Roles of Toll-like Receptors on Islets in Xenogenic Islet Transplantation

Abstract

Background: Stimulation of innate immunity through toll-like receptors (TLRs) interfered with tolerance induction in allogeneic transplantation. Recently, roles of TLRs have been reported in parenchymal cells such as renal tubular epithelial cells beyond antigen presenting cells. Here, we tried to elucidate the roles of TLRs on porcine islets in xenogeneic islet transplantation. Methods: After isolation, adult porcine islets were stimulated by poly I:C, lipopolysaccharide (LPS) and CpG. Induction of cytokines and chemokines in islets in response to TLR stimulation was assessed using RT-PCR and ELISA. Transmigration assays were performed in order to measure the chemotactic activity of islet culture supernatant. Impacts of TLR stimulation on both apoptosis and insulin secretion of islets were assessed using Annexin-V/7-AAD staining and glucose-induced insulin stimulation test, respectively. Procoagulant induction was also assessed using RT-PCR and tissue factor assay. Porcine islets were transplanted to renal subcapsular space of MyD88 knockout, TLR4 knockout and wild type C57BL6/J mice. TLR agonists (LPS, Poly I:C) were injected together with anti-CD154 antibodies (MR-1) after pig to mouse islet transplantation. Results: Porcine islets expressed TLRs at resting status. In vitro TLR stimulation upregulated expression of both cytokines (IL-6, type I interferon) and chemokines (MCP-1, RANTES, IP-10, IL-8). Islet culture supernatant after stimulation by TLR agonists induced transmigration of THP-1 and human peripheral blood mononuclear cells across the species barrier. However, TLR stimulation influenced neither apoptosis nor insulin secretion. Both expression and functional activity of tissue factor in islets were increased by TLR stimulation. Poly I:C treatment decreased graft survival in pig to wild type mouse islet transplantation. Moreover, LPS interfered with long-term graft survival induced by MR-1 in pig to TLR4 knockout or MyD88 knockout mouse islet transplantation. Conclusion: Stimulation of TLRs on porcine islets induced both proinflammatory and procoagulant responses and thereby interfered with long-term graft survival in xenogeneic islet transplantation.