Keywords
NETs - infectious diseases - autoimmune diseases - metabolic disorders - cancer
Introduction
Neutrophil extracellular traps (NETs) have been recently recognized as a part of the
wide defence strategy of neutrophils. In 2004, Brinkmann et al found that the induction
of NETs by interleukin (IL)-8 and lipopolysaccharide (LPS) supported their formation
during inflammation and bacterial infection, as seen in the course of appendicitis.[1] From this pivotal work, neutrophil alternative death pathway, named NETosis,[2] has been indicated as a further way to act in innate immune defence even after their
death. In recent years, NETs have been suggested to play a role also in non-infectious
diseases, such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), vasculitis,
diabetes, atherosclerosis and cancer.
In the present review, based on papers released on PubMed and MEDLINE up to July 2017
(searched terms in combination: NETs, neutrophils, infections, autoimmunity, cancer,
diabetes, cardiovascular [CV] diseases; articles have also been retrieved through
searches of reference lists and authors' files), we aimed at updating knowledge on
NETs, from their structure to their roles in infectious diseases as well as in CV
disease, autoimmunity, metabolic disorders, and cancer, finally concentrating on future
perspectives in terms of clinical usefulness for therapeutic purposes.
Structure and Formation of NETs
Structure and Formation of NETs
NETs are constituted by extracellular strands of unwound DNA usually in complex with
histones and proteins from neutrophil primary, secondary and tertiary granules, including
components with inflammatory and bactericidal activity, such as neutrophil elastase
(NE), myeloperoxidase (MPO), cathepsin G, lactoferrin, pentraxin 3, gelatinase, proteinase
3 and peptidoglycan-binding proteins. Generally, most of the neutrophil DNA is transcriptionally
inactive and condensed into heterochromatin within the nucleus, with DNA wrapped around
histones to form nucleosomes. Chromatin decondensation begins NETosis and is mediated
by peptidyl arginase deaminase 4 (PAD4) catalyzing the conversion of histone arginines
to citrullines, which weakens histone–DNA binding and consequently unwraps nucleosomes.
A central role in NET regulation is played by intracellular calcium as a second messenger
of neutrophil activation,[3] particularly PAD4 is activated by calcium itself.[4] NE is essential for NET production, too, as it cleaves histones during NET formation
([Fig. 1]).[5]
Fig. 1 Viable neutrophil extracellular trap (NET) formation. After neutrophil activation,
peptidyl arginase deaminase 4 (PAD4) citrullinates some histone arginines to weaken
the tight electrostatic binding between histones and DNA in nucleosomes. The immediate
consequence is that both nuclear and granule membranes dissolved. At this moment,
DNA is decondensed and meets citrullinated histones and granule proteins, which are
all expelled from neutrophils as NETs ready to catch and even kill microbes. The last
step sees the surface membrane coming intact leaving a viable neutrophil without nucleus.
Starting from in vitro studies, distinct activation pathways for NET formation have
been identified and are believed to be active in vivo as well. These pathways include
activation by integrins and toll-like receptors (TLRs)[6]
[7] as signals triggering NETosis in response to bacterial infections.[8] In this setting, L-selectin-mediated signals have also been described to elicit
NETs in vitro.[8] Most in vitro studies to identify mechanisms of formation of NETs used phorbol 12-myristate
13-acetate (PMA), despite this being an artificial trigger bypassing membrane receptors
and their specific intracellular pathways. Actually, the widely described critical
role of nicotinamide adenine dinucleotide phosphate oxidase (NOX) and MPO for NET
formation may be linked to the in vitro activation by PMA. In chronic granulomatous
disease (CGD) patients, a critical role of NOX in NET formation was confirmed in vivo.[9]
To date, three different models of NET formation have been identified. The best described
one is called “suicidal NETosis” and lasts from 2 to 4 hours.[10] After neutrophil activation, NOX increases its activity via the protein kinase C
(PKC)/rapidly accelerated fibrosarcoma (Raf)/mitogen-activated protein kinase ERK
kinase (MERK)/extracellular signal-regulated kinase (ERK) complex leading to cytosolic
calcium intake, PAD4 activation and chromatin decondensation. Once the increase in
cytosolic calcium takes place, PAD4 activation and chromatin decondensation occur.[11] Then, ROS acts as second messengers by promoting the loss of the nuclear membrane.
In this way, chromatin spreads throughout the cytoplasm mixing with cytoplasmic proteins
and granule mediators and is finally released outside the cell through membrane pores
and cellular lysis. Differently, during vital NET generation, neutrophils release
NETs with no loss of nuclear or plasma membrane within 5 to 60 minutes independently
of ROS and Raf/MERK/ERK pathway. This process evolves through different stages characterized
by morphological changes: (1) nuclear envelope growth and vesicle release, (2) nuclear
decondensation and (3) nuclear envelope disruption.[12] Typically, this type of NET production is stimulated by the recognition of stimuli
via TLRs and the C3 complement receptor[6]
[13] as well as via the interaction between glycoprotein Ib in platelets with β2 integrin
in neutrophils by the activation of ERK.[14] Finally, another type of vital, ROS-dependent NET generation has been described,
in which mitochondrial DNA is released instead of nuclear DNA with NET formation within
15 minutes by the recognition of C5a or LPS.[15]
By definition, neutrophils are widely recognized as the only producers of NETs, although
only a limited part of them (around 20% depending on the stimulus) owns this skill
contributing to the rising concept of neutrophils being a heterogeneous class of cells.[16] Anyway, it is not known whether these phenotypic differences could be relevant to
the ability of these neutrophils to undergo NETosis. Actually, the formation of extracellular
traps (ETs) is far known to be restricted to cells of myeloid origin, even if the
classical requirements for NET formation in PMA-stimulated neutrophils—such as respiratory
burst, NE and MPO—are mainly met by neutrophils. However, eosinophils have also been
described to generate a respiratory burst and their peroxidase can convert hydrogen
peroxide into oxidizing halogen derivatives.[17] These eosinophil ETs contain intact eosinophil granules, can catch bacteria and
have been demonstrated in vivo in eosinophil-rich secretions.[18]
[19] Basophils, too, can form ETs killing bacteria, but independently from NOX activity.[20]
[21] On the contrary, mast cells were shown to release NOX-dependent ETs in vitro when
stimulated by either Staphylococcus pyogenes or PMA.[22] In mice, monocytes and macrophages can form ETs when stimulated by PMA.[23]
NETs and Infections
NETs have been demonstrated to own a broad effectiveness against different pathogens,
such as bacteria, viruses, fungi, and parasites. Anyway, experimental data indicate
that NETs trigger is restricted to specific microorganisms. To date, the precise role
of NETs in sepsis has not been completely elucidated. NETs have been suggested to
reduce bacterial spreading, especially in the early phase of infection,[24] but their role appears to be limited: neither the lack of PAD4 nor the treatment
with deoxyribonuclease (DNAse) impacted on bacterial load in animals subjected to
sepsis.[25]
[26] Furthermore, the excessive formation of NETs during sepsis is associated with organ
damage. Interactions among platelets, neutrophils and activated endothelial cells
lead to an increased formation of NETs, which interact with vascular endothelium ultimately
leading to endothelial damage and organ injury in a histone- and MPO-dependent manner.[27] Besides, histones can stimulate TLR2 and TLR4 to enhance the production of proinflammatory
mediators via MyD88 signaling.[28]
[29] Considering all these findings, it is clear that NETs might have a detrimental role
in sepsis. Accordingly, both antibiotic therapy and anti-NETosis conditions, such
as DNAse treatment or abrogation of PAD4, have been described to increase the survival
rate of animals[26]
[30]
[31] and humans[32] with sepsis by reducing NET burden. Microorganisms inducing NET production are indicated
in [Table 1].
