Abstract
Neutrophils form neutrophil extracellular traps (NETs), which have been implicated in microcirculatory plugging. NET formation (NETosis) involves the fusion of granule and nuclear contents, which are then released in the extracellular space. Myeloperoxidase (MPO) plays a major role in NETosis leading to the dissociation of DNA from histones. During neutrophil activation, MPO is released and activated to convert hydrogen peroxide and chloride to hypochlorous acid (HOCl). HOCl targets plasmalogens leading to the production of the chlorinated lipids, 2-chlorofatty aldehyde and 2-chlorofatty acid (2-ClFA). Here, we tested the hypothesis that 2-ClFAs are important lipid mediators of NETosis. Human neutrophils treated with physiological levels of 2-ClFAs formed NETs, characterized by MPO association with DNA and neutrophil elastase (NE) redistribution to the perinuclear area. 2-ClFA-induced NETs reduced Escerichia coli colony forming units. 2-ClFA-induced NETosis is calcium- and protein arginine deiminase 4-dependent. Interestingly, unlike PMA, 2-ClFA initiates the NETosis process without neutrophil activation and degranulation. Furthermore, 2-ClFA elicits NETosis in bone-marrow derived neutrophils from MPO-deficient mice. Taken together, these findings suggest 2-ClFA as an MPO product that triggers the NETosis pathway following neutrophil activation.
Highlights
Neutrophils form neutrophil extracellular traps (NETs), which have been implicated in microcirculatory plugging
As efficient as NETs are considered to be in trapping bacteria, they are detrimental in several pathological conditions in which the formation of NETs mediate, in part, inflammatory sequelae leading to tissue damage and potentially contribute to the formation of thrombi with subsequent tissue ischemia
Because MPO has an important role in NET formation (NETosis) and 2-chlorofatty acid (2-ClFA) levels increase in activated neutrophils, these novel lipids were investigated as potential triggers of NETosis. 2-ClFAs are produced during inflammation as a consequence of MPO-derived hypochlorous acid (HOCl) targeting plasmalogen phospholipids
Summary
Materials 2-ClFAs were synthesized and prepared as described previously using palmitic or stearic acid as precursor [38]. Isolated human neutrophils were suspended in Hank’s Buffer (2×106 cells/ml) to a final volume of 2 ml and seated on coverslips in the presence of the described conditions for the indicated time. Neutrophils were suspended in Hank’s Buffer (106 cells/ml) and incubated with either vehicle (0.1% EtOH), PMA, palmitic acid (PA), or the 2-ClFA, 2-chloropalmitic acid (2-ClPA) in the presence of 200 M of NE chromogenic substrate (MeOSuc-AAPV-pNA, Cayman chemical, catalog #: 70967-90-7). Surface marker expression Human neutrophils were isolated, suspended in HBSS (106 cells/ml) with 1% BSA, and incubated in the described conditions at 37°C for 15 min. Human peripheral blood neutrophils were suspended in HBSS (106 cells/ml) and incubated with the described conditions at 37°C for 10 min. Trapping assay Human neutrophils (2×106 cells/ml HBSS) were plated in 12-well plates in the presence of the indicated treatments and centrifuged at 300 gmax for 5 min.
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