Netosis Proceeds by Cytoskeleton and Endomembrane Disassembly and PAD4-Mediated Chromatin Decondensation and Nuclear Envelope Rupture

NETosis proceeds by cytoskeleton and endomembrane disassembly and PAD4-mediated chromatin decondensation and nuclear envelope rupture

Hawa Racine Thiama,1, Siu Ling Wongb,c,1,2, Rong Qiud, Mark Kittisopikuld,e, Amir Vahabikashid, Anne E. Goldmand, Robert D. Goldmand, Denisa D. Wagnerb,c,f,1, and Clare M. Watermana,1,3

aCell and Developmental Biology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892; bProgram in Cellular and Molecular Medicine, Boston Children’s Hospital, Boston, MA 02115; cDepartment of Pediatrics, Harvard Medical School, Boston, MA 02115; dDepartment of Cell and Molecular Biology, Northwestern University Feinberg School of Medicine, Chicago IL 60611; eDepartment of Biophysics, University of Texas Southwestern Medical Center, Dallas, TX 75390; and fDivision of Hematology/Oncology, Boston Children’s Hospital, Boston, MA 02115

Edited by Gregory P. Downey, National Jewish Health, Denver, CO, and accepted by Editorial Board Member Carl F. Nathan January 21, 2020 (received for review June 3, 2019)

Neutrophil extracellular traps (NETs) are web-like DNA structures lipopolysaccharides, or can be pharmacologically induced with decorated with histones and cytotoxic proteins that are released phorbol ester (9). Regardless of the stimulus, NETosis requires by activated neutrophils to trap and neutralize pathogens during convergence of signaling pathways to mediate the cellular process the innate immune response, but also form in and exacerbate of chromatin decondensation, which is necessary for NET release sterile inflammation. Peptidylarginine deiminase 4 (PAD4) citrulli- (10). Two mechanisms are thought to promote histone release nates histones and is required for NET formation (NETosis) in from DNA to mediate decondensation: Neutrophil elastase and mouse neutrophils. While the in vivo impact of NETs is accumu- other proteases in granules may cleave histones to dissociate them lating, the cellular events driving NETosis and the role of PAD4 in from DNA (11), or PAD4, an enzyme that converts arginine to these events are unclear. We performed high-resolution time-lapse citrulline, may citrullinate histones, reducing their charge-based microscopy of mouse and human neutrophils and differentiated interaction with DNA to promote chromatin decondensation HL-60 neutrophil-like cells (dHL-60) labeled with fluorescent markers (10). The relative importance of proteases and PAD4 for com-

of organelles and stimulated with bacterial toxins or Candida albicans pletion of NETosis may be dictated by the cellular stimulus (12) or IMMUNOLOGY AND INFLAMMATION to induce NETosis. Upon stimulation, cells exhibited rapid disassem- the species. Indeed, neutrophil elastase is required downstream of the NADPH pathway when NETosis is induced in human neu- bly of the actin cytoskeleton, followed by shedding of plasma mem- trophils by phorbol esters or Candida albicans (12), while PAD4 is brane microvesicles, disassembly and remodeling of the microtubule critical for NETosis in mouse neutrophils stimulated with calcium and vimentin cytoskeletons, ER vesiculation, chromatin decondensa- ionophore or bacteria (13, 14). However, whether different cellular tion and nuclear rounding, progressive plasma membrane and nu- mechanisms are engaged during NETosis in mouse and human clear envelope (NE) permeabilization, nuclear lamin meshwork neutrophils and whether PAD4 is required for NET release in and then NE rupture to release DNA into the cytoplasm, and human neutrophils remains unclear. finally plasma membrane rupture and discharge of extracellular DNA. Inhibition of actin disassembly blocked NET release. Mouse Significance and dHL-60 cells bearing genetic alteration of PAD4 showed that chromatin decondensation, lamin meshwork and NE rupture and extracellular DNA release required the enzymatic and nuclear Neutrophils are white blood cells specialized as the first line of localization activities of PAD4. Thus, NETosis proceeds by a step- host defense in the immune system. One way they protect wise sequence of cellular events culminating in the PAD4- organisms is through NETosis, in which they expel their DNA to mediated expulsion of DNA. form a web-like trap that ensnares pathogens and promotes clotting. However, NETs also mediate sterile inflammation, neutrophil | innate immunity | microscopy causing damage to the body. We used high-resolution live-cell microscopy to characterize the timing of dynamic cellular events leading to NETosis in human and mouse neutrophils and a eutrophils deploy a variety of machineries to fight infection neutrophil-like cell line. We discovered that NETosis proceeds by Nand neutralize pathogens, including phagocytosis and de- a stepwise sequence of cellular events that is conserved across granulation, as well as the more recently characterized release of species and requires the activity of the PAD4 enzyme for DNA to neutrophil extracellular traps (NETs) termed NETosis (1). NETs be released from the nucleus and cell membrane. are web-like DNA structures decorated with histones and anti- microbial proteins that are released from stimulated neutrophils. Author contributions: H.R.T., D.D.W., and C.M.W. designed research; H.R.T., S.L.W., R.Q., NETs can trap and neutralize or kill pathogens, including bac- M.K., A.V., and A.E.G. performed research; R.D.G. and D.D.W. contributed new reagents/ teria (1), fungi (2), and viruses (3), and propagate inflammatory analytic tools; H.R.T., S.L.W., M.K., A.V., A.E.G., and R.D.G. analyzed data; and H.R.T. and C.M.W. wrote the paper. and immune responses (4). However, NETosis also conveys The authors declare no competing interest. detrimental effects, including tissue damage during sepsis (5, 6) This article is a PNAS Direct Submission. G.P.D. is a guest editor invited by the Editorial and thrombosis (7). Furthermore, several autoimmune diseases Board. are associated with high rates of NETosis and/or defects in NET This open access article is distributed under Creative Commons Attribution-NonCommercial- clearance (6), and there is evidence that NETosis promotes NoDerivatives License 4.0 (CC BY-NC-ND). cancer (6, 8). Thus, understanding the mechanisms mediating 1H.R.T., S.L.W., D.D.W., and C.M.W. contributed equally to this work. NETosis could facilitate either therapeutic improvement of in- 2Present address: Lee Kong Chian School of Medicine, Nanyang Technological University, nate immunity or mitigation of its damaging effects. Singapore 308232. The molecular requirements for NETosis have begun to be 3To whom correspondence may be addressed. Email: [email protected]. elucidated. NETosis can be stimulated with a variety of factors, This article contains supporting information online at https://www.pnas.org/lookup/suppl/ including bacteria or yeast, monosodium urate crystals associ- doi:10.1073/pnas.1909546117/-/DCSupplemental. ated with gout, platelet activating factor, bacterial ionophores or

