Mer regulates microglial/macrophage M1/M2 polarization and alleviates neuroinflammation following traumatic brain injury

doi:10.1186/s12974-020-02041-7

. 2021 Jan 5;18(1):2.

doi: 10.1186/s12974-020-02041-7.

Mer regulates microglial/macrophage M1/M2 polarization and alleviates neuroinflammation following traumatic brain injury

Haijian Wu^{1

2

3}, Jingwei Zheng¹, Shenbin Xu¹, Yuanjian Fang¹, Yingxi Wu³, Jianxiong Zeng³, Anwen Shao¹, Ligen Shi¹, Jianan Lu¹, Shuhao Mei¹, Xiaoyu Wang¹, Xinying Guo³, Yirong Wang², Zhen Zhao⁴, Jianmin Zhang^{5

6

7}

Affiliations

¹ Department of Neurosurgery, Second Affiliated Hospital, School of Medicine, Zhejiang University, 88 Jiefang Road, Hangzhou, 310009, Zhejiang, China.
² Department of Neurosurgery, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, China.
³ Center for Neurodegeneration and Regeneration, Zilkha Neurogenetic Institute and Department of Physiology and Biophysics, Keck School of Medicine, University of Southern California, 1501 San Pablo Street, Los Angeles, CA, 90089, USA.
⁴ Center for Neurodegeneration and Regeneration, Zilkha Neurogenetic Institute and Department of Physiology and Biophysics, Keck School of Medicine, University of Southern California, 1501 San Pablo Street, Los Angeles, CA, 90089, USA. zzhao@usc.edu.
⁵ Department of Neurosurgery, Second Affiliated Hospital, School of Medicine, Zhejiang University, 88 Jiefang Road, Hangzhou, 310009, Zhejiang, China. zjm135@zju.edu.cn.
⁶ Brain Research Institute, Zhejiang University, Hangzhou, China. zjm135@zju.edu.cn.
⁷ Collaborative Innovation Center for Brain Science, Zhejiang University, Hangzhou, China. zjm135@zju.edu.cn.

PMID: 33402181
PMCID: PMC7787000
DOI: 10.1186/s12974-020-02041-7

Mer regulates microglial/macrophage M1/M2 polarization and alleviates neuroinflammation following traumatic brain injury

Haijian Wu et al. J Neuroinflammation. 2021.

. 2021 Jan 5;18(1):2.

doi: 10.1186/s12974-020-02041-7.

Authors

Affiliations

¹ Department of Neurosurgery, Second Affiliated Hospital, School of Medicine, Zhejiang University, 88 Jiefang Road, Hangzhou, 310009, Zhejiang, China.
² Department of Neurosurgery, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, China.
³ Center for Neurodegeneration and Regeneration, Zilkha Neurogenetic Institute and Department of Physiology and Biophysics, Keck School of Medicine, University of Southern California, 1501 San Pablo Street, Los Angeles, CA, 90089, USA.
⁴ Center for Neurodegeneration and Regeneration, Zilkha Neurogenetic Institute and Department of Physiology and Biophysics, Keck School of Medicine, University of Southern California, 1501 San Pablo Street, Los Angeles, CA, 90089, USA. zzhao@usc.edu.
⁵ Department of Neurosurgery, Second Affiliated Hospital, School of Medicine, Zhejiang University, 88 Jiefang Road, Hangzhou, 310009, Zhejiang, China. zjm135@zju.edu.cn.
⁶ Brain Research Institute, Zhejiang University, Hangzhou, China. zjm135@zju.edu.cn.
⁷ Collaborative Innovation Center for Brain Science, Zhejiang University, Hangzhou, China. zjm135@zju.edu.cn.

PMID: 33402181
PMCID: PMC7787000
DOI: 10.1186/s12974-020-02041-7

Abstract

Background: Traumatic brain injury (TBI) is a leading cause of death and disability worldwide. Microglial/macrophage activation and neuroinflammation are key cellular events following TBI, but the regulatory and functional mechanisms are still not well understood. Myeloid-epithelial-reproductive tyrosine kinase (Mer), a member of the Tyro-Axl-Mer (TAM) family of receptor tyrosine kinases, regulates multiple features of microglial/macrophage physiology. However, its function in regulating the innate immune response and microglial/macrophage M1/M2 polarization in TBI has not been addressed. The present study aimed to evaluate the role of Mer in regulating microglial/macrophage M1/M2 polarization and neuroinflammation following TBI.

