Due to the large surface area of the gut and its continual exposure to a wide variety of agents including dietary constituents, commensal bacteria and pathogens, the intestinal arm of the immune system has evolved to be the largest component of the immune system. It must be able to discriminate between harmless and harmful antigens, so that it can induce tolerance to harmless commensal, self or dietary antigens, but active immunity against pathogens. As the sentinels of the immune system, intestinal dendritic cells (DCs) are central to these processes, continually sampling antigen in the environment and migrating to the mesenteric lymph nodes (MLNs), where they present the antigen to naïve T cells and induce appropriate T cell responses. However the nature and functions of DCs in the intestine remains a topic of debate. Their characterisation has been hampered by the use of non-specific and overlapping markers which has led to intestinal DCs being confused with other cells of the mononuclear phagocyte system, especially macrophages (mφs) which vastly outnumber DCs in the intestinal mucosa. While considerable progress has been made in recent years with the identification of CD103 and CX3CR1 as mutually exclusive markers of DCs and mφs respectively, it has become assumed that CD103+ DCs are intrinsically tolerogenic and thus it remains unclear how DCs contribute to active immunity in the intestine. Furthermore it is unknown whether CD103 is sufficient to define all intestinal DCs, or whether bona fide CD103- DCs may also exist. Thus a major aim of my project was to develop methods that allowed precise characterisation of the mononuclear phagocytes in the intestinal lamina propria (LP) and examine the functions of phenotypically defined subsets. As part of this, I also examined the contribution of the inhibitory signalling receptor, signal regulatory protein alpha (SIRPα) co-expressed by CD11b+ DCs, in regulating intestinal DC function. In Chapter 3, I set out to examine the phenotype of mononuclear phagocytes in the small intestine lamina propria (SI LP). Initially I confirmed previous studies that CD103 and CX3CR1 were mutually exclusive markers of DCs and mφs respectively. This identified 2 populations of DCs separated on the basis of CD11b expression, together with two populations of mφs distinguished by their levels of CX3CR1. However, further experiments examining F4/80 expression in combination with the recently identified mφ-specific marker CD64 showed that the CX3CR1int CD103- MPs were heterogeneous. Although the majority were F4/80+CD64+CD11b+ mφs, I could also identify two additional populations that were F4/80-CD64- and could be separated on the basis of CD11b expression. I hypothesised these were DCs and this was supported by the fact that all 4 subsets of putative DCs could also be found amongst CD11c+MHCIIhi migratory DCs in the MLNs and in pseudo-afferent intestinal lymph. All the subsets also expressed genes and markers of DCs but not mφ and were dependent on Flt3L in vivo. Unlike CD64+ mφs, the DC subsets had no ability to phagocytose E. coli particles. Four similar subsets were also identified in the colonic LP, however the proportions of the subsets in this location were distinct from those seen in the SI LP. While the CD103+CD11b+ DCs were the main subset in the SI LP, in the colonic LP the CD103+CD11b- DCs dominated. Having identified two novel populations of genuine CD103- DCs in the intestinal LP, in Chapter 4 I went on to examine their origin. Previous reports had shown that CD103+ LP DCs were derived from DC-committed precursors (pre-DCs), whereas CD103- MPs were reported to be of monocyte origin. However as I had shown the CD103- MPs to include both DCs and mφs, it was necessary to re-examine their origin using appropriate gating strategies. Adoptive transfer of pre-DCs from the BM into resting WT mice generated all subsets of DCs in the LP, including the two novel populations of CD103- DCs I had identified. In contrast adoptive transfer of Ly6Chi monocytes into monocytopenic CCR2-/- recipients generated mφs exclusively. By comparing the small intestine, colon and spleen, I could show that the development of pre-DCs was determined by the tissue they entered, as the progeny took on the same subset profiles as seen in the endogenous DC populations. Thus the differentiation of pre-DCs appears to be driven by the local microenvironment. By tracking the appearance of donor-derived DCs over time, I could monitor their differentiation in situ. These studies and experiments using BrdU incorporation showed that all DC subsets turned over much more rapidly in vivo than mφs and that a significant proportion were actively dividing in situ. No clear