Neutrophil Activation and Neutrophil Extracellular Traps (Nets) in COVID-19 ARDS and Immunothrombosis

Abstract

Acute respiratory distress syndrome (ARDS) is an acute inflammatory condition with a dramatic increase in incidence since the beginning of the coronavirus disease 19 (COVID-19) pandemic. Neutrophils play a vital role in the immunopathology of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection by triggering the formation of neutrophil extracellular traps (NETs), producing cytokines including interleukin-8 (CXCL8), and mediating the recruitment of other immune cells to regulate processes such as acute and chronic inflammation, which can lead to ARDS. CXCL8 is involved in the recruitment, activation, and degranulation of neutrophils, and therefore contributes to inflammation amplification and severity of disease. Furthermore, activation of neutrophils also supports a prothrombotic phenotype, which may explain the development of immunothrombosis observed in COVID-19 ARDS. This review aims to describe hyperinflammatory ARDS due to SARS-CoV-2 infection. In addition, we address the critical role of polymorphonuclear neutrophils, inflammatory cytokines, and the potential targeting of CXCL8 in treating the hyperinflammatory ARDS population. This article is protected by copyright. All rights reserved.

Full text

Eur. J. Immunol. 2023;53:2250010 Maria Candida Cesta et al.
DOI: 10.1002/eji.202250010 1of10
Clinical
HIGHLIGHTS
REVIEW
Neutrophil activation and neutrophil extracellular traps
(NETs) in COVID-19 ARDS and immunothrombosis
Maria Candida Cesta1, Mara Zippoli2, Carolina Marsiglia1,
Elizabeth M. Gavioli3, Giada Cremonesi4, Akram Khan5,
Flavio Mantelli1, Marcello Allegretti1and Robert Balk6
1Dompé farmaceutici SpA, L’Aquila, Italy
2Dompé farmaceutici SpA, Napoli, Italy
3Dompé U.S. Inc., Boston, USA
4Dompé Farmaceutici S.p.A., Milano, Italy
5Division of Pulmonary, and Critical Care Medicine, Oregon Health and Science University,
Portland, Oregon, USA
6Division of Pulmonary and Critical Care Medicine, Department of Medicine, Rush Medical
College and Rush University Medical Center, Chicago, Illinois, USA
Acute respiratory distress syndrome (ARDS) is an acute inammatory condition with a
dramatic increase in incidence since the beginning of the coronavirus disease 19 (COVID-
19) pandemic. Neutrophils play a vital role in the immunopathology of severe acute respi-
ratory syndrome coronavirus 2 (SARS-CoV-2) infection by triggering the formation of neu-
trophil extracellular traps (NETs), producing cytokines including interleukin-8 (CXCL8),
and mediating the recruitment of other immune cells to regulate processes such as acute
and chronic inammation, which can lead to ARDS. CXCL8 is involved in the recruitment,
activation, and degranulation of neutrophils, and therefore contributes to inammation
amplication and severity of disease.Furthermore, activation of neutrophils also supports
a prothrombotic phenotype, which may explain the development of immunothrombosis
observed in COVID-19 ARDS. This review aims to describe hyperinammatory ARDS due
to SARS-CoV-2 infection. In addition, we address the critical role of polymorphonuclear
neutrophils, inammatory cytokines, and the potential targeting of CXCL8 in treating the
hyperinammatory ARDS population.
Keywords: ARDS rCOVID-19 rCXCL8 rimmunomodulators
Introduction
Acute respiratory distress syndrome (ARDS) is an acute inflam-
matory and life-threatening respiratory condition with a mortality
rate that may exceed 40% [1]. Before the emergence of the coro-
navirus disease 2019 (COVID-19) pandemic, caused by severe
acute respiratory syndrome coronavirus-2 (SARS-CoV-2), there
were an estimated 190,000 cases of ARDS annually in the United
Correspondence: Robert Balk
e-mail: Robert_Balk@rush.edu
States, associated with 74,500 deaths per year [2]. However, in
the context of COVID-19, 29–42% of patients with COVID-19
have developed ARDS, with a mortality of 15–52% [3]. Common
causes of ARDS include pneumonia and nonpulmonary sepsis.
In addition, risk factors such as older age may place patients at
higher risk for developing ARDS [4, 5]. ARDS is characterized
by acute onset of severe hypoxemia, diffuse bilateral pulmonary
infiltrates, and extensive pulmonary edema induced by increased
vascular permeability, reduced compliance, and protein-rich fluid
in the alveolar space combined with accumulation of activated
immune cells such as neutrophils [6].
© 2022 The Authors. European Journal of Immunology published by Wiley-VCH GmbH www.eji-journal.eu
This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License,
which permits use, distribution and reproduction in any medium, provided the original work is properly cited
and is not used for commercial purposes.

2of10 Maria Candida Cesta et al. Eur. J. Immunol. 2023;53:2250010
Neutrophil extracellular trap (NET) formation may lead to
compromised gas exchange and profound hypoxemic respiratory
failure [7]. NETs are extracellular structures composed of granule,
nuclear, and mitochondrial constituents assembled on a scaffold
of decondensed chromatin with antimicrobial proteins and pep-
tides [8]. NETs play a crucial role in host defense and pathogens
clearance during infection; however, dysregulation of NETs can
lead to autoimmune and inflammatory disorders [9]. The for-
mation of NETs occur through various molecular mechanisms
related to neutrophil death. This process is mainly stimulated by
the induction of mitogen phorbol 12-myristate 13-acetate (PMA),
which results in the activation of PKC, leading to reactive oxy-
gen species (ROS) production through the activation of different
enzymes such as NAD phosphate (NADPH), oxidase, myeloperox-
idase (MPO), and through the release of neutrophil elastase (NE)
from granules into the cytoplasm. NE then migrates to the nucleus
of neutrophils where it leads to nuclear membrane disintegration.
Moreover, various proteins have also been found to prime neu-
trophils leading to the release of NETs [10–12].
Recent attempts to define precision management strategies for
the heterogeneous population of patients with ARDS have empha-
sized defining hyper-inflammatory versus hypo-inflammatory
phenotypes of ARDS [13]. The hyperinflammatory phenotype is
characterized by higher plasma levels of inflammatory biomark-
ers and is more common in patients with sepsis or pneumonia and
on vasopressors. Because of the high concentrations of cytokines
and chemokines present in patients with severe COVID-19, they
are likely to be classified as having a hyper-inflammatory pheno-
type [14]. This review focuses on hyperinflammatory ARDS, par-
ticularly due to SARS-CoV-2 infection, and addresses the critical
role of the polymorphonuclear neutrophils and the inflammatory
cytokine storm caused by an accumulation of high plasma levels
of inflammatory cytokines [15].
Role of neutrophils in cytokine storm in COVID-19
ARDS
Patients with COVID-19 ARDS tend to have higher levels of neu-
trophils in the plasma and BALF, which have been correlated with
poor outcomes and illness severity [16, 17]. Neutrophil infiltra-
tion of the lungs is a hallmark of ARDS [18]. Activated neu-
trophils trigger oxidative stress, release proteases, and form NETs,
resulting in lung damage [19]. Infiltration of neutrophils at the
site of infection, and their degranulation and release of NETs in
response to microbial stimuli, produces an increase of cytokines
and chemokines that might result in cytokine storm and con-
tribute to ARDS [20, 21]. The unregulated excess of cytokines
further causes lung inflammation resulting in diffuse alveolar
damage, septic shock, and multiple organ dysfunction [22]. Neu-
trophils are pivotal effector cells in the human innate immune
defense against infections that migrate to infected tissues in mul-
tiple ways including rolling, adhesion, crawling, and transmigra-
tion [23]. They are known to have protective roles against intra-
cellular pathogens such as viruses and mycobacteria. But, they are
also involved in the immunopathology of SARS-CoV-2 infection
by triggering NETosis, the process of NET formation, to result in
severe tissue damage and immunological and inflammatory pro-
cesses (Figure 1) [24]. Based on these findings, targeting neu-
trophils could represent a new therapeutic strategy in acute lung
injury and inflammation.
