Dendritic Cells Induce Clec5a-mediated Immune Modulation in MPTP-induced Parkinson’s Disease Mouse Model

Abstract

Background:

Parkinson’s disease (PD) is characterized by a progressive decline in dopaminergic neurons within the substantia nigra (SN). Although its underlying cause has yet to be fully elucidated, accumulating evidence suggests that neuroinflammation contributes substantially to disease development. Treatment strategies targeting neuroinflammation could improve PD outcomes. Monocyte-derived tolerogenic dendritic cells (tolDCs) modulate immune responses and induce regulatory T cells (Tregs) during various inflammatory diseases. However, the mechanisms underlying tolDC-mediated immunoregulation in PD remain unclear.

Methods:

We investigated the immune modulatory role of tolDCs by analyzing gene expression patterns and identified that the C-type lectin domain family 5 member A (Clec5a) was highly induced in tolDCs. To assess its function, we generated Clec5a-knockdown tolDCs and measured cytokine production, including interleukin (IL)-10 and IL-6, forkhead box protein P3 (Foxp3)⁺ Treg induction, and nuclear factor kappa B (NF-κB) signaling activity. Furthermore, we evaluated the therapeutic effects of Clec5a-expressing dendritic cells (DCs) in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model. Dopaminergic neuron survival, α-synuclein (α-syn) accumulation, neuroinflammatory markers, and locomotor behavior were analyzed following DC administration.

Results:

Clec5a-knockdown tolDCs exhibited reduced immunomodulatory function and IL-10 levels but enhanced IL-6 levels. In addition, these cells induced fewer Foxp3⁺ Tregs and showed significantly enhanced NF-κB signaling activity. In the MPTP-induced PD model, administration of Clec5a-expressing DCs ameliorated dopaminergic neuron loss and α-syn accumulation. Furthermore, Clec5a-expressing DCs reduced the number of CD45^highCD11b⁺CD86⁺ macrophages in the brain, reduced brain inflammatory cytokine expression, and improved locomotor activity.

Conclusions:

These findings suggest that Clec5a plays a critical role in the immunomodulatory function of tolDCs. The administration of Clec5a-expressing DCs effectively reduced neuroinflammation and protected dopaminergic neurons in an MPTP-induced PD model. Therefore, Clec5a-expressing tolDCs may demonstrate therapeutic potential by managing PD symptoms by suppressing inflammatory responses associated with neurodegeneration.

Graphical abstract

Keywords

dendritic cells / immune tolerance / Clec5a / regulatory T cells / Parkinson disease

1. Introduction

Parkinson’s disease (PD) ranks as the second most common neurodegenerative disease globally and predominantly affects individuals of advanced age [1, 2, 3]. The clinical profile of PD encompasses both motor dysfunctions, such as resting tremors, bradykinesia, rigidity, and impaired postural reflexes, and various non-motor symptoms, all of which substantially contribute to morbidity [4]. These manifestations are largely driven by the progressive degeneration of nigrostriatal dopaminergic neurons and the intracellular aggregation of misfolded \alpha-synuclein (\alpha-syn) protein, leading to Lewy body pathology [5], and may also be exacerbated by genetic mutations such as leucine-rich repeat kinase 2 (LRRK2), which promotes endoplasmic reticulum (ER) stress through the thrombospondin 1 (THBS1)/transforming growth factor beta 1 (TGF-\beta1) axis [6]. In addition to neuronal mechanisms, recent insights have underscored a substantial role for immune-related processes in PD pathogenesis, involving crosstalk between innate and adaptive immunity [3, 7]. Among these, microglial activation has been shown to facilitate neuroinflammatory cascades by releasing cytokines such as interleukin (IL)-6, tumor necrosis factor (TNF)-\alpha, and interferon (IFN)-\gamma, thereby aggravating neuronal injury [8, 9, 10, 11]. Moreover, aberrant infiltration of cluster of differentiation 4 (CD4)⁺ T cells into the substantia nigra (SN) contributes to immune imbalance [12, 13]. This occurs particularly by shifting the ratio of T helper type 1 (Th1)/T helper type 17 (Th17) cells relative to regulatory T cells (Tregs), which correlates with disease severity [14, 15, 16, 17, 18]. These findings collectively highlight the therapeutic potential of modulating CD4⁺ T cell-mediated immune responses to improve neuroinflammation in PD [19, 20].

