Wu et al. Journal of Neuroinflammation(2021) 18:2
https://doi.org/10.1186/s12974-020-02041-7

RESEARCH

Open Access

Mer regulates microglial/macrophage M1/M2 polarization and alleviates
neuroinflammation following traumaticbrain injury
Haijian Wu1,2,3†, 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 Zhao3* andJianmin Zhang1,4,5*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 andfunctional 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 wasintracerebroventricularly administered, and recombinant protein S (PS) was intravenously applied for intervention.
The neurobehavioral assessments, RT-PCR, Western blot, magnetic-activated cell sorting, immunohistochemistry andconfocal microscopy analysis, Nissl and Fluoro-Jade B staining, brain water content measurement, and contusion
volume assessment were performed.
(Continued on next page)* Correspondence: zzhao@usc.edu; zjm135@zju.edu.cn†
Haijian Wu and Jingwei Zheng contributed equally to this work.
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
1
Department of Neurosurgery, Second Affiliated Hospital, School of Medicine,
Zhejiang University, 88 Jiefang Road, Hangzhou 310009, Zhejiang, China
Full list of author information is available at the end of the article© The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,
which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give
appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate ifchanges were made. The images or other third party material in this article are included in the article's Creative Commonslicence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain
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The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to thedata made available in this article, unless otherwise stated in a credit line to the data.

Wu et al. Journal of Neuroinflammation(2021) 18:2

Page 2 of 20(Continued from previous page)

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: Mer, Microglia/macrophage, M1/M2 polarization, Neuroinflammation, TBI

Background
Traumatic brain injury (TBI) is a devastating neurological disease and a major cause of death and disability
worldwide, not only young people but also the elderly [1,2]. It is estimated that approximately 70 million individuals suffer a TBI annually [3]. Notably, the pathophysiological processes of TBI are rather complex, which occur
in the acute phase and yet continue to evolve over time,leading to persistent, sometimes life-long, consequences[4]. TBI is an important risk factor for a variety ofchronic neurological disorders, such as epilepsy, stroke,
psychiatric illness, and neurodegenerative diseases [5].
There is no doubt that TBI not only severely affects thedaily life of survivors and their families, but also resultsin substantial socio-economic impacts [6]. However, acomprehensive understanding of this notorious diseaseis still lacking, and the need for therapeutics that effectively improve long-term functional outcomes in TBI survivors remains unmet.
Mechanistically, both primary and secondary injury
mechanisms are involved in the neuropathology of TBI.
Secondary brain injury, which occurs from initial insults
to the brain, is an important determinant of TBI outcomes [7]. It consists of ionic homeostasis disturbance,
excitotoxicity, oxidative stress, inflammation, and cell
death, among others. In particular, neuroinflammation is
one of the most common and important cellular events
following TBI [8]. It can evolve over minutes to days,
months, and even years after the initial injury and contributes to the development of both acute and chronic
neurological consequences post-insult [9, 10]. Microglia,
the major resident immune cells in the brain, are presumed to be key players in neuroinflammation after TBI[11]. Also, peripherally derived macrophages are actively
involved in acute neuroinflammatory responses after
TBI [12]. It has reported that microglia/macrophages arehighly plastic and assume their phenotypes dependent
on different microenvironmental cues, such as proinflammation and anti-inflammation, among others [13].
The pro-inflammatory M1-like phenotype expressing
signature markers such as CD16 and CD32 tends torelease destructive mediators, including tumor necrosis
factor-α (TNF-α), interleukin-1β (IL-1β), and inducible
nitric oxide synthase (iNOS). In contrast, the antiinflammatory M2-like phenotype, characterized by themolecular signatures of CD206 and arginase 1 (Arg-1),
produces beneficial mediators such as interleukin-10(IL-10) and transforming growth factor-β (TGF-β) [14].
Notably, the M1-like phenotype is more likely associatedwith uncontrolled neuroinflammation often observed inneurodegenerative diseases, while the M2-like phenotype
promotes inflammation resolution and tissue repair [15].
Thus, a balanced response between polarized microglial/
macrophage phenotypes is essential for immune homeostasis in the brain.
Currently, the dualistic roles of distinctly polarized
microglial/macrophage populations have been demonstrated in major central nervous system diseases (CNS),
including TBI [16, 17]. Studies from animal models indicated that despite both M1-like and M2-like polarized
microglial/macrophage being activated after TBI, the activity of M2-like response declines over time, whereas
the pathological M1-like effect can persist for a long
period of time post-insult [16, 17], and modulating theM1-M2 polarization balance has been shown to be beneficial for functional outcomes [18, 19]. However, the molecular mechanisms underlying microglial/macrophage
polarization in TBI are still not fully understood. Remarkably, the neuroinflammatory responses following
TBI are highly complex and cannot be so easily delineated by using a binary M1/M2 nomenclature [20]. For
instance, in a controlled cortical impact (CCI) mousemodel of TBI, Morganti et al. found that activatedmicroglia/macrophages adjacent to the ipsilateral perilesional cortical tissue simultaneously express both “M1like” and “M2-like” phenotypic markers across multiple
time points after injury [21]. Instead of switching to apolarized “M1-only” or “M2-only” phenotype, these cells
shape their responses in complex and mixed phenotypes
during CNS injury and disease, including TBI [12, 20,21]. And the polarization responses of these cells areheterogeneous and overlapping after injury, such that

Wu et al. Journal of Neuroinflammation(2021) 18:2

pro-inflammatory responses are occurring concurrently
with the expression of anti-inflammatory and tissue repair mediators [12].
Myeloid-epithelial-reproductive tyrosine kinase (Mer),a member of the Tyro-Axl-Mer (TAM) family of receptor tyrosine kinases [22], plays a critical role in the regulation of inflammatory responses in peripheral
macrophages [23–26] and brain microglial cells [27–29].
Specifically,Mersignalingstiresmacrophage
polarization towards an M2-like phenotype [30]. For instance, it reported that protein S (PS)-mediated activation of Mer inhibited M1-like polarization of peritoneal
and tumor-derived macrophages [31]. Besides, growtharrest-specific protein 6 (Gas6)-mediated Mer signaling
increases the expression of M2-associated genes such as
Arg-2 and VEGF in bone marrow-derived macrophages[32]. Mer signaling is also required for the efficient
clearance of early apoptotic cells by M2c-like macrophages [33]. However, the function of Mer in regulating
the innate immune response and microglial/macrophage
M1/M2 polarization in TBI has not been addressed. In
the present study, we hypothesized that Mer upregulation is a key modulator of microglial/macrophage M1/M2 polarization and neuroinflammation in the acutestage of TBI. We found that inhibition of Mer shifts
microglial/macrophage polarization toward the M1-likephenotype via regulating the signal transducer and activator of transcription 1 (STAT1)/suppressor of cytokine
signaling 1/3 (SOCS1/3) pathway, which exacerbates
neuroinflammation and secondary brain damage following TBI. In contrast, activation of Mer signaling increases microglial/macrophage M2-like polarization
while decreases M1-like polarization and confers neuroprotection after TBI.

Page 3 of 20

efforts were made to minimize animal suffering and thenumber of animals sacrificed.
Randomization and blinding

All animals were randomized for group allocation andsurgical procedures and included in the analysis. The
operators responsible for the experimental procedures
and data analysis were blinded and unaware of group allocation throughout the experiments.
TBI model

TBI was induced in C57BL/6 mice using a CCI model(Suppl. Fig. 1A-C), as described previously [34]. Briefly,
mice were anesthetized by an intraperitoneal injection ofpentobarbital sodium (50 mg/kg) and placed in thestereotaxic frame. A 4-mm-diameter craniotomy was
performed using a portable drill over the right parietal
cortex between bregma and lambda, 2 mm lateral to themidline. The dura mater was kept intact over the cortex.
The CCI was performed perpendicular to the brain surface using a PinPoint™ Precision Cortical Impactor (Cary,
NC, USA) with a 3-mm-diameter impact tip. The impact
velocity is 3 m/s, the impact duration is 150 ms, and theimpact depth is 2 mm. After TBI, the bone flap was immediately replaced and sealed, and the scalp was sutured
closed. The animal’s core body temperature was maintained at 37 ± 0.5 °C with a thermostatically controlled
heating pad during surgery. Sham animals were subjected to all aspects of the protocol (surgery, anesthesia,
craniotomy, injection, and recovery) except for CCI. Recombinant PS (0.2 mg/kg) (9489-PS, R&D Systems) was
administered via the tail vein at 1 h, 1 day, and 2 days
after the CCI [35, 36] (Suppl. Fig. 2).
Intracerebroventricular (i.c.v.) injection

