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Cited by 128Neuroinflammation and Neurodegeneration MechanismsPhagocytosis and Immune RegulationAlzheimer's disease research and treatments

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Differential Phagocytic Properties of CD45low Microglia and CD45high Brain Mononuclear Phagocytes—Activation and Age-Related Effects

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Rangaraju, Srikant, Raza, Syed Ali, Li, Noel Xiang’An, Betarbet, Ranjita, Dammer, Eric B., Duong, Duc, Lah, James J., Seyfried, Nicholas T., Levey, Allan I.·2018·Frontiers in Immunology
DOI 10.3389/fimmu.2018.00405PMC PMC5840283
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Abstract

In the central nervous system (CNS), microglia are innate immune mononuclear phagocytes (CNS MPs) that can phagocytose infectious particles, apoptotic cells, neurons and pathological protein aggregates such as Aβ in Alzheimer’s disease (AD). While CD11b+CD45low microglia account for the majority of CNS MPs, a small population of CD11b+CD45high CNS MPs is also recognized in AD that surround Aβ plaques. These transcriptionally-distinct and pathologically-relevant CD45high cells have unclear origin and undefined phagocytic characteristics. We have comprehensively validated rapid flow cytometric assays of bulk-phase and amyloid β fibril (fAβ) phagocytosis and applied these to study acutely isolated CNS MPs. Using these methods, we provide novel insights into differential abilities of CD11b+ CD45low and CD45high CNS MPs to phagocytose macroparticles and fAβ under normal, acute and chronic neuroinflammatory states. CD45high CNS MPs also highly upregulate TREM2, CD11c and several disease-associated-microglia (DAM) signature genes, and have a higher phagocytic capacity for Aβ as compared to CD45low microglia in the 5xFAD mouse model of AD that becomes more apparent with ageing. Our data suggest an overall pro-phagocytic and protective role for CD11b+CD45high CNS MPs in neurodegeneration, which if promoted, could be beneficial.

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Figures

Figure 1 Validation of flow cytometric phycoerythrin (PE)-microsphere assay in BV2 microglia. (A) Forward scatter (FSC) and side scatter (SSC) profile of microspheres. (B) PE fluorescence vs. SSC. (C) Live cell gating strategy of BV2 microglia based on FSC and SSC. (D) Profile of PE-microsphere phagocytosis in resting BV2 cells. Four populations were identified for cell sorting. Gray histogram is fluorescence observed in untreated BV2 cells not exposed to microspheres. Immunofluorescence microscopy imaging of live fluorescence activated cell sorting-sorted BV2 cells. Blue: Hoescht nuclear stain, green: CD45-FITC, red: PE-microspheres. (E) Effect of CytoD treatment on overall cell viability. (F,G) Representative flow cytometric histograms and analyses comparing PE-microsphere phagocytosis with or without 50 µM CytoD pre-incubation. (H,I) Comparison of proportions of PE-microsphere phagocytosing cells after lipopolysaccharide (LPS) stimulation (100 ng/ml) for 24 h and inhibition by CytoD (50 µM). CytoD was added to BV2 cells after LPS stimulation but 30 min prior to addition of fAβ42-488 (10 µM).
