International Immunopharmacology 22 (2014) 522–525
Contents lists available at ScienceDirect
International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Regulatory T cells inhibit microglia activation and protect against inflammatory injury in intracerebral hemorrhage Zhao Yang a,1, Anyong Yu b,1, Yongping Liu c, Hanchao Shen c, Chuangan Lin c, Li Lin c, Shousen Wang a, Bangqing Yuan c,⁎ a b c
Department of Neurology, Yongchuan Hospital of Chongqing Medical University, Chongqing 400016, China Department of Emergency, The First Affiliated Hospital of Zunyi Medical College, Guizhou 563003, China Department of Neurosurgery, The 476th Hospital of PLA, Fuzhou, Fujian 350025, China
a r t i c l e
i n f o
Article history: Received 21 March 2014 Received in revised form 14 June 2014 Accepted 23 June 2014 Available online 4 July 2014 Keywords: Regulatory T cells Microglia Inflammatory injury Intracerebral hemorrhage
a b s t r a c t Numerous evidence demonstrate that microglia mediated inflammatory injury plays a critical role in intracerebral hemorrhage (ICH). Therefore, the way to inhibit the inflammatory response is greatly needed. Treg cells have been shown to play a critical role in immunologic self-tolerance as well as anti-tumor immune responses and transplantation. In the current study, we transfered Treg cells in the ICH model, and investigated the effect. The cytokines of microglia were measured by ELISA, JNK/ERK and NF-κB were measured by Western blot and EMSA (Electrophoretic Mobility Shift Assay), animal behavior was evaluated by animal behavioristics. We found that Treg cells could inhibit microglia mediated inflammatory response through NF-κB activation via the JNK/ERK pathway in vitro, and improve neurological function in vivo. Our findings suggest that Treg cells could suppress inflammatory injury and represent a novel cell-based therapeutical strategy in ICH. © 2014 Published by Elsevier B.V.
1. Introduction
2. Materials and methods
Intracerebral hemorrhage (ICH), estimated to affect over 1 million people worldwide each year, is the least treatable form of stroke and contributes substantially to the burden of cerebrovascular disease [1–3]. After ICH, a lot of changes occur in the brain including hematoma formation, brain edema, inflammation and microglia activation [4–6]. Therefore, there is an urgent need to develop new treatments for ICH. Treg cells are anti-inflammatory T cells able to suppress immune activation and inflammation [7–9]. They not only can suppress the activation and proliferation of other CD4+ and CD8+ T cells, but also can directly suppress B cell Ig production without having to suppress Th cells [10–12]. However, the effect of Treg cells in the ICH has not been reported. Therefore, we made a hypothesis that adoptive transfer of Treg cells might protect against ICH induced inflammatory injury. To identify that, we transfered Treg cells in the ICH model, and investigated the effect to anti-inflammatory response.
2.1. Animals
⁎ Corresponding author. Tel./fax: +86 591 88780603. E-mail address: [email protected] (B. Yuan). 1 Zhao Yang and Anyong Yu equally contributed to this work.
http://dx.doi.org/10.1016/j.intimp.2014.06.037 1567-5769/© 2014 Published by Elsevier B.V.
Male C57BL/6 mice (8–10 weeks) were obtained from the Animal Center of the Third Military Medical University and bred under specific pathogen-free conditions. Experiments were conducted in accordance with animal care guidelines approved by the Animal Ethics Committee of the Third Military Medical University. 2.2. Mouse model of intracerebral hemorrhage C57BL/6 mice were anesthetized with intraperitoneal chloral hydrate (40 mg/kg) and placed in a stereotaxic frame (Stoelting, Kiel, WI, USA). A micro-sample instrument was lowered into the center of the striatum by craniotomy under stereotactic guidance at the following coordinates relative to bregma: 1 mm anterior, 2.5 mm lateral, and 4 mm deep. A volume of 25 μl of autologous whole blood was infused at 2.5 μl/min over a period of 10 min. The needle was held in place for another 10 min after the infusion to prevent leakage. Craniotomy was then sealed with bone wax, and the scalp was closed with suture. Control mice were infused with 25 μl of 0.9% saline. We maintained the room temperature at about 25 °C during and after surgery, and exposed the animals to incandescent lighting to keep their rectal temperature at 37 ± 1 °C until palinesthesia.
