Vascular and cellular dynamics in pancreatic islets during type 1 diabetes – BETA-DYN

Type 1 diabetes (T1D) is an autoimmune disease usually diagnosed at young age and characterized by the destruction of pancreatic beta-cells, the only cells capable of producing insulin. Although treatments may normalize blood glycemia, diabetes complications can still occur, especially because of the early age onset. Therefore, T1D therapies aiming at stopping the disease at early stage or delaying diabetes onset are very much needed. Early steps of pathology development involve vascular modifications in the islets, followed by infiltration of immune cells inducing beta-cell destruction. Islet failure therefore implicates two major players: the islet vessels, no longer providing a supportive niche for beta-cell, and the auto-reactive lymphocytes, providing the fatal blow. However, mechanisms involved in the modification of vascular properties, molecules involved in the vessel-lymphocyte cross-talk, and dynamics leading to beta-cell killing are still largely unexplored. The objective of this project is to investigate the role of the islet vascular network in beta-cell function and T1D, and mechanisms involved in beta-cell destruction in single islets and in vivo.
Our first main aim is to determine how modifications of the islet micro-vasculature in the course of diabetes impact endocrine pancreas function. Blood flow dynamics may influence intercellular communication and hormone secretion. However, the role of blood flow dynamics in beta-cell function has been little investigated. Furthermore, it is unclear whether hemodynamic modifications in islets during T1D onset coincide with beta-cell function defect or immune cell attack. Three parameters will therefore be simultaneously analyzed: immune cells infiltration, blood flow velocity, and beta-cells metabolic activity. This will allow us to determine the precise sequence and interdependence of these 3 parameters during T1D development.
Our second main aim is to investigate by transcriptome analysis early molecular factors differentially expressed by vascular cells in normal or pre-diabetic islets that may exacerbate disease progression. The exact role of islet endothelial cells in immune cells recruitment still needs to be clarified. Identification of factors specifically expressed by islets vascular cells that may be involved in pro-inflammatory responses and immune cells entry may help understanding the role of endothelial cells in beta-cell death, and unveiling new therapeutic targets.
Our third main aim is to investigate the dynamics of immune cells infiltration and beta-cell destruction in vivo. Immune destruction of beta-cells involves both CD8+ and CD4+ lymphocytes. The dynamics of beta-cells destruction and interactions involved in vivo have however not been characterized and are essential to understand the mechanisms involved in beta-cell killing by immune cells. Here, we will investigate the dynamics of efficient beta-cell killing by either CD8+ or CD4+ T cells, and cooperation between these cells, by directly visualizing the interactions between individual CD4+/CD8+ T cells and beta-cells.
Given the complexity of genetic and environmental components involved in T1D development, we will use both transgenic mice models providing rapid and synchronous diabetes, and the non-obese diabetic (NOD) mouse model that in many aspects recapitulates the human disease. To assess vascular and cellular dynamics in vivo at the level of single islets in real time, we will use novel 2-photon microscopy and surgery methods pioneered at the host laboratory. Our prototype system, consisting of a 2-photon microscope adapted with long working distance objectives, provides the unique advantage of allowing access to metabolic sensing micropipettes while imaging. This approach will provide the opportunity to study mechanisms involved in islet vessels contribution to disease establishment and beta-cell destruction that will be valuable for the development of new intervention strategies.