M2 Microglial Extracellular Vesicles Attenuated Blood-brain Barrier Disruption via MiR-23a-5p in Cerebral Ischemic Mice

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.

Figure 1.

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.

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.

Figure 3.

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.

Figure 4.

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.

Figure 5.

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.