate ASIC3. Homology models of rASIC3 were constructed based on published 3D structures of chicken ASIC1a solved at various states. These models were then used for blind docking with some known small molecule modulators of ASIC3 that notably included the NSAID diclofenac. The resultant poses of these ligands were then subjected to further refinement using a focused docking approach. Altogether, this led to a prediction of a potential binding site and mode of binding for the ASIC3 selective NSAID inhibitors near the acidic pocket domain of rASIC3. A 2D-ligand similarity approach was undertaken to identify scaffolds possessing key functional groups and physico-chemical properties that were similar to those known ASIC3 modulating NSAIDs, and subsequently docked to predict binding interactions. Using these criteria, three molecules (diflunisal, fenbufen and tolmetin) were chosen from a number of hits and were then tested for their ability to modulate the function of rASIC3 transiently transfected in Chinese hamster ovary cells using whole-cell patch-clamp electrophysiology. This *in silico* approach was also conducted for pro-inflammatory mediators known to activate/enhance ASIC3 activity, which identified potential physiological modulators. Upon activation, the ASIC3 current showed two characteristic phases: a rapid transient phase followed by a prolonged and smaller sustained phase in the presence of continued stimulation, and this was in complete agreement with existing literature. Diclofenac significantly inhibited the sustained, but not the transient phase of the current at pH 4, but no effect on either phase was observed at pH 5. Conversely, the three hits identified *in silico* showed a varying degree of inhibition on the sustained phase at pH 4 and 5. Finally, site-directed mutagenesis was conducted to validate those amino acids computationally predicted to be involved in NSAID modulation of ASIC3. This thesis outlines a method to predict binding regions of ASIC3 ligands and identifies a possible functional region of ASIC3 by which these ligands interact. These results provide a workflow for identifying novel modulators of ASIC3, which may be of analgesic application.

Background: The inflammatory response relies on the well-coordinated trafficking of leukocytes from the circulation to sites of tissue injury. The endothelial expression of cell adhesion molecules, namely the selectin family, mediates the initial adhesive interactions between leukocytes and the endothelium. Immune cell infiltration is often involved in the initiation and progression of several vascular diseases, including atherosclerosis. Plasma Membrane Calcium ATPase 4 (PMCA4) belongs to a family of transmembrane ion transporters that extrude calcium from the cytosol to the extracellular environment (Strehler, 2015). They are the only high-affinity Ca2+ extrusion system in mammalian cells encoded by the ATP2B1-4 genes (PMCA1-4) (Strehler, 2015). PMCA4 acts as a structural protein providing a scaffold to interacting proteins at the plasma membrane of endothelial cells. Several of these interactions demonstrate functional significance, accounting for its emerging role in signal transduction. This work contributes to the delineation of PMCA4 in the physiology of the vascular endothelium and characterises a role in leukocyte adhesion and trafficking. Methods and Results: The effect of the pro-inflammatory cytokine IL-1β on the expression of PMCA4 was examined. A time and dose-dependent downregulation of PMCA4 mRNA and protein expression in human Aortic Endothelial Cells (AoEC) was observed. Differential gene expression was determined in PMCA4 silenced AoEC using an ECM-CAM array and RNA-sequencing. The siRNA-mediated knockdown of PMCA4 induced the expression of cell adhesion molecules, Selectin P (SELP) and SELL. The knockdown enhanced the IL-1β-induced expression of the metalloproteinases ADAMTS1 and -4. Ingenuity pathway analysis (IPA) revealed a role in immune cell trafficking with a propensity to develop aneurysms (activation z-score,-1.177). Similarly, silencing PMCA4 enhanced the VEGF-induced expression of ADAMTS1, VCAM-1 and SELE. Cyclosporin A partially ablated this enhancement through VEGF, implicating CN/NFAT transcriptional activity. Consistent with the IPA, a significant decrease in PMCA4 mRNA expression was observed in abdominal aortic lesions from hypercholesterolaemic ApoE-deficient mice infused with angiotensin II. Conclusion: The downregulation of PMCA4 is associated with a state of endothelial activation, indicated by the induction of cell adhesion molecules. Our results demonstrate that PMCA4 regulates intracellular signalling in the endothelium in response to leukocyte-derived inputs. Reinforced by bioinformatic analysis, our findings suggest that PMCA4 may influence leukocyte adhesion and trafficking on the endothelium.

Inflammation is a central process in homeostasis, and dysfunctional inflammatory responses have important implications in disease, for instance in severe COVID-19 pathogenesis and cancer. Inflammation is a metabolically demanding process, and targeting immune cell metabolism may open novel avenues for treatment of aberrant inflammatory diseases and malignancies. Production of high quantities of nitric oxide (NO) by the enzyme inducible NO synthase (iNOS) is found in chronic inflammation and cancer and is implicated in pathology. Here, we demonstrate that iNOS expression and activity are also enhanced in colon and prostate cancer and in severe COVID-19 disease. iNOS is expressed, although at low percentages, by human macrophages from tumours and from bronchoalveolar lavage fluid of severe COVID-19 patients. While macrophages do not comprise the main source of iNOS in tissue, we demonstrate that NO produced by neighbouring cells is sufficient to induce nitrotyrosine (NT) formation in macrophages, thus mediating its actions on these cells. Moreover, we show that both endogenous and exogenous NO promote alterations in macrophage effector function, favouring a pro-inflammatory phenotype and contributing to enhanced and sustained pro-inflammatory cytokine secretion. NO also reduces antigen presenting function of macrophages and impairs several M2 functions. NO mediates its effects by mitochondria-dependent mechanisms, through inhibition of respiration, and mitochondria-independent actions by HIF-1-alpha stabilisation. We also highlight a relationship between iNOS, COX-2 and HIF-1-alpha in macrophages, which may be relevant in the context of inflammation and cancer. Our results suggest that NO favours exaggerated pro-inflammatory responses, subverts adaptive immunity responses and impairs healing, which may contribute to inflammation that favours tumour development and growth and that is characteristic of severe COVID-19. Targeting inflammation and metabolism through NO signalling may be beneficial in such diseases. Systemic NO generation, evidenced by levels of protein-bound NT, may also predict COVID-19 severity and outcome, and further investigations are needed to evaluate its prognostic value in cancer.

Witch hazel (Hamamelis) bark and leaves have been known to ease inflammation. Some studies have demonstrated its effectiveness against dermatitis, but its anti-inflammatory and antihistamine mechanisms and comparative efficacy to a steroid or commonly used antihistamines drugs were unknown. Little is known about the anti-inflammatory properties of Witch hazel in HaCaT cells (cultured human keratinocytes) and mast cells (that release histamine and other allergic substances). In this thesis, the anti-inflammatory activity of Hamamelis (0.25%, 0.5%, and 1%) was investigated in TNF-α activated HaCaT cells using hydrocortisone as a benchmark control for ameliorating inflammation throughout in this thesis. The cytokines TNF-α, IL-6, IL-1β, IL-4, IL-13, TSLP, and chemokine CCL-17, CCL-5 were measured in activated HaCaT cells using ELISA (enzyme-linked immunosorbent assay). The amounts of nitrite were calculated utilizing a Griess assay. TNF-α produced ROS (reactive oxygen species) in HaCaT cells were analysed using DCFDA (dichlorofluorescein diacetate) assay. Protein concentrations in NF-κB (Nuclear factor kappa B) pathway and MAPK (mitogen-activated protein kinase) pathway were also quantified using ELISA. The effect of NRF2 (nuclear factor erythroid 2-related factor 2) pathway in mediating the anti-inflammatory properties of Hamamelis in HaCaT cells was measured by Western blot examination of HO-1, NQO-1, and ELISA for NRF2. The impact of Hamamelis on mast cells was compared with commonly used antihistamines (chlorpheniramine maleate and loratadine) to assess its role in allergic mediated inflammatory reactions on the skin. The results confirmed that the Hamamelis reduced NO productivity in TNF-α provoked HaCaT cells. Hamamelis further diminished the release of other pro-inflammatory factors IL-6, TNFα, IL-4, IL-13, IL-1β, TSLP, CCL-17, CCL-5, NO, and ROS. However, it had minimal effect on IL-1β and IL-13 in TNF-α stimulated HaCaT cells. The mechanistic studies concluded that the anti-inflammatory properties of Hamamelis was mediated through the inhibition of the MAPK or NF-κB pathways. Furthermore, the Hamamelis modulated antioxidant proteins HO1 and NQO-1, suggesting that the activity was mediated through NRF2 signaling pathway. Tea tree oil (TTO) was also considered as a potential natural substance for its anti-inflammatory properties due to its abundance in skin products and history of use on human skin. However, we found that it was not well tolerated by the cultured keratinocytes even at a very low dose. Moreover, TTO did not inhibit the production of proinflammatory cytokine, such as IL-1 β. Due to lack of anti-inflammatory properties and observed cytotoxic effects in HaCaT cells, TTO was not considered for further studies in this thesis. Considering the safety and anti-inflammatory efficacy of Hamamelis, it was also formulated as a topical ointment for potential application on the skin using conventional pharmacopeial methods. The formulation and excipients were also assessed for cytotoxicity to ensure safety and formulated product was tested for anti-inflammatory markers to ascertain the formulated product's efficacy compared to a typical steroid hydrocortisone as a control. The other formulation components, the olive and coconut oils (virgin, mechanically pressed) were also investigated individually to assess their impact on cell viability and sensitivity to keratinocytes resulting in a synergistic effect against inflammatory cascade. Both oils have been famous for their emollient properties on the skin. Therefore, the coformulation of coconut and olive oils with Hamamelis was deemed beneficial for topical application on the skin. The thesis provided material evidence confirming Hamamelis prevented TNF-α mediated inflammation through NRF2/ARE (antioxidant response element) antioxidant pathways in HaCaT cells. The antihistaminic effects of the Hamamelis on mast cells also hinted a potential anti-inflammatory role on skin via ameliorating processes triggered by histamine-mediated skin allergy. The formulated topical product of Hamamelis successfully retained its anti- inflammatory properties on a 15-month storage. A comprehensive stability evaluation of the formulated product as per ICH stability guidelines (ICH Q1A), is however, part of future works. The natural formulation offered a steroid-free natural alternative for topical application in patients with clinical and sub-clinical conditions of skin, offering the benefits of steroid and emollients in a single preparation. Despite a variety of emollients available in the market for soothing inflammatory conditions of the skin such as eczema; there is none available with strong anti-inflammatory and antipruritic properties except topical steroids which are often not preferred due to their obvious side effects and safety concerns over long term use. It is, therefore, remarkable to conclude that a topical formulation containing natural plant extracts from Hamamelis in combination with olive and coconut oils could be offered in prevention and treating skin inflammation for clinical and subclinical inflammatory conditions of the skin such as eczema or dermatitis. The results from anti-inflammatory screening in cultured human keratinocytes in this thesis are very promising and the product is worth taking forward for a formal clinical trial to confirm its clinical efficacy. Once clinically validated, the formulation can be scaled-up easily for commercial production using conventional manufacturing methods using commercially available "Hamamelis water" that is already compliant to the BPC (British Pharmaceutical Codex) or US NF (United States National Formulary) monographs, that was also investigated in this thesis alongside Hamamelis methanolic extract.

