Pharmaceuticals 17 00605

pharmaceuticals
Review
Targeting Neutrophil Extracellular Trap Formation: Exploring
Promising Pharmacological Strategies for the Treatment
of Preeclampsia
Leticia Lorena Hernández González 1,2,† , Laura Pérez-Campos Mayoral 3,† , María Teresa Hernández-Huerta 4 ,
Gabriel Mayoral Andrade 3 , Margarito Martínez Cruz 1 , Edgar Ramos-Martínez 5 , Eduardo Pérez-Campos Mayoral 3 ,
Víctor Cruz Hernández 6 , Ismael Antonio García 6 , Carlos Alberto Matias-Cervantes 4 ,
Miriam Emily Avendaño Villegas 1 , Carlos Mauricio Lastre Domínguez 1 , Carlos Romero Díaz 1,3 ,
Juan de Dios Ruiz-Rosado 7,8, *,‡ and Eduardo Pérez-Campos 1,9, *,‡
1 National Technology of Mexico/IT Oaxaca, Oaxaca de Juárez, Oaxaca 68030, Mexico;

leticialorenahg@gmail.com (L.L.H.G.); mcruz@itoaxaca.edu.mx (M.M.C.);
carlos.lastre@itoaxaca.edu.mx (C.M.L.D.); carlosrom74@hotmail.com (C.R.D.)
2 Faculty of Biological Systems and Technological Innovation, Autonomous University “Benito Juárez” of
Oaxaca, Oaxaca 68125, Mexico
3 Research Center, Faculty of Medicine UNAM-UABJO, Autonomous University “Benito Juárez” of
Oaxaca (UABJO), Oaxaca 68020, Mexico; lperezcampos.fmc@uabjo.mx (L.P.-C.M.);
gmayoral.fmc@uabjo.mx (G.M.A.); eperezcampos.fmc@uabjo.mx (E.P.-C.M.)
4 CONAHCyT, Faculty of Medicine and Surgery, Autonomous University “Benito Juárez” of Oaxaca (UABJO),
Oaxaca 68020, Mexico; mthernandez@conahcyt.mx (M.T.H.-H.); cmatias@conahcyt.mx (C.A.M.-C.)
5 School of Sciences, Autonomous University “Benito Juárez” of Oaxaca (UABJO), Oaxaca 68020, Mexico;
eramos.ciencias@uabjo.mx
6 “Dr Aurelio Valdivieso” Hospital General. SS., Oaxaca 68040, Mexico; is_angar6@yahoo.com (I.A.G.)
7 Kidney and Urinary Tract Research Center, Abigail Wexner Research Institute, Nationwide Children’s
Citation: Hernández González, L.L.;
Hospital, Columbus, OH 43215, USA
Pérez-Campos Mayoral, L.; 8 Division of Nephrology and Hypertension, Nationwide Children’s Hospital, Columbus, OH 43205, USA
Hernández-Huerta, M.T.; Mayoral 9 Clinical Pathology Laboratory, “Eduardo Pérez Ortega”, Oaxaca 68000, Mexico
Andrade, G.; Martínez Cruz, M.; * Correspondence: juandedios.ruizrosado@nationwidechildrens.org (J.d.D.R.-R.); pcampos@itoaxaca.edu.mx or
Ramos-Martínez, E.; Pérez-Campos perezcampos@prodigy.net.mx (E.P.-C.)
† These authors contributed equally to this work and shared first authorship.
Mayoral, E.; Cruz Hernández, V.;
‡ These authors contributed equally to this work and share senior authorship.
Antonio García, I.; Matias-Cervantes,
C.A.; et al. Targeting Neutrophil
Extracellular Trap Formation: Abstract: Neutrophils, which constitute the most abundant leukocytes in human blood, emerge
Exploring Promising Pharmacological as crucial players in the induction of endothelial cell death and the modulation of endothelial cell
Strategies for the Treatment of responses under both physiological and pathological conditions. The hallmark of preeclampsia
Preeclampsia. Pharmaceuticals 2024, 17, is endothelial dysfunction induced by systemic inflammation, in which neutrophils, particularly
605. https://doi.org/10.3390/ through the formation of neutrophil extracellular traps (NETs), play a pivotal role in the development
ph17050605
and perpetuation of endothelial dysfunction and the hypertensive state. Considering the potential of
Academic Editor: Gary J. Stephens numerous pharmaceutical agents to attenuate NET formation (NETosis) in preeclampsia, a compre-
hensive assessment of the extensively studied candidates becomes imperative. This review aims to
Received: 23 April 2024
identify mechanisms associated with the induction and negative regulation of NETs in the context of
Accepted: 4 May 2024
preeclampsia. We discuss potential drugs to modulate NETosis, such as NF-κβ inhibitors, vitamin
Published: 9 May 2024
D, and aspirin, and their association with mutagenicity and genotoxicity. Strong evidence supports
the notion that molecules involved in the activation of NETs could serve as promising targets for the
treatment of preeclampsia.
Copyright: © 2024 by the authors.
Licensee MDPI, Basel, Switzerland. Keywords: neutrophil extracellular traps; NETs; preeclampsia; pregnancy; neutrophils; oxidative
This article is an open access article stress; hypertension; NF-κβ inhibitors; vitamin D; aspirin
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Pharmaceuticals 2024, 17, 605. https://doi.org/10.3390/ph17050605 https://www.mdpi.com/journal/pharmaceuticals

