COVID-19 and Neutrophils: The Relationship between Hyperinflammation and Neutrophil Extracellular Traps

Abstract

Coronavirus disease 2019 (COVID-19) is a virus-induced respiratory disease that may progress to acute respiratory distress syndrome (ARDS) and is triggered by immunopathological mechanisms that cause excessive inflammation and leukocyte dysfunction. Neutrophils play a critical function in the clearance of bacteria with specific mechanisms to combat viruses. The aim of this review is to highlight the current advances in the pathways of neutrophilic inflammation against viral infection over the past ten years, focusing on the production of neutrophil extracellular traps (NETs) and its impact on severe lung diseases, such as COVID-19. We focused on studies regarding hyperinflammation, cytokine storms, neutrophil function, and viral infections. We discuss how the neutrophil's role could influence COVID-19 symptoms in the interaction between hyperinflammation (overproduction of NETs and cytokines) and the clearance function of neutrophils to eliminate the viral infection. We also propose a more in-depth investigation into the neutrophil response mechanism targeting NETosis in the different phases of COVID-19.

Full text

Review Article
COVID-19 and Neutrophils: The Relationship between
Hyperinflammation and Neutrophil Extracellular Traps
Leandro Borges ,
1
Tania Cristina Pithon-Curi,
1
Rui Curi,
1,2
and Elaine Hatanaka
1
1
Instituto de Ciências da Atividade Física e Esportes (ICAFE), Universidade Cruzeiro do Sul, São Paulo, SP, Brazil
2
Instituto Butantan, São Paulo, SP, Brazil
Correspondence should be addressed to Leandro Borges; sbleandro@yahoo.com.br
Received 8 September 2020; Revised 15 November 2020; Accepted 25 November 2020; Published 4 December 2020
Academic Editor: Juliana Vago
Copyright © 2020 Leandro Borges et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Coronavirus disease 2019 (COVID-19) is a virus-induced respiratory disease that may progress to acute respiratory distress
syndrome (ARDS) and is triggered by immunopathological mechanisms that cause excessive inflammation and leukocyte
dysfunction. Neutrophils play a critical function in the clearance of bacteria with specific mechanisms to combat viruses. The
aim of this review is to highlight the current advances in the pathways of neutrophilic inflammation against viral infection over
the past ten years, focusing on the production of neutrophil extracellular traps (NETs) and its impact on severe lung diseases,
such as COVID-19. We focused on studies regarding hyperinflammation, cytokine storms, neutrophil function, and viral
infections. We discuss how the neutrophil’s role could influence COVID-19 symptoms in the interaction between
hyperinflammation (overproduction of NETs and cytokines) and the clearance function of neutrophils to eliminate the viral
infection. We also propose a more in-depth investigation into the neutrophil response mechanism targeting NETosis in the
different phases of COVID-19.
1. Introduction
The World Health Organization (WHO) established the
coronavirus disease 2019 (COVID-19) as a pandemic on
March 11, 2020. Severe acute respiratory syndrome corona-
virus 2 (SARS-CoV-2) is a member of the coronavirus family,
a class of enveloped viruses with a positive-sense single-
stranded RNA genome. This virus can cross species barriers
and induce illnesses ranging from the usual cold to severe
interstitial pneumonia, respiratory failure, and septic shock
[1]. While there is a global effort in the development of
vaccines and improvement of diagnostic methods [2, 3] and
therapies that relieve the symptoms and prognosis of
COVID-19 patients under severe infection [4], there remain
gaps in our understanding of the pathophysiology of
COVID-19 related to innate immunity.
In a scenario where patients with severe COVID-19 could
develop dysfunction of the immune response that aggravates
the hyperinflammation [5, 6], it is hypothesized that neutro-
phils can amplify pathological damage or control other cell
subsets depending on the infection features. Therefore, to
use the potential of NETs with minimal damage to the hosts,
there must be a right balance of NET formation and reduc-
tion of the amount of NETs that accumulate in tissues [7].
Notwithstanding the rapid progress in the field, there are
many critical unknown features of neutrophils in fighting
viral infections. We highlighted the current progress in the
pathways of neutrophilic inflammation in viral infection,
with a focus on the release of NETs and its influence on lung
disease. The knowledge summarized in this study should
benefit researchers in integrating neutrophil biology to
design new and more efficient virus-targeted interventions
concerning COVID-19.
2. Hyperinflammation
Although a well-regulated innate immune process is the first
protection action against viral infections [8], in severe
COVID-19 condition occurs hyperinflammation (“cytokine
Hindawi
Mediators of Inflammation
Volume 2020, Article ID 8829674, 7 pages
https://doi.org/10.1155/2020/8829674

storm”) that might lead to the acute respiratory distress
syndrome (ARDS) [6, 9].
