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Keshavan et al. Cell Death and Disease (2019) 10:569
https://doi.org/10.1038/s41419-019-1806-8 Cell Death & Disease
REVIEW ARTICLE Open Access
Nano-bio interactions: a neutrophil-centric
view
Sandeep Keshavan
1
, Paolo Calligari
2
, Lorenzo Stella
2
,LauraFusco
3
,LuciaGemmaDelogu
4,5
and Bengt Fadeel
1
Abstract
Neutrophils are key components of the innate arm of the immune system and represent the frontline of host defense
against intruding pathogens. However, neutrophils can also cause damage to the host. Nanomaterials are being
developed for a multitude of different purposes and these minute materials may find their way into the body through
deliberate or inadvertent exposure; understanding nanomaterial interactions with the immune system is therefore of
critical importance. However, whereas numerous studies have focused on macrophages, less attention is devoted to
nanomaterial interactions with neutrophils, the most abundant leukocytes in the blood. We discuss the impact of
engineered nanomaterials on neutrophils and how neutrophils, in turn, may digest certain carbon-based materials
such as carbon nanotubes and graphene oxide. We also discuss the role of the corona of proteins adsorbed onto the
surface of nanomaterials and whether nanomaterials are sensed as pathogens by cells of the immune system.
Known facts
●Nanomaterials are inevitably cloaked with proteins
giving rise to a bio-corona.
●Nanomaterials can trigger inflammation with
activation of the inflammasome.
●Carbon-based materials may undergo digestion by
macrophages or neutrophils.
Open questions
●Does the innate immune system sense engineered
nanomaterials as pathogens?
●Are nanomaterial-induced neutrophil extracellular
traps or NETs good or bad?
●Can nanomaterials elicit exosome-mediated pro- or
anti-inflammatory signals?
Introduction
Inflammation is a complex biological response involving
soluble factors and cells that arises in a tissue in response
to harmful stimuli including pathogens, toxicants, or dead
cells. The process normally leads to recovery and healing.
However, inflammation can also lead to persistent tissue
damage and may even promote neoplastic transforma-
tion
1,2
. Indeed, the distinction between acute and chronic
inflammation is important, not least in toxicology.
Inflammation is fundamentally a normal, protective phy-
siological response to injury or infection. However, if
inflammatory responses are persistent due to an exag-
gerated or dysregulated response, including failure of
resolution of inflammation, a pathological response
occurs
3
.
Understanding interactions of engineered nanomater-
ials with the immune system is of considerable relevance
both from a toxicological and biomedical perspective
4
.
However, whereas numerous publications have focused
on nanomaterial interactions with macrophages, less
attention is devoted to neutrophils, despite the fact that
neutrophils are key factors in inflammation. In fact,
research in recent years has revealed that these cells may
also inform and shape adaptive immune response, in
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Correspondence: Bengt Fadeel (bengt.fadeel@ki.se)
1
Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden
2
Department of Chemical Sciences and Technologies, University of Rome Tor
Vergata, Rome, Italy
Full list of author information is available at the end of the article.
Edited by H.-U. Simon
Official journal of the Cell Death Differentiation Association
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addition to their traditional roles as hunters and killers of
microbes
5
. Furthermore, although this has previously
been overlooked, neutrophils also express a rich reper-
toire of so-called pattern recognition receptors or PRRs
6
.
We will focus here on the interactions of engineered
nanomaterials with neutrophils, the most abundant of the
white blood cells.
Nanomaterial effects on neutrophils
Neutrophils are key factors in inflammation and
numerous studies have shown that engineered nanoma-
terials may elicit acute and/or chronic inflammation in
different animal models
7
. However, despite numerous
studies showing tissue infiltration of neutrophils upon
exposure to nanoparticles, it can be argued that neu-
trophils are a somewhat neglected cell in nanotoxicology,
as there are relatively few studies on direct interactions of
nanomaterials with these cells. Nevertheless, neutrophils
are normally the first responders in an inflammatory
reaction while macrophages arrive in the second wave of
inflammation and serve mainly to remove cell debris and
to promote tissue healing
8
. Similarly, it is worth noting
that macrophages are not the only cells that are involved
in the clearance of nanoparticles from the blood; in fact, a
recent study showed that neutrophils also play a major
role in nanoparticle clearance, at least in some mouse
strains
9
. Notably, although neutrophils are cleared from
the circulation via the liver and spleen, evidence has been
put forward that the bone marrow is a major site of
neutrophil clearance
10
. It follows that nanoparticles that
are cleared from the circulation by neutrophils could end
up in the bone marrow and yet the bone marrow is fre-
quently overlooked as a possible site for the sequestration
of nanoparticles, as particle uptake by macrophages in the
liver or spleen is usually in focus
11
.
Girard and colleagues
12–15
have published a series of
papers in which various metal and metal oxide nano-
particles including nanoparticles of titanium dioxide, zinc
oxide, and silver were shown to activate neutrophils and/
or to inhibit neutrophil apoptosis. In contrast, gold
nanoparticles were found to activate or accelerate neu-
trophil apoptosis
16
. Needless to say, careful attention to
endotoxin contamination of the tested particles is
required
17
. Other authors have shown that silver nano-
particles differently affect distinct subpopulations of
neutrophils
18
. Fromen et al.
19
documented interactions
between injected nanoparticles and circulating neu-
trophils, which could drive particle clearance but could
also alter neutrophil responses in a mouse model of acute
lung injury. The attachment of poly(ethylene glycol)
(PEG) onto the surface of nanoparticles is commonly
thought to prevent particle opsonization and macrophage
uptake. However, a recent study suggested, instead, that
neutrophils preferentially internalized PEGylated particles
(i.e., polystyrene microspheres) in the presence of human
plasma
20
. Notably, when the authors used model cell lines
such as HL-60 or THP-1 cultured in standard cell med-
ium supplemented with 10% fetal bovine serum, PEGy-
lation reduced uptake of the particles. However, when
these cells were cultured in human plasma, the PEGylated
particles were more avidly taken up, in line with the
results obtained with primary cells
20
. This suggests that
nanomedicine approaches based on PEGylation of nano-
particles need to be reconsidered—perhaps the adminis-
tered particles with their “shield”of surface-attached
polymers are being sequestered by neutrophils in the
blood? Bisso et al.
21
conducted an in-depth study of
nanomaterial interactions with human neutrophils
focusing on polymeric and liposomal particles ranging in
size from 20 nm to 5 µm. The authors found that nano-
particles were readily internalized by neutrophils ex vivo
in the absence of serum proteins, and that the inter-
nalization was size-dependent insofar as a significant
increase in uptake of the 200 nm particles was observed
over particles < 100 nm in diameter. The inclusion of
albumin in the cell culture medium prevented uptake of
polystyrene particles and reduced the uptake of liposomal
nanoparticles, but enhanced neutrophil uptake of poly
(lactic-co-glycolic acid) (PLGA) particles. Notably,
particle-laden neutrophils (i.e., 1 µg/mL of polystyrene
particles or 5 µg/mL of liposomes) were found to undergo
normal degranulation upon stimulation with conventional
agonists
21
.
Carbon-based nanomaterials, including carbon nano-
tubes (CNTs), are widely studied in nanotoxicology
22
and
these materials were shown to trigger apoptosis and/or
autophagic cell death in macrophages (Box 1). Less is
known in regards to neutrophils. In a recent study con-
ducted in the frame of the Horizon2020 project BIOR-
IMA, the toxicity of three multi-walled CNTs
(MWCNTs) with varying physicochemical properties was
evaluated in neutrophils vs. macrophages. Macrophages
were susceptible only to the fiber-like MWCNTs, but
neutrophil cell viability was significantly affected by all
three CNTs, both long and tangled (Keshavan et al.,
manuscript in preparation). Thus, although macrophages
are capable of ingesting nanomaterials and are widely
used as a model in nanotoxicology, neutrophils should
not be ignored.
Bio-corona formation on nanoparticles
Nanomaterials promptly adsorb biomolecules leading to
the formation of a so-called bio-corona
23
. The binding of
proteins or other biomolecules to nanoparticle surfaces
may thereby afford a new “identity”to the nanoparticle
24
.
Deng et al.