Pharmaceuticals 2024, 17, 605 2 of 19
1. Introduction
Preeclampsia (PE) is a multifactorial and multisystem syndrome specific to human
pregnancy [1]. It manifests as hypertension that emerges at 20 weeks of gestation in
previously normotensive women [2,3].
PE is categorized into subtypes, including early and late-onset, as well as severe, mild,
and mild to severe [4]. From a haemodynamic perspective, based on the value of cardiac
output (CO) at rest, PE is divided into two types, hypo- or hypervolemic. The hypov-
olemic type also denoted as “classic” or placental early onset, is characterized by placental
hypertension induced by vasoconstriction [5]. It presents inadequate placental perfu-
sion, endothelial damage, placental insufficiency, and elevated levels of anti-angiogenic
molecules such as soluble fms-like tyrosine kinase 1 (sFlt-1) [6] and soluble endoglin (sEng).
The hypervolemic, maternal, or term type is more prevalent. This variant is associated with
elevated CO, increased oedema due to water retention, augmented placental perfusion,
higher foetal birth weight, relaxed vasculature, and maternal obesity [7].
Severe preeclampsia can lead to the development of HELLP syndrome (Haemoly-
sis, Elevated Liver enzyme, and Low Platelets). This syndrome is characterised by mi-
croangiopathic haemolytic anaemia, hypertension, proteinuria, oedema, liver dysfunction,
thrombocytopenia [8], increased CO, and inotropy [9].
Furthermore, endothelial cells, platelets, and neutrophils play pivotal roles in the
inflammatory, hypertensive, and prothrombotic processes observed in preeclampsia [10–12].
Although distinct neutrophil subpopulations have been characterized in different human
pathologies and experimental models [13–18], their specific roles in preeclampsia remain
under investigation. Numerous groups of molecules orchestrate the relationships between
neutrophils, platelets, and endothelial cells, such as the P-selectin-PSGL-1 axis [19] via
phosphodiesterase type-4 or Src family kinases and the vWF-glycoprotein Ibα axis [20].
Studies have demonstrated alterations in the quantity and functionality of phagocyto-
sis, degranulation, extracellular vesicle release, and the release of neutrophil extracellular
traps (NETs) in preeclampsia. These changes are intricately linked to the systemic in-
flammatory and immune state. Furthermore, there is compelling evidence indicating
increased elastase [21] and nucleosomes [22] release, particularly in early-onset PE. These
findings suggest that alterations in neutrophil functions, such as NETosis, may contribute
to inflammation and endothelial dysfunction, thereby promoting the development of PE.
In this review, we discuss the role of neutrophil activation and NETosis in the context
of preeclampsia. Additionally, we examined pharmacological compounds that exhibit
a negative regulatory influence on NETs, which could potentially be employed in the
treatment of preeclampsia. Additionally, we explored the mutagenicity and genotoxicity
associated with the use of these compounds.
To complete this review, we conducted a focused search and discussed the selection
of drugs for analysis with a panel of experts using a Delphi exercise. We searched on
PubMed, Google Scholar, and the Cochrane Library from 1 January 2023 to 31 January
2024, using the search terms “preeclampsia”, “neutrophils”, “neutrophil extracellular
traps”, “NETs”, “NETosis”, “preeclampsia and oxidative stress”, and “preeclampsia and
NF-κβ inhibitors”; we also cross-referenced these terms with the following: “aspirin”,
“acetylsalicylic acid”, “dexamethasone”, “glucocorticoids”, “resveratrol”, “cyclosporine
A”, “azithromycin”, “chloramphenicol”, “metformin”, “hydroxychloroquine”, “heparin”,
“vitamin D”, “disulfiram”, “curcumin”, “roflumilast, “apremilast”, “rolipram, “Glycyrrhiza
glabra”, “activated protein C”, and “recombinant human DNase I”.
2. Activation of Neutrophils in PE
Neutrophil activation in preeclampsia is characterized by distinct molecular and func-
tional changes. An analysis of the expression of 40 inflammation-related genes in leukocytes
has revealed a significant increase in the mRNA expression of the nuclear factor of Kappa
light chain (NFKβ 1A), Cyclin-dependent kinase inhibitor 1A (CDKN-1A), Interleukin-
8 (IL)-8, and IL-1b genes in leukocytes of pregnant women with PE compared to their
healthy counterparts. This heightened expression favours the polarization of neutrophils

towards an inflammatory profile [23]. Additionally, peripheral blood neutrophils in PE
show lower expression of apoptosis markers compared to neutrophils from healthy preg-
nant women. Higher expression of these markers of apoptosis was observed in neutrophils
from non-pregnant women, suggesting that delayed neutrophil apoptosis contributes to
complications during pregnancy, such as PE [24,25].
In PE, neutrophil granule release and ROS production cause direct damage to the
cell membrane through peroxidation of membrane lipids, leading to cell death [26]. This
phenomenon triggers endothelial activation characterized by an increased expression of vas-
cular cell adhesion molecule-1 (VCAM-1) and inactivation of factors such as endothelium-
derived relaxing and prostacyclin (PGI2). In addition, there is an increase in the number
of activated neutrophils, which is associated with a programmed neutrophil extracellular
traps formation (NETosis) [27–29].
Neutrophils from pregnant women with PE exhibit increased expression of the adhe-
sion molecule CD11b, which serves as a marker of cell activation [30]. Furthermore, there is
a notable reduction in the surface expression of L-selectin (CD62L) on CD15+ neutrophils,
which correlates with immune activation in PE [31]. Studies have shown that NETs can
induce platelet activation and a prothrombotic state during PE [28,32]. This body of evi-
dence suggests that neutrophils are activated and potentially primed for NET release in
PE, both in the microcirculation and periphery, contributing to a feedback loop involving
inflammation and endothelial dysfunction.
3. Neutrophils in the Uteroplacental Microvasculature in PE

