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The adaptor ASC has extracellular and 'prionoid' activities that propagate inflammation

Nature Immunology volume 15, pages 727–737 (2014)Cite this article

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Abstract

Microbes or danger signals trigger inflammasome sensors, which induce polymerization of the adaptor ASC and the assembly of ASC specks. ASC specks recruit and activate caspase-1, which induces maturation of the cytokine interleukin 1β (IL-1β) and pyroptotic cell death. Here we found that after pyroptosis, ASC specks accumulated in the extracellular space, where they promoted further maturation of IL-1β. In addition, phagocytosis of ASC specks by macrophages induced lysosomal damage and nucleation of soluble ASC, as well as activation of IL-1β in recipient cells. ASC specks appeared in bodily fluids from inflamed tissues, and autoantibodies to ASC specks developed in patients and mice with autoimmune pathologies. Together these findings reveal extracellular functions of ASC specks and a previously unknown form of cell-to-cell communication.

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Figure 1: ASC specks accumulate in the extracellular space after the activation of inflammasomes.
Figure 2: Extracellular specks remain active in the extracellular space.
Figure 3: Extracellular specks represent a cell-derived danger signal.
Figure 4: ASC has prionoid features.
Figure 5: ASC specks propagate inflammation in vivo.
Figure 6: Extracellular ASC specks are formed in vivo and accumulate during human chronic inflammatory disease.
Figure 7: Antibodies to ASC specks develop in autoinflammatory diseases.

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Acknowledgements

We thank C. Tiberi and P.-I. Chiang for technical support. Supported by Deutsche Forschungsgemeinschaft (SFB670 to E.L.), the US National Institutes of Health (R01-HL093262, R01-HL112661 and R01-AI083713 to E.L.), the Alexander von Humboldt Foundation (B.S.F.), intramural BONFOR research support at the University of Bonn (B.S.F.), the European Research Council (Advanced Grant to A.A.), the European Union (NEURINOX to A.A.), the Swiss National Foundation (Sinergia grant to A.A.), the Novartis Research Foundation (A.A.), the Clinical Research Priority Programs “Small RNAs” and “Mechanisms of Human Hemato-Lymphatic Diseases” of the University of Zurich (A.A.), the ImmunoSensation cluster of Excellence in Bonn (E.L.) and the German Center for Infection Research (E.L.).

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Authors and Affiliations

  1. Institute of Innate Immunity, University Hospitals, University of Bonn, Bonn, Germany

    Bernardo S Franklin, Dominic De Nardo, Jacqueline M Ratter, Andrea Stutz, Gudrun Engels, Matthew S Mangan, Brian G Monks & Eicke Latz

  2. Department of Immunology and Rheumatology, Hannover Medical School, Hannover, Germany

    Lukas Bossaller, Martin Fricke & Reinhold E Schmidt

  3. Department of Infectious Diseases and Immunology, University of Massachusetts Medical School, Worcester, Massachusetts, USA

    Lukas Bossaller, Brian G Monks, Patricia Busto, Ann Marshak-Rothstein & Eicke Latz

  4. German Center for Neurodegenerative Diseases, Bonn, Germany

    Christoph Brenker, Mark Nordhoff, Sandra R Mirandola, Ashraf Al-Amoudi, Matthew S Mangan & Eicke Latz

  5. Department of Medicine/Cardiology, University Hospitals, University of Bonn, Bonn, Germany

    Sebastian Zimmer

  6. Department of Cancer Research and Molecular Medicine, Center of Molecular Inflammation Research, Norwegian University of Science and Technology, Trondheim, Norway

