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. 2016 Sep 27;37(5):919–929. doi: 10.1007/s10571-016-0429-1

Adoptive Regulatory T-cell Therapy Attenuates Perihematomal Inflammation in a Mouse Model of Experimental Intracerebral Hemorrhage

Lei-Lei Mao 1, Hui Yuan 1,2, Wen-wen Wang 1, Yu-jing Wang 1, Ming-feng Yang 1, Bao-liang Sun 1,2, Zong-yong Zhang 1,✉, Xiao-yi Yang 1,✉
PMCID: PMC11482213  PMID: 27678140

Abstract

The CD4+CD25+ regulatory T cells (Tregs), an innate immunomodulator, suppress cerebral inflammation and maintain immune homeostasis in multiple central nervous system injury, but its role in intracerebral hemorrhage (ICH) has not been fully characterized. This study investigated the effect of Tregs on brain injury using the mouse ICH model, which is established by autologous blood infusion. The results showed that tail intravenous injection of Tregs significantly reduced brain water content and Evans blue dye extravasation of perihematoma at day (1, 3 and 7), and improved short- and long-term neurological deficits following ICH in mouse model. Tregs treatment reduced the content of pro-inflammatory cytokines interleukin (IL)-1β, IL-6, tumor necrosis factor-α, and malondialdehyde, while increasing the superoxide dismutase (SOD) enzymatic activity at day (1, 3 and 7) following ICH. Furthermore, Tregs treatment obviously reduced the number of NF-κB+, IL-6+, TUNEL+ and active caspase-3+ cells at day 3 after ICH. These results indicate that adoptive transfer of Tregs may provide neuroprotection following ICH in mouse models.

Keywords: Intracerebral hemorrhage, Regulatory T cells, Neurological deficits, Pro-inflammatory cytokines

Introduction

Intracerebral hemorrhage (ICH) is a fatal subtype of hemorrhagic stroke that accounts for about 10–15 % of all strokes with high morbidity and mortality, and occurs when the weakened blood vessels of brain parenchyma suddenly rupture. When ICH occurs, the key factor that affects ICH outcome is hemorrhagic volume and hematoma expansion that causes the physical trauma and mass effect, increases intracranial pressure and affects blood flow. Primary brain injury is mainly due to hematoma formation, which induces mechanical damage to the neurovascular architecture surrounding the hematoma. The intraparenchymal blood components, such as red blood cells, immunoglobulins, complement components and coagulation factors, contribute to secondary brain injury after ICH, which subsequently activates cytotoxic, oxidative stress, excitotoxic and inflammatory pathways and play an essential role in blood–brain barrier (BBB) breakdown, brain edema and neurological deterioration (Keep et al. 2012; Zhou et al. 2014). Currently, authoritative opinions show that inflammation is a key contributor of ICH-induced secondary brain injury, aggravates the ICH-induced primary brain injury, and ultimately leads to BBB disruption, brain edema, and massive brain cell death (Zhou et al. 2014; Chen et al. 2015). The inflammatory response begins after the presence of blood in brain parenchyma, causes the resident microglia and astrocyte rapid activation. The activated microglia clears the hematoma and apoptotic cells through phagocytosis. However, excessive microglial activation aggravates ICH-induced primary brain injury by releasing a variety of pro-inflammatory cytokines, free radicals, chemokines, and other toxic chemicals. Release of cytokines and chemokines subsequently causes infiltration of blood-derived inflammatory cells (leukocytes and macrophages) (Aronowski and Hall 2005; Wu et al. 2008). The activated blood-derived inflammatory cells in hemorrhagic parenchyma contributes to ICH-induced brain injury by releasing pro-inflammatory mediators, producing reactive oxygen species (ROS), and increasing BBB permeability (Joice et al. 2009; Nguyen et al. 2007). Hence, anti-inflammatory therapy may be a potential strategy against ICH.

