Abstract Neutrophil extracellular traps (NETs) are web-like structures of DNA coated with cytotoxic proteins and histones released by activated neutrophils through a process called NETosis. NETs release occurs through a sequence of highly organized events leading to chromatin expansion and rupture of nuclear and cellular membranes. In calcium ionophore-induced NETosis, the enzyme peptidylargine deiminase 4 (PAD4) mediates chromatin decondensation through histone citrullination, but the biochemical pathways involved in this process are not fully understood. Here we use live-imaging microscopy and proteomic studies of the neutrophil cellular fractions to investigate the early events in ionomycin-triggered NETosis. We found that before ionomycin-stimulated neutrophils release NETs, profound biochemical changes occur in and around their nucleus, such as, cytoskeleton reorganization, nuclear redistribution of actin-remodeling related proteins, and citrullination of actin-ligand and nuclear structural proteins. Ionomycin-stimulated neutrophils rapidly lose their characteristic polymorphic nucleus, and these changes are promptly communicated to the extracellular environment through the secretion of proteins related to immune response. Therefore, our findings revealed key biochemical mediators in the early process that subsequently culminates with nuclear and cell membranes rupture, and extracellular DNA release. Keywords: Neutrophil extracellular traps, Nucleus, Actin remodeling, Citrullination, Proteomics, Neutrophils Graphical abstract [33]Image 1 [34]Open in a new tab Highlights * • Ionomycin-treated neutrophils rapidly change their nucleus morphology. * • Neutrophils communicate with the environment in early NETosis. * • Cytoskeleton reorganization occurs in and around nucleus of NETotic neutrophils. * • Protein redistribution across cellular fractions precedes DNA release. * • Nuclear structural and cytoskeleton-related proteins are citrullinated in NETosis. Abbreviations NETs – Neutrophil extracellular traps PAD4 – Peptidylarginine deiminase 4 ELANE – Neutrophil elastase MPO – Myeloperoxidase LPS – Lipopolysaccharides ROS – Reactive oxygen species PR3 – Proteinase 3 CTSG – Cathepsin G fMLP – N-formil-methionyl-leucyl-phenylanine AZU1 – Azurocidin ACTC1/ACTA1/2 – Alpha-cardiac actin, alpha-actin-1, alpha-actin-2 ACTB – Beta-actin VIM – Vimentin CFL1 – Cofilin 1 ANXA1 – Annexin A1 ARHGDIB Rho GDP-dissociation inhibitor 2 GAPDH – Glyceraldehyde-3-phosphate dehydrogenase TMSB4X – Thymosin beta-4 S100P – Protein S100P ANXA3 – Annexin A3 OLFM4 – Olfactomedin-4 ANXA6 – Annexin A6 PFN1 – Profilin-1 CAP1 – Adenylyl cyclase-associated protein 1 TALDO1 – Transaldolase PKM – Pyruvate kinase PGD – 6-phosphogluconate dehydrogenase G6PD – Glucose-6-phosphate 1-dehydrogenase AIF1 – Allograft inflammatory factor 1 CNN2 Calponin-2 CORO1A – Coronin HCLS1 – Hematopoietic lineage cell-specific protein LASP1 LIM and SH3 domain protein 1 LSP1 Lymphocyte-specific protein 1 VASP – Vasodilator-stimulated phosphoprotein TLN1 – Talin-1 ADSS Adenylosuccinate synthetase isozyme 2 H1FX – Histone H1.10 LBR – Lamin-B receptor LMNB1 – Lamin-B1 IF – Intermediate filament 1. Introduction Neutrophils constitute the host's first line of defense [[35]1], employing several defense strategies to neutralize viruses, fungi, and bacteria [[36]2]. Thus, generation of reactive oxygen species, degranulation and phagocytosis are well-known mechanisms used by neutrophils. Another killing strategy discovered more recently involves the release of extracellular DNA networks known as neutrophil extracellular traps (NETs) [[37]3]. NETs consist of chromatin fibers coated with cytotoxic proteins released from the cytosol, nucleus and granules, including histones, neutrophil elastase (ELANE) and myeloperoxidase (MPO) [[38]4,[39]5]. NETs are the result of a programmed type of cell death named NETosis, distinct from necrosis or apoptosis [[40]3,[41]6]. A variety of stimuli can trigger NETosis, such