UCL Discovery
UCL home » Library Services » Electronic resources » UCL Discovery

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

Bell, OH; Copland, DA; Ward, A; Nicholson, LB; Lange, CAK; Chu, CJ; Dick, AD; (2020) Single Eye mRNA-Seq Reveals Normalisation of the Retinal Microglial Transcriptome Following Acute Inflammation. Frontiers in Immunology , 10 , Article 3033. 10.3389/fimmu.2019.03033. Green open access

[thumbnail of fimmu-10-03033.pdf]
Preview
Text
fimmu-10-03033.pdf - Published Version

Download (4MB) | Preview

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: Cx3cr1CreER: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 Cx3cr1CreER: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.

Type: Article
Title: Single Eye mRNA-Seq Reveals Normalisation of the Retinal Microglial Transcriptome Following Acute Inflammation
Open access status: An open access version is available from UCL Discovery
DOI: 10.3389/fimmu.2019.03033
Publisher version: https://doi.org/10.3389/fimmu.2019.03033
Language: English
Additional information: This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) (https://creativecommons.org/licenses/by/4.0/). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
Keywords: microglia, transcriptome, uveitis, retina, resolution, mRNA-Seq, lipopolysaccharide (LPS), heterogeneity
UCL classification: UCL
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences > Institute of Ophthalmology
URI: https://discovery.ucl.ac.uk/id/eprint/10092773
Downloads since deposit
29Downloads
Download activity - last month
Download activity - last 12 months
Downloads by country - last 12 months

Archive Staff Only

View Item View Item