Medicine (St Vincent's) - Theses

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    The role of Trig: a novel toll-like receptor induced gene, in dendritic cell function and autoimmune disease
    Ashton, Michelle Pauline ( 2013)
    The immune system is comprised of a complex network of cells and signalling pathways that must be tightly regulated to maintain immune homeostasis. Defective negative regulation results in enhanced immunogenicity, loss of immune tolerance and, eventually, the development of autoimmune disease. Toll-like receptors (TLRs) are an essential component of the immune system as they act as early sensors of microbial pathogens and play a critical role in linking the innate and adaptive arms of the immune response. There is also increasing evidence that aberrant TLR signalling and TLR-mediated immune responses contribute to the development of autoimmune diseases, such as type 1 diabetes (T1D), although investigating these abnormalities and the underlying genetic defects in humans is often difficult. Instead, the non-obese diabetic (NOD) mouse strain, which spontaneously develops T1D, has proven to be a useful animal model for investigating genetic variants that contribute to autoimmune disease by altering TLR-mediated immune responses. Predisposition to T1D in the NOD mouse is due to allelic variation at multiple loci across the genome. More than 30 susceptibility loci have been linked to T1D development in the NOD mouse. Positional cloning of one of these loci, termed Idd11, has led to the identification of a novel gene termed Trig (AK005651). Allelic variation for Trig is associated with T1D development and this gene is differentially expressed in immune-related tissues between diabetes-resistant and diabetes-susceptible mouse strains. Preliminary experiments also revealed that Trig is upregulated in a dendritic cell (DC) line in response to TLR9 stimulation. It was therefore hypothesised that genetic variation for Trig alters TLR-mediated immune responses that affect the development of autoimmune disease. The first aim of this thesis was to perform a preliminary characterisation of a novel mouse strain deficient for Trig. Trig-deficient mice were observed to be viable and fertile, were indistinguishable from wildtype littermates for weight and gross anatomical development, and did not develop any observable signs of ill health. Furthermore, no statistical differences between Trig-deficient and wildtype littermates were identified for immune cell number or frequency in the peripheral blood, thymus or spleen. This study indicates that Trig is not an essential gene for the basic development and viability of B6 mice housed in specific pathogen-free conditions. The second aim was to investigate the experimental conditions that alter expression of Trig. Trig was found to be upregulated in an immortalised DC line, primary DCs and bone-marrow-derived macrophages, after exposure to ligands that activate MyD88-dependent TLR signalling pathways. This upregulation was abrogated by interferon (IFN)γ signalling, indicating that TLR/IFNγ signalling cross-talk regulates the expression of Trig. These studies suggest that Trig might act as a negative feedback regulator of TLR-mediated immune responses. The third aim was to determine which TLR-mediated immune responses were regulated by Trig in DC subsets. A series of in vitro assays were performed to assess the capacity of TLR9-stimulated Trig-deficient DC subsets to produce cytokines, upregulate cell-surface molecules and present antigen to T cells. This revealed that Trig-deficiency leads to enhanced cytokine production in specific DC subsets. Subsequent analysis of DC subsets isolated from NOD, B6 and Idd11 congenic mice revealed that strain variation for Trig also affects TLR9-mediated cytokine production. Collectively, these findings indicate that Trig negatively regulates TLR9-mediated cytokine responses in specific DC subsets. The fourth aim of this thesis was to explore the role of Trig in systemic inflammation and autoimmune disease. The serum cytokine levels of Trig-deficient mice treated with a TLR9 agonist were similar to those of wildtype littermates, indicating that Trig is not an essential negative regulator of TLR9-mediated cytokine production under the experimental conditions tested. Instead, Trig-deficiency had a subtle affect on the onset and/or severity of three inducible models of autoimmune disease. Therefore, this thesis presents cumulative evidence that supports a role for Trig in attenuating TLR-mediated immune responses that contribute to immune dysregulation and the development of autoimmune disease.
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    Characterisation of susceptibility to Listeria monocytogenes infection in the non-obese diabetic (NOD) mouse
    WANG, NANCY ( 2012)
    The immune system has evolved the ability to prevent infection by a wide range of pathogens while maintaining tolerance to self-tissues. Due to the strong selective pressure imposed by microbial pathogens, susceptibility to infection can be modulated by a large number of genetic loci. It is postulated that, at least in some cases, allelic variants for particular loci confer increased resistance to pathogens while simultaneously increasing the risk of developing autoimmune diseases. A number of recently discovered disease loci appear to act as “genetic pivot points” between pathogen defence and autoimmune pathogenesis. It is anticipated that characterising these loci will provide novel insights regarding the interplay between immune and autoimmune responses, as well as reveal potential therapeutic targets for treating both infectious and autoimmune diseases. Murine models provide a complementary approach to human studies for investigating genetic and cellular mechanisms that underlie susceptibility to infectious and autoimmune diseases. The non-obese diabetic (NOD) mouse strain is one of the best-characterised models of type 1 diabetes (T1D), an autoimmune disease caused by the destruction of insulin-producing pancreatic β cells. Similar to humans, predisposition to T1D is attributed to multiple genetic loci in NOD mice. Intriguingly, non-diabetogenic mouse strains can also harbour diabetogenic alleles for some T1D susceptibility loci. Using congenic mice, our laboratory confirmed that non-diabetogenic C57BL/6 (B6) mice harbour a diabetogenic allele for a T1D susceptibility locus on chromosome (Chr) 13, termed Idd14. Coincidentally, the Idd14 locus overlapped with Listr2, a proposed susceptibility locus for Listeria monocytogenes, which is an extensively studied intracellular bacterial pathogen. Notably, B6 mice are resistant to L. monocytogenes infection, whereas NOD mice are reported as susceptible. We therefore hypothesised that allelic variation for this interval, which increases T1D risk, would confer resistance against L. monocytogenes in NOD mice. The goal of this study was to investigate the biological and genetic effects of allelic variation for Idd14/Listr2 in NOD mice for L. monocytogenes infection and T1D. Towards this goal, the first aim was to investigate the immunological basis of susceptibility to L. monocytogenes in NOD mice. In comparison to infected B6 mice, infected NOD mice exhibited reduced antigen-specific CD8+ T-cell responses, which was associated with apparent deficiencies in dendritic cells. Infected NOD mice also exhibited exacerbated neutrophilia, a potential compensatory mechanism in susceptible hosts. The second aim was to determine the role of neutrophils during L. monocytogenes infection. Specific depletion of neutrophils impaired bacterial clearance in susceptible NOD mice, but not in resistant B6 mice, demonstrating that neutrophils are essential for controlling L. monocytogenes infection in susceptible hosts. In addition, NOD macrophages exhibited impaired antimicrobial function. These results indicate that neutrophils may compensate for deficient T-cell and macrophage responses to ensure host survival during L. monocytogenes infection. The third aim was to confirm the Listr2 locus using congenic NOD mouse strains, which harbour different B6-derived Chr 13 interval, and determine if the refined intervals for Listr2 and Idd14 continue to co-localise. Concurrently, it was found that the Idd14 locus could be dissected into two sub-loci, Idd14.1 and Idd14.2. Listr2 was confirmed and co-localised with Idd14.1 to an overlapping interval (~ 18 Mb) on Chr 13. This co-localisation raises the intriguing possibility that allele variation for the same gene(s) within this interval affects both infection and autoimmune disease susceptibility.