Treg Cells Attenuate Neuroinflammation And Protect Neurons In A Mouse Model Of Parkinson’s Disease

Objectives:CD4⁺CD25⁺regulatory T（Treg）cells are one of the subsets of helper T（Th）cells.Treg cells have high affinity to autoantigen and can suppress proliferation and activation of effector T cells by direct contact or by secreting anti-inflammatory cytokines such as transforming growth factor（TGF）-βand interleukin（IL）-10.Therefore,Treg cells are committed to maintain immune homeostasis and tolerance.Recently,peripheral and central immune dysfunction has been reported to participate in pathogenesis of the neurodegenerative disease--Parkinson’s disease（PD）.Mounting evidence suggests that microglial cells excessive activation and peripheral effector T cells through the blood brain barrier into the brain result in the progression of dopaminergic cell death.However,the role and mechanism of the adaptive immune cells,Treg cells,in PD remain enigmatic.Herein,we explore the effects of Treg cells on neuroinflammatory processes and dopaminergic neurons loss by adoptive transfer of Treg cells,and reveal the underlying mechanisms involved.Further,our data may provide a rationale for targeting the adaptive arm of the immune system as a therapeutic strategy in PD.Methods:1.Make the PD model mice:C57BL/6 mice received 4 i.p.injections of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine（MPTP）（20 mg/kg）at 2-hour intervals to induce PD.Saline-injected and naive mice were used as controls.2.The functions of adoptive transfer of Treg cells in PD neuroinflammation were detected:CD4⁺CD25⁺Treg cells were purified by using a Treg cell isolation kit.12 h after MPTP injection,CD3/CD28-actived Treg cells were adoptively transferred to MPTP-lesioned mice by the tail vein injection.On day 3 after MPTP injection,rotarod test,pole test and open-field test were employed to estimate balance and coordination performance;immunofluorescence histochemical staining was applied to detect the number of tyrosine hydroxylase（TH）-positive neurons in the substantia nigra compact part（SNpc）;dopamine（DA）content in the striatum was measured by high performance liquid chromatography（HPLC）.Real-time PCR,Western blot and ELISA were applied to analyze the relative expression levels and the secretion of Th17 cell-related cytokines（IL-17 and IL-22）,Treg cell-related cytokines（TGF-βand IL-10）,inflammatory mediators（IFN-γ,TNF-αand IL-1β）and neurotrophic factors IGF-1.Besides,immunofluorescence was applied to determine the expression of CD11b in microglia and GFAP in astrocytes,to observe the activation of microglias and astrocytes in the SNpc of PD mice.3.The functions of adoptive transfer of Treg cells in PD peripheral inflammation were detected:On 7 after the last injection,mononuclear cells were isolated from the spleen or mesenteric lymph nodes,and serum from inner canthus vein blood was collected.Expressions of the cytokines including IFN-γ,IL-2,IL-4,IL-17,IL-22,IL-10,TGF-βat mRNA and protein levels and concentrations of cytokines in serum were measured.Expressions of the specific-transcription factors including T-bet,GATA3,ROR-γt and Foxp3,for Th1,Th2,Th17 and Treg cells,respectively,were determined.Besides,the percentages of CD3⁺CD4⁺T lymphocytes,CD4⁺IFN-γ⁺（Th1）cells,CD4⁺IL-4⁺（Th2）cells,CD4⁺IL-17⁺（Th17）cells and CD4⁺CD25⁺（Treg）cells in lymphoid tissues of PD mice were determined.In addition,after adoptive transfer of Treg cells,spleen and lymph nodes were rapidly removed,real-time PCR and Western blot were applied to analyze the relative mRNA and protein expressions of the pro-inflammatory cytokines including IFN-γ,IL-2,IL-17 and IL-22.4.The mechanisms of Treg cells infiltration into the brain parenchyma in the PD mice was examined:12 h after MPTP injection,Treg cells were adoptively transferred to MPTP-lesioned mice by the tail vein