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Interaction between the beta-cell and the immune system in type 1 diabetes: role for post-translational modifications

Book - Dissertation

Diabetes mellitus is one of the most common endocrine diseases in the world and the incidence is still increasing rapidly. Clinical characteristic of diabetes include hyperglycemia and glucosuria, as a result of relative or absolute shortage in insulin. This is the hormone secreted by the beta-cell in the islets of Langerhans upon elevated blood glucose concentrations after a meal. Where the most prevalent type, T2D, is rather considered as a metabolic disease involving insulin resistance and beta-cell dysfunction, T1D, accounting for 5-10% of all diabetic patients, is an autoimmune disease where the body’s own immune system attacks the insulin-secreting pancreatic beta-cells. The mechanisms responsible for loss of tolerance against the beta-cells are not yet completely understood. The immune system is supposed to be of major importance, but more and more evidence point to the beta-cells themselves as active partners in their own destruction. Aspecific inflammation in the islets, indicated as the first trigger for T1D, results in upregulation of HLA-I molecules on the surface and release of chemokines and cytokines. In addition, ER and oxidative stress are induced, resulting in beta-cell dysfunction, apoptosis and the generation of molecular changes like alternative splicing and PTMs. This PhD project, with the general objective to study how the interaction between the beta-cell and immune system participates in the development of T1D, put specifically the importance of PTMs of beta-cell antigens in the spotlight. In the first project (Chapter 3) we investigated if at early age before onset of insulitis, there are differences in islets of Langerhans from NOD mice compared to two healthy mouse strains namely the NOR mice, showing 88% sequence similarity with NOD mice, and the more distant C57Bl/6 mice, that could potentially contribute to an intrinsic higher risk for auto-immune attack or dysfunction. Analysis of the transcriptome and proteome, combined with integrated pathway and network analyses, indicated that islets of NOD mice are subjected to higher levels of stress due to an inefficient supply of energy and by a higher probability that misfolded proteins will accumulate, mainly due to lower expression of PDIA3, PDIA4 and PDIA6, enzymes implicated in S-S bond formation. However, the most remarkable finding in NOD islets at this early pre-insulitic age was the high expression of Padi2 mRNA. Padi2 is one of the five Ca2+ dependent enzymes responsible for the modification of arginine to citrulline, known as citrullination. This modification that highly impacts protein conformation and HLA binding, is known to be implicated in the generation of autoantigens is several autoimmune diseases, most importantly RA, but also in T1D citrullination of GAD65 results in autoantigen generation. In order to examine both aspects of the interaction between the beta-cell and immune system, the aim of the second project (Chapter 4) was to investigate how inflammation affects the beta-cell proteome resulting in an active contribution to T1D development. We build further on a previous finding from our lab that demonstrated PTM of GRP78 in a rat beta-cell line (INS-1E) upon exposure to pro-inflammatory cytokines, a model that is used to mimic T1D ex-vivo. Further, we characterized this PTM as a citrullination on arginine 510 of GRP78. In addition, it was also shown that cytokines induce the translocation of GRP78 from the ER to the plasma-membrane of INS-1E cells and mouse islets, a process mediated by ER stress. While intracellularly we identified 3 isoforms of GRP78, much more modified isoforms of GRP78 were expressed on the cell surface. Finally, citrullination of GRP78 results in loss of immune tolerance in diabetic NOD mice by the presence of autoantibodies and auto-reactive T-cells. The aim of the third project (Chapter 5) was to translate these findings from INS-1E cells and NOD mice to the human situation. This pointed to a similar PTM of GRP78 in human islets exposed to pro-inflammatory cytokines in three out of five healthy donors. Furthermore, proteome analysis by LC-MS/MS indicated that inflammation in general induced citrullination and deamidation, another PTM known to be involved in autoantigen generation. The identification of two citrullinated and deamidated peptides of GRP78 showed evidence for the implication of these PTMs in human islets as well. To investigate the relevance in terms of autoantigen recognition, modified GRP78 epitopes were designed that were predicted to bind to the T1D susceptibility HLA-DR4 molecule and these peptides were further on used in immunological assays. The first indication for the implication of citrullinated GRP78 as autoantigen in human T1D patients came from the presence of reactive CD4+ T-cells in infiltrated T1D islets. Next, higher CD4+ T-cell frequencies recognizing a citrullinated epitope were also present in peripheral blood of T1D patients compared to healthy controls, indicating a possible application as biomarker for T1D. To conclude, this PhD project clearly demonstrated the importance of the interaction that connects beta-cells and immune system in the pathogenesis of T1D. PTMs of beta-cell antigens, especially citrullination, appeared to have substantial importance for the break of tolerance. Furthermore, the ability to detect autoreactive T-cells against citrullinated GRP78 in blood of NOD mice and T1D patients emphasizes its possible use as a biomarker and opens the road to the development of new therapeutic applications.
Number of pages: 222
Publication year:2017
Accessibility:Closed