Immunologic Research

https://doi.org/10.1007/s12026-021-09199-z

ORIGINAL ARTICLE

Structural differences of neutrophil extracellular traps induced

by biochemical and microbiologic stimuli under healthy
and autoimmune milieus
Sorely Adelina Sosa‑Luis1 · William de Jesús Ríos‑Ríos2 · Ángeles Esmeralda Gómez‑Bustamante2 ·
María de los Ángeles Romero‑Tlalolini3 · Sergio Roberto Aguilar‑Ruiz4   · Rafael Baltierez‑Hoyos3 ·
Honorio Torres‑Aguilar2 

Received: 29 September 2020 / Accepted: 23 April 2021

© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2021

Abstract
Neutrophil extracellular traps (NETs) are networks of decondensed chromatin loaded with antimicrobial peptides and
enzymes produced against microorganisms or biochemical stimuli. Since their discovery, numerous studies made separately
have revealed multiple triggers that induce similar NET morphologies allowing to classify them as lytic or non-lytic. How-
ever, the variability in NET composition depending on the inducer agent and the local milieu under similar conditions has
been scarcely studied. In this work, a comparative study was conducted to evaluate structural and enzymatic divergences in
NET composition induced by biochemical (phorbol myristate acetate [PMA] and hypochlorous acid [HOCl]) and microbio-
logic (Candida albicans, Staphylococcus aureus, and Pseudomonas aeruginosa) stimuli, along with the presence of plasma
from healthy donors or patients with systemic lupus erythematosus (SLE). The results showed a differential composition
of DNA and the antimicrobial peptide cathelicidin (LL37) and a variable enzymatic activity (neutrophil elastase, cathepsin
G, myeloperoxidase) induced by the different stimuli despite showing morphologically similar NETs. Additionally, SLE
plasma´s presence increased DNA and LL37 release during NET induction independently of the trigger stimulus but with
no enzymatic activity differences. This work provides new evidence about NET composition variability depending on the
inducer stimulus and the local milieu.

Keywords  Neutrophils · Neutrophil extracellular traps · Innate immune response · Autoimmunity · Systemic lupus
erythematosus

Introduction

Neutrophils are the most numerous white blood cells cru-

cial for the defense against infection in the innate immune
response. Phagocytosis, granule releasing, and reactive oxy-
* Honorio Torres‑Aguilar
qbhonorio@hotmail.com gen species (ROS) production were their initially described
microbicide mechanisms. In this regard, neutrophils may
1
Department of Molecular Biomedicine, Centro de perform directed and specialized functions according to the
Investigación y de Estudios Avanzados del Instituto recognized pathogens by selecting granular components and
Politécnico Nacional, Mexico City, Mexico
2
cytokines [1]. Additionally, NETosis was later described
Clinical Immunology Research Department of Biochemical as an alternative neutrophil antimicrobial role. Neutrophil
Sciences Faculty, Universidad Autónoma “Benito Juárez” de
Oaxaca, Oaxaca City, Mexico extracellular traps (NETs) are networks or filaments of
3 decondensed chromatin loaded with histones, enzymes,
CONACYT ‑ Medicine and Surgery Faculty, Universidad
Autónoma “Benito Juárez” de Oaxaca, Oaxaca City, Mexico and antimicrobial peptides. This process was defined as a
4 different type of programmed cell death for capturing, neu-
Molecular Immunology Research Department of Medicine
and Surgery Faculty, Universidad Autónoma “Benito Juárez” tralizing, and eliminating certain kinds of bacteria, fungi,
de Oaxaca, Oaxaca City, Mexico and some viruses, contributing to neutrophils’ functional

