Advertisement

Visualization and Quantification of Neutrophil Extracellular Traps

Protocol
First Online:
  • 789 Downloads
Part of the Methods in Molecular Biology book series (MIMB, volume 2255)

Abstract

Neutrophils are innate immune cells that play important roles in many physiological and pathological processes, including immune defense and cancer metastasis. In addition to the release of proinflammatory cytokines, chemokines, and cytoplasmic granules containing digestive proteins, in recent years, neutrophils have been observed to release neutrophil extracellular traps (NETs) that consist of extracellular DNA associated with antimicrobial proteins, such as histones and myeloperoxidase. These NETs are increasingly being recognized as an important mechanism of neutrophil host defense and function. This chapter will summarize the current literature on the known processes of NET formation and describe in detail an immunofluorescence approach that can be employed to visualize and quantify NETs in vitro.

Key words

NETs Extracellular DNA Activation Myeloperoxidase Sytox PicoGreen Citrullination 
This is a preview of subscription content, log in to check access.

Notes

Acknowledgments

Conflicts of interest: The authors have no conflicts of interest.

Funding: Supported by grant R01AI121183 (to VMA) from The National Institute of Allergy and Infectious Diseases (NIAID), the National Institutes of Health (NIH).

References

  1. 1.
    Kolaczkowska E, Kubes P (2013) Neutrophil recruitment and function in health and inflammation. Nat Rev Immunol 13(3):159–175.  https://doi.org/10.1038/nri3399CrossRefPubMedGoogle Scholar
  2. 2.
    Nathan C (2006) Neutrophils and immunity: challenges and opportunities. Nat Rev Immunol 6(3):173–182.  https://doi.org/10.1038/nri1785CrossRefPubMedGoogle Scholar
  3. 3.
    Brinkmann V, Reichard U, Goosmann C, Fauler B, Uhlemann Y, Weiss DS, Weinrauch Y, Zychlinsky A (2004) Neutrophil extracellular traps kill bacteria. Science 303(5663):1532–1535.  https://doi.org/10.1126/science.1092385CrossRefPubMedGoogle Scholar
  4. 4.
    Fuchs TA, Abed U, Goosmann C, Hurwitz R, Schulze I, Wahn V, Weinrauch Y, Brinkmann V, Zychlinsky A (2007) Novel cell death program leads to neutrophil extracellular traps. J Cell Biol 176(2):231–241.  https://doi.org/10.1083/jcb.200606027CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Mayadas TN, Cullere X, Lowell CA (2014) The multifaceted functions of neutrophils. Annu Rev Pathol 9:181–218.  https://doi.org/10.1146/annurev-pathol-020712-164023CrossRefPubMedGoogle Scholar
  6. 6.
    Tong M, Abrahams VM (2019) Neutrophils in preterm birth: friend or foe? Placenta 495(1):60–63.  https://doi.org/10.1016/j.placenta.2019.12.010CrossRefGoogle Scholar
  7. 7.
    Yipp BG, Kubes P (2013) NETosis: how vital is it? Blood 122(16):2784–2794.  https://doi.org/10.1182/blood-2013-04-457671CrossRefPubMedGoogle Scholar
  8. 8.
    Douda DN, Khan MA, Grasemann H, Palaniyar N (2015) SK3 channel and mitochondrial ROS mediate NADPH oxidase-independent NETosis induced by calcium influx. Proc Natl Acad Sci U S A 112(9):2817–2822.  https://doi.org/10.1073/pnas.1414055112CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Pilsczek FH, Salina D, Poon KK, Fahey C, Yipp BG, Sibley CD, Robbins SM, Green FH, Surette MG, Sugai M, Bowden MG, Hussain M, Zhang K, Kubes P (2010) A novel mechanism of rapid nuclear neutrophil extracellular trap formation in response to Staphylococcus aureus. J Immunol 185(12):7413–7425.  https://doi.org/10.4049/jimmunol.1000675CrossRefPubMedGoogle Scholar
  10. 10.
    Rochael NC, Guimaraes-Costa AB, Nascimento MT, DeSouza-Vieira TS, Oliveira MP, Garcia e Souza LF, Oliveira MF, Saraiva EM (2015) Classical ROS-dependent and early/rapid ROS-independent release of neutrophil extracellular traps triggered by Leishmania parasites. Sci Rep 5:18302.  https://doi.org/10.1038/srep18302CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Chapman EA, Lyon M, Simpson D, Mason D, Beynon RJ, Moots RJ, Wright HL (2019) Caught in a trap? Proteomic analysis of neutrophil extracellular traps in rheumatoid arthritis and systemic lupus erythematosus. Front Immunol 10:423.  https://doi.org/10.3389/fimmu.2019.00423CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Petretto A, Bruschi M, Pratesi F, Croia C, Candiano G, Ghiggeri G, Migliorini P (2019) Neutrophil extracellular traps (NET) induced by different stimuli: a comparative proteomic analysis. PLoS One 14(7):e0218946.  https://doi.org/10.1371/journal.pone.0218946CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Tong M, Potter JA, Mor G, Abrahams VM (2019) Lipopolysaccharide-stimulated human fetal membranes induce neutrophil activation and release of vital neutrophil extracellular traps. J Immunol 203(2):500–510.  https://doi.org/10.4049/jimmunol.1900262CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Yousefi S, Mihalache C, Kozlowski E, Schmid I, Simon HU (2009) Viable neutrophils release mitochondrial DNA to form neutrophil extracellular traps. Cell Death Differ 16(11):1438–1444.  https://doi.org/10.1038/cdd.2009.96CrossRefPubMedGoogle Scholar
