RANTES-induced invasion of Th17 cells into substantia nigra potentiates dopaminergic cell loss in MPTP mouse model of Parkinson’s disease

Abstract

Although Parkinson’s disease (PD) is a progressive neurodegenerative disease, the disease does not progress or persist in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model, the most common animal model of PD. Recently, we have described that supplementation of regulated on activation, normal T cell expressed and secreted (RANTES), a chemokine known to drive infiltration of T cells, induces persistent nigrostriatal pathology in MPTP mouse model. However, which particular T cell subsets are recruited to the substantia nigra (SN) by RANTES is not known. Here, by adoptive transfer of different subset of T cells from tomato red transgenic mice to MPTP-intoxicated immunodeficient Rag1^−/− mice, we describe that invasion of Th17 cells into the SN is stimulated by exogenous RANTES administration. On the other hand, RANTES supplementation remained unable to influence the infiltration of Th1 and Tregs into the SN of MPTP-insulted Rag1^−/− mice. Accordingly, RANTES supplementation increased MPTP-induced TH cell loss in Rag1^−/− mice receiving Th17, but neither Th1 nor Tregs. RANTES-mediated aggravation of nigral TH neurons also paralleled with significant DA loss in striatum and locomotor deficits in MPTP-intoxicated Rag1^−/− mice receiving Th17 cells. Finally, we demonstrate that levels of IL-17 (a Th17-specific cytokine) and RANTES are higher in serum of PD patients than age-matched controls and that RANTES positively correlated with IL-17 in serum of PD patients. Together, these results highlight the importance of RANTES-Th17 pathway in progressive dopaminergic neuronal loss and associated PD pathology.

1. Introduction

Parkinson’s disease (PD) is the most common movement disorder caused by progressive loss of dopaminergic (DAergic) neurons present in the substantia nigra pars compacta (SNpc) region of midbrain (Kish et al., 1988). Clinically, the disease is characterized by tremor, bradykinesia, rigidity and postural instability (Olanow and Tatton, 1999; Vila and Przedborski, 2004). Studies indicate that intracytoplasmic inclusions (Lewy bodies), mitochondrial dysfunction, and inflammation are the important pathological features of the disease (Alam et al., 1997; Borah and Mohanakumar, 2010; Dexter et al., 1989; Gonzalez-Hernandez et al., 2010; Przedborski, 2004; Schapira et al., 1989; Schulz-Schaeffer, 2010).

Recently we have described that infiltration of T cells to the injured site of the brain causes progressive loss of DAergic neurons and striatal dopamine (DA) in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-intoxicated mice (Chandra et al., 2016). We and others have also found that chemokines like RANTES are significantly up-regulated in both SNpc and serum of PD patients and hemiparkinsonian mice and monkeys (Chandra et al., 2016; Mondal et al., 2019; Roy et al., 2015b; Tang et al., 2014). However, in case of mice, the drastic up-regulation of these chemokines starts falling down after 1 day of MPTP administration eventually becoming normal on 7d of MPTP insult (Chandra et al., 2017). Accordingly, nigrostriatal pathology also vanishes in MPTP mouse model within a few weeks of insult (Chandra et at, 2017). However, supplementation of recombinant RANTES twice in a week causes persistent infiltration of peripheral T cells into the SN, potentiating glial activation and demise of tyrosine hydroxylase (TH) positive DAergic neurons in a progressive manner (Chandra et at, 2017). Consistent to an important role of RANTES, neutralization of RANTES by antibodies protects DAergic neurons against MPTP toxicity and corrects behavioral deficits in mice (Chandra et al., 2016). Numerous studies have also demonstrated the effect of particular subsets of CD4⁺ T cells on PD pathology. For example, Reynolds and co-workers have shown that Th17 cells exacerbate nigrostriatal pathology and that regulatory T cells (Tregs) suppress the effect of Th17 on DAergic cell death (Reynolds et al., 2010). The same group also demonstrates that invasion of Th2 cells does not protect the nigral DAergic neurons against MPTP-induced toxicity. It emphasizes that not all types of CD4⁺ T cells have roles in accelerating or resisting DAergic cell loss in SN of parkinsonian brains. Another report (Brochard et al., 2009) has shown that inhibition of CD4⁺ T cells, but not CD8⁺ cells, reduces the cell loss induced by MPTP in mice. Moreover, few findings describe that Th17 level and the proportion of Th1 and Th17 cells are markedly increased in peripheral circulation of PD patients than the other subtypes of Th cells (Chen et al., 2015; Yang et al., 2017). These studies support that higher production of particular T cell subtypes along with the infiltration into the brain are correlated with the demise of DAergic neurons in SN and that this event might be controlled by RANTES.

Therefore, here, we have investigated the role of RANTES in the recruitment of T cell subtypes in the SN of MPTP-injected Rag1^−/− mice. The findings reveal that RANTES preferentially helps the invasion of Th17 cells in the SN of MPTP-intoxicated animals. Abundance of Th17 cells in the SN potentiated MPTP-induced death of nigral DAergic neurons and DA deficit in striatum. Furthermore, levels of RANTES and Th17 cytokine IL-17 were significantly higher in serum of PD patients as compared to age-matched control subjects. A positive correlation between RANTES and IL-17 was also seen in serum of PD patients. These results identify Th17 cells as the primary T cell type playing an important role in RANTES-induced progressive death of DAergic neurons.

2. Materials and Methods

2.1. Reagents:

Roswell Park Memorial Institute (RPMI) medium were purchased from Mediatech (Washington, DC). Fetal bovine serum (FBS) was obtained from Atlas Biologicals (Fort Collins, CO). Antibiotic-antimycotic and MPTP were purchased from Sigma-Aldrich (St. Louis, MO). Recombinant mouse RANTES was purchased from R&D Systems (Minneapolis, MN). Anti-CD4 antibody was purchased from eBioscience. Rabbit anti-tyrosine hydroxylase (TH) antibody was purchased from Pel-Freeze biologicals (Rogers, AR). Cy2- and Cy5-conjugated antibodies were obtained from Jackson Immuno-Research Laboratories (West Grove, PA).

