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Molecular definition of the endogenous Toll-like receptor signalling pathways

Nature volume 631, pages 635–644 (2024)Cite this article

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Abstract

Innate immune pattern recognition receptors, such as the Toll-like receptors (TLRs), are key mediators of the immune response to infection and central to our understanding of health and disease1. After microbial detection, these receptors activate inflammatory signal transduction pathways that involve IκB kinases, mitogen-activated protein kinases, ubiquitin ligases and other adaptor proteins. The mechanisms that connect the proteins in the TLR pathways are poorly defined. To delineate TLR pathway activities, we engineered macrophages to enable microscopy and proteomic analysis of the endogenous myddosome constituent MyD88. We found that myddosomes form transient contacts with activated TLRs and that TLR-free myddosomes are dynamic in size, number and composition over the course of 24 h. Analysis using super-resolution microscopy revealed that, within most myddosomes, MyD88 forms barrel-like structures that function as scaffolds for effector protein recruitment. Proteomic analysis demonstrated that myddosomes contain proteins that act at all stages and regulate all effector responses of the TLR pathways, and genetic analysis defined the epistatic relationship between these effector modules. Myddosome assembly was evident in cells infected with Listeria monocytogenes, but these bacteria evaded myddosome assembly and TLR signalling during cell-to-cell spread. On the basis of these findings, we propose that the entire TLR signalling pathway is executed from within the myddosome.

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Fig. 1: Endogenous myddosomes are long-lived and dynamic signalling organelles.
Fig. 2: Mapping components of TLR-free myddosomes by proximity labelling and proteomics.
Fig. 3: Myddosomes are signalling assemblies with a defined architecture.
Fig. 4: The role of IRAKs in myddosome dynamics, signalling and plasticity.
Fig. 5: Myddosomes recruit and activate functionally distinct signalling clusters.
Fig. 6: TLR–myddosome activities during Listeria infection.

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Data availability

All data supporting the findings of this study are available within the Article and its Supplementary Information, except for the raw proteomics data, which were deposited in the PRIDE repository with the identifier PXD047416.

Change history

  • 11 July 2024

    In the version of the article initially published, Fig. 3c erroneously included the number “2” in the top right image, which has now been removed in the HTML and PDF versions of the article.

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Acknowledgements

We thank the members of the Kagan laboratory for discussions; S. De, S. Hur, H. Wu and E. Frickel for providing reagents; E. Anastasakou for designing the summary figure; the staff at the HDDC core facility (Boston Children’s Hospital), for support with flow cytometry, cell sorting and microscopy; and P. M. Llopis, P. V. Anekal and the members of the HMS MicRoN facility for support with TIRF-M. This study was supported by NIH grants AI167993, AI116550 and DK34854 to J.C.K. and NIH grant R01AI146102 to D.E.H. D.F. was supported by a Human Frontier Science Program (HFSP) long-term postdoctoral fellowship (LT0006/2022-L) and an EMBO postdoctoral fellowship (ALTF 491-2022). The Nikon TIRF microscope was purchased with the support of the National Institutes of Health (S10 RR027344-01). T.Z. was supported by NCI grant R00CA273170.

Author information

Authors and Affiliations

  1. Division of Gastroenterology, Boston Children’s Hospital and Harvard Medical School, Boston, MA, USA

    Daniel Fisch, Weiyi Ma, Yunhao Tan & Jonathan C. Kagan

  2. Department of Cell Biology, Harvard Medical School, Boston, MA, USA

    Tian Zhang & Steven P. Gygi

  3. Department of Biochemistry and Molecular Genetics & Comprehensive Cancer Center, University of Virginia School of Medicine, Charlottesville, VA, USA

    Tian Zhang

  4. Department of Microbiology, Blavatnik Institute, Harvard Medical School, Boston, MA, USA

    He Sun & Darren E. Higgins

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  1. Daniel Fisch
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Contributions

D.F. and J.C.K. conceived the study. D.F. performed experiments. T.Z. performed MS analysis. T.Z., S.P.G., H.S. and D.E.H. provided essential reagents. H.S. assisted with Listeria experiments. Y.T. generated the mouse-specific TLR4 antibody, which was validated for the endogenous protein by W.M.; S.P.G., D.E.H. and J.C.K. acquired and contributed funding and experimental oversight. D.F. and J.C.K. analysed the data and wrote the manuscript, with input from all of the authors. All of the authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to Jonathan C. Kagan.

