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Functional differences between microglia and monocytes after ischemic stroke.

Ritzel RM, Patel AR, Grenier JM, Crapser J, Verma R, Jellison ER, McCullough LD - J Neuroinflammation (2015)

Bottom Line: The lack of discriminating markers between these two myeloid populations has led many studies to generate conclusions based on the grouping of these two populations.We found that at 72 h after a 90-min middle cerebral artery occlusion (MCAO), microglia populations decrease whereas monocytes significantly increase in the stroke brain compared to sham.In summary, the resident microglia population is vulnerable to the effects of severe ischemia, show compromised cell cycle progression, and adopt a largely pro-inflammatory phenotype after stroke.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT, 06030, USA. rritzel@uchc.edu.

ABSTRACT

Background: The brain's initial innate response to stroke is primarily mediated by microglia, the resident macrophage of the CNS. However, as early as 4 h after stroke, the blood-brain barrier is compromised and monocyte infiltration occurs. The lack of discriminating markers between these two myeloid populations has led many studies to generate conclusions based on the grouping of these two populations. A growing body of evidence now supports the distinct roles played by microglia and monocytes in many disease models.

Methods: Using a flow cytometry approach, combined with ex-vivo functional assays, we were able to distinguish microglia from monocytes using the relative expression of CD45 and assess the function of each cell type following stroke over the course of 7 days.

Results: We found that at 72 h after a 90-min middle cerebral artery occlusion (MCAO), microglia populations decrease whereas monocytes significantly increase in the stroke brain compared to sham. After stroke, BRDU incorporation into monocytes in the bone marrow increased. After recruitment to the ischemic brain, these monocytes accounted for nearly all BRDU-positive macrophages. Inflammatory activity peaked at 72 h. Microglia produced relatively higher reactive oxygen species and TNF, whereas monocytes were the predominant IL-1β producer. Although microglia showed enhanced phagocytic activity after stroke, monocytes had significantly higher phagocytic capacity at 72 h. Interestingly, we found a positive correlation between TNF expression levels and phagocytic activity of microglia after stroke.

Conclusions: In summary, the resident microglia population is vulnerable to the effects of severe ischemia, show compromised cell cycle progression, and adopt a largely pro-inflammatory phenotype after stroke. Infiltrating monocytes are primarily involved with early debris clearance of dying cells. These findings suggest that the early wave of infiltrating monocytes may be beneficial to stroke repair and future therapies aimed at mitigating microglia cell death may prove more effective than attempting to elicit targeted anti-inflammatory responses from damaged cells.

No MeSH data available.


Related in: MedlinePlus

Phagocytic activity of microglia and monocytes at 24 and 72 h and 7 days following stroke. Representative histogram showing a relative increase in side scatter (granularity) properties of microglia at 72 h after stroke (red) compared to sham (blue; a). Mean side scatter intensity of microglia was quantified at different time points after MCAO (N = 5/group; b). Phagocytic activity after stroke was measured by bead assay using flow cytometry (c). The percentages and mean fluorescence intensity (MFI) of bead+ cells were quantified at 0, 24, and 72 h and 7 days (N = 6/group; d, e). A representative histogram shows higher expression of the phagocytic marker SIRPα on monocytes (blue) compared to microglia (red) at 72 h in the ischemic hemisphere (f). The MFI of SIRPα+ cells are quantified (N = 5/group; g). Cell-specific FMO controls were used to determine positive gating. Error bars show mean SEM. Abbreviation: SEM standard error of the mean, SH sham, ST stroke, SSC side scatter, YG yellow green, a.u.i. arbitrary units of intensity
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Fig4: Phagocytic activity of microglia and monocytes at 24 and 72 h and 7 days following stroke. Representative histogram showing a relative increase in side scatter (granularity) properties of microglia at 72 h after stroke (red) compared to sham (blue; a). Mean side scatter intensity of microglia was quantified at different time points after MCAO (N = 5/group; b). Phagocytic activity after stroke was measured by bead assay using flow cytometry (c). The percentages and mean fluorescence intensity (MFI) of bead+ cells were quantified at 0, 24, and 72 h and 7 days (N = 6/group; d, e). A representative histogram shows higher expression of the phagocytic marker SIRPα on monocytes (blue) compared to microglia (red) at 72 h in the ischemic hemisphere (f). The MFI of SIRPα+ cells are quantified (N = 5/group; g). Cell-specific FMO controls were used to determine positive gating. Error bars show mean SEM. Abbreviation: SEM standard error of the mean, SH sham, ST stroke, SSC side scatter, YG yellow green, a.u.i. arbitrary units of intensity