2.2. CD4⁺ T cell polarization into Th1, Th17 and Tregs and mouse treatment

T cell polarization into Th1, Th17 and Tregs was performed according to the protocol of Brüstle and colleagues (Brustle et al., 2012). Briefly, whole splenocytes were isolated from the spleen of adult Tomato red mice (Jackson Laboratory) and initially suspended in complete RPMI-1640 medium (Sigma-Aldrich) containing 10% FBS, 50 μM β-mercaptoethanol, 100 U/ml penicillin, and 100 mg/ml streptomycin. After 2 h of incubation at 37°C the non-adherent cells were taken out and plated in new culture dishes coated with 1 μg/ml anti-CD3 antibodies (BD Bioscience, San Jose, CA) to stimulate the T cells. The cells were further primed with anti-CD28 antibodies (BD Bioscience) added at a concentration of 1 μg/ml. The cells were maintained for 48 h in the culture dishes followed by polarization to Th1 or Th17 or Tregs by adding particular set of cytokines. Naive cells were induced to differentiate into Th1 type by adding 4 ng/ml IL-12 (eBioscience) plus 50 U recombinant human IL-2 (rhIL2, eBioscience); into Th17 cells by addition of 5 μg/ml anti-IFN-γ (R&D Systems), 30 ng/ml rhIL-6, 2 ng/ml rhTGF-β (eBioscience), and 50 U rhIL-2; into Tregs by adding 3 ng/ml rhTGF-β (eBioscience), 50 U rhIL-2, and 5 μg/ml anti-IFN-γ antibodies. Following the differentiation period, cells were collected and centrifuged at 500 xg for 10 min to pellet the cells. Cells were washed with sterile PBS twice and counted in hemocytometer. The polarized T cells were finally suspended in sterile PBS in such a way so that 10⁷ cells are present in 200 μl volume of PBS. This volume of PBS consisting of 10⁷ cells was adoptively transferred into each Rag1^−/− mice through the tail vein.

2.3. Flow cytometry

Two-color flow cytometry was performed as described previously (Mondal et al., 2017) to characterize the polarized Th1, Th17 and Tregs. For stimulation of cytokine production, Th cell subtypes were stimulated with 20 ng/ml PMA and 1 mM ionomycin (Sigma-Aldrich) for 5 h, cells were washed with PBS and kept in flow staining buffer containing diluted FITC-labeled anti-CD4 antibody for 30 min at 4°C. The cells were then washed and resuspended in fixation and permeabilization buffer. After 30 min of incubation in dark, cells were further washed, blocked with test Fc block (anti-mouse CD16/32) in permeabilization buffer, and subsequently incubated with appropriately diluted PE-labeled antibodies specific to Th1 specific marker IFN-γ, Th17 specific marker IL-17A and Treg specific marker Foxp3 at 4°C in the dark. After incubation, the cell suspension was centrifuged and washed thrice, and further suspended in flow staining buffer. The cells then were analyzed through FACS (BD Biosciences). Cells were gated based on morphological characteristics. Apoptotic and necrotic cells were not accepted for FACS analysis.

2.4. Animals

Tomato red transgenic (B6;129-Gt(ROSA)26Sor^{tm7(CAG-tdTomato*)Nat}/J) and Rag1^−/− (B6.129S7-Rag1^tm1Mom/J) mice were purchased from the Jackson Laboratory. Animal maintenance and experiments were done in accordance with National Institutes of Health guidelines and were approved by the Institutional Animal Care and Use committee of the Rush University Medical Center (Chicago, IL).

2.5. MPTP intoxication

Mice were intoxicated with MPTP (acute) as described earlier (Chandra et al., 2017; Ghosh et al., 2009; Khasnavis and Pahan, 2014; Khasnavis et al., 2013). Briefly, the mice were injected intraperitoneally (i.p.) with MPTP (18 mg/kg, Sigma-Aldrich, St. Louis, MO) four times at 2-h intervals. Saline was given as controls.

2.5. Treatment of MPTP-intoxicated mice with recombinant RANTLS

After 3 days of the last dose of MPTP treatment, Rag1^−/− mice were injected with 100 ng of recombinant mouse RANTES i.p. at 3 days interval. Recombinant RANTES was dissolved in sterile PBS in such a way so that each mouse received 100 ng of RANTES present in 100 μl of PBS (Chandra et al., 2017). Another MPTP-intoxicated mice group was also treated with only 100 μl of PBS/mouse.

2.6. Western blot analysis

Western blotting of TH was performed as described earlier (Khasnavis and Pahan, 2014; Patel et al., 2019). Briefly, tissues were homogenized in radioimmunoprecipitation assay (RIPA containing 50 mM Tris-HCl, 1 mM EDTA sodium salt, 150 mM NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate) buffer mixed with protease inhibitor cocktail. The homogenate was centrifuged at 17500 xg for 10 min at 4°C, supernatant was collected and protein was estimated. Equal amount of proteins were electrophoresed in 12% SDS-PAGE and transferred onto nitrocellulose membrane. The blot was probed with anti-TH (1:2000, Pel-freeze Biologicals) and anti-actin antibodies (1:10000, Abcam, Cambridge, MA) overnight at 4°C. Then corresponding infrared fluorophore-tagged secondary antibodies (1:10,000; Jackson Immuno-Research) were added at RT. Finally blots were scanned with an Odyssey infrared scanner (Li-COR, Lincoln, NE). Band intensities were quantified using ImageJ software (NIH, USA).

2.7. Immunostaining and counting of TH neurons in SN

Briefly, animals were perfused with 4% paraformaldehyde, and the brains were kept in 30% sucrose solution at 4°C. Using a cryotome, 30 μm coronal sections were cut from the midbrain region and processed for immunostaining. Sections were blocked with either 4% BSA in PBST containing 0.5% Triton X-100 (Sigma-Aldrich) and 0.05% Tween 20(Sigma-Aldrich) for 1 h. Then the samples were kept in primary antibodies for TH (1:1000) and CD4 (1:100) and incubated at 4°C temperature overnight under shaking conditions. Next day, the samples were washed with PBST for 30 min and further incubated with Cy2- or Cy5-labeled secondary antibodies (all 1:500; Jackson Immuno-Research) for 1 h under similar shaking conditions. Following four 15-min washes with PBST, sections were incubated for 5 min with 4’,6-diamidino-2-phenylindole(DAPI, 1:10,000; Sigma-Aldrich). The samples were run in an ethanol and xylene (Fisher) gradient, mounted, and observed under Olympus BX41 fluorescence microscope or by confocal microscope (Zeiss). TH immunohistochemistry in both SN and striatum was performed according to the protocol mentioned earlier (Chandra et al., 2017; Ghosh et al., 2009). Counting of TH neurons in SN of each hemisphere of brain was performed by using STEREO INVESTIGATOR software (MicroBrightfield, Williston, VT) having an optical fractionator. Every 6^th section from the starting of nigra (around 2.8 mm from bregma) was considered for counting of TH neurons (Naskar et al., 2015). TH fiber density in the striatum was measured by using Fiji software containing ImageJ 2 version.