Studies have revealed the role of inflammation in the onset, progression and pathogenesis of gingivitis and periodontitis. Nigella sativa (NS) is known for its anti-inflammatory activities. This study therefore investigated the role of NS methanol extract and its bioactive component, thymoquinone (TQ) on gingival inflammation in Porphyromonas gingivalis lipopolysaccharide (P.g.LPS)-stimulated human gingival fibroblasts (HGFs). HGFs in culture were stimulated with P.g.LPS (100 ng/mL) following pre-treatment with NS (12.5, 25 and 50 μg/mL) or TQ (2.5, 5 and 10 μM). Levels of NO., PGE₂, IL-1β, IL-6, IL-8 and TNF-α production were measured from culture supernatants by ELISA. Protein levels of COX-2, iNOS, phosphorylated-p38, Nrf2, HO-1 and NQO-1 were determined by western blot. Phosphorylation of NF-κB-p65 sub-unit protein expression was determined by In-cell western and ELISA. Phosphorylated-IκB-α, total-IκB, phosphorylated-ERK1/2, phosphorylated-JNK1/2, pro-and active-MMP-9 from extracts were also determined by ELISA. HGFs were transfected with Promega pGL4.32[luc2P/NF-κB-RE/Hygro] vector and reporter gene assays were employed to explore the effect of NS on NF-κB luciferase activity in the nucleus. NS methanol extract blocked the production of NO., PGE₂, IL-6 and TNF-α in P.g.LPS-stimulated HGFs. Protein levels of phosphorylated-NF-κB-p65 sub-unit as well as phosphorylated-IκB-α were significantly suppressed by NS while an elevation in total-IκB protein level was observed. NF-κB binding as well as its transactivational activities were markedly inhibited by NS in P.g.LPS-stimulated HGFs. TQ significantly inhibited the production of IL-1β, IL-6, IL-8 and TNF-α. COX-2 and iNOS mediated production of PGE2and NO. respectively were significantly blocked by TQ in P.g.LPS-stimulated HGFs. Increases in P.g.LPS-mediated phosphorylation of ERK1/2, p38 and JNK1/2 MAPKs was significantly blocked by TQ. Pro-MMP-9 protein as well as the secretion of active enzyme was also significantly suppressed by TQ in P.g.LPS-stimulated HGFs. Protein levels of Nrf2, HO-1 and NQO-1 were significantly elevated by TQ in HGFs. These results suggest that N Sand TQ suppressed gingival inflammation through mechanisms involving NF-κB and MAPK and activation of antioxidant defense mechanisms. This study also demonstrates that bioactive TQ and NS are potential adjunctive therapeutic candidates in the prevention and treatment of gingivitis.

Chronic inflammation is prevalent in society with a wide range of inflammatory diseases, such as rheumatoid arthritis and inflammatory bowel disease, as well as association with cancer, strokes, and neurodegenerative conditions. Due to the complexity in the inflammatory pathways and a lack of understanding of those processes, there are limited therapeutic options available that aim to resolve the causes of inflammation, with most treatments instead aiming to manage symptoms. More bespoke imaging agents are required to help advance our understanding of the fundamental processes governing these disease states. To overcome current imaging limitations such as quantification, correlation, depth penetration and resolution quality, this work takes a multimodal molecular imaging approach to study one type of immune cells, macrophages, that are of particular importance in rheumatoid arthritis. Such methods aim to aid the non-invasive characterisation of disease phenotypes and therapeutic response in inflammatory diseases such as IBD and rheumatoid arthritis. The general structure of selective imaging probes is an imaging moiety, a targeting group to localise at a specific site and a linker group to attach the two. The work within this thesis focusses on designing a toolkit of imaging probes that target macrophages by using folate and tuftsin derivatives to target receptors with macrophage selective expression. A variety of imaging modalities are explored in this work such as lanthanide luminescence, magnetic resonance and photoacoustic, with the aim to permit the choice of the most appropriate modality required for a given spatial resolution, scale, and depth penetration. In addition, different bioconjugation strategies have been investigated, such as amide linkages and azide-alkyne cycloadditions, to enable access to a wider variety of macrophage imaging agents. By using a toolkit of molecular components that can be easily combined, this work demonstrates preliminary work for luminescent microscopy, magnetic resonance, and photoacoustic imaging to enable the study of macrophage populations in tissue and model systems. Chapter 1 outlines the processes governing inflammation and the current methods of imaging inflammation. It provides a general overview of the different imaging modalities, with a specific focus on lanthanide chemistry and its photophysics, magnetism and stability in relation to its in vivo imaging. Moreover, the suitability of the targeting moieties - folate and tuftsin - are discussed and the prior work in these areas. Chapter 2 outlines the synthesis, paramagnetic NMR spectroscopy and photophysical characterisation of alkyne appended DO3A derivatives. The purpose of these alkynes is for use in bioconjugation to the targeting moieties with an azide functionality for use in CuAAC and SPAAC. Chapter 3 describes the synthesis of multiple terbium-folate conjugates using solid-phase synthesis, as well as the generation of folate-azides for conjugation to the alkynes discussed in chapter 2. The NMR spectroscopy and photophysical properties of these terbium-folate conjugates are discussed. Chapter 4 details the synthesis of lanthanide-tuftsin conjugates for use in optical and magnetic resonance imaging. The relaxivity and cell diffusion NMR spectroscopy of the gadolinium complexes were studied in blood plasma and whole blood and was compared to a commercially available non-specific gadolinium complex, DOTAREM. Chapter 5 discusses the preparation of macrophage-targeted photoacoustic probes and their optical properties. The probes are investigated for their photoacoustic properties and stability. Chapter 6 provides a summary of work and conclusions that can be drawn, with opportunities for future work proposed. Finally, chapter 7 contains the experimental procedures.

Macrophages are heterogeneous and plastic cells, which populate almost every organ in the body and migrate towards sites of inflammation. However, tools designed for studying macrophage biology, particularly genetic models and chemotaxis assays, are standardised to investigate a broad range of leukocyte populations. They fail to capture the unique properties of macrophages and distinguish them from other ontologically related populations. In this thesis, I set out to create novel in vitro and in vivo tools that are adjusted for studying macrophage biology. To address the in vivo arm of this project, I phenotyped a novel tamoxifen-inducible hCD68-CreERT2 mouse line. Having established an optimal protocol for tamoxifen dosing, I detected robust expression of Cre recombinase activity in tissue-resident macrophage populations in peritoneum, spleen and liver. Importantly, the expression was restricted to tissue-resident macrophages with marginal effects in blood leukocyte populations and splenic non-macrophage populations. When compared to other 'macrophage-specific' lines, hCD68-CreERT2 mice had fewer 'off-target' effects. Additionally, I was able to successfully use hCD68-CreERT2 mice to target macrophages present in atherosclerotic lesions. To address the in vitro arm of this project, I used the Freestyle Fluidics (FF) platform to design and optimise circuits that can be used to study macrophage migration. I created a passive diffusion-based system and an active pumping system that created stable controllable gradients. I showed that both chemotaxis and chemokinesis can be readily detected and quantified in both systems. Using the FF chemotaxis assay, I observed novel properties of macrophage behaviour such as population-level coordination of chemotaxis and the ability to sense the gradients both spatially and temporally. In summary, this project advanced the macrophage-specific technology platforms available to researchers.

Heart failure (HF) associated with diabetes is a distinct clinical entity known as diabetic cardiomyopathy (DCM) without the prevalence of other risk factors such as hypertension. The human heart is a pool of different cell types, including cardiomyocytes, fibroblasts, endothelial cells and immune cells, interacting via various secretory factors. The cardiomyocyte acting as an active mediator of the inflammatory response is an emerging concept with limited mechanistic understanding. Superimposed effects of hyperglycaemia and hyperlipidaemia dysregulate the intercellular links, contributing to diabetes-induced cardiac dysfunction. DCM is instigated by various pathological insults, including endoplasmic reticulum (ER) stress response and ectopic fibroblast growth factor receptor 1 (FGFR1) signalling, resulting in detrimental impacts on cardiac structure and function. Hence, elucidating the paracrine and autocrine mediators of cardiac intracellular signalling can provide new mechanistic and therapeutic strategies for HF in diabetes. First, we demonstrate the opposing roles of the cardiac ER stress response in our time-course model of high fat high-sugar diet (HFHSD)-induced diabetes. Using cardiac P21-activated kinase 2 (PAK2) deficient mice, we found that PAK2, an ER-localised signalling kinase, decelerates the transition from an adaptive to a maladaptive ER response in DCM. A concurrent examination of the myocardial inflammation, particularly macrophage response, revealed chronic cardiac ER stress to be the initiating cascade of the alarmin high-mobility group box 1 (HMGB1) in DCM. HMGB1 expression and subsequent secretion into the circulation was promoted via C/EBP homologous protein (CHOP), an ER stress responder. Our in vivo DCM models of ER dysfunction, the remediated ER response and pharmacological treatments established the functional and therapeutic relevance of targeting cardiac ER stress-mediated inflammation for alleviating DM-induced cardiac complications. Secondly, we determined that the FGFR1 cascade is the upstream facilitator of the PAK2 response. In diabetes, we observed a lack of cardiac FGFR1 and beta-Klotho (β-KL) signalling. We found that restoring cardiac FGFR1/β-KL expression results in the increased secretion of anti-inflammatory and proangiogenic factors from cardiomyocytes. Using proteomic analyses, we identified key cardiac-derived signals that regulate angiogenesis and macrophage polarisation upon FGFR1/β-KL activation in diabetes. This provides a proof-of-concept to explore the novel therapeutic avenue of myocardial cell interactions to prevent cardiac dysfunction under metabolic stress. Type 2 diabetes is a chronic disorder; therefore, our time-course analyses provide a fascinating insight into the early mechanistic events in DCM. We address the knowledge gap of cardiomyocyte-mediated macrophage and endothelial cell response and reveal mechanistic links behind cardiac inflammation and vascular damage in response to diet-induced metabolic stress. Hence, further dissection of the identified molecular web between cardiomyocytes and endothelial cells or macrophages in DCM may lead to the development of efficacious treatments for diabetes-induced HF.

Angiotensin II (Ang II) is a peptide associated with the regulation of blood pressure, though the elevated presence of Ang II in the inflamed bowel - and other inflamed tissues - and the presence of receptors for Ang II on sensory neurons may point to additional roles in nociception in inflammatory disease. Work in this Thesis sought to examine the identity of sensory neurons with which Ang II interacts, the mechanisms underpinning these interactions, and the consequences on neuronal properties and function. Ca2+ imaging revealed that Ang II stimulated a rise in cytosolic Ca2+ in small-diameter sensory neurons which expressed the nociceptive markers TRPV1 and Nav1.8. This population of Ang II-sensitive neurons could be divided in two, with one subpopulation expressing Tmem45b and binding the non-peptidergic marker isolectin-B4 (IB4), while the other subpopulation lacked both Tmem45b and IB4 binding. The response of IB4-binding sensory neurons to Ang II was mediated by the Type I Ang II receptor (AT1R). Conversely, the response of IB4-negative neurons to Ang II did not require AT1R but did require the presence of non-neuronal satellite cells. Ang II-evoked Ca2+ signals downstream of AT1R were mediated by Ca2+ release from intracellular stores via the activation of IP3 receptors, followed by store-operated Ca2+ entry (SOCE). SOCE downstream of Ang II application was found to be mediated by the non-selective cation channel, TRPC3, activated by the endoplasmic reticulum Ca2+ sensor, STIM. Incubation of sensory neurons with Ang II induced to nuclear translocation of the transcription factor, nuclear factor of activated T-cells 5 (NFAT5). This translocation was dependent on Ca2+ influx through TRPC3 and the Ca2+-sensitive phosphatase, calcineurin. Translocation of NFAT5 may indicate prolonged changes in nociceptor properties induced by Ang II, as evidenced by an increase in electrical excitability of IB4-binding neurons following overnight Ang II incubation. In summary, experiments detailed in this Thesis have revealed that Ang II stimulated nociceptive sensory neurons in vitro. Ang II-evoked Ca2+ signals were mediated by store depletion and subsequent SOCE through TRPC3. These Ca2+ signals drove translocation of NFAT5 to the nucleus in a manner dependent on calcineurin. Finally, non-peptidergic neurons exposed to Ang II displayed elevated excitability. These data highlight a mechanism through which Ang II may drive prolonged changes in nociceptor function.

Introduction: Cerebral small vessel disease (SVD) is increasingly recognised as a key factor in dementia and cognitive impairment, although its role in the pathogenesis of Alzheimer's disease (AD) remains poorly understood. Through the investigation of how SVD relates and interacts with (1) molecular AD pathologies and (2) early midlife risk factors of dementia, this body of work aims to advance our understanding of SVD in relation to AD. Methods: In two separate cohorts, SVD was quantified on 3T MRI through the assessment of white matter hyperintensities (WMH), enlarged perivascular spaces (EPVS), cerebral microbleeds (CMB), and lacunes. Regional data on SVD location was used to derive composite scores for global SVD burden, and the SVD subtypes of hypertensive arteriopathy and cerebral amyloid angiopathy. The first objective was investigated in the Neuroimaging of Inflammation in Memory and Other Disorders (NIMROD) study, which consisted of multimodal PET imaging of older adults with varying degrees of clinical severity. Here, we investigated whether SVD was associated and interacted with the hallmark AD pathologies of amyloid, tau, and neuroinflammation (microglial activation) - these were quantified with PET imaging of [11C]PiB, [18F]AV-1451, and [11C]PK11195, respectively. For insights into the pre-clinical stages of AD pathogenesis, we examined cognitively healthy midlife adults from the PREVENT-Dementia study. We investigated whether well-established risk factors of late-life dementia were related to SVD burden much earlier on, at midlife. Results: SVD was associated with PET markers of neuroinflammation and tau, and plasma Aβ. Notably, associations with amyloid and tau markers were largely limited to WMH. In healthy middle-aged adults, inherited predisposition to dementia (APOE4, parental family history of dementia) was related to longitudinal SVD progression, but not baseline SVD severity. On the other hand, modifiable risk factors of late-life dementia were associated with midlife SVD burden. The two SVD subtypes were differentially related to AD pathologies and risk factors - neuroinflammation and modifiable risk factors were more closely associated with hypertensive arteriopathy, while amyloid and tau related more closely to cerebral amyloid angiopathy. Conclusions: Together, our findings implicate SVD in the early pathogenesis of AD and suggest that lifestyle modifications as early as midlife may reduce the vascular-driven component of AD. Findings highlight the need to further inspect the biological underpinnings of WMH and fluid markers of AD pathology.