Table 1
Microorganisms inducing NETosis
|
Authors
|
Microorganism
|
Microbe peptides inducing NETs
|
Effects
|
Type of NETosis
|
|
Bacteria
|
|
Pilsczek et al[10]
|
S. aureus
|
Leukotoxin GH; Panton-Valentine leukocidin
|
ROS-independent induction with nuclear DNA liberation, TLR2- and C3-dependent
|
Vital
|
|
Mori et al[39]
|
S. pneumoniae
|
EndA; α-enolase
|
ROS-independent induction
|
Suicidal
|
|
Carestia et al,[14] Marin-Esteban et al,[226] Pieterse et al[227]
|
E. coli
|
Unknown
|
Platelet-free or platelet-dependent induction, TLR4-dependent or independent, and
ROS-dependent or independent
|
Suicidal (in absence of platelets) or vital (in presence of platelets)
|
|
Brinkmann et al[1]
|
S. flexneri
|
IcsA and IpaB
|
Induction
|
Suicidal
|
|
Brinkmann et al[1]
|
S. typhimurium
|
Unknown
|
Induction
|
Suicidal
|
|
Möllerherm et al,[49] Gillenius and Urban[228]
|
Yersinia spp.
|
Yops proteins and invasion protein
|
ROS-dependent induction, PI3K signaling, β-integrin pathway
|
Suicidal
|
|
Braian et al[51]
|
M. tuberculosis
|
Adhesins, ESAT/6;
hsp72 is released after M. tuberculosis phagocytosis
|
Phagocytosis-, elastase, and ROS-dependent induction
|
Suicidal
|
|
Seper et al[17]
|
V. cholerae
|
Dns and Xds
|
ROS-dependent induction
|
Suicidal
|
|
Viruses
|
|
Tripathi et al[54]
|
Influenza virus
|
Unknown
|
ROS- and PAD4-dependent induction
|
Suicidal
|
|
Moreno-Altamirano et al[55]
|
Dengue virus
|
Unknown
|
NET inhibition and Glut-1 decreasing glucose capture
|
Unknown
|
|
Saitoh et al[52]
|
HIV
|
Unknown
|
ROS-dependent induction
|
Suicidal
|
|
Raftery et al
[57]
|
Hantavirus
|
Viral particles, Src kinase, and β2 integrin
|
ROS-dependent induction
|
Suicidal
|
|
Fungi
|
|
Byrd et al[229]
|
C. albicans
|
Unknown
|
C3R- and fibronectin-dependent and ROS-independent induction
|
Vital
|
|
Bruns et al[60]
|
A. fumigatus
|
RodA
|
ROS-dependent induction; spores containing RodA do not induce NETs
|
Suicidal
|
|
Rocha et al[61]
|
C. neoformans
|
Unknown
|
ROS and NETs inhibition
|
Unknown
|
|
Parasites
|
|
Waisberg et al[230]
|
P. falciparum
|
Agaphelin
|
Induction through P. falciparum and inhibition through agaphelin
|
Suicidal
|
|
Abi Abdallah et al[65]
|
T. gondii
|
Unknown
|
MEK-ERK-dependent induction
|
Suicidal
|
Abbreviations: C3R, C3 receptor; ESAT, early secretory antigen target; HIV, human
immunodeficiency virus; hsp, heat shock protein; MEK-ERK, mitogen-activated protein
kinase/extracellular signal regulated kinase; NET, neutrophil extracellular trap;
PAD, protein arginine deiminase; PI3K, phosphatidylinositol 3-kinase; ROS, reactive
oxygen species; TLR, toll-like receptor.
Bacteria
A great number of Gram-positive and Gram-negative bacteria have been demonstrated
to trigger NET formation. Brinkmann et al have used Staphylococcus aureus in 2004 in their seminal work as a stimulus to investigate NETosis.[1] Some years later, Pilsczek et al deepened the previous finding by describing a faster,
ROS-independent NET production in response to Staphylococcus-related infection, named vital NET release.[10] S. aureus carries several virulence factors, among which leukotoxin GH and Panton–Valentine
leukocidin can promote NET formation via an oxidative mechanism.[33] Despite this, the bacterium has evolved different mechanisms to escape NET killing.
For example, S. aureus can express pore-forming virulence factors neutralizing neutrophils by inducing necrosis
at the expense of NETosis.[34] Besides, its catalase expression blocks the stack of hydrogen peroxide, thus protecting
the bacterium from intracellular oxidation and NETs.[35] Furthermore, methicillin-resistant S. aureus (MRSA) has been found to express extracellular nucleases for biofilm dispersal and
degradation of NETs. As a proof of this, mice infected by MRSA presented with a higher
mortality with respect to controls infected with a nuclease-deficient strain that
is more susceptible to extracellular killing by neutrophils.[36]
Streptococcus pneumoniae and S. pyogenes can induce NETosis, but they also developed some escape mechanisms. S. pyogenes virulence factor M1 modulates NET formation via an association with fibrinogen ultimately
leading to a complex, which stimulates neutrophils.[37] Furthermore, overexpression of M1 in susceptible strains of S. pyogenes confers resistance to extracellular killing because of the sequestration and neutralization
of the LL37 (also known as cathelicidin), which is a neutrophil antimicrobial peptide
significantly decreasing bacterial colonization.[38] Mutant forms of M1 have been found with a decreased ability of NET induction and
deletion of M1 increases the tendency toward NET killing.[7] Similarly, α-enolase from S. pneumoniae can increase neutrophil-migrating activity and induce their death by releasing NETs;
however, genetic ablation of α-enolase has not blocked NETosis.[39] S. pneumoniae can escape NETs in a passive manner through its polysaccharide capsule reducing NET
binding[40] or trough active strategies. S. pneumoniae can express the DNase EndA, which facilitates the escape from NETs increasing the
virulence in vivo.[41] In a similar way, the nuclease Sda1 in S. pyogenes degrades NETs and confers high virulence in vivo.[42] Some strains of Streptococci can express the protease SpeB, which degrades Sda1 blocking the possibility to escape
NETs, as found in mouse models.[43] Interestingly, Sda1 has been demonstrated to degrade bacterial DNA, thus preventing
the alert of the immune system via TLR9.[44] This witnesses the virulence attributed to Sda1 which does not depend merely on
NET escape, but probably on an intrinsic capacity.
NETs have been described to be significantly induced when neutrophils are stimulated
with the serum of patients suffering from septic shock by Escherichia coli and this is likely to depend on TLR or complement receptor activation.[45] The enteropathogenic strain WS2572 of E. coli can trigger NET formation in neutrophils from the bone marrow of wild-type mice.
Interestingly, NET synthesis is abolished in neutrophils from glutathione reductase
(GSR)-deficient mice suggesting a role for oxidative levels in the formation of NETs.[46] As LL37 is associated with NET synthesis, it may play a relevant role in pathogen
elimination by cooperating with NETs.[47]
Clostridium difficile is likely to stimulate NET formation, which may act to reach the injured areas of
the intestinal epithelium and effectively hinder bacterial dissemination.[48] Brinkmann et al demonstrated that Shigella flexneri is trapped by NETs in vitro and described the ability of DNA-associated elastase
to abolish virulence factors IcsA and IpaB.[1] Salmonella typhimurium has been shown to trigger NETs. S. typhimurium is usually trapped and eliminated by components of NETs, including granular proteins
and H2A histone.[1] In 2015, Möllerherm et al demonstrated that some serotypes of Yersinia enterocolitica could induce NETs in vitro within 1 hour of incubation, but induction diminished
as the incubation time increased, maybe related to the effects of calcium- and magnesium-dependent
nucleases.[49] Vibrio cholerae can induce NETs in vitro after the contact with neutrophils. However, it is able
to release the nucleases Dns and Xds as an evasion mechanism, thus feeding the infectious
process.[17] Finally, the facultative intracellular bacterium Mycobacterium tuberculosis has also been demonstrated to induce NETs when co-cultured with neutrophils. Although
NETs effectively trap and hinder the spread of M. tuberculosis, NET-derived components do not kill them.[50] Braian et al proposed a role for the heat shock protein 72 sequestered in NETs in
the interaction between neutrophils and macrophages during the early innate immune
phase of an infection by M. tuberculosis.[51]
Viruses
Neutrophils are slightly involved in viral infections and only few studies investigated
this issue. After neutrophils recognize human immunodeficiency virus (HIV)-1 nucleic
acids through TLR7 and TLR8, they release ROS inducing NETs, which capture and neutralize
HIV virions by MPO and α-defensins.[52] At the same time, HIV-1 is also able to suppress NET formation being recognized
by CD209 on dendritic cells (DCs) and leading them to the production of anti-inflammatory
IL-10.[52] Influenza A virus stimulates NETs via PAD4, while NET-associated α-defensin-1 blocks
its replication by abrogating protein kinase C pathway.[53] Also LL37 is involved in NET production in response to influenza A virus in vitro,
while arginine-rich H3 and H4 histones are important for viral aggregation and neutralization.[54] A role for NETs has also been demonstrated for dengue virus[55] and respiratory syncytial virus, although for the latter NETs can contribute to
airway obstruction, thus exerting a dual protective and pathogenic role.[56] NETs have also been detected in kidney biopsies and sera of patients infected by
hantavirus.[57] Hantaan virus (HTNV), the prototype member of the genus Hantavirus, uses the β2 integrin complement receptor (CR)3 and CR4 as entry receptors, but can
concurrently induce both ROS production and NET formation through the same β2 integrin
signaling. This systemic NET overflow is accompanied by the production of autoantibodies
towards nuclear antigens. Moreover, HTNV has been found to stimulate human NETs in
a more efficient way and at lower titres compared with vaccinia virus or LPS.[57]
Fungi
Neutrophils play a crucial role in the control of fungal infections, with NETs behaving
as a precious weapon for this purpose. Candida albicans can change from yeast to hyphae; since hyphae are too big to be phagocytosed, extracellular
killing by NETs is a perfect strategy to block this form as well as an efficient way
to kill C. albicans as a single cell.[58] Calprotectin has been identified as a major antifungal agent toward C. albicans and in fact is found to be associated with it in NETs; anyway, direct contact with
the fungus is not required as calprotectin can chelate magnesium and zinc ions requested
for Candida growth.[59]
Aspergillus fumigatus induces NET release in vitro requiring NOX.[35] Although NETs are fundamental for the elimination of A. fumigatus hyphae, they are not induced by spores because of the presence of RodA in the wall
of spore cells.[60] RodA-deficient A. fumigatus conidia induce NETs better than wild-type hyphae, so that RodA may have an inhibitory
effect on NET formation by shielding yet unidentified NET-inducing elements in conidia.
