Research ArticleInnate Immunity

NF-κB signaling dynamics is controlled by a dose-sensing autoregulatory loop

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Science Signaling  30 Apr 2019:
Vol. 12, Issue 579, eaau3568
DOI: 10.1126/scisignal.aau3568
  • Fig. 1 The TNFR and TLR or IL-1R elicit qualitatively different dose-dependent NF-κB dynamics.

    (A) Schematic representation of the innate immune signaling network. TNFα, LPS, and IL-1β activate innate immune signaling through different receptors and signaling proteins. Although TLR4 (LPS) and IL-1R (IL-1β) share most signaling components, TNFR (TNFα) signals through different proteins. Both cascades lead to activation of NF-κB and MAPK signaling via TAK1. (B) Parental strain (PS) cells (NIH3T3, RelA−/−, p65-dsRed, H2B-EGFP) were treated with increasing concentrations (top to bottom) of indicated stimuli, imaged, and quantified as described in Materials and Methods (see sections on live-cell imaging and segmentation and tracking). Five randomly selected traces from >2000 are shown per condition. Traces have been aligned to their first peak for clarity. (C) Peak counting quantification (see Materials and Methods for details) of the data presented in (B). Fractions of cells with more than one, two, or three peaks are shown to highlight population distribution. Data are representative of three independent experiments. High versus Low, ***P < 0.001 by χ2 test. For TNFα, LPS, and IL-1β, >6700, 9600, and 11,600 cells, respectively, were analyzed across concentration.

  • Fig. 2 TLR and IL-1R stimuli render cells cross-tolerant to further stimulation.

    (A) Schematic representation of the experimental timeline. During tolerance experiments, cells were stimulated with a primary (1°) and secondary (2°) stimulation for 30 min with an intervening rest period of 3 hours. Traces of NF-κB activity during the secondary response were converted to heatmaps, where rows indicate individual cells, columns indicate time, and the grayscale colormap represents nuclear to cytoplasmic median intensity ratio of p65-DsRed. (B and C) PS cells (RelA−/−, p65-DsRed, H2B-EGFP) expressing JNK-KTR-mCerulean3 were stimulated with different combinations of primary and secondary inputs as indicated [TNFα (10 ng/ml), LPS (5 μg/ml), IL-1β (1 ng/ml), IL-1βLow (0.1 ng/ml)] and monitored for NF-κB (B) and JNK (C) activity. Time period for secondary stimulation only is shown for clarity. Purple lines over heatmaps indicate time period when cells were in secondary stimulus. Cells were filtered to include only those responding to the primary stimulus. Data represent two independent experiments (n > 300 cells per condition, >9000 cells total).

  • Fig. 3 Optogenetic control of signaling at MyD88 and TRAF6 nodes maps the cross-tolerance mechanism to IRAK proteins.

    (A and B) Schematic representation of OptoMyD88 (A) or OptoTRAF6 (B) activation of innate immune signaling. Upon light stimulation, Opto tools activate their respective downstream proteins labeled in light green. (C and D) Cells (NIH3T3, p65-mRuby, H2B-iRFP) expressing OptoMyD88 (C) or OptoTRAF6 (D) under the Tet-responsive element third-generation (TRE3G) promoter were incubated in doxycycline (2 μg/ml) overnight and stimulated with either IL-1β (1 ng/ml) or light (470/24 nm, five 250-ms pulses with 5-min intervals). Representative NF-κB localization images before and after indicated stimuli are shown. Experiment was performed with a 10× objective. Scale bars, 50 μm. (E and F) Secondary response heatmaps of NF-κB nuclear translocation in OptoMyD88 (E) or OptoTRAF6 (F) cells with varied primary (1°) and secondary (2°) stimulations (experimental timeline as in Fig. 2A). Cells were incubated in doxycycline (2 μg/ml) overnight and treated with TNFα (10 ng/ml), IL-1β (1 ng/ml), light (470/24 nm, 250-ms pulses), or medium for 30 min (purple bars). Data represent three independent experiments (n > 100 cells per condition). Cells were filtered to include only those responding to the primary stimulus, except for those with no primary stimulus.