    Terje Espevik & Eicke Latz

  7. Centre for Asthma and Respiratory Disease, The University of Newcastle, Newcastle, Australia

    Bernadette Jones, Andrew G Jarnicki & Philip M Hansbro

  8. University Hospital Zürich, Institute of Neuropathology, Zürich, Switzerland

    Simone Hornemann & Adriano Aguzzi

  9. Institute of Molecular Medicine and Experimental Immunology, University Hospitals, University of Bonn, Bonn, Germany

    Wolfgang Kastenmüller

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Contributions

B.S.F., L.B. and W.K. designed and did experiments and analyzed data; J.M.R., A.S., G.E., M.S.M., B.G.M., P.B. and S.H. did experiments; C.B., M.N., S.R.M., A.A.-A., S.H. and A.A. did and analyzed data from electron microscopy and cryo–electron microscopy; D.D.N., T.E., P.B., A.M.-R., A.A. and W.K. analyzed data and provided critical suggestions and discussions throughout the study; S.Z. provided BALF samples from patients with COPD; L.B., M.F. and R.E.S. provided serum samples from patients with autoimmune disease; B.J., A.G.J. and P.M.H. provided the mouse model of COPD; B.S.F. and E.L. designed the study; and B.S.F., D.D.N. and E.L. wrote the paper.

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Correspondence to Eicke Latz.

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Integrated supplementary information

Supplementary Figure 1 ASC forms speck-like clusters in various cells.

(a) Confocal imaging of unstimulated (–) or LPS-primed (200 pg/ml), nigericin-activated (10 μM) human PBMCs (upper panel) or THP-1 monocytes (LPS 1 μg/ml, nigericin 10 μM, lower panel). Scale bars: 22 μm. (b) Flow charge and flow cytometry analysis and fluorescence imaging of ASC-mCerulean specks that were spiked into supernatants of LPS-primed (1 μg/ml), nigericin-activated (10 μM) THP-1 monocytes. Data is representative of two independent experiments.

Supplementary Figure 2 Molecular composition of extracellular ASC specks.

(a) Immunoblot of ASC and NLRP3 in ASC specks purified from cell-free supernatants or from whole cell lysates of LPS-primed, ATP activated Wt, Pycard–/–, Casp1–/– or Nlrp3–/– iMøs. (b) Flow cytometry analysis showing in vitro assembly of ASC-mCerulean specks from cytosolic lysates (S100) of untreated ASC-mCerulean expressing cells. (c) Flow cytometry analysis of in vitro-assembled ASC specks prepared as in b, that were left unstained (Uns) or stained with anti-ASC or anti-Caspase1 Abs followed by staining with A488-direclty conjugated secondary Abs. Samples were also stained with the caspase-1 inhibitor z-YVAD-FMK-FITC and compared to ASC specks generated from Casp1–/– iMøs. Beads with defined sizes (μm) were used as reference. (d) Flow cytometry analysis showing the purification with a 50% percoll cushion and immunoblot for ASC, NLRP3 and Caspase-1 in in vitro-assembled ASC-mCerulean specks prepared as in b. (e) Confocal imaging of LPS-primed and nigericin-activated WT THP-1 monocytes. Cells were fixed and stained with anti-ASC or anti-NLRP3 Abs coupled to fluorochrome-conjugated secondary Abs. Arrows show intra- and extracellular specks containing both endogenous ASC and NLRP3 proteins. Scale bar: 3.2 μm. (f) Confocal imaging of LPS-primed NLRP3-mCitrine expressing iMøs left untreated (UT) or activated with nigericin (10 μm). Cells were fixed and stained with anti-ASC Abs followed by staining with fluorescence-conjugated secondary Abs. The mean fluorescence intensity (MFI) of ASC or NLRP3 was compared in the cytosol (soluble) and on the specks. Data are from one representative out of two (a-f) independent experiments.

Supplementary Figure 3 ASC specks recruit pro-caspase-1 from cell-free supernatants.

(a) Immunoblot for IL-1β and Caspase-1 in cell-free supernatants from untreated or LPS-primed (500 ng/ml, 3h), ATP activated (2.5 mM) iMøs before and after addition of fluorescent recombinant ASC-mCerulean specks generated from WT or Casp1–/– iMøs. (b) Immunoblot for Caspase-1 and GFP in in recombinant ASC-mCerulean specks from WT or Casp1–/– iMøs that were incubated with cell-free supernatants of unstimulated (None), or LPS-primed and ATP, or nigericin-activated iMøs. Data are from one representative of two independent experiments.

Supplementary Figure 4 ASC has 'prionoid' features.

