Investigating type 1 diabetes pathogenesis using the live pancreas tissue slice platform

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Type 1 diabetes (T1D) is an autoimmune disease that results in the destruction of the insulin-producing β cells of the pancreas. The timeline of T1D development and progression is heterogenous with diagnosis occuring at a few years of age in some patients versus other patients having autoantibodies for years before diagnosis. In T1D, immune dysregulation occurs leading to autoantibody production, immune cell infiltration into the islets (insulitis), and eventual loss of β cells. The events that occur within the β cell during this time and the resulting impacts on β cell function remain unknown. The goal of this proposal is to determine how β cells function during the course of T1D development, particularly during the critical early, asymptomatic stages. Understanding these functional changes within β cells or islets is critical for a complete understanding of disease progression and for the identification of possible therapeutic targets. The overall objective of my proposal is to use live pancreas tissue slices to explore how β cells function during T1D development, specifically I will identify the role immune cell infiltration plays in β cell dysfunction and loss. Live pancreas tissue slices are ideal for these studies because these samples keep the islet in its native microenvironment and preserve the extant tissue pathologies, allowing for studies of both β cell function and immune cell populations. I hypothesize that immune dysregulation early in T1D progression results in dysfunction of the β cell glucose metabolism pathway primarily mediated by mitochondrial stress, resulting in MHC class I hyperexpression and increased β cell visibility to the immune system. To test this hypothesis, live pancreas tissue slices generated from human organ donor tissue will be used and complemented by slices made from pancreata of mouse models of T1D. Confocal microscopy techniques will be used to determine the degree of insulitis within the tissue while islet function is simultaneously assessed. Additional functional assessments such as perifusion experiments followed by insulin ELISAs and gene expression will be employed to look at function in greater detail. The first part of this proposal focuses on assessments conducted to determine the impacts of in situ immune cells on β cell function. To gain more control over the disease timeline, particularly to investigate the earliest stages of disease, HLA-matched T cell avatars will be introduced to control human pancreas tissue slices, creating an insulitic environment. Disease initiation and progression will be studied further through adoptive transfer experiments with splenocytes from NOD mouse models. Progression to T1D will be monitored with live pancreas tissue slices being made as disease develops. Functionality assessments and immune cell studies will be conducted as discussed above. I expect the contribution of the proposed research will be the identification of the role that immune dysregulation plays in β cell dysfunction as well as the mechanistic origination of β cell dysfunction. This will provide critical information about how T1D develops and will indicate possible therapeutic targets to halt disease progression.