www.pnas.org/cgi/doi/10.1073/pnas.1909546117 PNAS Latest Articles | 1of12 Downloaded by guest on September 23, 2021 Despite advancing knowledge of the molecular requirements and shall be used as an easily visualized temporal initiation point for NETosis, less is known about its cellular mechanisms (15). for the remainder of this study. For DNA to be released to the cell exterior during NETosis, it We then analyzed DNA and membrane dynamics in the cells’ must escape from the nucleus, pass through the cytoplasm con- central confocal plane. Prior to stimulation, nuclei of all three taining a network of membranous organelles and cytoskeletal cell types were lobulated with spatially heterogeneous DNA systems, and finally breach the plasma membrane (PM). While it staining indicative of regions of varied levels of chromatin con- is generally thought that decondensed chromatin is expelled via densation. Quantification of timing in movies showed that within nuclear envelope (NE) and plasma membrane rupture resulting ∼10 min after ionomycin-induced vesiculation, DNA fluores- in neutrophil death (1, 15), some evidence for “vital NETosis” cence became homogeneous throughout the nucleus (Fig. 1J and suggests that vesicles containing DNA might be exocytosed to Movie S1). Similar fluorescence homogenization was observed allow neutrophils to survive and retain the capacity for phago- during NETosis in dHL-60 cells expressing mEmerald-tagged cytosis and induction of adaptive immunity after NET release linker histone H1 (SI Appendix, Fig. S1C and Movie S2), sug- (16, 17). However, little is known about how chromatin breaches gesting that these changes correspond to chromatin deconden- organelles and the cytoskeleton to pass through the cytoplasm. sation. DNA decondensation was followed ∼10 min later by loss There is evidence that actin filaments (18–20) and microtubules of nuclear lobulation and rounding of the nuclear surface in both (MTs) (19, 21) disassemble during NETosis, yet pharmacological mouse PMN and dHL-60, while in human PMN, nuclei delo- perturbations that either disassemble or stabilize actin can im- bulated but did not fully round up (Fig. 1 E and F). At 20 to pair NETosis (15, 18, 19), while MT disruption has little effect 60 min after vesiculation, the sharp DIC contrast band at the rim (19). Thus, an in-depth understanding of the cellular events of the nucleus was lost and DNA expanded rapidly into the cy- driving NETosis requires the detailed visualization of various tosol, indicating nuclear rupture (Fig. 1J and Movie S1). After cellular compartments during the entire process. nuclear rounding and before nuclear rupture, there was also a Here we used time-lapse high-resolution multimode micros- sudden reduction in DIC contrast in the cell periphery (Movie copy of living human and mouse blood-derived polymorpho- S3), indicating a decrease in cytoplasmic refractive index that nuclear neutrophils (PMNs) as well as differentiated human likely corresponded to loss of cellular contents. Despite this leukemia (dHL-60) neutrophil-like cells bearing fluorescent leakage of cytosol, the peripheral cell membrane appeared in- markers of organelle and cytoskeletal systems to quantitatively tact, and DNA was not released extracellularly until many mi- characterize the cellular events mediating NETosis after stimula- nutes later, marking the completion of NETosis (Fig. 1 B, D, F, tion with bacterial toxins (ionomycin, lipopolysaccharide [LPS]) and J). Although not all stimulated cells that exhibited DNA or C. albicans. We demonstrate that NETosis proceeds by a well- decondensation and leakage of cytosol completed NETosis, defined and conserved sequence of cellular events. Character- 100% of cells that completed NETosis had previously com- ization of neutrophils derived from PAD4 knockout mice or gene- pleted these cellular processes. However, while all cells that edited dHL-60 cells demonstrates that PAD4 enzymatic activity completed NETosis exhibited nuclear rupture prior to extracellular and nuclear localization signal are required for efficient DNA DNA release, a substantial portion of cells with ruptured nuclei decondensation, nuclear lamin meshwork, and NE rupture and did not go on to release DNA extracellularly (Fig. 1 B, D,and extracellular DNA release. F). Thus, nuclear rupture may be required but not sufficient for NETosis. Results To determine whether the sequence of cellular events ob- NETosis Starts with Vesiculation Prior to DNA Decondensation, DNA served after ionomycin-induced NETosis occurred with other Release into the Cytoplasm, and Extracellular DNA Expulsion in Mouse, pathogen-derived stimuli, we exposed human PMNs to C. albicans Human PMNs, and dHL60 Cells. To characterize the cellular events (multiplicity of infection [MOI] of 2) or mouse PMNs to lipopoly- mediating NETosis, we performed high-resolution time-lapse im- saccharide (LPS, 25 μg/mL). 38% of C. albicans-stimulated hu- aging of mouse and human PMNs isolated from fresh blood, as man PMNs and 34% of LPS-stimulated mouse PMNs completed well as dHL-60 cells, upon NETosis stimulation. Cells stained with NETosis. Time-lapse analysis showed that human PMNs exposed the DNA-specific vital dye, SiR-DNA, were plated on coverslips to C. albicans phagocytosed actively growing yeast prior to initiating for imaging and stimulated with ionomycin. Pairs of differential NETosis, and completion of NETosis was very rapid. Quantification interference contrast (DIC) and spinning-disk confocal images indicated that cells that phagocytosed C. albicans or were stimulated were acquired at two confocal planes (at the coverslip surface and with LPS and that completed NETosis exhibited similar membrane cell center) at each 1- to 2-min time interval for 4 h. Under these and nuclear dynamics as those seen in ionomycin-stimulated cells conditions, 17.0%, 44.6%, and 55.6% of ionomycin-stimulated (Fig. 1 G–J, SI Appendix,Fig.S2A and B,andMovie S1). Together, mouse PMNs, human PMNs, and dHL-60 cells observed went on these results show that NETosis proceeds by a dynamic, stepwise to complete NETosis, respectively, as evidenced by extracellular sequence of cellular events that is conserved, independent of the expulsion of DNA (Fig. 1 B, D,andF). Importantly, unstimulated species or NETosis stimulant. mouse PMNs did not exhibit DNA expulsion (SI Appendix,Fig. S1A), and the NETosis efficiencies attained during live-cell im- Microvesicles Containing Cytosolic Components Are Shed from the aging of dHL-60 cells were similar to those measured in a fixed- Plasma Membrane at NETosis Onset. Given the bioactive nature timepoint assay (31% in dHL-60; SI Appendix,Fig.S1B) and those of extracellular vesicles (23), we sought to better characterize the previously published for mouse (22) and human (12) PMNs, in- origin and content of the vesicles that appeared in the early stage dicating that our cell isolation and long-term imaging treatments of NETosis. Immunostaining for granule components in ionomycin- are minimally perturbative to NETosis. stimulated dHL-60 cells showed that vesicles were positive for We then examined time-lapse movies to analyze cell dynamics markers of primary (neutrophil elastase and myeloperoxidase), during NETosis. DIC and confocal movies of membrane dy- secondary (Lactoferrin and CD11b) and tertiary granules (MMP9 namics in the optical plane at the ventral cell surface of all three and CD11b), as well as secretory vesicles (tetranectin) (Fig. 2A). cell types revealed that within minutes after ionomycin stimula- However, single vesicles often contained markers of multiple tion, cells rapidly formed many small (∼<2 μm) vesicles that granule types, suggesting that vesicles containing cytosolic com- were devoid of fluorescent DNA (Fig. 1 A, C, E, and I and Movie ponents, including granules, may be shed from cells. To determine S1). Following vesiculation, cells rounded up and eventually whether vesicles contained cytosol and were physically discon- expelled extracellular DNA, marking completion of NETosis. nected from the cell, we expressed soluble mEmerald and per- Quantification revealed that although not all cells that vesicu- formed fluorescence recovery after photobleaching (FRAP). We lated went on to expel DNA, 100% of cells that completed found that mEmerald localized stably to vesicles, and when their NETosis had previously formed vesicles. Thus, vesiculation is a contents were photobleached, the fluorescence did not recover, hallmark of cellular entry into NETosis (Fig. 1 B, D, F, and I), indicating a lack of continuity between vesicles and the cell body.

2of12 | www.pnas.org/cgi/doi/10.1073/pnas.1909546117 Thiam et al. Downloaded by guest on September 23, 2021 Ms PMN + 4 μM Ionomycin Ms PMN + Ionomycin; n = 427 A B 120 -8 min-2 min 0 min4 min 18 min 20 min 92 min 100 80 60 μ m 40

% of Cells 20 Z = 0

SiR-DNA 0

μ m -20

DIC / NETosis MV Shedding DNA release Z = + 3 Nuclear Rounding dHL60 + 4 μM Ionomycin DNA DecondensationPM Permeabilization CD-3 min-2 min 0 min 4 min 43 min 55 min 145 min dHL60 + Ionomycin; n = 260 120 100

m 80 μ 60

Z = 0 40 % of Cells SiR-DNA 20 0 μ m DIC /

NETosis MV Shedding DNA release Z = + 3 Nuclear Rounding DNA DecondensationPM Permeabilization EFHm PMN + 4 μM Ionomycin Hm PMN + Ionomycin; n = 306 120 -8 min-2 min 0 min 4 min12 min 68 min 92 min 100 80 μ m

60 IMMUNOLOGY AND INFLAMMATION 40 % of Cells Z = 0

SiR-DNA 20

μ m 0 DIC / A release NETosis

Z = + 3 MV Shedding DN Nuclear Rounding PM Permeabilization Hm PMN + dTomato expressing Candida albicans (MOI=2) DNA Decondensation GH -12 min-10 min 0 min 4 min8 min 14 min 170 min Hm PMN + C. albicans; n = 143 120 100 μ m 80 60 Z = 0 40 % of Cells m

μ 20 0

Z = + 3 NETosis

DIC/ SiR-DNA / Sytox Green MV Shedding DNA release Ms PMN + Ionomycin Hm PMN + Ionomycin Nuclear Rounding IJMV Shedding dHL60 + Ionomycin Hm PMN + C. albicans DNA DecondensationPM Permeabilization n= 286 228 306 99 n=282 223 306 97 257 211 12 39 193 195 305 96 253 181 200 95 42 142 134 49 240 240 120 200 60 160 120 30 80 0 40 0 Time relative to Time

Time relative to Time -30 2 15 4 106 Stimulation (min) -40 11.2 8 2 7 20 10 32 11 61 61 23 12 82 47 118 14 78 102 134 52 +/-1 +/-33 +/-10 +/-39 MV Shedding (min) -60 -80 +/-12 +/-24 +/-2 +/-15 +/-19 +/-13 +/-13 +/-21 +/-49 +/-50 +/-30 +/-17 +/-36 +/-38 +/-46 +/-19 +/-48 +/-60 +/-48 +/-24 DNA Nuclear DNA Release PM NETosis N 60 N N PM L PM M Decondensation Rounding Permeabilization s dH m P M H Hm . albicans + Ionomycin + C

Fig. 1. NETosis proceeds by vesiculation, DNA decondensation, DNA release from the nucleus into the cytoplasm, and extracellular DNA expulsion in mouse and human blood neutrophils and dHL-60 cells. Mouse (Ms; A, B, I, and J) or human (Hm; E–J) PMNs or dHL-60 cells (C, D, I, and J) stained with far-red SiR-DNA were stimulated with ionomycin (4 μM, A–F, I, and J) or dTomato-expressing C. albicans (C. albicans, MOI of 2, G–J, dTomato shown in blue), and imaged by DIC and confocal microscopy at the coverslip–cell interface (Z = 0 μm) and in the cell center (Z =+3 μm) at 1- to 2-min intervals for 4 h. Time (min) relative to plasma membrane microvesicle (MV) shedding noted. Double-arrow-headed lines indicate presence of ionomycin (A, C, and E) or phagocytosed C. albicans (G); colored boxes around images correspond to color coded cellular events in the graphs. C. albicans experiments (G and H) included Sytox green to highlight extracellular DNA. (A, C, E, and G) Time series of image overlays of DIC (grayscale) and fluorescence of cell, C. albicans (blue in G) and DNA (red in A, C, E,red and green in G) dynamics. (B, D, F, and H) Percent of cells exhibiting MV shedding, DNA decondensation, nuclear rounding, DNA release into the cytoplasm, loss of DIC contrast (PM permeabilization), and extracellular DNA release (NETosis) after stimulation. n = total number of cells, each point = percent of cells in one experiment. (I and J) Timing of MV shedding relative to ionomycin or C. albicans stimulation (I) or of cellular event initiation relative to MV shedding (J). N = total number of cells observed, points = individual cells. In B, D, F, H, I, and J: long bar = mean, short bars = SD. (Scale bars In A, C, E, and G:10μm.)

