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.