Figure 1 Validation of flow cytometric phycoerythrin (PE)-microsphere assay in BV2 microglia. (A) Forward scatter (FSC) and side scatter (SSC) profile of microspheres. (B) PE fluorescence vs. SSC. (C) Live cell gating strategy of BV2 microglia based on FSC and SSC. (D) Profile of PE-microsphere phagocytosis in resting BV2 cells. Four populations were identified for cell sorting. Gray histogram is fluorescence observed in untreated BV2 cells not exposed to microspheres. Immunofluorescence microscopy imaging of live fluorescence activated cell sorting-sorted BV2 cells. Blue: Hoescht nuclear stain, green: CD45-FITC, red: PE-microspheres. (E) Effect of CytoD treatment on overall cell viability. (F,G) Representative flow cytometric histograms and analyses comparing PE-microsphere phagocytosis with or without 50 µM CytoD pre-incubation. (H,I) Comparison of proportions of PE-microsphere phagocytosing cells after lipopolysaccharide (LPS) stimulation (100 ng/ml) for 24 h and inhibition by CytoD (50 µM). CytoD was added to BV2 cells after LPS stimulation but 30 min prior to addition of fAβ42-488 (10 µM).CC BY · PubMed Central
Figure 2 Validation of flow cytometric fluorescent fAβ42 phagocytosis assay in BV2 microglia. (A) TEM imaging showing Aβ42 fibril formation over a 10-day time course. (B,C) Representative flow cytometric histograms and quantitative analysis showing uptake of fAβ42-Hilyte488 by BV2 microglia and inhibition by Cyto D (Left); non-phagocytic binding of monomeric Aβ42-Hilyte488 shown on the right. Dotted line represents quantitation of data from five independent experiments. # in panel (C) represents statistical comparison of mAβ42 to the negative control (p = 0.01). (D,E) Comparison of proportions of fAβ42-phagocytosing cells after lipopolysaccharide (LPS) stimulation (100 ng/ml) for 24 h and inhibition by CytoD (50 µM). CytoD was added to BV2 cells after LPS stimulation but 30 min prior to addition of fAβ42-488 (10 µM). N = 5 independent experiments per condition (*p < 0.05, **p < 0.01, ***p < 0.005).
Figure 2 Validation of flow cytometric fluorescent fAβ42 phagocytosis assay in BV2 microglia. (A) TEM imaging showing Aβ42 fibril formation over a 10-day time course. (B,C) Representative flow cytometric histograms and quantitative analysis showing uptake of fAβ42-Hilyte488 by BV2 microglia and inhibition by Cyto D (Left); non-phagocytic binding of monomeric Aβ42-Hilyte488 shown on the right. Dotted line represents quantitation of data from five independent experiments. # in panel (C) represents statistical comparison of mAβ42 to the negative control (p = 0.01). (D,E) Comparison of proportions of fAβ42-phagocytosing cells after lipopolysaccharide (LPS) stimulation (100 ng/ml) for 24 h and inhibition by CytoD (50 µM). CytoD was added to BV2 cells after LPS stimulation but 30 min prior to addition of fAβ42-488 (10 µM). N = 5 independent experiments per condition (*p < 0.05, **p < 0.01, ***p < 0.005).CC BY · PubMed Central
Figure 3 Identification of phagocytic receptors that regulate microsphere and fAb42 phagocytosis in BV2 microglia. (A) Differentially expressed phagocytic proteins identified by quantitative proteomics in BV2 microglia that were exposed to M1 [lipopolysaccharide (LPS)], M2a (IL4), and M2c (IL10 + TGFβ) polarizing stimuli for 48 h. Please refer to Table S1 in Supplementary Material for additional information. (B) Relative expression of BV2 phagocytic proteins at the transcriptomic level in acutely isolated CNS MPs, comparing LPS-treated vs. untreated CD45low CNS MPs and CD45low vs. CD45high CD11b+ CNS MPs. Red indicates relative upregulation, Blue indicates relative downregulation and Gray indicates genes/proteins not identified by RNAseq. These data were obtained from publicly available RNAseq data. (C) Comparison of phycoerythrin (PE)-microsphere and fAβ42 phagocytic profiles of classically activated (LPS), alternatively activated (IL4), and deactivated (TGFβ + IL10) BV2 microglia. (D,E) Effects of functional blockade of selected phagocytic receptors on PE-microsphere (D) and fAβ42 phagocytosis (E) by BV2 microglia. N = 5 independent experiments; *p < 0.05, **p < 0.01, ***p < 0.005.