Thiam et al. PNAS Latest Articles | 3of12 Downloaded by guest on September 23, 2021 A dHL60 + 4 μM Ionomycin; Z = 0 μm; immunostaining dHL60 + 4 μM Ionomycin; Z = 0 μm; immunostaining Phase C. Elastase Lactoferrin CD11b PC/Elastase/ Phase C. Elastase MMP9 PC/Elastase/MMP9 CD11b/Lactoferrin

Fig. 2. Microvesicles containing cytosolic components are shed from the plasma membrane to initiate NETosis. (A–D) dHL-60 cells stimulated with ionomycin. Phase C. Elastase Tetranectin CD11b PC/Elastase/ Phase C. Elastase MPO PC/Elastase/MPO (A) Phase contrast (grayscale) and immunolocalization CD11b/Tetranectin of neutrophil elastase (Elastase, red), myeloperoxidase 0min (MPO, green), lactoferrin (magenta), CD11b (green), MMP9 (green), and tetranectin (magenta), fixed 30 min after stimulation. (B, Left) DIC and confocal images of a living dHL-60 cell expressing soluble mEmerald (green) before (Upper row), and after (time dHL60 + 4 μM Ionomycin; Z = 0 μm B C noted, Middle and Lower row) photobleaching (PB) of DIC mEmrld mEmrld/DIC -5 min -2 min 0 min 12 min 45 min the vesicle (arrowhead). (Scale bar: 2 μm.) (Right) Photobleaching (PB) 1.4 Normalized fluorescence intensity of the bleached t = 0s 1.2 region over time in regions in the cell body (black) or 1.0 in microvesicles (MVs) (green), error bar = SD, ar-

0.8 DIC 0.6 rows = time of photobleaching. (C and D) Cells im-

t = 1.4s 0.4 Cell Body (n = 25) aged by DIC and confocal microscopy at the coverslip– 0.2 Microvesicles (n = 38) 0.0 cell interface (Z = 0 μm) and in the cell center (Z = Normalized intensity (AU) AnnexinV- 012345678910 AF488 +3 μm) at 1- to 2-min intervals for 4 h. Time (min) Time (sec) t = 10.1s DIC / AnnexinV relative to plasma membrane MV shedding noted. 0min 8min 22min 42min 49min Double-arrow-headed lines indicate presence of ion- dHL60 + 4 μM Ionomycin; Z = 0 μm D omycin; boxes around images indicate different -1 01 min 20 min 29 min 75 min cellular events (green: MV shedding; yellow: DNA decondensation; blue: DNA release to the cytosol, red: extracellular DNA release). (C and D)Time series of DIC (Upper) and fluorescence of cells in media containing annexin V-Alexa Fluor 488 (AF488, C) or expressing CAAX-mApple (Lower row, D). C, Bottom row: DIC (grayscale) and annexin V-AF488 (red-hot color scale) overlay. (Scale bar: 10 μm.) (A and C) Yellow boxes (Upper rows, Scale bars: 10 μm.)

CAAX-mApple DIC shown zoomed below (Scale bars: 1 μm.).

This was in contrast to bleaching a region of mEmerald in the cell by live-cell microscopy. To analyze DNA extrusion relative to lamin body, which rapidly recovered from the unbleached pool (Fig. 2B meshwork structure, we imaged SiR-DNA-stained dHL-60 cells and Movie S4). To differentiate between microvesicles and exo- expressing either lamin A- or B1-mEmerald (Movie S6). Prior to somes, we used plasma membrane markers that would be pre- stimulation, lamins formed punctate meshworks at the nuclear sent on microvesicles and absent from exosomes. Imaging dHL- periphery (Fig. 3 D and I and SI Appendix, Fig. S3 B and C). 60 cells either expressing the farnesylation signal sequence fused After ionomycin-induced microvesicle shedding, DNA decon- to mApple (CAAX-mApple) or with a fluorescent analog of the densation, and nuclear rounding, the lamin meshwork formed phosphatidylserine (PS)-binding protein annexin V added to the holes, often at multiple points (Fig. 3 D, E, and I), followed media showed that vesicles were positive for both plasma within 1 to 2 min by DNA extrusion through the holes and membrane markers (Fig. 2 C and D and Movies S2 and S5). disassembly of the lamin meshwork as the DNA rapidly ex- Thus, NETosis is initiated by shedding of microvesicles con- panded throughout the cytoplasm (Fig. 3 D and I and Movie S6). taining cytosolic contents that form by budding from the We analyzed NE dynamics using either a fluorescent endoplas- plasma membrane. mic reticulum (ER) vital dye (Movie S7) or expressed mEmerald fusions of either the ER-retention signal sequence (ER-5- ER Vesiculation Is Followed by Nuclear Lamin Meshwork Disassembly, mEmerald) or the inner nuclear envelope protein, Lap2β (Movie NE Rupture, and Release of DNA into the Cytoplasm, Leading to S8). Prior to stimulation, ER markers localized to a reticular net- NETosis. We next focused on how DNA is released from the nu- work in the cell periphery as well as the NE, and Lap2β-mEmerald cleus into the cytoplasm, which requires passing first through the localized only to the NE. Upon ionomycin stimulation of dHL- nuclear lamina and then breaching both membranes of the NE. 60cellsorLPSstimulationofmousePMNsandsoonafter We first confirmed the expression and localization of lamin microvesicle shedding, the ER network broke into vesicles (Fig. 3 isoforms in dHL-60 (24) and human and mouse PMNs (Fig. 3 A F–H and J, SI Appendix, Figs. S2C and S4A, and Movie S7) with and C and SI Appendix,Fig.S3A–D). Fixation and immunos- the NE remaining intact during nuclear rounding and chromatin taining of lamin B1/B2 in mouse PMNs and lamin B1 in human decondensation (Fig. 3 G and H, SI Appendix, Figs. S2C and PMNs after ionomycin stimulation of NETosis showed dis- S4A, and Movie S8). Subsequent to nuclear rounding, the NE continuities in the lamin meshwork detectable at 5 min after suddenly became discontinuous and rapidly retracted from sites stimulation, with extrusion of DNA from the nucleus through of DNA extrusion into the cytoplasm, signifying NE rupture (Fig. the discontinuities and into the cytoplasm visible at 10 min, and 3 G and H, SI Appendix, Figs. S2C and S4A, and Movie S8). disassembly of the lamin meshwork at later timepoints (Fig. 3 Coexpression of Lap2β-mEmerald and lamin B1-mApple in A–C). The apparent lag between lamin meshwork disruption dHL-60 cells showed that the lamin meshwork became discon- and DNA extrusion suggests that the NE may remain intact tinuous slightly before NE rupture (Fig. 3 I and J and Movie S9). longer than the lamina. We never observed DNA release from the nucleus or cell in We next sought to determine the sequence of lamin meshwork NE-derived membrane vesicles (16) or without prior lamin disruption, NE breakage, and chromatin release into the cytosol meshwork and NE rupture, and NE or lamin meshwork resealing

4of12 | www.pnas.org/cgi/doi/10.1073/pnas.1909546117 Thiam et al. Downloaded by guest on September 23, 2021 Fig. 3. ER vesiculation is followed by A Ms PMN + 4 μM Ionomycin; Immunostaining B nuclear lamina disassembly, NE rup- Ms PMN DMSO5 min 10 min 60 min 240 min 100 n= ture, and release of DNA into the cy- 56 52 52 51 51 Lamin B1/2 toplasm, leading to NETosis. Mouse 80 (Ms; A and B) or human (Hm; C and H) 60 – PMN or dHL60 cells (D G, I, and J) 40 stimulated with ionomycin and fixed Lamin B1/2 / DNA % 20 % % of Cells with 2 8 4% 6 83 % 7 (A and C, time noted) or imaged live 90 % 0