as bacteria, fungi, platelets, or small compounds such as lipopolysaccharides (LPS), monosodium urate crystals, bacterial ionophores or phorbol esters [[42]7,[43]8]. Evidence shows that NETosis mechanisms are stimulus-dependent [[44]9,[45]10], and the main difference between them is the dependence on NADPH oxidase activation and reactive oxygen species (ROS) production [[46]11,[47]12]. Regardless of the mechanism, the decondensation of chromatin is a requirement for NETosis [[48]13]. The chromatin decondensation during NETosis has been described to occur due to the loss of attractive forces between the DNA and modified histones, caused by neutralization of positive charges in histone tails. Two suggested mediators for this effect are the enzyme protein-arginine deiminase type-4 (PAD4), which catalyzes the deimination of arginine residues in citrulline [[49]14], and the joint action of NE, proteinase 3 (PR3), and cathepsin G (CTSG), proteases which can cleave histones [[50]15]. However, there is no consensus regarding which one, or even if both mediators are the drivers of chromatin decondensation and its expansion through the cytosol after neutrophil activation with different stimuli [[51]16]. Moreover, an increase in intracellular calcium, either by mobilization of intracellular calcium stores [[52]11,[53]17] or via influx from the extracellular medium [[54]11,[55]13] was also proven to be crucial for NETosis, once the chelation of intracellular or extracellular calcium impaired the process. In this context, treatment of neutrophils with calcium ionophores promote an influx of calcium from the extracellular medium similar to the intracellular calcium oscillations triggered by bacterial pore-forming toxins [[56]18,[57]19]. Furthermore, neutrophil activation by ionomycin, a calcium ionophore derived from the species Streptomyces, leads to PAD4 activation unraveling the ROS-independent mechanism of NETosis [[58]11,[59]17]. After the in vitro activation of neutrophils, a series of morphological changes are observed, such as the disassembly of actin filaments (f-actin) [[60]20,[61]21], shedding of microvesicles [[62]21], remodeling of vimentin [[63]21], microtubules disassembly [[64]21,[65]22], endoplasmic reticulum vesiculation [[66]21], granules disintegration [[67]15,[68]18], chromatin decondensation [[69]3,[70]20], nuclear envelope permeabilization [[71]21,[72]23], DNA release in the cytosol [[73]6], plasma membrane permeabilization and rupture [[74]21,[75]24], and NETs release [[76]3]. Nonetheless, Neubert et al [[77]23], showed that the biochemical active phase of NETosis occurs before chromatin expansion, and the morphological changes seen after that phase are driven by mechanical forces, with the active processes being secondary. Thus, the investigation of the biochemical pathways leading to chromatin swelling are key to understand NETosis. Yet, the key mediators of these morphological changes are not fully understood. Therefore, to investigate NETosis in the context of high intracellular calcium concentration, human neutrophils treated with the calcium ionophore ionomycin were tracked by live-imaging microscopy and subjected to proteomic studies after cellular fractionation. We have shown that soon after treatment, biochemical changes occur in their nuclei, and this active process is immediately communicated to the extracellular environment. Also, we revealed altered protein remodeling across cell fractions, and citrullination of multiple proteins involved in cytoskeleton organization, as well as in chromatin and nuclear structure, thus providing potential mediators for the nucleoskeleton and cytoskeleton organization process taking place in and around the nucleus. 