Abstract
Protecting the integrity of the blood-brain barrier (BBB) is crucial
for maintaining brain homeostasis after ischemic stroke. Previous
studies showed that M2 microglial extracellular vesicles (EVs) played a
neuroprotective role in cerebral ischemia. However, the role of M2
microglial EVs in maintaining BBB integrity is unclear. Therefore, we
explored the mechanisms of M2 microglial EVs in regulating BBB
integrity. To identify microglial EVs, we used nanoparticle tracking
analysis, transmission electron microscopy, and western blot analysis.
Adult male ICR mice were subjected to 90-min middle cerebral artery
occlusion (MCAO), followed by the injection of PKH26-labeled M2
microglial EVs via the tail vein. After MCAO, we assessed brain infarct
and edema volume, as well as modified neurological severity score. BBB
integrity was measured by assessing IgG leakage. The effects of M2
microglial EVs on astrocytes and endothelial cells were also examined.
To investigate the molecular mechanisms, we performed RNA sequencing,
miR-23a-5p knockdown, and luciferase reporter assays. Our results
showed that PKH26-labeled microglial EVs were mainly taken up by
neurons and glial cells. M2 microglial EVs treatment decreased brain
infarct and edema volume, modified neurological severity score, and IgG
leakage, while increasing the ZO-1, occludin, and claudin-5 expression
after MCAO. Knockdown of miR-23a-5p reversed these effects. RNA
sequencing revealed that the TNF, MMP3 and NFκB signaling pathway
involved in regulating BBB integrity. Luciferase reporter assay showed
that miR-23a-5p could bind to the 3’ UTR of TNF. M2 microglial
EVs-derived miR-23a-5p decreased TNF, MMP3 and NFκB p65 expression in
astrocytes after oxygen-glucose deprivation, thereby increasing ZO-1
and Claudin-5 expression in bEnd.3 cells. In conclusion, our findings
demonstrated that M2 microglial EVs attenuated BBB disruption after
cerebral ischemia by delivering miR-23a-5p, which targeted TNF and
regulated MMP3 and NFκB p65 expression.
Keywords: blood-brain barrier, extracellular vesicles, microglia,
miR-23a-5p, stroke
INTRODUCTION
Ischemic stroke is a disease with high morbidity, high mortality, and
high disability, which has been receiving more and more attention
[[51]1]. Thrombolysis and arterial thrombectomy therapies could save
many acute ischemic stroke patients; however, ischemia-induced
secondary brain injury could cause serious brain edema and neurological
dysfunction in survivors [[52]2]. Therefore, we focused on exploring a
new therapeutic approach for ischemic stroke.
The blood-brain barrier (BBB) is a dynamic structure that consists of
endothelial cells, pericytes, astrocyte end-feet, and the basement
membrane. It regulates the exchange of substances between the blood and
the brain [[53]3, [54]4]. Maintaining BBB integrity is essential for
brain homeostasis [[55]5]. However, in the acute phase of ischemic
stroke, the BBB is severely disrupted, leading to damage tight
junctions and BBB leakage [[56]6]. This damage allows blood components
to enter the brain parenchyma, resulting in irreversible brain injury
[[57]7]. Therefore, protecting BBB integrity is a promising strategy
for ischemic stroke therapy.
After ischemic stroke, microglia in the brain were activated and
polarized into M1 or M2 phenotypes [[58]8]. M1 microglia increased the
expression of pro-inflammatory cytokines such as IL-β and TNFα, while
M2 microglia were involved in tissue repair, phagocytosis of debris,
and activation of neurotrophic pathways [[59]3]. Extracellular vesicles
(EVs) are small vesicles that contain various substances, including
proteins, lipids, and nucleic acids [[60]9]. EVs derived from
IL4-stimulated microglia, also known as M2 microglia, have been shown
neuroprotective role following middle cerebral artery occlusion (MCAO)
mice [[61]10-[62]12]. This indicated that the M2 microglial EVs could
be a promising therapeutic approach for ischemic stroke. To
comprehensively understand the role of M2 microglial EVs in ischemic
stroke, we investigated whether M2 microglial EVs protect BBB integrity
in MCAO mice and explored the underlying mechanisms.
In this study, we detected the BBB integrity in M2 microglial
EVs-treated mice 3 days after MCAO. We found that M2 microglial EVs
treatment increased the expression of tight junction proteins in brain
endothelial cells. When we injected PKH26-labeled microglial EVs into
the tail vein, they were primarily taken up by Tuj-1^+ neurons, Iba-1^+
microglia, and GFAP^+ astrocytes. However, only a small number of
PKH26-labeled microglial EVs were taken up by CD31^+ endothelial cells,
demonstrating that the high expression of tight junction proteins by M2
microglial EVs could not occur directly through endothelial cells.
Further analysis using bulk RNA sequencing revealed that MMP3 was the
most downregulated gene in M2 microglial EVs-treated mice compared to
the PBS-treated mice. Since MMP3 is primarily expressed in astrocytes
[[63]13], we hypothesized that M2 microglial EVs could regulate the
expression of tight junction proteins in endothelial cells by
decreasing MMP3 expression in astrocytes. To test this hypothesis, we
conducted in vitro assays using primary astrocytes and bEnd.3 cells.
