Background
The hippocampus, an area under the medial temporal lobe of the mammalian brain, plays a pivotal role in the neurobiology of learning and memory. It is one of the first regions damaged in Alzheimer’s disease (AD) [
1,
2]. Hippocampal neurodegeneration accounts for the cognitive impairments observed in neurodegenerative disorders, such as AD [
3]. There is a clinical association between hippocampal neurogenesis and cognition and microglia are important effectors of hippocampal neurogenesis. Activated pro-inflammatory microglia have a negative effect on hippocampal neurogenesis and cognitive processes [
4].
Trimethyltin (TMT) is an organotin compound that is considered a potent neurotoxicant and causes behavioral alterations as well as learning and memory impairment in mammals [
5,
6]. Cognitive impairment, including memory loss and learning impairment, developed in experimental animals exposed to TMT, indicating severe hippocampal damage [
7]. It was reported that the levels of activated microglia and pro-inflammatory factors, such as TNFα, IL-1β, and NO were elevated in the hippocampus prior to neuronal death by TMT treatment in rodents. Consistently, previous studies have indicated that microglial activation by TMT exacerbates neuronal death in vivo and in vitro [
8‐
10].
Regulatory T cells (Tregs) act as immune suppressors, playing a role in self-tolerance and immune homeostasis. Immune balance in the central nervous system (CNS) is tightly controlled by Tregs. Previous studies suggested that the Tregs suppress the microglial inflammation by promoting polarization toward anti-inflammatory M2 rather than pro-inflammatory M1 phenotype [
11‐
13]. However, the suppressive activity of Tregs is dysregulated in neurodegenerative diseases, leading to neuroinflammation in these diseases. For these reasons, Tregs are emerging as an attractive therapeutic strategy against neurodegenerative diseases [
14,
15]. Therefore, adoptive cell therapy using Tregs has attracted attention as an individualized medicine for inflammatory diseases [
16]. Treg cell therapy has been attempted in mouse models of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and Parkinson’s disease (PD) to evaluate its neuroprotective effects [
17,
18]. In our previous study, we adoptively transferred Tregs into 3×Tg-AD mice containing three mutations associated with familial Alzheimer’s disease (APP Swedish, MAPT P301L, and PSEN1 M146V) and demonstrated the inhibitory effects of Tregs on the accumulation of amyloid-beta (Aβ) and activation of microglia in the hippocampus [
19]. Since TMT-treated animal models are used to study hippocampus-specific neurodegeneration that accompanies microglial activation, similar to that seen in AD, we aimed to confirm that Treg cell therapy is also effective in TMT-induced hippocampal neurodegeneration. Based on the effects of Treg therapy in other neurodegenerative diseases, it is expected that TMT-induced neuronal loss and behavior disorders will prevent through microglial activation by Treg transfer.
There is some evidence indicating that antigen-specific Tregs may be more efficient, so the generation and expansion of antigen-specific Tregs are important in Treg cell therapy [
20]. To generate antigen-specific Tregs, we presented fibrillar Aβ to bone marrow-derived dendritic cells (Aβ-DCs) and performed ex vivo Treg expansion in the presence of Aβ-DCs. In addition, to increase the efficiency of Treg expansion, we treated cells with bee venom phospholipase A2 (bvPLA2), a Treg expansion inducer [
21]. We previously demonstrated that bvPLA2 induced the Treg population by suppressing apoptosis [
22]. Moreover, we reported that administration of bvPLA2 had neuroprotective effects on AD and PD mouse model [
23,
24].
In the present study, we attempted to expand Aβ-specific Tregs and examine the effects of the adoptive transfer of these Tregs on behavioral deficits, memory formation, and neuronal loss in TMT-induced neurodegenerative mice. Furthermore, we sought to determine whether the effects of Tregs are associated with microglial activation, which induces pro-inflammatory responses. Our findings would be helpful in developing a new treatment strategy for neurodegenerative diseases.