2.8. HPLC analysis for measurement of striatal DA and its metabolite levels

Striatal level of DA, 3, 4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) was measured as mentioned earlier (Chandra et al., 2016; Chandra et al., 2017). In brief, after 7 days of MPTP treatment, mice were sacrificed by cervical dislocation and striatum was isolated from each mouse and sonicated in 0.2 M ice cold perchloric acid. The homogenate was centrifuged at 17500 xg for 15 min at 4°C. The supernatant was collected in a fresh tube and 10 μl of supernatant was injected in an Eicompak SC-3ODS column (Complete Stand-Alone HPLC-ECD System EiCOMHTEC-500; JM Science, Grand Island, NY). The level of neurotransmitters was analyzed according to the manufacturer’s protocol.

2.9. Behavioral analyses

Two major types of behavioral analyses were conducted after 7 days of the last dose of MPTP treatment. These are rotarod test for feet movement as described earlier (Ghosh et al, 2009) and open field experiment for locomotor activity of different groups of animals. Locomotor activity was monitored with a camera linked to Noldus system and EthoVisionXT software (Netherlands). The instrument records the overall movement abilities of the animals including total distance moved, velocity, total moving time, resting time, center time and frequencies of movement. Before any treatment, mice were placed inside the open-field arena for 10 min daily and on rotarod for 10 min daily for 3 consecutive days to train them and record their baseline values. Following the treatments, each mouse was taken from the cage and gently placed in the middle of the open field arena. After releasing the animal, data acquisition was started by the software for the next 5 min and the parameters related to the locomotor activities were collected by the software. For rotarod analysis, mouse was placed on the rotating rod, which rotates with a gradual increasing speed of 4-40 rpm. The latency time to fall from the rod onto the base of the instrument was monitored for each mouse (Prorok et al., 2019).

2.10. Serum samples of PD patients and healthy controls

Serum samples of PD patients and age-matched healthy controls were obtained from the Movement Disorders Clinic of the Rush University Medical Center.

Inclusion criteria for PD patients:

a) PD as defined by the UK PD Research Society Brain Bank criteria, b) Not on NSAIDs or other anti-inflammatory medications, c) Not on statins (cholesterol-lowering drugs) as we have found that statins are anti-inflammatory (Pahan et al., 1997; Roy et al., 2015a) and that statins protect dopaminergic neurons in mouse models of PD (Ghosh et al., 2009; Roy and Pahan, 2011), d) Not on melatonin as melatonin is also anti-inflammatory (Esposito and Cuzzocrea, 2010), e) Not received any hormone replacement therapy, f) Informed consent signed, g) No history of drug abuse, h) Not pregnant or lactating (if female).

Inclusion criteria for age-matched control subjects:

a) No clinical or laboratory supported signs of infection, b) No major illness within the last six months, c) Not on NSAIDs, other anti-inflammatory medications or sleep medication (melatonin), d) Not on statins, e) Not received any hormone replacement therapy, f) No history of drug abuse, f) Not pregnant or lactating (if female).

2.10. ELISA

Levels of RANTES and IL-17A in serum of patients with PD and control subjects were measured using human CCL5/RANTES ELIS A kit (R&D System, Minneapolis, MN) and IL-17A ELISA kit (Thermo Fisher Scientific, Waltham, MA) following manufacturers’ protocol.

2.11. Statistical Analysis

Statistics were performed using GraphPad Prism v7.0. Values are expressed as mean ± S.E.M. Statistical analyses for differences between two different samples were performed using Student’s t test. One way ANOVA followed by Tukey’s multiple comparison was performed while comparing multiple samples. This criterion for statistical significance was p < 0.05.

3. Results

3.1. Rag1^−/− animals resist MPTP-induced pathology

Acute MPTP treatment caused more than 50% loss of DAergic neurons in SN of WT C57BL6 mice (Fig 1A, B). The number of TH positive neurons in SN of each hemisphere of control WT mice was found to be 6877.40 ± 555.13, whereas it is reduced to 3716.43 ± 193.95 in MPTP-treated mice brain. Loss of TH positive neurons is also accompanied with significant reduction in the total level of TH protein level in SN of these mice (Fig 1C, D). In case of Rag1^−/− mice, loss of TH neurons was significantly less severe compared to WT mice. There was around 30% cell loss in SN of MPTP-intoxicated Rag1^−/− mice compared to the respective control (Fig 1A, B). Therefore, TH level in nigra is comparatively less depleted in Rag1^−/− mice following 7 days of toxin administration. The data exhibit that even after 7 days of MPTP treatment, Rag1^−/− mice have significantly higher level of nigral DAergic neurons (Fig 1B) as well as TH level (Fig 1D) than that of the MPTP-treated WT mice. Demise of SN DAergic neurons resulted in drastic loss of TH neuronal fibers in the striatum of MPTP-intoxicated WT animals, but remarkable protection of neuronal fibers in the striatum was observed in Rag1^−/− mice exposed to MPTP. Optical density (O.D.) of neuronal fibers showed almost 80% loss of TH fiber intensity in case of toxin treated WT mice compared to the control (Fig 1E, F). However, TH fibers in striatum were not significantly protected in case of Rag1^−/− animals following MPTP insult (Fig 1E, F). Death of TH neurons in the nigra and consequent loss of TH fibers in striatum resulted in more than 80% depletion in striatal DA level (Fig 1G), which is also concurrent with depleted level of DA metabolites DOPAC (Fig 1H) and HVA (Fig 1I). Expectedly, MPTP-induced Rag1^−/− mice resisted the loss of DA and its metabolites in the striatum. DA level was found to be depleted by 50% (Fig 1G), although it is significantly higher than that of the MPTP-treated WT animals (Fig 1H, I). Altogether the data suggest that immunodeficient Rag1^−/− mice are remarkably less susceptible to MPTP-induced toxicity.

Fig. 1.

Rag1^−/− mice resist MPTP-induced dopaminergic (DAergic) neuronal death. (A, B) 8-10 weeks old C57BL6 (WT) and Rag1^−/− mice were treated with MPTP (18 mg/kg; 4 doses, 2 h apart) and after 7 days mice were sacrificed. Ventral midbrain sections from each brain were immunostained for (A) tyrosine hydroxylase (TH) and (B) TH neurons were counted using the Stereo Investigator. TH immunostaining images were captured under a bright field microscope at 5X magnification, scale bar was 200 μm. (C, D) Total TH protein content in SN of control and MPTP-treated mice was assessed by immunoblotting. Ratio of band intensities of TH to Actin is shown. (E, F) Striatal sections of control and MPTP-treated groups of WT and Rag1^−/− mice were immunostained for TH. At least 3 sections per brain were used for immunostaining. Optical density (O.D.) of stained sections was measured by Fiji software. Relative O.D. as percentage of the respective control was plotted in the bar diagram. (G, H, I) Level of dopamine (DA), 3, 4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) was measured from the striatal tissues of different groups of mice by HPLC-ECD method. Content of DA and its metabolites are presented as ng/mg of tissue. *, ** and *** signify p< 0.05, p< 0.01 and p < 0.001 respectively. Statistical analyses were performed using one way ANOVA followed by Tukey’s multiple comparison tests. Data are represented as mean ± SEM (n=4 animals per group for each experiment).