Methods: The controlled cortical impact (CCI) mouse model was employed. Mer siRNA was intracerebroventricularly administered, and recombinant protein S (PS) was intravenously applied for intervention. The neurobehavioral assessments, RT-PCR, Western blot, magnetic-activated cell sorting, immunohistochemistry and confocal microscopy analysis, Nissl and Fluoro-Jade B staining, brain water content measurement, and contusion volume assessment were performed.

Results: Mer is upregulated and regulates microglial/macrophage M1/M2 polarization and neuroinflammation in the acute stage of TBI. Mechanistically, Mer activates the signal transducer and activator of transcription 1 (STAT1)/suppressor of cytokine signaling 1/3 (SOCS1/3) pathway. Inhibition of Mer markedly decreases microglial/macrophage M2-like polarization while increases M1-like polarization, which exacerbates the secondary brain damage and sensorimotor deficits after TBI. Recombinant PS exerts beneficial effects in TBI mice through Mer activation.

Conclusions: Mer is an important regulator of microglial/macrophage M1/M2 polarization and neuroinflammation, and may be considered as a potential target for therapeutic intervention in TBI.

Keywords: M1/M2 polarization; Mer; Microglia/macrophage; Neuroinflammation; TBI.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

**Fig. 1**
Dynamic changes in mRNA expression of microglial/macrophage M1-like and M2-like phenotypic markers following TBI. a Representative photographs of whole brains in the sham and different traumatic brain injury (TBI) groups (at 3 h, 12 h, 1 day, 3 days, and 7 days post-insult, respectively). Scale bar = 1 mm. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to assess the mRNA expression levels of microglial/macrophage M1-like and M2-like phenotypic markers in the injured cortex at 3 h, 12 h, 1 day, 3 days, and 7 days after TBI or the equivalent area of the sham-operated brains. b–d Expressions of mRNA of M1-like phenotypic markers, including CD16 (b), CD32 (c), and iNOS (d), were gradually increased over time from 12 h onward and remained elevated for at least 7 days after injury. e–g Expressions of mRNA of M2-like phenotypic markers, including CD206 (e), Arg-1 (f), and IL-10 (g), were significantly upregulated at 1 day, 12 h, and 1 day after TBI, respectively, and all peaked around day 3 post-injury, then declined dramatically on day 7, even though they did not return to baseline levels. In b–g, data are expressed as fold change. n = 6 mice per group. ns, nonsignificant, p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001; versus sham-operated controls; NS, nonsignificant, p > 0.05; ###, p < 0.001; versus the TBI group on day 3. One-way ANOVA followed by Bonferroni’s post hoc tests

**Fig. 2**
Expression patterns and cellular localization of Mer following TBI. a Representative immunoblots and quantification showing the expression level of Mer protein in the injured cortex at 3 h, 12 h, 1 day, 3 days, and 7 days after TBI or the equivalent area of the sham-operated brains. Data are expressed as fold change compared to sham-operated controls. n = 6 mice per group. b Quantitative RT-PCR was used to assess the mRNA expression level of Mer in the injured cortex at 3 h, 12 h, 1 day, 3 days, and 7 days after TBI or the equivalent area of the sham-operated brains. Data are expressed as fold change compared to sham-operated controls. n = 6 mice per group. Double immunofluorescent staining of Mer with the microglial/macrophage marker Iba-1 in the ipsilateral cerebral cortex was performed at 3 days post-TBI or sham operation. c Fluorescence intensity quantification of Mer expression in activated microglia/macrophages in the impacted cortical area at 3 days after TBI, compared to that in resting microglia/macrophages from sham-operated brains. n = 6 mice per group. d Low-magnification images of the brain (left) indicate the region of interest. Representative confocal images from the ipsilateral cortex (right) showing Mer was abundantly expressed in the plasma membrane of microglia/macrophages and substantially upregulated in activated microglia/macrophages following TBI. The dotted white area indicates contusion region. Scale bar = 15 μm. e Confocal microscopy analysis at a single-cell resolution showing Mer expression was substantially upregulated in the activated microglia/macrophages with phagocytotic morphology at 3 days post-TBI. Orthogonal views demonstrated the colocalization of Mer and Iba-1 in microglia/macrophages. Scale bar = 5 μm. In a, b, data are presented as mean ± SD; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, nonsignificant, p > 0.05; versus sham-operated controls; one-way ANOVA followed by Bonferroni’s post hoc tests. In c, data are presented as mean ± SD; ***, p < 0.001 by Student’s t test