Effectors of neutrophil activation in COVID-19 ARDS
Inappropriate activation of neutrophils can lead to tissue or organ
injury, thrombus formation, vascular leakage or necrosis that may
lead to the development of a wide range of diseases, including
ARDS and COVID-19 ARDS [25, 26]. Specifically, this inappropri-
ate hyperactivation is caused by a cascade of cellular processes
(oxidation, degranulation, NETosis and/or cytokine overproduc-
tion) and it is characterized by the production of various neu-
trophils effectors, ultimately leading to host tissue/organ dam-
age. The most commonly reported effectors are ROS, granular
enzymes such as MPO, NE, cathepsin G (CatG), NETs and various
pro-inflammatory cytokines (Figure 2) [9, 27, 28]. Additionally,
increasing evidence shows that oxidative stress plays an essen-
tial role in endothelial dysfunction and can lead to coagulation,
thrombosis, and atherosclerosis [29, 30]. Patients with COVID-19
ARDS have damage to pulmonary microvascular endothelial cells
due to higher levels of ROS that contribute to pulmonary vascular
injury, permeability defects, and thrombotic complications [30].
In support of this hypothesis, ROS produced by the NADPH oxi-
dase complex (Nox2) has been associated with thrombotic events
in COVID-19 patients [31].
Increased serum levels of NE, a proteolytic enzyme, in patients
with COVID-19 and fatal ARDS have been reported, suggesting
a key role of neutrophil serine proteinases (NSPs) in COVID-19
pneumonia-driven ARDS [32]. This observation deserves further
investigation to define the role of NSPs, and to evaluate if NSPs’
elimination, by using cathepsin C inhibitors which block NSP mat-
uration, could be a potential therapeutic strategy to prevent the
pulmonary damage in patients with COVID-19 [33]. Myeloperox-
idase, the principal enzyme of peroxidases that catalyzes H2O2
to produce toxic ROS, was demonstrated to be a local media-
tor of alveolar damage [34]. COVID-19 disease severity is corre-
lated with MPO-DNA complexes as a measure of NETs in plasma
and tracheal aspirates of patients with COVID-19 [35]. Exagger-
ated NET formation and high concentrations of NET markers have
been observed in hospitalized patients, including circulating free
DNA (cfDNA), DNA-MPO and DNA-NE complexes [36, 37].
Several neutrophil subpopulations exert different biological
functions [38]. Among them, high density neutrophils (HDNs)
are abundantly represented both in physiological and pathological
settings. Whereas, low-density neutrophils (LDNs), an immature
neutrophil population, and low-density granulocytes (LDGs), a
distinct set of pro-inflammatory granulocytes, differ from LDNs in
surface marker expression and are mainly associated with patho-
logical conditions [39, 40]. LDN subpopulations that expresses
intermediate levels of CD16 (CD16Int) have been identified in
© 2022 The Authors. European Journal of Immunology published by
Wiley-VCH GmbH www.eji-journal.eu

Eur. J. Immunol. 2023;53:2250010 HIGHLIGHTS 3of10
Figure 1. Inltration of neutrophil in the lung contribute to ARDS. In ARDS, activated alveolar macrophages recruit neutrophils, and induce the
secretion of IL-8, into the lungs; this process leads to alveolar pathology associated with the development and severity of the condition. Excessive
accumulation of neutrophils in the alveolar space may trigger release of reactive oxygen species, and neutrophil extracellular traps (NETs) which
can result in epithelial injury. An excess of inammatory cytokines further causes immune cell inltration into inamed lungs resulting in the
pathologic hallmark of diffuse alveolar damage. ARDS, Acute respiratory distress syndrome; NETs, neutrophil extracellular traps; CXCL8, Interleukin
8; IL-1b, interleukin 1 beta; IL-2R, Interleukin 2 receptor; IL-6, interleukin 6; TNF-α, tumor necrosis factor alfa.
blood samples of patients with severe COVID-19, and exhibit a
pro-inflammatory phenotype with phagocytic capacity, NET for-
mation, and interactions with platelets leading to a hypercoag-
ulable state [41]. Additionally, a significant increase of the LDG
subpopulation, the generally less characterized neutrophil sub-
population in viral infections, has been observed in the blood
of patients with COVID-19, thus demonstrating an increase in
neutrophil recruitment and activation of this disease [42]. These
findings support the hypothesis that hyperactivation of specific
neutrophilic subpopulations is an essential feature of inflam-
matory progression and pathogenesis of COVID-19 ARDS, and
that neutrophil effectors produced by the over-activated pro-
cess can consequently lead to tissue/organ damage and disease
progression.
Role of NETs in COVID-19 ARDS
The neutrophilic infiltration into lung tissue in COVID-19 ARDS
is associated with NETs release and surrogate markers of NETs,
such as DNA-MPO and DNA-citrullinated histone-3 complexes,
linked with disease severity [36]. Further evidence suggests that
SARS-CoV-2 can directly stimulate human neutrophils to release
NETs, through the stimulation of angiotensin-converting enzyme
2 (ACE2) and transmembrane serine protease 2 (TRPMSS2) axis
[43]. ACE2 is a key mediator of viral entry into the host cells in
SARS-CoV-2 infection due to its interaction with the glycoprotein
Spike (S) and its expression in several host cells, including lung
pneumocytes, epithelial cells, and endothelial cells, and it is cru-
cial in release of NETs by neutrophils [43]. ACE2-mediated SARS-
CoV-2 cell entry causes a reduction of ACE2 levels on the cell
surface, leading to worse disease severity from neutrophil infiltra-
tion, vascular permeability, and lung edema [44].
NETs cause endothelial damage and necroinflammation via
complement activation linked to the release of thrombogenic
NETs combined with Tissue Factor (TF) which may lead to mul-
tiorgan failure and death [45, 46]. In parallel, NETs have been
proposed to promote thrombus formation during COVID-19 that
leads to multiple organ system dysfunction [47]. Severe disease is
associated with markers of increased coagulopathy, thus suggest-
© 2022 The Authors. European Journal of Immunology published by
Wiley-VCH GmbH www.eji-journal.eu

4of10 Maria Candida Cesta et al. Eur. J. Immunol. 2023;53:2250010
Figure 2. Neutrophil hyperactivation in ARDS COVID-19. Neutrophil hyperactivation causes an enhancement of cellular processes including oxi-
dation, degranulation, NETosis and increases in the production of various neutrophils effectors, ultimately leading to host tissue/organ damage.
