Correlates of circulating extracellular vesicle cargo with key clinical features of type 1 diabetes

Type 1 diabetes (T1D) is a heterogeneous disease with a slower evolution in individuals diagnosed at older ages. There are no validated clinical or laboratory biomarkers to predict the rate of insulin secretion decline either before or after the clinical onset of the disease, or the rate of progression to chronic complications of the disease. This pilot study aimed to characterize the proteomic and phosphoproteomic landscape of circulating extracellular vesicles (EVs) across a range of obesity in carefully matched established T1D and control subjects. We used archived serum samples from 17 human subjects (N=10 with T1D and N=7 normal healthy volunteers) from the ACME study (NCT03379792). EVs were isolated using EVtrap(R) technology (Tymora). Mass spectrometry-based methods were used to detect the global circulating EV proteome and phosphoproteome. Differential expression, coexpression network (WGCNA), and pathway enrichment analyses were implemented. The detected proteins and phosphoproteins were highly enriched (75%) in exosomal proteins cataloged in the ExoCarta database. A total of 181 differentially expressed EV proteins and 15 differentially expressed EV phosphoproteins were identified, with 8 upregulated EV proteins (i.e., CD63, RAB14, VCP, BSG, FLNA, GNAI2, LAMP2, and EZR) and 1 downregulated EV phosphoprotein (i.e., TUBA1B) listed among the top 100 ExoCarta proteins. This suggests that T1D could indeed modulate EV biogenesis and secretion. Enrichment analyses of both differentially expressed EV proteins and EV phosphoproteins identified associations of upregulated features with neutrophil, platelet, and immune response functions, as well as prion disease and other neurodegenerative diseases, among others. On the other hand, downregulated EV proteins were involved in MHC class II signaling and the regulation of monocyte differentiation. Potential novel key roles in T1D for C1q, plasminogen, IL6ST, CD40, HLA-DQB1, and phosphorylated S100A9, are highlighted. Remarkably, WGCNA uncovered two protein modules significantly associated with pancreas size, which may be implicated in the pathogenesis of T1D. Similarly, these modules showed significant enrichment for membrane compartments, processes associated with inflammation and the immune response, and regulation of viral processes, among others. This study demonstrates the potential of EV proteomic and phosphoproteomic signatures to provide insight into the pathobiology of type 1 diabetes and its complications.