Nasal administration of CTB-insulin induces active tolerance against autoimmune diabetes in non-obese diabetic (NOD) mice

Nasal administration of beta cell-derived auto-antigens has been reported to suppress the development of autoimmune diabetes. We investigated the tolerogenic effects of insulin conjugated to the B subunit of cholera toxin (CTB). Nasal administration of 1 mu g of CTB-insulin significantly delayed the incidence of diabetes in comparison to CTB treated mice. However, administration of 4 or 8 mu g of the conjugate had no protective effect. Protection induced by CTB-insulin was transferred to naive recipients by splenic CD4(+) T cells. This result favours an active cellular mechanism of regulation, which was lost using higher (4-8 mu g) or lower (0.1-0.5 mu g) amounts of the conjugate. When co-administered with diabetogenic T cells, splenic T cells from CTB-insulin-treated mice reduced the lymphocytic infiltration of the islets. Reverse transcription-polymerase chain reaction analysis of recipients' pancreatic glands revealed an increase of TGF-beta and IL-10 transcripts after donor mice tolerization, while levels of IFN-gamma and IL-4 RNAs were unchanged. We observed a significant increase of T cell proliferation after unspecific stimulation in the spleen and pancreatic lymph nodes 24 h after CTB-insulin administration in -comparison to control treatment. Higher amounts of IL-4 and IFN-gamma were noticed in pancreatic lymph nodes of tolerized mice upon in vitro stimulation. Antigen-specific unresponsiveness after immunization and upon subsequent in vitro exposure to homologous antigen was obtained in nasally treated animals. Our results underlined the importance of nasal mucosa as an inducing site of tolerance and provided evidence for similar mechanisms of action to what has been described for the oral route, which favoured a CTB-insulin specific effect.