– Lamin B1/2 Discontinuity (D and F I) by DIC and confocal mi- 0 5 10 60 240 croscopy at the coverslip–cell interface Time after Ionomycin stimulation (min) (Z = 0 μm) and in the cell center (Z = Hm PMN + 4 μM Ionomycin C ; Immunostaining DEdHL60 + 4 μM Ionomycin; Z = + 3 μm +3 μm) at 1- to 2-min intervals for 4 h. dHL60 DMSO 5 min 10 min 60 min 240 min Time (min) relative to MV shedding -8 min 0 min 3 min 5 min 15 min 100 n=53 Phase Contrast (Z = 0 μm) DIC noted. Double-arrow-headed lines in- 80 dicate presence of ionomycin. Arrow- 60 heads, lamina or NE rupture points. (D Lamin B1 and G–I) Green boxes around images LaminA-mEmerald % of Cells 40 indicate MV shedding. (A and C)Im- 20 munofluorescence of lamin B1/B2 in 0 Ms PMN (A, Upper row and Bottom LaminB1 / DNA LaminA / DNA row, green) or of lamin B1 in Hm PMN 1 Rupture Point (C, Middle row and Bottom row, Z =+1μm Z =+3 μm >1 Rupture Points green) and staining of DNA with DAPI F dHL60 + 4 μM Ionomycin; Z = 0 μm G dHL60 + 4 μM Ionomycin; Z = + 3 μm (A, 10 min, C) or SiR-DNA (A, DMSO, 5, -18 min -4 min-1 min 2 min -2 min0 min 17 min 18 min 20 min 60, and 240 min) (Bottom row, red) DIC DIC fixed after ionomycin stimulation at the time noted. (C, Top row) Phase contrast images at Z = 0 μm; Middle and Bottom rows: confocal images at ER-mEmerald ER-mEmerald Z = 0 μm and Z =+1 μm, Z =+3 μm. (B) Percent (numbers in bar) of Ms PMN

exhibiting lamin meshwork disconti- IMMUNOLOGY AND INFLAMMATION nuities, 0 = DMSO control. n = total Hm PMN + 4 μM Ionomycin H ER / DNA cells. (D) Time-series of DIC (Top row) -4 min0 min 24 min 26 min 88 min and confocal (Lower rows) images DIC (Z=0) of dHL60 cells expressing lamin A-mEmerald (Middle row and Bottom row, green) and stained with SiR-DNA dHL60 + 4 μM Ionomycin ER-Tracker Red dye (Z = 0 μm) I (Bottom row, red). (E) Percent of cells -4 min 0 min 16 min 17 min 31 min with one or more rupture points. n = DIC (Z = 0 μm) total number of cells, each point = percent of cells in one experiment. ER-Tracker Red dye (Z = + 3 μm) (F–I) Time series of DIC (Upper rows) and confocal (Lower rows) images of Lap2B-mEmerald (Z = + 3 μm) dHL60 cells (F, G,andI) expressing ER- ER-Tracker Red dye / DNA (Z = + 3 μm) mEmerald (F, Bottom row; G, Middle and Bottom row, green) or coexpress- Lamin B1-mApple (Z = + 3 μm) ing Lap2β-mEmerald (I, second row and bottom row, green) and lamin dHL60 B1-mApple (I, third row and bottom J n=671355510921 200 ns row, blue), or live Hm PMN (H) 160 **** ns * Lap2B / Lamin B1 / DNA (Z = + 3 μm) stained with ER-Tracker Red (H, Mid- 120 dle rows and Bottom row, green) and 80 stained with SiR-DNA (G–I Bottom 40 0

rows red). (J) Timing of initiation of relative to Time -40 0.8 21 23 32 28 MV Shedding (min) +/-1.7 +/-20 +/-18 +/-24 +/-36 ER vesiculation or Lamin B1 or Lamin -80 A rupture, Lap2β rupture or outer NE rupture (ER [ONM]) relative to MV Rupture ER VesiculationLamin A Lap2B Rupture shedding in dHL-60 cells. n = total Lamin B1 Rupture ER (ONM) Rupture number of cell observed, points = individual cells. (E and J) Mean (long bar) and SD (short bar), shown below each plot. ****P < 0.0001; *P < 0.1; ns, nonsignificant. Statistical test: Mann– Whitney U test. (Scale bars in A, C, D, F–I:5μm.)

was never observed. Thus, cells vesiculate their ER networks actin, we stained mouse or human PMNs with SiR-actin (Movies before decondensing their DNA and rupturing their lamin S10 and S11) or expressed either F-tractin-mApple (Movie S12)or meshwork, followed by NE rupture to allow chromatin release actin-mEmerald (Movie S13) in dHL-60 cells. MT dynamics were into the cytoplasm. analyzed in mouse and human PMNs stained with SiR-tubulin (Movies S10 and S11) or dHL-60 expressing either the ensconsin The Actin, MT, and Peripheral Vimentin Cytoskeletons Disassemble MT-binding domain fused to eGFP (Movie S12) or tubulin- Prior to NETosis, and F-Actin Disassembly Is Important for NET mEmerald (Movie S14). Vimentin IFs were visualized in dHL- Release. We next sought to examine how chromatin breaches 60 cells expressing vimentin-mEmerald (Movie S12)orbyimmu- the actin, MT, and vimentin intermediate filament (IF) cyto- nofluorescence in human and mouse PMNs. Prior to stimulation skeletal networks to allow its extracellular expulsion. To visualize in spread neutrophils, actin concentrated in ruffles, uropods and in

Thiam et al. PNAS Latest Articles | 5of12 Downloaded by guest on September 23, 2021 punctate structures at the cells’ ventral surface, MTs radiated to- NETosis, the plasma membrane becomes permeable to in- ward the cell periphery from the centrosome, and vimentin IF creasingly larger macromolecules over time, allowing their entry formed around the nuclear surface and extended into the cyto- into and leakage from cells prior to plasma membrane rupture and plasm. Ionomycin (dHL60, mouse, and human PMNs) or LPS expulsion of DNA. (mouse PMNs) stimulation induced rapid redistribution of actin, tubulin, and vimentin from these structures into diffuse cytosolic PAD4 Is Critical to DNA Decondensation, NE Rupture, and Extracellular fluorescence, indicating filament disassembly (Fig. 4 A–G, SI Ap- DNA Expulsion in Mouse PMN. We next sought to determine the pendix, Figs. S4 B and C and S2 D and E,andMovies S10–S14). role of the arginine deiminase PAD4 in mediating the cellular Quantitative analysis of dHL-60 cells showed that actin disas- events of NETosis. PMNs from wild-type (WT) and PAD4 sembly occurred just before microvesicle shedding, while MT and knockout (KO) mice were stained with SiR-DNA, SiR-Actin, SiR- vimentin IF disassembly was initiated nearly concurrent with Tubulin, or ER-dye to visualize the DNA, actin, and MT cyto- shedding (Fig. 4G). Cytoskeletal disassembly was then followed skeletons, or endoplasmic reticulum and NE, respectively, and many minutes later by DNA decondensation, nuclear rounding, stimulated with ionomycin. Time-lapse microscopy showed that and NETosis completion (Movies S13 and S14). Although diffuse similartoWTPMNs,PAD4KOPMNsinitiatedNETosisby vimentin fluorescence after disassembly masked visualization of disassembling their actin cytoskeleton before shedding micro- perinuclear vimentin structures in live cells (Fig. 4F and Movie vesicles (SI Appendix, Fig. S4F,Fig.6A and B,andMovie S18), S12), immunostaining showed that while peripheral vimentin IFs followed by ER vesiculation, MT disassembly, nuclear rounding, were largely disassembled within 5 min after stimulation, at 10 min and plasma membrane permeabilization (Fig. 6 A–D, H,andI, SI some particulate perinuclear vimentin remained (Fig. 4E and SI Appendix,Fig.S4G and H,andMovies S18 and S19). However, in Appendix,Fig.S4I and J). Thus, NETosis is initiated by actin contrast to WT, PAD4 KO cells were deficient in DNA decon- disassembly, followed by microvesicle shedding, disassembly of densation, NE rupture, DNA release into the cytoplasm, and ex- vimentin IFs, and MTs prior to DNA decondensation, nuclear tracellular NET release (Fig. 6 B–D and I and Movies S18 and rounding, nuclear lamin meshwork and NE rupture, DNA release S19). The few PAD4 KO cells that completed NETosis had tem- into the cytoplasm, and extracellular DNA expulsion. poral delays in DNA decondensation and extracellular DNA re- We then determined the requirement for actin and MT disas- lease (Fig. 6D). Immunostaining of vimentin IFs and lamin B1/ sembly in NETosis using jasplakinolide or taxol to stabilize actin or B2 showed that PAD4 KO cells exhibited delays in the disassembly MTs, respectively. Pretreatment of dHL-60 cells expressing actin- of peripheral vimentin IFs (SI Appendix,Fig.S4I and J)and or tubulin-mEmerald prior to ionomycin stimulation showed that formation of lamin meshwork discontinuities (Fig. 6 E–G). Thus, jasplakinolide caused formation of a thick cortical actin network PAD4 is required for efficient remodeling of the vimentin IF (Movie S13), while taxol (Movie S14) induced peripheral MT network, DNA decondensation, lamin meshwork and NE rupture, bundles, and both structures remained mostly intact throughout and extracellular DNA release. NETosis. Quantification revealed that neither treatment affected microvesicle shedding, DNA decondensation, nuclear rounding, PAD4 Localizes Predominantly to the Nucleus and Its Enzymatic and DNA release into the cytoplasm, or loss of cytosolic contents. Nuclear Localization Activities Mediate Efficient DNA Decondensation, However, while taxol had no effect on extracellular DNA expulsion, NE Rupture, and Extracellular DNA Expulsion in Human dHL-60. While jasplakinolide treatment reduced the fraction of cells that com- the importance of PAD4 in mouse PMN NETosis is well docu- pleted NETosis (Fig. 4 H and I). Examination of actin dynamics in mented (13, 14), the role of PAD4 in human neutrophils remains the subset of jasplakinolide-treated cells that released extracellular controversial (14, 25). We thus first analyzed PAD4 localization by DNA showed that DNA escaped through a local disruption in the immunostaining in dHL-60 and human PMNs and its dynamics by cortical actin network (Movie S13). Thus, actin disassembly is expression of PAD4-mEmerald in dHL-60 cells. This showed that critical to extracellular DNA release during NETosis. PAD4 primarily localized to the nucleus of resting cells as assessed by colocalization with Hoechst- or SiR-stained DNA (Fig. 7 A–C) Plasma Membrane Permeability Is Progressively Increased Prior to Its or with coexpressed mCherry fused to the nuclear localization Rupture and NET Release. We next sought to determine how DNA signal sequence (NLS-mCherry) which served as a soluble nu- breaches the plasma membrane to be released into the extracellu- cleoplasmic marker (Fig. 7D and Movie S20). Imaging SiR- lar environment. We analyzed plasma membrane dynamics using DNA-stained dHL-60 cells expressing PAD4-mEmerald after CAAX-mApple in ionomycin-stimulated dHL-60 cells that were ionomycin stimulation showed that PAD4 remained largely in either stained with SiR-DNA or coexpressing histone H2B- the nucleus through microvesicle shedding, DNA deconden- mEmerald. This showed that the plasma membrane appeared to sation, and nuclear rounding. However, prior to nuclear rup- remain intact during microvesicle shedding, nuclear rupture, and ture, PAD4 began accumulating in the cytoplasm, followed by DNA release into the cytoplasm, after which a large hole was rapid nuclear release along with DNA at nuclear rupture (Fig. formed concomitant with extracellular DNA expulsion (Fig. 5A and 7 D and F). Coexpression of PAD4-mEmerald with NLS- Movie S15). Such plasma membrane and DNA release occurred mCherry showed that the two initially colocalized in the nucleus much later than the decrease in contrast in the cell periphery vi- and simultaneously accumulated in the cytosol after DNA decon- sualized by DIC microscopy (Movie S3), suggesting that leakage of densation but prior to nuclear rupture (Fig. 7 D–F and Movie S20). cytosol may occur by permeabilization of the plasma membrane This indicates that loss of NE integrity, rather than regulated prior to extracellular DNA release. To test this, we added different PAD4 export caused its release from the nucleus. In addition, both sized membrane-impermeant fluorescent markers to the imaging PAD4-mEmerald and NLS-mCherry were released to the extra- media and monitored their entry into cells stained with SiR-DNA cellular environment prior to plasma membrane rupture and DNA and stimulated with ionomycin (human PMNs or dHL-60) or LPS expulsion (Fig. 7 D and F). Thus, during NETosis, nuclear PAD4 is (mouse PMNs). We found that calcein (0.6 kDa), 10 kDa dextran, first released into the cytoplasm by NE permeabilization after and 70 kDa dextran all entered cells much earlier than extracellular DNA decondensation but before nuclear rupture, then released DNA release (Fig. 5 B–E and H, SI Appendix,Figs.S4D and E and extracellularly by plasma membrane permeabilization, and finally S2 F and G,andMovies S16 and S17). Quantification of dHL- the remaining PAD4 exits the cell along with NET expulsion. 60 cells showed that calcein, 10 kDa dextran, and 70 kDa dextran To address the requirement for PAD4 in NETosis in human entered cells on average at ∼20, ∼30, and ∼50 min after micro- neutrophils, we generated an HL-60 cell line, PAD4 CR dHL-60, vesicle shedding. Addition of either calcein together with 10 kDa using CRISPR-Cas9 technology. This resulted in near complete dextranor10kDadextrantogetherwith70kDadextranshowed loss of PAD4 protein (SI Appendix,Fig.S5A and B), and had that calcein entered cells before 10 kDa dextran (human PMNs, no effect on HL-60 differentiation into neutrophil-like cells, as Fig. 5 I and J and Movie S17),whichpreceded70kDadextranentry assessed by reactive oxygen species production after PMA stimulation (dHL-60cells,Fig.5F and G and Movie S17). Thus, during (20 nM, ref. 26), SI Appendix, Fig. S5 C and D). Stimulation of