differences suggesting a precursor-product relationship between any of the DC subsets could be seen in these kinetic experiments. To gain a better idea of how the DC subsets might develop, I also examined them in neonatal animals and examined the effects of administering broad-spectrum antibiotics. These studies demonstrated that the CD103+CD11b+ DCs were likely regulated by the presence of specific microbiota as they did not develop in the neonatal animals until day 7 after birth and were increased in proportion following administration of antibiotics. In Chapter 5, I examined how the DC populations might behave during inflammation, using DSS colitis, post-operative ileus and infection with Citrobacter rodentium as models. DSS-colitis caused considerable inflammatory infiltrate and the number of DCs was increased, however there were no subset specific differences. Post-operative ileus also caused inflammation characterised by monocyte and neutrophil infiltration, but had few effects on the DC populations. Infection with C. rodentium resulted in a selective increase in the number of CD103- DCs in the colonic LP, suggesting these may be involved in modulating the Th17 response which characterises the protective immune response in this infection. By transferring pre-DCs into colitic mice, I found that these still gave rise to all the DC subsets during inflammation. In Chapter 6, I examined the functions of the phenotypically defined subsets of LP DCs by pulsing them with ovalbumin (OVA) protein in vitro and culturing them with OVA-specific CD4+ or CD8+ T cells. Consistent with their expression of CD8α and XCR1, I found the CD103+CD11b- DCs to be the most efficient at cross-presenting antigen to naïve CD8+ T cells and they were also the most efficient inducers of IFNγ-producing CD4+ T cells. All populations of DCs could induce FoxP3+ TReg cells, but consistent with their ability to produce retinoic acid as measured by the ALDEFLUOR assay, the CD103+ DC subsets were most efficient at this. The CD103+CD11b- subset also expressed the TGFβ-activating integrin αvβ8. In contrast, induction of IL17a-producing CD4+ T cells was a function of CD103+CD11b+ and CD103-CD11b+ DCs, with the latter being the most efficient. In Chapters 7 and 8, I examined the role of the inhibitory molecule SIRPα in intestinal DC behaviour by examining the DC populations in SIRPα mutant (mt) mice, which have a truncated cytoplasmic domain and hence cannot signal intracellularly. Despite being expressed by most myeloid cells including all CD11b+ DC subsets and CD64+ mφs, SIRPα mt mice had a selective defect in the number of CD103+CD11b+ DCs in the LP and MLN. This correlated with a reduction in the number of Th17 cells in the LP of steady state SIRPα mt mice and these mice showed reduced levels of Th17 cell induction after antigen-specific immunisation and infection by C. rodentium. In parallel, SIRPα mt mice had impaired clearance of C. rodentium infection. T cells from SIRPα mt mice did not have an intrinsic defect in their ability to be polarised to the Th17 phenotype and CD103+CD11b+ DCs from SIRPα mt LP were fully capable of priming Th17 cells in vitro.

Serum autoantibodies directed towards a wide range of single glycosphingolipids, especially gangliosides, in humans with autoimmune peripheral neuropathies have been extensively investigated since the 1980s and these are widely measured both in clinical practice and research. It has been recently appreciated that glycosphingolipid and lipid complexes, formed from 2 or more individual components, can interact to create molecular shapes capable of being recognised by autoantibodies that do not bind the individual components. Conversely, 2 glycosphingolipids may interact to form a heteromeric complex that inhibits binding of an antibody known to bind one of the partners. As a result of this, previously undiscovered autoantibodies have been identified, providing substantial new insights into disease pathogenesis and diagnostic testing. In particular, this newly-termed ‘combinatorial glycomic’ approach has provided the impetus to redesigning the assay methodologies traditionally used in the neuropathy-associated autoantibody field. Combinatorial glycoarrays can be readily constructed in house using any lipids and glycosphingolipids of interest, and as a result many new antibody specificities to gangliosides and other glycosphingolipid complexes are being discovered in neuropathy subjects. Herein we also highlight the role of the neutral lipids cholesterol and galactocerebroside in modifying glycosphingolipid orientation as two critical components of the molecular topography of target membranes in nerves that might favour or inhibit autoantibody binding.

