Toll-like receptors (TLRs) recognize specific pathogen-associated molecular patterns and initiate innate immune responses through signaling pathways that depend on the adaptor proteins MyD88 (myeloid differentiation marker 88) or TRIF (TIR domain-containing adaptor protein-inducing interferon-β). TLR4, in particular, uses both adaptor proteins to activate the transcription factor nuclear factor κB (NF-κB); however, the specificity and redundancy of these two pathways remain to be elucidated. We developed a mathematical model to show how each pathway encodes distinct dynamical features of NF-κB activity and makes distinct contributions to the high variability observed in single-cell measurements. The assembly of a macromolecular signaling platform around MyD88 associated with receptors at the cell surface determined the timing of initial responses to generate a reliable, digital NF-κB signal. In contrast, ligand-induced receptor internalization into endosomes produced noisy, delayed, yet sustained NF-κB signals through TRIF. With iterative mathematical model development, we predicted the molecular mechanisms by which the MyD88- and TRIF-mediated pathways provide ligand concentration-dependent signaling dynamics that transmit information about the pathogen threat.

The innate immune response is largely initiated by pathogen-responsive activation of the transcription factor IRF3. Among other target genes, IRF3 controls the expression of IFN-β, which triggers the activation of the transcription factor ISGF3 via the IFNAR. IRF3 and ISGF3 have been reported to control many of the same target genes and together, control the antimicrobial innate-immune program; however, their respective contributions and specificities remain unclear. Here, we used genomic technologies to characterize their specificity in terms of their physical DNA-binding and genetic function. With the use of ChiP-seq and transcriptomic measurements in WT versus ifnar(-/-) versus ifnar(-/-)irf3(-/-) macrophages responding to intracellular dsRNA, we confirmed the known ISGF3 DNA-binding motif and further specified a distinct IRF3 consensus sequence. The functional specificity of IRF3 is particularly pronounced in cytokine/chemokine regulation; yet, even in the control of IFN-β, that specificity is not absolute. By mathematically modeling IFN-β production within an abstracted tissue layer, we find that IRF3 versus ISGF3 specificity may be critical to limiting IFN-β production and ISGF3 activation, temporally and spatially, but that partial overlap in their specificity is tolerable and may enhance the effectiveness of the innate-immune response.