Six distinct NF?B signaling codons convey discrete information to distinguish stimuli and enable appropriate macrophage responses

Macrophages initiate in?ammatory responses via the transcription factor NF?B. The temporal pattern of NF?B activity determines which genes are expressed and thus, the type of response that ensues. Here, we examined how information about the stimulus is encoded in the dynamics of NF?B activity. We generated an mVenus-RelA reporter mouse line to enable high-throughput live-cell analysis of primary macrophages responding to host- and pathogen-derived stimuli. An information-theoretic work?ow identi?ed six dynamical features—termed signaling codons—that convey stimulus information to the nucleus. In particular, oscillatory trajectories were a hallmark of responses to cytokine but not pathogen-derived stimuli. Single-cell imaging and RNA sequencing of macrophages from a mouse model of Sjögren's syndrome revealed inappropriate responses to stimuli, suggestive of confusion of two NF?B signaling codons. Thus, the dynamics of NF?B signaling classify immune threats through six signaling codons, and signal confusion based on defective codon deployment may underlie the etiology of some in?ammatory diseases. Overall design: Single cell RNAseq from Wildtype and Sjögren's Syndrome mouse BMDMs stimulated with 10ng/ml TNF, 100ng/ml LPS, or 50ug/ml p(I:C) for 8hours.