Male C57BL/6N mice (aged 6–8 weeks, weighing 14–16 g) were obtained from Orient Bio (Seongnam, Gyeonggi-do, Republic of Korea). They were kept in a controlled environment with a 12/12 h light/dark cycle and regulated temperature and humidity.

To modulate Clec5a expression, a short hairpin RNA (shRNA) construct targeting the gene was synthesized by GenePharma (Shanghai, China). The sense sequence was inserted into the plasmid lentiviral knockout (pLKO)-shRNA-green fluorescent protein (GFP) vector. A control vector lacking the shRNA insert (pLKO-GFP) was used for comparison. The inserted shRNA sequence was as follows: 5^′-CACCGGAACAGTCTGTCCCAGAAACTTCAAGAGAGTTTCTGGGACAGACTGTTCCTTTTTTG-3^′.

The 293T cells (ATCC, CRL-3216, Manassas, VA, USA) were co-transfected with recombinant vectors and packaging plasmids (pRSV.Rev, pMDLg/pRRE, and pMD.G) using Lipofectamine™ 3000 Transfection Reagent (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA, Lot#2237579) for 4 h to generate lentivirus. Short tandem repeat (STR) profiling was used to validate 293T cells and confirm that they were free of mycoplasma. Post-transfection, the culture medium was replaced with fresh medium supplemented with GlutaMAX™ (Gibco, Thermo Fisher Scientific, Waltham, MA, USA, Lot#2360312) and sodium pyruvate (Gibco). After 24 h, the lentiviral supernatant was harvested, purified via ultracentrifugation, aliquoted, and stored at –80 °C. To determine viral titers, GFP-positive 293T cells were quantified by flow cytometry following exposure to serially diluted lentiviral vectors.

Bone marrow-derived DCs were prepared from C57BL/6 mice euthanized with CO₂, following established protocols [42]. Bone marrow cells were isolated, dispersed into single-cell suspensions, and plated at 2 $\times$ 10⁶ cells per 10 mL of culture medium. The medium contained RPMI 1640 (Cytiva, Hyclone Laboratories, Logan, UT, USA) with 10% fetal bovine serum (FBS; Corning, NY, USA), GlutaMAX™, antibiotic-antimycotic, 2-mercaptoethanol (Gibco), and 20 ng/mL recombinant mouse (rm) GM-CSF and IL-4 (JWCreaGene, Gwacheon, Gyeonggi-do, Republic of Korea). On day 3, an equal amount of fresh medium was added.

On day 5, the cells were transduced with lentiviral particles at a multiplicity of infection (MOI) of 6 in the presence of 8 µg/mL polybrene (Santa Cruz Biotechnology, Dallas, TX, USA). After 24 h, the culture medium was replaced. For generation of tolDCs and Clec5a-knockdown tolDCs, the cells were stimulated on day 8 with 10 ng/mL TNF-$\alpha$ (BD Biosciences, San Jose, CA, USA) and 10 µg/mL keyhole limpet hemocyanin (KLH; Sigma-Aldrich, St. Louis, MO, USA) for 4 h. Conventional mDCs were produced by treating immature DCs (imDCs) with 1 µg/mL lipopolysaccharide (LPS) and 10 µg/mL KLH (Sigma-Aldrich) for 24 h. For in vivo use, KLH was replaced with 10 µg/mL rm $\alpha$-syn (rPeptide, Watkinsville, GA, USA). All DC subsets were collected simultaneously, and their supernatants were harvested for cytokine analysis.