In the uteroplacental microvasculature of pregnant women with PE, there is a marked
infiltration of neutrophils associated with increased expression of the cell adhesion molecule
ICAM-1 and a positive gradient of IL-8. In addition, expression of the β-2 integrins
CD11a, CD11b, and CD11c is increased in neutrophils from women with PE compared to
their healthy counterparts [33]. This heightened presence of neutrophils and associated
molecules in the maternal tissue vasculature can cause vasoconstriction, ischemia, oxidative
stress, inflammation, and subsequent endothelial dysfunction [34].
Analyses of biopsies obtained by caesarean section in pregnant women with PE have
shown that the number of neutrophils attached to the vasculature is elevated [35,36]. A
higher concentration of neutrophils in the vasculature potentially contributes to endothelial
dysfunction [37].
The syncytiotrophoblast, the membrane layer that covers the placenta and is in di-
rect contact with maternal blood, releases microvesicles (placental syncytiotrophoblast
microvesicles, STBMs) [38] that can stimulate ROS production in neutrophils [39]. Syn-
cytiotrophoblast cells and serum from pregnant women with PE contain higher levels of
IL-32β compared to healthy pregnant women. IL-32 exerts multiple functions such as NET
induction [40].
4. NETs
NETs are structures consisting of nuclear or mitochondrial DNA with proteins such
as lactoferrin, actin, histones, neutrophil elastase (NE), and myeloperoxidase (MPO) [41].
The main function of NETs is the containment and elimination of pathogens [42,43]; how-
ever, their participation in thrombosis, autoimmune diseases, cancer, diabetes [44], and
cardiovascular diseases such as hypertension [45] has also been reported.
NETosis can be triggered by various stimuli, including microbial pathogens, inflamma-
tory cytokines, and chemical agents. The formation of NETs relies on histone citrullination,
catalysed by elevated levels of neutrophil peptidyl-arginine deaminase-4 (PAD4). Addi-
tionally, NET formation depends on the production of ROS and activation of MPO and
NE [46], leading to nucleosome histone digestion, chromatin decondensation, and release
of DNA and antimicrobial molecules [47].
NETosis can be triggered by various stimuli, including microbial pathogens, inflam-
matory cytokines, and chemical agents. The formation of NETs relies on histone citrulli-
nation, catalysed by elevated levels of neutrophil peptidyl-arginine deaminase-4 (PAD4).
Additionally, NET formation depends on the production of ROS and activation of MPO
Pharmaceuticals 2024, 17, 605 and NE [46], leading to nucleosome histone digestion, chromatin decondensation, 4and of 19
release of DNA and antimicrobial molecules [47].
There are several signalling pathways involved in NET formation [48]; one pathway
involves
Therethe generation
are of ROS through
several signalling pathways the nicotinamide
involved in NETadenine dinucleotide
formation phosphate
[48]; one pathway
(NADPH) oxidase. Activation of the NADPH oxidase complex 2 (NOX2)
involves the generation of ROS through the nicotinamide adenine dinucleotide phosphatetriggers the
Raf/MEK/ERK pathway and augments cytosolic ROS levels [49], resulting
(NADPH) oxidase. Activation of the NADPH oxidase complex 2 (NOX2) triggers the in citrullinated
histone H3-dependent
Raf/MEK/ERK pathway(Cit-Histone
and augmentsH3)cytosolic
lytic or ROS
suicidal NETosis,
levels Figure in
[49], resulting 1. citrullinated
These lytic
NETs are
histone released by the
H3-dependent presence of
(Cit-Histone extracellular
H3) microbes,
lytic or suicidal fungi,Figure
NETosis, viruses,
1. interferon,
These lytic
phorbol
NETs aremyristate
released acetate
by the (PMA),
presenceIL-8, antibody–antigen
of extracellular complexes,
microbes, fungi,autoantibodies, and
viruses, interferon,
concanavalin A.
phorbol myristate acetate (PMA), IL-8, antibody–antigen complexes, autoantibodies, and
concanavalin A.
Figure 1. Mechanisms of NET formation. Neutrophils generate NETs in response to various stimuli.
Four types of NETs have been described based on structural changes during their formation. These
Figure 1.involve
changes Mechanisms of NET of
the activation formation.
differentNeutrophils generate NETs
signalling pathways. In thein response
case to various stimuli.
of mitochondrial NETosis,
Four types
nuclear DNA of remains
NETs have beenthe
inside described
cell and based on structural
has been changes
observed in during
specific cases.their formation. These
changes involve the activation of different signalling pathways. In the case of mitochondrial NETo-
sis, nuclear DNA remains inside
NOX2-independent the cell and
pathways can has
leadbeen
toobserved in specific
the formation ofcases.
non-lytic NETs, also
known as vital NETs [50]. These NETs are released in the presence of Staphylococcus
aureusNOX2-independent
(S. aureus), Escherichiapathways can damage-associated
coli (E. coli), lead to the formation of non-lytic
molecular NETs,
patterns also
(DAMPs),
known asToll-like
platelets, vital NETs [50]. These
receptors 2/4NETs are released
(TLR2/4), in the presence
and lymphocytes of Staphylococcus
associated aureus
with a function
(S. aureus), antigen
neutrophil Escherichia coli (E. coli), damage-associated molecular patterns (DAMPs), plate-
1 (LFA1).
lets, Other
Toll-like receptors 2/4
less-studied (TLR2/4),that
pathways andinduce
lymphocytes associated
NETs include withinvolving
those a functionsignal
neutro-in-
phil antigen 1 (LFA1).
hibitory receptor 1 (SIRL1) [51] and extracellular cold-inducible RNA-binding protein
(eCIRP) through PD4 [52]. In addition, the formation of mitochondria-dependent vital NETs
by silent information regulator 1 (SIRT1) that has been reported in both tumour-associated
aged neutrophils (Naged, CXCR4+ CD62L low) in breast cancer lung metastasis [53] and
non-tumour pathologies [54] has not been studied in preeclampsia.
NETs can be identified and quantified by distinct visualisation methods or through
markers of their released products. Visualisation methods use molecules that are inter-
calated into DNA, such as propidium iodide and SYTOX Orange. On the other hand,
the markers include those that detect DNA-histone complexes (nucleosomes), MPO [55],