Tregs are a specialized T-cell population, which can suppress inflammatory response and maintain immune homeostasis, exert suppressive roles on effector T cells and monocytes/macrophages activation through either release of the immunosuppressive cytokines, transforming growth factor-β and IL-10 or by direct contact with the suppressed immune cells (Vignali et al. 2008). Interestingly, the number of Tregs of peripheral blood system increases and remains at an elevated level for several weeks after stroke in patients and experimental animals (Yan et al. 2009; Shi et al. 2015; Offner et al. 2006). Increasing number of studies show that Tregs inhibit cerebral inflammation following ischemic stroke (Liesz et al. 2009; Li et al. 2013a, b) and subarachnoid hemorrhage (Wang et al. 2016), whereas depletion of Tregs exacerbates brain injury and deteriorates functional outcome (Liesz et al. 2009). Recent study suggests that Tregs inhibit microglia activation and against inflammatory injury at 72 h following ICH (Yang et al. 2014). However, it is not known whether Tregs affect delaying BBB disruption, cell apoptosis and long-term neurological function after ICH.

We hypothesized that Tregs treatment after ICH would inhibit the cerebral inflammation, reduce cerebral damage and improve functional outcome in ICH model. Therefore, we measured the delaying brain edema, BBB permeability, cerebral inflammation, cell apoptosis, short- and long-term neurological function after experimental ICH in mouse treated with Tregs or vehicle.

Materials and Methods

Animals and Experimental Design

Male C57/BL6 mice (weighing 25–30 g, 12 weeks old) were purchased from the Animal Center of Lukang (Jining, China), and were group-housed in individual cage at a constant humidity and temperature on a 12 h light/dark cycle. All animal experiments were approved by the Institutional Animal Care and Use Committee of Taishan Medical University and conducted in accordance with the National Institutes of Health (NIH) guideline for the Care and Use of Laboratory Animals. The mice were randomly assigned to three groups: the sham-operated group (n = 18), vehicle (PBS)-treated ICH group (n = 18), and the Tregs-treated ICH group (n = 18). All experiments were performed following the experimental design, as shown in Fig. 1.

Fig. 1.

Fig. 1

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Scheme for experimental design. a Schematic representation of experimental design and the method for inducing ICH model in the present study. Tregs were isolated from lymph nodes and splenocyte of donor, and injected into tail vein of recipients after ICH which is established by autologous blood striatum infusion. b Timeline for experimental parameters is indicated

Mouse ICH Model

Mouse ICH model was produced by the autologous blood infusion as previously described (Rynkowski et al. 2008), which proved to be a reproducible and reliable animal model of ICH in acute and chronic study. In brief, we randomly selected mouse and mounted in the stereotaxic frame. Under isoflurane (3–4 % for induction and 1–2 % for maintenance, evaporated in an oxygen-air mixture, 20:80 %) anesthesia, the skin midline incision was made between the eye and the lambda, scraped the underlying muscular attachments plus periosteum and dry the skull. After drilling a burr hole, 30 µL of autologous blood was injected into the striatum (anterior, 0.2 mm; lateral, 2.3 mm; ventral, 3.5 mm from bregma) using a 26-gauge needle attached to a Hamilton syringe; 5 µL of blood was injected at a rate of 2 µL/min, and the injection was stopped for 7 min, then the remaining portion of blood was injected at a rate of 2 µL/min. The needle was held in place for 10 min and then slowly removed. The warmed bone wax sealed the drilled hole, and the scalp was closed with suture. Then, the mouse were placed under an incandescent lighting until palinesthesia.

Isolation, Identification and Adoptive Transfer of Tregs

The CD4+CD25+ Tregs were isolated, identified and adoptive-transferred according to our previous study (Li et al. 2013a, b; Wang et al. 2016). Briefly, single-cell suspensions were prepared from axillary and inguinal lymph nodes and spleens of mice using the gentleMACS Dissociator (Miltenyi Biotec, Germany). The CD4+CD25+ Tregs were collected by CD4-negative selection and CD25-positive selection with a Regulatory T cell Isolation kit (Miltenyi Biotec, Germany) according to the manufacturers’ instructions. Flow cytometric analysis was performed to identify the enriched CD4+CD25+ Tregs populations; cells were stained with anti-mouse CD4 FITC and anti-mouse CD25 PE (Miltenyi Biotec, Germany) and were then analyzed by flow cytometer (BD FACSCCalibur, USA). The cell dose and time of Tregs were based upon prior investigation in cerebral ischemia and subarachnoid hemorrhage model (Li et al. 2013a, b; Wang et al. 2016). The recipient mouse received 2 × 106 freshly enriched Tregs (in 0.1 mL PBS, tail intravenous injection) at 30 min post ICH surgery.