Materials and methods
Animals

Adult male and female C57BL/6 mice (22–27 g, aged 8–
10 weeks) which were purchased from Slac LaboratoryCo., Ltd. (Shanghai, China) were used for this study.
Notably, male mice were used in this study except that
14 female mice were tested to compare the effect of sex
differences on Mer expression and neurofunctional outcomes after TBI. All mice were housed in filter-top cages
and fed a standard diet, with a 12-h light/dark cycle.
Free access to food and water as well as controlledtemperature and humidity were provided. All animal experiments were performed according to the Institutional
Animal Care and Use Committee of Zhejiang University.
The procedures were conducted according to the National Institutes of Health’s Guide for the Care and theUse of Laboratory Animals and the ARRIVE (Animal
Research: Reporting In Vivo Experiments) guidelines. All

Previous studies demonstrated that i.c.v. delivery ofsiRNA can efficiently silence target gene expression in
the brain with a range of 50–80% [37–39]. The i.c.v. administration of siRNA was performed as previously described [40] (Suppl. Fig. 1D-F). A 1-mm-diameter burrhole was drilled into the right side of the skull (1.0 mm
lateral and − 0.25 mm anterior-posterior (AP) to
bregma). Mer siRNA or scrambled siRNA (500 pmol,
Thermo Fisher Scientific) mixed with the transfection
reagent (Engreen Biosystem Co., Ltd.), for a total volumeof 2 μl, was delivered into the ipsilateral ventricle (1.0mm lateral and − 0.25 mm AP to bregma, 2.5 mm dorsoventral (DV) below the skull). The injection was performed at a rate of 0.5 μl/min, then the needle stayed in
the brain for another 5 min after injection to avert leakage, and then the burr hole was sealed with bone wax,
and the incision was closed with sutures. Mice wereplaced in individual recovery cages. The i.c.v. injection of

Wu et al. Journal of Neuroinflammation(2021) 18:2

siRNA was performed 1 day before and 10 min after
TBI.
Neurobehavioral function assessment
Modified neurological severity scores (mNSS)

The mNSS score was assessed as previously reported[34]. It includes sensory, motor, balance, and reflex tests.
The neurological function was graded on a scale of 0–18
(normal score, 0; maximal deficit score, 18) and recorded
before TBI, as well as at 1, 3, and 7 days after TBI.
Foot-fault test

It was performed to evaluate the motor function similarly to that described previously [40]. Mice were placedon hexagonal grids of different sizes. Mice placed theirpaws on the wire while moving along the grid. With
each weight-bearing step, the paw may fall or slip between the wire. This was recorded as a foot fault. The
total number of steps (movement of each forelimb) thatthe mouse used to cross the grid was counted, and thetotal number of foot faults for each forelimb was recorded. The percentage of forelimb faults to the total
number of steps was calculated.
Rotarod test

It was performed to test the motor coordination and thelimb strength as previously described with the Rota-Rod
Treadmills (BW-ZH600, Shanghai Bio-will Co., Ltd.)[40]. Test sessions consist of six trials at a variable speed(an initial velocity of 5 rpm was used for the first 10 s, alinear increase from 5 to 10 rpm for the next 30 s, and alinear increase from 10 to 20 rpm between 40 and 90 s).
The final score was determined as the mean time that amouse was able to remain on the rod over six trials.

Page 4 of 20

percentage that was calculated by the use of the following formulae: [(contralateral hemispheric volume − ipsilateral hemispheric volume)/(contralateral hemisphericvolume) × 100%].
Quantitative RT-PCR

Total RNA was isolated from sham and injured brainsusing the TRIzol reagent (Sigma-Aldrich, St. Louis, MO,
USA) according to the manufacturer’s instructions.
Then, RNA (1 μg) from each sample was reversetranscribed to cDNA by PrimeScriptTM RT reagent kit(Takara Bio Inc, Shiga, Japan). Afterward, quantitative
reverse transcription-polymerase chain reaction (RTPCR) was conducted with SYBR® Premix Ex Taq™
(Takara Bio Inc, Shiga, Japan) on a 7300 Plus Read-Time
PCR System (Thermo Fisher Scientific). The cDNA wasused as a template in a 20 μl reaction volume (10 μl of
PCR mix, 5 pmol of forward and reverse primers, 1 μl
cDNA template and proper volume of water). The PCR
reaction was performed as follows: Cycling conditions
began with an initial DNA denaturation step at 95 °C for
20 s, followed by 40 cycles at 94 °C for 15 s, 56 °C for 30s, and 72 °C for 25 s. Each sample was examined in triplicate. The threshold cycle (CT) readings were collected,
and the relative expression of mRNA of target genes wascalculated with the 2−ΔΔCT method and was normalized
to the glyceraldehyde 3-phosphate dehydrogenase(GAPDH) level in all samples. The expression levels of
the mRNA were then reported as fold changes versus
sham controls. The sequences of the primer pairs fortarget genes are as shown below:
Gene

Forward primer sequence(5′–3′)

Reverse primer sequence(5′–3′)CD16

TTTGGACACCCAGATGTTTCAG

GTCTTCCTTGAGCACCTGGATC

Brain water content measurement

CD32

AATCCTGCCGTTCCTACTGATCGTGTCACCGTGTCTTCCTTGAG

Brain water content was measured as previously reported [34]. Briefly, mice were sacrificed 72 h after TBI,
and the brains were obtained without transcardiac perfusion. Tissue samples of injured hemispheres were dissected and weighed on an electric analytic balance toobtain the wet weight and then dried at 100 °C for 48 h
to obtain the dry weight. Brain water content was calculated using the following formula: brain water content(%) = (wet weight − dry weight)/wet weight × 100%.

iNOSCAAGCACCTTGGAAGAGGAG

Contusion volume assessment

To measure the contusion volume in the ipsilateral cortex 72 h after TBI, cresyl violet-stained sections weredigitized and analyzed by using ImageJ (National Institutes of Health, Bethesda, MD, USA) as previously reported [41]. The volume was computed by adding the
injury areas and multiplying with the inter-slice distance(500 μm). Hemispheric tissue loss was expressed as a

CD206 CAAGGAAGGTTGGCATTTGT

AAGGCCAAACACAGCATACC
CCTTTCAGTCCTTTGCAAGC

Arg-1

TCACCTGAGCTTTGATGTCGCTGAAAGGAGCCCTGTCTTG

IL-10

CCAAGCCTTATCGGAAATGATTTTCACAGGGGAGAAATCG

Mer

CCTCTGCTTCGCCACATCTGTATG

GACCAGCCAATCTCATTCCGACAGSOCS1

CCTCGTCCTCGTCTTCGTCCTCGAAGGTGCGGAAGTGAGT
GTCG

SOCS3

GACCAAGAACCTACGCATCCAG GCACCAGCTTGAGTACAC
TG
AGTCG

GAPD
H

TTCAACGGCACAGTCAAGG

CACCAGTGGATGCAGGGAT

Western blot

Western blot was performed as previously described[34]. Briefly, tissue samples from the ipsilateral cortex ofsham/TBI mouse brains were homogenized in RIPA

Wu et al. Journal of Neuroinflammation(2021) 18:2

buffer (50 mM Tris-HCl at pH 7.4,150 mM NaCl, 1%
Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1
mM EDTA) with protease and phosphatase inhibitors,
followed by denaturation at 95 °C for 10 min. Then theprotein samples were separated by SDS-PAGE andtransferred onto polyvinylidene fluoride (PVDF) membranes (Millipore). Next, the PVDF membranes were
blocked with 5% bovine serum albumin for 1 h and incubated with the primary antibodies overnight, including
anti-Mer antibody (1:1000, Abcam, ab184086), anti-pMer antibody (1:500, Abcam, ab192649), anti-STAT1
antibody (1:1000, CST, 14994), anti-p-STAT1 antibody(1: 1000, CST, 9167), anti-SOCS-1 (1:1000, Abcam,
ab62584), anti-SOCS-3 (1:1000, Abcam, ab16030), andanti-GAPDH (1:5000, Abcam, ab8245). After that, the
PVDF membranes were disposed of with the relevant
secondary antibodies (1:5000) for 1 h at room
temperature and observed using the ECL kit chemiluminescence reagents (Millipore, Billerica, MA, USA).
The signals of protein bands were detected with theChemidoc detection system and quantified using Quantity One software (Bio-Rad).
CD11b-positive cell isolation