Z. Yang et al. / International Immunopharmacology 22 (2014) 522–525
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2.3. Isolation and adoptive transfer of Tregs Single-cell suspensions were prepared from spleen of normal C57BL/ 6 mice. CD4+CD25+ Treg populations were enriched by negative selection and positive selection with a regulatory T-cell isolation kit (Miltenyi Biotec) according to the manufacturer's instructions. The recipient mouse received a tail vein injection of 1 × 106 freshly enriched Tregs in 0.2 ml PBS at 24 h after ICH. 2.4. Enzyme-linked immunosorbent assay (ELISA) ELISA was performed as per the manufacturer's instructions (Dakewe Biotech, Shenzhen, China) to assess the concentrations of TNF-α, IL-1β and MMP-2 in brain tissues obtained from the perihematoma region. Brain tissues (80 mg) were centrifuged at 12,000 g and the supernatant was collected for analysis. 2.5. Microglia culture and treatment Cerebral hemispheres of 1-day old postnatal C57BL/6 mice were digested with 0.1% trypsin. The cells were seeded into a six-well plate coated with poly-L-lysine and fed with Dulbecco's Modified Eagle Media (DMEM; Sigma, St. Louis, MO, USA) containing 10% fetal bovine serum (FBS; Hyclone, Logan, UT/USA). Culture media were refreshed twice per week for 2 weeks. Microglia were detached by gentle shaking and filtered through a nylon mesh to remove astrocytes. After centrifugation at 100 g for 10 min, the cells were resuspended in fresh DMEM supplemented with 10% FBS and plated at a final density of 5 × 105 cells/ml on a poly-L-lysine coated 6-well culture plate. The following day, cells were subjected to the experiments. The cell purity was determined by immunohistochemical staining using microglia specific antibody CD11b. The microglia cultures used were N95% pure. 2.6. Tregs and microglia mixed reactions Tregs (1 × 105) were co-cultured with microglia (1 × 105), and microglia activation in response to stimulus with 10 μl erythrocyte lysis or 10 μl PBS were evaluated. After 3 days, the supernatants were removed and further analyzed for cytokine production with ELISA (Fig. 1). 2.7. Western blot analysis After 3 day post ICH modeling, mice were randomly selected from each group and euthanized. The perihematoma tissues (80 mg) were digested by 0.005% trypsin/0.002% EDTA (10 min, 37 °C), mechanically dissociated, and centrifuged at 1000 g for 5 min. The proteins were separated from perihematoma tissues (80 mg) by SDS polyacrylamide gel electrophoresis with a quantity of 15μl (100 mg/ml) loaded per gel and transferred onto polyvinylidene fluoride (PVDF) membranes (Amersham Pharmacia). The PVDF membranes were incubated with the primary antibodies, including: rabbit anti-mouse phospho-NF-κB p65 (Abcam, UK), rabbit anti-mouse phospho-JNK (Abcam, UK), and rabbit anti-mouse phospho-ERK followed by incubation with peroxidaseconjugated secondary antibodies (1:2000, Jingmei, China). The signals were detected with an ECL system (Amersham Pharmacia). The same membranes were probed with antibody for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) after being washed with stripping buffer. The signals were quantified by scanning densitometry and computerassisted image analysis.
Fig. 1. Detection of cytokine secretion of microglia. Tregs (1 × 105) were co-cultured with microglia (1 × 105), and microglia activation in response to stimulus with 10 μl erythrocyte lysis or 10 μl PBS control furtherly were evaluated. After 3 days, the supernatants were removed and further analyzed for cytokine production of TNF-α, IL-1β and MMP-2 with ELISA. Experiments performed in triplicate showed consistent results. Compared with controls, P b 0.05.
2.9. Measurement of cerebral water content of mice To measure cerebral water content after ICH, mice were randomly selected from each group and euthanized. We levered the skull within 1 min to take out brain tissues, blotted up the water on the surface of the left hemisphere with filter paper, and took the humid weights (GW) on an electronic balance. We then dried them for 24–48 h at 95–100 °C in an Electro-Thermostatic Blast Oven and took their dry weights (DW). Cerebral water contents were calculated by the formula: cerebral water content% = (GW − DW) / GW × 100%. 2.10. Statistical analysis The statistical significance of differential findings between experimental groups and controls was determined by t-test and considered significant if P b 0.05.
2.8. Neurological deficit scores
3. Results
After 3 day post ICH modeling, the mice were in stable condition, the neurological deficit tests were performed by behavioral measurement, including postural flexing test, circling or sidewalk, forelimb placing and foot fault test and repeated 3 times.
