Introduction: Streptococcus pneumoniae is an important cause of morbidity and mortality worldwide and a leading cause of pneumonia, bacteraemia and meningitis. Nasal colonisation is a vital prerequisite to disease. Memory T cells and the cytokine IL-17 has been reported to be required for the control of murine nasal colonisation and humans have pneumococcus specific memory T cells but the role of IL-17 in invasive pneumococcal disease remains poorly understood. Methods: Human pneumococcus specific T cells from peripheral blood using a co-culture model. Young and old adults as well as patients with invasive pneumococcal disease were included. A mouse model of colonisation and disease was used to assess the role of IL-17 and the related cytokine IL-22 in naïve mice using transgenic mice deficient in IL-17 and IL-22 signalling. Data from this model was supplemented with next generation sequencing to investigate the microbiome and prokaryotic transcriptome during colonisation and infection respectively . Results: Pneumococcal specific memory T cells were identified in human peripheral blood samples and some of these expressed IL-17, IL-22 and interferon-γ following clonal stimulation. IL-17 expressing T cells arose from the CCR6+ subset of T cells. The memory T cell responses of young and old adults were compared. Young adults had greater T cell proliferative in response to pneumococci than old adults and also less expressed more IL-17, IL-22 and interferon-γ. Two principal models of pneumococcal disease were investigated in animal models. TIGR4 is a highly invasive strain which causes bacteraemia while SRL1 causes severe pneumonia and empyema. RNA-Seq was used to identify differential gene expression in infection relative to growth in broth. Peak IL-17 and IL-22 are production occurs within hours of induction of infection and the likely source of IL-17 was identified as γδ lymphocytes. When IL-17RA-/- mice were infected either strain they demonstrated decreased neutrophil recruitment to blood and lung. TIGR4 infected IL-17RA-/- mice had decreased survival 4 whereas SRL1 infected IL-17RA-/- mice had increased survival. TIGR4 is significantly more susceptible to neutrophil phagocytosis than SRL1 in vitro and experiments with mice depleted of neutrophils suggested that neutrophils were important in the survival difference demonstrated between strains. Subsequent studies with a further pneumonic strain (serotype 6B) also demonstrated increased survival in IL-17RA-/- mice. IL-17 was important in the control of nasal colonisation with both TIGR4 and SRL1. The nasal microbiome of IL-17RA-/- mice was significantly less diverse than that of wild type mice with the microbiome dominated by a small number of bacterial species. IL-22-/- mice had impaired resolution of inflammation and increased collagen deposition in lungs following treatment of pneumonia compared to wild type. Conclusions: IL-17 is an important component of the immune response to pneumococcal disease although it can be either beneficial or harmful depending on the pneumococcal strain and site of infection. Further work will expand on the role of this cytokine in colonisation and disease.

Coping with oxidative stress is vital for survival of the intracellular parasite Leishmania, but the complex biochemical mechanisms involved are not fully understood. This study focused on enzymes of cysteine metabolism in Leishmania and the parts they play. Mercaptopyruvate sulfurtransferase (EC 2.8.1.2) of Leishmania major and L. mexicana and serine acetyltransferase (EC 2.1.3.30), cysteine synthase (EC 4.2.99.8) and cystathionine b-synthase (EC 4.2.1.22) of Leishmania major have been cloned, expressed as active enzymes in Escherichia coli, and characterised. The leishmanial mercaptopyruvate sulfurtransferase is structurally peculiar in possessing a C-terminal domain of some 70 amino acids. Homologous genes of T. cruzi and T. brucei encode enzymes with a similar C-terminal domain, which suggests that the feature, not known in any other sulfurtranferase, is a characteristic of trypanosomatid parasites. Short truncations of the C-terminal domain resulted in misfolded, inactive proteins, demonstrating that the domain plays some key role in facilitating correct folding of the enzymes. The recombinant sulfurtransferase exhibit high activity towards 3-mercaptopyruvate and catalyse the transfer of sulfane to cyanide to form thiocyanate and sulfide. The sulfide can react with O-acetyl serine to yield cysteine through the action of cysteine synthase. They also use thiosulfate as a substrate and mercaptoethanol, glutathione, cysteine or reduced thioredoxin as the accepting nucleophile, the latter being oxidised. Mercaptopyruvate sulfurtransferase and cysteine synthase are expressed in all life cycle stages of Leishmania and the expression levels are increased under hypo-sulfur stress. The expression level of mercaptopyruvate sulfurtransferase is also increased under oxidative stress whereas overexpression of serine acetyltransferase, cysteine synthase and cystathionine beta-synthase in Leishmania promastigotes produced cell lines resistant to the oxidants hydrogen peroxide (0.5 mM), tert-butyl hydroperoxide (10 mM) and cumene hydroperoxide (10 mM).