25
showed that poly(acrylic acid)-coated gold
nanoparticles bind fibrinogen, a protein involved in blood
clot formation, in a charge-dependent manner, inducing
Keshavan et al. Cell Death and Disease (2019) 10:569 Page 2 of 11
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unfolding of the protein, and that binding to integrin
receptors on the surface of the monocytic cell line, THP-1
leads to activation of the nuclear factor-κB pathway and
secretion of the pro-inflammatory cytokine tumor
necrosis factor-α. In a comprehensive study using a panel
of silica and polystyrene nanoparticles of various sizes and
surface modifications, Tenzer et al.
26
could show that
plasma protein adsorption occurs very rapidly, and that it
affects hemolysis, thrombocyte activation, cellular uptake,
and endothelial cell death. Vlasova et al.
27
reported that
adsorbed plasma proteins influenced neutrophil responses
caused by polymer-coated, single-walled CNTs
(SWCNTs). Specifically, the adsorption of IgG resulted in
neutrophil activation, as determined by degranulation and
release of myeloperoxidase (MPO). Similarly, protein
adsorption modulated neutrophil responses toward car-
boxylated, non-PEGylated SWCNTs
28
. Lara et al.
29
employed an immuno-mapping technique to study epi-
tope presentation of two major proteins in the serum
corona, low-density lipoprotein (LDL) and
immunoglobulin G. The authors could show that both
proteins displayed functional motifs allowing for recog-
nition of the bio-corona on silica nanoparticles by LDL
receptor and Fc-γreceptor I, respectively. On the other
hand, others have pointed out that the bio-corona could
shield targeting ligands on nanoparticles
30
. However,
recent studies suggested clever ways in which to cir-
cumvent this problem
31,32
. Furthermore, purposeful sur-
face modification of CdSe/ZnS quantum dots to induce a
protein misfolding event in the bio-corona enabled
receptor-mediated endocytosis of the particles
33
. Bisso
et al.
21
reported that the presence of serum reduced the
ex vivo uptake of poly(styrene) nanoparticles and lipo-
somes by neutrophils and enhanced the uptake of micro-
and nanosized PLGA particles. However, the composition
of the bio-corona and the role of specific proteins for
neutrophil uptake, or lack thereof, was not examined.
Furthermore, neutrophils were found to preferentially
internalize PEGylated particles
20
. The authors noted that
this is linked to factor(s) in human plasma and provided
some evidence for a role of complement. Complement
factors also facilitate phagocytosis of apoptotic cells and
microbes
4
.
Viruses are natural nanoparticles and it is not surprising
that a bio-corona of proteins may form on viruses in
various biological fluids, or that the bio-corona may affect
immune responses to viruses
34
. Indeed, the adsorbed bio-
corona could be considered as part of the motifs that are
sensed by immune cells. Hence, the boundaries between
pathogen-associated molecular patterns (PAMPs) and
damage-associated molecular patterns (DAMPs) appar-
ently begin to dissolve at the nano-bio interface, as both
engineered and natural nanoparticles (i.e., viruses) adsorb
host proteins on their surface. This topic is discussed in
more detail below.
Exosomes: message in a bottle
Exosomes are nanosized extracellular vesicles that are
naturally secreted by cells and they may play a particularly
important role in conveying information between
immune cells
35
. Exosomes were initially thought to fulfill
a janitorial function by providing the cell with a means of
getting rid of non-functional proteins and other mole-
cules. Subsequent studies suggested important roles of
exosomes in intercellular communication and the interest
in exosomes and other microvesicles has escalated in the
past two decades due to their emerging roles in health and
disease
36
. Exosomes thus harbor specific proteins and
nucleic acids, mostly small RNAs, such as ribosomal RNA,
transfer RNA, microRNA, and mRNA molecules
36
. Exo-
somes from activated neutrophils were recently reported
to acquire surface-bound neutrophil elastase (NE) and the
exosomes were shown to degrade extracellular matrix
components causing the hallmarks of chronic obstructive
Box 1 Cell death: implications for nanotoxicology
The Nomenclature Committee on Cell Death recently provided
guidelines for molecular definitions of various cell death
modalities
128
, although this is hardly the first nor the last attempt
at defining cell death
129
. It is important to bear in mind that
nanomaterials can elicit different modes of cell death depending
on the material as well as on the cell type, and that the mitigation
of the adverse effects of such materials requires the correct
diagnosis of cell death
130
.
Apoptosis: Regulated cell death precipitated by caspases, either
through plasma membrane receptor ligation (extrinsic apoptosis)
or via perturbation of mitochondria (intrinsic apoptosis). Nano-
materials such as CNTs trigger apoptosis, for instance in lung
cells
131
, but chronic, low-dose exposure may result in apoptosis
resistance and oncogenic transformation
132
.
Pyroptosis: Regulated cell death associated with the formation of
pores in the plasma membrane by gasdermin proteins, usually
provoked by caspase-1 activation
133
. Rare earth metal nanopar-
ticles were found to trigger pyroptosis in macrophages and
apoptosis in hepatocytes
134
.
Necroptosis: Regulated cell death that transpires with RIP1/RIP3
activation and subsequent plasma membrane permeabilization
by MLKL (mixed-lineage kinase domain-like pseudokinase)
135
.
Few, if any, studies have reported evidence of nanoparticle-
induced necroptosis.
Ferroptosis: Novel, iron-dependent cell death characterized by
lipid peroxidation that is subject to regulation by GPX4
(glutathione peroxidase 4) and tightly linked to glutathione
synthesis
136
. Importantly, a recent study revealed that nanopar-
ticles trigger different forms of cell death (i.e., apoptosis or
ferroptosis) depending on subtle variations in nanoparticle
surface properties
137
.
Autophagic cell death: Cell death that requires components of the
autophagic machinery (note that autophagic cell death should
not be confused with autophagy, a cell survival mechanism).
Nanoparticles with a high propensity to release toxic ions may
harness autophagy to trigger cell death
138
.
Keshavan et al. Cell Death and Disease (2019) 10:569 Page 3 of 11
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pulmonary disease
37
. Exosomal NE was much more
potent than free NE. In the latter study, neutrophils were
activated with the bacterial peptide, formyl-methionine-
leucine-phenylalanine (fMLP), a known neutrophil ago-
nist. Whether or not such exosome-mediated pathological
responses occur following nanomaterial exposure merits
close attention; several studies have shown that CNTs
cause pulmonary inflammation as well as airway remo-
deling
38
. In a seminal study, Zhu et al.
39
reported that
exosomes were generated in significant numbers in the
lungs of mice exposed to iron oxide nanoparticles, and
noted that the exosomes were quickly transferred to the
systemic circulation, thereby conveying immune activa-
tion in extrapulmonary organs. The authors inferred that
the exosomes were of macrophage origin, although fur-
ther studies are warranted to discern the source of
nanoparticle-induced exosomes and whether neutrophils
are also involved. Studies have shown that different metal
or metal oxide nanoparticles may elicit varying cellular
patterns of inflammation and one cannot a priori assume
that macrophages are the only cell type at play
40
.In
another recent study, ZnO nanoparticles were found to
trigger neutrophilic inflammation in rats and numerous
microRNAs were shown to be selectively up- or down-
regulated in serum exosomes from ZnO-exposed animals
when compared with controls
41
. Using single-particle
inductively coupled plasma-mass spectrometry and other
techniques, Logozzi et al.
42
reported that primary human
macrophages are capable of endocytosis of gold nano-
particles (20 nm) with subsequent discharge of the
nanoparticles via exosomes. Further studies are required
to determine whether this is a general phenomenon and
to what extent the exosomal content of nanoparticles
correlates with the delivered dose. Nevertheless, it is
conceivable that exosomes could be exploited as bio-
markers of exposure to nanoparticles
42
.
Neutrophil traps: a necessary nuisance?
Brinkmann et al.
43
reported 15 years ago that neu-
trophils kill pathogens extracellularly by releasing so-
called neutrophil extracellular traps or NETs. NETs are
comprised a backbone of nuclear chromatin decorated
with antimicrobial proteins such as MPO and NE. In
addition to the classical or most commonly studied form
of NADPH oxidase-dependent NETs, which contain
nuclear chromatin, some studies have shown that neu-
trophils under certain conditions release NETs compris-
ing mitochondrial DNA
44,45
.