Brain Water Content and Blood–Brain Barrier Permeability

The brain water content of each group was measured according to the wet/dry method at day (1, 3 and 7) after ICH. After isoflurane anesthesia, the hemisphere (infusion side) of brain was taken within 1 min and weighed immediately to determine the wet weight (WW), then dried at 100 °C for 2 days and was weighed to determine the dry weight (DW). Perihematomal edema was determined, where % brain water content = (WW − DW)/WW × 100 %.

The blood–brain barrier (BBB) permeability was measured by Evans blue extravasation at day (1, 3 and 7) after ICH according to our previous study (Zhang et al. 2015a). Briefly, the 2 % (w/v) Evans blue (Sigma, USA) was injected intravenously through the tail vein and allowed to circulate for 60 min. After isoflurane anesthesia, mouse was perfused transcardially with equal volume PBS buffer using a 50-ml syringe at the same rate. The hemisphere (infusion side) of brain was taken within 1 min, weighed immediately, and homogenized using the power-driven Polytron handheld homogenizers for 1 min. The homogenates were mixed with an equal volume of ethanol plus trichloroacetic acid (3:1) and incubated for 12 h. Then, the standards and samples were centrifuged at 12,000 rpm for 10 min, and the supernatant was measured at OD610 nm using a SpectraMax microplate reader (Molecular Devices, USA). The Evans blue content IS represented as μg/g tissue protein.

Corner Test, Cylinder Test, and Foot Fault Test

The neurological score of each group was analyzed by corner test, cylinder test, and foot fault test at day (1, 3, 7, 14, 21, and 28) after ICH. Two ‘blinded’ researchers evaluated the scoring.

The corner test was originally to evaluate neurological deficits of cerebral ischemia (Zhang et al. 2002), is correlated with the magnitude of brain edema and used to evaluate sensorimotor deficits after ICH (Krafft et al. 2014). In brief, mice were permitted to place in middle of open side and trained to walk toward the 30° angle corner that is made by two attached Plexiglas boards, and had to turn either to the right or to the left. The choice of turning the left or right in each mouse was recorded for 10 trials (at least 30 s breaks between the trials). The left turn (%) as indices of the sensorimotor asymmetry was calculated as left turns/total number of turn × 100 %.

The cylinder test was used to evaluate the forelimb asymmetry following ICH according to previous study (Wakai et al. 2015). Briefly, mice were individually placed in a transparent Plexiglas cylinder (9 cm diameter, 15 cm high) and were video-recorded for 5 min. The number of ipsilateral forelimb (I), contralateral forelimb (C), and bilateral forelimb (B) wall contacts was determined. The asymmetry score was calculated as (I − C)/(I + C + B) × 100 %.

The foot fault test was used to evaluate the motor precision of experimental animals after ICH according to previous study (Galho et al. 2016). Mice were placed in an elevated metallic grid with parallel bars 1.5 cm apart and raised 60 cm above the floor, and allowed to freely move for 5 min. The number of failed attempts to place a paw on the grid was counted. Performance was defined by the percentages of error rate for the lesion on contralateral side of the body.

Immunofluorescence and TUNEL Staining

Immunofluorescence and TUNEL staining were performed at 72 h after sham- or ICH-operated surgery according to our previous study (Zhang et al. 2015b). After isoflurane anesthesia, mouse was perfused transcardially with PBS followed by 4 % paraformaldehyde. The harvested brains were fixed in 4 % paraformaldehyde/PBS for 6 h followed by 30 % sucrose/PBS buffer for 24 h at 4 °C. The 10-µm-thick coronal brain sections were obtained from a Leica CM1950 cryostat (Leica, Germany), permeabilized with 0.1 % Triton X-100 for 5 min and blocked in 5 % donkey serum for 2 h, and were incubated with primary antibodies (IL-6, 1:200; NF-κB p65, 1:100; active caspase-3, 1:200, Abcam, USA) for 16 h at 4 °C. Coronal sections were washed with PBS buffer, and incubated with fluorescently labeled secondary antibody (1:500, Jackson ImmunoResearch, USA) for 2 h at room temperature. Coronal sections were washed with PBS and cover-slipped with anti-fading buffer. Also, TUNEL staining of coronal sections was performed with In Situ Cell Death Detection Kit with Fluorescein (TUNEL, Roche, Germany) according to the manufacturers’ instructions. Images were captured using a fluorescent microscope (Olympus BX51, Japan) using the constant parameters.

