June 2021
Volume 62, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2021
Single-cell RNA-seq reveals a dynamic shift of engrafted peripheral macrophages in the CNS towards a microglia signature.
Author Affiliations & Notes
  • Fengyang Lei
    Cornea, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts, United States
    Harvard University John A Paulson School of Engineering and Applied Sciences, Cambridge, Massachusetts, United States
  • Naiwen Cui
    Harvard University John A Paulson School of Engineering and Applied Sciences, Cambridge, Massachusetts, United States
  • Chengxin Zhou
    Cornea, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts, United States
  • Yamei Cai
    Harvard University John A Paulson School of Engineering and Applied Sciences, Cambridge, Massachusetts, United States
  • Claes H Dohlman
    Cornea, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts, United States
  • James Chodosh
    Cornea, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts, United States
  • Demetrios G. Vavvas
    Massachusetts Eye and Ear Infirmary, Boston, Massachusetts, United States
  • Huidan Zhang
    Harvard University John A Paulson School of Engineering and Applied Sciences, Cambridge, Massachusetts, United States
  • David A Weitz
    Harvard University John A Paulson School of Engineering and Applied Sciences, Cambridge, Massachusetts, United States
  • Eleftherios I Paschalis
    Cornea, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts, United States
  • Footnotes
    Commercial Relationships   Fengyang Lei, None; Naiwen Cui, None; Chengxin Zhou, None; Yamei Cai, None; Claes Dohlman, None; James Chodosh, None; Demetrios Vavvas, None; Huidan Zhang, None; David Weitz, None; Eleftherios Paschalis, None
  • Footnotes
    Support  Boston KPro Research Fund
Investigative Ophthalmology & Visual Science June 2021, Vol.62, 918. doi:
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      Fengyang Lei, Naiwen Cui, Chengxin Zhou, Yamei Cai, Claes H Dohlman, James Chodosh, Demetrios G. Vavvas, Huidan Zhang, David A Weitz, Eleftherios I Paschalis; Single-cell RNA-seq reveals a dynamic shift of engrafted peripheral macrophages in the CNS towards a microglia signature.. Invest. Ophthalmol. Vis. Sci. 2021;62(8):918.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Ocular injuries can result in permanent remodeling of the neuroglia system. This has been associated with increased susceptibility to neuroinflammation and subsequent glaucoma. Here, we employed a high throughput in-drop single-cell RNAseq to assess the dynamic changes in the transcriptional profile of myeloid cells of the retina after ocular injury.

Methods : C57BL/6J mice received corneal alkali burn to induce sterile retinal inflammation. Retinal tissues were collected 1, 4, and 7 days after the injury, CD45+CD11b+ cells were isolated using FACS sorting and processed for high-throughput microfluidic-based single cell sequencing. Bioinformatic analysis was performed with the Seurat V2 program and key findings were further assessed using a CX3CR1+/EGFP bone marrow chimera model.

Results : One day post injury, we identified two distinct clusters of myeloid cells, representing the resident microglia (Siglec1-) and the peripheral monocytes (Siglec1+) that infiltrate into the parenchyma after injury. Acetylation genes Eif5a, Eif3k, Naa38 were highly upregulated in microglial cells as compared to peripherally engrafted monocytes. However, 4 days post injury, the signatures of the two populations started to overlap, with peripheral monocytes expressing a subset of microglia genes, such as P2ry12, Tmem119, Aif1, Fcrls. At 7 days post injury, the signatures of the two populations appeared to converge further. Further validation in bone marrow chimeras revealed a morphometric convergence between infiltrated and resident CX3CR1+ cells, further corroborating the RNAseq findings. The transition of peripheral monocytes to microglia was supported by gradual change from CD45hiCX3CR1lo monocyte to CD45loCX3CR1hi microglia phenotype. Further, 4 days after injury, P2ry12 was found to be expressed on infiltrated monocytes.

Conclusions : Our results suggested that infiltrated monocytes in the retina undergo in situ phenotypical changes that allow them to adapt to the new environment. This is associated with de novo expression of P2ry12 and Tmem119 “microglia specific genes” and phenotypical changes that promote their long-term engraftment into the tissue.

This is a 2021 ARVO Annual Meeting abstract.

 

Myeloid cells (t-SNE plot) are grouped into 4 major clusters. Clusters are circled (A), and their relationships to their harvest time (B). Gene expression in clustered cells (C) and violin plots of those genes in different clusters.

Myeloid cells (t-SNE plot) are grouped into 4 major clusters. Clusters are circled (A), and their relationships to their harvest time (B). Gene expression in clustered cells (C) and violin plots of those genes in different clusters.

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