Macrophages (mφ) are one the most numerous leukocytes present in the healthy gut and contribute to both harmful and beneficial immune reactions. In the colon, mφ are exposed continuously to large amounts of material from the environment, including harmful agents such as invasive bacteria, viruses and parasites, as well as harmless materials such as food proteins and the commensal bacteria which inhabit the healthy intestine. As a result, mφ play an important role in helping defend the intestine against harmful invaders. However if these cells make similar reactions to harmless food proteins or commensal bacteria, it would be both wasteful and detrimental, likely leading to inflammatory diseases such as coeliac disease and Crohn’s disease. Several genes, which underlie susceptibility to Crohn’s disease are involved in controlling how macrophages respond to the microbiota, with considerable evidence indicating that this reflects a loss of the normal unresponsiveness that characterises intestinal macrophages in the healthy intestine. One of the most significant aspects of the epidemiology of Crohn’s disease is a particularly rapid increase in its incidence in childhood, suggesting that the first encounters between the microbiota and intestinal macrophages may be of critical importance in determining disease susceptibility. Given this link, it is essential that we elucidate the processes controlling macrophage seeding and development in the intestine and this was an aim of this thesis. In the adult healthy colon, two main mφ subsets can be identified: A dominant and homogenous one, made up of mature mφ, which express high levels of F4/80, MHC II, CX3CR1, are CD11bint/+, highly phagocytic and produce high amounts of IL10. The second mφ group is relatively smaller and is much more heterogeneous. These cells express intermediate levels of F4/80 and CX3CR1, are CD11b+ and can be divided into 3 subsets based on their levels of Ly6C and MHC II. These subsets represent a maturation continuum towards the mature mφ phenotype. Recent reports have suggested that resident macrophages in healthy tissues may be derived from yolk-sac and/or foetal liver precursors that seed tissues during development and subsequently self-renew locally. In contrast, it is proposed that macrophages in inflammation are generated by recruitment of blood monocytes, raising the possibility that these different origins could be exploited in therapy. However none of these studies have examined macrophages in the intestine and recent work in our laboratory has suggested that monocytes may be the precursors of macrophages in both healthy and inflamed gut of adult mice. Therefore, the aims of this thesis were to investigate the development of murine colonic mφ from birth until adulthood, examining the relative roles of the yolk sac, foetal liver and bone marrow monocytes, exploring their functions and comparing them with the well-characterised adult mφ. In addition, I also examined how mφ phenotype and functions are influenced by the microbiota using broad-spectrum antibiotics and germ free mice. Lastly, I examined the role of fractalkine and its receptor CX3CR1 in defining the development and functions of intestinal macrophages. Development of macrophages in early life The initial characterisation and comparison of colonic mφ subsets is included in Chapter 3. In this chapter, I describe a series of experiments adapting existing protocols and techniques used for examining the adult murine intestine in order to analyse the origin, phenotype and functions of murine colonic macrophages from late foetal life through to adulthood. These studies found that intestinal mφ are present before birth, with similar levels of phagocytic ability and IL10, TNFα and CD163 mRNA expression to the adult. However, the numbers and phenotype of mφ in the intestine do not reach the adult level until the 3rd week of postnatal life. This phenomenon appears to reflect the de novo recruitment of blood monocytes in a CCR2-dependent fashion at this time and throughout adult life, but not at early stages of life. In the colon of newborn mice, two macrophage populations can be observed and are clearly differentiated based on their F4/80 and CD11b expression: F4/80hi CD11bint/+ and F4/80lo CD11b+. Interestingly, unlike adult colonic F4/80hi mφ, the majority of F4/80hi neonatal cells do not express MHC II, however they gradually express this molecule as they age. In addition to acquiring MHC II expression, the two populations in the newborn colon gradually merge and from the 3rd week of life it is difficult to discriminate them reliably. My experiments show that both mφ subsets proliferate actively during the first 2 weeks of life, but this is later reduced and maintained at low levels indicating that there is no self-renewal of mature mφ. Moreover, fate-mapping analysis carried out in collaboration with Professor Frederic Geissmann, showed that yolk sac-derived precursors contribute only minimally to the pool of colonic mφ, even at early life stages. Conversely, additional fate mapping studies suggested that most intestinal macrophages are derived from Flt3+ progenitors. Taken together, the results in this chapter demonstrate that blood monocytes are vital in replenishing the intestinal macrophage pool in the steady state, setting them apart from other tissue macrophages, which derive from primitive progenitors. Investigating the effect of the microbiota on intestinal macrophage subsets In Chapter 4, I assessed the effects of the commensal microbiota on intestinal mφ, using two different approaches: First, I assessed the function and gene expression of colonic macrophages following administration of broad-spectrum antibiotics. My results showed that this did not alter the numbers, phenotype, intracellular cytokine production or mRNA expression by macrophages. Several reasons may account for this, including dose/nature of antibiotics, length of administration or lifespan of macrophages. To overcome these issues, I compared the phenotype of colonic mφ in germ free (GF) and conventionally (CNV) reared mice of different ages in collaboration with Dr David Artis. Absolute absence of microbiota in GF mice severely impacted Ly6Chi monocyte recruitment to the colon, suggesting that constant recruitment of monocytes to the gut is at least in part due to the microbial burden. The biggest differences between GF and CNV mice were evident at 3 weeks of age, when GF mice had a much lower number and frequency of monocyte-derived cells than their CNV counterparts. By 12 weeks of age, Ly6Chi mφ populations from GF mice were partially restored, although the expression of MHC II by F4/80hi mφ remained reduced. Additionally, I FACS-purified F4/80hi cells from GF and CNV adults and sent RNA for microarray analysis, the results of which we are waiting to receive. This data will provide further information regarding how GF intestinal mφ differ from those found in conventional animals. Role of the CX3CL1-CX3CR1 axis in mφ development and function As mature colonic mφ express high levels of the chemokine receptor CX3CR1 (fractalkine), finally, in Chapter 5 I went on to investigate the role of CX3CL1-CX3CR1 axis in colonic lamina propria. In addition to the high expression of CX3CR1 by colonic mφ, its ligand, CX3CL1 has been reported to be expressed at high levels by the intestinal epithelium. Furthermore, as there is strong evidence that the CX3CL1-CX3CR1 axis may be involved in inflammation in several tissues, we hypothesised this axis might play a role in mφ function in the gut. To this end, I examined mφ phenotype, activation status and survival following in vitro co-culture of WT or CX3CR1-deficient bone marrow-derived mφ with an epithelial cell line modified to express either the soluble or membrane-bound forms of CX3CL1. I also examined the development of chemically induced colitis in CX3CR1-deficient mice.

