Recent progress in the mechanistic understanding of NET formation in neutrophils

Abstract

Neutrophils are the most abundant circulating white blood cells and one of the major cell types of the innate immune system. Neutrophil extracellular traps (NETs) are a result of the extracellular release of nuclear chromatin from the ruptured nuclear envelope and plasma membrane. The externalized chromatin is an ancient defense weapon for animals to entrap and kill microorganisms in the extracellular milieu, thus protecting animals ranging from lower invertebrates to higher vertebrates. Although the externalized chromatin has the advantage of acting as anti-infective to protect against infections, extracellular chromatin might be problematic in higher vertebrate animals as they have an adaptive immune system that can trigger further immune or autoimmune responses. NETs and their associated nuclear and/or cytoplasmic components may induce sterile inflammation, immune and autoimmune responses, leading to various human diseases. Though important in human pathophysiology, the cellular and molecular mechanisms of NET formation (also called NETosis) are not well understood. Given that nuclear chromatin forms the backbone of NETs, the nucleus is the root of the nuclear DNA extracellular traps. Thus, nuclear chromatin decondensation, along with the rupture of nuclear envelope and plasma membrane are required for nuclear chromatin extracellular release and NET formation. So far, most of the literature focuses on certain signaling pathways which are involved in NET formation but without explanation of cellular events and morphological changes described above. Here, we have summarized emerging evidence and discuss new mechanistic understanding, with our perspectives, in NET formation in neutrophils.

Graphical Abstract

The nucleus is the root of nuclear DNA extracellular traps in neutrophils. Nuclear envelope rupture is required for chromatin release and extracellular NET formation. Several recent works indicate that kinase-mediated nuclear lamina disassembly is crucial to nuclear envelope rupture which enhances nuclear entry of decondensation enzymes, nuclear chromatin decondensation and extracellular release for NET formation. The new mechanistic understanding is helpful for identification of molecular targets to control NET formation.

Introduction - NETosis and extracellular NET formation

Neutrophils are the most abundant white blood cells in the circulation. As one of the major cell types of the innate immune system, neutrophils are known to be the primary effectors of acute inflammation, either due to infection or tissue injury (1–6). In addition to their classical role in acute infection, recent evidence from many (7–11), including our own (12, 13), studies demonstrate the importance of neutrophils in chronic and sterile inflammation, and in various human diseases, including autoimmune diseases (9, 10, 14–18).

In 1996, Takei and colleagues first observed a unique type of neutrophil cell death in which phorbol 12-myristate 13-acetate (PMA)-treated neutrophils showed morphological changes with the release of nuclear chromatin from the broken nuclear envelope and cell membrane (19). Several years later, Brinkman et al conducted a more detailed seminal investigation on this interesting cellular phenomenon (20), and showed that the released nuclear chromatin can form neutrophil extracellular traps (NETs) which can kill bacteria (20). Thereafter, this unique type of neutrophil cell death (21) has been termed “NETosis” (22) based on the term of “NET” (21) and the Greek suffix “-osis” (23). In contrast, the cellular phenomenon of nuclear DNA (24) or mitochondrial DNA (25) release from viable neutrophils has also been described. However, the coexistence of dead vs viable, two types of NET formation, remains controversial. Thus, a recent consensus review article published by tens of investigators in NET formation research field suggested using the term “NET formation” instead of NETosis to cover the phenomenon of DNA release from neutrophils with or without cell death (26, 27). Here, we will mainly focus on the dead NETotic neutrophils that release their nuclear chromatin to form extracellular NETs.

It has been reported that extracellular NETs are associated or decorated by over 30 bioactive cellular components from nucleus, cytoplasm and cell membrane, i.e. DNA, histone, HMGB1, nuclear lamin B, elastase, myeloperoxidase, cathepsin G, lactoferrin, proteinase 3, gelatinase/ matrix metalloproteinase 9 (MMP9), LL-37, complements, tissue factor etc. (13, 28–31). NETs can also serve as platform for processing or activation of proinflammatory cytokines (32). Given that these extracellular NET-associated components are bioactive, NETs have been reported to be important in sterile inflammation and involved in various noninfectious diseases (2), including cardiovascular (33–35), metabolic (36–38), digestive or gastroenterological (26, 39–41), renal (42–44), autoimmune diseases (18, 45–49), as well as cancer (50–52). On the other hand, NETs have been shown to be important in infectious diseases and infection-induced pathologic conditions or complications (20, 24, 53, 54), including coronavirus disease 2019 (Covid-19) infection and its related severe pathological changes and complications (13, 31, 55).

