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Annual Review of Cell and Developmental Biology

Cellular Mechanisms of
NETosis
Hawa Racine Thiam,1 Siu Ling Wong,2
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Denisa D. Wagner,3,4,5 and Clare M. Waterman1

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1
Cell and Developmental Biology Center, National Heart, Lung, and Blood Institute (NHLBI),
National Institutes of Health, Bethesda, Maryland, USA; email: [email protected],
[email protected]
2
Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore 308232
3
Program in Cellular and Molecular Medicine, Boston Children’s Hospital, Boston,
Massachusetts 02115, USA
4
Department of Pediatrics, Harvard Medical School, Boston, Massachusetts 02115, USA
5
Division of Hematology/Oncology, Boston Children’s Hospital, Boston, Massachusetts 02125,
USA

Annu. Rev. Cell Dev. Biol. 2020. 36:6.1–6.28 Keywords

The Annual Review of Cell and Developmental Biology
neutrophil, cell biology, nucleus, PAD4, innate immunity, cytoskeleton,
is online at cellbio.annualreviews.org
neutrophil extracellular trap
https://doi.org/10.1146/annurev-cellbio-020520-
111016 Abstract
Copyright © 2020 by Annual Reviews.
Neutrophils are critical to innate immunity, including host defense against
All rights reserved
bacterial and fungal infections. They achieve their host defense role by
phagocytosing pathogens, secreting their granules full of cytotoxic enzymes,
or expelling neutrophil extracellular traps (NETs) during the process of
NETosis. NETs are weblike DNA structures decorated with histones and
antimicrobial proteins released by activated neutrophils. Initially described
as a means for neutrophils to neutralize pathogens, NET release also occurs
in sterile inflammation, promotes thrombosis, and can mediate tissue dam-
age. To effectively manipulate this double-edged sword to fight a particular
disease, researchers must work toward understanding the mechanisms driv-
ing NETosis. Such understanding would allow the generation of new drugs
to promote or prevent NETosis as needed. While knowledge regarding the
(patho)physiological roles of NETosis is accumulating, little is known about
the cellular and biophysical bases of this process. In this review, we describe
and discuss our current knowledge of the molecular, cellular, and biophysical
mechanisms mediating NET release as well as open questions in the field.

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Contents
1. INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2
1.1. Neutrophil Extracellular Traps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3
1.2. NETosis Is a Double-Edged Sword . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3
2. NETOSIS INITIATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3
2.1. Neutrophil Activation Is a Prerequisite for NETosis . . . . . . . . . . . . . . . . . . . . . . . . . 6.4
2.2. Calcium Spikes in NETosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4
2.3. The Role of Kinases in NETosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.5
2.4. NADPH Oxidase: The Role of Reactive Oxygen Species in NETosis . . . . . . . . 6.5
2.5. Cell Morphodynamics on Initiation of NETosis: Increased
Cell–Extracellular Matrix Adhesion and Shedding of Plasma
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Membrane Microvesicles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.5

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2.6. NETosis Initiation: Open Questions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.6

3. MECHANISM OF DNA DECONDENSATION DURING NETOSIS . . . . . . . . . 6.6
3.1. PAD4, Histone Citrullination, Chromatin Decondensation, and NETosis . . . . 6.8
3.2. The Role of Proteases in Chromatin Decondensation During NETosis . . . . . . 6.11
3.3. The Role of Myeloperoxidase in Chromatin Decondensation
and NETosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.13
4. DNA RELEASE FROM THE NUCLEUS TO THE CYTOSOL . . . . . . . . . . . . . . . 6.13
4.1. Lamin Dynamics During NETosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.13
4.2. Nuclear Membrane Dynamics During NETosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.14
5. REMODELING OF CYTOSKELETAL AND MEMBRANOUS
ORGANELLES DURING NETOSIS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.15
5.1. Cytoskeletal Disassembly During NETosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.16
5.2. Membranous Organelles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.18
6. EXTRACELLULAR DNA RELEASE: BREACHING
THE PLASMA MEMBRANE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.19
6.1. Increase in Permeability of the Plasma Membrane During NETosis . . . . . . . . . . 6.19
6.2. Plasma Membrane Rupture Driven by Chromatin Swelling . . . . . . . . . . . . . . . . . . 6.20
7. CONCLUSION AND PERSPECTIVES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.20

1. INTRODUCTION
Neutrophils are crucial to host defense. For instance, patients with a low number of neutrophils
have recurrent bacterial and fungal infections and defects in clearing such infections (Dale &
Hammond 1988). Differentiated in the bone marrow from the granulocyte–monocyte progen-
itor cells (Akashi et al. 2000, Görgens et al. 2013), mature neutrophils are characterized by the
presence of granules in their cytosol and a multilobulated nucleus (Campbell et al. 1995). Neu-
trophils are released into the circulation, where they can be recruited to sites of both sterile and
pathogen-induced injury by activated endothelial cells, resident immune cells, and injured epithe-
lial cells (de Oliveira et al. 2016). Once they reach the site of injury, engagement of neutrophil
surface receptors with proinflammatory signals, such as bacterial components, fungal β-glucan,
or cytokines, induces a signaling cascade resulting in enhanced neutrophil effector functions (i.e.,
activation) (Mayadas et al. 2014). Activated neutrophils can neutralize invaders by releasing their
granules by degranulation, internalizing and degrading pathogens by phagocytosis, and releasing

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neutrophil extracellular traps The mechanisms of neutrophil differentiation, migration to injury

sites, degranulation, and phagocytosis have been extensively reviewed (de Oliveira et al. 2016,
Hidalgo et al. 2019, Ley et al. 2018). Here we focus on the more recently characterized process of
the release of NETs.

1.1. Neutrophil Extracellular Traps

NETs are made of decondensed chromatin that forms weblike DNA structures with approximately
30-nm pores (Pires et al. 2016). They are coated with nuclear proteins including histones, granule
proteins (such as neutrophil elastase and myeloperoxidase), and cytosolic proteins (such as S100
calcium-binding proteins A8, A9, and A12, as well as actin and α-actinin) (Chapman et al. 2019,
Petretto et al. 2019, Urban et al. 2009). In addition to neutrophils, eosinophils (Yousefi et al. 2008),
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basophils (Morshed et al. 2014), mast cells (von Köckritz-Blickwede et al. 2008), and macrophages
(Chow et al. 2010) have all been observed to release extracellular DNA upon stimulation. Initially
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described as a programmed cell death pathway distinct from apoptosis and necrosis, the release
of extracellular traps is also thought to occur without cell lysis in a process termed vital NETosis
(Adrover et al. 2020, Clark et al. 2007, Pilsczek et al. 2010, Yipp & Kubes 2013, Yipp et al.
2012).

1.2. NETosis Is a Double-Edged Sword

The seminal work of Brinkmann and colleagues (2004) first reported the ability of activated
neutrophils to release nuclear DNA into the extracellular environment, where it can trap and
neutralize pathogens in a process termed NETosis. The ability of various pathogens to induce
NETosis (Abi Abdallah et al. 2012, Brinkmann et al. 2004, Saitoh et al. 2012, Urban et al. 2006),
together with the demonstration that NETs can trap and kill bacteria (Berends et al. 2010,
Brinkmann et al. 2004, Lappann et al. 2013, Pilsczek et al. 2010) and fungi (Urban et al. 2006)
as well as limit HIV-1 virus infectivity (Saitoh et al. 2012), suggests that NETosis could be
important for innate immunity. However, NETs also form in sterile inflammation (Sorvillo et al.
2019, Wong & Wagner 2018). Moreover, the ability of the cytotoxic proteins associated with
NETs to damage host cells (Clark et al. 2007) and activate platelets (Fuchs et al. 2010), together
with the presence of autoantibodies against proteins released during NETosis in patients with
autoimmune diseases (Hakkim et al. 2010, Jorch & Kubes 2017), illustrates the double-edged
sword effect of NETosis (Thanabalasuriar et al. 2019).
While the (patho)physiological relevance of NETosis has been demonstrated, the cellular
mechanisms of NETosis have just begun to be revealed. For decondensed DNA decorated with
nuclear, granular, and cytosolic contents to be released extracellularly, the process has to be ini-
tiated either extracellularly or intracellularly; the chromatin has to be decondensed and released
from the nucleus; and the cytoskeleton, organelles, and intracellular and nuclear as well as plasma
membranes must be remodeled. In the following, we review our current knowledge of these cel-
lular steps.