Although the central nervous system (CNS) was once considered an immunologically privileged site, in recent years it has become increasingly evident that cross talk between the immune system and the CNS does occur. As a result, patients with chronic inflammatory diseases such as rheumatoid arthritis, inflammatory bowel disease or psoriasis are often further burdened with neuropsychiatric symptoms such as depression, anxiety and fatigue. Despite the recent advances in our understanding of neuroimmune communication pathways, the precise effect peripheral immune activation has on neural circuitry remains unclear. Therefore, the primary aim of this thesis was to develop a better understanding of the bidirectional relationship, and communication pathways, that exist between the immune system and the nervous system. By utilising transcriptomics in a well-characterised murine model of systemic inflammation, I have investigated the molecular mechanisms by which inflammation originating in the periphery can induce transcriptional modulation in the brain. Systemic inflammation was induced in male C57BL/6 mice via intraperitoneal injection of lipopolysaccharide (LPS). After 48 hours, whole brain transcriptional profiles were assessed, and compared to that of a vehicle- treated control group, using Affymetrix GeneChip microarrays. Target gene induction, identified by microarray analysis was validated independently using QPCR. Expression of the same panel of target genes was then investigated, in the brains of mice, following the induction of different sterile, and TLR- dependent, models of peripheral inflammation. Microarray analysis of whole brains collected 48hr after LPS challenge revealed increased transcription of a range of interferon-stimulated genes (ISGs) in the brain, including a significant upregulation of the classic interferon-induced chemokine CXCL10. This transcriptional profile could not be reproduced by the systemic administration of TNFα, or following lipoteichoic acid-induced systemic inflammation. However, target genes remained induced in the brain following daily LPS injections, in the absence of a detectable inflammatory cytokine response in the periphery. 1 The central induction of CXCL10 suggests that acute exposure to LPS in the periphery may prime the brain for T cell infiltration. This prompted an investigation into whether leukocytes infiltrated the brain following daily systemic LPS injections. First, the inflammatory chemokine repertoire in the brains of LPS treated mice was systematically characterised. In addition to Cxcl10, repeated injection of LPS in the periphery triggered a transient increase in the transcription of a number of other inflammatory chemokines in the brain. Chemokine induction was associated with an influx of leukocytes from the periphery, and an increase in mRNA encoding the relevant chemokine receptors. Therefore, chemokine induction in the brain following daily systemic LPS injections may mediate the recruitment of leukocytes from the periphery. The transcriptional response in the brain following systemic LPS challenge is indicative of a peripherally triggered inflammatory response in the brain. The data described in this thesis highlight a potential mechanism of gene modulation in the brain which may be dependent on a TLR-induced type I interferon response. Considerable evidence links type I interferons to psychiatric disorders, and consequently, interferon production in the brain could represent an important mechanism linking peripheral TLR-induced inflammation with behavioural changes. In addition, the data described in this thesis demonstrate that chronic exposure to LPS in the periphery may remotely modulate the recruitment of leukocytes to the brain. This highlights a potential protective mechanism that could prevent a chronic bacterial infection from spreading from the periphery to the brain.

Apoptotic cells (AC) are able to modulate the immune system, dampening inflammation and triggering anti-inflammatory responses by various immune cells as a consequence of interaction and uptake. Rituximab (RTX) is an anti-CD20 monoclonal antibody used as a treatment in several autoimmune diseases, including rheumatoid arthritis (RA). Treatment results in B cell depletion, with B cell apoptosis known to contribute to RTX-mediated B cell death. However the simple removal of B cells from the system does not seem to account for all the beneficial effects of this biologic. We propose that RTX treatment in RA results in the re-establishment of temporary tolerance to the system, through an apoptotic B cell-dependent mechanism. Initial in vitro and in vivo investigations were undertaken to explore the validity of this hypothesis. The present work sought to examine the immunomodulatory capacity of apoptotic B cells and to determine whether the potential anti-inflammatory effects of apoptotic B cells are modulated by RTX, with both in vitro methods and an in vivo model of autoimmunity utilized in these studies. The results presented in this thesis demonstrate that apoptotic B cells have comparable effects on bone marrow derived macrophage (BMDM) phenotype and function in vitro as previously described AC from other cellular sources. Surprisingly, in the in vitro assay system used, viable cells had the same immunomodulatory effects on BMDM as AC, for all criteria investigated. Preliminary studies indicate this may be a promising avenue of inquiry, however further work is needed before a conclusion can be reached as to the relative level of involvement of apoptotic B cell-mediated tolerance in the improvement seen on RTX treatment in RA.