The most commonly reported effectors are ROS, MPO,NE, CatG, NETs and various pro-inammatory cytokines. IL-1b, interleukin 1beta; IL-2R, Inter-
leukin 2 receptor; IL-6, interleukin 6; CXCL8, Interleukin 8; TNF-a, tumor necrosis factor alfa; ROS, reactive oxygen species; NE, neutrophil elastase;
CatG, cathepsin G; MPO, myeloperoxidase; NETs, neutrophil extracellular traps; ARDS, Acute respiratory distress syndrome
ing a potential interplay between the coagulation system and NET
formation [48]. Occlusion of small pulmonary vessels, by aggre-
gated NETs, have been observed in lungs of patients who died
from COVID-19 [35]. Interestingly, the recent use of a combina-
tion of high-dimensional single-cell analysis and ex vivo functional
assays of neutrophils from patients with COVID-19 ARDS, com-
pared with patients with non-COVID ARDS, was used to identify
a distinct landscape of neutrophil activation in COVID-19 ARDS
that was intrinsically programmed during SARS-CoV-2 infection
[49]. Thus, demonstrating that neutrophils in COVID-19 ARDS
are functionally primed to produce high amounts of NETs. This
molecular mechanism of neutrophil priming escapes conventional
therapy with corticosteroids, like dexamethasone, paving the way
to consider neutrophil priming in COVID-19 as a promising new
target for adjunctive treatments against severe COVID-19.
The concept of activated neutrophils that produce various
cytotoxic products, NETS, and pro-inflammatory cytokines (IL-
1β, IL-6, IL-8, TNF-α, MCP-1, and GM-CSF) leading to enhanced
inflammation and tissue damage and lung dysfunction is estab-
lished in ARDS and COVID-19 ARDS [50–52]. A prospective study
investigated the presence of NETs in blood and lung samples of
patients with critical COVID-19, and demonstrated that plasma
NET levels peaked early after intensive care unit (ICU) admission
and correlated with plasma levels of chemokines and inflamma-
tory markers [53]. NET quantity correlated with disease sever-
ity and infection duration, with continued detection of NETs in
bronchial and alveolar spaces [37]. All these findings support the
hypothesis of the crucial role of NETs in severe COVID-19 pneu-
monia and ARDS, and their potential contribution to the patho-
logic changes observed in severe cases.
Neutrophil role in immunothrombosis in COVID-19
ARDS
COVID-19 acutely causes lung injury, primarily affecting the vas-
cular endothelium, as evident by the finding of endothelitis and
capillaritis in patients with diffuse severe lung alveolar dam-
age and ARDS [54]. Venous thromboembolism (VTE) has been
reported to occur in up to one-third of COVID-19 cases and
is associated with severe disease, worse clinical outcomes, and
an increase in mortality [55, 56]. Higher VTE rates in patients
© 2022 The Authors. European Journal of Immunology published by
Wiley-VCH GmbH www.eji-journal.eu

Eur. J. Immunol. 2023;53:2250010 HIGHLIGHTS 5of10
Figure 3. Microvascular coagulopathy and immunothrombosis processes in patients with severe COVID-19. A dysregulated inammatory condi-
tion contributes to a prothrombotic phenotype characterized by activation of platelets, endothelial dysfunction and complement activation. This
inammatory induced coagulopathy can result in excessive formation of immunologically mediated thrombi primarily in the microvasculature, a
process known as immunothrombosis. The microvascular coagulopathy and immunothrombosis observed in patients with severe COVID-19 may
be responsible for severe multiple organ dysfunction like ARDS. IL-1b, interleukin 1β; IL-2, Interleukin 2; IL-6, interleukin 6; CXCL8, Interleukin 8;
TNF-a, tumor necrosis factor α; INF – γ, interferon gamma; NETs, neutrophil extracellular traps; ARDS, Acute respiratory distress syndrome.
with severe COVID-19, compared to matched cohorts with other
forms of ARDS, suggest that additional mechanisms beyond typi-
cal risk factors of hospitalized critically ill patients (e.g. immobil-
ity and severe illness) exist [57]. Microvascular coagulopathy and
immunothrombosis observed in patients with severe COVID-19
are partially responsible for the hypercoagulation consequences
from severe COVID-19 (Figure 3) [58]. This cascade can be acti-
vated by SARS-CoV-2 itself, but also by hypoxemia, activated com-
plement and pro-inflammatory cytokines (such as IL-1βand IL-6)
released as part of the cytokine storm that can trigger endothelial
cells injury [59–62].
Neutrophil activation induced by thrombin and factor Xa,
through fibrinogen, fibrin, and C5a, can lead to increased pro-
duction of IL-6 and CXCL8, to result in a strong procoagulant
response and neutrophil-derived TF and NETs formation [63, 64].
As previously reported, in severe COVID-19, high-dimensional
flow cytometric analysis of circulating neutrophils revealed a
highly activated phenotype, LDN, that is more prone to spon-
taneously forming NETs and is associated with microthrombosis
and organ damage [35, 37, 65]. NET-driven thrombosis is largely
platelet-dependent, and have been identified as a major contribu-
tor to neutrophil-related thrombo-inflammation. Von Willebrand
Factor (VWF), released by endothelial cells and platelets, can
result in increased platelet adhesion and fibrin formation, and can
contribute to immunothrombosis. Histone proteins, present in the
DNA fragments of NETs, are potent Damage-Associated Molecu-
lar Patterns (DAMP) molecules also capable of initiating a positive
inflammatory feedback loop [66–68].
Several cell types of the immune system can contribute to
cytokine storm, including polymorphonuclear neutrophils (PMN),
through NET formation [69]. Cytokine storm, through the release
of proinflammatory cytokines and chemokines can exaggerate the
immunothrombotic and coagulation response to create immune
thrombi formation [70, 71]. Both IL-6 and IFN-γincrease platelet
production and activity, while TF expression on endothelial cells
and monocytes increases endothelial dysfunction augmenting
coagulopathy. In addition, IL-2 can further promote coagulation
by decreasing fibrinolysis as a consequence of plasminogen acti-
vator inhibitor-1 (PAI-1) release [72]. CXCL8 has a key role
in the production of the prothrombotic phenotype by attract-
ing neutrophils to the site of infection and causing NETs forma-
tion [63]. Dysregulated signaling in the CXCL8/CXCR1/2 axis
may initiate and perpetuate a self-sustaining loop leading to an
activated, prothrombotic neutrophil phenotype, characterized by
intense degranulation and NET formation [73, 74]. Serum and
plasma from patients with COVID-19 demonstrated NET release
and increased adhesion to activated platelets from healthy neu-
trophils. [65, 75]
Petito et al. found thrombotic complications in 22.2% of 36
patients with COVID-19 and suggested a crucial role of neutrophil
activation as compared to platelet activation, because NET forma-
tion was the biomarker most associated with increased thrombo-
sis and overall disease severity. Thrombotic complications were
observed more often in severe disease, including patients admit-
ted to the ICU and requiring mechanical ventilation, as compared
to patients with mild COVID-19 [76]. Thus, this suggests that NET
© 2022 The Authors. European Journal of Immunology published by
Wiley-VCH GmbH www.eji-journal.eu

6of10 Maria Candida Cesta et al. Eur. J. Immunol. 2023;53:2250010
Table 1. Targeted Immunological Therapies for COVID-19 ARDS
Medication Mechanism PHASE Patient Population NCT
Lenzilumab Inhibitory GMCSF
mAb
III Severe and Critical COVID-19 Pneumonia NCT04351152
Mavrilimumab Inhibitory GMCSF
mAb
II/III Severe COVID-19 Pneumonia NCT04447469
Otilimab Inhibitory GMCSF
mAb
II Severe COVID-19 Pneumonia NCT04376684
Anakinra IL-1 receptor
antagonist
III Severe COVID-19 Pneumonia NCT04680949
Canakinumab Inhibitory IL-1
mAb
III Severe COVID-19 Pneumonia NCT04362813
Sarilumab Inhibitory IL-6
receptor mAb
II Moderate and Severe COVID-19 Pneumonia NCT04357808
Tociluzumab Inhibitory IL-6
receptor mAb
III Severe COVID-19 Pneumonia NCT04320615
Siltuximab Inhibitory IL-6
mAb
III Acute Respiratory Failure by COVID-19 Pneumonia NCT04330638
Reparixin IL-8–CXCR1/2
pathway
inhibitors
III Severe COVID-19 Pneumonia NCT04878055
Dornase alfa Aerosolized
DNAse
III Acute Respiratory Failure by COVID-19 Pneumonia NCT04402970
DNase I II Acute Respiratory Failure by COVID-19 Pneumonia NCT04541979
rh-DNase II Acute Respiratory Failure by COVID-19 Pneumonia NCT04445285
Mesenchymal
stem cell
therapy
I/IIa Acute Respiratory Failure by COVID-19 Pneumonia NCT04524962
https://clinicaltrials.gov/ct2/home last access 03.03.2022
formation is a possible biomarker to predict disease severity and
the potential for thrombotic complications [76].
