Research ArticleInflammation

Quantitative analysis of competitive cytokine signaling predicts tissue thresholds for the propagation of macrophage activation

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Science Signaling  24 Jul 2018:
Vol. 11, Issue 540, eaaf3998
DOI: 10.1126/scisignal.aaf3998
  • Fig. 1 TLR4-induced macrophage activation is graded.

    (A) Tissue-level propagation of inflammatory signals: nuclear NF-κB activation (green) and TNF-α production (red) by a single macrophage. (B) Confocal microscopy images of RAW264.7 cells expressing the EGFP-p65 reporter and stimulated with lipid A (500 ng/ml) for the indicated times. (C) Density plots of individual RAW264.7:Gp65 cell traces (total of 432 cells from 91, 76, 59, 87, and 119 cells going from left to right) in response to the indicated lipid A doses (average trajectory indicated by the green line). (D) Analysis of the single-cell traces from (C). Data are mean values of the area under the curve (AUC), maximum peak amplitude and timing, and coefficient of variation (CV) of the AUC of the nuclear NF-κB. The last graph shows the percentage of responding cells. (E) Population-level response of RAW264.7:κB nls-luc cells stably expressing an NF-κB luciferase reporter. Data are means ± SD of triplicate samples per dose of lipid A. a.u., arbitrary units. (F) The NF-κB–dependent production of the indicated mRNAs by RAW264.7 cells stimulated with lipid A (500 ng/ml) for 3 hours was determined by qRT-PCR analysis. Data are means ± SDs of triplicate samples per lipid A dose. (G) The amount of TNF-α in the culture medium of RAW264.7 cells stimulated with the indicated concentrations of lipid A for 3 hours (2.66 × 105 cells in 1 ml) were determined by enzyme-linked immunosorbent assay (ELISA). Data are means ± SD of three replicate experiments per dose of lipid A. *P < 0.05 by Kruskal-Wallis analysis of variance (ANOVA) using Dunn’s correction for multiple comparisons. In (D) and (F), comparison was made to untreated controls. In all other panels, comparison is as indicated.

  • Fig. 2 Heterogeneous NFKBIA and TNFA mRNA abundance is correlated in single cells.

    (A) smFISH analysis of TNFA and NFKBIA mRNA expression. Deconvolved microscopy image of RAW264.7 cells stimulated with lipid A (500 ng/ml) for 3 hours. TNFA mRNA is in red, NFKBIA mRNA is in green, and 4′,6-diamidino-2-phenylindole (DAPI) is in blue. (B) Analysis of the dose-dependent changes in TNFA (left) and NFKBIA (right) mRNA abundance from the experiments represented in (A). Data are present as log10, with median and 25% quartiles for a total of 264, 319, 401, and 270 cells pooled from at least three replicate experiments per dose that were untreated (U) or stimulated with lipid A (10, 100, and 500 ng/ml, respectively). *P < 0.05 by Kruskal-Wallis ANOVA with Dunn’s correction for multiple comparisons. (C) Fano factor calculations of the indicated mRNA numbers from the experiments shown in (B). (D) Correlation between TNFA and NFKBIA mRNA numbers from the experiments shown in (B) depicted with a fitted regression line and Pearson correlation coefficient r (and associated P value). (E) Joint distribution of mRNA numbers from the data shown in (D). (F) Different modes of mRNA regulation: Lipid A dose regulates the range of the response. Inset: Constant response range per given dose. (G) Decomposition of noise in the TNFA and NFKBIA mRNA numbers: extrinsic noise (gray) versus NFKBIA (green) and TNFA (red) noise. Inset: schematics of noise decomposition method. Trunk noise represents extrinsic variability between cells (potentially due to TLR4/NF-κB signaling or generic gene transcription machinery), whereas branch noise corresponds to gene-specific regulation.

  • Fig. 3 TNF-α activates NF-κB–mediated signaling in macrophages.

