Reviews

The role of neutrophil extracellular

traps in rheumatic diseases
Falko Apel, Arturo Zychlinsky* and Elaine F. Kenny*
Abstract | Rheumatic diseases are a collection of disorders defined by the presence of
inflammation and destruction of joints and internal organs. A common feature of these diseases
is the presence of autoantibodies targeting molecules commonly expressed in neutrophils.
These preformed mediators are released by neutrophils but not by other immune cells such as
macrophages. Neutrophils, major players in the host innate immune response, initiate a cell death
mechanism termed neutrophil extracellular trap (NET) formation as a way to ensnare pathogens.
NETs are also a source of released self-​molecules found in rheumatic diseases. Subsequently ,
research on the role of NETs in the onset, progression and resolution of inflammation in rheumatic
diseases has intensified. This Review has two aims. First, it aims to highlight the mechanisms
required for the generation of NETs, the research landscape of which is rapidly changing. Second,
it aims to discuss the role of neutrophils and NETs in systemic lupus erythematosus, vasculitis
(specifically anti-​neutrophil cytoplasmic autoantibody-associated vasculitis), rheumatoid arthritis
and gout. Our goal is to clarify the field of NET research in rheumatic diseases in the hope of
improving the therapeutic approaches utilized for these diseases.

Department of Cellular
Microbiology, Max Planck
Institute for Infection Biology,
Berlin, Germany.
*e-​mail: zychlinsky@
mpiib-​berlin.mpg.de;
kenny@mpiib-​berlin.mpg.de
https://doi.org/10.1038/
s41584-018-0039-z
Rheumatic diseases are a set of inflammatory disor-
ders that affect tissues and joints1. A hallmark of these
diseases is the generation of antibodies that recognize
self-​molecules usually residing within cells. These
autoantibodies are produced as a result of a break-
down in self-​tolerance, and they cause inflammation
and tissue destruction in affected areas of the body2.
Much research has been geared towards identifying the
source of the self-​molecules against which these auto­
antibodies are generated. The discovery that neutrophils
undergo NETosis3 (a defence and/or cell death mecha-
nism by which neutrophils release their contents) led
to the identification of these innate immune cells as a
potential major source of self-​molecule release in rheu-
matic disease development4. As research into both neu-
trophil signalling mechanisms, including NETosis, and
the development and progression of rheumatic disease
is an ever changing landscape, we hope to clarify the
current state of the field and aid in the advancement of
our knowledge of and ability to diagnose and treat these
diseases.
Neutrophils
Neutrophils are the most abundant white blood cell in
the circulation. They develop in the bone marrow and
once released into the circulation they patrol for patho-
gens and are considered the host’s first line of immune
defence5. These myeloid cells have a richly granular
cytoplasm and a lobulated nucleus; hence, along with
other granulocytic immune cells such as eosinophils
and basophils, they are often defined as granulocytes.
Neutrophil development is well characterized (reviewed
elsewhere6). Owing to the unusual form of their nucleus
they are also known as polymorphonuclear cells but we
will use the term neutrophil throughout. Neutrophils
have a particularly short lifespan, which is a surprising
and unexplained trait; this short lifespan and their gen-
eral abundance mean that a healthy adult is estimated to
produce one billion neutrophils per day7,8.
Neutrophils produce cytokines (for example, IL-8)
that recruit more neutrophils or further inflammatory
mediators, such as TNF9. They also secrete cytokines
that dampen the inflammatory response, presumably
when the microbial threat has abated9. Neutrophils
induce cytokine production to a lower level than other
myeloid cells, such as macrophages or dendritic cells;
however, as neutrophils are so abundant in inflamma-
tory sites, individual small contributions are potentially
amplified9.
Neutrophils receive pro-​inflammatory instructions
from cytokines and chemokines and rapidly leave the
circulation and arrive at inflammatory sites by a care-
fully orchestrated process. Upon arrival, these cells
deploy their effector functions to kill microorganisms
and signal to the immune system to promote or dimin-
ish the influx of cells to the site6. At the inflammatory

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Reviews
Key points
• Molecules released during neutrophil extracellular trap (NET) formation can often
become autoantigens in systemic lupus erythematosus, small vessel vasculitis,
rheumatoid arthritis and gout.