Cytokines play a relevant function in immunopathology
during virus infections. The host-viral interactions are estab-
lished via host identification of pathogen-associated molecu-
lar patterns (PAMPs) of the virus [10]. This identification
occurs through host pattern recognition receptors (PRRs)
manifested on innate immune cells (e.g., neutrophils,
dendritic cells, epithelial cells, and macrophages) [11], and
the recognition of PAMPs and viral danger-associated
molecular patterns (DAMPs) by conserved PRRs marks the
first line of defense against pathogens, involving toll-like
receptors (TLRs) [11].
TLR stimulation activates the nuclear factor-κB (NF-κB)
signaling cascade, causing the production of inflammatory
markers from monocytes (interleukin- (IL-) 1, tumor necro-
sis factor-alpha (TNF-α), and IL-6) to control virus infec-
tions [8] by direct antiviral pathways and the recruitment
of other leukocytes [10]. Moreover, the exacerbated oxidative
stress induced by elevated concentrations of cytokines, along
with reduced concentrations of interferon αand interferon β
(IFN-α, IFN-β), influences the severity of COVID-19 [12].
Several mediators control the release of chemoattractants
and neutrophil activity [10], and studies have demonstrated
that higher values of proinflammatory markers are related
to extensive lung damage and pulmonary inflammation in
MERS-CoV [13] and ARDS infection [14]. COVID-19 in
the severe state exhibits a cytokine storm with elevated
plasma levels of chemokine ligand 2 (CCL2), IFNγ, IFNγ-
inducible protein 10, G-CSF, chemokine C-C motif ligand 3
(CCL3), IL-1β, IL-2, IL-6, IL-7, IL-8, IL-10, IL-17, and
TNF-α[12, 15]. Nucleotide-binding oligomerization
domain- (NOD-) like receptor and increased plasma levels
of chemokines and cytokines in COVID-19 patients relate
to the severity of the disease rather than did those nonsevere
patients [5]. In this sense, Huang et al. [15] found that
patients in the intensive care unit (ICU) with laboratory-
confirmed COVID-19 infection had higher plasma levels of
IL-2, IL-7, IL-10, interferon-inducible protein 10, granulo-
cyte colony-stimulating factor, CCL2, CCL3, and TNF-α
when compared with non-ICU patients [15].
3. Neutrophils: The First Cell Recruitment
Neutrophils are innate immune cells with a brief lifespan
after leaving the bone marrow and exist in a quiescent,
primed, or active state. These leukocytes are the leading
players in innate immunity since they are among the first
innate leukocytes recruited during infections [16]. The pri-
mary function of neutrophil is clearance of pathogens and
debris through phagocytosis [17]. They also have a distinct
array of other immune roles, such as the liberation of NETs
for viral infection inactivation [18] and cytokine production
to restrict virus replication [16].
The release of neutrophil-chemoattractive elements and
the resulting recruitment of neutrophils are a global host
response to viral infection [19]. In this scenario, the neutro-
phil cell membrane also expresses a complex array of recep-
tors and adhesion molecules for various ligands, including
immunoglobulins, membrane molecules on other cells, and
cytokines [20].
In addition to the trafficking to infection places to phago-
cytize viruses, the neutrophils can initiate, enlarge, and/or
repress adaptive immune effector processes by promoting
bidirectional cross-talk with T cells [21, 22]. Following the
acute inflammation arising from immunological processes,
such as viral infections, neutrophils with decreased expres-
sion of CD62L weaken T cell migration via the CXCL11 che-
mokine gradient by releasing H
2
O
2
into an immunological
synapse [23]. Thus, neutrophils that uncovered viral antigens
can home to draining lymph nodes, acting as antigen-
presenting cells (APC) [24]. Hufford et al. [25] evidenced
that neutrophils expressing viral antigen as an outcome of
direct infection by influenza A virus (IAV) display the most
potent APC activity and that viral antigen-presenting neutro-
phils infiltrating the IAV-infected lungs act as APC for effec-
tor CD8(+) T lymphocytes in the infected lungs [25].
Neutrophils recruit the T cell molecular mechanism during
the influenza virus infection and associate to CXCL12 reser-
voirs left behind. CD8+ T cells follow the chemoattractant
trail left behind by neutrophil uropods to the influenza virus
infection site [26].
Decreased cell number or impaired leukocyte function
can play a part in advance of mild to severe clinical disease
conditions [16]. Regarding the new coronavirus, the
neutrophil-to-lymphocyte ratio (NLR), a well-known marker
of infection and systemic inflammation, has evidenced an
enhanced inflammatory response in COVID-19 patients
[5]. Since the ARDS is the primary cause of mortality in
patients with COVID-19, the elevated NLR values suggest a
poor prognosis in COVID-19 disease [27], especially severe
COVID-19 compared to mild patients. Sun et al. [28] studied
116 patients with COVID-19 and showed a higher NLR [28].
The authors compared severe COVID-19 patients admitted
to the ICU with others or severe patients not admitted to
the ICU. They reported that COVID-19 patients have the
lowest count of lymphocytes and the highest neutrophil
count and NLR [28]. Wang et al. [29] also showed that sev-
eral COVID-19 patients have a rising neutrophil count and
a falling lymphocyte count during the severe phase [29].