Previous study has shown that miR-23a-5p is highly enriched in M2
microglial EVs compared to M0 microglial EVs [[64]12], indicating that
it may play an essential role in the function of M2 microglial EVs.
However, the role of miR-23a-5p in regulating the expression of tight
junction proteins is still unclear. Therefore, we performed miR-23a-5p
knockdown experiments to validate its role in regulating BBB integrity.
Our study demonstrated that M2 microglial EVs could attenuate BBB
disruption in MCAO mice by delivering miR-23a-5p, which targeted TNF
and regulated MMP3 and NFκB p65 expression.
MATERIALS AND METHODS
Animal Experimental Design
The animal experimental procedures were approved by the Institutional
Animal Care and Use Committee (IACUC) of Shanghai Jiao Tong University,
Shanghai, China, and were conducted in accordance with the US National
Research Council’s Guide for the Care and Use of Laboratory Animals.
The animal studies were reported following the ARRIVE guidelines. Adult
male Institute of Cancer Research mice (2 months old; Jiesijie,
Shanghai, China) weighing 25 to 30 grams were used in this study. To
investigate the role of M2 microglial EVs in BBB protection after MCAO,
twenty-six mice were randomly divided into PBS and M2 groups. The mice
were injected with PBS (200 μl/d) or M2 microglial EVs (100 μg in 200
μl/d) for 2 d after MCAO. To clarify whether M2 microglial EVs
attenuated BBB disruption via miR-23a-5p, twenty-seven mice were
randomly divided into the NC, M2 EVs, and k/d groups. The mice received
MCAO surgery 3 d after stereotactic injection of negative control (NC)
antagomiR or miR-23a-5p antagomir. Then, the mice in the NC, M2 EVs,
and k/d groups received PBS (200 μl/d) or M2 microglial EVs (100 μg in
200 μl/d) for 2 d after MCAO. The mice were sacrificed 3 d after MCAO
for further detection.
M2 Microglia Culture
BV2 microglia cells were purchased from Shanghai Zhong Qiao Xin Zhou
Biotechnology Company (Zhong Qiao Xin Zhou, Shanghai, China). The cells
were grown in Dulbecco’s Modified Eagle’s Medium (DMEM, #11965126,
Gibco) supplemented with 10% EVs-depleted FBS (#10099141, Gibco), 100
units/ml penicillin, and 100 μg/ml streptomycin (#15-140-122, Gibco) at
37°C in a humidified incubator with 5% CO2 and 95% air. To eliminate
EVs, the FBS was centrifuged at 100,000 g for 18 h and then incubated
at 56°C for 30 min to inactivate. The BV2 microglia were stimulated
with 20 ng/ml IL-4 (#CK74, Novoprotein) for 48 h, and the cell culture
supernatants were collected for EVs isolation [[65]10].
M2 Microglial EVs Isolation, Analysis, and Labeling
Differential ultracentrifugation was performed to isolate and purify
EVs, as previously described [[66]14]. Briefly, the supernatants from
M2 microglia culture were first centrifuged at 300 g for 10 min,
followed by centrifugation at 2000 g for 10 min to remove cells and
dead cells. Subsequently, the remaining supernatants were centrifuged
at 10,000 g for 30 min to remove debris. The final supernatants were
ultracentrifuged at 100,000 g for 70 min to obtain the EVs. To
eliminate contaminating proteins, the EVs were washed once with PBS and
subjected to ultracentrifugation at 100,000 g for 70 min. Finally, the
EVs were resuspended in PBS and stored at -80°C. The protein
concentration of the EVs was determined using a BCA protein kit
(#ZJ102, Epizyme). The particle diameter and concentration of EVs were
assessed using Nanoparticle Tracking Analysis and Transmission Electron
Microscopy. To label the EVs, the PKH26 Red Fluorescent Cell Linker Kit
(#PKH26GL-1KT, Sigma-Aldrich) was utilized with minor modifications
[[67]15]. In brief, EVs and 2 μl of PKH26 dye were added to 500 μl of
Diluent C, and the mixture was incubated for 4 min at room temperature
in the dark. To eliminate excess dye, 200 μl of FBS was added, and the
labeled EVs were washed with PBS at 100,000 g for 70 min. The EVs
pellet was resuspended in PBS for further in vitro and in vivo assays.
A Mouse Model of MCAO
The MCAO surgery was performed according to previously reported methods
[[68]10]. Briefly, mice were anesthetized using 1.2% isoflurane with a
mixture of 60% nitrous oxide and 40% oxygen. The common carotid artery,
external carotid artery, and internal carotid artery were isolated, and
a silicone-coated 6-0 nylon suture was inserted into the internal
carotid artery. The blood flow was then occluded for 90 min, followed
by reperfusion. Cerebral blood flow was monitored using a laser Doppler
perfusion and temperature monitor (Moor Instruments, Devon, UK). The
criteria for confirming a successful MCAO model were a decrease in
cerebral blood flow to 10% of the baseline level.
Neurological Severity Assessment
The investigator blinded to the experiment performed the modified
neurological severity score at 3 d after MCAO as previously reported
[[69]16]. Briefly, the investigator assessed the motor, sensory,
reflex, and balance abilities of the mice and recorded the scores. A
score of 1-6 indicated mild injury; 7-12 indicated moderate injury;
13-18 indicated severe injury.