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heterogeneity [1]. Two main processes may produce NETs:
(a) a “suicidal” lytic or NADPH oxidase 2 (NOX2)-depend-
ent NETosis, beginning with arrest and actin depolarization.
Simultaneously, the nuclear membrane is disassembled, and
the chromatin is decondensed in the cytoplasm to be mixed
with granular components. The plasma membrane is then
permeabilized, and the complete content is released to the
extracellular space involving the neutrophil death [2]. (b) A
“vital” non-lytic or NOX2-independent NETosis in which
DNA is packed in vesicles subsequently expelled into the
extracellular space. This mechanism occurs rapidly, and
neutrophils maintain their viability remaining as anucleated
cytoplasts able to roll and engulf bacteria even after NET
expulsion [3]. Additional to NOX2 activity, the neutrophil
elastase (NE) and myeloperoxidase (MPO) requirements
have been described for the later NET formation stages,
primarily for chromatin decondensation. Nevertheless, it is
now clear that some forms of NET formation can occur inde-
pendently of NOX2 (for vital NETosis) or MPO [4, 5]. NET
activation processes may differ on the inducing stimulus and
the released DNA quantity and protein composition [6].
Because not all NET generation pathways elicit cell death,
the term “NET formation” instead of “NETosis” is more
accurately used. Additionally, given that diverse stimuli acti-
vate different signaling, current statements avoid describing
specific protein requirements for NET composition [7].
Numerous stimuli such as inflammatory cytokines and
chemokines, immune complexes, contact with activated
platelets, calcium influx, and biochemical and microbiologic
components have been described as in vitro and in vivo NET
inducers. For instance, PMA (phorbol myristate acetate) is
a specific activator of protein kinase C (PKC) and therefore
activates the nuclear factor-kappa B (NF-κB) in an NADPH
oxidase 2 (NOX2)-dependent mechanism. Hence, PMA is
characterized by inducing a lytic NET formation depending
on ROS production by PKC activation, and several pathways
of NET formation converge at the level of PKC [2]. Alterna-
tively, HOCl (hypochlorous acid) activates NET formation’s
non-lytic pathway independently of ROS since HOCl is a prod-
uct of myeloperoxidase activity [8]. Additional to biochemical
stimuli, microbial agents such as Candida albicans (non-lytic)
[9], Staphylococcus aureus (non-lytic) [10], and Pseudomonas
aeruginosa (lytic) [11] have also been described and separately
characterized as NET inducers. Immunocytochemistry is the
most widely accepted method for NET detection by identifying
extracellular DNA structures colocalizing with granule-derived
proteins and nuclear components [1, 7].
Despite the NET’s relevant role during the innate immune
response against microorganisms, an overproduction and
lack of clearance might be harmful, as described in sys-
temic lupus erythematosus (SLE) [12, 13]. In SLE, NET-
derived DNA–protein complexes activate plasmacytoid
dendritic cells (pDCs) by facilitating self-DNA recognition
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Immunologic Research
via TLR-7/9 and promoting IFN-I production. Then, IFN-I
activates neutrophils and induces more NET release contrib-
uting to a pDC-neutrophil activation loop [14]. The antimi-
crobial activity of cathelicidin (LL37) [15] and cathepsin
G (CG) [16] in nucleic acid complexes produced by NET
formation may vary depending on the microbiological stimu-
lus and have been identified as potent IFN-I triggers for B
lymphocyte activating and autoantibody production [17].
Several studies have evidenced the extensive heteroge-
neity of NET composition, which varies according to the
inducer stimulus and investigation methods [7]. This study
evaluates the NET morphology and composition (DNA
quantification, LL37, NE, CG, and MPO) employing a
comparative analysis performed under similar experimental
conditions, evaluating NET features induced by biochemi-
cal inducers (PMA and HOCl) and microbiological stimuli
such as fungi (C. albicans), and a Gram-positive (S. aureus)
and a Gram-negative (P. aeruginosa) bacterium, as well as
the effect of soluble factors derived from an autoimmune
process (SLE plasma) on NET formation.
Materials and methods
Neutrophil’s isolation and plasma samples
Neutrophils were purified from 10 mL of EDTA blood sam-
ples of healthy donors by Percoll gradients (GE Healthcare).
Blood samples were initially diluted (ratio 1:3) with Dul-
becco’s phosphate-buffered saline (DPBS), and a 1.079 g/
mL Percoll density was used to isolated peripheral blood
mononuclear cells (PBMC). Then, PBMC were washed with
DPBS, and a 1.098 g/mL Percoll density was performed to
obtain the granulocyte layer. Granulocytes were collected
and brought under an osmotic shock with 0.2% and 0.65%
saline solution to remove residual erythrocytes. Differential
centrifugation was performed to eliminate platelets, and then
purified neutrophils were resuspended in Hank´s balanced
salt solution (HBSS) buffer. Cell numbers and viability were
quantified by trypan blue exclusion test (0.4%, Sigma), and
neutrophil purity was evaluated by flow cytometry (MAC-
SQuant 10) with FSC/SSC analysis by the FlowJo software
(Tree Star, Inc.), and morphology was observed by Wright
staining (Hycel).
The project was approved by the research and ethical
