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. 2014 Aug 7;8(3):883-96.
doi: 10.1016/j.celrep.2014.06.044. Epub 2014 Jul 24.

A myeloperoxidase-containing complex regulates neutrophil elastase release and actin dynamics during NETosis

Kathleen D Metzler  1 , Christian Goosmann  1 , Aleksandra Lubojemska  2 , Arturo Zychlinsky  1 , Venizelos Papayannopoulos  3
Affiliations

A myeloperoxidase-containing complex regulates neutrophil elastase release and actin dynamics during NETosis

Kathleen D Metzler et al. Cell Rep. .

Abstract

Neutrophils contain granules loaded with antimicrobial proteins and are regarded as impermeable organelles that deliver cargo via membrane fusion. However, during the formation of neutrophil extracellular traps (NETs), neutrophil elastase (NE) translocates from the granules to the nucleus via an unknown mechanism that does not involve membrane fusion and requires reactive oxygen species (ROS). Here, we show that the ROS triggers the dissociation of NE from a membrane-associated complex into the cytosol and activates its proteolytic activity in a myeloperoxidase (MPO)-dependent manner. In the cytosol, NE first binds and degrades F-actin to arrest actin dynamics and subsequently translocates to the nucleus. The complex is an example of an oxidative signaling scaffold that enables ROS and antimicrobial proteins to regulate neutrophil responses. Furthermore, granules contain protein machinery that transports and delivers cargo across membranes independently of membrane fusion.