Similarly, Barnes et al. [30] found extensive neutrophil infil-
tration in pulmonary capillaries from a COVID-19 patient
[30]. Nevertheless, even though severe cases of COVID-19
appear to be related to increased NLR levels [5], whether
NLR could be an independent predictor of mortality in
COVID-19 patients still requires investigation.
4. Neutrophil Extracellular Traps (NETs) and
Viral Infection
Neutrophils can develop a sophisticated network of DNA
called NETs through NETosis, a liberation of web-like struc-
tures of nucleic acids wrapped with histones that detain viral
particles [31]. Upon discovery, the researchers believed that
the production of NETs defended only against fungi and bac-
teria [32]. However, the NETosis process plays an important
function in the response to viral diseases [33], thereby
2 Mediators of Inflammation

protecting the host during the virus response by trapping and
eliminating distinct pathogens [31].
The formation of NETs is a controlled process, even
though the related signals remain unknown. NETosis is con-
ditional on the production of reactive oxygen species (ROS)
by nicotinamide adenine dinucleotide phosphate oxidase
(NADPH oxidase) [34]. There is evidence of NETosis pro-
duced in a ROS-independent mechanism [35]. In general,
the NETosis process includes the release of nuclear chroma-
tin lined with effector proteins and peptidyl arginine deimi-
nase type IV (PAD4) activation [36]. After stimulation, the
neutrophil nuclear envelope disintegrates to enable the
mixing of chromatin with granular proteins [37]. Myeloper-
oxidase (MPO) and neutrophil elastase (NE) stimulate
chromatin condensation and deteriorate histones [38]. In
the presence of histone hypercitrullination, PAD4 mediates
chromatin decondensation, and the DNA-protein complexes
are released extracellularly as NETs [37]. Therefore, differ-
ently from apoptosis or necrosis, both the granular mem-
brane and nuclear membrane deteriorate during NETosis,
whereas plasma membrane integrity remains [36].
The overproduction of NETs induces lung tissue damage
by NETosis-related enzymes such as NE and MPO [39].
Uncontrolled NET production correlates with disease gravity
and lung injury extension. For instance, NETosis markers
are related to bacterial burden and local inflammation in the
lung [40] and patients with pneumonia-associated ARDShave
neutrophils in a “primed”condition to generate NETs [41].
During chronic obstructive pulmonary disease aggrava-
tion, the production of NETs increases in people with acute
respiratory failure [39] and in ARDS patients [40, 42]. The
elevated NET production, as noted in patients with severe
IAV infection [43], increased injury to the pulmonary
endothelial and epithelial cells [44], directing to severe pneu-
monia. Zhu et al. [43] also noted that the production of NETs
positively correlates with multiple organ dysfunction
syndromes [43].
The inflammatory process is a trigger for thrombotic
complications usually noted in COVID-19 patients, and the
immunothrombotic dysregulation seems to be an important
key marker for the disease severity [45]. Skendros et al. [46]
found that complement activation potentiates the platelet/-
NET/tissue factor/thrombin axis during SARS-CoV-2 infec-
tion [46]. In contrast, Nicolai et al. [47] noted that, in
COVID-19, inflammatory microvascular thrombi are found
in the kidney, lung, and heart, containing NETs related to
the fibrin and platelets. In blood, Nicolai et al. also show that
COVID-19 patients have neutrophil-platelet aggregates and
adifferent platelet and neutrophil activation pattern, which
alters with the disease severity [47]. Middleton et al. [48] also
found that plasma MPO-DNA complexes increased in
COVID-19 and that the elevated NET formation correlates
with COVID-19-related ARDS. Together, these findings sug-
gest the timely application of therapeutic strategies that can
disrupt the vicious cycle of COVID-19 immunothrombosis/-
thromboinflammation by targeting neutrophil activation and
NET formation.
In addition to the physical containment promoted by
NETosis [33], NETs contain DNA, modified extracellular
histones, proteases, and cytotoxic enzymes that allow neutro-
phils to centralize lethal proteins at infection sites [7]. The
mechanisms of NETs’release in the viral response seem to
Recognition mechanism
PAMP/DAMP activation
Neutrophil inltration
IL-6
IL-8
Hyperinammation
TNF
IL-1
ROS
Cytokine storms
Lung thromboinammation
SARS-CoV-2
Viral infection
Alveolar
injury
SARS-CoV-2
NETosis
NET overproduction
MPO
Histones
NE
Figure 1: The interaction hypothesis between neutrophil and hyperinflammation in COVID-19. After the host-viral interaction, the virus
signaling leads to a cascade of interactions between the virus recognition mechanism, neutrophil activation, and inflammatory stimuli.
The NETosis process can protect the host during the virus response or exacerbate lung hyperinflammation in COVID-19 patients. The
figure is made with BioRender (https://app.biorender.com/). Abbreviations: SARS-CoV-2: severe acute respiratory syndrome coronavirus
2; PAMP: pathogen-associated molecular pattern; DAMP: danger-associated molecular pattern; TNF: tumor necrosis factor; IL-6:
interleukin-6; IL-1: interleukin-1; IL-8: interleukin-8; ROS: reactive oxygen species; NE: neutrophil elastase; MPO: myeloperoxidase.