Osteoporosis is a condition that results from substantially weakened bone, increasing an individual’s risk of fracture. Post-menopausal osteoporosis is the most common form of the condition, affecting 30% of post-menopausal women over the age of 50. Following the menopause, female oestrogen levels decline and this perturbs bone homeostasis by promoting an environment that is biased towards bone erosion. Osteoclasts are the cells responsible for eroding bone and are normally inhibited by oestrogen. However, the decline in oestrogen production results in increased osteoclast differentiation and activity. This rapidly decreases the bone mineral density and results in fracture-prone bone. Osteoclasts are derived from mononuclear myeloid progenitors found in the blood and bone marrow, which fuse to form large multinucleated cells that reside in the bone cavity. These progenitor cells are also responsible for replenishing monocytes, macrophages and dendritic cells. One class of receptors present on the surface of these cells, which are capable of dictating a cells function, are Fcγ receptors and modulation of Fcγ receptors has been shown to inhibit the differentiation of human monocytes to osteoclasts. This thesis investigates Fcγ receptor modulation on murine osteoclastogenesis and in order to stimulate Fcγ receptors, both IgG and IgG complexes were used. IgG complexes were generated using Staphylococcus aureus Protein A (SpA) in combination with IgG to form SpA-IgG complexes (SIC). We show that IgG and SIC are capable of engaging with Fcγ receptors resulting in the inhibition of osteoclast differentiation. Furthermore, both IgG and SIC inhibit the transcription of mRNA essential for the fusion of progenitors and enzymes for the erosion of bone matrix. Therefore, IgG and SIC are capable of inhibiting murine osteoclastogenesis. The murine model of osteoporosis was used to further investigate the ability of SIC to inhibit murine osteoclast differentiation. Previous studies have shown that when SpA is administered in vivo it is capable of binding circulating IgG to form SIC. We used this property to test the ability of SpA to bind to the surface of monocytes. SpA was found to bind with highest affinity to blood Ly6Chigh monocytes, which are known to differentiate in vitro to OCs. IgG and SIC were also able to inhibit the in vitro osteoclastogenesis of Ly6Chigh monocytes. It was hypothesised that SpA would co-opt IgG and inhibit the in vivo differentiation of progenitors to osteoclasts in the ovariectomy model of osteoporosis. To generate this animal model the ovaries were removed from the mice in order to simulate the menopause and induce bone loss. To assess the percentage of bone present after ovariectomy, we used micro-computer tomography and discovered that SpA was unable to prevent bone loss associated with ovariectomy. Therefore, SpA can bind to the surface of osteoclast progenitors but is unable to inhibit bone loss in the model of osteoporosis. In addition to studying the role of Fcγ receptor modulation of osteoclastogenesis, the role of Bcl-3 (a negative regulator of NF-κB) in osteoclast differentiation and bone remodelling was also investigated. NF-κB is an essential signalling molecule and transcription factor involved in osteoclast differentiation. Previous research has shown that in the absence of Bcl-3 (Bcl-3-/-) aberrant cytokine responses to LPS and TNF- occur. Therefore, RANKL stimulation of WT and Bcl-3-/- osteoclast precursors was done to determine whether Bcl 3 /- animals responded aberrantly to RANKL. WT and Bcl-3-/- animals were able to generate in vitro osteoclasts, which were phenotypically and transcriptionally similar. However, comparison of in vivo osteoclast progenitors revealed that Bcl-3-/- animals had reduced CD115+ osteoclast progenitors compared to WT animals. Examination of the trabecular bone present in the proximal tibia revealed that Bcl-3-/- animals had a higher percentage of bone present that WT controls. Therefore, Bcl-3 does not effect in vitro osteoclast differentiation but further work needs to be done to understand the role of Bcl 3 in bone remodelling. This thesis aimed to investigate whether SpA-IgG complexes or Bcl-3 could represent a novel avenue of therapeutic intervention in osteoporotic disease. In summation, SpA is able to form IgG complexes that can inhibit the differentiation of OCs in vitro; however, treatment of osteoporotic animals with SpA was unable to halt bone loss. This suggests that SpA-IgG complexes are able to modulate Fcγ receptors in vitro and skew progenitors from differentiation into osteoclasts but cannot overcome the prevailing pro-osteoclastogenic environment that results from ovariectomy. The presence of osteoclast progenitors was also shown to be partially dependent on Bcl-3 and as such Bcl-3 may be a novel target for therapeutic agents to target osteoclast progenitors in diseases like osteoporosis. However, the role of Bcl-3 in bone remodelling requires further investigation.