Enzyme-Linked Immunosorbent Assay (ELISA) and Measurement of MDA Content and SOD Activity of Perihematoma

The segments of perihematoma of hemispheres were harvested at 72 h after ICH, and homogenized in 0.9 % saline using the power-driven Polytron handheld homogenizers on ice for 30 s. The homogenates were centrifuged at 13,000 rpm for 10 min at 4 °C, and protein concentration of the supernatants was measured using a BCA Protein Assay Kit (Tiangen, China). The concentrations of IL-1β, IL-6 and TNF-α were quantified with specific ELISA kits for mouse (IL-1β, IL-6, TNF-α ELISA kit, Life Technologies, USA) according to the manufacturer’s instructions and represented as (ng/g tissue protein). The MDA content (nmol/mg tissue protein) and SOD activity (U/mg tissue protein) of the supernatant were directly used for measurement following the manufacturer’s instructions (Jiancheng Bioengineering Institute, China).

Statistical Analysis

All values were presented as mean ± SEM (std. error of mean). Statistical analysis was performed by one-way ANOVA (and nonparametric) of Bonferroni’s multiple-comparisons test using the GraphPad Prism software 6.0. A value of P < 0.05 represented statistical significance.

Results

Physiological Variables of ICH

There were no statistical differences in physiological parameters (blood pressure, and arterial pH, pO2 pCO2) between the sham-operated group, vehicle-treated ICH group and Tregs-treated ICH group (Table 1). Blood clots can be found on striatum after ICH at day (1, 3 and 7) (Fig. 2a).

Table 1.

Physiological parameters of the experimental groups

Before ICH (n = 6) Sham (n = 6) Vehicle (n = 6) Tregs (n = 6)
Blood pressure (mmHg) 108.9 ± 5.3 107.5 ± 5.6 111.3 ± 5.7 110.0 ± 7.2
Arterial pH 7.17 ± 0.04 7.15 ± 0.04 7.18 ± 0.05 7.20 ± 0.05
Arterial pO2 (mmHg) 114.2 ± 5.1 113.6 ± 8.3 107.3 ± 12.8 108.4 ± 10.9
Arterial pCO2 (mmHg) 40.9 ± 6.0 41.2 ± 5.6 42.4 ± 6.1 41.9 ± 5.7
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The values are expressed as mean ± SEM. Mean physiological parameters were not statistically significant (P > 0.05) between the experimental groups

Fig. 2.

Fig. 2

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Tregs treatment attenuates perihematomal edema and BBB permeability after ICH. a Representative photograph of coronal section at day (1, 3, and 7) after ICH surgery, the segments of perihematoma (pentagram indicated) were taken for assay. b Representative flow cytometry plot of CD4+CD25+ Tregs after CD4-negative selection and CD25-positive selection. c Brain water content as indices of brain edema, d Evans blue content as indices of BBB permeability, in the ICH + vehicle group and ICH + Treg group at day 1, day 3, and day 7 after surgery. Values were expressed as mean ± SEM (n = 6 in each group). *P < 0.05 compared to ICH + vehicle group and assessed by unpaired t test

Effect of Tregs on Perihematomal Edema, BBB Permeability and Neurological Score After ICH

To investigate the effect and therapeutic value of Tregs on ICH, we isolated Tregs from the donor mice and injected them into the recipient mice after ICH. First, the CD4+CD25+ Tregs were collected by CD4-negative selection and CD25-positive selection from the lymph nodes and spleens of donor mice, and analyzed by flow cytometry using anti-mouse CD4 and CD25 antibodies. The representative figure of the isolated CD4+CD25+ Tregs (>91 % enriched) is shown in Fig. 2b.