As a further proof of it, A. fumigatus conidia are killed primarily by phagocytosis and not by NETs.[60]
Cryptococcus neoformans can modulate NET production. Particularly, neutrophils incubated with strains whose
capsules contained glucuronoxylomannan and galactoxylomannan have been demonstrated
to produce neither ROS nor NETs, but they did in non-capsulated strains. NET-associated
antimicrobial peptide, such as MPO, elastase and collagenases, are needed to kill
the fungus.[61]
Parasites
The number of studies investigating the role of NETs in immune responses toward protozoan
parasites is increasing, although most of them have been conducted using animal-derived
(e.g. goat or seal) neutrophils. Plasmodium falciparum induces NET formation. NETs entangle parasitized red blood cells and trophozoites
together with antinuclear antibodies are involved in the pathophysiology of malaria
in children and in the development of autoimmune phenotypes.[62]
The two main parasite stages of Leishmania, amastigotes and promastigotes, have been found to induce NETs ex vivo, which acts
as a mechanism of defence against the infection. The induction of NETs is independent
of NOS activity and ROS release. The entanglement of parasites within NETs decreased
the viability of parasites, even if some authors state that the main role for NETs
is immobilization of parasites and control of the infection.[63]
[64]
Toxoplasma gondii can stimulate NET formation. The presence of the parasite in the bloodstream is not
necessary to trigger NET release. NETs kill around 25% of the entangled parasites,
thus controlling the infection. In human blood-derived neutrophils, the production
of NETs has been demonstrated by the activation of the Raf/MEK/ERK pathway in response
to T. gondii.[65]
Finally, some animal parasites, such as Eimeria bovis and Besnoitia besnoiti, were reported to participate to NET formation.[66]
[67]
[68]
[69]
NETs in Cardiovascular Diseases
NETs in Cardiovascular Diseases
Dysregulation of neutrophil function has been recently indicated to play a pivotal
role throughout the pathogenesis of most representative vascular diseases, such as
acute coronary syndrome (ACS), stroke and venous thrombosis. Among these disorders,
ACS and stroke are dramatic complications of advanced atherosclerosis. In the earliest
phases of atherogenesis, neutrophils are recruited by upregulated adhesion molecules
on the dysfunctional endothelial surface, where they exacerbate the oxidative stress
and invade the vessel wall. After extravasation, and later in advanced atherosclerotic
lesions, these cells sustain a vicious cycle leading to chronic inflammation and increased
plaque vulnerability by releasing oxidative enzymes, ROS and chemokines.[70] On the contrary, during venous thrombosis, neutrophils are largely recruited from
activated endothelium through von Willebrand factor (vWF)-, tissue factor (TF)- and
adhesion molecule-mediated pathways accounting for the large part of inflammatory
cells found in thrombus during the early stage of the disease.[71] Thus, after the description of NETosis in 2004, questions raised about its specific
contribution to the pathogenesis of CV disease and, consequently, its feasibility
as marker of disease or therapeutic target.
NET Activity in CV Risk Factors and Early Endothelial Dysfunction
Supporting the pathophysiological link between NETosis and vascular diseases, some
known CV risk factors are associated with exacerbated or dysfunctional NET production.
Diabetic patients showed high plasma levels of NET-associated proteins (e.g. elastase,
mono-oligonucleosomes and double-strand DNA)[72] and impaired NET release in response to bacterial infections.[73] The constitutive NET formation described during hyperglycaemia can blunt their formation
in response to proper stimuli.[74] Interestingly, this phenomenon does not appear as a consequence of an impaired glycaemic
control, rather it seems related to the chronic proinflammatory environment accompanying
diabetes.[75] Recently, age-related CV dysfunction has also been directly linked to NETosis in
mice. While wild-type old mice showed a high rate of heart fibrosis and a decline
in systolic and diastolic ventricular function, these features are considerably reduced
in age-matched PAD4−/− animals, in which NETosis is abolished.[76] Similarly, hypertension has been demonstrated to induce NETosis and platelet recruitment
in wild type, but not in PAD4−/− or DNase-treated myocardium.[76] On the contrary, endothelial dysfunction is widely described as primum movens of atherogenesis. NET-associated proteins have already shown their ability to induce
endothelial toxicity and increase its thrombogenicity. Both circulating cell-free
ds-DNA and histones are released by NETs upon the effect of inflammation and, acting
as danger-associated molecular patterns (DAMPs), they can exacerbate inflammation
itself, thus creating a deleterious vicious cycle at the endothelial level.[77]
[78] In particular, histones have been shown to stimulate the exocytosis of proinflammatory
and procoagulant Weibel-Palade bodies by endothelial cells.[79] Moreover, matrix metalloproteinase (MMP)-9 contained within the NET web can reduce
aortic endothelium-dependent vasorelaxation and trigger endothelial dysfunction through
the activation of MMP-2.[80] Interestingly, interactions between NET and dysfunctional endothelium are not unidirectional:
activated endothelial cells are able to induce NET formation in a C-X-C motif chemokine
(CXCL)8-dependent way and extensive neutrophil co-culture with endothelial cells results
in endothelial damage, which could be abrogated by DNase or through the inhibition
of NOX.[81]
The Role of NETs in Atherosclerosis and Atherothrombosis
NETs have been identified within atherosclerotic lesions and arterial thrombi in both
human samples and atherosclerotic animals.[82]
[83] The pathophysiological relevance of NETosis in atherosclerosis has been underscored
by inhibiting PAD4 in ApoE−/− mice with chloramidine.[84] In particular, chloramidine-treated mice showed reduced vessel inflammation (e.g.
interferon [IFN]-α levels), plaque size and thrombotic attitude.[84] Recently, interesting insights in this field came from a research by Warnatsch et
al.[85] In this study, cholesterol crystals were able to induce NETosis in vivo and NETs
could prime macrophage to produce pro-IL-1β, thus activating a strong pro-inflammatory
Th17 response.[85] In atherosclerotic-prone mice fed with high cholesterol diet, intraplaque NETs co-localized
with cholesterol crystals and macrophages near the necrotic core, while they did not
in more stable zones. The same atherosclerosis model, when lacking NE and proteinase-3,
developed dramatically smaller and less inflamed atherosclerotic lesions with respect
to ApoE−/− control mice, together with lower plasma IL-1β levels.[85] Another mechanism by which NETs are supposed to increase atherogenesis has been
described by Döring et al and involves plasmacytoid DC.[86] In this paper, complexes formed by extracellular DNA and neutrophil-derived proteins
have been shown to increase the plaque burden by stimulating a DC-driven strong type
I IFN response. On the contrary, the absence of these cells leads to decreased atherogenesis
and reduced inflammatory response.[86]
Preclinical studies specifically investigating the effect of NET modulation on atherothrombosis
are scarce and the current knowledge largely derives from immunohistochemical analysis
of human specimens. The blockage of neutrophil-derived nucleosomes by anti-histones
antibodies treatment has been demonstrated to prolong the time to occlusion in the
ferric chloride arterial injury model.[87] This effect was completely reversed in elastase/cathepsin G double knockout mice;
under a mechanistic point of view, nucleosomes are able to facilitate thrombosis by
allowing NE to digest the TF pathway inhibitor.[87] Moreover, neutrophil-derived serine proteases and nucleosomes, two main components
of NETs, may contribute to arterial thrombosis in the context of sterile inflammation
supporting dramatic atherosclerosis complications, such as myocardial infarction and
ischemic stroke. In a mouse model of myocardial ischaemia/reperfusion (I/R) injury,
NETosis targeting via DNase I injection has shown cardioprotective features by reducing
both plasma levels of nucleosomes and citrullinated histone 3 (citH3) presence at
the site of injury.[88] Similar features have been found after cardiac ischemic damage in PAD4−/− mice.[88] Finally, concurrent DNase I and recombinant-tissue plasminogen activator (r-TPA)
therapy has been demonstrated to reduce infarct size, no-reflow area and postischaemic
left ventricle remodelling in an animal myocardial I/R injury model, whereas these
beneficial effects have not been observed in rats treated with DNase and r-TPA alone,[89] thus suggesting a potential role for anti-NET agents as therapeutic strategy during
ischaemic heart diseases.