    (A) Single-cell analysis of NF-κB translocation in RAW264.7:Gp65 cells stimulated with TNF-α (30 ng/ml) for the indicated times. Data are confocal microscopy images of the cells showing the EGFP-p65 signal in green. (B) Density plots of nuclear NF-κB trajectories in RAW264.7:Gp65 cells stimulated with the indicated concentrations of lipid A over time. Data are from 91, 131, 108, 83, and 111 cells for untreated cells and cells treated with the indicated concentrations of TNF-α, respectively, and are pooled from at least three replicate experiments. (C) Characteristics of the single-cell traces from the data shown in (B). AUC values of nuclear NF-κB, maximum peak amplitude and timing, the CV of AUC, and the percentage of responding cells to each concentration of lipid A were determined. *P < 0.05 by Kruskal-Wallis ANOVA against untreated controls and using Dunn’s correction for multiple comparisons. (D) Left: Time-lapse microscopy images of BMDM:p65-DsRedxp cells that were treated with TNF-α (30 ng/ml) for the indicated times. NF-κB p65 is in red, whereas Hoechst nuclear staining is in blue. Right: Corresponding density plots of individual p65-DsRedxp BMDM cell traces across different conditions (average single-cell trajectory in red line). A total of 37 and 39 cells for TNF-α and lipid A stimulation pooled from three replicate experiments were analyzed (49 untreated controls; representative of three replicate experiments). (E) WT BMDMs were treated for 3 hours with the indicated concentrations of TNF-α, and the abundances of the indicated mRNAs were then determined by qRT-PCR analysis. Data are means ± SD of three replicate experiments. (F and G) Analysis of the abundances of the indicated mRNAs in BMDMs that were untreated or treated with TNF-α (30 ng/ml) or lipid A (500 ng/ml) for the indicated times. (F) TNFA mRNA was measured by smFISH [shown as log10(mRNA + 1)]. (G) The abundances of the indicated mRNAs were measured by qRT-PCR analysis. Data are means ± SD of three replicate experiments. *P < 0.05 by Kruskal-Wallis ANOVA with Dunn’s correction for multiple comparisons.

  • Fig. 4 The amount of available TNF-α is regulated by competitive uptake.

    (A) Analysis of TNF-α internalization. Representative confocal images of MEFs treated with FITC–TNF-α (20 ng/ml, left) and RAW264.7 cells treated with FITC–TNF-α (20 and 200 ng/ml, right) for 30 min. (B) Analysis of the total cellular fluorescence signal of the cells represented in (A) and treated with the indicated concentrations of FITC–TNF-α. Data are means ± SD of three replicate experiments. (C) Time-lapse microscopy analysis of TNF-α internalization. Data are means ± SD of the total fluorescence in MEFs (n = 10) and RAW264.7 cells (n = 13) treated for the indicated times with FITC–TNF-α (30 ng/ml). (D and E) Flow cytometry analysis of FITC–TNF-α internalization and TNFR1 abundance [with phycoerythrin (PE)–labeled antibody] for WT MEFs (D) and TNFR1 knockdown MEFs (E) treated with FITC–TNF-α (10 ng/ml). (F) Confocal microscopy image of WT and TNFR1 knockdown MEFs treated with Texas Red–labeled TNF-α (30 ng/ml). (G) Analysis of the loss of mouse TNF-α (mTNF-α; pg/ml) from the culture medium of RAW264.7 cells or MEFs (5 × 104 cells in 1 ml) treated with human TNF-α (hTNF-α; 1 ng/ml). Cell culture medium was assayed by ultrasensitive ELISA at the indicated times. Data are means ± SD of three replicate experiments. (H) Loss of TNF-α from the culture medium is cell density–dependent. A range of MEF densities were simulated experimentally by passing the culture medium across one and up to a total of six cell cultures (each with 2 × 105 cells in 1 ml) every 10 min. The initial culture was stimulated with human TNF-α (1 ng/ml). Zero density indicates no cell control (passed over six different cell-free dishes). The amount of human TNF-α after each step was measured by ultrasensitive ELISA. Data are means ± SD of three replicate experiments. (I) Loss of lipid A–induced TNF-α in the coculture medium of RAW264.7 cells and MEFs. Cocultured RAW264.7 cells and MEFs or RAW264.7 cells alone were stimulated for the indicated times with lipid A (500 ng/ml) before the concentration of TNF-α in the culture medium was measured by ultrasensitive ELISA. For the coculture experiments, a 1:3 ratio of RAW264.7 cells (5 × 104 cells) to MEFs (1.5 × 105 cells) in 1 ml of medium was used. Left: Time-course experiments. Data are means ± SD of three replicate experiments. Right: Pooled individual data points. *P < 0.05 by Mann-Whitney test.

