Regulatory T cells attenuate Th17 cell-mediated nigrostriatal dopaminergic neurodegeneration in a model of Parkinson's disease

Abstract

Nitrated alpha-synuclein (N-alpha-syn) immunization elicits adaptive immune responses to novel antigenic epitopes that exacerbate neuroinflammation and nigrostriatal degeneration in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of Parkinson's disease. We show that such neuroimmune degenerative activities, in significant measure, are Th17 cell-mediated, with CD4(+)CD25(+) regulatory T cell (Treg) dysfunction seen among populations of N-alpha-syn-induced T cells. In contrast, purified vasoactive intestinal peptide induced and natural Tregs reversed N-alpha-syn T cell nigrostriatal degeneration. Combinations of adoptively transferred N-alpha-syn and vasoactive intestinal peptide immunocytes or natural Tregs administered to MPTP mice attenuated microglial inflammatory responses and led to robust nigrostriatal protection. Taken together, these results demonstrate Treg control of N-alpha-syn-induced neurodestructive immunity and, as such, provide a sound rationale for future Parkinson's disease immunization strategies.

FIGURE 1. N-α-syn adaptive immunity accelerates MPTP-nigrostriatal degeneration

Photomicrographs and enumeration of Mac-1⁺, FJ-C⁺, TH⁺ neurons and CD4⁺ T cells in the SN of mice treated with PBS, MPTP, or MPTP and N-4YSyn SPCs. Shown are midbrains from mice 2 days after MPTP treatment and immunostained for (A) Mac-1⁺ reactive microglia (scale bar, 200 μm) with estimated numbers of Mac-1⁺ microglia per mm²; and (B) stained for FJ-C⁺ neurons (scale bar, 200 μm) with estimated total numbers of FJ-C⁺ neurons. (C) From the midbrains of mice 7 days post MPTP treatment, dopaminergic neurons in the SN were identified as TH⁺Nissl⁺ neurons, while non-dopaminergic neurons were identified as TH⁻Nissl⁺ neurons (scale bar, 200 μm). Total numbers of nigral neurons were determined and represented as mean number of TH⁺Nissl⁺ neurons (black bars and TH⁻Nissl⁺ neurons (gray bars). (D) Infiltration of CD4⁺ T cells (arrow heads) within the SN of mice 7 days following MPTP-intoxication (scale bar, 100 μm) and total numbers of CD4⁺ T cells within the SN were estimated. (A, B, C, and D) Differences in means (± SEM, n=7 mice per group) were determined, where P<0.05 compared with groups treated with ^aPBS or ^bMPTP. Concentration of cytokines in culture supernatants of (E) anti-CD3 stimulated naïve (black bars) and N-4YSyn T cells (white bars) and (F) N-4YSyn-stimulated N-4YSyn effector CD4⁺ T cells. (G) Assessment of Treg-mediated inhibition of effector T cell proliferation by Treg sorted from naïve (black bars), PBS/adjuvant- (PBS/Adj, gray bars), or N-4YSyn/adjuvant- (N-4YSyn/Adj, white bars) immunized FoxP3-GFP transgenic mice. (E and G) Differences in means (± SEM) were determined where P < 0.05 compared with Treg from ^anaïve mice or ^bPBS/Adj immune mice, (n=4).

FIGURE 2. Th17 immunity amplifies N-α-syn nigrostriatal degeneration

(A) Experimental design for in vitro polarization and adoptive transfers of CD4⁺ T cells of a Th1, Th2 or Th17 phenotype. Donor mice were immunized with N-4YSyn in adjuvant and CD4⁺ T cells were polarized in culture with indicated cytokine/anti-cytokine cocktails in the presence of N-4YSyn to yield N-4YSyn Th1-, Th2-, or Th17-enriched T cells. Recipient mice were intoxicated with MPTP and polarized T cell subsets were adoptively transferred to separate recipient groups at 12 hours post-MPTP treatment. Survival of nigrostriatal dopaminergic neurons and termini were evaluated 7 days post MPTP-treatment. (B) Concentration of cytokines produced by CD4⁺ T cells isolated from donor N-4YSyn immunized mice and cultured for 5 days under nonpolarizing control conditions (Thc) or polarized to Th1, Th2 or Th17 T cells. (C) Photomicrographs of TH⁺ neurons in the SN (scale bar 500 μm) and striatal termini (scale bar 1000 μm) of mice treated with PBS, MPTP, or MPTP and polarized Th1, Th2 or Th17 CD4+ effector cells. (D) Total numbers of TH⁺ Nissl⁺ dopaminergic neurons in the SN (red bars) and non-dopaminergic neurons (TH⁻ Nissl⁺, green bars). (E) TH densitometry of dopaminergic neuronal termini within the striatum. (D and E) Differences in means (± SEM, n=7 mice per group) were determined where P<0.05 compared with groups treated with ^aPBS, ^bMPTP, ^cMPTP/N-4YSyn Th1 and ^dMPTP/N-4YSyn Th2.