2. NETOSIS INITIATION
While the primary signal that initiates NETosis instead of other neutrophil responses remains
unknown, NET release starts with the activation of surface receptors followed by changes in in-
tracellular calcium concentration, activation of kinase signaling cascades, and production of re-

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active oxygen species (ROS). These first signaling events translate into morphological changes,

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particularly an increase in cell spreading and changes in cell shape. In the following, we discuss
these early signaling and cellular events.

2.1. Neutrophil Activation Is a Prerequisite for NETosis

NETosis requires neutrophil activation. Therefore, resting neutrophils in noninflammatory con-
ditions do not undergo NETosis. Moreover, neutrophils of mice deficient in Toll-like receptor 2
(TLR2) or complement component 3 and, more generally, mice defective in Interleukin-1 (IL-1)-
receptor/TLR signaling [MyD88 knockout (KO)] do not release NETs upon Staphylococcus aureus
stimulation (Yipp et al. 2012). Ligands of G-protein-coupled receptors (GPCRs) (Gupta et al.
2014, Rossaint et al. 2014) tumor necrosis factor (TNF) (Keshari et al. 2012) and Fc (Rossaint
et al. 2014) receptors have been reported to induce NETosis, suggesting that these receptors
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might be involved in NETosis initiation. Although CD18 (β2 integrin) is dispensable for NETosis
induced by calcium influx (Wong et al. 2015), phorbol 12-myristate 13-acetate (PMA), saliva
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mucin (Mohanty et al. 2015), or exogenous H2 O2 (Raftery et al. 2014), it is required for activated-
platelet- (McDonald et al. 2012, Rossaint et al. 2014), S. aureus– (Mohanty et al. 2015), and
hantavirus-induced NET release (Raftery et al. 2014). In addition to plasma membrane surface
receptors, NETosis can also be triggered by activation of the nucleotide oligomerization domain
(NOD)-like receptor protein 3 (P. Munzer, R. Negro, S. Fukui, L. di Meglio, C. Cherpokova,
et al., unpublished manuscript). Bacterial toxins such as ionomycin (Liu & Hermann 1978, Wang
et al. 2009) and nigericin (Kenny et al. 2017, Lardy et al. 1958), as well as reactive oxygen species
(ROS) (Fuchs et al. 2007), can induce NETosis, presumably without activating surface receptors.
Thus, neutrophil activation, via either bacterial toxins or surface receptor engagement, initiates
NETosis.

2.2. Calcium Spikes in NETosis

Neutrophil activation leads to an increase in the intracellular calcium concentration (Krause et al.
1990). For instance, ligand binding on GPCRs, Fcγ receptors, TLR4, and complement recep-
tors (β2 integrin) triggers a release of endoplasmic reticulum (ER)-stored calcium followed by
the opening of plasma membrane calcium channels (Dixit & Simon 2012, Gennaro et al. 1984,
Hellberg et al. 1996, Immler et al. 2018, Kandasamy et al. 2013, Schappe et al. 2018, Schorr
et al. 1999). In the context of NETosis, an increase in intracellular calcium was observed in neu-
trophils stimulated by lipopolysaccharides (LPSs), IL-8, and PMA, and treatment with the calcium
ionophores ionomycin and A23187 (de Bont et al. 2018, Gupta et al. 2014) leads to NET release
(Wang et al. 2009). More importantly, extracellular calcium chelation inhibits IL-8-, PMA-, Pseu-
domonas aeruginosa–, ionomycin-, and nigericin-stimulated NETosis (Gupta et al. 2014, Kenny
et al. 2017, Parker et al. 2012), while intracellular calcium chelation impairs IL-8-, PMA-, and
nigericin-induced but not Candida albicans– or Group B Streptococcus–induced NETosis (Kenny
et al. 2017, van der Linden et al. 2017). Thus, an increase in intracellular calcium, via either re-
lease of intracellular stores or influx from the extracellular environment, is important for NETosis.
However, the cellular processes requiring calcium during NETosis are poorly understood. One
pathway known to be mediated by calcium is via peptidyl arginine deiminase 4 (PAD4), an enzyme
critical for NETosis (Li et al. 2010, Wong & Wagner 2018) that requires a high concentration of
calcium for its activation (Kearney et al. 2005). Thus, further identification of the mechanisms
of calcium increase and the cellular targets of calcium during NETosis will be critical for under-
standing how calcium regulates NET release.

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2.3. The Role of Kinases in NETosis

Both kinases activated downstream of calcium influx or cytokine engagement and regulators of
the cell cycle have been implicated in NETosis. The phospholipid-dependent, phorbol ester- and
calcium-activated key cell cycle regulator kinase, protein kinase C (PKC), in particular PKCα,
PKCβ1, and PKCζ, mediates PMA-, ionomycin-, IL-8-, platelet-activating factor–, C. albicans–,
and Group B Streptococcus–induced NETosis (Gupta et al. 2014, Hakkim et al. 2011, Kenny et al.
2017, Radic & Neeli 2013). Cyclin-dependent kinase 6, which regulates the G1 /S transition of the
cell cycle, and the Raf-MEK-ERK MAP kinase pathway are critical to PMA-induced NETosis
(Amulic et al. 2017), while the SYK-PI3K-mTorc2 pathway mediates monosodium urate crystal–
and S. aureus–induced NETosis (van der Linden et al. 2017). Finally, the nonreceptor tyrosine ki-
nase Janus kinase 2 ( JAK2), which transduces cytokine-mediated signaling and controls cell pro-
liferation, has recently been implicated in NETosis. Indeed, an activating mutation in JAK2 that
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causes cancer also enhances the propensity for NETosis (Wolach et al. 2018). However, similar to
calcium, the requirement of PKC, the Raf-MEK-ERK and SYK-PI3K-mTorc2 pathways depend
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on the NETosis stimulant (Hakkim et al. 2011, van der Linden et al. 2017). Thus, downstream of
receptor activation and/or calcium influx, several kinases implicated in cell cycle regulation medi-
ate NETosis. Determination of their downstream effectors will be crucial for designing drugs for
NETosis.

2.4. NADPH Oxidase: The Role of Reactive Oxygen Species in NETosis

Although the production of ROS has traditionally been regarded as a direct mechanism of killing
pathogens via oxidative damage (Nguyen et al. 2017), ROS are also central to the signaling impli-
cated in NETosis. The two main sources of ROS in neutrophils are NADPH oxidase and mito-
chondria. Stimulation of neutrophils with PMA, A23187, C. albicans, S. aureus, or Group B Strepto-
coccus leads to a rapid (within 20 min) increase in ROS production. Treatment of neutrophils with
ROS scavengers inhibits PMA- and C. albicans–induced NETosis (Fuchs et al. 2007, Kenny et al.
2017). Importantly, neutrophils from chronic granulomatous disease patients that lack functional
NADPH oxidase fail to undergo NETosis upon S. aureus or PMA stimulation (Bianchi et al. 2009).
However, NADPH oxidase–mediated ROS are not required for NETosis stimulated by Leishmania
donovani (Gabriel et al. 2010), C. albicans (Byrd et al. 2013), Paracoccidioides brasiliensis (Mejía et al.
2015), soluble immune complexes (Chen et al. 2012), A23187, the potassium ionophore nigericin,
Group B Streptococcus (Kenny et al. 2017), S. aureus, or monosodium urate crystals (van der Linden
et al. 2017). Thus, similar to the requirement for calcium and specific kinases, the importance of
ROS and NADPH oxidase in NETosis seems to depend on the stimulus. The exact mechanism
by which NADPH oxidase drives NET release remains unclear.

2.5. Cell Morphodynamics on Initiation of NETosis: Increased

Cell–Extracellular Matrix Adhesion and Shedding of Plasma
Membrane Microvesicles
The initiation of NETosis is accompanied by profound cellular rearrangements. Upon stimu-
lation of NETosis in vitro, neutrophils spread on substrates before shedding plasma membrane
microvesicles and rounding up (Neubert et al. 2018, Thiam et al. 2020). The initial increase in
cell spreading suggests activation of cell surface extracellular matrix (ECM) receptors such as
integrins. While integrin activation occurs during neutrophil activation and promotes NETo-
sis, depending on the stimulus (Byrd et al. 2013, 2016; McDonald et al. 2012; Mohanty et al.