Brain Infarct Volume and Edema Measurements
Brain infarct and edema volume were measured following the previously
described protocol [[70]6]. A series of 20-μm-thick coronal brain
slices were stained using cresyl violet (# MA0129, Meilunbio). The
infarct volume was then measured using Image J software (National
Institutes of Health, Bethesda, MD). The formula used for calculating
the infarct volume was V =
[MATH:
∑1n[Sn+S
n*Sn<
/mi>+1+Sn+1<
/mrow>*h/3]
:MATH]
, where V represents the infarct volume, Sn indicates the infarct area,
and h represents the distance between two adjacent brain slices. To
calculate the edema ratio, the volume of the ipsilateral side was
divided by the volume of the contralateral side.
BBB Leakage Determination
To detect IgG leakage, brain slices were incubated in 0.3% hydrogen
peroxide for 30 minutes, followed by serum blocking. After incubating
the brain slices with biotinylated universal antibody (#PK-7200, Vector
Laboratories) for 30 min, the slices were then incubated with DAB
reagents (#SK-4105, Vector Laboratories) and hematoxylin (#C0105S,
Beyotime Biotechnology). The integrity of tight junctions was assessed
using double staining with ZO-1/CD31, Occludin/CD31, and
Claudin-5/CD31. Brain sections were fixed by pre-cooled methyl alcohol
for 10 min, followed by a 10-minute incubation with 0.3% Triton X-100.
The slices were then blocked with 1% BSA for 1 h at room temperature.
Subsequently, the brain slices were incubated overnight at 4°C with
primary antibodies: anti-ZO-1 (1:200, #61-7300, Invitrogen),
anti-Occludin (1:200, #33-1500, Invitrogen), anti-Claudin-5 (1:200,
#35-2500, Invitrogen), anti-CD31 (1:200, #AF3628, R&D Systems), goat
IgG isotype control (1:200, #02-6202, Invitrogen), rabbit IgG isotype
control (1:200, #ab172730, Abcam), and mouse IgG isotype control
(1:200, #MAB002, R&D Systems). After incubating the slices with
fluorescence-conjugated secondary antibodies (1:500, #A21432, #A21206,
#A21202, Invitrogen), they were mounted with coverslips for imaging
using a confocal microscope (Leica, Solms, Germany). The leakage of IgG
and the breakage of tight junction proteins were assessed using Image J
software following previously described methods [[71]6]. In each slice,
four fields in peri-infarct areas were imaged, and a total of four
slices were counted for each mouse brain. The quantification of gap
formation for ZO-1 and occludin was calculated using the following
formula: Gap formation (%) = (Gap length/Whole tight junction staining
length) ×100%.
RNA Sequencing (RNA-seq)
RNA-seq was performed as previously reported [[72]6]. Mouse brain
tissues in the peri-ischemic region were used for total RNA isolation
using TRIzol reagent (#15596026, Thermo Scientific). The qualified RNA,
with a RNA Integrity Number of ≥ 7, as evaluated by Agilent 2100
Bioanalyzer (Agilent Technologies, Santa Clara, CA), was used for
further RNA-seq analysis. The libraries were constructed using the
TruSeq Stranded mRNA LTSample Prep Kit (Illumina, San Diego, CA) for
Illumina sequencing. Differential expression analysis and KEGG pathway
enrichment analysis were performed between the PBS and M2 EVs groups.
Real-time PCR Assay
Mouse brain tissues in the peri-ischemic region and primary astrocytes
were collected to isolate total RNA using TRIzol reagent (Thermo
Scientific). The cDNA was synthesized using a cDNA synthesis kit
(#11123ES60, Yeasen) following the manufacturer’s protocol. The mRNA
expression levels were then quantified using qPCR SYBR Green master mix
(#11203ES08, Yeasen). The real-time PCR amplification parameters
consisted of an initial denaturation step at 95°C for 30 s followed by
40 cycles of denaturation at 95°C for 5 s and annealing/extension at
60°C for 30 s. The expression level of mRNA was calculated using the
2^-ΔΔCt method and normalized to the expression of the endogenous
control GAPDH [[73]6]. The primers utilized in this study are detailed
in [74]Table 1.
Table 1.
The primers used in the real-time PCR assay.
