Abstract
Purpose :
Microglia exist in different functional states to maintain the health and homeostasis of the retina. However, the mechanisms regulating microglia state transition remain poorly understood. This study aims to test whether manipulation of signal dependent transcription factors, RelA and Stat3 can alter the expression of microglia genes associated with their different functional states.
Methods :
To test our hypothesis, we treated C57BL6J mice with either Tnf, Lif, or equal concentrations of both cytokines. We measured gene expression on whole retina RNA from these mice using RT-qPCR analysis. We performed Transcription Factor Chromatin Immunoprecipitation qPCR (TF ChIP-qPCR) in the Sim A9 microglia cell line to identify RelA and Stat3 binding changes to target genes under different stimuli. We used RT-qPCR to quantify known proinflammatory and anti-inflammatory gene expression in these cells. To test whether Stat3’s interaction with RelA can inhibit inflammatory gene expression, a Lif dose response was completed in a luciferase reporter cell line expressing the NF-kB response element.
Results :
Stimulation of retinas with Tnf and Lif caused a 2.5-fold increase in classical proinflammatory genes macrophage receptor with collagenous structure (Marco) and p91-phox (Cybb). TF ChIP-qPCR studies in Sim A9 microglia cells showed gene-specific antagonistic and synergistic effects of RelA and Stat3 binding to Tnf, Il6, P2ry12, and Cdk1 DNA sequences under different cytokine stimulation. RT-qPCR of cells treated with Tnf showed a 2.5-fold increase in the expression of Tnf and Il6 genes. Similarly, Lif treatment significantly increased the expression of P2ry12 in microglia cells. Finally, increasing doses of Lif in Tnf-treated cells caused a decrease in luciferase activity.
Conclusions :
Our data suggests that Lif-dependent Stat3 signaling promotes microglia homeostatic gene expression in the retina, while Tnf-dependent RelA signaling increases proinflammatory gene expression. These effects appear to be mediated by the selective binding of RelA and Stat3 to DNA sequences within these target genes. These results suggest that RelA and Stat3 activation can regulate microglia gene expression, and potentially their activity. Future studies will elucidate the role of these transcription factors in microglia functional states by integrating microglia-specific conditional KO mouse models and transcriptomics.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.