Neutrophils and NETs have been previously implicated in the
pathophysiology of thrombosis in VTE, as well as ARDS and sep-
sis [77, 78]. In this context, ARDS is defined as a “NETopathy” in
which higher levels of NETs in the plasma and BALF are a charac-
teristic feature of transfusion-associated and pneumonia-related
ARDS [79–81]. There remains an unmet need in the context
of immunothrombosis in COVID-19 and ARDS-related syndrome.
Future and ongoing clinical trials may help to identify agents
with “immunothrombosis targeted interventions” that modulate
the immune response and the cytokine cascade.
Targeting neutrophils and NETosis in COVID-19
There are several investigational anti-inflammatory strategies
under evaluation as potential immunological therapies for hyper-
inflammatory COVID-19 ARDS (Table 1). Corticosteroids are cur-
rently recommended in severe COVID-19 patients due to their
ability to inhibit cytokine release by blocking the NF-κB pathway.
Other commonly utilized agents include anti-IL-6 monoclonal
antibodies, tocilizumab or sarilumab, and agents targeting the
JAK-STAT pathway, such as baricitinib. A recent prospective meta-
analysis of 10,930 patients hospitalized for COVID-19, demon-
strated that IL-6 antagonists were associated with lower 28-day
all-cause mortality [82]. Still, many current immunomodulators
have unknown benefits in early cytokine release syndrome.
Recognizing the important role of neutrophils in severe
COVID-19 has led to the investigation of novel potential thera-
peutic strategies targeting neutrophil recruitment and/or activa-
tion. CXCL8, a key neutrophil chemotactic factor and its bind-
ing to the cognate CXC receptor types 1 and 2 (CXCR1/2),
mediates neutrophil recruitment, activation, and NETs release to
potentiate inflammation and multiple organ system dysfunction
in severe COVID-19 [83, 84]. Elevated CXCL8 levels have been
found in patients at risk for ARDS, and found useful as a prognos-
tic biomarker in patients with severe COVID-19 and ARDS [85].
In the setting of COVID-19, CXCL8 has been significantly associ-
ated with time until death and time from hospital admission until
death [86]. In a study of 24 critically ill adults, CXCL8 was shown
to be significantly correlated with 30-day mortality (r =0.50),
respiratory failure (r =-0.68), acute kidney injury (r =0.73),
and elevated CRP levels [87]. Li et al. demonstrated a correlation
of CXCL8 levels and survival after 14 days from their ICU admis-
sion [88]. It was hypothesized that IL-6 levels spike first and pro-
mote the recruitment of CXCL8 later, resulting in the continuous
cytokine cascade [88]. Elevated admission CXCL8 plasma levels
(>30 pg/mL) have been shown to have a sensitivity of 54.90%
and specificity of 90.26% in predicting mortality in patients with
© 2022 The Authors. European Journal of Immunology published by
Wiley-VCH GmbH www.eji-journal.eu

Eur. J. Immunol. 2023;53:2250010 HIGHLIGHTS 7of10
COVID-19, or can be utilized to predict the length of hospital stay
until death [89]. This data lends further support that CXCL8 may
be a prognostic biomarker for hospital mortality along with other
poor outcomes, and supports new clinical trials targeting CXCL8
as strategy to treat severe COVID-19.
Several CXCL8 inhibitors have now entered clinical evaluation
in patients with severe COVID-19 pneumonia (Table 1). Repar-
ixin, a CXCR1/2 allosteric inhibitor has completed a multicenter,
randomized, adaptive phase 2/3 clinical study (NCT04794803).
The phase 2 portion demonstrated positive results in patients
with severe COVID-19 pneumonia who received oral reparixin
and had a lower incidence of the primary composite clinical
outcome relative to those receiving standard of care [90]. A
larger phase 3 clinical study is ongoing to confirm the role of
reparixin in limiting disease progression in patients with severe
COVID-19 (NCT05254990). Another pro-inflammatory cytokine,
granulocyte-macrophage colony stimulating factor (GM-CSF),
plays a major role in neutrophil differentiation and activation
leading to downstream inflammation caused by cytokines, and
activation of myeloid cells. LIVE-AIR, a phase 3 randomized,
double-blind, placebo-controlled trial, demonstrated a survival
benefit due to lenzilumab, an anti-GM-CSF monoclonal anti-
body, in patients without invasive mechanical ventilation and
with early-stage COVID-19 disease [91]. IL-1βis a major inducer
of CXCL8 expression and release, as well as other neutrophil
recruiting chemotactic factors. The SAVE-MORE trial investigated
anakinra, a recombinant human IL-1 receptor antagonist, in a
Phase 3 double-blind, randomized, controlled trial in 594 patients
with moderate to severe COVID-19 and demonstrated a significant
improvement in clinical status (World Health Organization Clin-
ical Progression Scale) and mortality when compared to placebo
[92]. However, other conflicting studies have demonstrated that
blockade of IL-1 has no effect in clinical improvement in patients
with COVID-19 [93].
The role of NETs in the development of ARDS complications
has prompted the investigation of the therapeutic potential of
agents designed to reduce NETs formation or accumulation. Pro-
tein arginine deiminase 4 (PAD-4) inhibitors, such as CL-Amidine,
are being evaluated in COVID-19 due to their potential role in
thrombotic complications and ability to attenuate NET formation
[94]. ROS may lead to activation of PAD-4, an enzyme involved
in histone citrullination, leading to decondensed chromatin, “net-
like” occlusions, and thrombus formation [94]. Further modula-
tion of NETosis is being investigated by administration of DNase
agents to suppress cytokine levels in patients with COVID-19 by
targeting cell-free DNA from NETs [95]. Recombinant human
DNase, is being investigated in patients presenting with COVID-19
to reduce sputum NETs and improve oxygenation [96, 97]. Sive-
lestat, a NE inhibitor, has been shown to improve pulmonary func-
tion and ventilator free days in patients with acute lung injury by
inhibiting neutrophil chemotaxis and downregulating the NF-kB
pathway [98, 99]. Sivelestat may also be beneficial in preventing
the development of disseminated intravascular coagulation (DIC),
a coagulopathic complication contributed to excess NE, as it has
shown to reduce DIC scores [100].
Conclusions
The COVID-19 pandemic has resulted in a dramatically greater
number of patients with ARDS over the past several years.
The complex relationship of neutrophils, the coagulation system,
NETosis, and the hyperimmune response has provided clinical
challenges for its management. This composite pattern supports
the evaluation of potential therapeutic agents targeting the dys-
regulated neutrophil response in SARS-CoV-2 infection to miti-
gate disease complications. The observation that elevated levels
of CXCL8 are associated with poor prognosis in severe COVID-
19 suggests a potential role in targeting CXCL8 in the future to
impact hyperinflammation and improve patient outcomes.