3Mediators of Inflammation

involve neutrophil NE production attributed to the change of
macrophage role by the cleavage of TLRs [49]. A range of
stimuli, including toxic factors, viruses, and proinflammatory
cytokines, such as TNF-αand IL-8, can lead neutrophils to
release NETs [7, 33]. Mechanisms that determine strain spec-
ificity to induce NETosis formation during viral infection are
still unknown.
Lung inflammation is the leading cause of the life-
threatening respiratory complication at the severe levels of
COVID-19 [50]. Veras et al. [51] investigated the potentially
detrimental function of NETs in the pathophysiology of 32
hospitalized severe COVID-19 patients and found that the
levels of NETs increase in tracheal aspirate and plasma from
patients with COVID-19 and their neutrophils naturally pro-
duced more significant concentrations of NETs [51]. The
authors also reported NETs in the lung tissue specimens
from autopsies of COVID-19 patients. In vitro, they noted
that viable SARS-CoV-2 cause NET production by healthy
neutrophils through a PAD-4-dependent manner and that
NETs produced by SARS-CoV-2-activated neutrophils
instigated lung epithelial cell death [51]. Zuo et al. [52] also
investigated sera from COVID-19 patients and found higher
cell-free DNA, myeloperoxidase-DNA (MPO-DNA), and
citrullinated histone H3 (Cit-H3) [52]. In vitro, they also
noted that sera from COVID-19 patients trigger NET release
from control neutrophils [52].
Although the literature does not report direct evidence
linking NETs and SARS-CoV2 clearance, virus entrapping
by NETs was already found in syncytial respiratory virus
infection [53] or influenza [54]. Furthermore, in virus infec-
tion, NETs are efficient to block viruses at the infection site,
entrapping them in a DNA web [22]. Therefore, the NETosis
process induced by the virus could operate as a double-edged
sword: on the one hand, there are essential and efficient
mechanisms for trapping the virus [55], and on the other,
there are highly intense immunological and inflammatory
processes triggered by NET release causing damage to the
organism [7]. These interactions could influence the
COVID-19 symptoms in the relationship between hyperin-
flammation (overproduction of NETs and cytokine storm)
and the function of neutrophils to destroy the viral infection
(Figure 1).
5. Concluding Remarks and Future Directions
The exacerbated NET formation can drive to a cascade of
inflammatory reactions that destroys surrounding tissues,
favors microthrombosis, contributes to the progress of can-
cer cell metastasis, and results in permanent damage to the
pulmonary, cardiovascular, and renal systems [56]. Whether
by coincidence or a cause-and-effect relationship, these
organs are affected in the severe state of the COVID-19
Table 1: Interventional studies registered at the ClinicalTrials.gov database relating the treatment of COVID-19 with NET inhibitors.
NCT identifier Status Location Study type Condition
or disease
Intervention
and phase Primary outcome
Estimated
completion
date
NCT04409925 Not yet
recruiting Canada Nonrandomized
pilot study COVID-19 Dornase Alfa
Phase: 1 (1) Rate of all adverse events January 2021
NCT04359654 Not yet
recruiting
United
Kingdom
Randomized
clinical trial
COVID-19
Hypoxia
Drug:
Dornase Alfa
Phase: 2
(1) Change in inflammation
(C-reactive protein)
November
2020
NCT04445285 Recruiting United
States
Randomized
clinical trial COVID-19 Dornase Alfa
Phase: 2
(1) All-cause mortality
(2) Systemic therapeutic response
February
2021
NCT04432987 Recruiting Turkey Randomized
clinical trial COVID-19 Dornase Alfa
Phase: 2
(1) Clinical improvement and
inflammatory markers in blood
(2) Intubation or extubation
September
2020
NCT04402944 Not yet
recruiting
United
States
Randomized
clinical trial COVID-19 Dornase Alfa
Phase: 2 (1) Ventilator-free days December
2021
NCT04322565 Recruiting Italy Randomized
clinical trial
COVID-19
Pneumonia
Colchicine
Phase: 2
(1) Clinical improvement
(2) Hospital discharge
December
2020
NCT04326790 Recruiting Greece Randomized
clinical trial COVID-19 Colchicine
Phase: 2
(1) Time to clinical deterioration
(2) Concentration of cardiac
troponin
September
2020
NCT04402970 Recruiting United
States
Nonrandomized
clinical trial
COVID-19
ARDS
Dornase Alfa
Phase: 3
(1) Improvement in partial pressure
of O
2
to fraction of inspired O
2
ratio May 2022
NCT04355364 Recruiting France Randomized
clinical trial
COVID-19
ARDS
Dornase Alfa
Phase: 3
(1) Occurrence of at least one grade
improvement (ARDS scale severity) August 2020
NCT04322682 Recruiting United
States
Randomized
clinical trial COVID-19 Colchicine
Phase: 3
(1) Number of participants who die
or require hospitalization
December
2020
NCT04328480 Recruiting Argentina Randomized
clinical trial COVID-19 Colchicine
Phase: 3
(1) Number of participants who die
(all-cause mortality) August 2020
Retrieved October 30, 2020, from https://www.https://www.clinicaltrials.gov/ct2/home. Abbreviations: NCT: National Clinical Trial; O
2
: oxygen; ARDS: acute
respiratory distress syndrome.