A magnetic-bead conjugated anti-CD11b antibody wasused to isolated microglia/macrophages from injured
brain tissue using magnetic-activated cell sorting(MACS) technology as previously described [18]. Ipsilateral cortical tissues from sham and CCI mice were dissociated using Neural Tissue Dissociation Kit (Miltenyi
Biotec, 130-093-231) according to the manufacturer’s instructions. The dissociated brain samples were resuspended and applied to a Strainer (70 μm). Debris in
the sample was removed using Debris Removal Solution(Miltenyi Biotec, 130-109-398). Red blood cells werelysed by Red Blood Cell lysis Solution (Miltenyi Biotec,
130-094-183). Myelin was removed by Myelin Removal
Beads (Miltenyi Biotec, 130-096-733). Finally, cell sample
was incubated with anti-CD11b microbeads (Miltenyi
Biotec, 130-049-601) and loaded onto a magnetic LS column ((Miltenyi Biotec, 130-042-401) placed in the magnetic field of a MACS Separator (Miltenyi Biotec, 130090-976). After washing, the magnetically labeled cells
were flushed out immediately by firmly pushing theplunger into the column to get CD11b positive cells,
which can be used for PCR analysis.
Immunohistochemistry and confocal microscopy analysis

Mice were anesthetized and transcardially perfused with
0.1 mmol PBS and 4% paraformaldehyde (PFA) at 3 days
post-TBI. Twenty-micrometer coronal cryosections werepermeabilized and incubated in 5% Donkey Serum for 1
h for blocking. Then the brain tissues were incubated inprimary antibody overnight at 4 °C. The primary

Page 5 of 20

antibody information is as follows: anti-Mer antibody (1:400, AF591, R&D Systems), anti-CD16/32 antibody (1:200, Abcam, ab25235), anti-CD206 antibody (1:400,
Abcam ab64693), anti-Iba-1 antibody (1:500, Wako, 01919741 or 1:500, Abcam, ab5076). After the incubation
overnight, the cryosections were incubated with the secondary antibodies (1:500, Jackson Immunoresearch Laboratories) for 1 h at room temperature. After that, thesections were rinsed with PBS and covered with fluorescence mounting medium with 4',6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, H-1200).
For
image
analysisandquantificationofimmunofluorescent, Nissl, and Fluoro-Jade B (FJB) staining, coronal sections from bregma − 1.0 to − 3.0 mm
were collected. In each animal, 5 randomly selected
fields from 5 nonadjacent sections with intervals of
100 μm in the ipsilateral cortex were analyzed. The region of interest (ROI) is ~ 200 μm adjacent to the lesion
boundaries in the brain lesions. For immunofluorescenceanalysis of ROI (450 × 350 μm2 per field) (as indicated inFig. 2d, left and Suppl. Fig. 3A), the fluorescence intensity and the number of Mer+, CD16/32+, and CD206+
cells, as well as their colocalization with Iba-1 staining,were calculated by using ImageJ software and averaged
per mouse, and each group included 6 animals [42].
Nissl staining

Nissl staining was performed according to the
manufacturer’s instructions. After 20-μm coronal sections had been deparaffinized and rehydrated, the slides
were stained in Nissl Staining Solution (C0117, Beyotime) for 5 min at 37 °C. A large cell body, with abundant cytoplasm and with substantially significant levelsof Nissl body, represents a normal neuron. However,some other cell forms such as a shrunken cell body, condensed nuclei, and reduced or disappearance of Nissl
body represents a damaged cell. In each animal, 5 randomly selected fields (450 × 350 μm2 per field) in the ipsilateral cortex (Suppl. Fig. 3B) were analyzed in 5
nonadjacent sections (100 μm apart) and averaged permouse. Cell counting was performed by using the ImageJ software, and a total of 6 mice from each group wereused for quantification.
Fluoro-Jade B (FJB) staining

The FJB staining was performed as previously described[34]. Twenty-micrometer coronal sections were first
immersed in a solution containing 1% sodium hydroxide
in 80% alcohol for 5 min, followed by 2 min in 70% alcohol and 2 min in distilled water. The slides were then incubated in a solution of 0.06% potassium permanganate
for 10 min, rinsed in distilled water for 2 min, and incubated in a 0.0004% solution of FJB (Chemicon, Temecula, CA, USA) made in 0.1% acetic acid for 30 min. The

Wu et al. Journal of Neuroinflammation(2021) 18:2

slides were rinsed, air-dried, cleared in xylene for a minute, and coverslipped with fluorescence mounting
medium with DAPI. All sections were observed andphotographed under a fluorescence microscope withblue (450–490 nm) excitation light. In each animal, 5randomly selected fields (640 × 480 μm2 per field) in theipsilateral cortex (Suppl. Fig. 3C) were analyzed in 5
nonadjacent sections (100 μm apart) and averaged permouse. Cell counting was performed by using the ImageJ software, and a total of 6 mice from each group wereused for quantification.
Statistical analysis

All data were shown as means ± standard deviation. For
comparison between two groups, the F test was
conducted to determine the similarity in the variances
between the groups that are statistically compared, andstatistical significance was analyzed by Student’s t test.
For multiple comparisons, Bartlett’s test for equalvariances was used to determine the variances between
the multiple groups and one-way or two-way analysis ofvariance (ANOVA) followed by Bonferroni’s post hoc
test which was used to test statistical significance. Allanalyses were performed using GraphPad Prism 8 software by an investigator blinded to the experimental conditions. A p value < 0.05 was considered as statisticallysignificant.

Results
Dynamic changes in mRNA expression of microglial/
macrophage M1/M2 polarization markers following TBI

Polarized microglia/macrophages can be commonly
distinguished by their expression profiles of signaturesurface markers and cytokines/chemokines. To evaluate
the microglial/macrophage activation states following
TBI (Suppl. Fig. 2A), RT-PCR was performed using total
RNA extractions from the perilesional cortical tissues at
3 h, 12 h, 1 day, 3 days, and 7 days after TBI or sham operation (Fig. 1a). Levels of M1-associated markers, including CD16 (Fig. 1b), CD32 (Fig. 1c), and iNOS (Fig.
1d), were gradually increased over time from 12 h onward and remained elevated for at least 7 days after injury. The mRNA levels of tested M2-like markers,
including CD206 (Fig. 1e), Arg-1 (Fig. 1f), and IL-10(Fig. 1g), began to be significantly upregulated at 1 day,
12 h, and 1 day after TBI, respectively, and all peakedaround day 3 post-injury, then declined dramatically on
day 7, even though they did not return to baseline levels.
These results indicated that both M1-like and M2-likepolarized microglia/macrophages were activated during
the acute and subacute phase response after TBI. TBI induces transient upregulation in the M2-like phenotype
but causes sustained upregulation in the M1-likePage 6 of 20

phenotype post-insult, which is consistent with recent
reports [16, 17].
Expression patterns and cellular localization of Mer
following TBI

Mer plays a critical role in regulating microglial/
macrophage phenotypes and properties underphysiological and pathological conditions [29]. We first
analyzed the levels of Mer protein and mRNA in the
injured cortex at different time points after TBI (Suppl.
Fig. 2A). As shown in Fig. 2a, b, both Mer protein andmRNA levels were significantly increased 12 h after TBI,
peaked around day 3 and returned to the pre-injury
levels around day 7. We further investigated the cellular
localization and expression of Mer in microglia/macrophages in the ipsilateral cerebral cortex at 3 days after
TBI, using double immunofluorescent staining of Mer
and microglial/macrophage marker Iba-1. As demonstrated in Fig. 2c, e, Mer was abundantly expressed in
the plasma membrane of microglia/macrophages in thesham group and substantially upregulated in the activated microglia/macrophages with phagocytotic morphology (Fig. 2d, e) near the lesion at 3 days post-TBI.
Inhibition of Mer worsened the functional outcomes after
TBI