2. Materials and methods 2.1. Materials Dextran, Hystopaque, ammonium bicarbonate, tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), sodium deoxycholate (SDC), dimethylsulfoxide (DMSO), ionomycin, N-formil-methionyl-leucyl-phenylanine (fMLP), paraformaldehyde, ethylene-bis(oxyethylenenitrilo)tetraacetic acid (EGTA), phenylmethanesulfonyl fluoride (PMSF), 1,4-piperazinediethanesulfonic acid (PIPES), adenosine 5’ – triphosphate disodium salt (ATP(Na)[2]), cOmplete protease inhibitor cocktail, benzonase nuclease, and trifluoroacetic acid (TFA) were obtained from Sigma (St. Louis, MO, USA). SYTOX™ green, Hoechst 33342, Prolong Diamond, and Pierce™ BCA protein assay kit were purchased from Invitrogen (Waltham, MA, USA). Dithiothreitol (DTT) and iodoacetamide were purchased from Bio-Rad laboratories (Hercules, CA, USA). Poly-d-Lysine, acetonitrile, acetone and 0.1% formic acid were obtained from Merck (Darmstadt, Germany). Trypsin was obtained from Promega (Madison, WI, USA). 2.2. Neutrophil isolation Human neutrophils were obtained from heparinized blood of healthy volunteers. Neutrophils were separated from blood by Dextran sedimentation followed by density centrifugation with Hystopaque 1.077 g/mL. Contaminating erythrocytes were lysed with a hypotonic solution, and neutrophils were resuspended in PBS with 5.5 mM glucose or RPMI medium without phenol red [[78]25]. Bright-field microscopy with May-Grünwald-Giemnsa stain was used to check for the presence of eosinophils, and the sample was considered appropriate if eosinophils were <5% of the cells. Blood collection was approved by the Research Ethics Committee of the Faculty of Pharmaceutical Sciences at University of São Paulo (CAAE 60860016.5.0000.0067). 2.3. Fluorescence microscopy Neutrophils were allowed to settle on 0.001% poly-d-lysine coated glass coverslips for 20 min at 37 °C. Then, cells were treated with 0.005% v/v DMSO (vehicle), fMLP 1 μM or ionomycin 6.7 μM for 90 min at 37 °C, and subsequently fixed with 4% paraformaldehyde. After fixation, cells were washed with Tris-HCl buffer pH 7.4, and stained with 500 nM SYTOX Green. Coverslips were mounted over the glass slides using Prolong Diamond, and visualized with a fluorescence microscope (Zeiss Axiovert 200) with a 20x/0.4 objective using excitation and emission wavelengths of 485 nm and 520 nm, respectively. Images were taken by an Axiocam HR R3 camera device. 2.4. Live-imaging microscopy Neutrophils (1,5 × 10^5) in phenol-red free RPMI 1640 medium supplemented with 1% of antibiotic and 2 μM Hoechst 33342 were allowed to settle on a 24-well plate coated with 0.001% of poly-d-lysine for 20 min at 37 °C. Next, neutrophils were treated with 6.7 μM ionomycin or the vehicle followed by addition of 500 nM SYTOX Green [[79]12]. Cells were monitored using a 20x objective on a Leica DMi8 epifluorescence microscope coupled with the LASX Application Software (Leica Microsystems). Fluorescent images for Hoechst 33342 and SYTOX Green were automatically acquired every 2 min, for a total of 120min at 37 °C with 5% CO[2]. 2.5. Quantification of nuclei and NETs The method of quantification described here was based on previously established methods [[80]13,[81]26]. Images obtained from the live-imaging cell microscopy were loaded into the ImageJ software (version 1.52p) and converted to an 8-bit binary image. The local threshold was adjusted by the Phansalkar or Bernsen functions, and the cells that touched each other were separated by the function watershed. Then, the total number of cells was counted, and classified according to size in μm^2, circularity and loss of plasma membrane integrity (SYTOX Green positive cells) ([82]Table 1). Based on these criteria, cells were divided in those containing a polymorphic nucleus, a spherical nucleus, those with a decondensed nucleus, and those with NETs, following the quantification criteria described in [83]Table 1. The percentage of cells with decondensed nuclei but without loss of cellular membrane integrity (Hoechst 33342 positive and SYTOX Green negative) was obtained by counting all cells >94 μm^2 and subtracting those cells presenting NETs (Hoechst 33342 positive and SYTOX Green positive). Polymorphic cells correspond to the subtraction of cells with a