Research ArticleImmunology

Single-cell analysis shows that paracrine signaling by first responder cells shapes the interferon-β response to viral infection

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Science Signaling  10 Feb 2015:
Vol. 8, Issue 363, pp. ra16
DOI: 10.1126/scisignal.2005728
  • Fig. 1 Single-cell measurement of individual mRNA molecules in situ.

    Schematic representation of the measurement of single molecules of mRNA in situ. DCs were attached to precoated coverslips before being infected with NDV. The in situ hybridization assay was performed with fluorescently tagged oligonucleotide probes to measure the abundances of Ifnb1 mRNA (probes indicated in red) and HN mRNA (probes indicated in blue) simultaneously in each cell. The fluorescent, spot-like signals developed in the cell for each of the target mRNAs were imaged as z-stacks with an epifluorescence microscope. Each image (z-stack) was processed computationally to determine fluorescent spot counts and the numbers of each target mRNA per cell as described in Materials and Methods.

  • Fig. 2 Measurement of Ifnb1 and HN mRNA amounts at the single-cell level after viral infection.

    (A and B) Representative digital interference contrast (DIC) and high-resolution images showing the intracellular distribution of Ifnb1 mRNAs (red spots) and viral HN mRNAs (purple spots) in individual (circled) DCs (A) 12 hours after they were infected with NDV or (B) that were left uninfected (mock). The fluorescent probe images shown are flattened merges of z-stacks of deconvolved fluorescent images. Scale bars, 5 μm. (C) Scatter plot of the distribution of the numbers of Ifnb1 and HN mRNAs per cell measured 12 hours after NDV infection. Note that single-cell, single-molecule measurements yield numbers of mRNA molecules per cell and, thus, discrete values. Circles on the x and y axes represent the overlap of multiple cells, and the numbers in parentheses indicate the number of overlapping cells that are represented by the same symbol. Data are from 220 cells of a representative experiment out of three independent experiments. HN values when Ifnb1 = 0 were binned for clarity of presentation. Inset: The same scatter plot analysis was performed with uninfected control DCs (Mock) in the same experiment. (D) Percentages of DCs expressing the indicated mRNAs when left uninfected (0 hour) or 12 hours after infection with NDV. (E) Average numbers of the indicated mRNAs per cell in DCs that were left uninfected (0 hour) or that were infected for 12 hours with NDV. Data in (D) and (E) are means ± SEM of 670 cells from three independent experiments.

  • Fig. 3 Temporal dynamics of single-cell Ifnb1 responses to viral infection.

    (A) Distribution of Ifnb1-expressing DCs at the indicated times after NDV infection. Data are the percentages of DCs that had the indicated numbers of Ifnb1 mRNAs at each time point. Underlined tick labels in the x axis indicate the center of histogram bins with a bin width of 10 mRNA molecules. Inset: A similar analysis of the distribution of HN mRNA–expressing cells over time was performed. For clarity, only results at 10 hours are shown. Data are from 750 cells analyzed in a representative experiment out of three independent experiments. (B) Distribution of the numbers of Ifnb1 and HN mRNAs per cell measured at the indicated times after NDV infection. Time points are represented by the same colors used in (A). The squares of the Pearson correlation coefficients between both mRNAs (r2) at 2, 4, 6, and 10 hours after infection are 0.00, 0.05, 0.04, and 0.05, respectively. Circles on the x and y axes represent overlapping cells, and the number next to each of these circles represents the number of cells per circle. Data are from 750 cells analyzed in the representative experiment used in (A). HN values when Ifnb1 = 0 were binned for clarity of presentation. Inset: A similar distribution analysis was performed with uninfected (mock) control DCs (shown at 12 hours). (C) Percentages of Ifnb1-expressing cells (black), HN-expressing cells (light gray), and cells expressing both Ifnb1 and HN (dark gray) as a function of time after NDV infection (black line). The pink dotted line represents the mean percentage of the total cells that were infected with NDV, which was estimated on the basis of the numbers of cells that had detectable HN mRNA at 10 hours after infection. (D) Average numbers of Ifnb1 mRNAs per cell calculated only for cells expressing Ifnb1 (black line) or for all cells (gray line) at the indicated times after infection. Data in (C) and (D) are means ± SEM of 2500 cells from three independent experiments.

