Mechanisms Of Abnormal Immunotolerance In The Type 1 Diabetes Model Induced By STZ

Autoimmune Disease (AD) is caused by the loss of self-tolerance. The development of AD reflected the disorder of central tolerance and/or peripheral tolerance. At present, it had been confirmed that the central tolerance mechanism was the base to assure the normal selection of T and B cells in central immune organs. The promiscuous genes expression of self-antigen by thymus is related with the consequence of T cells negative selection and is crucial for maintaining central tolerance mechanism. However, CD4+CD25+ regulatory T cells (CD4+CD25+Treg) could actively suppress or modulate the self reactive T cells (SRT) in peripheral and was a important regulator for maintaining peripheral tolerance, and it was a key issue in self-tolerance research work. Type 1 Diabetes Mellitus (T1DM) is a common organ specific AD endangering human health, the precise pathogenesis of which is still not clear. Now it could be considered T1DM occurrence is the result of activaton of autoimmune response, which requires a genetic susceptibility and is triggered by the virus infectious or toxicity of chemical materials. Streptozotocin (STZ) is synthesized by Streptomycetes achromogenes and is able to induce T1DM and Type 2 diabetes mellitus. Multiple injection of Low Dose STZ Induced DM (MLDS-DM) was a common used model to study T1DM, their pathogenesis was not confirmed. In this study, the MLDS-DM mice were used as the model to research the effects of the self-tolerance mechaniam disorder on the development of autoimmune diabetes triggered by STZ. The results would provide more foundation for the further application of MLDS-DM model, moreover, provide the new way for the clinical therapy of T1DM. Two parts of the study are included as followed: Part 1: MLDS-DM is an AD mediated by cellular immune To determide whether MLD-STZ injection could trigger the diabetes, and whether immune response involved in the development of diabetes and what immune response character involved in the desease. Study on the model injected with MLD-STZ and mesure indexes associated with diabetes and the cellular and humoral immune function in peripheral. Ⅰ. Changes related with diabetes on model mice To evaluate the state of the disease, the general symptom and morphological alterations had been analysed. The results show that MLDS-DM model exhibited typical T1DM symptom and insulitis, which are high glucose level in blood and urine, It was evident that MLDS-DM could mimic the symptom and morphologic alterantion as T1DM patients. Ⅱ. Changes of humoral immune function in peripheral of model mice For the comprehension on the immunologic mechanism in the development of MLDS-DM, we determined the level of anti-insulin antibody by ELISA in serum and found that the autoantibody level increased oberviously. It .indicated the function of autoreactive B cells enhanced in the model and the development of disease is a process of autoimmune response. Ⅲ. Changes of cellular immune function in peripheral of model mice 1,Changes of the frequency and the proliferation of CD4+T cells in spleen of diabetes mice triggered by STZ To investigate the immune function state of MLDS-DM mice further, we measured the frequency of CD4+T cells in spleen by FACS; and tested the nonspecific proliferation and specific proliferation ability of splenocyte to the stimulation with ConA and Insulin respectively. Results show that the level of CD4+T cells increased, while the proliferation of insulin specific T cells increased relatively. It suggested that cellular immunity involved in thedevelopment of autoreactive diabetes. But similar with NOD mice and T1DM sufferer, the model was deficient in proliferation of T cells to polyclonal stimulation in peripheral. 