committees of the Hospital Regional de Alta Especialidad
de Oaxaca, Mexico (approval number: HRAEO/CIC/CEI
013/16). Blood samples were obtained after informed con-
sent from recently diagnosed and before treatment SLE-sta-
ble patients to avoid interference effects on NET production
likely attributable to the immunosuppressive therapy. Hence,
due to the ethical committee guidelines, three samples from
SLE patients and three healthy individuals matched in age
Immunologic Research
and gender could be provided by the rheumatology depart-
ment during this study’s approval period.
NET induction
The 2 × ­10 5 neutrophils were placed and homogenized
on 0.001% poly-L-lysine-treated coverslips in 400 µL of
RPMI 1640 medium without phenol red (supplemented
with 10% heat decomplemented autologous plasma, 2 mM
L-glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential
amino acids, 100 U/mL penicillin, 100 mg/mL streptomycin,
and 50 mM 2-ME) for fluorescence microscopic analysis, or
in HBSS buffer for enzymatic activity quantification. Neu-
trophils purified from the same subject were used for each
group of ten independent experiments (ten analyzed indi-
viduals) to avoid experimental variation owed to individual
cell responses between individuals and were included in the
following culture conditions.
Neutrophils were kept in the absence of a stimulus
(HBSS), or NET formation was induced by biochemi-
cal stimuli: PMA [200  nM] (Sigma-Aldrich) or HOCl
[4.5 mM] (NaOCl, Sigma-Aldrich); or by bacterial stimuli:
Gram-positive, S. aureus (ATCC 25,923) or Gram-negative,
P. aeruginosa (ATCC 10,145) at MOI 100; or by a fungal
stimulus: pseudohyphae of C. albicans (ATCC 10,231) at
MOI 1.0. The NET formation was analyzed in the presence
of every single stimulus, or every stimulus plus autologous
(from neutrophil donor), healthy allogenic or allogeneic SLE
plasma (10%). After adding the corresponding stimulus and
plasma, cells were incubated for 4 h at 37 °C and 5% CO2.
NET characterization by fluorescence microscopy
After NET induction, cells were fixed (4% paraformal-
dehyde/30 min), blocked (10% decomplemented autolo-
gous plasma/30  min), and permeabilized (0.2% Tri-
ton X-100/10 min). Then, DNA was stained with DAPI
Fluoroshield (Sigma) (blue color) to describe extracellular
DNA structures as “lytic-like” or “non-lytic-like” NET
morphologies, and with anti-LL37 Alexa Fluor 594 (red
color) or isotype control (Santa Cruz) overnight at 4 °C.
Staphylococcus aureus, P. aeruginosa, and pseudohyphae
of C. albicans were previously labeled with CFSE (5 µM,
Sigma) (green color). Five images (four extremes and center)
per well, for each condition of ten experiments performed
by triplicate (150 analyzed images per condition), were
acquired by fluorescence microscopy (Axio observer Z1,
ZEISS). The fluorescence background readings were estab-
lished with unstained cells for DAPI or the isotype control
for LL37 to analyze the mean gray value of signal per area
for each color with the ImageJ software. The combined anal-
ysis (merge) of DNA/LL37 was performed by colocalizing
independent images after image thresholding, dividing each
image into the two classes of pixels, defined as background.
Cell aggregation was defined based on cell distances, and it
was quantified as a Cell Aggregation Index = the number of
isolated cells at every stimulus/number of isolated cells at
every stimulus plus plasma.
Enzymatic activity quantification
Basal and maximum enzymatic quantification was ana-
lyzed in 2 × ­106 neutrophils kept in HBSS buffer or lysed
by freeze and defrost at − 70 °C, respectively. Additionally,
to analyze the enzymatic activity produced by the NET for-
mation, NETs were induced by the mentioned stimuli in
HBSS. Supernatants were discarded, wells were washed,
and enzymatic activity was evaluated by colorimetric reac-
tions in supernatants obtained after disengaging DNA–pro-
tein structures using DNAse (1 U/mL, Sigma) for 10 min
at 37 °C to assess the enzymatic activity due to just NET-
bound proteins. The enzymatic activities of NE, CG, and
MPO were analyzed as described by White PC and cowork-
ers [18] by using 0.5 M, N-methoxysuccinyl-Ala-Ala-Pro-
Val-p-nitro aniline (Sigma), 1 mM, N-succinyl-Ala-Ala-Pro-
Phe-p-nitroanilide (Sigma), or 3,3′,5,5′ tetramethylbenzidine
(Sigma) as substrates respectively, and quantified by photo-
metric analysis in comparison to the corresponding calibra-
tion curves. Results are presented as the percentage of each
condition in contrast to the maximum enzymatic activity.
Statistical analysis
Triplicate measurements were performed in each one of the
ten independent experiments. Dates were analyzed by the
Minitab software for ANOVA statistical analysis to per-
formed comparisons between groups of experimental con-
ditions with a confidence level of 95%.
Results
Morphologic, structural, and enzymatic
differences in NETs induced by the biochemical
and microbiologic stimulus
Morphologic features observed by Wright staining
(Fig. 1a), FSC-SSC profile by flow cytometry (Fig. 1b),
and viability quantification by trypan blue exclusion
allowed to obtain a cell purity of 98.9% ± 0.06 and cell
viability of 95.5% ± 4.2 of purified neutrophils in ten per-
formed experiments. Morphologic and fluorescent features
of DNA-DAPI staining of freshly purified neutrophils
(Fig. 1c) and CFSE staining of C. albicans (Fig. 1d), S.
aureus (Fig. 1e), and P. aeruginosa (Fig. 1f) were initially
13

