The Therapeutic Effect Of Th17/Tregs Immune Balance Regulated By Dendritic Cells On Epilepsy Rats

Epilepsy is a chronic brain disease characterized by recurrent and susceptible seizure activity and is associated with a variety of neurological diseases and other systemic diseases.Fifty million people worldwide suffer from epilepsy,and about 30%of epilepsy patients are drug-resistant and are accompanied by progressive cognitive impairment.Temporal lobe epilepsy（TLE）affects the limbic system and is the most common form of drug-resistant epilepsy.The epilepsy model induced by Li Cl-Pilocarpine shows hippocampal changes similar to those in TLE patients,and is accompanied by drug resistance.Although the cellular and molecular mechanisms of epilepsy are unclear,it is speculated that focal or systemic uncontrolled inflammation can lead to abnormal neuronal connections and over-excited neuronal networks,which mediated seizures.Experimental research and clinical evidence on brain specimens obtained from human and model animals with various drug-resistant epilepsy indicate that the activation of innate and adaptive immune responses and the induction of related inflammatory processes have played an important role in epileptic foci,but the key molecular mechanisms involved in the onset and progression of epilepsy are still elusive.More and more evidences support that neuroinflammation may mediate the pathological process during the development of epilepsy,and this process can be intensified by oxidative stress,and the termination of the process depends on an effective homeostatic anti-inflammatory mechanism in the body.Therefore,enhancing the endogenous anti-inflammatory response may be a clinically feasible treatment option,which can improve the disease process by re-establishing immune homeostasis.Dendritic cells（DCs）,as the most powerful antigen-presenting cells（APCs）in the body,can control the activation and polarization of T cells to a specific lineage,and play an important role in the regulation of immunity and tolerance.In pathogenic infection,DCs are activated,and express costimulatory molecules and cytokines that promote effector T-cell responses,such as interleukins IL-12p70 and IL-23,which are important for the differentiation of Th1 and Th17 cells,respectively.DCs can also establish and maintain immune tolerance by inducing anergy and the deletion of effector T cells,while promoting the differentiation of regulatory T cells（Treg cells）.This tolerogenic phenotype was initially associated with immature DCs.However,increasing evidence suggests that mature DCs can also perform tolerogenic functions following,for example,activation with IL-27,IL-10,or ligands of the aryl hydrocarbon receptor.Based on the previous studies results,this study makes a hypothesis that IL-27-treated DCs can induce the immune tolerance,and then rebuild the immune homeostasis,and achieve the therapeutic effect.In this study,TLE rats were induced by pilocarpine through intraperitoneal injection.First,to explore the relationship between immune homeostasis and the development of epilepsy,the changes in the numbers of Th17 cells,Treg cells and Tr1 cells and the expressions of related cytokines in the hippocampus of TLE rats at different time points were explored and analyzed.Second,to explore the therapeutic effect of IL-27-treated DCs on epileptic rats,IL-27-treated DCs were intravenously intervene in TLE rats,IL-27 treated DCs were injected into TLE rats by tail vein for intervention.Finally,to explore the relevant mechanism of the therapeutic effect of IL-27-treated DCs on TLE rats.Part One The changes of Th17 cells and Treg cells and their related cytokines in the hippocampus of TLE rats during the development of epilepsyObjective:To explore the relationship between the occurrence and development of epilepsy and the Th17 cells/Tregs cells immune balance in the hippocampus of pilocarpine-induced TLE rats.Methods:In this study,we took pilocarpine-induced epileptic rats as the research object.1.Using Morris Water Maze（MWM）,we explored the spatial learning and memory ability of the TLE rats.2.The hippocampus tissue of TLE rat was collected,using H.E staining and Nissle staining,we explored the pathological changes at different time points in TLE rats hippocampus3.Using immunohistochemical staining and western blotting,we explored the changes of the numbers of Th17 cells and Tregs cells and the exprssions of the related cytokines in TLE rats hippocampus.Results:1.In the MWM,the escape latency in TLE group was significantly prolonged,the number of platform crossing,the swimming speed and the target quadrant time percentage were significantly less than those in normal group.2.In H.E staining and Nissle staining,the number of neurons was significantly reduced in TLE group,the cell bodies were swollened and deformed,and the nuclei were solidified.These changes appeared on D1 after modeling,and peaked on D5,then gradually recovered.3.The results of immunohistochemistry and Western blotting showed that the numbers of Th17 cells and the expression of its related cytokine IL-17A began