Previous studies reported that i.c.v. delivery of siRNAcan efficiently silence specific genes in the brain [37–39].
To determine the role of Mer in the pathophysiology of
TBI, we administrated Mer siRNA into the ipsilateral
ventricle at 1 day before and 10 min after TBI to inhibit
its expression in the brain [43] (Suppl. Fig. 2B). Data
from the Western blot and RT-PCR demonstrated theknockdown efficacy of Mer siRNA was > 70% (Fig. 3a,b). Besides, we evaluated the functional outcomes following TBI using the mNNS score and foot-fault androtarod tests performed before and 1, 3, and 7 days after
TBI. When compared to the sham group, TBI mice exhibited significantly worse in all neurobehavioral tests(Fig. 3c–e). More importantly, administration of Mer
siRNA significantly aggravated the neurobehavioral deficits post-injury when compared with the group that received control siRNA at days 1, 3, and 7 after TBI, basedon mNNS scores (Fig. 3c), foot-fault test (Fig. 3d), androtarod test (Fig. 3e). Specifically, research studies reported that sex differences exist in the pathophysiologyof CNS injuries and diseases [44]. To evaluate sexdimorphic responses after TBI, neurobehavioral function
assessment was performed before and after TBI. Our
preliminary data showed that there is no difference inbehavioral tests between male and female mice at days 1,
3, and 7 after TBI, based on the mNSS score (Suppl. Fig.
4A), foot-fault test (Suppl. Fig. 4B), and rotarod test
(Suppl. Fig. 4C). Also, the expression levels of Mer inWu et al. Journal of Neuroinflammation(2021) 18:2

Page 7 of 20

A

C

8

8

NS
***

6

***

***

4
**
ns

2

Sham

3h

12h
1d
Groups

3d

5

6

***
***

4
**
ns

2

3h

12h
1d
Groups

3
ns

*

ns

1
0
3h

12h
1d
Groups

3d

***
**

2

ns
ns

1

Sham

7d

3h

12h
1d
Groups

3d

12

###

30
***
15

**

*

7d

7d

G

###

ns
0

Sham

3

***

**

2

3d

45

***

4

NS
***

0
Sham

F

###

7d

D 4

NS
***

7d

Arg-1 mRNA Ratio

CD206 mRNA Ratio

3d

0

0

E

1d

IL-10 mRNA Ratio

CD16 mRNA Ratio

12h

CD32 mRNA RatioB

3hiNOS mRNA Ratio

Sham

***

9
6
3

ns

ns

**

*

0
Sham

3h

12h
1d
Groups

3d

7d

Sham

3h

12h
1d
Groups3d

7dFig. 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), andiNOS (d), were gradually increased over time from 12 h onward and remained elevated for at least 7 days after injury. e–g Expressions of mRNAof 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 shamoperated 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

the injured cortex between male and female mice wereanalyzed on day 3 post-injury. As shown in Suppl. Fig.
4D-E, no difference was detected in Mer protein (Suppl.
Fig. 4D) and mRNA (Suppl. Fig. 4E) expression between
male and female mice on day 3 after TBI.
To assess the gross pathological changes, we
compared brain contusion volume and tissue edema
among sham and TBI groups at 3 days post-injury. As
demonstrated, TBI caused significant loss of brain tissue(Fig. 3f) and increased brain edema in the ipsilateralhemisphere (Fig. 3g). Interestingly, although Mer siRNA
did not affect the brain contusion volume on day 3 after
TBI (Fig. 3f), it significantly elevated brain edema levelat 3 days post-injury, when compared to both the vehicle
and control siRNA group (Fig. 3g). These findings suggested that Mer presence and its upregulation after injury is beneficial for the recovery after TBI.
Mer modulated microglial/macrophage M1/M2
polarization after TBI

To investigate the role of Mer in regulating microglial/
macrophage M1/M2 polarization and neuroinflammation
following TBI, we evaluated the level of Mer in both M1like and M2-like polarized microglia/macrophages in TBIaffected cortical regions. Results from double immunofluorescent staining demonstrated that Mer was at a

Wu et al. Journal of Neuroinflammation(2021) 18:2

A Mer

140kDa

Sham

3h

12h

1d

3d

Mer

Iba-1

DAPI

Merge

Mer

Iba-1

DAPI

Merge

7d
Sham

***
Mer Relative density

D

36kDa

GAPDH
4

Page 8 of 20

3
***
2

ns

*

ns

TBI

1
0
Sham

3h

12h
1d
Groups

3d

7d

B

C
1.5
***

4
***
3
**
2

ns

ns

1

Mer/Iba-1 intensity

5
Mer mRNA Ratio

E

***

Sham

1.0

0.5

TBI
0.0

0
Sham

3h

12h
1d
Groups3d

7d

Sham TBI

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 withthe microglial/macrophage marker Iba-1 in the ipsilateral cerebral cortex was performed at 3 days post-TBI or sham operation. c Fluorescenceintensity quantification of Mer expression in activated microglia/macrophages in the impacted cortical area at 3 days after TBI, compared to thatin resting microglia/macrophages from sham-operated brains. n = 6 mice per group. d Low-magnification images of the brain (left) indicate theregion 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 substantiallyupregulated in the activated microglia/macrophages with phagocytotic morphology at 3 days post-TBI. Orthogonal views demonstrated thecolocalization 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 arepresented as mean ± SD; ***, p < 0.001 by Student’s t test

relatively lower level in CD16/32-positive M1-like cells(Fig. 4a and Suppl. Fig. 5A), but highly expressed in
CD206-positive M2-like cell population (Fig. 4b andSuppl. Fig. 5A), in the perilesional area of cortex at 3 days
post-TBI.
To specifically evaluate the effect of Mer siRNA on theM1/M2 polarization state of microglia/macrophages
after TBI, CD16/32 or CD206 was co-labeled with themicroglia/macrophage marker Iba-1 in the cortex surrounding the lesion cavity at 3 days post-insult, respectively. As demonstrated, the percentage of CD16/32 andIba-1 double-positive M1-like cells was significantly increased in the cortex on day 3 after TBI, which was

further elevated in the Mer siRNA group (Fig. 4c, d). On
the other hand, the percentage of CD206 and Iba-1
double-positive M2-like cells was also increased significantly in the ipsilateral cortex on day 3 after TBI whencompared with the sham group (Fig. 4e, f), but it wassignificantly decreased following Mer siRNA administration when compared with the TBI + vehicle or TBI +control siRNA group (Fig. 4e, f). In addition, the ratio
between CD16/32+Iba-1+ M1-like cells and CD206+Iba1+ M2-like cells was nearly doubled after Mer siRNA administration (Fig. 4g). Taken together, these findings indicated that Mer plays an important role in regulating
microglial/macrophage M1/M2 polarization following

A(2021) 18:2

Page 9 of 20

C
140kDa

GAPDH

36kDa
TBI+V

TBI+C

***

ns

*

F

TBI+Mer siRNA
Sham

**

Sham

***

5

0
Before TBI

1d

3d

7d

D
40

Foot-fault (%)

TBI+M

ns

20

10

0
*

0.0

TB TB
I+
I+
V
C
eh
on
ic
tr
l
TB ol s e
i
I+
R
N
M
A
er
s iR
N
A

TBI+C

30

1.0

0.5

TBI+V

10

30

Sh
am
TB
I+
TB
V
I+
eh
co
ic
nt
le
ro
l
TB
siR
I+
N
A
M
er
siR
N
A

Mer Relative density

1.5

TBI+M

TBI+Vehicle
TBI+Control siRNA

15

mNSS score

Mer

Lesion volume (%)

Wu et al. Journal of Neuroinflammation

***
***

20
10

G

B

TB TB
I+
I+
V
C
eh
on
ic
tr
l
TB ol s e
iR
I+
N
M
A
er
siR
N
A

0.0

3d

E
100
80
***

60
**

40
*

0
1d

***
85

#

ns

80
75
70

20

Before TBI

90

7d

3d

7d

Sh
am
TB
I+
V
eh
+c
ic
on
le
tr
ol
TB
siR
I+
N
A
M
er
siR
N
A

0.5

1d

TB
I

1.0

Before TBI

Latency to fall (s)ns
Mer mRNA Ratio

0***

Brain water content (%)

1.5

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 theexpression 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, whencompared 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