Fig. 1  Initial purity and morphologic features of neutrophils and
microorganisms. After isolation, neutrophil purity was confirmed by
Wright staining, × 100 (a), and quantified by flow cytometry, FSC
vs. SSC dot blot (b). Initial morphologic and fluorescent features
of freshly purified neutrophils, DNA-DAPI stained, × 40 (c) and
established by fluorescence microscopy to characterize
morphologic changes due to NET induction.
After stimulating with PMA, HOCl, or CFSE-stained
microorganisms, DNA-DAPI and LL37-Alexa Fluor
594 were visualized by fluorescence microscopy (Fig. 2,
panel I), and mean gray values of signal per area for the
indicated colors were quantified by the ImageJ software
(Fig. 2, panel II and III) as described in the “Materials
and methods” section. Unstimulated neutrophils showed
condensed chromatin (Fig. 2a) with LL37 in a cytoplasmic
location (Fig. 2b and 2c). In comparison, the morphologi-
cal characteristics induced with PMA were profuse extra-
cellular DNA structures, looking like a cloudy appear-
ance, observing a large amount of DNA emitted toward
extracellular space, with delocalized lysis out the plasma
membrane, favoring a diffuse DNA dispersion as described
for lytic NET formation (Fig. 2d). LL37 was located in the
extracellular space attached to the entire NET structure
(Fig. 2e and 2f). On the other hand, HOCl induced a mor-
phology of elongated emitted DNA filaments still attached
to membrane cell as described for non-lytic NET forma-
tion (Fig. 2g). In this case, LL37 does not colocalize with
13
Immunologic Research
CFSE stained, × 40, C. albicans (d), S. aureus (e), and P. aeruginosa
(f). Small right-down squares show × 100 amplification into panel
c, × 100 optic amplification into panel d, and × 100 g staining ampli-
fication in panels e and f. Pictures are representative of one of the ten
experiments
the formed filamentous NETs, yet it is located close to the
whole remainder nucleus (Fig. 2h and 2i).
In comparison to the morphologic characteristics of
DNA, LL37, and microorganisms before NET formation (C.
albicans (Fig. 2j), S. aureus (Fig. 2o), and P. aeruginosa
(Fig. 2t)), C. albicans induced a non-lytic-like morphology
with prominent DNA filaments and anucleated cytoplast-like
structures (Fig. 2k). Preserved whole structures of pseudo-
hyphae were still present (Fig. 2m), while LL37 kept a pre-
dominant cytoplasmic location with a weak dragging toward
DNA filament structures (Fig. 2l) but scarcely colocalized
with pseudohyphae (Fig. 2n). Alternatively, although S.
aureus also triggered non-lytic-like NET features (Fig. 2p),
the bacteria seen throughout the microscopic fields were
grouped (Fig. 2r) and caught by DNA filaments and LL37
(Fig. 2q) as observed by an almost whole colocalization
(Fig. 2s). However, although NETs induced by C. albicans
and S. aureus showed non-lytic-like characteristic as those
displayed by HOCl induction, only S. aureus induced a sta-
tistically significant higher DNA staining (Fig. 2, panel II),
but with no significant differences in LL37 participation
(Fig. 2, panel III) in comparison to unstimulated neutrophils.
Immunologic Research
Fig. 2  DNA and LL37 composition of NETs induced by biochemi-
cal and microbiologic triggers. After NET induction described in
the “Materials and methods” section, cells were stained with DAPI
for DNA visualization (blue) and anti-LL37 Alexa Fluor 594 (red) or
isotype control. Staphylococcus aureus, P. aeruginosa, and pseudo-
hyphae of C. albicans were visualized in green color due to CFSE
staining before NET induction. MERGE images show colocalizations
of the mentioned colors. (I) Images show representative results of
fluorescence microscopy of ten independent experiments performed
by triplicate. Graphics show averages ± SD of mean gray value of sig-
nal per area for the indicated colors of five images (four extremes and
center) per well in ten independent experiments performed by tripli-
cate and analyzed with the ImageJ software for (I) DAPI-DNA and
(II) Alexa Fluor 594-LL37 staining. Statistical analysis was achieved
by the Minitab software by ANOVA to perform comparisons between
groups of experimental conditions with a confidence level of 95%.
* = p ≤ 0.05. HBSS, Hank´s balanced salt solution; PMA, phorbol
myristate acetate; HOCl, hypochlorous acid
13

