Single Eye mRNA-Seq Reveals Normalisation of the Retinal Microglial Transcriptome Following Acute Inflammation

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: Cx3cr1 CreER :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 Cx3cr1 CreER :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.

Applications of single-cell sequencing in cancer research: progress and perspectives

“…Moreover, SMART-Seq v4 is more efficient in template switching, which shortens the time of cDNA synthesis and cDNA library construction, and has a higher sensitivity for low input and higher reproducibility [ 28 ]. SMART-Seq v2 and v4 are widely utilized in cancer research, such as research on hepatocellular carcinoma (HCC) [ 29 – 31 ]. In addition, SMART-Seq does not require additional equipment, which means that it largely depends on experienced researchers.…”

Sectoral activation of glia in an inducible mouse model of autosomal dominant retinitis pigmentosa

“…Vol. :(0123456789) Scientific Reports | (2020) 10:16967 | https://doi.org/10.1038/s41598-020-73749-y www.nature.com/scientificreports/ Interestingly, microglia are capable of defaulting to the baseline, homeostatic transcriptome weeks after acute inflammation in endotoxin uveitis 47 . On the other hand, microglia in slowly progressive forms of RP may remain pro-inflammatory even after significant photoreceptor loss, which has been demonstrated in the P23H RHO rat 48 .…”

“…We found that microglia in the I307N Rho mouse reduce in number, move towards homeostatic positioning among the retinal layers, and exhibit a more ramified phenotype in the weeks following light exposure. Interestingly, microglia are capable of defaulting to the baseline, homeostatic transcriptome weeks after acute inflammation in endotoxin uveitis 47 . On the other hand, microglia in slowly progressive forms of RP may remain pro-inflammatory even after significant photoreceptor loss, which has been demonstrated in the P23H RHO rat 48 .…”

Quantitative Assessment of Experimental Ocular Inflammatory Disease

“…One essential tool for advancing understanding of these different tissue states is a rigorous method of clinical assessment that separates healthy tissue from the vulnerable and that also distinguishes between different states of the vulnerable tissue. Such a scheme could then complement studies describing gene expression in different forms of ocular inflammation (13,40). Recent advances in the range and quality of techniques that can be applied to quantify ocular inflammatory disease make such objective and transferrable assessments increasingly feasible.…”

“…experimental autoimmune uveitis; EAU), inspired in the mouse by early work identifying susceptible strains (3,4) and used widely to probe important aspects of immune function including tolerance (5,6), regulation (7,8), microbiome (9), lymphocyte dynamics (10) and macrophage/monocyte function (11). But other models of ocular inflammation are also important, including endotoxin induced uveitis (EIU) (12)(13)(14) and primed mycobacterial uveitis (PMU) (15). Ocular infectious disease can also be studied and has proven to be an informative model of inflammation (16)(17)(18).…”