Neutrophil Extracellular Traps
Neutrophil extracellular traps (NETs) are made of a network of extracellular strings of DNA that bind pathogenic microbes. Histones and several neutrophil granule proteins associated with the DNA framework damage entrapped microorganisms. Reactive oxygen species generated by the neutrophil NADPH oxidase have been shown to be essential to mediate NET release by several stimuli including numerous pathogenic bacteria. Although several methods have been used in the literature to detect NETs in vitro and in vivo, a consensus is urgently needed on the field to standardize the best NET-specific assays. In this chapter, two methods are described in details that can be used to detect NETs and to distinguish them from other mechanisms of neutrophil cell death. While NET-specific, these assays are also relatively simple and straightforward enabling their potential use by a wide audience.
Key wordsNETs Neutrophil NADPH oxidase MPO-DNA complex NE-DNA complex Citrullinated histone Peptidylarginine deiminase 4 Nuclear decondensation
This work was supported by the NIH grant 1 R01 HL136707-01A1 awarded to B. Rada. I thank previous graduate students Dae-goon Yoo, Madison Floyd, and Payel Sil, for taking the immunofluorescence images presented here and for optimizing and recording detailed protocols that helped writing the “Notes” section of this book chapter.
- 3.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
- 4.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
- 5.Yipp BG, Petri B, Salina D, Jenne CN, Scott BN, Zbytnuik LD, Pittman K, Asaduzzaman M, Wu K, Meijndert HC, Malawista SE, de Boisfleury Chevance A, 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
- 8.Leffler J, Gullstrand B, Jonsen A, Nilsson JA, Martin M, Blom AM, Bengtsson AA (2013) Degradation of neutrophil extracellular traps co-varies with disease activity in patients with systemic lupus erythematosus. Arthritis Res Ther 15(4):R84. https://doi.org/10.1186/ar4264CrossRefPubMedPubMedCentralGoogle Scholar
- 9.Kuhns DB, Alvord WG, Heller T, Feld JJ, Pike KM, Marciano BE, Uzel G, DeRavin SS, Priel DA, Soule BP, Zarember KA, Malech HL, Holland SM, Gallin JI (2010) Residual NADPH oxidase and survival in chronic granulomatous disease. N Engl J Med 363(27):2600–2610. https://doi.org/10.1056/NEJMoa1007097CrossRefPubMedPubMedCentralGoogle Scholar
- 13.Yoo DG, Winn M, Pang L, Moskowitz SM, Malech HL, Leto TL, Rada B (2014) Release of cystic fibrosis airway inflammatory markers from Pseudomonas aeruginosa-stimulated human neutrophils involves NADPH oxidase-dependent extracellular DNA trap formation. J Immunol 192(10):4728–4738. https://doi.org/10.4049/jimmunol.1301589CrossRefPubMedPubMedCentralGoogle Scholar
- 15.Wang Y, Li M, Stadler S, Correll S, Li P, Wang D, Hayama R, Leonelli L, Han H, Grigoryev SA, Allis CD, Coonrod SA (2009) Histone hypercitrullination mediates chromatin decondensation and neutrophil extracellular trap formation. J Cell Biol 184(2):205–213. https://doi.org/10.1083/jcb.200806072CrossRefPubMedPubMedCentralGoogle Scholar
- 18.Kraaij T, Tengstrom FC, Kamerling SW, Pusey CD, Scherer HU, Toes RE, Rabelink TJ, van Kooten C, Teng YK (2016) A novel method for high-throughput detection and quantification of neutrophil extracellular traps reveals ROS-independent NET release with immune complexes. Autoimmun Rev 15(6):577–584. https://doi.org/10.1016/j.autrev.2016.02.018CrossRefPubMedGoogle Scholar
- 22.Yoo DG, Floyd M, Winn M, Moskowitz SM, Rada B (2014) NET formation induced by Pseudomonas aeruginosa cystic fibrosis isolates measured as release of myeloperoxidase-DNA and neutrophil elastase-DNA complexes. Immunol Lett 160(2):186–194. https://doi.org/10.1016/j.imlet.2014.03.003CrossRefPubMedGoogle Scholar
- 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). https://doi.org/10.3791/52779
- 24.Brinkmann V, Laube B, Abu Abed U, Goosmann C, Zychlinsky A (2010) Neutrophil extracellular traps: how to generate and visualize them. J Vis Exp (36). https://doi.org/10.3791/1724