(a) Cryo-EM of in vitro-assembled ASC-mCerulean specks. Scale bars: 100 nm and 200 nm. (b) Time-lapse confocal imaging of the in vitro nucleation of soluble ASC-mCherry (red) by ASC-mCerulean specks (green). Scale bar: 4.4 μm. Graph shows mean fluorescence intensity over time of ASC-mCerulean specks after addition of soluble ASC-mCherry. (c) Rendered 3D confocal image of z-stack sections of ASC-mCerulean specks after incubation with soluble ASC-mCherry. (d) Flow cytometry of pre-assembled ASC-mCerulan specks incubated with soluble ASC-mCherry for the indicated time. (e) Confocal imaging of the nucleation of soluble ASC-mCherry, or FC-mCherry protein by ASC-mCerulean specks. Images were recorded every 30 seconds at 63X magnification. Data are from one representative of two (a,d,e) or three (b,c) independent experiments.

Supplementary Figure 5 ASC specks are resistant to proteases in vivo.

(a) Confocal images showing whole mount staining of ears from LysMgfp/+ transgenic mice at 24, 48, 72 or 96 hours post injection with 1 μg of ASC-mCerulean specks in 10 μl of PBS. (b) Confocal images of whole skin mount of wild-type mice injected in the ear with 1μg of ASC-mCerulean specks. Tissues were stained with anti-ASC (AL177) and anti-Rb Alexa Fluor 568. Scale bars: 20 μm. Data show one representative of two independent experiments.

Supplementary Figure 6 Autoimmune serum contains antibodies to ASC specks.

(a) ELISA of anti-ASC specks Abs in sera from autoimmune patients (n = 14), or healthy donors (n = 7). (b) ELISA of anti-ASC specks Abs in sera from autoimmune (n = 10), or control (n = 10) mice. ELISA plates were coated with 50 μg/ml of ASC specks or PBS. Anti-ASC specks Abs in sera were detected with specific anti-human or anti-mouse HRP conjugated secondary Abs. The absorbance (OD) at 450 nm was normalized against the OD obtained in wells coated with PBS and assayed with anti-human or mouse-HRP Abs. The reactivity of secondary Abs against ASC specks without human or mouse sera is shown.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6 (PDF 9670 kb)

Supplementary Video 1

Time-lapse confocal imaging of ATP-activated (5 mM) inflammasome reporter iMΦs expressing ASC-mCerulean (green). Images were recorded every 5 min. Scale bar: 48 μm. (MOV 9434 kb)

Supplementary Video 2

Time-lapse fluorescence imaging of LPS-primed and nigericin-activated THP-1 cells expressing ASC-mCerulean (green). Nuclei (blue) was stained with Draq5. Images were recorded every 5 sec. Scale bar: 25 μm. (MOV 1128 kb)

Supplementary Video 3

Time-lapse confocal imaging of LPS-primed and nigericin-activated THP-1 cells expressing ASC-mCerulean (red). Plasma membrane (green) was stained with CTxB-Alexa Fluor 555. Images were recorded every 5 sec. Scale bar: 25 μm. (MOV 5148 kb)

Supplementary Video 4

Time-lapse confocal imaging of LPS-primed and nigericin-activated THP-1 cells expressing ASC-mCerulean (green) in the presence of propidium iodide (red). Nuclei (blue) was stained with Draq 5. Images were recorded every 5 min. Scale bar: 30 μm. (MOV 4348 kb)

Supplementary Video 5

Time-lapse confocal imaging of ATP activated ASCmCerulean expressing iMΦs in the presence of 1 μg of Alexa Fluor 555-conjugated anti-GFP mAb. Images were recorded every 5.5 min, scale bar: 80 μm. (MOV 4434 kb)

Supplementary Video 6

Time-lapse confocal imaging of ASC-mCerulean specks-mediated nucleation of soluble ASC-mCherry. ASC-mCerulean specks were seeded into glass-bottom dishes and cytosols from ASC-mCherry expressing cells were added during imaging. Images were recorded every 3 seconds. (MP4 11570 kb)

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Franklin, B., Bossaller, L., De Nardo, D. et al. The adaptor ASC has extracellular and 'prionoid' activities that propagate inflammation. Nat Immunol 15, 727–737 (2014). https://doi.org/10.1038/ni.2913

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