**Fig. 3**
In vivo knockdown of Mer worsened the functional outcomes after TBI. a Western blot was used to assess the knockdown efficacy of Mer siRNA. Representative immunoblots and quantification showing Mer siRNA inhibited the expression of Mer protein in the injured cortex at 3 days post-TBI. TBI + vehicle: TBI + V, TBI + control siRNA: TBI + C, TBI + Mer siRNA: TBI + M. GAPDH: loading control. Data are expressed as fold change compared to the TBI + V group; n = 6 mice per group. b Quantitative RT-PCR analysis also showing Mer siRNA significantly inhibited the expression of Mer mRNA in the injured cortex at 3 days post-TBI. Data are expressed as fold change compared to the TBI + V group; n = 6 mice per group. c–e Modified neurological severity scores (mNSS) (c), foot-fault test (d), and rotarod test (e) were performed before and 1, 3, and 7 days after TBI. n = 8 mice per group. f Quantification of TBI-induced lesion volume at 3 days post-insult. n = 6 mice per group. Scale bar = 1 mm. g Cerebral edema was measured by brain water content. Mer siRNA significantly elevated brain edema level at 3 days post-injury, when compared to both the vehicle and control siRNA group. n = 8 mice per group. In a–g, data are presented as mean ± SD; *, #, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, nonsignificant, p > 0.05. In a, b, f, g, one-way ANOVA followed by Bonferroni’s post hoc tests. In c–e, two-way ANOVA followed by Bonferroni’s post hoc tests

**Fig. 4**
Mer modulated microglial/macrophage M1/M2 polarization after TBI. a Representative images of immunofluorescent staining for Mer (red), CD16/32 (green), and DAPI (blue) showing Mer was expressed in CD16/32-positive cells in the perilesional area of cortex at 3 days post-TBI. Scale bar = 10 μm. b Representative images of immunofluorescent staining for Mer (red), CD206 (green), and DAPI (blue) showing Mer was expressed in CD206-positive cells in the perilesional area of cortex at 3 days post-TBI. Scale bar = 10 μm. c Representative images of immunofluorescent staining for CD16/32 (red), Iba-1 (green), and DAPI (blue) in the ipsilateral cortex at 3 days post-TBI. Scale bar = 50 μm. d Quantification showing the percentage of CD16/32 and Iba-1 double-positive cells was significantly increased in the cortex on day 3 after TBI, which was further elevated in the Mer siRNA group. n = 6 mice per group. e Representative images of immunofluorescent staining for Mer (red), CD206 (green), and DAPI (blue) in the ipsilateral cortex at 3 days post-TBI. Scale bar = 50 μm. f Quantification showing the percentage of CD206 and Iba-1 double-positive cells increased significantly in the ipsilateral cortex on day 3 after TBI; however, it significantly decreased following Mer siRNA administration. g The ratio between CD16/32⁺ Iba-1⁺ M1-like cells and CD206⁺ Iba-1⁺ M2-like cells nearly doubled after Mer siRNA administration. In d, f, and g, data are presented as mean ± SD; #, p < 0.05; ***, p < 0.001; ns, nonsignificant, p > 0.05. One-way ANOVA followed by Bonferroni’s post hoc tests