In ICH patients, perihematomal edema and expansion are associated with worse functional outcome, and may represent an attractive therapeutic target for secondary injury after ICH (Murthy et al. 2015; Urday et al. 2016). In this study, perihematomal edema was evaluated at day (1, 3 and 7) after post ICH surgery via the wet/dry weight method. Mean value of brain water content was significantly decreased in Tregs-operated group as compared with that observed in vehicle-treated ICH group (Fig. 2c). Similarly, BBB permeability was assessed by EB dye extravasation of perihematoma at day (1, 3 and 7) following ICH. The amount of extravasated EB dye was significantly decreased in Tregs-treated ICH group as compared with that observed in vehicle-treated ICH group (Fig. 2d). To evaluate the functional outcome of Tregs on ICH, the corner test, cylinder test, and foot fault test were conducted at day (1, 3, 7, 14, 21, and 28) after ICH. As shown in Fig. 3, Tregs-treated ICH group showed the improved corner test scores (at day1, 3, 7, 14 and 21), cylinder test scores (at day 1, 3, 7 and 14), and foot fault test (at day 1, 3, 7 and 14) compared with those of vehicle-treated ICH group.

Fig. 3.

Fig. 3

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Tregs treatment improves the neurofunctional deficit after ICH. Neurological examination scores a corner test, b cylinder test, and c foot fault test for the sham, ICH + vehicle, and ICH + Treg group at day (1, 3, 7, 14, 21, and 28). Values were expressed as mean ± SEM (n = 10 in each group). *P < 0.05 compared to ICH + vehicle group and assessed by unpaired t test

Effect of Tregs on NF-κB p65 Expression and the Content of IL-1β, IL-6, and TNF-α of Perihematoma After ICH

The nuclear factor kappa B (NF-κB) is a predominant regulator for pro-inflammatory cytokines and relative to the cell death after ICH (Hickenbottom et al. 1999). Immunofluorescent staining for NF-κB p65 and IL-6 was performed after ICH. The number of NF-κB p65- or IL-6-positive cells was increased in perihematoma at 72 h after ICH compared with that of the sham-operated group (Fig. 4), which indicates robust inflammatory cells following ICH. However, Tregs treatment significantly reduced the NF-κB p65- or IL-6-positive cells in comparison with the vehicle-treated ICH group (Fig. 4). Next, we measured the protein levels of IL-1β, IL-6, and TNF-α that are known to be the most important pro-inflammatory cytokines. As shown in Fig. 5, the content of IL-1β, IL-6, and TNF-α increased at day (1, 3 and 7) after ICH compared with that of the sham-operated group, while Tregs treatment significantly attenuates this increase. These results indicate that Tregs treatment attenuates the cerebral inflammation after ICH.

Fig. 4.

Fig. 4

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Tregs treatment decreases the IL-6- or NF-κB p65-positive cells after ICH. a Representative immunofluorescence staining slices of IL-6 or NF-κB p65 in the sham, ICH + vehicle, and ICH + Treg group at 72 h after surgery, images were captured at ×20 magnifications. b Quantitative analysis of the IL-6- or NF-κB p65-positive cells were expressed as mean ± SEM (n = 6 in each group). *P < 0.05 compared to ICH + vehicle group and assessed by unpaired t test

Fig. 5.

Fig. 5

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Tregs treatment attenuates the concentration of pro-inflammatory cytokines after ICH. The concentration of a IL-6, b TNF-α and c IL-1β in the sham, ICH + vehicle, and ICH + Treg group at day (1, 3 and 7) after surgery. Values were expressed as mean ± SEM (n = 6 in each group). *P < 0.05 or **P < 0.01 compared to ICH + vehicle group and assessed by unpaired t test

Effect of Tregs on the SOD Enzymatic Activity and MDA Content of Perihematoma After ICH

To explore the effect Tregs have on the antioxidant defense system and lipid peroxidation, we assessed the SOD enzymatic activity and MDA content in perihematoma after ICH. As shown in Fig. 6, the SOD enzymatic activity decreased at day (1, 3 and 7) after ICH compared with that of the sham-operated group, while Tregs treatment significantly restores this decrease (Fig. 6a). Contrarily, the MDA content increased at day (1, 3 and 7) after ICH compared with that of the sham-operated group, while Tregs treatment significantly inhibited this increase (Fig. 6b).

Fig. 6.

