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Exploring Neutrophil Extracellular Traps (NETs) in Lung Transplantation

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Lindsay Caldarone
University Health Network
Toronto, Canada

A "suicide bomber" that undergoes "cellular kamikaze" to produce a "spider's web" - and these are just a few of the many colorful descriptions used to explain the process of neutrophils undergoing NETosis [1,2,3]. NETosis was first described in 2004 by Brinkmann et al. as an innate immune response to bacterial invasion [4]. Since that time, research into the mechanisms and consequences of NETosis has exploded. NETs have been described as beneficial agents that fend off viruses and fungi, in addition to bacteria. They have also been implicated in a wide variety of inflammatory disease states, from autoimmune disease to acute lung injury.

The specific mechanisms of NETosis are not yet fully described, but there is a general consensus that key steps include the activation of peptidylarginine deiminase 4 (PAD4) that assists in the citrullination of histones resulting in decondensation of nuclear chromatin. This chromatin co-localizes with granular proteins such as neutrophil elastase and myeloperoxidase, before being actively extruded into the extracellular space. NETosis can be NADPH oxidase-dependent or -independent, and can be either "suicidal," wherein the neutrophil dies after producing NETs, or "vital," where the NET is extruded through vesicles, the neutrophil membranes are preserved and the resulting anuclear neutrophil retains phagocytic function[2, 5].

Of special note to our community is the discovery by Sayah et al. that NETs are pathogenic in primary lung graft dysfunction [6]. This group showed that NETs were detectable in mouse lungs after both a hilar clamp model as well as orthotopic lung transplantation, indicating that NETosis occurs as a result of the ischemia and reperfusion of the lung, as well as in the more clinically applicable scenario of orthotopic transplant. In fact, many steps in the NETosis pathways are well described as mediators in ischemia-reperfusion injury (IRI); for instance, generation of reactive oxygen species, autophagy and influx of intracellular calcium [7, 8].

IRI occurs in the recipient; however, donor lungs may also sustain various types of injury. Many of these injuries trigger inflammatory pathways that may also lead to NETosis; for instance, brain death can lead to increased levels of the neutrophil activator interleukin-8[9]. NETs are thought to propagate neutrophil recruitment and activation, thus contributing to a positive feedback cycle of NETosis at the site of injury. NETs found in the donor lung could be an artifact of the injury sustained by the donor lung and presumably, if these donor NETs are transplanted into the recipient they could amplify the IRI post-transplant. Examining the donor lung for NETs could a) give a quantifiable assessment of inflammatory injury in the donor lung and b) provide a potential therapeutic target so NETs could be removed or degraded prior to transplant, resulting in reduced IRI.

How could this be done? Ex vivo lung perfusion (EVLP) provides the unique opportunity to assess the viability and functionality of donor lungs prior to transplantation. EVLP has been successfully implemented clinically in our center and others around the globe. Furthermore, the perfusate from the circuit can be analyzed for markers of biological processes occurring in the donor lung. Understanding what is happening in the donor lung on a cellular level offers an entirely new insight into how a lung will perform post-transplant. More precise, objective donor lung assessment can lead to an expansion of the donor pool and alleviate the intense shortage of donor lungs, as lungs that may otherwise be too risky to transplant can be proven to be safe. Likewise, donor lungs that may seem acceptable based on the current practice of visual, physiological and functional assessment can be rejected if cellular markers of injury are apparent, thus preventing adverse outcomes in the recipient.

If we can assess NETs in the donor lung, we can understand more about the type of injury sustained by each individual lung as well as learn more about how this novel, programmed neutrophil response impacts lung injury pre- and post-transplant. EVLP provides the ideal opportunity for quantifying NETs in the donor lung. Just two decades ago, the use of "clinical ex vivo lung perfusion" or the phrase "neutrophil extracellular traps" did not exist; by continuing to build on the rapid advancements in research in this field, we can continue to have a greater and greater impact on clinical outcomes and patient lives. ■

Disclosure Statement: The author has no conflicts of interest to disclose.


  1. Wilhelm, K. The tangled NETs of the immune system. 2011.
  2. Yipp, B.G. and P. Kubes, NETosis: how vital is it? Blood, 2013. 122(16): p. 2784-94.
  3. Cooper, P.R., L.J. Palmer, and I.L. Chapple, Neutrophil extracellular traps as a new paradigm in innate immunity: friend or foe? Periodontol 2000, 2013. 63(1): p. 165-97.
  4. Brinkmann, V., et al., Neutrophil extracellular traps kill bacteria. Science, 2004. 303(5663): p. 1532-5.
  5. Douda, D.N., et al., SK3 channel and mitochondrial ROS mediate NADPH oxidase-independent NETosis induced by calcium influx. Proc Natl Acad Sci U S A, 2015. 112(9): p. 2817-22.
  6. Sayah, D.M., et al., Neutrophil extracellular traps are pathogenic in primary graft dysfunction after lung transplantation. Am J Respir Crit Care Med, 2015. 191(4): p. 455-63.
  7. Kalogeris, T., et al., Cell biology of ischemia/reperfusion injury. Int Rev Cell Mol Biol, 2012. 298: p. 229-317.
  8. Remijsen, Q., et al., Dying for a cause: NETosis, mechanisms behind an antimicrobial cell death modality. Cell Death Differ, 2011. 18(4): p. 581-8.
  9. Avlonitis, V.S., et al., Pulmonary transplantation: the role of brain death in donor lung injury. Transplantation, 2003. 75(12): p. 1928-33.

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