TBI, and inhibition of Mer expression tips the balance of
TBI-induced microglial/macrophage activation towards
the pro-inflammatory M1-like phenotype.
Downregulation of Mer aggravated neuronal damage anddegeneration following TBI

To further evaluate the pathological effects of Mer
knockdown following TBI, Nissl and FJB staining were
employed to access the neuronal damage and degeneration
following TBI. As shown, TBI caused a significant decreasein the number of Nissl-positive neurons in the perilesional
cortex at 3 days post-insult (Fig. 5a). Moreover, in vivo
knockdown of Mer aggravated neuronal damage following
TBI, as demonstrated by the significant decrease in thenumber of Nissl-positive neurons in the injured cortex in the
TBI + Mer siRNA group compared to the TBI + vehicle or
TBI + control siRNA group (Fig. 5a). Besides, FJB staining
showed that TBI induced a significant increase in the number of degenerating neurons in the injured cortex, and Mer
siRNA application further increased the number of degenerating neurons in the injured cortex after TBI (Fig. 5b). These
data indicated inhibition of Mer expression aggravated neuronal damage and degeneration following TBI.
Inhibition of Mer reduced STAT1 activation and SOCS
expression following TBI

Previous studies indicated that Mer is a pleiotropic
regulator of the innate immune system, and it regulatesWu et al. Journal of Neuroinflammation

C

Page 10 of 20

Mer

CD16/32

DAPI

Merge

CD16/32

Iba-1

DAPI

Merge

B

E

Sham

Sham

TBI+Vehicle

TBI+Vehivle

TBI+Control siRNA

TBI+Control siRNA

TBI+Mer siRNA

TBI+Mer siRNA

D

#

***

F
40

***

ns

60
40
20

#

30
20

Merge

CD206

Iba-1

DAPI

Merge

G
6

***

4

ns

2

10
0

Sh
am
TB
I+
V
eh
C
ic
on
le
tr
ol
TB
s iR
I+
N
A
M
er
s iR
N
A
TB
I+

Sh
am
TB
TB
I+
V
I+
eh
C
ic
on
le
tr
ol
TB
siR
I+
N
A
M
er
siR
N
A

DAPI

ns

0

0

CD206

M1/M2 Ratio

80

CD206 +Iba-1+ cells/
Iba1+ cells (%)

CD16/32 +Iba-1+ cells/
Iba-1+ cells (%)

100

Mer

TB TB
I+
I+
V
C
eh
on
ic
tr
l
TB ol s e
iR
I+
N
M
A
er
siR
N
A

A

(2021) 18:2

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. Scalebar = 10 μm. b Representative images of immunofluorescent staining for Mer (red), CD206 (green), and DAPI (blue) showing Mer was expressedin 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 elevatedin 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

inflammatory responses through modulating multiple
downstream cellular pathways such as STAT1/SOCS
signaling [22, 25]. Activation of Mer signaling is
associated with phosphorylation of the tyrosine residue
749 in the kinase domain [45]. To clarify the mechanism
by which Mer interferes with microglial/macrophage
M1/M2 polarization, we determined the expression of
the suppressor of cytokine signaling SOCS-1 and SOCS3 after TBI, which are key mediators of Mer signaling in
regulating inflammatory responses [46, 47]. As demonstrated, the levels of phosphorylated Mer (p-Mer) (Fig. 6a)and phosphorylated STAT1 (p-STAT1) (Fig. 6b) were significantly inhibited by Mer siRNA administration on day 3
following TBI. More importantly, the protein and mRNA
expression of SOCS-1 (Fig. 6c, d) and SOCS-3 (Fig. 6e, f)were significantly reduced after TBI in the Mer siRNAtreatment group when compared with the vehicle or control siRNA group. These findings indicated that Mer signaling is required for activation of STAT1 and SOCS
signaling pathway in the perilesional area of the cortex following TBI.
Mer activation alleviated functional deficits following TBI

To further determine the role of Mer following TBI,
administration of recombinant PS (a ligand and activatorof Mer) was performed at 1 h, 1 day, and 2 days after
TBI, respectively (Suppl. Fig. 2C and 2D) [48]. Western
blot assay revealed PS treatment caused a significant
increase of p-STAT1 (Fig. 7a), SOCS-1 (Fig. 7b), andSOCS-3 (Fig. 7c) expression when compared with theWu et al. Journal of Neuroinflammation(2021) 18:2

Page 11 of 20

A

B

TBI+C

TBI+V

TBI+M

Sham

TBI+V

TBI+M

200
***

FJB-positive cells/field

#
100
ns
50

0

#

***

150

ns

100
50

A

A
N

er

iR
TB
I+
M

ol
s

I+
C

siR
N

eh
ic

le

am
Sh

I+
V
TB

A

er

s iR
N

N
A
iR

ol
s

I+
M
TB

on
tr

TB

I+
C

TB

I+
V

eh
ic
l

e

Sh
am

0

TB

Nissl-positive cells/field

150

TBI+C

on
tr

Sham

Fig. 5 Inhibition of Mer aggravated neuronal damage and degeneration following TBI. a Representative images of Nissl staining in the ipsilateralcortex 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, andMer 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

vehicle group. PCR assay revealed that expression of
M1-associated markers, including CD16 (Suppl. Fig. 5B),
CD32 (Suppl. Fig. 5C), and iNOS (Suppl. Fig. 5D), washeavily suppressed by PS treatment when compared withthe vehicle group. By contrast, the tested M2-likemarkers, including CD206 (Suppl. Fig. 5E), Arg-1 (Suppl.
Fig. 5F), and IL-10 (Suppl. Fig. 5G), were further upregulated after PS administration. Consistently, PCR assay of
MACS-sorted CD11b-positive cells revealed that expression levels of M1-associated markers, including CD16
(Fig. 7d), CD32 (Fig. 7e), and iNOS (Fig. 7f), were largely
suppressed by PS treatment when compared with vehicle
treatment. By contrast, the tested M2-associatedmarkers, including CD206 (Fig. 7g), Arg-1 (Fig. 7h), andIL-10 (Fig. 7i), were further increased after PS administration. Furthermore, TBI-induced brain edema was reduced by PS treatment at 3 days post-injury (Suppl. Fig.
6A). Also, TBI-induced neuronal damage was attenuatedby PS treatment, as demonstrated by the significant increase in the number of Nissl-positive neurons (Suppl.
Fig. 6B-C) and the significant decrease in the number of
FJB-positive degenerating neurons (Suppl. Fig. 6D-E) in
the injured cortex in the TBI + PS group compared to
the TBI + vehicle group. Moreover, PS treatment significantly alleviated neurobehavioral deficits on day 3 post-

injury, based on the mNNS score (Fig. 7j), foot-fault test
(Fig. 7k), and rotarod test (Fig. 7l). However, Mer siRNA
administration abolished PS-induced changes in pSTAT1 level (Fig. 8a) and expression levels of SOCS-1(Fig. 8b) and SOCS-3 (Fig. 8c) in TBI mice. In addition,
the mNNS score (Fig. 8d), foot-fault test (Fig. 8e), androtarod test (Fig. 8f) showed worsened neurological outcomes in mice from the PS + Mer siRNA group whencompared with the PS + control siRNA treatment group.
Taken together, these findings suggested that Mer promoted STAT1-mediated upregulation of SOCS expression, alleviated secondary brain injury, and improved
functional outcomes following TBI.