In human coronary specimens from infarcted patients, NETs were most frequently found
in the early stages of thrombus evolution (e.g. fresh and lytic thrombi), while these
cells missed in advanced, organized thrombi.[83] Recently, a study analysing coronary thrombectomy from patients with ST-elevated
myocardial infarction showed that the thrombus NET burden correlates in a positive
fashion with the infarct size and negatively with ST-segment resolution.[90] In the same work, the plasma collected from the culprit lesion site contained higher
concentrations of NET-related biomarkers when compared with femoral samples from the
same patients as well as DNase activity at the culprit lesion site negatively correlated
with thrombus NET burden and infarct size. Moreover, recombinant DNase accelerated
the coronary thrombi lysis in vitro.[90] In particular, Stakos et al recently demonstrated that neutrophils at culprit lesion
site can release thrombogenic signals through NET formation and subsequent delivery
of active TF.[91]
Even if most of the currently available evidence links NETs to atherothrombosis, they
may also participate to earlier superficial erosion of the plaque by inducing endothelial
cell apoptosis.[92] Of importance, biomarkers of NETosis already showed to be positively associated
with the severity of atherosclerosis and to predict future CV events ([Table 2]). Under this point of view, Borissoff et al gave fundamental insights by correlating
the disease severity in patients with coronary disease assessed by computed tomographic
angiography and markers of NETosis.[93] Although these data underline the importance of such death mechanism in atherosclerosis-related
diseases, more studies are needed to overcome the low specificity of some NET-related
biomarkers (e.g. histones and double strain [ds]DNA).
Table 2
Recent studies investigating NET biomarkers in CV diseases
|
Author
|
Year
|
Patients
|
Biomarkers
|
Results
|
|
Atherothrombosis
|
|
Shimony et al[231]
|
2010
|
16 patients with STEMI and 47 healthy subjects
|
dsDNA
|
dsDNA levels were significantly higher in patients compared with controls (p = 0.001) and positively correlated with levels of CK and TnT (r = 0.79 and 0.65, p < 0.001 and p = 0.006, respectively)
|
|
Borissoff et al[93]
|
2013
|
282 patients with suspected coronary heart disease undergoing coronary CTA were grouped
according to the presence and severity of CAD
|
dsDNA, nucleosomes, citH4 and MPO-DNA complexes
|
dsDNA, nucleosomes and MPO-DNA complex levels were significantly higher in patients
with severe CAD (p = 0.003, p < 0.001 and p < 0.05, respectively) or abundant coronary artery calcification (p < 0.001 for all) with respect to healthy controls. Their levels correlate with the
severity of luminal stenosis (p ≤ 0.001 for all) and with number of diseased coronary artery segments (p ≤ 0.001 for all).
Baseline values higher than the total group median of dsDNA (OR, 3.12; 95% CI, 1.27–7.63;
p = 0.013), nucleosomes (OR, 2.59; 95% CI, 1.09–6.14; p = 0.030) and MPO–DNA (OR, 3.53; 95% CI, 1.38–9.03; p = 0.009) were significantly associated with the occurrence of MACEs
|
|
Cui et al[232]
|
2013
|
137 ACS patients (51 with unstable AP, 37 with NSTEMI, and 49 with STEMI), 13 stable
AP patients, and 60 healthy controls
|
dsDNA
|
ACS patients showed higher dsDNA levels compared with stable AP patients and control
group (p < 0.05 for both). Significant differences in dsDNA concentrations were observed in
ACS group among unstable AP, NSTEMI, and STEMI sub-groups (p < 0.05 for all).
dsDNA levels were different among ACS patients divided into three groups according
to Gensini score with increasing levels of dsDNA concurrent with increasing Gensini
score (p < 0.05, for all)
|
|
Mangold et al[90]
|
2015
|
111 patients with STEMI undergoing PCI
|
Nucleosomes and dsDNA
|
Nucleosomes and dsDNA levels were significantly higher at the culprit lesion site
than to the femoral artery (p = 0.0002 and p < 0.0001, respectively)
|
|
Helseth et al[233]
|
2016
|
30 patients with CAD undergoing PCI (20 with STEMI and 10 with stable AP)
|
dsDNA and nucleosomes
|
dsDNA and nucleosome levels were higher in patients with STEMI compared with patients
with AP (p < 0.03 for both). dsDNA significantly correlated with peak TnT and CK-MB at day 5
(p = 0.03) and with lesion size assessed by MRI at days 5 and 7 (p = 0.01 and 0.04, respectively). Nucleosomes correlate with infarct size at day 5
(p = 0.02)
|
|
Venous thrombosis
|
|
van Montfoort et al[106]
|
2013
|
150 patients with symptomatic DVT and 195 patients with clinical suspicion of DVT
in whom the pathology was excluded by US examination
|
Nucleosomes
|
Nucleosome levels were significantly higher in patients with DVT (p < 0.001) and positively correlated with neutrophil activation in both case and control
samples (p < 0.001 for both). An increase in nucleosome levels >80th percentile carried an increased
risk of DVT than levels ≤80th percentile after adjustment for potential confounders
(OR: 3.0, 95% CI: 1.7–5.0)
|
|
Diaz et al[107]
|
2013
|
47 patients with symptomatic DVT, 28 patients with clinical suspicion of DVT not confirmed
by US examination, and 19 healthy volunteers
|
dsDNA
|
dsDNA levels were higher in DVT group with respect to both negative control groups
(p < 0.01 for both). dsDNA levels showed a positive correlation with CRP (p < 0.01), D-dimer (p < 0.01), and vWF (p < 0.01) and the Wells score (p < 0.01). A negative correlation was found with ADAMST13 (p < 0.01)
|
Abbreviations: ADAMST13, a disintegrin and metalloproteinase with thromboSpondin-1 motifs (13th member of the family); CAD, coronary artery disease;
citH4, citrullinated histone H4; CK-MB, creatine kinase, muscle and brain; CRP, C-reactive
protein; CTA, computed tomography angiography; CV, cardiovascular; ds, double strain;
DVT, deep venous thrombosis; HR, hazard ratio; LV, left ventricle; MACEs, major cardiovascular
events; MI, myocardial infarction; MPO, myeloperoxidase; MRI, magnetic resonance imaging;
NSTEMI, non–ST-elevated myocardial infarction; N/L, neutrophil to lymphocyte; OR,
odds ratio; PCI, percutaneous coronary intervention; RR, relative risk; SINTAX, Synergy
between Percutaneous Coronary Intervention with Taxus and Cardiac Surgery; STEMI,
ST-elevated myocardial infarction; TnT, troponin T; US, ultrasound; vWF, von Willebrand
factor.
NETs have also been investigated in the setting of acute ischemic stroke (AIS) highlighting
the role of thrombo-inflammation as a pivotal player in the pathophysiology of ischaemic
stroke[94] along with thrombosis and inflammation, which constitute a loop of bidirectional
regulation contributing to ischaemic damage in brain or other tissues.[95] Recently, Vallés et al described for the first time that three markers of NETs—dsDNA,
nucleosomes and citH3—are significantly elevated in patients with AIS when compared
with healthy subjects; in particular, the greatest proportional increase was found
for the most specific NET marker citH3.[96] Levels of the above-mentioned NET markers have been correlated with stroke severity
at onset and discharge by the clinical National Institutes of Health Stroke Scale
and modified Rankins scale scores as well as by significant elevations of citH3, dsDNA
and nucleosomes. Authors also found that citH3 and dsDNA levels were higher in patients
with cardioembolic stroke, this being related to a higher inflammatory activation.
This issue was in touch with the significant increase in NET markers for patients
with a history of atrial fibrillation (AF), too. Finally, citH3 has been found elevated
especially in older patients, with higher fasting glucose, and with prior AF and independently
associated with all-cause mortality at 1-year follow-up.[96] These results indicate that citH3 might represent a useful prognostic marker in
patients with AIS, warranting new researches for neuroprotective therapies in this
field. The results of other studies investigating NETs in AIS are listed in [Table 3].
