Alternatively activated brain-infiltrating macrophages facilitate recovery from collagenase-induced intracerebral hemorrhage

Abstract

Background: Intracerebral hemorrhage (ICH) is one of the major causes of stroke. After onset of ICH, massive infiltration of macrophages is detected in the peri-hematoma regions. Still, the function of these macrophages in ICH has not been completely elucidated.

Results: In a collagenase-induced ICH model, CX3CR1(+) macrophages accumulated in the peri-hematoma region. Characterization of these macrophages revealed expression of alternatively activated (M2) macrophage markers. In the macrophage-depleted mice, ICH-induced brain lesion volume was larger and neurological deficits were more severe compared to those of control mice, indicating a protective role of these macrophages in ICH. In the ICH-injured brain, mannose receptor-expressing macrophages increased at a delayed time point after ICH, indicating M2 polarization of the brain-infiltrating macrophages in the brain microenvironment. To explore this possibility, bone marrow-derived macrophages (BMDM) were co-cultured with mouse brain glial cells and then tested for activation phenotype. Upon co-culture with glia, the number of mannose receptor-positive M2 macrophages was significantly increased. Furthermore, treatment with glia-conditioned media increased the number of BMDM of M2 phenotype.

Conclusions: In this study, our data suggest that brain-infiltrating macrophages after ICH are polarized to the M2 phenotype by brain glial cells and thereby contribute to recovery from ICH injury.

Keywords: Immune response; Macrophages; Wound healing.

Fig. 1

CX3CR1⁺ cells increase in the peri-hematoma region after ICH. a CX3CR1⁺ cells were detected in peri-hematoma regions of hemorrhagic brain sections. b–e Representative images on brain sections were obtained from Cx3cr1 ^+/GFP mice at 1, 3, and 7 days after ICH or sham operation. Three days after ICH, amoeboid cells were detected in the peri-hematoma region (arrows) (Scale bar, 100 μm). f Myeloid cells (F4/80⁺) in the lesion were analyzed and further divided into CD45-low microglia and CD45-high macrophage populations according to the expression level of CD45 according to flow cytometry. The flow cytometry data are representative of three independent experiments

Fig. 2

M2 macrophages are increased in the ipsilateral brain after ICH. a-e mRNA expressions of M2 markers (Arg-1 and Ym1) or M1 markers (CD16, CD86 and iNOS) in the ICH mice brains were measured using real-time RT-PCR. Total RNA was isolated from ipsilateral hemorrhagic tissue and used for quantitative real-time RT-PCR (n = 3 per group, **p < 0.01, ***p < 0.001). f Representative images of Arg-1 immunofluorescence (red) in the CX3CR1 ^+/GFP mice brain sections obtained from ICH or sham-control mice. Arg-1 immunofluorescence merged with myeloid cell-specific GFP signals is denoted (arrows) (Scale bar, 100 μm). g Dissociated cells from the injured tissue were obtained 1, 3, and 7 days after ICH injury and were used for flow cytometry with anti-CD11b-FITC, −CD45-PE, and -CD206-APC antibodies. Representative flow cytometry histograms show CD206⁺ cells gated on CD11b⁺/CD45⁺ populations. The flow cytometry data are representative of three independent experiments

Fig. 3

Depletion of peripheral monocytes in ICH injury. a Schematic diagram of the myeloid cell depletion protocols using Cl₂MDP. b Blood cells obtained from saline (black line)- or Cl₂MDP-treated ICH mice (gray line) were stained with anti-CD11b-FITC antibody and analyzed with flow cytometry. c Cell suspensions obtained from saline- or Cl₂MDP-treated mouse brain were stained with anti-CD11b-FITC and anti-CD45-PE antibody and analyzed with flow cytometry. Representative flow cytometry dot plots are shown. d ICH injured brain sections were collected and stained with anti-CD68 antibody. Representative images of three independent experiments are shown (Scale bar, 50 μm). e One day after Cl₂MDP injection, mice were subjected to ICH, and the brains were sectioned and stained with cresyl violet at 3 dpi. Representative pictures are shown (Scale bar, 1 mm). f Hemorrhagic injured volumes were quantified and presented in a graph (n = 4 per group, *p < 0.05). g, h After Cl₂MDP or saline i.p. injection, mice were subjected to ICH, and neurological outcomes were evaluated by focal deficit and sticky tape removal time at 1 and 3 days after ICH (n = 5 for saline and Cl₂MDP groups in ART; n = 4 for saline group, n = 3 for Cl₂MDP group in focal deficit, *p < 0.05). Data are expressed as mean ± SEM

Fig. 4

Soluble factors from mouse brain glial cells induce M2 polarization of BMDM. a BMDM from Cx3cr1 ^+/GFP mice were cultured with or without primary glial cells from C57BL/6 mice in a 1:1 ratio for 1 or 3 days. Then the expression of CD206 (a M2 marker) was analyzed using flow cytometry. Representative histograms of CD206 expression in GFP⁺/CD45⁺ populations are shown. b The percentages of CD206⁺ populations in BMDM were calculated. Mean ± SEM of 3 independent experiments are shown (*p < 0.05). c BMDM were incubated in primary glia-conditioned media for 1 and 3 days. Then, the cells were harvested and stained with CD45-PE, CD11b-FITC, and CD206-APC for flow cytometry analysis. The percentages of CD206⁺ cells in CD45⁺/CD11b⁺ populations are analyzed. Mean ± SEM of 3 independent experiments are shown (*p < 0.05, ***p < 0.001). d–g Total RNA was isolated from BMDM at 1 or 3 days after glia-conditioned media treatment. Arg-1, Ym1, iNOS, and TNF-α mRNA expressions were measured using real-time RT-PCR. Mean ± SEM of at least 3 independent experiments are shown (*p < 0.05)