Dendritic cells (DCs), as pivotal regulators of immune surveillance, specialize in presenting antigens (Ags) and initiating immune responses within both the peripheral and the central nervous system (CNS), where they continuously monitor and respond to immunological cues. Located near the cerebrospinal fluid, DCs can migrate to the draining cervical lymph nodes (LNs), where they may either exhibit tolerogenic capabilities or elicit immunogenic T-cell responses [21]. The objectives of DC-based therapy for PD are to promote Treg responses and induce tolerance against certain brain Ags [22]. By modulating immune responses against self-Ags, tolerogenic dendritic cells (tolDCs) serve as key modulators in immune-mediated diseases, including autoimmune and chronic inflammatory disorders such as rheumatoid arthritis [23, 24], Type 1 diabetes [25, 26], multiple sclerosis [27, 28], and myocardial infarction [29, 30]. Despite their therapeutic potential, tolDC-based therapies are challenging as the functionally distinct DC subsets need to be identified. Furthermore, due to the opposing functional characteristics of tolDCs and immunogenic mature DCs (mDCs), it is vital to identify the functional markers that can help distinguish between them. We previously performed gene profiling on mouse-derived tolDCs [31], which suggested that the C-type lectin domain family 5 member A (Clec5a) is uniquely overexpressed in tolDCs compared with other DC subsets.

Predominantly expressed on cells of the myeloid lineage, Clec5a, also known as myeloid DNAX-activating protein of 12 kDa (DAP12)-associating lectin 1 (MDL-1), interacts with the adaptor molecule DAP12 to initiate downstream signaling, which may result in either immunostimulatory or immunosuppressive responses depending on the cellular context [32, 33, 34]. Therefore, the dual functions of DAP12 may help explain the diverse roles of Clec5a in different cell types. Notably, some studies have suggested that Clec5a is involved in various immune responses, such as calcium mobilization, immunosuppressive leukocyte accumulation, and inflammatory cytokine production [35, 36, 37, 38]. However, its specific role in DCs remains unclear. Moreover, the role of Clec5a expression in mediating the therapeutic effects of DCs in PD has not yet been investigated.

Therefore, this study evaluated the therapeutic effects of Clec5a-expressing tolDCs in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mice, which is the most widely used animal model for PD [39, 40, 41]. We confirmed that tolDCs suppress neuroinflammation and attenuate PD-related dopaminergic neurodegeneration. We further observed that tolDCs enhanced forkhead box protein P3 (Foxp3)⁺ Tregs and reduced the levels of inflammatory cytokines, including IL-1\beta and IL-6, in a Clec5a-dependent manner. These findings suggest that Clec5a-expressing tolDCs may serve as a potential Ag-specific immunotherapy for inflammatory diseases.

2. Materials and Methods

2.1 Animals

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.

2.2 Lentiviral Constructs for Regulating Clec5a Gene Expression

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^′.

2.3 Lentiviral Vector Production and Titration

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.

2.4 Generation of DCs and Transduction Using 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.

2.5 Generation of an MPTP-induced PD Mouse Model and tolDC Administration

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.

2.6 Clinical Parkinson’s Disorder Score

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).

2.7 Behavioral Analysis

2.7.1 Stepping Test

On day 4, three types of behavioral experiments were performed. The forepaw stepping adjustment test is a widely accepted method for evaluating forelimb akinesia in mice. During the procedure, the mouse’s hindlimbs were gently elevated by lifting its tail, ensuring that only the forepaws remained in contact with the surface. The animal was then guided backward by the tail for a distance of 1 m at a consistent speed over approximately 3–4 s. The entire process was recorded on video, and the number of adjusting steps made by each forepaw was subsequently analyzed.