4 Mediators of Inflammation

disease [57, 58]. The uncontrolled and poorly acknowledged
host response regarding the cytokine storm is one of the
major causes of severe COVID-19 conditions [12]. In this
pandemic scenario, there is a compelling need to investigate
the mechanisms associated with hyperinflammation process
and NET production in response to COVID-19.
The NLR is an independent risk factor for severe
COVID-19 [27], and neutrophilia forecasts poor outcomes
in COVID-19 patients [29]. In this sense, new frontiers in
NET assessment regarding COVID-19 may be expressed by
analyzing NETosis directly after sputum induction or after
bronchoscopy using the bronchial alveolar fluid of COVID-
19 patients [42]. Since patient samples usually become acces-
sible at the hospital, it could investigate whether the existence
of NETs is associated with the severity of COVID-19.
Treatments using NET-targeting approaches, although
would not directly target the new coronavirus, could reduce
the damage caused by hyperinflammation [59], thereby
decreasing the disease’s severity and avoiding invasive
mechanical ventilation, consequently diminishing mortality.
Drugs that target NETs include inhibitors of the molecules
necessary for NET formation, such as gasdermin D [60],
PAD4 [61], and NE [62]. Studies on treatment of inflamma-
tory state in COVID-19 patients with NET inhibitors are still
in development (please see Table 1).
Caution is needed to define which people would advan-
tage from suppressing the neutrophil response and which
would help more from a strengthened neutrophil action dur-
ing viral infections. Despite prior studies linking pulmonary
diseases to aberrant NET formation
[
3, 4
]
, our understanding
of NETosis mechanisms in viral infection is still limited.
The hyperinflammation is related to the severity of
COVID-19 by influencing the pulmonary inflammation
[12]. Neutrophils exhibit an intense response to virus infec-
tion, promoting bidirectional cross-talk with T cells [21].
Neutrophils also express a complex array of receptors and
adhesion molecules for various ligands, including immuno-
globulins and inflammatory markers [20]. In this sense,
severe cases of COVID-19 appear to be related to increased
NLR levels [5], and treatments using NET-targeting
approaches have the potential to decrease the damage caused
by hyperinflammation [40, 41]. The researchers should
consider hyperinflammation in the different phases of
COVID-19, neutrophil response mechanisms, and NETosis.
Data Availability
The data supporting this narrative review are from previously
reported studies and datasets, which have been cited.
Conflicts of Interest
The authors declare that there is no conflict of interest
regarding the publication of this paper.
Acknowledgments
This work was supported by the CAPES (Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior) (grant
number 88882.314890/2013-01); FAPESP (Fundação de
Amparo a Pesquisa do Estado de São Paulo); and CNPq
(Conselho Nacional de Desenvolvimento Científico e
Tecnológico).
References
[1] G. Kutti-Sridharan, R. Vegunta, R. Vegunta, B. P. Mohan, and
V. R. P. Rokkam, “SARS-CoV2 in different body fluids, risks of
transmission, and preventing COVID-19: a comprehensive
evidence-based review,”International Journal of Preventive
Medicine, vol. 11, p. 97, 2020.
[2] L. L. Fernandes, V. B. Pacheco, L. Borges et al., “Saliva in the
diagnosis of COVID-19: a review and new research direc-
tions,”Journal of Dental Research, vol. 99, no. 13, pp. 1435–
1443, 2020.
[3] L. L. Fernandes, L. Borges, V. B. Pacheco et al., “SARS-CoV-2 :
a promising path in salivary diagnosis,”The Open Dentistry
Journal, vol. 14, no. 1, pp. 343-344, 2020.
[4] A. C. L. Fernandes, A. J. M. Vale, F. P. Guzen, F. I. Pinheiro,
R. N. Cobucci, and E. P. de Azevedo, “Therapeutic options
against the new coronavirus: updated clinical and laboratory
evidences,”Frontiers in Medicine, vol. 7, p. 546, 2020.
[5] C. Qin, L. Zhou, Z. Hu et al., “Dysregulation of immune
response in patients with coronavirus 2019 (COVID-19) in
Wuhan, China,”Clinical Infectious Diseases, vol. 71, no. 15,
pp. 762–768, 2020.
[6] Q. Ruan, K. Yang, W. Wang, L. Jiang, and J. Song, “Clinical
predictors of mortality due to COVID-19 based on an analysis
of data of 150 patients from Wuhan, China,”Intensive Care
Medicine, vol. 46, no. 5, pp. 846–848, 2020.
[7] O. Z. Cheng and N. Palaniyar, “NET balancing: a problem in
inflammatory lung diseases,”Frontiers in Immunology, vol. 4,
p. 1, 2013.
