Medicine (St Vincent's) - Theses
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ItemInvestigation of Serinc1 and Apics in immune cell function and autoimmune diabetes( 2016)Type 1 diabetes (T1D) is an autoimmune disease in which immune cells mediate specific destruction of the insulin producing β cells in the pancreatic islets. The events that trigger this autoimmune response are still not clear, but genetic studies in humans have identified more than 50 loci that affect the risk for T1D. However, it is often difficult to pinpoint the underlying genes for these loci, let alone identify and characterise causative alleles that affect immune cell function and increase disease susceptibility. Instead it has proven useful to complement human genetic studies with strategies in mouse models that not only aim to discover "naturally" occurring alleles, but also engineer "artificial" alleles in putative and novel candidate genes to determine their effect on disease pathogenesis. In particular, investigation of natural and artificial alleles in non-autoimmune-prone mouse strains and in the non-obese diabetic (NOD) mouse strain, which spontaneously develops T1D, has provided important genetic and immunological insights into T1D. Using this strategy, two candidate genes, Serinc1 and Apics, were investigated for potential roles in immune cell function and the development of autoimmune disease, in particular T1D. Serinc1 encodes a carrier protein that facilitates synthesis of serine-derived lipids. These lipids are important in the formation of highly ordered membrane domains that mobilise receptors and adaptor molecules required for appropriate immune cell activation and responses. Altered membrane domain formation in immune cells, particularly lymphocytes, has been implicated in the development of autoimmune disease. The mechanisms, however, that modulate lipid composition in immune cell membrane domains are poorly understood. It was therefore hypothesised that disruption of Serinc1 expression would affect lymphocyte function and the development of autoimmunity. Two different Serinc1 mutant mouse strains were used to further investigate this gene: a transposon mutation on the NOD genetic background (NOD-Serinc1sb/sb) and a gene-trap insertion mutation on the B6;129S5 genetic background (B6;129S5-Serinc1gt/gt). The aims of this part of the thesis were to determine if altered Serinc1 expression in these mouse strains affected: 1) lymphocyte activation, 2) lymphocyte membrane lipid composition and 3) susceptibility to autoimmune disease. Our studies confirmed that Serinc1 is expressed in lymphocytes and that its expression is either reduced (NOD-Serinc1sb/sb) or absent (B6;129S5-Serinc1gt/gt) in the two mutant mouse strains. Disruption of Serinc1 expression in both strains, however, did not result in a robust difference for lymphocyte development and proliferation, or the incidence of autoimmune disease compared to wildtype mice. Furthermore, lymphocyte lipid membrane composition was not affected by altered Serinc1 expression. These results suggest that Serinc1 is dispensable in lymphocytes, as well as in the development of autoimmunity. Apics was previously identified as a putative long noncoding RNA (lncRNA) for which sequence variation is associated with the development of T1D in the NOD mouse strain. Preliminary studies indicated that C57BL/6 (B6) mice, which do not develop T1D, exhibit greater expression of this gene than NOD mice. Moreover, Apics was induced by toll-like receptor (TLR) activation in immune cells. TLRs serve as early molecular sensors of invading pathogens and tissue injury, as well as linking innate and adaptive immune responses. Accumulating evidence suggests aberrant TLR signaling in immune cells contributes to autoimmunity. It was therefore hypothesised that genetic variation for Apics alters TLR-mediated immune responses that affect the development of autoimmune disease. To further investigate this gene, an Apics-deficient B6 mouse strain (B6-Apics-/-) was established. The aims of this part of the thesis were to characterise the role of Apics in TLRmediated immune responses and determine how it affects systemic inflammation and autoimmune disease. Notably, B6-Apics-/- mice showed increased susceptibility to induction of autoimmune disease in three different models. Apics was detected in the nucleus and cytoplasm of dendritic cells (DCs) after induction by TLR activation, and binds to SHIP1, an inositol phosphatase that can regulate DC function. Upon TLR activation, Apics-deficient DCs exhibited enhanced TLR-mediated cytokine production compared to wildtype. Moreover, Apics-deficient mice when injected with TLR ligands also have increased serum cytokine concentrations. These results indicate that lncRNAs, such as Apics, can serve as TLR-inducible repressors that attenuate the magnitude of immune responses to reduce the risk of developing autoimmune disease.