2,Changes of the level of cytokine secreted by T cells in spleen of diabetes mice triggered by STZ The development of T1DM related with the imbalance of Th1 and Th2. To further clarify the immunologic factors associated with diabetes, we determined the level of cytokine IFNγand Il-4 secreted by splenocyte activated in vitro .The results show that and the cytokine IFNγsecreted predominantly by Th1 and illuminate that STZ induced the direct damage on islet cells and result in the release of self-antigens which induced the immune system participating in the development of diseases, lead to the enhancement of Th1 and Th2 function simultaneity and the repolarization of peripheral T cells. the function of IFNγproducing cells was over-increased and play crucial role in the process of islet βcells damage. In this report we show that MLDS-DM is a AD mediated by cellular immune, Th1 and cytokine IFNγsecreted by Th1 play especially important role in the development of diseases; The comparative decrease of both Th2 response and the secretion of IL-4 by Th2 can`t suppress Th1 response, result in the imbalance between Th1 and Th2 and the predominance response of Th1, which is the direct reason for the disease occurring. As well, autoimmune B cells pay a definite role in the disease, various autoantibody could be detected in the MLDS-DM mice, including anti-insulin, anti-GAD67, anti-islet cells and anti-HSP antibodies. However, it had not been determined the role of B cells, promote or protect. Nonspecific immune response mediated by macrophage and inflammatory cytokine perhaps involved in the development of diabetes. Above all, the activation of an antigen specific autoimmune process is the direct reason for the disease, which induced the infiltration of islets by lymphocyte and insulitisoccurrence, caused the damage on islet βcells and made their secrete function damaged and impaired, lead to the raise of blood glucose ultimately. Part2: Deficiency in both central and peripheral tolerance is essential for the development of MLDS-DM To further study the role of abnormal immunotolerance mechanism in the development of diabetes , the makeup and function of cells in thymus was observed and the frequency and function of CD4+CD25+Treg with immunoregulatory function in the spleen was detected. Ⅰ. Mechanism of peripheral tolerance is impaired in the model 1,The level of CD4+CD25+Treg is decreased comparatively in spleen of MLDS-DM mice CD4+CD25+Treg is a population existing naturally in the normal bodies, and it has anergy phenotype and suppress function, which can maintain peripheral tolerance and prevent the development of AD. To determine the immunoregulatory ability in peripheral of MLDM-DM mice, we examine the level of CD4+CD25+Treg in the CD4+ splenocyte by FACS. We found the frequency of CD4+CD25+Treg decreased obviously, indicated that the imbalance between CD4+T cells and CD4+CD25+Treg existed in the model in peripheral. There are two subpopulations with opposite function in CD4+ population, CD4+CD25+Treg and CD25-effector T cells (Te). Te help and induce the development of autoimmune, while CD4+CD25+Treg inhibit Te and prevent the pathogenic immune response. Only the two subpopulations balanced in a proper ratio, immune response could be limited in a safe extent and avoid the pathogenic immune response. This study showed a disorder of peripheral tolerance in MLDS-DM model manifesting the imbalance between CD4+CD25+Treg and Te. The enhanced function of Te compared with CD4+CD25+ Treg lead to CD4+CD25+Treg lossing their efficient regulatory function and the continued occurrence of autoimmune response.2,Expression of Foxp3 mRNA increased in splenocytes Foxp3 is a transcription factor highly expressed in CD4+CD25+Treg specifically and is related with the development and maturation of CD4+CD25+Treg closely. The level of Foxp3 could reflect the function state of CD4+CD25+Treg in some degree. To further determine the functional condition of peripheral CD4+CD25+Treg, RT-PCR analyse the expression of Foxp3 mRNA in splenocytes of MLDS-DM mice. The results show that the expression of Foxp3 mRNA in splenocytes of the model is higher than the control prevalently. It indicated that the function of CD4+CD25+Treg is promoted, which does not conflict with the decrease of CD4+CD25+Treg in CD4+T cells. Several studies show that proliferation and activation of CD4+CD25+Treg are two separate processes. Although CD4+CD25+Treg is anergy generally, it is prone to be activated by TCR stimulation. It suggested that self-antigen could trigger the activation of Te and cause autoimmune, furthermore, it could induce the activation and proliferation of CD4+CD25+ Treg with suppress function simultaneously. But CD4+CD25+Treg can`t inhibit the development of AD mediated by Te and which lead to the convertible pathogenic damage of islet, because of their lower proliferation than Te. Ⅱ. Mechanism of central tolerance is impaired in the model 1. Thymus is atrophied in the model In the experiment, we found thymus in the model mice is atrophied compared with the control prevalently. Consistent with the result of Nichols`s studies that STZ can induce the bone marrow cells and precursors of T lymphocytes from STZ-diabetic mice damaged or impaired and induce involution of thymus of those strains mice being sensitive to STZ extremely. Involution of thymus might be caused by STZ directly or by the lessened insulin indirectly, in that, the loss of thymocytes and nucleated spleen cells is only partially reversed by insulin treatment. It should be still clarified therelationship between thymus atrophy and diabetes. 