NET formation is frequently viewed as a specialized
form of neutrophil cell death that is distinct from apop-
tosis and necrosis
46
, and this cell death has been dubbed
NETosis. This has led to some confusion in the literature,
as the term NETosis is commonly equated with NET
formation, and to further compound the situation, some
authors refer to “vital NETosis”
47,48
. Indeed, as the terms
suicidal and vital NETosis are controversial, it is advisable
to simply refer to neutrophil formation of NETs with or
without attendant cell death. On the other hand, it is well
established that the stimulation of neutrophils with
phorbol 12-myristate 13-acetate (PMA) leads to a cas-
pase-independent, non-apoptotic form of cell death
49,50
.
Recent studies suggest some commonalities between NET
formation and other forms of programmed cell death
(Box 1). Hence, gasdermin D, a pore-forming protein and
a key executor of pyroptosis, is required for NET forma-
tion in neutrophils stimulated with PMA
51,52
. Further-
more, anti-neutrophil cytoplasmic antibodies trigger
NETs via receptor-interacting protein kinase 1/3 and
MLKL, key factors in necroptosis
53
, although it is
important to note in this context that different stimuli
may trigger different pathways of NET formation
54,55
.
NETs are thought to play a role during infection by
allowing neutrophils to capture and kill pathogens
extracellularly
43,56
. However, mounting evidence suggests
that uncontrolled or excessive production of NETs, or
defective degradation or removal of NETs, is related to the
exacerbation of inflammation and the development of
several diseases
57
. Hakkim et al.
58
reported that impair-
ment of DNaseI-mediated NET degradation is associated
with systemic lupus erythematosus. Excessive formation
of NETs, on the other hand, could clog blood vessels and
provide a scaffold for thrombus formation
59
, whereas a
recent study has shown that both DNase1 and DNase1-
like 3 are capable of degrading NETs in circulation
60
.We
found that NETs are handled differently by macrophages
and dendritic cells with LL-37-dependent uptake followed
by intracellular degradation in the former case, and
extracellular, DNase1L3-mediated degradation of NETs in
the latter case (Lazzaretto et al., manuscript in prepara-
tion). NETs were shown to prime T cells and reduce the
activation threshold to specific antigens
61
. TLR9, an
intracellular sensor that functions to alert the immune
system of viral and bacterial infections by binding to
DNA, was not involved. Nevertheless, this suggests that
NETs may serve as a link between the innate and adaptive
immune system. Furthermore, and in support of this
notion, a recent study showed that deposition of cell-free
DNA through neutrophil formation and ejection of NETs
occurs at the site of immunization and drives the activity
of aluminum adjuvant (alum), thereby enhancing
adjuvant-induced adaptive immune responses
62
.
Can nanomaterials trigger NETs? Early work suggested
that rod-shaped gold nanoparticles, as well as cationic lipid
nanoparticles, are capable of triggering NETs, but compel-
ling evidence was not presented as it is difficult to distin-
guish between neutrophil cell death with (passive) release of
intracellular contents vs. the production of NETs
63,64
.
Naturally, endotoxin contamination also needs to be
Keshavan et al. Cell Death and Disease (2019) 10:569 Page 4 of 11
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excluded. More recently, agglomerates of endotoxin-free
superparamagnetic iron oxide nanoparticles (SPIONs) were
shown to elicit NETs, albeit at a very high concentration
(200 µg/mL)
65
. Importantly, stabilization of the SPIONs
with human serum albumin prevented NET formation. The
authors also found that agglomerates of SPIONs triggered
NET formation in vivo in an animal model, and that the
particles were “glued”together by the NETs, and they
suggested that such SPION-NET co-aggregates might
occlude blood vessels
65
. The study highlights the need for
careful particle design and passivation strategies to make
nanoparticles safe for intravenous use. Muñoz et al.
66
reported that nanodiamonds (10nm)causeplasmamem-
brane damage and signs of lysosomal instability in neu-
trophils, and found that these nanoparticles triggered the
formation of NETs at high concentrations of nanoparticles
(200 µg/mL). In contrast, larger particles (100–1000 nm)
were relatively inert. The smaller particles also triggered
inflammation following subcutaneous injection of a high
dose (1 mg) into the foot pads of mice, but the swelling was
resolved after 1–2weeks
66
.Therelevanceofthesefindings
is difficult to judge due to the high doses that were applied.
It is worth noting that nanodiamonds were recently shown
to be well-tolerated in sub-acute and chronic duration
studies in rodents and non-human primates following
intravenous injections
67
.
We recently provided first evidence that graphene oxide
(GO) can trigger NETs in primary human neutrophils,
indicating that the immune system can “sense”even a
two-dimensional material (Fig. 1). Hence, we could show
that low doses of micrometer-sized GO sheets triggered
NETs in primary human neutrophils more effectively than
small GO with nanosized lateral dimensions
68
. Both
materials were produced under sterile conditions and
were proven to be endotoxin-free. Interestingly, the large
GO sheets initiated the oxidation of cholesterol in the
plasma membrane of neutrophils as evidenced by time-of-
flight secondary ion mass spectrometry (ToF-SIMS) fur-
thermore, the release of NETs was reduced by Trolox, a
potent lipid antioxidant
68
. Neumann et al.
69
previously
reported that methyl-β-cyclodextrin (MβCD), a
cholesterol-depleting agent, triggered the formation of
NETs in a manner that was independent of the NADPH
oxidase (i.e., insensitive to pharmacological inhibition
using diphenylene iodonium [DPI]). To study the signal-
ing pathway underlying the formation of NETs in GO-
exposed cells, we explored the effect of DPI on NET
formation in neutrophils exposed to GO, PMA, or MβCD.
DPI was found to block PMA-induced production of
NETs, as expected, and blocked the production of NETs
in neutrophils exposed to the small GO sheets
68
. How-
ever, NET formation in MβCD-treated cells and in cells
incubated with large GO was unaffected by DPI, indicat-
ing that NADPH oxidase activation is not required.
Interestingly, the mitochondria-targeted antioxidant
MitoTEMPO significantly reduced the production of
NETs by GO. Mitochondrial reactive oxygen species
(ROS) are also required for calcium ionophore-induced
NETs
70
. In a companion paper, we showed that small and
large GO are degraded in an MPO-dependent manner in
NETs purified from activated neutrophils
71
. Neutrophils
can also enzymatically digest CNTs (discussed below).
Thus, it appears that neutrophils are capable of handling
at least some carbon-based nanomaterials as pathogens,
leading to the destruction of the offending agents
72
.
Neutrophil degradation of nanomaterials
The membrane-bound NADPH oxidase generates ROS
that are instrumental for the killing of ingested pathogens.
Degranulation with the release of MPO is also an
important feature of the microbicidal actions of neu-
trophils
73
. In addition to its role in antimicrobial defense,
MPO is also reported to be involved in the degradation of
CNTs and the clearance of CNTs from the lungs was
markedly less efficient in MPO-deficient mice when
GO
NET
GO
A
B
C
D
E
Fig. 1 Neutrophils capture graphene oxide sheets in extracellular
traps. a–cConfocal images of neutrophils incubated in the presence
of large GO sheets (12.5 μg/mL). Cells were stained with antibodies to
neutrophil elastase (NE) followed by a secondary FITC-labeled
antibody (green) and counterstained with DAPI (blue) for visualization
of cell nuclei. dLight and fluorescence microscopy images
superimposed to show the presence of GO. eScanning electron
microscopy (SEM) image of neutrophils exposed to GO (12.5 μg/mL).
The arrow points to a large GO sheet that has been “captured”in a
network of chromatin fibers (i.e., NETs). Reproduced from Mukherjee
et al.
68
, with permission from Elsevier
Keshavan et al. Cell Death and Disease (2019) 10:569 Page 5 of 11
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compared with wild-type mice
74
. Neutrophil-mediated
destruction of CNTs was first described by Kagan et al.
75
.
Subsequently, MPO-dependent degradation of PEGylated
CNTs was reported and this was suggested to occur in a
two-step process whereby the polymers were first
removed from the CNTs by NE followed by the degra-
dation of the CNTs themselves by MPO
76
. Eosinophils
can also digest CNTs
77
. Furthermore, CNTs and GO were
shown to undergo acellular degradation in NETs purified
from activated neutrophils
71,78
(Fig. 2). In a recent study,
GO functionalized with fMLP was shown to stimulate
neutrophil degranulation leading to degradation (Martin
et al., manuscript in preparation). Even graphene can
undergo neutrophil-mediated degradation, although the
process is considerably slower when compared with GO
79
.