Conversely, P. aeruginosa generated vast cloudy DNA struc-
tures described as lytic NET formation (Fig. 2u) but much
more extended than those observed with PMA (Fig. 2d).
Bacterial particles observed all over the microscopic fields
(Fig. 2w) were covered by DNA networks, but mainly by
LL37 (Fig. 2v) as detected by a whole bacteria-LL37 colo-
calization (Fig. 2x). Interestingly, both PMA and P. aer-
uginosa showed statistically significant higher DNA (Fig. 2,
panel II) and LL37 levels (Fig. 2, panel III) compared to
initial staining.
NET supernatants were discarded, and colorimetric
assays were used to evaluate the enzymatic action after
disengaging DNA–protein structures using DNAse to ana-
lyze the activity due to just NET-bound proteins described
by White PC and coworkers [18]. The results revealed a
remainder NE (2.3 ± 0.6%), CG (6.2 ± 2.7%), and MPO
(21.4 ± 2.0%) enzymatic activity in NET-derived DNA,
regarding the maximum activity obtained from lysed neu-
trophils. NE activity was detected consistently low in NETs
induced by all stimuli but showing higher significant activ-
ity facing S. aureus. CG activity was also present under all
conditions but significantly higher in PMA-induced NETs.
Meanwhile, MPO was still present in NETs induced with all
stimuli showing significantly higher activity facing micro-
biological triggers in comparison to biochemical stimuli,
but with no statistically significant differences among them
(Fig. 3). These results provide descriptive evidence about the
Fig. 3  NET-remaining enzymatic activity induced by biochemical
and microbiologic triggers. After NET induction, supernatants were
discarded, and colorimetric assays were used to evaluate the enzy-
matic activity after disengaging DNA–protein structures by DNAse
to analyze just NET-bound proteins. Graphics show averages ± SD of
the percentage of NET-remaining enzymatic activity induced by each
biochemical or microbiologic stimuli compared to the MAXIMUM
enzymatic activity (supernatants of neutrophils lysed by freeze and
defrost at − 70  °C). BASAL shows the enzymatic activity in super-
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Immunologic Research
specificity in neutrophil recognition and response, revealed
by the differential morphologic and structural characteris-
tics and the variable enzymatic activity observed in NETs
induced by different stimuli.
SLE plasma induces neutrophil aggregation
and increases DNA/LL37 release during NET
induction
Neutrophils were stimulated under the conditions as men-
tioned earlier plus HBSS, autologous (from neutrophil
donor) or allogeneic (healthy) plasma as controls, as well as
SLE (allogeneic) plasma in age and gender, paired study to
evaluate the effect of soluble factors present in the plasma
of SLE patients on NET production. NETs were analyzed
by DNA/LL37 quantification as described in the “Materials
and methods” section. Plasmas from recently diagnosed and
before treatment SLE patients were used to these experi-
ments to avoid interference effects on NET production likely
attributable to immunosuppressive therapy.
The results showed that in comparison to neutrophils
maintained in the absence of plasma and stimulus (Fig. 2,
panel I, a), the presence of autologous (Fig. 4, panel I, a)
or allogeneic (Fig. 4, panel I, g) plasma produced no mor-
phologic changes neither variations in DNA/LL37 loca-
tion (Fig. 2, panel I). Nevertheless, neutrophils in SLE
plasma’s presence displayed an evident cell aggregation
natants of neutrophils kept in HBSS buffer. Statistical analysis was
performed with data of ten independent experiments and achieved by
the Minitab software by ANOVA to perform comparisons between
groups of experimental conditions with a confidence level of 95%. *
and ** = p ≤ 0.05 compared to all other stimuli. *** = p ≤ 0.05 com-
paring the signaled groups of stimuli. NE, neutrophil elastase; cath-
epsin G; MPO, myeloperoxidase; PMA, phorbol myristate acetate;
HOCl, hypochlorous acid
Immunologic Research
Fig. 4  Effect of autologous, healthy allogeneic, and SLE allogeneic
plasma on structural differences induced by biochemical and micro-
biologic triggers. The NET formation was induced by each described
stimulus separately (NO PLASMA) or each stimulus plus autologous
(from neutrophil donor), healthy allogenic or SLE allogeneic plasma
at 10%. Then, NETs were stained with DAPI for DNA visualiza-
tion (blue) and anti-LL37 Alexa Fluor 594 (red) or isotype control.
Staphylococcus aureus, P. aeruginosa, and pseudohyphae of C. albi-
cans were visualized in green color due to CFSE staining before NET
induction. (I) MERGE images show colocalizations of the mentioned
colors of representative results of three independent experiments per-
formed by triplicate. (II) The graphic shows the Cell Aggregation
Index quantified as the number of isolated cells at every stimulus/
and spontaneous NET-like tiny DNA fibers and LL37 dif-
fuse staining (Fig. 4, panel I, m).
On the other hand, the presence of autologous (Fig. 4,
panel I, a-f) or allogeneic (Fig. 4, panel I, g-l) plasma dur-
ing NET induction by either biochemical or microbiologi-
cal stimuli induced no substantial changes in DNA/LL37
structures and locations when compared to the absence of
plasma (Fig. 2, MERGE column). Reproducibly, the pres-
ence of SLE plasma during all stimuli induced a statisti-
cally significant neutrophil aggregation (Fig. 4 II). How-
ever, after DNA and LL37 quantification by image analysis
and statistics, significant differences were not found when
comparisons were performed to analyze the effect of each
plasma on the same stimulus (e.g., PMA/no plasma vs.
number of isolated cells at every stimulus plus plasma and normal-
ized to the data obtained with the HBSS control. (III) The graphics
show the statistical analysis of the mean gray value of signal per area
for each color (DAPI-DNA, Alexa Fluor 594-LL37, and MERGE
staining) of five images (four extremes and center) per well, for each
condition of three experiments performed by triplicate. Results were
obtained through the Minitab software by ANOVA analysis to per-
form comparisons between the absence of plasma and the presence
of autologous, healthy allogeneic, or SLE allogeneic plasma with
all stimuli, and between the presence of the different plasmas as
described in the Y-axis, with a confidence level of 95%. * = p ≤ 0.05.
HBSS, Hank´s balanced salt solution; PMA, phorbol myristate ace-
tate; HOCl, hypochlorous acid; SLE, systemic lupus erythematosus
PMA/autologous vs. PMA/allogeneic vs. PMA/SLE) (not
shown).
Interestingly, the statistical analysis performed between
the presence of the different plasmas or no plasma with
all stimuli (e.g., all stimuli with no plasma vs. all stimuli
with autologous plasma vs. all stimuli with allogeneic
plasma vs. all stimuli with SLE plasma) revealed sig-
nificant remarkable differences (Fig. 4, panel III). Hence,
under the last statistical analysis, DNA, LL37, and DNA/
LL37 merge quantification showed no statistical differ-
ences between autologous vs. no plasma, allogeneic vs.
no plasma, and allogeneic vs. autologous. These results
indicated that any healthy plasma’s presence did not affect
the DNA/LL37 composition of NETs induced with all
13

