Roles Of Th17and Treg Cells In Neuroinflammation And Dopaminergic Neuron Loss Of Parkinson’s Disease Model Mice And Mechanisms Involved

Objectives:T helper (Th)17cells and regulatory T (Treg) cells are two subsets of CD4+Tlymphocytes newly defined until the end of the last century. Th17cells are highlyproinflammatory and play a vital role in chronic inflammatory and autoimmunity diseases.Treg cells, known to suppress immune activation, are committed to maintain immunehomeostasis and tolerance. Recently, the immune dysfunction has been reported toparticipate in pathogenesis of the neurodegenerative disease--Parkinson’s disease (PD).Mounting evidence suggests that innate neuroinflammatory processes associated withmicroglial cells excessive activation result in the progression of dopaminergic cell death.However, the role and mechanism of the adaptive immune cells, Th17cells and Treg cells,in PD remain enigmatic. Herein, we explore the effects of Th17cells and Treg cells onneuroinflammatory processes and dopaminergic neurons loss and reveal the underlyingmechanisms of these two subpopulations of CD4+T cells from glial cells activation anddirect contact medicated by membrane molecules or transmembrane molecules. Further,our data may provide a rationale for targeting the adaptive arm of the immune system as atherapeutic strategy in PD.Methods:1. Make the PD model mice: C57BL/6mice received4i.p. injections of1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)(20mg/kg) at2-hour intervals to induce PD.Saline-injected and naive mice were used as controls.2. The PD mice models were evaluated: On days2and7after the last injection,rotarod test, pole test and open-field test were employed to estimate balance andcoordination performance; immunofluorescence histochemical staining was applied todetect the number of tyrosine hydroxylase (TH)-positive neurons in the substantia nigra compact part (SNpc); dopamine (DA) content in the striatum was measured by highperformance liquid chromatography (HPLC).3. Th17and Treg cells infiltration into the brain parenchyma in the PD mice wasexamined: On days2and7after the last injection, FITC-labeled albumin was slowlyinfused into the carotid artery. In the SNpc, blood-brain barrier (BBB) integrity wasassessed by detection of parenchymal extravasation of FITC-albumin. Besides, Ventralmidbrain sections were double-immunofluorescence stained for CD4and RORγt or Foxp3,a specific transcriptional factor of Th17and Treg cells, respectively.4. The functions of Th17cells and Treg cells were detected: On days2and7afterthe last injection, the expression of Th17cells-related transcriptional factors, RORγt, andpro-inflammatory cytokines, interleukin (IL)-17and IL-22, and Treg cells-relatedtranscriptional factors, Foxp3, and anti-inflammatory cytokines, transforming growthfactor (TGF)-β and IL-10, in the substantia nigra (SN) were assessed by real-time PCR andWestern blot. Besides, double-immunofluorescence was applied to determine theexpression and co-localization of IL-17and TGF-β in RORγt+Th17cells, Foxp3+Tregcells, CD11b+microglia, GFAP+astrocytes, or NeuN+neurons, respectively, in the SNpcof PD mice.5. Neuroinflammation mediated by glial cell activation in PD mice was evaluated:On days2and7after the last injection, the expression of inflammatory mediators--tumornecrosis factor (TNF)-α, IL-1β, interferon (IFN)-γ, and inducible nitric oxide synthase(iNOS), and neurotrophic factors--glial cell derived neurotrophic factor (GDNF) and brainderived neurotrophic factor (BDNF) in the SN was detected by real-time PCR and Westernblot assay. Besides, double-immunofluorescence was applied to determine the expressionand co-localization of TNF-α and GDNF in microglia and astrocytes, tagged with CD11band GFAP, respectively, in the SNpc of PD mice.6. Make PD cell model in vitro: Fetuses obtained from pregnant C57BL/6mouse onthe13th gestational day were used for preparation of ventral mesencephalic neuron-glialcultures. The cultures were exposed to the neurotoxin1-methyl-4-pheryl pyridinium ion(MPP+)5μM for24h, the number of TH-immunreactive dopaminergic neurons andNeuN-positive non-dopaminergic neurons was tested by immunocytochemical staining.7. The effects of Th17cells and Treg cells on neuroinflammation anddopaminergic neurons loss induced by MPP+were determined: Th17cells were enriched with polarization of CD4T cells in vitro, and CD4CD25Treg cells werepurified by using a Treg cell isolation kit. The polarized Th17cells or isolated Treg cellswere added into the primary