  • Fig. 4 The temporal dynamics of single-cell Ifnb1 responses to LPS differ from those to viral infection.

    (A to C) DCs were left untreated or were treated with LPS (100 ng/ml) for the indicated times. The numbers of Ifnb1 mRNAs per cell were then measured by single-molecule imaging of RNA in situ. (A) Distribution of Ifnb1-expressing cells at the indicated times after LPS treatment. Underlined tick labels in the x axis indicate the center of histogram bins with a bin width of 10 mRNA molecules. Data are from 1000 cells analyzed in a representative experiment out of three independent experiments. (B) Percentages of Ifnb1-expressing cells quantified as a function of time after LPS treatment. (C) Average numbers of Ifnb1 mRNA molecules per cell calculated only for cells expressing Ifnb1 (black line) or for all cells (gray line) as a function of time after LPS treatment. Data in (B) and (C) are means ± SEM of 2400 cells analyzed in three independent experiments.

  • Fig. 5 Stochastic features and propagation modes studied to fit single-cell Ifnb1 responses.

    (A) We explored four stochastic features: (i) whether an individual cell is infected by virus, which is probabilistic, or exposed to a pathogenic component, which is set to 1 for LPS (infectivity, I); (ii) whether an infected or exposed cell exhibits Ifnb1 expression (the “Ifnb1 response”), which is probabilistic (response, R); (iii) cell-to-cell variability in the rate of expression of Ifnb1 (strength, S); and (iv) cell-to-cell variability in the time of initiation of the Ifnb1 response after infection or exposure (time, T). (B) Top: Representative simulations of 250 cells for the three indicated combinations of stochastic features or propagation modes of interest: IRS, IRT, and IRST. (Bottom) Schematic for the propagation modes IRS, IRT, and IRST.

  • Fig. 6 Viral infection elicits a single-cell Ifnb1 response pattern that is characterized by temporal dispersion.

    (A) Flow diagram describing the stochastic feature screening approach applied to analyze experimental single-cell Ifnb1 response data. (B) Characteristic propagation mode of the single-cell Ifnb1 response to viral infection. Cost function representing the goodness of fit of the IRS, IRT, and IRST modes to the single-cell Ifnb1 responses obtained experimentally from DCs infected for 2, 4, 6, and 10 hours with NDV at MOIs of 1 (left) and 4 (right). Similar results were obtained for three independent data sets. Box plots depict the result of 20 simulations for each propagation mode. Error bars represent the maximum and minimum values of all data. (C) Characteristic propagation mode of the single-cell Ifnb1 response to LPS. Cost function representing the goodness of fit of the IRS, IRT, and IRST models to the single-cell Ifnb1 responses obtained experimentally from DCs treated for 1, 2, and 4 hours with LPS at concentrations of 10 ng/ml (left) and 100 ng/ml (right). Similar results were obtained for three data sets. Box plots depict the result of 20 simulations for each propagation mode. Error bars represent the maximum and minimum values of all data. The stochastic feature time (T) in the propagation modes IRT and IRST was strictly enforced. See Supplementary Materials for the range of parameters explored for each stochastic feature. ***P < 0.001 by analysis of variance (ANOVA) on ranks, because cost function values were not normally distributed, followed by Tukey test.

  • Fig. 7 Paracrine signaling drives the temporal dispersion of single-cell Ifnb1 responses to viral infection.