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2015; Rossaint et al. 2014), neutrophils deposited on surfaces lacking integrin ligands still undergo
NETosis upon PMA stimulation (Neubert et al. 2018). This suggests that cell spreading and in-
tegrin engagement might not be required for NETosis per se.
After spreading, NETosis in adherent cells proceeds by shedding of annexin V–positive plasma
membrane microvesicles (Figure 1) containing cytosolic components including granules (Thiam
et al. 2020). In addition to microvesicle shedding, neutrophils also round up after their initial
spreading (Neubert et al. 2018), similar to adherent cells undergoing mitosis. While plasma mem-
brane microvesicle shedding and rounding up could lead to a decrease in effective cell surface
area, whether these morphological changes are required for NETosis progression remains un-
clear. Nevertheless, the microvesicles that are shed at NETosis onset are of great interest: They
could be messengers that induce systemic effects, such as promoting thrombosis (Hrachovinová
et al. 2003). In addition, annexin V–positive microvesicles could be subjected to the same fate as
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annexin-positive apoptotic cells and be internalized by macrophages without inducing inflamma-

tion (Gordon & Plüddemann 2018). It is also possible that granule proteins in these microvesi-
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cles could exert toxic effects on both pathogens and host cells. Neutrophil microvesicles have
been shown to limit bacterial growth (Timár et al. 2013), induce cytokine secretion from en-
dothelial cells (Mesri & Altieri 1998), activate platelets (Pluskota et al. 2008), and downmodulate
macrophage activation (Gasser et al. 2003), although whether such neutrophil-derived vesicles
arise via NETosis is unclear. However, it is tempting to speculate that microvesicle shedding at
NETosis onset may participate in the bactericidal function of NETosis while dampening inflam-
mation. Further studies will be required to reveal the roles of microvesicles formed in NETosis
under infection and sterile inflammation and to shed light on the molecular mechanisms under-
lying their release.

2.6. NETosis Initiation: Open Questions

It remains unclear why activation of the same surface receptors that trigger phagocytosis or de-
granulation also induces NETosis. The size of the pathogen has been proposed as a key deter-
minant in NETosis decision-making: When the pathogen is too large to engulf, neutrophils re-
lease NETs (Branzk et al. 2014). However, a multitude of small extracellular or even intracellular
pathogens, including viruses, can also induce NETosis (Delgado-Rizo et al. 2017). Indeed, C. al-
bicans can trigger NETosis after it has been phagocytosed by a neutrophil (Thiam et al. 2020).
It has recently been proposed that the higher granule count in younger neutrophils makes them
more prone to NETosis than their older counterparts that have fewer granules (Adrover et al.
2020), suggesting that the number of granules might determine neutrophils’ susceptibility to un-
dergoing NETosis. However, this does not agree with the observation that aged neutrophils in
the circulation are more prone to form NETs (Zhang et al. 2015). Moreover, low-density granu-
locytes found in patients with systemic lupus erythematosus (SLE) or psoriasis are characterized
by their low granule content but spontaneously undergo NETosis (Lood et al. 2016). Thus, the
number of granules might not be a key factor for NETosis decision-making. Investigations into
what determines when NETosis occurs in response to stimulation, whether the decision depends
on the concentration of the stimulus or the activation status of the cells, would provide insights
that would allow us to better define the physiological significance of NETosis.

3. MECHANISM OF DNA DECONDENSATION DURING NETOSIS

Chromatin decondensation is the main defining feature of NETosis compared with other
cell death processes such as apoptosis, necrosis, or pyroptosis, in which chromatin either is

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not changed or becomes condensed (de Vasconcelos et al. 2019, Goldmann & Medina 2013).

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Stimulation
Initial state Time

1 Actin disassembly

F-actin ΔT ≈ 2 min

2 PM microvesicle shedding

DIC/
ΔT ≈ 1 min
annexin V
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3 Vimentin remodeling, MT disassembly, ER vesiculation

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Vimentin

ΔT ≈ 2–11 min
MT

ER

4 Chromatin decondensation

Histone (H1) ΔT ≈ 5–50 min

5 DNA release into the cytosol

Lamin B1/
DNA
ΔT ≈ 2–77 min

ER/DNA

6 PM permeabilization

Extracellular
dextran ΔT ≈ 4–46 min
10 kDa

7 PM rupture/NET release

PM/H2B

(Caption appears on following page)

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Figure 1 (Figure appears on preceding page)

NETosis proceeds via a well-defined sequence of cellular events. Representative images are shown of living neutrophil-like HL60 cells
expressing fluorescent protein–tagged cytoskeletal (actin, MT, and vimentin), nuclear [histones (H1 and H2B) and lamin B1], and
membranous (ER and PM) marker probes, before (initial state) and after NETosis stimulation. Fluorescently tagged annexin V and
10-kDa dextran were added to the imaging media before NETosis stimulation. DNA was stained with SiR-DNA. (● 1 ) Actin

disassembly. The fluorescent intensity of the F-actin probe (F-tractin-mApple) decreases. (● 2 ) PM microvesicle shedding. Shows the

appearance of annexin V–positive microvesicles (green meaning exposed phosphatidylserine) while the cell body remains annexin V
negative. (●3 ) Vimentin remodeling, MT disassembly, and ER vesiculation. Illustrates the solubilization of the vimentin intermediate

filament probe (vimentin eGFP) and then the MT probe (ensconsin MT-binding domain fused to eGFP). Finally, the tubular ER
network is lost and vesiculates, as assessed by the ER probe (ER-5-mEmerald: KDEL sequence combined with the ER-retention signal
sequence from calreticulin fused to mEmerald). (● 4 ) Chromatin decondensation. Histone (H1-mEmerald) and DNA fluorescent signal

heterogeneity decreases. (● 5 ) DNA release into the cytosol. Shows the appearance of discontinuities in the lamin B1 (lamin B1–mApple)

and outer nuclear membrane (visualized with ER-5-mEmerald) from where the DNA is released. (● 6 ) PM permeabilization. The

intracellular fluorescent intensity of membrane-impermeable, 10-kDa dextran from the extracellular media increases. This increase
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corresponds with a contrast decrease shown by DIC microscopy at the cell periphery. (● 7 ) PM rupture/NET release. Illustrates the

appearance of discontinuities in the plasma membrane (CAAX-mApple) from where the DNA is released. All scale bars are 5 μm. T
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represents the time between two consecutive events. Abbreviations: DIC, differential interference contrast; eGFP, enhanced green
fluorescent protein; ER, endoplasmic reticulum; MT, microtubule; NET, neutrophil extracellular trap; PM, plasma membrane.

Chromatin decondensation during NETosis is characterized by the loss of chromatin heterogene-

ity (Figure 2; see also Figure 1), suggesting a loss in heterochromatin, as well as by an increase in
the cellular space occupied by DNA. Similar to chromatin decondensation for DNA transcription,
chromatin decondensation in NETosis is thought to be mediated by histone posttranslational
modifications. Although recent evidence suggests the involvement of acetylation (Hamam et al.
2019), the best-characterized histone modification during NETosis are citrullination mediated
by peptidyl arginine deiminase 4 (PAD4) (Wang et al. 2009) and cleavage mediated by serine
proteases (Papayannopoulos et al. 2010). In the following, we discuss these unusual histone
modifications and how they could drive chromatin decondensation and NETosis.

3.1. PAD4, Histone Citrullination, Chromatin Decondensation, and NETosis

PAD4 is currently thought to drive NETosis by citrullinating histones. This induces chromatin
decondensation by decreasing the electrostatic interaction between histone and DNA.

3.1.1. PAD4 and histone citrullination. Citrullination is a posttranslational modification that

converts an arginine to a citrulline, resulting in the loss of a positive charge, a small decrease in
mass (<1 Da), and the release of ammonia (Smith & Denu 2009). PADs constitute a family of
proteins that catalyze citrullination (Thompson & Fast 2006). Among the five types of PADs ex-
pressed in mammals (van Beers et al. 2013), PAD4 is mainly expressed in granulocytes (Nakashima
et al. 1999) and is the only type that contains a conventional nuclear localization signal (Nakashima
et al. 2002). Although PAD4 cytosolic activity has been reported (Sun et al. 2017), direct visual-
ization has shown that PAD4 primarily localizes to the nucleus in resting neutrophils (Nakashima
et al. 2002, Thiam et al. 2020). PAD4 mediates citrullination of the nucleosome histones H3 at
arginines 2, 8, 17, and 26 (Cuthbert et al. 2004) and H4 and H2A at arginine 3 (Hagiwara et al.
2005), as well as the linker histone H1 at arginine 54 (Christophorou et al. 2014). The ability
of PAD4 to citrullinate histone and to modify histone–DNA interactions suggested its role in
transcription regulation (Thompson & Fast 2006). As such, PAD4 was reported to associate with
promoters (Cuthbert et al. 2004), and its expression level correlates with the transcription of sev-
eral key pluripotency genes, including Tcl1 and Nanog in mouse embryonic stem cells (mESCs)
(Christophorou et al. 2014). The ability of PAD4 to citrullinate histones and its specific expres-
sion in granulocytes makes it a perfect candidate for mediating chromatin decondensation during

·.•�-
NETosis.