  15. 15.
    McIlroy DJ, Jarnicki AG, Au GG, Lott N, Smith DW, Hansbro PM, Balogh ZJ (2014) Mitochondrial DNA neutrophil extracellular traps are formed after trauma and subsequent surgery. J Crit Care 29(6):1133 e1131–1133 e1135.  https://doi.org/10.1016/j.jcrc.2014.07.013CrossRefGoogle Scholar
  16. 16.
    Wang H, Li T, Chen S, Gu Y, Ye S (2015) Neutrophil extracellular trap mitochondrial DNA and its autoantibody in systemic lupus erythematosus and a proof-of-concept trial of metformin. Arthritis Rheumatol 67(12):3190–3200.  https://doi.org/10.1002/art.39296CrossRefPubMedGoogle Scholar
  17. 17.
    Onouchi T, Shiogama K, Mizutani Y, Takaki T, Tsutsumi Y (2016) Visualization of neutrophil extracellular traps and fibrin meshwork in human Fibrinopurulent inflammatory lesions: III. Correlative light and electron microscopic study. Acta Histochem Cytochem 49(5):141–147.  https://doi.org/10.1267/ahc.16028CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Yipp BG, Petri B, Salina D, Jenne CN, Scott BN, Zbytnuik LD, Pittman K, Asaduzzaman M, Wu K, Meijndert HC, Malawista SE, de Boisfleury CA, Zhang K, Conly J, Kubes P (2012) Infection-induced NETosis is a dynamic process involving neutrophil multitasking in vivo. Nat Med 18(9):1386–1393.  https://doi.org/10.1038/nm.2847CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    de Buhr N, von Kockritz-Blickwede M (2016) How neutrophil extracellular traps become visible. J Immunol Res 2016:4604713.  https://doi.org/10.1155/2016/4604713CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Clark SR, Ma AC, Tavener SA, McDonald B, Goodarzi Z, Kelly MM, Patel KD, Chakrabarti S, McAvoy E, Sinclair GD, Keys EM, Allen-Vercoe E, Devinney R, Doig CJ, Green FH, Kubes P (2007) Platelet TLR4 activates neutrophil extracellular traps to ensnare bacteria in septic blood. Nat Med 13(4):463–469.  https://doi.org/10.1038/nm1565CrossRefPubMedGoogle Scholar
  21. 21.
    Barr FD, Ochsenbauer C, Wira CR, Rodriguez-Garcia M (2018) Neutrophil extracellular traps prevent HIV infection in the female genital tract. Mucosal Immunol 11(5):1420–1428.  https://doi.org/10.1038/s41385-018-0045-0CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Gonzalez AS, Bardoel BW, Harbort CJ, Zychlinsky A (2014) Induction and quantification of neutrophil extracellular traps. Methods Mol Biol 1124:307–318.  https://doi.org/10.1007/978-1-62703-845-4_20CrossRefPubMedGoogle Scholar
  23. 23.
    Sil P, Yoo DG, Floyd M, Gingerich A, Rada B (2016) High throughput measurement of extracellular DNA release and quantitative NET formation in human neutrophils in vitro. J Vis Exp 112:52779.  https://doi.org/10.3791/52779CrossRefGoogle Scholar
  24. 24.
    Zhao W, Fogg DK, Kaplan MJ (2015) A novel image-based quantitative method for the characterization of NETosis. J Immunol Methods 423:104–110.  https://doi.org/10.1016/j.jim.2015.04.027CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Mohanty T, Sorensen OE, Nordenfelt P (2017) NETQUANT: automated quantification of neutrophil extracellular traps. Front Immunol 8:1999.  https://doi.org/10.3389/fimmu.2017.01999CrossRefPubMedGoogle Scholar
  26. 26.
    van Breda SV, Vokalova L, Neugebauer C, Rossi SW, Hahn S, Hasler P (2019) Computational methodologies for the in vitro and in situ quantification of neutrophil extracellular traps. Front Immunol 10:1562.  https://doi.org/10.3389/fimmu.2019.01562CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Granger V, Faille D, Marani V, Noel B, Gallais Y, Szely N, Flament H, Pallardy M, Chollet-Martin S, de Chaisemartin L (2017) Human blood monocytes are able to form extracellular traps. J Leukoc Biol 102(3):775–781.  https://doi.org/10.1189/jlb.3MA0916-411RCrossRefPubMedGoogle Scholar
  28. 28.
    Doster RS, Rogers LM, Gaddy JA, Aronoff DM (2018) Macrophage extracellular traps: a scoping review. J Innate Immun 10(1):3–13.  https://doi.org/10.1159/000480373CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2021

Authors and Affiliations

  1. 1.Department of Obstetrics, Gynecology and Reproductive SciencesYale School of MedicineNew HavenUSA

Personalised recommendations

Cite protocol

Buy options