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Competing interests

J.C.K. consults and holds equity in Corner Therapeutics, Larkspur Biosciences, MindImmune Therapeutics and Neumora Therapeutics. None of these relationships impacted this study. The other authors declare no competing interests.

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Extended data figures and tables

Extended Data Fig. 1 Generation of MyD88 knock-in macrophages.

(a) Schematic detailing the generation of THP-1 or iBMDM MyD88-AGF cells using an adapted version of CRISPaint. In brief, cells were electroporated with plasmids carrying the necessary sgRNAs and tag-donor sequences, pre-selected with Puromycin and sorted for homogenous GFP-fluorescence to obtain homozygous knock-in cells. (b) Graphs showing cell sorting for selection of homozygous THP-1 or iBMDM MyD88-AGF knock-in cells. Scatter plot shows gating for a GFPhigh-population (top) and histograms show difference in GFP fluorescence between wild type (WT) and obtained homozygous knock-in cells (bottom). (c) Schematic depicts CRISPaint procedure and integration of the tag into the target site within the MyD88 C-terminal exon on the macrophage genome (top, left). Successful integration was analysed by analytical PCR using primer pairs binding within the MyD88 C-terminal exon and the 3′ UTR (WT probes) or the AGF-tag (AGF probes; top, middle) and performed on genomic DNA from WT cells or MyD88-AGF cells pre- and post-sorting for a GFPhigh, homozygous population (top, right). Obtained amplicons were analysed by Sanger sequencing to confirm in-frame integration of the tag-sequence (bottom). Extend of the Myd88 coding sequences (CDS), frame 1 sgRNA binding-site, cut-site and protospacer adjacent motif (PAM) are indicated. (d) Immunoblots confirming successful integration of the AGF-tag into the THP-1 or iBMDM genome and C-terminal tagging of endogenous MyD88 protein. Data information: Images and graphs in (b-d) representative of n = 2 experiments. For gel source data, see Supplementary Data 1.

Extended Data Fig. 2 Functional validation of MyD88 knock-in macrophages.

(a) RT-qPCR analysis of MYD88/Myd88 gene expression in THP-1 and iBMDM WT or Myd88-AGF cells. Plotted as ∆Ct relative to HPRT1/Hprt1 housekeeping gene controls. (b) Immunoblots for TLR signalling pathway activation in THP-1 or iBMDM wild type (WT) or MyD88-AGF cells following treatment with LPS or Pam3CSK4 (P3C) for 0.5 or 24 hrs. (c) Flow cytometry analysis of TLR2/4 surface localization following treatment of THP-1 or iBMDM WT or MyD88-AGF cells with LPS or P3C for 6 h. Plotted relative to untreated (UT) controls (indicated by the dashed line). (d) RT-qPCR analysis of TLR response gene expression in THP-1 or iBMDM WT or Myd88-AGF cells treated with LPS or P3C for 6 hrs. Plotted as fold change relative to UT controls. (e) ELISA analysis of IL-6 and TNFα secretion from THP-1 or iBMDM WT or MyD88-AGF cells treated with LPS or P3C for 24 hrs. Data information: Images in (b) representative of n = 3 experiments. Box plots in (a) show 25th to 75th percentiles (box), minimum to maximum value (whisker) and median from n = 6 experiments. Graphs in (c-e) show mean ± SEM from n = 3 experiments. P-values for indicated comparisons in (a) from unpaired, two-sided t-tests; ns: not significant. For gel source data, see Supplementary Data 1.

Source data

Extended Data Fig. 3 TIRF-M shows proto-myddosome formation at the plasma membrane.

