2-Chlorofatty acids: lipid mediators of neutrophil extracellular trap formation

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.

Keywords: cell signaling; fatty acids; myeloperoxidase; neutrophils; oxidized lipids; plasmalogens.

Fig. 1.

ecDNA quantification in response to 2-ClFA treatment. Human neutrophils were isolated from peripheral blood and treated at 37°C with either vehicle (0.1% EtOH, CTRL), 100 nM phorbol myristate acetate (PMA), 10 μM palmitic acid (PA), 10 μM 2-chloropalmitic acid (2-ClPA), 10 μM stearic acid (SA), or 10 μM 2-chlorostearic acid (2-ClSA) as indicated with extracellular DNA (ecDNA) detection with Sytox Green as described in the Materials and Methods. ecDNA accumulation was monitored by measuring fluorescence for up to 4 h of treatment as indicated in A, and after 3 h, in B. Fluorescence values represent mean ± SEM, n = 3.

Fig. 2.

MPO colocalization with DNA in 2-ClFA-treated neutrophils. Human neutrophils were isolated from peripheral blood, suspended in HBSS, and seeded on coverslips in 6-well plates. Cells were then treated with either vehicle (0.1% EtOH, CTRL), 100 nM PMA, 10 μM PA, or 10 μM 2-ClPA for 90 or 180 min at 37°C. After incubation, cells were fixed, permeabilized, and analyzed via immunofluorescence for MPO (green) and DAPI (nuclei, blue). A shows images acquired with confocal microscope, and B and C show the quantification of samples at 90 and 180 min, respectively (mean ± SEM). ****P < 0.0001 for comparisons with CTRL treatment.

Fig. 3.

2-ClFA induces functional NETs. Human neutrophils were stimulated with either PMA or 2-ClFA for 3 h and then treated with or without DNase. The stimulated neutrophils were then incubated with an equal number of E. coli for 1 h. A shows colony forming unit (CFU) count, n = 4. B represents the average of four different experiments (mean ± SEM). Bacterial trapping was measured as described in the Materials and Methods. *P < 0.05 for t-test comparisons between conditions with and without DNase treatment.

Fig. 4.

Dissection of the canonical NETosis pathway in the presence of 2-ClFA. Human neutrophils were isolated from peripheral blood and treated with either vehicle (0.1% EtOH, CTRL), 100 nM PMA, 10 μM PA, or 10 μM 2-ClPA in the presence or absence of 10 μM diphenyleneiodonium chloride [DPI, NADPH oxidase inhibitor (NOX inhibitor)], 50 μM chloro-amidine (Cl-Am, PAD4 inhibitor), or 100 μM of 2- aminoethyldiphenyl borate (2-APB, calcium release inhibitor). Neutrophils were incubated at 37°C in the presence of Sytox Green. Extracellular DNA (ecDNA) accumulation was measured by fluorescence analysis after 3 h of treatment. Fluorescence values represent mean ± SEM, n = 3, as described in the Materials and Methods. *P < 0.05, **P < 0.005, ***P < 0.001 for t-test comparisons between conditions with and without inhibitor treatment.

Fig. 5.

Neutrophil activation marker analysis in response to PMA and 2-ClFA treatment. Human neutrophils were isolated from peripheral blood and treated with either vehicle (0.1% EtOH, CTRL), 100 nM phorbol myristate acetate (PMA), 10 μM palmitic acid (PA) or 10 μM chloropalmitic acid (2-ClPA) and incubated at 37°C for 15 min. Neutrophils were then fixed and stained with antibodies against CD11b and CD62L as described in the Materials and Methods and the expression level of the markers was analyzed by flow cytometry (A, B). In C, after the incubation with the same conditions, neutrophils were pelleted, and the supernatant was tested via ELISA for lysozyme release. In D, after neutrophils were incubated in the same conditions as above, neutrophil elastase (NE) activity was measured as indicated in the Materials and Methods. Values are expressed in arbitrary units and represent mean ± SEM, n = 3 (A–C) or n = 5 (D). ***P < 0.001, ****P < 0.0001 for comparisons with CTRL treatment.

Fig. 6.

Murine WT- and MPO^−/−-neutrophil NETosis. Murine neutrophils were isolated from bone marrow from either WT or MPO^−/− mice as described in the Materials and Methods. Neutrophils were suspended in HBSS and treated with either vehicle (0.1% EtOH, CTRL), 100 nM PMA, 10 μM PA, or 10 μM 2-ClPA at 37°C. A: ecDNA accumulation using Sytox Green was measured by fluorescence analysis after 3 h of treatment. Fluorescence values represent mean ± SEM, n = 3, as described in the Materials and Methods. B: after an incubation interval of 1 h, lipids were extracted in the presence of internal standard, and 2-ClFA levels were quantified as described in the Materials and Methods. In C, human neutrophils were isolated from peripheral blood. After 1 h incubation, lipids were extracted in the presence of internal standard, and 2-ClFA levels were quantified as described in the Materials and Methods. Values represent the mean ± SEM (n = 3). **P < 0.005, ****P < 0.0001 for comparisons with CTRL treatment.

Fig. 7.

Canonical pathway for NETosis. Known mediators and mechanisms of NETosis are shown by solid arrows. Proposed mechanistic refinement of the model is shown in the broken arrows.