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

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

JOURNAL=Frontiers in Immunology

VOLUME=10

YEAR=2020

URL=https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2019.03033

DOI=10.3389/fimmu.2019.03033

ISSN=1664-3224

ABSTRACT=<p><bold>Background:</bold> 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).</p><p><bold>Experimental Approach:</bold><italic>Cx3cr1</italic><sup><italic>CreER</italic></sup><italic>:R26-tdTomato</italic> (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 <italic>p</italic>-value was ≤0.05 and the fold-change was ≥±2.</p><p><bold>Results:</bold> Flow cytometric analysis indicates that the <italic>Cx3cr1</italic><sup><italic>CreER</italic></sup><italic>:R26-tdTomato</italic> strain is both sensitive (&gt;95% tagging) and specific (&gt;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.</p><p><bold>Conclusion:</bold> 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.</p>