In addition to humans, the release of extracellular traps has been reported in neutrophils or other immune cells in many different organisms, such as earthworm, fish, mouse, ox, horse, and cat neutrophils, as well as chicken heterophils (56–59). Thus, the externalized chromatin is an ancient defense weapon that has been conserved through evolution. Therefore, extracellular chromatin release seems to be a well-conserved primordial defense mechanism for animals to entrap and kill microorganisms in the extracellular milieu (60). This primitive innate immune mechanism is beneficial for protection against infections in lower animals who do not possess an effective adaptive immune system (61). However, the externalized chromatin might be a double-edge sword in higher vertebrate animals, and it could be problematic, given the presence of an adaptive immune system in these advanced animals (60). Thus, the phylogenetic history of extracellular traps as a primitive host defense mechanism may account for their paradoxical effects in higher vertebrates, as the extracellular nuclear chromatin is involved in sterile inflammation and autoimmune responses (2, 18, 62–65).

Due to the importance in innate immune responses in different organisms and in various human diseases, the numbers of publications involving NET formation have been greatly increased since the seminal work published by Brinkman and colleagues (20), particularly in the past few years. Since its discovery, numerous characteristics and physiological functions of NETs have been investigated, although a lot of unansewered questions remain to be addressed (26). Given that the nucleus is the root of nuclear DNA extracellular traps, decondensation of the nuclear chromatin, and rupture of the nuclear envelope and plasma membrane, are required for extracellular release of nuclear chromatin and NET formation. However, cellular and molecular mechanisms that regulate the key morphological changes of neutrophils during NET formation have not yet been well understood. The classical concept and signaling pathways for induction of NET formation have been extensively described elsewhere (66). Here we will only give a brief discussion about it, but mainly focus on the emerging evidence from the latest publications to discuss recent conceptual advances and new mechanistic understanding of the key morphological changes that are necessary for NET formation, namely nuclear envelope disintegration/rupture, nuclear entry of enzymes that regulate chromatin decondensation, extrusion of the decondensed nuclear chromatin, and plasma membrane rupture for chromatin extracellular release and NET formation.

Signaling pathways that have been shown to be involved in NET formation

NET formation is a well-controlled process of cell death in neutrophils (67). Under stimulation by various stimuli, neutrophils generate reactive oxygen species (ROS) by NADPH oxidase-2 (NOX-2) (35, 66). ROS modulates granule enzyme myeloperoxidase (MPO) (68), and both are required for release of neutrophil elastase from granules and its translocation to the nucleus (35, 66). In the nucleus, neutrophil elastase contributes to decondensation of chromatin by proteolysis of histones (21, 66, 69), resulting in nuclear extrusion of DNA, further extracellular release, and NET formation (35, 66). In addition to the above NOX-dependent pathways for induction of NET formation (22, 70), NOX-independent NET formation has also been reported, in which mitochondrial ROS, but not NOX-derived ROS, drives NET formation when neutrophils are stimulated by certain stimuli, including immune complexes (71–74). Based on the findings from early studies (21, 35, 66, 69, 75), the hypothesis of “lytic NETosis” has been proposed (76) to explain the cellular phenomenon of plasma membrane rupture and extracellular release of nuclear chromatin and cytoplasmic contents of neutrophils (35, 66). Lytic NET formation is considered a suicidal neutrophil cell death that serves as an innate immune response to infections (66, 76, 77).

