Due to their anti-inflammatory and immunomodulatory properties, mesenchymal stromal cells (MSCs) are under intense investigation in many pre-clinical and clinical trials as a potential cellular therapy to be used in an array of clinical settings. The majority of the literature surrounding MSC phenotype and function is derived from studies focusing on bone marrow (BM) derived MSCs. Recently however, it has become apparent that MSCs can be isolated in a less invasive manner, from the majority of tissues in the human body. In light of this, many studies have been published promoting the use of alternative tissue sources for MSC isolation with no thorough standardised comparison of the phenotype or potential in vivo function of these MSCs. The advanced therapeutics department within the Scottish National Blood Transfusion Service (SNBTS) is involved in the development and optimisation of several cellular therapies including the use of MSCs within various clinical settings. SNBTS has access to fully consented human tissues rich in MSCs including; pancreatic islets, visceral adipose tissue, liposuction aspirate, bone marrow and umbilical cord. Therefore this study aimed to objectively compare the phenotype and potential in vivo function of MSCs isolated from the aforementioned tissues in a stringent, standardised manner in order to assess if MSCs isolated from one specific tissue source might be optimal for use within the clinic. The beneficial therapeutic effect of MSCs often depends on their ability to migrate to target tissues and interact with residing or migratory immune and non-immune cells, frequently within an inflammatory environment. Therefore this study focussed on how MSCs might migrate in vivo by assessing and comparing MSC chemokine receptor expression, whilst also assessing and comparing MSC chemokine secretion profiles to understand which immune cells MSCs might attract, and therefore potentially interact with, in vivo. This study found that chemokine receptor expression by MSCs isolated from islet, visceral adipose, adipose, bone marrow and umbilical cord tissues was very low, with CXCR4, CCR7 and ACKR3 expression being restricted to visceral adipose and bone marrow derived MSCs. Inflammatory chemokines were secreted at very high levels by MSCs isolated from all of the aforementioned tissues, which induced migration of target immune cells towards all MSCs tested in vitro and in vivo, importantly however, the tissue origin of MSCs dictated the quantities of immune cells attracted. This study highlighted that the tissue origin of MSCs could affect MSC in vivo migratory capacity and their ability to chemoattract surrounding immune cells, thereby potentially influencing their clinical performance.

CD4 T cells play an important role in the initiation and maintenance of inflammation in numerous inflammatory diseases. Rheumatoid arthritis (RA) is one such autoimmune inflammatory condition where inflammation of the joint occurs. CD4 T cells are one of the key cells in RA pathogenesis due to their ability to activate or influence other cells in the joint including B cells, macrophages and osteoclasts, which collectively lead to joint destruction. The recruitment and function of CD4 T cells at inflamed tissues has been studied extensively. However, the signals that regulate CD4 T cell accumulation and persistence at peripheral inflamed sites are poorly understood. In this study, a novel in vivo model of inflammation was designed in the murine ear pinnae to study the signals which regulate CD4 T helper 1 (Th1) cell persistence at inflamed tissues. Congenically marked, in vitro polarised CD4 Th1 cells were adoptively transferred directly into inflamed or non-inflamed ear pinnae and their persistence and survival were studied using flow cytometry. Higher numbers of CD4 Th1 cells were found at the inflamed as compared to the non-inflamed site. Intravital microscopy was used to further study the behaviour of these cells. Th1 cells were found to be more mobile in inflamed compared to non-inflamed ear pinna. To investigate the molecular mechanism of this, either the ear pinnae or the T cells themselves were manipulated. Introducing cognate antigen at the inflamed site did not alter the number of recovered T cells, nor did the T cells proliferate at the site. Next, the survival of persistent CD4 Th1 cells was examined by investigating their expression of active caspases. Lower proportion of Th1 cells recovered from inflamed tissues were found to express active caspases compared to those from a non-inflamed site. Together these data suggest that local T cell activation is not required for persistence but rather, the increase in T cells at inflamed sites may be due to a combination of persistence and survival signals. The sphingolipid sphingosine-1-phosphate (S1P) has been implicated in driving both egress of T cells out of secondary lymphoid organs and their survival. To investigate whether S1P affects Th1 cell persistence and/or survival at inflamed tissues, Th1 cells were treated with S1PR agonists or antagonists, prior to transfer. Fewer Th1 cells were recovered from the inflammatory site of mice injected with antagonist treated cells. Additionally, S1PR agonism was sufficient to induce Th1 cell persistence at non-inflamed tissues. A trend towards increased expression of active caspases was also found in S1PR antagonist treated T cells recovered from inflamed ear pinnae compared to untreated controls. Finally, elevated levels of the S1P metabolising enzyme, SPHK1, was found in human RA joints compared to OA joints. In sum, I propose a novel function for S1P and its receptors in regulating the persistence of activated CD4 Th1 cells at inflamed tissue sites. Moreover, targeting S1P and its receptors at peripheral inflamed tissues could provide a novel target for the development of more effective anti-inflammatory therapeutics.