The latter studies were conducted ex vivo using human
neutrophils. Notably, a recent in vivo study has shown
that GO (20 mg/kg) administered subcutaneously elicits
an inflammatory reaction in mice in response to
implantation consistent with a foreign body reaction
80
.
The latter study did not evaluate biodegradation of the
implanted materials. Girish et al.
81
, on the other hand,
explored biodegradation after intravenously injecting
graphene (20 mg/kg) into mice by using a Raman confocal
imaging approach. The authors noted graphene engulf-
ment by tissue-bound macrophages and found that
degradation was prominent after 90 days. Tuning the
properties of graphene-based materials to achieve optimal
performance while maintaining an acceptable degree of
biocompatibility and biodegradability remains an impor-
tant challenge in the field
82
.
Inflammasomes: double-edged swords
How do nanomaterials and other exogenous substances
trigger inflammation? Numerous studies have shown that
the inflammasome, originally described by Tschopp and
colleagues
83
, is a key signaling hub that regulates innate
immunity and inflammation
84
. The inflammasomes are
multiprotein complexes that are activated in response to
diverse pathogen- and host-derived danger signals leading
to the activation of caspase-1 with processing of cytosolic
pro-IL-1βand secretion of pro-inflammatory IL-1β
85
.
NLRP3, in particular, responds to a diverse array of dif-
ferent stimuli including crystalline and particulate matter
such as uric acid crystals, silica, asbestos, and alum, as well
as to pathogens
86–89
. NLRP3 is also a sensor of various
nanomaterials
90
. Indeed, not only long and rigid CNTs
but also ultrathin GO sheets and spherical carbon parti-
cles are able to trigger NRLP3-dependent IL-1βsecretion
in human macrophages
91–93
(Fig. 2). However, less is
known regarding inflammasome activation in neutrophils.
Neutrophils are capable of activating the NLRC4 inflam-
masome in response to bacterial challenge, but this occurs
without the induction of pyroptosis
94
. On the other hand,
NE cleaves gasdermin D in neutrophils
95
, thus providing
an alternative route to pyroptosis in these cells. Indeed, it
should be noted that the production of IL-1βis not
exclusively dependent on caspase-1
96
. Thus, neutrophil-
derived serine proteases are also involved in the proces-
sing of IL-1 family cytokines, and serine proteases and/or
caspases may be involved in neutrophils depending upon
the stimulus
97
.
How big is a speck? ASC (apoptosis-associated speck-like
protein containing a CARD) is a CARD (caspase
cytokines
exosomes
PEG
cytokines
ASC
NLRP3
pro-casp-1
pro-IL-1β IL-1β
NETs
MPO
PC
NE
O2O2·-
NADPH NADP+ + H+
e-
H2O2HOCl
MPO
NADPH oxidase
inflammasome
NOX
NOX
iNOS
NO ONOO-
iNOS
Fig. 2 Nano-bio interactions: from coronation to degradation.
This schematic diagram depicts nanoparticles with or without a
protein corona (PC) and/or a polymer coating (i.e., poly(ethylene
glycol) or PEG) interacting with neutrophils (left) vs. macrophages
(right). Neutrophils release neutrophil extracellular traps (NETs)
consisting of nuclear chromatin decorated with granule proteins such
as myeloperoxidase (MPO), and recent studies have shown that
carbon nanotubes (CNTs) and graphene oxide (GO) are captured and
digested in NETs in an MPO-dependent manner
71,78
. The NADPH
oxidase (commonly abbreviated as NOX) is a multiprotein complex
expressed in phagocytes that catalyzes the generation of superoxide.
Superoxide, in turn, dismutates to form hydrogen peroxide and MPO
catalyzes the formation of hypochlorous acid, a freely diffusible
oxidant that is microbicidal and also is responsible for the degradation
of carbon-based nanomaterials
124
. In addition, superoxide and nitric
oxide, produced by inducible nitric oxide synthase (iNOS), react to
form peroxynitrite, which was shown to digest nanomaterials in
macrophages
125
. Macrophages emit pro-inflammatory IL-1βthrough
an inflammasome-dependent mechanism (a cytosolic protein
complex shown outside the cell for clarity). Neutrophils and
macrophages release exosomes, thus providing a further means of
cell-to-cell communication and propagation of inflammation. Recent
work has shown that neutrophil-derived exosomes express neutrophil
elastase (NE) on the surface; these exosomes degrade extracellular
matrix more readily when compared with free NE
37
Keshavan et al. Cell Death and Disease (2019) 10:569 Page 6 of 11
Official journal of the Cell Death Differentiation Association
recruitment domain) carrying protein of 22 kDa that is
involved in the recruitment of pro-caspase-1 to the
inflammasome. ASC was described 20 years ago as a protein
that could be visualized as a small spot or speck in the
cytosol of apoptotic cells
98
. Intriguingly, although inflam-
masome activation was originally believed to take place in
the cytosol of cells, subsequent studies have shown that cells
may transmit inflammation in a prion-like manner via
extracellular ASC. These micrometer-sized clumps of ASC
proteins continued to stimulate caspase-1 activation extra-
cellularly and stimulated further inflammasome activation
in neighboring macrophages that had ingested the ASC
oligomers
99,100
. Extracellular ASC was found in tissues of
patients with inflammatory diseases and autoantibodies to
ASC developed in some patients with autoimmune
pathologies. Thus, as pointed out previously, danger signals
come in many shapes and sizes
101
.Itshouldthereforenot
come as a surprise that the immune system is capable of
responding to synthetic (nano)particles.
Decoding danger at the nanoscale
Cells of the immune system are equipped with PRRs
that monitor the extracellular or intracellular environ-
ment for signs of infection or “danger”. Toll-like receptors
(TLRs) are present both on cell surfaces and in endosomal
compartments, whereas retinoid acid-inducible gene-I-
like receptors and nucleotide-binding and oligomerization
domain-like receptors (NLRs) are present in the cyto-
sol
102
. Furthermore, soluble scavenging receptors have
been described
103,104
. PRRs presumably evolved to dis-
criminate between foreign intruders and “self,”but they
also recognize DAMPs released from stressed or damaged
cells
105
. It has been estimated that a single cell may
express as many as 50 distinct PRRs, thus testifying to the
importance of sensing “danger”
106
. Nanomaterials—with
or without a corona of proteins or other biomolecules—
may be considered as a particular case of danger signals
that are able to trigger sterile inflammatory responses.
Indeed, we have previously postulated that engineered
nanomaterials may present nanomaterial-associated
molecular patterns or NAMPs to cells of the immune
system
107
. Thus, in analogy with microorganisms (bac-
teria, viruses) displaying PAMPs and damaged or stressed
cells releasing DAMPs, we postulated that engineered
nanomaterials coated with a corona of biomolecules may
act as nanoparticle-associated molecular patterns or
NAMPs
107
. The notion of nanomaterial-associated
molecular patterns has captured the attention of several
other authors
108–111
. The fact that nanoparticles with an
adsorbed corona of proteins may display epitopes that are
sensed by the immune system is of considerable interest,
as this may point toward a systematic understanding of
nano-bio interactions
112
. It may also be worthwhile to
explore whether nanoparticles per se present molecular
patterns that are decoded by the immune system. Indeed,
emerging studies suggest that some nanoparticles could
act as protein mimics capable of engaging with intra- or
extracellular receptors
113
, and the combination of
experimental and theoretical studies promises to shed
light on this exciting topic
114,115
. Using a proteomics
approach, He et al.
116
found that carbon-based nanoma-
terials (i.e., single-walled carbon nanohorns, SWCNTs,
and MWCNTs) bound to glycoprotein nonmetastatic
melanoma protein B (GPNMB, also known as osteoacti-
vin) in macrophages. The authors suggested that GPNMB
serves as an intracellular PRR for these nanomaterials. We
have recently demonstrated, by using a transcriptomics
approach, that SWCNTs prompted the upregulation and
secretion of chemokines in primary human macrophages,
and we provided evidence for direct binding of CNTs to
TLR2/4
117
. The nanomaterials used were endotoxin-free.