Death Receptor 3 (DR3) is a death domain containing member of the TNF Receptor Superfamily (TNFRSF), refereeing a range of cellular responses from differentiation and proliferation to cell death, depending upon the context of receptor activation. DR3 has been reported to have a role in many inflammatory diseases, including inflammatory arthritis and inflammatory bowel disease. The aim of this study was to determine the contribution of DR3 in a mouse model of acute and chronic allergic lung inflammation. Mice genetically deficient in the DR3 gene (DR3ko) were resistant to cellular accumulation within the lungs and bronchoalveolar lavage following acute lung inflammation, induced by priming with ovalbumin (OVA) and the adjuvant aluminium hydroxide (Alum) prior to 2 OVA aerosol exposures. To discern the role of DR3 in a more physiologically relevant chronic model of allergic lung inflammation, mice underwent repeated inhalation challenges with OVA subsequent to priming with OVA and Alum. Whilst cellular accumulation did not differ, DR3ko mice displayed reduced immuno-histopathology, and goblet cell hyperplasia, hallmarks of the asthmatic phenotype. Intriguingly, DR3ko mice exhibited reduced accumulation of various cell types into the spleen in both models. Early priming events were therefore investigated, prior to aerosolised antigenic challenge to decipher the effects of DR3. One sensitisation injection was sufficient to induce decreased DR3ko splenocyte accumulation, though T and B cell responses were observed to be comparable between DR3ko and DR3wt controls. DR3ko mice had depleted CXCL10 levels, suggesting cellular recruitment in response to inflammation is DR3 dependent. The underlying DR3 dependent mechanisms concerning the DR3ko splenic defects are under further investigation and may have impact on the use of the DR3/TL1A pathway as a therapeutic target, either as an anti-inflammatory or as a booster of the immune response to pathogens.

Background: Interleukin (IL)-33 is a newly identified member of the IL-1 cytokine family. Multiple cell types are able to produce or respond to IL-33, including non-haematopoietic structural cells, innate and adaptive immune cells. The biological activity of IL-33 was initially described as being associated with the promotion of type 2 immune responses which were characterized by the induction of CD4+ T helper (Th) 2 cells. For example, exogenous administration of IL-33 in experimental models caused pathological changes in mucosal tissues such as the lung and gastrointestinal tracts; early studies reported that IL-33 can activate Th2 cells, mast cells, eosinophils or basophils to produce type 2 cytokines such as IL-4, IL-5, and IL-13. This was associated with pathological changes reminiscent of asthma, fibrosis and ulcerative colitis. Recently, a newly recognised cell population which was inducible by IL-33 and referred to as ‘type 2 innate lymphoid cells’ was identified and these were thought to be important for initiating type 2 immunity. However, the underlying mechanism by which IL-33 was involved in the inflammation and remodelling of diseases of the respiratory and gastrointestinal tracts remains to be fully understood. Hypothesis: My hypothesis is that IL-33 is induced in the gut and lung mucosa by inflammatory signals and mediates both early inflammation and late fibrosis by amplifying the innate immune response. Aims: To address this hypothesis I set out the following aims: i) to investigate the induction and effect of IL-33 via its receptor ST2 on cellular pathogenic pathways in the development of lung fibrosis (chapter 3); ii) to unravel the mechanism by which IL-33 promotes lung fibrosis (chapter 4); iii) to understand the involvement of the IL-33/ST2 pathway in ulcerative colitis (chapter 5). Methods: To address these aims I used two experimental murine models. To investigate the effect of IL-33 in the fibrosis phase of airway mucosal inflammation I used the bleomycin (BLM)-induced lung fibrosis (chapters 3 and 4). To investigate the effect of IL-33 in the acute phase of mucosal inflammation in the gut, I used dextran sulphate sodium (DSS)-induced colitis (chapter 5). These disease models are widely accepted for laboratory investigation and I acknowledge that they do not reflect the full complexity of the human conditions. However they are extremely useful for hypothesis generation. Results: My results showed i) that IL-33 promotes the pathogenesis of bleomycin-induced lung fibrosis. This was indicated by IL-33 being constitutively expressed in lung epithelial cells but induced in macrophages by bleomycin. The specificity of this response was confirmed by using either ST2-deficient mice, or neutralising anti-IL-33 antibody treatment, which both attenuated lung fibrosis (chapter 3). ii) that IL-33 promotes the initiation and progression of pulmonary fibrosis by recruiting and directing inflammatory cell function, and enhancing the production of pro-fibrogenic cytokines IL-13 and TGF-β in an ILC2- and M2-macrophages (chapter 4). iii) that IL-33 signalling via ST2 induces an IL-4-dependent immune response that is pathogenic in the early stage of ulcerative colitis. I found that the clinical