The infectivity of three different clones of the human malaria parasite Plasmodium falciparum to two different natural vector mosquito species, Anopheles albimanus and Anopheles stephensi was investigated. Two of the P. falciparum clones studied (HB3B-B2 and 7G8) established relatively low levels of mature oocyst infection in both mosquito species. In contrast, the third P. falciparum clone investigated (3D7A) did not produce mature oocyst infections in An. albimanus but infected stephensi at a relatively high level. These observations demonstrate the existence of differences between the three malaria parasite clones iii the ability to infect the mosquitoes, and the two mosquito species in their susceptibility to malaria parasite infection. Direct immunofluorescence microscopy and examination of Giemsa-stained histological sections by light microscopy were used to investigate further the development of the P. falciparum 3D7A clone in An. albimanus and An. stephensi. The P. falciparum 3D7A clone formed mature ookinetes within the bloodmeals of both albimanus and An. stephensi. In An. albimanus, these malaria parasite stages subsequently failed to migrate from the bloodmeal and invade the surrounding midgut epithelium suggesting that ookinetes were destroyed within the endoperitrophic space of this mosquito species. The reasons for the disappearance of ookinetes within the endoperitrophic space of An. albimanus were uncertain but were possibly related to the faster time to completion of bloodmeal digestion in this mosquito species compared to An. stephensi. Simultaneous investigation of P. falciparum 3D7A development within An. Stephensi revealed that in this mosquito species ookinetes entered the midgut epithelium via an intracellular route, causing destruction of invaded midgut epithelial cells, and subsequently exited the midgut epithelium by an intercellular route. A general model for the route of ookinete invasion across the midgut epithelium is proposed based upon these observations. Examination of the Giemsa-stained histological sections also provided evidence that regenerative cells within the midgut epithelium of An. stephensi imdergo proliferation and differentiation into midgut epithelial cells following infection with P. falciparum 3D7A, presumably as a mechanism to replace the midgut epithelial cells destroyed as a consequence of ookinete invasion of the midgut epithelium.

The intestine is exposed to a vast array of harmless dietary antigen as well as an enormous community of commensal bacteria. Alongside this harmless antigen, pathogens can enter the body via the intestinal mucosal surface. The intestinal immune system must discriminate between pathogens and innocuous antigens. Dendritic cells (DCs), present in the small intestine (SI) and colon, are fundamental in controlling intestinal immune responses; they migrate to the mesenteric lymph node (MLN) and prime effector or regulatory T cells. Furthermore, DCs direct the immune response in the gut-associated lymphoid tissues (GALT), the Peyer’s patches (PP) and isolated lymphoid follicles (ILFs). The aim of this work was to determine the anatomical origins of DCs in the MLN. First, the anatomical organisation of lymphatic vessels draining to the MLN from the SI and colon was investigated. Second, DC migration in mice lacking specific GALT was explored. Finally, the migration of DCs from PPs to the MLN was investigated. To achieve these aims, DC migration was studied using a variety of labelling methods, mice that lacked specific GALT were employed, and surgical procedures were used to collect DCs from the thoracic duct. Here, I demonstrate the anatomical segregation of DCs that migrate to the MLN from the SI and colon. This was reflected in differences in DC subset composition and antigen presentation in the SI and colon-draining nodes of the MLN. Separate analysis of MLN nodes will allow a more refined understanding of intestinal immune responses. All but one DC subset, CD103-CD11b- DCs, were still present in pseudo-afferent lymph from mice lacking both PPs and ILFs. This subset is therefore likely to originate from either PPs or ILFs. Surprisingly, CD103+CD8α+ DCs were present in these mice, showing that many CD8α+ DCs were resident in the lamina propria and are not limited to lymphoid tissue. Four DC subsets are able to migrate from the intestine in PP-null mice, suggesting that CD103-CD11b- DCs migrate specifically from ILFs. I then demonstrated that DCs migrate from PPs to the MLN. These migrating PP DCs expanded in response to a DC specific growth factor and their migration depended on CCR7 and S1P. These cells may play an important role in driving immune responses in the MLN and their manipulation could lead to advances in controlling intestinal immune responses.