3.2. RANTES does not affect DAergic neuronal loss in MPTP-challenged Rag1^−/− mice

Previous studies reported that RANTES aggravates MPTP-induced DAergic cell death in mice by promoting continuous T cell infiltration and enhanced gliosis in nigra of WT mice (Chandra et al., 2017). However, Rag1^−/− mouse lacks mature T lymphocytes and therefore it was rational to find out whether RANTES exhibits deleterious effect in promoting MPTP-induced PD pathology in Rag1^−/− mice. Rag1^−/− mice were intoxicated with MPTP followed by RANTES supplementation twice in a week. The findings demonstrated that similar to our earlier finding, MPTP caused around 30% loss of DAergic neurons in SN of Rag1^−/− mice (Fig 2A, B), but RANTES supplementation following MPTP intoxication could not aggravate DAergic cell death in SN. There was no significant difference in the number of DAergic cells in SN of MPTP and MPTP+RANTES treated groups of mice (Fig 2A, B). Similarly the level of nigral TH level was also found to be unaltered by RANTES supplementation in MPTP-intoxicated animals (Fig C, D). Demise of neurons in SN resulted in decrease of TH fibers in striatum of MPTP-treated animals (Fig 2E, F), although no further difference was observed in TH fiber density in RANTES supplemented animals. Consequently the level of striatal DA and its metabolites were also not deteriorated by RANTES supplementation compared to only MPTP-intoxicated group (Fig G-I). Motor performance of experimental animals was monitored initially by open field locomotor test, where no significant difference in locomotor activities was observed between control and MPTP-intoxicated groups (Fig 2J–M). But rotarod test exhibited compromised feet movement of MPTP-treated animals compared to the control Rag1^−/− mice. However, RANTES supplemented MPTP-intoxicated animals performed similar to the only MPTP-treated animals (Fig 2N). The findings revealed that RANTES could not influence MPTP-induced cell death and associated pathologies in Rag1^−/− mice.

Fig. 2.

RANTES does not affect MPTP-induced DAergic neuronal death in Rag1^−/− mice. Adult Rag1^−/− mice were injected with MPTP (18 mg/kg; 4 doses, 2 h apart) and those mice received two doses of 100 ng of RANTES via I.P. at 3 days interval. (A) Immunostaining shows TH neurons in SN of control, MPTP and MPFP+RANTES-treated mice. (B) TH neurons of every 6^th section from each brain were counted by Stereo Investigator. (C) Immunoblotting demonstrates TH protein level in SN of all groups of mice. Actin was used as the loading control. (D) Band intensities of TH and actin were measured by ImageJ and TH to actin ratio is shown in the diagram. (E) Striatal sections from each brain were immunostained for TH. (F) Relative O.D. of TH immunostaining with respect to the control is presented. (G, H, I) Levels of striatal DA, DOPAC and HVA were measured by HPLC-ECD method. (J) Heatmaps show locomotor activities of experimental animals over 5 min time period in the open field arena. (K, L, M) Velocity, distance and cumulative duration of movement of experimental animals are shown. (N) Rotarod exhibits the latency time taken by mice of each group to fall from the rotating rod. Statistical analyses were conducted using one way ANOVA followed by Tukey’s multiple comparison tests. *, ** and *** signify p< 0.05, p< 0.01 and p< 0.001 respectively. Data are represented as mean ± SEM (n=4 animals per group for each experiment).

3.3. Th1, Th17 and Treg polarization

Total splenocytes were isolated from Tomato red mice followed by polarization into Th1, Th17 and Treg cell rich population as described above. To further characterize the polarized T cells, flow cytometry was performed using CD4⁺ T cell subtype specific antibodies. Antibodies against IFN-γ, IL-17A and Foxp3 were used as the markers for Th1, Th17 and Treg populations, respectively. The data showed that Th1 specific polarization resulted in generation of 8.75 ± 0.56% CD4⁺IFN-γ⁺ cells from the naive splenocytes where the amount of Th1 cells was found to be only 0.48 ± 0.12% (Fig 3A–C). Similarly Th17 polarization resulted in generation of 16.74 ± 1.95% of CD4⁺IL-17A⁺T cells, whereas naive splenocytes contain only 0.19 ± 0.05% of Th17 cells (Fig 2D–F). Lastly, Treg polarization from the naïve splenocytes generated 19.5 ± 2.52% CD4⁺Foxp3⁺ cell population from the initial value of 6.01 ± 0.56% (Fig 2G–I). The data suggested successful enrichment of Th1, Th17 and Treg cells using the mentioned procedures. Each individual T cell enriched population was adoptively transferred into Rag1^−/− mice. To establish the effect of individual T cell subset on MPTP-induced toxicity, Rag1^−/− mice were reconstituted with each polarized T cell subset followed by acute MPTP insult and then these mice were treated with RANTES at 3 days interval (twice in a week). The objective was to understand that which particular T cell subset or subsets are recruited in the affected region of brain following RANTES supplementation and whether there is any effect of that T cell infiltration over MPTP-induced TH neuronal vulnerability in nigra. The whole experimental paradigm is given schematically in Fig 4A.

Fig. 3.

Th1, Th17 and Treg cell polarization assessed by flow cytometry. Splenocytes isolated from Tomato red (Tm red) mice were incubated under (B) Th1 (anti-CD3, anti-CD28, IL-12, and IL-2), (E) Th17 (anti-CD3, anti-CD28, IL-6, and TGF-β), and (F) Treg (anti-CD3, anti-CD28, TGF-β, and IL-2) specific differentiating conditions for 72 hours. (A, D, G) Control cells were kept with only anti-CD3 and anti-CD28 antibody containing medium. Following polarization, cells were stained with anti-CD4, and anti-IFN-γ (for Th1), with anti-CD4, and anti-IL-17A (for Th17), and with anti-CD4, and anti-Foxp3 (for Treg) antibodies for flow cytometric analysis. (C, F, I) Levels of IFN-γ, IL-17 and Foxp3 among CD4⁺ T cells are shown by representative FACS analysis and mean fluorescence intensity (MFI). Significance of mean between groups was tested with paired t-test with equal variances. *** indicates p<0.001 compared to respective control groups (n=2 replicates/dose in 3 independent experiments), data are presented as mean ± STDEV.

Fig. 4.