Table 3
Studies investigating NET biomarkers in acute ischemic stroke
|
Author
|
Year
|
Patients
|
Biomarkers
|
Results
|
|
Rainer et al[234]
|
2003
|
88 patients with stroke-like symptoms presenting to the ED
|
nDNA
|
nDNA concentrations within 3 h of symptom onset were higher in died patients than
in those who survived at discharge (p = 0.03) as well as in died patients with NIHSS scores >8 who survived to 6 mo (p = 0.002). nDNA concentrations correlated with the volume of cerebral hematoma (p = 0.03). nDNA concentrations predict 6-mo mortality (OR: 1.6, 95% CI: 1.1–2.4; p = 0.03) and 6-mo RS score >2 (OR: 1.8, 95% CI: 1.0–3.3; p = 0.05)
|
|
Geiger et al[235]
|
2006
|
63 patients with stroke observed daily during the first week
|
Nucleosomes
|
In patients with BI score ≥50, the increase in nucleosomes is prolonged until day
5. Patients with BI score <50 showed a steeper initial increase with a maximum on
day 3. Both days after stroke and BI score significantly influenced nucleosome concentrations
(p < 0.001 for both). Nucleosome concentration showed a significant correlation on day
3 with infarction volume (p = 0.001)
|
|
Lam et al[236]
|
2006
|
44 patients aged ≥18 y presenting to the ED with a stroke-like syndrome but negative
neuroimaging results
|
nDNA
|
Patients with post-stroke mRS grades 3–6 have been shown with a nDNA concentration
significantly higher than that of patients with post-stroke mRS grades 0–2 (p = 0.01). nDNA concentration could predict post-stroke morbidity and mortality in
patients with negative neuroimaging
|
|
Geiger et al[237]
|
2007
|
63 patients with stroke observed daily during the first week
|
Nucleosomes
|
Levels of nucleosomes at days 3 and 6 correlated significantly with initial BI (p = 0.0023 and 0.0284, respectively) and with infarction volume only at day 3 (p = 0.0001). Strong correlations have been shown between BI at admission and BI at
discharge and between BI at admission and infarction volume (p < 0.0001 for both). In patients with initially severe defects (BI <50), nucleosomes
at day 3 have been found to be prognostically relevant (p = 0.014). In multivariate analysis, nucleosomes and BI at admission showed independent
prognostic relevance (p = 0.039)
|
|
Tsai et al[238]
|
2011
|
50 AIS patients and 50 control subjects
|
nDNA and mDNA
|
Levels of nDNA and mDNA were higher in patients with AIS than in controls (p < 0.05). Elevated circulating nDNA in plasma persisted until 1 mo after AIS. Levels
of nDNA positively correlated with the clinical severity of stroke according to NIHSS
score
|
|
Hirose et al[239]
|
2014
|
49 critically ill patients admitted to ICU, of whom 8 with stroke
|
DNA, citH3
|
DNA has been found elevated in patients with stroke with respect to controls. citH3
has been detected in blood smears by immunofluorescence
|
|
Thålin et al[240]
|
2016
|
31 patients with ischemic stroke
|
DNA, citH3
|
Patients with concurrent hsTnT elevation revealed cerebral micro-thrombosis with citH3
in thrombi in a higher rate than controls (p < 0.001). citH3 correlated positively with thrombin–antithrombin complex (p = 0.004) and soluble P-selectin (p < 0.001)
|
|
Vallés et al[96]
|
2017
|
243 patients with AIS followed up for 12 mo after the event
|
dsDNA, nucleosomes, citH3
|
dsDNA, nucleosomes and citH3 were significantly higher among patients with AIS compared
with healthy subjects (p < 0.05; p < 0.001 for the latter). These parameters were increased in patients who were older
than 65 y (p < 0.001), in those with a history of AF (p = 0.013 for dsDNA, p = 0.007 for citH3, p = 0.02 for nucleosomes), CE stroke (p < 0.05 for dsDNA and citH3), high glucose levels (p < 0.05 for all markers) and severe stroke scores at admission (p < 0.001 for all markers) and discharge (p < 0.001 for citH3 and nucleosomes, p = 0.038 for dsDNA). In multivariate analysis, elevated levels of citH3 at onset was
independently associated with AF (OR: 6.704, 95% CI: 1.4–32.1; p = 0.017) and with all-cause mortality at 1-year follow-up (OR: 7.055, 95% CI: 1.631–30.50;
p = 0.009)
|
|
Laridan et al[241]
|
2017
|
68 ischemic stroke patients undergoing endovascular treatment
|
DNA, citH3
|
citH3 was observed in almost all thrombi and co-localized with extracellular DNA released
from neutrophils. citH3 was more abundant in thrombi of CE origin compared with other
etiologies (p < 0.05). Older thrombi contained significantly more neutrophils and citH3 compared
with fresh thrombi (p < 0.001 and p < 0.05, respectively)
|
Abbreviations: AF, atrial fibrillation; AIS, acute ischemic stroke; AUC, area under
the curve; BI, Barthel index; CE, cardioembolic; CI, confidence interval; citH3, citrullinated
histone H3; CRP, C-reactive protein; ds, double strain; ED, emergency department;
hsTnT, high sensitive troponin T; mRS, modified Rankin scale; NIHSS, National Institutes
of Health Stroke Scale; m, mitochondrial; n, nuclear; NET, neutrophil extracellular
trap; NSE, neuron-specific enolase; OR, odds ratio.
NETs and Venous Thrombosis
Although arterial and venous thromboses are different syndromes with different main
causes, they found in NETs a common pathogenic pathway. Several key components of
NETs, such as nucleic acids, histones and enzymes, have shown venous procoagulant
features.[97]
[98]
[99] Histone infusion leads to vWF release and has been found to accelerate the thrombus
formation in inferior vena cava (IVC) stenosis model of deep venous thrombosis (DVT).[100] Thrombi from the same animal models are characterized by an important presence of
NETs associated with vWF, especially in the earliest stages.[100]
[101] NETs interact with vWF through histone A1 domain,[102] but they can also bind other thrombosis-related proteins (e.g. fibronectin) containing
several DNA-binding domains.[101] Not only vWF but also TF sustains the strong relation between venous thrombosis
and NETs. During the generation of NET, neutrophils produce both TF and NE. NE is
critical to further increase TF activity by the cleavage of TF inhibitory molecules.[71]
[91] Thus, after platelet and neutrophil activation by procoagulant factors, the latter
generates NETs acting as trap and framework for thrombus elements, such as red blood
cells, leukocytes, platelets and activated coagulation factors.[71]
[103] The crucial contribution of NETosis in venous thrombosis has been underlined by
PAD4−/− mice, in which the lack of NETs results in fewer thrombi after IVC stenosis compared
with wild-type ones; similar effects have also been demonstrated by the administration
of DNase I.[104] Although some controversies still remain,[105] these results encouraged scientists to shift their attention from preclinical setting
to the bedside. In 2013, two papers showed an association between increased plasma
markers of NETs and DVT ([Table 2]). The first article stated that in patients with DVT circulating nucleosomes and
elastase-α1-antitrypsin complexes, levels are increased with respect to patients with
first clinical suspicion of DVT, which has been then excluded by ultrasonography.[106] The second study focused on plasma DNA level showing higher concentrations of this
marker in DVT patients and its correlation with D-dimer, Wells score and MPO, the
third indicating neutrophil as the source of the nucleic acid.[107] Recently, the immunohistochemical analysis of thrombi at different stages of development
from human surgery or autopsy showed high presence of NETs (indicated by the association
of citrullinated histone H3 with MPO and DNA) in thrombi during the phase of organization.[108]
NETs in Autoimmune and Autoinflammatory Diseases
NETs in Autoimmune and Autoinflammatory Diseases
Systemic autoimmune diseases develop as multistep processes through a complex interplay
between genetic and environmental factors leading to cellular damage and the consequent
exposure of immune cells to autoantigens. The imbalance of different cell death mechanisms
(apoptosis, necroptosis, pyroptosis, NETosis and autophagy) has been proposed as a
critical source of modified and/or externalized autoantigens.[109] Especially nuclear material released from NETs seems to be more immunogenic than
the apoptotic one. Both native and oxidized self-DNA bound to NETs activate DCs to
synthetize IFN-α in a TLR-dependent manner.[110]
[111] In mice, the immunization with NET-loaded DCs promotes the development of autoimmunity
better than apoptotic neutrophil debris.[112] NETs also increase T-cell response to antigens and activate B cells to induce immunoglobulin
(Ig) class switching and antibody production.[113] Once externalized, oxidized DNA is also more resistant to degradation and this behaviour
contributes to sustain a dysregulated immune response.[114] In addition, NET-mediated activation of the inflammasome further amplifies the inflammatory
response through a feed-forward loop. The inflammasome stimulation triggers synthesis
and release of IL-18 and IL-1β, which in turn induces NET formation.[115] Activation of classic and alternative pathways of complement system as well as coagulation
cascade is additional immunogenic mechanisms linking NETs to autoimmune/autoinflammatory
diseases.[116] Noteworthy, many of the proteins found in NETs are recognized as major autoantigenic
targets in rheumatologic diseases: dsDNA and histones in SLE, vimentin and enolase
in RA, MPO and proteinase-3 in vasculitis associated with anti-neutrophil cytoplasmic
antibodies (ANCAs). Different autoantibody profiles may then be influenced by the
cargo protein within NETs and detailed analysis revealed differences in protein content
and posttranslational modifications associated with different autoimmune diseases.[117]
[118]
[119]
Systemic Lupus Erythematosus
Neutrophils from patients with SLE showed various abnormalities in their phenotype
and function.[120] Circulating levels of apoptotic neutrophils, which may provide excess autoantigen
such as dsDNA, are increased in patients with SLE and correlate with disease activity.[121] Furthermore, patients with SLE are characterized by a distinct neutrophil subpopulation