3.1. Tregs inhibited cytokine secretion of microglia ICH could induce microglia mediated inflammatory response, and Tregs might inhibit the reaction. To explore the effect of Tregs to
524
Z. Yang et al. / International Immunopharmacology 22 (2014) 522–525
Fig. 2. Analysis of phospho-NF-κB p65 and phospho-JNK/ERK activity of microglia. Tregs (1 × 105) were co-cultured with microglia (1 × 105), and microglia activation in response to stimulus with 10 μl erythrocyte lysis or 10 μl PBS control furtherly were evaluated. After 3 days, phospho-NF-κB p65 and phospho-JNK/ERK activity of microglia were analyzed. Experiments performed in triplicate showed consistent results. Compared with controls, P b 0.05.
microglia, after in response to stimulus with erythrocyte lysis, the cytokine production was analyzed. The results demonstrated that Tregs could inhibit TNF-α, IL-1β and MMP-2 expression of microglia. The cytokine expression was only about half of the control group. 3.2. Tregs inhibited microglia activation through JNK/ERK pathway and NF-κB activation Previous evidence demonstrated that neuronal JNK/ERK signaling and NF-κB were emerging regulator of cell fate and function in the nervous system. In this study, we also evaluated JNK/ERK and NF-κB activation 24 h after stimulus with erythrocyte lysis. As shown in Fig. 2, erythrocyte lysis induced a significant increase of JNK/ERK and NF-κB. However, Tregs could inhibit JNK/ERK and NF-κB expression. The results demonstrated Tregs inhibited microglia activation through JNK/ERK pathway and NF-κB activation.
Fig. 4. Analysis of cytokine of perihematoma region. After 3 day post ICH modeling, the concentrations of TNF-α, IL-1β and MMP-2 in brain tissues (n = 10 per group) obtained from the perihematoma region were analyzed with ELISA. Brain tissues (80 mg) were centrifuged at 12,000 g and the supernatant was collected for analysis. Experiments performed in triplicate showed consistent results. Compared with controls, P b 0.05.
3.3. Tregs inhibited inflammation and neurological impairment following ICH To further assess the role of Tregs in the inflammatory response to ICH, we transfered Tregs into mice and then evaluated brain damage and neurological impairments 3 days after ICH. The results demonstrated that Tregs could inhibit brain water content and neurological deficit scores. The brain water content and neurological deficit scores were only about half of the control group (Fig. 3). 3.4. Tregs inhibited microglia activation in vivo To identify whether Tregs could inhibit microglia activation, we also analyzed inflammatory cytokines expression in mice after ICH. The results showed that Tregs could significantly inhibit expression of TNF-α, IL-1β and MMP-2 in perihematoma tissues 3 days after ICH. The cytokine expression was only about half of the control group (Fig. 4). 4. Discussion Fig. 3. Analysis of inflammation and neurological impairment following ICH. (Top) After 3 day post ICH modeling, the mice (n = 10 per group) were in stable condition, the neurological deficit tests were performed by behavioral measurement, including postural flexing test, circling or sidewalk, forelimb placing and foot fault test and repeated 3 times. (Bottom) After 3 day post ICH modeling, the cerebral water content of mice (n = 10 per group) were also analyzed. Experiments performed in triplicate showed consistent results. Compared with controls, P b 0.05.
Intracerebral hemorrhage (ICH) is a common type of fatal stroke, accounting for about 15% to 20% of all strokes [13–15]. Hemorrhagic strokes are associated with high mortality and morbidity, and increasing evidence shows that innate immune responses and inflammatory injury play a critical role in ICH-induced neurological deficits [16–18].