2.7.2 Pole Test

The pole test was conducted by positioning the mice head-upward at the top of a vertical wooden pole (50 cm in length and 1 cm in diameter) situated within their home cages. Before testing, mice underwent a 2-day training period, during which they completed three trials per day. On the test day, each mouse performed three additional trials, and the mean time required to execute a complete 180° turn (T-turn) and descend to the cage floor was measured. To prevent physical fatigue, cutoff times were established at 90 s for the T-turn and 180 s for the total descent. In cases where the mice fell while turning, the maximum time was recorded.

2.7.3 Tremor Test

The tremor assessment was performed by a blinded researcher. To measure the tremor amplitude, brief video recordings (8–10 s) of mice positioned on a small platform were obtained, consisting of approximately 60 consecutive frames. These frames were merged into a smart object, and the “mean” stack mode in Adobe Photoshop software v.25.12.3 (Adobe Inc., San Jose, CA, USA) was used to integrate the motion into a single composite image. This approach allowed for the visualization of the displacement of the spine across frames, with the images inverted to enhance clarity. The image contrast was then analyzed using pixel intensity measurements in ImageJ v.1.53t (National Institutes of Health, Bethesda, MD, USA, https://imagej.net) to determine the degree of sharpness.

2.8 Immunofluorescence Staining of Tyrosine Hydroxylase (TH)-positive Neurons

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.

2.9 Immunofluorescence Staining for Clec5a

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.

2.10 Flow Cytometric Immunophenotyping of DCs

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.

2.11 Cytokine Measurement

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.

2.12 Carboxyfluorescein Succinimidyl Ester (CFSE) T Cell Proliferation Assay

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.

2.13 Intracellular Cytokine Staining of the CD4⁺ T-cell Subsets

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.

2.14 Quantitative Real-time PCR (qRT-PCR)

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).

2.15 Western Blotting

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-\kappaB) p65, NF-\kappaB 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.

2.16 CNS Leukocyte Isolation

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 \timesg 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.

2.17 Statistical Analysis

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.

3. Results

3.1 Neuroprotective Effect of tolDCs in MPTP-intoxicated Mice

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.

3.2 Clec5a is Upregulated in tolDCs and Affects the T Cell-mediated Immune Response

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).

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.

3.3 Changes in NF-\kappaB and STAT3 Activation via the Regulation of Clec5a Expression

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-\kappaB 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-\kappaB and STAT3 signaling pathways.

3.4 Neuroprotective Effect of Clec5a-expressing tolDCs in MPTP-intoxicated Mice

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.

3.5 Clec5a-expressing tolDCs Modulate Brain Immune Cells in MPTP-intoxicated Mice

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.

4. Discussion

PD is a progressive neurodegenerative disease characterized by dopaminergic neuron loss, motor dysfunction, and \alpha-syn accumulation. Although current treatments provide only symptomatic relief, they do not prevent or reverse neurodegeneration. As inhibition of CD4⁺ T cell proliferation via immune tolerance induction may suppress immune responses in PD [12, 13, 14, 15], we hypothesized that tolDCs may also play a crucial role in CNS immunity. However, a major challenge in tolDC-based therapies is the lack of reliable functional markers to distinguish them from immunogenic mDCs, as both share surface molecules. This study investigated the applicability of Clec5a as a marker of the tolerogenic phenotype.