In contrast to the lytic NET formation, the neutrophils under non-suicidal vital NET formation keep their plasma membrane integrity but release NETs without affecting their life span (24, 25, 76, 78, 79). Yousefi et al have described (25) that neutrophils can generate NETs with mitochondrial DNA release when cells are first primed with granulocyte/macrophage colony-stimulating factor (GM-CSF) for 20 min following by subsequent 15 min stimulation of receptors of toll-like receptor 4 (TLR4) or complement factor 5a (C5a) (25). This type of NET formation with the release of mitochondrial DNA does not require neutrophil death (25). In addition, Pilsczek et al introduced another type of “vital NETosis” in which NET formation is achieved via a novel process when neutrophils exposed to Staphylococcus aureus in vitro for 10–60 min (24). In this type of rapid NET formation, the nuclear DNA is released by nuclear envelope blebbing and vesicular exportation (24). Interestingly, the viable NET formation with nuclear DNA blebbing is ROS-independent (24), while the viable NET formation with mitochondrial DNA release is considered ROS-dependent (25). It remains controversial how the suicidal lytic and the vital NET formation coexist (80, 81), indicating that this field still have many unclarified questions. In contrast to the short period of stimulation (10–60 min) in the above vital NET formation studies (24, 25), most studies on NET formation with neutrophil cell death and nuclear/plasma membrane rupture need to stimulate cells for 2–3 hours or even longer period of time. More studies are needed to clarify the relationship between the rapid vital NET formation vs NET formation with nuclear/plasma membrane rupture.

So far, both nuclear vs mitochondrial DNA have been reported to contribute to NET formation (24, 25, 35, 66, 82). Regarding their contributions to NET formation, the fact is that every single cell contains only 16,569 nucleotides of mitochondrial DNA, but 3,200,000,000 (3.2 billion) nucleotides of nuclear DNA (83). Therefore, nuclear chromatin from the ruptured nuclear envelope forms the backbone of NETs (28, 82), while mitochondrial DNA is found to be decorated on the surface of extracellular NET structure (28). However, mitochondria-derived ROS can mediate oxidative modification of mitochondrial DNA which is potent in induction of type I interferon (28).

Given that nuclear DNA forms the backbone of NETs, the nucleus is the root of nuclear DNA extracellular traps. Thus, nuclear envelope rupture is a hallmark and a prerequisite for release of nuclear chromatin and NET formation (82). Most literature focuses on certain signaling pathways that are involved in NET formation, but without an explanation of cellular events or morphological changes that are required for chromatin extracellular release and NET formation. The existing literature (21, 69, 84, 85) and emerging evidence (82, 86–88) suggest that the following key cellular events and morphological changes are crucial for nuclear chromatin extracellular release and NET formation in neutrophils: 1) nuclear envelope disintegration/rupture, 2) nuclear chromatin decondensation, 3) plasma membrane breakdown. In the following, we will discuss the relevant cellular mechanisms that regulate the above morphological changes in order to provide insights into the cellular and molecular mechanisms, suggesting targets for novel therapeutic strategy on NET-related human diseases.

Emerging evidence and new understanding about NET formation

Nuclear envelope disintegration/rupture is regulated by NETotic lamin kinases

The nuclear envelope consists of outer and inner lipid nuclear membranes (ONM and INM) and nuclear lamina, which is a fibrous protein meshwork underneath the INM that reinforces nuclear envelope integrity (82, 89–91). Nuclear lamin proteins are categorized as lamin A/C or lamin B (82, 90, 91). Lamin B forms thin but highly organized meshworks that are crucial to the integrity of the nuclear envelope (89). Several studies with microscopic analyses indicate the involvement of lamin B in NET formation (82, 88), and lamin B proteins are released with the extruded chromatin from the ruptured nuclear envelope, and thus can be decorated on the surface of extracellular NETs (82).

To explore how nuclear lamin B is involved, our study found that nuclear lamin B remained as intact full-length molecules, but not cleaved fragments, in immunoblot analysis of either platelet activating factor (PAF)- or PMA-stimulated neutrophils (82). Nuclear envelope rupture is a common cellular phenomenon seen in nuclear division during mitosis (92, 93), and in viral nuclear access during viral infection (94). Kinase-mediated lamin disassembly is crucial in these processes. Similarly, further study demonstrated that PKCα-mediated lamin B phosphorylation and disassembly, but not caspase-3-mediated proteolytic cleavage, is responsible for nuclear envelope disintegration/rupture (82). In line with this finding, Remijsen et al reported that NET formation is a caspase-independent process in neutrophils (70). Therefore, PKCα serves as a NETotic lamin kinase that mediates lamin B phosphorylation, driving the consequent nuclear lamina disassembly (Figure 1) (82). The causal role of PKCα-mediated lamina phosphorylation in nuclear envelope disintegration/rupture has been confirmed using several approaches, including pharmacological inhibition or genetic deletion of PKCα, and mutation of PKC consensus phosphorylation sites on lamin B1 (82). In addition, strengthening nuclear envelope by overexpression of nuclear lamin B can also attenuate NET formation vitro and in vivo, and decrease the display of NET-associated proinflammatory cytokines in UVB-irradiated skin of lamin B transgenic mice as compared to the UVB-irradiated skin of wild type mice. All of these results suggested that nuclear envelope integrity is crucial to NET formation.