Fig. 6

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Tregs treatment attenuates SOD enzyme activity and MDA content after ICH. The SOD enzyme activity (a) and b MDA content in the sham, ICH + vehicle, and ICH + Treg group at day (1, 3 and 7) after surgery. Values were expressed as mean ± SEM (n = 6 in each group). *P < 0.05 or **P < 0.01 compared to ICH + vehicle group and assessed by unpaired t test

Effect of Tregs on the Cell Apoptosis of Perihematoma After ICH

To investigate the effect of Tregs on the cell apoptosis of perihematoma after ICH, we assessed brain sections with ICH treated with vehicle or Tregs by TUNEL staining and active caspase-3 immunofluorescent staining. The number of TUNEL or active caspase-3-positive cells was increased in perihematoma at 72 h after ICH compared with that of the sham-operated group (Fig. 7). However, Tregs treatment significantly reduced the TUNEL or active caspase-3-positive cells in comparison with the vehicle-treated ICH group (Fig. 7).

Fig. 7.

Fig. 7

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Tregs treatment attenuates cell apoptosis following ICH. a Representative immunofluorescence staining slices of TUNEL or active caspase-3 in the sham, ICH + vehicle, and ICH + Treg group at 72 h after surgery, images were captured at ×20 magnification. b Quantitative analysis of the TUNEL or active caspase-3 positive cells were expressed as mean ± SEM (n = 6 in each group). **P < 0.01 compared to ICH + vehicle group and assessed by unpaired t test

Discussion

This study shows that tail intravenous injection of Tregs significantly improves both short- and long-term neurological function, attenuates perihematomal edema and BBB permeability at day (1, 3 and 7) after ICH. Also, Tregs obviously reduce the number of NF-κB p65- and IL-6-positive cells (at day 3), and the content of pro-inflammatory cytokines IL-1β, IL-6 and TNF-α at day (1, 3 and 7) after ICH. Moreover, Tregs significantly increase the SOD enzyme activity and decrease the MDA content at day (1, 3 and 7) following ICH. Furthermore, Tregs obviously reduce the TUNEL and active caspase-3-positive cells at day 3 after ICH. These findings suggest that Tregs treatment attenuates the secondary brain injury induced by ICH, in part, through modulating cerebral inflammation and cell apoptosis.

Brain edema is a major independent risk factor for poor outcome, which is classified into vasogenic edema (reflects extracellular fluid accumulation as the consequence of BBB disruption) and cytotoxic edema (refers to intracellular water accumulation as a result of failure of extrusion) (Nag et al. 2009). The expression and activation of NF-κB p65 occurs within minutes and persists over a week following ICH (Zhao et al. 2007), and leads to upregulation of pro-inflammatory cytokines and contributes to brain injury (Zhou et al. 2014). IL-1β and TNF-α have been shown to be increased after ICH in animal model (Hua et al. 2006; Lin et al. 2012) and ICH patients (Castillo et al. 2002), and contribute to post-ICH perihematomal edema formation. Consistent with a previous study (Yang et al. 2014), our results show that Tregs inhibit the release of IL-1β, IL-6, and TNF-α, decrease the expression of NF-κB p65, and attenuate perihematomal edema at day 3 after ICH. Of note, we also found that Tregs inhibit ICH-elevated IL-1β, IL-6, and TNF-α concentration and brain water content at day 1 and day 7. In view the Treg inhibits the cerebral inflammation and perihematomal edema at a similar time, so we speculate that Tregs attenuate the perihematomal edema via reducing inflammatory response. Earlier animal experiment hasshown that injection of IL-1 and TNF-α into brain causes BBB disruption and vasogenic edema (Megyeri et al. 1992; Holmin and Mathiesen 2000). Our results suggest that Tregs reduce the ICH-induced increase of extravasated Evans blue dye, which is a reliable way to assess the BBB permeability in animal model. Although it is not investigated in present study, the mechanism of Tregs against BBB disruption might be similar to that demonstrated by previous reports, where Tregs can prevent BBB disruption via an inhibition of peripheral neutrophil-derived matrix metallopeptidase 9 (MMP9) (Li et al. 2013a). Therefore, Tregs may attenuate the vasogenic perihematomal edema via BBB protection and inhibition of cerebral inflammation after ICH.