Infiltration of Th1 cells in brain of Rag1^−/− animals. Adult 8-10 weeks old Rag1^−/− mice received 10⁷ number of Th1 or Th17 or Treg enriched cells. Next day, those mice were injected with four doses of MPTP at 18 mg/kg dose I.P., 2 h apart. RANTES (100 ng/mouse) was administered into MPTP-induced mice twice in a week at 3 days interval. Behavioral experiments were conducted on 7^th day after the last dose of MPTP intoxication. Mice of all groups were sacrificed and neurotransmitter analysis was done from striatal tissues, TH immunostaining and immunoblotting were conducted from SN tissues and infiltration of T cells in SN was evaluated by CD4 staining. (B) Immunofluorescence staining of TH (green) in nigra of Th1 cell supplemented control, MPTP and MPTP+RANTES treated mice demonstrates presence of red tagged T cells (Tm red) in the brain parenchyma. Arrows show the red signals emanating from the surface of DAPI positive nucleus of the T cells. Merged pictures represent 40X magnification images, which was further zoomed to 80X for clear visualization of T cells. Scale bars were set as 20 μm and 10 μm for smaller and higher magnification images respectively. (C) Immunofluorescence images show presence of CD4 +ve (green) and Tm red +ve T cells in SN of experimental mouse brains. Counting of CD4 +ve cells was performed in control, MPTP and MPTP+RANTES treated brain sections and plotted as number of T cells per mm² of tissue. Total 10 sections (2 sections per brain) from each group of mice were counted. One way ANOVA followed by Tukey’s multiple comparison tests was performed for statistical analysis. *** indicates p< 0.001, data are represented as mean ± SEM (n=5 animals per group).

3.4. MPTP-induced infiltration of Th1 cells in nigra is independent of RANTES

Rag1^−/− mice were injected with 10⁷ Th1 polarized cells and the following day these mice received acute MPTP insult. RANTES was administered twice in a week at 3 days interval. After 7 days of MPTP insult, the mice were sacrificed and presence of Tomato (Tm) red cells in brain parenchyma of SN was visualized by fluorescence immunostaining. Tomato mice derived cells are demarcated as red fluorescence positive cells where the fluorescence signal come from the membrane of these cells identified around the DAPI positive nucleus of each cell (arrows Fig 4B). The data showed that saline treated Rag1^−/− mice contained very low level of Tm red cells in SN and the fluorescence signal appears to be very weak. But there was considerable increase in the number of Tm red cells in SN of MPTP treated mice compared to the saline treated control group. Even after RANTES supplementation similar abundance of Tm red positive cells were found in SN of MPTP-intoxicated mice. The brain sections were also stained with anti-CD4 antibodies to validate the presence of T cells in SN of all the experimental groups of mice. The result demonstrated that the number of CD4 +ve cells in nigra was increased by more than 2-fold in nigra (81.06 cells/mm²vs 39.39 cells/mm² in control) after MPTP intoxication (Fig 4C, D). However, RANTES supplementation in MPTP-intoxicated mice did not influence Th1 cell infiltration in the brain (90.9 cells/mm²) as similar number of CD4 +ve cells were found in SN of this group of animals as that of the only MPTP-intoxicated mice (Fig 4D). The data reveal that MPTP-induced pathology is accompanied by trafficking of peripheral Th1 cells in nigra, but further RANTES supplementation perhaps does not help in more Th1 cell invasion in the brain.

3.5. Invasion of Th17 cells in SN increases following RANTES supplementation

Th17 cells are another major subtype of inflammatory T cell that has the propensity of invading CNS and exacerbating disease pathology in autoimmune diseases like multiple sclerosis. Therefore, similar experiment was conducted with Th17 polarized cells to find out whether exogenous RANTES treatment has any effect in facilitating infiltration of these cells in the brain of MPTP-intoxicated mice. Our results showed that MPTP-treated mice had significantly higher number of Tm red cells in SN of MPTP-treat mice than the control (Fig 5A). Higher Th17 cell invasion was also confirmed by CD4 staining, which exhibited 2-fold increase in number of T cells in nigra of MPTP-intoxicated mice (75.10 cells/mm² vs 30.75 cells/mm² in control; Fig 5B, C). Interestingly, RANTES supplementation further increases the number of infiltrating T cells in brain of MPTP-induced animals. The number of existing Th17 cells in nigra of RANTES treated group of animals was found to be 107.37 cells/mm² (Fig 5B, C). The data indicate that RANTES has significant influence in brain infiltration of Th17 cells following the injury caused by MPTP.

Fig. 5.

RANTES-induced increased Th17 invasion in brain of MPTP-intoxicated Rag1^−/− animals. (A) TH immunoreactive DAergic neurons (green) were colocalized with Tm red cells in SN of Th17 cell supplemented control, MPTP and MPTP+RANTES treated mouse brains. White arrows indicate Tm red cells present in the vicinity of TH neurons (green). Smaller and higher magnification images were captured at 40X and 80X magnification and the scale bars are 20 μm and 10 μm respectively. (B) CD4 +ve cells are shown in SN of control, MPTP and MPTP+RANTES treated mice. Counting of CD4 +ve cells in nigra was performed by ImageJ and individual values per mm² of tissue from each brain are presented. Total 10 sections (2 sections per brain) from each group of mice were counted. One way ANOVA followed by Tukey’s multiple comparison tests was performed for statistical analysis. ** and *** indicate p< 0.01 and p< 0.001 respectively, whereas ‘ns’ stands for statistically nonsignificant . Data are represented as mean ± SEM (n=5 animals per group).

3.6. Infiltration of Tregs into brain is not effected by either MPTP or by RANTES supplementation

It was previously shown that Tregs are protective against parkinsonian neurotoxins like MPTP and these cells can even salvage the deleterious effects of other inflammatory immune cells for attenuating disease progression in PD. However, in the present study no considerable up-regulation of Tregs was observed in nigra of MPTP-intoxicated animals. The number of existing Tm red cells found in SN of toxin-induced animals was almost similar to that of the untreated control mice (Fig 6A). Moreover, RANTES supplemented MPTP-intoxicated mice also showed no alteration in the number of Tregs present in nigra (Fig 6A). CD4 staining also validated the previous observation that the number of Tregs present in nigra of only MPTP-induced mice or RANTES supplemented MPTP-intoxicated mice is same as the control mice (Fig 6B, C). It signifies that invasion of Tregs is not influenced in any way by MPTP-induced toxicity.

Fig. 6.