  • Fig. 5 TLR4 signal propagation is restricted in the tissue.

    (A) Two-cell model of tissue-level macrophage signaling. Lipid A activates the secretion of TNF-α by a producing cell. TNF-α can activate a receiving cell but can also be removed by competitive uptake. (B) Distribution of first-peak nuclear NF-κB amplitude in a receiving cell at different distances from the producing cell. We performed 100 simulations with lipid A (500 ng/ml) at each of the indicated distances. TNFR1 abundance was distributed lognormally with a mean of 500; mean TNFR1 abundance is shown in blue. (C) Global sensitivity analysis of the two-cell model. The sensitivity index was calculated with respect to the AUC of nuclear NF-κB in a receiving cell 40 μm from the producing cell. The producing cell was stimulated with lipid A (500 ng/ml) for 3 hours. We assumed the mean TNFR1 abundance on the receiving cells. Parameters describing the producing and target cells are shown in red and gray, respectively. Spatial parameters corresponding to TNF-α diffusion are shown in green. Sensitivity index values <0.5 and >−0.5 are indicated by the gray area. (D) Correlation between the sensitivity indexes calculated for distances of 20 and 40 μm. Simulations were performed as described in (C). (E) The probability of TNF-α signal propagation depends on system parameters. The probability of signal propagation was calculated for changes in TNF-α uptake (c5), production (c1t), diffusion (D), as well as target cell TNFR1 receptor abundance (Rt) and A20 transcription (c1). Nominal parameter values are indicated in red. Activation was defined by a first-peak nuclear NF-κB amplitude >104 molecules across 300 cells simulated for each distance. Producing cells were stimulated with lipid A (500 ng/ml) for 180 min. Receiving cell TNFR1 abundance was distributed lognormally with a mean equal to Rt. (F) Propagation distance depends on tissue thresholds. A local sensitivity analysis of the propagation distance with respect to TNFA transcription (c1t), as well as tissue uptake (c5) and diffusion (D), is shown. Sensitivity was calculated on the basis of the data in (E) by averaging the changes in distance versus twofold parameter changes (up and down, in comparison to the nominal parameter values). Distances were calculated for 0.5 and 0.75 propagation probability (P). (G) Schematics of the proposed tissue-level signaling: TLR4 signal propagation depends on the competitive uptake of TNF-α (right), rather than on the variability in the TLR4-induced production of TNF-α (left).

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/540/eaaf3998/DC1

    Fig. S1. NF-κB reporter cell lines used in the study.

    Fig. S2. FCS calibration of live-cell imaging data.

    Fig. S3. Single-cell NF-κB p65 responses.

    Fig. S4. Proteomic analysis of TLR4-induced secretion.

    Fig. S5. Temporal analysis of TNF-α production.

    Fig. S6. Larger cells exhibit a stronger TNFA response.

    Fig. S7. NF-κB signaling does not affect the TNFA response.

    Fig. S8. Analysis of NF-κB signaling in primary macrophages.