(a) Images from live cell total internal reflection microscopy (TIRF-M) of THP-1 or iBMDM MyD88-AGF cells treated with LPS showing formation and subsequent disappearance of proto-myddosomes. Grey: MyD88-AGF. Scale bar 5 µm. (b) Higher magnification images from live cell TIRF-M of LPS-treated iBMDM MyD88-AGF cells showing formation of a larger proto-myddosome complex. Grey: MyD88-AGF. Scale bar 2 µm. (c) Frequency distribution of the maximum GFP fluorescence intensity of MyD88-AGF clusters in LPS-treated iBMDM or THP-1 MyD88-AGF cells compared with single molecules of GFP from calibration images. Fluorescence ranges of proto-myddosomes and the larger clusters are highlighted. (d) Quantification of cellular response and proto-myddosome formation at the plasma membrane of LPS-treated THP-1 or iBMDM MyD88-AGF cells from live cell TIRF-M as depicted in (a). (e) Quantification of average lifetimes of proto-myddosomes from live cell TIRF-M of LPS-treated THP-1 or iBMDM MyD88-AGF cells, depending on observed sizes. Data information: Images in (a-b) representative of n = 10 imaging experiments. Graph in (c) shows frequency quantification from n = 10 imaging experiments and GFP calibration images. Graphs in (d) show mean ± SD from n = 10 imaging experiments. Violin plot in (e) shows distribution of average myddosome lifetimes from n = 10 experiments, mean and SD indicated in the figure. P-values for indicated comparisons in (e) from ordinary one-way ANOVA.

Source data

Extended Data Fig. 4 Quantification of NF-κB and myddosome dynamics.

(a) Immunoblots validating the Doxycycline (Dox)-inducible expression of mPlum-p65 in iBMDM-MyD88-AGF+Tet-mPlum-p65 cells. Cells were treated with Dox for 16 hrs. (b) Validation of the NF-κB reporter assay using LPS-treatment of iBMDM-MyD88-AGF+Tet-mPlum-p65 cells. Images of resting or LPS-treated (1 hr) cells and higher magnification insets depicting p65 nuclear translocation in the highlighted cell (left). Quantification of average nuclear:cytosolic mPlum-p65 fluorescence ratio per cell (right). Cells with a ratio > 1.0 are considered to have active NF-κB signalling. Magenta: mPlum-p65; Blue: nuclei. Scale bars 20 µm. (c) Quantification of myddosome formation and NF-κB signalling activation in LPS or Pam3CSK4 (P3C)-treated iBMDM MyD88-AGF+Tet-mPlum-p65 cells from live cell imaging over 12 hrs. (d) Quantification of time delay between appearance of first myddosome and NF-κB-activation in LPS- or P3C-treated iBMDM MyD88-AGF+Tet-mPlum-p65 cells. (e) Quantification of average time to NFκB-activation in LPS- or P3C-treated iBMDM MyD88-AGF+Tet-mPlum-p65 cells stratified on whether the cell contains a major myddosome or not. (f) Quantification of myddosome numbers and p65 nuclear:cytosolic ratio (NF-κB activity) in LPS- or P3C-treated iBMDM MyD88-AGF+Tet-mPlum-p65 cells at indicated times from live cell imaging over 12 hrs. Each dot represents one cell. Data information: Images in (a-b) representative of n = 2 experiments. Graph in (b) shows mean p65 nuclear:cytosolic ratio from N = 10 fields of view (FoV) from one representative of n = 2 total experiments. Graph in (c) shows mean ± SD from N = 10 FoV from one representative of n = 3 total experiments. Graphs in (d-e) show quantification of n = 3 experiments with N > 800 cells for each condition. Graphs in (f) shows N > 740 cells for each condition from n = 3 total experiments. P-values for indicated comparisons in (d-e) from ordinary one-way ANOVA. For gel source data, see Supplementary Data 1.

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Extended Data Fig. 5 MyD88 expression levels ensure sensitive responses to TLR ligands.

