Neuroprotective effects of ex vivo-expanded regulatory T cells on trimethyltin-induced neurodegeneration in mice

Abstract

Background: Trimethyltin (TMT) is a potent neurotoxicant that leads to hippocampal neurodegeneration. Regulatory T cells (Tregs) play an important role in maintaining the immune balance in the central nervous system (CNS), but their activities are impaired in neurodegenerative diseases. In this study, we aimed to determine whether adoptive transfer of Tregs, as a living drug, ameliorates hippocampal neurodegeneration in TMT-intoxicated mice.

Methods: CD4⁺CD25⁺ Tregs were expanded in vitro and adoptively transferred to TMT-treated mice. First, we explored the effects of Tregs on behavioral deficits using the Morris water maze and elevated plus maze tests. Biomarkers related to memory formation, such as cAMP response element-binding protein (CREB), protein kinase C (PKC), neuronal nuclear protein (NeuN), nerve growth factor (NGF), and ionized calcium binding adaptor molecule 1 (Iba1) in the hippocampus were examined by immunohistochemistry after killing the mouse. To investigate the neuroinflammatory responses, the polarization status of microglia was examined in vivo and in vitro using real-time reverse transcription polymerase chain reaction (rtPCR) and Enzyme-linked immunosorbent assay (ELISA). Additionally, the inhibitory effects of Tregs on TMT-induced microglial activation were examined using time-lapse live imaging in vitro with an activation-specific fluorescence probe, CDr20.

Results: Adoptive transfer of Tregs improved spatial learning and memory functions and reduced anxiety in TMT-intoxicated mice. Additionally, adoptive transfer of Tregs reduced neuronal loss and recovered the expression of neurogenesis enhancing molecules in the hippocampi of TMT-intoxicated mice. In particular, Tregs inhibited microglial activation and pro-inflammatory cytokine release in the hippocampi of TMT-intoxicated mice. The inhibitory effects of TMT were also confirmed via in vitro live time-lapse imaging in a Treg/microglia co-culture system.

Conclusions: These data suggest that adoptive transfer of Tregs ameliorates disease progression in TMT-induced neurodegeneration by promoting neurogenesis and modulating microglial activation and polarization.

Keywords: Cell therapy; Hippocampal neurodegeneration; Microglia; Regulatory T cells; Trimethyltin.

Fig. 1

Isolation and ex vivo expansion of Tregs. CD11c⁺ dendritic cells and CD4⁺ T cells were isolated from bone marrow leukocytes and splenocytes, respectively. CD4⁺CD25⁺ Tregs were isolated after 4 days of CD11c⁺ DC and CD4⁺ T cell co-culture and expanded for 2 weeks. For the in vivo study, TMT was injected into all groups except the non-treated control group. The TMT group consisted of only TMT-intoxicated mice. Aricept group was treated with Aricept as a positive control. For the Treg group, 4 × 10⁴, 2 × 10⁵, or 1 × 10⁶ expanded Tregs were injected per mouse. After the behavioral test, mice were killed (A). The purity (B) and phenotype (C) of the isolated cells were analyzed by flow cytometry

Fig. 2

Treg improved the behavioral disorder of TMT-intoxicated mice. The MWM test was performed 10 days after Treg injection. Latency time (s) on a hidden platform (A) and time in quadrant (%) (B) were measured (n = 10–13 mice/group). The EPM test was performed 10 days after Treg injection. Number of closed arm entries (C) and number of open arm entries (D) were recorded (n = 10–13 mice/group). Error bars represent the mean ± SEM. Significance was determined by Tukey’s HSD test (*p < 0.05, ***p < 0.001 vs. the Con group and ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. the TMT group)

Fig. 3

Treg increased the expression of CREB and PKC in the brain of TMT-intoxicated mice. Immunohistochemistry was performed for CREB expression in the hippocampi of TMT-intoxicated mice (A). The number of CREB-positive cells among CA1 and CA3 (B) cells was measured (n = 10–13 mice/group). PKC expression was assessed in the hippocampi (C), and the intensity of PKC in CA1 and CA3 (D) was measured using ImageJ software (n = 5–8 mice/group). Data are presented as the mean ± SEM. Significance was determined by Tukey’s HSD and t-test (*p < 0.05, ***p < 0.001 vs. the Con group and ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. the TMT group)

Fig. 4

Treg increased the expression of NeuN and NGF in the brain of TMT-intoxicated mice. Immunohistochemistry was performed for NeuN expression in the hippocampi of TMT-intoxicated AD mice (A). The number of NeuN-positive cells among CA1 and CA3 cells (B) was measured (n = 5–8 mice/group). Immunohistochemistry was performed for NGF expression in the hippocampi of TMT-intoxicated AD mice (C). The intensity of NGF-positive cells in CA1 and CA3 (D) was measured (n = 5–8 mice/group). Data are presented as the mean ± SEM. Significance was determined by Tukey’s HSD (***p < 0.001 vs. the Con group and ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. the TMT group)

Fig. 5

Treg inhibited pro-inflammatory factors via microglial activation in the brain of TMT-intoxicated. The expression of Iba1 was observed in the hippocampi of TMT-intoxicated mice using immunostaining (A). The number of Iba1-positive cells in CA1 and CA3 (B) was measured (n = 10–13 mice/group). The protein levels of pro-inflammatory cytokines, including TNFα, IL-1β, and IL-6, in the brain were measured using ELISA and calculated as a relative for Con (C) (n = 3–5 mice/group). The relative mRNA levels of pro-inflammatory microglia-associated markers, including TNFα, NOS2, IL-1β, and IL-6, and anti-inflammatory markers, TGFβ, Mrc1, Arg1, and BDNF in the brain were analyzed (D) (n = 3–5 mice/group). Data are presented as the mean ± SEM. Significance was determined by Tukey’s HSD (*p < 0.05, **p < 0.01 vs. the Con group and ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. the TMT group)

Fig. 6

Treg modulates TMT-induced microglial activation and polarization. Time-lapse live imaging of BV2 microglial cells was monitored for 30 min with a CDr20 live microglia-specific probe (A). Representative images show microglial activity at 0 and 30 min with TMT treatment in the presence of CDr20 under the red fluorescent channel (excitation at 570 nm and emission at 600 nm). BV2 microglial cells were co-cultured with Tregs and stimulated with TMT for 24 h. The secreted levels of TNF-α and TGF-β (B) were measured using ELISA with supernatants. Relative mRNA expression of M1 cytokines, TNF-α and IL-1β; M1 microglial maker, NOS2; M2 cytokine TGFβ; M2 microglial makers, Mrc1 and Ym1 was examined and normalized to actin (C). Data are presented as the mean ± SEM. Significance was determined by Bonferroni’s correction and Tukey’s HSD (*p < 0.05, **p < 0.01, ***p < 0.001). Scale bar = 50 μm

Fig. 7

Treg has neuroprotective effects by modulating microglial polarization in TMT-intoxicated mice. Ex vivo expanded and adoptively transferred Tregs ameliorates hippocampal neurodegeneration in TMT-intoxicated mice. Tregs promotes microglial phenotype shift from pro-inflammatory M1 to anti-inflammatory M2, resulting in a neuroprotective effects on behavioral deficits, memory formation, neuronal loss, and neuroinflammation