Attenuation of microglial RANTES by NEMO-binding domain peptide inhibits the infiltration of CD8(+) T cells in the nigra of hemiparkinsonian monkey

Abstract

Parkinson's disease (PD) is a progressive neurodegenerative disease characterized by the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc). Despite intense investigations, little is known about its pathological mediators. Here, we report the marked upregulation of RANTES (regulated on activation, normal T cell expressed and secreted) and eotaxin, chemokines that are involved in T cell trafficking, in the serum of hemiparkinsonian monkeys. Interestingly, 1-methyl-4-phenylpyridinium (MPP(+)), a Parkinsonian toxin, increased the expression of RANTES and eotaxin in mouse microglial cells. The presence of NF-κB binding sites in promoters of RANTES and eotaxin and down-regulation of these genes by NEMO-binding domain (NBD) peptide, selective inhibitor of induced NF-κB activation, in MPP(+)-stimulated microglial cells suggest that the activation of NF-κB plays an important role in the upregulation of these two chemokines. Consistently, serum enzyme-linked immuno assay (ELISA) and nigral immunohistochemistry further confirmed that these chemokines were strongly upregulated in MPTP-induced hemiparkinsonian monkeys and that treatment with NBD peptides effectively inhibited the level of these chemokines. Furthermore, the microglial upregulation of RANTES in the nigra of hemiparkinsonian monkeys could be involved in the altered adaptive immune response in the brain as we observed greater infiltration of CD8(+) T cells around the perivascular niche and deep brain parenchyma of hemiparkinsonian monkeys as compared to control. The treatment of hemiparkinsonian monkeys with NBD peptides decreased the microglial expression of RANTES and attenuated the infiltration of CD8(+) T cells in nigra. These results indicate the possible involvement of chemokine-dependent adaptive immune response in Parkinsonism.

Keywords: Parkinson’s disease; RANTES; T cell infiltration; hemiparkinsonian monkeys; microglia; neuroinflammation.

Figure 1. Schematic presentation of experimental schedule

Fifteen naïve rhesus monkeys received intracarotid injection of MPTP. Only 10 monkeys displayed classical parkinsonian postures within 7d of the first MPTP intoxication. These monkeys were treated with NBD peptides (1 mg/kg body wt/2d) for 30 days via i.m. injection (n=4 for MPTP; n=4 for MPTP+ wtNBD; n=2 for MPTP+ mNBD). After 30 days of treatment, blood was collected for chemokine measurement.

Figure 2. ELISPOT analyses of different chemokines in monkey serum

Naïve female rhesus monkeys received a right intracarotid injection of MPTP. After 37 d of injection, concentrations of chemokines were determined in the serum using commercially available ELISPOT kit (Raybiotech). Briefly, serum was diluted to 1:1 with assay diluents, incubated with the blot, and detected with the biotin-labeled antibodies by chemiluminescent detection method (A, Control before MPTP injection; B, MPTP). (C) Heatmap analyses were presented after the measurement of pixel density of each spot. Results are mean of four monkeys per group. RNTS, RANTES; ETX, eotaxin.

Figure 3. MPP⁺ stimulates the expression of RANTES and eotaxin in BV-2 microglial cells

(A) BV-2 microglial cells were treated with 2μM of MPP⁺ for 0, 2, 6, and 12 h under serum-free condition followed by semi-quantitative RT-PCR analyses for RANTES and eotaxin. GAPDH gene was analyzed as control. (B) Quantitative real-time PCR analyses of these genes were analyzed under similar condition. Results are mean ± SD of three independent experiments. *p<0.001 vs. control. Promoter analyses of (C) RANTES and (D) eotaxin were performed in MatInspector, a Genomatix online promoter scan module. E) BV-2 microglial cells pre-treated with 5μM wtNBD or mtNBD for 30 min were stimulated with 2μM of MPP⁺ for 30 min followed by monitoring the levels of phospho-IKKβ (p-IKK) and total IKKβ (t-IKK) by Western blot. F) Bands were scanned and values of p-IKK/Actin and tIKK/Actin presented as relative to control. *p<0.001 vs. control; **p<0.001 vs. MPP⁺. BV-2 microglial cells pre-treated with 5μM wtNBD or mtNBD for 30 min were stimulated with 2μM of MPP⁺ for 5 h followed by analysis of RANTES and eotaxin mRNA by RT-PCR (G) and real-time PCR (H). (I) After 24 h of stimulation with MPP⁺, supernatants were analyzed for RANTES production using RANTES ELISA kit (eBioscience). *p<0.001 vs. control; **p<0.001 vs. MPP⁺. GAPDH= glyceraldehyde-3-phosphate dehydrogenase. J) After 24 h of stimulation with MPP⁺, cells were double-immunolabeled for Iba-1 and RANTES.