• Damaged tissues and organs in these rheumatic diseases are prone to containing
NET-​derived material.
• Neutrophils from patients with some rheumatic diseases spontaneously undergo
NETosis in vitro, reflecting the pre-​activation of neutrophils during chronic
inflammation.
• To date, whether NET formation is deleterious to progression of all rheumatic diseases
is unclear.
site, neutrophils are further activated and can degran-
ulate, phagocytose or produce cytokines. These cells
contain several types of granules that store defined and
overlapping cargoes of enzymes and antimicrobial pep-
tides. Granule fusion with the plasma membrane exter-
nalizes the cargo in a process termed degranulation.
This process is well characterized in vitro, although
confirming it in vivo is challenging. Neutrophils are
extraordinarily efficient phagocytes; they recognize
microorganisms directly or through receptors that rec-
ognize complement or antibodies and engulf particles
by classical phagocytosis into vacuoles. Granules then
fuse to these vacuoles and unload their contents. The
NADPH oxidase complex is also recruited to the surface
of the vacuole, where it produces reactive oxygen spe-
cies (ROS), for example, superoxide. The combination
of ROS, acidification and the many enzymes and anti-
microbial peptides that come from the granule makes
the phagolysosome an inhospitable environment for
microorganisms10.
Neutrophil extracellular traps
Another aspect to the defensive arsenal of neutrophils is
neutrophil extracellular trap (NET) formation3 (Fig. 1).
NETs are defined as extracellular structures that contain
DNA, histones and neutrophil proteins such as myelo­
peroxidase, neutrophil elastase or calgranulin. During
NET formation, neutrophils undergo chromatin remod-
elling via processing of histones by proteases, and the
remodelled chromatin subsequently binds antimicrobial
proteins and enzymes. The modified chromatin is then
released through a process termed NETosis. NETs occur
in inflammatory sites and probably have a short lifespan
owing to degradation by local DNases. Hence, detecting
and quantifying NETs is tricky (Box 1).
Several pathways result in NET release11. Classically,
NETosis requires proteases and the generation of ROS.
A complex of proteins, called the ‘azurosome’, senses
hydrogen peroxide through myeloperoxidase, which
converts this ROS into halic acids12. Myeloperoxidase
activation leads to the permeabilization of neutro-
phil granules and the dissociation of the azurosome.
This process results in the release of serine proteases,
including neutrophil elastase, which translocate to the
nucleus where they clip histones13. This proteolytic activ-
ity decondenses the chromatin, which is necessary for
NET formation. Neutrophil elastase, cathepsin G and
proteinase 3 (PR3) belong to the same family of serine
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proteases and recognize a variety of substrates. After
initial activation of the neutrophils, cyclin-​dependent
kinase 6 (CDK6) is also activated, probably permitting
the vesiculation of the nuclear membrane14. Importantly,
NETs expose several enzymes such as myeloperoxidase,
neutrophil elastase, cathepsin G, PR3 and LL37 as well as
histones to the extracellular space. These, and other pro-
teins, enable NETs to fulfil their function in microbial
defence, coagulation and immune activation. ROS are
also released during NETosis.
Not all NET inducers utilize this classical pathway to
NETosis. For example, NET production in response
to the calcium ionophore A23187 and nigericin lacks
the requirements of classical NET formation (described
elsewhere11).
Citrullination of histones via protein arginine deimi-
nase (PAD) enzymes occurs during some forms of
NETosis. However, the role of citrullination is unclear
in NET production. Human neutrophils do not require
PADs to generate NETs, but proteins in them are often
citrullinated in response to many stimuli11; however,
further work is needed to definitively identify the role
PADs have in NETosis. These citrullinated proteins are
common in rheumatic disease, so the source of these
modified proteins is of great therapeutic interest11.