The experimental design is summarized in Fig. 1. MPTP-HCl (18 mg/kg of free base; Sigma-Aldrich, #M0896) was intraperitoneally injected into the mice four times at 2 h intervals for 5 days [43]. The control animals received only phosphate-buffered saline (PBS). On days 0 and 2, 2 h after the last MPTP injection, the mice were subcutaneously injected at the back of the neck with 1 $\times$ 10⁶ DCs. These cells were validated by STR profiling and verified to be mycoplasma-free before use in animal experiments. On day 5 following MPTP injection, Avertin (500 µL/mouse; 20 mg/mL) was intraperitoneally injected and the mice were subsequently CO₂ euthanized. Euthanasia was performed using the gradual-fill method. CO₂ was administered at a flow rate equivalent to approximately 10% of the 57 L chamber volume per minute (5.7 L/min), allowing for a gradual increase in CO₂ concentration. Upon confirmation of loss of consciousness, the flow rate was increased to 8 L/min (~14%/min) to complete the euthanasia process. This procedure was conducted in accordance with the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals. To remove blood and leukocytes from the brain’s vasculature, the spleen and LNs were removed, and the mice were transcardially perfused with ice-cold 0.9% saline.

Fig. 1.

Scheme of the experimental protocol. MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

The clinical symptoms of PD, which include chronic neurodegeneration with features indicating core motor function disruption, as well as behavioral symptoms, are challenging to reproduce in a mouse model [39]. Thus, we established a score based on the motor dysfunctions described in an MPTP-induced monkey model [44]. PD progression was assessed through daily observations in the home cage. The PD score was calculated by summing the scores of each of the four components (Supplementary Table 1).

On day 5, following MPTP injection, mouse brains were collected post-perfusion, as previously described. The brain tissue was immersed overnight in 4% paraformaldehyde (PFA) in PBS for fixation. Samples were subsequently equilibrated in 15% and 30% sucrose solutions for at least 16 h. Using a cryostat Leica CM3050 S (Leica Biosystems, Nussloch, Germany), the brains were coronally sliced into 30 µm-thick sections and preserved at –20 °C in a cryoprotectant medium until further analysis. For immunofluorescence staining, every sixth section encompassing the SN was selected. To prevent nonspecific interactions, frozen sections were pretreated in PBS containing 3% goat serum and 1% Triton™ X-100 (MilliporeSigma, Burlington, MA, USA) for 30 min. The tissue sections were then incubated overnight at 4 °C with a primary mouse anti-TH antibody (Invitrogen). The next day, the sections were incubated for 2 h at ambient temperature with a fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA). Afterward, the sections were washed three times with PBS (5 min per wash). Fluorescent signals were acquired using a Leica fluorescence microscope (Leica Microsystems, Wetzlar, Germany), and TH-positive cells were quantified using LAS AF imaging software v.4.0 (Leica Microsystems). Antibody information is provided in Supplementary Table 2.

DCs were seeded onto coverslips pre-coated with poly-L-lysine (MilliporeSigma) and subsequently fixed in 1% PFA in PBS for 10 min. Following fixation, the cells were permeabilized with 0.1% Triton™ X-100 in PBS, followed by washing with 0.05% Tween 20 in PBS (PBST; Daejung Chemicals & Metals, Siheung, Gyeonggi-do, Republic of Korea). To minimize nonspecific interactions, the cells were incubated with 5% normal goat serum for 30 min at room temperature. Subsequently, an anti-Clec5a antibody (R&D Systems, Minneapolis, MN, USA) was applied for an overnight incubation at 4 °C. The next day, the cells were treated with an Alexa Fluor 594-conjugated anti-mouse IgG antibody (Jackson ImmunoResearch) in antibody diluent (Agilent Technologies, Santa Clara, CA, USA) for 2 h at room temperature. After multiple washes with PBST, the coverslips were mounted using Fluoroshield™ containing DAPI (Sigma-Aldrich). Imaging was conducted using a TCS NT/SP confocal microscope (Leica) equipped with argon, krypton, and helium/neon lasers. Supplementary Table 2 summarizes the antibodies used in this study.

