Protective Effects Of Regulatory T Cells On Myelin Injury In Central Nervous System Disorders

Objective:Demyelination occurs in multiple diseases in central nervous system(CNS),which is tightly associated with neuroinflammation.As the major immunosuppressive cells in the body,regulatory T cells(Tregs)play an essential role in maintaining homeostasis and have afforded protection in diverse diseases via the effects of anti-inflammation and proregeneration.This study aims to investigate the roles of Tregs in myelin injury induced by chronic cerebral hypoperfusion and lysophosphatidylcholine(LPC),and to uncover the underlying mechanisms.Methods:This study applied bilateral carotid artery stenosis(BCAS)and LPCdemyelination model to imitate chronic cerebral hypoperfusion and focal demyelination in corpus callosum(CC)respectively.To confirm the successful hypoperfusion of BCAS,laser speckle contrast imaging was used to monitor cerebral blood flow.Flow cytometry was applied to detect the dynamic alterations of lymphocytes in spleens and brains.For Tregs depletion,Foxp3-diphtheria toxin receptor(DTR)mice were treated with diphtheria toxin(DT),wild type(WT)C56BL/6 mice were treated with anti-CD25 antibody,respectively.Immunofluorescence,Western blot,quantitative real-time PCR(qRT-PCR),luxol fast blue(LFB)staining and electron microscopy were used to evaluate the effects of Tregs depletion on myelin injury and neuroinflammation in BCAS and LPC-induced demyelination.Meanwhile,Morris water maze and novel object recognition tests were applied to test the effects of Tregs depletion on cognitive defects after demyelination induced by BCAS and LPC.In LPC-induced demyelination,RNA-sequencing was further used to uncover the mechanism how Tregs regulate myelin injury.Results:The results of laser speckle contrast imaging confirmed BCAS was successfully established.The proportions of lymphocytes in spleen and brain after BCAS were comparable to those in sham group.BCAS induced decreased mature oligodendrocytes,microglial activation,increased expressions of inflammatory cytokines,myelin injury and cognitive dysfunction.Compared with mice without Tregs depletion,depletion of Tregs further decreased the number of mature oligodendrocytes and anti-inflammatory microglia,and upregulated the expressions of inflammatory cytokines in CC while had no effects on myelin injury and cognitive function.On the other side,LPC induced focal demyelination successfully.Peripheral immune cells infiltrated into demyelinated lesion induced by LPC.At LPC10d,great loss of mature oligodendrocytes,microglial activation and pyroptosis,largely increased inflammatory cytokines in demyelinated lesion were observed,and mice manifested cognitive dysfunction.Tregs depletion augmented peripheral immune cells infiltration,expressions of pro-inflammatory cytokines,microglial activation,microgliosis as well as microglial pyroptosis,and exacerbated the severity of myelin injury and cognitive defects in LPC-induced demyelination.However,the pyroptosis inhibitor VX765 partially reversed myelin injury and cognitive defects which were exacerbated by Tregs ablation.Further RNA-sequencing suggested an important role of Toll-like receptor 4(TLR4)/myeloid differentiation marker 88(MyD88)in regulation of pyroptosis by Tregs,and inhibiting TLR4 by TAK-242 verified that refraining TLR4/MyD88/NF-κB pathway in mice with Tregs ablation alleviated microglial pyroptosis.Conclusions:Tregs regulate neuroinflammation in BCAS and LPC-induced demyelination.However,Tregs have marginal effects on myelin injury in BCAS while could protect against myelin injury and cognitive defects by alleviating microglial pyroptosis via TLR4/MyD88/NF-κB pathway in LPC-induced demyelination.Tregs may be the potential therapeutic target of myelin injury in CNS disorders.Part Ⅰ:Effects of Tregs on chronic cerebral hypoperfusion and the related mechanismsObjective:To establish BCAS model and explore the immune responses after chronic cerebral hypoperfusion.To investigate the effects of Tregs on myelin injury,neuroinflammation,construction and function of white matter after BCAS and the possible molecular mechanisms.Methods:BCAS was performed in C57BL/6 mice.Dynamic alterations of CD3+T cells,CD4+T cells and Tregs in spleen and CNS were examined by flow cytometry at 14 and 28 days after BCAS.Next,BCAS was performed after Tregs depletion either by DT treatment in Foxp3-DTR mice or anti-CD25 antibody treatment in WT C57BL/6 mice.Immunofluorescence,Western blot,qRT-PCR,LFB staining and electron microscopy were applied to explore the effects of Tregs depletion on microglial activation and phenotypes,expressions of pro-inflammatory and anti-inflammatory cytokines,the number of oligodendrocytes and severity of myelin injury after BCAS.Moreover,Morris water maze and novel object recognition tests were performed to investigate the effects of Tregs depletion on cognitive function of mice at BCAS28d.Results:Laser of speckle contrast imaging confirmed BCAS was successfully established by showing that cerebral blood flow after BCAS decreased to 53%of the baseline.Compared with sham