Neutrophil extracellular traps (NETs) are part of the innate immune response to infections. NETs are a meshwork of DNA fibers comprising histones and antimicrobial proteins. Microbes are immobilized in NETs and encounter a locally high and lethal concentration of effector proteins. Recent studies show that NETs are formed inside the vasculature in infections and noninfectious diseases. Here we report that NETs provide a heretofore unrecognized scaffold and stimulus for thrombus formation. NETs perfused with blood caused platelet adhesion, activation, and aggregation. DNase or the anticoagulant heparin dismantled the NET scaffold and prevented thrombus formation. Stimulation of platelets with purified histones was sufficient for aggregation. NETs recruited red blood cells, promoted fibrin deposition, and induced a red thrombus, such as that found in veins. Markers of extracellular DNA traps were detected in a thrombus and plasma of baboons subjected to deep vein thrombosis, an example of inflammation-enhanced thrombosis. Our observations indicate that NETs are a previously unrecognized link between inflammation and thrombosis and may further explain the epidemiological association of infection with thrombosis.neutrophils | platelets | histones | red blood cells | chromatin

Hyper–inflammatory responses can lead to a variety of diseases including sepsis1. We now report that extracellular histones released in response to inflammatory challenge contribute to endothelial dysfunction, organ failure and death during sepsis. They can be targeted pharmacologically by antibody to histone or by activated protein C (APC). Antibody to histone reduced the mortality of mice in lipopolysaccharide (LPS), tumor necrosis factor (TNF) or cecal ligation and puncture models of sepsis. Extracellular histones are cytotoxic toward endothelium in vitro and are lethal in mice. In vivo, histone administration resulted in neutrophil margination, vacuolated endothelium, intra–alveolar hemorrhage and macro and microvascular thrombosis. Histone was detected in the circulation of baboons challenged with E. coli and the increase in histone levels accompanied the onset of renal dysfunction. APC cleaves histones and reduces their cytotoxicity. Co–infusion of APC with E. coli in baboons or histones in mice prevented lethality. Blockade of protein C activation exacerbated sublethal LPS challenge into lethality which was reversed by antibody to histone. We conclude that extracellular histones are potential molecular targets for therapeutics for sepsis and other inflammatory diseases.

There is emerging evidence that platelets are major contributors to inflammatory processes through intimate associations with innate immune cells. Here, we report that activated platelets induce the formation of neutrophil extracellular traps (NETs) in transfusion-related acute lung injury (TRALI), which is the leading cause of death after transfusion therapy. NETs are composed of decondensed chromatin decorated with granular proteins that function to trap extracellular pathogens; their formation requires the activation of neutrophils and release of their DNA in a process that may or may not result in neutrophil death. In a mouse model of TRALI that is neutrophil and platelet dependent, NETs appeared in the lung microvasculature and NET components increased in the plasma. We detected NETs in the lungs and plasma of human TRALI and in the plasma of patients with acute lung injury. In the experimental TRALI model, targeting platelet activation with either aspirin or a glycoprotein IIb/IIIa inhibitor decreased NET formation and lung injury. We then directly targeted NET components with a histone blocking antibody and DNase1, both of which protected mice from TRALI. These data suggest that NETs contribute to lung endothelial injury and that targeting NET formation may be a promising new direction for the treatment of acute lung injury.

We previously reported that extracellular histones are major mediators of death in sepsis. Infusion of extracellular histones leads to increased cytokine levels. Histones activate TLR 2 and 4 in a process that is enhanced by binding to DNA. Activation of TLR4 is responsible for the histone dependent increase in cytokine levels. To study the impact of histone release on pathology we used two models: a concanavalin A (ConA) triggered activation of T cells to mimic sterile inflammation and acetaminophen (APAP), to model drug induced tissue toxicity. Histones were released in both models and anti-histone antibodies were protective. TLR 2 or TLR 4 null mice were also protected. These studies imply that histone release contributes to death in inflammatory injury and in chemical induced cellular injury, both of which are mediated in part through the toll-like receptors.

Mer tyrosine kinase (MerTK) is a major macrophage apoptotic cell (AC) receptor. Its functional impairment promotes autoimmunity and atherosclerosis, while overexpression correlates with poor prognosis in cancer. However, little is known about mechanisms regulating MerTK expression in humans. We found that MerTK expression is heterogenous among macrophage subsets, being mostly restricted to anti-inflammatory “M2c” (CD14+CD16+CD163+CD204+CD206+CD209−) cells, differentiated by M-CSF or glucocorticoids. Small numbers of MerTK+ “M2c-like” cells are also detectable among circulating CD14brightCD16+ monocytes. MerTK expression levels adapt to changing immunological environment, being suppressed in M1 and “M2a” macrophages, and in dendritic cells. Remarkably, while glucocorticoid-induced differentiation is IL-10-independent, M-CSF-driven M2c polarization and related MerTK up-regulation require IL-10. However, neither IL-10 alone nor TGFβ are sufficient to fully differentiate M2c (CD16+CD163+MerTK+) macrophages. M-CSF and IL-10, both released by T lymphocytes, may thus be required together to promote regulatory T cell-mediated induction of anti-inflammatory monocytes-macrophages. MerTK enables M2c macrophages to clear early ACs more efficiently than other macrophage subsets, and mediates AC clearance by CD14brightCD16+ monocytes. Moreover, M2c cells release Gas6, which in turn amplifies IL-10 secretion via MerTK. IL-10-dependent induction of the Gas6/MerTK pathway may, therefore, constitute a positive loop for M2c macrophage homeostasis and a critical checkpoint for maintenance of anti-inflammatory conditions. Our findings give new insight into human macrophage polarization and favor a central role for MerTK in regulation of macrophage functions. Eliciting M2c polarization can have therapeutic utility for diseases such as lupus, in which a defective AC clearance contributes to initiate and perpetuate the pathological process.