Introduction: Intrahepatic islet transplantation represents an effective therapeutic strategy to restore physiological insulin delivery in patients with Type 1 Diabetes. However, transplantation currently requires islets isolated from multiple donors, and results in an estimated 70% loss of transplanted β-cell mass acutely post-transplant. Necroptosis, a programmed and regulated form of necrotic cell death, occurs following cell damage or inflammation. The signaling cascade requires the involvement of receptor interacting protein kinase 1 and 3 (RIPK1 and RIPK3, respectively), which form the necrosome. Previous research has examined the function of necroptosis in other organ systems such as renal ischemia/reperfusion injury, however, the role of necroptosis in islets remains unexplored. Herein, we hypothesize that inhibition of RIPK1 and RIPK3 in islets, and subsequently, inhibition of the necrosome formation, will prevent necroptosis from occurring following transplant.

Methods: Necrostatin-1s is an inhibitor of RIPK1 and necrostatin-1 is an inhibitor of both RIPK1 and RIPK3. Human islets and MIN6 cells were co-cultured in ± necrostatin-1s and ± necrostatin-1 for 24 and 48 hours at 37°C and 5% CO2, to determine their safety, efficacy, and optimal dose. Cells and islets were also co-cultured in ± Z-VAD-FMK, a pan-caspase inhibitor, to shunt cell death events away from apoptosis and towards necroptosis. In-vitro islet function was assessed by oxygen consumption rate, glucose stimulated insulin secretion, and cell membrane integrity. In-vivo islet function was assessed through human islet marginal mass transplantation (500 islet equivalents) under the kidney capsule of diabetic immunodeficient Rag-/- mice. Human islets were cultured for 48 hours in 100μM necrostatin-1s or necrostatin-1 ± Z-VAD-FMK prior to transplant. Graft function was assessed via measurement of non-fasting blood glucose, and intraperitoneal glucose tolerance testing (IPGTT).

Results: When paired with a pan-caspase inhibitor, at 48 hours, cell membrane integrity of cells treated with necrostatin-1 is significantly increased, as compared with controls (p<0.001). Furthermore, post-transplant non-fasting blood glucose means and blood glucose area under the curve (AUC) calculated following IPGTT are lowest in mice transplanted with subtherapeutic doses of human islets treated with necrostatin-1 + ZVAD (p<0.05), and ZVAD alone (p<0.01), and are highest in mice transplanted with subtherapeutic doses of untreated human islets.  

Conclusions: The present results indicate the therapeutic potential of administration of combination therapy of an apoptosis and necroptosis inhibition in improving islet survival. Inhibition of necroptosis and apoptosis in conjunction may improve human marginal islet engraftment, leading to an increased rate of cell survival. Improvement of transplant outcomes could potentially lead to improved rates of single donor islet transplant success rates and increase graft durability. Furthermore, pre-treatment of islets alone, as opposed to systemic delivery of inhibitors, provides a safer translational pathway to clinical therapeutic use.