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Figures

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Graphical abstract
Figure 1
Figure 1
ROS and MPO Are Required for NE Translocation during NETosis (A) Single confocal microscopy images of neutrophils from control, CGD, and ΔMPO donors. The neutrophils were stimulated for 60 min with C. albicans, immunolabeled for NE (red), and stained for DNA (Draq5, blue). Arrows indicate nuclear NE. Scale bars, 20 μm. (B) NE release into the cytosol during NETosis measured by ELISA in cytosolic extracts derived from naive neutrophils alone (N) or PMA-activated neutrophils (NP) from control and ΔMPO donors. NE in the cytosol normalized to the total amount of NE in the cytoplasmic extract of each sample at time 0. Error bars indicate SD in triplicate samples; ∗∗∗p < 0.001 between control and ΔMPO samples at 60 min. Cytoplasmic extracts were made by nitrogen cavitation, without detergent, to keep the granule membranes intact. Cytosolic extracts were made by ultracentrifugation of cytoplasmic extracts. (C) Immunoblot of the degradation of exogenous histone H4 by cytoplasmic extract from naive neutrophils alone (N) or PMA-activated (NP) control and ΔMPO neutrophils. The cells were activated for the indicated time durations and H4 was incubated with the cytoplasmic extracts for 3 hr. (D) Immunoblot against endogenous histone H4 in total cell lysates of naive neutrophils alone (N) from control or ΔMPO donors. Naive neutrophils (N) or stimulated with PMA (NP) or C. albicans (NC) for the indicated durations in the presence (+NEi) or absence of NEi. Full-length (H4, arrow) and proteolytically processed H4 (H4∗, red arrow). C, C. albicans alone. See also Figure S1.
Figure 2
Figure 2
Azurophilic Granules Contain the Azurosome Complex (A) β-galactosidase activity against X-gal after incubation with azurophilic granules from a control and a ΔMPO donor in the absence or presence of H2O2 and/or detergent. Error bars indicate SD in triplicate samples; ∗∗∗p < 0.001 between the indicated samples. (B) CASY impedance cell counter analysis of azurophilic granules, either untreated or treated with H2O2 or with detergent. (C) Coomassie stain of azurophilic granule lysates before immunoprecipitation (IP, left), or proteins immunoprecipitated with anti-NE (IP α-NE) or control anti-MMP9 antibody (mock) from isolated azurophilic granules untreated or treated with hydrogen peroxide (H2O2) (IP, right). LTF, lactoferrin; MPO, myeloperoxidase; AZU, azurocidin; CG, cathepsin G; ECP, eosinophil cationic protein; LYZ, lysozyme; HD1, defensin-1. (D) IP of solubilized azurophilic granule extract with rabbit anti-BPI or rabbit anti-NE, followed by immunoblotting with a mouse anti-BPI antibody. (E) Coomassie stain of purified azurosome complex (fractions 19–22 from Figure S3B, pooled and concentrated). PR3, proteinase 3. See also Figure S2.
Figure 3
Figure 3
NE and MPO Localize to the Membrane in a Subpopulation of Granules (A) Immunoelectron micrographs of granules from control or ΔMPO neutrophils, naive or stimulated for 60 min with PMA and labeled with antibodies against MPO and NE coupled to 6 and 12 nm particles. Arrows indicate the membrane localization of NE and MPO. (B) Representative immunoelectron micrographs of azurophilic granules exhibiting localization of MPO and NE in the granule lumen. Control neutrophils were either left untreated or stimulated with PMA for 60 min before fixing and immunogold labeling for MPO and NE. (C) Average distribution per single cell of granules with the indicated MPO and NE localization in electron micrographs from eight naive and 13 PMA activated neutrophils; 114 and 153 granules, respectively, were counted. Error bars indicate SD within each granule group. Nonparametric ANOVA for median differences, p = 0.003. (D) Azurophilic granules incubated with rabbit IgG against MPO or BPI, fractionated by centrifugation over a discontinuous Percoll gradient of 1.05, 1.09, and 1.12 g/ml density. The soluble (S) top layer and the 1.09/1.12 interface that contains the intact azurophilic granules were collected to detect primary antibodies by SDS-PAGE electrophoresis and western immunoblotting with anti-rabbit IgG. Arrows point to MPO antibody that cofractionates with azurophilic granules. (E) Intact azurophilic granules from naive neutrophils alone or treated with proteinase K (0, 1, 10, and 100 μg/ml) and immunoblotted for MPO, AZU, and BPI. Arrow indicates the cleavage product of AZU.
Figure 4
Figure 4
H2O2 Drives the Dissociation of the Azurosome (A) NE release into the cytosol by untreated or PEG-catalase-treated neutrophils at the indicated time points, measured by proteolytic activity against a chromogenic NE substrate, since catalase interferes with the peroxide-based ELISA readout. Data were normalized to the amount of NE in the cytoplasm of each sample at the start of the time course. Error bars indicate SD in triplicate samples; ∗∗∗p < 0.001 between 60′ PMA untreated versus catalase-treated triplicates. (B) Immunoprecipitation from a control azurophilic granule lysate with α-NE or α-MMP9 antibody (mock), followed by immunoblotting against MPO. Granules were left untreated or treated with H2O2 in the absence or presence of the protease inhibitors (PIs) NEi, CGi, PMSF and Roche cocktail, or MPO inhibitor (ABAH). The input lysate prior to immunoprecipitation is shown in the bottom lane. (C) Immunoprecipitation from a control azurophilic granule lysate, untreated or treated with H2O2, using