The Role Of Damage Associated Molecular Patterns(DAMPs)in Experimental Autoimmune Encephalomyelitis

Part 1The role of IL-33 in experimental autoimmune encephalomyelitisBackground:Interleukin(IL)-33,a member of the IL-1 cytokine family,induces innate and adaptive immune responses and plays a protective role in many diseases,including autoimmune diseases.IL-33 is highly expressed in the central nervous system(CNS),suggesting its potential role in the CNS.Experimental autoimmune encephalomyelitis(EAE),an autoimmune disease in the central nervous system(CNS),is the most common animal model of multiple sclerosis(MS).It is characterized by demyelination,axonal damage and T-cell as well as monocyte infiltration in the CNS.In the present,more studies have paid an attention to the role of IL-33,among which the role of IL-33 in CNS is poorly investigated.Objective:To explore the effect of IL-33 knockout on the the main cells participating in experimental autoimmune encephalomyelitisMethods:1、Preparation of experimental autoimmune encephalomyelitis model:female C57BL/6J mice(8-9--week-old)including wild type and IL-33 knockout mice were used for active induction of EAE.Briefly,the mice were subcutaneously(s.c.)immunized with 200μg of MOG35-55,a part of myelin protein,and scored daily on day 7 after immunized.During the peak stage of EAE,the spleen,lymph nodes as well as the center nerve system were collected for the next experiment2、Luxol Fast Blue(LFB)staining:Mice were intraperitoneally anesthetized with 300μL of 0.5%pentobarbital sodium and perfused first with saline,and then with 4%paraformaldehyde in PBS.Thoracic spinal cords of the mice were isolated,post-fixed(in 4%paraformaldehyde),dehydrated(in 30%sucrose),frozen,and sectioned at a thickness of 20-μm.The sections were stained with LFB for myelin detection.Lesions were identified as shadow areas in LFB stained sections and scored in a blinded fashion for demyelination:0,none;1,rare foci;2,a few areas of demyelination;3,large(confluent)areas of demyelination3、Acquisition of single-cell suspensions from the inguinal lymph nodes,spleen,and CNS:the fresh inguinal lymph nodes,spleen,spinal cords and brains were isolated and single-cell suspensions were obtained by triturated or separation.Then the cells were measured by flow cytometry to observe the change of the percentage of pathogenic-effector T cells,Foxp3+regulatory T cells,non-pathogenic Th2 cells as well as the antigen presenting cells.4、The obtaining of primary astrocytes and microglia:on day 1 to day 2 after born,the neonatal C57BL/6J mice were used to isolate and culture primary astrocytes and microglia.After the purity were confirmed by flow cytometry or immunofluorescence,the expression of ST2 in the cells was detected by western blot.The primary astrocytes/microglia were stimulated by brain interstitial fluid from the peak stage of EAE(100μg/mL)together with or without IL-33 antibody(2.5μg/mL)and recombinant IL-33(200ng/mL)for 24 hours,then the level of chemokine MCP-1/CCL2 and TNF-αwere detected by ELISA5、The detection of spinal cord sections by immunofluorescence:the thoracic spinal cords sections from EAE were labeled by the special marker for astrocytes(GFAP)and microglia(Ibal)to detect the activation of the resident cellsResults:1、The establishment of the experimental autoimmune encephalomyelitis model is successful and IL-33 knockout exacerbated the progress of EAE,accompanied by more serve demyelination2、The effect of IL-33 on effector T cells(Thl,Th17,Tc1,Tc17)in EAE:IL-33 knockout increased the percentage of effector T cells in spleen,lymph nodes and CNS,compared to the wild-type(WT)EAE mice3、The effect of IL-33 on Foxp3+regulatory T cells in EAE:IL-33 knockout decreased the percentage of Tregs in spleen and lymph nodes,but not in CNS,compared to the wild-type(WT)EAE mice4、The effect of IL-33 on Th2 cells in EAE:IL-33 knockout significantly decreased the percentage of Th2 cells in CNS,but had no obvious effect on them in spleen and lymph nodes,compared to the wild-type(WT)EAE mice5、The effect of IL-33 on NK cells in EAE:IL-33 knockout significantly decreased the percentage of NK cells in CNS,but had no obvious effect on them in spleen and lymph nodes,compared to the wild-type(WT)EAE mice6、The effect of IL-33 on antigen presenting cells in EAE:IL-33 knockout increased the percentage of CD11B+CD11C+dendritic cells(DC)and macrophages in CNS,compared to the wild-type(WT)EAE mice.7、The effect of IL-33 on the phenotype shift of macrophages/microglia in EAE:In CNS,the percentage of M1 macrophages/microglia increased in IL-33 knockout mice while that of M2 macrophages/microglia producing TGF-β or IL-10 seemed to be no different between two groups8、The effect of IL-33 on the resident glia cells in CNS in EAE:Astrocytes and microglia were more activated in the IL-33 knockout mice with EAE.ST2,the receptor of IL-33,was expressed by resident glia cells especially astrocytes.Furtherly,in primary