Infiltration of Tregs is unaltered by MPTP treatment. (A) Immunofluorescence pictures show presence of Tm red cells near TH neurons in SN of Treg supplemented control, MPTP and MPTP+RANTES groups of mice. (B) CD4+ve T cells merged with Tm red signal are shown in SN of mice brains. Number of CD4 +ve cells in nigra was counted by ImageJ and individual values per mm² of tissue from each brain are presented. Total 10 sections (2 sections per brain) from each group of mice were counted. Difference in CD4 +ve cell number among the groups is shown as ‘ns’. Data are represented as mean ± SEM (n=5 animals per group).

3.7. Increased infiltration of Th17 cells in brain by RANTES aggravates DAergic cell death

We evaluated the number of TH positive DAergic neurons in SN of different groups of mice by immunohistochemistry followed by counting of surviving neurons in one hemisphere of brain. Rag1^−/− mice injected with Th1 cells were found to have around 40% loss of nigral DAergic neurons following MPTP treatment (6775.21 ± 189.64 for saline-treated animals and 4050.172 ± 248 for MPTP-treated animals, Fig 7A, B). Animals treated with RANTES following the MPTP administration also showed around 40% loss of nigral TH neurons compared to the saline-treated control mice (4158.522 ± 302.62, Fig 7B). But in case of mice receiving Th17 polarized T cells, RANTES was found to increase MPTP-induced DAergic neuronal death. In only MPTP-induced mice, there was around 25% cell loss in SN (5474.862 ± 471.94) compared to the control mice (7347.65 ± 525.16, Fig 7A, C). But mice treated with MPTP followed by RANTES exhibited more than 50% loss of TH neurons in SN (3591.242 ± 405.49), which was even remarkably higher than the only MPTP-intoxicated animals (Fig 7C). Lastly, the experiment was also performed in Treg polarized cells treated animals. However, in these groups of mice including only MPTP and MPTP+RANTES treated animals, DAergic neuronal loss in SN was much less compared to the other groups of mice. Only MPTP and MPTP+RANTES treated mice did show 26% (5224.892 ± 292.49) and 30% (4970.21 ± 290.22) loss of nigral TH neurons with respect to the saline treated control animals (7145.738 ± 314.64, Fig 7A, D). It signifies that the extent of MPTP-induced TH neuronal loss is considerably less severe in Rag1^−/− mice receiving exogenous Treg polarized cells and certainly RANTES supplementation did not exert further toxicity on the DAergic neurons.

Fig. 7.

RANTES exacerbates MPTP-induced DAergic cell death in Rag1^−/− mice receiving Th17 cells. Th1, Th17 and Treg cell supplemented Rag1^−/− mice were treated with MPTP followed by RANTES administration twice in a week at 3 days interval. (A) Ventral midbrain sections were immunostained for TH and (B-D) every 6^th section from starting of SN (total 5 sections per brain) was counted from each brain. (E-G) TH protein level from nigra of Th1, Th17 and Treg supplemented group of animals was monitored by immunoblotting. (H-J) Normalized TH band intensity with respect to actin is shown in bar diagrams. Statistical analysis was performed using one way ANOVA followed by Tukey’s multiple comparison tests. *, ** and *** signify p< 0.05, p< 0.01 and p< 0.001, respectively. Data are represented as mean ± SEM (n=5 animals per group).

The whole findings were further substantiated by monitoring TH protein level in SN by immunoblotting. Mice receiving Th1 cells had significant loss of nigral TH level after MPTP treatment and this loss was persistent in animals administered with both MPTP and RANTES (Fig 7E, H). Down-regulation of TH level was also found in MPTP-intoxicated mice receiving Th17 polarized cells (Fig 7F, I). However, following RANTES supplementation, these animals experienced much more pronounced TH loss in SN compared to the only MPTP-treated animals (Fig 7F, I). This finding also correlates well with the data obtained from TH neuronal counting in these groups of mice emphasizing that RANTES potentiates MPTP-induced DAergic cell loss in Rag1^−/− animals receiving Th17 cells. Tregs were found to be comparatively protective against MPTP-induced nigral TH loss. Only MPTP or MPTP+RANTES treated mice receiving prior administration of Tregs had subtle loss in TH level compared to the saline treated control animals (Fig 7G, J).

3.8. RANTES-induced exaggerated DA depletion in striatum of MPTP-treated animals receiving Th17 cells

DAergic neurons of SN project the neuronal terminals to the striatum (caudate and putamen) of basal ganglia. Therefore we have measured the level of striatal DA and its metabolites to corroborate the data obtained from nigral TH loss in different groups of animals. The data demonstrated that Th1 cells treated mice had almost 80% loss of DA level in striatum following MPTP insult. RANTES treatment did not further perturb reduced level of DA and its metabolites in MPTP-intoxicated animals (Fig 8A, D, G). Depletion of DA in striatum is well correlated with the TH neuronal loss, as MPTP and (MPTP+RANTES)-treated animals exerted similar extent of degeneration in SN. However the effect of RANTES on MPTP-induced toxicity was much more pronounced in case of animals receiving Th17 cells. Here, MPTP caused 50% loss in striatal DA level (Fig 8B), accompanied with reduced level of DOPAC and HVA (Fig 8E, H). Interestingly, RANTES treatment following MPTP increased the extent of DA loss to 80% in striatum and it was significantly less compared to the only MPTP-intoxicated animals (Fig 8B). On the contrary, in case of Treg polarized cells treated group, there was around 45% loss in striatal DA level in MPTP-treated and MPTP+RANTES treated animals (Fig 8C). Although DOPAC and HVA level were reduced following MPTP insult in these groups of animals, the values were statistically insignificant compared to the saline treated control (Fig 8F, I). It signifies that presence of increased Tregs could make these mice less susceptible to MPTP, where RANTES treatment did not have any additive effect on the toxin-induced neuronal death.

Fig. 8.

Enhanced loss of striatal DA and its metabolites by RANTES in MPTP-intoxicated Rag1^−/− mice supplemented with Th17 cells. Th1, Th17 and Treg supplemented control, MPTP and MPTP+RANTES treated animals were sacrificed on 7^th day after MPTP treatment, striatum was isolated from each brain and the level of (A-C) DA, (D-F) DOPAC and (G-I) HVA was measured by HPLC-ECD method and presented as ng/mg tissue. Statistics was conducted by one way ANOVA followed by Tukey’s multiple comparison tests. *, ** and *** signify p< 0.05, p< 0.01 and p< 0.001, respectively. Data are represented as mean ± SEM (n=5 animals per group).