circulating MPO complexes (MPO)-DNA [56], surface citrullinated histone (H3cit), double-
stranded DNA (ds), myeloid-related protein (MRP), DNase, and elastase [57,58].
5. NETs in PE
In PE, there is an increased trafficking of foetal cells into the maternal circulation. The
elevated concentration of DNA in blood from foetal-derived cells could activate the immune
response and induce cytokine production, potentially leading to pregnancy complications
and an increased risk of foetal rejection.
Higher concentrations of NET components (DNA, histones, and MPO) have been
reported in the plasma of pregnant women with preeclampsia compared to healthy preg-
nant women [55]. The first molecule associated with neutrophil activation in the blood of
women with PE was neutrophil elastase [59].
Concordantly, in vitro experiments have shown that neutrophils from pregnant women
with PE are more likely to form NETs compared to healthy pregnant and non-pregnant
women [28]. Also, women with PE and obesity have a higher presence of MPO in their
systemic vasculature. MPO catalyses the formation of ROS and RNS in PE by consuming
circulating nitric oxide [60,61].
Marder et al. analysed placental samples from women with PE, healthy pregnant
women, and abdominal tissue from non-pregnant women, revealing that MPO expression
is elevated in tissues from pregnant women compared to non-pregnant women. Notably,
NETs were found in the intervillous space in PE [62].
Using anti-NE, Gupta et al. demonstrated that NETs were present in the vicinity of
the syncytiotrophoblastic layer in healthy pregnant and preeclamptic women. However,
the intervillous space was frequently infiltrated by numerous NETs, and their presence
increased in PE [63]. Anti-histone 2A (H2A) staining showed that H2A was significantly
increased in the intervillous space of women with PE [64]. Gupta et al. showed that
inflammatory syncytiotrophoblast microparticles (STBM) and IL-8-induced neutrophil
extracellular DNA extrusion increased CD11b expression and ROS generation in PE [65].
PE is associated with markers of autoimmunity, including anti-β2 glycoprotein-I
(ab2GPI); anticardiolipin antibodies (aCL); lupus anticoagulant (LA); β1, β2, and α1
adrenoreceptors; prothrombin; endothelin-1 receptor type A (ETA-AA); and Angiotensin II
receptor type 1(AT1-AA) antibodies [66,67] as well as antibodies with different specificity
than AT1-AA [68]. Furthermore, the level of antineutrophil cytoplasmic autoantibodies
(ANCA) in PE patients was significantly elevated compared with a healthy pregnancy [69].
Although these autoantibodies may induce NETosis during PE, this hypothesis remains to
be addressed.
Altogether, the studies mentioned above provide a compelling association between
preeclampsia and the increased presence of NETs in pregnant women, Figure 2. Strategies
targeting NETs in preeclampsia may hold promise for mitigating immune responses and
complications during pregnancy.
Pharmaceuticals 2024,17,
Pharmaceuticals2024, 17,605
x FOR PEER REVIEW 6 6ofof20
19
Figure 2. NETs in preeclampsia. In preeclampsia, neutrophils can be activated in two different envi-
ronments. On the one hand, systemic inflammation with a consequent release of cytokines, DAMPs,
Figure
and 2. NETsof
activation inplatelets
preeclampsia. In preeclampsia,
and macrophages neutrophils
stimulates can be activated
neutrophils, promoting in two different en-
the expression of
vironments. On the one hand, systemic inflammation with a consequent release of cytokines,
selectins and their migration to the site of damage, such as the uteroplacental microvasculature where
DAMPs, and activation of platelets and macrophages stimulates neutrophils, promoting the expres-
NETs
sion ofwill be released.
selectins In addition,
and their migrationneutrophil activation
to the site of damage, in theasperipheral
such vasculature
the uteroplacental can lead to
microvascula-
the formation of NETs. These networks and their components promote thrombosis, inflammation,
ture where NETs will be released. In addition, neutrophil activation in the peripheral vasculature
damage,
can lead and endothelial
to the formationdysregulation.
of NETs. TheseLikewise,
networksinflammation and ischemia
and their components activate
promote neutrophils
thrombosis, in-
resident in the
flammation, uteroplacental
damage, tissue, dysregulation.
and endothelial promoting theLikewise,
formationinflammation
of NETs. These NETs perpetuate
and ischemia activate
neutrophils
and increaseresident in the damaging
inflammation, uteroplacental
localtissue,
tissuespromoting the formation
and promoting of NETs.
the formation These NETs
of inflammatory
perpetuatethat
mediators andreach
increase inflammation,
the circulation, damaging
attracting local
more tissues and promoting the formation of in-
neutrophils.
flammatory mediators that reach the circulation, attracting more neutrophils.
6. Pharmacological Modulation of NETs
6. Pharmacological Modulation
NETs can be targeted through of NETs drugs with diverse mechanisms of action, Table 1.
different
Several
NETs can pharmacological agents different
be targeted through are currently
drugsbeing
withstudied
diversefor the treatment
mechanisms of of mod-
action,
erate and
Table 1. severe preeclampsia (Figure 3), including aspirin, sildenafil citrate, hydrox-
ychloroquine, pravastatin, metformin,
Several pharmacological agents are magnesium
currently being sulphate,
studiednifedipine, labetalol,
for the treatment and
of mod-
nitro-glycerine.
erate and severe However, only one
preeclampsia trial has
(Figure 3),been reported,
including on clinicaltrials.gov,
aspirin, that was
sildenafil citrate, hy-
designed to inhibit NET
droxychloroquine, formation
pravastatin, in PE usingmagnesium
metformin, Toll-like receptor (TLR)
sulphate, blocking antibodies.
nifedipine, labetalol,
Some agents mitigateHowever,
and nitro-glycerine. NETs by only
inhibiting ROS,
one trial hasincluding flavonoids
been reported, like epicatechinthat
on clinicaltrials.gov, and
rutin, along with
was designed vitamin
to inhibit NETC, formation
5-aminosalicylic acid (5-ASA),
in PE using N-acetyl-L-cysteine
Toll-like receptor (TLR) blocking (NAC),
an-
dexamethasone, azithromycin, and Glycyrrhiza glabra [70,71]. Others inhibit