The pathogen Streptococcus pneumoniae encounters different levels of oxygen during the infection cycle including colonisation, pneumonia, bateraemia and meningitis. These different anatomical niches require high levels of genome changes to sense and respond to those external environmental stimuli. The bacterial gene expression is known to be affected by oxygen, and it must react properly for survival and for developing invasive pneumococcal desiseases (IPDs). Microarray techniques have allowed scanning the whole pneumococcal genome during growth in different tensions of oxygen mimicking in vivo conditions. It was found that oxygenated growth conditions have significantly elevated several key virulence genes. This was further confirmed with qRT-PCR for a selection of genes implicated in pathogenicity. Moreover, post-transcriptional stages have been also investigated such as protein production, biofilm formation, biological activities and adherence assays for several virulence factors performed under the effect of presence or absence of oxygen. The data illustrate that 420 out of 2,236 genes (17 % of the entire TIGR4 genome) were differentially expressed in the presence of oxygen compared to its absence. 262 genes (11 %) were over-expressed when pneumococci were grown in oxygenated conditions relative to transcriptional profile in anaerobic growth conditions, indicating the magnitude of roles played by oxygen on pneumococcal gene expression. Anaerobic growth of TIGR4 showed down-regulation of 158 genes (7 %). Oxygen modulates induction of ply, pspC and other seven genes involved in pili structuring subunits (rlrA, rrgA, rrgB and rrgC) and assembling enzymes (srtB, srtC and srtD). This may suggest that the pneumococcal population grown under atmospheric environment is equipped with greater capability to progress IPDs compared to anaerobically grown bacteria. In addition to this, pneumococcal adhesion in vitro for TIGR4 grown in oxygenated or anaerobic growth conditions revealed a significant increase in those grown in oxygenated growth conditions, indicating that oxygen may play a key role in bacterial-host attachment. Interestingly, ablation of pspC has resulted in similar adhesion percentages of TIGR4 grown under both conditions, oxygenated and anaerobic. Furthermore, several genes involved in metabolism were up-regulated in oxygenated environment, particularly transporters, which are considered highly important for a bacterium that lacks an electron transport chain, catalase and tricarboxylic acid. Additionally, the results showed phenotypic characterisation and changes in cells morphology from pneumococcal growth curves for several strainswith different genome backgrounds grown under different levels of oxygen concentrations. Further investigation of the pathogen biology revealed differences in pneumolysin production and activity. These findings highlight that virulence genes expression is induced once the micro-organism is exposed to oxygenated environment, and data analysis has demonstrated potential links between pneumococcal metabolism and their ability to cause diseases.