Much work on citrullination and the role of PAD4
has used the PAD4-deficient mouse; however, major
differences seem to exist in the way human and mouse
neutrophils induce NETosis7. For example, although
neutrophil elastase is required for NET formation in
humans, mice deficient in neutrophil elastase can make
NETs15, but mice in which both neutrophil elastase
and PR3 are deleted can not14. Another major differ-
ence between human and mouse neutrophils is the
total number in circulation, with neutrophils making
up 70% of white blood cells in the human circulation
compared with only 30% in mice7. Differences also exist
in the binding abilities of the Fc receptors expressed on
human and mouse neutrophils16 and in their ability to
react to infection. All of these factors could contribute
to the difference seen in NETosis induction between
the species.
Neutrophils are deployed to inflammatory sites
where they release molecules that are toxic to micro-
organisms but also to the host17. Neutrophils are cul-
pable for inducing collateral damage in the process of
antimicrobial defence. It is well documented that neu-
trophil proteases and ROS can destroy healthy tissue18.
Interestingly, extracellular histones are potent antimicro-
bials, as described more than 50 years ago by Hirsch19,
and can also kill host cells, and they are implicated in
many pathologies. Furthermore, the externalization
of neutrophil proteins as well as histones and DNA, as
occurs in NETosis, provides a source for antigen pres-
entation in the development of different rheumatic dis-
eases. NETs are also immune-​stimulants that exacerbate
inflammation and activate the acquired immune system.
Furthermore, the role of NETs in thrombus formation, a
complication of rheumatic diseases20, adds another link
between NETosis and autoimmunity.
In the following sections we focus on the role of NETs
in the development and progression of systemic lupus

C D
Fig. 1 | Neutrophil extracellular trap induction in human neutrophils. a | Unstimulated
primary human neutrophils. b | Primary human neutrophils treated with 50 nM phorbol
myristate acetate for 3 hours. Cells were then fixed and stained for neutrophil elastase
(green) and chromatin (red) with antibodies and for DNA (blue) with Hoechst. Scale bar is
20 µm. Image courtesy of V. Brinkmann, Max Planck Institute for Infection Biology , Germany.
erythematosus (SLE), vasculitis, rheumatoid arthritis
(RA) and gout.
Systemic lupus erythematosus
SLE is a multifactorial autoimmune disease affecting
different organs, and is most prevalent in women of
childbearing age21. Its pathogenesis combines dysregu-
lated innate and adaptive immune responses, culminat-
ing in a lack of tolerance to self-​antigens. The molecular
hallmarks of SLE are the formation of autoantibodies
(commonly antinuclear antibodies) and upregulation
of type I interferon-​regulated genes. Patients with SLE
are at increased risk of developing atherosclerosis and
cardiovascular symptoms compared with the general
population22,23. Another major complication is lupus
nephritis (LN), which can lead to symptoms ranging
from mild proteinuria to acute kidney failure24.
Dating back to the 1940s, it was observed that neu-
trophils and macrophages phagocytose nuclear mate-
rial opsonized by autoantibodies. This so-​called LE
(lupus erythematosus) cell phenomenon was used to
diagnose SLE25 and might reflect an increase in NET
formation in these patients or reduced clearance of
nuclear material. Although 40–60% of patients with
SLE are neutropenic26, increasing evidence shows that
neutrophils and NETs contribute to SLE pathogenesis.
NETs are detectable in the skin and in the glomeruli
of patients with SLE27–29. In vitro experiments suggest
that SLE neutrophils make NETs spontaneously28,30 and
upon stimulation with antibodies against LL37 (ref.30)
or ribonuclear particles31.