Phenotypic analysis involved direct immunofluorescence staining of the DC surface markers. The cells were stained using the following antibodies at 4 °C for 20 min: allophycocyanin (APC)-conjugated anti-CD11c (BioLegend, San Diego, CA, USA) and phycoerythrin (PE)-conjugated anti-CD11c (Invitrogen), PE-conjugated anti-CD40 (BD Biosciences), anti-CD80 (Invitrogen), anti-major histocompatibility complex (MHC) class II (BD Biosciences), anti-IL-10 (BD Biosciences), FITC-conjugated anti-CD14 (BioLegend), anti-CD54 (BD Biosciences), anti-CD86 (BioLegend), anti-MHC class I (BioLegend), propidium iodide (PI; BD Biosciences), and corresponding isotype controls. The cells were analyzed using a NovoCyte 3005 (ACEA Biosciences, Agilent Technologies, San Diego, CA, USA), and the data were analyzed using the FlowJo v.10 software (FlowJo, Ashland, OR, USA). All antibody information is presented in Supplementary Table 3.

On day 5, following MPTP injection, the perfused mouse brains were harvested as described above. For in vivo experiments, the brain tissue was lysed using the PRO-PREP™ protein extraction kit (iNtRON Biotechnology, Seongnam, Gyeonggi-do, Republic of Korea, #17081). The total protein concentration of the lysate was measured using a Pierce™ Bradford Plus Protein Assay Kits (Thermo Fisher Scientific, #23236). Cytokine levels were quantitated using commercial enzyme-linked immunosorbent assay (ELISA) kits following the manufacturer’s protocols. For in vitro experiments, cytokine levels—including IL-1$\beta$, IL-4, IL-6, and TNF-$\alpha$ (BioLegend); IL-10, IL-12p40, and IFN-$\gamma$ (BD Biosciences); and TGF-$\beta$ (R&D Systems)—secreted by DCs or T cells were measured using commercially available ELISA kits.

Spleens from mice were harvested and dissociated in RPMI 1640 medium. To enrich CD3⁺ T cells, splenocytes were applied to nylon wool fibers (Polysciences, Inc., Warrington, PA, USA) according to the manufacturer’s protocol. The purified T cells were labeled with a CFSE labeling kit (65-0850-84, Invitrogen, Waltham, MA, USA) and subsequently co-cultured with DCs at a 1:10 ratio (DC:T cells). CD3⁺ T cells (1 $\times$ 10⁶ cells/mL) and DCs (1 $\times$ 10⁵ cells/mL) were incubated in 2 mL of RPMI 1640 medium supplemented with 10% FBS at 37 °C for 72 h. In this assay, T cells acted as responder cells, with DCs serving as stimulators.

To assess T cell subsets, surface markers were stained with APC-conjugated anti-CD4 (BioLegend) and FITC-conjugated anti-CD25 (BD Biosciences), followed by incubation at 4 °C for 20 min. The cells were subsequently fixed and permeabilized using either the BD Intracellular Staining Kit (554722/554723, BD Biosciences, San Jose, CA, USA) or the Foxp3/Transcription Factor Staining Buffer Set (00-5523-00, Invitrogen, Waltham, MA, USA). Intracellular staining was performed with PE-conjugated anti-IFN-$\gamma$ (BD Biosciences), PE-conjugated anti-IL-17A (BioLegend), PE-conjugated anti-Foxp3, and Alexa Fluor 488-conjugated anti-IL-4 (Invitrogen). Flow cytometric analysis was conducted on a NovoCyte 3005, and data were processed using FlowJo software v.10. The antibodies used in the flow cytometry are listed in Supplementary Table 3.

Total RNA was extracted using Labozol (CMRZ001, Cosmogenetech, Seoul, Republic of Korea), and cDNA was synthesized with the LaboPass cDNA synthesis kit (CMRTK002, Cosmogenetech) according to the manufacturer’s instructions. Quantitative real-time PCR was performed using Clec5a-specific primers and SensiFast™ SYBR® Hi-Rox Mix (Bioline, Memphis, TN, USA). The primer sequences were: Clec5a forward, 5^′-TCTGCTGTATTTCCCACAGG-3^′; Clec5a reverse, 5^′-TTCGTCATTTCTCCCAAAGA-3^′; glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forward, 5^′-AACAGCAACTCCCACTCTTC-3^′; and GAPDH reverse, 5^′-CCTGTTGCTGTAGCCGTATT-3^′. Gene expression was normalized to GAPDH and analyzed in triplicate (Relative expression levels were calculated using the 2^{-Δ⁢Δ⁢CT} method with GAPDH as the reference gene).