a α-NE antibody, followed by immunoblotting against MPO, CG, AZU, or LYZ. (D) Immunoprecipitation with anti-NE (IP α-NE) from cytoplasmic neutrophil lysate of naive (N, 0′) or PMA-activated (NP, 60′) control neutrophils. Left lanes: total protein in the cytoplasmic lysate before immunoprecipitation. Right lanes: proteins immunoprecipitated with an α-NE antibody from cytoplasm and immunoblotted with antibodies against CG, AZU, or MPO. Images are from the same exposed blot, but were separated to remove irrelevant lanes. (E) Single confocal microscopy sections of control neutrophils stimulated with PMA for 60 min and immunolabeled for CG (red) and NE (green). The nucleus was labeled with the DNA stain Draq5 (blue). Arrows indicate nuclear NE and CG. Upper panels: a neutrophil during the early stage of NETosis. Lower panels: a neutrophil in a later stage, exhibiting a large decondensed nucleus. Scale bars, 10 μm. Right: quantitation of the percentage of neutrophils that contained more than 10% of total NE in the nucleus and the percentage of nuclear NE that colocalized with CG. Error bars indicate SD in duplicate samples.
Figure 5
Figure 5
The Azurosome Promotes Translocation across Granule Membranes (A and B) Calcein release from synthetic PC/PS liposomes. Calcein fluorescence was measured after 15 min of incubation and normalized to liposomes alone and liposomes permeabilized with NP-40. Error bars indicate SD in duplicate samples. (A) Calcein release from synthetic liposomes incubated with control or ΔMPO azurosome, monitored by fluorescence dequenching of released calcein. The azurosome was quantified based on NE content as measured by ELISA and expressed in moles (x axis). Black and white squares: flowthrough buffer from the purification of control (black) and ΔMPO (white) azurosome. Black and white rhombuses: boiled samples (black) and ΔMPO azurosome (white) at the highest concentration. Fitting was used to calculate the concentration of azurosome required for 50% release (R50) and the apparent cooperativity coefficient (nH). (B) Calcein release from synthetic liposomes incubated with control azurosome or purified MPO, monitored by fluorescence. The azurosome was quantified based on MPO content as measured by ELISA and expressed in moles (x axis). Fitting was used to calculate the concentration of azurosome required for 50% release (R50). (C) CASY impedance cell counter analysis of calcein-loaded synthetic PC/PS liposomes, either untreated or incubated with azurosome from a control donor or NP-40. (D) Release of LYZ from specific and gelatinase granules incubated with control azurosome, ΔMPO azurosome, or NP-40. Samples were separated into soluble (S) and total (T) fractions by ultracentrifugation and immunoblotted against LYZ. Complexes without granules were used as controls for the background levels of LYZ from azurosomes alone. (E) NE release by azurophilic granules as it was captured and detected by NE ELISA. Duplicate reactions of intact azurophilic granules, untreated or treated with NEi and activated with 100 μM H2O2 for 30 min. Additional reactions in the same conditions but treated with NP-40 were used for total to calculate the fraction of NE released. Error bars indicate SD in duplicate samples. (F) AZU and MPO release from isolated native azurophilic granules alone or after incubation with H2O2 in the absence or presence of NEi. Samples were incubated for 30 min and insoluble granules were removed by centrifugation to yield soluble (S) protein. Total protein (T) prior to centrifugation. See also Figures S4 and S5.
Figure 6
Figure 6
NE Regulates Actin Dynamics during Translocation to the Nucleus (A) Time lapse of live-cell microscopy depicting neutrophils depolarizing prior to the onset of nuclear decondensation in response to C. albicans (moi = 50). Scale bars, 10 μm. (B) NE release by control neutrophils either left naive or activated with PMA in the absence or presence of NEi (+NEi). ∗∗∗p < 0.001 comparing control versus NEi-treated at 30 min and 60 min. Error bars indicate SD in triplicate samples. (C) Untreated and NEi-treated neutrophils immunostained for NE (red), MPO (green), and DNA (DAPI, blue) 60 min after PMA stimulation. Arrows point to cytoplasmic areas containing NE in the absence of MPO. Scale bars, 5 μm. (D) Untreated and NEi-treated neutrophils immunostained for F-actin (phalloidin, red), NE (green), and DNA (DAPI, blue) 120 min after exposure to C. albicans. Arrows point to areas where NE and F-actin colocalize in the cytoplasm. Scale bars, 10 μm. (E) NE binding to F-actin in vitro by cosedimentation, showing NE alone or treated with NEi in the absence or presence of polymerized F-actin filaments. Reactions were incubated for 30 min at 37C and centrifuged at 100,000 g to generate supernatant containing soluble unbound protein supernatant (Sup) and actin-bound pellet. (F) Anti-Actin and anti-MPO immunoblots of whole-cell extracts of naive neutrophils or stimulated with LPS or C. albicans (moi = 10) at the indicated times after activation. (G) Mechanism of ROS-mediated NE translocation. In resting neutrophils, azurosome complexes are associated with a subset of azurophilic granule membranes. Upon oxidative activation (1), H2O2 triggers the release and activation of NE/CG/AZU protease complex into the cytoplasm (2). The complex binds to F-actin (3). The degradation of F-actin by active NE liberates the protease complex, allowing it to enter the nucleus.
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