IL-33/ST2 signaling in monocyte-derived macrophages maintains blood-brain barrier integrity and restricts infarctions early after ischemic stroke

Abstract

Background: Brain microglia and infiltrating monocyte-derived macrophages are vital in preserving blood vessel integrity after stroke. Understanding mechanisms that induce immune cells to adopt vascular-protective phenotypes may hasten the development of stroke treatments. IL-33 is a potent chemokine released from damaged cells, such as CNS glia after stroke. The activation of IL-33/ST2 signaling has been shown to promote neuronal viability and white matter integrity after ischemic stroke. The impact of IL-33/ST2 on blood-brain barrier (BBB) integrity, however, remains unknown. The current study fills this gap and reveals a critical role of IL-33/ST2 signaling in macrophage-mediated BBB protection after stroke.

Methods: Transient middle cerebral artery occlusion (tMCAO) was performed to induce ischemic stroke in wildtype (WT) versus ST2 knockout (KO) male mice. IL-33 was applied intranasally to tMCAO mice with or without dietary PLX5622 to deplete microglia/macrophages. ST2 KO versus WT bone marrow or macrophage cell transplantations were used to test the involvement of ST2⁺ macrophages in BBB integrity. Macrophages were cocultured in transwells with brain endothelial cells (ECs) after oxygen-glucose deprivation (OGD) to test potential direct effects of IL33-treated macrophages on the BBB in vitro.

Results: The ST2 receptor was expressed in brain ECs, microglia, and infiltrating macrophages. Global KO of ST2 led to more IgG extravasation and loss of ZO-1 in cerebral microvessels 3 days post-tMCAO. Intranasal IL-33 administration reduced BBB leakage and infarct severity in microglia/macrophage competent mice, but not in microglia/macrophage depleted mice. Worse BBB injury was observed after tMCAO in chimeric WT mice reconstituted with ST2 KO bone marrow, and in WT mice whose monocytes were replaced by ST2 KO monocytes. Macrophages treated with IL-33 reduced in vitro barrier leakage and maintained tight junction integrity after OGD. In contrast, IL-33 exerted minimal direct effects on the endothelial barrier in the absence of macrophages. IL-33-treated macrophages demonstrated transcriptional upregulation of an array of protective factors, suggesting a shift towards favorable phenotypes.

Conclusion: Our results demonstrate that early-stage IL-33/ST2 signaling in infiltrating macrophages reduces the extent of acute BBB disruption after stroke. Intranasal IL-33 administration may represent a new strategy to reduce BBB leakage and infarct severity.

Keywords: Blood brain barrier; IL-33/ST2 signaling; Macrophage; Stroke.

Fig. 1

Global ST2 deficiency enhances brain infarct volumes and BBB damage following experimental stroke. (A-C) MAP2 and IgG staining were used to determine the infarct volume and BBB damage, respectively, in WT and ST2 KO mice 3 days after 60 min–40 min tMCAO. Shown are representative coronal sections stained with anti-MAP2 (A, green) or anti-IgG (B, red) antibodies. (C) Top: Surface plot images generated from IgG immunostaining. Middle and Bottom: Representative images of IgG, and IgG/CD31 double staining in the peri-infarct areas. (D) Quantification of infarct volumes. (E) Quantification of IgG intensity in the ipsilesional hemisphere. (F) Representative double labeling of CD31 (red) and ZO-1 (green). Red inset boxes in the top left image illustrate the imaging areas for the quantification of CD31 and ZO-1 staining. Areas in the white insets were enlarged and reconstructed in 3D, as shown on the right column. (G) Quantification of the ratio of CD31⁺ vessels covered by ZO-1 staining (top) and diameters of CD31⁺ vessels (bottom) 3 days after tMCAO. (H) Representative immunoblots and quantification of ZO-1 protein levels in the ischemic hemisphere and contralateral hemispheres (Contra) collected 3 days after 60 min tMCAO. β-actin was used as the loading control. *P < 0.05, **P < 0.01, ***P < 0.001. Student’s two-tailed t test (G bottom), one-way ANOVA followed by post hoc Bonferroni test (D, E, G top), or Brown-Forsythe and Welch ANOVA test (H)