The human kidney maintains whole organism homeostasis through tightly regulated filtration, transport, and endocrine functions that are subserved by a wide array of specialised cell types with distinct developmental origins and anatomical organisation. Amongst these cells lies a network of tissue-resident immune cells with incompletely characterised roles in tissue homeostasis, repair, and defence. Furthermore, different regions of the kidney are affected by distinct immunologically relevant insults - ascending bacterial infection in the medullary-pelvic region, and immune complex-mediated inflammation within the cortex. We hypothesised that the kidney-resident immune network is adapted to respond to and counter these immunological challenges. Recently the advent of high-throughput single-cell genomics methods has permitted the atlasing of human organs in unprecedented molecular detail. These techniques permit the unbiased characterisation of cell types and cell states, the prediction of functional programmes, and the mapping of intercellular interaction networks underpinning tissue organisation. In this thesis, we used single-cell RNA sequencing to generate the first detailed and spatio-temporally resolved atlas of the human kidney in development and maturity. This study has identified the transcriptional signatures of mature kidney parenchymal cell types from nephron epithelial, vascular, and stromal compartments, and allowed the reconstruction of the developmental trajectories of early nephrogenesis. Within the immune compartment, we charted the landscape of immune cells, and the seeding of these cells through early development, identifying a macrophage subset in mature kidneys with transcriptional similarity to macrophages seeded early in fetal life, and likely representing a long-lived self-renewing subset. This work also identified a transcriptional signature mediating antimicrobial defence in the pelvis of the kidney. Here pelvic epithelial cells express antimicrobial peptides, and neutrophil- and monocyte-recruiting chemokines, orienting the immune response towards the dominant infectious threat. We later optimised and refined the single-cell genomics approach to the human kidney, generating high quality data on a larger panel of donors in a compartmentally-resolved fashion. In this work we were able to assess the immune composition of glomerular and tubulointerstitial compartments, finding a strikingly divergent immune landscape, with glomeruli occupied by monocytes, dendritic cells, and NK cells, and the tubulointerstitial compartment housing populations of resident macrophages, T cells, and B cells. Finally we have begun to explore the dynamic transcriptional responses of kidney immune and parenchymal cells to IgG immune-complex stimulation using an ex vivo normothermic normoxic perfusion model. These experiments have uncovered cell type-specific signalling responses in stimulated monocytes, NK cells, and glomerular endothelial cells, with evidence for abundant chemokine and cytokine signalling coordinating the early response to this highly disease-relevant perturbation.

Neutrophils are innate immune cells and the first to be recruited to sites of injury, where they are essential for host defence. Defective neutrophil recruitment can inhibit pathogen clearance, while impaired neutrophil resolution can lead to chronic inflammation. Therefore, it is essential that neutrophil migration is tightly controlled and self-resolving. It was originally thought that neutrophilic inflammation was resolved through apoptosis. However, more recent studies have revealed that neutrophils can actively depart (reverse migrate) from target sites, which is believed to support resolution of the response. Immune cell migration is regulated by chemoattractants, including the chemokine family. Chemokines bind to cognate G protein-coupled receptors to induce cell migration and are upregulated in many chronic inflammatory diseases characterised by aberrant immune cell migration. While chemokines represent an attractive therapeutic target, the development of drugs targeting them in chronic inflammatory diseases has been hampered by the complexity of the chemokine system. It is thought that a greater understanding of the context-specific roles of chemokines will aid identification of suitable targets for the treatment of chronic inflammatory diseases. Here, using a zebrafish model of inflammation, I investigated the ligand preferences and subcellular trafficking of two chemokine receptors, Cxcr1 and Cxcr2, which were previously shown to mediate clustering and dispersal of neutrophils, respectively. I demonstrate that Cxcr1 recognises Cxcl8a and is rapidly internalised, whereas Cxcr2 recognises Cxcl8b and traffics in a manner consistent with recycling. These differences explain, in part, how Cxcr1 and Cxcr2 mediate opposing neutrophil behaviours, ensuring a balanced response. To investigate whether different spatial or temporal expression of the chemokines Cxcl8a and Cxcl8b also contributes to this balanced response, I explored CRISPR/Cas9 technology to generate a fluorescent reporter line of endogenous Cxcl8a expression. While I introduced mutations in the target gene, the knock-in approach was ultimately not successful, and I discuss possible approaches which could be applied in the future to achieve this technically challenging goal. The observation that neutrophils can reverse migrate provided a potential new mechanism for the resolution of sterile inflammation. However, the role of reverse migration in the resolution of infected wounds remained unclear. By generating a neutrophil-specific photoconvertible line, I determined the fate of neutrophils recruited to sterile and infected wounds, finding that reverse migration plays an important role in the resolution of infected wounds. A potential regulator of neutrophil reverse migration is desensitisation of chemokine receptors. Previous work in our lab showed that desensitisation of Cxcr1 is required for neutrophils to stop and cluster. Combining a Cxcr1-desensitisation mutant with the photoconvertible line, I showed that receptor desensitisation is required for reverse migration. Finally, I sought to investigate the functional impacts of Cxcr1 desensitisation on wound healing and responses to secondary wounds and infections. I generated new protocols to study such impacts, which will form the basis of future studies with Cxcr1-desensitisation mutants.

Antimicrobial resistance is a major global health threat, and there is growing interest in how modulation of the host immune response can enhance pathogen killing and reduce reliance on antimicrobials. One target cell is the macrophage; a key innate immune cell that possesses a range of microbicidal mechanisms and can combine responses for optimal pathogen killing. Streptococcus pneumoniae and Staphylococcus aureus are important gram-positive pathogens that represent differing intracellular burdens for the macrophage. A key macrophage microbicidal mechanism relevant to the killing of these pathogens is production of reactive oxygen species (ROS). While NADPH oxidase-derived ROS is an early response to infection, mitochondrial ROS (mROS) production is a later response and is enhanced during infection by alterations in mitochondrial dynamics. ROS and mROS can combine with other macrophage responses to facilitate pathogen killing, therefore the significance and potential for such interplay with other host defence mechanisms to enhance macrophage killing of pathogens such as S. pneumoniae and S. aureus is the focus of this thesis, with specific attention to mitochondrial-associated responses and the microbicidal and immunomodulatory host defence peptide cathelicidin. The data presented in this thesis show that expression of the CAMP gene, encoding cathelicidin, was upregulated by vitamin D in macrophages, was synergistically enhanced by bacterial infection or phenylbutyrate and was impaired by pro-inflammatory cytokines. Cathelicidin directly killed extracellular S. pneumoniae and contributed to early macrophage killing of intracellular S. aureus when bacterial burden was high. Mitochondrial adaptations to S. pneumoniae were more prevalent in macrophages during later stages of bacterial challenge and included increased mitochondrial fission and increased mROS production. Mitochondrial adaptations to S. aureus, which stresses macrophage microbicidal responses to a greater extent than S. pneumoniae, were observed during early stages of bacterial challenge. The regulators of canonical fission, dynamin-related protein 1 (Drp1) and mitochondrial fission factor (Mff), failed to influence overall levels of fission in the initial response to S. aureus. In contrast, Drp1 regulated localisation of mROS to intracellular S. aureus in a subset of macrophages, suggesting roles in mROS delivery to bacterial-containing phagolysosomes. In regard to mechanisms of mROS production, I have provided evidence that reverse electron transport (RET) occurs as an early response to S. pneumoniae challenge, but not late S. pneumoniae, or S. aureus challenge. S. aureus enhanced mROS production in macrophages, and while NADPH oxidase-derived ROS was the greater contributor to early killing of S. aureus, mROS also contributed to killing. Cathelicidin enhanced microbicidal responses against S. aureus particularly when NADPH oxidase-derived ROS generation was impaired, but also appeared to function as a brake on alterations in mitochondrial dynamics and mROS production in the presence of bacteria, therefore potentially regulating mitochondrial homeostasis. Results in this thesis demonstrate that macrophages use ROS, alterations in mitochondrial dynamics and mROS, and cathelicidin to combat S. pneumoniae and S. aureus infections with pathogen-dependent kinetics. Macrophages adapt responses to different pathogens to ensure a multi-layered immune response to clear pathogens. The work in this thesis provides greater insight into macrophage microbicidal responses to S. pneumoniae and S. aureus infection and could inform future therapeutic strategies to enhance macrophage microbicidal responses.

Inflammatory bowel disease (IBD) defines two chronic inflammatory diseases of the gastrointestinal tract, ulcerative colitis and Crohn's disease. The prevalence of these diseases is increasing in the western world, and there is currently no cure for either. Biological therapy is widely used to treat IBD; however, patients often lose response to these treatments. There is a need to stratify patients into further disease subsets, which require biomarkers that could act as read-outs of disease progression and severity. Granzymes (A, B, H, K and M) are released by cytotoxic T cells and NK cells, with granzyme A and B the most abundant forms found in humans. Granzyme A has previously been reported to be elevated in tissue biopsies from IBD patients who responded well to anti-integrin therapy (etrolizumab) and subsequently achieved clinical remission, in comparison to those who did not respond. Granzyme A has also recently been reported to have a pro-inflammatory role in other inflammatory diseases including rheumatoid arthritis. Granzyme B, typically thought of as a cytotoxic protease, has been reported to be elevated in the serum of IBD patients. Based on these data, in chapter 3, we sought to characterise the expression of granzymes A and B in IBD tissue samples and to determine whether the expression of granzymes A or B could be used as blood and/or stool-based readout of IBD activity. We also investigated the source of granzyme A in IBD tissue, using fresh tissue biopsies taken from patients undergoing research colonoscopies. Granzymes typically must be activated before they exert their effects on target cells. We therefore hypothesised that activity of an enzyme would be a more informative biomarker than expression of the enzyme, as only when active are these granzymes known to have cytotoxic effects. To have a read-out of granzyme A activity, in Chapter 4, we sought to create substrate-based probes that would fluoresce when cleaved by active granzyme A but would remain intact in the presence of inactive granzyme A. Lastly, in Chapter 5, we also sought to understand the pro-inflammatory role of granzyme A and identified that it could induce the secretion of pro-inflammatory cytokines from monocytes. We investigated the receptors responsible for the pro-inflammatory effects on monocytes and used an inhibitor to block the pro-inflammatory effects of granzyme A. Finally, we synthesised a new granzyme A inhibitor, which was shown to be efficacious in vitro. We performed preliminary in vivo experiments in a mouse model of colitis using a PAR-1 receptor inhibitor and the granzyme A inhibitor but observed no efficacy at the concentrations used. Altogether, the work in this thesis characterised the expression of granzymes A and B in gut tissue from IBD patients and created new substrate-based probes for granzyme A that could act as a read-out of enzyme activity rather than expression. We also began to investigate how granzyme A could induce inflammation in an IBD setting and lastly created a granzyme A inhibitor to attenuate the pro-inflammatory effects of the enzyme.