known as low-density granulocytes (LDGs).[122] Those cells are prone to release proinflammatory cytokines and show enhanced NET
formation. NETs released by LDGs contain high levels of autoantigens and immunostimulatory
molecules, such as LL37, MMP-9, and α- and β-defensins.[123] LDGs are also enriched of dsDNA and oxidized nucleic acids, which are strong inducers
of IFN-α and NOD-like receptor family pyrin domain-containing (NLRP)3 inflammasome.[115]
[124] Finally, patients with SLE exhibit impaired NET clearance correlating with disease
activity.[125] As a result of overproduction and defective clearance of NETs, the presentation
of autoantigens to autoreactive B cells is enhanced.[116]
[126] A leading role in SLE pathogenesis may be played by an alternative form of NETs,
called mitochondrial DNA NETs. In fact, more severe forms of SLE have been observed in mice after injection of
oxidized mitochondrial DNA and in humans carrying NOX-deficient genes (typically in
CGD).[124]
[127] Although the role of NETs in SLE requires further investigations, their high levels
found in the skin, kidney and bone marrow support a direct role in SLE-associated
organ dysfunction[123] and preliminary clinical studies support a potential association between circulating
levels of NETs and disease activity ([Table 4]).[116]
[123]
[125]
[128]
[129]
[130]
[131]
Table 4
Clinical evidence linking circulating NETs to clinical activity of autoimmune diseases
|
Author
|
Year
|
Disease
|
Patients
|
Outcome
|
Result
|
|
Hakkim et al[125]
|
2010
|
SLE
|
Lupus nephritis vs. healthy controls
|
Activity index on renal biopsy
|
Poor NET degradation was associated with lupus nephritis and increased serum levels
of anti-dsDNA antibodies
|
|
Villanueva et al[123]
|
2011
|
SLE
|
III or IV class GN
|
Activity index on renal biopsy
|
Patients with class IV GN had higher activity index (% of netting neutrophils) and
higher circulating levels of anti-dsDNA antibodies
|
|
Leffler et al[116]
|
2012
|
SLE
|
Patients with disease remission and flare vs. healthy controls
|
Disease activity
|
Low NET degradation was associated with complement activation/deposition, high levels
of circulating autoantibodies and renal involvement (GN)
|
|
Leffler et al[128]
|
2013
|
SLE
|
69 patients followed up for a median of 784 d
|
Disease activity
|
Decreased ability to degrade NETs was associated with clinical manifestations in SLE
according with the SLEDAI-2K score
|
|
Nakazawa et al[129]
|
2014
|
MPA
SLE
|
38 patients with MPA vs. 23 SLE vs. 8 control subjects
|
BVAS
SLEDAI-2K score
|
Both MPA and SLE patients showed reduced NET degradation. In MPA patients, this ability
correlated with disease activity, while no correlation was shown between NETs and
disease activity (SLEDAI-2K score) in SLE patients
|
|
Zhang et al[130]
|
2014
|
SLE
|
54 patients vs. 43 control subjects
|
Lupus nephritis
|
High circulating levels of cfDNA (marker of NET dysregulation) was found in patients
with SLE and were associated with markers of renal injury, such as 24-h urinary protein
content (r = 0.350; p = 0.013), serum albumin (r = − 0.500; p < 0.001), and creatinine clearance (r = − 0.354; p = 0.044)
|
|
Pérez-Sánchez et al[131]
|
2017
|
RA
|
106 patients vs. 40 control subjects
|
RF, ESR, CRP,
NO, cIMT
|
RA patients exhibited enhanced NET generation and impaired DNase activity. Furthermore,
NETosis-derived products, such as cfDNA, correlated with autoimmune parameters, inflammatory
mediators, oxidative stress markers as well as early atherosclerosis
|
Abbreviations: BVAS, Birmingham vasculitis activity score; cfDNA, cell-free DNA; cIMT,
carotid intima media thickness; CRP, C-reactive protein; dsDNA, double-stranded DNA;
ESR, erythrocyte sedimentation rate; GN, glomerulonephritis; NET, neutrophil extracellular
trap; NO, nitric oxide; MPA, microscopic polyangiitis; RA, rheumatoid arthritis; RF,
rheumatoid factor; SLE, systemic lupus erythematosus; SLEDAI-2K, Systemic Lupus Erythematosus
Disease Activity Index 2000.
Rheumatoid Arthritis
In RA, activated neutrophils are the most abundant cells in the synovial fluid. In
addition, a wide number of anti-citrullinated proteins/peptides antibodies (ACPA)
is produced in RA, representing specific disease markers.[132] Both endogenous and exogenous antigens become target of ACPA after deimination (or
citrullination), which is a post-translational modification largely catalysed by the
PAD2 and 4, usually overexpressed in RA patients.[133]
[134] A major contribution to the citrullination comes from NET generated from activated
neutrophils, especially those belonging to the LDG subpopulation.[118]
[135] Furthermore, deiminated histones have been recognized as key mechanism leading to
ACPA generation in RA patients, especially those with Felty syndrome.[59]
[136] Noteworthy, the ectopic lymphoid structures localized in the RA joint synovium may
also contribute to the NET generation. They represent functional structures supporting
the clonal selection of autoreactive B cells and then their differentiation to plasma
cells producing antibodies against citrullinated antigens.[137]
[138]
[139] Therefore, a delay in the clearance of NETs might form a reservoir of citrullinated
antigens, which sustain the autoimmune response in RA as already described for SLE.[125]
ANCA-Associated Vasculitis
ANCA-associated vasculitis (AAV) is referred to as a group of pauci-immune vasculitis
characterized by neutrophil-rich necrotizing inflammation of small vessels and the
presence of ANCAs. In this context, NETs have been recently found at the sites of
vasculitic lesions (kidney and skin) and in thrombi, both as co-localizations of DNA
and granule proteins and as mere citrullinated histones.[112]
[140]
[141]
[142]
[143]
[144]
[145] Some cross-sectional studies compared NET levels during remission and active disease,
although results remain inconclusive.[146]
[147]
[148] Neutrophils from patients with AAV are less prone to undergo apoptosis and show
spontaneous NET formation. Even though the high fraction of LDGs observed in AAV may
explain this behaviour, normal-density neutrophils have also been found to spontaneously
release more NETs as compared with healthy blood donors.[149] A growing body of data indicates ANCAs not only as neutrophil activators but also
as promoters of NET generation. As neutrophil activation is epitope specific, epitope
specificity and affinity are found increased during active disease.[150]
[151]
[152] In turn, the overproduction of NETs enhanced the exposition of epitopes (MPO end
proteinase-3), further perpetuating the generation of ANCAs.[129]
[140] Alongside, elevated levels of NETs in AAV patients may be explained by a reduced
NET clearance as confirmed by in vitro experiments.[129]
Other Autoimmune Diseases
Recent evidence links NETs and antiphospholipid (aPL) antibody syndrome (APS). High
levels of dsDNA and NETs have been found in patients with APS and the serum of those
patients display defective NET degradation.[153]
[154] More specifically, aPL antibodies from patients with APS may induce the release
of NETs from control neutrophils and especially LDGs.[154] Also, thrombi from mice treated with APS IgG have been recently found to be enriched
of citrullinated histone H3, thus indicating a role for NETs in thrombotic complications
of APS.[155] Further supporting a direct role in thrombosis, growing data linked NETs to preeclampsia,
also in aPL-negative patients.[156]
[157]
[158] Finally, NETosis has also been observed in psoriatic skin lesions. By inducing the
expression of human β-defensin-2 in keratinocytes, NETs amplify the local inflammation
leading to DC activation and consequent development of Munro's abscess.[159]
[160] Finally, a direct correlation between the amount of NETs in the peripheral blood
and disease severity has also been demonstrated.[160]
Gout and Inflammatory Bowel Diseases
Actually, very little is known about the link between NETs and gout and inflammatory
bowel diseases (IBDs), such as Crohn's disease (CD) and ulcerative colitis (UC). In
gout, NETs have been described not only to feed inflammation,[161] but also to regulate the inflammatory process and block gout episodes.[162] NET generation is responsible for the reduction of neutrophil density, then DNA
nets encapsulate monosodium urate crystals protecting them from further phagocytosis,
and finally NET-derived proteases block cytokines.[163]
[164]
NET formation has been poorly studied in IBDs. As ROS levels are elevated, it is likely
that neutrophils could produce NETs. In UC, NETs have been observed and correlated
with inflammation by proteomic studies, but more studies are warranted to clarify
the NET involvement in both CD and UC.[165] In 2016, He et al evaluated the role of NETs in IBDs. The authors found that NETs
were generated by peripheral blood neutrophils from patients with active IBD; however,
stimulation with sera from patients with active disease also induced significantly
NET release on neutrophils isolated from healthy subjects. Moreover, NETs along with
phosphatidylserine (PS) exposure on platelets, leukocytes and endothelial cells are
involved in the hypercoagulability characterizing active IBD. The researchers also
proved an inhibitory effect of lactadherin and DNase I on PS and NETs, separately,
suggesting new targets for IBD drugs in next studies.[166]
NETs and Lung Diseases
In the last decade, the formation of NETs has drawn great attention concerning lung
diseases. Components of NETs have been shown to damage both epithelial and endothelial
cells as well as connective tissue worsening the lung pathology.[167] Indeed, NETs have been identified in the lung of patients suffering from cystic
fibrosis (CF), transfusion-related acute lung injury (TRALI), asthma, chronic obstructive
pulmonary disease (COPD), and in lungs infected with bacteria, virus, or fungi.[168] In these pulmonary diseases, NETs have been described as emerging pathophysiological
players of potential therapeutic interest.