Under normal conditions, reactive oxygen species (ROS) keep at a low level that is regulated by antioxidant system. However, excessive ROS derived from the malfunctioning mitochondria, metabolic product of hemoglobin, and inflammatory cells, leads to oxidative stress in the form of lipid peroxidation, DNA damage and protein oxidation, and initiates the BBB disruption and apoptosis after ICH (Hu et al. 2016). ROS can cause the expression of pro-inflammatory cytokines directly such as TNF-α. On the other hand, pro-inflammatory mediators can induce the ROS production (Duan et al. 2016). In addition to the release of pro-inflammatory mediators, excessive activated microglia aggravates ICH-induced brain injury by inducing ROS production, and inhibition of microglial activation reduces the brain injury following ICH (Taylor and Sansing 2013). In the present study, our findings show that Tregs treatment obviously reduces MDA content, while increasing the SOD enzymatic activity following ICH, indicating that Tregs restore the antioxidant system and attenuate lipid peroxidation. Previous studies have shown that Tregs can inhibit microglia activation (Yang et al. 2014) and shift the polarization of microglia toward the M2 phenotype after ICH (Zhou et al. 2016). Therefore, these evidences plus our findings suggest that Tregs may attenuate oxidative stress after ICH via the inhibition of microglia activation and pro-inflammatory response.

Evidence indicates that the mechanisms of cell apoptosis following ICH are highly complex and divided into the extrinsic pathway [induced by death receptor of tumor necrosis factor (TNF) family] and intrinsic pathway (triggered by many cytotoxic stimuli) (Zhou et al. 2014). In this study, our results shows that Tregs reduce the content of TNF-α at day (1, 3, and 7) and apoptosis cells at day 3, indicating Tregs against ICH-induced cell apoptosis via inhibition of TNF-α. Metabolic product of hemoglobin, excessive activated microglia, infiltrated neutrophils and macrophages, plus activation of complement cascade mediate neuronal death following ICH (Hwang et al. 2011). These Tregs-mediated anti-apoptosis effects should be further investigated.

Tregs maintain peripheral tolerance and immune homeostasis, exert immunomodulatory effect by either direct contact with effector T cells and other immune cells or release of immunosuppressive cytokines, transforming growth factor-β (TGF-β) and IL-10 (Wing and Sakaguchi 2010). Tregs can accumulate in the ischemic hemisphere after MCAO (Stubbe et al. 2013; Liesz et al. 2009), and can also accumulate in the hemorrhagic hemisphere after ICH (Zhou et al. 2016), and are pivotally involved in maintaining immune homeostasis in primary inflammation of stroke (Liesz and Kleinschnitz 2016). Infusion of Tregs is protective after ischemic stroke or ICH, indicating the similar function of Tregs in stroke. Tregs adoptive therapy may benefit post-ICH immune status via restricting inflammatory over-activation and maintaining immune homeostasis.

It becomes clear that exogenous Tregs quickly distribute into bone marrow, blood, spleen, and lymph nodes after injection following cerebral ischemia (Li et al. 2013a). However, it is important to note that Tregs adoptive therapy is technically demanding and time-consuming, and exerts predominant protective effect within the surrounding inflammatory milieu and the neurovascular unit. Further immunological investigation would be necessary to evaluate the effect of Tregs treatment on post-ICH immune functions.

Conclusion

Our results showed that tail intravenous injection of Tregs significantly improves neurological deficit and BBB permeability and attenuates perihematomal edema after ICH. Moreover, Tregs significantly reduce the content of IL-1β, IL-6, TNF-α, and MDA, while increasing the SOD activity following ICH. Furthermore, Treg treatment obviously reduces the number of IL-6+, NF-κB+, TUNEL+ and active caspase-3+ cells after ICH.

Acknowledgments

The national natural science foundation of China (NSFC) (Nos. 81471212 and 81301018) and natural science foundation of Shandong province of China (No. ZR2014HQ027) supported this work.

Authors’ contributors

ZZ, XY and BS conceived the project and designed experiments. LM, HY, WW, YW, and MY performed the experiment. LM, ZZ and BS analyzed the results. ZZ wrote the manuscript with contributions from LM, BS and XY. XY revised the manuscript.

Compliance with Ethical Standards

Conflict of interest

All authors declare no conflict of interest.

Contributor Information

Zong-yong Zhang, Email: zongyongzhanghust@163.com.

Xiao-yi Yang, Email: xyyang@tsmc.edu.cn.

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