IFNB1-DDX58 Feedback Loop Cell-to-Cell Variability Figure 4. Experimental results in individual DCs with and without blocking antibodies. The scatter plots display IFNB1 (X axis) and DDX58 (Y axis) copy numbers in individual cells, as determined by the hemi-nested single cell qRT-PCR, and normalized to ACTB. 4A. IFNB1 and DDX58 expression in single DCs with no NDV infection and no antibody blockage. 4B. IFNB1 and DDX58 expression in single DCs following NDV infection. In the same order as in Figure 3B, experiments without blocking antibodies (upper panels) or with blocking antibodies (lower panels) are shown at 6 hrs (left) and 11 hrs (right) post infection. Note that the dataset for 6 hours was obtained from donor 1, while the datasets for 11 hours and uninfected control were obtained from donor 2. doi:10.1371/journal.pone.0016614.g004 shows the original simulation results and the results of a simulation with initial conditions with 10 times less variability in the initial DDX58 distribution. As the variance is decreased, the sensitivity of IFNB1 becomes large enough for small fluctuations in the concentration of RIG-I to significantly push a cell closer to activation. As a result, many cells are activated by their internal levels of RIG-I (which is not significantly different from the average) and so the average activation of IFNB1 occurs earlier. Figure 7 confirms that decreasing the variability results in similar activation times for IFNB1 and DDX58, supporting our claim that variability is essential in order to induce the dynamics seen in the system. We also note that the simulation with reduced variability results in significantly higher levels of interferon, which can be harmful. As such, the early responder dynamics allows an efficient response to the viral infection while maintaining controlled levels of interferon induction. Discussion The general picture that emerges from our experimental and modeling studies of the early immune response to viral infection provides a fine grain, mechanistic view of how a sub-population of early responder cells can facilitate the activation of a positive feedback loop in the whole population of cells. Two waves of Type I IFN induction by NDV infection of murine fibroblasts were previously observed and attributed to a positive feedback loop of IFNb and IFNa4 inducing IRF7 [13]. Our single cell level study results are consistent with a positive feedback loop at the population level [13]. At early time points after viral infection, only a few infected cells respond to the infection, while all others remain unaltered. The early responder cells produce and secrete interferon that – after binding to interferon receptors – induces DDX58 both in themselves and in neighboring cells. If a neighboring cell is already infected, the increased amount of DDX58 will facilitate viral detection and subsequent IFNB1 production. This process of IFNB1 induction is stochastic, depending on many sources of noise, among which are the time to signaling component activation and subsequent time to IFNB1 enhanceosome formation. Even if a neighboring cell is not infected it will be primed through the induction of DDX58 to better resist subsequent viral infection. The result is that in all cells DDX58 is increased beyond the control level (Figure 2B) a few hours after Figure 5. Single cell simulations of early responder DCs. Simulation scatter plots of IFNB1 (X axis) and of DDX58 (Y axis) copy numbers in individual DCs at 1, 2, 3 and 4 hours post infection. The simulations were performed with 1000 cells in order to show a sufficient number of cells responding to infection at early time points. doi:10.1371/journal.pone.0016614.g005 PLoS ONE | www.plosone.org 6 February 2011 | Volume 6 | Issue 2 | e16614