6of12 | www.pnas.org/cgi/doi/10.1073/pnas.1909546117 Thiam et al. Downloaded by guest on September 23, 2021 ABHm PMN + 4 µM Ionomycin; Z = 0 µm dHL60 + 4 µM Ionomycin; Z = 0 µm -8 min-6 min-4 min - 2 min 0 min -14 min -6 min-4 min -2 min 0 min DIC DIC Fig. 4. The actin, MT, and vimentin cytoskeletons disassemble prior to NETosis, and F-actin disassembly is important for NET release. Human (Hm) PMN (A, C, and E) or dHL-60 cells (B, D, F, and G–I) stimulated with ionomycin and fixed (time noted, E) or imaged F-Tractin-mApple SiR-Actin(F-Actin) (F-Actin) by DIC and confocal microscopy at the coverslip–cell C Hm PMN + 4 µM Ionomycin; Z = 0 µm D dHL60 + 4 µM Ionomycin; Z = 0 µm interface (Z = 0 μm) and in the cell center (Z =+3 μm) -2 min 0 min 4 min 12 min 24 min -54 min-4 min0 min 2 min 5 min at 1- to 2-min intervals for 4 h, time relative to

DIC DIC microvesicle (MV) shedding noted. Double-arrow- headed lines indicate presence of ionomycin. (A–F) Time-series of DIC (A–D and F) or phase contrast (E) (Top rows) and confocal (Lower rows) images of live (A and C) or fixed (E) Hm PMN or live dHL-60 cells (B, D, and F). (A and B) Actin visualized with far red SiR- Ensconsin-eGFP (MT) SiR-Tubulin (MT) SiR-Tubulin Hm PMN + 4 µM Ionomycin actin (A) or expression of F-Tractin-mApple (B). (C E ; Immunostaining; Z = 0 µm F dHL60 + 4 µM Ionomycin; Z = 0 µm and D) MTs visualized with far red SiR-tubulin (C)or DMSO 5 min 10 min 60 min 240 min -9 min-3 min-2 min -1 min 0 min expression of Ensconsin-eGFP (D). (E and F) Immu-

DIC nolocalized vimentin (E) or expression of vimentin- mEmerald (F). (A–D and F) Green boxes around im- Phase C. ages indicate MV shedding. (G) Timing of actin, MT, and vimentin disassembly (Dis) relative to MV shedding. n = total number of cell observed, points rep- Vimentin- mEmerald Vimentin resent individual cells, mean (long bar) and SD (short

dHL60 dHL60 dHL60 bar) shown below each plot. (H and I) Percent of cells GI40 H n=133 124 50 120 Control (n=246) Jasplakinolide 1µM (n=271) 120 Control (n=172) Taxol 10 µM (n=167) exhibiting actin disassembly, MV shedding, DNA 100 100 decondensation, nuclear rounding, DNA release into 20 **** 80 80 the cytoplasm, loss of DIC contrast (plasma mem- 0 60 60 brane (PM) permeabilization, and extracellular DNA 40 IMMUNOLOGY AND INFLAMMATION % of Cells

% of Cells 40 release (NETosis) after stimulation in cells treated -20 Time relative to to relative Time -1.7 -0.13 0.9 20 μ MV shedding (min) 20 with vehicle (control), jasplakinolide (1 M, H), or +/- 4 +/-1.5 +/-7.7 0 -40 0 taxol (10 μM, I), n = total of cell observed, points = percent of cells in one experiment, long bar = mean, Actin Dis. NETosis NETosis Vimentin Dis. DNA release DNA release Encons-MT Dis. MV Shedding MV Shedding short bars = SD. Statistical test: Fisher’s exact test, Actin DisassemblyA DecondensationNuclear Rounding Nuclear Rounding DN PM Permeabilization DNA Decondensation PM Permeabilization ****P < 0.0001. (Scale bars in A–F:5μm.)

SiR-DNA-stained WT and PAD4 CR dHL-60 cells with ion- and mouse primary blood neutrophils, as well as in neutrophil- omycin showed that PAD4 CR dHL-60 cells exhibited a signif- like dHL-60 cells. We show that NETosis proceeds by a stepwise icant delay in DNA decondensation and largely failed in nuclear sequence of cellular events that is conserved between mouse rupture and extracellular DNA expulsion (Fig. 8 A, B, and G and and human primary neutrophils, as well as in dHL-60 cells, thus Movie S21). Analysis of PAD4 CR dHL-60 cells expressing establishing this cell line as an important model system for in Lap2β-mEmerald or laminB1-mEmerald showed that for the vitro studies of the cellular mechanisms of NETosis. We demon- small fraction of cells that completed NETosis, both NE and strate that NETosis begins with the rapid disassembly of the actin lamina rupture was delayed (Fig. 8 H–L). Importantly, reex- cytoskeleton, followed by shedding of plasma membrane micro- pressing PAD4-mEmerald in PAD4 CR dHL-60 cells rescued the vesicles containing granules and cytosolic components, disassembly defects, while expressing mEmerald alone did not (Fig. 8 C, D,and and remodeling of the MT and vimentin IF cytoskeletons, ER G and Movie S22). Thus, PAD4 is critical for DNA decondensa- vesiculation, chromatin decondensation and nuclear rounding, tion, nuclear lamina, and NE rupture, and DNA expulsion in both progressive plasma membrane and NE permeabilization, nuclear mouse and human neutrophils. lamin meshwork and then NE rupture to release chromatin into the cytoplasm, and finally PM rupture and discharge of To test if the citrullination or nuclear localization activities SI Appendix were required for the roles of PAD4 in NETosis, we rescued extracellular chromatin ( ,Fig.S6). We find that PAD4 CR HL-60 cells with mEmerald-tagged PAD4 mutants certain cellular events always precede other events, suggesting a requirement for a precise sequence for progression through specifically defective in these functions. PAD4 is rendered en- NETosis. For example, cytoskeletal disassembly, microvesicle zymatically dead by a C645A substitution (27), while its nuclear shedding and ER vesiculation always occurred before nuclear localization is disrupted by the triple K59A/K60A/K61A sub- rupture, while plasma membrane permeabilization could occur stitution (28). Expressed PAD4-C645A-mEmerald localized pre- before or after nuclear rupture but always preceded extracel- dominantly to the nucleus (Fig. 8E), while PAD4-K59A/K60A/ F lular DNA release. We found no evidence of extracellular K61A-mEmerald was mostly cytosolic (Fig. 8 ). In ionomycin- DNA release in shed microvesicles or without catastrophic stimulated PAD4 CR dHL-60 cells, expression of neither PAD4- rupture of the nucleus or plasma membrane, and we never C645A-mEmerald nor PAD4-K59A/K60A/K61A-mEmerald res- observed cell viability after NET release, independent of the cued the defects in DNA decondensation, nuclear rupture, and stimulus or cell system examined. Analysis of mouse PMN and extracellular DNA release (Fig. 8 E–G and Movie S22)inducedby human dHL-60 cells lacking PAD4 demonstrate its required loss of PAD4. This indicates that efficient DNA decondensation, role in efficient DNA decondensation, NE rupture, and extracel- nuclear rupture, and DNA expulsion requires PAD4 enzymatic lular DNA release, and prompt timing of lamin meshwork and activity in the nucleus in human neutrophils. vimentin IF disassembly, and mutant add-back experiments show that the nuclear localization and enzymatic activities of PAD4 are Discussion critical to these roles. In this study we performed the systematic characterization of the The conservation of cellular events between mouse and hu- timing of dynamic cellular events leading to NETosis in human man PMNs and neutrophil-like dHL-60 cells with multiple

