Mechanisms Of Immunotolerance Disorder In Mouse Model For Autoimmune Diabetes

Autoimmune disease (AD) is a kind of disease caused by harmfulimmune responses between autoantigens and potentially pathogenicautoreactive cells, which result in tissues and/or organs destruction and lossof physical functions. Breakdown of self-tolerance is the critical factor forAD. While self-tolerance, referred as immune system's specific anergystate to autoantigens, is the essential mechanism to maintain immunehomeostasis. Self-tolerance is controlled by central and peripheralmechanisms. Therefore, an ideal strategy to treat AD is to induceself-tolerance of target autoantigens.Type 1 diabetes is an AD mediated by T cells, with geneticbackgrounds. Under certain circumstances, βcells in pancreatic islet areinvolved in autoimmune responses, which pathologically features asinsulitis, damagingβcells and dysfunction of insulin synthesizing andsecreting, to cause glycometabolism disorders. The prevalence of type 1diabetes has a trend to rise in recent years. However, the exactpathogenesis is still unknown.Up to date, no effective therapeutic strategy to cure type 1 diabetesEarly determining high-risk population and practicing immune interferenceprocedures for prevention is an important method. During the past severaldecades, with help of various diabetes animal models, knowledge inpathogenesis of type 1 diabetes, especially that about immune tolerance,has been rapidly and continuously broaden. Some researchers doubtwhether results of pathogenesis studies from animal model are suitable forhuman beings, though, more researchers have proved that, abnormalinsulin level, breakdown of immune tolerance and disorder of immuneresponse appearing in diabetes animal models, can also be verified indiabetes high-risk populations. Thus, Pathogenesis researches based onanimal models will eventually provide valid approaches for prevention andtreatment of type 1 diabetes.In this resaerch, by choosing diabetes mouse model induced bymultiple injection of low dose STZ and NOD mouse model of ten weeksold,we have studied influences of self-tolerance abnormality inpathogenesis of autoimmune diabetes completely (from central andperipheral aspects) and systematically.This research was composed of two parts:Part I Mechanisms of Immunotolerance Disorder in Type 1diabetes Model Induced by STZSection 1 Establishment and Assessment of Diabetes ModelInduced by STZIn this part, diabetes related indexes, cellular and humoral immunitywere detected, in order to study the mechanisms of STZ at different dosesinducing diabetes model, then to verify the optimal dose for establishingthe model, and to observe the characters of immune responses in type 1diabetes.1. Detection of diabetes related indexes in the mouse modelAssessment and detection of normal condition, body weight, blood glucose,urine glucose and pathological state of pancreatic islet was practiced tomodel mice, for the purpose of evaluating diabetes model induced by STZof different doses. Results indicated that STZ can destroy β cells inpancreatic islet through certain mechanisms, causing functional damages topancreatic islet and increases in blood glucose concentration. However,different dose of STZ follows different cell damage mechanism, to causepancreatic islet cell demonstrating different pathological courses andchanges. Mouse groups of STZ 20mg/kg·bw and 40mg/kg·bw can be bettermimic natural course of type 1 diabetes.2. Changes of humoral immune state in model mice Mouse insulinautoantibody (IAA) levels were detected through indirect ELISA toevaluate whether the diabetes induced by STZ of different doses is a resultof autoimmunity. Results showed that no significance can be observedbetween STZ model groups and control group, in a short period (4 days)after injection.7 days after the injection, and later, IAA concentrationsincreased in STZ model groups of 20mg/kg·bw and 40mg/kg·bw, while theSTZ model group of 80mg/kg·bw showed no significant differencescompared with control group. We concluded that high dose STZ injectioncan destroy pancreatic isletβcells to cause diabetes. While multiple lowdose STZ injections can gradually release autoantigen, to activateautoimmune to pancreatic gland and produce autoantibody.3.Changes of cellular immune state in model mice Functionalchanges of mature and immature T cells were detected through lymphocyteproliferation test (MTT method) to further verify whether the diabetes11induced by STZ of different doses is a result of autoimmunity. For