tibodies. Some agents mitigate NETs by inhibiting ROS, including flavonoids like epicat- the JAK/STAT
pathway
echin andsuch
rutin,asalong
tofacitinib, ruxolitinib,
with vitamin and baricitinib
C, 5-aminosalicylic [72].
acid Furthermore,
(5-ASA), interleukin
N-acetyl-L-cysteine
antagonists, such as tocilizumab,
(NAC), dexamethasone, canakinumab,
azithromycin, anakinra,
and Glycyrrhiza and[70,71].
glabra rilonacept, alsoinhibit
Others modulate
the
NETs [73]. pathway such as tofacitinib, ruxolitinib, and baricitinib [72]. Furthermore, in-
JAK/STAT
terleukin antagonists, such as tocilizumab, canakinumab, anakinra, and rilonacept, also
modulate NETs [73].
Pharmaceuticals 2024, 17, x FOR PEER REVIEW 7 of 20
Figure 3. Drugs and molecules that reduce neutrophil extracellular traps (NETs). Signalling path-
Figure 3. Drugs and molecules that reduce neutrophil extracellular traps (NETs). Signalling pathways
ways involved in reducing NETs include Raf-MEK-ERK signalling, calcineurin signalling, phos-
involved in reducing
phorylation of the p65NETs include
subunit Raf-MEK-ERK
of NF-κβ via Rac2, signalling,
PAD4, andcalcineurin signalling,
block receptors with phosphorylation
monoclonal an-
of the p65
tibodies andsubunit
DNAases.of NF-κβ via Rac2, PAD4, and block receptors with monoclonal antibodies
and DNAases.
Amid the COVID-19 pandemic, there has been a surge in accelerated investigations
Amid
on drugs andthemolecules
COVID-19 pandemic,for
repurposed there has
their been a surge
potential in accelerated
to inhibit investigations
NETs, as summarized in
on drugs and molecules repurposed for their potential to inhibit NETs,
Table 1. Some of them have garnered attention for their well-established anti-inflamma- as summarized in
Table 1. Some of them have garnered attention for their well-established
tory effects. Some have not only been utilized in inflammation management but also in anti-inflammatory
effects. Some have not only been utilized in inflammation management but also in aiding
aiding lung maturation during preterm births and mitigating preeclampsia [74–81]. Aspi-
lung maturation during preterm births and mitigating preeclampsia [74–81]. Aspirin,
rin, vitamin D, and recombinant human DNase I have emerged as candidates worthy of
vitamin D, and recombinant human DNase I have emerged as candidates worthy of study
study due to their reported lower risk or absence of mutagenicity and genotoxicity.
due to their reported lower risk or absence of mutagenicity and genotoxicity.
The Raf-MEK-ERK signalling pathway is involved in NET formation through the ac-
The Raf-MEK-ERK signalling pathway is involved in NET formation through the
tivation of NADPH oxidase. Drugs that reduce NET formation through this pathway in-
activation of NADPH oxidase. Drugs that reduce NET formation through this pathway
clude curcumin, GW5074 [82], and Celastrol [83]. While drugs that inhibit NETs by inhib-
include curcumin, GW5074 [82], and Celastrol [83]. While drugs that inhibit NETs by
iting the phosphorylation of the p65 subunit of NF-κβ are anti-inflammatory drugs such
inhibiting the phosphorylation of the p65 subunit of NF-κβ are anti-inflammatory drugs
as acetylsalicylic acid (ASA), BAY 11-7082, and Ro 106-9920 [84].
such as acetylsalicylic acid (ASA), BAY 11-7082, and Ro 106-9920 [84].
On the other hand, cyclosporine A uses the calcineurin pathway to reduce NETs [85],
On the other hand, cyclosporine A uses the calcineurin pathway to reduce NETs [85],
while the antioxidants N-acetylcysteine, ethotrexate, trolox, tempol [86], epigallocatechin-
while the antioxidants N-acetylcysteine, ethotrexate, trolox, tempol [86], epigallocatechin-
3-gallate
3-gallate [87],
[87], and
and diphenyleneiodonium
diphenyleneiodoniumchloride chloride(DPI)
(DPI)[88]
[88] decrease
decrease NETs
NETs byby reducing
reducing
mitochondrial
mitochondrial ROS ROS formation
formation [85].
[85]. DPI
DPIalso
also reduces
reduces NET
NET formation
formation through
through thethe PKC-βII
PKC-βII
pathway
pathway [89]
[89] like
like metformin.
metformin. Other
Otherdrugs
drugs that
that have
have been
been shown
shown to to significantly
significantly inhibit
inhibit
ROS-dependent
ROS-dependent NET NET production
production areare propofol
propofol andand lipid
lipid emulsion
emulsion [90].
[90].
Furthermore,
Furthermore, drugs
drugs such
such as
as Hydroxychloroquine
Hydroxychloroquine (HCQ) (HCQ)[91],
[91], PF3758309,
PF3758309,and andIPA-3
IPA-3
inhibit NETs through Rac2 [92]. Chloroquine (CQ) and HCQ can prevent
inhibit NETs through Rac2 [92]. Chloroquine (CQ) and HCQ can prevent NET formation by NET formation
by inhibiting
inhibiting PAD4 PAD4
[93] [93]
or TLRor signalling
TLR signalling
as withasHCQ,
with CQ,
HCQ, andCQ, and enpatoran
enpatoran [94]. In
[94]. In addition,
PDE4 inhibitors such as apremilast, rolipram, and crisaborole can reduce the formation of
NETs [95].
Table 1. Molecules that downregulate NETosis.
Evaluated for
NOX-Dependent (NOX-D) Clinical Mutagenicity and
Drugs Substance Mechanism of Action
and ROS-Independent Trials/Models/Examples Genotoxicity In Vitro
(ROX-I) NET Formation
Drugs
Human neutrophils were
stimulated with Phorbol
In isolated neutrophils
12-myristate 13-acetate
stimulated with sodium
(PMA) or TNF-α. In
hydroxide, ASA can
addition, ASA, BAY ASA can protect against
Aspirin (ASA) NOX-D enhance the migration of
11-7082, and Ro 106-9920 genotoxicity [97].
corneal epithelial cells
prevented the formation
(HCEs) and reduce the
of NETs by reducing the
formation of NETs [96].
phosphorylation of the
p65 subunit of NF-κβ [84].
Inhibits neutrophil Neutrophils cultured with It can induce significant
functions such as dexamethasone showed DNA damage in human
Dexamethasone intracellular ROS, NOX-D reduced NET formation, cells; however, it passes the
degranulation, after stimulation with Ames/Salmonella assay
and NETosis. PMA [98]. [99,100].
During in vitro tests in the
presence of PMA, it was Despite its genotoxic effects,
shown that resveratrol it does not cause
Decreases the release of
decreases the formation of mutagenesis and is used for
Resveratrol free DNA from NOX-D
NETs and cytokine its genotoxic activity against
neutrophils and NETosis
production in healthy gastric cell adenocarcinoma
controls and with [102,103].
COVID-19 [101].
In isolated neutrophils, It is not genotoxic in