Z. Yang et al. / International Immunopharmacology 22 (2014) 522–525
Although there is much research on the events following ICH, the initial cellular events trigger innate immune and inflammatory responses are still complex. Therefore, the strategy for inhibiting the inflammatory responses is greatly needed. Much evidence suggests that microglia mediated inflammation plays a vital role in the ICH-induced brain injury. After ICH, various stimuli could activate microglia and initiate inflammatory response, and subsequently release proinflammatory cytokines and chemokines to increase brain injury. Tregs have been under focus since they could abrogate the pathogenic functions of reactive immune cells and maintain tolerance to self antigens [19–21]. The first mechanism involves inhibition of cytokine production and/or proliferation of pathogenic T (Teff) cells [22–24]. The second mechanism involves modulation of the cytokine environment at the site of inflammation through direct secretion of cytokines such as TGF-β and IL-10 [25–27]. The third mechanism involves physical elimination of cytotoxic cells, which is considered to have a relatively minor contribution to physiological immune regulation [28–30]. However, whether Tregs could play the role to inhibit the immune response in ICH has not been reported. The most straightforward method for the introduction of blood and hematoma formation in the brain is singular injection. In rodents, blood injection was first used by Ropper et al. in 1982. The authors used Sprague–Dawley rats as experimental subjects. They permanently implanted a 27-gauge needle in the right basal ganglia, allowed the animals to awake, and infused 0.24–0.28 ml of whole or centrifuged blood from another rat, without anesthesia, over 1 s. In the control animals, they inserted plastic polymer. In the current study, we firstly cocultured the Tregs and microglia together, to stimulus with erythrocyte lysis, and analyzed the cytokine production. The results demonstrated that Tregs could inhibit TNF-α, IL-1β and MMP-2 expression of microglia. The data suggested that Tregs could inhibit ICH induced microglia mediated inflammation. In addition, we also explore the pathway of microglia activation 24 h after stimulus with erythrocyte lysis. The results demonstrated that erythrocyte lysis induced a significant increase of phospho-NF-κB p65 and phospho-JNK/ERK. However, Tregs could inhibit phospho-NF-κB p65 and phospho-JNK/ERK expression. The results demonstrated Tregs inhibited microglia activation through JNK/ERK pathway and NF-κB activation. Lastly, we transfered Tregs in ICH model to identify whether Tregs could inhibit inflammation and neurological impairment following ICH. We evaluated brain damage and neurological impairments 3 days after ICH. The results demonstrated that Tregs could inhibit brain water content and neurological deficit scores. Additionally, the results showed that Tregs could significantly inhibit expression of TNF-α, IL-1β and MMP-2 in perihematoma tissues 3 days after ICH. All the data suggested that Tregs could inhibit microglia activation in vivo. Taken together, our study suggests that Treg adoptive therapy is a promising strategy to inhibit inflammation and neurological impairment in ICH, and might be utilized in the clinic.
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Contents lists available at ScienceDirect
International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Regulatory T cells inhibit microglia activation and protect against inflammatory injury in intracerebral hemorrhage Zhao Yang a,1, Anyong Yu b,1, Yongping Liu c, Hanchao Shen c, Chuangan Lin c, Li Lin c, Shousen Wang a, Bangqing Yuan c,⁎ a b c
Department of Neurology, Yongchuan Hospital of Chongqing Medical University, Chongqing 400016, China Department of Emergency, The First Affiliated Hospital of Zunyi Medical College, Guizhou 563003, China Department of Neurosurgery, The 476th Hospital of PLA, Fuzhou, Fujian 350025, China
a r t i c l e
i n f o
Article history: Received 21 March 2014 Received in revised form 14 June 2014 Accepted 23 June 2014 Available online 4 July 2014 Keywords: Regulatory T cells Microglia Inflammatory injury Intracerebral hemorrhage
a b s t r a c t Numerous evidence demonstrate that microglia mediated inflammatory injury plays a critical role in intracerebral hemorrhage (ICH). Therefore, the way to inhibit the inflammatory response is greatly needed. Treg cells have been shown to play a critical role in immunologic self-tolerance as well as anti-tumor immune responses and transplantation. In the current study, we transfered Treg cells in the ICH model, and investigated the effect. The cytokines of microglia were measured by ELISA, JNK/ERK and NF-κB were measured by Western blot and EMSA (Electrophoretic Mobility Shift Assay), animal behavior was evaluated by animal behavioristics. We found that Treg cells could inhibit microglia mediated inflammatory response through NF-κB activation via the JNK/ERK pathway in vitro, and improve neurological function in vivo. Our findings suggest that Treg cells could suppress inflammatory injury and represent a novel cell-based therapeutical strategy in ICH. © 2014 Published by Elsevier B.V.
