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Aging and disease    2018, Vol. 9 Issue (2) : 235-248     DOI: 10.14336/AD.2017.0328
Orginal Article |
TOPK Promotes Microglia/Macrophage Polarization towards M2 Phenotype via Inhibition of HDAC1 and HDAC2 Activity after Transient Cerebral Ischemia
Han Ziping1, Zhao Haiping1, Tao Zhen1, Wang Rongliang1, Fan Zhibin1, Luo Yumin1, Luo Yinghao1,3,*, Ji Xunming1,2,3,*
1Cerebrovascular Diseases Research Institute and Department of Neurology, Xuanwu Hospital of Capital Medical University, Beijing 100053, China.
2Beijing Institute for Brain Disorders, Beijing 100053, China.
3Beijing Geriatric Medical Research Center, Beijing 100053, China.
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Abstract  

T-LAK-cell-originated protein kinase (TOPK) is a newly identified member of the mitogen-activated protein kinase family. Our previous study has showed that TOPK has neuroprotective effects against cerebral ischemia-reperfusion injury. Here, we investigated the involvement of TOPK in microglia/ macrophage M1/M2 polarization and the underlying epigenetic mechanism. The expression profiles, co-localization and in vivo interaction of TOPK, M1/M2 surface markers, and HDAC1/HDAC2 were detected after middle cerebral artery occlusion models (MCAO). We demonstrated that TOPK, the M2 surface markers CD206 and Arg1, p-HDAC1, and p-HDAC2 showed a similar pattern of in vivo expression over time after MCAO. TOPK co-localized with CD206, p-HDAC1, and p-HDAC2 positive cells, and was shown to bind to HDAC1 and HDAC2. In vitro study showed that TOPK overexpression in BV2 cells up-regulated CD206 and Arg1, and promoted the phosphorylation of HDAC1 and HDAC2. In addition, TOPK overexpression also prevented LPS plus IFN-γ-induced M1 transformation through reducing release of inflammatory factor of M1 phenotype TNF-α, IL-6 and IL-1β, and increasing TGF-β release and the mRNA levels of TGF-β and SOCS3, cytokine of M2 phenotype and its regulator. Moreover, the increased TNF-α induced by TOPK siRNA could be reversed by HDAC1/HDAC2 inhibitor, FK228. TOPK overexpression increased M2 marker expression in vivo concomitant with the amelioration of cerebral injury, neurological functions deficits, whereas TOPK silencing had the opposite effects, which were completely reversed by the FK228 and partially by the SAHA. These findings suggest that TOPK positively regulates microglia/macrophage M2 polarization by inhibiting HDAC1/HDAC2 activity, which may contribute to its neuroprotective effects against cerebral ischemia-reperfusion injury.

Keywords T-LAK-cell-originated protein kinase      microglia/macrophage      polarization      ischemia-reperfusion      histone deacetylase     
Corresponding Authors: Luo Yinghao,Ji Xunming   
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These authors contributed equally to this work.