Materials and methods
Animals
Seven-week-old male C57BL/6 mice were purchased from Taconic Farms, Inc. (Samtako Bio Korea, Kyunggi, Korea) and Deahan Biolink (Chungbuk, Korea). The mice were maintained under a 12-h light/dark cycle and temperature-controlled conditions, with food and water ad libitum. All experiments were performed in accordance with the approved animal protocols and guidelines established by Kyung Hee University (KHUAP(SE)-18-073).
Regulatory T cell preparation
To prepare fibrillary Aβ, 5 mM Aβ1–42 peptide (Genscript, NJ, USA) in dimethyl sulfoxide (DMSO) was diluted with 10 mM HCl to a final concentration of 100 µM Aβ and incubated overnight (O/N) at 37 °C. Bone marrow (BM)-leukocytes from femurs and tibiae of mice were resuspended in a medium containing 20 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF; R&D Systems, Minneapolis, MN, USA) [
25]. After 7 days, BM-leukocytes were washed with magnetic-activated cell sorting buffer (Miltenyi Biotec Inc., CA, USA) and dendritic cells (DCs) were isolated using CD11c
+ MicroBeads (Miltenyi). The DCs were resuspended at a density of 2 × 10
5/mL and seeded in 96-well U-bottom plates. For antigen presentation, DCs were treated with 0.5 µM fibrillated Aβ for 24 h. CD4
+ T cells from splenocytes were isolated using CD4 (L3T4) MicroBeads (Miltenyi), resuspended at a density of 2 × 10
6/mL, and added to the DC culture at a ratio of 10: 1 (CD4
+ T cells: DCs) with 0.4 µg/mL bvPLA2 (Sigma-Aldrich, MO, USA). Four days after CD4 T cell–DC co-culture, CD4
+CD25
+ T cells (Tregs) were isolated using MACS, according to the manufacturer’s protocol (CD4
+CD25
+ Regulatory T Cell Isolation Kit; Miltenyi). CD4
+CD25
+ regulatory T cells were stimulated using the Treg Expansion Kit (Miltenyi) for 2 weeks. To confirm the purity of isolated cells and the change in phenotype, cells were stained with fluorescently labeled antibodies and analyzed using flow cytometry. The following antibodies were used (1:1000): PE-CD11c (eBioscience, San Diego, CA, USA) for DC purity, PE-CD4 (BD Pharmingen, CA, USA) for CD4 T cell purity, and PE-CD127 (eBioscience), PE-Cy7-CD4 (Invitrogen, CA, USA), APC-CD62L (Invitrogen), and APC-Cy7-CD25 (BD Pharmingen) for Treg phenotype. Samples were washed with the BD FACS stain buffer (BD Bioscience, CA, USA) and stained for 30 min at 4 °C in the dark. After staining, the cells were washed 2 times with the stain buffer. The data were acquired using a BD FACSlyric™ flow cytometer (BD Bioscience) and analyzed using BD FACSuite software (BD Bioscience).
BV2 microglia and Treg co-culture
BV2 microglia were incubated at 37 °C with 95% humidity and 5% CO
2 for all experiments. To examine the effects of Tregs on microglial polarization, 1 × 10
6 BV2 microglia in Dulbecco’s modified Eagle’s medium (DMEM; Welgene Daegu, Korea) 500 µL were seeded into 12-well plates. After 2–3 h, Tregs were co-cultured with BV2 cells (BV2:Treg = 10:1) and the cells were immediately stimulated with 3 µM TMT for 24 h according to previous study [
26]. The cell culture supernatants were collected for ELISA, and the remaining adherent cells were harvested for mRNA extraction.
Animal experiments
For TMT (Sigma-Aldrich, Steinheim, Germany) treatment, the mice were intraperitoneally (i.p.) administered TMT (2.6 mg/kg) and randomly divided into five groups of 18 to 25 mice, except for the control group (n = 21) that did not receive TMT. After 7 days, Treg cells (4 × 104, 2 × 105, or 1 × 106) were intravenously injected (i.v.) into the tail vein of TMT-treated mice. Aricept (3 mg/kg; Eisai Co. Ltd, Tokyo, Japan) was orally administered once daily for 2 weeks from day 7.
