Expansion microscopy of cytokine-induced alterations in pancreatic cells and tissues

Type 1 Diabetes (T1D) is a chronic disease characterized by inflammation and consequent dysfunction of pancreatic β-cells. Despite the efforts, the molecular determinants underlying β-cell failure are not fully clarified, hindering the efforts to prevent and/or cure the disease. Thus far, β- cell failure has been investigated using a combination of genomic, transcriptomic, proteomic, and biochemical approaches. Based on such “omics” data it appears clear that proinflammatory cytokines (including IL-1β, TNF-α, and IFN-γ) play a key role in β-cell failure in T1D, inducing alterations at the molecular level (e.g. by upregulation of selected genes and/or proteins), which then, in turn, promote β-cell failure, mainly by apoptosis. Yet, a direct observation of the subcellular signatures of cytokine-induced β-cell damage is lacking.This thesis addresses that gap by exploiting a combination of advanced optical-microscopy tools and fluorescently labeled molecular targets in rat Insulinoma 1E (INS-1E) β-cells exposed to IL-1β and IFN-γ cytokines. In detail, expansion microscopy (ExM) was used to achieve the spatial resolution (~50 nm) needed to analyze the structural features and of key subcellular targets; time-lapse live-cell microscopy, on the other hand, provided complementary information on key dynamic and metabolic subcellular parameters.It was found that 24-hours exposure to proinflammatory cytokines induces a neat decrease in the number of ISGs and alteration in the dynamics of the residual pool, marked depolymerization of microtubules, change in mitochondrial morphology (increased circularity) and metabolic activity (priming towards oxidative phosphorylation), decreased cell responsiveness to glucose stimulation and fragmentation of Golgi apparatus and Endoplasmic reticulum.  This is accompanied by the observation of clear signatures of the production of reactive oxygen species (ROS) under cytokine exposure. Overall, reported results provide direct evidence that proinflammatory cytokines act as potent stimulators of insulin secretion and, concomitantly, as cell stressors.The study further extends these findings to human pancreatic tissues, documenting diabetes-related structural changes in critical organelles such as insulin granules and mitochondria through the use of Magnify, an innovative variant of ExM, applied to FFPE samples.These findings underscore the crucial role of these organelles in β-cell dysfunction and open the door for future research into the molecular mechanisms driving this process, as well as the development of targeted therapies aimed at restoring insulin production in diabetes.