African trypanosomes, the causative agent of sleeping sickness in humans, and nagana in cattle, are typically transmitted by the bite of an infected tsetse fly. The nature of the mammalian innate immune response during and immediately after the bite of an infected tsetse fly remains poorly understood. Previous studies characterising the events occurring in the skin post-infected tsetse fly bite have mainly focussed on the development of the chancre, which occurs from day 5 post-infection. Additionally, most immunopathological studies on trypanosomes have used intravenous or intraperitoneal injections of blood stage parasites, therefore bypassing relevant inoculation routes (tsetse fly), site (skin), and parasite life cycle stages (metacyclics). It is known that following tsetse fly bites, trypanosomes leave the skin via the host lymphatic system in order to initiate a blood stage infection. However, how the host responds to this challenge and how the parasite negotiates the anatomy of the host immune system remains unclear. In the present study, I have built on existing intravital microscopy tools to visualise T. b. brucei infections in the dermis and lymphatics of an infected mouse ear after transmission. I have also characterised by flow cytometry, taqman low density arrays and depletion studies the magnitude and kinetics of the early innate immune response in the skin, as well as the functional role of neutrophils, by examining infections in the context of the natural route of infection- the bite of a tsetse fly. Neutrophils were identified to be the predominant responders at the bite site, the neutrophil response was rapid, and they were recruited independent of the infection status of the tsetse flies. Taqman low-density arrays, which measured expression levels of inflammation-associated genes, suggested that neutrophil recruitment was mediated by CXCL1/CXCL2 release in the skin following mechanical trauma by the tsetse fly, in addition to the release of pro-inflammatory cytokines- IL-1β and IL-6. Following the identification of neutrophils by flow cytometry, I then applied intravital microscopy to visualise influx of neutrophils, which was rapid, directed at the bite site, and did not form dynamic clusters. To further test the functional role of neutrophils very early in infection, neutrophils were depleted using a monoclonal antibody and mice infected via tsetse fly bites. Neutrophil depleted mice had no effect on pathogenesis in vivo. Using Prox-1 mOrange reporter mice, I also examined the interaction of bloodstream trypanosomes with lymphatic vessels in the skin in the period immediately following inoculation using intravital imaging. I imaged metacyclic trypanosomes in situ and demonstrated that they had significantly higher velocity in the extravascular matrix compared to bloodstream forms. Additionally, my data showed bloodstream parasites actively migrating towards lymphatic vessels, and intra- lymphatic T. b. brucei were also observed, enabling comparison of trypanosome motility in the extravascular matrix and lymphatic vessels; in lymph vessels trypanosomes were moving in a more directional and rapid manner. This work revealed the early cellular and molecular responses to T. b. brucei infection and investigated interactions of parasites with the anatomy and cells of the host immune system. These studies demonstrate that furthering our understanding of the very early events in trypanosome infections is essential to understand how a systemic trypanosome infection is established.

Anti-ganglioside antibodies have been implicated in autoimmune neuropathies for several decades. They are thought to elicit injury through binding to sites in the peripheral nervous system, where they activate the complement pathway to induce cell death. Patient serum is therefore regularly screened for these antibodies to aid in the diagnosis of various conditions. Recent work has found that complexes composed of gangliosides and other glycolipids can improve the detection of these antibodies beyond the signals detected to the single ganglioside species. In MMN research, complexes comprised of GM1 and GalC have been found to significantly enhance antibody detection in patient sera. In certain patients, however, antibody binding was only detected against these complexes and not the single antigens. This led some researchers to hypothesise that an unidentified class of antibody may have arisen that binds specifically to a neoepitope formed by the combination of the two glycolipids. It has also been hypothesised that that this complex may be the true target of immune mediated attack in MMN. This thesis sought to address this hypothesis by either cloning these antibodies directly from patient serum or through active immunisations with mice. Analysis of previously generated human monoclonal antibodies indicated that their behaviours were modified by complexes containing particular gangliosides or glycolipids. Furthermore, the antibodies behaviours were found to diverge, when they were screened against complexes comprised of gangliosides and different concentrations of accessory lipids. These findings suggested that the accessory lipids were interacting with the ganglioside headgroups to modify the presentation of different binding epitopes. This indicated that conformational modulation, rather than neo-epitope formation, may be responsible for complex enhancement Cloning antibodies from patient sera was unsuccessful but examination of the screening techniques suggested that the appearance of complex-dependent antibodies may have been an artefact. Attempts to induce complex-specific responses in mice were similarly unsuccessful but several anti-ganglioside and anti-sulfatide antibodies were created. The subsequent chapters focused on the characterisation of these antibodies and indicated that most of them bound well to solid-phase assays, cells and tissue and may therefore be of use in future studies. Taken together, the data from this thesis suggests that complex-dependent antibodies may not exist but are merely low concentrations of anti-ganglioside antibodies that are cis-enhanced by particular lipids. Future work should therefore focus on assessing how the ganglioside microenvironment modifies epitope presentation and how this affects the binding capabilities of antiganglioside antibodies.