Neutrophils form part of the innate immune system, they are the most abundant circulating leukocytes in humans and play a crucial role in host defence and inflammation. Upon stimulation, neutrophils leave the blood stream and chemotax to sites of inflammation after which they employ a range of effectors functions to eliminate the threat. It is key that these functions are tightly controlled to ensure pathogen clearance and prevent a suboptimal neutrophil response, as is observed in immunosenescence, defined by the deterioration of the immune system with old age. However, it is also essential to avoid the inappropriate activation of the immune response which could lead to autoimmune diseases. Through a range of receptors, the activation of phosphoinositide 3-kinase (PI3K) signalling regulates many aspects of neutrophil biology. Phosphatidylinositol 3,4,5-trisphosphate (PIP3), the lipid second messenger product of PI3K is subjected to dephosphorylation by several 5'phosphatases, converting it into PI(3,4)P2, a lipid second messenger in its own right. The SHIP family 5'phosphatases include the well characterised leukocyte restricted SHIP1, and the ubiquitously expressed SHIP2. SHIP1-deficient neutrophils spread extensively on the substratum, and in response to chemoattractant stimulation fail to chemotax efficiently towards a chemoattractant. Here, SHIP2 is characterised using mice with catalytically inactive SHIP2, revealing an in vivo recruitment defect to sites of sterile inflammation. Isolated neutrophils from catalytically inactive SHIP2 mice were also identified to have chemotaxis, directionality, and polarisation defects in vitro. Several other effector functions were also tested including phagocytosis and swarming but portrayed a lack of any major defects. The effects of catalytically inactive SHIP2 are mechanistically explained by a subtle increase in PIP3 and an larger decrease in PI(3,4)P2. The effects of immunosenescence have been well documented in the adaptive immune system, however, its effects on in the innate immune system have yet to be robustly explored. Therefore, I studied the effects of neutrophil ageing on its effector functions and identified a defect in neutrophil swarming in the absence of defects in elastase release and the formation of reactive oxygen species This is congruent with results from other groups identifying a chemotaxis defect in neutrophils from older donors. In summary, my work identifies a non-redundant role for SHIP2 in neutrophil chemotaxis and trafficking, further enhancing our understanding of the PI3K signalling pathways in neutrophils. I also studied the phenotype of immunosenescent neutrophils, identifying a defect in neutrophil swarming.

For over a century, it has been apparent that tumours behave somewhat like wounds that fail to heal. A number of studies have established several cellular and molecular parallels between wound healing and cancer, and a key one of these is inflammation, with both wounds and tumours triggering an inflammatory response that plays an essential role in the outcome of these tissue lesions. As a consequence, any potential therapy with the capacity to alter the wound/tumour inflammatory response is now considered clinically relevant. In this regard, infection is known to modulate the inflammatory response to both cancer and wound healing. Serendipitous findings extending back to those of Coley in the early 1900s, have shown cancer remissions occurring upon treatment with heat-killed bacteria, suggesting that infection can enhance, or prime, the host immune response to better recognise and eradicate cancers. During tissue repair, there is evidence that some aspects of the wound inflammatory response may be activated by microbial antigens, although, of course, if infection becomes overwhelming, this can lead to chronic, non-healing wounds. Whilst the longer term consequences of infection on cancer and wound healing have been partially explored, rather little is known about the precise cellular and molecular mechanisms by which bacteria-induced inflammation can influence these consequences, for example, how infection alters the behaviour of inflammatory cells and their cellular interactions with the tissue insult. Here I used the translucent larval zebrafish (Danio rerio), which has relatively recently become a powerful model of inflammatory-related human conditions, in combination with high-resolution confocal imaging and a novel bespoke automated cell tracking and cell-cell interaction software, to investigate how inflammatory cells behave and interact with repairing wounds and early cancer cells in sterile versus infection conditions. Using this approach, I showed that infection, delivered either systemically or locally, induced changes in the number and behaviour of inflammatory cells recruited to acute wounds and to pre-neoplastic cells, and these alterations led to a significant delay in wound healing, and to a reduction in the number of pre-neoplastic cells, which correlated with increased numbers of pro-inflammatory (tnfα-positive) macrophages. I expanded these studies into adult zebrafish where I observed that infection-induced inflammation inhibits the growth of established human-like melanomas. Finally, I tested a more therapeutically viable anti-cancer strategy by using a novel particle-based protocell system to selectively load macrophages and neutrophils, via their natural foreign body phagocytic abilities, with immunostimulatory, tumour-suppressing molecules (anti-miR223 or R848). I showed that anti-miR223-loaded protocells or R848-loaded protocells can effectively "reprogramme" macrophages (and neutrophils) towards a more pro-inflammatory, and thus anti-cancer state, in zebrafish (and cultured human macrophages), which led to a significant reduction in both larval pre-neoplastic burden and in the size and number of adult melanomas also.

Osteoarthritis (OA) is a highly prevalent condition that affects more than 40% of individuals over the course of their lifetime. There are currently no disease-modifying OA drug (DMOAD) licensed for the treatment of this disease. An Icelandic genome wide association study (GWAS) study identified common polymorphic variants in ALDH1A2, the gene which encodes the enzyme involved in the synthesis of all-trans retinoic acid (atRA), with severe hand OA. Subsequent data from our lab showed that atRA-responsive genes are downregulated on cartilage injury. Mechanical injury is one of the principal risk factors in the development of OA. Cartilage injury is known to activate downstream inflammatory signalling that is driven in a transforming growth factor activated-beta 1 (TAK1) dependent manner and thereby increase inflammatory gene regulation, in a process known as mechanoflammation. Talarozole (TLZ), a CYP26 inhibitor, which prevents the breakdown of atRA, suppresses the upregulation of inflammatory genes on cartilage injury. My project sought to identify the mechanism by which atRA-responsive genes were downregulated and determine how atRA suppressed mechanoflammation on cartilage injury. The downregulation of atRA-responsive genes on cartilage injury was mediated in both cytosolic phospholipase A2 (cPLA2) - and reactive oxygen species (ROS)-sensitive manner. TAK1 phosphorylation on cartilage injury was partially mediated in a nicotinamide adenine dinucleotide phosphate oxidase (NOX)-sensitive manner, whereas TAK1 was responsible for the partial phosphorylation of cPLA2 on cartilage injury. Apoptosis signal regulating kinase 1 (ASK1) was the key driver of mitogen activated protein kinases (MAPKs) (Jun N terminal kinase (JNK) and extracellular signal regulated kinase (ERK)) on cartilage injury and downstream inflammatory gene regulation. atRA suppressed mechanoflammation in a peroxisome proliferator activated receptor gamma (PPARG)-sensitive manner. These findings suggest that both boosting atRA level and ASK1 inhibition can suppress mechanoflammation in articular cartilage and identifies each pathway as targets for potential disease modifying drugs in the treatment of OA.

Type 1 diabetes (T1DM) is a chronic autoimmune pathology driven by loss of pancreatic beta cells as a consequence of T-cell mediated inflammatory responses and/or dedifferentiation of beta cells. Numerous genetic markers have been identified that carry risk for T1D development, worse secondary complications, and poor beta cell function. However, the mechanism by which risk is conferred is poorly understood. This thesis aimed to unravel the molecular basis for T1D risk by examining candidate markers identified in microarray and Genome Wide Association Studies (GWAS) and to identify and explore novel pathways of interest in beta cell function and survival. Our initial focus for T1D risk wasTNF Alpha Induced Protein 3 (TNFAIP3/A20), a negative regulator of the NF-kB pathway. TNFAIP3 has been widely cited as the most highly regulated antiapoptotic gene in the pancreatic islet and a risk locus for T1D development (in addition to other autoimmune conditions). We have shown that silencing of TNFAIP3 in human pancreatic islets led to increased levels of apoptosis, reduced insulin secretion in response to glucose stimulation and a reduction in the expression of beta cell markers and transcription factors. These effects were driven by cytokine production and found to be partly, although not exclusively, dependent on the P65 subunit of NF-kB. We next focused on two variants- rs10517086 located at chr4:26083889 and rs1534422 located at chr2:12500615 - identified in The Environmental Determinants of Diabetes in the Young (TEDDY) study as being associated with T1D risk, worse outcomes for residual beta cell function and strong correlations with islet autoantibodies. Using clinical data from the DARE (Diabetes Alliance for Research in England) study, we confirmed the association of rs10517086, but not rs1534422, with low c-peptide concentrations in T1D. The variants were introduced into the BRIN-BD11 beta cell line using CRISPR/Cas9 and consistently showed effects on glucose-stimulated insulin secretion, gene expression and cytokine secretion that were similar to the impact of TNFAIP3 deficiency. However, there was one notable exception in gene expression: HNF1A expression was significantly upregulated in the presence of either variant, and we did not find a role for pro -inflammatory cytokines in this process. Subsequent silencing ofHNF1A in variant-expressing cells largely normalised gene expression and we propose that these variants inflict a double insult on the beta cell through increased cytokine production and excessive upregulation of HNF1A. These data provide insights into the mechanisms by which T1D risk occurswith low levels of TNFAIP3 or the presence of rs10517086 and rs1534422, which may be clinically useful in the prediction of outcomes in T1D. Although these markers have prognostic value, they are not readily targetable. Pathway analysis on microarray data from TNFAIP3 deficient beta cells identified tumor necrosis factor-like weak inducer of apoptosis (TWEAK) signalling as novel pathway that could be targeted for therapy. TWEAK signalling appears to be largely protective in the beta cell since TWEAK silencing reduced glucose-stimulated insulin secretion and expression of beta cell markers and transcription factors. Again, there was one notable exception inNGN3,which was significantly upregulated upon TWEAK silencing. Overexpression of NGN3 is associated with hormone negative beta cells and shunting towards an alpha cell phenotype early in embryogenesis. We propose that novel therapies targeting TWEAK and its receptors may prove useful in the context of diabetes and other chronic inflammatory pathologies. Overall, the work presented in this thesis sheds light on how established risk factors for T1D development act within the beta cell and has identified a novel and targetable pathway that regulates beta cell function warranting further attention.

Cystic fibrosis (CF) is a hereditary disease caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The lung disease is, however, also associated with dysregulation of the epithelial sodium channel (ENaC) which leads to dehydrated airways predisposing the individual to chronic cycles of infection and inflammation. ENaC is activated by trypsin-like channel activating proteases (TLPs), inhibition of which increases airway surface liquid volume and improves mucociliary function of primary human airway epithelial cells (hAECs) grown at air-liquid interface (ALI).

Pulmonary exacerbations (PEx) are clinically impactful events that accelerate Cystic Fibrosis (CF) lung disease progression. Current PEx treatment approaches have suboptimal outcomes and the frequency of PEx experienced by people with CF (pwCF) is variable and poorly predicted by demographic or clinical factors. There is an unmet need to improve the prediction, treatment, and management of PEx, however this has been hampered by a lack of understanding of the pathophysiology of PEx. Characterisation of the underlying host immune and microbiological mechanisms underlying PEx could improve therapeutic management of PEx and inform a more individualised approach to treatment. In this thesis we provide novel insights into the pathophysiological mechanisms underlying infrequent and frequent exacerbations which we anticipate will be highly informative for improving the personalisation of treatment of PEx through targeting the underlying disease processes.

Alzheimer's disease (AD) is a devastating neurodegenerative condition which affects millions of people throughout the world. It is the most common form of dementia and manifests with progressive memory impairment and cognitive decline. While a small proportion of AD cases are inherited, a majority of patients have sporadic disease. Thus, it is a combination of one's genes, environment and lifestyle which ultimately determine whether they develop dementia. Two of the defining hallmarks of AD include deposits of extracellular amyloid-β (Aβ) plaques and intraneuronal neurofibrillary tau tangles. These coincide with reduced hippocampal and cortical volume, synapse loss, and signs of inflammation. Neuropathology and some known genetic and environmental risk factors help give clues as to the pathogenesis of AD, but we still lack a fundamental knowledge of how exactly this disease comes to fruition. Many environmental risk factors have now been associated with an increased risk of developing AD. Amongst others, these include activators of inflammation, cardiovascular disease, sedentary life style, pollution, smoking, diabetes, and stress. Inflammation which can occur due to infection and/or trauma is closely related to AD risk, and a common denominator between many of the other environmental risk factors. Vascular impairment, resulting in illnesses such as stroke and heart disease, is also closely linked to dementia, and in fact shares many risk factors with AD. Organotypic hippocampal slice cultures (OHSCs) are an underused tool in dementia research. They contain a variety of cell types including neurons, astrocytes, microglia, and oligodendrocytes, and maintain some of the structure and connections of the hippocampus. Yet, slice cultures are an in vitro system, and thus are easy to work with, without the constraints of the blood brain barrier. The major aim of this work was to use OHSCs as a means of studying sporadic AD. This was performed by carrying out experiments where environmental risk factors and sporadic genetic risk factors were modelled in the slice culture system. While multiple individual risk factors resulted in alterations in either Aβ, tau or synaptic proteins, there are several key findings within this thesis. First, modelling inflammation in wild type OHSCs results in a microglia-dependent decrease in the presynaptic protein synaptophysin. Next, lowering glucose concentration to model hypoglycaemia, causes an increase in Aβ in wild type OHSCs, which can be rescued with a variety of alternative fuel sources. And finally, OHSCs cultured from a mouse transgenic line show that the presence of the human APOE4 allele results in a decrease in synaptophysin levels relative to cultures with humanised APOE3. These results show how environmental and genetic risk factors can directly result in AD biomarker pathology in an in vitro system, without the need for the presence of a dominantly inherited causal mutation of AD, and they provide a basis for testing combinatorial risks to model disease in more sporadic cases.