STZmodel groups of 20mg/kg·bw and 40mg/kg·bw, results showed that spleencells had an enhanced proliferation to ConA stimulation, while thymuscells decreased, compared with control group. For STZ model group of80mg/kg·bw, no significance can be obtained compared with control group.The results indicated that enhancement of spleen cells proliferation forSTZ model groups of 20mg/kg·bw and 40mg/kg·bw, proved that diabetesinduced by multiple low dose injection of STZ involves immune responses.Functional inhibition of thymus cells maybe a result of interference undercertain mechanism.Section 2 Mechanisms of Abnormal Central and peripheralImmunotolerance in Autoimmune Diabetes Model induced by STZFor determining STZ direct effect or indirect effect on thymus damagein diabetes model, insulin-treated procedure was practiced while adoptingSTZ inducing diabetes model, which will help to study abnormal centraland peripheral immunotolerance mechanisms in diabetes model induced bySTZ.1.Changes of diabetes related indexes in model mice afterinsulin-treated Blood glucose, urine glucose, serum insulin autoantibodyand panereatic pathological specimen were detected to determine whethertype 1 diabetes in model mice was induced by multiple low dose injectionof STZ and insulin-treated. Results demonstrated that blood glucose andurine glucose concentrations increased in model mice, which signifiesdiabetes. Serum autoantibody concentration was higher than that in controlgroup, and different grades of pathological changes can be observed inpancreatic islet specimen, which indicate existence of type 1 diabetes andautoimmune responses.2.Abnormal changes of central immunotolerance afterinsulin-treated in model mice We detected thymus size, pathologicalstate, cell number of CD4+ T cellsand CD4+CD25+T cells ,thymus cellimmunal function, cytokine level and autoantigen promiscuous geneexpression for discussing mechanism of abnormal central immunotolerancein model mice. No significance was observed in thymus weight/bodyweight ratios between insulin supplement group and control group, whilediabetes group lower than control group. For diabetes group, thymus cortexbecame thinner and the region of cortico-medullary junction was unclear.Those conditions were better for insulin-treated group. Amounts of CD4+and CD4+CD25+ Treg were higher than those in diabetes group, though,lower than normal level.The proliferation of lymphocyte in thymus washigher in insulin-treated group than diabetes group,which was lower thancontrol group.Th1/Th2 tends to equilibrium. No significance was observedin thymus promiscuous gene expression for both groups. The resultsshowed that, STZ can directly damage thymus tissues. Meanwhile,hypoinsulinemia caused by direct STZ effect on pancreatic gland can alsoaffect thymus function. Thymus damage is caused by the two factors.Moreover, low promiscuous gene expression in STZ inducing diabetesmodel, as well as decrease in CD4+CD25+ regulatory T cell amount, whichcauses central immunotolerance abnormality, can induce diabetes.3.Abnormal changes of peripheral immunotolerance after insulinsupplement in T1DM mice In order to study the condition of peripheralimmunotolerance of insulin-treated T1DM mice, we detected the cellimmune function of spleenocytes and the levels of cytokines secreted bythem, as well as the amount of CD4+CD25+Tr cell and the expression ofFoxp3 in spleenocytes. The results showed that in insulin-treated group theproliferation ability of spleenocytes was higher than control group,meanwhile slightly higher than diabetes group. Both insulin-treated groupand diabetes group the ratio of IFN-g/IL-4 were higher than those incontrol group, and the insulin-treated groups was slightly higher thandiabetes groups. At the same time CD4+CD25+Tr/ CD4+ T cells in the twogroups were higher than in control group, and in insulin-treated group wasweakly higher than diabetes group. However in insulin-treated and diabetesgroups, the expression of Foxp3 in spleenocytes were higher than incontrol group, and in insulin-treated group is higher than diabetes group.Considering these results, insulin as an ecdemic antigen can enhance theimmune response. On the other hand CD4+CD25+Treg play a critical rolein regulation of peripheral immunotolerance.Conclusions:1. Low dose STZ(20mg/kg.bw,40mg/kg.bw) induced T1DM, a kindof autoimmune disease involved cell and humoral response. And the doseof 40mg/kg.bw is better.High dose STZ(80 mg/kg.bw)induced TIIDM.2. STZ may directly injury the thymus with unknown