Objective
There is a critical gap in knowledge of our understanding regarding the role of the b cell in the development of autoimmunity and immune cell activation during the development of type 1 diabetes. To address these gaps in knowledge, we will pursue the following objectives: (1) Determine how activation of the integrated stress response in human islets during early T1D leads to dysregulated mRNA translation and production of defective and novel proteins that the immune system may interpret as foreign; (2) Define whether pharmacological modulation of the ISR is sufficient to prevent T1D development, and (3) Identify RNA and protein species that are derived from dysregulated mRNA translation and packaged as cargo within extracellular vehicles (EVs), which we will detect in culture media from human islets and plasma samples of mice and human individuals with T1D.
Background Rationale
The pathogenesis of type 1 diabetes (T1D) encompasses a spectrum that ranges from predominantly aggressive autoimmunity against islet β cells to genetic/acquired defects inherent to β-cell function and survival. Accumulating evidence from recent studies suggests the nature and tempo of this dialogue varies by disease stage with distinct cytokine (protein mediators) signatures and pathophysiologic mechanisms predominating in early disease, which are distinct from those responsible for terminal β cell decline in late-stage disease. At present, the precise molecular mechanisms leading to T1D are not understood fully; however, clinical and pre-clinical studies implicate interferon alpha (IFN-α) mediated signaling as an integral part of early T1D pathogenesis and suggest that the pro-inflammatory cytokines IFNg) + IL-1b might be involved in later disease stages. Previous and preliminary studies indicate that both IFN-α and IFNg + IL-1b activate downstream signaling pathways within the β cell that create a feed-forward cycle of inflammation and endoplasmic reticulum (ER) stress. Moreover, activation of chronic ER stress leads to the induction of the integrated stress response (ISR), which is a cytoprotective mechanism that maintains cellular protein homeostasis in response to environmental and cellular stress signals transduced intracellularly to activate a host of eIF2α kinases. Phosphorylation of eIF2α causes inhibition of mRNA translation and reduction in global protein synthesis. Data from our preliminary studies suggest that ISR activation in islets during early T1D leads to dysregulated mRNA translation, which could be a major determinant of whether aberrant protein isoforms that trigger immune targeting of β cells are produced. To shed further light upon the gap in knowledge surrounding the pathogenesis of T1D, this research proposal will dissect how the integrated stress response can be harnessed to prevent the development of T1D.
Description of Project
Type 1 diabetes (T1D) is an autoimmune disease that causes progressive destruction of insulin
producing cells (β-cells) of the pancreas, leading to insulin deficiency and chronic hyperglycemia. Accumulating evidence suggests the nature and tempo of this dialogue varies by disease stage with distinct cytokine (protein mediators) signatures and pathophysiologic mechanisms predominating in early disease, which are distinct from those responsible for terminal β cell decline in later-stage disease. At present, the precise molecular mechanisms leading to T1D are not understood well; however, clinical and pre-clinical studies implicate interferon-alpha (IFN-α), a type I interferon (cytokine) as an integral part of early T1D pathogenesis and suggest that the pro-inflammatory cytokines (IFNg + IL-1b) might be involved the early insulitis or later stage of the disease. Published and preliminary studies indicate that both IFN-α and IFNg + IL-1b activate downstream signaling pathways within the β cell that create a feed-forward cycle of inflammation and dysfunction within a key cellular biosynthetic compartment the endoplasmic reticulum (ER). Moreover, activation of chronic ER stress leads to the induction of a complex signaling program called the integrated stress response (ISR). This is a cytoprotective mechanism that maintains cellular protein homeostasis in response to environmental cues, and it includes signal transduction to activate kinase activity upon the translation initiation factor, eIF2α. Phosphorylation of eIF2α on residue Ser51 causes inhibition of mRNA translation and reduction in global protein synthesis. Data from our preliminary studies suggest that there is an activation of the ISR in human islets during early T1D that leads to dysregulated mRNA translation. We hypothesize this may lead to the production of aberrant (defective) protein isoforms that trigger immune cell targeting of β cells. Interestingly, in murine T1D models (NOD and RIP-LCMV-GP), pharmacological modulation (TYK2 inhibition and ISRIB) of these pathways resulted in the prevention of T1D. Thus, we hypothesize that activation of ISR-mediated dysregulation of mRNA translation is an early b cell stress pathway in T1D that determines cell survival and that it can be monitored in pre-and early T1D individuals to detect disease initiation. We aim to test this hypothesis by, 1) determine the role of inflammation-induced integrated stress response in the modulation of global RNA translation and generation of defective protein isoforms in human islets; 2) To evaluate how pharmacological reconstitution of mRNA translation using small molecular inhibitors of TYK2 and ISR (ISRIB) could prevent T1D development, and (3) identifying RNA and protein signatures in islet-derived extracellular vesicles that are generated as a result of defective translation as a putative biomarker of β cell stress and T1D risk. Together, the work described in this proposal will significantly advance our gap in knowledge and shed a new perspective on how β cells “invite their own demise” during early T1D pathogenesis. Results from these studies represent a multidisciplinary “bench to bedside” approach with a goal to develop (1) risk-stratification/disease prediction, (2) tailor therapeutic interventions, and (3) monitor therapeutic responses.
Anticipated Outcome
We anticipate the following outcomes: (1) Cytokine stressed human islets studies would provide critical insights on how integrated stress response (ISR)-mediated deranged mRNA translation might amplify β cell autoimmunity during early pathogenesis of T1D; (2) Pre-clinical studies using mouse models of T1D mouse models using specific inhibitors of TYK2 and the ISR (ISRIB) will allow us to develop stage (early vs. late) specific therapeutic interventions, and (3) The identification of defective RNA and protein species in extracellular vesicles that arise as a result of islet cell stress and ISR activation will allow us to (a) perform risk-stratification/disease prediction; (b) tailor therapeutic interventions, and (c) monitor therapeutic responses.
Relevance to T1D
Type 1 diabetes (T1D) develops in the setting of a complex dialogue that is established between cytokine producing immune cells that invade the islet and putative immunogenic signals released by injured or dying β cells. Accumulating evidence suggests the nature and tempo of this dialogue varies by disease stage with distinct cytokine signatures and pathophysiologic mechanisms predominating in early disease, which are distinct from those responsible for terminal β cell decline in later stage disease. A perspective that has been gaining traction in recent years recognizes the involvement of β cells in triggering autoimmunity. However, the exact mechanism whereby β cells accelerate immune activation is still not well understood. To address this knowledge gap and identify β cell specific therapeutic targets, we aim in the present proposal to interrogate the role of integrated stress response in human islets during early T1D. Our preliminary studies have documented activation of the ISR in human islets, leading to dysregulated mRNA translation that may produce aberrant (defective) protein isoforms as a trigger for the immune cells to target β cells during early T1D. Pharmacological modulation of this pathway using specific pharmacological inhibitors of IFNa signaling (TYK2 inhibitor) and eIF2α phosphorylation (ISRIB) will be tested as a novel means to prevent the development of T1D.