The presence of diabetes autoantigens in microparticles derived from human islets
Craig Hasilo1,2, Sarita Negi1,2, Marco Gasparrini1,2, Nathalie Cloutier3, Eric Boillard3, Eric Bonneil4, Pierre Thibault4, Steven Paraskevas1,2.
1Department of Surgery, McGill University, Montréal, QC, Canada; 2Human Islet Transplantation Laboratory, McGill University Health Centre, Montréal, QC, Canada; 3Centre de Recherche en Rhumatologie et Immunologie, Centre de Recherche du Centre Hospitalier Universitaire de Québec, Québec, QC, Canada; 4Institut de Recherche en Immunologie et en Cancérologie, Université de Montréal, Montréal, QC, Canada
Background: The signaling and injury pathways associated with onset of autoimmune diabetes are poorly understood. Microparticles (MP) are nano-sized vesicles produced by most mammalian cells under normal and stressed physiological states. Neo-antigens may be contained within MP that are not normally exposed, except in times of injury, and may act as accelerators of autoimmune stimulation. It is not known whether human βcells release MP following stress, and whether these MP could present βcell antigens to the immune system. Here we report the production of MP by human islets, and the characterization of βcell-specific markers of islet injury within the MP isolated from islet conditioned media (ICM) using a microFACS and proteomic-based approach.
Methods: Human ICM was collected from enriched human islet preparations (n=5), purified from multi-organ donor pancreases. Islets were cultured up to 72hrs at 37°C with 5% CO2 for analysis of MP production. MicroFACS analyses were performed using a FACS Canto II with small particle analyzer. Samples of ICM (n=5) were labeled with the following antibodies: diabetes autoantigens anti-GAD65-PE, anti-PTPRN-APC, and anti-ZnT8-FITC; βcell markers anti-Glut2-PE and anti-HSC70-APC; membrane vesicle or exosome markers AnnexinV-V450, AnnexinV-FITC, anti-CD9-BV421 and anti-CD14-PE. For proteomic analyses by mass spectrometry, MP fractions were isolated from ICM (n=5) through sequential centrifugation at 50 000g and 200 000g for microvesicles (MV) and exosomes (EX), respectively, then separated by SDS-PAGE. Tryptic digests were analyzed by LS-MS/MS on an LTQ-Orbitrap Elite, and compared to that of the islet cell lysate (ICL). Statistical analyses were performed on the comparison of means by one-way ANOVA with Bonferroni’s post-hoc test (p<0.05).
Results: AnnexinV+ (AnnV+) MP were produced by human islets, with highest levels at 24hrs and with a greatest mean density of 100-840 nm-sized particles. Diabetes autoantigens, GAD65 and ZnT8, and βcell-specific glucose transporter, Glut2, were detected in ICM MP. We identified 174, 102, and 1642 proteins in 50,000 g, 200,000 g fractions and ICL, respectively. Enrichment analysis showed that the majority of the proteins in the MV and EX fractions displayed an exosome lineage, and were associated with diabetes pathways and the metabolism of lipids and lipoproteins.
Discussion: Cultured human islets produced high levels of AnnV+ MP. Diabetes autoantigens, GAD65 and ZnT8, were identified in ICM, although they are normally contained within the ß-cell and only externalized following injury. Characterization of MP revealed biomarkers in common with islet cells, indicating that they may be strong targets for early diagnostic markers and interventional strategies in diabetes and ß-cell injury following islet transplantation.
Canadian National Transplant Research Program. Transplant Québec.
15:30 - 17:00
|Getting Beta Cells to Survive: Beta Cell Survival and Injury Mechanisms||The presence of diabetes autoantigens in microparticles derived from human islets||Plenary Room 1|