Cystic Fibrosis
In CF, patients with high levels of free DNA in the sputum have been shown with a
diminished lung function as compared with those with mild disease, because the airway
obstruction is mainly a consequence of the amount of NETs and DNA.[161] Marcos et al recently confirmed this finding, showing that free CF airway DNA levels
correlated with pulmonary obstruction both in CF patients and mice.[162] The importance of NETs in CF is highlighted by the fact that the elimination of
free DNA from patient's airways is considered as an important therapeutic option.
In fact, in both early and mild stages of the disease, NETs can provide extracellular
antibacterial and antifungal host defences. At this time, recombinant inhaled DNase
should be used more cautiously, being potentially responsible for the delivery of
encaptured pathogens. Differently, in moderate to severe stages of CF, the amount
of mucus and DNA was responsible for airway obstruction, which can be efficiently
resolved by DNase by cleaving DNA traps.[162]
[169] Moreover, isolates of Pseudomonas aeruginosa can trigger a great respiratory burst and NET release in CF.[170] P. aeruginosa-mediated NET formation was found responsible for the bactericidal permeability-increasing
(BPI) protein cleavage by P. aeruginosa elastase, suggesting a novel mechanism in the development of autoimmunity to BPI.[171] Moreover, the authors also provided a role for autoimmunity in CF disease severity,
as autoantibody levels have been associated with diminished lung function.
Chronic Obstructive Pulmonary Disease
Neutrophil elastase has been detected in the airway mucosa of COPD patients during
severe exacerbations showing a proinflammatory role via the secretion of CXCL8, which
is a powerful NET inducer.[172] In the sputum from acutely exacerbated COPD patients, NETs and NETotic neutrophils
have been found in a great amount by confocal laser microscopy and electron microscopy;
an abundance of NE and citH3 has also been demonstrated.[173] These findings witness that NETosis can be considered as a part of COPD pathology,
relevant for new therapeutic options. Anyway, NETs have been described in the airways
of stable COPD patients as a marker of neutrophils in the sputum.[174]
[175] In particular, a positive correlation between the abundance of NETs in the sputum
of COPD patients and disease severity has been described; in fact, more than 90% of
exacerbated COPD patients presented with a higher number of NETs in their sputum with
respect to stable COPD patients. Indeed, the NET amount directly correlated with the
disease severity in terms of airflow limitation.[176] Recently, levels of NETs in the sputum of COPD patients have been directly associated
with the severity of the disease and the number of exacerbations as well as with the
loss of microbiota diversity and impaired ex vivo neutrophil phagocytosis.[177] In this view, NETs could be an interesting therapeutic target in the future, considering
that the only effective drugs are currently long-acting bronchodilators, which do
not affect inflammation.
Asthma
Asthma has been classically considered as an eosinophilic disease. However, recent
data claim that some asthmatic patients show an important neutrophilic inflammation
in the lungs. Patients with “neutrophilic” asthma have shown a reduced response to
the classical therapy with glucocorticoids, which in turn aggravate local inflammation
by increasing neutrophil survival.[178]
In addition, glucocorticoid administration to neutrophilic asthmatics could aggravate
lung inflammation, since glucocorticoids can prolong neutrophil survival. Since the
hypothesis of autoimmune involvement in asthma has gained great interest recently,
NETs have been demonstrated as key players in the stimulation of airway epithelial
cells to produce autoantigens, especially in severe asthma, as antibodies against
NE or MPO attenuated these effects.[179] Dworski et al have shown that eosinophils in the airways of atopic asthmatic individuals
could release eosinophil ETs and co-localized with eosinophil granule proteins, such
as major basic protein and eosinophil cationic protein.[180] In this case, DNA was of mitochondrial origin, and not nuclear.[181] Interestingly, allergens did not show any increase in eosinophil ET or NET formation
in the airways of asthmatic patients.[180] Recently, eosinophils from asthmatic mice have been demonstrated to release eosinophil
ETs co-localizing with eosinophil peroxidase aggravating pulmonary impairment, which
was reversed by DNase therapy.[182] Recombinant human DNase treatment improved resistance and decreased oxidative stress
in the lungs of asthmatic mice.[183] In light of this, a combined use of recombinant human DNase therapy along with inhaled
glucocorticoids may provide a reduction in sputum viscosity and improve the quality
of life and prognosis of these patients.
Transfusion-Related Acute Lung Injury
TRALI is the leading cause of blood transfusion-related death developing within 6
hour of transfusion and presenting with hypoxemia, respiratory distress, and pulmonary
infiltrates. In 2012, Thomas et al demonstrated that NETs form during TRALI both in
humans and in mice and that their degradation by DNase 1 inhalation improved the condition
of mice with TRALI.[184] Also, platelets can accumulate in the lungs of mice with TRALI and have been described
to induce NET formation.[185] In turn, histones expressed by NETs may activate platelets, thus feeding a vicious
cycle.[186] As a further proof of it, the pre-treatment of mice with a histone-blocking antibody
decreased NET generation as well as lung oedema, lung vascular permeability, and mortality.[185]
NETs and Cancer
Neutrophils are known to be present inside and around solid cancers since long time,
thus being the most representative tumour-infiltrating immune cells. Anyway, controversies
have been raised about their role in this setting and two subsets of tumour-associated
neutrophils (TANs) have been found to develop depending on the influences of the tumour
microenvironment: the N1 phenotype displays proinflammatory and antitumourigenic functions,
while the N2 phenotype has protumorigenic activity with transforming growth factor-β
produced by the local tumour microenvironment playing a central role in polarizing
mature neutrophils to adopt a pro-tumour N2 phenotype.[187] Many questions are open about NET's function in tumour growth modulation, even if
a role for NETs has been recognized in the local tumour development and especially
in tumour-associated thrombosis.
The first finding of a role for NETs in tumours dates back to 2013 based on a small
number of Ewing's sarcoma samples suggesting that patients with intratumoral NETs
experienced a poorer prognosis.[188] A stronger suggestion for a role of NETs in tumour progression comes from studies
investigating NET-associated proteins, such as NE and MPO. NE can directly impact
on tumour growth, progression, and cell migration by inducing cell proliferation in
both human and mouse adenocarcinoma cell lines.[189] In addition, NE deficiency has been found to blunt tumour burden in mice.[189] Although these results mainly referred to the soluble NE, they suggest a potential
role for NETs, which need to be clarified by targeted study. Sangaletti et al found
that NETs could stimulate the proliferation and malignant transformation of B cells
toward malignant lymphoma via the NF-κB signaling.[190] A role for NETs has also been tested in pancreatic ductal adenocarcinoma.[191] Another NET-associated protein involved in the pathophysiology of cancers is MMP-9,
which contributes to carcinogenesis, tumour growth and progression, and metastasis.