2. Changes of cells amounts in thymus Thymus atrophy might lead to changes in it`s structure and function. To further recognize the influence on thymus function by thymus atrophy, we use CD4 and CD25 antibody labeled with fluorescence to mark cells in thymus, and then determined thymocytes constitution by FACS. The result show that the level of CD4+T cells in thymus is not changed, while the level of CD4+CD25+ thymocytes in CD4+ cells decreased distinctly, and it suggested that the thymic selection of CD4+CD25+Treg was abnormal in the model mice, which lead to the loss of both CD4+CD25+ thymocytes and CD4+CD25+Treg, and lead to the abnormal of CD4+CD25+Treg function in peripheral. 3. Alteration to proliferation and cytokine secretion of thymocytes To further evaluate the immunologic function of thymocytes in the model, we detected non-specific and insulin-specific proliferation of thymocytes stimulated with ConA and insulin respectively, Analysis the level of cytokine IL-4 and IFNγsecreted by thymocytes stimulated ConA for 37h by ELISA in vitro. The result show that cytokine secretion of thymocyts and proliferation of thymocytes with ConA were not changed, while proliferation of thymocytes stimulated with insulin was enhanced dramatically, it suggest that the imbalance betweenTh1 and Th2 is induced by antigen stimulation in peripheral, but not the consequence of the abnormal differentiation of thymocytes. The remarkable augment of autoreactive thymocyte clones cause potentially enhanced activity of peripheral autoimmune T cells. 4. Analysis of the promiscuous expression of self-antigen by thymus The promiscuous expression of self-antigen by thymus is known as a phenotype that various tissue specific antigen out of thymus express widely on the thymic epithelia cells (TEC), especially the thymic medullar epithelia cells(mTEC). The recent studies verified that it is associated with the thymicselection of T cells and as central tolerance to autoimmune closely. To determine whether thymic atrophy effect on the promiscuous expression by TEC and confirm what blockade the development of CD4+CD25+Treg in thymus and what promote the function of autoreactive clone, RT-PCR analyze the expression of Insulin and PLP in thymus. Insulin is a self-antigen associated with T1DM while PLP is not associated with T1DM. The result show that the expression of Insulin and PLP by thymus is diminished and indicated the lack of promiscuous expression of self-antigen by thymus in MLDS-DM mice, and which lead to two results. Either autoreacive cells clone increased for the abnormity of negative selection, or CD4+CD25+Treg clone diminished for the blockade of CD4+CD25+Treg thymic selection. The abnormity of promiscuous expression by thymus is potentially correlate with the the conceivable alteration of structure and microenviroment of thymus induced by thymic atrophy. In a word, deficiency of central tolerance in MLDS-DM mice might be a possible reason for the development of disease. The conclusions of the study: 1 MLDS-DM is an AD mediated by cellular immune, especially Th1 dominant immune response result in the pathogenic immunologic damage. 2 MLDS-DM mice are deficient in peripheral tolerance, which involved in the development of disease. CD4+CD25+Treg descend compared with Te and unable to inhibit the development of AD effectively and lead to the disease occurrence. 3,MLDS-DM mice lack of central tolerance, which is related with the development of diabetes. The proliferation of autoreactive thymocytes upregulate and the CD4+CD25+ thymocytes declined. It demonstrated that the deficiency of peripheral tolerance may partially owing to the abnormity of central tolerance. 4 ,The blockade of thymic selection of autoreactive T cells and CD4+CD25+Treg is caused by the diminution of the promiscuous expression...