Gene name Forward primers Reverse primers
TNF ATGTCTCAGCCTCTTCTCATTC GCTTGTCACTCGAATTTTGAGA
TLR4 GCCATCATTATGAGTGCCAATT AGGGATAAGAACGCTGAGAATT
ICAM-1 CTGAAAGATGAGCTCGAGAGTG AAACGAATACACGGTGATGGTA
MYD88 CGGAACTTTTCGATGCCTTTAT CACACACAACTTAAGCCGATAG
RIPK1 TACTTGCAAAGAAGAGTCGACT ATAGGGTTCAGGTGTTCATCAG
BLNK GAAGTGTTGTGGAAATCGTCAA CACAGCATATTTCAGTCTCGTG
TNFSF11 GGAAGCGTACCTACAGACTATC AAAGTGGAATTCAGAATTGCCC
TRAF1 CAAACATTGTTGCTGTCCTCAA TCTTCCACAGGAAAGTACCATC
CARD14 GGTGAATGGCTCCTGCTACTTGTC TCTCCGTCCCCTCCCCTCTG
MMP2 ACTTTGAGAAGGATGGCAAGTA CTTCTTATCCCGGTCATAGTCC
MMP3 TGTCACTGGTACCAACCTATTC TCTCAGGTTCCAGAGAGTTAGA
MMP9 CAAAGACCTGAAAACCTCCAAC GACTGCTTCTCTCCCATCATC
MMP10 ACAAATGTGATCCTGCTTTGTC ATCAAATGAAATTCAGGCTCGG
MMP27 CCCCAAATCCATCCACACACTCG TCTGTCCATTGCTTGTGCCATCTC
GAPDH GGTTGTCTCCTGCGACTTCA TGGTCCAGGGTTTCTTACTCC
[75]Open in a new tab
Western Blot Analysis
Brain tissues in the peri-ischemic area and primary astrocyte were used
for western blot analysis. The protein concentration was measured using
a BCA protein kit (Epizyme). The proteins (30 μg/group) were separated
into a 5-10% gradient SDS-PAGE gel and then transferred onto PVDF
membranes. After blocking with 5% milk, the bands were incubated
overnight at 4°Cwith primary antibodies, including anti-CD63 (1:1000,
#sc-15363, Santa Cruz Biotechnology), anti-TSG101 (1:1000, #ab83,
Abcam), anti-TNF (1:500, #sc-52746, Santa Cruz Biotechnology),
anti-MMP3 (1:1000, # 17873-1-AP, ProteinTech Group), anti-NFκB p65
(1:1000, #8242T, Cell Signaling Technology), or anti-β-actin (1:1000,
#66009, ProteinTech Group). Then the membranes were incubated with
HRP-conjugated secondary antibody (1:5000, #HA1006 or #HA1001, HUABIO)
at room temperature for 1 h. Visualization analysis was performed using
an enhanced chemiluminescence substrate (#SQ201, Epizyme) and an
imaging system (Bio-Rad, Hercules, CA).
Primary Astrocyte Culture
Primary astrocytes were isolated from postnatal ICR mice (Jiesijie)
using previously described methods [[76]11]. The cortex was isolated
and trypsinized at 37°C for 12 min. The cell suspension was filtered
through 70-μm strainers (#CLS431751-50EA, Millipore). After
centrifugation at 1000 rpm for 5 min, the cells were seeded onto a
6-well plate and cultured in an incubator at 37 °C with 5% CO2. After
24 h, the DMEM (Gibco) supplemented with 10% FBS (Gibco), 100 U/mL
penicillin, and 100 mg/mL streptomycin (Gibco) was replaced, and the
medium was subsequently replaced every 3 d.
Oxygen-glucose Deprivation and Reoxygenation (OGD)
The OGD assay was conducted following the previously reported method
[[77]17]. The cell culture medium was substituted with glucose-free
DMEM (#11966025, Gibco). Then the oxygen-glucose deprivation
experiments were carried out in a chamber filled with a gas mixture
containing 95% N2 and 5% CO2. The primary astrocytes were kept in the
chamber for 5 h, while the bEnd.3 cells were exposed for 14 h.
Afterward, the cells were removed from the chamber and the medium were
replaced with DMEM supplemented with 10% FBS for a reoxygenation period
of 3 h.
Stereotactic Injection of MiR-23a-5p AntagomiR
The stereotactic injection of miR-23a-5p antagomiR was carried out
following the previously described method [[78]1]. ICR mice under
anesthesia were immobilized on a stereotactic apparatus (RWD Life
Science, Shenzhen, China). Next, miR-23a-5p antagomiR
(#miR30017019-4-5, RIBOBIO) or NC antagomiR (#miR3N0000001-4-5,
RIBOBIO) was injected into the left striatum region at a 2 mm lateral
to the sagittal suture and 3 mm beneath the dura, with a pumping rate
of 0.2 μl/min using a pump (WPI, Sarasota, FL). After 3 d, these mice
were utilized for MCAO surgery.
Dual-luciferase Reporter Assay
The dual-luciferase reporter assay was performed following a previously
described protocol [[79]12]. Briefly, luciferase vectors containing
wild-type or mutated binding sites of the TNF 3’-untranslated region
(3’-UTR) were constructed by OBiO Technology in Shanghai, China. 293T
cells were cultured in 96-well plates for 24 h and then transfected
with luciferase vectors and miR-23a-5p or NC mimics. After 48 h of
transfection, luciferase activities were measured using the
Dual-Luciferase Reporter Assay System (#E1910, Promega). The relative
luciferase activity was calculated as the ratio of firefly luciferase
activity to renilla luciferase activity.
Statistical Analysis
Statistical analyses were performed using GraphPad Prism 8. The
Shapiro-Wilks normality test was conducted to assess the normal
distribution of the data. For normally distributed data, we utilized an
unpaired two-tailed Student t-test or one-way ANOVA followed by
Newman-Keuls or Tukey’s post-hoc test. For non-normally distributed
data, we replaced the t-test with the Mann-Whitney test and the one-way
ANOVA with the Kruskal-Wallis test followed by Dunn’s multiple
comparisons test. The data were presented as mean±SD. P<0.05 was
considered statistically significant. The figures displayed
representative images that accurately represented the average data of
all samples. The P values were indicated as asterisks in the figures
and presented as ranges in the figure legends.