1. Introduction
2. Materials and methods
Intracerebral hemorrhage (ICH), estimated to affect over 1 million people worldwide each year, is the least treatable form of stroke and contributes substantially to the burden of cerebrovascular disease [1–3]. After ICH, a lot of changes occur in the brain including hematoma formation, brain edema, inflammation and microglia activation [4–6]. Therefore, there is an urgent need to develop new treatments for ICH. Treg cells are anti-inflammatory T cells able to suppress immune activation and inflammation [7–9]. They not only can suppress the activation and proliferation of other CD4+ and CD8+ T cells, but also can directly suppress B cell Ig production without having to suppress Th cells [10–12]. However, the effect of Treg cells in the ICH has not been reported. Therefore, we made a hypothesis that adoptive transfer of Treg cells might protect against ICH induced inflammatory injury. To identify that, we transfered Treg cells in the ICH model, and investigated the effect to anti-inflammatory response.
2.1. Animals
⁎ Corresponding author. Tel./fax: +86 591 88780603. E-mail address: [email protected] (B. Yuan). 1 Zhao Yang and Anyong Yu equally contributed to this work.
http://dx.doi.org/10.1016/j.intimp.2014.06.037 1567-5769/© 2014 Published by Elsevier B.V.
Male C57BL/6 mice (8–10 weeks) were obtained from the Animal Center of the Third Military Medical University and bred under specific pathogen-free conditions. Experiments were conducted in accordance with animal care guidelines approved by the Animal Ethics Committee of the Third Military Medical University. 2.2. Mouse model of intracerebral hemorrhage C57BL/6 mice were anesthetized with intraperitoneal chloral hydrate (40 mg/kg) and placed in a stereotaxic frame (Stoelting, Kiel, WI, USA). A micro-sample instrument was lowered into the center of the striatum by craniotomy under stereotactic guidance at the following coordinates relative to bregma: 1 mm anterior, 2.5 mm lateral, and 4 mm deep. A volume of 25 μl of autologous whole blood was infused at 2.5 μl/min over a period of 10 min. The needle was held in place for another 10 min after the infusion to prevent leakage. Craniotomy was then sealed with bone wax, and the scalp was closed with suture. Control mice were infused with 25 μl of 0.9% saline. We maintained the room temperature at about 25 °C during and after surgery, and exposed the animals to incandescent lighting to keep their rectal temperature at 37 ± 1 °C until palinesthesia.
Z. Yang et al. / International Immunopharmacology 22 (2014) 522–525
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2.3. Isolation and adoptive transfer of Tregs Single-cell suspensions were prepared from spleen of normal C57BL/ 6 mice. CD4+CD25+ Treg populations were enriched by negative selection and positive selection with a regulatory T-cell isolation kit (Miltenyi Biotec) according to the manufacturer's instructions. The recipient mouse received a tail vein injection of 1 × 106 freshly enriched Tregs in 0.2 ml PBS at 24 h after ICH. 2.4. Enzyme-linked immunosorbent assay (ELISA) ELISA was performed as per the manufacturer's instructions (Dakewe Biotech, Shenzhen, China) to assess the concentrations of TNF-α, IL-1β and MMP-2 in brain tissues obtained from the perihematoma region. Brain tissues (80 mg) were centrifuged at 12,000 g and the supernatant was collected for analysis. 2.5. Microglia culture and treatment Cerebral hemispheres of 1-day old postnatal C57BL/6 mice were digested with 0.1% trypsin. The cells were seeded into a six-well plate coated with poly-L-lysine and fed with Dulbecco's Modified Eagle Media (DMEM; Sigma, St. Louis, MO, USA) containing 10% fetal bovine serum (FBS; Hyclone, Logan, UT/USA). Culture media were refreshed twice per week for 2 weeks. Microglia were detached by gentle shaking and filtered through a nylon mesh to remove astrocytes. After centrifugation at 100 g for 10 min, the cells were resuspended in fresh DMEM supplemented with 10% FBS and plated at a final density of 5 × 105 cells/ml on a poly-L-lysine coated 6-well culture plate. The following day, cells were subjected to the experiments. The cell purity was determined by immunohistochemical staining using microglia specific antibody CD11b. The microglia cultures used were N95% pure. 2.6. Tregs and microglia mixed reactions Tregs (1 × 105) were co-cultured with microglia (1 × 105), and microglia activation in response to stimulus with 10 μl erythrocyte lysis or 10 μl PBS were evaluated. After 3 days, the supernatants were removed and further analyzed for cytokine production with ELISA (Fig. 1). 2.7. Western blot analysis After 3 day post ICH modeling, mice were randomly selected from each group and euthanized. The perihematoma tissues (80 mg) were digested by 0.005% trypsin/0.002% EDTA (10 min, 37 °C), mechanically dissociated, and centrifuged at 1000 g for 5 min. The proteins were separated from perihematoma tissues (80 mg) by SDS polyacrylamide gel electrophoresis with a quantity of 15μl (100 mg/ml) loaded per gel and transferred onto polyvinylidene fluoride (PVDF) membranes (Amersham Pharmacia). The PVDF membranes were incubated with the primary antibodies, including: rabbit anti-mouse phospho-NF-κB p65 (Abcam, UK), rabbit anti-mouse phospho-JNK (Abcam, UK), and rabbit anti-mouse phospho-ERK followed by incubation with peroxidaseconjugated secondary antibodies (1:2000, Jingmei, China). The signals were detected with an ECL system (Amersham Pharmacia). The same membranes were probed with antibody for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) after being washed with stripping buffer. The signals were quantified by scanning densitometry and computerassisted image analysis.