Discussion
Neuroinflammation is conceived as an importantmanipulable aspect of secondary brain injury following
TBI [13]. Here, we demonstrate that Mer upregulation
provides neuroprotective effects in a CCI mouse modelof TBI via regulating microglial/macrophage M1/M2
polarization
andneuroinflammation
(Fig.
9).
Mechanistically, Mer mediates activation of the STAT1/
SOCS signaling pathway. Inhibition of Mer markedly
decreases microglial/macrophage M2-like polarization
while it increases M1-like polarization, thus aggravating

Wu et al. Journal of Neuroinflammation

A(2021) 18:2

Page 12 of 20

B
p-Mer

140kDa

GAPDH

36kDa

SOCS-1

STAT1

91kDa

GAPDH

TBI+M

TBI+V

***

TBI+C

ns
1.0

0.5

e
V
I+
TB

c
hi

le
on
C
I+
TB

t

l
ro

N
siR

A

I
TB

er
+M

N
s iR

***

1.0

0.5

I
TB

le

I
TB

ol
tr
on
+C

N
s iR

A

I
TB

er
+M

***

TB

A

I
TB

o
+C

nt

l
ro

N
siR

I
TB

er
+M

N
siR

t

A
N
iR
TB

er
M
I+

A
N
s iR

1.5

ns

SOCS-3 mRNA Ratio

SOCS-3 Relative density

SOCS-1 mRNA Ratio

I
TB

e
+V

e

on
C
I+
TB

ls
ro

***

***

1.0

0.5

0.0

cl
hi

e

TBI+M

1.5

0.0

l
ic
eh
V
+
I

A
N
s iR

36kDa
TBI+C

TBI+V

0.5

0.5

30kDa

GAPDH

1.0

1.0

F

SOCS-3

ns

ns

0.0

ic
eh
+V

E

1.5

TBI+M

***

1.5

ns

A

D

36kDa
TBI+C

TBI+V

0.0

0.0

26kDa

TBI+M

1.5

p-STAT1 Relative density

1.5

p-Mer Relative density

91kDa

SOCS-1 Relative density

TBI+C

TBI+V

C

p-STAT1

ns
1.0

0.5

0.0

A
I
TB

i
eh
+V

cl

e

I
TB

tr
on
+C

ol

N
siR

A

I
TB

er
+M

A
N
siR
I
TB

eh
+V

ic

le

I
TB

o
+C

ro
nt

ls

A
N
iR
I
TB

er
+M

N
siR

A

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. GAPD
H: 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

secondary brain damage and functional deficits after
TBI.
TBI is an evolving neurological event, and it heavily
contributes to both acute injuries and chronic
neurodegenerative cascades post-insult [49, 50]. Thisdisease is challenging to be treated because of its heterogeneous nature and complex pathogenic cascades [51,52]. Of note, microglial/macrophage activation and neuroinflammation are hallmark features of TBI pathophysiology [8]. As the resident innate immune cells in the
CNS, microglia usually respond within minutes toward
the sites of damage, where they can even sustain formany years following TBI [13]. Initially, these responses
allow them to scavenge debris, promote tissue remodeling and repair, and protect the brain from secondary

injury after TBI [53]. However, abnormal activation of
microglia can interfere with endogenous repair mechanisms and drives inflammatory damage after TBI [53]. In
addition, peripherally derived macrophages actively participate in acute neuroinflammatory responses after TBI[12]. Indeed, microglia/macrophages have multidimensional activation states in CNS diseases depending
on different pathophysiological conditions [54]. They
can become polarized ranging from the classic M1-likephenotype to an alternative M2-like phenotype after
CNS injuries including TBI [8, 55]. The M1-like response is presumed to be pro-inflammatory [15],whereas the M2-like phenotype owns anti-inflammatory
effects [56]. Multiple molecular pathways, such as STAT,
nuclear factor-κB (NF-κB), and interferon regulatory(2021) 18:2

B

TBI+V

6
SOCS-1 Relative density

***
2

0

***
***

***

8
6
4
2
0

9
6
***
3

***

20

40

***

20

0

15

***

0

20

10

+P
S
TB
I
***

80

40

0

PS
TB
I+

+V
TB
I

m
Sh
a

TB
I+
PS

+V
TB
I

m

***
***

0

Sh
a

m
120
Latency to fall (s)

5

L

***

***
Foot-fault (%)

mNSS score

***

5

Sh
a

TB
I

TB
I
***

30
***

10

***

10

+P
S

+V

m

K

J

15

0

Sh
a

+P
S
TB
I

TB
I

Sh
a

+V

m

0

***

***
IL-10 mRNA Ratio

***

Sh
am

***

60
Arg-1 mRNA Ratio

CD206 mRNA Ratio

I

***

12

1

TB
I+
PS

TB
I+

V

m

H

G

2

0

Sh
a

PS
TB
I+

TB
I

Sh
a

+V

m

0

***

3

+P
S

3

***

4
***
iNOS mRNA Ratio

***

6

m

TB
I+

F
10
CD32 mRNA Ratio

CD16 mRNA Ratio

***

Sh
a

PS

V
TB
I+

E
***

9

***
2

TB
I

D

4

0

Sh
am

PS
TB
I+

TB
I

+V

Sh
am

0

***

+P
S

2

4

***

TB
I

***

***

TBI+PS

V

4

6

***

TB
I+

***

TBI+V

Sham

+V

***

36kDa

GAPDH

TBI+PS

TB
I+
PS

6
p-STAT1 Relative density

36kDa
Sham

+V

GAPDH

30kDa

SOCS-3TB
I

91kDa
TBI+PS

C

TB
I

TBI+V

26kDa

+V

Sham

SOCS-1

TB
I

STAT1

91kDa

SOCS-3 Relative density

p-STAT1m

A

Page 13 of 20

Sha

Wu et al. Journal of Neuroinflammation

Fig. 7 Mer activation alleviated functional deficits following TBI. a–c Representative immunoblots and quantification showing protein expressionof 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 analysisof 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–lModified 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

(2021) 18:2

C

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3

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Page 14 of 20

TB
I

Wu et al. Journal of Neuroinflammation

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

factor (IRF), are involved in the regulation of M1/M2
phenotypic transitions [57–59]. Preclinical evidence indicated that mixed phenotypes are present in the pathological processes of TBI, which offer opportunities fortherapeutic interventions [60]. In the current study, we
also found that TBI induces transient upregulation in
the M2-like phenotype but causes sustained upregulation in the M1-like phenotype post-insult, which is consistent with other findings [16, 17]. Modulating
microglial/macrophage M1/M2 polarization via regulating those intrinsic molecular switches will facilitate therepair activity of microglia/macrophages and promote
neurovascular network restoration after TBI. Notably,
microglia/macrophages are endowed with spectacular
plasticity, allowing them to acquire multiple phenotypes
and thereby fulfill numerous activities in health and disease [61]. The existing M1/M2 paradigm, based on expression analysis of a small subset of genes (labeled M1
or M2), is inadequate for accurate description of the true
diversity of microglia/macrophages during complex disease environments such as TBI [13]. Indeed, existing evidence suggests that microglia/macrophages do notstrictly polarize to an “M1-only” or “M2-only” phenotype
in response to the multifaceted inflammatory milieu following TBI [20]. In contrast, these cells are dually labeled with “M1/M2” markers and display a mixed
phenotype in both the microenvironment and within thesame cell after injury [12, 21]. Thus, a binary M1/M2
polarization paradigm is not adequate to define the inflammatory profiles following TBI [21]. And it is not
feasible to classify the cells (e.g., M1, M2a, M2b, M2c)
depending on few selectively chosen inflammatory
markers [21, 56], while exploring the role and underlying
regulatory mechanisms of these markers in the context
of TBI-induced neuroinflammation would be more favorable [20, 62]. Also, multiple methodological approaches that can reveal transcriptomic and proteomic
profiling of microglia/macrophages are indeed needed infuture studies, which will help tremendously in our understanding of microglial/macrophage diversity and development of better-targeted therapies for a variety ofneurological disease conditions including TBI [13, 61].
Mer, an important member of the TAM family, ispredominantly
expressedonmyeloid-derived