No description available.

No description available.

Campylobacter jejuni represents a major foodborne pathogen causing gastroenteritis worldwide. The infection route to the consumer is primarily associated with improper handling of raw meat of poultry and porcine origin. Biocontrol of this pathogen through conventional biosecurity measures remains challenging. Alternative bacteriophage-based strategies for the reduction of campylobacters in primary production and post-harvest have gained attention in recent years, as a number of promising biocontrol studies have emerged. However, the effective application of bacteriophage cocktails relies on an extensive characterization of suitable bacteriophage candidates, as well as detailed information related to host-phage interactions during the phage infection process. In this project, bacteriophages were isolated from numerous environmental samples and characterized with respect to host interactions. Inclusion of source material from the porcine intestinal tract revealed that the genetic diversity of bacteriophages exhibits similar limitations to that observed from poultry. All bacteriophage isolates were found to be members of either the CP220like or CP8unalike genera of the Eucampyvirinae subfamily of bacteriophages. The screening of isolated bacteriophages against a panel of isogenic single gene knock-out mutants of C. jejuni PT14 revealed that the phages were either flagellotropic or dependent on capsular polysaccharides (CPS). In one case, both factors showed a fundamental impact on bacteriophage infection. Further, it was discovered that the minor flagellin gene product (FlaB) had profound influence on the infection process of novel isolated CP8unalike phage CP_F1. Disruption of the flaB gene resulted in alterations in a number of parameters (burst size and adsorption rate) connected with bacteriophage infection, and in essence rendered the affected mutant strain more susceptible towards phage infection. Sequence analysis of CP220like bacteriophage genomes demonstrated variability in the length and frequency of regions of repetitive non-coding DNA. These are a characteristic of CP220like phages and are absent in CP8unalike phages. Deeper in silico analysis of these areas revealed potential binding sequences for all three sigma factors of C. jejuni. Through electro mobility shift assays, it was shown that a PCR amplified repeat area binds to components from a protein extract isolated from a phage infected culture of C. jejuni. This finding raises questions concerning novel control mechanisms of the host during bacteriophage infection.

Recent years have witnessed virus disease outbreaks that have caused considerable morbidity and mortality. As 80% of human viruses are zoonotic, outbreaks can be prevented by active surveillance of wildlife for pathogens with a potential for cross-species transmission. Rodents serve as important reservoirs for many zoonoses and epizooses. In this thesis, a cDNA library of 976 rodents, shrews, moles and birds was constructed. Molecular methods using inosine-containing degenerate primers were developed for the screening of novel viruses of the families, Arenaviridae and Filoviridae. Filovirus RNA was absent in all specimen screened. Lymphocytic choriomeningitis mammarenavirus (LCMV) RNA was detected in 4 Mus musculus (19%). Further screening of specimens obtained from a zoo outbreak resulted in the detection of LCMV RNA in a total of 20 M musculus, 2 Geoffroy’s marmoset, 1 black and white colobus and 1 Black-crested gibbon from two separate sampling sample sets. Viral RNA was detected in non-human primates (NHP) not previously reported. Rodents screened at the zoo after the outbreak were negative for LCMV RNA. Phylogenetic analyses of full-length glycoprotein precursor (GPC) of 4 NHP-derived and 4 rodent-derived LCMV revealed a clustering of the former with other lineage-I LCMV GPC sequences. Molecular characterisation of the novel LCMV strains uncovered a consistency in amino acid substitutions across primate-derived and rodent-derived LCMV strains at position 211 of GPC which may be a possible determinant for cross-species transmission. Analysis of the alpha-dystroglycan receptor of rodents and NHPs revealed residues that might influence GPC binding affinity and therefore drive virus evolution. Pseudoviruses constructed from GPC of rodent-derived LCMV displayed higher infectivities in hepatocellular carcinoma cells than their NHP-derived LCMV counterpart which is indicative of the existence of another unidentified host receptor. Together, the results establish a framework for further investigation of the molecular basis for cross-species transmission of LCMV between rodent and primate hosts.