Fig. 2

ST2 expression in endothelial cells, microglia, and macrophages in the ischemic brain early after experimental stroke. (A-B) ST2 expression on ECs and microglia/macrophages was elevated after tMCAO. (A) Top: Representative gating strategy for CD31⁺Ly6G⁻ EC, CD31⁻Ly6G⁻CD11b⁺CD45^intermediate microglia, and CD31⁻Ly6G⁻CD11b⁺F4/80⁺CD45^high macrophages in the brain 3d after tMCAO. Bottom: Histograms showing ST2 expression on ECs, macrophages, and microglia in sham and stroke brains. (B) Quantification of the percentage of ST2⁺ ECs, macrophages, or microglia in sham or ischemic brains. (C) ST2 staining (magenta) in the ischemic brains from WT and ST2 KO mice 3d after tMCAO. Cell nuclei are counterstained with DAPI (blue). (D) Co-immunostaining of ST2 (magenta) and Iba1 (green) in the peri-infarct area of WT mice 3d after tMCAO. Areas in the white insets are enlarged. (E) Co-immunostaining of ST2 (magenta) and CD31 (green) in the brain of CCR2-RFP reporter mice 3d after tMCAO. Areas in the white inset were enlarged and reconstructed in 3D. White arrows: ST2 expression in CD31⁺ ECs. Yellow arrows: ST2 expression in CCR2⁺ infiltrating macrophages. (F) Immunostaining of IL-33 and quantification of the number of IL-33⁺ cells and intensity of IL-33 staining in the peri-infarct area of WT and ST2 KO mice 3d following tMCAO. *P < 0.05. Two-tailed Student’s t test (B) or Welch’s t test (F)

Fig. 3

Intranasal IL-33 treatment protects against BBB damage in a microglia/macrophage-dependent manner. Mice were maintained on PLX5622 diet or control diet for 7 days and subjected to 60 min tMCAO. IL-33 or PBS was intranasally applied 2 h after ischemia and repeated daily for 3d. Brains were collected 3 days after stroke. (A) Representative coronal sections stained with antibodies against MAP2. (B) Quantification of infarct volumes on MAP2-stained sections. (C) Representative coronal sections immunolabeled for infiltrating IgGs. (D) Quantitative analysis of IgG intensity. (E) Representative images of CD31 (green) and Iba1 (white) immunostaining in the peri-infarct area. (F) Representative images of CD31 (red) and ZO-1 (green) double staining in the peri-infarct area. Cell nuclei are counterstained with DAPI (blue). (G) Quantification of the percentages of CD31⁺ vessels covered by ZO-1 staining. *P < 0.05, **P < 0.01. Two-way ANOVA followed by post hoc Bonferroni test

Fig. 4

Transplantation of ST2 KO bone marrow into irradiated WT mice exacerbates BBB damage following experimental stroke. (A) Schematic illustrating bone marrow chimera mouse construction. Bone marrows from WT or ST2 KO donors were injected intravenously (5 × 10⁶ cells/animal) into irradiated WT mice. After 6 weeks of reconstruction, the chimera mice (WT/WT or WT/ST2KO) were subjected to 60 min tMCAO. (B) Flow cytometry to confirm loss of ST2 expression in blood CD11b⁺F4/80⁺ macrophages and CD11b⁺F4/80^- monocytes of WT/ST2 KO chimera mice. (C) Representative coronal sections stained with anti-MAP2 antibodies. (D) Quantification of infarct volume on MAP2 stained sections. (E) Representative coronal sections stained with anti-IgG antibodies. (F) Representative images of CD31 (green) and IgG (red) immunostaining in the peri-infarct area. (G) Quantification of infarct area in 6 levels of MAP2-stained coronal sections, spaced 1 mm apart, throughout the MCA territory. (H) Quantitative analysis of IgG intensity in 6 levels of IgG-stained coronal sections, spaced 1 mm apart, throughout the MCA territory. (I) The ratio of IgG intensity and infarct volume. (J) Representative images of CD31 (red) and ZO-1 (green) double staining in the peri-infarct area. Cell nuclei are counterstained with DAPI (blue). Areas in the white inset were enlarged and reconstructed in 3D. (K) Quantification of the percentages of CD31⁺ vessels covered by ZO-1 staining. *P < 0.05, ***P < 0.001. Two-tailed Student’s t test (D, I), one-way (K) or two-way (G, H) ANOVA followed by post hoc Bonferroni test