In healthy individuals, phagocytes clear apoptotic neu-
trophils32 and NETs33. This clearance is immunologically
silent and without the production of pro-​inflammatory
cytokines33. However, in SLE, an increased number of
apoptotic neutrophils and NETs is observed, while their
clearance by macrophages is impaired31,34,35. This lack
of clearance provides antigens for autoantibodies recog-
nizing neutrophil proteins36. How NET material reaches
the germinal centres to contribute to antibody-​dependent
autoimmunity is not fully understood. One potential
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mechanism might be the opsonization of NETs by
complement factors37 and their further complement-​
receptor-mediated uptake and presentation by follicular
dendritic cells to B cells38. Neutrophils themselves might
serve as antigen-​presenting cells; in mice, neutrophils
have been shown to transport antigens to the sites of
T cell activation39,40 and function as antigen-​presenting
cells to T cells41. Although none of the above-​mentioned
mechanisms have been described for SLE specifically,
they are potential explanations for how NETs could
contribute to antibody-​dependent autoimmunity.
In patients with SLE, autoantibodies are directed
to nuclear antigens, such as double-stranded DNA
(dsDNA) and chromatin, and to proteins such as his-
tones, myeloperoxidase, PR3 and neutrophil elastase42–47;
all these antigens are NET components. Interestingly,
anti-​NET autoantibodies induce NET formation in
IFNα-​primed neutrophils30,31,48 and can perpetuate
immune activation. Furthermore, immune complexes,
probably formed between autoantibodies and NET com-
ponents, directly contribute to the development of LN.
The complexes deposit along the glomerular capillary of
the kidney, inducing necrosis and kidney destruction, as
seen in vasculitis49.
DNase1. Normally, NETs are degraded by endo­
nucleases27 and DNase1-like proteins in the circula-
tion. DNase1 activity negatively correlates with disease
activity and the concentration of serum markers such as
antinuclear antibodies, anti-​dsDNA antibodies and anti-​
nucleosome antibodies in SLE50. Missense mutations in
the genes encoding DNase1 (ref.51) and DNase1L3 (also
known as DNase gamma52) cause familial forms of SLE,
whereas mutations in DNases lead to lupus-like disease
in mice53,54.
Additional DNase1-related mechanisms reduce NET
degradation in SLE. NETs contain oxidized DNA in the
form of 8-hydroxyguanosine, which protects nucleic
acids from degradation, increasing exposure of the NETs
to autoantigens and thus triggering an autoimmune
response55. NET-​binding proteins, such as antibodies
or complement component C1q, protect the NETs from
degradation, possibly by inhibiting DNase1 (refs27,37).
Additionally, sera from patients with SLE contain pro-
teins that inhibit DNase1, including globular actin
(G-​ a ctin)56 and anti-​DNase1 antibodies57,58. Taken
together, the evidence suggests that in SLE NET degra-
dation is hampered, leading to prolonged exposure of
autoantigens to the immune system.
Type I interferons. Type I interferons have an important
role in SLE pathogenesis59–62. In vitro, NETs induce pro-
duction of type I interferons in plasmacytoid dendritic
cells. The NETs are endocytosed with the help of LL37
and high mobility group B1 (HMGB1), and activate
Toll-​like receptor 9 (TLR9)31. In addition, both oxidized
mitochondrial DNA and genomic NET DNA stimulate
interferons31,48,63,64. Interestingly, TLR9-deficient mice, in
an MRL.FASlpr mouse model of SLE, produce increased
amounts of autoantibodies and display a severe renal
phenotype65,66. NETs and their components also induce
IL-1β production by activating the inflammasome67.
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Box 1 | Looking for neutrophil extracellular traps
The detection of neutrophil extracellular traps (NETs) in vivo at sites of infection or injury
is aided by DNA dyes143,144. This technique is, however, restricted to experimental animals
and is limited by a lack of markers for other NET components. Hence, animal models
in which other NET markers can be observed are needed to aid our understanding
of NETosis in vivo.
NETs can be readily detected in tissue sections of both experimental animals and
pathological samples from patients. Indeed, standard paraffin sections, commonly used
in clinical pathology laboratories, can be used to detect NETs by immunofluorescence.
Standard technologies combining a DNA dye and commercially available antibodies
that detect histones and neutrophil proteins (including neutrophil elastase and
myeloperoxidase) are useful for detecting the presence of NETs145. Furthermore,
automatic microscopy methods can be used to quantify the juxtaposition of the three
elements of NETs (DNA, histones and neutrophil proteins) and determine the level of
NET formation in a specific sample.