Acknowledgments: This work was supported by Dompé farma-
ceutici S.p.A. All figures have been created with BioRender.com.
Conflicts of interest: R.B. and A.K. are consultants for Dompé
U.S. Inc. E.M.G., M.C.C., M.Z., C.M., F.M., and M.A. are full-time
employees of Dompé farmaceutici S.p.A.
Author contributions: All authors contributed equally in editing
and proofreading of the manuscript. M.C.C., C.M., E.G., and M.Z.
wrote the manuscript and G.C. prepared the figures. R.B., A.K.,
M.A., and F.M. provided critical analysis and overall guidance of
the science.
Data availability statement: Data sharing is not applicable to
this article as no new data were created or analyzed in this study.
References
1Pham, T. and Rubenfeld, G.D., Fifty years of research in ARDS. The
epidemiology of acute respiratory distress syndrome. A 50th birthday
review. Am J Respir Crit Care Med. 2017. 195(7): 860-70.
2Hendrickson, K.W.,Peltan, I.D. and Brown, S.M., The Epidemiology of
acute respiratory distress syndrome before and after coronavirus dis-
ease 2019. Crit Care Clin. 2021. 37(4): 703-16.
3Peltan, I.D.,Caldwell, E.,Admon, A.J.,Attia, E.F.,Gundel, S.J.,Mathews,
K.S.,et al., Characteristics and outcomes of US patients hospitalized
With COVID-19. Am J Crit Care. 2022. 31(2): 146-57.
4Meyer, N.J.,Gattinoni, L. and Calfee, C.S., Acute respiratory distress syn-
drome. Lancet. 2021. 398(10300): 622-37.
5Johnston, C.J.,Rubenfeld, G.D. and Hudson, L.D., Effect of age on the
development of ARDS in trauma patients. Chest. 2003. 124(2): 653-9.
6Forc e, A.D.T.,Ranieri, V.M.,Rubenfeld, G.D.,Thompson, B.T.,Ferguso n,
N.D.,Caldwell, E.,et al., Acute respiratory distress syndrome: the Berlin
Denition. JAMA. 2012. 307(23): 2526-33.
7Bourenne, J.,Carvelli, J. and Papazian, L., Evolving denition of acute
respiratory distress syndrome. JThoracDis. 2019. 11(Suppl 3): S390-S3.
© 2022 The Authors. European Journal of Immunology published by
Wiley-VCH GmbH www.eji-journal.eu

8of10 Maria Candida Cesta et al. Eur. J. Immunol. 2023;53:2250010
8Brinkmann, V.,Reichard, U.,Goosmann, C.,Fauler, B.,Uhlemann, Y.,
Weiss, D.S.,et al., Neutrophil extracellular traps kill bacteria. Science.
2004. 303(5663): 1532-5.
9Kaplan, M.J. and Radic, M., Neutrophil extracellular traps: double-edged
swords of innate immunity. J Immunol. 2012. 189(6): 2689-95.
10 Pruchniak, M.P. and Demkow, U., Potent NETosis inducers do not show
synergistic effects in vitro. Cent Eur J Immunol. 2019. 44(1): 51-8.
11 Kirchner, T.,Moller, S.,Klinger, M.,Solbach, W.,Laskay, T. and Behnen,
M., The impact of various reactive oxygen species on the formation of
neutrophil extracellular traps. Mediators Inflamm. 2012. 2012:849136.
12 Papayannopoulos,V.,Metzler, K.D.,Hakkim, A. and Zychlinsky, A.,Neu-
trophil elastase and myeloperoxidase regulate the formation of neu-
trophil extracellular traps. JCellBiol. 2010. 191(3): 677-91.
13 Calfee, C.S.,Delucchi, K.,Parsons, P.E.,Thompson, B.T.,Ware, L.B.,
Matthay, M.A.,et al., Subphenotypes in acute respiratory distress syn-
drome: latent class analysis of data from two randomised controlled tri-
als. Lancet Respir Med. 2014. 2(8): 611-20.
14 Bos, L.D.J.,Artigas, A.,Constantin, J.M.,Hagens, L.A.,Heijnen, N.,Laffey,
J.G.,et al., Precision medicine in acute respiratory distress syndrome:
workshop report and recommendations for future research. Eur Respir
Rev. 2021. 30(159).
15 Ruan, Q.,Yan g, K .,Wan g, W.,Jiang, L. and Song, J., Clinical predictors of
mortality due to COVID-19 based on an analysis of data of 150 patients
from Wuhan, China. Intensive Care Med. 2020. 46(5): 846-8.
16 Voiriot, G.,Dorgham, K.,Bachelot, G.,Faja c, A.,Morand-Joubert, L.,Pari-
zot, C.,et al., Identication of bronchoalveolar and blood immune-
inammatory biomarker signature associated with poor 28-day out-
come in critically ill COVID-19 patients. Scientific reports. 2022. 12(1):
9502.
17 Dentone, C.,Ven a, A. ,Loconte, M.,Grillo, F.,Brunetti, I.,Barisione, E.,
et al., Bronchoalveolar lavage uid characteristics and outcomes of inva-
sively mechanically ventilated patients with COVID-19 pneumonia in
Genoa, Italy. BMC Infect Dis. 2021. 21(1): 353.
18 Zemans, R.L. and Matthay, M.A., What drives neutrophils to the alveoli
in ARDS? Thorax. 2017. 72(1): 1-3.
19 Zhu, Y.,Chen, X. and Liu, X., NETosis and Neutrophil Extracellular Traps
in COVID-19: Immunothrombosis and Beyond. Frontiers in Immunology.
2022. 13.
20 Shah, R.D. and Wunderink, R.G.,Viral Pneumonia and Acute Respiratory
Distress Syndrome. Clin Chest Med. 2017. 38(1): 113-25.
21 Eworuke, E.,Major, J.M. and Gilbert McClain, L.I. National incidence
rates for Acute Respiratory Distress Syndrome (ARDS) and ARDS cause-
specic factors in the United States (2006-2014). JCritCare. 2018. 47:192-
7.
22 McElvaney, O.J.,McEvoy, N.L.,McElvaney, O.F.,Carroll, T.P.,Murphy,M.P.,
Dunlea, D.M.,et al., Characterization of the Inammatory Response
to Severe COVID-19 Illness. Am J Respir Crit Care Med. 2020. 202(6):
812-21.
23 Niedzwiedzka-Rystwej, P.,Grywalska, E.,Hrynkiewicz, R.,Bebnowska,
D.,Wolacewicz, M.,Majchrzak, A.,et al., Interplay between Neutrophils,
NETs and T-Cells in SARS-CoV-2 Infection-A Missing Piece of the Puzzle
in the COVID-19 Pathogenesis? Cells. 2021. 10(7).
24 Muraro, S.P.,De Souza, G.F.,Gallo, S.W.,Da Silva, B.K.,De Oliveira, S.D.,
Vinolo, M.A.R.,et al., Respiratory Syncytial Virus induces the classical
ROS-dependent NETosis through PAD-4 and necroptosis pathways acti-
vation. Sci Rep. 2018. 8(1): 14166.
25 Kruger, P.,Saffarzadeh, M.,Webe r, A. N.,Rieber, N.,Radsak, M.,von
Bernuth, H.,et al., Neutrophils: Between host defence, immune mod-
ulation, and tissue injury. PLoS Pathog. 2015. 11(3): e1004651.
26 Reyes, L.,AS-G, M.,Morrison, T.,Howden, A.J.M.,Watts, E.R.,Arienti, S.,
et al., A type I IFN, prothrombotic hyperinammatory neutrophil signa-
ture is distinct for COVID-19 ARDS. Wellcome Open Res. 2021. 6:38.