    Fig. S9. NF-κB signaling in MEFs does not lead to TNF-α amplification.

    Fig. S10. TNF-α uptake and production across different cell types.

    Fig. S11. IFN-γ enhances TNFR1 abundance in macrophages.

    Fig. S12. Measurements of TNF-α production and loss.

    Fig. S13. Theoretical analysis of TNF-α uptake in small volumes.

    Fig. S14. Proposed model of the NF-κB signaling pathway in macrophages.

    Fig. S15. Simulated NF-κB model outputs.

    Fig. S16. Bifurcation analysis of the NF-κB system.

    Table S1. Summary of TNF-α production measurements.

    Table S2. Single-cell NF-κB model variables.

    Table S3. Parameterization of the single-cell NF-κB model.

    Table S4. Parameterization of the two-cell NF-κB model.

    Movie S1. Untreated RAW264.7 cells.

    Movie S2. Lipid A–stimulated RAW264.7 cells.

    Movie S3. TNF-α–stimulated RAW264.7 cells.

    Movie S4. Lipid A–stimulated BMDMs.

    Movie S5. TNF-α–stimulated BMDMs.

    Model S1. TNF-α half-life in a local tissue environment.

    Model S2. Mathematical model of NF-κB signaling in macrophages.

    Model S3. Propagation of signaling between producing cells and target cells.

    Model S4. Analysis of autocrine TNF-α feedback.

    File S1. Tabulated smFISH data.

    File S2. Mathematical model files.

    References (6987)

  • The PDF file includes:

    • Fig. S1. NF-κB reporter cell lines used in the study.
    • Fig. S2. FCS calibration of live-cell imaging data.
    • Fig. S3. Single-cell NF-κB p65 responses.
    • Fig. S4. Proteomic analysis of TLR4-induced secretion.
    • Fig. S5. Temporal analysis of TNF-α production.
    • Fig. S6. Larger cells exhibit a stronger TNFA response.
    • Fig. S7. NF-κB signaling does not affect the TNFA response.
    • Fig. S8. Analysis of NF-κB signaling in primary macrophages.
    • Fig. S9. NF-κB signaling in MEFs does not lead to TNF-α amplification.
    • Fig. S10. TNF-α uptake and production across different cell types.
    • Fig. S11. IFN-γ enhances TNFR1 abundance in macrophages.
    • Fig. S12. Measurements of TNF-α production and loss.
    • Fig. S13. Theoretical analysis of TNF-α uptake in small volumes.
    • Fig. S14. Proposed model of the NF-κB signaling pathway in macrophages.
    • Fig. S15. Simulated NF-κB model outputs.
    • Fig. S16. Bifurcation analysis of the NF-κB system.
    • Table S1. Summary of TNF-α production measurements.
    • Table S2. Single-cell NF-κB model variables.
    • Table S3. Parameterization of the single-cell NF-κB model.
    • Table S4. Parameterization of the two-cell NF-κB model.
    • Legends for movies S1 to S5
    • References (6987)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mp4 format). Untreated RAW264.7 cells.
    • Movie S2 (.mp4 format). Lipid A–stimulated RAW264.7 cells.
    • Movie S3 (.mp4 format). TNF-α–stimulated RAW264.7 cells.
    • Movie S4 (.mp4 format). Lipid A–stimulated BMDMs.
    • Movie S5 (.mp4 format). TNF-α–stimulated BMDMs.
    • Model S1 (Microsoft Word format). TNF-α half-life in a local tissue environment.
    • Model S2 (Microsoft Word format). Mathematical model of NF-κB signaling in macrophages.
    • Model S3 (Microsoft Word format). Propagation of signaling between producing cells and target cells.
    • Model S4 (Microsoft Word format). Analysis of autocrine TNF-α feedback.
    • File S1 (Microsoft Excel format). Tabulated smFISH data.
    • File S2 (.zip format). Mathematical model files.

    [Download Models S1 to S4]

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