stimulated with PMA or humans. It inhibits the
Inhibits IL-8-induced
ionomycin, treated with protein phosphatase
Cyclosporine A NETosis by inhibiting the NOX-D
Cyclosporine A or calcineurin and can induce
calcineurin pathway.
ascomycin, the formation lymphoma in Xpa/p53
of NETs decreases [104]. mice [105].
Pre-treatment with
Azithromycin decreases
NETosis in neutrophils It does not induce mutations
Decreases the production isolated from or chromosomal aberrations
Azithromycin NOX-D
of ROS PMA-stimulated healthy in microbial or mammalian
subjects. This effect is cells [107].
observed at low
doses [106].
Reduces the formation of Pre-treatment with
In rodents and human cells,
NETs, possibly chloramphenicol reduces
Chloramphenicol NOX-D it is a pro-mutagenic
by inhibiting PMA-induced NET
compound [108].
myeloperoxidase (MPO) release [94].
There is conflicting evidence
about the effects of
metformin. Micronucleus
Metformin decreases
assay suggests it may be
Affects nuclear dynamics NETosis and its
genotoxic; however,
(delobulation and components such as
analyses using chromosomal
decondensation) as well as elastase, proteinase-3,
aberration (CA) and
Metformin PKC-βII membrane NOX-D histones, and
cytokinesis-block
translocation and NADPH double-strand DNA in
micronucleus (CBMN) assay
oxidase activation in PMA-stimulated
report that it has a
neutrophils [109]. neutrophils in in vitro and
radioprotective effect on
clinical trial samples [109].
DNA damage and apoptosis
in human lymphocytes
[110,111].
Table 1. Cont.
Evaluated for
Hydroxychloroquine
alleviates hepatic It induces both oxidative
Inhibits the expression of ischemia/reperfusion (IR) DNA damage detected by
Hydroxychloroquine PAC4, Rac2, and the NOX-D injury in severe combined 8-oxodG and the induction
formation of NETs immunodeficiency (SCID) of mutants in mouse
mice and C57BL/6 mice embryonic fibroblasts [112].
by inhibiting NETosis [91].
In vitro studies have
shown that heparinized
adsorbents such as
heparin sepharose
deplete PF4,
histones/nucleosomes,
NET molecules, such as and HMGB1 [114].
neutrophil elastase, Heparin pre-treatment
interact with heparin and decreased serum and lung
heparin oligomers to form NETs in a C57BL/6J mice
molecular complexes that model [115].
can regulate NETosis [113]. Circulating histones LMWH does not show any
Heparin --
Low molecular weight bound to H3 and H4 mutagenic activity [116,117].
heparin (LMWH) nucleosomes are increased
can repair in patients with
nucleosome/histone preeclampsia and
3-mediated damage in intrauterine growth
trophoblasts [69]. restriction. H3 affects
extravillous trophoblast
migration, invasion, and
survival. This effect can be
reversed in vitro by
LMWH, but not with
ASA [69].
PMA-stimulated
neutrophils from patients
Vitamin D
with systemic lupus In cancer rodents treated
supplementation has been
erythematosus (SLE) and with cyclophosphamide, it
shown to reduce the risk
hypovitaminosis D were reduced the frequency of
of preeclampsia [118], as
treated with chromosomal aberrations in
Vitamin D well as decrease the NOX-D
calcitriol/1,25(OH)2 D3. Chinese hamster lung cells
generation of NETs,
The authors reported a and reduced micronuclei
particularly when
dose-independent and lymphocyte damage in
combined with omega-3
decrease in externalised mice [121].
PUFAs [119].
neutrophil elastase (NE)
during NETosis [120].
It reduced NETs and
perivascular fibrosis and
downregulated innate
Disulfiram Inhibits NETs [122]. NOX-D --
immune and
complement/coagulation
pathways [111].
High concentrations are
In a mouse model,
cytotoxic and increase the
curcumin was shown to
Inhibits the generation of frequency of micronuclei in
reduce hepatic
Curcumin NETs by suppressing the NOX-D PC12 cells; at low doses, it
ischemia-reperfusion
MEK/ERK pathway [123]. reduces the number of
injury by inhibiting NET
micronuclei induced by
formation [91].
cisplatin [124].
Table 1. Cont.
Evaluated for
Roflumilast blocks PDE4
and reduces in vitro and
Phosphodiesterase in vivo NETosis in animal Clinical trials have been
Type-4 (PDE4) models [125]. conducted with
inhibitors such as Inhibition of PDE4 by Roflumilast for severe
Roflumilast rolipram prevents the ROX-I chronic obstructive --
(Daliresp), adhesion of platelets and pulmonary disease
Apremilast (Otezla), neutrophils, which (COPD) and with Otezla
and Rolipram involves members of the for psoriasis [128].
Src family kinase (SFK)
[126,127].
In an animal model,
Inhibits ROS,
Glycyrrhiza glabra was
mitochondrial ROS
Glycyrrhiza glabra -- proven to decrease --
(mtROS), NET generation,
COVID-19 pathology by
and cytokine release.
reducing NETosis [129].
Protein molecules
A clinical trial was
conducted evaluating the
safety and efficacy
Cleaves and detoxifies of recombinant
extracellular histones and human-activated protein
its effect on reducing NETs C (rhAPC; drotrecogin alfa
dependent on endothelial [activated]) in Drotrecogin alfa (activated)
Activated protein C
protein C receptor (EPCR), -- preeclampsia, but further has not been studied for
(APC)
protease-activated studies are needed [131]. carcinogenicity [133].
receptor 3 (PAR3), and APC variants have been
macrophage antigen-1 designed to have a greater
(Mac-1) [130]. ability to destroy histone
H3 with fewer
anticoagulant
properties [132].
Intravenous
Recombinant administration of
human DNase I rhDNase to mice
Hydrolyses extracellular It does not show cytotoxicity
(rhDNase I, degraded NETs and
DNA released by -- in human peripheral blood
rhDNase, attenuated coagulopathy
neutrophils [134]. mononuclear cells [136].
Pulmozyme® , in the acute respiratory
dornase alfa) distress syndrome (ARDS)
model [135].
Other molecules such as PGE2, which has been used in labour induction, decrease
NET formation through cAMP production. However, there are also no reports of trials with
PGE2 to reduce NETs in preeclampsia [137–139].
Others, although they reduce NET formation, are not considered due to the risk of
mutagenicity or genotoxicity during pregnancy. A mutagenic agent causes damage to DNA
and results in mutation, while a genotoxic agent can cause damage to DNA or chromosomes
but without necessarily resulting in mutation [140], such as 5-fluorouracil (5FU) [141].
7. Therapeutic Approaches to Inhibit NET Formation in PE