People with bronchiectasis and cystic fibrosis (CF) frequently suffer from recurrent infective pulmonary exacerbations (PEx) which lead to a progressive loss of lung function, reduced quality of life and increased morbidity and mortality. Determining how bacterial load and community composition changes with time may improve our understanding of the natural history of these chronic airway diseases. The aims of this thesis were to (i) to determine the psychometric properties of HRQol questionnaires in bronchiectasis, (ii) to measure the longitudinal change in bacterial load and community composition in patients with bronchiectasis when clinically stable and following antibiotic treatment of a PEx, (iii) to determine the antimicrobial susceptibility of P. aeruginosa and H. influenzae isolates cultured from sputum samples collected from patients with bronchiectasis and (iv) to determine changes in total bacterial and P. aeruginosa load following antibiotic treatment of a PEx in people with CF who received either microbiome directed orempirical antibiotic treatment. Results showed that all HRQol questionnaires had good internal consistency and short-term test-retest reliability in at least some domains. Responsiveness was reasonable with the meta-analysis showing a difference between the treatment and placebo effect. In people with bronchiectasis, no statistical difference was apparent in total bacterial, P. aeruginosa and H. influenzae load and microbiome composition during periods of clinical stability and following antibiotic treatment of a PEx. However, P. aeruginosa load was associated with lower lung function and H. influenzae load was associated with increased systemic inflammation .P. aeruginosa isolates were susceptible to tobramycin, ceftazidime and ciprofloxacin, while H. influenzae isolates were susceptible to ampicillin, ciprofloxacin and co-amoxiclav but resistant to azithromycin. In the CF study, no difference was observed in total bacterial and P. aeruginosa load between patients who received microbiome directed and patients who received empirical antibiotic treatment. Resilience in bacterial load and microbiome composition was observed in the lungs of patients with chronic airway diseases with no change observed during periods of clinical stability and following antibiotic treatment of a PEx.

Chronic Obstructive Pulmonary Disease (COPD) is a progressive and preventable disease associated with loss of lung function following exposure to noxious particles or gases. Exacerbations of COPD are acute periods associated with an increase in hyperinflation and gas trapping and characterised by reduced expiratory flow and increased airway inflammation. Although exacerbations are commonly associated with respiratory infections from bacteria or viruses, there remains an incomplete understanding on the interaction between pathogens and airway epithelial cells (AECs) in the triggering and progression of these events. The initial aim of the study was to assess the responses of airway epithelial cells to different pathogenic stimulus associated with infections in COPD. Treatment of primary bronchial epithelial cells (PBEC), from a healthy and COPD donor, with heat-inactivated strains of Streptococcus pneumoniae and Haemophilus influenzae led to an increase in a number of inflammatory mediators as analysed using the Bioplex human inflammatory mediator panel (37-plex). Of particular interest was thymic stromal lymphopoietin (TSLP). TSLP is a cytokine that has been associated with airways disease including allergic asthma but its role in bacterial infections is less established. In the PBEC model, S. pneumoniae stimulation increased TSLP levels to a greater extent than H. influenzae in both healthy and COPD cells. Further investigation using COPD PBEC indicated a similar increase in TSLP expression following 6-hour treatment with clinical isolates of both H. influenzae and S. pneumoniae. Another aim of the project was to investigate the effect of pathogen-associated immune cell stimulus on AEC responses. Sterile-filtered cell-conditioned media from the differentiated macrophage-like THP-1 cells, infected with strains of H. influenzae (TCCM), was used to treat cells. Optimisation of the model on 16HBE14o- cells (HBE cells) led to significant increases in IL-8 levels (p<0.01) following treatment with TCCM. PBEC from two healthy and COPD donors were treated with TCCM, with levels of 36 inflammatory mediators analysed using the Proteome Profiler Human Cytokine Antibody Array screen, with subsequent validation in changes in expression of genes of interest by qPCR. TCCM treatment stimulated both the production and expression of a number of inflammatory mediators in PBEC from all donors including IL-8. Two potential targets for further work were identified, G-CSF and SERPIN E1. There was a reduction in G-CSF production and lower levels expression of the gene coding for G-CSF from the COPD donor PBEC compared to the levels produced by the healthy PBEC following treatment with TCCM. SERPIN E1 levels and SERPINE1 gene expression levels were reduced in PBEC from both healthy and COPD donors treated with TCCM, compared to the untreated and vehicle controls. Treatment for 96 hours of COPD PBEC grown at air-liquid interface with TCCM from cells infected with H. influenzae ELB 008 at MOI 50 led to increase mucociliary clearance (MCC), with a decrease in CXCL8 and MUC5AC expression. The final aim of this study was to investigate the effect of first-in-class, small molecule furin inhibitors, BOS-857 and BOS-318 on ICAM-1, a cell-surface adhesion molecule used to facilitate initial adherence of pathogens, including H. influenzae and the human rhinovirus strain, RV-A16. There was an indication of reduced ICAM-1 expression in PBEC treated with BOS-857 and BOS-318 but no clear reduction in ICAM-1 protein levels. Whilst treatment of HBE cells with furin inhibitors did not reduce ICAM-1 levels in those cells infected with H. influenzae (shown to stimulate a pro-inflammatory response, increasing levels of IL-8 and ICAM-1) they did seem to lead to a reduction in IL-8 levels. Furin inhibition was not shown to have an effect on RV-A16 replication in HBE cells or within PBEC from a healthy and COPD donor. In conclusion, this project increased our understanding of the inflammatory responses of airway epithelial cells to pathogenic stimulus which included the identification of a number of inflammatory mediators of interest for future studies. Studies using infected immune cells showed that inflammation within airway epithelial cells could be driven above levels seen from direct bacterial stimulation alone and that this inflammatory response may lead to unexpected increases in MCC and reductions in MUC5AC expression, priming future studies. Whilst there was no clear change in ICAM-1 levels following furin inhibition, IL-8 was identified as an inflammatory mediator of interest for further studies. Future work will build upon the results within project, expanding it to include PBEC from a greater number of healthy donors and COPD donors and to further refine models developed within this project to give further insight into the inflammatory responses of airway epithelial cells in COPD.

Sedentary behaviour is an emerging risk factor for cardiovascular disease (CVD), but there is a clear gap in the literature with regards to the mechanisms that might underlie this association. Psychobiological stress reactivity testing is a paradigm that could be used to investigate potential mechanisms linking sedentary behaviour with CVD, including those that may not be evident under resting conditions. The pre-existing sedentary behaviour and stress reactivity literature was systematically reviewed (Chapter 2) and it was found that no studies had examined postural and metabolic components of sedentary behaviour in the context of psychobiological stress reactivity. Therefore, Chapters 4 and 5 explored the link between habitual sedentary behaviour (using inclinometry) and psychobiological responses to acute psychological stress. Sedentary behaviour was positively associated with cardiovascular, inflammatory, and cortisol reactivity to stress (Chapter 4). Sedentary behaviour was also related with respiratory responses (as an index of metabolic regulation) to stress (Chapter 5), with hyperventilatory and metabolically dysregulated responses observed in those with higher volumes of sedentary behaviour. Chapter 6 extended the observational findings from Chapters 4 and 5 (focusing on habitual sedentary behaviour) to a randomised crossover trial, which examined whether frequent interruptions to prolonged sitting could impact stress reactivity measures. Breaking up 4 hours of prolonged sitting every 30 minutes with light body-weighted resistance exercise altered cardiovascular, inflammatory, cortisol and respiratory responses to acute psychological stress, relative to 4 hours of uninterrupted prolonged sitting. In conclusion, the findings from Chapters 4 and 5 suggest that dysregulated psychobiological stress reactivity is a possible mechanism linking habitual sedentary behaviour with CVD, which therefore helps to fill an important gap in the literature. As shown in Chapter 6, altering psychobiological responses to stress by reducing sedentary behaviour (e.g., by regularly breaking up prolonged sitting) could be a clinically relevant strategy to reduce CVD risk, but further research is needed to explore the longer-term implications of our acute findings.

Acute liver failure is a rare and devastating condition. In the Western world, paracetamol overdose is the leading cause of acute liver failure. Although there have been improvements in the management of patients with acute liver failure - most notably the acceptance of emergency liver transplantation as a therapeutic option - mortality rates remain high. The studies in this thesis focus on advancing our understanding of the short and long-term outcomes in patients with acute liver failure. In Scotland, all patients with acute liver failure are managed in the Scottish Liver Transplant Unit (SLTU). This thesis describes changes in the aetiology of acute liver failure, changes in the management of acute liver failure and changes in short-term outcomes in patients admitted to the SLTU over a 22-year period. In acute liver failure, the generally accepted paradigm is that if the patient survives to hospital discharge without requiring emergency liver transplantation, then they return to a level of health similar to that of the general population. The studies in this thesis disprove this theory, and demonstrate that spontaneous (transplant-free) survivors of acute liver failure have a significantly increased risk of death after discharge compared with the general population. Spontaneous survivors of acute liver failure also have a significantly increased risk of readmission to hospital. These observations challenge current thinking in the field of acute liver failure, and suggest that formal follow up strategies for these patients should be devised. It remains challenging for clinicians to determine prognosis in patients with acute liver failure, and to be confident in identifying which patients will survive without emergency liver transplantation. New prognostic scoring systems have been devised. Work in this thesis externally validates an American prognostic tool designed to predicted short-term transplant-free survival in a UK population of patients with acute liver failure. Although of some value in predicting prognosis, no single tool should be used in isolation to make clinical decisions about an individual patients' need for liver transplantation. Novel treatments, accurate prognostic scoring tools and formal follow up strategies are needed to improve short and long-term outcomes in patient surviving acute liver failure with and without emergency liver transplantation.

In recent years, e-cigarettes have been marketed as an alternative to cigarette smoking that is less damaging to health. However, there is a lack of scientific evidence to support this assertation that they are a safer alternative to cigarettes. This study aimed to evaluate the effect of e-cigarettes on key respiratory pathogens which included S. aureus (SA), P. aeruginosa (PA), H. influenzae (HI), S. pneumoniae (SP) and M. catarrhalis (MCAT). Results from this study have shown that neat e-liquids can reduce bacterial viability with particular flavours causing significant reductions in bacterial viability. It was found that e-liquids contained numerous chemical compounds some of which had antimicrobial properties. The greater the number of chemical compounds present in the e-liquid the greater the reduction in bacterial viability. E-liquid induced antimicrobial pressure affected HI, MCAT and SP isolates to a greater degree than PA and SA isolates. It was discovered that both vaped and neat e-liquids increased biofilm formation in PA and SA isolates whilst decreasing biofilm formation in HI, SP and MCAT isolates. The vaping of the e-liquids indicated that PGVG and flavours had significant effects on biofilm formation in bacteria. Additionally, e-cigarette vapour extract (ECVE) exposure modified the expression of two PA virulence factors, pyoverdine and pyocyanin. The chemical components of e-liquids components can act as environmental stressors resulting in modification of important bacterial virulence factors. Exposing respiratory bacteria to ECVE or cigarette smoke extract (CSE) before infection was found to result in significant increases and decreases in the release of IL-8 and IL-6 and activation of NF-κB in human cells. The degree of effect on cell inflammation appeared to be dependent on the bacterial isolate and whether it was exposed to ECVE or CSE. The exposure of respiratory isolates to ECVE or CSE can modulate the host immune response to infection. Overall, the study shows that e-cigarette exposure can induce changes in key respiratory pathogens associated with chronic lung diseases. These changes could impact host-bacterial interactions and may increase bacterial persistence and inflammatory potential.