Protein quantification of whole-cell lysates from DCs was performed using the Pierce™ Bradford Plus Protein Assay Kits following lysis with the PRO-PREP™ protein extraction solution (iNtRON Biotechnology). Equal amounts of protein were loaded and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes (Thermo Fisher Scientific). The membranes were blocked in 10% skim milk (w/v) prepared in PBST and subsequently incubated overnight at 4 °C with the following primary antibodies: Clec5a, phosphorylated (p)-extracellular signal-regulated kinase (Erk)1/2, Erk1/2, p-nuclear factor kappa B (NF-$\kappa$B) p65, NF-$\kappa$B p65, p-c-Jun N-terminal kinase (JNK), JNK, p-p38, p38, p-signal transducer and activator of transcription (STAT)3, STAT3, p-spleen tyrosine kinase (Syk), and Syk (all purchased from Cell Signaling Technology, Danvers, MA, USA); DAP12 (Santa Cruz Biotechnology); and GAPDH (Bioss, Woburn, MA, USA). After washing, the membranes were incubated at room temperature for 2 h with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies, such as goat anti-mouse, mouse anti-goat, or mouse anti-rabbit IgG (Santa Cruz Biotechnology). Protein signals were detected using the SuperSignal™ Chemiluminescence Substrate (Thermo Fisher Scientific) and captured with a luminescence imaging system (ImageQuant LAS 4000; GE Healthcare, Chicago, IL, USA). Detailed antibody information is provided in Supplementary Table 2.

On day 5, after MPTP injection, the perfused mouse brains were harvested as described above. Percoll® (Sigma-Aldrich) was used for leukocyte isolation, according to the manufacturer’s instructions. Briefly, the brains were gently homogenized and pelleted. The homogenates were resuspended in 37% isotonic Percoll®. A discontinuous isotonic Percoll® density gradient consisting of 70%, 37%, 30%, and 0% layers was prepared. The gradient was centrifuged at 300 $\times$g for 40 min at room temperature. Microglia and leukocytes were collected from the 37%/70% interface. The isolated cells were subjected to flow cytometric analysis to verify cellular identity based on their surface marker expression. All cell preparations tested negative for mycoplasma contamination. Detailed products information is provided in Supplementary Table 4.

All experimental procedures were performed in at least three independent replicates. Data are expressed as the mean $\pm$ standard deviation (SD). To determine whether the data followed a normal distribution, the Shapiro–Wilk test was conducted. For comparisons between the two groups, the Student’s t-test was applied when the data met the parametric assumptions, while the Mann–Whitney U test was used for nonparametric datasets. When comparing three or more independent groups, one-way ANOVA with a subsequent Tukey–Kramer post hoc test was used for normally distributed data. In contrast, for nonparametric comparisons involving multiple groups, the Kruskal–Wallis test was utilized, followed by pairwise testing adjusted with the Bonferroni correction. Survival analyses were performed using the Kaplan–Meier method (InStat; GraphPad Software, La Jolla, CA, USA). A p-value of less than 0.05 was considered to indicate statistical significance.