group,the proportions of CD3+T cells,CD4+T cells and Tregs in spleen after BCAS were not statistically significant,and the proportions of CD4+T cells and Tregs in brain were comparable between sham group and BCAS group at 28 days after surgery.BCAS induced decrease in mature oligodendrocytes,microglial activation,increased expressions of inflammatory cytokines,damage to myelin integrity,reduced thickness of the myelin sheath in CC,and finally impaired the cognitive function in mice.Depletion of Tregs further decreased the number of mature oligodendrocytes as well as antiinflammatory microglia,and augmented the expressions of inflammatory cytokines including tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),interleukin-6(IL-6)and interferon-γ(IFN-γ),while had no effects on pro-inflammatory microglia in CC.On the other side,Tregs ablation had marginal effects on the expressions of myelin-associated glycoprotein(MAG)and myelin basic protein(MBP),the thickness of myelin sheath and severity of white matter injury as well as the cognitive defects after BCAS indicated by neurobehavioral assessments.Conclusions:Depletion of Tregs decreases the number of mature oligodendrocytes and anti-inflammatory microglia in CC,and aggravates neuroinflammation while has no effects on myelin injury and cognitive defects after BCAS.Part Ⅱ:Tregs protect against LPC-induced demyelination by regulating pyroptosisObjective:To establish LPC-induced focal demyelination model and study the characteristic of immune responses after focal demyelination in CC.To explore the roles of Tregs in myelin injury,neuroinflammation,pyroptosis and cognitive function and to study the roles of pyroptosis in myelin injury regulated by Tregs in LPC-induced demyelination.Methods:Focal demyelination in CC was performed in Foxp3-DTR transgenic mice by stereotactic injection of LPC.Immunofluorescence was used to detect infiltration of CD45+leukocytes,CD3+T cells and Tregs in lesion.DT was injected intraperitoneally to deplete Tregs.Immunofluorescence,Western blot,qRT-PCR and LFB staining were applied to investigate the effects of Tregs depletion on myelin injury,neuroinflammation and pyroptosis in LPC-induced demyelination.The influence of Tregs depletion on cognitive function was assessed by Morris water maze and novel object recognition tests.Pyroptosis inhibitor VX765 was used to identify the role of pyroptosis in myelin injury regulated by Tregs.Results:LPC triggered focal demyelination and cognitive defects successfully.Peripheral CD45+leukocytes,CD3+T cells and Tregs infiltrated into demyelinated lesion induced by LPC.At LPC10d,decrease in mature oligodendrocytes,proliferation of oligodendrocyte precursor cells(OPCs),microglial activation,abundant inflammatory cytokines in demyelinated lesion were observed,accompanied by cognitive defects in mice.Results of Western blot and qRT-PCR showed increased expressions of diverse inflammasomes and pyroptosis-related proteins in demyelinated lesion.Results of immunofluorescence showed caspase-1,GSDMD,NLRP3 and IL-1β were mainly localized in the cytoplasm of microglia,implying microglia underwent pyroptosis in demyelinated lesion.Depletion of Tregs aggravated infiltration of peripheral immune cells,expressions of pro-inflammatory cytokines,microglial activation,microgliosis as well as microglial pyroptosis,finally aggravated the severity of myelin injury and cognitive defects in LPC-induced demyelination.Moreover,treatment with pyroptosis inhibitor VX765 inhibited microglial pyroptosis successfully and partially reversed myelin injury and cognitive defects which were exacerbated by Tregs ablation.Conclusions:Tregs protect against myelin injury and improve cognitive function by alleviating microglial pyroptosis in LPC-demyelination.Part Ⅲ:The related mechanisms how Tregs regulate pyroptosis in LPCinduced demyelinationObjective:To establish LPC-induced focal demyelination model and explore the underlying mechanisms how Tregs regulate pyroptosis in focal demyelination in CC.Methods:Stereotactic injection of LPC in CC was performed in Foxp3-DTR transgenic mice to induce focal demyelination.DT was injected intraperitoneally to deplete Tregs.RNA-sequencing in demyelinated lesions was further applied.Kyoto Encyclopedia Of Genes and Genomes(KEGG)and Gene Ontology(GO)enrichment analyses were performed in differentially expressed genes(DEGs).Protein-protein interactions(PPI)and key driver analysis(KDA)were applied to identify the key molecules,and corresponding molecular inhibitor was further used to verify the underlying mechanisms how Tregs regulate pyroptosis.Results:RNA-sequencing revealed that DEGs were mainly enriched in immune system and inflammatory response,with Nod-like receptor signaling pathway top ranked.PPI and KDA analyses implied that TLR4/MyD88 played a central role in regulation of pyroptosis by Tregs.Moreover,TLR4 inhibitor TAK-242 prohibited TLR4/MyD88/NF-κB pathway and alleviated microglial pyroptosis in LPC-demyelinated mice with Tregs depletion.Conclusions:Tregs inhibit microglial pyroptosis via TLR4/MyD88/NF-κB pathway in focal demyelination induced by LPC.