Fig. 1. Detection of cytokine secretion of microglia. Tregs (1 × 105) were co-cultured with microglia (1 × 105), and microglia activation in response to stimulus with 10 μl erythrocyte lysis or 10 μl PBS control furtherly were evaluated. After 3 days, the supernatants were removed and further analyzed for cytokine production of TNF-α, IL-1β and MMP-2 with ELISA. Experiments performed in triplicate showed consistent results. Compared with controls, P b 0.05.
2.9. Measurement of cerebral water content of mice To measure cerebral water content after ICH, mice were randomly selected from each group and euthanized. We levered the skull within 1 min to take out brain tissues, blotted up the water on the surface of the left hemisphere with filter paper, and took the humid weights (GW) on an electronic balance. We then dried them for 24–48 h at 95–100 °C in an Electro-Thermostatic Blast Oven and took their dry weights (DW). Cerebral water contents were calculated by the formula: cerebral water content% = (GW − DW) / GW × 100%. 2.10. Statistical analysis The statistical significance of differential findings between experimental groups and controls was determined by t-test and considered significant if P b 0.05.
2.8. Neurological deficit scores
3. Results
After 3 day post ICH modeling, the mice were in stable condition, the neurological deficit tests were performed by behavioral measurement, including postural flexing test, circling or sidewalk, forelimb placing and foot fault test and repeated 3 times.
3.1. Tregs inhibited cytokine secretion of microglia ICH could induce microglia mediated inflammatory response, and Tregs might inhibit the reaction. To explore the effect of Tregs to
524
Z. Yang et al. / International Immunopharmacology 22 (2014) 522–525
Fig. 2. Analysis of phospho-NF-κB p65 and phospho-JNK/ERK activity of microglia. Tregs (1 × 105) were co-cultured with microglia (1 × 105), and microglia activation in response to stimulus with 10 μl erythrocyte lysis or 10 μl PBS control furtherly were evaluated. After 3 days, phospho-NF-κB p65 and phospho-JNK/ERK activity of microglia were analyzed. Experiments performed in triplicate showed consistent results. Compared with controls, P b 0.05.
microglia, after in response to stimulus with erythrocyte lysis, the cytokine production was analyzed. The results demonstrated that Tregs could inhibit TNF-α, IL-1β and MMP-2 expression of microglia. The cytokine expression was only about half of the control group. 3.2. Tregs inhibited microglia activation through JNK/ERK pathway and NF-κB activation Previous evidence demonstrated that neuronal JNK/ERK signaling and NF-κB were emerging regulator of cell fate and function in the nervous system. In this study, we also evaluated JNK/ERK and NF-κB activation 24 h after stimulus with erythrocyte lysis. As shown in Fig. 2, erythrocyte lysis induced a significant increase of JNK/ERK and NF-κB. However, Tregs could inhibit JNK/ERK and NF-κB expression. The results demonstrated Tregs inhibited microglia activation through JNK/ERK pathway and NF-κB activation.
Fig. 4. Analysis of cytokine of perihematoma region. After 3 day post ICH modeling, the concentrations of TNF-α, IL-1β and MMP-2 in brain tissues (n = 10 per group) obtained from the perihematoma region were analyzed with ELISA. Brain tissues (80 mg) were centrifuged at 12,000 g and the supernatant was collected for analysis. Experiments performed in triplicate showed consistent results. Compared with controls, P b 0.05.