[8] G. Zhu, Y. Xu, X. Cen, K. S. Nandakumar, S. Liu, and
K. Cheng, “Targeting pattern-recognition receptors to dis-
cover new small molecule immune modulators,”European
Journal of Medicinal Chemistry, vol. 144, pp. 82–92, 2018.
[9] C.-K. Min, S. Cheon, N.-Y. Ha et al., “Comparative and kinetic
analysis of viral shedding and immunological responses in
MERS patients representing a broad spectrum of disease sever-
ity,”Scientific Reports, vol. 6, no. 1, article 25359, 2016.
[10] R. Channappanavar and S. Perlman, “Pathogenic human coro-
navirus infections: causes and consequences of cytokine storm
and immunopathology,”Seminars in Immunopathology,
vol. 39, no. 5, pp. 529–539, 2017.
[11] D. Kolli, T. S. Velayutham, and A. Casola, “Host-viral interac-
tions: role of pattern recognition receptors (PRRs) in human
pneumovirus infections,”Pathogens, vol. 2, no. 2, pp. 232–
263, 2013.
[12] P. Mehta, D. F. McAuley, M. Brown, E. Sanchez, R. S. Tatter-
sall, and J. J. Manson, “COVID-19: consider cytokine storm
syndromes and immunosuppression,”The Lancet, vol. 395,
no. 10229, pp. 1033-1034, 2020.
[13] W. H. Mahallawi, O. F. Khabour, Q. Zhang, H. M. Makhdoum,
and B. A. Suliman, “MERS-CoV infection in humans is associ-
ated with a pro-inflammatory Th1 and Th17 cytokine profile,”
Cytokine, vol. 104, pp. 8–13, 2018.
[14] M. Prete, E. Favoino, G. Catacchio, V. Racanelli, and F. Perosa,
“SARS-CoV-2 inflammatory syndrome. Clinical features and
5Mediators of Inflammation

rationale for immunological treatment,”International Journal
of Molecular Sciences, vol. 21, no. 9, article 3377, 2020.
[15] C. Huang, Y. Wang, X. Li et al., “Clinical features of patients
infected with 2019 novel coronavirus in Wuhan, China,”The
Lancet, vol. 395, no. 10223, pp. 497–506, 2020.
[16] P. P. Lamichhane and A. E. Samarasinghe, “The role of innate
leukocytes during influenza virus infection,”Journal of Immu-
nology Research, vol. 2019, Article ID 8028725, 17 pages, 2019.
[17] C. Rosales, “Neutrophils at the crossroads of innate and adap-
tive immunity,”Journal of Leukocyte Biology, vol. 108, no. 1,
pp. 377–396, 2020.
[18] F. D. Barr, C. Ochsenbauer, C. R. Wira, and M. Rodriguez-
Garcia, “Neutrophil extracellular traps prevent HIV infection
in the female genital tract,”Mucosal Immunology, vol. 11,
no. 5, pp. 1420–1428, 2018.
[19] J. Bordon, S. Aliberti, R. Fernandez-Botran et al., “Under-
standing the roles of cytokines and neutrophil activity and
neutrophil apoptosis in the protective versus deleterious
inflammatory response in pneumonia,”International Journal
of Infectious Diseases, vol. 17, no. 2, pp. e76–e83, 2013.
[20] C. D. Russell, S. A. Unger, M. Walton, and J. Schwarze, “The
human immune response to respiratory syncytial virus infec-
tion,”Clinical Microbiology Reviews, vol. 30, no. 2, pp. 481–
502, 2017.
[21] S. Costa, D. Bevilacqua, M. A. Cassatella, and P. Scapini, “Recent
advances on the crosstalk between neutrophils and B or T lym-
phocytes,”Immunology, vol. 156, no. 1, pp. 23–32, 2019.
[22] V. D. Giacalone, C. Margaroli, M. A. Mall, and
R. Tirouvanziam, “Neutrophil adaptations upon recruitment
to the lung: new concepts and implications for homeostasis
and disease,”International Journal of Molecular Sciences,
vol. 21, no. 3, p. 851, 2020.
[23] J. Pillay, V. M. Kamp, E. van Hoffen et al., “A subset of neutro-
phils in human systemic inflammation inhibits T cell
responses through Mac-1,”The Journal of Clinical Investiga-
tion, vol. 122, no. 1, pp. 327–336, 2012.
[24] M. V. Lukens, A. C. van de Pol, F. E. J. Coenjaerts et al., “A sys-
temic neutrophil response precedes robust CD8
+
T-cell activa-
tion during natural respiratory syncytial virus infection in
infants,”Journal of Virology, vol. 84, no. 5, pp. 2374–2383,
2010.
[25] M. M. Hufford, G. Richardson, H. Zhou et al., “Influenza-
infected neutrophils within the infected lungs act as antigen
presenting cells for anti-viral CD8(+) T cells,”PLoS One,
vol. 7, no. 10, article e46581, 2012.