Figure 1. Kinase-mediated nuclear envelope disintegration/rupture.

1) nuclear translocation of PKCα or CDK4/6 induces 2) PKCα-mediated lamin B phosphorylation or CDK4/6-mediated lamin A/C phosphorylation, which consequently results in 3) disassembly of nuclear lamin B or lamin A/C.

Besides the nuclear lamin B, A-type lamins form thick filament bundles and are also substantial components of nuclear lamina (89, 95, 96). Amulic et al reported the involvement of lamin A/C in NET formation (86, 88). They found that CDK4/6 can control NET formation through modulation of lamin A/C phosphorylation (86), which may result in lamina disassembly and nuclear envelope disintegration (97, 98), although the causal role of lamin A/C phosphorylation in nuclear envelope disintegration was not examined (86). Importantly, the study showed that myeloid-specific Cdk6^−/− mice have impaired neutrophil NET formation in vivo to infection by Candida albicans (86). Taken together, these important studies provide a novel cellular and molecular explanation for nuclear envelope disintegration/rupture during NET formation (82, 86). Therefore, NETotic lamin kinase-mediated nuclear lamina disassembly (82, 86), but not proteolytic cleavage (82), is responsible for nuclear envelope disintegration/rupture (Figure 1). Since these kinases are mainly localized in the cytoplasm, the nuclear translocation of PKCα or CDK4/6 in the early stage induction of NET formation may require the intact actin cytoskeleton (99, 100). Studies from our and other groups indicate that pharmacologic (87, 101, 102) or genetic (103) inhibition of actin assembly or its upstream regulating molecule Rho kinase (104, 105), decreases NET formation, suggesting the involvement of the functional actin cytoskeleton in induction of NET formation (100).

In addition to the role of actin cytoskeleton in nuclear translocation of lamin kinases from cytoplasm, it has been shown that mechanical forces from actin cytoskeleton can also directly promote nuclear rupture in other scenarios (106–108). It is not clear if this is the case in NET formation. However, Neubert et al recently reported that chromatin decondensation and nuclear swelling provides the major physical force to drive nuclear envelope rupture (87). It might be that the lamin kinase-mediated nuclear lamina disassembly provides a molecular basis for disintegration of the highly organized protein meshwork of the nuclear envelope (82, 86). At the same time, nuclear swelling forms the inside-out physical forces (87) that drive the continuous disintegration of the nuclear envelope and enlarge the rupture size until the complete nuclear envelope ruptures, making it possible for extrusion of the decondensed nuclear chromatin, DNA discharge, and extracellular NET formation. Therefore, kinase-mediated nuclear lamina disassembly together with the inside-out physical forces from nuclear swelling, drives nuclear lamina disintegration and nuclear envelope rupture. On the other hand, nuclear chromatin is tethered and associated with the nuclear lamina, and their interaction is important for maintaining their individual organization (109, 110). Therefore, chromatin decondensation may also contribute to nuclear envelope rupture (35, 111), and vice versa.

Decondensation of nuclear chromatin is required for its extrusion from the ruptured nuclear envelope

Nuclear chromatin is tightly packed and surrounded by the nuclear lamina/nuclear envelope. To make it possible for nuclear chromatin extrusion from the nucleus, the thick chromatin needs to be decondensed or solubilized. Studies reported that peptidyl arginine deiminase 4 (PAD4) can convert the positively charged arginine into a neutral citrulline in histones, leading to their loss of positive charges and the disruption of electrostatic interaction in DNA-histone, thus resulting in chromatin decondensation and extracellular release (84, 85, 101, 112). PAD4 has nuclear localization sequences (NLS) (35, 113), but this enzyme mostly resides in cytoplasm in the resting neutrophils (114). Although NLS was thought to mediate the nuclear translocation of PAD4, our unpublished data with time course study indicated that nuclear chromatin extrusion did not occur immediately after nuclear envelope disintegration/rupture, suggesting that chromatin decondensation has not yet been achieved when the nuclear envelope is ruptured. In addition, it is unknown if kinase-mediated nuclear envelope disintegration/rupture (82, 86) is involved in nuclear entry of PAD4. However, it is known that nuclear lamina disassembly can help viral nuclear access during viral infection (94, 115). In addition to arginine citrullination, other post-translational modifications of histone, i.e. histone acetylation, may also contribute to chromatin decondensation during NET formation (116–119). It is known that acetylation (e.g., acetylated histone H4) removes the positive charge of histone, thus altering interaction between histone and negatively charged DNA, resulting in chromatin decondensation and NET formation (117).

