In this study, first, we have identified that platelet activation can induce NET formation in the absence of infection and that NETs increase endothelial permeability. Second, we found that NETs are present in abundance in the lungs of mice undergoing experimental TRALI, and NETs and circulating NET components are strongly increased in clinical cases of TRALI. Third, inhibiting platelet activation reduced NET formation and ALI in vivo. Fourth, by targeting NETs directly with a histone blocking antibody or dismantling NETs with DNase1 in preinjury or postinjury models, there was strong protection from lung endothelial injury in TRALI. The discovery of the important role of platelet-induced NET formation in ALI represents a paradigm shift that may lead to new therapeutic approaches.
NETs were described less than a decade ago (11
), but there is a growing list of diseases that are associated with NET formation. NETs have classically been linked with severe infections, such as sepsis, in which the release of chromatin decorated with neutrophil granular proteins serves as an additional weapon of the innate immune system against circulating bacteria. However, NETs are also found in noninfectious diseases in which the presence of NETs may be a maladaptive response that leads to tissue injury. We now add ALI to the list of diseases that involve NETs, and indeed, in our model of noninfectious ALI, TRALI, we cannot postulate an obvious beneficial role of NET formation.
A major mechanism by which platelets promote inflammation and injury is through critical interactions with neutrophils. For example, in sepsis, activation of platelet TLR4 is a potent stimulus for neutrophils residing in the sinusoids of the liver and other capillary beds to release NETs (16
). We also found that activation of platelets with PAR-1 agonists (TRAP, thrombin) was equally efficient in producing NETs, which suggests that a variety of platelet agonists may be capable of promoting NET formation. The mechanism by which platelets promote NETosis is still not clear, but we propose that platelet activation primes neutrophils for NET formation. It is known that neutrophil-platelet aggregates have greater adhesive capacity, greater reactive oxygen species production, and phagocytic potential (35
). Reactive oxygen species production is tied closely to NET formation (36
). Experiments have shown that MEK signaling, which is upstream of the NADPH oxidase, is critical to NET formation resulting from PMA-induced activation (26
). Interruption of MEK signaling in our platelet-activation experiments also efficiently decreased NET formation, suggesting that MEK signaling is a common pathway through which different stimuli can induce NETosis. We also found that inhibition of TXA2
signaling in our system reduced NET formation, even though neutrophils are not known to possess thromboxane receptors. However, TXA2
has been previously shown to augment the respiratory burst of neutrophils, and this effect can be blocked with aspirin (37
We found that in TRALI, within minutes after antibody challenge, there was an increase in platelet aggregation on neutrophils in the lungs, which supports the hypothesis that critical neutrophil-platelet interactions are promoting NET formation. The speed by which these interactions develop after MHC class I antibody injection is remarkable and is proximal to the onset of overt ALI in our model. We also observed ultrastructural evidence of neutrophil-platelet aggregates in the lung microcirculation that included close interactions with the lung endothelium and red blood cells. Similar findings have been reported in TRALI and are dependent on polarized domains of CD11b/CD18 (38
). However, using the same MHC class I mAb, another group has recently reported the observation of neutrophil- and platelet-independent lung injury (39
). This report contrasts with our findings of neutrophil- and platelet-dependent ALI and our current findings of platelet-mediated NET formation. There are fundamental differences in the animal models that likely explain the divergent results, including (a) production and purification of mAb, (b) dosing of mAb, and (c) recipient immune status. Delivery of large doses of mAb in mainly unprimed animals (39
) could concentrate mAb on endothelial surfaces, which potentially could produce a more complement- and monocyte-dependent lung injury. Our results showing intense neutrophil and platelet sequestration in the lung, intimate neutrophil-platelet interactions, abrogation of injury with nonantibody-based platelet inhibitors (aspirin, tirofiban) and inhibitors of NETs (DNase1) or NET components (histone antibody), and the presence of NETs in human TRALI cases all strongly point to the importance of platelets and neutrophils in TRALI. Additionally, recent results from a prospective TRALI case-control study found that thrombocytopenia before transfusion (platelet count < 50 × 109
/l) was associated with protection from TRALI, and there was a trend for neutropenia before transfusion also being protective (34
We found abundant NET formation in the lung microcirculation and plasma in experimental TRALI. A postulated function of NETs is to skim the plasma for intravascular pathogens; however, other intravascular components may also be efficiently trapped. We found intense platelet sequestration in areas of NET formation. Platelets may bind especially well to NETs through interactions with charged extracellular histones (29
). Platelet accumulation on NETs may perpetuate platelet aggregation and activation and promote coagulation in the lung microcirculation that could have ischemic consequences (18
). However, using fibrinogen immunostaining, we were unable to detect increased clot formation in NET-containing areas of TRALI lungs (data not shown), but ischemic conditions could still be induced by NETs through physical trapping of platelets, red blood cells, and other leukocytes. Consistent with the role of TXA2
in our in vitro experiments, treatment of mice with aspirin decreases plasma TXB2
levels, ALI, and mortality (5
). Here, we found decreased NET formation in the lung microcirculation and plasma and decreased lung deposition of platelets with aspirin treatment. When we blocked platelet-platelet interactions with a glycoprotein IIb/IIIa inhibitor, mice were also protected from TRALI and mortality.
NETs likely induce injury to the lung through direct toxicity to endothelial cells, as we showed in our in vitro model of endothelial permeability, perhaps by concentrating histones and granular proteins to reach high local concentrations that enhance their toxicity. We also propose that there is a substantial contribution from the DNA scaffold that may trap cells and impede forward flow, leading to microcirculatory injury. However, to move the field forward beyond the association of NETs with disease, evidence is needed showing that NET-specific treatment approaches ameliorate disease manifestations.
Effective therapeutic agents for ALI are desperately needed. We propose that NETs are a new target for pharmaceutical development in ALI. One treatment strategy focuses on inhibiting the initial formation of NETs; we propose that antiplatelet agents (aspirin, tirofiban) are effective approaches. Targeting NET components is an alternative approach and potentially more clinically feasible, since NETs may be formed early in diseases like ALI and sepsis and already producing injury upon initial clinical presentation. We targeted NET components using a blocking antibody against H2A and H4 histones and also by disrupting the NET scaffold with DNase1. Both strategies were effective in reducing lung injury and mortality. DNase1 was also effective when given 5 minutes after initiation of TRALI when NETs are already present and lung injury is developing. DNase1 is naturally occurring in human blood (40
) and is also produced as a defense mechanism by bacteria attempting to escape entanglement by NETs (41
). Intrapulmonary DNase is already an effective therapy in cystic fibrosis, targeting the extracellular DNA that interferes with mucociliary clearance (42
). Delivering therapeutic agents to patients with TRALI will be a challenge, given the abrupt onset of the condition, but targeting NET components (histones, DNA) is an attractive option that is downstream of the events that initiate transfusion-induced lung injury.
In conclusion, NETs are present and pathogenic in TRALI, and targeting platelet activation or NET components (histones, DNA) strongly protects mice from lung injury. Therapeutic advances in ALI will likely come from a better understanding of pathogenesis, and we propose that NETs are a worthy target for future preclinical and clinical testing.