injection.On day 3 after MPTP injection,FITC-labeled dextran was slowly infused into the carotid artery.Leakage FITC-linked dextran was observed in the fixed frozen SNpc sections under a fluorescent microscope.Western blot was applied to analyze the expressions levels of two tight junction protein,ZO-1 and occluding in the SNpc.Blood-brain barrier（BBB）integrity was assessed by detection of parenchymal extravasation of FITC-dextran and the two tight junction protein levels in the SNpc.Besides,Treg cells,with or without anti-LFA-1 antibody（5μl/ml）,were adoptively transferred into PD mice,which were achieved anti-ICAM-1 antibody i.v.injection or not.Glial cells and leukocytes were seperated from the SN tissue and were subjected to measure the percentage of CD4⁺CD25⁺Foxp3⁺Treg cells by flow cytometry analysis.5.The transwell co-culture of Treg cells on dopaminergic neurons loss induced by MPP⁺were determined:Activated Treg cells were rinsed and resuspended in complete DMEM medium at a density of 5×10⁵ cells/ml,which was transferred into a transwell cell culture chamber,bottom of which was comprised of a membrane with0.4-μm pore size.The transwell chamber containing Treg cells was placed into the 24-well culture plate containing seven-day-old ventral mesencephalic neurons.The ratio of Treg cells to ventral mesencephalic neurons was 1:1.After co-culture for 1 h,MPP⁺（5μM）was applied to ventral mesencephalic neurons in the lower 24-well plate.After 24 h of incubation,the transwell chamber was removed,and the number of dopaminergic neurons and non-dopaminergic neurons was counted by immunocytochemical staining;the secretion of Treg cell-related cytokines,IL-10 and TGF-β1 in neuron cultures was measured by ELISA.6.The alleviative effects of Treg cells interaction with MN9D DA neuronal cell line on neurotoxicity induced by MPP⁺were evaluated:MN9D cells,a fusion of embryonic ventral mesencephalic and neuroblastoma cells,are extensively used as a model of DA neurons because it expresses TH and synthesizes and releases DA.The MN9D cells were cultured in RPMI-1640 medium with 15%fetal bovine serum in a humidified 5%CO₂ atmosphere at 37°C.The isolated Treg cells were prepared as described above.Double-immunofluorescent staining for CD45 and galectin1 with Foxp3 and TH,and live cell imaging of cell-cell interactions were processed in the Treg cell-MN9D cell cocultures.Treg cells,with or without CD45 inhibitor,were added into the MN9D cell cultures,which were transfected with galectin1-miRNA plasmid vector or not.MPP⁺（50μM）stimulated the co-cultures 1 h later.TH immunostaining was employed at 48 h after MPP⁺administration to detect MN9D cell loss.Annexin V/PI assay,TUNEL staining and the LDH release assay were employed to determine cell apoptosis and necrocytosis.Besides,Western blot was applied to analyze the expressions levels of cleaved-caspase-3 and cleaved-caspase-9.Results:1.Adoptive transfer of Treg cells to PD mice preventes decrease in DA content in the striata,the loss of TH positive neurons in SNpc and the behavior changes in PD mice.The MPTP injection led to decreases in latency to fall off rotarod,in number of moving through grids and the average velocity in open field,in number of TH-positive cells in the SNpc,and in content of DA in the striatum when compared with intact or saline-treated animals.More importantly,adoptive transfer of Treg cells attenuated these effects of MPTP.2.Adoptive transfer of Treg cells to PD mice inhibits activation of microglias and astrocytes in SNpc,preventes changes of cytokine expression and secretion in the substantia nigra:There was an activation of microglias and astrocytes in SNpc in PD mice.The mRNA and protein expressions and protein secretion of Th17 cell-related cytokines（IL-17 and IL-22）and inflammatory mediators（IFN-γ,TNF-αand IL-1β）were increased,while the