NETs can also be detected in fluids such as serum or sputum146. Different techniques
can be used to achieve this detection, many of which are quantitative. A useful tool that
can be implemented in most laboratories is a ‘sandwich’ enzyme-​linked immunosorbent
assay. An antibody against histones is used to capture NETs, and a second antibody,
which detects a neutrophil protein, is used to identify the presence of NETs in the
sample. Given that only NETs contain both histones and neutrophil proteins, specific
detection of NETs is guaranteed147. The automation capacity and precision of this
approach could be exploited in the clinic to determine NET formation in samples
from patients.
Low-​density granulocytes. Interestingly, most patients
with SLE present a subset of neutrophil-​like cells termed
low-​density granulocytes (LDGs)68 that separate with
the peripheral blood mononuclear cell fraction in a
density gradient69,70. These cells serve as a marker of
juvenile SLE71, and their occurrence correlates with
skin involvement and vasculitis in patients with SLE68.
LDGs produce increased amounts of IL-6, IL-8, TNF
and type I interferons68 and spontaneously make NETs,
which damage the endothelium28. Although the origin
and maturity state of these cells require further investi-
gation, these findings suggest that LDGs are important
contributors to the pathogenesis of SLE.
Mouse studies. Further evidence for the role of NETs in
SLE progression also comes from mouse studies. DNase1
injection ameliorates disease severity in the NZBW
mouse model of SLE72 but not in human patients73. We
suggest that this discrepancy results from the timing of
the initiation of treatment or the presence of DNase1
inhibitors in humans. Mice deficient in NADPH oxidase 2
(NOX2) present with increased autoantibody produc-
tion and a severe renal phenotype74–76. Furthermore,
NOX2 inhibitors mitigate pristine-​induced lupus, a dis-
ease model in which LDGs are also observed77. A poten-
tial explanation for these seemingly paradoxical results
might be either that autoimmune-​related NETs are pro-
duced independently of NOX2 or that the blockage of
ROS-​dependent NET formation results in another form
of cell death, providing the required antigens.
The effect of PAD4 inhibition on SLE progression
in mouse models is still under debate. Although two
studies78,79 reported an ameliorated disease outcome in
Cl-​amidine (a PAD inhibitor)-treated lupus-​prone mice,
a study combining Cl-​amidine treatment and genetic
depletion of PAD4 in MRL.Faslpr lupus-​prone mice did
not observe an effect80. This discrepancy might reflect
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different treatment regimens, and further studies are
needed to achieve a conclusive result.
In summary, the ineffective clearance of NETs in
patients with SLE exposes self-​molecules to the immune
system and contributes to the development of autoan-
tibodies and pro-​inflammatory cytokines, driving the
pathogenesis of SLE (Fig. 2a).
Vasculitis
The term vasculitis is used for a group of rare chronic
diseases that present with inflammation of blood
vessels81, which is sometimes accompanied by necro­
tizing cell death. Although the direct cause of these
diseases is unclear, the presence of autoantibodies82 and
pro-​inflammatory events (for example, infections83) have
an important role. A proportion of these vasculitides are
termed small vessel vasculitis, in which neutrophils have
a detrimental role and damage organs84,85.
Small vessel vasculitis is a relapsing-​remitting
chronic autoimmune disease characterized by dam-
age to small blood vessels. A portion of patients with
small vessel vasculitis present with anti-​neutrophil
cytoplasmic auto­antibody (ANCA)-associated vascu-
litis (AAV). More than 90% of these patients produce
autoantibodies against proteins located in the cytoplasm
of neutrophils85. These ANCAs are mainly generated
against PR3 and myeloperoxidase86–88. AAV can be
further divided into granulomatosis with polyangiitis,
microscopic poly­angiitis and eosinophilic granulomato-
sis with polyangiitis89. In these diseases, ANCA damage
is present in small-​sized and medium-​sized blood vessels
and other tissues including the lung, upper respiratory
tract and kidney.