3.3. Tregs inhibited inflammation and neurological impairment following ICH To further assess the role of Tregs in the inflammatory response to ICH, we transfered Tregs into mice and then evaluated brain damage and neurological impairments 3 days after ICH. The results demonstrated that Tregs could inhibit brain water content and neurological deficit scores. The brain water content and neurological deficit scores were only about half of the control group (Fig. 3). 3.4. Tregs inhibited microglia activation in vivo To identify whether Tregs could inhibit microglia activation, we also analyzed inflammatory cytokines expression in mice after ICH. The results showed that Tregs could significantly inhibit expression of TNF-α, IL-1β and MMP-2 in perihematoma tissues 3 days after ICH. The cytokine expression was only about half of the control group (Fig. 4). 4. Discussion Fig. 3. Analysis of inflammation and neurological impairment following ICH. (Top) After 3 day post ICH modeling, the mice (n = 10 per group) were in stable condition, the neurological deficit tests were performed by behavioral measurement, including postural flexing test, circling or sidewalk, forelimb placing and foot fault test and repeated 3 times. (Bottom) After 3 day post ICH modeling, the cerebral water content of mice (n = 10 per group) were also analyzed. Experiments performed in triplicate showed consistent results. Compared with controls, P b 0.05.
Intracerebral hemorrhage (ICH) is a common type of fatal stroke, accounting for about 15% to 20% of all strokes [13–15]. Hemorrhagic strokes are associated with high mortality and morbidity, and increasing evidence shows that innate immune responses and inflammatory injury play a critical role in ICH-induced neurological deficits [16–18].
Z. Yang et al. / International Immunopharmacology 22 (2014) 522–525
Although there is much research on the events following ICH, the initial cellular events trigger innate immune and inflammatory responses are still complex. Therefore, the strategy for inhibiting the inflammatory responses is greatly needed. Much evidence suggests that microglia mediated inflammation plays a vital role in the ICH-induced brain injury. After ICH, various stimuli could activate microglia and initiate inflammatory response, and subsequently release proinflammatory cytokines and chemokines to increase brain injury. Tregs have been under focus since they could abrogate the pathogenic functions of reactive immune cells and maintain tolerance to self antigens [19–21]. The first mechanism involves inhibition of cytokine production and/or proliferation of pathogenic T (Teff) cells [22–24]. The second mechanism involves modulation of the cytokine environment at the site of inflammation through direct secretion of cytokines such as TGF-β and IL-10 [25–27]. The third mechanism involves physical elimination of cytotoxic cells, which is considered to have a relatively minor contribution to physiological immune regulation [28–30]. However, whether Tregs could play the role to inhibit the immune response in ICH has not been reported. The most straightforward method for the introduction of blood and hematoma formation in the brain is singular injection. In rodents, blood injection was first used by Ropper et al. in 1982. The authors used Sprague–Dawley rats as experimental subjects. They permanently implanted a 27-gauge needle in the right basal ganglia, allowed the animals to awake, and infused 0.24–0.28 ml of whole or centrifuged blood from another rat, without anesthesia, over 1 s. In the control animals, they inserted plastic polymer. In the current study, we firstly cocultured the Tregs and microglia together, to stimulus with erythrocyte lysis, and analyzed the cytokine production. The results demonstrated that Tregs could inhibit TNF-α, IL-1β and MMP-2 expression of microglia. The data suggested that Tregs could inhibit ICH induced microglia mediated inflammation. In addition, we also explore the pathway of microglia activation 24 h after stimulus with erythrocyte lysis. The results demonstrated that erythrocyte lysis induced a significant increase of phospho-NF-κB p65 and phospho-JNK/ERK. However, Tregs could inhibit phospho-NF-κB p65 and phospho-JNK/ERK expression. The results demonstrated Tregs inhibited microglia activation through JNK/ERK pathway and NF-κB activation. Lastly, we transfered Tregs in ICH model to identify whether Tregs could inhibit inflammation and neurological impairment following ICH. We evaluated brain damage and neurological impairments 3 days after ICH. The results demonstrated that Tregs could inhibit brain water content and neurological deficit scores. Additionally, the results showed that Tregs could significantly inhibit expression of TNF-α, IL-1β and MMP-2 in perihematoma tissues 3 days after ICH. All the data suggested that Tregs could inhibit microglia activation in vivo. Taken together, our study suggests that Treg adoptive therapy is a promising strategy to inhibit inflammation and neurological impairment in ICH, and might be utilized in the clinic.
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