Schematics
The schematic cartoons in [80]Figures 1E and [81]4B were created using
[82]BioRender.com.
Figure 1.
[83]Figure 1.
[84]Open in a new tab
Identification of extracellular vesicles (EVs) and EV distribution in
mouse brains after cerebral ischemia. (A) Representative images of
microglia under the bright-field microscope and immunostaining of the
microglia marker Iba-1. Scale bar, 100 (left)/50(right) μm. (B)
Relative mRNA levels of Arg-1, CD206, IL-1β, TNF, and iNOS in control
and IL-4 stimulated microglia. **P<0.01, ****P<0.0001 (n=3, unpaired
two-tailed Student’s t-test). (C) Nanoparticle tracking analysis and
the ultrastructural image showed the diameter of M2 microglial EVs.
Scale bar, 100 nm. (D) Protein levels of TSG101, CD63, and β-actin in
cells and EVs. (E) M2 microglial EVs were injected into middle cerebral
artery occlusion (MCAO) mice via the tail vein. (F) Immunostaining of
Tuj-1, Iba-1, GFAP, and CD31 in ischemic mouse brains after injection
of PKH26-labeled M2 microglial EVs. Scale bar, 25 μm. Enlarged images
showed the square area in the left images. The number of PKH26^+ cells
per field and the percentage of PKH26^+ cells were measured (below).
**P<0.01, ***P<0.001, ****P<0.0001 (n=3, one-way ANOVA with Tukey’s
post-hoc test). ns indicates not significant; CON indicates control
group; IL4 indicates IL4 stimulated group.
Figure 2.
[85]Figure 2.
[86]Open in a new tab
M2 microglial EVs attenuated brain injury and blood-brain barrier (BBB)
disruption. (A) Cresyl violet staining of coronal brain sections in PBS
and M2 microglial EVs-treated mice at 3 d after MCAO (left). The dashed
line areas indicate infarction. Infarct volume and edema volume were
calculated from brain sections (right). *P<0.05, **P<0.01 (n=6,
unpaired two-tailed Student’s t-test for statistical analysis of
Infarct Volume, Mann-Whitney test for statistical analysis of Ipsi.
/Contra. Volume). (B) The modified neurological severity score. *P<0.05
(n=9, Mann-Whitney test). (C) Immunostaining of IgG, ZO-1, occludin,
claudin-5, and CD31 in the perifocal area at 3 d after MCAO. Arrows
indicate gaps caused by the breakage of tight junction proteins. Scale
bar (left to right), 25/10/10/100 μm. **P<0.01, ***P<0.001 (n=6,
unpaired two-tailed Student’s t-test). PBS indicates PBS-treated mice;
M2 EVs indicates M2 microglial EVs-treated mice; Contra indicates
contralateral; Ipsi indicates ipsilateral.
RESULTS
Identification of EVs and EV distribution in mouse brains after cerebral
ischemia
BV2 microglia were stained with anti-Iba-1 and DAPI ([87]Fig. 1A). The
results showed that all BV2 microglia were Iba-1^+. After 24 h of IL-4
stimulation, we observed an upregulation of the mRNA expression levels
of the M2 microglial markers Arg-1 and CD206, while the M1 microglial
marker TNF was downregulated ([88]Fig. 1B). Nanoparticle Tracking
Analysis and ultrastructure imaging indicated that the diameter of M2
microglial EVs was mainly between 30-150 nm ([89]Fig 1C). Western blot
analysis revealed that M2 microglial EVs, isolated through differential
ultracentrifugation, exhibited high expression of the EV markers TSG101
and CD63, but low expression of β-actin ([90]Fig 1D).
PKH26-labeled M2 microglial EVs were injected into MCAO mice via the
tail vein ([91]Fig. 1E). Neurons, microglia, astrocytes, and
endothelial cells were stained with anti-Tuj-1, anti-Iba-1, anti-GFAP,
and anti-CD31, respectively. The results showed that PKH26-labeled M2
microglial EVs localized in Tuj-1^+ neurons, Iba-1^+ microglia, GFAP^+
astrocytes, and CD31^+ endothelial cells ([92]Fig. 1F). The statistical
results indicated that M2 microglial EVs were mainly taken up by
Tuj-1^+ neurons, Iba-1^+ microglia, and GFAP^+ astrocytes ([93]Fig.
1F), indicating that M2 microglial EVs may regulate BBB integrity
through astrocytes, which are critical components of the BBB.
M2 microglial EVs attenuated brain injury and BBB disruption
Cresyl violet staining was performed on mouse coronal sections to
evaluate neuronal injury. The infarct volume and edema volume decreased
in the M2 microglial EVs-treated mice compared to the PBS-treated group
after MCAO ([94]Fig. 2A). Furthermore, modified neurological severity
score decreased in the M2 EVs group compared to the PBS group at 3 d
after MCAO ([95]Fig. 2B). Additionally, IgG leakage and breakage of
tight junction proteins (ZO-1, Occludin, and Claudin-5) decreased in
the M2 EVs group compared to the PBS group at 3 d after MCAO ([96]Fig.
2C). Therefore, treatment with M2 microglial EVs may be effective in
attenuating BBB disruption in the acute stage of MCAO.