FIGURE 3. Microglial activation and nigrostriatal responses

Recipient mice were treated with MPTP. SPC from donor mice treated with VIP or immunized with N-4YSyn, or equal numbers of pooled SPC were adoptively transferred 12 hours post-MPTP treatment. Mice were sacrificed on day 2 post-MPTP treatment for evaluation of the substantia nigra for reactive Mac-1⁺ microglia and neuronal cell injury by Fluorojade C (FJ-C) stain. (A) Photomicrographs (scale bars, 200 μm) of midbrain immunostained for Mac-1 (top panels) to identify reactive microglia or FJ-C to identify dead or dying neurons (bottom panels). (B) Mean numbers of Mac-1⁺ microglia and (C) FJ-C⁺ neurons within the SN were determined. (B and C) Differences in means (± SEM, n=5 mice per group) were determined where P<0.05 compared with groups treated with ^aPBS, ^bMPTP, ^cMPTP/VIP SPC, or ^dMPTP/N-4YSyn SPC.

FIGURE 4. Treg mediated nigrostriatal neuroprotection in immunized MPTP-intoxicated mice

Recipient mice were treated with MPTP, and SPC from donor mice immunized with N-4YSyn were adoptively transferred with SPC from naïve mice, VIP-treated mice, naïve Treg, Treg from donor mice treated with VIP, or pooled SPC populations were adoptively transferred 12 hours post-MPTP treatment. Mice were sacrificed on day 7 post-MPTP treatment and evaluated for surviving dopaminergic nigral neurons and striatal termini. (A) Midbrain sections (top panels, scale bar 200 μm) and striatum (bottom panel, scale bar 1000 μm) immunostained for TH. (B) Dopaminergic neurons in the SN were identified as TH⁺Nissl⁺ neurons (red bars), while non-dopaminergic neurons were identified as TH⁻Nissl⁺ neurons (green bars). (C) Mean densities of striatal dopaminergic termini were determined by digital image analysis. (B and C) Differences in means (± SEM, n=7 mice per group) were determined where P<0.05 compared with groups treated with ^aPBS, ^bMPTP, ^cMPTP/VIP SPC, ^dMPTP/N-4YSyn SPC (D) Dopaminergic neuronal survival in the SN and (E) density of striatal dopaminergic termini following adoptive transfer of 5 × 10⁷ N-4YSyn SPC without other cells or in combination with 5 × 10⁷ naïve SPC (Naïve), 5 × 10⁷ SPC from VIP-treated mice (VIP), 1 × 10⁶ Treg from naïve mice (Treg), or 1 × 10⁶ Treg from mice treated with VIP (VIP Treg). (D and E) Differences in means of neuron number and striatal TH density (± SEM, n=7 mice per group) were determined where P<0.05 compared with groups treated with ^aPBS, ^bMPTP, ^cMPTP/N-4YSyn SPC, dMPTP/N-4YSyn SPC + naive SPC, or ^eMPTP/N-4YSyn SPC + naïve Treg. (E) Mean percentages of survival for nigral dopaminergic neuronal bodies or striatal termini based on PBS controls are presented parenthetically in white on each bar.

FIGURE 5. Phenotypic and functional characterization of Treg

SPC, CD4⁺ T cells and Treg from naïve mice, mice immunized with N-4YSyn, or mice treated with VIP were evaluated for B and T cell frequencies, ability to inhibit T cell proliferation, and expression of Teff and Treg genes. (A) Flow cytometric analysis of cell subsets within splenic lymphocytes from naïve mice (black bars), mice immunized with PBS/adjuvant (white bars) or N-4YSyn/adjuvant (dark gray bars), or treated with VIP (light gray bars). (B) Inhibition assay to assess the suppressive function of Treg isolated from each donor group on proliferation of anti-CD3 stimulated naïve CD4⁺CD25⁻ T cells. (C) Table of relative fold-differences in expression of CD4⁺ T cell related genes from T cells isolated from N-4YSyn immunized, VIP-treated, and N-4YSyn+VIP T cells compared with T cells from naïve mice (Supplemental data 1,2 and 3). (D) Cytokine production assayed from T cell supernatants, normalized to absorbance obtained from supernatants of naïve T cells. (E) T cell proliferation cultured in the absence of antigen (media) or presence of nonnitrated 4YSyn or nitrated N-4YSyn. (F) Dose response of N-4YSyn on T cell proliferation. (G) Fold difference of gene expression for T cells from N-4YSyn-immunized mice treated with or without VIP. Values shown are differences in means (± SEM, n=4).