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Updated: May 21, 2025
Analysis of Microglia and Monocyte-derived Macrophages from the Central Nervous System by Flow Cytometry
Published on: June 22, 2017
Differential Roles of Macrophages and Microglia in Subretinal Fibrosis Secondary to Neovascular Age-Related Macular
Manon Szczepan1, María Llorián-Salvador1, Caijiao Yi2
1Queen's University Belfast, School of Medicine Dentistry and Biomedical Sciences, Belfast, Belfast, United Kingdom.
Purpose:
To investigate the differential role of infiltrating CCR2+ macrophages and CX3CR1+ microglia in neovascular AMD (nAMD)-mediated subretinal fibrosis.
Methods:
Subretinal fibrosis was induced using the two-stage laser protocol in C57BL/6J or CX3CR1gfp/+ mice. The fibrotic lesion was detected using collagen-1 staining in retinal pigment epithelial /choroidal flatmounts. Infiltrating macrophages and microglial were identified using F4/80, CCR2, and CX3CR1 markers at one, three, six, and 10 days after the second laser. Circulating CCR2+ monocytes were depleted using the MC-21 antibody, whereas CX3CR1+ microglia were depleted using PLX5622. BV2 microglia were treated with TGF-β1 for 96 hours, and their profibrotic potential was examined by quantitative PCR and immunocytochemistry.
Results:
Subretinal fibrosis lesions developed three days after the second laser, accompanied by persistent CCR2+F4/80+ macrophage and CX3CR1+ cell infiltration. Inflammation in the first three days after the second laser was dominated by filtrating CX3CR1+ cells, and the number increased until day (D) 10 post-second laser. Depletion of CCR2+ monocytes from D5-10 significantly reduced the vascular and fibrotic components of the lesion, while CX3CR1+ cell depletion reduced Isolectin B4+ but not collagen-1+ lesion size. Bone marrow-derived macrophages from D6 and D10 mice expressed significantly higher levels of α-smooth muscle actin (α-SMA) and collagen-1 compared to cells from D1 and D3. TGFβ1 treatment increased TMEM119, CX3CR1, IL1b and iNOS gene expression but did not affect Acta2 and Col1a1 gene expression in BV2 cells.
Conclusions:
CCR2+ monocytes, but not CX3CR1+ microglia, critically contribute to the development of subretinal fibrosis in nAMD.
Insights
CCR2+ monocytes, not CX3CR1+ microglia, drive subretinal fibrosis in neovascular AMD (nAMD). Depleting these monocytes reduced fibrosis, highlighting their critical role in nAMD pathogenesis.
Area of Science:
- Ophthalmology
- Immunology
- Cell Biology
Background:
- Neovascular age-related macular degeneration (nAMD) is a leading cause of vision loss.
- Subretinal fibrosis is a major complication of nAMD, leading to irreversible vision impairment.
- The specific roles of infiltrating immune cells in nAMD-mediated fibrosis remain incompletely understood.
Purpose of the Study:
- To elucidate the distinct contributions of CCR2+ macrophages and CX3CR1+ microglia to subretinal fibrosis development in nAMD.
- To investigate the therapeutic potential of targeting these specific immune cell populations.
Main Methods:
- Subretinal fibrosis was induced in mice using a two-stage laser protocol.
- Immune cell infiltration was analyzed using specific markers (F4/80, CCR2, CX3CR1) at various time points.
- Depletion strategies targeted circulating CCR2+ monocytes (MC-21 antibody) and CX3CR1+ microglia (PLX5622).
- Profibrotic gene expression in microglia was assessed after TGF-β1 stimulation.
Main Results:
- Subretinal fibrosis lesions formed by day 3 post-laser, with infiltration of CCR2+ macrophages and CX3CR1+ cells.
- Depletion of CCR2+ monocytes significantly reduced both vascular and fibrotic components of the lesion.
- Depletion of CX3CR1+ cells primarily affected the vascular component, not collagen deposition.
- Bone marrow-derived macrophages showed increased expression of fibrotic markers (α-SMA, collagen-1) over time.
Conclusions:
- CCR2+ monocytes are critical drivers of subretinal fibrosis in nAMD.
- CX3CR1+ microglia play a less significant role in collagen-1 deposition during nAMD-related fibrosis.
- Targeting CCR2+ monocytes may offer a therapeutic strategy for managing nAMD-associated fibrosis.