3.9. Loss of DA in striatum results in movement deficit in MPTP and RANTES treated animals receiving Th17 cells

Behavioral tests were performed to find out the functional aspect of DA loss in striatum of different groups of animals. For that purpose, horizontal locomotor activities and rotarod tests were conducted for all these experimental groups of mice. It was found that mice treated with Th1 cells exerted reduced velocity and total distance covered in the open field arena following only MPTP and MPTP+RANTES administration (Fig 9A, D, G). Rotarod analysis exhibited significant movement deficit in MPTP-treated animals as these mice were unable to perform on the rotating rod for longer time and felt down much earlier compared to the saline treated control animals (Fig 9J). In case of mice receiving Th17 enriched cells, MPTP treatment could not cause significant motor deficits compared to the control. This group of mice, although experiencing 50% striatal DA loss, performed well in the open field locomotor test (Fig 9B, E, H) as well as during the rotarod test (Fig 9K). However, following RANTES supplementation in this group of mice, drastic deterioration of motor performance was observed. The velocity of movement and the total distance covered in the arena were significantly lower in the MPTP+RANTES treated group compared to only MPTP-treated groups (Fig 9E, H). In addition, these mice showed poor performance in rotarod indicating significant motor deficits exerted by RANTES treatment (Fig 9K). In case of mice treated with Treg enriched cells, there was no significant deficit in behavioral activities found in only MPTP or MPTP+RANTES treated animals. The protection of TH neurons and striatal DA level in Tregs-treated animals are perhaps responsible for better performance in both open field and rotarod tests (Fig 9C, F, I, L).

Fig. 9.

RANTES treatment compromises motor performance of Th17 supplemented MPTP-intoxicated mice. Th1, Th17 and Treg supplemented control, MPTP and MPTP+RANTES groups of mice were subjected to open field locomotor activities and rotarod tests on 7^th day after MPTP intoxication. (A-C) Heatmaps exhibit the horizontal locomotor activities of experimental animals in the open field arena as captured by the Noldus software. Parameters related to movement of animals were obtained from the software and presented as (D-F) velocity, (G-I) distance moved for all the groups of mice. (J-L) Rotarod test exhibits the feet movement of animals on the rotating rod and the latency time taken by each mouse to fall on the base was monitored. Statistics was conducted by one way ANOVA followed by Tukey’s multiple comparison tests. *, ** and *** signify p< 0.05, p< 0.01 and p< 0.001, respectively. Data are represented as mean ± SEM (n=5 animals per group).

3.10. Increased level of RANTES and IL-17A in serum of PD patients

Next, to understand the significance of our finding in PD, we measured levels of RANTES and Th17 specific cytokine IL-17A by ELISA in serum of PD patients (n=9) and age-matched controls (n=9) (Table 1). There was no significant difference between PD patients and healthy controls in terms of age and race (Table 1). Details of the PD patients and the control subjects including the UPDRS score and H & Y are also provided in Table 1. Results showed that levels of both RANTES [F_1,16=14.25 (>F_c=4.49) and **p<0.01 (=0.0016543)] and IL-17A [F_1,16=7.28 (>F_c=4.49) and *p<0.05 (=0.0157)] were significantly up-regulated in serum of PD patients as compared to age-matched controls (Fig 10A, B). Moreover, Pearson correlation analysis showed that there was a positive correlation (R=0.4903; p=0.0240) between RANTES and IL-17A in serum of PD patients (Fig 10C), suggesting a possible crosstalk between RANTES and Th17 cells in the disease process.

Table 1.

Levels of RANTES and IL-17A in serum of patients with Parkinson’s disease and control subjects

Samples	Gender	Age	Ethnicity	UPDRS	H & Y Stage	RANTES (pg/ml)	IL-17A (pg/ml)
PD-1	M	79	Hispanic	21	2	1081±128.26	5.40±0.54
PD-2	M	42	White	9	1	832.75±132.9	5.40±0.47
PD-3	F	73	White	25	2	821±148.14	8.77±0.63
PD-4	M	52	African American	25	2	727.75±132	5.94±0.81
PD-5	M	65	White	12	0	833±207.85	3.90±0.41
PD-6	M	60	White	15	2	679±342.44	4.53±0.47
PD-7	F	58	White	16	2	835±289.3	8.12±0.13
PD-8	M	54	White	40	2	659±283.62	8.45±0.13
PD-9	F	61	White	13	2	1209±329.5	6.38±0.64
Cont-1	F	54	White	NA	NA	649±193.43	5.40±0.79
Cont-2	F	34	White	NA	NA	331±141	5.40±0.23
Cont-3	M	65	White	NA	NA	825±192.17	5.08±0.23
Cont-4	M	61	White	2	1	589±81.56	4.31±0.41
Cont-5	M	44	White	1	0	331±55.51	3.66±1.58
Cont-6	F	71	White	5	0	637.75±132.19	6.38±0.91
Cont-7	F	70	White	1	0	349±63.29	2.25±0.25
Cont-8	F	NA	White	NA	NA	901±60.46	3.55±0.56
Cont-9	F	74	White	6	1	787±252.87	3.01±0.27

Serum samples were obtained from the Movement Disorders Clinic of the Rush University Medical Center. Each sample was analyzed for RANTES and IL-17A three times by ELISA. UPDRS, Unified Parkinson’s disease Rating Scale; H&Y, Hoehn and Yahr; NA, not available; PD, Parkinson Disease; Cont, Control.

Fig. 10.

Up-regulation of RANTES and IL-17A in serum of PD patients. Level of (A) RANTES and (B) IL-17A in serum of 9 PD patients and 9 age-matched controls was measured by ELISA. (C) Correlation was drawn between the levels of these cytokines. * indicates p< 0.05 compared to the control. Pearson coefficient (r) refers to the linear correlation between the level of IL-17A and RANTES.

4. Discussion

The role of adaptive immune response as an important player in promoting PD pathologies was first convincingly shown in MPTP-intoxicated mice (Brochard et al., 2009). It states that brain infiltration of peripheral immune cells has major effect in nigral DAergic cell loss. Moreover, only CD8⁺ and CD4⁺ T cells, but not B lymphocytes invade the brain during disease progression in MPTP mouse model and also in human PD brains. The study by these researchers also confirmed that only CD4⁺ T cells significantly influences nigrostriatal pathology in PD. Although CD8⁺ T cells are also found to invade the brain upon MPTP insult, these are comparatively less in number and CD8a^−/− mice show comparable loss of TH neurons upon MPTP insult like the WT mice (Brochard et al., 2009). However, the role of CD4⁺ T cell infiltration in SN in terms of disease progression and persistence was unknown. Our previous finding on adaptive immune response in PD provided the first indication that chemokines such as RANTES and eotaxin are markedly up-regulated in SN and serum of MPTP-intoxicated mice as well as hemiparkinsonian monkeys (Roy et al., 2015b). It was also found that microglia are the primary cells producing these chemokines in brain following MPTP insult. These chemokines facilitate the brain infiltration of peripheral T cells and blockade of these chemokines by neutralizing antibodies was found to remarkably reduce the number of infiltrating T cells in the brain of MPTP-treated mice, eventually attenuating nigral DAergic neuronal loss and associated PD pathologies (Chandra et al., 2016).

