SINGLE-CELL MECHANISMS OF DYNAMIC NF-κB SIGNALING THAT ORCHESTRATE TISSUE LEVEL INNATE IMMUNITY

To coordinate multicellular processes, individual cells receive and release signals that are interpreted via signaling networks. Single-cell live imaging techniques have revealed that cells use signaling dynamics, i.e. temporal patterns of activity, to encode specificity of input to output. Specificity is particularly important in the context of innate immunity because either hyper- or hypo-inflammation can result in disease. In this study we define molecular mechanisms that shape signaling dynamics in the NF-κB network, a major regulator of the inflammatory response. We find that NF-κB oscillations shared by the Toll Like and IL1 receptor pathways dampen with increasing input concentration. We use novel optogenetic tools to demonstrate that Interleukin-1 Receptor-Associated Kinase1 (IRAK1) autophosphorylation controls dose-dependent termination of signal and tolerance to subsequent inputs. We conclude that IRAK1 kinase activity is dispensable for signaling but critical to NF-κB signaling dynamics. To further define the physiological role of this feedback mechanism, we expanded the study to include tissue-level models. Thus, we develop a mammary epithelial cell line that reports both the translocation and gene expression of NF-κB and tested a panel of inflammatory inputs at different concentrations. In agreement with our previous findings, we establish that pathways signaling through the MyD88-IRAK4-IRAK1 complex generate a single pulse of NF-κB translocation, while others create more oscillatory dynamics. The screen also revealed that bacterial MAMPs activate only a small fraction of the epithelial monolayer in an all-or-nothing (i.e. digital) manner. We show that the fraction of responsive cells is regulated by a bimodal epigenetic switch that permits TLR2 expression and increases with prolonged, high basal inflammatory signaling. This mechanism confers spatially localized adjustment of immune responses depending on the local inflammatory environment. We propose that epigenetic modifications in individual cells enables long-term, quantitative fine-tuning of population-level responses. In summary, we define molecular mechanisms that shape signaling dynamics and demonstrate how single cells impact tissue-level parameters. Moreover, our work highlights the value of live, single-cell imaging to measure the quantitative aspects of the inflammatory response. Future studies will continue to illuminate the purpose of dynamic NF-κB signaling, its importance to the immune system, and general human health