Aim of Study: The development of steroid-free protocol by the Edmonton group revived islet transplant as a potential therapy to cure type-1 Diabetes. However, long-term dysfunction in islet transplantation is a major challenge that needs to be addressed. Recent immunosuppression protocols are toxic to the beta cells resulting in chronic graft failure. There is an unmet need for a more suitable immunosuppressive regimen that maintains long term beta cell function. Withaferin A (WA) is a plant-derived molecule which has been used as an anti-inflammatory molecule because of its ability to block Nuclear Factor Kappa B (NFκB) pathway. Since, immune cell activation is regulated by NFAT, AP-1 and NFκB, we hypothesized that WA can be used to suppress this activation. The purpose of this study to demonstrate the immunosuppressive ability of WA in vitro.
Methods: Human and mouse islets were cultured with or without WA at various concentrations and viability was measured to demonstrate safety. Splenocytes were extracted from C57BL/6 mice and stained with 10uM CFSE for 10 mins. Stained cells were stimulated with equal numbers of CD3/CD28 beads in the presence or absence of 1ug/mL WA and cultured for 5 days. T cell proliferation and surface marker analysis for CD4, CD8 and CD25 was determined using flow cytometric analysis. Experiments were also repeated using mouse T cells and human T cells purified from PBMCs.
Results: There was no significant change in the viability of islets up to a concentration of 1ug/ml WA. Flow cytometric analysis revealed that the proliferation of T cells was significantly inhibited by WA in mouse splenocytes, purified T cells and human T cells [Figure 1]. Further analysis of markers revealed significant reduction in CD4 and CD8 T cell numbers but the percentage was unaffected.
Conclusion: A low dose of WA showed no toxic effect on Islets. T cell activation was significantly inhibited by WA. The use of WA with no beta cell toxicity has the potential to become a favorable immunosuppressive agent for islet transplantation.