(a) Images from live cell imaging of THP-1 or iBMDM MyD88-AGF cells treated with indicated PAMPs for 3 hrs (top), quantification of TNFα and IL-6 secretion by ELISA after 24 hrs (middle) and quantification of myddosome numbers per cell and proportion of cells forming myddosomes by fixed fluorescence imaging after 3 hrs of treatment with indicated concentrations of PAMPs (bottom). Scale bars: 10 µm. (b) Cartoon depicting the experimental setup (left) and graph showing cell sorting into six fractions based on GFP fluorescence as proxy for MyD88 protein levels in iBMDM MyD88-AGF cells (middle). Flow cytometry followed by mean fluorescence intensity (MFI) analysis confirmed successful sorting into cell fractions with different MyD88 protein levels (right). (c) ELISA analysis of IL-6 and TNFα secretion from unsorted pools or sorted iBMDM Myd88-AGF cell fractions left untreated or treated with LPS for 24 hrs. (d) Immunoblots assessing TLR-signalling pathway activation in sorted iBMDM MyD88-AGF cell fractions or unsorted pools. Pooled cells treated with LPS for 2 hrs served as positive control. (e) Images from live cell imaging (left) and quantification of proportion of cells with myddosomes (right) of sorted iBMDM Myd88-AGF cell fractions at 2 hrs after sorting illustrating myddosome formation in the absence of TLR-activation. Scale bars: 10 µm. (f) RT-qPCR analysis of TLR response gene expression in sorted iBMDM Myd88-AGF cell fractions or unsorted pools. Pooled cells treated with LPS for 6 hrs served as positive control. Plotted as ∆Ct relative to Hprt1 control. (g) Immunoblots (bottom) and densitometry quantification (top) of MyD88-AGF protein levels in sorted iBMDM Myd88-AGF cell fractions or unsorted pools at indicated times post sorting. Data information: Images in (a), (d-e) and (g) representative of n = 3 experiments. Graphs in (a), (c), (e) and (g) show mean ± SEM from n = 3 experiments. Graphs in (b) show mean ± SEM from N = 3 technical replicates from one representative sorting run of a total of n = 9 sorts. Box plots in (f) show 25th to 75th percentiles (box), minimum to maximum value (whisker) and median from n = 4 experiments. P-values for comparisons to pool in (c) and (f) from two-way ANOVA; ns: not significant. For gel source data, see Supplementary Data 1.

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Extended Data Fig. 6 Multiplexed mass spectrometry analysis for myddosome protein mapping.

(a) Schematic depicting the myddosome mapping assay using spatiotemporally-resolved, multiplexed mass spectrometry (MS). (b) Immunoblots showing biotinylation of (myddosome-) proteins in LPS or P3C-treated THP-1 or iBMDM MyD88-AGF cells in which biotinylation was activated with H2O2 for 30 s. (c) Quantification of average MyD88 peptide intensities in the mass spectrometry samples from indicated conditions. Average enrichment comparing to non-H2O2-treated samples noted in the figure. (d) Principal component analysis (PCA) of proteomics data from the indicated conditions showing good precision of the replicates and clustering based on treatments. Variance (var.) of the principal components (PC) as displayed. (e) MS data normalization, filtering, and enrichment analysis for myddosome proteins. Number of discarded and remaining proteins after each step as indicated. Data information: Images in (b) representative of n = 3 experiments. Graphs in (c-d) show data from N = 3 technical replicates as analysed by multiplexed TMT mass spectrometry. Graphs in (c) show mean ± SD and individual values of the replicates. For gel source data, see Supplementary Data 1.

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Extended Data Fig. 7 TLR signalling effectors are activated within myddosomes.

(a) Flag-immunoprecipitation of MyD88-AGF from THP-1 or iBMDM MyD88-AGF cells treated with LPS or Pam3CSK4 (P3C) for the indicated times to validate myddosome-associated proteins by co-immunoprecipitation. (b) Immunoblots following Flag-immunoprecipitation of MyD88-AGF from LPS or P3C-treated iBMDM MyD88-AGF (top) or THP-1 MyD88-AGF (bottom) to divide cells into cytosolic and myddosome fractions. Input lysate (Ly), unbound (Ub; cytosol), and elution (El; myddosomes) fractions were analysed (left) and quantified by densitometry to determine the proportion of activated TLR signalling effector proteins within myddosomes (right). (c) Immunofluorescence images of iBMDM MyD88-AGF cells treated with LPS for the indicated times and stained for IRF5, p-p65 or p-p38α (left). Graphs show quantification of protein recruitment into myddosomes and translocation to the nucleus (plotted as nuclear:cytoplasmic ratio; right). Data information: Images in (a-b) representative of n = 3 and in (c) of n = 2 experiments. Graphs in (b) shows mean ± SEM from n = 3 and in (c) from n = 2 experiments. For gel source data, see Supplementary Data 1.

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Extended Data Fig. 8 Imaging myddosome proteins and nanoscale architecture.

