New Publication!
Congratulations to Raela Ridley; Sydney Lawton Cristhian Idelfonso, MD on the publication of “RelA Regulates a Specialized Microglial Subset by Suppressing Homeostatic Genes and Driving IL-β Expression,” which appears in the June edition of IOVS.
Abstract
Purpose : RelA(p65) is a key regulator of the NF-kB pathway and inflammatory gene expression. Our previous work has indicated that RelA is essential for the inflammatory response in microglia; however, the underlying mechanisms remain unclear. In this study, we aim to define how RelA modulates microglial inflammation through transcriptomic and epigenetic analysis of enriched microglial cells.
Methods : We knocked out RelA in the microglia of Cx3cr1CreERT2:RelAflx (microgliaRelAcKO) using five daily tamoxifen injections (75 mg/kg IP) followed by a 30-day washout. Tamoxifen-treated microgliaRelAflx mice served as controls. We employed the inducible EAU and EIU models to study the effects of RelA loss on retinal inflammation. Immune cell infiltration and retinal structure were evaluated via OCT, and IL-1β levels were measured with ELISA. Microglia morphology was analyzed using Sholl analysis of retinal flat mounts, while transcriptional changes were assessed with bulk and single-cell RNA sequencing of CD11b-enriched microglia treated with TNF or saline. Lastly, we used CUT & RUN analysis on flow-sorted microglia to examine H3K4me3 modifications.
Results : Our results show that RelA can only be knocked out in ~10% of microglial cells, despite expectations of widespread expression. OCT results indicate that RelA loss does not affect immune cell infiltration in EIU and EAU models, suggesting it’s not required for recruiting inflammatory cells in uveitis. However, IL-1β expression, usually elevated in EAU, was undetectable in microgliaRelAcKO retinal lysates. Sholl analysis showed that EAU microglia lacking RelA were less ramified, while those in EIU were more ramified. Gene expression analysis of CD11b-enriched microglia from TNF-treated retinas showed increased homeostatic microglia markers, such as Tmem119 (5.7-fold change) and P2ry12 (3.5-fold change).
Conclusions : RelA is expressed in a subset of microglial cells, and its deletion halts IL-1β production without affecting the recruitment of inflammatory cells in uveoretinitis. This deletion also increases the expression of homeostatic markers in microglia, regardless of the presence of TNF. We have identified RelA-positive microglia in the retina that mediate the endogenous inflammatory response. Future studies will explore RelA’s role in chronic inflammatory mouse models and examine other co-regulating transcription factors like Stat3 and SPi1.