spherical nucleus, decondensed nucleus, or with NETs from total cells. Table 1. Criteria for quantification of neutrophils based on nuclear shape by live-imaging. Total cells Polymorphic nucleus Spherical nucleus Decondensed nucleus NETs Hoechst 33342 Positive Positive Positive Positive Positive SYTOX Green Positive Negative Negative Negative Positive Auto-local threshold Phansalkar Phansalkar Bernsem Phansalkar Phansalkar Size (μm^2) >26 26–94 26–50 >94 >94 Circularity (0.0–1.0) 0.0–1.0 0.0–1.0 0.94–1.00 0.0–1.0 0.0–1.0 [84]Open in a new tab 2.6. Cell lysis and fractionation Neutrophils (5 × 10^6) were incubated with 0.005% v/v DMSO (vehicle), 6.7 μM ionomycin, or fMLP 1 μM for 2 min at 37 °C, kept on ice, and the secretome was collected and concentrated to 30% of the initial volume at 4 °C. Subsequently, the cells were resuspended in ice-cold disruption buffer (100 nM KCl, 3 mM NaCl, 3.5 mM MgCl[2], and 10 mM PIPES, pH 7.2). Next, 1 mM ATP(Na)[2] and 0.6 mM PMSF were added to the cells, and the cells were lysed by nitrogen cavitation [[85]27] at 4 °C and 375 psi for 5 min, followed by addition of 1.5 mM EGTA. To minimize sample processing time, each replicate was individually processed up to the cell lysis. The cell lysate obtained after nitrogen cavitation was centrifuged in 3 cycles of 5 min at 400×g at 4 °C to obtain the nuclear fraction (pellet). Supernatants were used to obtain the organelles and soluble protein fractions. The organelles fraction was separated from soluble proteins through centrifugation at 79,000×g for 30 min at 4 °C [[86]28]. The remaining supernatant, containing the soluble proteins fraction, was concentrated to 30% of the initial volume at 4 °C using a vacuum concentration system. Nuclear and organelles fractions were lysed with 0.2% SDC in 100 mM ammonium bicarbonate in the presence of 0.5x (v/v) cOmplete protease inhibitor. cOmplete was also added to the secretome and soluble proteins fractions. Proteins were precipitated with 1:4 v/v acetone and NaCl 100 mM overnight at – 20 °C, centrifuged at 13,000×g for 10 min at 4 °C, and resuspended in 100 mM ammonium bicarbonate with 0.2% SDC. DNA aggregates were digested twice with 0.1 U/μL benzonase nuclease for 45 min at 37 °C. 2.7. Sample preparation for mass spectrometry The concentration of proteins in each fraction (secretome, nucleus, organelles and soluble proteins) was measured by the bicinchoninic acid assay (BCA). Ten μg of proteins from each fraction were reduced with 5 mM DTT for 1h at 37 °C, alkylated with 15 mM iodoacetamide for 30 min in the dark at 25 °C, and the excess of iodoacetamide was quenched with 2.5 mM DTT for 15 min at 25 °C. Next, proteins were digested with two sequential additions of trypsin (1:40 w/w), for 4 h at 37 °C, and overnight at 37 °C. Digestion was stopped with 0.5% v/v TFA at 37 °C for 30 min, followed by centrifugation at 14,000×g for 30 min. Half of the volume of the supernatant was used for desalting the samples using the stage tip protocol [[87]29]. Samples were lyophilized and stored at −80 °C until the injection into the mass spectrometer. Before injection, each sample was resuspended in 100 μL of formic acid 0.1%. 2.8. LC-MS/MS measurements The peptides were separated and analyzed in a Nano EASY-nLC 1200 (Thermo Fisher Scientific, Bremen, Germany) coupled to an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). First, each sample was injected into a trap column (nano Viper C18, 3 μm, 75 μm × 2 cm, Thermo Scientific) with 12 μL of solvent A (0.1% formic acid) at 500 bar. Then, the peptides were eluted onto a C18 column (nano Viper C18, 2 μm, 75 μm × 15 cm, Thermo Scientific) at a flow rate of 300 nL/min. Peptides were eluted from the column using a linear gradient of solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in 20:80 water: acetonitrile, v/v), starting with 5–28% B for 80 min, followed by an increase to 40% B for 10 min. Next, column wash was accomplished with an increase of solvent B percentage to 95% in 2 