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Neutrophil
Actin
Chromatin filaments
Nuclear envelope
Lamin meshwork Stimulant

Vimentin intermediate Actin filament

filaments disassembly
MT
ER

Chromatin expansion
into the cytoplasm
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PAD4-, serine protease–,

gasdermin D–,
Annu. Rev. Cell Dev. Biol. 2020.36. Downloaded from www.annualreviews.org

calpain-dependent

Plasma membrane
microvesicle shedding,
ER vesiculation, MT
disassembly, and loss of
peripheral vimentin
filaments

ER vesicles
Plasma membrane
rupture and NET release
PAD4-, serine protease–, MPO-,
CDK6-, NLRP3-, chromatin
swelling–, actin
disassembly–dependent

Nuclear envelope and

lamin meshwork rupture
PAD4-, NLRP3-, lamin
phosphorylation–dependent
Chromatin
decondensation
PAD4-, serine protease–,
calpain-dependent
Nuclear rounding and plasma
membrane and nuclear
envelope permeabilization
Gasdermin D–dependent?

Figure 2
Model of the cellular events driving NETosis and their molecular regulators. Figure adapted from Thiam et al. (2020). Abbreviations:
CDK6, cyclin-dependent kinase 6; ER, endoplasmic reticulum; MPO, myeloperoxidase; MT, microtubule; NET, neutrophil
extracellular trap; NLRP3, nucleotide oligomerization domain (NOD)-like receptor protein 3; PAD4, peptidyl arginine deiminase 4.

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3.1.2. The role of PAD4 in chromatin decondensation and NETosis. The findings of
various studies converge toward PAD4 having a critical role in NETosis (Wong & Wagner 2018).
Neutrophils from PAD4 KO mice do not show citrullinated histone H3, nor do they undergo
NETosis induced by PMA, LPSs, Shigella flexneri (Li et al. 2010), calcium ionophores (Martinod
et al. 2013), methicillin-resistant S. aureus infection (Kolaczkowska et al. 2015), or exposure to
P. aeruginosa biofilms (Thanabalasuriar et al. 2019). PAD4 was also shown to be required for
NETosis during sterile inflammation, such as deep vein thrombosis and cancer (Demers et al.
2012, Martinod et al. 2013), and its expression level is upregulated in chronic diseases such as dia-
betes (Wong et al. 2015). Pharmacological inhibition of PADs abrogates histone citrullination and
diminishes NET release in unopsonized C. albicans or ionomycin-stimulated mouse neutrophils
(Lewis et al. 2015, Wu et al. 2019), nicotine, fMLP, granulocyte–macrophage colony-stimulating
factor (GM-CSF), TNF-α, and PMA-stimulated primary human neutrophils (Hosseinzadeh et al.
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2016, Tatsiy & McDonald 2018), as well as ionomycin-stimulated HL60-derived neutrophils

(Wang et al. 2009). However, the specificity of these inhibitors is subject to question ( Jones et al.
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2012, Muth et al. 2017). More recently, it was shown that neutrophil-like human HL60 cells
with a disrupted PAD4 gene failed to rapidly decondense their chromatin or release NETs upon
ionomycin stimulation (Thiam et al. 2020), indicating that PAD4 may be crucial to NETosis in
humans as well as mice.
Although much evidence implicates PAD4 in NETosis, the idea that it is a universal require-
ment has been challenged. PMA-induced NETosis has been reported to occur without histone H3
citrullination (Kenny et al. 2017, Radic & Neeli 2013). In addition, pharmacological inhibition of
PADs has been reported to have no effect on NETosis upon PMA, A23187, nigericin, C. albicans,
or Group B Streptococcus stimulation (Kenny et al. 2017). And while opsonized C. albicans–induced
NETosis correlates with histone citrullination, it still occurs in PAD4 KO mice (Guiducci et al.
2018). These data suggest that the requirement of PAD4 for NETosis may vary depending on the
NETosis stimulus.

3.1.1.1. How is PAD4 activated during NETosis? PAD4 is a calcium-specific enzyme con-
taining five calcium-binding sites; its activation in vitro requires high calcium concentration
(>100 μM to mM range) at the optimal pH of 7.6 (Kearney et al. 2005, Nakayama-Hamada et al.
2005). However, the concentration of intracellular calcium in activated neutrophils was reported
to be in the low micromolar range (0.7 μM) (Krause et al. 1990), much below the level needed
for in vitro PAD4 activation, suggesting that either NETosis-specific mechanisms for increasing
intracellular calcium or alternative PAD4 activation pathways must exist. ROS were proposed to
activate PAD4 (Neeli et al. 2008); however, direct evidence is lacking. Thus, the understanding of
PAD4 activation during NETosis requires better insight into the cellular mechanisms of calcium
regulation in neutrophils.

3.1.1.2. How does PAD4 mediate chromatin decondensation and NETosis? The current model
in the field is that a reduction in histone positive charge induced by PAD4-mediated citrullina-
tion decreases the affinity between histones and the negatively charged DNA, resulting in histone
dissociation from DNA and so leading to the loss of the compacted chromatin structure and de-
condensation (Wang et al. 2009). In line with this concept, PAD4-deficient neutrophils are less
efficient at decondensing their chromatin (Thiam et al. 2020). Rescue experiments showed that
PAD4 nuclear localization and enzymatic activity are required for efficient chromatin decondensa-
tion, indicating that PAD4 citrullination activity in the nucleus mediates NETosis. Several lines of
evidence indicate that PAD4 can directly decondense chromatin. DNA from PAD4-activated cells

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is degraded faster by micrococcal nuclease, suggesting that the linker DNA is more accessible for

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cleavage and, thus, chromatin is less condensed (Wang et al. 2009). PAD4-citrullinated histone H1
in mESCs is dissociated from chromatin and affects chromatin condensation (Christophorou et al.
2014). These data suggest that PAD4 mediates chromatin decondensation by inhibiting linker
histone–mediated compaction. Linker histone citrullination has been demonstrated in mESCs
(Christophorou et al. 2014), but whether it occurs during NETosis requires further investigation.
More importantly, experiments with non-citrullinatable histone mutants are required to directly
demonstrate that histone citrullination mediates chromatin decondensation during NETosis.
Although PAD4 appears to be crucial for chromatin decondensation, other factors may con-
tribute to achieving complete chromatin decondensation during NETosis. While heterologous
expression of PAD4 in nonneutrophil cell types causes them to expel decondensed chromatin
upon ionomycin stimulation (Leshner et al. 2012), isolated neutrophil nuclei treated with PAD4
protein exhibited only a mild (20%) increase in nuclear area (Gößwein et al. 2019). In addition,
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chromatin decondensation is delayed but not inhibited in PAD4-deficient neutrophil-like HL60

cells (Thiam et al. 2020). This suggests that additional factors may be required to achieve the ex-
Annu. Rev. Cell Dev. Biol. 2020.36. Downloaded from www.annualreviews.org

tent of chromatin decondensation observed during NETosis. Thus, in human neutrophils PAD4
may not be fully sufficient for chromatin decondensation or may be required for a specific phase
of decondensation, such as initiation, with complete decondensation requiring cooperation with
other cellular factors.

3.1.1.3. PAD4 in NETosis: open questions. The significance of PAD4 in NETosis is now well
recognized in both mouse and human cells; however, many questions remain. How is PAD4 ac-
tivated during NETosis in (patho)physiological conditions in vivo? What is the required calcium
concentration and its mechanism of production for PAD4 activation in vivo? Does PAD4 undergo
posttranslational modifications to regulate its threshold of activation? Does PAD4 drive chro-
matin decondensation via histone citrullination alone, or do other PAD4 substrates contribute to
the process? Answers to these questions will not only shed light on the process of chromatin de-
condensation during NETosis but also reveal novel insights into the fundamental mechanisms of
the regulation of histone–DNA interaction.

3.2. The Role of Proteases in Chromatin Decondensation During NETosis

Proteases are central to neutrophil function. For instance, they mediate bacterial killing both in-
side the phagosome and in the extracellular environment, and they facilitate neutrophil migration
by cleaving the ECM (Belaaouaj et al. 1998, Okada 2017, Weinrauch et al. 2002). The proteases
neutrophil elastase, proteinase 3 (PR3), and cathepsin G (which are all contained in primary gran-
ules), as well as calpain (a cytosolic protease), have all been implicated in NETosis.

