Regulatory T Cells In The Peripheral Blood Of Patients With Fulminant Type1Diabetes

Part one Immunological features in the peripheral blood of patients with fulminant type1diabetesObjective With the development of technologies of immunological detections, growing evidences indicated that autoimmunity was involved in part of FT1DM patients. In this part, the immunological features including autoimmunity and regulatory T cells related functional molecules were explored in peripheral blood mononuclear cells (PBMCs) from patients with FT1DM.Methods GADA (glutamic acid decarboxylase autoantibody), IA-2A (protein tyrosine phosphatase antibody) and ZnT8A (zinc transporter8autoantibody) were determined with radioligand assay in22patients with FT1DM. GAD, insulin and C peptide responsive T cells were determined with enzyme linked immunospot assay in10patients with FTIDM and healthy subjects. mRNA level of IFN-y, IL-4, RORC, IL-17, Foxp3, CTLA-4, IL-10and TGF-β were determined by real-time PCR.Results Nine of twenty FTIDM patients were autoantibody-positive, including five patients with GADA, two patients with IA-2A and three patients with ZnT8A. Eight of ten FTIDM patients were found at least one type of islet antigen reactive T cells, including seven patients with GAD reactive T cells,4patients with C-peptide reactive T cells and4patients with insulin reactive T cells. IFN-γ was increased and Foxp3was decreased significantly in patients with FT1DM relative to healthy controls (P=0.02,P<0.01, respectively)Conclusion Autoimmunity and abnormal regulatory T cells are involved in part of FT1DM patients. Part two Aberrant methylation pattern of Foxp3gene in peripheral blood mononuclear cells from patients with fulminant type1diabetesObjective Epigenetics was involved in many autoimmune diseases. However, the epigenetic modifications in FTIDM were unknown. In this part, we determined the methylation status of PBMCs from patients with FT1DM and investigated the relationship between DNA methylation and the deficiency of regulatory T cells.Methods PBMCs were isolated from peripheral venous blood from10patients with FT1DM and10healthy controls. Global methylcytosine levels in PBMCs were quantified using the MethylampTM global DNA methylation quantification kit. mRNA levels of DNA methyltransferases (DNMTs) were measured by real-time polymerase chain reaction (real-time PCR). Foxp3protein was determined by western-blot. Bisulfite sequencing was used to determine the methylation status of promoter region of Foxp3.Results Global DNA methylation was significantly increased in PBMCs from patients with FT1DM relative to healthy controls (P=0.017). DNMTl mRNA levels were significantly higher in PBMCs from patients with FTIDM than that in healthy controls(P=0.032). Foxp3protein levels were significantly lower in PBMCs from patients with FT1DM. The mean methylation levels of CG pairs within promoter region of Foxp3in PBMCs from FTIDM patients were higher than healthy controls(P< 0.01). Moreover, Foxp3mRNA expression negatively correlated with PBMCs DNA methylation within FTIDM patients(r=-0.709, P<0.01).Conclusion Hypermethylation of Foxp3promoter contributes to low Foxp3expression in PBMCs from patients with FTIDM. Part3CpG ODN increases Foxp3expression via TLR9/IRF7pathway in peripheral blood mononuclear cells from healthy subjectsObjective TLR9and Foxp3were downexpressed in PBMCs from patients with FT1DM, whether TLR9pathway correlated with Foxp3expression were unknown. In this part, the effects of the TLR9/IRF7pathway on the expression of Foxp3were explored in PBMCs from healthy subjects, so that they provided evidences for the further study in FT1DM patients.Methods TLR9mRNA levels were measured by real-time polymerase chain reaction (Real-time PCR). Foxp3protein was determined by western-blot. The transcriptional factors of the Foxp3gene were predicted with bioinformatics. real-time PCR and Western blot were employed to detect the expression of Foxp3and IRF7in PBMCs with IRF7overexpression or silencing treatment. Luciferase assay was used to assess the effect of IRF7on the transcription of Foxp3. The binding site of IRF7on the promoter of Foxp3was confirmed by ChIP. Real-time PCR and Western blot were also utilized to evaluate the role of IRF7in the expression of Foxp3and its targeting genes; Immunofluorescence and siRNA interference were used to demonstrate the mechanisms of CpG ODN induced Foxp3expression.Results TLR9was down-regulated at mRNA level in PBMCs from FTIDM patients (P<0.05). Foxp3was increased and decreased at both mRNA and protein levels when IRF7overexpressed and downexpressed respectively in PBMCs from healthy subjects (P<0.05). Transcription of Foxp3gene was induced by transcription factor IRF7predicted by online software and confirmed by ChIP (P<0.05). Foxp3was increased via CpG ODN initiated TLR9/IRF7pathway (P<0.05)Conclusion Foxp3is increased via CpG ODN initiated TLR9/IRF7pathway in PBMCs from healthy subjects.