Issue Date: 01 April 2018
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Han Ziping
Zhao Haiping
Tao Zhen
Wang Rongliang
Fan Zhibin
Luo Yumin
Luo Yinghao
Ji Xunming
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Han Ziping,Zhao Haiping,Tao Zhen, et al. TOPK Promotes Microglia/Macrophage Polarization towards M2 Phenotype via Inhibition of HDAC1 and HDAC2 Activity after Transient Cerebral Ischemia[J]. Aging and disease, 2018, 9(2): 235-248.
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http://www.aginganddisease.org/EN/10.14336/AD.2017.0328     OR     http://www.aginganddisease.org/EN/Y2018/V9/I2/235
Figure 1.  Time-dependent changes and localization of TOPK, M1, and M2 phenotype markers in the ipsilateral hemisphere of mouse brains after 45 min of ischemia with different reperfusion durations of 0.5 h, 12 h, 24 h, 3 days, 7 days, 14 days, and in the sham-operated group. (A-C) Western blot and corresponding quantitative analysis of protein expression of the M1 markers CD16 and iNOS, the M2 markers CD206 and Arg1, and TOPK. β-actin was used as the loading control. Values are expressed as the mean ± SEM. *p < 0.05 versus sham. N = 5 per group. S, sham group. (D) Representative immunofluorescence images stained for CD16 (red) or CD206 (red), TOPK (green), Iba1 (white) and DAPI (blue) in the ipsilateral cortex of mouse brain after 45 min ischemia and 3 days reperfusion. DAPI, nuclei. Scale bar = 20 µm. Statistics: Tukey’s honest significance test.
Figure 2.  TOPK overexpression upregulated M2 marker protein expression in BV2 cells. (A) Western blot analysis verified the overexpression and knockdown TOPK after tranfection with TOPK overexpressed and siRNA1, 2, 3, 4 vector. (B-C) Western blot analysis of the expression of the M1 markers CD16 and iNOS, and the M2 markers CD206 and Arg1 at 48 h after TOPK overexpression and siRNA4 transfection, with quantification of the results shown below. β-actin was used as the loading control. Data are expressed as the mean ± SEM. *p < 0.05 versus control. N = 6. control=control vector. Statistics: Tukey’s honest significance test.
Figure 3.  Changes in the expression of p-HDAC1, HDAC1, p-HDAC2, and HDAC2, and colocalization of p-HDAC1 and p-HDAC2 with TOPK following cerebral ischemia-reperfusion. (A and B) Changes in the protein expression of HDAC1, p-HDAC1, HDAC2, and p-HDAC2 in the ipsilateral hemisphere of mouse brains after 45 min ischemia with different reperfusion durations of 0.5 h, 12 h, 24 h, 3 days, 7 days, and 14 days, and in the sham-operated group. Quantification of the results is shown below. β-actin was used as the loading control. Data are expressed as the mean ± SEM. *P < 0.05 versus control. N = 5. (C and D) Representative double-staining immunofluorescence of p-HDAC1/TOPK and p-HDAC2/TOPK in the ischemic cortex of ipsilateral brain at 3 days after ischemia-reperfusion. Scale bar: 20 μm. N = 5. *p < 0.05 versus sham. Statistics: Tukey’s honest significance test.
Figure 4.  TOPK binds to HDAC1 and HDAC2 in brain tissues under normal conditions and following brain ischemia-reperfusion. (A) Immunoprecipitation was performed with a TOPK antibody, followed by immunoblotting with HDAC1 antibody or TOPK antibody (top panel). Bands of 55 and 50 kDa represent HDAC1 and TOPK, respectively, with quantification of the results (bottom panel). (B) Immunoprecipitation was performed with TOPK antibody, followed by immunoblotting with HDAC2 antibody or TOPK antibody (top panel). Bands of 55.3 and 50 kDa represent HDAC2 and TOPK, respectively, with quantification of the results (bottom panel). Data are expressed as the mean ± SEM. *p < 0.05 versus sham, N = 3. Statistics: Tukey’s honest significance test.
Figure 5.  Effect of TOPK overexpression and TOPK siRNA on HDAC1 and HDAC2 phosphorylation, and Histone 3 and Histone 4 acetylation in BV2 cells. (A-D) The expression of p-HDAC1/HDAC1, p-HDAC2/HDAC2, ac-H3/H3, and ac-H4/H4 in BV2 cells was determined by western blotting after transfection with TOPK overexpressing and TOPK siRNA vector for 48 h. Data are expressed as the mean ± SEM. * p < 0.05 versus control, N = 6. Statistics: Tukey’s honest significance test.
Figure 6.  TOPK influences inflammatory response in BV2 cells following LPS plus IFN-γ stimulation. (A) ELISA analysis of TNF-α, IL-6 and IL-1β levels in the culture medium of LPS (100 ng/ml) plus IFN-γ (20 ng/ml)-induced BV2 cells for 24 h. (B) ELISA analysis of TGF-β level in the culture medium of LPS (100 ng/ml) plus IFN-γ (20 ng/ml)-induced BV2 cells for 24 h. (C) Determination of mRNA expression of M2 phenotype, TGF-β and SOCS3 by RT-PCR. LPS, lipopolysaccharide. Data are expressed as the mean ± SEM. N=6. *p < 0.05 versus control. #p < 0.05 versus LPS. Statistics: Tukey’s honest significance test.
Figure 7.  Effects of TOPK overexpression, TOPK siRNA, the HDAC1/2 specific inhibitor FK228, and the broad-spectrum HDACi SAHA on microglia/macrophage M1/M2 polarization and cerebral injury in mice following 45 min ischemia/14 days reperfusion. (A) The western blotting verified the overexpression and knockdown of TOPK in ipsilateral brain tissue of mice intracerebroventricularly injected with TOPK overexpressing and siRNA4 letivirus. (B) Representative images of HE-stained sections (upper panels) and assessment of cerebral atrophy (lower panel). (C) Western blot (left) and corresponding quantitative analysis (right) of the expression of M1 markers (CD16 and iNOS) and M2 markers (CD206 and Arg1) in the ipsilateral brain of ischemic mice. *p < 0.05 versus sham, #p < 0.05 versus MCAO. (D) Immunofluorescence images stained for the M2 marker CD206 (red, first row) or M1 marker CD16 (red, third row), the microglia/macrophage marker Iba1 (green) and DAPI (blue), which showed the changes in CD206 and CD16 positive microglia/macrophage in the ipsilesional brain at 14 days following tMCAO (vehicle, TOPK overexpression, TOPK siRNA, TOPK siRNA+FK228 and TOPK siRNA +SAHA groups) or sham surgery (sham group). Images were taken from the red-boxed area in the brain slice of TTC staining. Scale bar: 20 μm. con, control letivirus. N=6. Statistics: Tukey’s honest significance test.
Figure 8.  Effects of TOPK overexpression, TOPK siRNA, the HDAC1/2 specific inhibitor FK228, and the broad-spectrum HDACi SAHA on the sensorimotor functions after MCAO. (A and B) Rotarod test and Balance beam test showed the changes in sensorimotor functions up to 14 days following tMCAO (vehicle, TOPK overexpression, TOPK siRNA, TOPK siRNA+FK228 and TOPK siRNA+SAHA groups) or sham surgery (sham group). Shown are the mean ± SEM. *p < 0.05 versus sham, #p < 0.05 versus MCAO. N=6. Statistics: Tukey’s honest significance test.
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