[26] K. Lim, Y.-M. Hyun, K. Lambert-Emo et al., “Neutrophil trails
guide influenza-specific CD8
+
T cells in the airways,”Science,
vol. 349, no. 6252, article aaa 4352, 2015.
[27] F. A. Lagunas-Rangel, “Neutrophil-to-lymphocyte ratio and
lymphocyte-to-C-reactive protein ratio in patients with severe
coronavirus disease 2019 (COVID-19): a meta-analysis,”Jour-
nal of Medical Virology, vol. 92, no. 10, pp. 1733-1734, 2020.
[28] S. Sun, X. Cai, H. Wang et al., “Abnormalities of peripheral
blood system in patients with COVID-19 in Wenzhou,
China,”Clinica Chimica Acta, vol. 507, pp. 174–180, 2020.
[29] D. Wang, B. Hu, C. Hu et al., “Clinical characteristics of 138
hospitalized patients with 2019 novel coronavirus-infected
pneumonia in Wuhan, China,”JAMA, vol. 323, no. 11,
pp. 1061–1069, 2020.
[30] B. J. Barnes, J. M. Adrover, A. Baxter-Stoltzfus et al., “Target-
ing potential drivers of COVID-19: neutrophil extracellular
traps,”The Journal of Experimental Medicine, vol. 217, no. 6,
article e20200652, 2020.
[31] C. N. Jenne, C. H. Y. Wong, F. J. Zemp et al., “Neutrophils
recruited to sites of infection protect from virus challenge by
releasing neutrophil extracellular traps,”Cell Host & Microbe,
vol. 13, no. 2, pp. 169–180, 2013.
[32] V. Brinkmann, U. Reichard, C. Goosmann et al., “Neutrophil
extracellular traps kill bacteria,”Science, vol. 303, no. 5663,
pp. 1532–1535, 2004.
[33] T. Saitoh, J. Komano, Y. Saitoh et al., “Neutrophil extracellular
traps mediate a host defense response to human immunodefi-
ciency virus-1,”Cell Host & Microbe, vol. 12, no. 1, pp. 109–
116, 2012.
[34] V. Delgado-Rizo, M. A. Martínez-Guzmán, L. Iñiguez-Gutier-
rez, A. García-Orozco, A. Alvarado-Navarro, and M. Fafutis-
Morris, “Neutrophil extracellular traps and its implications
in inflammation: an overview,”Frontiers in Immunology,
vol. 8, p. 81, 2017.
[35] O. Tatsiy and P. P. McDonald, “Physiological stimuli induce
PAD4-dependent, ROS-independent NETosis, with early and
late events controlled by discrete signaling pathways,”Fron-
tiers in Immunology, vol. 9, article 2036, 2018.
[36] S. Yousefi, D. Stojkov, N. Germic et al., “Untangling “NETosis”
from NETs,”European Journal of Immunology, vol. 49, no. 2,
pp. 221–227, 2019.
[37] T. A. Fuchs, U. Abed, C. Goosmann et al., “Novel cell death
program leads to neutrophil extracellular traps,”The Journal
of Cell Biology, vol. 176, no. 2, pp. 231–241, 2007.
[38] V. Papayannopoulos, K. D. Metzler, A. Hakkim, and
A. Zychlinsky, “Neutrophil elastase and myeloperoxidase
regulate the formation of neutrophil extracellular traps,”
The Journal of Cell Biology, vol. 191, no. 3, pp. 677–691,
2010.
[39] F. Grabcanovic-Musija, A. Obermayer, W. Stoiber et al.,
“Neutrophil extracellular trap (NET) formation charac-
terises stable and exacerbated COPD and correlates with
airflow limitation,”Respiratory Research, vol. 16, no. 1,
p. 59, 2015.
[40] C. Mikacenic, R. Moore, V. Dmyterko et al., “Neutrophil extra-
cellular traps (NETs) are increased in the alveolar spaces of
patients with ventilator-associated pneumonia,”Critical Care,
vol. 22, no. 1, p. 358, 2018.
[41] I. Bendib, L. de Chaisemartin, V. Granger et al., “Neutrophil
extracellular traps are elevated in patients with pneumonia-
related acute respiratory distress syndrome,”Anesthesiology,
vol. 130, no. 4, pp. 581–591, 2019.
[42] J. J. M. Wong, J. Y. Leong, J. H. Lee, S. Albani, and J. G. Yeo,
“Insights into the immuno-pathogenesis of acute respiratory
distress syndrome,”Annals of Translational Medicine, vol. 7,
no. 19, p. 504, 2019.
[43] L. Zhu, L. Liu, Y. Zhang et al., “High level of neutrophil extra-
cellular traps correlates with poor prognosis of severe influ-
enza A Infection,”The Journal of Infectious Diseases,
vol. 217, no. 3, pp. 428–437, 2018.
[44] T. Narasaraju, E. Yang, R. P. Samy et al., “Excessive neutro-
phils and neutrophil extracellular traps contribute to acute
lung injury of influenza pneumonitis,”The American Journal
of Pathology, vol. 179, no. 1, pp. 199–210, 2011.