Clostridium difficile infection is the leading cause of antibiotic-associated diarrhoea in North America and Europe. The disease is most commonly treated with only a handful of antibiotics, amongst those vancomycin, a so-called last resort therapy. Growing concerns over antibiotic resistance have led to the desire for more targeted therapies. In this study, we report the isolation and characterisation of four novel bacteriophages, ΦCD08011, ΦCD2301, ΦCD418 and ΦCD1801, able to infect a range of clinically relevant C. difficile PCR ribotypes. The four phages belong to the Myoviridae family of tailed phages, established through the use of transmission electron microscopy and the identification of key genes within their genomes. Host range analysis of the four phages showed that phage ΦCD1801 had the broadest host range, able to infect and lyse all but one of the PCR ribotype 078 isolates tested. Further growth rate characterisation, rate of attachment and determination of burst size were also completed. Finally, SlpA, the main component of C. difficile’s S-layer, was confirmed as the bacterial receptor for this phage. The presence of slpA from the S-layer cassette of PCR ribotype 078 C. difficile strains conferred ΦCD1801 binding to an otherwise resistant strain. These findings will be instrumental in the ability to expand C. difficile phage host range to allow more targeted phages for the treatment of C. difficile infections.

Introduction: Liver X receptors (LXRα and LXRβ) are members of the nuclear receptor superfamily of ligand-activated transcription factors that regulate many biological and physiological processes. LXRs are important regulators of cholesterol and lipid metabolism and this is mediated by regulating a wide range of genes such as ABCA1 and ABCG1 that are involved in lipogenesis, cholesterol efflux and absorption, and bile acid synthesis. Since there is a relationship between chronic inflammatory diseases and lipid metabolic dysfunction, the role of LXRs has been investigated in different inflammatory diseases and disease models. Generally, LXRs have an anti-inflammatory effector function, however occasionally pro-inflammatory effects have also been reported. MicroRNAs (MiRNAs) are small, evolutionary conserved, single-stranded, non-coding RNA molecules with 20- 22 nucleotide base pairs. They regulate mRNA translation by fine tuning the production of proteins involved in the initiation or maintenance of inflammation. MiR-155 is one of the most studied members of miRNAs, and it has a regulatory role in certain inflammatory diseases such as collagen induced arthritis, lung fibrosis, and cardiovascular diseases. Idiopathic pulmonary fibrosis (IPF) is a devastating inflammatory disease of unknown aetiopathogenesis characterised by progressive breathlessness. IPF is characterised by approximately 50% survival of around 3 years after diagnosis, and there is no effective treatment. The main imperative for pulmonary fibrosis research is to identify potential causal inflammatory and remodelling pathways that contribute to IPF initiation and progression in order to determine possible candidate pathways for therapeutic intervention. Hypothesis: LXRs play an important role in lipid metabolism and cholesterol homeostasis and because there is a strong relationship between metabolic disease and chronic inflammatory and fibrotic diseases, e.g. LXR agonists may be beneficial for the treatment of RA. We proposed the following hypothesis ‘’ Liver X Receptors can modulate bleomycin-induced pulmonary fibrosis and therapeutic intervention with LXR agonists may be beneficial