Colorectal cancer (CRC) is a heterogenous disease of the intestinal epithelium that is characterised by the accumulation of mutations and a dysregulated immune response. A growing body of literature now also illustrates that this is accompanied by an "oncogenic" CRC-associated microbiota, but whether this dysbiosis is a functional hallmark of CRC or merely a bystander effect of disease is unclear. Therefore, the aim of this DPhil project was to identify changes within the microbiome during cancer progression and the functional consequences. To achieve this, we induced dysbiosis in genetically susceptible mice by colonisation with the pathobiont Helicobacter hepaticus (Hh) followed by the treatment with the carcinogen azoxymethane (HhAOM). Using a range of unbiased longitudinal approaches, we found a distal colon-specific neutrophil signature during tumorigenesis and identified 118 genes that were differentially expressed in HhAOM versus control mice, indicative of putative tumour-promoting properties. Amongst these, the enzyme peptidyl arginine deaminase 4 (PAD4) was upregulated in epithelial cells of early and late tumorigenesis. PAD4 was verified to play a functional role in cancer progression because therapeutic in vivo inhibition reduced tumour area. Alongside this observation, we discovered that Hh resides within established tumours but lacks the capability to drive CAC in gnotobiotic settings. Collectively, our findings suggest that Hh-driven colitis triggers an early neutrophil influx into the distal colon which fosters a microenvironment that promotes tumour progression characterised by the expression of PAD4. These observations open new potential avenues to explore tumour-promoting function of neutrophil subsets in more detail and the role of the citrullination enzymes like PAD4 in CRC progression.

Inflammation is a critical immune response that protects the host against infection and tissue damage. However, dysregulated inflammation can be damaging to host tissue and can exacerbate disease. The NOD-like receptor pyrin-domain-containing protein 3 (NLRP3) inflammasome is a key orchestrator of inflammation, activating caspase-1-mediated processing and release of the pro-inflammatory cytokines interleukin (IL)-1beta and IL-18, as well as driving gasdermin D (GSDMD)-dependent pyroptotic cell death. In addition to pathogenic stimuli, NLRP3 senses perturbations in cellular and tissue homeostasis. As such, aberrant NLRP3 activation is frequently associated with low-grade, sterile inflammation that contributes to the pathogenesis of many non-communicable diseases, including Alzheimer's, atherosclerosis and diabetes. As such, the NLRP3 inflammasome represents an attractive therapeutic target. In order to facilitate the development of new treatments for diseases in which NLRP3 is implicated, the primary aim of this thesis was to gain further insight into the mechanisms governing NLRP3 inflammasome activation and mature IL-1beta secretion. Growing evidence suggests that chloride (Cl-) efflux is an important cellular event during NLRP3 inflammasome activation. The work conducted in this thesis demonstrates that the volume regulated anion channel (VRAC) is essential for hypotonicity-induced NLRP3 inflammasome activation but is dispensable for NLRP3 inflammasome activation in response to classical NLRP3 agonists such as adenosine triphosphate (ATP), nigericin and silica, suggesting that alternative Cl- channels are involved in the response to these stimuli. Following on from this work, Cl- channels were then explored as therapeutic targets within the NLRP3 inflammasome pathway. Using rational chemical design and iterative screening, a new series of urea-based Cl- channel inhibitors were developed that demonstrate potency and selectivity for the potassium (K+)-dependent pathway of NLRP3 inflammasome activation. In developing these molecules, a specific role for Cl- efflux during the K+-dependent pathway of NLRP3 inflammasome activation was revealed. Finally, we present an inflammasome-free model of unconventional IL-1beta secretion that allows detailed study of the mechanisms underlying IL-1beta release in the absence of GSDMD pore formation and/or pyroptosis. A new proximity labelling method to explore the IL-1beta interactome was also established with the aim of identifying mediators of IL-1beta release in our cell model. In summary, the findings of this thesis provide new insights into the mechanisms governing NLRP3 inflammasome activation and IL-1beta release, and excellent tools to study this inflammatory pathway. In doing so, these studies reveal new therapeutic avenues for selective inhibition of NLRP3/IL-1beta, which may pave the way for new treatments for inflammatory disease.

Background: Exercise may reduce inflammation in coronary artery disease (CAD) patients. However, the molecular mechanisms that orchestrate this cardioprotective effect are yet to be elucidated. Moreover, CAD patient participation rates in cardiac rehabilitation (CR) and long-term exercise are poor. Thus, this thesis aimed to further scientific understanding of the role of exercise in the secondary prevention of CAD by investigating these areas. In addition, the factors that influence CAD patient participation in CR and long-term exercise training were qualitatively explored. Methods: The evidence generated by randomised studies that investigated the effect of exercise on inflammatory biomarkers in CAD patients was examined by a systematic review and meta-analysis (Study 1). A pilot prospective cohort study (Study 2) was performed in post-acute myocardial infarction (AMI) patients who had been invited to a phase-III CR programme. Finally, Study 3 comprised semi-structured interviews with post-AMI patients and their significant others. Results: Study 1 demonstrated an anti-inflammatory effect of exercise in CAD patients, as indicated by a reduction in C-reactive protein, fibrinogen, and von Willebrand factor post-intervention. The outcome of Study 2 resulted in a future prospective cohort study being deemed feasible with minor amendment (recruitment strategy). Moreover, preliminary evidence for a beneficial effect of exercise on sirtuin-1 in post-AMI patients was generated. Regarding Study 3, post-AMI patients and their significant others reported that health benefits were the primary motive for participating in CR and long-term exercise, with aspects related to motivation, extrinsic influences, and CR experience underpinning the decision. Conclusion: The results of Study 1 and Study 2 further scientific understanding of the role of exercise in the secondary prevention of CAD, and offer future directions to stimulate progress in this area. Finally, the novel qualitative findings of Study 3 may inform future strategies to promote patient participation in CR and long-term exercise.

Diseases associated with unhealthy diets and lifestyles, such as type 2 diabetes, atherosclerosis, and fatty liver disease, are driven by sterile tissue inflammation that is driven in part by the NLRP3 inflammasome. This macromolecular complex responds to diverse danger signals by inducing secretion of inflammatory IL-1 family cytokines and promoting a type of cell death called pyroptosis. Saturated fatty acids (SFAs) are known to activate the NLRP3 inflammasome but the intracellular pathways by which this activation occurs are incompletely described. I hypothesized that SFAs activate the NLRP3 inflammasome through different intracellular pathways than "classical" NLRP3 activators. Motivated by preliminary data on the kinetics of NLRP3 activation by the SFA palmitic acid (PA) and the weak effect of the NLRP3 inhibitor MCC950, I found that this process requires not only the canonical inflammasome pathway but also the non-canonical inflammasome components caspase-4/5/11. Disruption of either pathway leads to partial impairments in the inflammasome response to PA, though the canonical pathway generally accounts for more IL-1 secretion while the non-canonical pathway is the primary driver of cell death. PA also has a weaker dependence on reactive oxygen species (ROS) compared to other NLRP3 activators. While classical activators engage multiple ROS sources including both mitochondrial and cytosolic enzymes, PA activation of NLRP3 specifically requires ROS from NADPH oxidases. PA exposure leads to activation of the MAP kinase JNK in the cytosol of macrophages and interference with JNK signaling blocks PA-induced NLRP3 activation. While SFAs activate the NLRP3 inflammasome, unsaturated fatty acids such as arachidonic acid (AA) inhibit NLRP3 activation. AA counteracts the effect of PA on multiple intracellular signaling pathways, especially the JNK pathway. My findings further characterize the mechanisms by which SFAs activate the NLRP3 inflammasome and describe a novel role of JNK as a key mediator for opposing effects of saturated and unsaturated fatty acids on NLRP3. These mechanistic insights highlight pathways that may be targeted to delay or reverse the progression of sterile inflammation in many "lifestyle diseases".

Inflammation, the body's response to tissue damage or infection, involves a number of cell types and is often associated with localised tissue acidosis. The resulting decrease in extracellular pH can exacerbate inflammation, additionally protons may directly depolarise or sensitise sensory neurones to drive the heightened nociception associated with chronic inflammatory diseases such as rheumatoid arthritis and inflammatory bowel disease. Extracellular protons can coordinate cellular responses through a number of proton-sensitive receptors including both ion channels and G protein-coupled receptors, many of which are expressed by immune cells and sensory neurones, the principal drivers of inflammation and nociception respectively. While proton-sensitive ion channels, such as the acid-sensing ion channel family have established roles in inflammatory pain, the contribution of proton-sensing G protein-coupled receptors (PS-GPCRs) is less clear. The overarching aim of this work was to further basic pharmacological understanding of the PS-GPCR family and assess their role in inflammatory processes and nociception. The pain associated with chronic inflammation is severely debilitating and represents major unmet clinical need worldwide, there is thus considerable interest in elucidating the molecular mechanisms underlying inflammatory pain. Animal studies represent a valuable tool for studying the complexities of inflammation, which involves a concert of cell types. However, the interplay between changes at the cellular level and the manifestation of hypersensitive states in rodent models is often poorly assessed. Here, combining evoked and spontaneous behavioural assays, target innervating electrophysiology and gene expression studies, a comprehensive characterisation of sensory neurones and pain behaviour was made for the intraplantar carrageenan model of inflammatory pain in mice. Results highlighted a dynamic relationship between physical inflammation, changes in sensory neurone function and rodent pain experience, and suggested a potential role of PS-GPCRs in the observed pain. To further explore the possibility that PS-GPCRs might mediate proton-induced pain, attention was turned to the naked mole-rat (Heterocephalus glaber), a species which does not perceive acid as painful. Cloning and characterisation of PS-GPCRs from naked mole-rats and mice revealed differences in the abilities of protons to coordinate signalling at PS-GPCRs between the two species, furthering the idea that PS-GPCRs might contribute to proton-induced pain. The lack of pharmacological and physiological understanding of PS-GPCRs may be attributed to the fact that protons activate a number of other receptors, as well as technical difficulties arising from the high sensitivity of many pharmacological assays to perturbations in pH. Using a uniform cellular background, the signalling and intracellular trafficking of the PS-GPCR GPR65 was interrogated in response to stimulation with protons as well as two other reported agonists. These studies highlighted that a synthetic agonist (BTB09089), with higher selectivity for GPR65 over other PS-GPCRs, was able to recapitulate many of the proton- induced signalling responses of GPR65. Injecting BTB09089 into the knee joint of mice, resulted in joint swelling and a concomitant increase in nociceptive behaviours. Electrophysiological studies of knee-innervating neurones isolated from mice injected with BTB09089 revealed higher excitability, offering some explanation to the observed pain. However, acute stimulation of naïve neurones with BTB09089 alone could not reproduce the changes in excitability, suggesting the involvement of another cell type. Fibroblast-like synoviocytes, non-neuronal cells resident in the joint, were shown to express GPR65 and secrete pro-inflammatory mediators in response to BTB09089 stimulation, which might be responsible for the sensory neurone sensitisation observed. Taken together data presented here support a central hypothesis that PS-GPCRs, in particular GPR65, are critical integrators of neuroimmune responses to tissue acidosis in inflammation.

Iron deficiency is a common complication of colorectal cancer, being present in ~60% of patients and often leading to the manifestation of anaemia. Preoperative anaemia in colorectal cancer patients is associated with inferior clinical outcomes, hence this leads to the requirement of iron supplementation to treat anaemia. The current standard treatment for iron deficiency anaemia is oral iron supplementation. However, this can contribute to an increased gut luminal iron concentration, which has the potential to alter the gut microbiota and mucosal immune system, potentially leading to inferior oncological outcomes. Previous animal studies have supported this association; however, here is provided the first human clinical studies to assess the microbiological and immunological outcomes of iron supplementation, through a colorectal cancer randomised control trial comparing oral and intravenous iron therapy. The results of these studies suggest that oral iron leads to differential bacterial populations, potentially contributing to a more procarcinogenic microbiota, as well as leading to greater tumour immune cell activity and increased pro-inflammatory cytokine production, compared to intravenous iron therapy. Furthermore, this research provides an insight into the differences between patients with right- and left-sided colorectal cancer; showing the non-tumour microbiota is significantly different between right- and left-sided colorectal cancer patients, whereas the tumour microbiota is more consistent. Furthermore, the results show that right-sided colorectal tumours are more immunogenic, showing an increase in inflammatory cytokines compared to patients with left-sided colorectal tumours. Finally, presented is the long term clinical data from this cohort of patients, assessing differences in tumour location and iron therapy on survival outcomes. Collectively the results of these studies support the use of intravenous iron therapy preoperatively in colorectal cancer patients with iron deficiency anaemia, in order to limit the potential microbial perturbations and inflammatory outcomes associated with oral iron therapy. As well as supporting the stratification of colorectal cancer based upon tumour location, particularly in regard to studies of probiotic and immune therapies.