(a) Immunofluorescence images of LPS-treated iBMDM MyD88-AGF immunostained for the respective candidate myddosome proteins, Proteostat rotamer dye or markers for cellular compartments. Green: MyD88-AGF; Magenta: stains; Blue: nuclei. Scale bars: 5 µm. (b) 3D-STED imaging based classification and frequency determination of myddosomes. Four classes were identified based on myddosome morphology. Images show representative myddosome cross-sections in the middle plane. Grey: Flag-antibody stain for MyD88-AGF. Scale bars: 500 nm. (c) Example fixed cell immunofluorescence images of myddosomes in iBMDM-Myd88-AGF cells treated with LPS for 3 hrs. Myddosomes were stained with an anti-Flag antibody and imaged using stimulated emission depletion (STED) super resolution microscopy. Scale bars: 500 nm. Data information: Images in (a) representative of n = 2 and in (b-c) of n = 3 experiments. Frequency of classes in (b) based on imaging and annotation of N = 100 myddosomes from n = 3 experiments.

Extended Data Fig. 9 IRAK4 kinase activity only regulates myddosome signalling.

(a) Immunoblots for IRAK4 trans-autophosphorylation at Thr345/Ser346 in LPS-treated iBMDM WT cells. (b) Images of iBMDM MyD88-AGF WT, ∆IRAK4 or IRAK4 kinase inhibitor (IRAK4i; Zimlovisertib/PF-06650833)-treated cells stimulated with LPS for 3 hrs to assess myddosome formation (black clusters). Scale bars: 5 µm. (c) Quantification of myddosome numbers per cell, average size, and proportion of cells responding to LPS stimulation of iBMDM MyD88-AGF WT, ∆IRAK4 or IRAK4i-treated cells from live cell imaging. (d) Violin plots depicting the average myddosome lifetime in LPS-treated iBMDM MyD88-AGF WT, ∆IRAK4 or IRAK4i-treated cells. (e) Measurement of myddosome plasticity in iBMDM MyD88-AGF WT, ∆IRAK4 or IRAK4i-treated cells using fluorescence recovery after photobleaching (FRAP) assays following treatment with LPS. Plotted as %recovery of fluorescence per minute. (f) Immunoblots assessing TLR4-signalling pathway activation in iBMDM MyD88-AGF WT, ∆IRAK4 or IRAK4i-treated cells. (g) RT-qPCR analysis of TLR4 response gene expression in iBMDM MyD88-AGF WT, ∆IRAK4 or IRAK4i-treated cells. Plotted as fold change relative to untreated (UT) controls. (h) ELISA analysis of IL-6 and TNFα secretion from iBMDM MyD88-AGF WT, ∆IRAK4 or IRAK4i-treated cells, following treatment with LPS for 24 hrs. (i) Analysis of myddosome composition at 3 or 18 hrs post LPS-treatment of iBMDM MyD88-AGF ∆IRAK4 or IRAK4i-treated cells. Plotted relative to iBMDM MyD88-AGF WT cells and average labelled in the heatmap. Data information: Images in (a-b) and (f) representative of n = 3 experiments. Graph in (c) shows mean from N = 4 fields of view (FoV) from one representative of n = 3 total experiments. Graph in (d) shows myddosome lifetimes from n = 3 experiments and mean ± SD. Graph in (e) shows FRAP of N = 5 myddosomes per time point and condition. Graphs in (g-h) show mean ± SD from n = 4 experiments and heatmap in (i) shows mean from N = 24 FoV from n = 2 experiments. P-values for comparisons of ∆IRAK4 to WT cells in (e) and for comparisons to WT cells in (h) from two-way ANOVA; ns: not significant. For gel source data, see Supplementary Data 1.

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Extended Data Fig. 10 Autophagy targets myddosome remnants for cytosolic clearance.