Fig. 5

Depletion of ST2 on macrophages exacerbates BBB damage 3 days following experimental stroke. (A) Schematic diagram for macrophage depletion and adoptive transfer experiments. WT recipient mice received intravenous (i.v.) clodronate liposome injections to deplete monocyte/macrophage cells 48 h before tMCAO. Bone marrow-derived macrophages (MΦ) prepared from WT or ST2 KO mice were injected (i.v.) into recipient WT mice immediately after tMCAO. (B) Adoptive transfer of CX3CR1-GFP macrophages was followed by infiltration of donor GFP⁺IBA1⁺ cells (yellow arrows) in the peri-infarct area 3d post tMCAO. (C) Quantification of brain infarct volumes on MAP2-stained sections. (D) Representative images of CD31 and ZO-1 immunostaining in the peri-infarct area. Areas in the white insets were enlarged and reconstructed in 3D. Cell nuclei are counterstained with DAPI (blue). (E) Quantification of the percentages of CD31⁺ vessels covered by ZO-1 staining. (F) Fluorescent Cadaverine tracer was injected 60 min before sacrifice to quantify BBB leakage. Representative images showing leakage of exogenous tracer (red) or endogenous IgG (turquoise) outside of CD31⁺ vessels (green) in the peri-infarct area. (G) Representative high magnifications of tracer leakage (red), CD31 (white), and ZO-1 (green) in the peri-infarct area 3d following tMCAO. 3D-reconstructed images acquired from brains of ST2 KO macrophage-transplanted mice. (H-I) Quantification of tracer leakage (H) and IgG intensity (I). *P < 0.05, ***P < 0.001. Two-tailed Student’s t test (C, H, I), or one-way ANOVA followed by post hoc Bonferroni (E)

Fig. 6

IL-33/ST2 signaling in macrophages protects against BBB leakage in vitro in response to oxygen-glucose deprivation. (A) Primary mouse brain microvascular endothelial cells (BMECs) in cell culture inserts were exposed to 6 h of OGD. Reperfused BMECs were then cocultured with PBS-treated or IL-33-treated (25 µg/mL) WT or ST2 KO macrophages for 6 h. The diffusion of 40 kDa FITC-dextran from the luminal to abluminal chamber was measured over time. (B-C) Primary mouse BMECs in cell culture plates were exposed to 6 h of OGD. Reperfused BMECs were then treated for 4 h with conditioned media collected from PBS-treated or IL-33-treated WT or ST2 KO macrophages. (B) Representative western blot images and quantification of ZO-1. β-Actin was used as the loading control. (C) Representative images of BMECs immunolabeled for VE-cadherin (green) or ZO-1(magenta), and counterstained with DAPI (blue) for nuclear labelling. Images are representative of 3 independent experiments. (D) PCR arrays were used to measure immune regulatory changes in IL-33 or PBS-treated macrophages. The black line indicates fold-changes (2 ^ ^(-ΔCt)) relative to one. The purple lines indicate two-fold or negative two-fold changes in gene expression. Two arrays/group in two independent experiments. (E) RT-PCR was used to quantify the expression of IL10 and IL13ra2 in PBS or IL-33 treated macrophages. (F) BMECs in cell culture inserts were exposed to 6 h of OGD followed by reperfusion and treatment with PBS or IL-33 for 6 h. The diffusion of FITC-dextran from the luminal to abluminal chamber was measured over time. (G-H) BMECs in cell culture plates were exposed to 6 h of OGD followed by reperfusion and treatment with PBS or IL-33 for 4 h. (G) Representative western blot images and quantification of ZO-1. (H) Representative images of BMECs immunolabeled for VE-cadherin (green) or ZO-1(magenta), and counterstained with DAPI (blue) for nuclear labelling. Images are representative of 3 independent experiments. *P < 0.05. **P < 0.01. Two-tailed Student’s t test (E), one-way (B, G) or two-way (A, F) ANOVA followed by post hoc Bonferroni test