mesencephalic neuron-glial cultures1h before MPP+(5μM)was applied, which were incubated for24h. The number of dopaminergic neurons andnon-dopaminergic neurons was counted by immunocytochemical staining; the expressionof the pro-inflammatory cytokines, TNF-α and IL-1β, and the neurotrophic factors,insulin-like growth factor (IGF)-1, GDNF, and BDNF, was measured by real-time PCR andWestern blot.8. The aggravating effects of Th17cells interaction with dopaminergic neuronson neurotoxicity induced by MPP+were evaluated: The ventral mesencephalicneuron-enriched cultures were prepared from the midbrains of embryonic day (E)13mice.Double-immunofluorescent staining for LFA-1and ICAM-1with RORγt and TH wasdetected in the Th17cell-neuron cocultures. The neurons, in the presence or absence ofneutralizing anti-ICAM-1antiboby, were treated with Th17cells, which was neutralizedwith LFA-1monoclonal antibody or not, for1h followed by MPP+(5μM) administration,the number of TH+NeuN+dopaminergic neurons and TH-NeuN+non-dopaminergicneurons was counted by immunocytochemical staining; the levels of phosphorylated JNKand the expression of activator protein-1(AP-1, such as c-Jun and c-Fos), MMP-9andcaspase-3were detected by Western blot assay. Besides, live cell imaging of cell-cellcontacts was processed in the Th17cell-neuron cocultures.9. The alleviative effects of Treg cells interaction with dopaminergic neurons onneurotoxicity induced by MPP+P were evaluated: The ventral mesencephalicneuron-enriched cultures and the isolated Treg cells were prepared as described above.Double-immunofluorescent staining for CD47and signal regulatory protein α (SIRPA)with Foxp3and TH and live cell imaging of cell-cell interactions were processed in theTreg cell-neuron cocultures. Treg cells, with or without CD47gene knockdown, wereadded into the neuron cultures, which were transfected with SIRPA-shRNA lentiviralvector or not. MPP+(5μM) stimulated the co-cultures1h later. Immunocytochemical stainwas employed to detect the number of dopaminergic and non-dopaminergic neurons,respectively; GST pull-down assay was used to examine the expression of activeGTP-Rac1; Western blot was applied to estimate the levels of phosphorylated Akt and theexpression of Bcl-xl and activated caspase-3. In addition, the neuron cultures, pretreated with Rac1activation inhibitor NSC23766for30min, were exposure to Treg cells for1hfollowed by MPP+(5μM) application. The number of dopaminergic and non-dopaminergic neurons and the expression of phosphorylated Akt, Bcl-xl and caspase-3with various treatments were evaluated as above.Results:1. MPTP induces PD-like changes of mice: The MPTP injection led to decreases inlatency to fall off rotarod, in number of moving through grids in open field, in number ofTH-positive cells in the SNpc, and in content of DA in the striatum, and increase in poletest time when compared with intact or saline-treated animals. And these effects werelarger on day7than on day2post-MPTP injection. Therefore, degeneration of thenigrostriatal dopaminergic pathway in PD can be recapitulated in MPTP-intoxicated mice.2. Disruption of BBB and Th17and Treg cells invaded into the brainparenchyma: The PD mice showed an evident BBB leakage and CD4+T cells’ infiltrationinto brain parenchyma in the SNpc on day7after MPTP treatment. CD4/RORγt andCD4/Foxp3double-stained cells were seen in the SNpc in PD mice.3. The dysfunction of Th17and Treg cells in PD mice: There was an increase inRORγt, IL-17, and IL-22, mRNA and protein expression expression in the SN on day7after MPTP compared with that on day2after MPTP and of controls; the expression ofTGF-β and IL-10was markedly decreased within the SN in MPTP-treated mice; but therewas no notable change in Foxp3expression. Besides that, upregulated IL-17wasco-expressed in RORγt+Th17cells and GFAP+astrocytes in the SNpc of PD mice;Co-localization of TGF-β with Foxp3+Treg cells, NeuN+neurons, CD11b+microglial, andGFAP+astrocytes in the SNpc was observed, which was decreased after MPTP exposure.4. There was neuroinflammation in the PD mice, which was induced byactivation of glial cells: The mRNA and protein expression of neurotrophic factors,GDNF and BDNF, was markedly decreased within the SN in MPTP-treated mice, andMPTP intoxication enhanced a broad spectrum of pro-inflammatory mediators, includingTNF-α, IL-1β, and iNOS. Pro-inflammatory cytokine TNF-α principally overlapped withactivated microglial and astrocytes following MPTP exposure. GDNF expression was alsovisible on microglia and astrocytes.5. In