Low-dose aspirin (75 mg) is used in the clinic to prevent preeclampsia; its best-known
action is the inhibition of prostaglandin and thromboxane A2 synthesis by cyclooxygenases
COX-1 and COX-2.
Preeclampsia is characterized by elevation of TXA2 and decreased prostacyclin. TXA2
is a procoagulant molecule related to thrombosis observed in preeclampsia; prostacyclin
PG1 is a vasodilator molecule. In PE, there is an increase in TXA and a decrease in PGI;
aspirin acts on both, although platelets are the main producers of TXA2 [142].
PE is related to an inflammatory and prothrombotic state, and thus the effect of aspirin
in inhibiting inflammation and platelet aggregation is considered to prevent and treat
it; however, beyond its antithrombotic action, the effect of aspirin on the activation and
modulation of the inflammatory response of neutrophils has recently been studied.
During pregnancy, neutrophils express PAR-1 and F2R, which are thrombin receptors.
Aspirin prevented p65 translocation of NF-kβ to the nucleus and TXA2 production in neu-
trophils from pregnant women via PAR-1. In addition, aspirin inhibits lipid peroxidation
by COX-2 in pregnant neutrophils [143]. These PAR-1 and F2R receptors are not expressed
in neutrophils from pregnant women.
Both aspirin and glucocorticoids reduce NF-κβ activity. It is a family of transcrip-
tion factors, phosphorylated p65 (RelA), RelB, c-Rel, p105/p50 (NF-κβ1) and p100/52
(NF-κβ2), that regulate the immune response, inflammatory response, apoptosis and prolif-
eration [144].
In PE, neutrophil adhesion to the vasculature increases. This adhesion could be
reduced in the presence of an intermediate in aspirin metabolism and aspirin-activated
lipoxin A4 (ATL, 15-epi-LXA4) [145]. Aspirin generally inhibits NETosis, but more studies
are needed to analyse its usefulness in preeclampsia.
Some meta-analyses and individual cases have shown that low-dose aspirin can
prevent preeclampsia in people with a risk factor, but aspirin crosses the placental barrier
and can inhibit foetal platelet aggregation, increasing the risk of spontaneous abortion;
thus, its use is under medical supervision. The use of aspirin has been empirical, but more
data are needed to implement it as a treatment [146].
Furthermore, the therapeutic use of glucocorticoids in PE is primarily indicated in cases
of foetal growth restriction. It has been proposed that single doses after week 24 (2 doses
of 12 mg betamethasone 24 h apart or 6 mg dexamethasone 12 h apart before delivery)
help foetal lung maturation in PE [147,148]. Dexamethasone has been shown to transiently
restore absent end-diastolic flow in the umbilical artery in early-onset preeclampsia [149].
However, dexamethasone treatment for PE should be carefully considered, as the use of
high doses and repeated doses should be avoided for fear of possible long-term adverse
effects on the foetal brain [150].
On the other hand, glucocorticoids such as dexamethasone, prednisone, and rimex-
olone interact through glucocorticoid receptor antagonism in PE [151]. Glucocorticoid
receptors are expressed in the trophoblast, and their inhibition favours the expression
of angiotensin 2 receptor antagonist (ART2), which has vasodilatory effects. In normal
pregnancy, ART2 predominates, reducing hypertension, while ART1, which has a vaso-
constrictor effect, increases in PE [152]. Numerous studies show that dexamethasone in
early pregnancy has harmful effects such as deficient trophoblast development, increased
trophoblast invasion inhibitor SERPINE1, and increased systolic blood pressure [153,154],
even causing DNA damage (Table 1). Dexamethasone caused abnormal mitochondrial
morphology and mitochondrial dysfunction in the placentas of pregnant rats, in addition
to altering placental signalling pathways such as oxidative phosphorylation, inflammation,
and the insulin-like growth factor system [155].
PE is characterized by a state of systemic inflammation where platelet abnormalities
occur. It has been shown that dexamethasone significantly inhibited degranulation, intra-
cellular ROS production, CXCL8 release, and neutrophil NETosis in patients with severe
COVID-19 pneumonia [98]. Dexamethasone has been shown to inhibit Staphylococcus au-
reus-induced NET formation via upregulation of TLR2 and TLR4 receptors [156], this could
mean that Dexamethasone induces vital NETs. In PE, dexamethasone has been shown to
delay neutrophil apoptosis in early-onset preeclampsia; however, it does not affect the rate
of neutrophil apoptosis between late-onset preeclampsia and normal pregnancy [157].
Another drug with a potential therapeutic effect, particularly in preeclampsia as-
sociated with severe obesity [158], is metformin. This is a hypoglycemic agent that at
therapeutic doses has a reduced impact on trophoblast differentiation [159]. Metformin
activates AMPK signalling pathways involved in the regulation of NF-κβ/sFlt-1, and
Nrf2/HO-1 signalling pathways, thus inhibiting the inflammatory response and oxidative
stress [160].
Finally, several systematic reviews have reported that 25-hydroxyvitamin D supple-
mentation before 20 weeks of gestation could reduce NETosis (Table 1) and the risk of
PE [161,162], through inhibition of lysosome-associated membrane glycoprotein 3 [163].
8. Conclusions
The pathophysiology of preeclampsia is significantly aligned with the activation
of neutrophils and the release of mediators, especially those associated with neutrophil
extracellular traps (NETs). Consequently, understanding the role of neutrophils and NETs
becomes imperative in the context of preeclampsia treatment. Currently, certain drugs are
already under review in clinical trials; these include azithromycin, hydroxychloroquine,
aspirin, and metformin. Additionally, promising results have already been observed in
the use of vitamin D for preeclampsia. This underscores the importance of exploring and
considering neutrophil and NET-targeted treatments in the comprehensive approach to
managing preeclampsia.
Author Contributions: Conceptualization, J.d.D.R.-R. and E.P.-C.; investigation, L.L.H.G., L.P.-C.M.,

