IFNB1-DDX58 Feedback Loop Cell-to-Cell Variability Figure 3. Single DC simulations with and without IFN-blocking antibodies. 3A. Time course of the average copy number of IFNB1 (solid line) and of DDX58 (dashed line) obtained from the simulation with the ABM model. 3B. Scatter plots display IFNB1 (X axis) and DDX58 (Y axis) mRNAs copy numbers in each single DC as simulated with the ABM model. Simulations without blocking antibodies (upper panel) or with blocking antibodies (lower panels) are shown at 6 hrs (left) and 11 hrs (right) post infection. doi:10.1371/journal.pone.0016614.g003 there is first IFNB1 induction followed by increasing numbers of bound receptors and IFNB1 through early autocrine and later paracrine signaling. For late responder cells (Figure 6B) the behavior at early times is opposite to that of early responders with infinite slope for the trajectory instead of zero slope: at first the number of bound receptors increases without any production of IFNB1 message, indicating paracrine signaling. Later in the simulation this leads to IFNB1 production with numbers increasing rapidly through the positive feedback loop. Notably, the ratio between early and late responders is 7:124 in the simulation, supporting the hypothesis that a small percentage of the population is responsible for activating the whole culture of cells. Figure 6D follows the same cell in two simulations, one without antibodies (solid line), and one with antibodies (dashed line), and highlights the effect of suppressing paracrine signaling on late responder cells. In the simulation with no antibodies the cell exhibits a clear late responder trajectory. However, when the paracrine signaling is suppressed the trajectory becomes similar to that of an early responder, where IFNB1 induction starts before any receptors are bound. This induction occurs late in the simulation and results in lower steady state values, due to partial blocking of the feedback loop. Thus far we have shown that the proposed mechanism (of a sub population of early responders efficiently activating the rest of the cells) is consistent with the experimental results. Cell-to-cell variations are essential for the process to occur so that interferon- induced genes will seem to be activated prior to significant interferon activation. To test this conjecture, we ran the simulation with decreasing levels of cell-to-cell variability, while maintaining the same average initial RIG-I concentration, and the same percentage of early responding cells To this end, the sensitivity of IFNB1 induction to the concentration of RIG-I (parameter c) was increased (see Supplementary Text S2, Changing the Variance and Maintaining Early Responder Percentage, for details). Figure 7 PLoS ONE | www.plosone.org 4 February 2011 | Volume 6 | Issue 2 | e16614