Purpose: Pancreatic islets undergo enormous stress during isolation and peri-transplant periods which causes a significant release of stressomes (exosomes released during stress). Uncovering the biological role of such vesicles is vital for long-term graft function and survival. Here, we sought to identify the global protein content and significance of stressomes isolated during isolation in autologous islet transplantation (AIT).  

Methods: Post-transplant stress was mimicked in vitro by subjecting human islets to hypoxia and cytokines (Cyt+Hyp). Global protein contents within the stressomes were characterized by mass spectrometry and validated by immunoblotting. In this study, we sought to find its function and correlation with post-transplant outcomes. Recipients receiving an islet dose of >4000IEQ/kg were divided into insulin-dependent and insulin-free groups. The stressomes were isolated from the final islet product (infusion bags) used for transplantation (n=20). The immunogenic proteins (HSPA1B, H2B) in the stressomes were quantified using ELISA and data were correlated with 1-year TPIAT outcomes.  Finally, to evaluate the functional significance of the islet stressomes, they were used to stimulate allogeneic high-purity human islets and macrophages and to assess their uptake and immunological response.

Results: The mass spectrometry analysis has revealed the islet stressomes contain Damage Associated Molecular Patterns (DAMPs) like histones (H2B, H3, and H4) and heat shock proteins (HSPs). The ER stress signal seems to play a crucial role in the biogenesis of islet-derived stressomes induced by stimulation with Cytokines and Hypoxia. Stressomes from the final prep samples showed elevated chaperone levels (HSPA1B, p=0.042) and DAMP (H2B, p=0.023) in the insulin-dependent group. Moreover, high levels of HSPA1B (r=0.4185, n=18, p=0.083) and H2B (r=0.6184, n=18, p=0.010*) in the infusion bags were positively correlated with the recipient's insulin requirements at 1-yr post-transplant. Treatment of THP-1 human macrophage cell line with islet stressomes caused immediate uptake of islet exosomes as shown by fluorescent confocal microscopy analysis. Exosomes from islets exposed to stress (CYT+HYP) caused an inflammatory phenotype in THP-1 cells compared to control islet exosomes. Further, the THP-1 cells stimulated with stressomes from islet bag samples showed a strong inflammatory phenotype (CD11b+CD68+CD80+) and an increased expression for innate immune receptors (TLR3 and TLR7). The stressome-treated islets demonstrated elevated expression of innate immune receptors like TLR2 and AGTR1.

Conclusion: The study has evaluated the global protein content of islet stressomes and assessed the functional impact on inflammatory cells that may reflect poor long-term AIT graft outcomes.