RNA-Seq Reveals Alterations in Homeostatic Microglial Function during Inflammation and Resolution

Purpose: The status of retinal microglia during inflammation and resolution is currently poorly understood. We performed RNA-sequencing on microglia at different timepoints following a single inflammatory stimulus (early, peak infiltrate, and “resolved”) to observe whether they maintain a microglial transcript to subserve homeostatic function, and whether this was altered after resolutionMethods: CX3CR1CreER:R26-tdTomato (C57BL/6) male heterozygotes were administered tamoxifen via different regimes at 4-5 weeks of age. Four weeks post-tamoxifen, they were injected intravitreally with 10 ng lipopolysaccharide (EIU). Six-hundred microglia were isolated from individual retinas by FACS in the following groups: naïve eyes, 4 hours, 18 hours, and 2 weeks post-injection (n = 4).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 of retina, spleen, blood, bone marrow, liver, and kidney at four-weeks post-tamoxifen administration (and of the retinas during EIU) indicates 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 (n = 3); with the subcutaneous route 2.2 times more splenic cells were tdTomatohi, and the specificity during EIU dropped to 93.2% (from >99%).Quantification indicated that, for most samples (22/24), the protocol resulted in good quality cDNA appropriate for obtaining high-quality sequencing runs, with unique alignments of >85% on average (>95% alignment average). At peak cellular infiltrate (18h), 1,068 DEGs were identified (compared to naive controls); in contrast, 12 DEGs were identified at 2 weeks. Significantly-enriched pathways included the IL-17 signalling, cytokine-cytokine receptor interaction, and multiple infectious and autoimmune/autoinflammatory disease pathways.Conclusions: We characterised acute transcriptional changes in microglia, which largely resolve to their original state after 2 weeks; however, during inflammation, large reductions in expression of many “homeostatic” microglial genes, corresponding to loss of partial homeostatic function, was also observed.