Double Knock-In Reporter Mice Reveal NF-κB Trajectories in Signaling, Immune Cell Development, and Aging

Abstract

NF-κB is a transcription factor whose regulatory control reaches far beyond the immune system. In vitro studies suggest that mapping the spatiotemporal complexity of NF-κB signaling in primary cells and in vivo is essential to understanding its function in health and disease, but the lack of tools to directly monitor NF-κB protein components has hindered such efforts. To address this need, we generated reporter mice with the endogenous RelA (p65) or c-Rel labeled with distinct fluorescent proteins and a double knock-in with both labeled subunits. Overcoming hurdles in simultaneous live cell imaging of RelA and c-Rel in cells from the reporter mice, we found that the quantitative features of signaling reflect the identity of activating ligands, differ between primary and immortalized cells of the same type, and shift toward c-Rel in microglia from aged brains. We also identified an unexpected depletion of nuclear RelA:c-Rel heterodimers in stimulated cells. Quantitative same-cell measurements in these mice revealed a trajectory of subunit expression in several immune lineages, with a surprising downregulation at key cell maturation stages. These data begin to reveal the power of these reporters in gaining deeper insights to NF-κB-linked biology, with the spectral complementarity of the labeled NF-κB proteins enabling diverse applications from single-molecule analyses to in situ identification of cells in active inflammatory states.

Keywords: NF-κB, RelA, c-Rel, fluorescent fusion reporter mice, endogenous knock-in, live microscopy, fluorescence correlation spectroscopy, intravital imaging, inflammatory signaling.