Pseudomonas aeruginosa is an opportunistic pathogen that represents a major burden to the healthcare system as it accounts for a vast number of hospital-acquired infections. P. aeruginosa is a particular threat in Cystic Fibrosis (CF) patients. This bacterium expresses an arsenal of secreted virulence factors that play a fundamental role in the invasion of the damaged physical barriers of the host and abrogate immune responses which aid the persistence of P. aeruginosa infections. The aim of this study is to elucidate the role of P. aeruginosa secretions in the modulation of the immune system and determine the key factors and their specific impact on immune cells such as T helper cells proliferation and monocytes viability. The present study employed peripheral blood mononuclear cells from healthy donors as a source of immune cells and bacterial supernatants as a source of exoproducts. In this context, the P. aeruginosa strain PAO1 was used throughout this project and two sublines of this strain were mainly involved in this work; the PAO1 sublines Lausanne (PAO1-L) and Nottingham (PAO1-N). Different sublines of PAO1 are genetically diverse and this variation was utilised as a tool to identify the key molecules produced by P. aeruginosa that are responsible for the observed effect. The present study demonstrated that P. aeruginosa supernatants obtained from PAO1-L has an inhibitory effect on T cell proliferation in response to Staphylococcus Enterotoxin B (SEB), but not α-CD3 and α-CD28 stimulation in peripheral blood mononuclear cells cultures. This effect appears to be mediated by the cytotoxic effect of P. aeruginosa products on monocytes, a precursor of antigen presenting cells. Analysis of different P. aeruginosa mutants showed that the cytotoxic activity is controlled by toxR. ToxR is a regulatory protein which enhances the production of one of the most important virulence factors of P. aeruginosa, exotoxin A (ToxA; ETA). The impact of ETA production by P. aeruginosa on immune cells was investigated using a newly generated ETA-deficient mutant ΔtoxA in PAO1-L (Lausanne background) in comparison with its isogenic wild type (WT). Preliminary observations showed that the generation of apoptotic monocytes correlated with the high expression of ETA in the bacterial supernatants as both PAO1-LΔtoxR and PAO1-LΔtoxA are less toxic strains than PAO1-L. These findings suggest that the presence of monocytes is crucial to mediate ETA down regulation of T cell proliferation. However, the effect of PAO1-LΔtoxA supernatants on T cell proliferation in response to SEB could not be fully assessed because SEB stopped inducing T cell proliferation as before. To overcome this obstacle, phytohaemagglutinin (PHA) was employed as a promoter of T cell division. However, the suppressor effect was only clear on the CD3+/CD4+ T cell subset after stimulation with PAO1-L (ETA+) supernatant in response to PHA and full T cell proliferation was not recovered in the absence of ETA (PAO1-LΔtoxA). Finally, P. aeruginosa and Candida albicans are typical opportunistic respiratory tract pathogens and the nature of the relationship between these two pathogens is still not entirely clear. Thus, in this work, an in vitro assay to evaluate the impact of P. aeruginosa secretions on C. albicans was developed. Our results suggest that P. aeruginosa secreted products promote C. albicans hyphal growth and the filament formation is not correlated with either the presence of the toxR gene or the production of ETA and rhamnolipids. Interestingly, a supernatant prepared from PAO1-L mutant with a 58 Kb deletion induces the growth of the yeast form suggesting that the gene that encodes the key element, which is responsible of inducing the hyphal growth, is located within this region. This work might deliver new understandings on the interactions between P. aeruginosa and C. albicans in polymicrobial infections particularly in susceptible individuals like those with Cystic Fibrosis and burn victims. Further work is currently being carried out in our group to identify the active molecules that trigger filamentous growth in C. albicans and to determine the contribution of this agent on the interaction between P. aeruginosa and C. albicans during co-infection.