Taken together, these results give credence to the idea
that nanomaterials may act as NAMPs
107
. Interestingly,
computational studies predicted that the binding of
carbon-based nanostructures to proteins is guided mainly
by hydrophobic interactions
114
. More specifically, we and
others have analyzed the association of CNTs and other
carbon nanostructures to TLRs
117,118
. Turabekova et al.
118
predicted that the hydrophobic pockets of some TLRs
might be capable of binding pristine SWCNTs and C
60
fullerenes (Fig. 3). Furthermore, we suggested that ion-
pair interactions with positively charged residues might
strengthen the binding of carboxylated CNTs to TLRs
117
.
It will certainly be important to study whether engineered
nanomaterials also engage with neutrophil PRRs.
It is worth noting that nanoparticles can also be
exploited for the removal of DAMPs such as cell-free
DNA that is expelled from dying cells to ameliorate
inflammatory diseases initiated by the inappropriate
activation of TLR signaling. Hence, Liang et al.
119
pre-
pared cationic nanoparticles composed of the block
copolymer of PLGA and poly(2-(diethylamino)ethyl
methacrylate), and found that these particles had a high
DNA-binding capacity. Furthermore, when injected
intravenously the cationic nanoparticles could alleviate
symptoms in animal models of arthritis. These results,
along with previous work by other investigators, suggest
that cationic nanoparticles may act as nucleic acid sca-
vengers
120,121
. Further studies are needed to formally
address whether the acquisition of a protein corona on
scavenger particles navigating the blood stream would
interfere with or promote nucleic acid binding.
Concluding remarks
Neutrophils are key effector cells of the innate arm of
the immune system and play important roles in host
defense against pathogens, and yet, paradoxically, they are
also involved in numerous pathological conditions
Keshavan et al. Cell Death and Disease (2019) 10:569 Page 7 of 11
Official journal of the Cell Death Differentiation Association
characterized by chronic inflammation. Studies in recent
years have shown that nanomaterials can modulate and
activate neutrophils and other immune cells. Moreover,
activated neutrophils may capture and digest certain
carbon-based nanomaterials. Neutrophils also play a role
in particle clearance in the systemic circulation (at least in
mice). Understanding the interactions between nanoma-
terials and neutrophils is important for the development
of safe and effective nanomaterials for biomedical
applications.
The nanotoxicology literature is replete with publica-
tions on the negative impact of nanomaterials, often
referencing the pro-inflammatory effects of the materials,
even when studies are performed in cell culture where
coordinated immune reactions cannot occur. However, it
is important to note that inflammation as such is not a
detrimental response. Therefore, one should not seek to
prevent (acute) inflammation at every cost. Instead,
careful design of nanomaterials is required in order to
avoid chronic, adverse reactions. Furthermore, nanoma-
terials may be exploited to harness immune responses to
ameliorate chronic inflammation and/or autoimmune
diseases, and leverage immune responses toward cancer
cells
122,123
.
Acknowledgements
The work is supported by the European Commission’s H2020 program through
BIORIMA (grant agreement number 760928) and the Graphene Flagship (grant
agreement number 785219), and the Swedish Research Council (grant
agreement number 2016-02040). L.D. acknowledges the support of the Marie
Skłodowska-Curie Actions Individual Fellowship IMM-GNR (grant agreement
number 797914).
Author details
1
Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden.
2
Department of Chemical Sciences and Technologies, University of Rome Tor
Vergata, Rome, Italy.
3
Department of Chemical and Pharmaceutical Sciences,
University of Trieste, Trieste, Italy.
4
Department of Biomedical Sciences,
University of Padua, Padua, Italy.
5
Fondazione Istituto di Ricerca Pediatrica Città
della Speranza, Padua, Italy
Author contributions
B.F. wrote the paper with input from S.K., P.C., L.S., L.F., and L.D. P.C. and L.S.
performed docking studies. L.F. prepared the schematic figure. All co-authors
approved the final version of the paper.
Conflict of interest
The authors declare that they have no conflict of interest.
TLR3
RNA
CNT
TLR4TLR4
BA
DC
TLR1
TLR2
Fig. 3 Macrophage sensing of single-walled carbon nanotubes as pathogens. Computational modeling of a SWCNT bound to the extracellular
domains of TLR1/TLR2 (a). The nanotube is surrounded by amino acids of mostly hydrophobic nature giving rise to strong van der Waals interactions
(b). The results shown in cdepict one of the best binding poses obtained by molecular docking of carboxylated SWCNTs with TLR4
117
. Interestingly,
the highest scoring binding mode of SWCNT and TLR4 shared several similarities with the experimentally resolved structure of TLR3 in complex with
double-stranded RNA (d)
126
. These findings suggest that TLR4 homodimers may engage with SWCNTs through a tweezer-like mechanism. It is noted
that these modeling results were derived in the absence of a protein corona in order to elucidate direct binding to TLRs. The efficiency of protein
adsorption is well-known to be proportional to the diameter of the nanotubes
127
. Therefore, the small diameter of these SWCNTs may limit protein
adsorption, thus leaving a sufficient surface for the direct interaction with TLRs
117
. Panel aand bare from Turabekova et al.
118
with permission from
The Royal Society of Chemistry. Results shown in panel care from ref.
117
while results in panel d were generated based on ref.
126
Keshavan et al. Cell Death and Disease (2019) 10:569 Page 8 of 11
Official journal of the Cell Death Differentiation Association
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
Received: 7 June 2019 Revised: 4 July 2019 Accepted: 9 July 2019
References
1. Nathan, C. Points of control in inflammation. Nature 420,846–852 (2002).
2. Medzhitov, R. Origin and physiological roles of inflammation. Nature 454,
428–435 (2008).
3. Bhattacharya,K.,Andón,F.T.,El-Sayed,R.&Fadeel,B.Mechanismsofcarbon
nanotube-induced toxicity: focus on pulmonary inflammation. Adv. Drug
Deliv. Rev. 65,2087–2097 (2013).
4. Boraschi, D. et al. Nanoparticles and innate immunity: new perspectives on
host defence. Semin. Immunol. 34,34–51 (2017).
5. Nathan,C.Neutrophilsandimmunity: challenges and opportunities. Nat. Rev.
Immunol. 6,173–182 (2006).
6. Thomas,C.J.&Schroder,K.Patternrecognitionreceptorfunctioninneu-
trophils. Trends Immunol. 34,317–328 (2013).
7. Leso, V., Fontana, L. & Iavicoli, I. Nanomaterial exposure and sterile inflam-
matory reactions. Toxicol. Appl. Pharmacol. 355,80–92 (2018).
8. Underhill, D. M. & Goodridge, H. S. Information processing during phago-
cytosis. Nat. Rev. Immunol. 12,492–502 (2012).
9. Jones, S. W. et al. Nanoparticle clearance is governed by Th1 / Th2 immunity
and strain background. J. Clin. Invest. 123, 3061–3073 (2013).
10. Furze,R.C.&Rankin,S.M.Theroleofthebonemarrowinneutrophil
clearance under homeostatic conditions in the mouse. FASEB J. 22,
3111–3119 (2008).
11. Tavares,A.J.etal.Effectofremoving Kupffer cells on nanoparticle tumor
delivery. Proc. Natl Acad. Sci. USA 114, E10871–E10880 (2017).
12. Gonçalves, D. M., Chiasson, S. & Girard, D. Activation of human neu-
trophils by titanium dioxide (TiO
2
) nanoparticles. Toxicol In Vitro.24,
1002–1008 (2010).
13. Babin,K.,Antoine,F.,Goncalves,D.M.&Girard,D.TiO
2
,CeO
2
and ZnO
nanoparticles and modulation of the degranulation process in human
neutrophils. Toxicol. Lett. 221,57–63 (2013).
14. Goncalves, D. M. & Girard, D. Zinc oxide nanoparticles delay human
neutrophil apoptosis by a de novo protein synthesis-dependent and
reactive oxygen species-independent mechanism. Toxicol In Vitro. 28,
926–931 (2014).
15. Babin, K., Goncalves, D. M. & Girard, D. Nanoparticles enhance the ability of
human neutrophils to exert phagocytosis by a Syk-dependent mechanism.