Another well-characterized mechanism for chromatin decondensation is neutrophil elastase-mediated histone cleavage (21, 66, 69). Since neutrophil elastase does not have NLS, it is still unclear how neutrophil elastase is translocated into the nucleus. Amulic et al demonstrated that CDK4/6-mediated nuclear envelope disintegration may be important for nuclear entry of neutrophil elastase, as they found that inhibition of CDK4/6 can block nuclear translocation of neutrophil elastase and attenuate NET formation (86). In this vein, our unpublished studies indicated that inhibition of PKCα can also decrease nuclear translocation of neutrophil elastase. PKCα-mediated nuclear envelope disintegration/rupture (82) may enhance the nuclear entry of neutrophil elastase in the early stage of NET formation. Therefore, these results from our and other groups (86) suggest a crucial role of lamin kinase-mediated nuclear envelope disintegration/rupture in nuclear entry of neutrophil elastase that contributes to chromatin decondensation, allowing discharge of solubilized chromatin from the ruptured nuclear envelope and extracellular NET formation.

In addition, pore-forming gasdermin D has recently been shown to be involved in NET formation (120–122). Gasdermin D is classically thought to be an executor of pyroptosis (123–125), during which several caspases (caspase-1, 4, 5, 8, 11) process gasdermin D. Then the N-terminus of gasdermin D translocate to plasma membrane to form pore for interleukin-1β secretion (126). However, the studies from our and other groups demonstrated that NET formation is a caspase-3-independent processes (70, 82). Interestingly, several groups reported that neutrophil elastase and caspase-11 can process gasdermin D, allowing the pore-forming N-terminus of gasdermin D to potentially form pores in membranes of nucleus and granules, as well as the plasma membrane of the cells (120, 121, 127). As suggested by Sollberger et al, the gasdermin D pore in the granule membrane may help release neutrophil elastase from granules, and then further translocate to the nucleus with the help of the gasdermin D pore in the nuclear membrane (120).

All in all, nuclear chromatin decondensation can be achieved either by post-translational modification of histone, i.e. PAD4-mediated citrullination (84, 85, 112), or acetylation (116–119); or by neutrophil elastase-mediated proteolytic cleavage (21, 66, 69). However, there are still many unanswered questions about the process of chromatin decondensation, both PAD4 (128) and neutrophil elastase (129) have been questioned for their role in NET formation. More studies are needed for their involvement and their detailed processes of nuclear translocation in order to contribute to chromatin decondensation. Although formation of a gasdermin pore at the plasma membrane has been very well established (126), while docking of gasdermin D on membranes of the nucleus and granules needs more investigation (130). Therefore, the lamin kinase-mediated nuclear envelope disintegration/rupture might be important not only for nuclear entry of neutrophil elastase in the early stage (86), but also for nuclear chromatin extrusion from the broken nuclear envelope (82) in the late stage of NET formation. Since chromatin is associated with the nuclear lamina, which also regulates chromatin organization and nuclear structure (111), nuclear lamina disintegration thus may also contribute to nuclear chromatin disorganization and decondensation.

Plasma membrane rupture is required for discharge of nuclear chromatin and extracellular NET formation

Although the cell plasma membrane itself is a double layer lipid membrane, the cortical actin cytoskeletal networks are attached to the cell membrane by bivalent membrane-microfilament binding proteins (131–134). The cortical actomyosin networks are beneath the plasma membrane, and the former reinforce the latter (131). The connections of the two and the myosin-generated contractile forces are important for maintenance of plasma membrane integrity (131).