[45] Y. Zuo, M. Zuo, S. Yalavarthi et al., “Neutrophil extracellular
traps and thrombosis in COVID-19,”Journal of Thrombosis
and Thrombolysis, vol. 1-8, 2020.
6 Mediators of Inflammation

[46] P. Skendros, A. Mitsios, A. Chrysanthopoulou et al., “Comple-
ment and tissue factor-enriched neutrophil extracellular traps
are key drivers in COVID-19 immunothrombosis,”The Jour-
nal of Clinical Investigation, vol. 130, no. 11, pp. 6151–6157,
2020.
[47] L. Nicolai, A. Leunig, S. Brambs et al., “Immunothrombotic
dysregulation in COVID-19 pneumonia is associated with
respiratory failure and coagulopathy,”Circulation, vol. 142,
no. 12, pp. 1176–1189, 2020.
[48] E. A. Middleton, X.-Y. He, F. Denorme et al., “Neutrophil
extracellular traps contribute to immunothrombosis in
COVID-19 acute respiratory distress syndrome,”Blood,
vol. 136, no. 10, pp. 1169–1179, 2020.
[49] H. Domon, K. Nagai, T. Maekawa et al., “Neutrophil elastase
subverts the immune response by cleaving toll-like receptors
and cytokines in pneumococcal pneumonia,”Frontiers in
Immunology, vol. 9, p. 732, 2018.
[50] W.-J. Guan, Z.-Y. Ni, Y. Hu et al., “Clinical characteristics of
coronavirus disease 2019 in China,”The New England Journal
of Medicine, vol. 382, no. 18, pp. 1708–1720, 2020.
[51] F. P. Veras, M. Pontelli, C. Silva et al., “SARS-CoV-2–triggered
neutrophil extracellular traps mediate COVID-19 pathology,”
The Journal of Experimental Medicine, vol. 217, no. 12, article
e20201129, 2020.
[52] Y. Zuo, S. Yalavarthi, H. Shi et al., “Neutrophil extracellular
traps in COVID-19,”JCI Insight, vol. 5, no. 11, article
e138999, 2020.
[53] G. A. Funchal, N. Jaeger, R. S. Czepielewski et al., “Respira-
tory syncytial virus fusion protein promotes TLR-4-
dependent neutrophil extracellular trap formation by human
neutrophils,”PLoS One, vol. 10, no. 4, article e0124082,
2015.
[54] L. A. Perrone, J. K. Plowden, A. García-Sastre, J. M. Katz, and
T. M. Tumpey, “H5N1 and 1918 pandemic influenza virus
infection results in early and excessive infiltration of macro-
phages and neutrophils in the lungs of mice,”PLoS Pathogens,
vol. 4, no. 8, article e1000115, 2008.
[55] S. P. Muraro, G. F. De Souza, S. W. Gallo et al., “Respiratory
syncytial virus induces the classical ROS-dependent NETosis
through PAD-4 and necroptosis pathways activation,”Scien-
tific Reports,vol. 8, no. 1, article 14166, 2018.
[56] S. K. Jorch and P. Kubes, “An emerging role for neutrophil
extracellular traps in noninfectious disease,”Nature Medicine,
vol. 23, no. 3, pp. 279–287, 2017.
[57] R. O. Bonow, G. C. Fonarow, P. T. O’Gara, and C. W. Yancy,
“Association of coronavirus disease 2019 (COVID-19) with
myocardial injury and mortality,”JAMA Cardiology, vol. 5,
no. 7, pp. 751–753, 2020.
[58] N. Chen, M. Zhou, X. Dong et al., “Epidemiological and clini-
cal characteristics of 99 cases of 2019 novel coronavirus pneu-
monia in Wuhan, China: a descriptive study,”The Lancet,
vol. 395, no. 10223, pp. 507–513, 2020.
[59] S. Cicco, G. Cicco, V. Racanelli, and A. Vacca, “Neutrophil
extracellular traps (NETs) and damage-associated molecular
patterns (DAMPs): two potential targets for COVID-19 treat-
ment,”Mediators of Inflammation, vol. 2020, Article ID
7527953, 25 pages, 2020.
[60] J. J. Hu, X. Liu, S. Xia et al., “FDA-approved disulfiram
inhibits pyroptosis by blocking gasdermin D pore forma-
tion,”Nature Immunology, vol. 21, no. 7, pp. 736–745,
2020.
[61] C. C. Yost, H. Schwertz, M. J. Cody et al., “Neonatal NET-
inhibitory factor and related peptides inhibit neutrophil extra-
cellular trap formation,”The Journal of Clinical Investigation,
vol. 126, no. 10, pp. 3783–3798, 2016.
[62] T. Tagami, R. Tosa, M. Omura et al., “Effect of a selective neu-
trophil elastase inhibitor on mortality and ventilator-free days
in patients with increased extravascular lung water: a post hoc
analysis of the PiCCO Pulmonary Edema Study,”Journal of
Intensive Care, vol. 2, no. 1, p. 67, 2014.
7Mediators of Inflammation