Biochim. Biophys. Acta 1850, 2276–2282 (2015).
16. Noël, C., Simard, J. C. & Girard, D. Gold nanoparticles induce apoptosis,
endoplasmic reticulum stress events and cleavage of cytoskeletal proteins in
human neutrophils. Toxicol In Vitro. 31,12–22 (2016).
17. Gonçalves, D. M. & Girard, D. Evidence that polyhydroxylated C
60
full-
erenes (fullerenols) amplify the effect of lipopolysaccharides to induce
rapid leukocyte infiltration in vivo. Chem. Res. Toxicol. 26, 1884–1892
(2013).
18. Fraser, J. A. et al. Silver nanoparticles promote the emergence of hetero-
geneic human neutrophil sub-populations. Sci. Rep. 8,1–14 (2018).
19. Fromen, C. A. et al. Neutrophil-particle interactions in blood circulation drive
particle clearance and alter neutrophil responses in acute inflammation. ACS
Nano. 11, 10797–10807 (2017).
20. Kelley,W.J.,Fromen,C.A.,Lopez-Cazares,G.&Eniola-Adefeso,O.PEGylation
of model drug carriers enhances phagocytosis by primary human neu-
trophils. Acta Biomater. 79, 283–293 (2018).
21. Bisso, P. W., Gaglione, S., Guimarães, P. P. G., Mitchell, M. J. & Langer, R.
Nanomaterial interactions with human neutrophils. ACS Biomater. Sci. Eng. 4,
4255–4265 (2018).
22. Bhattacharya, K. et al. Biological interactions of carbon-based nanomaterials:
from coronation to degradation. Nanomedicine 12,333–351 (2016).
23. Barbero, F. et al. Formation of the protein corona: the interface between
nanoparticles and the immune system. Semin. Immunol. 34,52–60
(2017).
24. Fadeel, B. Hide and seek: nanomaterial interactions with the immune system.
Front. Immunol. 10, 133 (2019).
25. Deng,Z.J.,Liang,M.,Monteiro,M.,Toth,I.&Minchin,R.F.Nanoparticle-
induced unfolding of fibrinogen promotes Mac-1 receptor activation and
inflammation. Nat. Nanotechnol. 6,39–44 (2011).
26. Tenzer, S. et al. Rapid formation of plasma protein corona critically affects
nanoparticle pathophysiology. Nat. Nanotechnol. 8,772–781 (2013).
27. Vlasova, I. I. et al. Adsorbed plasma proteins modulate the effects of single-
walled carbon nanotubes on neutrophils in blood. Nanomedicine 12,
1615–1625 (2016).
28. Lu,N.,Sui,Y.,Tian,R.&Peng,Y.Y.Adsorptionofplasmaproteinsonsingle-
walled carbon nanotubes reduced cytotoxicity and modulated neutrophil
activation. Chem. Res. Toxicol. 31, 1061–1068 (2018).
29. Lara, S. et al. Identification of receptor binding to the biomolecular corona of
nanoparticles. ACS Nano. 11,1884–1893 (2017).
30. Salvati, A. et al. Transferrin-functionalized nanoparticles lose their targeting
capabilities when a biomolecule corona adsorbs on the surface. Nat.
Nanotechnol. 8,137–143 (2013).
31. Tonigold, M. et al. Pre-adsorption of antibodies enables targeting of
nanocarriers despite a biomolecular corona. Nat. Nanotechnol. 13,862–869
(2018).
32. Oh, J. Y. et al. Cloaking nanoparticles with protein corona shield for targeted
drug delivery. Nat. Commun. 9,1–9(2018).
33. Prapainop,K.,Witter,D.P.&Wentworth,P.Achemicalapproachforcell-
specific targeting of nanomaterials: small-molecule-initiated misfolding of
nanoparticle corona proteins. J. Am. Chem. Soc. 134,4100–4103 (2012).
34. Ezzat, K. et al. The viral protein corona directs viral pathogenesis and amyloid
aggregation. Nat. Commun. 10, 2331 (2019).
35. Théry, C., Ostrowski, M. & Segura, E. Membrane vesicles as conveyors of
immune responses. Nat. Rev. Immunol. 9,581–593 (2009).
36. van Niel, G., D’Angelo,G.&Raposo,G.Shedding light on the cell biology of
extracellular vesicles. Nat. Rev. Mol. Cell Biol. 19,213–228 (2018).
37. Genschmer, K. R. et al. Activated PMN exosomes: pathogenic entities causing
matrix destruction and disease in the lung. Cell 176,113–126 (2019).
38. Shvedova, A. A., Pietroiusti, A., Fadeel, B. & Kagan, V. E. Mechanisms of carbon
nanotube-induced toxicity: focus on oxidative stress. Toxicol. Appl. Pharmacol.
261,121–133 (2012).
39. Zhu, M. et al. Exosomes as extrapulmonary signaling conveyors for
nanoparticle-induced systemic immune activation. Small 8,404–412 (2012).
40. Cho,W.S.etal.Metaloxidenanoparticlesinduceuniqueinfammatory
footprints in the lung: important implications for nanoparticle testing.
Environ. Health Perspect. 118, 1699–1706 (2010).
41. Qiao, Y. et al. Identification of exosomal miRNAs in rats with pulmonary
neutrophilic inflammation induced by zinc oxide nanoparticles. Front. Physiol.
9,1–11 (2018).
42. Logozzi, M. et al. Human primary macrophages scavenge AuNPs and
eliminate it through exosomes. A natural shuttling for nanomaterials. Eur. J.
Pharm. Biopharm. 137,23–36 (2019).
43. Brinkmann,V.etal.Neutrophilextracellular traps kill bacteria. Science 303,
1532–1535 (2004).
44. Yousefi, S., Mihalache, C., Kozlowski, E., Schmid, I. & Simon, H. U. Viable
neutrophils release mitochondrial DNA to form neutrophil extracellular traps.
Cell Death Differ. 16, 1438–1444 (2009).
45. Lood, C. et al. Neutrophil extracellular traps enriched in oxidized mito-
chondrial DNA are interferogenic and contribute to lupus-like disease. Nat.
Med. 22,146–153 (2016).
46. Fuchs,T.A.etal.Novelcelldeathprogram leads to neutrophil extracellular
traps. J. Cell Biol. 176, 231–241 (2007).
47. Clark,S.R.etal.PlateletTLR4activates neutrophil extracellular traps to
ensnare bacteria in septic blood. Nat. Med. 13,463–469 (2007).
48. Yipp, B. G. et al. Infection-induced NETosis is a dynamic process involving
neutrophil multitasking in vivo.Nat. Med. 18,1386–1393 (2012).
49. Takei, H., Araki, A., Watanabe, H., Ichinose, A. & Sendo, F. Rapid killing of
human neutrophils by the potent activator phorbol 12-myristate 13-acetate
(PMA) accompanied by changes different from typical apoptosis or necrosis.
J. Leukoc. Biol. 59,229–240 (1996).
50. Fadeel,B.,Åhlin,A.,Henter,J.I.,Orrenius,S.&Hampton,M.B.Involvementof
caspases in neutrophil apoptosis: regulation by reactive oxygen species.
Blood 92,4808–4818 (1998).
51. Chen, K. W. et al. Noncanonical inflammasome signaling elicits gasdermin D-
dependent neutrophil extracellular traps. Sci. Immunol. 3, eaar6676 (2018).
52. Sollberger, G. et al. Gasdermin D plays a vital role in the generation of
neutrophil extracellular traps. Sci. Immunol. 3, eaar6689 (2018).
Keshavan et al. Cell Death and Disease (2019) 10:569 Page 9 of 11
Official journal of the Cell Death Differentiation Association
53. Schreiber, A. et al. Necroptosis controls NET generation and mediates
complement activation, endothelial damage, and autoimmune vasculitis.
Proc. Natl Acad. Sci. USA 114, E9618–E9625 (2017).
54. Kenny,E.F.etal.Diversestimuliengagedifferent neutrophil extracellular trap
pathways. Elife 6, e24437 (2017).
55. VanDerLinden,M.,Westerlaken,G.H.A.,VanDerVlist,M.,VanMontfrans,J.&
Meyaard, L. Differential signalling and kinetics of neutrophil extracellular trap
release revealed by quantitative live imaging. Sci. Rep. 7, 6529 (2017).