the evaluated stimuli. Strikingly, SLE plasma’s presence
revealed higher DNA quantification, LL37, and DNA/
LL37 merge than no plasma and autologous. This signifi-
cant difference is maintained when LL37 and DNA/LL37
are analyzed by comparing SLE vs. allogeneic, but not
when only DNA is analyzed (Fig. 4, panel III). Therefore,
these results revealed that SLE plasma’s presence sig-
nificantly increases DNA and LL37 release during NET
induction independently of the trigger stimulus.
As DNA and LL37, the statistical analysis of NE, CG,
and MPO enzymatic activity in each plasma’s presence
on the same stimulus did not display significant differ-
ences (not shown). However, the analysis of the existence
of SLE plasma with all stimuli compared to autologous,
allogeneic, or no plasma did reveal significant, impres-
sive results (Fig. 5). NE activity was found significantly
reduced in the presence of either autologous, allogeneic,
or SLE plasma in comparison to the absence of plasma
(no plasma). Nevertheless, no differences were found in
NE activity when comparing any plasma between them,
and the analysis detected no changes between any plasma
with its absence regarding CG activity. Interestingly, sig-
nificant differences were found when comparing alloge-
neic or SLE with autologous plasmas. Finally, MPO enzy-
matic activity was found significantly increased in the
presence of either autologous, allogeneic, or SLE plasma
compared to no plasma, but with no differences between
them. Hence, the results of this study revealed no differ-
ences in NET-remaining NE, CG, and MPO enzymatic
activity due to the presence of SLE plasma-derived solu-
ble factors.
Fig. 5  Effect of autologous, healthy allogeneic, and SLE allogeneic
plasma on NET-remaining enzymatic activity induced by biochemi-
cal and microbiologic triggers. Enzymatic activity was evaluated after
NET induction by each stimulus used individually (NO PLASMA),
or by each stimulus plus autologous (from neutrophil donor), healthy
allogenic, or SLE allogeneic plasma at 10%. Supernatants were dis-
carded, and after disengaging DNA–protein structures by DNAse,
enzymatic activity was evaluated as described in the “Materials and
methods” section just from NET-bound proteins. The graphics show
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Immunologic Research
Discussion
The presence of certain anticoagulants as heparin or chelat-
ing agents and calcium and magnesium during neutrophil
isolation for NET assays have been described as likely
inhibitors or clumping and adhesion inducers affecting NET
formation [19]. The characterization of the initial morphol-
ogy after purification (Fig. 1a-c) and before NET formation
(Fig. 2j, o, and t), along with the following up of resting
neutrophils during NET induction (Fig. 2a-c), allowed to
discard neutrophil changes owed to the isolation methods
and to coin such differences exclusively to the added stim-
uli. Since the NET discovery, many studies have revealed
multiple triggers that induce similar morphologic features
and composition, allowing to classify them as lytic or non-
lytic NET inducers [2]. Nevertheless, the heterogeneity and
specificity in NET morphology and design depending on
the inducer agent under equal methodologic conditions have
been scarcely studied.
Given that several studies have evidenced the extensive
heterogeneity of NET composition, current statements rec-
ommend identifying NETs as extracellular DNA structures
colocalizing with granule-derived proteins and nuclear
components [7]. Because LL37 is a protein found at a high
concentration in secondary (specific) granules of neutro-
phils, it was chosen to identify a neutrophil’s secondary
granule-derived protein in extracellular DNA structures.
Additionally, since LL37 has been strongly associated with
SLE immunopathology as a neutrophil-derived inflamma-
tion factor and an autoantigen [20], LL37 was chosen to
evaluate its likely variable presence facing different stimuli
under healthy and autoimmune environments. Current state-
ments for monitoring NET formation suggest analysis by
the statistical analysis of three independent experiments performed
by triplicate achieved by the Minitab software by ANOVA analy-
sis. Comparisons were performed between NET-remaining enzy-
matic activity in the absence of plasma and the presence of autolo-
gous, healthy allogeneic, or SLE allogeneic plasma with all stimuli
and between the presence of the different plasmas as described in
the Y-axis. The statistical analysis used a 95% confidence interval.
* = p ≤ 0.05. SLE, systemic lupus erythematosus
Immunologic Research
real-time live-cell methods such as intravital imaging. Nev-
ertheless, in addition to the NET formation, our study aimed
to quantify differential enzymatic activity exclusively due
to NET-bound proteins. Hence, it was necessary to discard
supernatants and analyze enzymatic activity only in disen-
gaged proteins from fixed DNA structures. Therefore, given
the used methodology for NET description by fluorescence
microcopy in this study, the results allowed to describe the
extracellular DNA structures only as lytic-like or non-lytic-
like NET morphologies.
The results obtained in this study showed that both PMA
and P. aeruginosa induced a cloud-like appearance of extra-
cellular DNA and just these stimuli presented significantly
higher DNA staining in comparison to resting neutrophils.
Some authors state that a cloudy morphology of nuclear
DNA might result from necrotic cell death due to prolonged
neutrophil incubation in vitro [21] and that NET appearance
might be altered from cloud-like to extended fibers due to
currents in the media [7]. The authors could discard this
artifactual effect because the methodology included internal
controls that allowed them to keep unchanged morphology
in resting neutrophils treated under the same conditions.
Likewise, the obtained NET morphologies were similar
to those separately reported for each stimulus by differ-
ent authors (lytic for PMA [2] and P. aeruginosa [11]; and
reproducibly non-lytic for HOCl [8], C. albicans [9], and S.
aureus [10]) (Fig. 2a).
Additionally, these cloudy features were reproduc-
ibly more extensive and colocalized against P. aeruginosa
(Fig. 2x). Although the analysis detected no differences in
DNA or LL37 quantification between these lytic stimuli,
both induced significantly higher LL37 staining than resting
neutrophils (Fig. 2 II and III). Different enzymatic activity
was found insomuch CG activity was markedly higher in
PMA-induced NETs. Likewise, MPO activity was signifi-
cantly superior in NETs induced by P. aeruginosa (Fig. 3).
Hence, beyond a mere cellular death, this variable enzymatic
activity in the NET-derived DNA shows a certain degree
of specialization attributable to different pathways’ activa-
tion. PMA-induced lytic NETs depend on ROS production
by PKC activation [7], while P. aeruginosa also induces
lytic NET formation but via an LPS-TLR4 recognition in
an ROS-dependent and autophagy-dependent pathway [11].
The discovery that neutrophils possess the ability to discrim-
inate between LPS from various Gram-negative bacteria and
selectively discern the NET formation pathway strengthens
this hypothesis [22]. Thus, morphologically similar NET
structures might be endowed with specialized enzymatic
activity depending on the inducer stimulus.
Alternatively, HOCl [8], C. albicans [9], and S. aureus
[10] triggered no-lytic fibrous characteristics in the released
DNA (Fig. 2). This morphology is sometimes described as
mainly composed of mitochondrial DNA [23]. Like the lytic
inducers, the analysis detected no differences in DNA and
LL37 quantification between them (Fig. 2 II and III). How-
ever, divergences were observed in their enzymatic activ-
ity. Both microbiologic stimuli (C. albicans and S. aureus)
induced significantly higher MPO activity than HOCl, and
S. aureus provoked higher NE activity than all its corre-
sponding counterparts (Fig. 3). HOCl is a product of MPO
activity; thus, the non-lytic DNA release induced by this
stimulus is ROS independent [7, 8], while bacteria generate
ROS via MEK–ERK signaling, triggering the MPO path-
way [11]. On the other hand, neutrophils might produce
higher NE enzymatic activity induced by S. aureus in NETs
because the peptidoglycan-TLR2 recognition activates the
arginine deiminase 4 (PAD4) signaling pathway. This signal-
ing modulates nuclear translocation of NE to the nucleus to
drive chromatin decondensation by processing histones [10];
meanwhile, the recognition of cell wall components of C.
albicans by dectin-2 in neutrophils leads to neutrophil-yeast
aggregation, filling of intracellular vesicles with DNA and to
an NADPH oxidase-independent NET formation involving
a quick DNA release in a non-lytic route [24].
Considering the NET composition induced only by
microbiological stimuli, DNA amount and LL37 were sig-
nificantly higher, challenging S. aeruginosa (Fig. 2 II and
III). DNA differences between microorganisms might be
explained by the different activated NET pathways and the
presence of nuclease activity in bacteria as described for S.
aureus, whose nuclease expression facilitates escape from
NETs [25]. Meanwhile, the more significant presence of
LL37 in P. aeruginosa-induced NETs might undoubtedly be
explained by the higher DNA release induced by lytic NET
stimuli (PMA and P. aeruginosa (Fig. 2, panel III)). The
bactericidal activity of LL37 has been previously reported
with more potent activity against P. aeruginosa due to its
capability to biofilm formation compared to other Gram-
positive and Gram-negative bacteria [26]. Hence, the dif-
ferent activated signaling pathways by recognizing specific
pathogen-associated molecular patterns (PAMPs) mediated
by neutrophil receptors might suitably produce the neces-
sary amount of DNA network and load specific enzymes and
antimicrobial peptides, to be later selectively released to act
against particular pathogens.
SLE plasma’s presence revealed a cell aggregation in
resting neutrophils with spontaneous NET-like tiny DNA
fibers (Fig. 4i, m). Interestingly, NET induction against all
stimuli in the presence of SLE plasma also presented sig-
nificant neutrophil aggregation (Fig. 4 II) with a substan-
tial increase of DNA/LL37 colocalization when comparing
with the presence of autologous, allogeneic, or no plasma
(Fig. 4, panel III, DNA/LL37 MERGE). Cell aggregation,
as an expected effect of neutrophil activation caused by
increased levels of IFN-α and immunocomplexes in SLE
patients, might boost NET formation [14] and, besides the
13

