@article{discovery10092773, note = {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.}, publisher = {FRONTIERS MEDIA SA}, volume = {10}, month = {January}, year = {2020}, title = {Single Eye mRNA-Seq Reveals Normalisation of the Retinal Microglial Transcriptome Following Acute Inflammation}, journal = {Frontiers in Immunology}, keywords = {microglia, transcriptome, uveitis, retina, resolution, mRNA-Seq, lipopolysaccharide (LPS), heterogeneity}, url = {https://doi.org/10.3389/fimmu.2019.03033}, author = {Bell, OH and Copland, DA and Ward, A and Nicholson, LB and Lange, CAK and Chu, CJ and Dick, AD}, 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 na{\"i}ve 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 {$\leq$}0.05 and the fold-change was {$\ge$}{$\pm$}2. / Results: Flow cytometric analysis indicates that the Cx3cr1CreER:R26-tdTomato strain is both sensitive ({\ensuremath{>}}95\% tagging) and specific ({\ensuremath{>}}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.} }