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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

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)
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Fig4: 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)

Mentions: Since the macrophages in the injured brain were M2 phenotype, it is conceivable that brain microenvironment polarizes brain-infiltrating macrophages to the M2 phenotype. Supporting this possibility, a previous study has shown that bone marrow-derived macrophages (BMDM) exhibit an M2-like phenotype after co-culture with an astroglioma cell line [25]. To test if glial cells affect macrophage polarization toward the M2 phenotype, BMDM from Cx3cr1+/GFP mice were co-cultured with primary cultured mouse brain glial cells, and mannose receptor expression was measured using flow cytometry. After co-culture with primary glia for 3 days, the percentage of CD206+ cells in the BMDM population (GFP+/CD45+) increased to 20 %, indicating polarization to the M2 phenotype (Fig. 4a and b). Notably, the percentage of CD206+ BMDM also increased to more than 60 % upon incubation in glia-conditioned media for 3 days (Fig. 4c), indicating that direct cell contact between BMDM and glia is not required for M2 polarization. In addition, the expression of other M2 genes Arginase-1 and Ym1 increased by 1.5- and 6-fold, respectively (Fig. 4d and e). However, expression of iNOS and TNF-α mRNA, M1 marker genes, was not significantly upregulated after glia-conditioned media treatment (Fig. 4f and g). These data suggest that glia-derived soluble factor(s) induce M2 polarization of the BMDM, which might account for the M2 polarization of macrophages observed in the ICH-injured brain.Fig. 4


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)

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)
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Related In: Results  -  Collection

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Fig4: 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)
Mentions: Since the macrophages in the injured brain were M2 phenotype, it is conceivable that brain microenvironment polarizes brain-infiltrating macrophages to the M2 phenotype. Supporting this possibility, a previous study has shown that bone marrow-derived macrophages (BMDM) exhibit an M2-like phenotype after co-culture with an astroglioma cell line [25]. To test if glial cells affect macrophage polarization toward the M2 phenotype, BMDM from Cx3cr1+/GFP mice were co-cultured with primary cultured mouse brain glial cells, and mannose receptor expression was measured using flow cytometry. After co-culture with primary glia for 3 days, the percentage of CD206+ cells in the BMDM population (GFP+/CD45+) increased to 20 %, indicating polarization to the M2 phenotype (Fig. 4a and b). Notably, the percentage of CD206+ BMDM also increased to more than 60 % upon incubation in glia-conditioned media for 3 days (Fig. 4c), indicating that direct cell contact between BMDM and glia is not required for M2 polarization. In addition, the expression of other M2 genes Arginase-1 and Ym1 increased by 1.5- and 6-fold, respectively (Fig. 4d and e). However, expression of iNOS and TNF-α mRNA, M1 marker genes, was not significantly upregulated after glia-conditioned media treatment (Fig. 4f and g). These data suggest that glia-derived soluble factor(s) induce M2 polarization of the BMDM, which might account for the M2 polarization of macrophages observed in the ICH-injured brain.Fig. 4

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