Thiam et al. PNAS Latest Articles | 7of12 Downloaded by guest on September 23, 2021 A dHL60 + 4 μM Ionomycin; Z = +3 μm dHL60 + 4 μM Ionomycin; Z = +3 μm -3 min 0 min 15 min 24 min 38 min -3 min 0 min 15 min 24 min 38 min mEmerald H2B- DIC H2B / CAAX CAAX -mApple dHL60 + 4 μM Ionomycin; Z = 0 μm C B dHL60 -7 min0 min 24 min 31 min 32 min 57 min 1.0 DNA Decondensation 0.9

0.8

DIC 0.7

Calcein Entry Calcein 0.6 kDa 0.6

Ratio Intra/Extracellular 0 0 0 0 2 4 6 E Time Relative to MV Shedding (min) dHL60 Calcein 0.6 kDa 1.0 DNA D dHL60 + 4 μM Ionomycin; Z = 0 μm Decondensation 0.9 -4 min0 min 22 min 77 min 85 min 86 min PM 0.8 Permeabilization 0.7 Dextran 10 kDa 0.6 DIC

Ratio Intra/Extracellular 0 0 0 0 0 0 0 Dextran 10 kDa 2 4 6 8 0 2 1 1 Entry Time Relative to MV Shedding (min) G dHL60 Ratio Intra/Extracellular Dextran 10 kDa 1.0 DNA 1.0 dHL60 + 4 μM Ionomycin; Z = 0 μm Decondensation Dextran 10 kDa F 0.9 0.9 -6 min0 min 3 min 46 min 52 min 161 min PM 0.8 0.8 Permeabilization 0.7 0.7

Dextran 70 kDa 0.6 0.6

DIC 0 0 0 0 0 0 0 Ratio Intra/Extracellular 2 4 6 8 0 2 Dextran 10kDa 1 1 Entry Time relative to MV Shedding (min) H dHL60 200 n= 106 88 67 Dextran 10 kDa Dextran 70kDa 160 Entry 120 80 40

Dextran 70 kDa 0 -40 18.5 27.2 49.3 Time relative to Time +/-14.5 +/-32.9 +/-32.1

Hm PMN + 4 μM Ionomycin MV Shedding (min) -80 I Calcein Dextran Dextran -2 min0 min 70 min 84 min 110 min 124 min 0.6 kDa 10 kDa 70 kDa J

Hm PMN Ratio Intra/Extracellular

Z = 0 μm 1.0 DNA 1.0 Decondensation Dextran 10 kDa Calcein Entry 0.9 0.9

0.8 0.8

Calcein 0.6 kDa Z DIC = + 3 μm Dextran 10kDa 0.7 0.7 Entry Calcein 0.6 kDa 0.6 0.6

Ratio Intra/Extracellular 0 0 0 0 0 2 4 60 8 0 1 120 Z = + 3 μm Time relative to MV Shedding (min) Dextran 10 kDa

Fig. 5. Plasma membrane becomes permeable to progressively larger molecules prior to plasma membrane rupture and NET release. dHL-60 (A–H) or human (Hm) PMN (I and J) stimulated with ionomycin imaged by DIC and confocal microscopy at the coverslip–cell interface (Z = 0 μm) and in the cell center (Z = +3 μm) at 1- to 2-min intervals for 4 h, time relative to MV shedding noted. Double-arrow-headed lines indicate presence of ionomycin; colored boxes around images correspond to color coded cellular events in the graphs in Fig. 1. (A) Time series of DIC (Upper Left) and fluorescence images of a dHL-60 cell coexpressing CAAX-mApple (Lower Left and Right, magenta) and H2B-mEmerald (Upper Right and Bottom Right, yellow). (B, D, F, and I) Time series of DIC (Upper row) and fluorescence (Lower rows) images of dHL-60 (B, D, and F)orHmPMN(I) cells and calcein (622 Da, B and I, grayscale), Alexa Fluor 647 (D and F, red) or Alexa Fluor 594 (I, red), 10 kDa dextran, or Oregon Green 488 70 kDa (F, green) added to the media. (C, E, G, and J) Normalized intracellular fluorescence of calcein (C and J, green), 10 kDa (E, G, and J, red) or 70 kDa (G, green) dextran over time (in min). Time points of DNA decondensation noted. (H) Onset of calcein, 10 or 70 kDa fluorescent dextran cellular entry relative to MV shedding in dHL-60 cells. n = total number of cell observed, points = individual cells, mean (long bar) and SD (short bar) shown below each plot. (Scale bars in A, B, D, F, and I:10μm.)

8of12 | www.pnas.org/cgi/doi/10.1073/pnas.1909546117 Thiam et al. Downloaded by guest on September 23, 2021 Fig. 6. PAD4 is critical to DNA A Ms PMN Padi4 WT + 4 μM Ionomycin; DIC / SiR-DNA B Ms PMN Padi4 KO + 4 μM Ionomycin; DIC / SiR-DNA decondensation, NE rupture, and -6 min 0 min 6 min 22 min 148 min -6 min 0 min 22 min 22 min 148 min extracellular DNA expulsion in mouse PMN. PMN from Padi4 WT or Padi4 KO mice (Ms) stimulated with ionomycin, stained with SiR- Z = 0 μm Z = 0 μm DNA, and imaged live (A, B, H,and I) by DIC and confocal microscopy at the coverslip–cell interface (Z = Z = + 3 μm Z = + 3 μm 0 μm)andinthecellcenter(Z= +3 μm) at 2-min intervals for 4 h, CDMsPMN WT (n = 451) Ms PMN Padi4 KO (n = 342) n= 444 78 90 36 288 29 120 240 ** time relative to MV shedding **** ns 200 noted, or fixed (E and F) after 100 160 stimulation (time noted). Double- 80 120 MsPMN WT arrow-headed lines above images 60 80 Ms PMN Padi4 KO indicate presence of ionomycin, 40 40 % of Cells colored boxes around images 20 Time relative to Time 0

correspond to color coded cel- MV Shedding (min) 10 74 58 59 61 103 0 -40 +/- +/- +/- +/- +/- +/- lular events in the graphs (C). 10 81 45 52 47 93 -80 (A, B, H,andI). Time series of DNA ER (ONM) DNA Release image overlays of DIC (Top release NETosis Decondensation Rupture MV Shedding DNA Nuclear Rounding rows, grayscale) and SiR-DNA DNA DecondensationPM PermeabilizationER (ONM) Rupture E Ms PMN Padi4 WT + 4 μM Ionomycin; Immunostaining (Bottom rows, red). (C) Percent DMSO5 min 10 min 60 min 240 min of cells exhibiting MV shed- G ding, DNA decondensation, nu- Ms PMN; Lamin B1/2 Discontinuity clear rounding, loss of DIC contrast 120 WT Padi4 KO Lamin B1/2 (PM permeabilization) and outer n= 100 56 54 52 53 52 51 51 52 51 58 nuclear membrane (ER(ONM)) rup- 80 LaminB1/2 / DNA ture in cells stained with ER-Tracker 60 Ms PMN Padi4 KO + 4 μM Ionomycin; Immunostaining Red, DNA release into the cyto- 40 F IMMUNOLOGY AND INFLAMMATION plasm, and extracellular DNA % of Cells 20 DMSO5 min 10 min 60 min 240 min release (NETosis) after ionomycin 0 = = 4% 0% 62% 15% 83% 37% 90% 78% 57% stimulation. n total cells, point 92% Lamin B1/2 percent of cells in one experi- 051060240 ment. (D) Timing relative to MV Time after Ionomycin Stimulation (min) shedding of DNA decondensa- LaminB1/2 / DNA tion, ER (ONM) rupture and DNA H Ms PMN Padi4 WT + 4 μm Ionomycin I Ms PMN Padi4 KO + 4 μm Ionomycin release. n = total cells observed, point = individual cells, mean -4 min 0 min 44 min 46 min 192 min -6 min 0 min 44 min 192 min 262 min (long bar) and SD (short bar) shown below each plot. **** and < < ** for P value 0.0001, and 0.01, DIC (Z = 0 μm) DIC (Z = 0 μm) respectively; ns, nonsignificant, Mann–Whitney U test. (E and F) Immunofluorescence of lamin B1/ B2 (Upper row and Bottom row, ER-Tracker Red dye (Z = 0 μm) ER-Tracker Red dye (Z = 0 μm) green) and DAPI (at 10 min) or SiR-DNA(inDMSO,andat5,60, and240min)stainingofDNA ER-TrackerER-R-Tracraaackerk r RedReReddd dyedyeye / DNDDNANA (Z( = ++3 3 μm)m) ER-TrackerER-ER-Tracraackere RedReR ddd dyedyyee / DNADND A (Z( = ++3 3 μm)m) (Bottom row, red) in fixed WT (E) or PAD4 KO Ms PMN (F). Arrowheads, rupture points in the lamina. (G) Percent (numbers below bars) of cells with discontinuities in lamin B1/B2 meshwork (time after stimulation noted, 0 = DMSO control). n = total cells. (H and I) Cells stained with ER-Tracker Red (Middle row and Bottom row, green) and SiR-DNA (Bottom row, red). (Scale bars in A, B, E, F, H,andI:5μm.)