RNA-seq revealed underlying mechanisms of M2 microglial EVs in regulating BBB
integrity
Our previous work performed microRNA sequencing to identify
differentially expressed microRNAs between M2 microglial EVs and M0
microglial EVs [[97]12]. The results demonstrated that miR-23a-5p was
the most upregulated miRNA in M2 microglial EVs compared to M0
microglial EVs, indicating its critical role in M2 microglial EVs
[[98]12]. However, it remains unclear whether M2 microglial EVs-derived
miR-23a-5p regulates BBB integrity. To investigate the underlying
mechanisms of M2 microglial EVs in BBB integrity regulation, we
collected perifocal mouse brain tissues from PBS and M2 EVs group mice
and performed RNA-seq analysis at 3d after MCAO. The heatmap displayed
the differentially expressed genes between the M2 EVs group and PBS
group at 3 d after MCAO ([99]Fig. 3A). We identified a total of 3154
differentially expressed genes (foldchange>2, P<0.05), including 862
upregulated genes and 2292 downregulated genes ([100]Table 1 in the
Data Supplement). Among the downregulated genes, we identified MMP3
(foldchange=106), MMP10 (foldchange=89), and MMP27 (foldchange=58) as
the top 3 downregulated genes (FPKM>2) marked in the volcano plot
([101]Fig. 3B). Furthermore, the downregulated differentially expressed
genes were enriched in inflammation-related signaling pathways, such as
the NFκB signaling pathway ([102]Fig. 3C). Real-time PCR results
confirmed the RNA-seq findings, showing downregulation of matrix
metalloproteinases (MMP3, MMP10, MMP27, MMP2, and MMP9) and molecules
associated with the NFκB signaling pathway (TLR4, TNF, ICAM-1, MYD88,
RIPK1, BLNK, TNFSF11, TRAF11, and CARD14) in the M2 EVs group compared
to the PBS group ([103]Fig.3D). Additionally, the miRWalk database
predicted that miR-23a-5p could bind to the 3’UTR of TNF ([104]Fig.
3E).
Figure 3.
Figure 3.
[105]Open in a new tab
RNA sequencing revealed underlying mechanisms of M2 microglial EVs in
regulating BBB integrity. The heatmap (A) and the volcano plot (B) show
differentially expressed genes in the perifocal region at 3 d after
MCAO. (C) KEGG pathway analysis of upregulated (red) genes and
downregulated (green) genes. (D) Real-time PCR assay to validate
several differentially expressed genes between the PBS (n=3) and M2 EVs
(n=5) groups. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (Mann-Whitney
test for statistical analysis of MMP9, MMP10, MYD88, and TNFSFSF11;
unpaired two-tailed Student’s t-test for statistical analysis of other
genes). (E) Predicted binding sites of miR-23a-5p in the 3’UTR of TNF.
NoDiff indicates No difference; up indicates upregulated genes; down
indicates downregulated genes; FC indicates fold change; 3’ UTR
indicates 3’ untranslated region.
M2 microglial EVs-derived miR-23a-5p downregulated TNF, MMP3 and NFκB p65 in
astrocytes
The immunostaining image demonstrated that primary astrocytes were
capable of internalizing PKH26-labeled M2 microglial EVs ([106]Figure
4A). After subjecting astrocytes to oxygen-glucose deprivation for 5 h,
the mRNA expression levels of TNF and MMP3 were assessed at 0, 1, 3, 5,
and 7 h after oxygen-glucose deprivation. We found that TNF was
initially upregulated at 0 h, but subsequently downregulated at 5 and 7
h, while MMP3 was upregulated at 3, 5, and 7 h ([107]Fig. 4A). The
experimental design was illustrated in a schematic diagram ([108]Fig.
4B). To investigate the potential regulatory effects of M2 microglial
EVs on TNF, MMP3, and NFκB p65 expression in astrocytes, primary
astrocytes were isolated. Astrocytes were pretreated with M2 microglial
EVs and miR-23a-5p antagomiR, followed by oxygen-glucose deprivation
for 5 h and reoxygenation for 3 h. The results demonstrated that the
mRNA and protein levels of TNF, MMP3, and NFκB p65 decreased in M2
microglial EVs-treated group compared to the NC group. However,
miR-23a-5p knockdown reversed this effect ([109]Fig. 4C). To
investigate whether M2 microglial EVs could regulate the expression of
endothelial tight junction proteins through astrocytes, the conditioned
medium from NC, M2 EVs, and k/d group astrocytes was collected and used
to treat bEnd.3 cells. Immunostaining results revealed that the
conditioned medium derived from M2 EVs group astrocytes increased the
expression of ZO-1 and Claudin-5 in bEnd.3 cells after 14 h of
oxygen-glucose deprivation. Conversely, the conditioned medium from k/d
group astrocytes reversed this protective effect ([110]Fig. 4D).
Figure 4.
[111]Figure 4.
[112]Open in a new tab
M2 microglial EVs-derived miR-23a-5p downregulated TNF, MMP3 and NFκB
p65 in astrocytes. (A) Relative mRNA expression of TNF and MMP3 in
primary astrocytes at 0, 1, 3, 5, and 7 h after OGD. The microscopic
image shows the uptake of PKH26-labeled M2 microglial EVs in primary
astrocytes. *P<0.05, **P<0.01 (n=3, unpaired two-tailed Student’s
t-test or Mann-Whitney test). (B) Schematic diagram of in vitro assay.