3.2.1. The role of primary granule resident proteases. Papayannopoulos and colleagues
(2010) were the first to propose that primary granule-resident serine proteases are critical for
NETosis. Indeed, they showed that neutrophil elastase and PR3 can induce chromatin deconden-
sation in isolated nuclei, as assessed by the increase in DNA surface area. Inhibition of neutrophil
elastase (with a nonselective inhibitor that impacts PR3 as well) impairs the release of extracellular
traps upon PMA or C. albicans stimulation. The importance of proteases in NETosis was confirmed
when neutrophils from mice deficient in the serine protease inhibitor SerpinB1 were shown to
release more NETs upon PMA, platelet-activating factor, chemokine ligand 2 (cxcl2), LPS, or
P. aeruginosa stimulation (Farley et al. 2012). Additionally, neutrophils from neutrophil elastase
KO mice were shown to be deficient in NETosis upon Klebsiella pneumoniae (Papayannopoulos
et al. 2010) or methicillin-resistant S. aureus infection (Kolaczkowska et al. 2015) or exposure to

·.•�-
P. aeruginosa biofilm (Thanabalasuriar et al. 2019).
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While the requirement for serine proteases in NETosis is well accepted, the specific require-
ment of neutrophil elastase for chromatin decondensation is controversial. Indeed, ionophore- or
saliva mucin–induced NETosis proceeds without neutrophil elastase activity (Kenny et al. 2017,
Mohanty et al. 2015). Moreover, neutrophils from neutrophil elastase–deficient mice still undergo
NETosis upon PMA or platelet-activating factor stimulation (Martinod et al. 2016).

3.2.1.1. How do primary granule proteases mediate chromatin decondensation in NETosis?

Proteases are proposed to mediate chromatin decondensation and NETosis via histone cleavage,
thus releasing DNA from histones (Papayannopoulos et al. 2010). Indeed, histones H1, H2A,
H2B, H3, and H4 can be degraded in vitro by neutrophil elastase and PR3. In addition, degrada-
tion products of H2B and H4 were observed in neutrophils stimulated with PMA, and inhibition
of neutrophil elastase prevents H4 degradation in PMA-stimulated neutrophils (Papayannopoulos
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et al. 2010). This suggests that H4 cleavage by neutrophil elastase is critical for chromatin decon-
densation during NETosis.
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Because neutrophil elastase, PR3, and cathepsin G localize to membrane-bound granules in

the cytoplasm, the mechanism by which they obtain access to histones inside the nucleus during
NETosis remains unclear. Transient activity of neutrophil elastase and cathepsin G but not PR3
is detected in the cytosol of neutrophils 30 min after PMA stimulation, indicating that these pro-
teases are released intracellularly from granules in their active form (Metzler et al. 2014). This
finding led to the hypothesis that active neutrophil elastase and cathepsin G are first released to
the cytosol before translocating to the nucleus during NETosis. How do proteases leave granules
without their permeabilization? Myeloperoxidase (MPO) and hydrogen peroxide (H2 O2 ) have
been proposed to mediate the release of proteolytically active neutrophil elastase from granules
(Metzler et al. 2014); however, the associated mechanism still needs to be revealed. The pore-
forming protein gasdermin D has been implicated in neutrophil elastase nuclear translocation
(Sollberger et al. 2018). Whether gasdermin D mediates neutrophil elastase exit from granules or
entry into the nucleus remains to be determined. Thus, the cellular mechanism by which serine
proteases mediate chromatin decondensation during NETosis remains to be investigated.

3.2.1.2. Primary granule resident proteases in NETosis: open questions. Although evidence
implicates proteases in NETosis, much is still unknown. How and when do neutrophil elastase
and cathepsin G co-localize with DNA during NETosis? Do they contain a nuclear localization
sequence that allows them to enter the nucleus? Do they enter the nucleus before or after nuclear
envelope permeabilization or rupture? More importantly, neutrophil elastase has a strong affinity
for DNA, which was shown to inhibit its protease activity (Duranton et al. 2000). This begs the
question, How does this protease mediate H4 cleavage in the presence of DNA? Live-cell imaging
of neutrophil elastase, PR3, and cathepsin G during NETosis could shed light on their roles during
this process.

3.2.2. Calpain: the new player in chromatin decondensation. An interesting emerging con-
cept is that chromatin decondensation during NETosis could result from the combinatorial effect
of PAD4-mediated citrullination and protease-mediated cleavage. As such, the calcium-activated
serine protease calpain was shown to enhance chromatin decondensation in the presence of PAD4
but not by itself. Moreover, ionomycin-induced NETosis was impaired upon calpain inhibition
(Gößwein et al. 2019). It is thus tempting to speculate that PAD4-mediated citrullination could
target nuclear proteins for calpain-mediated degradation, thus driving chromatin decondensation.

·.•�-
However, further studies are required to understand this synergistic effect.

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3.3. The Role of Myeloperoxidase in Chromatin Decondensation and NETosis

MPO, a primary granule resident protein that catalyzes chloride oxidation into hydrochloric acid
in the presence of hydrogen peroxide, is implicated in chromatin decondensation during NETosis.
Neutrophils from MPO-deficient patients fail to release NETs upon PMA, C. albicans, or Group
B streptococcus stimulation (Kenny et al. 2017, Metzler et al. 2011). Interestingly, pharmacologi-
cal inhibition of MPO only delays NETosis, suggesting that limited MPO activity is sufficient
for NETosis (Metzler et al. 2011). While MPO does not directly decondense chromatin in iso-
lated nuclei or degrade histones in vitro, it was shown to enhance neutrophil elastase–mediated
chromatin decondensation (Papayannopoulos et al. 2010). How does MPO facilitate chromatin
decondensation? Does it mediate oxidation of DNA and block its inhibitory effect on neutrophil
elastase? Live-cell imaging of MPO-deficient neutrophils stimulated for NETosis, would help
identify which NETosis step is inhibited and hence the role of MPO in NET release.
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4. DNA RELEASE FROM THE NUCLEUS TO THE CYTOSOL

For decondensed DNA to be released extracellularly, it must first escape from the nucleus (Fuchs
et al. 2007). Chromatin in the nucleus is isolated from the cytosol by the nuclear membrane, which
is made up of two lipid bilayers connected by multiprotein complexes including the nuclear pore
complex (NPC) and the linker of nucleoskeleton and cytoskeleton (LINC) complex ( Janota et al.
2017). Underneath the inner nuclear membrane (INM) lies a meshwork of lamin intermediate
filaments composed of lamin A/C, B1, and B2. Lamins play major roles in gene expression and
have also been described as molecular shock absorbers (Dahl et al. 2004), suggesting that they
can dampen mechanical forces generated outside the nucleus before they reach the chromatin.
Shielding the chromatin from nucleases and mechanical forces, the nuclear envelope was long
thought to be very stable except during mitosis, when the combined actions of mitotic kinase–
mediated lamin disassembly and pulling forces from microtubule (MT) motors drive its rupture
and subsequent release of compacted chromatin into the cytosol (Güttinger et al. 2009). How-
ever, mechanical forces alone were reported to be sufficient for nuclear envelope rupture during
immune and cancer cell migration under confinement (Denais et al. 2016, Raab et al. 2016).
Similar to the rearrangement described during cell division and migration, DNA release into
the cytosol during NETosis was shown to involve lamin remodeling as well as formation of holes in
the nuclear envelope prior to its rupture (Chen et al. 2018, Fuchs et al. 2007, Gößwein et al. 2019,
Li et al. 2019, Neubert et al. 2018, Thiam et al. 2020). Interestingly, vital NETosis is proposed
to occur without nuclear membrane disruption with decondensed DNA leaving the nucleus in
vesicles (Pilsczek et al. 2010). In the following, we describe the molecular players involved in DNA
release in the cytosol and discuss how the mechanics of the chromatin could drive this process.

4.1. Lamin Dynamics During NETosis

Dramatic changes in the lamin meshwork have been observed during NETosis (Figures 1 and
2). The appearance of local discontinuities in the lamin B1 and B2 networks has been reported in
human and mouse neutrophils stimulated with PMA or ionomycin (Li et al. 2019, Neubert et al.
2018, Thiam et al. 2020). Using high-resolution live cell imaging of HL60-derived neutrophils
stimulated with ionomycin, Thiam and colleagues (2020) found that decondensed DNA extruded
from these lamin discontinuities.
How does the lamin meshwork disassemble during NETosis? The formation of meshwork

·.•�-
discontinuities could be the result of lamin posttranslational modifications. As such, lamin A/C

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(Amulic et al. 2017) and PKCα-mediated lamin B1 (Li et al. 2019) phosphorylation was reported
in PMA-induced NETosis. Indeed, mutation of the PKC phosphorylation sites of lamin B1 im-
pairs extracellular trap release in RAW264.7 macrophages (Li et al. 2019). Additionally, lamin B1
degradation has been observed during ionomycin-induced NETosis (Gößwein et al. 2019), and
PAD4 deficiency has been shown to delay the formation of lamin discontinuities and to diminish
lamin B1 degradation in ionomycin-stimulated neutrophils (Gößwein et al. 2019, Thiam et al.
2020). This suggests that PKC-mediated lamin phosphorylation and/or PAD4-mediated lamin
degradation could drive lamin disassembly during NETosis. Whether PKC phosphorylates lamin
B1 in neutrophils and how PAD4 drives lamin B1 degradation await further studies.

