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.
Highlights
It has been challenging to investigate microglia during inflammatory contexts primarily because distinguishing them from infiltrating monocytes and macrophages has been unreliable due to similar expression of cell-surface markers [1,2,3,4,5,6]
We wished to determine if the microglial transcriptome resets after an acute and resolving insult, or if homeostatic thresholds have been reset or altered permanently
We show that the retinal microglia undergo acute transcriptional changes which resolve to their original homeostatic state by 2 weeks and support microglial heterogeneity in response to inflammatory signals
Summary
It has been challenging to investigate microglia during inflammatory contexts primarily because distinguishing them from infiltrating monocytes and macrophages has been unreliable due to similar expression of cell-surface markers [1,2,3,4,5,6]. Conflicting data remains on how microglia regulate or promote inflammation depending on insult [9, 10]. During inflammation microglia alter their homeostatic state [8, 11,12,13]. We wished to determine if the microglial transcriptome resets after an acute and resolving insult, or if homeostatic thresholds have been reset or altered permanently. 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). 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. Genes were considered differentially-expressed (DEG) if the FDR step-up p-value was ≤0.05 and the fold-change was ≥±2
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