56. Urban, C. F., Reichard, U., Brinkmann, V. & Zychlinsky, A. Neutrophil extra-
cellular traps capture and kill Candida albicans and hyphal forms. Cell.
Microbiol. 8,668–676 (2006).
57. Papayannopoulos, V. Neutrophil extracellular traps in immunity and disease.
Nat. Rev. Immunol. 18,134–147 (2018).
58. Hakkim, A. et al. Impairment of neutrophil extracellular trap degradation is
associated with lupus nephritis. Proc. Natl Acad. Sci. USA 107, 9813–9818
(2010).
59. Fuchs, T. A. et al. Extracellular DNA traps promote thrombosis. Proc. Natl Acad.
Sci. USA 107, 15880–15885 (2010).
60. Jiménez-Alcázar, M. et al. Host DNases prevent vascular occlusion by neu-
trophil extracellular traps. Science 358,1202–1206 (2017).
61. Tillack, K., Breiden, P., Martin, R. & Sospedra, M. T lymphocyte priming by
neutrophil extracellular traps links innate and adaptive immune responses. J.
Immunol. 188, 3150–3159 (2012).
62. Stephen, J. et al. Neutrophil swarming and extracellular trap formation play a
significant role in alum adjuvant activity. npj Vaccines 2, 1 (2017).
63. Bartneck, M., Keul, H. A., Gabriele, Z. K. & Groll, J. Phagocytosis independent
extracellular nanoparticle clearance by human immune cells. Nano Lett. 10,
59–64 (2010).
64. Hwang, T. L., Aljuffali, I. A., Hung, C. F., Chen, C. H. & Fang, J. Y. The impact of
cationic solid lipid nanoparticles on human neutrophil activation and for-
mation of neutrophil extracellular traps (NETs). Chem. Biol. Interact. 235,
106–114 (2015).
65. Bilyy, R. et al. Inert coats of magnetic nanoparticles prevent formation of
occlusive intravascular co-aggregates with neutrophil extracellular traps.
Front. Immunol. 9, 2266 (2018).
66. Muñoz, L. E. et al. Nanoparticles size-dependently initiate self-limiting
NETosis-driven inflammation. Proc. Natl Acad. Sci. USA 113, E5856–E5865
(2016).
67. Moore, L. et al. Biocompatibility assessment of detonation nanodiamond in
non-human primates and rats using histological, hematologic, and urine
analysis. ACS Nano. 10, 7385–7400 (2016).
68. Mukherjee, S. P. et al. Graphene oxide elicits membrane lipid changes and
neutrophil extracellular trap formation. Chem 4, 334–358 (2018).
69. Neumann, A. et al. Lipid alterations in human blood-derived neutrophils lead
to formation of neutrophil extracellular traps. Eur. J. Cell Biol. 93,347–354
(2014).
70. Douda,D.N.,Khan,M.A.,Grasemann,H.&Palaniyar,N.SK3channeland
mitochondrial ROS mediate NADPH oxidase-independent NETosis induced
by calcium influx. Proc. Natl Acad. Sci. USA 112, 2817–2822 (2015).
71. Mukherjee, S. P. et al. Graphene oxide is degraded by neutrophils and the
degradation products are non-genotoxic. Nanoscale 10, 1180–1188 (2018).
72. Mukherjee,S.P.,Bottini,M.&Fadeel,B.Grapheneandtheimmunesystem:a
romance of many dimensions. Front. Immunol. 8,1–11 (2017).
73. Klebanoff,S.J.,Kettle,A.J.,Rosen,H.,Winterbourn,C.C.&Nauseef,W.M.
Myeloperoxidase: a front-line defender against phagocytosed microorgan-
isms. J. Leukoc. Biol. 93, 185–198 (2013).
74. Shvedova, A. A. et al. Impaired clearance and enhanced pulmonary
inflammatory/fibrotic response to carbon nanotubes in myeloperoxidase-
deficient mice. PLoS ONE 7, e30923 (2012).
75. Kagan,V.E.etal.Carbonnanotubesdegradedbyneutrophilmyeloperox-
idase induce less pulmonary inflammation. Nat. Nanotechnol. 5,354–359
(2010).
76. Bhattacharya, K. et al. Enzymatic ‘stripping’and degradation of PEGylated
carbon nanotubes. Nanoscale 6, 14686–14690 (2014).
77. Andón, F. T. et al. Biodegradation of single-walled carbon nanotubes by
eosinophil peroxidase. Small 9, 2720–2729 (2013).
78. Farrera, C. et al. Extracellular entrapment and degradation of single-walled
carbon nanotubes. Nanoscale 6,6974–6983 (2014).
79. Kurapati, R., et al. Degradation of single-layer and few-layer graphene by
neutrophil myeloperoxidase. Angew. Chemie Int. Ed. Engl.57, 11722–11727
(2018).
80. Sydlik, S. A., Jhunjhunwala, S., Webber, M. J., Anderson, D. G. & Langer, R. In
vivo compatibility of graphene oxide with differing oxidation states. ACS
Nano. 9,3866–3874 (2015).
81. Girish, C. M., Sasidharan, A., Gowd, G. S., Nair, S. & Koyakutty, M. Confocal
Raman imaging study showing macrophage mediated biodegradation of
graphene in vivo.Adv. Healthc. Mater. 2,1489–1500 (2013).
82. Fadeel, B. et al. Safety assessment of graphene-based materials: focus on
human health and the environment. ACS Nano. 12,10582–10620 (2018).
83. Martinon, F., Burns, K. & Tschopp, J. The inflammasome: a molecular platform
triggering activation of inflammatory caspases and processing of proIL-β.
Mol. Cell. 10,417–426 (2002).
84. Broz,P.&Dixit,V.M.Inflammasomes: mechanism of assembly, regulation
and signalling. Nat. Rev. Immunol. 16,407–420 (2016).
85. Mangan, M. S. J. et al. Targeting the NLRP3 inflammasome in inflammatory
diseases. Nat. Rev. Drug Discov. 17,588–606 (2018).
86. Martinon, F., Pétrilli, V., Mayor, A., Tardivel, A. & Tschopp, J. Gout-associated
uric acid crystals activate the NALP3 inflammasome. Nature 440,237–241
(2006).
87. Dostert, C. et al. Innate immune activation through Nalp3 inflammasome
sensing of asbestos and silica. Science 320,674–677 (2008).
88. Cassel, S. L. et al. The Nalp3 inflammasome is essential for the development
of silicosis. Proc. Natl Acad. Sci. USA 105,9035–9040 (2008).
89. Hornung, V. et al. Silica crystals and aluminum salts activate the NALP3
inflammasome through phagosomal destabilization. Nat. Immunol. 9,
847–856 (2008).
90. Sun,B.,Wang,X.,Ji,Z.,Li,R.&Xia,T.NLRP3inflammasome activation induced
by engineered nanomaterials. Small 9,1595–1607 (2013).
91. Palomäki, J. et al. Long, needle-like carbon nanotubes and asbestos activate
the NLRP3 inflammasome through a similar mechanism. ACS Nano. 5,
6861–6870 (2011).
92. Mukherjee,S.P.,Kostarelos,K.&Fadeel,B.Cytokineprofiling of primary
human macrophages exposed to endotoxin-free graphene oxide: size-
independent NLRP3 inflammasome activation. Adv. Healthc. Mater.7,
1700815 (2018).
93. Andón, F. T. et al. Hollow carbon spheres trigger inflammasome-dependent
IL-1βsecretion in macrophages. Carbon 113, 243–251 (2017).
94. Chen, K. W. et al. The neutrophil NLRC4 inflammasome selectively promotes
IL-1βmaturation without pyroptosis during acute Salmonella challenge. Cell
Rep. 8,570–582 (2014).
95. Kambara,H.etal.GasderminDexertsanti-inflammatory effects by pro-
moting neutrophil death. Cell Rep. 22,2924–2936 (2018).
96. Netea,M.G.,vandeVeerdonk,F.L.,vanderMeer,J.W.M.,Dinarello,C.A.&
Joosten, L. A. B. Inflammasome-independent regulation of IL-1-family cyto-
kines. Annu. Rev. Immunol. 33,49–77 (2015).
