Streptococcus pneumoniae is a major cause of morbidity and mortality in HIV infected individuals. Mortality rates remain high despite modern antibiotics, therefore, successful vaccination is key to preventing infection. Vaccination strategies against pneumococcus include a pure polysaccharide vaccine, Pneumovax® (PPV-23), and a polysaccharide-conjugate vaccine, Prevenar-13 ®(PCV-13). PCV-13 is given as part of three vaccine series in infants but is currently recommended only as a single vaccine in adults. The evidence base for either vaccine in adults is limited and guidelines vary. In 2009, national guidelines recommended PPV-23 vaccination but a PCV-13+PPV-23 schedule is now recommended (BHIVA, 2008 & 2015). The Assessment of Immune Responses to Routine Immunisations (AIR) is an observational study that has examined the immune response to UK recommended vaccinations in adults with HIV. Firstly, the AIR study went on to examine the impact of HIV-infection on pneumococcal vaccination with PPV-23, as assessed by pre- and post- vaccine IgG antibodies against 12 pneumococcal (Pn) serotypes (Pn 1, 3, 4, 5, 6B, 7F, 9V, 14,18C, 19A, 19F, and 23F at the WHO (World Health Organization) protective threshold 0.35μg/mL in ≥ 8/12 serotypes threshold using a 19-plex Luminex-based assay. HIV-infected patients responded poorly to a single dose of PPV-23 compared to HIV-negative controls. AIR then established that PCV-13 could increase the percentage of patients that reach WHO protective thresholds compared to a single dose of PPV-23 and that immune responses to PCV-13 could be maintained for a longer period compared to PPV-23. Response rates could be further improved by booster doses of PCV-13. Low antigen-specific IgG concentrations are associated with impaired opsonophagocytic killing against pneumococcus, thus a novel opsonophagocytic assay was developed in order to further understand the relationship between the quantity and opsonic functionality of Pn-specific antibody. Furthermore, assessing responses to pneumococcal vaccination is important in determining immunogenicity. Thus, this thesis also explored the whole vaccine and serotype IgG subclass (IgG1-IgG4) response to PPV-23 and PCV-13 by developing novel Pn-specific IgG subclass assays. Lastly, HIV-infection is characterised by a dysregulated humoral system, therefore, we have examined the impact on different B cell populations at baseline and relationships with total and Pn- specific antibody post-vaccination with PCV-13 are described. In summary, this study aimed to examine the impact of HIV-infection on pneumococcal vaccination by investigating the quantity and quality of the Pn-specific IgG response, IgG subclass responses, and the effects of a dysfunctional humoral system on total and Pn-specific antibody. Findings would be informative in developing vaccination strategies in HIV-infected adults in the UK.

The ability of umbilical cord-derived mesenchymal stromal cells (UC-MSCs) to immunomodulate offers therapeutic potential in liver injury but the inherent heterogeneity of unsorted MSC populations may explain varied/reduced function as well as posing regulatory challenges. Thus, we aimed to evaluate the therapeutic potential of purified CD362+ MSC infusion in murine models of acute liver injury. UC-MSCs were injected intravenously into mice injured by single dose of Carbon tetrachloride (CCl4) & OVA-BIL mice. MSC used were either unsorted or sorted CD362+. The extent of liver damage was determined by liver histology, serum analysis, gene expression and FACS analysis 3 or 5 days after cell infusion. Homing and bio-distribution of stem cells was determined by whole mouse cryo-imaging of Q-dot labelled MSC following infusion of UC-MSC into injured mice. CD362+ MSC were as effective as unsorted MSC in ameliorating liver injury, with reductions in serum ALT seen in both models. In contrast heat-inactivated MSC had no effect on liver injury. MSC also led to a reduction in CD45+staining on liver sections in both models of liver injury corroborated by an accompanying reduction in hepatic CD45+ cells in (FACS analysis of liver digest). In addition, there was a significant reduction in hepatic CD19+ B cells in digested liver in CCl4 injury. CD362+ MSCs were found to have the ability to reduce the level of adhesion molecules (ICAM and VCAM) in Ova-Bil mice. Cryo-imaging of time-course in both animal models indicated that MSC had migrated to the lung within 1 hour and were then cleared rapidly, although there was a liverspecific increase in MSC 2-3 day in Ova-Bil mice. CD362+ human MSC exert potent anti-inflammatory activity in toxic and immune-mediated murine liver injury with demonstrable reductions in infiltrating inflammatory leucocytes and B cells.

Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) transcription factor family plays a key role in the regulation of the immune response and the transcriptional response to infection through transcriptional activation of genes involved in those processes. The NF-κB response is regulated in the nucleus by the balance between ubiquitination and deubiquitination processes. Ubiquitination of the p65 subunit of NF-κB terminates the NF-κB response by targeting p65 for proteasomal degradation. Nevertheless, the ubiquitin molecules can be removed from targeted proteins by the action of deubiquitinating enzymes (DUBs). Ubiquitin-specific protease 7 (USP7) is a deubiquitinase enzyme from the ubiquitin-specific protease (USP) family which deubiquitinates p65. Besides p65 deubiquitination, USP7 is involved in a huge variety of biological processes due to stabilisation or localisation of proteins involved in those processes. USP7 is a multidomain protein formed by an N-terminal Meprin and tumour necrosis factor receptor-associated factor homology (MATH) / tumour necrosis factor receptor-associated factor (TRAF) domain, a catalytic domain (CD) and five ubiquitin-like domains (UBLs) in the C-terminal region. p65 recognition by USP7 takes place through the C-terminal region, but the molecular determinants involved in the interaction are still unknown. New therapeutic compound design strategies are based on interrupting the interaction interface between both proteins involved in the interaction. Therefore, in order to design a specific inhibitor of the deubiquitinase activity of USP7 on p65 we performed a peptide array and subsequent alanine scan followed by site directed mutagenesis experiments. We concluded that UBL2 of USP7 is necessary for the interaction with p65. UBL2 deletion completely abolishes the interaction and the deubiquitinase activity of USP7 on p65. Specificity of this mutant was tested by immunoprecipitation assays with different USP7 substrates. In silico modelling revealed a putative binding pocket in USP7 UBL2 that may be targeted to inhibit the interaction with p65. Together our data suggest that a binding pocket present on UBL2 composed by amino-acids 627-ARSNGTK-633, 736-EEVKPNLTER-745 and 757-LDELMDGD-764 directs the interaction with p65, besides UBL2 when deleted inhibits the interaction with p65 and subsequently its deubiquitination in a substrate specific manner.