To evaluate the therapeutic effects of tolDCs, we administered either tolDCs or mDCs to MPTP-intoxicated mice. On day 5, the tolDC-treated mice (MPTP + tolDC) exhibited a higher survival rate compared with the mDC-treated (MPTP + mDC) or MPTP-intoxicated (MPTP) groups (Fig. 2A). Disease progression was monitored over time across all groups. Compared with the MPTP group, the MPTP + tolDC group displayed a lower Parkinson’s score, whereas the MPTP + mDC group showed the highest score (Fig. 2B). To investigate whether tolDCs exerted neuroprotective effects in the experimental PD model, dopaminergic neuronal degeneration in the substantia nigra pars compacta (SNpc) was analyzed using immunofluorescence staining. MPTP administration reduced TH-positive dopaminergic neuron numbers in the SN compared with the PBS-treated mice. While MPTP + tolDC significantly preserved dopaminergic neurons, the neuronal loss was comparable between the MPTP + mDC and MPTP groups (Fig. 2C,D). To determine whether the injected DCs modulated inflammation in the MPTP-intoxicated mouse brains, we measured the levels of IL-1$\beta$ and IFN-$\gamma$ in whole brain lysates. These cytokines are functionally crucial for the activation of microglia and the exacerbation of symptoms in experimental PD models [45, 46]. The expression of IL-1$\beta$ and IFN-$\gamma$ was lower in the MPTP + tolDC group, while the highest levels were detected in the MPTP + mDC group (Fig. 2E). Regarding behavioral tests, tolDC-treated mice performed better in the pole test, exhibiting shorter T-turn and total descent times (Fig. 2F). Improved performance in the stepping test (Fig. 2G) further supported the attenuation of motor deficits. Collectively, these findings indicate that tolDCs may confer neuroprotective benefits in MPTP-intoxicated mice.

Fig. 2.

Neuroprotective effect of tolerogenic dendritic cells (tolDCs). (A) Survival rates. Kaplan–Meier survival analysis in the phosphate-buffered saline (PBS) (n = 10), MPTP (n = 20), MPTP + tolDC (n = 10), and MPTP + mDC (n = 10) groups. tolDCs and mature DCs (mDCs) were generated by treatment with $\alpha$-synuclein ($\alpha$-syn). (B) Plots depicting disease severity. A scale from 0 to 10 was used to grade the severity of the disease (Supplementary Table 1) (^#p 0.05 and ^#⁢#p 0.01 vs. PBS; *p 0.05 vs. MPTP + tolDC). (C) Tyrosine Hydroxylase (TH)-expressing dopaminergic neurons in substantia nigra pars compacta (SNpc) were visualized via Immunofluorescence imaging (TH; a marker of dopamine neurons); Representative images are displayed. Scale bar: 500 µm. (D) Quantification of TH-positive neurons in the substantia nigra (SN) region (Tukey–Kramer ANOVA; n = 3; ^#⁢#⁢#p 0.001 vs. PBS; ⁺⁺p 0.01 vs. MPTP; *p 0.05 vs. MPTP + tolDC). (E) Cytokine profile of the brain. Bar plots illustrating the levels of interleukin (IL)-1$\beta$ and interferon (IFN)-$\gamma$ in the brain measured by enzyme-linked immunosorbent assay (ELISA) (Kruskal–Wallis ANOVA; n = 3; ⁺⁺p 0.01 vs. MPTP; *p 0.05 and **p 0.01 vs. MPTP + tolDC). (F) Pole test measuring descent time and reversal initiation (Kruskal–Wallis ANOVA; n $\ge$ 5; **p 0.01 vs. MPTP + tolDC). (G) Stepping test counting adjusted forelimb steps (Kruskal–Wallis ANOVA; n = 6; ^#⁢#p 0.01 vs. PBS). All data are expressed as the mean $\pm$ SD.

The therapeutic potential of tolDCs in MPTP-treated mice was assessed in comparison with that of mDCs. In vitro analyses of tolDC-mediated immune responses revealed reduced levels of co-stimulatory molecules and proinflammatory cytokines relative to mDCs (Supplementary Fig. 1A,B). Conversely, the expression of IL-10, a representative anti-inflammatory cytokine, was elevated in tolDCs compared with that in mDCs (Supplementary Fig. 1C). Moreover, tolDCs had a lower proliferative capacity than mDCs but promoted the Th2 and Treg populations in the co-culture experiments (Supplementary Fig. 1D–G). These results suggest that tolDCs efficiently induced immune tolerance by suppressing inflammatory cytokine production and enhancing the proportion of Tregs.