indices of DSS-induced experimental UC, diarrhoea and colon inflammation, were respectively impaired in ST2 knockout mice and exacerbated in WT mice by treatment with exogenous recombinant IL-33. These were associated respectively with reduced and enhanced expression of inflammatory chemokines and angiogenic cytokines in vivo. The exacerbation effect of treatment with recombinant IL-33 on DSS-induced acute colitis was abolished in IL-4 knockout mice (chapter 5). Conclusion and prospect: Together, my results demonstrated that IL-33 expression was up-regulated in the lung and colon epithelium/endothelium in experimental BLM-induced fibrosis and DSS-induced colitis respectively. Furthermore, IL-33 exacerbated both diseases through recruiting and activating inflammatory cells and increasing the production of type 2 cytokines. Finally, I discussed the pathological mechanisms of IL-33 in mucosal tissue based on my results and the current literature. I concluded that this insight into IL-33 biology is informative of a new potential pathogenic pathway and might be a useful biomarker of disease and that targeting IL-33 may provide a new biological therapeutic approach in these disorders (chapter 6).

The primary aim of this study was to investigate the potential of an aroA mutant of Pasteurella multocida B:2 (vaccine strain JRMT12) as a candidate for DNA vaccine delivery (bactofection). First, the invasive property of the vaccine strain was assessed for its interaction with different mammalian cell lines. Next, a eukaryotic expression plasmid that could be maintained in Pasteurella was modified to contain a prokaryotic reporter gene to help in determining the location and viability of the bacteria when moving from the extracellular environment into the intracellular compartment of the mammalian cells. This plasmid was further developed to function with a dual prokaryotic and eukaryotic reporter system in order to demonstrate expression of the plasmid DNA in the mammalian cells. During interaction of strain JRMT12 with mammalian cell lines, the ability of the bacterium to adhere, invade and survive intracellularly was monitored and assessed. Three mammalian cell lines were used: a mouse macrophage-like cell line, J774.2; a bovine-lymphoma cell line, BL-3; and an embryonic bovine lung cell line, EBL. The JRMT12 strain was compared with strains of the wild-type P. multocida B:2 (85020), bovine P. multocida A:3, Mannheimia haemolytica A:1 and Escherichia coli XL-1 BLUE. Both P. multocida B:2 strains were capable of adhering to and invading J774.2, BL-3 and EBL cells. All of the Pasteurella and Mannheimia strains tested were able to adhere to EBL cells but only B:2 strains were taken up intracellularly in significant numbers. The vaccine strain, JRMT12 was found to survive intracellularly in EBL cells for at least 7 h although a steady decline in the number of viable intracellular bacteria was noted with time. In an invasion inhibition assay, the use of the microfilament formation inhibitor cytochalasin D suggested that the entry into mammalian cells was by an actin-dependent process. Cell viability assessment by trypan blue staining indicated that none of the bacterial strains was toxic for the mammalian cells. Upon entry into the mammalian cells the JRMT12 strain resided in a vacuolar compartment, as demonstrated by transmission electron microscopy. However, P. multocida A:3 and M. haemolytica A:1 were only found loosely adhering to the cell surface of EBL cells and were not detected intracellularly. Further morphological assessment by TEM showed that only a low percentage of mammalian cells appeared to contain one or more JRMT12, suggesting that only certain cells in the population were capable of being invaded by, or taking up, the bacteria. Attempts were made to construct a Pasteurella eukaryotic expression plasmid using a gene sequence from the Pasteurella shuttle plasmid pAKA16, developed previously in this laboratory, and the commercial eukaryotic expression plasmid pCMV-sCRIPT, but these were only partially successful. The origin of replication gene (oriP) in the Pasteurella shuttle plasmid was isolated and sequenced. Analysis of oriP showed sequence similarity with the known origins of replication in other Pasteurella plasmids. The E. coli plasmid origin of replication (oriE) was removed from pCMV-sCRIPT and the oriP gene was ligated into the oriE-free pCMV-sCRIPT but attempts to transform the resulting plasmid into P. multocida B:2 were not successful. An alternative approach to plasmid development was made using another commercial eukaryotic expression vector, pEGFP-N1. This plasmid has the same properties as pCMV-sCRIPT but has an additional, fluorescent reporter gene under the control of a eukaryotic promoter. It was found to be able to replicate in P. multocida B:2 but positive transformants were only recovered after prolonged incubation after electroporation. The plasmid was stably maintained in strain JRMT12 for at least 14 days with or without antibiotic selection. It was also successfully transfected into EBL cells, as shown by expression of green fluorescent protein (GFP) in individual cells. The P. multocida vaccine strain JRMT12 