**Fig. 5**
Inhibition of Mer aggravated neuronal damage and degeneration following TBI. a Representative images of Nissl staining in the ipsilateral cortex from the sham, TBI + vehicle (TBI + V), TBI + control siRNA (TBI + C), and TBI + Mer siRNA (TBI + M) groups, respectively. Quantification analysis showing TBI caused a significant decrease in the number of Nissl-positive cells in the ipsilateral cortex at 3 days post-TBI, and Mer siRNA application further decreased the number of Nissl-positive cells in the injured cortex after TBI. n = 6 mice per group. Scale bar = 20 μm. b Representative images of Fluoro-Jade B (FJB) staining in the ipsilateral cortex from the sham, TBI + V, TBI + C, and TBI + M groups, respectively. Quantification analysis showing TBI caused a significant increase in the number of FJB-positive cells in the ipsilateral cortex at 3 days post-TBI, and Mer siRNA application further increased the number of FJB-positive cells in the injured cortex after TBI. n = 6 mice per group. Scale bar = 15 μm. In a, b, data are presented as mean ± SD; #, p < 0.05; ***, p < 0.001; ns, nonsignificant, p > 0.05. One-way ANOVA followed by Bonferroni’s post hoc tests

**Fig. 6.**
Inhibition of Mer reduced STAT1 activation and SOCS expression following TBI. a, b, c, e Representative immunoblots and quantification showing the expression of phosphorylated Mer (p-Mer) (a), phosphorylated STAT1 (p-STAT1) (b), SOCS-1 (c), and SOCS-3 (e) was significantly inhibited by Mer siRNA administration on day 3 following TBI. TBI + vehicle: TBI + V, TBI + control siRNA: TBI + C; TBI + Mer siRNA: TBI + M. GAPDH: loading control. Data are expressed as fold change compared to the TBI + V group; n = 6 mice per group. d, f Quantitative RT-PCR analysis showing Mer siRNA application significantly inhibited the mRNA expression of SOCS-1 (d) and SOCS-3 (f) in the injured cortex at 3 days post-TBI. Data are expressed as fold change compared to the TBI + V group; n = 6 mice per group. In a–f, data are presented as Mean ± SD; ns, nonsignificant, p > 0.05; *** p < 0.001. One-way ANOVA followed by Bonferroni’s post hoc tests

**Fig. 7**
Mer activation alleviated functional deficits following TBI. a–c Representative immunoblots and quantification showing protein expression of p-STAT1 (a), SOCS-1 (b), and SOCS-3 (c) in the injured cortex at 3 days post-TBI; TBI + vehicle: TBI + V, TBI + recombinant protein S: TBI + PS. GAPDH: loading control. Data are expressed as fold change compared to the sham group; n = 6 mice per group. d–i Quantitative RT-PCR analysis of MACS-sorted CD11b-positive cells showing mRNA expression of CD16 (d), CD32 (e), iNOS (f), CD206 (g), Arg-1 (h), and IL-10 (i) in the injured cortex at 3 days post-TBI. GAPDH: loading control. Data are expressed as fold change compared to the sham group; n = 6 mice per group. j–l Modified neurological severity scores (mNSS) (j), foot-fault test (k), and rotarod test (l) were performed at 3 days after TBI. n = 8 mice per group. In a–l, data are presented as mean ± SD; ***, p < 0.001. One-way ANOVA followed by Bonferroni’s post hoc tests

**Fig. 8**
Inhibition of Mer abolished PS-mediated functional improvements following TBI. a–c Representative immunoblots and quantification showing protein expression of p-STAT1 (a), SOCS-1 (b), and SOCS-3 (c) in the injured cortex at 3 days after TBI; TBI + vehicle: TBI + V, TBI + PS + control siRNA (si.Ctrl): TBI + S + C, TBI + S + Mer siRNA (si.Mer): TBI + S + M. GAPDH: loading control. Data are expressed as fold change compared to the TBI + V group; n = 6 mice per group. d–f Modified neurological severity scores (mNSS) (d), foot-fault test (e), and rotarod test (f) were performed at 3 days after TBI. n = 8 mice per group. In a–f, data are presented as mean ± SD; ***, p < 0.001. One-way ANOVA followed by Bonferroni’s post hoc tests

**Fig. 9**
The proposed mechanism of Mer in regulating microglial/macrophage M1/M2 polarization and neuroinflammation following TBI. In the setting of TBI, Mer is upregulated and exerts protective effects via modulating microglial/macrophage M1/M2 polarization and neuroinflammation after injury. Specifically, activation of Mer signaling facilitates STAT1-mediated SOCS expression, which increases microglial/macrophage M2-like polarization while decreases M1-like polarization following TBI. In contrast, inhibition of Mer by siRNA markedly suppresses STAT1/SOCS signaling, thus decreasing microglial/macrophage M2-like polarization while increasing M1-like polarization after TBI