Figure 3 Identification of phagocytic receptors that regulate microsphere and fAb42 phagocytosis in BV2 microglia. (A) Differentially expressed phagocytic proteins identified by quantitative proteomics in BV2 microglia that were exposed to M1 [lipopolysaccharide (LPS)], M2a (IL4), and M2c (IL10 + TGFβ) polarizing stimuli for 48 h. Please refer to Table S1 in Supplementary Material for additional information. (B) Relative expression of BV2 phagocytic proteins at the transcriptomic level in acutely isolated CNS MPs, comparing LPS-treated vs. untreated CD45low CNS MPs and CD45low vs. CD45high CD11b+ CNS MPs. Red indicates relative upregulation, Blue indicates relative downregulation and Gray indicates genes/proteins not identified by RNAseq. These data were obtained from publicly available RNAseq data. (C) Comparison of phycoerythrin (PE)-microsphere and fAβ42 phagocytic profiles of classically activated (LPS), alternatively activated (IL4), and deactivated (TGFβ + IL10) BV2 microglia. (D,E) Effects of functional blockade of selected phagocytic receptors on PE-microsphere (D) and fAβ42 phagocytosis (E) by BV2 microglia. N = 5 independent experiments; *p < 0.05, **p < 0.01, ***p < 0.005.CC BY · PubMed Central
Figure 4 Flow cytometric microsphere and fAβ42 phagocytosis studies of acutely isolated CNS MPs from wild-type mice. (A) Outline of acute isolation of CNS MPs and typical scatter plot of live-gated CNS MPs based on CD11b and CD45 expression. Majority of cells are CD11b+ CD45low while a small proportion of cells are CD11b+ CD45high. Contaminating lymphocytes (CD11bneg CD45high) and other non-immune cells are also seen. (B) Typical scatter plot of acutely isolated live-gated and Ly6G negative splenocytes, further gated on CD11b+ status to enrich for splenic macrophages/monocytes. (C,D) Representative flow cytometric histograms showing distinct peaks of phycoerythrin (PE)-microsphere phagocytosis in both CD45low and CD45high CNS MPs as well as CD11b+ splenocytes. Analysis from five mice per group is shown in panel (D). (E,F) Representative flow cytometric histograms showing fluorescent fAβ42 phagocytosis by CNS MPs and splenic CD11b+ cells. Analysis from five mice per group is shown in panel (F) (*p < 0.05, **p < 0.01, ***p < 0.005).
Figure 4 Flow cytometric microsphere and fAβ42 phagocytosis studies of acutely isolated CNS MPs from wild-type mice. (A) Outline of acute isolation of CNS MPs and typical scatter plot of live-gated CNS MPs based on CD11b and CD45 expression. Majority of cells are CD11b+ CD45low while a small proportion of cells are CD11b+ CD45high. Contaminating lymphocytes (CD11bneg CD45high) and other non-immune cells are also seen. (B) Typical scatter plot of acutely isolated live-gated and Ly6G negative splenocytes, further gated on CD11b+ status to enrich for splenic macrophages/monocytes. (C,D) Representative flow cytometric histograms showing distinct peaks of phycoerythrin (PE)-microsphere phagocytosis in both CD45low and CD45high CNS MPs as well as CD11b+ splenocytes. Analysis from five mice per group is shown in panel (D). (E,F) Representative flow cytometric histograms showing fluorescent fAβ42 phagocytosis by CNS MPs and splenic CD11b+ cells. Analysis from five mice per group is shown in panel (F) (*p < 0.05, **p < 0.01, ***p < 0.005).CC BY · PubMed Central
Figure 5 Characterization of microsphere and fAβ42 phagocytosis by CNS MPs in the lipopolysaccharide (LPS)-induced acute neuroinflammation mouse model. For these experiments, adult (3- to 4-months old) C57BL6/J mice were given four daily IP injections of LPS (10 µg) or PBS, after which CNS MPs were acutely isolated. (A) Comparison of proportions of CD11b+CD45high CNS MPs in untreated and LPS-treated mice. (B) Comparison of pro-inflammatory marker ICAM-1 expression within CD45high and CD45low CNS MPs isolated from PBS and LPS-treated mice. (C,D) Comparison of phycoerythrin-microsphere phagocytosis in CD45high and CD45low CNS MPs from PBS and LPS-treated mice. (E,F) Comparison of fAβ42 phagocytosis in and CD45low and CD45high CNS MPs from PBS and LPS-treated mice. Data from six mice per treatment group are shown (*p < 0.05, **p < 0.01, ***p < 0.005).