Dendritic cells (DCs) are key players in the induction and re-elicitation of T helper (Th) 2 immune responses to allergens. Recent data by our group have shown that different C-type lectin receptors (CLRs) on DCs play a major role in allergen recognition and uptake. Particularly, mannose receptor (MR), through modulation of Toll-like receptor (TLR) 4 signalling pathway, can regulate indoleamine 2,3 dioxygenase (IDO) activity favouring Th2 responses. IDO is the rate limiting enzyme involved in tryptophan (TRP) catabolism and it is well known for its role in modulating immune responses through TRP depletion and generation of immune-regulatory metabolites known as kynurenines (KYN). Interestingly, another CLR named dendritic cell-specific intracellular adhesion molecule 3-grabbing non-integrin (DC-SIGN) has been suggested to support Th1 responses; however, the mechanisms involved are unknown. Therefore, the main objective of this project was to evaluate the role of CLRs, with focus on MR and DC-SIGN, and TLRs in modulating the IDO pathway in order to understand their involvement in regulating Th2 immune responses. In addition, it was aimed to study how regulation of IDO levels could impact immune responses in order to understand the signalling pathways involved in this process. The data show that mannosylated allergens can down-regulate TLR4-induced IDO1 and IDO2 expressions as well as IDO activity in human DCs, most likely through binding to the MR. This correlates with data showing increased IDO activity in MRlow-DCs (generated by gene silencing) stimulated with mannan compared with control-DCs (CT-DCs). Conversely, DC-SIGN could exert a differential regulation on IDO levels depending on the antigen’s sugar moieties. Mannosylated ligands could up-regulate TLR4-induced IDO activity through DC-SIGN as evidence by a significant reduction in IDO activity in DC-SIGNlow-DCs upon stimulation with mannan compared with CT-DCs. In contrast, DC-SIGN-specific fucosylated ligands, such as Lewis-X (LeX), can down-regulate IDO activity in a TLR4-dependent manner. It was also found that allergens from diverse source can down-regulate IL-12p70 production by DC, which can further impact T helper cell polarization by reducing IFN-γ production by T cells in autologous co-cultures. Moreover, mannan was also shown to decrease CD86 expression in human DCs, which might impact the IDO pathway, as CD86 is one of the B7 family molecules involved in IDO induction. Furthermore, it was found that the LeX can potentially down-regulate IL-6 production by DCs. In addition, DCs stimulated with LeX and LPS induce lower levels of IFN-γ production by autologous T cells than DCs stimulated with LPS only. This effect was reversed in the presence of KYN, showing that the IDO pathway can regulates T helper cell polarization. TLR4 signalling and LPS exposure have been shown to play a pivotal role in Th2-mediated inflammation and asthma. In addition, it is known that CLRs can modulate TLR-induced responses in an allergy context. Therefore, we further study the role of the TLR signalling pathway in modulating IDO levels in human DCs. TLR4 engagement on human DCs was shown to induce high levels of both IDO isoforms. DCs primed with LPS induce much higher levels of IDO than single LPS controls, inducing a cellular re-programing. This was characterized by an induction of anti-inflammatory mediators such as IL-10 and the transcription factor aryl-hydrocarbon receptor (AhR); the last has been previously linked with the IDO pathway in mouse DCs. Intriguingly, TLR9 engagement with synthetic cytosine-phosphate guanosine (CpG) A motifs was able to down-regulate IDO and induce a pro-inflammatory phenotype in human DCs. Future studies should aim at evaluating their implications in immune responses under tolerance and during infection. Finally, the study was aimed at elucidating the intracellular molecules involved in IDO regulation in human DCs. The analysis was first focused on AhR, a ligand-dependent transcription factor that have been suggested to modulate IDO levels in mouse DCs. It was shown for the first time that TLR4 induction of IDO was dependent on AhR in human DCs. In addition, AhR gene expression as well as AhR activity were reduced in DCs stimulated with mannan and LPS compared with LPS only controls. This data suggest that MR can regulate IDO levels by interfering with AhR expression. Furthermore, it was shown that the NF-κB pathway is key in regulating IDO levels through CLRs and TLRs in human DCs. Particularly, RelB, a member of the non-canonical pathway, expression correlates with the levels of AhR, suggesting that a functional and/or physical association between them might be involved in regulating IDO levels in human DCs. Future studies should aim at elucidating such interactions. To sum up, this project has shown the pivotal role of CLRs and TLRs in modulating the IDO pathway in human DCs, which can affect DC phenotype and function and impact immune responses. In addition, we have gained some understanding into the molecular mechanisms of IDO modulation which might involve cooperation between different transcription factors such as AhR and RelB. These data give new insights into how glycosylated allergens can induce Th2 immune responses, which can pave the way to develop better therapeutic strategies to fight back allergy related diseases.

No description available.