Roles Of Dendritic Cells For Immune Tolerance Induction In Type 1 Diabetes

Objective: To investigate the preventive effect of autoantigen insulin treatment on type 1 diabetes (T1D) murine model and explore the underlying mechanism of the induction of immune tolerance; The roles of dendritic cell (DC) and CD4⁺CD25⁺ regulatory T cells in murine immune tolerance model induced by autoantigen insulin was studied; To explore the effect and underlying mechanism of prevention of STZ-induced T1D by adoptive transfer of 3 tolerogenic dendritic cells induced with various methods in vivo and in vitro.Methods: (1) Thirty-two of 8-week-old male BALB/c mice were randomly divided into 4 groups: blank control group, model control group, insulin s.c group and insulin p.o group. The T1D model was established by intraperitoneal injection of multiple low dose of streptozotocin (STZ) 40 mg.kg^-1 for 5 consecutive days. The bovine insulin was given subcutaneously with IFA or orally before the STZ injection, respectively. The blood glucose was examined weekly and all mice were killed after 6 weeks. Pancreas tissues were taken for histopathologic analysis by H&E dyeing and isletsβcells apoptosis evaluation by TUNEL method. Splenocytes were harvested to detect lymphocyte proliferation by MTT assay. The levels of cytokines IL-2, IFN-γ, IL-4 and IL-10 in serum were determined by ELISA analysis.(2) DC precursor cells from bone marrow and lymphocytes from spleens in these animals were isolated. The phenotypes of DC and CD4⁺CD25⁺ regulatory T cells were analyzed by FACS. T cell stimulating activity by DC was determined by allo-MLR. The cytokine IL-12 p70 production was analyzed by ELISA.(3) Bone marrow cells from BALB/c mice were used to generate DCs with GM-CSF+IL-4 (DC0). To generate iDC, DC precursor cells were cultured without IL-4 (DC1). To evaluate the effects of Paecilomyces Hepiali Chen Mycelium (PHC) on the differentiation of DC, GM-CSF+IL-4-treated DC were co-cultured with extract of PHC in vitro (DC2). DC which were isolated from autoantigen insulin induced T1D murine immune tolerance model were named as DC3. The phenotypes, IL-12 p70 production and the T-cell stimulatory capability of these DCs were identified. These cells were used to modulate autoimmune response in prediabetic mice. The blood glucose was examined weekly, pancreas tissues were taken for histopathologic analysis and isletsβcells apoptosis evaluation, and spleen lymphocytes were isolated to detect lymphocyte proliferation by MTT assay and the ratio of CD4⁺CD25⁺ T cells by FACS. The cytokines secretion was determined by ELISA analysis.Results: (1) Multiple low dose of STZ in BALB/c mice can develop a stable model of T1D, characterized by the beta cell destruction with inflammatory cells. The level of blood glucose was decreased significantly by giving Insulin subcutaneously (P <0.05) rather than orally. Less lymphocytes infiltration and no obviousβcell apoptosis in the pancreatic island were observed in the insulin s.c group. Insulin given subcutaneously can inhibit T cell proliferation remarkably, compared with the model control group (P<0.05). Moreover, increased level of IL-4 and IL-10, a lesser extent of IL-2 and IFN-γin serum were observed.(2) There are some defects in the differentiation of bone marrow-derived DC in T1D mice, which showed very low level of CD11c, and CD4⁺CD25⁺ T cells were decreased. Treatment with insulin could improve the status of DCs, and the expression of CD11c was much higher than the DCs from the model group. However, expression of CD86 and MHC-II was not increased, which suggested that an immature DC were obtained. These DCs also exhibited impaired proinflammatory cytokine IL-12 p70 production and poor stimulatory capacity, compared with those mature DC from the normal mice. CD4⁺CD25⁺ T cells from spleen were significantly enhanced by autoantigen.(3) DC cultured in the various systems were all positive for the DC marker (CD11c). DC1, DC2 and DC3 all showed an immature phenotype, characterized by decreased expression of CD86 and MHC-II costimulatory molecules, lower level of IL-12 p70 production and poor stimulatory capacity, compared with DC0. The level of blood glucose was decreased significantly by transferring those tolerogenic DC rather than DC0. Less lymphocytes infiltration and no obviousβcell apoptosis in the pancreatic island were observed. In vitro, proliferation of lymphocytes was decreased and the proportion of CD4⁺CD25⁺ T cells was increased remarkably, compared with DC0 transfer group, which were associated with the increased level of Th2 cytokines and a lesser extent of Th1 cytokines.Conclusion: (1) Subcutaneous administration of insulin can induce immune tolerance to confer protection from T1D, which may be related to inducing of regulatory Th2 responses, inhibiting of lymphocyte proliferation and protecting of isletsβcells;(2) Subcutaneous administration of insulin can confer protection to mice from T1D induced by STZ. The immune protection of autoantigen may be associated with establishing immune tolerance by improving the function of abnormal DC, maintaining DCs with a potentially tolerogenic state, and promoting the production of CD4⁺CD25⁺ T cells in vivo.(3) Tolerogenic DC with an immature phenotype could be propagated by GM-CSF only, GM-CSF+ IL-4 +extract of PHC in vitro and from autoantigen insulin induced T1D murine immune tolerance model in vivo. Adoptive transferring of these tolerogenic DC could confer protection to mice from TID. The immunoprotection may be related to establishing immunotolerance by promoting the production of CD4⁺CD25⁺ T cells and restoring the imbalance between Th1 and Th2 cytokines in vivo.