Mentions: Microglia in the ischemic hemisphere were significantly more activated based on side scatter (granularity) properties compared to sham, suggesting enhanced uptake of dying cells and debris (Fig. 4a, b). Phagocytosis is important in debris clearance and injury repair. Using a bead assay, we measured phagocytic activity of microglia and monocytes (Fig. 4c). After stroke, the percentage of microglia that phagocytosed beads significantly increased at 24 h (p < 0.001) and peaked at a near four-fold increase by 72 h (p < 0.01; Fig. 4d). The phagocytic activity of microglia is restored back to baseline levels by 7 days. Compared to microglia, however, infiltrating monocytes exhibited far greater capacity for phagocytosis. Significantly more monocytes were bead-positive as were the number of beads phagocytosed per cell as evidenced by MFI (p < 0.001; Fig. 4e). Monocytes at 72 h also expressed significantly higher levels of the phagocytic marker SIRPα than did microglia (p = 0.005; Fig. 4f, g). Interestingly, phagocytic microglia were more likely to express TNF and at higher levels than non-phagocytic microglia, indicating that pro-inflammatory (M1) markers may overlap with anti-inflammatory (M2) function (Fig. 5a, b). Moreover, a positive correlation was found between the level of TNF production and the number of beads phagocytosed by microglia after stroke (p = 0.0167; Fig. 5c). Taken together, these data imply that microglia do increase phagocytic activity following stroke stimulus, albeit at significantly lower levels than recruited monocytes.Fig. 4


Functional differences between microglia and monocytes after ischemic stroke.

Ritzel RM, Patel AR, Grenier JM, Crapser J, Verma R, Jellison ER, McCullough LD - J Neuroinflammation (2015)

Phagocytic activity of microglia and monocytes at 24 and 72 h and 7 days following stroke. Representative histogram showing a relative increase in side scatter (granularity) properties of microglia at 72 h after stroke (red) compared to sham (blue; a). Mean side scatter intensity of microglia was quantified at different time points after MCAO (N = 5/group; b). Phagocytic activity after stroke was measured by bead assay using flow cytometry (c). The percentages and mean fluorescence intensity (MFI) of bead+ cells were quantified at 0, 24, and 72 h and 7 days (N = 6/group; d, e). A representative histogram shows higher expression of the phagocytic marker SIRPα on monocytes (blue) compared to microglia (red) at 72 h in the ischemic hemisphere (f). The MFI of SIRPα+ cells are quantified (N = 5/group; g). Cell-specific FMO controls were used to determine positive gating. Error bars show mean SEM. Abbreviation: SEM standard error of the mean, SH sham, ST stroke, SSC side scatter, YG yellow green, a.u.i. arbitrary units of intensity
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Fig4: Phagocytic activity of microglia and monocytes at 24 and 72 h and 7 days following stroke. Representative histogram showing a relative increase in side scatter (granularity) properties of microglia at 72 h after stroke (red) compared to sham (blue; a). Mean side scatter intensity of microglia was quantified at different time points after MCAO (N = 5/group; b). Phagocytic activity after stroke was measured by bead assay using flow cytometry (c). The percentages and mean fluorescence intensity (MFI) of bead+ cells were quantified at 0, 24, and 72 h and 7 days (N = 6/group; d, e). A representative histogram shows higher expression of the phagocytic marker SIRPα on monocytes (blue) compared to microglia (red) at 72 h in the ischemic hemisphere (f). The MFI of SIRPα+ cells are quantified (N = 5/group; g). Cell-specific FMO controls were used to determine positive gating. Error bars show mean SEM. Abbreviation: SEM standard error of the mean, SH sham, ST stroke, SSC side scatter, YG yellow green, a.u.i. arbitrary units of intensity
Mentions: Microglia in the ischemic hemisphere were significantly more activated based on side scatter (granularity) properties compared to sham, suggesting enhanced uptake of dying cells and debris (Fig. 4a, b). Phagocytosis is important in debris clearance and injury repair. Using a bead assay, we measured phagocytic activity of microglia and monocytes (Fig. 4c). After stroke, the percentage of microglia that phagocytosed beads significantly increased at 24 h (p < 0.001) and peaked at a near four-fold increase by 72 h (p < 0.01; Fig. 4d). The phagocytic activity of microglia is restored back to baseline levels by 7 days. Compared to microglia, however, infiltrating monocytes exhibited far greater capacity for phagocytosis. Significantly more monocytes were bead-positive as were the number of beads phagocytosed per cell as evidenced by MFI (p < 0.001; Fig. 4e). Monocytes at 72 h also expressed significantly higher levels of the phagocytic marker SIRPα than did microglia (p = 0.005; Fig. 4f, g). Interestingly, phagocytic microglia were more likely to express TNF and at higher levels than non-phagocytic microglia, indicating that pro-inflammatory (M1) markers may overlap with anti-inflammatory (M2) function (Fig. 5a, b). Moreover, a positive correlation was found between the level of TNF production and the number of beads phagocytosed by microglia after stroke (p = 0.0167; Fig. 5c). Taken together, these data imply that microglia do increase phagocytic activity following stroke stimulus, albeit at significantly lower levels than recruited monocytes.Fig. 4