Background: Inflammation and angiogenesis are hypothesised to be key contributors to the tumour microenvironment (TME) in vestibular schwannoma (VS) but few in vivo studies of this microenvironment or its response to radiotherapy have been performed. In this thesis we sought to interrogate the inflammatory and microvascular microenvironment in sporadic and neurofibromatosis type II (NF2) related VSs through a combined PET, MRI and neuropathology based approach. Methods: 28 sporadic VS patients were recruited and underwent prospective imaging with the TSPO PET tracer 11C-(R)-PK11195 (19 patients) and a comprehensive MRI protocol including high temporal resolution dynamic contrast enhanced (DCE) MRI (24 patients). An intertumour comparison of 11C-(R)-PK11195 binding potential (BPND) across different tumour growth cohorts was undertaken and immunohistochemistry used to confirm the cellular source of TSPO expression. Diffusion MRI and DCE-MRI datasets in 24 sporadic VSs and 20 size matched NF2-related VSs were then compared and regression analysis used to evaluate the effect of tumour size, pre-treatment tumour growth rate and tumour NF2 status on each imaging parameter. Tissue from 17 imaged sporadic VSs and a separate cohort of 12 previously resected NF2-related VSs were examined with immunohistochemistry markers for vessels (CD31), vascular permeability (fibrinogen), macrophage density (Iba1), and proliferation (Ki67). VEGF /VEGF receptor 1 (VEGFR-1) expression in both tumour groups was also evaluated through immunohistochemistry, western blotting and double immunofluorescence. Finally changes in the TME of five growing sporadic VSs undergoing stereotactic radiosurgery (SRS) were evaluated through longitudinal multimodal MRI incorporating DCE-MRI, diffusion tensor imaging and a spiral 23Na-MRI acquisition for estimating tumour total sodium concentration (TSC). Results: Compared with static tumours, growing VSs displayed significantly higher mean 11C-(R)-PK11195 BPND (p=0.020). Immunohistochemistry demonstrated that within growing VSs TSPO is predominantly expressed within tumour associated macrophages (TAM) and that TAM rather than Schwann cells accounted for the majority of Ki67+ proliferating cells. DCE-MRI-derived microvascular characteristics were markedly similar across imaged cohorts of sporadic and NF2-related VSs with Ktrans (p < 0.001), ve (p=0.004) and tumoural free water content (p=0.003) increasing with tumour size and pre-treatment tumour growth rate. Tissue analysis confirmed the imaging metrics and demonstrated that amongst both resected sporadic and NF2-related VSs there was consistent TAM infiltration, a close association between tumour vascularity and Iba1+ TAM density (r=0.55, p=0.002) and VEGF/VEGFR-1 expression by TAM. Interrogation of changes in the VS TME post SRS demonstrated marked reductions in the tumoural microvascular metrics Ktrans (p < 0.001) and vp (p < 0.001) at 6 months post-treatment and demonstrated that changes in tumour microvasculature, microstructure and tumour TSC could be detected as early as 2 weeks following treatment. Conclusions: Inflammation is a key contributor to the tumour microenvironment in sporadic and NF2-related VS and is associated with tumour growth. Within growing VSs, TAM rather than tumour cells account for the majority of proliferating cells and are likely instrumental to angiogenesis. In vivo this inflammatory microenvironment can be imaged using 11C-(R)-PK11195 PET and DCEMRI, and following radiotherapy early changes in tumour microvasculature, microstructure and cellular viability can be quantified using advanced MRI techniques. The data presented within this thesis provide compelling evidence for consideration of inflammation as a therapeutic target in VS and provide early data in support of clinically applicable imaging biomarkers for quantifying and monitoring this intratumoural inflammation in vivo.

Over 150 million individuals worldwide are affected by impaired wound healing, which can lead to chronic wound pathologies that often require lower limb amputations. Impaired wound healing is particularly prevalent in two growing demographics: aged and diabetic populations. Impaired wound healing continues to persist due to a lack of viable treatment options, exacerbated by an incomplete understanding of the mechanisms of wound healing. Research in this area has highlighted a significant role of dysregulated immune cells in impaired wound healing. We aimed to investigate the role of a key immune cell in wound healing: macrophages and how a known regulator of macrophages in other contexts, ERK5, influences wound healing, using genetic and therapeutic manipulations targeted at ERK5, both in vitro and in vivo. Genetic in vivo hyperactivation of ERK5 causes a persistent inflammatory profile in wounds, which aligned with similar observations of aberrant ERK5 signalling contributing to inflammation driven cancer progression. In contrast, genetic inactivation caused a brief increase in inflammation followed by an earlier shift to the resolution phase of wound healing as confirmed by flow cytometry and histology. This increase in reparative wound signalling in response to targeting ERK5 was also observed in human systems, using an in vitro keratinocyte model treated with a novel compound that targets and degrades ERK5, called OS1. Furthermore, using OS1 we were able to prime M0 THP-1 macrophages towards a reparative M2-like phenotype. This OS1-mediated pro-healing wound phenotype was recapitulated in vivo, where topical treatment with OS1 (inducing ERK5 loss in the entire wound) increased the rate of wound closure and was associated with an influx of M2-like macrophages and an earlier switch to the resolution phase of wound healing. This work is the first ever evaluation of the in vivo role of ERK5 in wound healing. We discovered that ERK5 signalling drives macrophage polarisation and immune infiltration during wound healing. The findings have the potential to improve diagnosis, stratification and treatment of chronic wounds. This study also provides evidence of the need to deviate from the traditional approach to target ERK5 via kinase inhibitors, since OS1-mediated degradation of ERK5, unlike ERK5 kinase inhibitors, was able to recapitulate the immune phenotypes of ERK5 genetic ablation models. Overall, this work highlights the potential for OS1 to be used to treat patients with impaired wound healing, such as those associated with diabetes.

Autophagy is an evolutionary conserved lysosomal degradation pathway, which can control cell-intrinsic functions such as di erentiation and cellular metabolism. The cell-intrinsic role of autophagy is of particular importance to immune cells, but autophagy also has a cell-extrinsic role in the tumour environment. It remains unclear whether autophagy in the tissue environment can alter immune responses. The release of free fatty acids (FFA) from adipocytes in times of high energy demands (e.g. inflammation) classically depends on the function of cytosolic lipases, however, the contribution of autophagy to this process remains unknown. Here, we investigated the role of autophagy in mature adipocytes during colitis, and its impact on intestinal inflammation and the immune response. Induction of intestinal inflammation using dextran sulphate sodium (DSS) increased autophagic flux in adipose tissues. Furthermore, the adipocyte-specific deletion of Atg7, an essential autophagy gene, resulted in an exacerbation of intestinal inflammation upon DSS treatment. We found that loss of adipocyte autophagy impaired optimal lipolytic secretion and availability of FFA during intestinal inflammation. Adipose tissue macrophages react to local FFA availability by altering the expression of the FFA transporter CD36. In line with this, restricting FFA from LPS-stimulated macrophages blunted CD36 expression and reduced IL-10 secretion, while supplementation of the FFA linoleic acid rescued IL-10 expression. Accordingly, we found that during DSS colitis IL-10 secretion from adipose tissues and serum IL-10 levels were reduced in mice with adipocyte-specific autophagy loss. This study highlights an underappreciated anti-inflammatory function of adipose tissues during intestinal inflammation. Future studies will be required to investigate whether this anti-inflammatory pathway is conserved in human inflammatory bowel disease and whether it can be exploited therapeutically. Overall, results presented in this study revealed a novel cell-extrinsic function of autophagy in the metabolic regulation of immune responses, mediated by the provision of adipocyte-derived FFAs for IL-10 production.

Nutrient deprivation through caloric restriction or time-controlled fasting has a profound impact on reducing risk factors and symptoms of inflammatory disease. Studies have highlighted the immune-modulatory and anti-inflammatory effects of nutrient deprivation, suggesting a critical role for metabolic and mitochondrial function on immune activity. To explore the mechanisms underlying these effects, we have carried out a clinical study to assess the immunologic effects of 24 hours of fasting followed by a period of re-feeding on peripheral blood mononuclear cells (PBMCs), monocytes, and CD4+ T cells in normal volunteers. Preliminary results from these studies show (i) a fasting-state decrease in activation of the NLRP3 inflammasome, a component of the innate immune system and a mediator of sterile inflammation, as well as (ii) a decrease in activation of CD4+ T cells. Furthermore, serum collected in the fasting and re-feeding states recapitulates these immune-modulatory effects in human macrophages, monocytes, and CD4+ T cells. Together, these findings suggest that nutrient level-dependent circulating proteins or metabolites, through putative paracrine signaling, confer these effects on innate and adaptive immune cell functioning. We hypothesize that circulating metabolites play a pivotal role in regulating nutrient-dependent immune function. To identify these metabolites and elucidate the underlying immune-modulatory mechanisms, we have pursued a systems biological approach that uses multivariate modeling of metabolomics and lipidomics data to describe changes in the circulating metabolome of fasting. Further, integration with PBMC RNAseq data has revealed key metabolite-sensing G protein-coupled receptors involved in fasting-mediated immune modulation. Through functional characterization of identified metabolites and metabolite-sensing pathways, we have identified a novel signaling role for the fatty amide N-arachidonylglycine in decreasing the activation of pro-inflammatory Th1 and Th17 CD4+ T cells during fasting. Additionally, we have characterized the counter-regulatory role of the short-chain fatty acid propionate in blunting the CD4+ T cell inflammation associated with re-feeding. Finally, we have found that N-arachidonylglycine and propionate reduce the elevated inflammatory cytokines from CD4+ T cells collected from volunteers with obesity to the level of those from lean volunteers. Further exploration of these anti-inflammatory signaling effects will contribute to our understanding of the complex mechanisms of fasting-mediated immune modulation and shed new light on the role of nutrient intake on inflammatory pathophysiology and treatment of metabolic disease.

During tissue injury, inflammatory cells migrate towards the injury site to release mediators and allow the tissue to regain its function. In disease states, these mechanisms can be dysregulated resulting in exacerbated tissue damage. This thesis uses the larval zebrafish caudal fin injury model to study the migration of innate inflammatory cells, including neutrophils and macrophages, post-injury during the tissue repair process in the tailfin. The lipid mediator platelet-activating factor (PAF) is produced by many cells. For example, macrophages release PAF following efferocytosis (phagocytosis of apoptotic cells). This research is novel as it investigates the mechanisms involved in the regeneration process of zebrafish larvae, specifically showing how PAF plays a role in the regeneration process. In this thesis, I hypothesise that macrophages and PAF are involved in the mediation of tissue repair in zebrafish caudal fin injury. Transgenic zebrafish with fluorescently labelled neutrophils (tg(mpx:GFPi114) and macrophages (tg(mpeg1:mCherry) were used in vivo to image and to examine the role of the innate inflammatory cells in tissue regeneration. To explore the role of macrophages more extensively, wildtype (WT) and macrophage-depleted (IRF8-/-) larvae were used. Zebrafish larvae were tailfin transected at 3 days post-fertilisation (dpf) and subsequently imaged up to 48 hours post-injury (hpi) to examine their tailfin regeneration. Following transection of the caudal fin, neutrophil migration increased at 4 hours, decreased at 24 hours and increased again at 48 hours. Macrophages migrated towards the wound at 4 hours with a peak at 24 hours, which plateaued at 48 hours. Simultaneously to the immune cell migration, the regeneration of the tailfin was measured using time lapse imaging and quantified using ImageJ. Bathing larvae in PAF (1µM) resulted in an increased rate of tailfin regeneration. In addition, a synthetic stable analogue of PAF, carbamyl-PAF (c-PAF) (500nM), also caused an increase in the rate of tailfin regeneration. In contrast, the PAF receptor antagonist, WEB-2086 (20µM), caused a reduction in tailfin regeneration in WT zebrafish larvae. Macrophage-depleted zebrafish larvae showed significantly diminished regeneration following injury compared to WT controls. In macrophage-depleted larvae c-PAF rescued this inhibited regeneration of the tailfin. WEB 2086 had no impact on tailfin regeneration in macrophage depleted larvae. Zebrafish larvae can be genetically modified using stable anti-sense oligonucleotide morpholinos (MO). The PAF receptor MO resulted in the inhibition of tailfin regeneration, which could not be rescued by adding exogenous PAF. In macrophage-depleted larvae the PAF receptor MO also caused inhibition of tailfin regeneration, which could not be rescued using exogenous PAF. Upstream of the PAF receptor, several enzymes essential to PAF synthesis were targeted using MO for lysophosphatidylcholine acyltransferase 1 and 2. MO treatment caused decreased regeneration of the tailfin, which was rescued by bathing the larvae in exogenous PAF. MO treatment targeting the de novo pathway directed against cholinephosphotransferase, had no effect on the tailfin regeneration rate and exogenous PAF had no further effect. A novel protocol was developed to isolate both neutrophils and macrophages from transgenic adult and larval zebrafish with immune cells tagged with fluorescent proteins for downstream cellular and molecular analysis. The digested cells were sorted using fluorescence-activated cell sorting (FACS). In the adult zebrafish the kidney was used as this contains high quantities of immune cells. In larvae, either the whole embryo or tailfins were digested from several hundred animals. Once sorted the pure populations of immune cells were then processed further to determine morphological phenotypes or RNA sequencing to identify important pathways and genes involved in tissue repair processes. Patterns of immune cell transcriptional characteristics are presented. My thesis highlights the importance of the innate inflammatory cells and their mediators in tissue repair and regeneration. My work clearly suggests an important role for PAF in this process. The PAF pathway is therefore an important target to develop novel therapeutic strategies for acute and chronic inflammatory diseases.