M.T.H.-H. and G.M.A.; writing—original draft preparation, L.L.H.G., L.P.-C.M., M.T.H.-H., G.M.A.,
M.M.C. and E.P.-C.M.; writing—review and editing, L.L.H.G., L.P.-C.M., M.T.H.-H., G.M.A., V.C.H.,
I.A.G., C.A.M.-C., E.R.-M., C.R.D., C.M.L.D. and M.E.A.V.; supervision, C.A.M.-C., M.E.A.V., J.d.D.R.-R.
and E.P.-C.; project administration, M.M.C. and E.P.-C.M. All authors have read and agreed to the
published version of the manuscript.
Funding: This work was supported by the National Institutes of Health (NIDDK) K01 DK128379-01
(J.d.D.R.R.).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data sharing is not applicable.
Acknowledgments: Thanks to the Consejo Nacional de Humanidades, Ciencia y Tecnología (CONAH-
CyT) for the scholarship granted to L.L.H.G., CVU number (792544). Also, to the National Technology
of Mexico (TecNM)/IT Oaxaca 18501.23-P, and to Eli Cruz Parada and Charlotte Grundy for their
technical advice.
Conflicts of Interest: The authors declare no conflicts of interest.
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1 National Technology of Mexico/IT Oaxaca, Oaxaca de Juárez, Oaxaca 68030, Mexico;

Pharmaceuticals 2024, 17, 605. https://doi.org/10.3390/ph17050605 https://www.mdpi.com/journal/pharmaceuticals

healthy counterparts. This heightened expression favours the polarization of neutrophils

3. Neutrophils in the Uteroplacental Microvasculature in PE

Table 1. Molecules that downregulate NETosis.

7. Therapeutic Approaches to Inhibit NET Formation in PE

Author Contributions: Conceptualization, J.d.D.R.-R. and E.P.-C.; investigation, L.L.H.G., L.P.-C.M.,

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Pharmaceuticals 17 00605

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1 National Technology of Mexico/IT Oaxaca, Oaxaca de Juárez, Oaxaca 68030, Mexico;

Pharmaceuticals 2024, 17, 605. https://doi.org/10.3390/ph17050605 https://www.mdpi.com/journal/pharmaceuticals

healthy counterparts. This heightened expression favours the polarization of neutrophils

3. Neutrophils in the Uteroplacental Microvasculature in PE

Table 1. Molecules that downregulate NETosis.

7. Therapeutic Approaches to Inhibit NET Formation in PE

Author Contributions: Conceptualization, J.d.D.R.-R. and E.P.-C.; investigation, L.L.H.G., L.P.-C.M.,

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