(a) Immunoblots for validation of knockout cell lines in the iBMDM MyD88-AGF background and for validation of functional defects in LC3B turnover. (b) Quantification of myddosome numbers per cell in iBMDM MyD88-AGF WT or knockout cells from live cell imaging at indicated times post LPS-stimulation. (c) Images from live cell imaging of LPS-treated (3 hrs) iBMDM MyD88-AGF WT or knockout cells assessing myddosome formation. Scale bars: 5 µm. (d) Quantification of myddosome numbers per cell, average size, and proportion of cells responding to LPS stimulation of iBMDM MyD88-AGF WT or knockout cells from live cell imaging. (e) Violin plots depicting the average myddosome lifetime in LPS-treated iBMDM MyD88-AGF WT or knockout cells from live cell imaging. (f) Measurement of myddosome plasticity using fluorescence recovery after photobleaching (FRAP) assays in iBMDM MyD88-AGF WT or knockout cells treated with LPS. Plotted as %fluorescence recovery per minute. (g) Immunoblots assessing TLR4-signalling pathway activation in iBMDM MyD88-AGF WT or knockout cells. (h) RT-qPCR analysis of TLR4 response gene expression in iBMDM MyD88-AGF WT or knockout cells. Plotted as fold change relative to untreated (UT) controls. (i) ELISA analysis of IL-6 and TNFα secretion from iBMDM MyD88-AGF WT or knockout cells following treatment with LPS for 24 hrs. (j) Analysis of myddosome composition at 3 or 18 hrs post LPS-treatment of indicated cell lines. Plotted as relative to iBMDM MyD88-AGF WT cells and average labelled in the heatmap. Data information: Images in (a), (c) and (g) representative of n = 3 experiments. Graphs in (b) show mean ± SD from n = 3 experiments. Graph in (d) shows mean from N = 5 FoV from one representative of n = 3 total experiments. Graph in (e) shows myddosome lifetimes from n = 3 experiments and mean ± SD. Graph in (f) shows FRAP of N = 5 myddosomes per time point and condition. Graphs in (h-i) show mean ± SD from n = 3 experiments and heatmap in (j) shows mean from N = 24 FoV from n = 2 experiments. P-values for indicated comparisons to WT cells at 24 hrs in (b) from two-way ANOVA and for indicated comparisons in (e) and (i) from ordinary one-way ANOVA; ns: not significant. For gel source data, see Supplementary Data 1.

Source data

Supplementary information

Supplementary Information

Supplementary Figs. 1–5 and Supplementary Tables 1–8.

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Supplementary Data 1

Uncropped immunoblots and gels. Uncropped immunoblots and gel images. Marker size and crop area are indicated.

Supplementary Data 2

Myddosome mapping proteomics data. Table containing the MS analysis results for identification of candidate myddosome proteins in LPS- or P3C-treated THP-1 or iBMDM MYD88–AGF cells. Green highlighted cells indicate significant hits that are enriched as compared to untreated cells.

Supplementary Video 1

TIRF imaging of proto-myddosomes. Representative time course from TIRF imaging of THP-1 MYD88–AGF cells treated with LPS showing the formation of receptor- and plasma-membrane-proximal proto-myddosomes.

Supplementary Video 2

Myddosome formation and dynamics in iBMDM MYD88–AGF cells. Representative time course from live-cell imaging of iBMDM MYD88–AGF cells treated with LPS or P3C showing the formation and dynamics of cytosolic myddosomes.

Supplementary Video 3

Myddosome formation and dynamics in THP-1 MYD88–AGF cells. Representative time course from live-cell imaging of THP-1 MYD88–AGF cells treated with LPS or P3C showing the formation and dynamics of cytosolic myddosomes.

Supplementary Video 4

NF-κB activation in LPS-treated mouse macrophages. Representative time course showing p65 nuclear translocation in LPS-treated mouse macrophages (iBMDM+Tet-mPlum-p65 cells).

Supplementary Video 5

Imaging of myddosomes in LPS-bead-treated macrophages. Representative time course showing myddosome formation in iBMDM MYD88–AGF cells ‘infected’ with LPS-coated beads.

Supplementary Video 6

Correlative myddosome imaging. Video depicting imaging of myddosome formation in live cells and subsequent imaging with increasing resolution to dissect the nanoscale architecture of these protein complexes.

Supplementary Video 7

Myddosome fate mapping. Video showing generation of myddosome fate maps depending on the type of myddosome and plotting the myddosome size versus the IRAK4:MYD88 fluorescence ratio. Myddosomes were tracked from live-cell imaging of LPS-treated iBMDM MYD88-AGF+IRAK4-mScarlet cells.

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Fisch, D., Zhang, T., Sun, H. et al. Molecular definition of the endogenous Toll-like receptor signalling pathways. Nature 631, 635–644 (2024). https://doi.org/10.1038/s41586-024-07614-7

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