Treg cell-related cytokines（TGF-βand IL-10）and neurotrophic factors IGF-1 were decreased in the substantia nigra on day 3 after MPTP compared with controls.Adoptive transfer of Treg cells attenuated these effects of MPTP.3.Disruption of BBB and the mechanisms of Treg invaded into the brain parenchyma:The PD mice showed an evident BBB leakage and the decreased expressions of two tight junction proteins,ZO-1 and occludin.CD4/Foxp3 double-stained cells and the percentage of CD4⁺CD25⁺Foxp3⁺Treg cells by flow cytometry analysis were increased in the SNpc after adoptive transfer of Treg cells in PD mice.Interestingly,blockade the LFA-1 activity in Treg cells or the ICAM-1 activity in endothelial cells prevents Treg cell migration across the BBB.4.Treg cells do not significantly prevent MPP⁺-induced dopaminergic neuronal loss in transwell co-culture:In the lack of Treg cell treatment,the cytokines TGF-β1 and IL-10 were not able to be detected in the supernatants of lower ventral mesencephalic neuron cultures.In contrast,in Treg cell co-culture with ventral mesencephalic neurons via the transwell system,both TGF-β1 and IL-10 levels significantly increased.Importantly,MPP⁺-induced TH⁺NeuN⁺cell loss in the lower ventral mesencephalic neuron cultures was not significantly ameliorated by the Treg cell transwell treatment.For TH^–NeuN⁺cells,the treatments with MPP⁺or Treg cell transwell co-culture did not significantly alter the cell number compared with untreated control.5.Treg cells attenuated MPP⁺-induced neurotoxicity by direct interaction with dopaminergic neuron Th17 cells aggravated MPP⁺-induced neurotoxicity by direct contact with dopaminergic neuron:A novel finding was that CD45 and galectinq,transmembrane molecules known as the ligand and receptor,are all principally expressed in Treg cells and TH⁺dopaminergic MN9D cells,which could serve as a mechanistic link for Treg cells remission of neurotoxic responses.Interestingly,live cell imaging reveals close appositions between the MN9D cells and Treg cells.Actually,Treg cells treatment of MN9D cell cultures significantly increased the number of TH-positive MN9D cells,decreased the percentages of apoptotic and necrotic cells compared with the MPP⁺toxicity,but Treg cells could not rescue the less of TH-positive cells when we inhibit CD45 which exist in Treg cells or knockdown galectin1 in neurons.In addition,inhibition of CD45 in Treg cells or knockdown of galectin1 gene in MN9D cells suppresses the effects of Treg cells on depressed expression of pro-apoptotic protein cleaved-caspase-3 and cleaved-caspase-9.6.Adoptive transfer of Treg cells to PD mice inhibits the increase of pro-inflammatory cytokine expressions in spleen and lymph nodes:Expression of the pro-inflammatory cytokines,IFNγ,IL-2,IL-17 and IL-22,in the spleen and mesenteric lymph nodes was upregulated and their concentrations in serum were elevated in PD process.But,the anti-inflammatory cytokines,IL-4,IL-10 and TGF-βwere not altered in the two lymphoid tissues or serum of PD mice.In addition,expression of T-bet,the specific transcription factor of Th1 cells,was downregulated,but expression of Foxp3,the transcription factor of Treg cells,was upregulation.In support of the results,CD4⁺IFN-γ⁺cells（Th1 cells）was reduced but CD4⁺CD25⁺cells（Treg cells）was elevated in both the lymphoid tissues of PD mice.Conclusions:1.Adoptive transfer of Treg cells attenuated neuroinflammation and neurodegeneration in PD mice.2.PD mice showed the disruption of BBB and Treg cells invaded into the brain parenchyma after adoptive transfer.LFA-1 in Treg cells and the ICAM-1 in endothelial cells mediated Treg cells infiltrate.3.The neuroprotective response of Treg cells is exerted by direct action on galectin1in dopaminergic cells by CD45.4.Adoptive transfer of Treg cells inhibits peripheral inflammation in PD mice.