PD cell model, Th17cells exacerbated dopaminergic neurodegeneration,upregulated expression of inflammatory mediators, and downregulated expression of neurotrophic factors induced by MPP+, and Treg cells attenuated these effects ofMPP+: In vitro application of MPP+induced TH+neuron loss in primary ventralmesencephalic neuron-glial cultures, which was blocked by isolated CD4+CD25+Tregcells administration. In addition, co-culture with Tregs was effective in attenuating cellularexpression of a host of inflammatory mediators (TNF-α and IL-1β) in response to toxicantand counteracted MPP+-induced reduction in the expression of all neurotrophic factors(GDNF, BDNF, and IGF-1). In the presence of MPP+, Th17cells stimulation of culturedventral mesencephalic cells exacerbated the imbalance between pro-/anti-inflammatoryresponse and further decreased dopaminergic neuron survival. More importantly, Tregcells reversed IL-17cells’ induction effect on inflammation and dopaminergic neuron lossin MPP+-treated cultures.6. Th17cells aggravated MPP+-induced neurotoxicity by direct contact withdopaminergic neurons: We found that LFA-1was mainly co-localized with Th17cellsand ICAM-1was co-localized in TH+dopaminergic neurons and also had captured live cellimaging of interaction between a LFA-1-overlapped Th17cell with a ICAM-1-expressingmesencephalic neuron in the cocultivations. After the exposure of ventral mesencephalicenriched neurons to Th17cells, MPP+stimulation aggravated the dopaminergic neuronloss and enhanced the levels of phosphorylated JNK and the expression of AP-1, MMP-9and activated caspase-3. Blockade of LFA-1and ICAM-1activity in Th17cells andneurons, separately, reduced the neurotoxicity and the activation effects of Th17cells onJNK/AP-1signal pathway and pro-apoptotic proteins. These results show that Th17cellsare permissive for a potent underlying pro-apoptotic response to neurodegeneration.7. Treg cells attenuated MPP+-induced neurotoxicity by direct interaction withdopaminergic neurons: A novel finding was that CD47and SIRPA, transmembranemolecules known as the ligand and receptor, are principally expressed in Treg cells andTH+dopaminergic neurons, separately, which could serve as a mechanistic link for Tregcells remission of neurotoxic responses. Interestingly, live cell imaging reveals closeappositions between the soma or neurite of neurons and Treg cells. Actually, Treg cellstreatment of the ventral mesencephalic neuron-enriched cultures significantly increased thenumber of TH-positive neurons compared with the MPP+toxicity, but Treg cells could notrescue the less of TH-positive cells when we knockdown the CD47which exist in Tregcells or the SIRPA in neurons. In addition, Silencing of CD47gene in Treg cells or knockdown of SIRPA gene in neurons suppresses the effects of Treg cells on elevatingactivation levels of Rac1, phosphorylation levels of Akt, and expression of Bcl-xl, and ondeclining expression of caspase-3in neurons. Similarly, Rac1activation inhibitorNSC23766abolishes Treg cells prevention from MPP+-induced dopaminergic neuron lossand induction in prominent phosphorylation level of Akt and expression of anti-apoptoticprotein Bcl-xl and in depressed expression of pro-apoptotic protein caspase-3.Conclusions:Collectively, our findings suggest that Th17cells and Treg cells infiltrate the sites ofinjury in PD progression, where the dysfunction between the pro-and anti-inflammatoryadaptive immune cells may result in neuroinflammation and neurodegeneration; in vitro,Th17cells exacerbated and Treg cells attenuated dopaminergic neuron loss induced byMPP+, which is mediated through altering the expression of inflammatory andneurotrophic factors initiated by glial cells and by direct contact with neurons; underlyingthe contact mechanisms, the neurotoxicity response of Th17is,conferred by direct contactwith ICAM-1on dopaminergic neurons via LFA-1and the neuroprotective response ofTreg cells is exerted by direct action on SIRPA in dopaminergic neurons by CD47; Th17cells activate JNK/AP-1signal pathway in neurons by LFA-1interaction with ICAM-1onneurons, which products a pro-apoptotic activity, and Treg cell-mediated combination byCD47with SIRPA in neurons participates in prevention dopaminergic neurons fromapotosis via activating Rac/Akt signal pathway, these results suggest a direct cross talkbetween neurons and Th17/Treg cells. Altogether, these results obtained in a murine PDmodel may participate in the understanding of Th17and Treg cells’ function inneurodegenerative diseases.