Figure 4. ELISA analyses of chemokine in monkey serum

Naïve female rhesus monkeys received a right intracarotid injection of MPTP. After 7 d of injection, monkeys displaying classical parkinsonian postures received wtNBD peptide (1 mg/kg body wt/2d) via i.m. injection. After 30 d of treatment, blood was collected and serum concentration of RANTES (A), eotaxin (B), IL-8 (C), MIP-1α (D), MIP-1β (E), MIG (F), ITAC (G), IP10 (H), and MCP-1 (I) were determined by ELISA. Data are means ± SEM of triplicate assays from four monkeys per group. ^ap<0.001 vs. control before MPTP injection and ^bp<0.001 vs. MPTP.

Figure 5. Effect of wtNBD on the microglial activation of IKKβ and expression of RANTES in the nigra of MPTP-intoxicated monkey

Naïve female rhesus monkeys received a right intracarotid injection of MPTP. After 7 d of injection, monkeys displaying classical parkinsonian postures received wtNBD and mNBD peptides (1 mg/kg body wt/2d) via i.m. injection. After 30 d of treatment, nigral sections were double-labeled for IBA-1 (red) and phospho-IKKβ (green) (A). Cells positive for phospho-IKKβ were counted in three nigral sections (two images per slide) of each of four monkeys (n=4) per group (B). ^*p<0.001 vs. control and ^**p<0.001 vs. MPTP. Nigral sections were double-labeled for IBA-1 (red) and RANTES (green) (C). Regions selected inside white box were magnified and displayed in the bottom panel for each treatment group (D). Cells positive for RANTES were counted in three nigral sections (two images per slide) of each of four monkeys (n=4) per group (E). ^*p<0.001 vs. control and ^**p<0.001 vs. MPTP.

Figure 6. Effect of wtNBD on the microglial expression of RANTES in the striatum of MPTP-intoxicated monkey

Naïve female rhesus monkeys received a right intracarotid injection of MPTP. After 7 d of injection, monkeys displaying classical parkinsonian postures received wtNBD and mNBD peptides (1 mg/kg body wt/2d) via i.m. injection. After 30 d of treatment, striatal sections were double-labeled for IBA-1 (red) and RANTES (green) (A). Regions selected inside white box were magnified and displayed in the bottom panel for each treatment group (B). Cells positive for RANTES were counted in three striatal sections (two images per slide) of each of four monkeys (n=4) per group (C). *p<0.001 vs. control and **p<0.001 vs. MPTP.

Figure 7. Effect of wtNBD on the infiltration of CD3⁺ T cells in the nigra of MPTP-intoxicated monkey

Naïve female rhesus monkeys received a right intracarotid injection of MPTP. After 7 d of injection, monkeys displaying classical parkinsonian postures received wtNBD and mNBD peptides (1 mg/kg body wt/2d) via i.m. injection. After 30 d of treatment, nigral sections were double-labeled for tyrosine hydroxylase (TH; red) and CD3 (green) (A). Blood vessel of each nigral picture was spotted inside dotted box, magnified, and then displayed next to each original image (B). Results represent analysis of three nigral sections of each of four monkeys (n=4) per group.

Figure 8. Effect of wtNBD on the infiltration of CD4⁺ and CD8⁺ T cells in the nigra of MPTP-intoxicated monkey

Naïve female rhesus monkeys received a right intracarotid injection of MPTP. After 7 d of injection, monkeys displaying classical parkinsonian postures received wtNBD and mNBD peptides (1 mg/kg body wt/2d) via i.m. injection. After 30 d of treatment, nigral sections were double-labeled for CD4⁺ (DAB; light brown) and CD8⁺ (DAB-nickel; dark brown or black) (A). Infiltrations of CD8⁺ cells (dark brown) were also monitored in the deep parenchyma of ventral midbrain region (B). Cells positive for CD4 (blue bar) and CD8 (red bar) were counted around blood vessels in three nigral sections (two images per slide) of each of four monkeys (n=4) per group (C). Cells positive for CD8 were counted in the deep brain parenchyma in three nigral sections (two images per slide) of each of four monkeys (n=4) per group (D). ^*p<0.001 vs. control and ^**p<0.001 vs. MPTP.