    (A) Percentage of HN-expressing DCs as a function of the time of exposure to NDV. Data are means ± SD of duplicate samples from one experiment and are representative of 1200 cells from two independent experiments. (B) Percentage of Ifnb1-expressing cells (left) and the average number of Ifnb1 mRNAs per cell (right) as a function of time after NDV infection in the presence (red lines) or absence (blue lines) of brefeldin A (5 μg/ml), which was added 40 min after exposure of the cells to virus. Data are from two independent experiments. (C) Changes in characteristic propagation mode after treatment with brefeldin A. Cost functions representing the goodness of fit of the IRS, IRT, and IRST modes to the single-cell Ifnb1 responses obtained experimentally from A549 cells infected for 1, 2, or 4 hours with NDV (MOI = 1) in the absence (left) or presence (right) of brefeldin A. Similar results were obtained for two independent data sets. Box plots depict the results of 20 simulations for each propagation mode. Error bars represent the maximum and minimum values of all data. Temporal dispersion (T) in the propagation modes IRT and IRST was strictly enforced. See Supplementary Materials for the range of parameters explored for each stochastic feature. ***P < 0.001, **P < 0.05 as determined by ANOVA on ranks followed by Tukey test. (D) Probability of finding more than one Ifnb1-expressing cell in a single field (142 × 106 μm). Black data points and the solid line indicate values for all experiments. Gray data points and the dotted line indicate the average values expected if Ifnb1-expressing cells were spatially distributed at random. Values were obtained by reshuffling the experimental data at each time point 100,000 times and randomly reassigning cells regardless to different fields as described in Materials and Methods. *P < 0.00001. (E) Percentage of Ifnb1-expressing cells as a function of time after infection with NDV under conditions in which cells were plated at 100 × 103 cells per field (gray) or 800 × 103 cells per field (black). *P < 0.05, as determined by ANOVA on ranks followed by Tukey test.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/8/363/ra16/DC1

    Computational methods

    Fig. S1. Validation of the single-molecule imaging of mRNA in situ.

    Fig. S2.Temporal dynamics of single-cell Ifnb1 mRNA responses to viral infection at an MOI of 4.

    Fig. S3. The percentages of Ifnb1-expressing cells are independent of the concentration of LPS.

    Fig. S4. Temporal dispersion is a characteristic stochastic feature of the Ifnb1 response after viral infection.

    Fig. S5. The faster dynamics of Ifnb1 expression in response to LPS does not change its characteristic propagation mode.

    Fig. S6. Comparison between experimentally measured and simulated average numbers of Ifnb1 mRNAs per cell and the percentages of responding cells.

    Fig. S7. Temporal dynamics of single-cell Ifnb1 responses in response to viral infection of A549 cells.

    Fig. S8. Brefeldin A has no effect on the infectivity of A549 cells.

    Table. S1. Sequences of oligonucleotide FISH probes specific for viral HN mRNA and tagged with Quasar 670.

    Table. S2. Sequences of oligonucleotide FISH probes specific for Ifnb1 mRNA and tagged with Quasar 570.

    Table. S3. Summary model parameter values.

  • Supplementary Materials for:

    Single-cell analysis shows that paracrine signaling by first responder cells shapes the interferon-β response to viral infection

    Sonali Patil, Miguel Fribourg, Yongchao Ge, Mona Batish, Sanjay Tyagi, Fernand Hayot, Stuart C. Sealfon*

    *Corresponding author. E-mail: stuart.sealfon{at}mssm.edu

    This PDF file includes:

    • Computational methods
    • Fig. S1. Validation of the single-molecule imaging of mRNA in situ.
    • Fig. S2.Temporal dynamics of single-cell Ifnb1 mRNA responses to viral infection at an MOI of 4.
    • Fig. S3. The percentages of Ifnb1-expressing cells are independent of the concentration of LPS.
    • Fig. S4. Temporal dispersion is a characteristic stochastic feature of the Ifnb1 response after viral infection.
    • Fig. S5. The faster dynamics of Ifnb1 expression in response to LPS does not change its characteristic propagation mode.
    • Fig. S6. Comparison between experimentally measured and simulated average numbers of Ifnb1 mRNAs per cell and the percentages of responding cells.
    • Fig. S7. Temporal dynamics of single-cell Ifnb1 responses in response to viral infection of A549 cells.
    • Fig. S8. Brefeldin A has no effect on the infectivity of A549 cells.
    • Table. S1. Sequences of oligonucleotide FISH probes specific for viral HN mRNA and tagged with Quasar 670.
    • Table. S2. Sequences of oligonucleotide FISH probes specific for Ifnb1 mRNA and tagged with Quasar 570.
    • Table. S3. Summary model parameter values.

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    Citation: S. Patil, M. Fribourg, Y. Ge, M. Batish, S. Tyagi, F. Hayot, S. C. Sealfon, Single-cell analysis shows that paracrine signaling by first responder cells shapes the interferon-β response to viral infection. Sci. Signal. 8, ra16 (2015).

    © 2015 American Association for the Advancement of Science

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