Aims/hypothesis <p>Clinically actionable biomarkers that accurately reflect the health status of the beta cell are needed to improve risk stratification and optimise the timing of interventions in type 1 diabetes. We hypothesised that inflammatory stress elicits a reproducible microRNA (miRNA) program in human islets and islet-derived extracellular vesicles (EVs) that can be detected in plasma EVs to stratify diabetes risk, while also providing insight into molecular pathways linked to beta cell dysfunction.</p> Methods <p>Human islets were exposed to IL-1β+IFN-γ, and small RNA-seq was performed on islets and islet-derived EVs. Differentially expressed miRNAs were validated in islets, using RT-PCR, in plasma-derived EVs from individuals with autoantibody positivity (AAb⁺) or recent-onset type 1 diabetes and matched control individuals using ultrasensitive, label-free localised surface plasmon resonance (LSPR) biosensors, and in pancreatic sections from organ donors using in situ hybridisation and spatial feature analysis. Finally, beta cell-targeted in vivo inhibition of miR-155 was tested in the NOD mouse model.</p> Results <p>Inflammatory cytokine exposure altered a restricted subset of miRNAs, identifying 20 differentially expressed miRNAs in islets and 14 in islet-derived EVs. Only two miRNAs, miR-155-5p and miR-146a-5p, were concordantly upregulated in both compartments. Machine learning prioritised an EV miRNA panel for translational validation, and custom LSPR biosensors enabled quantification of these miRNAs in plasma EVs. This plasma EV miRNA signature, consisting of miR-155-5p, miR-146a-5p, miR-30c-1-3p, miR-802 and miR-124-3p, differentiated individuals with AAb⁺ and those with recent-onset type 1 diabetes from control individuals with good sensitivity and specificity. In pancreatic tissue, miR-155 abundance and beta cell spatial/subcellular distribution were altered in donors with AAb⁺ and type 1 diabetes compared with non-diabetic control individuals. Functionally, beta cell-targeted inhibition of miR-155 improved glucose tolerance and reduced insulitis in prediabetic NOD mice.</p> Conclusions/interpretation <p>Using an organ-based model system of inflammatory stress, we validated a signature of EV-associated miRNAs capable of stratifying type 1 diabetes risk. Furthermore, we provided new mechanistic and imaging insights into miRNA expression patterns in pancreatic sections from human organ donors with type 1 diabetes or AAb⁺, and we used a preclinical model of type 1 diabetes to demonstrate the potential therapeutic efficacy of targeting these miRNAs.</p> Data availability <p>The data from small RNA sequencig of human islets and islet-derived EVs have been deposited in the GEO database (accession no. GSE160391).</p> Graphical Abstract <p></p>