Figure 5 Characterization of microsphere and fAβ42 phagocytosis by CNS MPs in the lipopolysaccharide (LPS)-induced acute neuroinflammation mouse model. For these experiments, adult (3- to 4-months old) C57BL6/J mice were given four daily IP injections of LPS (10 µg) or PBS, after which CNS MPs were acutely isolated. (A) Comparison of proportions of CD11b+CD45high CNS MPs in untreated and LPS-treated mice. (B) Comparison of pro-inflammatory marker ICAM-1 expression within CD45high and CD45low CNS MPs isolated from PBS and LPS-treated mice. (C,D) Comparison of phycoerythrin-microsphere phagocytosis in CD45high and CD45low CNS MPs from PBS and LPS-treated mice. (E,F) Comparison of fAβ42 phagocytosis in and CD45low and CD45high CNS MPs from PBS and LPS-treated mice. Data from six mice per treatment group are shown (*p < 0.05, **p < 0.01, ***p < 0.005).CC BY · PubMed Central
Figure 6 Characterization of phagocytic properties of CD45low and CD45high CNS MPs in the amyloidogenic 5xFAD mouse model of Alzheimer’s disease pathology. Acutely isolated CNS MPs and splenic CD11b+ cells from WT and 5xFAD mice at different ages were used for these flow cytometric studies. (A) Comparison of proportion of CD11b+ CD45high CNS MPs in 6-month-old 5xFAD and WT mice (n = 9/group). (B) Comparison of phycoerythrin (PE)-microsphere phagocytosis in CD45high and CD45low CNS MPs from 6-month-old WT and 5xFAD mice (n = 6/group). (C) Comparison of fAβ42 phagocytosis in CD45high and CD45low CNS MPs from 6-month-old WT and 5xFAD mice (n = 6/group). (D) Comparison of fAβ42 phagocytosis in 5xFAD and WT CNS MPs at 3–4, 5–6, and 8–10 months of age (n = 3 mice per group). (E) Comparison of fAβ42 phagocytosis in CD45high and CD45low CNS MPs and Splenic CD11b+ cells from untreated WT, LPS-treated WT and age-matched 5xFAD mice (age 6 months, n = 3 mice per group); *p < 0.05, **p < 0.01, ***p < 0.005.
Figure 6 Characterization of phagocytic properties of CD45low and CD45high CNS MPs in the amyloidogenic 5xFAD mouse model of Alzheimer’s disease pathology. Acutely isolated CNS MPs and splenic CD11b+ cells from WT and 5xFAD mice at different ages were used for these flow cytometric studies. (A) Comparison of proportion of CD11b+ CD45high CNS MPs in 6-month-old 5xFAD and WT mice (n = 9/group). (B) Comparison of phycoerythrin (PE)-microsphere phagocytosis in CD45high and CD45low CNS MPs from 6-month-old WT and 5xFAD mice (n = 6/group). (C) Comparison of fAβ42 phagocytosis in CD45high and CD45low CNS MPs from 6-month-old WT and 5xFAD mice (n = 6/group). (D) Comparison of fAβ42 phagocytosis in 5xFAD and WT CNS MPs at 3–4, 5–6, and 8–10 months of age (n = 3 mice per group). (E) Comparison of fAβ42 phagocytosis in CD45high and CD45low CNS MPs and Splenic CD11b+ cells from untreated WT, LPS-treated WT and age-matched 5xFAD mice (age 6 months, n = 3 mice per group); *p < 0.05, **p < 0.01, ***p < 0.005.CC BY · PubMed Central
Figure 7 CD45high CNS MPs do not express peripheral origin marker Ly6c but highly express CD44 and phagocytic genes TREM2, CD11c, and disease-associated microglia (DAM)-signature genes in 5xFAD mice. (A) Scatter plot demonstrating the CD45high and CD45low subsets of CNS MPs in 8-month-old 5xFAD mice. (B) Expression of Ly6c (marker of peripheral origin) and Ly6G (neutrophil marker) in CD11b+CD45low, CD11b+ CD45high CNS MPs and CD11b+CD45+ splenocytes. (C) Comparison of CD44 expression in CD11b+CD45low and CD11b+ CD45high CNS MPs in WT and 5xFAD mice. (D) Comparison of cell surface expression of phagocytic receptor TREM2, CD11c and activation marker ICAM-1 on CD45high and CD45low CD11b+ CNS MPs. Quantification of data from three WT and three 5xFAD mice (ages 6–8 months) is shown below. (E) A comparative transcriptomic analysis of phagocytic receptor genes identified by RNAseq in CD11b+ CD45low and CD45high CNS MPs, LPS-activated CD45low microglia, and DAM and homeostatic microglia in WT and 5xFAD mice. Color scheme used indicates magnitude of log2-fold difference as shown in the scale bar.
