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

Min H, Jang YH, Cho IH, Yu SW, Lee SJ - Mol Brain (2016)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience and Dental Research Institute, School of Dentistry, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul, 08826, Republic of Korea.

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.

No MeSH data available.


Related in: MedlinePlus

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
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Fig2: 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

Mentions: To characterize the phenotype of the brain-infiltrating macrophages, we analyzed expression of typical M1 (CD16, CD86, and iNOS) and M2 (Arginase-1 and Ym1) markers in the injured brain. Upon initiation of ICH, the mRNA expression of Arginase-1 increased more than 300-fold in the injured brain at 7 dpi. Likewise, Ym1, another M2 marker, expression was increased 15-fold at 3 dpi (Fig. 2a and b). For M1 markers, only CD16 expression was increased 4.7-fold at 3 dpi (Fig. 2c), whereas other M1 markers such as CD86 and iNOS expressions were not significantly altered (Fig. 2d and e). Arginase-1 expression was mainly detected in CX3CR1+ macrophages/microglia in the peri-hematoma region (Fig. 2f, arrows), suggesting that macrophages/microglia in the injured brain are polarized to the M2 phenotype. To confirm these data, we analyzed the expression of mannose receptor (CD206), another M2 marker, in the CD11b+ monocyte cell population in the ipsilateral ICH-injured brain using flow cytometry (Fig. 2g). The percentages of mannose receptor-expressing macrophages (CD206+/CD11b+/CD45high) increased to 68.5 % at 7 days post-ICH, whereas the CD206+ microglia population (CD206+/CD11b+/CD45low) increased to only 16.7 %. Taken together, these data suggest that the majority of macrophages in the ICH-injured brain are polarized to the M2 phenotype at delayed time points (3 and 7 dpi).Fig. 2


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

Min H, Jang YH, Cho IH, Yu SW, Lee SJ - Mol Brain (2016)

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
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Related In: Results  -  Collection

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Fig2: 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
Mentions: To characterize the phenotype of the brain-infiltrating macrophages, we analyzed expression of typical M1 (CD16, CD86, and iNOS) and M2 (Arginase-1 and Ym1) markers in the injured brain. Upon initiation of ICH, the mRNA expression of Arginase-1 increased more than 300-fold in the injured brain at 7 dpi. Likewise, Ym1, another M2 marker, expression was increased 15-fold at 3 dpi (Fig. 2a and b). For M1 markers, only CD16 expression was increased 4.7-fold at 3 dpi (Fig. 2c), whereas other M1 markers such as CD86 and iNOS expressions were not significantly altered (Fig. 2d and e). Arginase-1 expression was mainly detected in CX3CR1+ macrophages/microglia in the peri-hematoma region (Fig. 2f, arrows), suggesting that macrophages/microglia in the injured brain are polarized to the M2 phenotype. To confirm these data, we analyzed the expression of mannose receptor (CD206), another M2 marker, in the CD11b+ monocyte cell population in the ipsilateral ICH-injured brain using flow cytometry (Fig. 2g). The percentages of mannose receptor-expressing macrophages (CD206+/CD11b+/CD45high) increased to 68.5 % at 7 days post-ICH, whereas the CD206+ microglia population (CD206+/CD11b+/CD45low) increased to only 16.7 %. Taken together, these data suggest that the majority of macrophages in the ICH-injured brain are polarized to the M2 phenotype at delayed time points (3 and 7 dpi).Fig. 2

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience and Dental Research Institute, School of Dentistry, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul, 08826, Republic of Korea.

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.

No MeSH data available.


Related in: MedlinePlus