different stimuli suggests that despite possible divergences in permeabilization mediated by different membrane pores that signaling pathways mediating the process (12), NETosis follows could allow escape of specific bioactive molecules like cytokines, a very strict cellular mechanism. Thus, targeting cellular pathways citrullinated peptides, or proteins prior to NET release. For ex- might be a better approach for controlling NETosis progression ample, release of PAD4 from cells prior to NETosis could mediate than targeting the various divergent signaling pathways. Despite ADAMTS13 citrullination, preventing clearance of von Wille- the similarities, the three cell types showed some differences; for brand factor from vessel walls, thus enhancing NET attachment instance, while mouse PMNs and dHL-60 cells exhibited loss of and thrombosis (7). The mechanism of permeability is also of in- nuclear lobularity and full nuclear rounding during DNA decon- terest, with gasdermin D, that forms large pores and is critical for densation, human PMN nuclear lobularity decreased without full NETosis (20, 33, 34), being a good candidate for mediating the rounding up. This could result from differences in lamin expres- late stages of plasma membrane permeability during NETosis. sion (SI Appendix, Fig. S3A) promoting differences in nuclear Our documentation of rapid disassembly of the actin, MT, and mechanical properties (29). vimentin cytoskeletons and remodeling of nuclear lamins after Our study shows that the plasma membrane undergoes dramatic stimulation of NETosis is reminiscent of the cytoskeletal re- changes during NETosis, including shedding of microvesicles and modeling at mitosis (35–37) and further supports a role for mi- regulated permeabilization. Microvesicles could act as important totic kinases in NETosis (21). However, NETosis stimulants also systemic messengers, signaling stress (30) or contributing to disease trigger intracellular calcium influx (9, 38, 39), and it is well known (31), including the promotion of thrombosis (32). The progressive that calcium can directly or indirectly promote the disassembly of increase in size of molecules entering the cell suggests a stepwise MTs (40), actin (41), and vimentin filaments (42), suggesting an

Thiam et al. PNAS Latest Articles | 9of12 Downloaded by guest on September 23, 2021 A dHL60; Pad4 immunostaining B Hm PMN; Pad4 immunostaining Phase C. α-Pad4; 1 DNA α-Pad4;1/DNA DIC α-Pad4; 1 DNA α-Pad4;1/DNA /α-Pad4;1/DNA /α-Pad4;1/DNA

Phase C. α-Pad4; 2 DNA α-Pad4;2/DNA DIC α-Pad4; 2 DNA α-Pad4;2/DNA /α-Pad4;2/DNA /α-Pad4;2/DNA Fig. 7. PAD4 localizes to the nucleus and enters the cytosol prior to nuclear rupture. dHL-60 (A, D, E,andF)orhuman(Hm)PMN D dHL60 + 4 μM Ionomycin (B and C)fixed(A and B)orstimulatedwith -14 min0 min 18 min 25 min 80 min C Hm PMN; ionomycin and imaged live by DIC and con- DIC Pad4 immunostaining focal microscopy (D)atthecoverslip–cell in- n= 149 31 20 terface (Z = 0 μm) and in the cell center (Z = + μ 15 3 m) at 2-min intervals for 4 h, time relative to MV shedding noted. (A and B)Phase- 10 Pad4-mEmerald contrast (A, phase C), or DIC (B) and confo- 5 cal images of DAPI staining of DNA (blue) and 0 immunolocalization of PAD4 by two differ- +/- +/-

Pad4 Intensity Ratio 5 3 3 2 Nuclear/Cytosolic Mean ent antibodies (α-Pad4; 1 [red] and 2 [green]). Pad4 / NLS-mCherry (C) Mean nuclear to cytosolic ratio of PAD4 Pad4; 1 Pad4; 2 intensity from immumostaining. n = total cells observed, points = individual cells, mean Pad4 / DNA (long bar) and SD (short bar) below each plot. (D) Time series of a dHL-60 coexpressing PAD4-mEmerald (Middle and Bottom,green), Z = 0 μm Z = + 3 μm and NLS-mCherry (third row, red) and SiR-DNA (Bottom row, blue). Double-arrow-headed E dHL60; Nuclear Pad4 and NLS F dHL60; Cytosolic Pad4 and NLS PM PM Normalized Mean Cytosolic lines indicate presence of ionomycin, boxes

Permeabilization Normalized Mean Nuclear Permeabilization NLS Intensity (AU/ μ m2) NLS Intensity (AU/ μ m2) 1.0 1.0 1.0 1.0 around images indicate cellular events (green: 0.8 0.8 0.8 0.8 MV shedding; blue: DNA release to the cyto- 0.6 0.6 0.6 0.6 sol). (E and F) Normalized mean nuclear (E) 0.4 0.4 0.4 0.4 and cytosolic (F) intensity over time of PAD4- 0.2 0.2 0.2 0.2 mEmerald and NLS-mCherry measured from 0.0 0.0 0.0 0.0 the cell in (D). Loss of DIC contrast (PM per-

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Pad4 Intensity (AU/ μ m2) Pad4 Intensity (AU/ μ m2) 3 2 1 1 2 3 4 5 6 7 8 meabilization) noted. (Scale bars in A, B,and Normalized Mean Nuclear 3 2 1 1 2 3 4 5 6 7 8 - - -

- - - Normalized Mean Cytosolic Time Relative to DNA Release (min) Time Relative to DNA Release (min) D:5μm.)

important role for calcium in progression through NETosis. Our 30 °C in YPD media with 1% P/S. Cells were resuspended in imaging media finding that actin disassembly was required for plasma membrane (RPMI 1640 lacking phenol red with 25 mM Hepes and 1% antibiotics) before rupture and extracellular NET release indicates that molecules imaging. For more details, see SI Appendix, Materials and Methods. mediating actin disassembly could serve as therapeutic targets for NETosis inhibition. cDNA Expression Vectors. cDNAs encoding CaaX-mApple, H1-mEmerald, ER-5- Our systematic analysis of the role of PAD4 in the cellular mEmerald, Lamin B1-mApple, Lamin A-mEmerald, F-tractin-mApple, G-actin- events of NETosis utilized genetically modified mouse PMN and mEmerald, Tubulin-mEmerald, Vimentin-mEmerald, H2B-mEmerald, and dHL-60 human cells, precluding the lack of specificity or re- mEmeral-C1 were the kind gift of the late Mike Davidson, Florida State versibility of PAD4 inhibitors (14, 25). We demonstrate that University, Tallahassee, FL; eGFP-Ensconsin (3X-GFP-EMTB) was a kind gift PAD4 is critical for efficient DNA decondensation, lamin mesh- from Chloe Bulinski, Columbia University, New York, NY; pSpCas9-2A-GFP work and NE rupture and extracellular DNA release in mouse vector was a kind gift from James Anderson’s laboratory, NHLBI/NIH, PMNs and dHL-60 cells. However, we found that PAD4 plays a Bethesda, MD. Human PADi4 mRNA (pCMV3-N-His-PADi4, HG11072-NH) was more prominent role in efficient DNA decondensation in mouse inserted into a mEmerald-C1 vector and the enzymatically dead (mEmerald- PMN than in dHL-60 cells. This could be attributed to the re- PAD4-C645A) and the NLS dead (mEmerald-PAD4-K59A/K60A/K61A) mu- sidual (∼5%) PAD4 in the gene-edited HL-60 cell line, or pro- tants were generated by in vitro site-directed mutagenesis. For more details, teases may be more important for DNA decondensation in human see SI Appendix, Materials and Methods. than in mouse cells (11). We also show that both the citrullination activity and nuclear localization signal of PAD4 are required for Generation of Gene-Edited HL-60 PAD4 Crispr Cell Line. The generation of the NETosis, strongly supporting the notion that PAD4 enzymatic PAD4 Crispr line was performed as described (45) using the guide RNA activity in the nucleus, potentially through histone citrullination Guide1: 3′→5′ TC ACA CGG ATC AAT GTC CCC TGG. For more details, see SI (10), mediates NETosis. Finally, since PAD4 depletion delays the Appendix, Materials and Methods. disassembly of lamins and vimentin, both of which are known to maintain nuclear mechanical integrity (43, 44), it is possible that Immunofluorescence. Cells in imaging media were incubated on cleaned citrullination may regulate intermediate filament disassembly to coverslips for 5 min at 37 °C and 5% CO2 before the addition of DMSO or promote nuclear rupture during NETosis. ionomycin (4 μM); further incubation and then indirect immunofluorescence were performed. For more details, see SI Appendix, Materials and Methods. Materials and Methods dHL-60 NETosis Endpoint Assay. NETosis endpoint assay was performed as al- Cells. HL-60 cells (ATCC CCL-240) were cultured at 37 °C and 5% CO2 in RPMI 1640 plus L-glutamine with 25 mM Hepes, 1% penicillin and streptomycin ready described (22). For more details, see SI Appendix, Materials and Methods. (P/S), and 15% heat-inactivated FBS and differentiated to neutrophil-like cells by addition of 1.3% of DMSO. Human and mouse neutrophils were Western Blot. Western blots were performed as previously described (46). For isolated from fresh blood on Percoll gradients. C. albicans were cultured at more details, see SI Appendix, Materials and Methods.