27 Goud, P.T.,Bai,D. and Abu-Soud, H.M., A Multiple-Hit Hypothesis Involv-
ing Reactive Oxygen Species and Myeloperoxidase Explains Clinical
Deterioration and Fatality in COVID-19. Int J Biol Sci. 2021. 17(1): 62-72.
28 Tamassia, N.,Bianchetto-Aguilera, F.,Arruda-Silva, F.,Gardiman, E.,
Gasperini, S.,Calzetti, F.,et al., Cytokine production by human neu-
trophils: Revisiting the “dark side of the moon”. Eur J Clin Invest. 2018.
48 Suppl 2:e12952.
29 Jin, Y.,Ji, W.,Yan g, H. ,Chen, S.,Zhang,W. and Duan, G., Endothelial acti-
vation and dysfunction in COVID-19: from basic mechanisms to poten-
tial therapeutic approaches. Signal Transduct Target Ther. 2020. 5(1): 293.
30 Cekerevac, I.,Turnic, T.N.,Draginic, N.,Andjic, M.,Zivkovic, V.,Simovic,
S.,et al., Predicting Severity and Intrahospital Mortality in COVID-
19: The Place and Role of Oxidative Stress. Oxid Med Cell Longev. 2021.
2021:6615787.
31 DiNicolantonio, J.J. and McCarty, M., Thrombotic complications of
COVID-19 may reect an upregulation of endothelial tissue factor
expression that is contingent on activation of endosomal NADPH oxi-
dase. Open Heart. 2020. 7(1).
32 Zerimech, F.,Jourdain, M.,Onraed, B.,Bouchecareilh, M.,Sendid, B.,
Duhamel, A.,et al., Protease-antiprotease imbalance in patients with
severe COVID-19. Clin Chem Lab Med. 2021. 59(8): e330-e4.
33 Korkmaz, B.,Lesner, A.,Marchand-Adam, S.,Moss, C. and Jenne, D.E.,
Lung Protection by Cathepsin C Inhibition: A New Hope for COVID-19
and ARDS? J Med Chem. 2020. 63(22): 13258-65.
34 Aratani, Y., Myeloperoxidase: Its role for host defense, inammation,
and neutrophil function. Arch Biochem Biophys. 2018. 640:47-52.
35 Middleton, E.A.,He, X.Y.,Denorme, F.,Campbell, R.A.,Ng, D.,Salvatore,
S.P.,et al.,Neutrophil extracellular traps contribute to immunothrombo-
sis in COVID-19 acute respiratory distress syndrome. Blood. 2020. 136(10):
1169-79.
36 Zuo, Y.,Yal avart hi , S. ,Shi, H.,Gockman, K.,Zuo, M.,Madison, J.A.,et al.,
Neutrophil extracellular traps in COVID-19. JCI Insight. 2020. 5(11).
37 Leppkes, M.,Knopf, J.,Naschberger, E.,Lindemann, A.,Singh, J.,Her-
rmann, I.,et al., Vascular occlusion by neutrophil extracellular traps in
COVID-19. EBioMedicine. 2020. 58:102925.
38 Silvestre-Roig, C.,Fridlender, Z.G.,Glogauer, M. and Scapini, P.,Neu-
trophil Diversity in Health and Disease. Trends Immunol. 2019. 40(7): 565-
83.
39 Villanueva, E.,Yalav ar thi, S. ,Berthier, C.C.,Hodgin, J.B.,Khandpur, R.,
Lin, A.M.,et al., Netting neutrophils induce endothelial damage, inl-
trate tissues, and expose immunostimulatory molecules in systemic
lupus erythematosus. J Immunol. 2011. 187(1): 538-52.
40 Denny, M.F.,Yal ava rth i, S. ,Zhao, W.,Tha cker, S.G.,Anderson, M.,Sandy,
A.R.,et al., A distinct subset of proinammatory neutrophils isolated
from patients with systemic lupus erythematosus induces vascular
damage and synthesizes type I IFNs. J Immunol. 2010. 184(6): 3284-97.
41 Morrissey, S.M.,Geller, A.E.,Hu,X.,Tie ri, D.,Ding, C.,Klaes, C.K.,et al.,A
specic low-density neutrophil population correlates with hypercoagu-
lation and disease severity in hospitalized COVID-19 patients.JCI Insight.
2021. 6(9).
42 Cabrera, L.E.,Pekkarinen, P.T.,Alander, M.,Nowlan, K.H.A.,Nguyen,
N.A.,Jokiranta, S.,et al., Characterization of low-density granulocytes
in COVID-19. PLoS Pathog. 2021. 17(7): e1009721.
43 Ver as, F.P.,Pontelli, M.C.,Silva, C.M.,Toller-Kawahisa, J.E.,de Lima, M.,
Nascimento, D.C.,et al., SARS-CoV-2-triggered neutrophil extracellular
traps mediate COVID-19 pathology. JExpMed. 2020. 217(12).
© 2022 The Authors. European Journal of Immunology published by
Wiley-VCH GmbH www.eji-journal.eu

Eur. J. Immunol. 2023;53:2250010 HIGHLIGHTS 9of10
44 Eguchi, S.,Kawai,T.,Scalia, R. and Rizzo, V.,Understanding Ang iotensin
II Type 1 Receptor Signaling in Vascular Pathophysiology. Hypertension.
2018. 71(5): 804-10.
45 Skendros, P.,Mitsios, A.,Chrysanthopoulou, A.,Mastellos, D.C.,Metal-
lidis, S.,Rafailidis, P.,et al., Complement and tissue factor-enriched neu-
trophil extracellular traps are key drivers in COVID-19 immunothrom-
bosis. J Clin Invest. 2020. 130(11): 6151-7.
46 Czaikoski, P.G.,Mota, J.M.,Nascimento, D.C.,Sonego, F.,Castanheira,
F.V.,Melo, P.H.,et al., Neutrophil Extracellular Traps Induce Organ Dam-
age during Experimental and Clinical Sepsis. PLoS One. 2016. 11(2):
e0148142.
47 Llitjos, J.F.,Leclerc, M.,Chochois, C.,Monsallier, J.M.,Ramakers, M.,
Auvray, M.,et al., High incidence of venous thromboembolic events in
anticoagulated severe COVID-19 patients. J Thromb Haemost. 2020. 18(7):
1743-6.
48 Connors, J.M. and Levy, J.H., COVID-19 and its implications for throm-
bosis and anticoagulation. Blood. 2020. 135(23): 2033-40.
49 Panda , R.,Castanheira, F.V.,Schlechte, J.M.,Surewaard, B.G.,Shim,
H.B.,Zucoloto, A.Z.,et al., A functionally distinct neutrophil landscape
in severe COVID-19 reveals opportunities for adjunctive therapies. JCI
Insight. 2022. 7(2).
50 Liu, Y.,Du, X.,Chen, J.,Jin, Y.,Peng, L .,Wang, H.H.X.,et al., Neutrophil-
to-lymphocyte ratio as an independent risk factor for mortality in hos-
pitalized patients with COVID-19. J Infect. 2020. 81(1): e6-e12.
51 Bhaskar, S.,Sinha, A.,Banach, M.,Mittoo, S.,Weissert, R.,Kass, J.S.,
et al., Cytokine Storm in COVID-19-Immunopathological Mechanisms,
Clinical Considerations, and Therapeutic Approaches: The REPROGRAM
Consortium Position Paper. Frontiers in immunology. 2020. 11: 1648.