Bone and joint erosion and fragility fractures are associated with osteoarthritis and osteoporosis, and represent a major unmet clinical problem. In health, the balance between chondrocytes, osteoblasts and osteoclasts is a dynamic process under tight regulation. In disease, regulation is uncontrolled resulting in overt skeletal dysfunction and bone loss. NF-κB (nuclear factor κ-light-chain-enhancer of activated B cells) is a master regulator of cellular function and is an essential element in the development and homeostasis of the skeletal system. As such it is a critical controller of chondrocyte, osteoblast and osteoclast differentiation and function. Bcl-3 (B-cell lymphoma 3-encoded protein) is an atypical IκB protein and via its selective interaction with p50 and p52 homodimers of NF-κB is a regulator of cellular function. As a regulator of NF-κB, the role of Bcl-3 was hypothesised to be critical in affecting skeletal health. With the aim of defining the role of Bcl-3 in the skeleton, mice deficient in Bcl-3 (Bcl-3-/-) were compared to wild-type (Wt) mice. Ex vivo phenotypic analysis of Bcl-3-/- mice and in vitro cellular differentiation assays of Bcl-3-/- chondrocytes, osteoblasts and osteoclasts were performed. In neonatal Bcl-3-/- mice, multiple phenotypes were discovered, including dwarfism and increased mineral density. Morphometric analysis of developing and adult mice showed that 20-week Bcl-3-/- mice had a profoundly divergent phenotype from Wt. Male 20-week Bcl-3-/- mice had increased bone density in the trabecular and cortical regions and increased biomechanical strength, compared to Wt, implicating increased bone formation. Female 20-week Bcl-3-/- mice did not differ from Wt in the trabecular region; however, they possessed decreased bone area in cortical bone. Bone turnover rates of 20-week Bcl-3-/- males were lower than in Wt, implicating an accelerated rate of bone resorption, following peak bone density. While no differences in chondrocyte differentiation were found, Bcl-3-/- osteoblasts were found to have accelerated osteogenesis. Complementarily, simulated overexpression with Bcl-3 mimetic peptide restricted osteoblast differentiation. An expression repertoire of marker genes and miRNAs reflected increased RUNX2 (Runt-related transcription factor 2) activity in early-differentiating Bcl-3-/- osteoblasts. Compared to Wt, Bcl-3-/- osteoblasts had an altered whole-transcriptomic profile in early osteogenesis, with multiple NF-κB-driven osteogenic gene clusters identified. Osteoblasts increased expression of RANKL (RANK ligand) and decreased OPG (osteoprotegerin) expression, involved in signalling cross-talk with osteoclasts. Bcl-3-/- osteoclasts had increased intrinsic osteoclastogenesis and resorption, while treatment with Bcl-3 peptide restricted osteoclast differentiation and function. The ex vivo phenotypic results were complementary to in vitro assays, showing increased bone turnover. The lack of Bcl-3 in both, osteoblasts and osteoclasts, increased their differentiation and activities. Thus, Bcl-3 functions as an inhibitor of NF-κB-driven early osteogenesis and osteoclastogenesis. These findings help identify Bcl-3 as a novel target for the treatment of diseases involving skeletal pathology.

Rheumatoid Arthritis (RA) is a chronic systemic inflammatory disease, which targets the synovial membrane and joints of patients, resulting in irreversible bone damage, and disability. The development of new treatments for RA is limited by our incomplete understanding of disease pathogenesis. Macrophages are critically important in the pathogenesis of RA due to their ability to initiate and amplify both systemic inflammation and local tissue damage through the production of cytokines such as Tumour necrosis factor (TNF) α and Interleukin (IL)-6. Our understanding of the molecular mechanisms underlying monocyte and macrophage activation in arthritis remains unclear. MicroRNAs (miRs) are a novel class of post-transcriptional regulators. They are short, non-coding RNA molecules that bind to complementary sequences, typically in the 3’untranslated region (UTR) of target genes, resulting in mRNA degradation or sequestration. miRs have been shown to impact various aspects of mammalian biology including cell proliferation, differentiation and the immune response. We identified a miR profile of SF CD14+ cells in RA. The majority of this study concentrated on miR-125a which was shown to be upregulated in SF CD14+ cells and PB CD14+ cells of patients who responded well to cDMARD therapy when compared to healthy controls. Using miR sponge technology we generated macrophage like, miR-125a null cells, and demonstrated that these cells display a hyper inflammatory response to LPS by producing significantly higher levels of TNFα, IL-6, CCL4 and CCL5 when compared to a reporter control. This phenotype was confirmed in primary human macrophages in which miR-125a was inhibited. These cells produced significantly higher levels of TNFα, IL-6, IL-12, CCL4 and lower levels of anti-inflammatory IL-10. Prediction algorithms identified members of the Toll Like Receptor 4 (TLR4) signalling pathway and inflammatory cytokines as potential miR-125a targets. Interestingly, overexpression of miR-125a in primary human macrophages resulted in increased IL-10 production, but also increased TNFα production, highlighting the complicated nature of miR regulation. This study has identified miR-125a as a potential negative regulator of macrophage activation, which may be impaired at sites of chronic inflammation. Future work will therefore aim to characterise the pathways regulated by this miR in macrophages, and the relationship to their activation within the RA joint.

No description available.

