Single Eye mRNA-Seq Identifies Heterogeneity in Retinal Microglial Function during Acute Inflammation

Background: Understanding the heterogeneity of microglial responses during inflammation is critical in identifying changes specific to responders to identify targets for therapies. We performed single eye mRNA-sequencing on retinal microglia at different timepoints following a single inflammatory stimulus (early, peak infiltrate, and at resolution) in endotoxin-induced uveitis (EIU) and characterised their transcriptome.Methods: CX3CR1CreER:R26-tdTomato (C57BL/6) male heterozygotes were administered tamoxifen via different regimes at 4-5 weeks of age. 4 weeks post-tamoxifen, they were injected intravitreally with 10 ng lipopolysaccharide (EIU). 600 microglia were isolated from individual retinas by FACS in the following groups: naïve eyes, 4 hours, 18 hours, and 2 weeks post-injection. 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. Key marker changes were validated by flow cytometry and fluorescence microscopy.Results: Flow cytometric analysis at 4 weeks post-tamoxifen administration (and of the retinas during EIU) indicates that the CX3CR1CreER:R26-tdTomato mouse line is both sensitive (>95% tagging) and specific (>95% specificity) for microglia when tamoxifen is administered topically to the eye. During “early” activation, 613 DEGs were identified; in contrast, 537 were found during peak cellular infiltrate and 0 at 2 weeks, compared to baseline controls (1,069 total unique DEGs). Flow cytometry identified a generalised microglial response of proteins involved in cell-cell adhesion and migration, but also a subset of changes relating to lymphocyte activation and inhibition which were exclusive to C5ar1-expressing microglia.Conclusion: mRNA-Seq identified changes in transcriptome in the microglial population during EIU that resolved to steady state by 2 weeks. C5ar1 expression delineates subtypes of microglia during an acute LPS response.