Single Eye mRNA-Seq Reveals Normalisation of the Retinal Microglial Transcriptome Following Acute Inflammation

Oliver H Bell; David A Copland; Amy Ward; Lindsay B Nicholson; Clemens A K Lange; Colin J Chu; Andrew D Dick

doi:10.3389/fimmu.2019.03033

Single Eye mRNA-Seq Reveals Normalisation of the Retinal Microglial Transcriptome Following Acute Inflammation

Front Immunol. 2020 Jan 9:10:3033. doi: 10.3389/fimmu.2019.03033. eCollection 2019.

Authors

Oliver H Bell¹, David A Copland¹, Amy Ward¹, Lindsay B Nicholson¹, Clemens A K Lange^{2

3}, Colin J Chu¹, Andrew D Dick^{1

4}

Affiliations

¹ Academic Unit of Ophthalmology, Translational Health Sciences, University of Bristol, Bristol, United Kingdom.
² Eye Clinic, Medical Centre, University of Freiburg, Freiburg, Germany.
³ Faculty of Medicine, University of Freiburg, Freiburg, Germany.
⁴ Institute of Ophthalmology and the National Institute for Health Research Biomedical Research Centre, Moorfields Eye Hospital and University College London, London, United Kingdom.

Abstract

Background: Whether retinal microglia can maintain or restore immune homeostasis during and after inflammation is unclear. We performed single-eye mRNA-sequencing on microglia at different timepoints following a single inflammatory stimulus to characterise their transcriptome during and after resolution of endotoxin-induced uveitis (EIU). Experimental Approach:Cx3cr1^CreER:R26-tdTomato (C57BL/6) male heterozygotes were administered tamoxifen via different regimes at 4-5 weeks of age. Four weeks post-tamoxifen, mice were injected intravitreally with 10 ng lipopolysaccharide (endotoxin induced uveitis, EIU). Six-hundred retinal microglia were obtained by FACS from individual naïve retinas and at 4 h, 18 h, and 2 weeks following EIU induction. Samples were sequenced to a depth of up to 16.7 million reads using the SMART-Seq v4 Ultra Low Input RNA kit. The data was analysed using Partek software and Ingenuity Pathway Analysis. Genes were considered differentially-expressed (DEG) if the FDR step-up p-value was ≤0.05 and the fold-change was ≥±2. Results: Flow cytometric analysis indicates that the Cx3cr1^CreER:R26-tdTomato strain is both sensitive (>95% tagging) and specific (>95% specificity) for microglia when tamoxifen is administered topically to the eye for 3 days. During "early" activation, 613 DEGs were identified. In contrast, 537 DEGs were observed during peak cellular infiltrate and none at 2 weeks, compared to baseline controls (1,069 total unique DEGs). Key marker changes were validated by qPCR, flow cytometry, and fluorescence microscopy. C5AR1 was identified and validated as a robust marker of differentiating microglial subsets during an LPS response. Conclusion: Using EIU to provide a single defined inflammatory stimulus, mRNA-Seq identified acute transcriptional changes in retinal microglia which returned to their original transcriptome after 2 weeks. Yolk-sac derived microglia are capable of restoring their homeostatic state after acute inflammation.

Keywords: heterogeneity; lipopolysaccharide (LPS); mRNA-Seq; microglia; resolution; retina; transcriptome; uveitis.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Cell Differentiation / drug effects
Cell Differentiation / genetics
Endotoxins / pharmacology
Inflammation / chemically induced
Inflammation / genetics*
Lipopolysaccharides / pharmacology
Male
Mice
Mice, Inbred C57BL
Microglia / drug effects
Microglia / physiology*
Neuroglia / drug effects
RNA, Messenger / genetics*
Receptor, Anaphylatoxin C5a / genetics
Retina / drug effects
Retina / physiology*
Signal Transduction / drug effects
Signal Transduction / genetics
Transcriptome / genetics*
Uveitis / chemically induced
Uveitis / genetics

Substances

Endotoxins
Lipopolysaccharides
RNA, Messenger
Receptor, Anaphylatoxin C5a