4.2. Nuclear Membrane Dynamics During NETosis

The nuclear envelope has been proposed to vesiculate during both suicidal (Fuchs et al. 2007)
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and vital NETosis (Pilsczek et al. 2010). Indeed, using electron microscopy, Fuchs and colleagues
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(2007) detected vesicles containing the lamin B receptor (LBR) that presumably emanated from
the nuclear membrane. This led to the hypothesis that vesiculation of the nuclear envelope
mediates its disintegration, thus allowing chromatin release into the cytosol during PMA-induced
NETosis. But these results are open to interpretation due to thin-section artifacts that cannot
distinguish between continuous and discontinuous networks. Moreover, LBRs are also present in
the ER (Ellenberg et al. 1997) that has been observed by live-cell microscopy to vesiculate during
NETosis (Thiam et al. 2020), raising the possibility that the observed LBR-positive vesicles might
originate from the ER. During vital NETosis, the nuclear envelope is thought to vesiculate but
not to rupture. Indeed, an electron microscopy study by Pilsczek and colleagues (2010) detected
vesicles containing small amounts of material reminiscent of strands of DNA with nucleosomes
that were present in the cytosol of neutrophils stimulated with S. aureus. They proposed that those
vesicles form by budding off of the outer nuclear membrane (ONM) and are then exocytosed
at the plasma membrane, leaving both the nuclear envelope and the plasma membrane intact
during NET release. However, it is unclear how a vesicle containing DNA would form from
the ONM without either breaking the inner nuclear envelope or forming a double-membrane
vesicle. High-resolution, live-cell imaging of the inner and ONMs of human, mouse, and HL60-
derived neutrophils stimulated with ionomycin showed that the nuclear membrane does not
disintegrate by vesiculation but rather ruptures at multiple points, from which the chromatin is
released into the cytosol (Figures 1 and 2) (Thiam et al. 2020). This rupture of the INMs and
ONMs occurs within a minute after the appearance of lamin discontinuities, and no subsequent
nuclear envelope resealing was observed. However, these studies were performed on isolated
cells in vitro, and how DNA is released from the nucleus in a physiological setting remains to be
determined.

4.2.1. How does the nuclear envelope rupture during NETosis? As discussed earlier, the
nuclear envelope can rupture by forces generated either by MT motors during mitosis or in the ex-
tracellular environment during cell migration under tight confinement. In contrast, in neutrophils
undergoing NETosis, the nuclear envelope ruptures after complete disassembly of the cytoskele-
ton (Thiam et al. 2020) in the absence of cell confinement. Thus, alternative mechanisms must be
at play during NETosis to drive nuclear envelope rupture. PAD4 deficiency was shown to impair
nuclear envelope rupture (Thiam et al. 2020). The dependence of both chromatin decondensation
and nuclear envelope rupture on PAD4 suggests that rupture could be driven by decondensation.
Whether entropic (Neubert et al. 2018) or osmotic pressure generated by chromatin deconden-

·.•�-
sation could be sufficient for bursting the nuclear envelope remains to be determined.

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4.2.2. Gasdermin D–mediated nuclear envelope permeabilization. Gasdermin D is a pore-

forming protein that can be cleaved by caspases 1, 4, 5, and 11 (in mice) (Shi et al. 2015) to release
the N-terminal domain (gasdermin N) that can insert in membranes and form 20-nm pores by
oligomerization (Ruan et al. 2018, Sborgi et al. 2016). Gasdermin D is proposed to be cleaved by
caspase 11 during cytosolic LPS-induced NETosis (Chen et al. 2018) and by neutrophil elastase
during PMA-induced NETosis (Sollberger et al. 2018). Under both stimuli, neutrophils from gas-
dermin D–null mice failed to extrude their DNA into the cytosol (Chen et al. 2018, Sollberger et al.
2018), suggesting a defect in nuclear envelope rupture. Nuclear envelope permeabilization prior to
DNA release into the cytosol was observed by live imaging (Thiam et al. 2020). Thus, it is tempt-
ing to speculate that during NETosis, gasdermin D cleavage mediates nuclear membrane perme-
abilization, allowing proteases to obtain access to histones and promote subsequent chromatin
decondensation. Gasdermin D preferentially inserts into phosphorylated phosphatidylinositol-
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and phosphatidylserine-positive membranes (Ding et al. 2016, Sborgi et al. 2016). Although these
lipids, particularly phosphatidylinositol(4,5)bisphosphate [PtdIns(4,5)P2 ], were reported at the nu-
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clear membrane (Fiume et al. 2012, Kleinig 1970, Smith & Wells 1983), whether they are present
in the INM or ONM, or both, is unclear. Thus, whether and how gasdermin D pores form at the
nuclear envelope during NETosis requires further investigation.

4.2.3. DNA release into the cytosol: open questions. Although it is well accepted that for
NETosis to occur decondensed chromatin must breach the lamin meshwork and the nuclear mem-
brane, not much is known about the mechanisms involved in this process. What mediates the
appearance of discontinuities in the lamin meshwork during NETosis? If they are the result of
local disassembly, what enzymes mediate the posttranslational modifications necessary for lamin
meshwork disassembly during NETosis? Could mechanical forces generated by chromatin de-
condensation mediate the rupture of both the lamin meshwork and the nuclear envelope? What
are the dynamics of the INMs and ONMs and of the lamin network during vital NETosis? If
decondensed chromatin is packed into vesicles emanating from the nuclear envelope, what is the
structure of those vesicles? And if these vesicles contain both the INMs and ONMs, how would
they fuse with the single membrane of the plasma membrane? High-resolution live imaging of
cells undergoing vital NETosis will be critical to understanding the mechanism of DNA release
during this process.

5. REMODELING OF CYTOSKELETAL AND MEMBRANOUS

ORGANELLES DURING NETOSIS
Once decondensed chromatin is released from the nucleus into the cytosol, it must breach the
crowded and interconnected network of the cytoskeletons and membranous organelles before
reaching the plasma membrane. The cytoskeletons and membranous organelles undergo drastic
remodeling during lytic NETosis (Figures 1 and 2). Indeed, the actin filaments, MTs, and vi-
mentin intermediate filaments disassemble prior to extracellular DNA release (Amulic et al. 2017,
Metzler et al. 2014, Neubert et al. 2018, Thiam et al. 2020). Similarly, the ER was shown to vesic-
ulate (Thiam et al. 2020), granules are proposed to disintegrate (Brinkmann et al. 2004, Metzler
et al. 2014, Okubo et al. 2016, Papayannopoulos et al. 2010), and mitochondria may release their
DNA (Caielli et al. 2016, Lood et al. 2016, McIlroy et al. 2014, Wang et al. 2015, Yousefi et al.
2019).
Mitosis is another cellular process during which similar drastic remodeling of the cytoskeleton
and membranous organelles occurs. Formation of the mitotic spindle first requires disassembly of

·.•�-
the preexisting interphase MT array (Belmont et al. 1990). Similarly, remodeling of the interphase

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actin (Kunda et al. 2008) and vimentin intermediate filament (Duarte et al. 2019) networks is
critical to the formation of the stiff cortex in metaphase. Golgi apparatus vesiculation (Guizzunti
& Seemann 2016) and mitochondrial fission (Mishra & Chan 2014) during mitosis ensure equal
distribution of these organelles into daughter cells. Therefore, in addition to nuclear envelope
rupture, cytoskeletal and membranous organelle disassembly is a common step between mitosis
and NETosis, although these structures do not reform in the latter.

5.1. Cytoskeletal Disassembly During NETosis

The three cytoskeletal systems of the cell form a dense, interconnected network that confers and
maintains the physical integrity of the cell. The stiff MT network regulates the transport and dis-
tribution of membranous organelles throughout the cell, the dynamic actin cytoskeleton at the
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cortex sets the plasma membrane tension and drives cell shape changes, and the dense network of
intermediate filaments allows cells to resist large deformations. Intriguingly, all of these cytoskele-
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tal networks rapidly disassemble during NETosis.