Various studies from our and other groups indicate involvement of the actin cytoskeleton in the early-stage of induction of NET formation (87, 100–102). Our work demonstrated that a functional actin cytoskeleton is important for nuclear translocation of lamin kinases, i.e. PKCα (100, 104, 105). Interestingly, time-lapse microscopy analysis by Thiam et al revealed that disassembly of cortical actomyosin cytoskeleton may be important for plasma membrane rupture during the mid- to late-stage of NET formation (88). It is likely that actomyosin cytoskeleton disassembly results in weakness of the plasma membrane, and then the inside-out expanding force due to the dynamic nuclear chromatin decondensation and nuclear swelling may cause the rupture of the plasma membrane at the sites with the weakness and actomyosin disassembly. Thus, the cortical actomyosin disassembly (88) may orchestrate with expanding forces from chromatin swelling (87) to contribute to plasma membrane rupture, discharge of the nuclear chromatin, and extracellular NET formation.

In addition to DNA extracellular discharge and NET formation, Thiam et al reported that cortical actin cytoskeleton disassembly may also result in membrane microvesicle release (88) at the sites of the plasma membrane with weakness, due to the elevated intracellular swelling pressure (87, 135). In the same vein, we also found the phenomenon of cell membrane budding and microvesicle release in the early stage induction of NET formation in human neutrophils stimulated by PMA (Figure 2). As described by Thiam et al, microvesicles bud from the plasma membrane prior to DNA decondensation, nuclear rounding, and the rupture of nuclear lamina (88). This is in line with our observation (Figure 2) in which the nucleus of neutrophils is still kept as polymorphic lobes, indicating microvesicle budding during the early stage induction of NET formation. These results from our and other groups suggested that NETotic neutrophils can release both membrane microvesicles (13, 136) and nuclear DNA extracellular traps (13, 35, 42, 64, 137, 138). Skendros et al reported that neutrophils from patients with COVID-19 infection can release complement- and tissue factor (TF)-containing NETs (31), contributing to the prothrombotic condition and other severe complications in patients with COVID-19 infection (31). On the other hand, microvesicles released from these neutrophils may also associate with complement and TF (13), and thus be involved in the pathophysiology of COVID-19 infection. Therefore, microvesicles can also be released as by-products from NETotic neutrophils. Both the NETs and microvesicles may be involved in the pathophysiology of various human diseases (13, 35, 136).

Figure 2. Confocal microscopy images of a neutrophil that was stimulated by PMA.

The activated neutrophil buds and releases membrane vesicles in the early stage induction of NET formation, before its polymorphonucleus fused as a single round nucleus.

Concluding remarks

NET formation is a dynamic process featured with nuclear chromatin extracellular release and NET formation in which nuclear envelope disintegration/rupture, nuclear entry of chromatin decondensation enzymes, nuclear chromatin extrusion, plasma membrane rupture, and chromatin/DNA discharge and extracellular NET formation are key sequential cellular events (35, 82). Several recent studies have helped to explain some of the above cellular events (82, 86–88), but more mechanistic studies about key morphologic changes are needed.

In addition, various signaling pathways have been found to be involved in induction of NET formation, i.e. NOX-dependent vs NOX-independent, ROS-dependent vs ROS-independent, suicidal vs viable NET formation, it is unclear how these signaling pathways affect the above key cellular events that are required for extracellular release of nuclear chromatin for NET formation. Furthermore, the diversity of the described signaling pathways makes it difficult to identify targets that control extracellular NET release. Currently, targeting of chromatin decondensation with inhibitors of PAD4 (139, 140) or neutrophil elastase (141, 142) are the major approaches for inhibition of NET formation both for animal studies or clinical trials in human studies. Given the importance of NET formation in various human diseases, more studies are needed to better understand the cellular mechanisms that regulate extracellular DNA NET formation in order to provide insights into novel therapeutic strategies and new treatment in NET-related human diseases.

Abbreviations

C5a

complement factor 5a

CDK4/6

cyclin-dependent kinase 4 and 6

COVID-19

coronavirus disease 2019

GM-CSF

granulocyte/macrophage colony-stimulating factor

HMGB1

High mobility group box 1

INM

inner lipid nuclear membranes

LL-37

Cathelicidin antimicrobial peptides (CAMP) LL-37

MMP9

Matrix metallopeptidase 9

MPO

myeloperoxidase

NETs

neutrophil extracellular traps

NOX-2

NADPH oxidase-2

NLS

nuclear localization sequences

ONM

outer lipid nuclear membranes

PAD4

peptidyl arginine deiminase 4

PKCα

Protein kinase C alpha

PMA

phorbol 12-myristate 13-acetate

ROS

reactive oxygen species

TF

tissue factor

UVB

Ultraviolet B