Microbiology & Immunology - Theses
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ItemRole of the chemokines CCL17 and CCL22 in the immune defence against Salmonella infection( 2019)The chemokines CCL17 and CCL22 are both ligands of the chemokine receptor CCR4, which is expressed on dendritic cells (DC) and a variety of different effector T cells including regulatory T cells (Treg). Both chemokines are mainly produced by DC, but also by macrophages. CCL17 promotes numerous inflammatory and allergic diseases, whereas CCL22 is rather associated with an immunosuppressive milieu. These differential roles are reflected by preferential recruitment of distinct subsets of T cells to site of inflammation. While CCL17 facilitates chemotaxis of effector T cells and supports DC-T cell interactions as well as DC migration towards CCR7-ligands, CCL22 induces chemotaxis of Treg cells. In addition, CCL22 signalling induces a more rapid desensitisation and internalisation of CCR4 than CCL17, suggesting biased agonism of CCL17 and CCL22. The functionality of CCL17 and CCL22 should, therefore, be considered in combination as well as individually in the context of immune-related diseases. The role of CCL17 and CCL22 in infectious diseases has not been well understood. The central hypothesis was that CCL17 and CCL22 play important but potentially different roles during bacterial infection. This was modelled using a well-studied bacterial pathogen, Salmonella enterica serovar Typhimurium (STM). It was hypothesised that CCL17 expression may direct the migration of STM-infected DC from the gut to draining lymph nodes a key bottleneck in early infection that controls bacterial dissemination to systemic sites. It was further hypothesised that CCL22 may play a role in immune regulation through the induction of Treg cells. These regulatory cells may have downstream effects on Th1 responses, which are critical for the control of Salmonella infection. In the first part of the thesis, the role of CCL17+ DC in the transmission of STM was investigated. Histological analysis of CCL17 reporter mice revealed that CCL17-expressing cells co-localised with Salmonella in the dome area of Peyer’s patches (PP). Further, CCL17-expressing DC contributed to dissemination of STM from PP to the mesenteric lymph nodes (mLN). Within the mLN, STM were found within CCL17+ DC as well as in other DC, monocytes and macrophages. Analysis of the STM+ DC subpopulations revealed that all DC subsets carried STM, but the CD103+ CD11b- DC could be identified as the main STM-containing population. STM infection triggered upregulation of CCL17 expression in specific intestinal DC subsets in a tissue-specific manner. Interestingly, the CD103+ DC subsets upregulated CCL17 in the PP, whereas CD103- DC subsets upregulated CCL17 in the mLN. In the second part of this thesis, the role of CCL17 and CCL22 in the induction of antigen-specific CD4+ T cell responses was investigated. CCL17/CCLL22 double-deficient, CCL17- and CCL22 single-deficient, and wild type mice were analysed after live-attenuated STM TAS2010 vaccination, vaccination/challenge and in steady-state. Mice deficient in both chemokines, CCL22 and CCL17, demonstrated a reduction of effector Treg cells. This promoted an enhanced STM-specific Th1 immune response characterised by an expansion of Th1 T cells, resulting in a more favourable effector Treg/activated Tconv ratio and a significantly improved vaccine efficacy to challenge with virulent Salmonella. In conclusion, the work presented within this thesis showed the contribution of CCL17+ DC in the dissemination of STM and identified CCL22 as a potential target to improve vaccine approaches.
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ItemSalmonella Typhimurium metabolism in the murine host and importance to virulence( 2014)The bacterial pathogen Salmonella enterica is responsible for considerable global morbidity and mortality, being the cause of several enteric and systemic diseases, including typhoid fever. Non- typhoidal Salmonella (NTS) serovars such as Typhimurium cause gastroenteritis in immunocompetent individuals, but in the context of HIV-co-infection can cause invasive disease with high fatality rates. Successive generations of antimicrobials have become ineffective against S. enterica pathogens due to the widespread development of resistance, and new drugs are critically needed. In addition, vaccines against typhoid fever have sub-optimal efficacy and no human NTS vaccines are currently available. Although the in vitro metabolism of S. enterica is relatively well defined, little is known about the specific nutrients that the pathogen consumes in its hosts, and the metabolic mechanisms by which S. enterica utilise these nutrients. Therefore, the central aim of this study was to investigate and characterise S. enterica serovar Typhimurium metabolism in the murine host. Attention was focused on two areas of S. Typhimurium metabolism which have been speculated to contribute to bacterial virulence: methylglyoxal detoxification and central carbon (sugar) catabolism. It was anticipated that this study may contribute to better defining the metabolic requirements of S. Typhimurium during infection and disease, and present opportunities for the identification of potential drug targets and metabolically-attenuated strains amenable to use as live vaccines. Several studies have ventured that methylglyoxal detoxification mechanisms are required for survival of bacterial pathogens in the mammalian host, but this hypothesis has not been thoroughly tested. In the current study, although S. Typhimurium mutants defective in the glutathione-dependent glyoxalase system (GDGS) or Kef-mediated potassium efflux were highly sensitive to methylglyoxal, they were not attenuated for intracellular replication and growth in mice, suggesting that these methylglyoxal detoxification mechanisms are not required for S.Typhimurium pathogenesis in the mammalian host and are not suitable targets for antimicrobial therapy against S. Typhimurium disease. While others have reported that the Embden-Meyerhof pathway (EMP) and the ability to utilise glucose are required for S. Typhimurium virulence in mice, the importance of other sugars and carbon catabolic pathways to S. enterica virulence is unclear. In this study, S. Typhimurium mutants blocked in several sugar catabolic pathways including the Entner-Doudoroff pathway (EDP) were found not to be attenuated for intracellular growth or fulminant infection of mice, demonstrating that gluconate, glucuronate, galacturonate are not essential carbon sources for S. Typhimurium in vivo. However, evidence was presented which suggested that the ability to utilise gluconate and glucose is required for optimal shedding of the pathogen in the murine faeces, revealing potential strategies for reducing the faecal-oral transmission of S. Typhimurium. EMP/EDP double mutants showed greater attenuation in mice than a EMP mutant, suggesting that the EMP mutant utilises the EDP in order to facilitate it’s modest growth in vivo. These findings demonstrated the functional redundancy of S. Typhimurium metabolism and suggested that combinational drug therapies targeting several bacterial pathways concurrently might be a viable option for treating S. Typhimurium disease. The S. Typhimurium EMP/EDP mutant TAS2010 was found to provide increased, long-term protection from virulent infection in a murine typhoid vaccination model than the prototypical aro-negative vaccine strain BRD509. Given that the lack of effective vaccines against S. enterica pathogens can be largely attributed to an insufficient understanding of the host immune response to the pathogen, the immunological response to the TAS2010 vaccine strain was characterised in mice to understand the mechanisms responsible for the increased protective capabilities of this strain. In comparison to BRD509, TAS2010 was found to replicate to higher numbers in murine organs and induce an increased immune response in the form of increased interferon-gamma secretion by splenic CD4+ T cells. Evidence was presented which suggested that the protection provided by TAS2010 is less reliant on T cells and more dependent on a CD4-CD8-Thy1+ lymphocyte subset, probably Thy1+ NK cells. In conclusion, this study has enhanced the understanding of S. Typhimurium metabolism in the murine host and introduced a live vaccine strain with improved protective and immunogenic properties.