Next, we examined the functional markers in DC subsets to determine the basis for the functional differences between tolDCs and mDCs. As Clec5a is upregulated in tolDCs [31], we generated tolDCs and mDCs to determine whether tolDCs expressed Clec5a by employing quantitative real-time PCR (qRT-PCR). The results indicated that Clec5a was significantly overexpressed in tolDCs compared with mDCs. In addition, western blot analysis of whole cell lysates showed enhanced Clec5a levels in tolDCs (Fig. 3A).

Fig. 3.

C-type lectin domain family 5 member A (Clec5a) expression is affected by the immune response in tolDCs. (A) Clec5a mRNA levels were quantified using quantitative real-time PCR (qRT-PCR) (left; Mann–Whitney U test; n = 6 independent DC samples; **p 0.01 vs. tolDC), and protein expression was assessed by western blotting (right; Student’s t-test; n = 6 independent DC samples; **p 0.01 vs. tolDC). Expression levels were normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH). (B) Clec5a expression in shRNA-transduced dendritic cells (DCs) was analyzed by qRT-PCR (left; Student’s t-test; n = 4 independent DC samples; ^†⁢†p 0.01 vs. shCon) and western blotting (right). The bar graph presents the fold changes relative to immature DCs (imDCs) from at least four independent samples (Student’s t-test; n = 4 independent DC samples; ^†p 0.05 vs. shCon). (C) Immunofluorescence imaging visualized CD11c (green) and Clec5a (red) expression in DCs, with nuclei counterstained using DAPI (blue) (Mann–Whitney U test; n = 5 independent DC samples; **p 0.01 vs. tolDC). Scale bar: 20 µm. (D) Immunofluorescence analysis confirmed Clec5a (red) and green fluorescent protein (GFP) (green) expression, indicating the transduction efficiency. Nuclei were counterstained using DAPI (blue), and all images were acquired under consistent microscope settings (Mann–Whitney U test; n = 6; ^†⁢†p 0.01 vs. shCon). Scale bar: 20 µm. (C) and (D) represent montages of representative single-cell confocal images, not continuous confocal fields. (E) shClec5a-mediated T cell proliferation was assessed by flow cytometry. Representative histograms and bar graphs show the percentage of proliferating CD3⁺ T cells (Mann–Whitney U test; n = 6 independent samples; ^†p 0.05 vs. shCon). (F,G) shClec5a-mediated T cell polarization was determined by flow cytometry. The proportions of Th (F) and Treg (G) are presented. Representative histograms and bar graphs indicate the proportion of each T cell subset (Mann–Whitney U test; n = 13; ^†⁢†p 0.01 vs. shCon). (H) Cytokine secretion profiles of T cells stimulated with shClec5a were analyzed (Student’s t-test; n $\ge$ 3 independent samples; ^†p 0.05 vs. shCon). All data are presented as the mean $\pm$ SD.

To investigate the functional significance of Clec5a in tolDCs, lentiviral transduction with Clec5a-targeting shRNA was employed. The Clec5a knockdown efficiency was verified through qRT-PCR and western blotting (Fig. 3B). Immunofluorescence analysis further revealed that Clec5a was strongly expressed on the surface of tolDCs (Fig. 3C) and was significantly downregulated in the Clec5a-knockdown tolDCs (shClec5a). Additionally, the transduction of lentiviral vectors was confirmed by GFP expression in DCs (Fig. 3D). We next evaluated whether Clec5a knockdown affected tolDC function. The results demonstrated that although Clec5a did not influence the expression of surface molecules, it markedly induced the production of IL-6 while decreasing the expression of IL-10 in tolDCs (Supplementary Fig. 2A–C). To evaluate the impact of shClec5a on T cell activation, DCs were co-cultured with CD3⁺ T cells. Compared with shCon, T cell proliferation (Fig. 3E) and the CD4⁺IFN-$\gamma$⁺ Th1 population were significantly increased, whereas the Th2/Th17 populations remained similar to those in the shCon group (Fig. 3F). Moreover, shClec5a reduced the Treg population and TGF-$\beta$ secretion (Fig. 3G,H). These results suggest that adaptive cellular immune responses, particularly Th1 and Treg-mediated immune responses, were affected by Clec5a levels.