5.1.1. Actin disassembly: an early NETosis event. Among the three cytoskeletal networks,
the actin cytoskeleton has been the most studied during NETosis. It is proposed to be degraded
during C. albicans–induced NETosis (Metzler et al. 2014). However, the absence of fluorescent
phalloidin staining (Sollberger et al. 2018) and the solubilization of both monomeric and filamen-
tous actin probes (Neubert et al. 2018, Thiam et al. 2020) has been reported in PMA-, ionomycin-,
and LPS-induced NETosis, suggesting that the actin cytoskeleton disassembles by filament de-
polymerization rather than by degradation. The disassembly of the actin cytoskeleton is one of
the earliest events in PMA-, ionomycin-, and LPS-induced NETosis, occurring within minutes
after NETosis stimulation (Neubert et al. 2018, Thiam et al. 2020). More importantly, stabiliza-
tion of the actin cytoskeleton using jasplakinolide impairs extracellular trap formation (Neubert
et al. 2018, Thiam et al. 2020). However, Neubert and colleagues (2018) reported that the early
addition of actin filament depolymerization drugs after PMA stimulation also impairs NETosis,
suggesting that a dynamic actin network is important for NETosis onset and that actin filament
depolymerization is temporally regulated.

5.1.1.1. Possible mechanisms of actin disassembly during NETosis. As actin disassembly is re-
quired for efficient NETosis, understanding this mechanism is important. The intracellular cal-
cium concentration increases during NETosis, and several actin-depolymerization factors includ-
ing myosin II and gelsolin are calcium sensitive (Downey et al. 1990, Larson et al. 2005, Yoshida
et al. 1984). Thus, intracellular calcium influx could be a main driver of actin disassembly dur-
ing NETosis. Actin oxidation by proteins from the molecule interacting with CasL (MICAL)
family drives filament disassembly and prevents its repolymerization (Hung et al. 2010, 2011).
Considering that ROS increase rapidly at NETosis onset and that oxidized actin is present at
high levels in calcium ionophore–induced NETosis (Petretto et al. 2019), MICAL is also a good
candidate for mediating actin disassembly during NETosis. Because actin filament disassembly
is rapid and global during NETosis, it is likely to be caused by a combinatorial effect of several
actin-disassembling factors.

5.1.1.2. Possible roles of actin disassembly during NETosis. How does actin cytoskeleton
disassembly contribute to NETosis? Actin degradation has been proposed to mediate neu-
trophil elastase translocation to the nucleus (Metzler et al. 2014). However, this concept has not

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has not been established. Live-cell imaging of jasplakinolide-treated neutrophils stimulated for
NETosis showed that actin filament stabilization impairs extracellular DNA release without af-
fecting DNA release from the nucleus into the cytosol. Moreover, in cases where NETosis did
occur in the presence of jasplakinolide, decondensed chromatin was released locally through holes
of the actin cortex (Thiam et al. 2020). These data suggest that the cortical actin filament network
acts as a physical impediment to plasma membrane rupture and DNA release into the extracellu-
lar environment, and early disassembly of the actin cytoskeleton acts to remove this impediment.
Whether actin filament disassembly facilitates plasma membrane rupture by modifying the cell
membrane composition and/or decreasing the required plasma membrane rupture force warrants
investigation.

5.1.2. Microtubule and vimentin intermediate filament disassembly. In addition to actin

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depolymerization, NETosis is accompanied by a drastic decrease in polymerized MTs and vi-

mentin intermediate filaments. Neutrophils undergoing NETosis disassemble their MT networks,
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as reflected by the decrease in α- and β-tubulin immunostaining (Amulic et al. 2017, Neubert et al.
2018) and rapid solubilization of MT markers during live imaging (Thiam et al. 2020). Similarly,
the vimentin intermediate filament network is remodeled during NETosis as assessed by a decrease
in vimentin immunostaining and solubilization of vimentin-enhanced green fluorescent protein
(eGFP) in live cells (Thiam et al. 2020). Interestingly, immunostaining suggests that peripheral
vimentin disassembles more than the perinuclear filaments, implying that vimentin disassembly
during NETosis may be a regulated process.

5.1.2.1. Possible mechanisms and roles of microtubule disassembly during NETosis. Current
data suggest that disassembling or stabilizing MTs pharmacologically does not impair NETosis
(Neubert et al. 2018, Thiam et al. 2020), indicating that neither an intact MT network nor its
disassembly is required for NETosis. However, the rapid and dramatic loss of MTs is nonetheless
interesting. MTs are well known to be calcium labile (Weisenberg & Deery 1981) and have been
shown to disassemble in cells at calcium concentrations of 1 to 4 μM (Schliwa et al. 1981). The
intracellular calcium concentration in activated neutrophils is in the micromolar range (Krause
et al. 1990). Thus, the intracellular calcium concentration in neutrophils undergoing NETosis
may be sufficient to induce MT disassembly. The mechanism of MT disassembly during NETosis
and the possible role of microtubule regulatory proteins awaits further study.

5.1.2.2. Possible mechanisms and roles of vimentin remodeling during NETosis. Similar to
the disassembly of actin filaments, the loss of vimentin networks could act to clear the way for
chromatin to mediate its more efficient release from the cell, which makes the mechanism of
vimentin remodeling of interest. PAD4 deficiency delays vimentin remodeling during NETosis,
suggesting that this process involves PAD4 functions (Thiam et al. 2020). In addition, a recent
report has shown that neutrophils from vimentin-null mice release fewer NETs upon ionomycin
and nigericin stimulation (P. Munzer, R. Negro, S. Fukui, L. di Meglio, C. Cherpokova, et al.,
unpublished manuscript). Interestingly, vimentin is citrullinated during NETosis (Khandpur et al.
2013), and vimentin citrullination in calcium ionophore–stimulated macrophages is thought to
induce vimentin redistribution around the nucleus (Asaga et al. 1998). Thus, vimentin remodeling
during NETosis could be induced by citrullination. Citrullinated vimentin is found in the synovial
fluid of rheumatoid arthritis (RA) patients and is involved in RA pathogenesis (Van Steendam
et al. 2011). Moreover, antibodies against citrullinated vimentin can induce NETosis (Khandpur
et al. 2013), suggesting that citrullinated vimentin might contribute to the detrimental effects

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of NETosis. However, similar to the actin cytoskeleton, kinases and proteases that regulate the

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disassembly or degradation of vimentin intermediate filaments are also sensitive to intracellular

calcium (Ando et al. 1991, Nelson & Traub 1982, Spruill et al. 1983, Yoshida et al. 1984), suggesting
an alternative mechanism to PAD4. Further studies are required to decipher the contribution of
citrullination and calcium in vimentin disassembly during NETosis.

5.1.3. Cytoskeletal disassembly: open questions. In addition to its requirement for the struc-
tural integrity of cells, the cytoskeleton is critical for signaling, including mechanotransduction,
cell polarity, and transport of organelles, as well as force generation. Thus, the extent of cytoskele-
tal disassembly during NETosis raises several questions: Is the plasma membrane of cells under-
going NETosis polarized? How are organelles positioned during NETosis? How are the forces
necessary for NETosis completion generated? How would NETosis proceed in physiological en-
vironments in which cells have to integrate ECM stiffness and topology? Alternative thinking and
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physical models for force generation will be critical to understanding how NETosis occurs in vivo.
Annu. Rev. Cell Dev. Biol. 2020.36. Downloaded from www.annualreviews.org

5.2. Membranous Organelles

The cytosol is crowded with not only the cytoskeleton but also organelles including granules, ER,
Golgi apparatus, endosomes, lysosomes, mitochondria, and ribosomes, among others. Although
many organelle systems have not been thoroughly examined during NETosis, there is evidence
that the ER, granules, and mitochondria undergo major changes during NETosis.