Neuroinflammation is becoming increasingly recognised as key to the pathogenesis of Alzheimer's disease and tauopthies. Epidemiological studies report a delay in the onset of Alzheimer's in subjects using nonsteroidal anti-inflammatory drugs (NSAIDs). NSAIDs inhibit enzymes in the cyclooxygenase-2 (COX-2) pathway which play a key role in synthesising prostaglandin E2 (PGE2) from arachidonic acid. PGE2 has been implicated in preclinical stages of Alzheimer's disease, where elevated levels of PGE2 in the cerebrospinal fluid can be found, as well as aberrant amyloid processing in experimental models of disease. PGE2 signals via 4 E-prostanoid (EP) receptors, EP1-EP4, all G-protein coupled receptors (GPCR). The EP3 receptor, the most abundant PGE2 receptor in the brain, is unique in that it is alternatively spliced giving rise to species specific isoforms. One of the EP3 receptor isoforms, EP3Re, is human specific and an incidental finding within a project to investigate its distribution in brain, suggested that it could be associated with tau tangles. The aim of this project was to further investigate the unknown distribution of EP3Re in human brain, to determine its signalling mechanism and explore whether any meaningful interaction between EP3Re, tau and its pathology exists. We use immunohistochemistry, proximity ligation assays and electron microscopy to map out the distribution of EP3Re in the human brain and explore the interaction between EP3Re and tau. We also use gene reporter and second messenger assays to characterise EP3Re signalling and what role if any this may be playing in tauopathies. We show that EP3Re is expressed throughout the brain, with strong expression in brain stem nuclei, and signals predominantly through a Gi coupling pathway. Moreover, using a combination of human tissue, primary cell lines and neurons derived from induced pluripotent stem cells, we show that EP3Re appears to be associated with tau neurofibrillary tangles in disease. We also show, using the EP3 agonist sulprostone, that signalling through the receptor increases tau phosphorylation in our cellular systems. Further work will be required to fully clarify the specificity of the interaction and understand the mechanism behind this and if targeting inflammatory EP3Re signalling has the potential to affect tau pathology in disease.

Cystic fibrosis (CF) is a life-shortening, autosomal recessive disease caused by loss-of-function mutations in the CFTR gene. The primary cause of morbidity and mortality in CF is respiratory failure following progressive loss of lung function and characterised by recurrent infections and an intense, chronic, neutrophil-dominated inflammatory airway response that is highly excessive relative to the weight of the actual infectious burden. As such, the inflammatory response has been one of the central focuses of CF research for the past 30 years, with an array of studies elucidating the dysregulation of several key immune processes in CF. Macrophages are the most plastic of all immune cells and their immense functional diversity allows them to play a part in orchestrating all major stages of the inflammatory response, from initiation, to resolution and repair. Macrophages, often overlooked in CF research despite their integral role in other chronic inflammatory pathologies, have increasingly become recognised as potential orchestrators of the excessive inflammatory response in CF. The discovery of functional CFTR expression in several immune cells, monocytes and macrophages in particular, has also opened the possibility that some immune cells, much like epithelial cells, are intrinsically dysregulated in CF and pre-programmed to contribute to an excessive inflammatory response. The aim of this project was to investigate the role of macrophages in driving progressive lung disease in CF and gain a better understanding of the mechanisms underlying dysregulation of CF macrophage immune function, with a focus also on the extent of the CFTR-dependent nature of such dysfunction. Another aim was to track longitudinally the effects of novel CFTR modulator therapies, which work to correct the basic underlying defect in CF, on patient inflammation and myeloid function. Whole-blood flow cytometry was used to show that patient circulating monocytes exist in a significantly higher state of activation than non-CF controls and that this correlates with predicted lung function (FVCpp). This activation phenotype was reversed with CFTR modulator (Elexacaftor/Tezacaftor/Ivacaftor) treatment suggesting that such treatment modulates levels of inflammation in patient circulation. CF monocyte-derived macrophages (MDMs) were found to exhibit a hyperinflammatory response to activation with P. aeruginosa LPS and produce elevated levels of several important proinflammatory cytokines by qRT-PCR and ELISA. Given that such cells were cultured for 9 days in the same conditions as non-CF controls, such a phenotype would point towards intrinsic immune dysregulation in the cells. In order to gain a more high-resolution, genome-wide view of transcriptional changes in CF MDMs responding to inflammatory challenge, a highly unbiased approach of global transcriptome profiling was carried out using the cap analysis of gene expression (CAGE) method, which provides additional layers of information regarding promoter usage, enhancer activity and other important regulatory elements that drive gene expression. CAGE sequencing revealed substantial differences in gene expression patterns of CF LPS-treated MDMs compared to non-CF controls, particularly involving genes involved in the immune response, and elucidated a significantly blunted type I IFN signalling response in CF macrophages at 24 hours post-LPS. Such results provide scope for investigating the impact of defective type I IFN signalling in macrophages on bacterial inflammation in CF and also on antiviral immunity, with acute respiratory viral infections representing important, and common, precipitants of pulmonary exacerbation in CF. These findings provide novel insights into macrophages dysfunction in CF and further underline the potential of these cells as a target for novel, and thus far elusive, anti-inflammatory interventional strategies to effectively slow CF lung function decline in combination with novel CFTR modulators and the standard treatment regimen.

Neuroinflammation can be considered a tightly coordinated network of events involving the engagement of CNS-resident microglia and the recruitment of leukocytes from the periphery following an injurious or noxious insult. This process involves an initial amplification of the inflammatory event transitioning to a pro-resolution response in order to restore a healthy brain parenchyma. Yet, an aberrant or sustained neuroinflammatory response underpins the pathological progression of a multitude of acute and chronic neurological conditions. Numerous studies have implicated the immunoglobulin receptor "Triggering receptor expressed on the surface of myeloid cells-2" (TREM2), most notably expressed on microglia and on monocyte-derived cell populations, as a master regulator of core myeloid cellular functions. Moreover, individuals with TREM2-variants exhibit an increased susceptibility to chronic neurodegenerative diseases including Alzheimer's disease (AD) and frontotemporal dementia (FTD). Thus, given a substantial role of these cell subsets in many forms of neuroinflammation, this project sought to determine the influence of TREM2 on microglial and monocyte-derived cell reactivity and how it regulates inflammation resolution following an acute CNS inflammatory insult. To interrogate aspects of the neuroinflammatory cascade, both in vitro and in vivo models were employed. Firstly, an in vivo model of acute CNS inflammation, involving localised stereotaxic injections of lipopolysaccharide (LPS) into the striatum, showed an enrichment of the Trem2 transcript during the resolution phase highlighting a potential engagement of TREM2 function at this time. A bilateral version of a previously characterised a model of self-limiting acute CNS inflammation confirmed a defined neutrophil influx during the initiation of the inflammatory cascade followed by clearance during resolution. Moreover, the model demonstrated a symmetrical inflammatory response in both hemispheres enabling dual flow cytometry and immunohistochemistry analyses. Neutrophils were effectively cleared from the brain parenchyma in both WT and Trem2-/- mice after intrastriatal LPS injection. A reduction in the number of microglia and Ly6Clo monocyte-derived cell populations during resolution was observed in the absence of TREM2. Furthermore, this model identified an influx of potential CD11b-CD45hi lymphocyte populations which are maintained in the system during resolution in both genotypes. Additional analysis of the systemic immune blood and bone marrow compartments did not show an effect of TREM2 deficiency on myeloid cell populations in either location but did, however, identify an increase in T-cell proportions in the blood in Trem2-/- mice prompting speculation of an involvement of TREM2 in connecting innate and adaptive arms of the immune system at later stages of resolution. An in vitro primary adult murine microglial model was optimised and validated as a suitable platform to assess transcriptional responses to inflammatory stimuli, LPS and interleukin-4 (IL- 4), which mimic conditions associated with pro-inflammatory and pro-resolution aspects of the cascade, respectively. Application of these stimuli, to wildtype (WT) primary microglial cultures showed stimulus dependent induction of gene expression and, notably, the suppression of Trem2 in response to LPS. Moreover, WT and TREM2-deficent primary microglia and bone-marrow derived macrophages (BMDMs) were stimulated similarly. Markedly, transcriptional alterations in Trem2-/- BMDM cultures suggested the induction of compensatory transcriptional mechanisms associated with phagocytosis and lipid metabolism at baseline and the impaired induction of genes associated with regulating autophagy and apoptosis following LPS treatment. Taken together, the results within this thesis have furthered understanding of the multifaceted and complex nature in which TREM2 may regulate myeloid cell activity. Furthermore, these data suggest that TREM2 may influence an innate-to-adaptive transition in the immune response during acute CNS inflammation - a theory that requires more targeted and in-depth investigation in future studies.

Cardiovascular disease (CVD) is a major cause of premature deaths and disability globally. Inflammation, the response of the immune system to damaging stimuli, was reported to contribute to CVD risk, with key inflammation-associated proteins (e.g. interleukin-6 [IL-6] signalling) targeted in recent clinical trials. I investigated whether components (IL-6 and IL-6 receptor [IL-6R]) and markers (C-reactive protein [CRP] and fibrinogen) of the IL-6 signalling were associated with CVD risk, with the aim of identifying relevant therapeutic targets for CVD. First, using a nested case-control study design in the prospective China Kadoorie Biobank (CKB), I reported that in Chinese adults higher plasma CRP concentrations, but not plasma fibrinogen levels, were log-linearly associated with higher risks of major coronary events, ischaemic stroke (IS), and intracerebral haemorrhage (ICH). However, prior genetic studies have reported null associations of plasma CRP- associated genetic variants with CVD risk, precluding CRP as a therapeutic target for CVD. To investigate potential therapeutic targets, I used genome-wide association studies of CRP in CKB and in Europeans to derive genetic instruments representing the IL-6R and the IL-6 ligand. The IL-6R instrument was a non-synonymous genetic mutation in the IL6R locus (rs2228145-C) and reportedly mimicked the effects of tocilizumab (an IL-6R antagonist). The IL-6 instrument was composed of three genetic variants in CKB and a single genetic variant in Europeans (rs1880241-G) near the IL6 locus. The CKB/EUR IL-6 genetic instruments were, therefore, constructed to broadly represent effects of lower IL-6 signalling. Using a two-sample Mendelian randomization approach, I showed that although the IL-6R instrument, associated with lower plasma CRP, was associated with a lower risk of coronary heart disease (CHD) phenotypes in both CKB and Europeans, there was no evidence of an association of the IL-6 instrument with CHD in either population. Additionally, on trans-ethnic meta-analysis, there was weak evidence that the IL-6R instrument was associated with a lower risk of IS and a higher ICH risk but with consistent estimates observed with the IL-6 instrument. In conclusion, although the observational study supported the role of inflammation in CVD, the null association of the IL-6 instrument with CHD in both the Chinese and Europeans with weak evidence of lower and higher risk of IS and ICH, respectively, questions the net therapeutic value of targeting IL-6 signalling for CHD.