Although MPTP-intoxicated mouse is the most widely-used animal model of PD, nigrostriatal pathology does not persist for long time in this model. However, exogenous RANTES and eotaxin supplementation in MPTP-intoxicated mice causes irreversible loss of nigral DAergic neurons along with depletion of striatal DA level. The persistent PD pathology also coincides with continuous T cell trafficking in the brain by exogenous RANTES and eotaxin supplementation. It signifies that chemokine-induced T cell brain infiltration is responsible for progressive nature of neuronal death in PD. There are four major subtypes of T cells present in the peripheral immune system, therefore, it was very important to see brain infiltration of which particular T cell subset is facilitated by exogenous RANTES supplementation.

The present study shows that infiltration of only Th17 polarized cells, but neither Th1 nor Treg, cells is increased in SN of MPTP-intoxicated mice following RANTES supplementation. RANTES, also known as chemokine (C-C motif) ligand 5 (CCL5), binds to its receptor CCR5 present on circulating immune cells in periphery and causes migration and homing of T cells, monocytes, basophils, eosinophils and NK cells (Appay and Rowland-Jones, 2001). Sato and coworkers initially reported that human Th17 cells are identified as CCR2⁺CCR5⁻ cells. It indicates that Th17 cells lack CCR5 required for responding to RANTES effect to infiltrate into different organs (Sato et al., 2007). Later, more in depth studies on identification of trafficking receptors expressed on Th17 cells revealed that this group of cells is present in the peripheral blood and in the secondary lymphoid organs in a heterogenous and diverse fashion. Their report stated that developing Th17 cells highly express several Th1 specific (CCR2, CCR5, CXCR3, CXCR6) and Th2-associated (CCR4) trafficking receptors (Lim et al., 2008). Our current study clearly suggests that trafficking of Th17 cells into the non-lymphoid organ (e.g. CNS) can be made possible under the effect of RANTES.

How Th17 cells may cause DAergic cell death? According to Liu and coworkers, Th17 cells can trigger DAergic cell death via crucial interaction of LFA-1 and ICAM (Liu et al., 2017). Other reports have suggested that Th17 cells can impart neurotoxicity in more diverse manner via secretion of different pro-inflammatory cytokines. In addition to IL-17, Th17 cells also produce IL-8, IL-21, IL-22, IL-26, TNF-α, GM-CSF, etc. that allow the recruitment of neutrophils into inflammatory sites (Annunziato et al., 2012; Maddur et al., 2012). Moreover, Th17-secreted IL-21 can promote differentiation and proliferation of brain residual CD8⁺ T cells, IL-8 production from other antigen presenting cells, which in turn secrete more pro-inflammatory molecules like IL-6 and IL-23 in the injured site of the CNS (Peters et al., 2011). Exaggerated generation of IL-6 and IL-23 further helps proliferation and survival of infiltrated Th17 cells, resulting in creation of more hostile environment for the damaged neurons in the brain (El-Behi et al., 2011). This diverse mode of action of Th17 cells made them a prime target for alleviating pathogenesis in several autoimmune diseases (Ghoreschi et al., 2011). Therefore, it might be a possibility that RANTES-induced infiltration of Th17 in SN induces the canonical Th17 specific pro-inflammatory pathways to cause neuronal death in an irresistible fashion.

On the other hand, infiltration of Th1 cells was not found to be further enhanced by exogenous RANTES supplementation. It is known that Th1 cells are the primary T lymphocytes, which express RANTES receptor CCR5 on its membrane. The endogenous generation of RANTES by brain microglia and the gratuitous up-regulation of RANTES in the blood of MPTP-treated mice might be sufficient to drive the peripheral CD4⁺ Th1 cells in the injured site of nigra at the initial phase and that causes acute and severe death of TH neurons during the early period of MPTP-intoxication. However, this population of Th1 cells starts dropping after 3 days MPTP intoxication. Therefore, further RANTES supplementation does not increase the Th1 cell population in SN, suggesting for the presence of other possible regulatory mechanisms. It should also be noted that in the present study, infiltration of Th2 polarized cells and its brain invasion with respect to RANTES supplementation have not been addressed. A study conducted by Gendelman and coworkers has shown that adoptive transfer of Th2 polarized cells neither increases nor prevents MPTP-induced nigral TH neuronal loss (Reynolds et al., 2010). Our findings also demonstrated that Rag1^−/− mice receiving Treg polarized cells were much resistant to MPTP toxicity. However, very few CD4⁺ cells were observed in SN of MPTP-treated mice that received adoptive transfer of Treg polarized cells. It gives rise to the possibility that the neuroprotective effect might be due to the suppressive effect of Tregs on other activated T cell-driven inflammation via secretion of TGF-β and IL10 at the early stage (Kipnis et al., 2004), which perhaps prevents pro- inflammatory glial response in MPTP-treated mice. In a study of invasion of Tregs in the brain over 1 month period after large hemispheric lesions in ischemia, it has been shown that Treg invasion in the brain does not happen immediately after injury, rather it starts increasing after 14 to 30 days following the injury (Stubbe et al., 2013). It is speculated that following any brain injury Tregs play important immunomodulatory roles in the peripheral immune system, which supports our present finding as no significant increase in Treg cell number was observed in SN of MPTP-intoxicated animals within 7 d time period of the experiment.

In summary, RANTES augments preferential accumulation of Th17 cells over other subtypes of T-helper cells in the brain of MPTP-induced animals. Furthermore, our finding is also substantiated by the observed positive correlation between higher level of RANTES and Th17 specific cytokine IL-17 in serum of PD patients. Therefore this study is the first report providing the indication that RANTES up-regulation can be one of the driving factors behind the enhanced level of Th17 cells in PD patients. Moreover, some recent studies have also detected higher percentage of Th17 cells in the peripheral system of newly-detected PD patients (Kustrimovic et al., 2018). Persistent infiltration of Th17 cells could be the immune factor responsible for progressive demise of DAergic neurons in SN of MPTP-intoxicated animals. Therefore, blocking of Th17 infiltration in brain or targeting Th17 initiated cell death pathways might be used as a therapeutic strategy to prevent progressive nature of DAergic neuronal death in PD.

Abbreviations:

RANTES

Regulated on activation, normal T cell expressed and secreted

Rag1

Recombination activating gene 1

MPTP

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

Th17

T-helper 17

Tregs

Regulatory T cells

TH

Tyrosine hydroxylase

SN

Substantia nigra