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.


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.

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


  • Endotoxins
  • Lipopolysaccharides
  • RNA, Messenger
  • Receptor, Anaphylatoxin C5a