5.2.1. Endoplasmic reticulum vesiculation and granule disintegration. The ER was shown
to vesiculate within minutes following the disassembly of the cytoskeleton during NETosis stimu-
lated by ionomycin and LPS (Thiam et al. 2020). Interestingly, ER vesiculation occurred much ear-
lier than nuclear membrane rupture, suggesting a loss of continuity between these two organelles.
Calcium ionophores are known to be inducers of ER vesiculation (Koch et al. 1988, Wilson et al.
1998), suggesting that the increase in intracellular calcium might mediate ER vesiculation during
NETosis.
Neutrophil granule resident proteins, including neutrophil elastase, MPO, PR3, cathepsin G,
and lactoferrin (Brinkmann et al. 2004, Metzler et al. 2014, Okubo et al. 2016, Papayannopoulos
et al. 2010), are found on NETs. While the release of neutrophil elastase and cathepsin G from
primary granules occurs without granule lysis, the mechanism by which MPO, PR3, or lactoferrin
localizes to NETs is unknown. Interestingly, while MPO shows a diffuse intracellular localization
on DNA before NET release (Papayannopoulos et al. 2010), lactoferrin has been reported to
localize to the plasma membrane (Okubo et al. 2016), suggesting that specific granular components
may be targeted to specific cellular compartments during NETosis. Whether neutrophil granules
become permeabilized or rupture during NETosis to allow the release of granule-resident proteins
on NETs awaits further study.

5.2.2. The role of mitochondria during NETosis. Mitochondria play two roles in NETosis.
First, mitochondria can mediate ROS production, and second, mitochondrial DNA (mtDNA) can
be released extracellularly.
An increase in mitochondrial ROS production was shown to be required for calcium
ionophore– or ribonucleoprotein immune complex (RNP IC)–induced NETosis as well as spon-
taneous NETosis by low-density granulocytes from SLE patients (Douda et al. 2015, Lood
et al. 2016). Thus, in addition to NADPH oxidase, ROS can be released during NETosis by

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Several studies have shown that mtDNA can be released extracellularly and can associate with
nuclear DNA on NETs (Caielli et al. 2016, Lood et al. 2016, McIlroy et al. 2014, Wang et al. 2015).
However, the underlying mechanism remains unclear. Lood and colleagues (2016) proposed that
during RNP IC–induced NETosis mitochondria localize to the plasma membrane, where they
release oxidized mtDNA. However, mitochondria have two membranes; thus, how this organelle
would fuse with the single membrane of the plasma membrane and release DNA is unclear. Is
mtDNA first released in the cytosol by mitochondrial rupture? Do mitochondria fuse with the
plasma membrane? These questions remain to be answered by careful analysis of mitochondrial
membrane and DNA dynamics.

6. EXTRACELLULAR DNA RELEASE: BREACHING

THE PLASMA MEMBRANE
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NETs are frequently observed as extracellular DNA stained with membrane-impermeable DNA
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dyes (Sytox green or Dapi) (Brinkmann et al. 2004, van der Linden et al. 2017), a finding indicative
of DNA access to the extracellular milieu. For DNA to be released extracellularly, decondensed
chromatin must breach the plasma membrane. Other than vital NETosis, during which decon-
densed DNA is thought to be released extracellularly via exocytosis (Pilsczek et al. 2010), most of
the current evidence indicates that extracellular DNA released during lytic NETosis occurs fol-
lowing plasma membrane lysis. However, the mechanisms underlying the changes in the plasma
membrane during vital or lytic NETosis remain largely unknown. Recent evidence indicates that
breach of the plasma membrane may be a multistep process during NETosis in which the per-
meability of the plasma membrane increases prior to its rupture and extracellular DNA release
(Figures 1 and 2).

6.1. Increase in Permeability of the Plasma Membrane During NETosis

During NETosis, the plasma membrane becomes permeable to increasingly larger molecules over
time after stimulation yet retains the ability to contain chromatin expanding from the ruptured
nucleus into the cytoplasm (Thiam et al. 2020). The plasma membrane was shown to first become
permeable to calcein, then to 10 kDa dextran, and finally to 70 kDa dextran before exhibiting a
large hole and releasing DNA extracellularly (Thiam et al. 2020). The progressive permeabiliza-
tion of the plasma membrane without drastic cellular swelling or rapid bursting after cells become
permeable to small molecules (calcein) suggests that several classes of membrane pores with vari-
ous pore sizes may be forming and/or sealing over time.
Neutrophils express several types of pore-forming proteins; among these, gasdermin D has
been implicated in NETosis (Chen et al. 2018, Sollberger et al. 2018). Is plasma membrane perme-
abilization during NETosis mediated by gasdermin D? The N-terminal domain of gasdermin D
has been shown to form pores of about 20 nm in diameter (Sborgi et al. 2016). As calcein (0.6 kDa)
and dextran 10 kDa and 70 kDa have hydrodynamic radii of <1 nm, 1.9 nm, and 6.8 nm, respec-
tively (Armstrong et al. 2004, Chouinard-Pelletier et al. 2012, Guo & Santschi 2007), they would
be able to fit through 20-nm gasdermin D pores all at once rather than progressively. Thus, gas-
dermin D pores might form only during the later stages of plasma membrane permeability. Bac-
terial toxins, such as the one released by S. aureus, have been shown to disrupt plasma membrane
permeability during NETosis (van der Linden et al. 2017). However, progressive plasma mem-
brane permeabilization during NETosis has been observed in the absence of toxins, ruling out
their requirement. Thus, how and why plasma membrane permeability gradually increases during

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NETosis await further study.

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6.2. Plasma Membrane Rupture Driven by Chromatin Swelling

Live-cell imaging of neutrophils stained with a plasma membrane dye or expressing fluorescent
plasma membrane markers has shown that the release of extracellular DNA occurs locally at sites
of plasma membrane rupture (Neubert et al. 2018, Thiam et al. 2020). Importantly, such mem-
brane rupture occurs minutes to hours after a gradual increase in its permeability (Thiam et al.
2020), suggesting that it is not merely a consequence of cell swelling and bursting. The molecular
mechanism of plasma membrane rupture during NETosis remains to be revealed.
Neubert and colleagues (2018) proposed a biophysical mechanism for plasma membrane rup-
ture in NETosis. They showed that extracellular DNA release at the end of NETosis is a passive
process independent of glycolysis, ATP, metabolism, and MPO. Estimating the entropic pressure
generated by the fully unraveled genomic DNA to be 100–200 Pa and the membrane pressure at
the end of NETosis to be about 20 Pa, they proposed that chromatin swelling could mechanically
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rupture the plasma membrane (Neubert et al. 2018). This concept brings a novel perspective
to understanding the biophysics of NETosis; however, it is challenged by other existing data.
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For instance, if chromatin swelling is a point of no return sufficient for plasma membrane
rupture, why would some cells release their DNA only in the cytosol but not extracellularly even
hours after induction for NETosis (Thiam et al. 2020)? Thus, further studies will be required
to understand the molecular and physical mechanisms of plasma membrane rupture during
NETosis.

7. CONCLUSION AND PERSPECTIVES

The field of NETosis is in an exciting stage in which evidence of the (patho)physiological relevance
of NET release has been accumulating but the molecular, cellular, and biophysical mechanisms
driving this process have just begun to be revealed. Most of the current literature focuses on which
stimuli can induce NETosis and which proteins can inhibit NET release without well-defined in-
sights into how these factors influence the cellular events of NETosis. As NETosis proceeds via
a specific sequence of events (Figures 1 and 2), determining how proteins that inhibit NETosis
perturb this sequence will help us to understand the basic mechanisms of NETosis and will in-
form us of new targets to modulate NETosis in diseases. Moreover, studying this unusual cellular
process may provide insights into other cellular mechanisms, including chromatin decompaction
for transcription, as well as into diseases such as laminopathies. Among all the questions that have
been raised in this review, several may merit particular attention to significantly further our un-
derstanding of the cell biology and biophysics of NETosis (see Futures Issues).

FUTURE ISSUES
1. How is NETosis initiated?
2. What makes a neutrophil undergo NETosis versus other forms of host defense?
3. What are the mechanisms and roles of cytoskeletal and organelle remodeling?
4. How are the necessary forces required for NETosis generated?
5. What is the mechanism of vital NETosis?
6. How do neutrophils integrate the molecular and physical complexity of the in vivo en-
vironment while undergoing NETosis?

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DISCLOSURE STATEMENT
The authors are not aware of any affiliations, memberships, funding, or financial holdings that
might be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS
We thank the Marine Biological Laboratory and Nikon Instruments, whose support allowed the
initial collaboration between our labs. Of note, each of the authors of this review was born in a
different continent, illustrating the power of science in crossing boundaries. Funded by the Na-
tional Heart, Lung, and Blood Institute Division of Intramural Research (C.M.W. and H.R.T.),
the National Institutes of Health (grants RO1 GM106023 and R35 HL135765) (D.D.W.), and a
Nanyang Assistant Professorship from Nanyang Technological University Singapore (S.L.W.).
Access provided by Auckland University of Technology on 07/14/20. For personal use only.
Annu. Rev. Cell Dev. Biol. 2020.36. Downloaded from www.annualreviews.org

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