Extracellular DNA Traps Are Associated with Pathogenesis of TRALI in Humans and Mice
Authors: Thomas Grace M. , Carbo Carla , Curtis Brian , Martinod Kimberly , Mazo Irina B. , Schatzberg Daphne , Cifuni Stephen M. , Fuchs Tobias A. , von Andrian Ulrich H. , Hartwig John H. , Aster Richard H. , Wagner Denisa D. 
Published: October 2019

Abstract
Abstract 37
Transfusion-related acute lung injury (TRALI) is a serious complication of blood transfusion occurring within 6 hours of transfusion. It is characterized by hypoxemia, respiratory distress and pulmonary infiltrates (Kleinman et al. Transfusion 2004) and is the leading cause of transfusion-induced death. The biological processes contributing to TRALI are still poorly understood. All blood products can cause TRALI, and to date no specific treatment is available. A “two-event model” has been proposed as the trigger (Silliman et al Blood 2007). The first event may include surgery, trauma or infection; the second involves the transfusion of anti-neutrophil antibodies or bioactive lipids within the blood product. Together these events induce neutrophil activation and sequestration in the pulmonary capillaries (Looney et al J Clin Invest 2006) resulting in endothelial damage and capillary leakage (Shaz et al Blood 2011). In response to pathogens or under stress, activated neutrophils have the ability to release neutrophil extracellular traps (NETs) (Brinkmann Science 2004; Fuchs et al J Cell Biol 2007) that are composed of DNA fibers decorated with histones and antimicrobial proteins (Urban PLoS Pathog 2009) originally contained in the neutrophil granules. Although protective against infection, these NETs are injurious to tissue (Xu et al Nat Med 2009, Xu et al J Immunol 2011), activate platelets (Fuchs et al PNAS 2010) and can contribute to the pathology of inflammatory diseases (Kessenbrock et al Nat Med 2009, Garcia-Romo Sci Transl Med 2011). The purpose of this study was to determine whether NETs are formed in patients during TRALI and whether they contribute to TRALI in a mouse model.
Here we show that NETs biomarkers are present in TRALI patients' blood and that the antibody responsible for the most severe TRALI cases and that is directed against human neutrophil antigen (HNA)-3a (Davoren et al Transfusion 2003; Greinacher et al Nat Med 2010) stimulates NETs generation from primed human neutrophils when compared to a control antibody. This effect was inhibited when FcgRIIa-binding sites were blocked prior to antibody incubation. Moreover, anti-HNA-3a F(ab')2 fragments had no effect on NETs generation. These results allowed us to conclude that FcgRIIa activation was required in anti-HNA-3a antibody-mediated NETosis.
We next used an in vivo model of antibody-induced TRALI in mice (Looney et al J Clin Invest 2006; Looney et al. J Clin Invest 2009; Hidalgo et al Nat Med 2009). In this two-event model, BALB/c mice are injected i.p. with a low-dose of LPS and 24 hours later are infused with an anti-MHC class I monoclonal antibody (anti-H-2Kd). Two hours later, we measured arterial blood oxygen saturation to document lung function and the lung injury markers were quantified. As observed in blood from TRALI patients, we detected a significant increase in the concentration of circulating NETs biomarkers in mice with TRALI compared to mice that were challenged only with LPS. Multiphoton analysis of fixed lung tissue showed strong staining for citrullinated histone H3 (a phenomenon already described to correlate with NETs formation (Wang et al J Cell Biol 2009; Li et al J Exp Med 2010)) collocalizing with DNA streaks in the alveoli of mice with TRALI. Moreover, we found NETs in abundance in the TRALI-affected alveoli of mice by transmission electron microscopy. We demonstrate that intranasal DNaseI pretreatment of the mice prior to antibody infusion prevented NETs accumulation in the alveoli and also improved the lung function of the mice experiencing TRALI by increasing their arterial blood oxygenation. These results support the hypothesis that NETs are indeed formed in the inflamed lungs during TRALI and contribute to the disease process and thus, could be targeted to prevent or treat TRALI.

Disclosures:
No relevant conflicts of interest to declare.