for the treatment of pulmonary fibrosis’. Methods & Results: Administration of the LXR agonist GW3965 to LXRα-/-β-/- or LXRαβ wild type mice given bleomycin to induce pulmonary fibrosis significantly exacerbated the severity of the disease only in LXRαβ wild type mice. The worsening of disease was seen as enhanced loss of body weight, increased inflammatory and fibrotic pathomorphological changes in the lung, increased inflammatory cells in the bronchoalveolar lavage, increased concentrations of several pro-inflammatory and pro-fibrotic mediators, and increased expression of genes that regulate inflammation and fibrosis, such as collagen and TGFβ, increased lung collagen content, and finally up-regulation of the expression of the alternative activated macrophages (M2)markers arginase 2 and IL-13 receptor. The effect of the LXR agonist was mediated specifically by LXRs because the severity of disease did not change in LXRα-/-β-/- mice given bleomycin and treated with GW3965, nor on similarly treated single LXRα or LXRβ gene-deleted mice. Furthermore, similar activation of LXRs in primary human or murine fibroblasts demonstrated up-regulation of the expression of collagen. The function of LXR agonist was directly on collagen gene expression and did not require de novo protein synthesis as demonstrated by the addition of cycloheximide as a translation inhibitor to murine primary fibroblasts activated with LXR agonist. This suggested that the LXR may have acted directly on the promoter region of the collagen gene. Also I investigated if the collagen genes have response elements for LXR in their promoter regions using a cell reporter system. I demonstrated that the collagen genes have response elements for LXR in their promoter regions. In a parallel set of experiments, administration of bleomycin or PBS to miR-155-/- and miR-155 wild type mice demonstrated a significantly stronger inflammatory and fibrotic process in the miR-155 gene-deleted mice. This worsening of disease was seen as an enhanced loss of body weight, increased inflammatory and fibrotic pathomorphological changes in the lung, increased inflammatory cells in the bronchoalveolar lavage, increased concentration of several pro-inflammatory and pro-fibrotic mediators, and increased expression of genes that regulate inflammation and fibrosis; such as collagen and TGFb, increased lung collagen content, and finally up-regulation of the expression of the alternative activated macrophages (M2)markers arginase 2 and IL-13 receptor. The effect was mediated specifically by miR-155 because the severity of disease was increased in miR-155-/- mice compared with miR-155 wild type mice given bleomycin. Also no differences were observed in miR-155-/- and miR-155 wild type mice given PBS. Conclusion: My results demonstrate that both the LXR and miR-155 have an important impact on the progression and extent of murine pulmonary fibrosis. Since completing the work for this thesis some of my additional pilot work has shown that LXR is a target for miR-155 and therefore both may have an important role in lung homeostasis. My results suggest the therapeutic approaches for IPF might include targeting the LXRs or LXR-regulated pathways, including potential fine tuning levels of LXRα with miR-155 antagonists. The aim would be to prevent excessive remodelling. Furthermore, any such therapeutic intervention would need to be done in a very careful way because LXRs are involved in many other physiological processes. Therefore, more targeted therapy perhaps controlling miR-155 may be of clinical relevance for therapeutic strategies.