to increase from D1 after modeling,reached a peak on D3,maintained at a high level,and decreased from D14 gradually;Foxp3⁺Treg cells,Tr1 cells and their related cytokines Foxp3,IL-10 began to increase on D3,reached the peak on D7,maintained at a high level,and decreased from D14 gradually,until D28,the number of Th17 cells,Foxp3⁺Treg cells,Tr1 cells and the expression of related factors stabilized.Conclusions:1.Pilocarpine-induced TLE rats showed deterioration of general life status,decreased learning and memory ability and neuron damage,which were most obvious on D5 after modeling,then gradually recovered and stabilized on D28.2.The severity of general life status,learning and memory ability,and neuron damage in pilocarpine-induced TLE rats is related to the balance between Th17 cells/Tregs cells,indicating that Th17 cells/Tregs cell immune balance plays a important role in the occurrence and development of epilepsy.Using the plasticity of these cells to adjust the balance is very important for the study of epilepsy pathogenesis,prognosis and treatment.Part Two The therapeutic effect of IL-27-treated DCs on pilocarpine induced TLE ratsObjective:To explore the therapeutic effect of IL-27-treated DCs on TLE rats.Methods:1.Using cell culture,we obtained bone marrow-derived immature DCs,which were incubated with IL-27,PBS and LPS respectively.Using microscope and flow cytometry,we explored the histological morphology and the phenotype of each group DCs.2.The TLE rats were injected different treated DCs through the tail vein,and devided into TLE group,TLE+LPS-treated DCs group,TLE+IL-27-treated DCs group and TLE+PBS-treated DCs group respectively.Using MWM,we compared the differences in spatial learning and memory ability of rats in each group.3.The hippocampus specimens of rats in each group were collected.Using H.E staining and Nissle staining,we compared the differences of the pathological changes in each group hippocampus.4.Using immunohistochemical staining and Western blotting,we compared the differences of the numbers of Th17 cells and Tregs cells and the exprssions of the related cytokines in each group hippocampus.Results:1.In microscope observation and flow cytometry,the IL-27-treated DCs showed the histological characteristics and phenotype of immature DCs(CD11c^hiCD86^mi).2.In MWM,IL-27-treated DCs shortened the escape latency significantly,increased the numbers of platform crossing,the swimming speed and the target quadrant time percentage of TLE rats.3.In H.E staining and Nissle staining,IL-27 treated DCs increased the numbers of neurons,and improved the hippocampus structure gradually.4.In immunohistochemistry and Western blotting,IL-27-treated DCs increased the numbers of Tregs cells and the expression of related cytokines significantly;however,reduced the numbers of Th17 cells and the expression of related cytokines significantly.Conclusions:1.IL-27-treated DCs showed the histological morphology of immature DCs and moderate expression of the costimulatory factor CD86.2.IL-27 treated DCs improved the general life status,learning and memory ability and neuron damage in hippocampus of TLE rats.3.L-27-treated DCs reduced the number of Th17 cells in the hippocampus of TLE rats and the expression of related cytokines,while increased the numbers of Tregs cells and the expression of related cytokines.4.The improvement of general life status,learning and memory ability and neuron damage is related to the rebalance of Th17/Tregs cells,which indicated that IL-27 treated DCs can adjust the Th17/Tregs cells immune balance,then achieve the purpose of treating epilepsy.Part There IL-27-treated DCs improved the balance of Th17cells/Tregs cells through the PD-L1/PD-1 pathwayObjective:To explore the mechanism of the therapeutic effect of IL-27-treated DCs on TLE rats.Methods:1.Using immunofluorescence staining,we compared the differences of the expression of PD-L1 on the surface of DCs in each group.2.Using double immunofluorescence labeling,we compared cells that co-expressed PD-1 and CD25 in each group hippocampus.3.Using western blotting,we compared the differences of the PD-1expression in each group hippocampus.Results:1.In immunofluorescence staining,the expression of PD-L1 in the IL-27treated DCs was higher than other treated DCs.2.In double immunofluorescence labeling,IL-27-treated DCs increased the cells that co-expressed PD-1 and CD25 in hippocampus.3.In western blotting,IL-27-treated DCs increased the expression of PD-1 in the hippocampus of TLE rats.IL-27 treated DCs consistent with double immunofluorescence labeling,the expression of PD-1 in hippocampus was higher in TLE+group than that in the other treated groups.Conclusions:1.IL-27 increased the expression of PD-L1 on DCs.2.IL-27-treated DCs increased the he cells that co-expressed PD-1 and CD25 in hippocampus of TLE rats,and increased the expression of PD-1protein.3.IL-27-treated DCs may through PD-L1/PD-1 pathway inhibit the proliferation of Th17 cells,and induce the differentiation of Tregs cells,then restore the Th17 cells/Treg cells immune balance,and lead to treat epilepsy.