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The NLRP3 inflammasome is released as a particulate danger signal that amplifies the inflammatory response

Nature Immunology volume 15, pages 738–748 (2014)Cite this article

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Abstract

Assembly of the NLRP3 inflammasome activates caspase-1 and mediates the processing and release of the leaderless cytokine IL-1β and thereby serves a central role in the inflammatory response and in diverse human diseases. Here we found that upon activation of caspase-1, oligomeric NLRP3 inflammasome particles were released from macrophages. Recombinant oligomeric protein particles composed of the adaptor ASC or the p.D303N mutant form of NLRP3 associated with cryopyrin-associated periodic syndromes (CAPS) stimulated further activation of caspase-1 extracellularly, as well as intracellularly after phagocytosis by surrounding macrophages. We found oligomeric ASC particles in the serum of patients with active CAPS but not in that of patients with other inherited autoinflammatory diseases. Our findings support a model whereby the NLRP3 inflammasome, acting as an extracellular oligomeric complex, amplifies the inflammatory response.

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Figure 1: The activation of inflammasomes induces the release of NLRP3 and ASC.
Figure 2: Extracellular inflammasomes are oligomeric particulate complexes.
Figure 3: ASC specks activate extracellular caspase-1.
Figure 4: Extracellular ASC specks activate caspase-1 in macrophages.
Figure 5: The NLRP3(p.D303N) mutant forms functional oligomeric specks that nucleate ASC.
Figure 6: Extracellular NLRP3(p.D303N) particles form functional inflammasomes in macrophages after being internalized.
Figure 7: Extracellular inflammasome particles are present in the serum of patients with CAPS.

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Acknowledgements

We thank E. Latz (Institute of Innate Immunity) for immortalized ASC-mCherry, ASC-deficient and NLRP3-deficient mouse bone marrow macrophages and for the expression vector encoding red fluorescent protein–tagged ASC; V. Dixit (Genentech) for Pycard−/− mice; M.C. Baños and J.J. Martínez for technical assistance with both molecular and cellular analyses; L. Martinez-Alarcón for extraction of blood from healthy donors; M. Martínez-Villanueva for analysis of CRP; and C. de Torre for support with proteomics. Supported by the Wellcome Trust (V.C.), Instituto Salud Carlos III (CD12/00523 and CD13/00059 to F.M.-S. and J.A.-I.), Instituto Salud Carlos III–Fondo Europeo de Desarrollo Regional (EMER07/049, PS09/00120 and PI13/00174 to P.P. and PS09/01182 to J.Y.), Fundación Séneca (11922/PI/09 to P.P.) and Fundación Mutua Madrileña (ID98FMM013 to A.B.-M.).

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Author notes

  1. Vincent Compan

    Present address: Present address: Department of Cell Biology, University of Geneva, Geneva, Switzerland.,

  2. Alberto Baroja-Mazo and Fatima Martín-Sánchez: These authors contributed equally to this work.

Authors and Affiliations

  1. Inflammation and Experimental Surgery Unit, CIBERehd, Institute for Bio-Health Research of Murcia, Clinical University Hospital Virgen de la Arrixaca, Murcia, Spain

    Alberto Baroja-Mazo, Fatima Martín-Sánchez, Ana I Gomez, Carlos M Martínez, Joaquín Amores-Iniesta, Maria Barberà-Cremades & Pablo Pelegrín

  2. Faculty of Life Sciences, University of Manchester, Manchester, UK

    Vincent Compan, David Brough & Pablo Pelegrín

  3. Department of Immunology-CDB, Hospital Clinic, Barcelona, Spain

    Jordi Yagüe, Estibaliz Ruiz-Ortiz & Juan I Arostegui

  4. Reumathology Pediatric Unit, Hospital Sant Joan de Deu, Barcelona, Spain

    Jordi Antón

  5. Department of Internal Medicine, Hospital Vall d'Hebron, Barcelona, Spain

    Segundo Buján

  6. Experimental and Molecular Immunology and Neurogenetics, CNRS UMR 7355, University of Orleans, Orleans, France

    Isabelle Couillin

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Contributions

A.B.-M., F.M.-S., A.I.G., C.M.M., J.A.-I., V.C. and M.B.-C., execution of experiments; J.Y., E.R.-O., J.A., S.B., I.C. and J.I.A., provision of human samples and mutant mice; A.B.-M., F.M.-S., A.I.G., V.C., D.B., J.I.A. and P.P., analysis and interpretation of data; A.B.-M., F.M.-S., V.C., D.B., J.I.A. and P.P., manuscript preparation; and P.P., conception, design and supervision of this study.

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Correspondence to Pablo Pelegrín.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5 (PDF 597 kb)

Supplementary Table 1

Genotypes and serum inflammatory markers of patients with inherited autoinflammatory syndromes. (PDF 195 kb)

NLRP3 activation induces the release of ASC particles.

Time-lapse fluorescence microscopy of immortalized mouse BMDMs expressing ASC-mCherry primed in coverslips with LPS (1 μg/ml, 2h) and then perfused with nigericin (20 μM) with a flow rate of 1 ml/min. Frames are recorded every 20 sec, the real length of the video corresponds to the 10 min where the ASC speck is formed and released. Nigericin flow was opened after 2 min of basal recording and from that point nigericin concentration gradually increased in the recording chamber at the flow rate used. In these conditions ASC speck formation was observed after 6 min of nigericin flow opening and the ASC speck release occurred 5 min after formation. (AVI 182 kb)

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Baroja-Mazo, A., Martín-Sánchez, F., Gomez, A. et al. The NLRP3 inflammasome is released as a particulate danger signal that amplifies the inflammatory response. Nat Immunol 15, 738–748 (2014). https://doi.org/10.1038/ni.2919

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