52 Hussman, J.P., Cellular and Molecular Pathways of COVID-19 and Poten-
tial Points of Therapeutic Intervention. Frontiers in pharmacology. 2020. 11:
1169.
53 Teluguakula, N., Neutrophils Set Extracellular Traps to Injure Lungs in
Coronavirus Disease 2019. J Infect Dis. 2021. 223(9): 1503-5.
54 Goshua, G.,Pine, A.B.,Meizlish, M.L.,Chang, C.H.,Zhang, H.,Bahel, P.,
et al., Endotheliopathy in COVID-19-associated coagulopathy: evidence
from a single-centre, cross-sectional study. Lancet Haematol. 2020. 7(8):
e575-e82.
55 Klok, F.A.,Kruip, M.,van der Meer, N.J.M.,Arbous, M.S.,Gommers, D.,
Kant, K.M.,et al., Incidence of thrombotic complications in critically ill
ICU patients with COVID-19. Thromb Res. 2020. 191: 145-7.
56 Middeldorp, S.,Coppens, M.,van Haaps, T.F.,Foppen, M.,Vlaar, A.P.,
Muller, M.C.A.,et al., Incidence of venous thromboembolism in hos-
pitalized patients with COVID-19. J Thromb Haemost. 2020. 18(8): 1995-
2002.
57 Helms, J.,Tacqua rd , C.,Severac, F.,Leonard-Lorant, I.,Ohana, M.,
Delabranche, X.,et al., High risk of thrombosis in patients with severe
SARS-CoV-2 infection: a multicenter prospective cohort study. Intensive
Care Med. 2020. 46(6): 1089-98.
58 Bradley, B.T.,Maioli, H.,Johnston,R.,Chaudhry, I.,Fink, S.L.,Xu, H.,et al.,
Histopathology and ultrastructural ndings of fatal COVID-19 infections
in Washington State: a case series. Lancet. 2020. 396(10247): 320-32.
59 Zhang, S.,Liu, Y.,Wang , X.,Ya ng , L.,Li, H.,Wa ng, Y.,et al., SARS-CoV-2
binds platelet ACE2 to enhance thrombosis in COVID-19.JHematolOncol.
2020. 13(1): 120.
60 Price, L.C.,McCabe,C.,Gareld, B. and Wort, S.J.,Thrombosis and COVID-
19 pneumonia: the clot thickens! EurRespirJ. 2020. 56(1).
61 Gupta, A.,Madhavan, M.V.,Sehgal, K.,Nair, N.,Mahajan, S.,Sehrawat,
T.S .,et al., Extrapulmonary manifestations of COVID-19. Nat Med. 2020.
26(7): 1017-32.
62 Magro, C.,Mulvey, J.J.,Berlin, D.,Nuovo, G.,Salvatore, S.,Harp, J.,et al.,
Complement associated microvascular injury and thrombosis in the
pathogenesis of severe COVID-19 infection: A report of ve cases. Tra nsl
Res. 2020. 220:1-13.
63 Iliadi, V.,Konstantinidou, I.,Aftzoglou, K.,Iliadis, S.,Konstantinidis,T.G.
and Tsigalou, C., The Emerging Role of Neutrophils in the Pathogenesis
of Thrombosis in COVID-19. International journal of molecular sciences. 2021.
22(10).
64 Fletcher-Sandersjoo, A. and Bellander, B.M., Is COVID-19 associated
thrombosis caused by overactivation of the complement cascade? A lit-
erature review. Thromb Res. 2020. 194:36-41.
65 Nicolai, L.,Leunig, A.,Brambs, S.,Kaiser, R.,Weinberger, T.,Wei gand,
M.,et al., Immunothrombotic Dysregulation in COVID-19 Pneumonia Is
Associated With Respiratory Failure and Coagulopathy. Circulation. 2020.
142(12): 1176-89.
66 de Bont, C.M.,Boelens, W.C. and Pruijn, G J M., NETosis, complement,
and coagulation: a triangular relationship. Cell Mol Immunol. 2019. 16(1):
19-27.
67 Zucoloto, A.Z. and Jenne, C.N., Platelet-Neutrophil Interplay: Insights
Into Neutrophil Extracellular Trap (NET)-Driven Coagulation in Infec-
tion. Front Cardiovasc Med. 2019. 6:85.
68 Fuchs, T.A.,Brill, A.,Duerschmied, D.,Schatzberg, D.,Monestier, M.,
Myers D.D., Jr.,et al., Extracellular DNA traps promote thrombosis. Pro-
ceedings of the National Academy of Sciences of the United States of America. 2010.
107(36): 15880-5.
69 Keshari, R.S.,Jyoti, A.,Dubey, M.,Kothari, N.,Kohli, M.,Bogra, J.,
et al., Cytokines induced neutrophilextracellular traps formation: impli-
cation for the inammatory disease condition. PLoS One. 2012. 7(10):
e48111.
70 Gorham, J.,Moreau, A.,Corazza, F.,Peluso, L.,Ponthieux, F.,Talamonti,
M.,et al., Interleukine-6 in critically ill COVID-19 patients: A retrospec-
tive analysis. PLoS One. 2020. 15(12): e0244628.
71 Kim, J.S.,Lee, J.Y.,Yang, J.W.,Lee, K.H.,Effenberger, M.,Szpirt, W.,et al.,
Immunopathogenesis and treatment of cytokine storm in COVID-19.
Theranostics. 2021. 11(1): 316-29.
72 Du, F.,Liu, B. and Zhang, S., COVID-19: the role of excessive cytokine
release and potential ACE2 down-regulation in promoting hypercoag-
ulable state associated with severe illness. J Thromb Thrombolysis. 2021.
51(2): 313-29.
73 Wan g, J.,Jiang, M.,Chen, X. and Montaner, L.J., Cytokine storm and
leukocyte changes in mild versus severe SARS-CoV-2 infection: Review
of 3939 COVID-19 patients in China and emerging pathogenesis and
therapy concepts. JLeukocBiol. 2020. 108(1): 17-41.
74 Kaiser, R.,Leunig, A.,Pekayvaz, K.,Popp, O.,Joppich, M.,Polewka, V.,
et al., Self-sustaining IL-8 loops drive a prothrombotic neutrophil phe-
notype in severe COVID-19. JCI Insight. 2021. 6(18).
75 Bonaventura, A.,Vecc hie , A. ,Dagna, L.,Martinod, K.,Dixon, D.L.,Van
Tassell, B.W.,et al., Endothelial dysfunction and immunothrombosis
as key pathogenic mechanisms in COVID-19. Nature reviews Immunology.
2021. 21(5): 319-29.
76 Petito, E.,Falcinelli, E.,Paliani, U.,Cesari, E.,Vau do, G.,Sebastiano, M.,
et al., Association of Neutrophil Activation, More Than Platelet Acti-
vation, With Thrombotic Complications in Coronavirus Disease 2019. J
Infect Dis. 2021. 223(6): 933-44.
77 Lefrancais, E.,Mallavia, B.,Zhuo, H.,Calfee, C.S. and Looney, M.R., Mal-
adaptive role of neutrophil extracellular traps in pathogen-induced lung
injury. JCI Insight. 2018. 3(3).
78 Denning, N.L.,Aziz, M.,Gurien, S.D. and Wang, P., DAMPs and NETs in
Sepsis. Frontiers in immunology. 2019. 10:2536.
© 2022 The Authors. European Journal of Immunology published by
Wiley-VCH GmbH www.eji-journal.eu

10 of 10 Maria Candida Cesta et al. Eur. J. Immunol. 2023;53:2250010
79 Mitsios, A.,Arampatzioglou, A.,Arelaki, S.,Mitroulis, I. and
NETopathies?, R.K., Unraveling the Dark Side of Old Diseases through
Neutrophils. Frontiers in immunology. 2016. 7:678.