Autoimmune inflammatory disorders such as systemic lupus erythematosus (SLE) remain debilitating conditions, as many patients are refractory to existing conventional and biologic therapies or suffer serious adverse effects, such as susceptibility to catastrophic infection. Therapies based on the actions of parasite- derived immunomodulators that dampen inflammation to promote the survival of the parasite without seriously immunocompromising the host, may therefore provide alternative strategies for the development of novel and safer drugs. One such molecule, ES-62, protects against disease in mouse models of rheumatoid arthritis and asthma; in both of these pathologies, suppression of disease is due to modulation of pathogenic IL-17A responses. As IL-17A has been implicated in the pathogenesis of SLE, in this thesis, the therapeutic potential of ES-62 is explored in the MRL/lpr mouse model of SLE. SLE is characterized by autoantibody responses to dsDNA, as well as other nuclear and cytoplasmic antigens, which result in the deposition of autoantibody- immune complexes that cause localized inflammation in tissues with dense capillary networks, most often the skin, joints and kidneys. The MRL/lpr mouse is genetically predisposed to develop lupus-like pathology displaying many of the characteristics of human disease, including the major cause of morbidity, glomerulonephritis. Twice weekly treatment of MRL/lpr mice with ES-62 significantly suppressed the development of proteinuria, a direct measure of renal dysfunction. Despite drastic improvement in renal function, the kidneys from ES-62 treated mice did not show substantial improvement in histopathology as indicated by the overall levels of glomeruloproliferation, cellular infiltration and complement or immunoglobulin deposition in the kidneys. However, exposure to ES-62 did reduce the expression of complement (C3aR and C5aR) and immunoglobulin receptors (FcγRI (CD64)), thus rendering renal cells hypo-responsive to these pro- inflammatory stimuli. Moreover, by modulating MyD88 signaling, ES-62 likely suppresses renal cell responsiveness to chronic DAMP and IL-1 signals as well as potentially promoting glomerular barrier stability. Consistent with their hypo-responsive phenotype, renal fibroblasts from ES-62 treated mice produced less MCP-1 in response to TLR stimulation and this was associated with reduced infiltration into the kidney by effector T and B cells and granulocytes; along with the ability of the parasite product to modulate the production of the pro-inflammatory cytokines IL-17A and IL-22. ES-62 suppressed the production of IL-22 both prior to and following onset of disease suggesting a key role for this cytokine in lupus pathogenesis, a proposal confirmed by neutralization studies which demonstrated that IL-22 played an essential role in the development of disease in the MRL/lpr mouse. This was further supported by studies showing that recombinant IL-22 significantly accelerated and exacerbated disease. By contrast, despite suppressing early IL-17A responses, the production of IL-17A was significantly increased in ES-62 treated mice during the established phase of disease, suggesting that IL-17A may promote pathogenesis during the initiation of pathology, yet act to resolve aberrant inflammation in the kidney. This potential dual role for IL-17A in the regulation of kidney inflammation was corroborated by studies using neutralizing antibodies and recombinant IL-17A, as the early neutralization of IL-17A production slowed the onset and severity of proteinuria and the late administration of rIL-17A suppressed disease severity. Aberrant B cell responses drive pathogenesis both in murine models of SLE and also in human disease: reflecting this, B cell depleting therapies have proved successful in the clinic. Thus, the effects of ES-62 on the population dynamics of effector and regulatory B cell subsets were investigated and these studies revealed that ES-62 induced a hypo-responsive B cell phenotype that was associated with modulated development, migration and/or activation of pathogenic effector B cells. Furthermore, the proportion of IL-10 producing ‘regulatory’ B cells were significantly elevated in the ES-62 treated MRL/lpr mice during the established phase of disease. Crucially, the protection afforded against the development of proteinuria by ES-62 was mimicked by the adoptive transfer of B cells from ES-62 treated MRL/lpr mice: moreover, such protection was associated with modulation of the IL-17A/IL-22 axis, as observed in MRL/lpr mice treated with ES-62. Together with previous reports on the therapeutic potential of ES-62 in arthritis and asthma, these studies suggest that therapies based on the parasite product have a future in the clinic. ES-62 itself is not suitable as a therapy, due to it immunogenic nature and the complexity of its biosynthesis: thus small molecular analogues (SMAs) of the parasite product have been synthesized. Two of these were tested in the MRL/lpr mouse and found to suppress the development of proteinuria, even when administered after the onset of pathology. This protection, as with that afforded by ES-62, was associated with a modulation of MyD88 signaling in the kidney and indicates that novel drugs, based on the safe modulation of the immune system by the parasite derived product, ES-62, have the potential to treat lupus nephritis in SLE patients.

While these data therefore show that these molecules cannot be used as markers for T cell anergy, and while the search for a bona fide T cell anergy marker continues, it also opens up new possibilities for their role(s) in modulation of T cell activation. By advancing knowledge of the key signalling events that take place during antigen recognition, more targeted approaches for enhancing or inhibiting immunity or tolerance can be devised.