min, followed by 12 min with this solvent proportion. Re-equilibration of the system with 100% solvent A was performed before each injection. After ionization under positive electrospray conditions, the eluted peptides were analyzed in a data-dependent acquisition mode. The most intense ions detected after a full scan (400–1600 m/z) at a 120,000 resolution, were filtered for fragmentation by the quadrupole with a transmission window of 1.2 m/z, followed by HCD fragmentation with a normalized collision energy of 30, and detection of the fragments by the orbitrap mass analyzer with a 30,000 resolution. A new cycle of MS followed by MS2 events occurred at every 3 s. Monocharged ions or ions with undetermined charges were excluded from fragmentation. 2.9. Data analysis Raw files of all proteomic experiments were processed using MaxQuant software [[88]30]. Proteins were identified through the Andromeda algorithm [[89]31] against the Homo sapiens Uniprot database (downloaded March 2022; 20,401 entries). Error mass tolerance for precursors and fragments was set to 4.5 ppm and 20 ppm, respectively. Cysteine carbamidomethylation was selected as a fixed modification and methionine oxidation, deimination of arginine residues (citrullination) and N-terminal acetylation were selected as variable modifications. The deimination of the arginine residues (R) was set as an increase of +0.98401 Da with a neutral loss of HCNO (43.0058 Da) [[90]32]. A semi-tryptic digestion mode was set, with a maximum of 2 missed cleavages allowed. A maximum FDR of 1% was allowed both for peptides and proteins identification, and for proteins, FDR was calculated using a decoy database created from the reverse ordination of the protein sequences in the Uniprot database. Protein abundances were obtained from normalized chromatographic peak integrations calculated by MaxQuant through LFQ algorithm [[91]33]. A given protein was considered present if at least two peptides (one of them being unique) were detected. Match between runs option was enabled and the other parameters were kept as default. Neutrophils from three different volunteers were used for proteomic analyses. For each subject, the neutrophils were divided in 4 different replicates for ionomycin treatment and 4 replicates for control (DMSO only). All the fractions (secretome, nucleus, organelles and soluble proteins) from each volunteer were run in MaxQuant simultaneously. Alternatively, for comparisons within a specific fraction, raw files from all the samples of the volunteers in the fraction of interest (secretome or nucleus) were run in MaxQuant at the same time (n = 4 for ionomycin treatment, and 4 for controls). The proteins were analyzed using Perseus software (version 1.6.15.0) [[92]34] and data was plotted using GraphPad Prism (version 6.01) or R (version 1.2.5019). Volcano plots were made with the web app VolcaNoseR ([93]https://huygens.science.uva.nl/VolcaNoseR/) [[94]35]. Pathway enrichment analysis was performed combining enriched and unique proteins of each group using the web app WebGestalt ([95]http://www.webgestalt.org/) under over-representative analysis with FDR <0.05 and default parameters [[96]36]. The enriched biological processes were summarized by weighted-set cover. Before statistical analysis, the LFQ intensities were loaded into the Perseus software, filtered for reverse peptides and potential contaminants, and log[2] transformed. Missing values were filtered using the criteria of at least 3 valid values in each group. Proteins were considered unique to a group in a given fraction if they had at least 3 valid values in one group and 0 or 1 valid value in the other. To compare protein abundance across different fractions, for each protein, we calculated the percentage in each fraction in relation to the total (sum of the protein abundance in all 4 fractions). Only proteins common to all 4 fractions were selected for this analysis. Before analysis of citrullinated proteins, false-positive