inadequate clearance of DNA described in this disease, it
has been proposed as a likely mechanism for loss immu-
nological tolerance and anti-DNA and anti-LL37 autoan-
tibody production in SLE and psoriasis [27]. Likewise,
some differences in enzymatic activity were evidenced
by comparing all stimuli in the presence of each plasma
or no plasma. The analysis revealed significantly reduced
NE activity; meanwhile, MPO activity was significantly
increased in all induced NETs indistinctly in the presence
of either autologous, allogeneic, or SLE plasma (Fig. 5),
suggesting that the activity of the protease inhibitor of NE
(α-1 antitrypsin) and MPO activity might be unaffected
in SLE plasma of the analyzed patients. Regarding CG
activity, a significant and equivalent increase was found in
NETs induced in the presence of both allogeneic plasma
(healthy and SLE) in comparison to autologous (Fig. 5).
This result suggests that the presence of CG inhibitors
(e.g., α1-antichymotrypsin, α1-antitrypsin) in allogeneic
plasmas might possess reduced allogeneic activity prob-
ably due to CG polymorphisms, but with unaltered activ-
ity in SLE plasma [28]. Finally, although the results of
this study revealed no differences in NET-remaining NE,
CG, and MPO enzymatic activity due to SLE plasma’s
presence, a remarkable outcome was evidenced in the
presence of any plasma (Fig. 5). Given that plasma from
SLE patients was obtained in the absence of immunosup-
pressive treatment, effects on NET formation are mainly
attributable to the autoimmune/inflammatory condition at
the moment of sampling. Hence, because in vivo NET
formation is produced under the influence of plasma-
derived factors, the authors suggest including autologous
plasma in NET induction assays and evaluating the effect
of plasma-derived factors from other inflammatory dis-
eases on NET formation.
Acknowledgements  The authors would like to thank MSc. Paola Ester
López Díaz for the valuable discussions and technical assistance and to
the Flow Cytometry Department of the Medicine and Surgery Faculty
of UABJO.
Author contribution  All authors contributed to the study conception
and design. Material preparation and data collection were performed by
María de los Ángeles Romero-Tlalolini, Sergio Roberto Aguilar-Ruiz,
and Rafael Baltiérrez-Hoyos. Experimental analysis was performed by
Sorely Adelina Sosa-Luis, William de Jesús Ríos-Ríos, and Ángeles
Esmeralda Gómez-Bustamante. Honorio Torres-Aguilar wrote the first
draft of the manuscript and all authors commented on previous versions
of the manuscript. All authors read and approved the final manuscript.
Funding  This work was supported by a basic science grant (#285480)
from CONACyT and by the Department of Clinical Immunology
Research of the Biochemical Sciences Faculty, Universidad Autónoma
“Benito Juárez” de Oaxaca. S.A.S.L. and W.R.R have doctoral fel-
lowships of CONACyT numbers #660793 and #827788, respectively.
Data availability  Not applicable.
13
Immunologic Research
Code availability  Not applicable.
Declarations 
Ethics approval  The project was approved by the research and ethical
committees of the Hospital Regional de Alta Especialidad de Oaxaca,
Mexico (approval number: HRAEO/CIC/CEI 013/16).
Consent to participate  Blood samples were obtained after informed
consent.
Consent for publication  Not applicable.
Conflict of interest  The authors declare no competing interests.
References
1. Mayadas TN, Cullere X, Lowell CA. The multifaceted functions
of neutrophils. Annu Rev Pathol. 2014;9:181–218. https://d​ oi.o​ rg/​
10.​1146/​annur​ev-​pathol-​020712-​164023.
2. Brinkmann V, Reichard U, Goosmann C, Fauler B, Uhlemann
Y, Weiss DS, et al. Neutrophil extracellular traps kill bacteria.
Science. 2004;303(5663):1532–5. https://​doi.​org/​10.​1126/​scien​
ce.​10923​85.
3. Yipp BG, Petri B, Salina D, Jenne CN, Scott BN, Zbytnuik LD,
et al. Infection-induced NETosis is a dynamic process involving
neutrophil multitasking in vivo. Nat Med. 2012;18(9):1386–93.
https://​doi.​org/​10.​1038/​nm.​2847.
4. Papayannopoulos V. Neutrophil extracellular traps in immunity
and disease. Nat Rev Immunol. 2018;18(2):134–47. https://​doi.​
org/​10.​1038/​nri.​2017.​10.
5. Papayannopoulos V, Metzler KD, Hakkim A, Zychlinsky A. Neu-
trophil elastase and myeloperoxidase regulate the formation of
neutrophil extracellular traps. J Cell Biol. 2010;191(3):677–91.
https://​doi.​org/​10.​1083/​jcb.​20100​6052.
6. Petretto A, Bruschi M, Pratesi F, Croia C, Candiano G, Ghig-
geri G, et al. Neutrophil extracellular traps (NET) induced by
different stimuli: a comparative proteomic analysis. PLoS One.
2019;14(7):e0218946. https://d​ oi.o​ rg/1​ 0.1​ 371/j​ ourna​ l.p​ one.0​ 2189​
46.
7. Boeltz S, Amini P, Anders HJ, Andrade F, Bilyy R, Chatfield S,
et al. To NET or not to NET: current opinions and state of the
science regarding the formation of neutrophil extracellular traps.
Cell Death Differ. 2019;26(3):395–408. https://​doi.​org/​10.​1038/​
s41418-​018-​0261-x.
8. Palmer LJ, Cooper PR, Ling MR, Wright HJ, Huissoon A, Chap-
ple IL. Hypochlorous acid regulates neutrophil extracellular trap
release in humans. Clin Exp Immunol. 2012;167(2):261–8. https://​
doi.​org/​10.​1111/j.​1365-​2249.​2011.​04518.x.
9. Urban CF, Reichard U, Brinkmann V, Zychlinsky A. Neutrophil
