Introduction: Islets undergo significant stress and damage during isolation and in the peritransplant period which results in the release of immunogenic factors called damage-associated molecular patterns (DAMPs) that can induce immune cell activation causing post-transplant islet damage. Exosomes are released by islets in response to stress and but the functional impact of these exosomes is still not clearly understood. Here, we sought to identify the DAMP proteins released by islets via exosomes in response to islet isolation stress and also study their impact on TPIAT outcomes.

Methods: Islet stress during isolation and peritransplant period was mimicked in vitro by exposing human islets to hypoxia and cytokines (Cyt+Hyp) simultaneously for 24h. Separately, human islets were also treated with thapsigargin for 24hrs to induce ER stress. Exosomes were isolated after stress treatment and the differentially expressed proteins were determined by mass spectrometry and western blotting. To validate the in vitro findings, exosomes were isolated from the transplant media of TPIAT patients before the infusion to measure DAMP protein levels in them. Patients receiving an islet dose of 4000IEQ/kg were divided into insulin-dependent (ID; n=10) and insulin-independent (IID; n=10) groups. The exosomal DAMP protein levels were correlated with 1-year TPIAT outcomes.

Results: The number of exosomes released was significantly elevated in (Cyt+Hyp) treated islets compared to untreated islets. The mass spectrometry analysis has revealed about 9 proteins including DAMP protein histones (H2A, H2B, H4) and Keratins (Type I and II) were differentially expressed. Islets treated with thapsigargin or Cyt+Hyp overexpressed histones and keratins intracellularly through the activation of the PERK/IRE1α pathway. This subsequently led to the increased release of histones and keratins via exosomes during stress. Exosomes isolated from transplant media of TPIAT patients showed elevated histone (p=0.0796) and decreased keratin (p=0.072) in ID compared to IID patients. Moreover, high exosomal histone/IEQ resulted in increased insulin requirement (r=0.527, p=0.029 n=17) and HBA1c%, and lower c-peptide (r=-0.494, p=0.085 n=13) and SUITO index at 1-year POD. In contrast, the exosomal keratin/IEQ showed the opposite effect on 1-year outcomes.

Conclusion: The study has identified the DAMP protein-like histones are produced by the stressed islets are released via exosomes and these have been shown to affect the graft outcomes in TPIAT patients. Future directions will focus on the mechanism by which exosomal DAMP proteins affect graft outcomes and strategies to reduce DAMP secretion via exosomes.