Behavior tests
Ten days after Treg injection, spatial learning and memory were examined in mice using the Morris water maze (MWM) test with minor modifications [
27]. The water maze was a circular pool with a 90-cm diameter and was filled with opaque water containing 1 kg of powdered skim milk (maintained at 22 ± 2 °C). During training, a 6-cm hidden platform was fixed 1 cm below the water surface. The pool was surrounded by different extra-maze cues. The maximal trial duration was 60 s, with 30 s on the platform at the end of the first trial. Each animal was trained for one of the different starting positions and swimming paths once per day for 4 days. All mice were subjected to three trials per day at intervals of 15 min for 4 consecutive days. For the probe trial, the platform was removed from the pool, and the mice were allowed to swim freely for 60 s to search for the previous location of the platform. Escape latency, time spent in the platform quadrant, and the number of platform crossings were recorded for each mouse.
The elevated plus maze (EPM) test was performed after the first MWM training to measure the anxiety levels in mice. The EPM equipment was a cross-shaped maze that was elevated to a height of 50 cm above the floor. It consisted of two opposite open arms and two closed arms. Mice were positioned on the central platform and allowed to explore the maze for 3 min.
Data were collected using a video camera connected to a video recorder and a tracking device (S-MART, Pan-Lab).
Immunohistochemistry
After the behavioral test, mice were anesthetized by pentobarbital (50 mg/kg, i.p.) and transcardially perfused with formalin and PBS. The brain was transferred into a 30% sucrose solution, and frozen-sectioned on a sliding microtome into 30-μm-thick coronal sections. The brain sections (3–5 sections/mice) were washed with phosphate-buffered saline (PBS) and incubated for 10 min with 3% hydrogen peroxide (Sigma-Aldrich) to quench endogenous peroxidase activity. Nonspecific binding was reduced by blocking the sections with 1.5% bovine serum albumin (BSA; Millipore, MA, USA) in PBS for 1 h. The sections were incubated with antibodies (1:500) for mouse CREB (Cell Signaling Technology, MA, USA), Iba1 (WAKO, Osaka, Japan), PKC (Abcam, MA, USA), NeuN (Abcam), or NGF (Invitrogen) for 24 h at RT. Brain sections were washed with PBS, incubated with a biotinylated secondary antibody (Vectastain ABC kit; Vector Laboratories, CA, USA) for 2 h, and processed using an avidin–biotin peroxidase complex kit (Vectastain ABC kit; Vector Laboratories) for 1 h. Each marker was visualized by incubation with 0.05% diaminobenzidine–HCl (DAB; Vector Laboratories). The labeled sections were mounted and analyzed under a bright-field microscope (Nikon) and the intensities were quantified using the ImageJ software (US National Institutes of Health; available at
http://rsb.info.nih.gov/ij/) as previously describe [
28,
29]. Data were analyzed under the same conditions by two observers for each experiment in blinded conditions to avoid the bias. Images were calibrated into an array of 512 × 512 pixels corresponding to a tissue area. Each pixel resolution had 256 Gy levels, and the intensity of immunoreactivity was evaluated based on the ROD, which was obtained after transformation of the mean gray level using the following formula: ROD = log
10 (256/mean gray level).
RT-PCR assay
Mice were transcardially perfused with PBS after anesthetization. RNA was isolated from the brain and BV2 cells using the easy-BLUE RNA extraction kit (iNtRON Biotechnology, Seoul, Korea), and cDNA was synthesized using Cyclescript reverse transcriptase (Bioneer, Seoul, Korea). The samples were prepared for real-time PCR using the SensiFAST SYBR no-Rox kit (Bioline, OH, USA). Real-time quantitative PCR was performed using CFX Connect (Bio-Rad, WA, USA) and the data were analyzed using CFX Maestro Software (Bio-Rad). The amplification conditions were 95 °C for 30 s, followed by 50 cycles at 95 °C for 10 s and 55 °C for 30 s. The expression levels of each target mRNAs, 2
−dCt values, were normalized to those of mouse β-actin, a housekeeping gene used as an endogenous control [
30]. Then the relative mRNA expression values were calculated as a fold change in which the mean value of the control group considered 1. The base sequences of the primers are shown in Table
1.
