IFNB1-DDX58 Feedback Loop Cell-to-Cell Variability Figure 2. DDX58 and IFNB1 mRNA expression level. 2A. Total mRNA copy number. DDX58 and IFNB1 mRNA were measured by quantitative real- time PCR and the relative IFNB1 and DDX58 expression levels were normalized to ACTB. The columns show the gene expression level 7 hrs post treatment [NDV only (NDV), NDV plus blocking antibodies (NDV+Abs), not infected (ni), not infected with blocking antibodies (ni+Abs)]. Left panel: IFNB1. Right panel: DDX58. Error bars represent measurement error. 2B. Single cell mRNA expression. DDX58 and IFNB1 mRNA in individual DCs were measured 7 hrs post treatment by the hemi-nested PCR protocol illustrated in Supplementary Figure S4. The copy numbers of both DDX58 and IFNB1 in single DCs (normalized to ACTB) were determined for all cells with detectable expression of the mRNAs. Each symbol shows the gene expression in a single cell. The treatments were same as those in Figure 2A, but with uninfected DCs labeled as control (Ctr). Left panel: IFNB1. Right panel: DDX58. doi:10.1371/journal.pone.0016614.g002 cell. The effect of receptor antibody was included by reducing the receptor binding rate. Uninfected cells can be roughly character- ized as those with less than ten copies of IFNB1. The RIG-I mRNA distribution in uninfected cells was considerably shifted downward when antibodies blocked the paracrine loop (Figure 3B), as the paracrine loop acts as an inducer of RIG-I. Furthermore, DDX58 and IFNB1 showed significant correlation within individual cells at 11 hours, as expected when each cell is activated only by autocrine signaling. In order to test the distributions predicted by these simulations, experiments were performed using single cell assays that simulta- neously measured both IFNB1 and DDX58. The single cell data presented in Figure 2B did not include measurement of both transcripts within each individual cell and could not determine whether the small number of cells showing increased IFNB1 and DDX58 levels in the presence of blocking antibodies were the same subset of cells. As had been seen in the simulations, the experimentally obtained co-expression measurements in the presence of blocking antibodies showed a significant correlation between IFNB1 and DDX58 (Figure 4). Moreover, in the presence of antibodies the level of RIG-I expression in cells drops overall (Figure 4), as can also be seen in the simulation results (Figure 3B). The interpretation of these experimental results that use antibodies to block interferon signaling is that while the paracrine signaling appears to be efficiently cancelled, the autocrine loop is leaky, leading to the observed correlation and higher copy numbers of IFNB1 at 11 hours. The persistence of control levels of DDX58 and of IFNB1 in some, presumably uninfected, cells at 11 hours when antibodies were present also confirmed the effectiveness of the blockade of paracrine signaling. Overall, the experimental results are in agreement with the salient features about single cell response distribution in the absence and presence of blocking antibodies that were predicted by the model simulations. This consistency between model and experiment at later times provides some assurance in using simulations to investigate the distribution of responses at early time points. The results of such simulations (Figure 5) support the hypothesis that the initiation of the positive feedback loop results from a very small number of cells that are activated and release interferon at early time-points. This is made clear in Figure 6, where we plot the number of bound receptors (which is connected to the rate of DDX58 induction) versus the IFNB1 copy number for specific cells at different times through the simulation. In the resulting trajectories each point represents the simulation result at time points separated by 10 minutes. Cellular responses fall into typical patterns for uninfected, infected early responder and infected late responder cells. For uninfected cells (Figure 6A) there is no IFNB1 induction despite increasing receptor binding. For early responder cells (Figure 6C) PLoS ONE | www.plosone.org 3 February 2011 | Volume 6 | Issue 2 | e16614