Stimulation of neutrophils with ANCAs induces
ROS production and degranulation90, and exposure of
neutrophils primed with TNF to ANCAs stimulates
NETosis91. Patients with AAV with anti-​myeloperoxidase
antibodies in the circulation also have evidence of NETs
in their glomeruli91. Neutrophils isolated from patients
with AAV produce NETs spontaneously92. This process,
in turn, increases the levels of NET products in the
circulation92. Indeed, dead and dying neutrophils on
the wall of damaged small blood vessels are a histological
marker of this disease85.
In 2010, two whole-​genome gene expression profiling
studies identified the presence of a granulocyte signature
in blood from patients with AAV93,94. Subsequent work
confirmed the source of this signature to be the presence
of LDGs (a similar finding to that in patients with SLE95).
These cells spontaneously produce NETs containing
PR3 and myeloperoxidase. The presence of LDGs in the
blood of patients with AAV is associated with increased
disease activity and a decreased response to treatment
with rituximab95.
Mouse models of vasculitis and kidney damage sup-
port the idea that preventing NETosis or neutralizing
the molecules released from NETs is beneficial in treat-
ing this disease. Co-​incubation of pro-​inflammatory
subcutaneously elicited neutrophils with myeloid
dendritic cells (mDCs) resulted in the uptake of PR3
and myeloperoxidase into the mDCs; immunization
of mice with these mDCs resulted in the generation of
 Reviews

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Fig. 2 | The role of neutrophil extracellular traps in rheumatic diseases. Neutrophil extracellular trap (NET)
components are detected in the circulation and joints of patients with rheumatic diseases. a | In systemic lupus
erythematosus (SLE), DNase1 fails to degrade NETs spontaneously produced by low-​density granulocytes (LDGs).
Increased exposure induces autoantibody production by autoreactive B cells. Autoantibodies form immune complexes
with their respective antigen, which accumulate in the glomeruli of the kidney , leading to kidney destruction. Furthermore,
immune complexes activate plasmacytoid dendritic cells (pDCs), potentially via Toll-​like receptor 9 (TLR9), to produce
type I interferons and drive an inflammatory feedforward loop, causing more NET formation. b | Patients with vasculitis
present with autoantibodies against NET proteins, and NETs are detected in kidney lesions. c | In rheumatoid arthritis (RA),
citrullinated NET proteins are recognized by autoantibodies, which can be utilized for diagnosis. Neutrophils are also
known to infiltrate into the affected joint. d | In gout, NETs are also found in the affected joint, where they may aggregate
and degrade cytokines. MPO, myeloperoxidase; MSU, monosodium urate, NE, neutrophil elastase.

ANCAs, anti-​dsDNA antibodies and renal damage96.
This finding also occurred in biopsy samples from skin
lesions of patients with microscopic polyangiitis96. In
another mouse model, glomerulonephritis (a common
feature of AAV) was reduced by blocking extracellular
histones, again suggesting the importance of NET com-
ponent release in disease development97. Finally, a PAD
inhibitor reduced the titre of anti-myeloperoxidase anti-
bodies in a mouse model of myeloperoxidase-ANCA-
associated vasculitis98. Further evidence for the detri-
mental role of NETs in AAV comes from a study99 in
which a mouse deficient in both DNase1 and DNase3
(also known as TREX1) had vascular occlusion owing
to the accumulation of NET components. In conclusion,
the role of NETs in AAV is detrimental, as diagnosis is
based on the presence of ANCAs, and NETs provide the
source of these antigens (Fig. 2b).
Rheumatoid arthritis
RA is a chronic immune-​mediated systemic disease
with both genetic and environmental contributing fac-
tors. RA is characterized by synovial joint inflammation,
bone destruction and the presence of autoantibodies.
Onset of RA can be separated into two stages. First,
there is a preclinical asymptomatic stage that can last
for several years. During this stage, biomarkers, such as

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Reviews
autoantibodies against rheumatoid factor (RF, an anti-
body targeting the Fc chain of immunoglobulin G (IgG)
molecules) and citrullinated proteins, increase in circu-
lation. In the second stage, clinical symptoms become
evident100–103.