was also able to deliver the plasmid into EBL cells, although the number of EBL cells expressing GFP after bacterial delivery was lower than by direct transfection of the plasmid. Next, plasmid pMK-Express, a Pasteurellaceae prokaryotic expression vector with a gfp reporter gene, was used. When this was electroporated into the vaccine strain, the strain was shown to express GFP maximally as measured by fluorimetry, during the early exponential phase of bacterial growth. The DsRed.M1 gene coding for red fluorescent protein (RFP) from plasmid pDsRed-Monomer was then used to replace the gfp gene in pMK-Express to make the construct pMK-RED. After electroporation of pMK-RED into the JRMT12, RFP expression was detected maximally during the early exponential phase of bacterial growth. The same strain expressing RFP could also be detected in the intracellular compartment of the EBL cells by fluorescence microscopy at 3 h post-invasion. Finally, plasmid pSRG, our so-called “traffic light” plasmid with a dual reporter system was constructed. This was made from plasmid pEGFP-N1 (with its existing eukaryotic expression system for GFP expression) and the sodRED fragment (with a Pasteurella promoter controlling the DsRED.M1 gene for RFP expression) isolated from plasmid pMK-RED. This plasmid was stable in strain JRMT12 with or without antibiotic selection for 14 passages. RFP expression from JRMT12 was detected maximally during the early exponential phase of bacterial growth. Transfection of pSRG into EBL cells gave individual cells expressing GFP. Invasion assays with EBL cells and P. multocida B:2 JRMT12 pSRG+ showed that RFP-expressing bacteria could be detected intracellularly at 3 h post-invasion. At this stage, some EBL cells harbouring RFP-expressing bacteria were observed to express GFP simultaneously. At 5 h post-invasion, some of the EBL cells were still harbouring RFP-expressing bacteria and at the same time expressing GFP themselves. Concurrently, some Pasteurella free-EBL cells were shown to express GFP. These experiments proved the functionality of the pSRG dual reporter system and the potential of P. multocida B:2 JRMT12 for bactofection and delivery of a DNA vaccine. An apparent immunosuppressive effect of P. multocida B:2 on the proliferative response to concanavalin A (ConA) of peripheral blood mononuclear cells (PBMC) had been reported by Ataei (2007). The PBMC had been taken from calves infected with P. multocida B:2 or from normal calves and treated in vitro with extracts of P. multocida B:2. In the present study, in vitro assays with PBMC from normal calves were undertaken in an attempt to confirm these findings. A cell-free extract (CFE) of the vaccine strain JRMT12 was found to suppress the subsequent proliferation of PBMC in response to ConA in a dose-dependent manner. However, the results were not consistently reproducible and the same effect could not be demonstrated with CFE from the wild-type strain 85020.

Epidemic disease outbreaks pose a significant risk to the stability and survival of many populations on earth. Current methods to understand how epidemic diseases transmit are often confounded by heterogeneity in infection rates amongst host populations. Endemic parasites, which are often less severe compared to epidemic diseases, may contribute to that variation by impacting on host biology and therefore altering the transmission of epidemic diseases. The effects of an endemic, gastrointestinal infection on host fitness and host interactions with an epidemic parasite were explored in a novel invertebrate system developed for this study. The chosen host was the German cockroach, Blattella germanica, which was infected with an endemic protozoan parasite, Gregarina blattarum alone or in co-infection with an entomopathogenic nematode, Steinernema carpocapsae, which causes epidemic outbreaks in host populations. There was evidence of density dependent regulation by the endemic parasite. Reductions in both host survival and fecundity during endemic infection both contributed to this regulation. The endemic parasite also had fitness costs for offspring from infected parents, who took longer to reach adulthood and were less likely to survive. Protozoan infected host populations had lower densities and showed less variance in population fluctuation compared to parasite free populations and the endemic parasite was generally found at high prevalence within the infected groups. When hosts infected with G. blattarum were exposed to S. carpocapsae, the resulting co-infection led to reduced host survival but also reduced emergence of nematode transmission stages. Hosts infected with G. blattarum also had differential immune responses to macro and microparasites which could alter host susceptibility to different types of infections. Infection with an endemic parasite caused substantial changes in the biology of the host, which may have important effects on host population ecology. Endemic parasites can also have considerable consequences for the transmission potential of more a virulent pathogen. These findings demonstrate the important of endemic infections which should be given greater consideration in future host-parasite studies.