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Aggregated neutrophil extracellular traps limit inflammation by degrading cytokines and chemokines

Nature Medicine volume 20, pages 511–517 (2014)Cite this article

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Abstract

Gout is characterized by an acute inflammatory reaction and the accumulation of neutrophils in response to monosodium urate (MSU) crystals. Inflammation resolves spontaneously within a few days, although MSU crystals can still be detected in the synovial fluid and affected tissues. Here we report that neutrophils recruited to sites of inflammation undergo oxidative burst and form neutrophil extracellular traps (NETs). Under high neutrophil densities, these NETs aggregate and degrade cytokines and chemokines via serine proteases. Tophi, the pathognomonic structures of chronic gout, share characteristics with aggregated NETs, and MSU crystals can induce NETosis and aggregation of NETs. In individuals with impaired NETosis, MSU crystals induce uncontrolled production of inflammatory mediators from neutrophils and persistent inflammation. Furthermore, in models of neutrophilic inflammation, NETosis-deficient mice develop exacerbated and chronic disease that can be reduced by adoptive transfer of aggregated NETs. These findings suggest that aggregated NETs promote the resolution of neutrophilic inflammation by degrading cytokines and chemokines and disrupting neutrophil recruitment and activation.

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Figure 1: Characterization of MSU-induced NETs in tophi and synovial fluid from subjects with gout.
Figure 2: MSU crystals trigger NETosis and aggregation of NETs in cultured human neutrophils.
Figure 3: MSU-induced NETosis and aggNET formation depend on ROS.
Figure 4: Aggregated NETs degrade inflammatory mediators via proteases.
Figure 5: Inability to form NETs results in chronic MSU-induced neutrophilic inflammation.

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Acknowledgements

This project was supported by the Interdisciplinary Center for Clinical Research (IZKF) at the University Hospital of the University of Erlangen-Nuremberg, projects A41 and J41 (C.S., L.E.M. and Martin Herrmann), the Masterswitch project of the European Union (G.S. and Martin Herrmann), SPP1468-IMMUNOBONE (Markus Hoffmann and Martin Herrmann), SFB 643 (L.E.M.), training grant GK SFB 643 from the Deutsche Forschungsgemeinschaft (DFG) (C.S. and C.J.), the Emerging Fields Initiative (EFI) of the Friedrich-Alexander-Universität (FAU) Erlangen-Nuremberg (L.E.M.) and the K. und R. Wucherpfennigstiftung (Martin Herrmann). E.N. was supported by the Deutsche Krebshilfe AZ 109510. B.F. was supported by the European Commissions (DoReMi, European Network of Excellence, contract number 249689) and the German Research Foundation (GA 1507/1-1). R.H. was supported by the Swedish Science Strategic foundation and the EU FP7 project Neurinox. Markus Hoffmann was funded by the Austrian Science Fund FWF (project J3102-B13). We thank K. Wing, Karolinska Institutet, for genotyping of mice.

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Authors and Affiliations

  1. Department of Internal Medicine 3, University of Erlangen-Nuremberg, Erlangen, Germany

    Christine Schauer, Christina Janko, Luis E Munoz, Yi Zhao, Deborah Kienhöfer, Bernhard Manger, Jürgen Rech, Thomas Harrer, Ivica Jeremic, Georg Schett, Markus Hoffmann & Martin Herrmann

  2. Department of Otorhinolaryngology, Head and Neck Surgery, Section for Experimental Oncology and Nanomedicine (SEON), University Hospital Erlangen, Erlangen, Germany

    Christina Janko

  3. Department of Rheumatology and Immunology, West China Hospital, Sichuan University, Chengdu, China

    Yi Zhao

  4. Department of Radiation Oncology, University Hospital Erlangen, Erlangen, Germany

    Benjamin Frey

  5. Department of Radiology, Friedrich-Alexander University of Erlangen-Nuremberg, Erlangen, Germany

    Michael Lell

  6. Division of Molecular and Experimental Surgery, Department of Surgery, University of Erlangen Medical Center, Erlangen, Germany

    Elisabeth Naschberger

  7. Division of Medical Inflammation Research, Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden

    Rikard Holmdahl

  8. Department of Pathology, MVZ of Pathology, Trier, Germany

    Veit Krenn

  9. Institute of Rheumatology, School of Medicine, University of Belgrade, Belgrade, Serbia

    Ivica Jeremic

  10. Institute of Cell Biology, National Academy of Sciences of Ukraine, Lviv, Ukraine

    Rostyslav Bilyy

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  1. Christine Schauer
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Contributions

C.S. planned and performed most of the in vitro and in vivo experiments, conducted data analysis and wrote the manuscript. C.J., L.E.M., B.F., Y.Z., D.K., I.J. and R.B. performed in vivo and in vitro experiments and conducted data analyses. R.H. provided the Ncf1** mice and discussed the strategy of the experiments. M.L., B.M. and J.R. arranged and conducted the DECT analyses. E.N. performed and evaluated immune histology. T.H. and V.K. contributed samples from patients with CGD or gout and controls and provided scientific input. B.M., R.H., L.E.M. and G.S. provided scientific input and wrote the manuscript. Markus Hoffmann planned the experiments, provided scientific input and wrote the manuscript. Martin Herrmann supervised the project, planned and conducted experiments and data analysis and wrote the manuscript. All authors read and approved the manuscript.

Corresponding author

Correspondence to Markus Hoffmann.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–13 (PDF 2140 kb)

Generation of NETs by neutrophils

Isolated human neutrophils (5 × 106 cells ml-1) were stained with Hoechst 33342, incubated with monosodium urate crystals, and analyzed by timelapse video microscopy. Arrows show neutrophils rearranging their internal structures and finally forming NETs in a rapid disintegration process. The original length of the video is 70 minutes. (AVI 332 kb)

Untreated isolated human neutrophils do not undergo NETosis

Isolated human neutrophils (5 × 106 cells ml-1) were stained with Hoechst 33342, incubated with PBS as control, and analyzed by time-lapse video microscopy. The original length of the video is 70 minutes. (AVI 351 kb)

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Schauer, C., Janko, C., Munoz, L. et al. Aggregated neutrophil extracellular traps limit inflammation by degrading cytokines and chemokines. Nat Med 20, 511–517 (2014). https://doi.org/10.1038/nm.3547

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