Advertisement

Imaging and Manipulation of Extracellular Traps by Atomic Force Microscopy

  • Ricardo H. PiresEmail author
  • Mihaela Delcea
  • Stephan B. Felix
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1886)

Abstract

Neutrophil extracellular traps (NETs) are part of an immunological response and one of the mechanisms by which neutrophils protect the host from pathogen invasion and proliferation. Notwithstanding their protective role, NETs have also been linked to the development of a variety of disorders, including cardiovascular and autoimmune diseases. Since the first reports on NETs in 2004 it has been possible to image NETs by a variety of imaging techniques. Despite this, such reports seldomly include contact probe methods, and therefore lack the unique insights such techniques typically provide. In fact, more than 10 years have passed since the discovery of NETs, and although their importance as part of a unique cellular response mechanism has become very clear, studies that attempt to address them by atomic force microscopy (AFM) remain very limited. Particularly striking is the almost absent information on the mechanical properties of NETs, and factors that may influence them. The fact that NETs are a particularly adhesive network of filaments poses a considerable technical challenge for contact probe methods and can limit advances involving either imaging or manipulation of NETs by AFM. The current set of protocols aims at aiding a knowledgeable AFM operator to obtain AFM images and to perform force spectroscopy experiments with such samples. A variety of different topics, including sample preparation and data analysis, are discussed.

Key words

Atomic force microscopy AFM Chromatin DNA Extracellular traps Force spectroscopy NETs Neutrophils Surface modification 