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References

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1. Capizzi A, Woo J, Verduzco-Gutierrez M. Traumatic brain injury: an overview of epidemiology, pathophysiology, and medical management. Med Clin North Am. 2020;104:213–238. doi: 10.1016/j.mcna.2019.11.001. - DOI - PubMed
1. Dewan MC, Rattani A, Gupta S, Baticulon RE, Hung Y-C, Punchak M, et al. Estimating the global incidence of traumatic brain injury. J Neurosurg. 2019;130:1080–97. doi: 10.3171/2017.10.JNS17352. - DOI - PubMed
1. Blennow K, Hardy J, Zetterberg H. The neuropathology and neurobiology of traumatic brain injury. Neuron. 2012;76:886–899. doi: 10.1016/j.neuron.2012.11.021. - DOI - PubMed
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[1] GBD 2016 Neurology Collaborators Global, regional, and national burden of neurological disorders, 1990-2016: a systematic analysis for the Global Burden of Disease Study 2016. Lancet Neurol. 2019;18:459–480. doi: 10.1016/S1474-4422(18)30499-X. - DOI - PMC - PubMed

[2] GBD 2016 Neurology Collaborators Global, regional, and national burden of neurological disorders, 1990-2016: a systematic analysis for the Global Burden of Disease Study 2016. Lancet Neurol. 2019;18:459–480. doi: 10.1016/S1474-4422(18)30499-X. - DOI - PMC - PubMed

[3] Capizzi A, Woo J, Verduzco-Gutierrez M. Traumatic brain injury: an overview of epidemiology, pathophysiology, and medical management. Med Clin North Am. 2020;104:213–238. doi: 10.1016/j.mcna.2019.11.001. - DOI - PubMed

[4] Capizzi A, Woo J, Verduzco-Gutierrez M. Traumatic brain injury: an overview of epidemiology, pathophysiology, and medical management. Med Clin North Am. 2020;104:213–238. doi: 10.1016/j.mcna.2019.11.001. - DOI - PubMed

[5] Dewan MC, Rattani A, Gupta S, Baticulon RE, Hung Y-C, Punchak M, et al. Estimating the global incidence of traumatic brain injury. J Neurosurg. 2019;130:1080–97. doi: 10.3171/2017.10.JNS17352. - DOI - PubMed

[6] Dewan MC, Rattani A, Gupta S, Baticulon RE, Hung Y-C, Punchak M, et al. Estimating the global incidence of traumatic brain injury. J Neurosurg. 2019;130:1080–97. doi: 10.3171/2017.10.JNS17352. - DOI - PubMed

[7] Blennow K, Hardy J, Zetterberg H. The neuropathology and neurobiology of traumatic brain injury. Neuron. 2012;76:886–899. doi: 10.1016/j.neuron.2012.11.021. - DOI - PubMed

[8] Blennow K, Hardy J, Zetterberg H. The neuropathology and neurobiology of traumatic brain injury. Neuron. 2012;76:886–899. doi: 10.1016/j.neuron.2012.11.021. - DOI - PubMed

[9] Wilson L, Stewart W, Dams-O’Connor K, Diaz-Arrastia R, Horton L, Menon DK, et al. The chronic and evolving neurological consequences of traumatic brain injury. Lancet Neurol. 2017;16:813–825. doi: 10.1016/S1474-4422(17)30279-X. - DOI - PMC - PubMed

[10] Wilson L, Stewart W, Dams-O’Connor K, Diaz-Arrastia R, Horton L, Menon DK, et al. The chronic and evolving neurological consequences of traumatic brain injury. Lancet Neurol. 2017;16:813–825. doi: 10.1016/S1474-4422(17)30279-X. - DOI - PMC - PubMed

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Mer regulates microglial/macrophage M1/M2 polarization and alleviates neuroinflammation following traumatic brain injury

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Mer regulates microglial/macrophage M1/M2 polarization and alleviates neuroinflammation following traumatic brain injury

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