The key downstream molecules were assessed to explore the signaling mechanisms underlying Clec5a-mediated immune regulation. In Clec5a-knockdown tolDCs, the expression of DAP12 and phosphorylated Syk was slightly reduced (Fig. 4A). While the phosphorylation of Erk, p38, and JNK remained unchanged between the shCon and shClec5a groups, STAT3 phosphorylation significantly declined, and NF-$\kappa$B phosphorylation was markedly elevated in the shClec5a group (Fig. 4B). These results indicate that Clec5a contributes to the tolDC-based immune response via the NF-$\kappa$B and STAT3 signaling pathways.

Fig. 4.

Cell signaling analysis of shClec5a. (A) Western blotting was conducted to assess the expression of DNAX-activating protein of 12 kDa (DAP12), p-spleen tyrosine kinase (Syk), and Syk. The p-Syk expression was normalized to that of Syk. The corresponding bar graphs indicate the fold changes relative to imDCs (Mann–Whitney U test, n = 3). (B) Western blot analysis was also performed for key signaling proteins, including nuclear factor kappa B (NF-$\kappa$B), signal transducer and activator of transcription (STAT)3, extracellular signal-regulated kinase (Erk)1/2, p38, and c-Jun N-terminal kinase (JNK). Phosphoprotein expression was normalized to the total expression of each protein. The bar graph represents the fold changes relative to imDCs (Mann–Whitney U test, n = 3; ^†p 0.05 vs. shCon). All data are presented as the mean $\pm$ SD.

To determine whether the modulation of Clec5a expression in tolDCs influenced their therapeutic efficacy against PD, we administered Clec5a-expressing tolDCs to MPTP-intoxicated mice. The experimental protocol was the same as described previously. Kaplan–Meier analysis indicated enhanced survival in the tolDC-treated mice (MPTP + tolDC) compared with that in the mDC-treated (MPTP + mDC), shClec5a-treated (MPTP + shClec5a), or MPTP-intoxicated (MPTP) mice on day 5 (Fig. 5A). Disease severity increased with time in all experimental groups, whereas the MPTP + shClec5a group exhibited the highest Parkinson’s score compared with the MPTP group (Fig. 5B). Compared with the MPTP group, although MPTP + tolDC ameliorated the loss of dopaminergic neurons, the loss was greater or similar in the MPTP + mDC and MPTP + shClec5a groups (Fig. 5C,D). In the SN region, $\alpha$-syn levels were highest in the MPTP + mDC group, followed by the MPTP and MPTP + shClec5a groups (Fig. 5E). We next measured the levels of proinflammatory cytokines in whole brain lysates. The lowest IL-1$\beta$ and IFN-$\gamma$ levels were identified in the MPTP + tolDC group (Fig. 5F). Although the results were consistent among individuals, the T-turn and pole test times and the forepaw step counts were similar in the MPTP + shClec5a and MPTP + mDC groups; the MPTP + tolDC group exhibited minimal bradykinesia or motor dysfunction (Fig. 5G,H). The MPTP + tolDC group exhibited the lowest degree of tremors, whereas the MPTP + shClec5a and MPTP groups showed similar results (Fig. 5I and Supplementary Fig. 3). Taken together, these results imply that the modulatory effects of tolDCs on inflammation and motor dysfunction in MPTP-intoxicated mice depend on Clec5a expression levels.

Fig. 5.