Mucosal addressin cell adhesion molecule-1 (MAdCAM-1) is a tissue–specific protein that promotes α4β7+ lymphocyte recruitment on gut mucosal endothelium, playing an important role in the development of inflammatory bowel disease (IBD). Recent studies have reported its expression in liver diseases such as primary sclerosing cholangitis (PSC) and autoimmune hepatitis (AIH) that complicate IBD, therefore understanding the factors that drive hepatic expression of MAdCAM-1 might elucidate the pathogenesis of these diseases. In vitro stimulation of HSEC with tumor necrosis factor-α (TNFα) and methylamine, the physiological substrate of vascular adhesion protein-1 (VAP-1), as well as with the end products of methylamine deamination by VAP-1, resulted in increased levels of secreted and cell surface MAdCAM-1 protein that was able to support binding of α4β7+ lymphocytes under flow conditions. In vivo stimulation of mice that expressed hVAP-1 as a transgene, with methylamine, induced expression of MAdCAM-1 in Peyer’s patches and mesenteric lymph nodes, validating the effect of VAP-1 enzyme activity. In conclusion, we report for the first time that MAdCAM-1 is normally present in human liver and is further induced upon TNFα and methylamine stimulation resulting in the recruitment of mucosal cells to the liver, thus sustaining a destructive inflammatoty influx responsible for the establishment of chronic inflammation.

Communication between the immune system and the central nervous system (CNS) is becoming increasingly topical as evidence suggests the two systems are intricately linked. Although the brain is considered an ‘immune-specialised’ tissue, it is not free from the influences of the periphery. Recent data indicate that peripheral immune stimulation can significantly affect the CNS, and patients with chronic inflammatory diseases, including rheumatoid arthritis (RA) and psoriasis, are often further burdened by the onset of neuropsychiatric conditions such as major depressive disorder (MDD), schizophrenia and anxiety. However, despite increases in our understanding, the precise mechanisms underpinning this relationship remain unclear. Therefore, the aim of this thesis is to investigate the communication pathways that exist between the immune system and the nervous system and to enhance our understanding of this bidirectional relationship. Using a well-characterised animal model of psoriasis-like skin inflammation, I have investigated the effects of cutaneous, peripheral inflammation on the brain. Psoriasis-like skin inflammation was induced in female C57BL/6 mice via the repeated application of Aldara cream to the shaved dorsal skin. Twenty-four hours after the fifth application, the transcriptional response in the brain was assessed and compared with mice treated with an aqueous control cream, using Affymetrix GeneChip arrays. The induction of target genes, identified using microarray analysis, was confirmed in an independent model using QPCR and was compared to the gene induction following a number of other inflammatory models, including a sterile model of cutaneous inflammation. Transcriptional profiling techniques allowed me to identify a number of differentially expressed genes in the brains of Aldara- and Imiquimod (IMQ)- treated mice when compared with the brains of control mice. This response included a range of interferon-stimulated genes (ISGs) and chemokines that were not induced in the peripheral blood leukocytes (PBL), and occurred independently of an overt cytokine response in the PBL. The brain ISG and chemokine response was not detected following a sterile model of cutaneous inflammation or following the intraperitoneal administration of Imiquimod.  The central induction of a number of chemokines prompted the evaluation of immune cell infiltration into the brain parenchyma. In addition, the functional consequences of topical Aldara treatment, and the involvement of inflammatory chemokines, were determined by assessing dentate neurogenesis and burrowing behaviour in wild-type and ACKR2-deficient mice. The transcriptional response following cutaneous IMQ-induced inflammation is indicative of a peripherally triggered inflammatory response in the brain. In addition, the data described in this thesis demonstrate a functional consequence of peripheral immune stimulation and suggest that cutaneous inflammation could modulate the recruitment of leukocytes to the brain. These data highlight a potential mechanism of TLR-dependent communication between the periphery and the brain that could be mediated through the activation of the afferent vagus nerve.