Table 1
The base sequence of primers for rtPCR
β-actin | GTG CTA TGT TGC TCT AGA CTT CG | ATG CCA CAG GAT TCC ATA CC |
NOS2 | CAG CTG GGC TGT ACA AAC CTT | CAT TGG AAG TGA AGC GTT TCG |
IL-1β | AAG CCT CGT GCT GTC GGA CC | TGA GGC CCA AGG CCA CAG G |
IL-6 | TTC CAT CCA GTT GCC TTC TTG | GGG AGT GGT ATC CTC TGT GAA GTC |
TNFα | GGC AGG TTC TGT CCC TTT CAC | TTC TGT GCT CAT GGT GTC TTT TCT |
TGFβ | GAG GTC ACC CGC GTG CTA | TGT GTG AGA TGT CTT TGG TTT TCT C |
BDNF | GGA ATT CGA GTG ATG ACC ATC CTT TTC CTT AC | CGG ATC CCT ATC TTC CCC TTT TAA TGG TCA GTG |
Mrc1 | TTC GGT GGA CTG TGG ACG AGC | ATA AGC CAC CTG CCA CTC CGG |
Ym1 | TGG AGG ATG GAA GTT TGG AC | GAG TAG CAG CCT TGG AAT GT |
Arg1 | CTC CAA GCC AAA GTC CTT AGA G | AGG AGC TGT CAT TAG GGA CAT C |
ELISA
After anesthetization, mice were transcardially perfused with PBS. Total protein was isolated from the brain using RIPA buffer (Biosesang, Seoul, Korea) with protease and phosphatase inhibitors (Thermo Fisher Scientific, CA, USA). Levels of pro-inflammatory cytokines were quantified using TNF-α, IL-1β, and IL-6 DuoSet ELISA (R&D Systems) and normalized to the levels of BSA. The cytokines secreted by BV2 cells were measured using BV2 cell culture media and TNFα and TGFβ DuoSet ELISA (R&D Systems). The optical density was measured at 450 nm using a microplate reader (Versamax Microplate Reader, USA). All fold changes were expressed relative to those in the control group.
Live cell imaging
CDr20 is a microglia-specific biofluorescence probe with high performance for visualizing live microglia both in vitro and in vivo [
31]. For time-lapse imaging, 5 × 10
4 BV2 microglia in 1 mL were seeded into 4-well chambers and cultured for 2–3 h before live-cell imaging was performed. Approximately 5 × 10
4 mouse Tregs were seeded onto each chamber containing microglia. Microglia were continuously observed from pre-activation to post-activation with TMT (3 µM) treatment in the presence of 0.5 mM CDr20 (1 µM) every 3 min for a total of 30 min under the red fluorescent channel (excitation at 570 nm and emission at 600 nm). The change in the region of intensity (ROI) of each cell was measured for 30 min. All observations were performed using a DeltaVision imaging system (GE, Boston, MA, USA). To assess the intensity of fluorescence live-cell imaging, the SoftWorX software (v.6.1.3, GE) was used. CDr20 was kindly provided by Dr. YT Chang (Pohang University of Science and Technology, Pohang, Korea).
Statistical analysis
All data were analyzed using GraphPad Prism 5.01 (GraphPad Software Inc., CA, USA). The data are presented as the mean and standard error of the mean (SEM) where indicated. All statistical significance of each variable was evaluated by one-way analysis of variance (ANOVA), followed by Tukey multiple comparison test for multiple comparisons except the intensity of PKC and time-lapse live imaging: *p < 0.05, **p < 0.01, ***p < 0.001. The intensity of PKC and time-lapse live imaging were analyzed using two-tailed Student’s t-test and two-way ANOVA followed by Bonferroni post-tests, respectively. All experiments were performed in a blinded manner and repeated independently under identical conditions.
