Medicine (St Vincent's) - Theses
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ItemSlc16a10’s role in amino acid supply, immune cell function and autoimmune diabetes( 2016)Immune cells depend on nutrients from the extracellular environment to support biochemical processes required for proper function. In particular, changing the intracellular concentration of amino acids can alter various immune cell responses. Amino acids are important not only for protein biosynthesis, but also act as signalling molecules that modulate cellular processes, including gene expression and cell growth. Amino acids in immune cells must therefore be tightly regulated to initiate proper immune responses and maintain immune tolerance. Transporters are especially critical for sensing and translocating amino acids across membranes during immune cell activation. Defects in amino acid transport could therefore either impair or enhance an immune response. While ~50 amino acid transporters have been identified, relatively few have been characterised for their role in the immune system. Fundamental research is still required to identify which amino acid transporters are critical for cell function and contribute to immune-related diseases, for example autoimmune disease, by regulating specific immune cell responses. Type 1 diabetes in both humans and the non-obese diabetic (NOD) mouse model is an autoimmune disease that results from immune cell mediated destruction of insulinproducing β cells in the pancreatic islets. Diabetes occurs once the majority of β cells are destroyed and the lack of insulin causes increased blood glucose concentration. As it is often difficult to study disease pathogenesis in human patients, the NOD mouse strain provides an excellent model for investigating the role of amino acid transporters in the development of autoimmune diabetes. We have generated a novel NOD mouse strain with a transposon insertion within the gene Slc16a10 that encodes MCT10, a protein that transports aromatic amino acids (AAA) in and out of cells. It was hypothesised that transposon disruption of Slc16a10 would affect gene expression and AAA transport, leading to abnormal immune cell responses that alter the development of autoimmune diabetes. The first aim of this thesis was to determine the effect of the transposon insertion on Slc16a10 expression and mouse viability. Analysis of gene expression confirmed that the Slc16a10 transcript is not detected in our transposon mutant NOD mice. Our results also indicated that disruption of Slc16a10 expression does not affect fertility or peripheral blood cell numbers, or cause gross abnormalities in these mutant NOD mice. However, Slc16a10-deficient NOD mice do have increased plasma AAA levels confirming that MCT10 protein expression and function were disrupted. Our findings show that Slc16a10 is not an essential gene for the basic development and viability of NOD mice housed in specific pathogen free conditions, but demonstrate a role for MCT10 in regulating amino acid homeostasis. The second aim was to characterise diabetes pathology in Slc16a10-deficient mice. Notably, Slc16a10-deficient NOD mice have an increased incidence of autoimmune diabetes. Pancreatic islet morphology, glucose tolerance and islet susceptibility to cell death were not different between Slc16a10-deficient and wildtype NOD mice, suggesting that Slc16a10 is not essential for proper islet physiology and function. Instead, disruption of Slc16a10 was associated with an increase in immune cell infiltration of the pancreatic islets, which is a hallmark of autoimmune diabetes. These results also validated the feasibility of our transposon mutagenesis approach for identifying new genes that affect disease pathogenesis in the NOD mouse model of type 1 diabetes. The third aim was to determine the effect of Slc16a10 deficiency in immune cells. Slc16a10 expression can be detected in a variety of immune cell types, with the greatest relative expression detected in macrophages. Our studies found that Slc16a10 is important for amino acid efflux because Slc16a10-deficient macrophages have increased intracellular concentrations of tryptophan and tyrosine. A series of in vitro assays were performed to assess the effect of Slc16a10 disruption upon macrophage phagocytosis, as well as cytokine and nitric oxide secretion, but no effect was detected in mutant macrophages. Instead, T-cell proliferation was enhanced in the presence of Slc16a10-deficient macrophages. This suggests a complex role for Slc16a10/MCT10 in macrophage and T-cell interaction and function. Subsequent analysis of T cell subsets in the islets of Slc16a10-deficient NOD mice also revealed an increase in effector T cells along with a decrease in regulatory T cells amongst the infiltrating immune cells. Collectively, the results presented in this thesis support a role for Slc16a10/MCT10 in modulating macrophage and T-cell responses that contribute to immune dysregulation and the development of autoimmune disease.