Apart from its role in different cancer types, it is still controversial whether NET-bound
MMP-9 conveys these effects or NETs can even protect from MMP-9 functions; the reason
of this controversy can be found in the fact that MMP-9 has been shown to be inactive
when bound to NETs.[192] Since IL-8 has been demonstrated to play a role in NET generation and angiogenesis,
a clear clinical relevance for IL-8 in tumour progression has been shown for many
tumours, too.[193]
[194] The cascade including IL-8 can be probably considered a vicious cycle started by
NETs themselves recruiting additional neutrophils, which in turn produce NETs, all
this ultimately concluding with tumour growth and angiogenesis. Demers et al have
recently linked tumour progression and NETosis by showing that priming of neutrophils
by tumour-associated granulocyte-colony stimulating growth factor (G-CSF) is able
to promote tumour growth. Indeed, PAD4-deficiency was protective only against tumour
progression when the implanted tumour cell line produced G-CSF.[195]
Various studies have confirmed a role for TANs in the enhancement of cancer cell survival,
migration and poor prognosis, but none of this has focused on tumour-associated NETs.[196]
[197]
[198] Interestingly, in vivo evidence from cutaneous melanoma has shown that it becomes
more aggressive and metastatic after ultraviolet radiation because of neutrophil recruitment.
In fact, neutrophil recruitment has been associated with a more migratory phenotype,
local angiogenesis and angiotropism of melanoma cells,[199] with a possible responsibility for NETosis, although NETs have not been specifically
focused.
CXCL8 can also play a role in tumour-associated thrombosis as it is contained in microparticles.
In the granulocytic subset of myeloid-derived suppressor cells (considered an important
T-cell immunosuppressive component in cancer-bearing hosts), IL-8 has been shown to
stimulate the formation of NETs.[200] Interestingly, tumour-derived microparticles can interact with macrophages, activating
them, and promote the production of tumour necrosis factor-α favouring the recruitment
of other inflammatory cells and NETs.[201]
vWF is a known actor in metastasis and tumour growth and the interaction with NETs
can result deleterious. In mice models, vWF null mice or mice treated with antiglycoprotein
Ibα experienced increased experimental lung metastasis.[202]
[203] NETs have been demonstrated to bind to vWF on the vessel wall and this is important
when considering that melanoma cells can stimulate endothelial cells to produce vWF
contributing to thrombosis.[204]
In 2016, Guglietta et al investigated about the role of complement in NET induction
and tumour growth. In a spontaneous small intestine cancer model, hypercoagulation
can directly affect neutrophil effector function and is linked to complement activation,
in particular C3a, showing an increased number of TANs and low-density neutrophils.
LDGs displayed features of N2 neutrophils and spontaneously underwent NETosis, which
was dependent on the involvement of the complement receptor C3aR. To block this reaction,
the immune system triggers the development of neutrophils with an N2 phenotype, which
are responsible for tumour growth in a mutation-dependent protumorigenic milieu.[205]
A role for NETs has been hypothesized for metastasis, too, even if specific studies
are still lacking. Neutrophils per se promote the blocking of circulating tumour cells,
especially under inflammatory conditions, and NETs are very important in this setting.[206] Anyway, even considering other studies with DNase I suppressing tumorigenesis, the
activation of peripherals cells as a result of tumour-induced intravascular NET formation,
which predisposes for metastasis, is still a matter of debate. Platelet–granulocyte
complexes with tumour cells not clearly identified as NETs have been proved to create
early metastatic niches, which are pivotal for later metastatic progression.[207] Upon the secretion of CXCL5 and CXCL7 by platelets due to contact with platelets
and the release of IL-8 from tumour cells recruiting neutrophils, the tethering of
tumour cells to endothelium takes place, leading to transendothelial migration first
and the following development of metastasis.[208] Main roles of NETs in cancer are summarized in [Fig. 2].
Fig. 2 The complex interplay between neutrophils and cancer. (A) Interactions between neutrophils and tumour cells lead to neutrophil extracellular
trap (NET) production, which influence tumour growth and progression, angiogenesis
and metastasis. In fact, tumour cells can recruit and activate neutrophils, acquiring
pro- and antitumoural properties. Neutrophils are also responsible for cytokine secretion
promoting the formation of NETs, which present a variety of antimicrobial and cytotoxic
substances relevant for tumour growth and progression. (B) NETs may influence the local growth and progression of cancer. Neutrophil elastase
(NE) is known to stimulate tumour growth, progression and spreading. Interleukin (IL)-8
is responsible for angiogenesis, which in turn is fed by the vascular endothelial
growth factor (VEGF) from the extracellular matrix enhanced by matrix metalloproteinase
(MMP)-9. NETs themselves can recruit other neutrophils amplifying local inflammatory
reaction. (C) NETs are known to trigger tumour-associated thrombosis. Indeed, cancer stimulates
the release of NETs via the production of granulocyte colony-stimulating factor (G-CSF)
or the so-called tumour microparticles with tissue factor (TF) bound on their surface.
Since NETs are prothrombotic, their increased production fuels this vicious circle
along with the interaction with von Willebrand factor anchored to the vessel wall.
Besides, NET-bound NE can inactivate TF pathway inhibitor, ultimately leading to TF-dependent
coagulation. (D) NETs have been described to have a role in haematogenous metastasis. NET formation
within capillaries may provide a scaffold for metastasizing tumour cells, although
early adhesive events represent only a part of known mechanisms.
Future Perspectives
Accumulating data on the role of NETs in highly prevalent inflammatory conditions
(such as sepsis, CV diseases, autoimmune and inflammatory diseases) pave the way to
novel biomarkers and treatments, which might readily become available for patient
care. However, their translation into the clinical practice requires further investigations
specifically focused on NET biology and measurement assay. First, molecules integrated
in NETs may vary according to environmental factors, so that the characterization
of NET proteome represents an exciting challenge for the next future. Second, the
low activation threshold of neutrophils allows a ready availability of NETs, but this
limits the development of strong, easy and cheap diagnostic assay.[209] The lack of validation of NET assay methods might then explain the contrasting results
observed so far in clinical studies. So far, (1) analytical assays (either fluorimetry
or enzyme-linked immunosorbent assay [ELISA]) for NET products, (2) confocal microscopy
of neutrophil enzymes and extracellular DNA networks and (3) flow cytometry based
on nuclear morphology or citrullinated histones and DNA are the main approaches used
for NET detection and (semi)-quantification.[209] Furthermore, a standardization of methods and thresholds identifying an increased
NET formation is needed. ELISA of plasma samples is likely the method meeting robustness,
reproducibility, and easiness criteria for NET quantification. However, ELISA is not
yet able to discriminate between increased generation and defective clearance of NETs.
Furthermore, caution should be paid to avoid post-sampling NET generation, as many
physical and chemical stimuli may impact on neutrophil activation.[210]
On the contrary, many concerns exist on considering NETs as a selective therapeutic
target. This is largely due to the critical role of NETs as first-line response to
infectious agents. Nonetheless, in experimental PAD4−/− mouse, blocking NETosis is indicated as a relatively safe approach, characterized
by normal survive in septic shock and better outcome after severe sterile inflammation.[26] According to experimental data, many conventional therapies used for inflammatory
diseases revealed inhibitory properties on NET cascade. Reduced NET generation may
occur during treatment with ROS scavengers, such as N-acetyl cysteine.[211] Two small studies reported a clinical benefit of treatment with N-acetyl cysteine
in patients with SLE.[212]
[213] Similarly, other conventional treatment used for the treatment of SLE, such as vitamin
D and chloroquine, have been found to prevent NET generation.[214]
[215] Alongside known agents, NET cascade might be inhibited by monoclonal antibodies
targeting B cells (rituximab),[216]
[217] complement system (eculizumab)[218]
[219]
[220] and potentially IFN-α (sifalimumab and rontalizumab).[221] More recently, Cl-amidine, a PAD inhibitor, has been tested in mice. The suppression
of NET generation induced by Cl-amidine was associated with improvement of immune
complex deposition and organ injury in mouse models of SLE and RA.[222]
[223]
[224] Similarly, the inhibitory effect of Cl-amidine on NET formation has been demonstrated
to reduce atherosclerosis burden, arterial thrombosis and ischaemia/reperfusion injury
in mice.[84]
[225]
Conclusion
As depicted in the present narrative review, a growing body of evidence is accumulating
on NETs. Although initially investigated in infectious diseases, research studies
have expanded their activity in other settings, such as CV diseases, autoimmunity
and cancer. Available findings on NETs let us think that they may become new biomarkers
of disease activity, prognosis and potentially therapeutic targets. Although a shroud
of mystery still surrounds NETs, we now have few answers to some topic questions concerning
NET behaviour and a wide overview on their significant roles. However, still many
efforts are requested to clarify different aspects of NETs in both infectious and
non-infectious diseases, as the mighty neutrophil surprises every day for its unique
features and protean biology.