Discussion
In this study, we investigated the effects of Tregs on TMT-induced hippocampal neurodegeneration. We found that Tregs not only improved cognitive function, but also reduced anxiety in TMT-intoxicated mice. Moreover, Tregs inhibited neuronal loss, and the neuroprotective effects of Tregs could potentially be attributed to suppression of microglia-mediated neuroinflammation. Compared with Aricept, a drug used for AD, adoptive transfer of Tregs was found to be similarly or more effective. Our study supports the potential of Treg therapy for hippocampal neurodegeneration.
Tregs are considered attractive therapeutic targets for attenuating inflammation. Tregs play roles in inhibiting pro-inflammatory cytokines and inducing neurotrophic factors and apoptosis of pro-inflammatory microglia, ultimately promoting neuroprotection [
35]. In a previous study from our laboratory, adoptive transfer of Tregs was attempted in 3×Tg-AD mice, upon which a clear delay in the onset of AD neuropathology was observed. In addition, the neuroprotective effect of Tregs was demonstrated, including reduction in Aβ deposition and microglial activation in the hippocampus. However, Treg adoptive transfer has never been attempted in the TMT-induced neurodegenerative model, although it has been considered as a model of AD-like disease in rats [
36]. Therefore, in this study, we transplanted Tregs into TMT-intoxicated mice to alleviate TMT-induced hippocampal neurodegeneration.
For clinical application, various strategies were proposed to improve the effects of Treg therapy. The most common method is developing antigen-specific Tregs instead polyclonal Tregs which may lead to off-target suppression. Antigen presentation could enhance the therapeutic utility of T cell transfer to induce target sites [
37,
38]. Aβ is also present in the normal brain; however, it is misfolded and deposited in the hippocampus in several pathological conditions such as AD. Therefore, it is regarded as one of the characteristics of these diseases [
39]. Moreover, since Aβ accumulation was detected in TMT-intoxicated mice, we chose Aβ as an antigen for presentation to adoptively transfer Tregs [
40]. Additionally, we treated bvPLA2 during antigen presentation to expand the Treg population. It was reported that bvPLA2 induces Treg population both in vivo and in vitro and significantly suppresses apoptosis in Tregs [
21,
22]. The combination of antigen presentation via DCs and bvPLA2 treatment for the generation and expansion of Aβ-specific Tregs is an important attempt of this study. The effects and mechanism of action of Aβ presentation and bvPLA2 treatment on the efficacy of Tregs remain unclear. This will be investigated in a future study.
For decades, TMT-induced neurodegenerative models, especially rats and mice, have been used as good research tools. In the rat model, TMT administration induces a progressive cell death accompanied by microglial activation in CA1 and CA3 like AD [
36,
41]. Notably, TMT injection into mice can also cause dentate gyrus (DG) granular cell apoptosis. Many studies on TMT-induced mouse model focused on neuropathology in DG [
42‐
46]. DG is the site where adult hippocampus neurogenesis occurs and most information of DG is sent to CA3 to CA1 according to the tri-synaptic pathway in the hippocampus [
47]. Some studies reported neuronal self-repair following TMT-induced neuronal loss in DG. These data indicated that neuronal regeneration occurs in DG approximately 7–10 days after TMT intoxication [
48‐
51]. Therefore, TMT has been considered as a toxicant that acts exclusively on DG in mice. However, there are several reports that TMT induced neurodegeneration was not restricted in DG. KR Reuhl and colleagues reported extensive degenerative and necrotic changes in CA3 after TMT intoxication [
52]. In another report, IB4, a microglial marker, and Fas, an apoptotic molecule, were increased in CA1 after TMT intoxication [
53]. Nevertheless, degenerative change in neurons of CA was unremarkable than DG, TMT has been considered as a neurotoxicant that selectively affects in DG [
54]. However, studies on TMT-induced cognitive dysfunction have emerged [
55,
56]. In recent studies, it was reported that memory impairment accompanied by neurodegeneration in CA induced by TMT intoxication in mice. According to these studies, neuronal loss was observed in CA1 even after 7 days of TMT intoxication, unlike in DG [
57‐
60]. This implied that the timing of TMT-induced neurotoxicity on DG and CA is different, probably it occurs later in CA than in DG. It is a very interesting topic that these events could affect the tri-synaptic circuit related disorders and will be revealed in further studies. In the present study, we focus on TMT-induced cognitive disorders and molecular change in CA1 and CA3.
