Researchers in the laboratory of Denisa Wagner at the Program in Cellular and Molecular Medicine and the Immune Disease Institute (PCMM/IDI) have made an important connection between transfusion-related acute lung injury (TRALI) and NETs (neutrophil extracellular traps), the webs of DNA cast by neutrophils to sequester and fight infection.
Reporting in Blood online May 17, 2012, an interdisciplinary team led by Dr. Wagner has determined that the usually beneficial NETs are an important culprit in TRALI, which is thought to affect more than 2% of cardiac surgery patients. TRALI also occurs in other patients requiring blood transfusions, with fatal outcomes in about 20% of cases.
Though TRALI has been recognized for more than 50 years, its etiology was unclear. The condition is often detected while the patient is still being transfused, sometimes as soon as 30 minutes after infusion begins. Signs and symptoms include hypoxemia, respiratory difficulty, and pulmonary infiltrates. The only treatment currently available is supportive, including oxygen and mechanical ventilation.
Scientists have known about NETs for less than a decade. In 2004, Dr. Arturo Zychlinsky's group in Germany first discovered that activated neutrophils continue to fight infection even after their death The nucleus dissolves and the nuclear chromatin decondenses, after which the loosened DNA, histones, and other proteins fill the cell. As the cell breaks open, the nuclear materials are expelled as a NET, which binds microorganisms to stop their spread and surrounds them with antimicrobial agents.
Though Dr. Wagner's lab has a track record with NETs, the connection to TRALI was somewhat serendipitous. Dr. Wagner was interested in exploring the area of transfusions, and asked a colleague what the hottest topic in that field was. Though Dr. Wagner was not familiar with TRALI, "When they told me that neutrophils play an important role, and that somehow excitation of neutrophils leads to acute lung injury, I made the connection with NETs! That was the start."
Drs. Grace Thomas and Carla Carbo of IDI began by setting up in the lab an existing mouse model of TRALI. In collaboration with Dr. John Hartwig of the BWH Department of Translational Medicine, they saw that the interior of the mouse alveoli was nearly covered by a dense fibrous mesh. Intranasal DNase 1 dissolved the mesh, identifying DNA as its main structural element. Another marker of NETs is modification of the amino acid arginine into citrulline within histones, which are protein molecules that help organize DNA within the nucleus of a cell. The team, in collaboration with Dr. Ulrich von Andrian, also of the PCMM/IDI, detected citrullination in the lungs of TRALI mice, along with neutrophils.
Dr. Wagner continues the story. "I serve on the Scientific Advisory Board of the Blood Center of Wisconsin in Milwaukee, where our coauthor Richard Aster is located. I asked Dr. Aster, a TRALI expert, if he had blood samples from patients with TRALI and from donors whose blood caused TRALI. They had already isolated antibodies that bound to patients' neutrophils and caused them to activate and agglutinate. He was happy to provide samples, and when Dr. Thomas analyzed the TRALI patients' blood samples, she found evidence of NETs degradation products that were not found in controls."
TRALI is believed to be triggered in a two-step process: first a person undergoes some type of surgery, trauma, or infection; then he or she receives a blood transfusion that contains anti-leukocyte antibodies or bioactive lipids, which activates lung neutrophils. Dr. Thomas reproduced this process in vitro. She first primed neutrophils with TNF (tumor necrosis factor)-alpha, which is produced in the lungs during injury. Only when the antibody purified from TRALI-producing donor blood was added were NETs produced, confirming that two triggers are required.
To verify that NETs were indeed involved in the pathophysiology of TRALI in the mouse model, Dr. Thomas administered intranasal DNase 1. The treatment did improve blood oxygenation (whether administered before or after the onset of TRALI), confirming that NETs were harmful to the lungs during TRALI. In addition, those results suggest possible treatment options in human patients.
Asked to speculate, Dr. Wagner responds that the FDA-approved drug Pulmozyme (a DNase 1 made by Genentech) is already being given to cystic fibrosis patients, who inhale it in powdered form using a nebulizer. CF patients' sputum is highly viscous, partly because of NETs, and dissolving the DNA thins the sputum, both easing their breathing and reducing the risk of infection. "We think it could be administered to patients who are in distress with TRALI, and inhalation means you could get it into the lungs very fast." Dr. Thomas adds, "You could also use it preventatively (before the transfusion) in patients at risk."
Dr. Thomas goes on to explain that their next step is to transfer their work in BALB/c mice (a fairly uncommon genetic background) to the much more widely used C57BL/6 mice. Because so much work has already been done on C57BL/6 animals, including the construction of many genetic knockouts, those mice will allow the Wagner lab to examine the cell biology involved: the interactions among platelets, neutrophils, and the vascular endothelium that trigger NET formation in the lungs.
The transfer of the Wagner lab's work to a more common mouse strain will undoubtedly yield more exciting insights into TRALI. Meanwhile, the clear identification of NETs as the main culprit may soon make blood transfusions safer, with positive repercussions in other cases of immune-mediated respiratory distress.
Thomas GM, Carbo C, Curtis BR, Martinod K, Mazo IB, Schatzberg D, Cifuni SM, Fuchs TA, von Andrian UH, Hartwig JH, Aster RH, Wagner DD. Extracellular DNA traps are associated with the pathogenesis of TRALI in humans and mice. Blood. 2012 May 17. [Epub ahead of print]