  • Fig. 4 Low IRAK1 abundance correlates with the cross-tolerant state.

    (A) Schematic detailing of stimulation and sample collection timeline for Western blotting. Cells were stimulated with primary stimuli as indicated [IL-1β (1 ng/ml), green; OptoTRAF6 (light, 488 nm), orange] and sampled at 0, 5, and 15 min after primary stimulation. Cells were washed after 30 min of primary stimulation, allowed to recover for 3 hours, challenged with secondary stimulation of IL-1β, and sampled at 0, 5, and 15 min. (B) Quantification of immunoblots for phosphorylated IRAK4 (pIRAK4) in cells treated with light (orange) or IL-1β (green) and subjected to a secondary IL-1β stimulation. pIRAK4 abundance was first normalized to a β-actin loading control, and then fold change compared to the unstimulated condition (first time point) was calculated for each sample and normalized between zero and one across all time points. Data are means ± SD of three independent experiments. n.s., not significant by a t test. Representative blots are shown for the last three time points for both IL-1β and OptoTRAF6 primary conditions. Full blots and additional stimulation combinations are presented in fig. S4. (C) Quantification of Western blots for IRAK1, as described in (B). IRAK1 expression was first normalized to a β-actin loading control, and then fold change to the unstimulated condition was calculated for each sample. Data are means ± SD of three independent experiments. *P < 0.05 by a t test. Representative blots are shown for the last three time points for both IL-1β and OptoTRAF6 primary conditions. In (B), full blots and additional stimulation combinations are shown in fig. S4.

  • Fig. 5 Expression of unmodified IRAK1 protein bypasses tolerance.

    (A) Induced expression of IRAK1 after primary stimulation. Cells containing TRE3G::IRAK1-Clover were incubated without doxycycline (No dox), with doxycycline (2 µg/ml) at the time of primary stimulation (Dox), or 24 hours overnight (24 hour dox). Cells were stimulated with IL-1β (1 ng/ml) and imaged as described in Materials and Methods. After 30 min, cells were washed and allowed to recover for 3 hours and stimulated again with IL-1β (1 ng/ml). Red arrows indicate sample collection points for the Western blot shown in (B). Two-dimensional histograms show the distribution of peak amplitudes of nuclear/cytoplasmic NF-κB median intensity during the primary versus secondary response in each cell. Black line indicates primary equals secondary NF-κB amplitude. Data represent three independent experiments with n > 100 cells. (B) Western blotting for IRAK1 protein abundance between primary and secondary stimuli in cells treated as in (A) was harvested at indicated times [S1 to S5, corresponding to those in (A)]. HSC70 was used as a loading control. Blot is representative of three independent experiments. (C) Representative confocal images of IRAK1-Clover cluster formation after NF-κB activation. Cells stably expressing IRAK1-Clover were imaged before and after IL-1β stimulation (1 ng/ml). Scale bar, 50 μm. (D) IRAK1-Clover cells were imaged for 8 hours and stimulated with IL-1β (0.1 ng/ml) 45 min into the time course. NF-κB nuclear/cytoplasmic intensity ratio (left) and IRAK1-Clover clustering dynamics (right) are displayed in tandem. Heatmap rows are ordered from top to bottom on the basis of increasing IRAK1 clustering (see Materials and Methods). Dashed red line represents an arbitrary IRAK1 clustering threshold (1.3-fold change). (E) Grouping of cells by IRAK1 clustering separates oscillatory versus non-oscillatory cells. Irak1-KO cells expressing IRAK1-Clover were stimulated with IL-1β (0.1, 1, and 10 ng/ml) or LPS (0.5 and 5 μg/ml). Peaks of NF-κB activity and IRAK1 clustering from single-cell traces obtained in (D) and fig. S6B were measured as described in Materials and Methods. An arbitrary threshold of 1.3-fold increase in IRAK1 clustering was used in all conditions to group high versus low IRAK1 clustering cells. Within each group, fractions of cells with more than one, two, or three peaks are shown to highlight population distribution (n > 100 cells; **P < 0.01 and ***P < 0.001 by χ2 test). Additional clustering quantification is provided in fig. S8A, and heatmaps of additional concentrations of IL-1β are provided in fig. S6B.