It is reported that TMT-intoxicated animals developed cognitive impairment and hyperactivity [
7]. The results of MWM and EPM test showed that high dose of Tregs improved these behavior changes. But Aricept, a positive control, showed no significant effect. It is probably because Aricept is not an effective drug for long-term treatment. In fact, it has been reported that Aricept treatment for 16 weeks did not improve cognitive function in APPswe/PS1dE9 mice [
61]. These results imply that Treg possess a sufficient potential as a more effective treatment option than Aricept.
CREB is a key molecule in synaptic strengthening, memory formation, and neurogenesis. It controls the transcription of genes involved in neuronal growth and survival and the lack of CREB gene results in neurodegeneration. Indeed, disruption of the CREB phosphorylation mechanism results in a reduction in CREB activation following memory impairment in AD [
62‐
64]. Likewise, TMT-induced memory impairment was observed upon inhibition of CREB activation and was alleviated by regulation of the CREB-signaling pathway in the hippocampus [
65]. Since CREB plays a critical role in short- to long-term memory, drugs targeting CREB itself have been proposed for memory modification [
66]. One of the molecules present the upstream of CREB and regulating it is PKC. Therefore, activation of PKC leads to CREB phosphorylation [
67,
68]. In addition, PKC itself performs neurogenesis-related functions, including cell differentiation and proliferation and immune-related processes. In a previous study,
Bacopa monnieri (L.) Wettst. extract prevented TMT-induced hippocampal damage via PKC [
69]. NGF is also a neurotrophic factor that enhances neurogenesis [
70]. We showed that TMT intoxication induced neuronal cell death, represented by the expression of NeuN, in both CA1 and CA3, whereas there was no difference in DG (data not shown). These results are consistent with the possibility of different TMT toxicity timing on DG and CA mentioned above. And Tregs, especially at high dosage, increased the expression of CREB, PKC, and NGF as well as NeuN. This suggests that adoptively transferred Tregs not only prevent neuronal loss, but also induce neurogenesis in the hippocampus.
Microglia are phagocytic macrophages that comprise 10–15% of the total cells in the CNS. Since they can be either beneficial or harmful depending on their activation status, their polarization is considered a potential therapeutic target in neurodegenerative diseases such as AD. Classically activated “M1” microglia contribute to inflammation by secreting free radicals, NOS2, and pro-inflammatory cytokines such as IL-1, IL-6, and TNFα. Neuroinflammation amplifies microglial activation and further worsens the disease. In contrast, alternatively activated “M2” microglia promote tissue repair by releasing neuroprotective cytokines such as IL-10, TGFβ, and IGF1. Therefore, microglial polarization is considered an attractive therapeutic strategy against cognitive disorders [
71,
72]. Indeed, there have been many studies that treat neurodegenerative diseases by shifting microglial phenotypes. Some studies reported behavior recovery following enhancing M2 microglia in not only AD, but also traumatic brain injury and spinal cord injury [
73‐
75]. It is well known that pro-inflammatory microglial activation and cytokine secretion are associated with TMT intoxication [
76‐
79]. Therefore, we confirmed the activation and polarization of microglia in vivo and in vitro. As expected, microglial activation and pro-inflammatory marker expression were increased upon TMT intoxication. However, Tregs inhibited the activation of M1 but enhanced M2 microglia in vivo. Additionally, we observed microglial activation over time using time-lapse live imaging in vitro. TMT treatment activated microglia for 30 min, but co-culture with Tregs suppressed this activation. ELISA and RT-PCR data showed that this inhibition by Tregs targeted M1 microglia. Based on these in vivo and in vitro results, Tregs could effectively inhibit microglial activations and covert microglial phenotype from M1 to M2. These changes in microglia phenotype lead to neurogenesis and ultimately improve cognitive impairment. It is in line with those of previous studies showing that Tregs modulate microglia and alleviate neurodegenerative disorders [
11,
72,
80,
81].
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