Neuroprotective effects of Clec5a-expressing DCs. (A) Survival rates. Kaplan–Meier survival analysis in PBS (n = 15), MPTP (n = 38), MPTP + tolDC (n = 22), MPTP + mDC (n = 19), and MPTP + shClec5a (n = 11) groups. tolDCs and mDCs were generated by treatment with $\alpha$-syn. (B) Plots depicting disease severity. Disease severity was graded on a scale of 0–10 (⁺⁺p 0.01 vs. MPTP; Supplementary Table 1). (C) Immunofluorescence staining of the TH-positive neurons in the SNpc of mice. Representative photomicrographs of TH (a marker of dopamine neurons) in the SN region. Scale bar: top, 500 µm; bottom 100 µm. (D) Quantification of TH-positive neurons within the SN region (Kruskal–Wallis ANOVA; n = 3; ^#p 0.05 vs. PBS). (E) Immunofluorescence staining of TH-positive neurons and $\alpha$-syn in the SNpc of mice. Representative photomicrographs of TH and $\alpha$-syn staining in the SN region. Scale bar: 50 µm. All microscopy images were obtained under consistent imaging parameters, and representative images are displayed. (F) Cytokine profile of the brain (Tukey–Kramer ANOVA; n $\ge$ 3; ⁺p 0.05 vs. MPTP; *p 0.05 vs. MPTP + tolDC). (G) Pole test measuring descent time and reversal initiation (Kruskal–Wallis ANOVA; n $\ge$ 8; ^#⁢#p 0.01, and ^#⁢#⁢#p 0.001 vs. PBS; *p 0.05 vs. MPTP + tolDC). (H) Stepping test counting adjusted forelimb steps (Kruskal–Wallis ANOVA; n $\ge$ 6; ^#p 0.05, ^#⁢#p 0.01 and ^#⁢#⁢#p 0.001 vs. PBS). (I) Tremor test evaluating tremor severity based on motion blur from stacked video frames (Kruskal–Wallis ANOVA; n $\ge$ 4 independent mice; ^#p 0.05 vs. PBS). All data are expressed as the mean $\pm$ SD.

To determine the in vivo effects of tolDC treatment on the brain and systemic immune systems, the mice were euthanized, and their spleens, LNs, and perfused brains were isolated. Flow cytometry analysis measured the proportions of Tregs (Fig. 6A) and M1/M2 macrophages (Fig. 6B) in the brain tissue after tolDC administration. The percentages of CD4⁺CD25⁺Foxp3⁺ Tregs and CD45⁺CD11b⁺CD206⁺ M2 macrophages were enhanced in the brains of PD mice following tolDC injection. Furthermore, in the MPTP + mDC group, the proportion of Tregs in the brain decreased, and that of CD45⁺CD11b⁺CD86⁺ M1 macrophages significantly increased compared with the MPTP + tolDC mice. Such effects were not observed in the spleen (Supplementary Fig. 4A), although the proportion of Tregs in the submandibular LNs increased (Supplementary Fig. 4B). These results suggest that tolDCs may significantly reduce the number of M1 macrophages in the brain, potentially suppressing the excessive immune response in the CNS.

Fig. 6.

tolDC-induced Treg infiltration and M2 macrophage differentiation in the brain. (A) The percentages of regulatory T cells (Tregs) (CD4⁺CD25⁺Foxp3⁺) in the brain (Kruskal–Wallis ANOVA; n $\ge$ 3). (B) The percentage of M1 (CD45⁺CD11b⁺CD86⁺) and M2 (CD45⁺CD11b⁺CD206⁺) macrophages in the brain (Kruskal–Wallis ANOVA; n $\ge$ 3; **p 0.01 vs. MPTP + tolDC; ⁺p 0.05 vs. MPTP). Leukocytes were isolated from perfused mouse brains on day 5 post-MPTP treatment and analyzed by flow cytometry. All data are expressed as the mean $\pm$ SD.