Clec5a was selectively upregulated in tolDCs, thereby increasing IL-10 and Treg differentiation, but decreasing IL-6 levels. Clec5a knockdown in tolDCs abrogated these effects, enhancing Th1 responses but impairing Treg generation, highlighting its essential role in shaping the immune balance. Clec5a mechanistically modulated two key signaling pathways: STAT3 and NF-\kappaB. Clec5a knockdown reduced STAT3 phosphorylation but enhanced NF-\kappaB p65 activation, without influencing the MAPK pathway components. Given the established roles of STAT3 in promoting tolerance and NF-\kappaB in driving inflammation, these data suggest that Clec5a orchestrates the immune profile of tolDCs through the coordinated regulation of these pathways, although this reflects a correlative association rather than direct mechanistic evidence. This regulatory role contrasts with the well-established proinflammatory functions of Clec5a in other myeloid cells and suggests that its function may be context-dependent, influenced by the tolerogenic signaling milieu of tolDCs.

Crucially, the functional relevance of Clec5a-expressing tolDCs was confirmed in vivo. In this study, we specifically identified Treg and M1/M2 cells among the various brain cell types when assessing the impacts of tolDC. Clec5a-expressing tolDCs improved the pathological and behavioral impairments in the MPTP-induced PD model by regulating immune responses in the brain and reducing M1 macrophages but increasing Treg populations, which were associated with anti-inflammatory changes. Although we did not directly assess Treg suppressive function, the accompanying cytokine profile and behavioral improvements suggest their functional relevance. However, the MPTP mice injected with the Clec5a-knockdown tolDCs exhibited more severe pathogenesis and an accelerated loss of dopaminergic neurons. These observations provide compelling evidence that Clec5a plays a vital role in the neuroprotective activity of tolDCs. Although we did not investigate the direct interaction between tolDCs and microglia, the observed effects are likely mediated by peripherally induced Tregs. Exploring how these Tregs influence CNS-resident immune cells, such as microglia, would be an important direction for future studies.

From a translational perspective, DCs offer a unique therapeutic advantage in neurodegenerative diseases such as PD due to their dual roles in immune regulation and Ag presentation. This is supported by recent findings indicating that metabolic risk factors can affect treatment outcomes in PD models [47]. Compared with microglia, DCs are more effective in priming T cells and can migrate to lymphoid organs, making them superior candidates for CNS immune modulation [48, 49]. Notably, \alpha-syn-sensitized DCs induce Ag-specific immune responses without provoking systemic inflammation, supporting the DC-based Ag-specific immunotherapy of PD [50, 51, 52, 53, 54]. The “self-adjuvant” nature of DCs, combined with their autologous origin, minimizes the risk of immune rejection or off-target effects [55]. For future clinical applications, autologous monocyte-derived tolDCs may be preferred due to their low immunogenicity and established use in immunotherapy. Subcutaneous delivery near the axillary LNs—such as in the upper arm—could provide a practical route for promoting peripheral tolerance while minimizing the risk of autoimmune responses. Furthermore, our preliminary observation of elevated CLEC5A expression in human monocyte-derived tolDCs (data not shown) supports the potential biological relevance of this approach in human settings, although further validation is needed.

5. Conclusions

Our study provides evidence that Clec5a-expressing tolDCs play an important role in modulating neuroinflammation and promoting neuroprotection in a PD model. We demonstrated that Clec5a enhances IL-10 production and Treg populations while inhibiting NF-\kappaB signaling, contributing to the tolerogenic function of tolDCs. The therapeutic potential of Clec5a-expressing tolDCs was further supported by observations in MPTP-intoxicated mice, where they alleviated pathological and behavioral impairments and preserved dopaminergic neurons. Notably, the knockdown of Clec5a accelerated disease progression, highlighting its functional relevance in PD pathogenesis. Taken together, these findings suggest that Clec5a may serve as a potential marker and immune modulator for DC-based strategies aimed at regulating neuroinflammation in PD.

Availability of Data and Materials

The data supporting the findings of this study are available within the article and Supplementary Material. All data was generated at the Department of Life Science of CHA University and is available from the corresponding authors upon reasonable request.

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Funding

National Research Foundation of Korea (NRF)(2021R1I1A2045723)