(C) Relative mRNA and protein expression of TNF, MMP3, and NFκB p65 in
astrocytes after treatment with M2 Microglial EVs and miR-23a-5p
antagomiR. *P<0.05, **P<0.01 (n=3, one-way ANOVA followed by the
Tukey’s post-hoc test for statistical analysis of TNF/β-actin and NFκB
p65/β-actin; one-way ANOVA followed by Newman-Keuls multiple
comparisons test for statistical analysis of TNF mRNA, MMP3 mRNA, and
MMP3/β-actin). (D) Immunostaining of ZO-1, Occludin, and Claudin-5 in
bEnd.3 cells after treatment with conditioned medium from astrocytes.
Scale bar, 50 μm. *P<0.05 (n=3, one-way ANOVA followed by the Tukey’s
post-hoc test for statistical analysis of ZO-1 and claudin-5;
Kruskal-Wallis test followed by Dunn’s multiple comparisons test for
statistical analysis of occludin). ns indicates not significant; NC
indicates negative control antagomiR-treated astrocytes; M2 EVs
indicates M2 microglial EVs-treated astrocytes; k/d indicates
miR-23a-5p antagomiR and M2 microglial EVs-treated astrocytes. CM NC
indicates bEnd.3 cells treated with NC group astrocytes-derived
conditioned medium; CM M2 EVs indicates bEnd.3 cells treated with M2
EVs group astrocytes-derived conditioned medium; CM k/d indicates
bEnd.3 cells treated with M2 EVs group astrocytes-derived conditioned
medium.
M2 microglial EVs-derived miR-23a-5p attenuated BBB disruption and
downregulated TNF, MMP3 and NFκB p65 in MCAO mice
The schematic diagram illustrates the in vivo experimental design
([113]Fig. 5A). Measurement of mouse coronal sections indicated that
infarct volume decreased in the M2 EVs group compared to the NC group
at 3 d after MCAO. However, miR-23a-5p knockdown reversed this effect
([114]Fig. 5B). Additionally, the modified neurological severity score
decreased in the M2 group compared to the NC group at 3 d after MCAO,
but miR-23a-5p knockdown reversed this effect ([115]Fig. 5C). In
addition, miR-23a-5p knockdown reversed the protective role of M2
microglial EVs in maintaining BBB integrity, showing increased IgG
leakage and disruption of tight junction proteins (ZO-1, Claudin-5,
[116]Fig. 5D). The mRNA and protein expression levels of TNF, MMP3, and
NFκB p65 were downregulated in the M2 EVs group compared to the NC
group, but miR-23a-5p knockdown reversed this effect ([117]Fig. 5E).
The dual-luciferase reporter gene assay showed that miR-23a-5p could
bind to the 3’-UTR of TNF ([118]Fig. 5F).
Figure 5.
[119]Figure 5.
[120]Open in a new tab
M2 microglial EVs-derived miR-23a-5p attenuated BBB disruption and
downregulated TNF, MMP3 and NFκB p65 in MCAO mice. (A) Schematic
diagram of the in vivo assay. (B) Cresyl violet staining of mouse brain
sections at 3 d after MCAO. *P<0.05, **P<0.01, ***P<0.001 (n=6, one-way
ANOVA followed by the Tukey’s post-hoc test for statistical analysis of
Infarct Volume; Kruskal-Wallis test followed by Dunn’s multiple
comparisons test for statistical analysis of Ipsi. /Contra. Volume).
(C) The modified neurological severity score. *P<0.05 (n=9, one-way
ANOVA followed by the Tukey’s post-hoc test). (D) Immunostaining of
IgG, ZO-1, Occludin, Claudin-5, and CD31 in the perifocal area at 3 d
after MCAO. Scale bar (left to right, top to bottom), 25/10/10/100 μm.
*P<0.05, **P<0.01, ***P<0.001 (n=6, one-way ANOVA followed by the
Tukey’s post-hoc test for statistical analysis of IgG, ZO-1 and
claudin-5; Kruskal-Wallis test followed by Dunn’s multiple comparisons
test for statistical analysis of occludin). (E) Relative mRNA and
protein expression of TNF, MMP3, and NFκB p65 in the perifocal region
at 3 d after MCAO. *P<0.05, **P<0.01 (n=3, one-way ANOVA followed by
Newman-Keuls multiple comparisons test for statistical analysis of TNF
mRNA, MMP3 mRNA; one-way ANOVA followed by the Tukey’s post-hoc test
for statistical analysis of TNF/β-actin, MMP3/β-actin and NFκB
p65/β-actin). (F) Dual-luciferase reporter assay showing miR-23a-5p
could bind 3’-UTR of TNF. *P<0.05, **P<0.01 (n=6, one-way ANOVA
followed by the Tukey’s post-hoc test). ns indicates not significant;
NC indicates negative control antagomiR-treated mice; M2 EVs indicates
M2 microglial EVs-treated mice; k/d indicates miR-23a-5p antagomiR and
M2 microglial EVs-treated mice. NC+WT indicates negative control
mimic+wild type 3’untranslated region; NC+MUT indicates negative
control mimic+mutant 3’untranslated region; miR+WT indicates miR-23a-5p
mimic+wild type 3’untranslated region; miR+MUT indicates miR-23a-5p
mimic+mutant 3’untranslated region.