  • Fig. 6 IRAK1 kinase activity is critical to regulate NF-κB signaling dynamics.

    (A) WT cells and Irak1-KO cells reconstituted with IRAK1WT or IRAK1KD were incubated with or without IL-1β (1 ng/ml) for 3 hours. Lysate was collected and immunoblotted against IRAK1. Arrows indicate posttranslationally modified and unmodified IRAK1 protein. HSC70 was used as a loading control. Blot is representative of three experiments. (B) Secondary response heatmaps of IRAK1WT or IRAK1KD cells show reduced tolerance in IRAK1KD. Irak1-KO cells reconstituted with IRAK1WT and IRAK1KD were stimulated with a 30-min pulse of IL-1β (1 ng/ml), washed, allowed to recover for 8 hours, and stimulated again with a secondary pulse of IL-1β (1 ng/ml). Two-dimensional histograms show the distribution of peak amplitudes of nuclear/cytoplasmic NF-κB median intensity during the primary versus secondary response in each cell. Black line indicates primary equals secondary NF-κB amplitude. Data represent three independent experiments with n > 100 cells. (C) Irak1-KO cells expressing IRAK1WT-Clover or IRAK1KD-Clover were imaged before and 20 min after stimulation with IL-1β (1 ng/ml). Representative images are shown. Scale bar, 50 μm. (D) IRAK1 clustering was quantified as described in Materials and Methods in IRAK1WT-Clover or IRAK1KD-Clover cells stimulated with IL-1β (0.1 ng/ml) or LPS (0.5 μg/ml). Data represent n > 100 cells; ***P < 0.001 by a Kolmogorov-Smirnov test. AU, arbitrary units. (E) IRAK1 kinase activity regulates oscillatory dynamics. PS cells expressing IRAK1WT-Clover or IRAK1KD-Clover were stimulated with IL-1β (0.1, 1, or 10 ng/ml) and imaged for 8 hours. Five randomly selected single-cell traces are presented for each condition. Peak counting of NF-κB oscillations was done as described in Materials and Methods. Fractions of cells with more than one, two, or three peaks are shown to highlight population distribution. Data represent three independent experiments with n > 100 cells (***P < 0.001, χ2 test). See fig. S8B for TNFα data. (F) Schematic model of the effects of IRAK1-dependent autoinhibitory loop in NF-κB signaling dynamics. When ligand is in low abundance, TLR and IL-1R signaling is not inhibited after the initial activation and continues to signal in an oscillatory pattern. When ligand concentration is high, IRAK1 kinase activity strongly inhibits signaling after the initial activation, and oscillations are not detected.

Supplementary Materials

  • stke.sciencemag.org/cgi/content/full/12/579/eaau3568/DC1

    Fig. S1. Graphical summary of the image analysis pipeline.

    Fig. S2. Additional analysis and TNF concentration for data presented in Fig. 1.

    Fig. S3. Additional stimulus combinations for Figs. 2 and 3.

    Fig. S4. Full gels and quantification of Western blots in Fig. 4.

    Fig. S5. Increased abundance of MyD88 or TRAF6 after primary stimulation cannot bypass tolerance.

    Fig. S6. IRAK1 modification and clustering with varied recovery periods.

    Fig. S7. IRAK1 mutant screen and KO characterization.

    Fig. S8. Additional IRAK1 WT and KD-Clover characterization.

  • This PDF file includes:

    • Fig. S1. Graphical summary of the image analysis pipeline.
    • Fig. S2. Additional analysis and TNF concentration for data presented in Fig. 1.
    • Fig. S3. Additional stimulus combinations for Figs. 2 and 3.
    • Fig. S4. Full gels and quantification of Western blots in Fig. 4.
    • Fig. S5. Increased abundance of MyD88 or TRAF6 after primary stimulation cannot bypass tolerance.
    • Fig. S6. IRAK1 modification and clustering with varied recovery periods.
    • Fig. S7. IRAK1 mutant screen and KO characterization.
    • Fig. S8. Additional IRAK1 WT and KD-Clover characterization.

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