80 Rebetz, J.,Semple, J.W. and Kapur, R., The Pathogenic Involve-
ment of Neutrophils in Acute Respiratory Distress Syndrome and
Transfusion-RelatedAcute Lung Injury. Transfus Med Hemother.2018. 45(5):
290-8.
81 Bendib, I.,de Chaisemartin, L.,Granger, V.,Schlemmer, F.,Maitre, B.,
Hue, S.,et al., Neutrophil Extracellular Traps Are Elevated in Patients
with Pneumonia-related Acute Respiratory Distress Syndrome. Anesthe-
siology. 2019. 130(4): 581-91.
82 Group WHOREAfC-TW, Shankar-Hari, M.,Vale, C.L.,Godolphin, P.J.,
Fisher, D.,Higgins, J.P.T.,et al., Association Between Administration of
IL-6 Antagonists and Mortality Among Patients Hospitalized for COVID-
19: A Meta-analysis. Jama. 2021. 326(6): 499-518.
83 Narasaraju, T.,Tang, B.M.,Herrmann, M.,Muller, S.,Chow, V.T.K. and
Radic, M., Neutrophilia and NETopathy as Key Pathologic Drivers of Pro-
gressive Lung Impairment in Patients With COVID-19. Frontiers in phar-
macology. 2020. 11:870.
84 Group, R.C.,Horby, P.,Lim, W.S.,Emberson, J.R.,Mafham, M.,Bell, J.L.,
et al., Dexamethasone in Hospitalized Patients with Covid-19. N Engl J
Med. 2021. 384(8): 693-704.
85 Wan g, Y.,Wan g, H.,Zhang, C.,Zhang, C.,Yan g, H .,Gao, R.,et al.,Lung
uid biomarkers for acute respiratory distress syndrome: a systematic
review and meta-analysis. Crit Care. 2019. 23(1): 43.
86 Li, H.,Zhang, J.,Fang, C.,Zhao, X.,Qian, B.,Sun, Y.,et al., The prognostic
value of IL-8 for the death of severe or critical patients with COVID-19.
Medicine (Baltimore). 2021. 100(11): e23656.
87 Bulow Anderberg, S.,Luther, T.,Berglund, M.,Larsson, R.,Rubertsson,S.,
Lipcsey, M.,et al., Increased levels of plasma cytokines and correlations
to organ failure and 30-day mortality in critically ill Covid-19 patients.
Cytokine. 2021. 138:155389.
88 Li, J.,Rong, L.,Cui, R.,Feng , J.,Jin, Y.,Chen, X.,et al., Dynamic changes
in serum IL-6, IL-8, and IL-10 predict the outcome of ICU patients with
severe COVID-19. Ann Palliat Med. 2021. 10(4): 3706-14.
89 Luo, Y.,Mao, L.,Yua n, X. ,Xue, Y.,Lin, Q.,Tang, G .,et al., Prediction
Model Based on the Combination of Cytokines and Lymphocyte Sub-
sets for Prognosis of SARS-CoV-2 Infection. J Clin Immunol. 2020. 40(7):
960-9.
90 Landoni, G.,Piemonti, L.,Monforte, A.D.,Grossi, P.,Zangrillo, A.,Bucci,
E.,et al.,A Multicenter Phase 2 Randomized Controlled Study on the Ef-
cacy and Safety of Reparixin in the Treatment of Hospitalized Patients
with COVID-19 Pneumonia. Infect Dis Ther. 2022.
91 Tem es gen , Z.,Burger, C.D.,Baker, J.,Polk, C.,Libertin, C.R.,Kelley, C.F.,
et al., Lenzilumab in hospitalised patients with COVID-19 pneumonia
(LIVE-AIR): a phase 3, randomised, placebo-controlled trial. Lancet Respir
Med. 2021.
92 Kyriazopoulou, E.,Poulakou, G.,Milionis, H.,Metallidis, S.,Adamis,
G.,Tsiakos, K.,et al., Early treatment of COVID-19 with anakinra
guided by soluble urokinase plasminogen receptor plasma levels: a
double-blind, randomized controlled phase 3 trial. Nat Med. 2021. 27(10):
1752-60.
93 Declercq, J.,Van Damme, K.F.A.,De Leeuw, E.,Maes, B.,Bosteels, C.,Tav-
ernier, S.J.,etal., Effect of anti-interleukin drugs in patients with COVID-
19 and signs of cytokine release syndrome (COV-AID): a factorial, ran-
domised, controlled trial. Lancet Respir Med. 2021. 9(12): 1427-38.
94 Elliott, W., Jr.,Guda, M.R.,Asuthkar, S.,Teluguakula, N.,Prasad, D.V.R.,
Tsung, A.J.,et al.,PAD Inhibitors as a Potential Treatment for SARS-CoV-
2 Immunothrombosis. Biomedicines. 2021. 9(12).
95 Le e, Y.Y.,Park, H.H.,Park, W.,Kim, H.,Jang, J.G.,Hong, K.S.,et al.,Long-
acting nanoparticulate DNase-1 for effective suppression of SARS-CoV-
2-mediated neutrophil activities and cytokine storm. Biomaterials. 2021.
267:120389.
96 Fisher, J.,Mohanty, T.,Karlsson, C.A.Q.,Khademi, S.M.H.,Malmstrom,
E.,Frigyesi , A.,et al.,Proteome Proling of Recombinant DNase Therapy
in Reducing NETs and Aiding Recovery in COVID-19 Patients. Mol Cell
Proteomics. 2021. 20:100113.
97 Holliday, Z.M.,Earhart,A.P.,Alnijoumi, M.M.,Krvavac, A.,Allen,L.H. and
Schrum, A.G., Non-Randomized Trial of Dornase Alfa for Acute Respira-
tory Distress Syndrome Secondary to Covid-19. Frontiers in immunology.
2021. 12:714833.
98 Tamakuma,S.,Ogawa, M.,Aikawa,N.,Kubo ta, T.,Hirasawa,H.,Ishizaka,
A.,et al.,Relationship between neutrophil elastase and acute lung injury
in humans. Pulm Pharmacol Ther. 2004. 17(5): 271-9.
99 Aikawa, N.,Ishizaka, A.,Hirasawa, H.,Shimazaki, S.,Yamamoto, Y.,
Sugimoto, H.,et al., Reevaluation of the efcacy and safety of the neu-
trophil elastase inhibitor, Sivelestat, for the treatment of acute lung
injury associated with systemic inammatory response syndrome; a
phase IV study. Pulm Pharmacol Ther. 2011. 24(5): 549-54.
100 Hayakawa, M.,Katabami, K.,Wada , T.,Sugano, M.,Hoshino, H.,Sawa-
mura, A.,et al., Sivelestat (selective neutrophil elastase inhibitor)
improves the mortality rate of sepsis associated with both acute res-
piratory distress syndrome and disseminated intravascular coagulation
patients. Shock. 2010. 33(1): 14-8.
Full correspondence: Robert Balk, Professor of Medicine, Division of
Pulmonary, Critical Care, and Sleep Medicine, Rush Medical College
and Rush University Medical Center, 1750 West Harrison St, Chicago, IL
60612, Email: Robert_Balk@rush.edu
Received: 20/5/2022
Revised: 11/9/2022
Accepted: 12/10/2022
Accepted article online: 18/10/2022
© 2022 The Authors. European Journal of Immunology published by
Wiley-VCH GmbH www.eji-journal.eu