97. Bakele, M. et al. Localization and functionality of the inflammasome in
neutrophils. J. Biol. Chem. 289,5320–5329 (2014).
98. Masumoto, J. et al. ASC, a novel 22-kDa protein, aggregates during apoptosis
of human promyelocytic leukemia HL-60 cells. J. Biol. Chem. 274,
33835–33838 (1999).
99. Franklin, B. S. et al. The adaptor ASC has extracellular and ‘prionoid’activities
that propagate inflammation. Nat. Immunol. 15, 727–737 (2014).
100. Baroja-Mazo, A. et al. The NLRP3 inflammasome is released as a particulate
danger signal that amplifies the inflammatory response. Nat. Immunol. 15,
738–748 (2014).
101. Rettig, L. et al. Particle size and activation threshold: a new dimension of
danger signaling. Blood 115,4533–4541 (2010).
102. Miyake, K. Innate immune sensing of pathogens and danger signals by cell
surface Toll-like receptors. Semin. Immunol. 19,3–10 (2007).
103. Litvack, M. L. & Palaniyar, N. Soluble innate immune pattern-recognition
proteins for clearing dying cells and cellular components: implications on
exacerbating or resolving inflammation. Innate Immun. 16,191–200 (2010).
104. Arai, S. & Miyazaki, T. A scavenging system against internal pathogens pro-
moted by the circulating protein apoptosis inhibitor of macrophage (AIM).
Semin. Immunopathol. 40, 567–575 (2018).
105. Farrera,C.&Fadeel,B.Ittakestwototango:understandingtheinteractions
between engineered nanomaterials and the immune system. Eur. J. Pharm.
Biopharm. 95,3–12 (2015).
106. Silva, A. L. et al. Nanoparticle impact on innate immune cell pattern-
recognition receptors and inflammasomes activation. Semin. Immunol. 34,
3–24 (2017).
Keshavan et al. Cell Death and Disease (2019) 10:569 Page 10 of 11
Official journal of the Cell Death Differentiation Association
107. Fadeel, B. Clear and present danger? Engineered nanoparticles and the
immune system. Swiss Med. Wkly. 142, w13609 (2012).
108. Pradeu, T. & Cooper, E. L. The danger theory: 20 years later. Front. Immunol. 3,
287–287 (2011).
109. Gallo,P.M.&Gallucci,S.Thedendriticcell response to classic, emerging, and
homeostatic danger signals. Implications for autoimmunity. Front. Immunol.
4, 138 (2013).
110. Shirasuna, K., Karasawa, T. & Takahashi, M. Exogenous nanoparticles and
endogenous crystalline molecules as danger signals for the NLRP3 inflam-
masomes. J. Cell. Physiol. 234, 5436–5450 (2019).
111. Vita, A. A., Royse, E. A. & Pullen, N. A. Nanoparticles and danger signals: oral
delivery vehicles as potential disruptors of intestinal barrier homeostasis. J.
Leukoc. Biol.106,95–103 (2019).
112. Nel, A. E. et al. Understanding biophysicochemical interactions at the nano-
bio interface. Nat. Mater. 8,543–557 (2009).
113. Gallud, A. & Fadeel, B. Keeping it small: towards a molecular definition of
nanotoxicology. Eur. J. Nanomed. 7,143–151 (2015).
114. Xiu, P., Zhou, R., Zhao, Y., Zuo, G. & Kang, S. Interactions between proteins and
carbon-based nanoparticles: exploring the origin of nanotoxicity at the
molecular level. Small 9,1546–1556 (2012).
115. Yanamala, N., Kagan, V. E. & Shvedova, A. A. Molecular modeling in structural
nano-toxicology: interactions of nano-particles with nano-machinery of cells.
Adv. Drug Deliv. Rev. 65, 2070–2077 (2013).
116. He, B. et al. Single-walled carbon-nanohorns improve biocompatibility over
nanotubes by triggering less protein-initiated pyroptosis and apoptosis in
macrophages. Nat. Commun. 9, 2393 (2018).
117. Mukherjee, S. P. et al. Macrophage sensing of single-walled carbon nano-
tubes via Toll-like receptors. Sci. Rep. 8,1–17 (2018).
118. Turabekova, M. et al. Immunotoxicity of nanoparticles: a computational study
suggests that CNTs and C
60
fullerenes might be recognized as pathogens by
Toll-like receptors. Nanoscale 6,3488–3495 (2014).
119. Liang, H. et al. Cationic nanoparticle as an inhibitor of cell-free DNA-induced
inflammation. Nat. Commun. 9, 4291 (2018).
120. Lee, J. et al. Nucleic acid-binding polymers as anti-inflammatory agents. Proc.
Natl Acad. Sci. USA 108, 14055–14060 (2011).
121. Holl, E. K. et al. Scavenging nucleic acid debris to combat autoimmunity and
infectious disease. Proc. Natl Acad. Sci. USA 113,9728–9733 (2016).
122. Hubbell, J. A., Thomas, S. N. & Swartz, M. A. Materials engineering for
immunomodulation. Nature 462,449–460 (2009).
123. Getts, D. R., Shea, L. D., Miller, S. D. & King, N. J. C. Harnessing nanoparticles for
immune modulation. Trends Immunol. 36,419–427 (2015).
124. Vlasova, I. I. et al. Enzymatic oxidative biodegradation of nanoparticles:
mechanisms, significance and applications. Toxicol. Appl. Pharmacol. 299,
58–69 (2016).
125. Kagan, V. E. et al. Lung macrophages ‘digest’carbon nanotubes using a
superoxide/peroxynitrite oxidative pathway. ACS Nano. 8,5610–5621 (2014).
126. Liu, L. et al. Structural basis of Toll-like receptor 3 signaling with double-
stranded RNA. Science 320,379–381 (2008).
127. Mu, Q. et al. Protein binding by functionalized multiwalled carbon nanotubes
is governed by the surface chemistry of both parties and the nanotube
diameter. J. Phys. Chem. C 112,3300–3307 (2008).
128. Galluzzi, L. et al. Molecular mechanisms of cell death: recommendations of
the Nomenclature Committee on Cell Death 2018. Cell Death Differ. 25,
486–541 (2018).
129. Clarke,P.G.&Clarke,S.Nineteenthcenturyresearchoncelldeath.Exp. Oncol.
3, 139–145 (2012).
130. Andón, F. T. & Fadeel, B. Programmed cell death: molecular mechanisms and
implications for safety assessment of nanomaterials. Acc. Chem. Res. 46,
733–142 (2013).
131. Hussain, S. et al. Carbon black and titanium dioxide nanoparticles elicit distinct
apoptotic pathways in bronchial epithelial cells. Part. Fibre Toxicol. 7, 10 (2010).
132. Luanpitpong, S., Wang, L., Castranova, V. & Rojanasakul, Y. Induction of stem-like
cells with malignant properties by chronic exposure of human lung epithelial
cells to single-walled carbon nanotubes. Part. Fibre Toxicol. 11, 22 (2014).
133. Shi,J.,Gao,W.&Shao,F.Pyroptosis:gasdermin-mediatedprogrammed
necrotic cell death. Trends Biochem. Sci. 42,245–254 (2017).
134. Mirshafiee,V.etal.Toxicologicalprofiling of metal oxide nanoparticles in liver
context reveals pyroptosis in Kupffer cells and macrophages versus apoptosis
in hepatocytes. ACS Nano. 12,3836–3852 (2018).
135. Wang, X., Yousefi, S. & Simon, H. U. Necroptosis and neutrophil-associated
disorders. Cell Death Dis. 9, 111 (2018).
136. Stockwell, B. R. et al. Ferroptosis: a regulated cell death nexus linking
metabolism, redox biology, and disease. Cell 171,273–285 (2017).
137. Szwed, M., et al. Small variations in nanoparticle structure dictate differential
cellular stress responses and mode of cell death. Nanotoxicology 13, 761–782
(2019).
138. Zhang, J. et al. Zinc oxide nanoparticles harness autophagy to induce cell
death in lung epithelial cells. Cell Death Dis. 8, e2954 (2017).
Keshavan et al. Cell Death and Disease (2019) 10:569 Page 11 of 11
Official journal of the Cell Death Differentiation Association