peptides with citrullinated arginine in the C-terminal were manually excluded from the data. A citrullinated peptide was included in the analysis if it belonged to a protein present in at least 2 out of 3 individuals. Similarly, a peptide was reported as differentially regulated if it was found significantly altered in at least 2 subjects. Peptides present in a single individual are identified in supporting tables. For clarity, peptides and their miscleavages were compiled in a single lane in supporting tables presenting citrullinations. Common proteins had to have at least 2 valid values in both groups, while exclusive proteins had to have at least 2 valid values in one group and none in the other. Protein symbols (all capital letters, not italicized) are based on gene symbolses as recommended by the Human Genome Organization Gene Nomenclature Committee [[97]37]. 2.10. Statistical analysis Control and ionomycin live-imaging quantification data were evaluated separately through analysis of variance (ANOVA), followed by post hoc Tukey test of each nucleus type over time using R. For proteomic analyses of nucleus and secretome, as well as the analysis of citrullinated peptides, the comparisons between groups (control and ionomycin) in each fraction was performed with an unpaired t-test. The P-value was adjusted for multiple comparisons applying an FDR of 0.05. These analyses were performed in R and Perseus. For percentages in each fraction, after a t-test, proteins with adjusted P-value < 0.05 and with mean ratio of ionomycin/control >2 or <0.5 in at least one fraction each, were considered significantly different. 2.11. Data statement The data supporting the findings of this study are available from the corresponding author upon reasonable request. 3. Results 3.1. Ionomycin treatment induces early chromatin changes culminating in neutrophil NETosis The neutrophil's response to ionomycin treatment was first assessed by fluorescence microscopy using SYTOX Green. Confirming previous studies [[98]12,[99]17], treatment of human neutrophils with 6.7 μM ionomycin for 90 min at 37 °C produced large scaffolds of extracellular DNA, known as NETs ([100]Fig. 1A). In contrast, neutrophils that received only vehicle (DMSO) got a few spontaneous NETs, but the majority of the cells were still polymorphonuclear ([101]Fig. 1A). Fig. 1. [102]Fig. 1 [103]Open in a new tab Ionomycin treatment leads to chromatin changes and NETs release. Human neutrophils were treated with vehicle (DMSO) or 6.7 μM ionomycin for 90 min and fixed (A) or monitored (live) for 120 min (B–E) at 37 °C. Images are representative of three independent experiments. Approximately 200–350 cells from a seeding density of 1,5 × 10^5 per well, were monitored per condition. A) After fixation, cells were stained with 500 nM SYTOX Green followed by fluorescence microscopy analysis. Scale bars = 50 μm. B – E). Cells stained with 2 μM Hoechst 33342 and 500 nM SYTOX Green were monitored by live-imaging microscopy. B) 8-bit binary images with Bernsen local threshold of three representative cells from control (top) and ionomycin (bottom) groups over time. C) Microscope images of control (upper panel) and ionomycin-treated neutrophils (bottom panel) at 8 min of incubation. From left to right: fluorescence images, 8-bit binary images with Bernsen local threshold of all cells >26 μm and circularity 0–1.0, and outlines of cells with 26–50 μm and circularity 0.94–1.0 (spherical nucleus). Scale bars = 20 μm. D) Percentage of control (n = 3) and E) Ionomycin-treated neutrophils (n = 3) quantified in Phansalkar thresholded images with spherical (26–50 μm and circularity 0.94–1.0), decondensed (only Hoechst 33342 (+), >94 μm and circularity 0–1.0) or polymorphic (>26 μm and circularity 0–1.0) nucleus, or NETotic cells (Sytox Green (+), >94 μm and circularity 0–1.0). * Denotes P-value < 0.05, and **P-value < 0.01, after one-way ANOVA followed by Tukey posttest. (For interpretation of the references to colour in this