DISCUSSION
Our study demonstrated that M2 microglial EVs could decrease the
infarct and edema volume, ameliorate BBB disruption, and improve
functional recovery 3 d after MCAO. The mechanistic study revealed that
M2 microglial EVs attenuated BBB disruption in the acute stage of
cerebral ischemia by delivering miR-23a-5p, which targeted TNF and
regulated MMP3 and NFκB p65 expression.
EVs play a crucial role in cell-to-cell communication, and several
studies have reported the essential role of EVs-derived microRNAs in
this process [[121]10, [122]18, [123]19]. In our previous study, we
found that miR-23a-5p was the most abundant microRNA in M2 microglial
EVs compared to the M0 microglial EVs. Furthermore, we confirmed that
M2 microglial EVs-derived miR-23a-5p promoted white matter repair and
functional recovery after cerebral ischemia in mice [[124]12]. However,
the role of miR-23a-5p in maintaining BBB integrity remains largely
unknown. Our study demonstrated that M2 microglial EVs-derived
miR-23a-5p improved BBB integrity by reducing IgG leakage and tight
junction breakage in cerebral ischemic mice 3 d after MCAO. However,
our study does not exclude the potential contribution of other miRNAs
or proteins present in M2 microglial EVs to BBB integrity, which needs
to be further investigated.
To identify the downstream targets of M2 microglial EVs, we conducted
bulk RNA-seq in PBS and M2 microglial EVs-treated mice 3 d after MCAO.
Bulk RNA-seq results showed several downregulated genes, with MMP3
being the most significantly downregulated gene among them. Real-time
PCR analysis further confirmed the downregulation of MMP3 in M2
microglial EVs-treated mice compared to PBS-treated mice. MMPs are a
group of proteolytic enzymes that are known to degrade extracellular
matrix and tight junction proteins [[125]20]. MMPs could exacerbate
brain injury during the acute stage and promote recovery during the
late stage [[126]21]. MMP3 is one of the main MMPs inducible in the
brain and activated MMP3 could directly activate MMP9 [[127]22,
[128]23]. Previous studies have shown that MMP3 contributes to the
tPA-induced hemorrhagic transformation and worsens functional outcomes
after hyperglycemic stroke, partially due to MMP3 degrading tight
junction proteins [[129]24]. Since MMP3 is mainly expressed in
astrocytes [[130]13], our in vitro results showed that M2 microglial
EVs decreased the mRNA and protein levels of MMP3 in astrocytes after
OGD. However, miR-23a-5p knockdown reversed this effect. MMP2, MMP3 and
MMP9 are critical MMPs involved in BBB opening after ischemic stroke
[[131]25, [132]26]. Thus, we believed that M2 microglial EVs regulated
BBB integrity through modulation of the multiple MMPs.
The miRWalk results indicated that miR-23a-5p did not have any
predicted 3’ UTR binding sites of MMP3, suggesting that miR-23a-5p may
indirectly regulate MMP3 expression and activity. To identify the
direct target of miR-23a-5p, we analyzed the downregulated genes in the
RNA-seq data and identified the enriched KEGG pathways. The results
showed that the NFκB signaling pathway was one of the most relevant
pathways in regulating BBB integrity. A previous study has reported
that activation of the NFκB pathway upregulated pro-inflammatory
factors, which can worsen brain injury after intracranial hemorrhage
[[133]27]. But downregulating NFκB has been shown to reduce brain edema
and neurological dysfunction after brain ischemia/reperfusion
[[134]28]. We found that TNF and TLR4 as potential targets of
miR-23a-5p in the NFκB signaling pathway. Furthermore, we found that
TLR4 is a validated target of miR-23a-5p through dual-luciferase
reporter assay [[135]29]. Our study demonstrated that miR-23a-5p could
bind to the 3’-UTR of TNF via dual-luciferase reporter assay. Moreover,
we detected that M2 microglial EVs decreased the mRNA and protein
levels of TNF in astrocytes and MCAO mice. But miR-23a-5p knockdown
reversed this effect. Previous studies reported that TNF induced MMP3
in astrocytes and human cerebral endothelial cells [[136]30, [137]31].
Hence, M2 microglia EVs-derived miR-23a-5p decreased MMP3 by inhibiting
TNF in MCAO mice.
Although we demonstrated that the M2 microglial EV was a promising
treatment for ischemic stroke, there were still some limitations in the
study. Firstly, the EV isolation method should be improved to obtain
purer and higher-yield EVs. The use of an iodixanol-based
high-resolution density step gradient has been reported for the
isolation of distinct EV species [[138]32]. Secondly, nonspecific
uptake of M2 microglial EVs allowed them to enter various brain cells
and regulate brain function. However, non-targeted therapy caused
unpredictable side effects. Thus, the M2 microglial EVs modification to
achieve targeted therapy is the future direction for ischemic stroke
therapy. Thirdly, a recent study recommended avoiding the use of M1 and
M2 labels to investigate microglial function [[139]33]. In our study,
to simplify the microglia nomenclature, we referred to IL4-stimulated
microglia as M2 microglia.
Acknowledgments