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Single eye mRNA-Seq reveals normalisation of the retinal microglial transcriptome following acute inflammation

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  • معلومة اضافية
    • الموضوع:
      2020
    • Collection:
      University of Freiburg: FreiDok
    • نبذة مختصرة :
      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.
    • File Description:
      pdf
    • Relation:
      https://freidok.uni-freiburg.de/data/155186
    • الرقم المعرف:
      10.3389/fimmu.2019.03033
    • الدخول الالكتروني :
      https://freidok.uni-freiburg.de/data/155186
      https://nbn-resolving.org/urn:nbn:de:bsz:25-freidok-1551868
      https://doi.org/10.3389/fimmu.2019.03033
      https://freidok.uni-freiburg.de/dnb/download/155186
    • Rights:
      free
    • الرقم المعرف:
      edsbas.B60D1E01