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

Research output: Contribution to conferenceAbstract

Original languageEnglish
DatePublished - 1 Jul 2019
EventARVO Annual Meeting 2019 - Vancouver, Canada
Duration: 28 Apr 20192 May 2019


ConferenceARVO Annual Meeting 2019


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.


ARVO Annual Meeting 2019

Duration28 Apr 20192 May 2019

Event: Conference



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