Introduction: Periodontal disease (PD) is the most common bone destructive chronic inflammatory disease in humans. Severe PD affects 8-15% of the population and impacts on the ability to chew and appearance, reduces quality of life, and is responsible for a substantial proportion of dental care costs. A dysbiotic oral biofilm is necessary but insufficient for development of PD. Rather, a dysregulated immune response to the disease-associated biofilm results in destruction of tooth supporting structures and eventual tooth loss. Despite the apparent involvement of the immune system in PD, clinical management focuses solely on the mechanical removal of the oral biofilm – with partial success and frequent recurrence. Therefore, a better understanding of the immune response in PD could highlight potential novel preventative and therapeutic strategies. T cells are present at sites of PD; however, there remains ambiguity regarding whether these T cells are protective or destructive in PD. The aim of these studies was to characterize CD4+ T cells in a P. gingivalis-induced murine model of PD. Results: P. gingivalis-infected mice displayed subtle changes in their CD4+ T cell compartment, predominantly in the draining lymph nodes (dLNs). Such changes included a suggested increase in T follicular helper cells, a trend towards a decrease in regulatory T cells and a trend towards increased production of IFN-γ. Elevated levels of IFN-γ were also noted in gingival CD8+ T cells and splenocytes, with similar trends in CD8+ T cells from dLNs. The transcriptome of CD4+ T cells isolated from gingivae and dLNs of P. gingivalis–infected suggested minimal changes in gene expression following infection; however, identified a profile of the mucosal oral CD4+ T cell compared with CD4+ T cells of the dLN. To investigate the response of CD4+ T cells specific for P. gingivalis, the bacteria were genetically manipulated to express ovalubumin (OVA) peptide 323-339. However, these OVA peptide expressing P. gingivalis failed to induce a response in OVA-specific T cells, both in vitro and in vivo. Conclusion: These data imply that CD4+ T cells do not substantially change upon P. gingivalis infection in a murine model. IFN-γ production, however, was elevated both locally and systemically. Together, the data presented in this thesis and data previously published warrant further investigations into the role of IFN-γ in PD and may point to IFN-γ as a biomarker or biological target for adjunctive PD therapy.

Rheumatoid arthritis (RA) is a chronic, inflammatory disorder, whereby synovial inflammation ultimately results in joint destruction. Although the joints are the main target tissues affected, RA is also associated with a number of co-morbidities - such as cardiovascular disease. A key cell type involved in the perpetuation of disease pathogenesis is the macrophage, but the mechanisms underlying inflammatory gene expression in these cells are not fully understood. One fascinating and rather novel area of research that could provide insights into the activation of macrophages in RA comprises the biology of microRNA. These small, non-coding RNA molecules are implicated in the post-transcriptional regulation of gene expression. Here, we show that the expression of one particular microRNA, miR-34a, is increased in synovial fluid CD14+ cells compared to matched peripheral blood cells from RA patients. We have demonstrated that miR-34a expression is increased in synovial tissues from RA patients compared to osteoarthritis comparators, and that a proportion of these miR-34a positive cells are CD68+ macrophages. Of particular interest, miR-34a was also up-regulated in peripheral blood CD14+ cells isolated from multiple drug-resistant RA patients compared to healthy controls. Using over and under-expression methodologies we were able to demonstrate that miR-34a over-expression reduces toll like receptor-induced cytokine production by macrophages, while miR-34a inhibition enhances cytokine production. The altered cytokine activities included TNFα and IL-6 that are both critically linked to disease pathogenesis, therefore we propose that miR-34a over-expression in RA macrophages represents a failed attempt to attenuate on-going inflammation. To further explore the mechanism of miR-34a action, a microarray was performed to investigate transcripts that were regulated in response to miR-34a over-expression in monocytes. This study uncovered several pathways, including interferon, metallothionein and chemokine pathways, wherein many members were down-regulated upon miR-34a over-expression. Future work will therefore aim to dissect the role of these pathways, and their relevance to miR-34a regulated macrophage and dendritic cell biology, and thus to the chronicity of synovitis.