extracellular traps capture and kill Candida albicans yeast and
hyphal forms. Cell Microbiol. 2006;8(4):668–76. https://​doi.​org/​
10.​1111/j.​1462-​5822.​2005.​00659.x.
10. Pilsczek FH, Salina D, Poon KK, Fahey C, Yipp BG, Sibley CD,
et al. A novel mechanism of rapid nuclear neutrophil extracellular
trap formation in response to Staphylococcus aureus. J Immunol.
2010;185(12):7413–25. https://​doi.​org/​10.​4049/​jimmu​nol.​10006​
75.
11. Young RL, Malcolm KC, Kret JE, Caceres SM, Poch KR,
Nichols DP, et  al. Neutrophil extracellular trap (NET)-medi-
ated killing of Pseudomonas aeruginosa: evidence of acquired
Immunologic Research
resistance within the CF airway, independent of CFTR. PLoS One.
2011;6(9):e23637. https://d​ oi.o​ rg/1​ 0.1​ 371/j​ ourna​ l.p​ one.0​ 02363​ 7.
12. Jeremic I, Djuric O, Nikolic M, Vlajnic M, Nikolic A, Rado-
jkovic D, et al. Neutrophil extracellular traps-associated mark-
ers are elevated in patients with systemic lupus erythematosus.
Rheumatol Int. 2019;39(11):1849–57. https://​doi.​org/​10.​1007/​
s00296-​019-​04426-1.
13. Torres-Aguilar H, Sosa-Luis SA, Aguilar-Ruiz SR. Infections as
triggers of flares in systemic autoimmune diseases: novel innate
immunity mechanisms. Curr Opin Rheumatol. 2019;31(5):525–
31. https://​doi.​org/​10.​1097/​BOR.​00000​00000​000630.
14. Båve U, Magnusson M, Eloranta ML, Perers A, Alm GV, Rönn-
blom L. Fc gamma RIIa is expressed on natural IFN-alpha-pro-
ducing cells (plasmacytoid dendritic cells) and is required for the
IFN-alpha production induced by apoptotic cells combined with
lupus IgG. J Immunol. 2003;171(6):3296–302. https://​doi.​org/​10.​
4049/​jimmu​nol.​171.6.​3296.
15. Xhindoli D, Pacor S, Benincasa M, Scocchi M, Gennaro R, Tossi
A. The human cathelicidin LL-37–A pore-forming antibacte-
rial peptide and host-cell modulator. Biochim Biophys Acta.
2016;1858(3):546–66. https://​doi.​org/​10.​1016/j.​bbamem.​2015.​
11.​003.
16. Skrzeczynska-Moncznik J, Wlodarczyk A, Banas M, Kwitniewski
M, Zabieglo K, Kapinska-Mrowiecka M, et al. DNA structures
decorated with cathepsin G/secretory leukocyte proteinase inhibi-
tor stimulate IFNI production by plasmacytoid dendritic cells. Am
J Clin Exp Immunol. 2013;2(2):186–94.
17. Gestermann N, Di Domizio J, Lande R, Demaria O, Frasca L,
Feldmeyer L, et al. Netting neutrophils activate autoreactive B
cells in lupus. J Immunol. 2018;200(10):3364–71. https://​doi.​org/​
10.​4049/​jimmu​nol.​17007​78.
18. White PC, Chicca IJ, Ling MR, Wright HJ, Cooper PR, Milward
MR, et al. Characterization, quantification, and visualization of
neutrophil extracellular traps. Methods Mol Biol. 2017;1537:481–
97. https://​doi.​org/​10.​1007/​978-1-​4939-​6685-1_​29.
19. Manfredi AA, Rovere-Querini P, D’Angelo A, Maugeri N. Low
molecular weight heparins prevent the induction of autophagy of
activated neutrophils and the formation of neutrophil extracellular
traps. Pharmacol Res. 2017;123:146–56. https://d​ oi.o​ rg/1​ 0.1​ 016/j.​
phrs.​2016.​08.​008.
20. Bosch X. Clinical implications of basic research systemic
lupus erythematosus and the neutrophil. N Engl J Med.
2011;365(8):758–60. https://​doi.​org/​10.​1056/​NEJMc​ibr11​07085.
21. Yousefi S, Simon HU. NETosis-does it really represent nature’s
“suicide bomber”? Front Immunol. 2016;7:328. https://​doi.​org/​
10.​1016/j.​phrs.​2016.​08.​008.
22. Pieterse E, Rother N, Yanginlar C, Hilbrands LB, van der Vlag J.
Neutrophils discriminate between lipopolysaccharides of different
bacterial sources and selectively release neutrophil extracellular
traps. Front Immunol. 2016;7:484. https://d​ oi.o​ rg/1​ 0.3​ 389/fi ​ mmu.​
2016.​00484.
23. Yousefi S, Mihalache C, Kozlowski E, Schmid I, Simon HU.
Viable neutrophils release mitochondrial DNA to form neutro-
phil extracellular traps. Cell Death Differ. 2009;16(11):1438–44.
https://​doi.​org/​10.​1038/​cdd.​2009.​96.
24. Wu SY, Weng CL, Jheng MJ, Kan HW, Hsieh ST, Liu FT,
et al. Candida albicans triggers NADPH oxidase-independent
neutrophil extracellular traps through dectin-2. PLoS Pathog.
2019;15(11):e1008096. https://​doi.​org/​10.​1371/​journ​al.​ppat.​
10080​96.
25. Berends ET, Horswill AR, Haste NM, Monestier M, Nizet V, von
Köckritz-Blickwede M. Nuclease expression by Staphylococcus
aureus facilitates escape from neutrophil extracellular traps. J
Innate Immun. 2010;2(6):576–86. https://​doi.​org/​10.​1159/​00031​
9909.
26. Pestrak MJ, Chaney SB, Eggleston HC, Dellos-Nolan S, Dixit
S, Mathew-Steiner SS, et al. Pseudomonas aeruginosa rugose
small-colony variants evade host clearance, are hyper-inflam-
matory, and persist in multiple host environments. PLoS Pathog.
2018;14(2):e1006842. https://d​ oi.o​ rg/1​ 0.1​ 371/j​ ourna​ l.p​ pat.1​ 0068​
42.
27. Herster F, Bittner Z, Archer NK, Dickhöfer S, Eisel D, Eigenbrod
T, et al. Neutrophil extracellular trap-associated RNA and LL37
enable self-amplifying inflammation in psoriasis. Nat Commun.
2020;11(1):105. https://​doi.​org/​10.​1038/​s41467-​019-​13756-4.
28. Herrmann SM, Funke-Kaiser H, Schmidt-Petersen K, Nicaud V,
Gautier-Bertrand M, Evans A, et al. Characterization of poly-
morphic structure of cathepsin G gene: role in cardiovascular
and cerebrovascular diseases. Arterioscler Thromb Vasc Biol.
2001;21(9):1538–43. https://​doi.​org/​10.​1161/​hq0901.​095555.
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