Effects And Mechanisms Of Syngeneic Hematopoietic Stem Cell Transplantation In Type 1 Diabetes Mice Modles

Objective: Autologous hematopoietic stem cell transplantation (AHSCT) has recently become a novel therapy for patients with new-onset type 1 diabetes (T1D). To investigate the efficiency, we studied the relations among time points, source of hematopoietic stem cell and the effects of syngeneic bone marrow transplantation for T1D in mice.The differences of lymphocyte subsets in peripheral blood, the ratio of DCs in spleen and function ofβcell of islet influenced by Syn-HSCT, the proportion of Treg in spleen, the related gene Foxp3 mRNA,Foxp3 protein,the severity of diabetic nephropathy before and after Syn-HSCT are also compared in order to investigate the mechanism of syngeneic hematopoietic stem cell transplantation (Syn-HSCT) therapy in T1D mice and their roles in tolerance induction.Methods: Type 1 diabetes models in male C57BL/6 mice were induced by injecting with 40-50 mg/kg streptozotocin (STZ) intraperitoneally for 5 consecutive days. Only those STZ-injected mice whose fasting blood glucose levels > 13.9mol/L for 2 consecutive days were diagnosed as diabetes and included in this study. The diabetic mice were randomLy divided into HSCT donor group (n = 19); HSCT receipt group (n = 50) and T1D control group (n = 25). 14 normal mice were divided as normal control group, 31 normal mice as normal donor group. Mice in HSCT receipt group got bone marrow from either diabetic mice or normal mice.Syn-HSCT was performed at different time points (day 3 (n = 12), day 10 (n = 19), day 20 (n = 12), and day 40 (n = 7), respectively) after T1D onset. The conditioning regimen was 8Gy 60Coγrays total-body irradiation (TBI). In six hours after TBI, prepared bone marrow cells from normal mice were infused via tail vein into the diabetic mice in HSCT group. T1D control group was divided into groups according to the time points after T1D onset (day 3 (n = 6), day 10 (n = 19), day 20 (n = 6), and day 40 (n = 13), respectively), and had no further treatment. 30 days after HSCT, 6 mice in normal control group and 6mice in HSCT on day 10 after T1D onset group were killed; while other receipt mice and T1D control mice were killed 120 days after HSCT.Blood and urine glucose of T1D control group and HSCT receipt group were observed every 2 weeks; the account of white blood cells and platelets of HSCT receipt group mice were observed every three days. Histological analysis was tested by HE staining, lymphocyte subsets in peripheral blood and of the ratio of DC1/DC2 in spleen by flow cytometry analysis, serum insulin by ELISA, islet insulin expression by immunohistochemisty, the proportion of Treg in spleen by flow cytometry, the level of Foxp3 mRNA by Real time PCR, and the relative expression of Foxp3 protein by western blotting. Renal pathology and levels of oxidative stress were also studied and expressions of angiotensinogen (AGT), monocyte chemoattractant protein-1 (MCP-1) and transforming growth factor, beta 1 (TGF-β1) mRNA in kidney were investigated by quantitative real-time PCR.Results: Two weeks after the first STZ-injection, the blood glucose level of STZ injected mice was significantly higher(21.24±6.14 mmol/L)than that of normal control (6.71±2.35 mmol/L). The histopathologic examination of diabetic mice showed diffuse lymphocyte infiltration in pancreas. It showed that Syn-HSCT on day 10 after T1D onset led to a significant clinical amelioration of blood glucose as compared with untreated diabetic mice. The count of WBC and PLT were reduced to nadir on six and nine days after Syn-HSCT, respectively. Then the count of WBC and PLT gradually became normal on thirty days after Syn-HSCT. In the intraperitoneal glucose tolerance test, the HSCT receipt group receiving Syn-HSCT on day 10 after T1D onset, which had a curve similar to the normal mice, showed improved tolerance to glucose whereas untreated diabetic mice remained glucose intolerant throughout the 120 min experiment. Serum insulin tested by ELISA and islet insulin expression tested by immunohistochemisty was significantly increased by Syn-HSCT, compared with T1D control group mice.After Syn-HSCT on day 10 after T1D onset, compared with mice on day 10 after T1D onset, the ratio of CD4/CD8 and DC1/DC2 and proportion of NK cells significantly decreased, and proportion of CD8+CD28- T cell significantly increased;While compared with T1D control group mice, the ratio of CD4/CD8 and DC1/DC2 and proportions of NK cells and CD5+CD19+ B significantly decreased.In the whole 120 days after HSCT, it showed that Syn-HSCT can lead to total loss of diabetes when performed on day 10, attenuate diabetes when done on day 3 or day 20, but can not ameliorate diabetes when done on day 40. Interestingly, there was no difference when Syn-HSCT was performed with either normal or STZ-diabetic mice as donors. Performed on day 10 after T1D onset, Syn-HSCT leading to recovery of T1D results in induction of Tregs, increased Foxp3 mRNA expression and increased Foxp3 protein expression in both the groups of diabetic mice receiving HSCT with either diabetic or normal donors. And when Syn-HSCT was performed on day 3 or day 20, there was no increment of Tregs. Moreover, a decrement of Tregs showed when Syn-HSCT was performed on day 40.Syn-HSCT on early disease stage reduced kidney enlargement, besides, if it was given on day 10, attenuated glomerular hypertrophy. Oxidative stress factors such as catalase (CAT) and superoxide radical anion O2- (O2-) were markedly maintained while superoxide dismutase (SOD) was not changed by Syn-HSCT. In diabetic mice receiving no treatment, there were increases in renal angiotensinogen (AGT) and monocyte chemoattractant protein-1 (MCP-1) mRNA which were effectively suppressed by Syn-HSCT. But after Syn-HSCT, it showed no changes or even increment in transforming growth factor beta 1 (TGF-β1) mRNA.Conclusion: (1) It is proved to be feasible to induce the type-1-diabetic mice through the consecutive intraperitoneal injection of STZ; (2) Syn-HSCT, if performed on certain time points, can result in loss or at least attenuation of diabetes and improvements of insulin secretion in mice models; (3) The percentage of Tregs in the spleen was correlated with the attenuation of hyperglycemia; (4) After Syn-HSCT, the ratio of CD4/CD8 and DC1/DC2 and proportion of NK cells decreased and proportion of CD8+CD28-T cell increased, which may be the immune mechanism of Syn-HSCT therapy in T1D; (5) there was no difference on both the effects of HSCT and the changes of Tregs when Syn-HSCT was performed with either normal or STZ-diabetic mice as donors; (6) Syn-HSCT, if applied on early disease stage, ameliorates diabetic renal injury, partly by maintaining oxidative stress and expression of AGT and MCP-1 in renal tissue in diabetic mice.