Figure 7 CD45high CNS MPs do not express peripheral origin marker Ly6c but highly express CD44 and phagocytic genes TREM2, CD11c, and disease-associated microglia (DAM)-signature genes in 5xFAD mice. (A) Scatter plot demonstrating the CD45high and CD45low subsets of CNS MPs in 8-month-old 5xFAD mice. (B) Expression of Ly6c (marker of peripheral origin) and Ly6G (neutrophil marker) in CD11b+CD45low, CD11b+ CD45high CNS MPs and CD11b+CD45+ splenocytes. (C) Comparison of CD44 expression in CD11b+CD45low and CD11b+ CD45high CNS MPs in WT and 5xFAD mice. (D) Comparison of cell surface expression of phagocytic receptor TREM2, CD11c and activation marker ICAM-1 on CD45high and CD45low CD11b+ CNS MPs. Quantification of data from three WT and three 5xFAD mice (ages 6–8 months) is shown below. (E) A comparative transcriptomic analysis of phagocytic receptor genes identified by RNAseq in CD11b+ CD45low and CD45high CNS MPs, LPS-activated CD45low microglia, and DAM and homeostatic microglia in WT and 5xFAD mice. Color scheme used indicates magnitude of log2-fold difference as shown in the scale bar.CC BY · PubMed Central
Figure 8 Summary of flow cytometric and phagocytic characteristics of CD45high and CD45low CNS MPs.
Figure 8 Summary of flow cytometric and phagocytic characteristics of CD45high and CD45low CNS MPs.CC BY · PubMed Central

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Authors
Rangaraju, S., Raza, S. A., Li, N. X., Betarbet, R., Dammer, E. B., Duong, D., Lah, J. J., Seyfried, N. T., & Levey, A. I.
Affiliations
Emory University (US)
Corresponding author
Rangaraju, Srikant
ORCID
Venue
Frontiers in Immunology
Publisher
Frontiers Media SA
Published
2018-03-02
Volume
9
Pages
405-405
ISSN
1664-3224 (ISSN-L)
DOI
10.3389/fimmu.2018.00405
PMC
PMC5840283
OpenAlex
W2792377614
Cited by
126
References
80
Citations by year
2026: 9 · 2025: 8 · 2024: 12 · 2023: 18 · 2022: 35 · 2021: 13 · 2020: 19 · 2019: 8 · 2018: 3
FWCI
4.82
Type
journal article
Language
en
Open access
Licence
Locations
  • Frontiers in Immunology (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • National Institutes of Health (P50 AG025688, NS099474-1, P30 NS055077, 1U01AG046161)
  • National Science Foundation (CHE-1507932, NSF/DMR-BSF 1610377)
  • American Brain Foundation
  • Alzheimer’s Association
Topics
  • Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
  • Life Sciences › Immunology and Microbiology › Immunology › Phagocytosis and Immune Regulation
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Alzheimer's disease research and treatments
Keywords
microglia, phagocytosis, amyloid-beta fibrils, Alzheimer's disease, disease-associated microglia, TREM2, CD11c, neuroinflammation, aging, 5xFAD mice, neurodegeneration
MeSH terms
Aging, Alzheimer Disease, Animals, Brain, Cell Line, Disease Models, Animal, Humans, Membrane Glycoproteins, Mice, Inbred C57BL, Mice, Mutant Strains, Phagocytes, Phagocytosis, Receptors, Immunologic, Mononuclear Phagocyte System, Amyloid beta-Peptides, Leukocyte Common Antigens, Microglia, Mice

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