mechanisms.The selection and differentiation of T cells were subsequently affected andresulted in the dysfunction of central immunotolerance and the occuranceof T1DM. Additionally STZ might directly destroy the β cells ofpancreatic islet, following the reduction of insulin production andtrophonosis of thymocytes. The indirect injury of construction and functionof thymus furtherly interrupt the central immunotolerance and develop theautoimmune disease.143. STZ may injury the thymus directly or indirectly, therefore reducethe amount of CD4+CD25+Tr and the expression of Foxp3 which mightresult in the dysfunction of peripheral tolerance and the development ofT1DM.Part II Mechanisms of Immunotolerance Disorder in NOD MiceDiabetes related symptoms of NOD mice were studied, as well asabnormal central and peripheral immunotolerance mechanisms, in order toget knowledge of immunotolerance deficiency's influences on diabetespathogenesis.1. Detection of diabetes related symptoms in NOD mice Bloodglucose, urine glucose and insulin autoantibody (through indirect ELISA)were detected, as well as pancreatic gland pathological state was assessed.No significance was observed in blood glucose and autoantibody betweenNOD mice and Balb/C mice of the same age. Urine glucose was negativein both groups. Pancreatic islets of NOD mice were decreased in amount,rarely distributed, and low in density. Pancreatic islet cells decreased, withlarge amount of inflammatory cells infiltrating. This proved thatinflammatory cells infiltrate in pancreatic islet before diabetes symptomsappears in ten-week-old NOD mice.2. Researches on abnormal central immunotolerance in NODmice Abnormal central immunotolerance in NOD mice were evaluatedthrough morphological, cellular and molecular levels. No thymus atrophywas observed. No obvious cortico-medullary junction can be viewed, andcell amount in medulla decreased. Compared with Balb/C control group,no significance can be concluded in ConA responses.The level of IFN-γwas no significent difference between NOD and Balb/C group,however thelevel IL-4 was lower than those in Balb/C group . Insulin expression inthymus and bone origin dendritic cells and MHCII molecules in NODgroup were lower than those in Balb/C group. GAD67 and PLP expressionin thymus and bone origin dendritic cells in NOD mice were nosignificance difference compared with Balb/C group .3. Researches on abnormal peripheral immunotolerance in NODmice We detected lymphocyte proliferation of spleen cells, cytokine andCD4+CD25+Treg level, as well as expression of Foxp3 mRNA, toevaluated peripheral cellular immunity in NOD mice. Multiclonalproliferation deficiency can be observed in NOD mice peripheral mature Tcells. IFN-γ remarkably increased while IL-4 obviously decreased, withdisequilibria in Th1/Th2 ratio. CD4+CD25+Treg cells increased greatly inNOD mice spleen. Foxp3 mRNA expression in peripheral spleen cellsshowed an increasing trend, without significance,however. Peripheraltolerance deficiency can be concluded in NOD mice, with a demonstrationof spleen lymphocyte proliferation deficiency, upregulation of IFN-γ/IL-4,which demonstrated that activity of autoreactive Th1 cells topancreatic islet β cells increases. Cell apoptosis can be directly orindirectly induced through Th1 and/or cytokine activating effecter cells.Comparatively increase of CD4+CD25+Treg can suppress T cell functions,which prevents NOD mice from diabetes.Conclusions:1. NOD mice were at the stage of prediabetes without diabetes.There were lots of inflammatory cells infiltrations in pancreatic islets.2. The central tolerance was abnomal in NOD mice. The disorder ofthymus construction and significant reduction of insulin expression inthymus and DCs derived from bone marrow resulted in abnormal negativeselection of self reactive thymocytes and the increase of cloning. Apotential pathogenic fator of diabetes was the low expression of MHC-IImolecule in Medullar thymus epithelia cell which resulted in the reductionof combination with peptides.3. The peripheral tolerance was also abnomal in NOD mice.Theabsence of proliferation of spleenocytes and the unballence of Th1/Th2were observed in NOD mice. However the increasing of CD4+CD25+ Tregcompared to the effective T cells could suppress their function whichprevent the NOD mice from diabetes.This research, focusing on abnormal changes in mouse model forautoimmue diabetes, is helpful for further understanding of diabetespathogenesis, and provides efficient strategy for prevention and therapy oftype 1 diabetes.