Abstract
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.
Original language | English |
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Publication status | Accepted/In press - 14 Oct 2019 |
Event | British Society for Immunology Congress 2019 - Liverpool , United Kingdom Duration: 2 Dec 2019 → 5 Dec 2019 |
Conference
Conference | British Society for Immunology Congress 2019 |
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Abbreviated title | BSI Congress 2019 |
Country/Territory | United Kingdom |
City | Liverpool |
Period | 2/12/19 → 5/12/19 |
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Dive into the research topics of 'Single Eye mRNA-Seq Identifies Heterogeneity in Retinal Microglial Function during Acute Inflammation'. Together they form a unique fingerprint.Student theses
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Transcriptional plasticity of microglia during intraocular inflammation
Bell, O. (Author), Copland, D. A. (Supervisor), Chu, C. (Supervisor) & Dick, A. D. (Supervisor), 21 Jan 2021Student thesis: Doctoral Thesis › Doctor of Philosophy (PhD)
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