Bottom Line: The lack of discriminating markers between these two myeloid populations has led many studies to generate conclusions based on the grouping of these two populations.We found that at 72 h after a 90-min middle cerebral artery occlusion (MCAO), microglia populations decrease whereas monocytes significantly increase in the stroke brain compared to sham.In summary, the resident microglia population is vulnerable to the effects of severe ischemia, show compromised cell cycle progression, and adopt a largely pro-inflammatory phenotype after stroke.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT, 06030, USA. rritzel@uchc.edu.

ABSTRACT

Background: The brain's initial innate response to stroke is primarily mediated by microglia, the resident macrophage of the CNS. However, as early as 4 h after stroke, the blood-brain barrier is compromised and monocyte infiltration occurs. The lack of discriminating markers between these two myeloid populations has led many studies to generate conclusions based on the grouping of these two populations. A growing body of evidence now supports the distinct roles played by microglia and monocytes in many disease models.

Methods: Using a flow cytometry approach, combined with ex-vivo functional assays, we were able to distinguish microglia from monocytes using the relative expression of CD45 and assess the function of each cell type following stroke over the course of 7 days.

Results: We found that at 72 h after a 90-min middle cerebral artery occlusion (MCAO), microglia populations decrease whereas monocytes significantly increase in the stroke brain compared to sham. After stroke, BRDU incorporation into monocytes in the bone marrow increased. After recruitment to the ischemic brain, these monocytes accounted for nearly all BRDU-positive macrophages. Inflammatory activity peaked at 72 h. Microglia produced relatively higher reactive oxygen species and TNF, whereas monocytes were the predominant IL-1β producer. Although microglia showed enhanced phagocytic activity after stroke, monocytes had significantly higher phagocytic capacity at 72 h. Interestingly, we found a positive correlation between TNF expression levels and phagocytic activity of microglia after stroke.

Conclusions: In summary, the resident microglia population is vulnerable to the effects of severe ischemia, show compromised cell cycle progression, and adopt a largely pro-inflammatory phenotype after stroke. Infiltrating monocytes are primarily involved with early debris clearance of dying cells. These findings suggest that the early wave of infiltrating monocytes may be beneficial to stroke repair and future therapies aimed at mitigating microglia cell death may prove more effective than attempting to elicit targeted anti-inflammatory responses from damaged cells.

No MeSH data available.


Related in: MedlinePlus