References

  1. 1.
    Brinkmann V, Reichard U, Goosmann C, Fauler B, Uhlemann Y, Weiss DS et al (2004) Neutrophil extracellular traps kill bacteria. Science 303(5663):1532–1535CrossRefGoogle Scholar
  2. 2.
    Wardini AB, Guimaraes-Costa AB, Nascimento MT, Nadaes NR, Danelli MG, Mazur C et al (2010) Characterization of neutrophil extracellular traps in cats naturally infected with feline leukemia virus. J Gen Virol 91(Pt 1):259–264CrossRefGoogle Scholar
  3. 3.
    Ermert D, Urban CF, Laube B, Goosmann C, Zychlinsky A, Brinkmann V (2009) Mouse neutrophil extracellular traps in microbial infections. J Innate Immun 1(3):181–193CrossRefGoogle Scholar
  4. 4.
    Chuammitri P, Ostojic J, Andreasen CB, Redmond SB, Lamont SJ, Palic D (2009) Chicken heterophil extracellular traps (HETs): novel defense mechanism of chicken heterophils. Vet Immunol Immunopathol 129(1–2):126–131CrossRefGoogle Scholar
  5. 5.
    Ng TH, Chang SH, Wu MH, Wang HC (2013) Shrimp hemocytes release extracellular traps that kill bacteria. Dev Comp Immunol 41(4):644–651CrossRefGoogle Scholar
  6. 6.
    Poirier AC, Schmitt P, Rosa RD, Vanhove AS, Kieffer-Jaquinod S, Rubio TP et al (2014) Antimicrobial histones and DNA traps in invertebrate immunity: evidences in Crassostrea gigas. J Biol Chem 289(36):24821–24831CrossRefGoogle Scholar
  7. 7.
    Barrientos L, Bignon A, Gueguen C, de Chaisemartin L, Gorges R, Sandre C et al (2014) Neutrophil extracellular traps downregulate lipopolysaccharide-induced activation of monocyte-derived dendritic cells. J Immunol 193(11):5689–5698CrossRefGoogle Scholar
  8. 8.
    Vossenaar ER, Zendman AJ, van Venrooij WJ, Pruijn GJ (2003) PAD, a growing family of citrullinating enzymes: genes, features and involvement in disease. BioEssays 25(11):1106–1118CrossRefGoogle Scholar
  9. 9.
    Wang S, Wang Y (2013) Peptidylarginine deiminases in citrullination, gene regulation, health and pathogenesis. Biochim Biophys Acta 1829(10):1126–1135CrossRefGoogle Scholar
  10. 10.
    Wang Y, Li M, Stadler S, Correll S, Li P, Wang D et al (2009) Histone hypercitrullination mediates chromatin decondensation and neutrophil extracellular trap formation. J Cell Biol 184(2):205–213CrossRefGoogle Scholar
  11. 11.
    Leshner M, Wang S, Lewis C, Zheng H, Chen XA, Santy L et al (2012) PAD4 mediated histone hypercitrullination induces heterochromatin decondensation and chromatin unfolding to form neutrophil extracellular trap-like structures. Front Immunol 3:307CrossRefGoogle Scholar
  12. 12.
    Cooper PR, Palmer LJ, Chapple IL (2013) Neutrophil extracellular traps as a new paradigm in innate immunity: friend or foe? Periodontol 2000 63(1):165–197CrossRefGoogle Scholar
  13. 13.
    Sorensen OE, Borregaard N (2016) Neutrophil extracellular traps—the dark side of neutrophils. J Clin Invest 126(5):1612–1620CrossRefGoogle Scholar
  14. 14.
    Obermayer A, Stoiber W, Krautgartner WD, Klappacher M, Kofler B, Steinbacher P et al (2014) New aspects on the structure of neutrophil extracellular traps from chronic obstructive pulmonary disease and in vitro generation. PLoS One 9(5):e97784CrossRefGoogle Scholar
  15. 15.
    Pires RH, Felix SB, Delcea M (2016) The architecture of neutrophil extracellular traps investigated by atomic force microscopy. Nanoscale 8(29):14193–14202CrossRefGoogle Scholar
  16. 16.
    de Buhr N, von Kockritz-Blickwede M (2016) How neutrophil extracellular traps become visible. J Immunol Res 2016:4604713PubMedPubMedCentralGoogle Scholar
  17. 17.
    Manzenreiter R, Kienberger F, Marcos V, Schilcher K, Krautgartner WD, Obermayer A et al (2012) Ultrastructural characterization of cystic fibrosis sputum using atomic force and scanning electron microscopy. J Cyst Fibros 11(2):84–92CrossRefGoogle Scholar
  18. 18.
    Gregurec D, Wang G, Pires RH, Kosutic M, Lüdtke T, Delcea M et al (2016) Bioinspired titanium coatings: self-assembly of collagen–alginate films for enhanced osseointegration. J Mater Chem B 4:1978–1986CrossRefGoogle Scholar
  19. 19.
    Wang H, Bash R, Yodh JG, Hager GL, Lohr D, Lindsay SM (2002) Glutaraldehyde modified mica: a new surface for atomic force microscopy of chromatin. Biophys J 83(6):3619–3625CrossRefGoogle Scholar
  20. 20.
    Pires RH, Saraiva MJ, Damas AM, Kellermayer MS (2011) Structure and assembly-disassembly properties of wild-type transthyretin amyloid protofibrils observed with atomic force microscopy. J Mol Recognit 24(3):467–476CrossRefGoogle Scholar
  21. 21.
    Pires RH, Karsai A, Saraiva MJ, Damas AM, Kellermayer MS (2012) Distinct annular oligomers captured along the assembly and disassembly pathways of transthyretin amyloid protofibrils. PLoS One 7(9):e44992CrossRefGoogle Scholar
  22. 22.
    Gyimesi M, Pires RH, Billington N, Sarlos K, Kocsis ZS, Modos K et al (2013) Visualization of human Bloom’s syndrome helicase molecules bound to homologous recombination intermediates. FASEB J 27(12):4954–4964CrossRefGoogle Scholar
  23. 23.
    Hutter J, Bechhoefer J (1993) Calibration of atomic-force microscope tips. Rev Sci Instrum 64(7):1868–1873CrossRefGoogle Scholar
  24. 24.
    Reis LA, Rocha MS (2017) DNA interaction with DAPI fluorescent dye: force spectroscopy decouples two different binding modes. Biopolymers 107(5):e23015CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Ricardo H. Pires
    • 1
    • 2
    • 3
    Email author
  • Mihaela Delcea
    • 1
    • 3
  • Stephan B. Felix
    • 2
    • 3
  1. 1.ZIK-HIKE, Center for Innovation and Competence—Humoral Immune Reactions in Cardiovascular DiseasesUniversity of GreifswaldGreifswaldGermany
  2. 2.Department of Internal Medicine B, CardiologyUniversity of GreifswaldGreifswaldGermany
  3. 3.DZHK, German Center for Cardiovascular ResearchGreifswaldGermany

Personalised recommendations