10 of 12 | www.pnas.org/cgi/doi/10.1073/pnas.1909546117 Thiam et al. Downloaded by guest on September 23, 2021 ABWT dHL60 + 4 μM Ionomycin PAD4 CR dHL60 + 4 μM Ionomycin -2 min 0 min 2 min 43 min 86 min -2 min 0 min 13 min 140 min 235 min

Z = 0 μm Z = 0 μm SiR-DNA SiR-DNA DIC / DIC / Z = + 3 μm Z = + 3 μm CDPAD4 CR dHL60 + mEmerald-Pad4 + 4 μM Ionomycin PAD4 CR dHL60+ mEmerald-C1 + 4 μM Ionomycin -6 min0 min 2 min 20 min 112 min -4 min0 min 16 min 140 min 232 min Fig. 8. PAD4 enzymatic and nuclear localization activities mediate efficient DNA decondensation, NE rupture and extracel- Z = 0 μm Z = 0 μm SiR-DNA SiR-DNA lular DNA expulsion in human dHL-60. dHL-60 (WT; A, G, H, J,andL)orPAD4CR DIC / DIC / – Z = + 3 μm Z = + 3 μm dHL-60 (B G, I, K,andL), stained with SiR- Pad4 / DNA mEmerald-C1 / DNA DNA, stimulated with ionomycin, and imaged by DIC and confocal microscopy at Z = + 3 μm Z = 0 μm Z = + 3 μm the coverslip–cell interface (Z = 0 μm) and PAD4 CR dHL60 + mEmerald-Pad4-C645A PAD4 CR dHL60 + mEmerald-Pad4- in the cell center (Z =+3 μm) at 2-min in- EF+ 4 μM Ionomycin K59A/K60A/K61A + 4 μM Ionomycin tervals for 4 h, time in min relative to MV -8 min 0 min 16 min 140 min 232 min -6 min 0 min 16 min 140 min 234 min shedding noted on images. Double-arrow- headed lines indicate presence of ionomycin, colored boxes around images cor- Z = 0 μm Z = 0 μm SiR-DNA

SiR-DNA respondtocolor-codedcellulareventsin the graphs. (A–F and H–K)Timeseriesof DIC / DIC / Z = + 3 μm Z = + 3 μm image overlays of DIC (grayscale) and Pad4-C645A / DNA Pad4-K59/60/61A / DNA SiR-DNA (red). (C–F) PAD4 CR dHL-60 cells expressing mEmerald-PAD4 (C; Bot- Z = + 3 μm Z = 0 μm Z = + 3 μm tom row, green), mEmerald (D; Bottom WT dHL60 (n=186) Pad4 dHL60 CR (+ Em-Pad4; n=163) row, green), mEmerald-PAD4-C645A (E; Bot- G Pad4 dHL60 CR (n=179) Pad4 dHL60 CR (+ Em-Pad4-C645A; n=110) n= 152 98 118 141 89 110 129 29 48 119 64 47 95 10 28 79 15 37 Pad4 dHL60 CR (+ Em-C1; n=172) Pad4 dHL60 CR (+ Em-Pad4-K59A/K60A/K61A; n=132) ns tom row, green) or mEmerald-PAD4-K59A/

120 240 **** **** K60A/K61A (F; Bottom row, green). (G, Left) IMMUNOLOGY AND INFLAMMATION 200 100 160 Percent of cells exhibiting MV shedding, 80 120 DNA decondensation, nuclear rounding, 60 80 40 loss of DIC contrast (PM permeabiliza- % of Cells 40 0 tion), DNA release to the cytoplasm, and 20 relative to Time 9 31 49 14 43 41 59 64 59 49 60 64 100 105 132 89 136 130

MV Shedding (min) -40 +/- +/- +/- +/- +/- +/- +/- +/- +/- +/- +/- +/- +/- +/- +/- +/- +/- +/- extracellular DNA release (NETosis); (G, -80 28 45 54 23 51 53 47 40 51 38 49 47 57 44 72 55 52 54 DNA DNA Release NETosis Right) Timing relative to MV shedding of NETosis Decondensation DNA decondensation, DNA release to the MV Shedding DNA release Nuclear Rounding DNA Decondensation PM Permeabilization cytoplasm, and NETosis after ionomycin WT dHL60 + mEmerald_Lap2B PAD4 CR dHL60 + mEmerald_Lap2B stimulation. n = total cells observed, HI+ 4 μM Ionomycin + 4 μM Ionomycin L point = percent of cells in one experiment n= 109 57 135 71 -4 min 0 min 22 min 216 min -12 min 0 min 60 min 226 min 150 ** **** DIC

DIC (Left), or individual cells (Right). Long 100 bar = mean, short bars = SD shown below – 50 each plot (Right). (H K) WT or PAD4 CR / Lap2B Z = 0 μm / Lap2B Z = 0 μm DNA DNA dHL-60 cells expressing Lap2β-mEmerald 0 32 43 21 37

Time relative to Time (H and I; Bottom rows, green) or lamin +/-24 +/-29 +/-20 +/-27

MV Shedding (min) -50 Z = + 3 μm Z = + 3 μm B1-mEmerald (J and K; Bottom rows, WT dHL60 + mEmerald_Lamin B1 PAD4 CR dHL60 + mEmerald_Lamin B1 WT WT green). (L) Timing relative to MV shed- JK+ 4 μM Ionomycin + 4 μM Ionomycin Pad4 CR Pad4 CR ding of the Lap2β or laminB1 rupture. n = -4 min0 min 24 min 84 min -2 min 0 min 56 min 228 min Lap2B Lamin B1 DIC DIC Rupture Rupture total cells observed, points = individual cells, mean (long bar) and SD (short bar) shown below each plot. (G and L) ****P < Z = 0 μm / Lamin B1 Z = 0 μm / Lamin B1 DNA DNA 0.0001; **P < 0.01; ns, nonsignificant; (G Left and L): Mann–Whitney U test. (Scale bars in A–F and H–K:10μm.)

Nitroblue Tetrazolium Assay for Reactive Oxygen Species Production. The confocal microscope, or a Zeiss Axiovert 200M wide-field fluores- nitroblue tetrazolium (NBT) assay was performed as described (27). For more cence microscope. For live cell imaging, microscopes were equipped details, see SI Appendix, Materials and Methods. with stage-top incubators. For more details, see SI Appendix, Materials and Methods. RT-PCR for PADi4 mRNA Detection. RNA from WT and PAD4 CR dHL-60 cells was extracted and RT-PCR was performed using Gapdh as loading control. Image Analysis. Image analysis was performed from DIC and fluorescence The following primers were used: PAD4 P1 Fw: 5′-ACT TCT TCA CAA ACC ATA overlay time-lapse movies. Single cells were followed over time to assess the CAC TGG-3′; PAD4 P1 Rev: 5′-CCT CGA GTT ACA TAG CCA AAA TCT-3′; PAD4 percentage and timing of events. A detailed description of the quantification P2 Fw: 5′-GTG TTC CAA GAC AGC GTG GT-3′; PAD4 P2 Rev: 5′-GTT TGA TGG procedure can be found in SI Appendix, Materials and Methods. GAA ACT CCT TCA G-3′; and GAPDH Fw: 5′-ACC CAG AAG ACT GTG GAT GG- 3′; GAPDH Rev: 5′-CCC CTC TTC AAG GGG TCT AC-3′. For more details, see SI Statistical Analysis. Details on statistical tests can be found in SI Appendix, Appendix, Materials and Methods. Materials and Methods.

Microscopy. Imaging was performed on a Nikon Eclipse Ti or Ti2 equipped Material and Data Availability. Raw and processed data (Movies, Excel, and with a Yokogawa CSU-X1 or CSU-W1 spinning disk scanhead, a Nikon A1R GraphPad files), cell lines, and cDNA expression vectors are available upon resonance-scanning confocal microscope, a Zeiss LSM 510 laser scanning request to the corresponding author.

Thiam et al. PNAS Latest Articles | 11 of 12 Downloaded by guest on September 23, 2021 ACKNOWLEDGMENTS. We thank the Marine Biological Laboratory Whitman facilities; and the NHLBI blood bank. This work was funded by the NHLBI Fellows program (D.D.W.); Nikon Instruments for loan of equipment Division of Intramural Research (C.M.W. and H.R.T.); NIH R01 GM106023 and (C.M.W.); the Nikon Fellows program (D.D.W.); Schwanna Thacker, William NIH P01 GM096971 (R.D.G., A.E.G., R.Q., M.K., and A.V.); NIH T32 Shin, Nihal Altan-Bonnet, Robert Fischer, and Ana Pasapera; the National Heart, CA08062160038140 (M.K. and A.V.); and NIH R35 HL135765 (D.D.W. and Lung, and Blood Institute (NHLBI) flow cytometry, light microscopy, and animal S.L.W.).

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