Wu et al. Journal of Neuroinflammation(2021) 18:2

Page 15 of 20

Fig. 9 The proposed mechanism of Mer in regulating microglial/macrophage M1/M2 polarization and neuroinflammation following TBI. In thesetting 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-likepolarization 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

hematopoietic cells and functions as a pleiotropic inhibitor of the innate immune response [63]. RNA sequencing data demonstrated that Mer mRNA is highly
enriched in microglia in the CNS [64]. In contrast, themRNA expression levels of the other two family members, namely Axl and Tyro3, are very low in microglia[64]. Moreover, PS and Gas6, two best-characterized ligands, can bind the Ig1 and Ig2 domains of Mer via their
C-terminal regions [65]. Upon ligand stimulation, Mer
can be activated and initiate classic ligand-inducible
dimerization, causing receptor autophosphorylation, recruitment of signaling proteins with SH2 or PTB domains, and activation of downstream pathways. As an
example, activation of Mer can induce SOCS expression
and inhibit inflammation through regulating type I interferon receptor (IFNAR)-associated STAT1 pathway [22].
Genetic targeting of Mer can lead to persistent

inflammation and tissue damage in a mouse model ofacetaminophen-induced acute liver failure [66]. Also,
Mer can mediate recognition and subsequent efferocytosis of apoptotic cells to prevent immune responses [67].
In an in vivo murine model of allergic airway inflammation, Mer-mediated apoptotic eosinophil clearance byphagocytes contributes to resolution of allergic airway
inflammation [68]. In a murine model of experimental
myocardial infarction, deficiency of Mer in a subset of
myocardial monocytes suppresses the removal of dying
cardiac cells, delays inflammation resolution, and reduces systolic performance after myocardial infarction[69]. Proteolytic cleavage of macrophage Mer reduces
efferocytosis and inhibits plaque resolution in atherosclerosis [70]. In contrast, in fat-fed low-density lipoprotein receptor-deficient mice whose myeloid cells
expressing a cleavage-resistant variant of Mer, higherWu et al. Journal of Neuroinflammation(2021) 18:2

expression of Mer in macrophage contributes to improved efferocytosis, smaller necrotic cores, thicker fibrous caps, and increased ratios of pro-resolving versus
pro-inflammatory lipid mediators in the atherosclerotic
lesions [70]. These findings indicate Mer plays a prominent role in the resolution of inflammation through multiple approaches. Furthermore, previous studies havedemonstrated that TAM receptors play complex roles inmodulating neuronal migration and neurogenesis, synaptic plasticity, microglial activation and efferocytosis,
and peripheral nerve repair [71, 72], resulting in potential
interests in neuroinflammatory and neurodegenerative
diseases such as Alzheimer’s disease [73], Parkinson’s disease [29], and multiple sclerosis [74]. Our study added
new knowledge about the role of this pathway in TBI andshows that Mer is upregulated acutely in the injured cortex after injury, which exerts an effect on microglial/
macrophage polarization towards an M2-like phenotype
after TBI. And proper stimulation of Mer signaling by its
recombinant ligands such as PS can regulate microglial/
macrophage activities and neuroinflammatory responses
after TBI, which contributes to alleviation of secondary
brain damage and sensorimotor impairments after injury.
On the other hand, due to the complex and multiscale nature of secondary injury cascades following TBI, therapies
designed to target multiple mechanisms of this disease will
likely be needed [75]. Thus, molecular targets like Mer,
which can regulate multiple cellular processes, may be targeted for therapy in TBI and deserve further investigation.
Noteworthily, some limitations in this study should
not be ignored. First, the available molecular markers
are not specific enough to sufficiently distinguishmicroglia from other closely related myeloid subsets,
particularly monocyte-derived macrophages [64]. It istrue that portions of peripheral macrophages can infiltrate into the injured brain after TBI [76, 77]. Thus, theimpact of Mer-mediated regulation of inflammatory responses in microglia and infiltrating macrophages following TBI was not distinctively evaluated during theresult interpretation, and it is possible that infiltrating
macrophages could be driving some of the effects. In
fact, the crucial roles of peripheral infiltrated myeloid
cells in TBI neuropathology have been investigated [12,78–80]. As an example, Morganti et al. reported that
TBI induces a robust response involving the recruitment
and accumulation of peripheral CCR2+ macrophages
into the injured brain parenchyma, which significantly
contributes to neuroinflammatory sequelae and cognitive
dysfunction after injury [12]. By contrast, targeting thesubset of CCR2+ macrophages with a selective antagonist CCX872 heavily reduces the accumulation of thesecells, decreases their inflammatory and neurotoxic profiles, and ameliorates hippocampal-related cognitive dysfunction after TBI [12]. Interestingly, it demonstrated

Page 16 of 20

that age can exaggerate the recruitment of peripheral
CCR2+ macrophages to the injured parenchyma after
TBI, which exerts a non-redundant and contributingrole to TBI-induced neuroinflammation in the aged
brain [78, 79]. Moreover, it reported that peripherally
derived myeloid cells (CD45hiCD11b+) play an importantrole in initiating acute neuroinflammatory responses in
the injured brain after TBI, which are sexually dimorphic
and exaggerated in males compared to female mice [81].
Noteworthily, the relative contributions of resident
microglia and infiltrating peripheral myeloid cells to TBI
pathology were not adequately discriminated in a number of experimental studies, which should be largely examined in future research [81]. Also, we used whole
cortical samples but not isolated microglia/macrophages
from sham and CCI mice for mRNA analysis of dynamicchanges, and those tested factors can partially be produced by other cells such as neurons and astrocytes
within the contused cortex. Thus, in order to demonstrate the selective upregulation of M1/M2-like markers
in microglia/macrophages, isolation of these cells fromthe sham-injured and TBI brain for flow cytometry andimage analyses will be favorable in future studies.
Additionally, several studies reported that sex
difference underlies variations of microglial/macrophageactivation and sexually dimorphic neuroinflammatory
responses following TBI [82, 83], while other studies
indicated that sex manipulation does not affectinflammatory responses and brain injury after TBI [84].
Of note, a vast majority of preclinical studies have been
conducted just using male animals to look at the
inflammatory responses to TBI, while increasing
evidence indicates that sex differences may haveimportant implications for TBI outcome and potential
therapeutic development [85]. Contrary to our
expectations, we failed to detect any differences inbehavioral tests between male and female mice after TBI
in the preliminary study, based on the mNSS score, footfault test, and rotarod test. The small animal numbers
used in the current study as well as potentially inadequate sensitivity of these behavioral tests may underlie
the negative results, which still needs in-depth investigation in further research. Besides, we did not find any difference in the levels of Mer protein and mRNA
expression in the perilesional cortex between male andfemale mice on day 3 after TBI. Also, Hilliard et al. examined the effect of male-female differences on the expression of Mer in the human blood samples and foundthat no significant difference in Mer expression wasreached between female and male subjects [86]. To date,
information regarding sexual dimorphism in Mer expression is still scarce and has yet to be explored. But itshould not ignore the sex difference between males andfemales, which would be an important biological variableWu et al. Journal of Neuroinflammation(2021) 18:2

in CNS injury and disease [83, 87]. And sexual dimorphism in sex hormone levels (e.g., estrogen, progesterone,
and testosterone), microglial/macrophage properties,
and neuroinflammatory responses may contribute to thedifferences between the two sexes after TBI [44, 88].
Thus, more studies are required to decipher how sex differences affect microglial/macrophage activities, neuroinflammation, and neurofunctional outcomes after TBI.
And more data are certainly warranted before any definite conclusion about the relationship among sex, sex
hormones, and microglia/macrophages after TBI can be
drawn [44].

Conclusions
In conclusion, our study demonstrated that Mer isupregulated and exerts beneficial effects in the acutestage of TBI via modulating microglial/macrophage M1/M2 polarization. Inhibition of Mer markedly decreases
microglial/macrophage M2-like polarization while increases M1-like polarization, which contributes to theaggravation of secondary brain damage and exaggerationof sensorimotor deficits after TBI.
Supplementary Information
The online version contains supplementary material available at https://doi.
org/10.1186/s12974-020-02041-7.
Additional file 1: Supplementary Figure 1. (A-B) The controlled
cortical impact (CCI) model of TBI and experimental parameters in mice.
(C) A schematic map showing the location of TBI in mice. The center ofimpact was located at 2 mm medial-lateral (ML) and -2 mm anteriorposterior (AP) to bregma. (D-E) The intracerebroventricular (i.c.v) injectionand experimental parameters in mice. (F) A schematic map showing coordinates of i.c.v. injection in mice. The stereotaxic coordinates are 1 mm
ML and -0.25 mm AP to bregma, and 2.5 mm dorsoventral (DV) below
the skull. LV: lateral ventricle.
Additional file 2: Supplementary Figure 2. Experimental design andanimal groups. (A) Western blot (WB) and Real-Time Polymerase Chain
Reaction (RT-PCR) were performed to evaluate the expression profiles of
Mer and M1/M2 polarization markers at different time points after TBI (including 3 h, 12 h, 1 d, 3 d, and 7 d), as well as in the sham group; Besides, immunofluorescence was performed on day 3 after injury. (B) An
in vivo knockdown of Mer siRNA was adopted to evaluate the role of Mer
in regulating microglial/macrophage M1/M2 polarization after TBI. Mice
were randomly distributed into sham, TBI + Vehicle, TBI + Control siRNA,and TBI + Mer siRNA groups. Intracerebroventricular injection (i.c.v.) ofsiRNA was performed 1 d before and 10 min after TBI. The neurobehavioral functions were assessed before as well as 1, 3, 7 d after TBI. The
peri-injured cerebral cortex from each group and the equivalent area inthe sham-operated mice was collected for RT-PCR, WB, and immunohistochemistry analysis at 3 d after TBI. Also, contusion volume, brain edema,
neuronal damage and degeneration were measured at 3 d after injury.
(C) To evaluate the effect of PS on regulating STAT1/SOCSs pathway after
TBI, mice were randomly distributed into Sham, TBI + Vehicle, and TBI +recombinant protein S (PS) groups. PS (0.2 mg/kg) was administered via
the tail vein at 1 h, 1 d, and 2 d after the CCI. WB, RT-PCR, MACS, neuronal damage and degeneration, brain edema, and neurobehavioral assessments were conducted on day 3 post-injury. (D) Mice were randomly
distributed into TBI + Vehicle, TBI + PS + Control siRNA, and TBI + PS +Mer siRNA groups. Mer siRNA (i.c.v.) was administrated 1 d before and 10
min after TBI, and PS was administered via the tail vein at 1 h, 1 d, and 2