astrocytes,the brain interstitial fluid from the peak stage of EAE promoted the release of MCP-1/CCL2 and TNF-α,and the treatment of IL-33 antibody promoted astrocytes releasing more MCP-1/CCL2 and TNF-α.On the other hand,recombinant IL-33 supressed the release of MCP-1/CCL2 and TNF-α from astrocytes.Silmilar to astrocytes,IL-33 also inhibited TNF-α releasing from microgliaConclusion:1 IL-33 play a protective role in EAE2 In spleen and lymph nodes from EAE,IL-33 may promote the percentage of Foxp3+regulatory T cells and inhibited the percentage of effector T cells to contribute its protective role.3 In CNS from EAE,IL-33 may play a protective role by promoting Th2,suppressing the activation of the resident glia cells.On the other hand,IL-33 can also supress the release of MCP-1/CCL2 from astrocytes which contributing to the recruitment of macrophages,as well as supress the shift of microglia/macrophages toward M1-like phenotype.What’s more,IL-33 can also inhibit TNF-α releasing from astrocytes and microglia,thus decreasing the damage to myelin sheathPart 2The role of HMGB1 in experimental autoimmune encephalomyelitisBackground:High-mobility group box 1 protein(HMGB1)is a highly conserved protein found in the nucleus of nearly all eukaryotic cells.It is passively released from necrotic cells or actively secreted by damaged cells,and then transferred to the outside of the cell by the modification(mainly acetylation)to act as a proinflammatory role.It is a typical damage associated molecular pattern(DAMP).On the other hand,HMGB1 is also called amphoterin,which can facilitate neurite outgrowth in early development and play a prominent role in recovery from central nervous system injury.Sonic hedgehog(Shh)is a kind of secreted protein with covalent binding cholesterol,which is not only closely related to animal development in the early stage,but also closely related to endothelial cell permeability in the later stage.In experimental autoimmune encephalomyelitis(EAE),the destruction of blood-brain barrier(endothelial cell permeability increase)is a major pathological feature of the disease,suggesting that shh also plays an important role in the EAE.In previous study,HMGB1 was found to release largely during the peak stage of EAE acting as an proinflammatory role,but the study about its role in repairing damaged spinal cord is limited.Objective:To explore the protective role of HMGB1 in experimental autoimmune encephalomyelitis by its effect on the release of shh.Methods:1、The obtaining of primary astrocytes and the detection of its purity:referring to part1.2、The detection of HMGB1 receptors on the surface of the primary astrocytes:After climbed to the coverslip,the receptors were detected by immunofluorescence;or after obtaining protein from primary astrocytes,the receptors were detected by immunoblot assay,or after the single cells were obtained,flow cytometry(FC)were used to detect the expression of HMGB1 receptors on astrocytes3、The detection of shh:ELISA,western blot or Realtime PCR were used to detect the shh in supernatant or cells4、The acquisition and identification of different receptor knockout mice:TLR2,TLR4 and RAGE knockout C57BL/6 mice were obtained by gift or self-made.Then gene or FC were used to detect if the receptors were mutted successfully or expressed in cells5、The changes of downstream signal pathway of shh during the peak phase of EAE The frozen sections of the spinal cord tissue from naive and peak phase of EAE mice were used to detect the translocation of the key nuclear transcription factor Gli in the downstream signaling pathway of shhResults:1 The primary astrocytes could express three main receptors of HMGB1-TLR2,TLR4 and RAGE2 HMGB1 could promote the synthesis and release of shh in astrocytes,and the ability to promote shh release was blocked when HMGB1 was neutralized in the EAE tissue interstitium.3 The TLR2,TLR4 and RAGE knockout mice were established and identified successfully.4 When TLR2,TLR4 and RAGE receptors were knockout,the ability of astrocytes to release shh decreased in general,but in the same group of receptor(TLR2,TLR4)knock out cells,there was still more shh release except for the group of RAGE knock out cells,the release of shh in which does not increase when HMGB1 stimulated5 When the TLR4 receptor was blocked by TAK-2421,HMGB1 stimulation could increase the release of shh while when the RAGE receptor was blocked by FPS-ZM1,the release of shh was the same after HMGB1 stimulation6 Shh changes dynamically at different times of EAE in the interstitial fluid of brain,which reached to the highest on the peak phase of EAE.With the increase of shh level at the EAE peak phase,the transcription factor Gli in the downstream signaling pathway of shh was obviously translocated,that is,from the nucleus to the nucleusConclusion:1 HMGB1 can play a protective role in EAE by promoting the release of shh in astrocytes2 HMGB1 can promote the release of shh by receptor RAGE...