Figure 7. Single DC simulation with high and low cell-to-cell variation. Time course of the average copy number of IFNB1 per infected cell (solid line) and of DDX58 (dashed line) obtained from the simulation with the ABM model. The lines marked by ‘‘Using Fitted Variance’’ are drawn from a simulation with parameters fitted to the experimental data, and are identical to the ones shown in Fig. 3A. The lines marked by ‘‘Using Reduced Variance’’ result from a simulation in which the variance of the initial DDX58 concentration was reduced 10- fold. In order to ensure a similar number of early responder cells, the sensitivity of IFNB1 to RIG-I concentration was increased by more than 30-fold. The reduction in variability leads to results that do not account for the observed delay in IFN induction relative to DDX58 induction. Furthermore the highly variable system generates low levels of early interferon signaling that can initiate antiviral responses without being prone to later high and potentially toxic levels of interferon secretion. doi:10.1371/journal.pone.0016614.g007 We are aware that, although DDX58 induction plays a role in IFNB1 induction through its contribution to viral detection, IRF7, which is induced at the same time as RIG-I, is important as well in enhancing interferon production. We concentrated on RIG-I because of its participation in the interferon feedback loop, because DDX58 induction averaged over the cell population occurs unexpectedly earlier than IFNB1 induction, and because in the model it appeared reasonable to assume that the distribution of RIG-I across single cells provides the variability that differentiates early from late responders. Eliminating our simplifying assump- tions would impose a huge computational burden for the first assumption, and force the introduction of additional reactions and unknown parameters for the second one. We have also performed simulations at lower DC concentration and checked that, although the activation of the late-responding cells was somewhat delayed, the results obtained are qualitatively similar. In the model, cellular heterogeneity results from a distribution of constitutive RIG-I across cells coupled to a threshold for viral detection. We do not know whether this mechanism is actually the one that distinguishes early responder from late responder cells. We have verified (Supplementary Figure S3) that the heteroge- neity is not due to the degree of DC differentiation, but other sources of cell-to-cell variability can of course exist. Our investigation illustrates some of the advantages of integrating experimentation with modeling for immunological studies. Formal modeling sharpens the development of hypotheses and the interpretations of data. It allows rapid computational experiments and leads to predictions to guide subsequent bench experiments. Obtaining representative single cell measurements in individual infected human dendritic cells at early time points is IFNB1-DDX58 Feedback Loop Cell-to-Cell Variability difficult. The experimental intractability of this problem is sharpened by the inability to detect early responder cells even with the extremely sensitive assay that we used. We note that this assay is more accurate than a reporter construct, and is a direct measurement of promoter activity in individual cells. Direct measurement of IFNb protein production in single cells would be ideal for testing our hypotheses. However, detection of protein level by antibody binding assay would require inhibition of IFNb secretion, which would alter the DC response to viral infection. The limited detection sensitivity at early time points is also a barrier to precisely measuring the single cell level of IFNb. Interestingly, in the key immune response system we have studied, the emergent system-wide behavior results from, and depends on, noise, i.e. the enormous response variability between cells. The noisiness of this system may have physiological importance beyond merely resolving an experimental population level paradox in the DDX58-IFNB1 loop. The high degree of variation in response and its role in initiating the antiviral feedback loop could play a role in contributing to effective antiviral responses in vivo to a wide variety of viral pathogens. The capacity for a few cells to respond early and their capacity to produce an interferon signal that primes other cells could be beneficial in helping mount a response to viruses expressing immune antagonists. Furthermore, the noisiness of the interferon response, in which a few cells are activated but most remain silent, may help in avoiding a cytokine storm while mounting an appropriate response to infection. Materials and Methods Differentiation of DCs All human research protocols for this work have been approved by the IRB of the Mount Sinai School of Medicine. The IRB of the Mount Sinai School specifically waived the need for consent due to use of discarded samples not traceable to source. Monocyte- derived conventional DCs were obtained from human blood donors following a standard protocol [20]. Briefly, human monocytes from buffy coats were isolated by Ficoll density gradient centrifugation (Histopaque, Sigma Aldrich) and CD14+ monocytes were immunomagnetically purified by using a MACS CD14 isolation kit (Miltenyi Biotech.). CD14+ monocytes (0.76106 cells/ml) were later differentiated into immature MDDCs after 5-6 day incubation in DC growth media [RPMI Medium 1640 (Gibco), 10% fetal calf serum (Hyclone), 2 mM of L-glutamine, 100 units/ml penicillin, 100 mg/ml streptomycin (Pen/Strep) (Invitrogen), 500 U/ml hGM-CSF (Preprotech) and 1000 U/ml hIL-4 (Preprotech)] at 37uC. Virus preparation and viral infection The recombinant Hitchner B1 strain of Newcastle disease virus (rNDV/B1) was prepared as previously described [8,14]. Using our established protocol [14], the titered NDV stock was diluted 40 times in Dulbecco’s Modified Eagle Medium (DMEM) and added directly into pelleted MDDCs at a multiplicity of infection (MOI) of 0.5. MOI was measured as previously described by determining the frequency of cells expressing the NDV L gene mRNA [14]. After incubation for 30 minutes at 37uC, fresh DC growth medium was added back to the infected MDDCs (16106cells/ml). Virus free allantoic fluid was added to additional tubes of cells to serve as a negative control. Antibody blockage To block IFN signaling, MDDCs (16106 cells/ml) were pretreated for 30 minutes at 37uC before NDV infection with a PLoS ONE | www.plosone.org 8 February 2011 | Volume 6 | Issue 2 | e16614