Page 17 of 20

d after the injury. WB and neurobehavioral assessments were conducted
on day 3 post-injury.
Additional file 3: Supplementary Figure 3. (A) Low-magnification images indicate the region of interest for immunofluorescent staining of
CD16/32 or CD206 (red), Iba-1 (Green) and DAPI (blue) in the ipsilateralcortex from the sham, TBI + Vehicle, TBI + Control siRNA and TBI+Mer
siRNA groups on day 3 post-injury, respectively. (B) Low-magnification
images indicate the region of interest for Nissl staining in the ipsilateralcortex from the sham, TBI + Vehicle, TBI + Control siRNA and TBI+Mer
siRNA groups on day 3 post-injury, respectively. (C) Low-magnification
images indicate the region of interest for Fluoro-Jade B (FJB) staining in
the ipsilateral cortex from the sham, TBI + Vehicle, TBI + Control siRNAand TBI+Mer siRNA groups on day 3 post-injury, respectively. In A-C, thered dotted area indicates contusion region. * indicates region of interest.
Scale bar = 1 mm.
Additional file 4: Supplementary Figure 4. (A) Modified neurologicalseverity scores (mNSS), (B) foot-fault test, and (C) rotarod test were performed before and 1, 3, and 7 d after TBI. n = 8 mice per group. (D) Representative immunoblots and quantification showing the expression levelof Mer protein in the injured cortex at 3 d after TBI or the equivalent areaof the sham-operated brains. Data are expressed as fold change compared to sham-operated controls. n = 6 mice per group. M: male; F: female. (E) Quantitative RT-PCR was used to assess the mRNA expression
level of Mer in the injured cortex at 3 d after TBI or the equivalent area ofthe sham-operated brains. Data are expressed as fold change compared
to sham-operated controls. n = 6 mice per group. In A-E, data are presented as mean ± SD; ***, p < 0.001; ns, non-significant, p > 0.05. twoway ANOVA followed by Bonferroni’s post-hoc tests.
Additional file 5: Supplementary Figure 5. (A) Fluorescence intensity
quantification of Mer expression in CD16/32- and CD206- positive cells in
the impacted ipsilateral cortical area at 3 d after TBI. n = 6 per group. (BG) Quantitative RT-PCR analysis showing mRNA expression of CD16 (B),CD32 (C), iNOS (D), CD206 (E), Arg-1 (F), IL-10 (G) in the injured cortex
at 3 d post-TBI. GAPDH: loading control. Data are expressed as foldchange compared to the sham group; n = 6 mice per group. In A, data
are presented as Mean ± SD; ***, p < 0.001 by Student’s t-test. In B-G,
data are presented as Mean ± SD; ***, p < 0.001. one-way ANOVA
followed by Bonferroni’s post-hoc tests.
Additional file 6: Supplementary Figure 6. (A) Cerebral edema wasmeasured by brain water content. Quantification analysis showing TBI
increased brain edema in the ipsilateral hemisphere on day 3 after TBI,
which is significantly alleviated after PS treatment. n = 8 mice per group.
(B) Representative images of Nissl staining in the ipsilateral cortex fromthe sham, TBI + Vehicle (TBI + V), TBI + protein S (TBI + PS) groups,
respectively. (C) Quantification analysis showing TBI caused a significant
decrease in the number of Nissl-positive cells in the ipsilateral cortex at 3
d post-TBI, and PS application significantly increased the number of Nisslpositive cells in the injured cortex after TBI. n = 6 mice per group. Scalebar = 20 μm. (D) Representative images of Fluoro-Jade B (FJB) staining in
the ipsilateral cortex from the sham, TBI + V, TBI + PS groups, respectively.
(E) Quantification analysis showing TBI caused a significant increase in
the number of FJB-positive cells in the ipsilateral cortex at 3 d post-TBI,
and PS application significantly decreased the number of FJB-positive
cells in the injured cortex after TBI. n = 6 mice per group. Scale bar = 15
μm. In A, C, E, data are presented as Mean ± SD; **, p < 0.01; ***, p <0.001. one-way ANOVA followed by Bonferroni’s post-hoc tests.

Abbreviations
ANOVA: Analysis of variance; Arg-1: Arginase 1; CCI: Controlled cortical
impact; CNS: Central nervous system diseases; DAPI: 4’,6-Diamidino-2phenylindole; FJB: Fluoro-Jade B; Gas6: Growth arrest-specific factor 6; IFNA
R: Type I interferon receptor; IL-1β: Interleukin-1β; IL-10: Interleukin-10;
iNOS: Inducible nitric oxide synthase; IRF: Interferon regulatory factor;
MACS: Magnetic-activated cell sorting; Mer: Myeloid-epithelial-reproductive
tyrosine kinase; mNSS: Modified neurological severity scores; NF-κB: Nuclear
factor-κB; PVDF: Polyvinylidene fluoride; PS: Protein S; RT-PCR: Reverse
transcription-polymerase chain reaction; SOCS: Suppressor of cytokine
signaling; STAT1: Signal transducer and activator of transcription 1;

Wu et al. Journal of Neuroinflammation(2021) 18:2

Page 18 of 20

TAM: Tyro-Axl-Mer; TBI: Traumatic brain injury; TGF-β: Transforming growth
factor-β; TNF-α: Tumor necrosis factor-α7.

Acknowledgements
Not applicable.

8.

Authors’ contributions
HW and JZ conducted the research and wrote the manuscript; SX, YF, YW,
JZ, JL, SM, and XW helped to complete the experiment; AS, LS, XG, and YW
performed the statistical analysis and edited the manuscript; ZZ and JZ
guided the entire study and provided the supervision and final check. All theauthors read the final version of this paper and approved it.

9.

10.

11.
Funding
This work was supported by the Young Scientists Fund of the National
Natural Science Foundation of China (No. 81701214), the National Natural
Science Foundation of China (No. 81870916), the National Key Research andDevelopment Program of China (No. 2017YFC1308500), the Key Program of
Science and Technology Development of Zhejiang (No. 2017C03021), andthe ZJU Scholarship for Outstanding Doctoral Candidates (No. 2019070). The
work of Z.Z. is supported by the BrightFocus Foundation grant A2019218S.

12.

13.

14.
Availability of data and materials
The authors confirm that all data underlying the findings are fully available
without restriction. All relevant data are within the paper and its supporting
information files.
Ethics approval and consent to participate
Human subjects or samples were not used in this study. All animal
experiments were approved by the Institutional Animal Care and Use
Committee of Zhejiang University.

15.
16.

17.

Consent for publication
Not applicable.
18.
Competing interests
The authors declare that they have no conflict of interest.
Author details
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. 3Center for
Neurodegeneration and Regeneration, Zilkha Neurogenetic Institute andDepartment of Physiology and Biophysics, Keck School of Medicine,
University of Southern California, 1501 San Pablo Street, Los Angeles, CA
90089, USA. 4Brain Research Institute, Zhejiang University, Hangzhou, China.
5Collaborative Innovation Center for Brain Science, Zhejiang University,
Hangzhou, China.

19.

1

20.
21.
22.
23.

Received: 2 October 2020 Accepted: 19 November 2020
24.
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