Neutrophils are implicated in RA progression and are
the most abundant cells in the synovial fluid, where they
are longer lived than normal104,105. Ex vivo, neutrophils
from patients with RA are pre-​activated by immune
complexes such as RF, resulting in excessive ROS release,
degranulation and NETosis106,107. Thus, the activation of
neutrophils in the synovium is linked to the character-
istic inflammation and damage in the joint. Neutrophil
granular proteins such as myeloperoxidase, neutrophil
elastase and cathepsin G are abundant in the synovial
fluid, probably because of excessive NETosis, and these
proteins cause damage to the joint108.
As anti-​citrullinated protein antibodies (ACPAs) are
a diagnostic hallmark of RA, the source of the modi-
fied self-​proteins that serve as autoantigens is of great
interest. ACPAs against vimentin, collagen II and his-
tones, among others, have been identified in serum from
patients with RA107–109. The source of these ACPAs in
patients with RA has been investigated110. Indeed, citrul-
linated vimentin is found on NETs isolated from healthy
donors and patients with RA108. NETs containing citrul-
linated proteins (such as histone H3, histone H4 and
vimentin) are ingested by fibroblast-like synoviocytes
(FLS) in a receptor for advanced glycation end-products
(RAGE)–TLR9-dependent manner. This process leads
to the upregulation of major histocompatibility complex
(MHC) class II expression by the FLS and the subse-
quent presentation of NET peptides to CD4+ T cells.
The outcome of this inflammatory response to NETs
by the FLS is the activation of B cells and the generation
of ACPAs110.
Another neutrophil feature in RA is the presence of
LDGs that express low levels of TNF receptor 1 (TNFR1)
and TNFR2 on their surface and thus are less respon-
sive to TNF stimulation111. This feature could potentially
explain why some patients with RA do not respond to
TNF interference treatment, as they contain a subpopu-
lation of cells that remain TNF-unresponsive. However,
unlike LDGs in patients with SLE, LDGs in RA do not
produce NETs excessively111.
Mouse models. Mouse models are used to investigate
the development and progression of RA. The F1 gener-
ation of K/BxN mice, produced by crossing T cell recep-
tor transgenic KRN mice on a C57Bl/6 background
with autoimmune-​prone non-​obese diabetic (NOD)
mice, develop severe arthritis by 3–5 weeks of age112,113.
Transfer of serum or purified IgG molecules from these
mice into mice deficient in B cells or T cells replicates
the RA-​like phenotype113. The phenotype is also repli-
cated by injection of IgG molecules into healthy mice114.
Neutrophil depletion in this model of serum transfer
arthritis abrogates disease onset115,116. Furthermore, in
a serum transfer model using a Gfi1-deficient mouse,
in which neutrophil maturation is reduced, arthritis
does not progress117,118. Notably, serum transfer failed
to induce arthritis in Nrf2-deficient mice (Nrf2 encodes
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a transcription factor involved in ROS generation),
indirectly implicating NETs in RA pathogenesis119.
However, despite much evidence of citrullination in
human RA, PAD4-deficient mice develop arthritis in the
serum transfer model, which suggests that citrullination
of NET components is not required in this model120. This
finding again suggests that disease progression and NET
induction vary greatly between mice and humans.
Therefore, although increasing evidence suggests that
the death of neutrophils via NETosis occurs in patients
with RA and that this activity is detrimental for RA pro-
gression, further work is needed to clarify the source of the
citrullinated proteins and subsequent autoantigens,
the mechanism by which they are modified and whether
they are actually important in RA pathogenesis (Fig. 2c).
Felty syndrome . Felty syndrome is a complication of
RA that affects 1–3% of patients. This syndrome typ-
ically presents with splenomegaly, neutropenia and
joint destruction121. A low absolute neutrophil count is
central to a diagnosis of Felty syndrome. Development
of Felty syndrome usually occurs 10–15 years after
the onset of RA, although simultaneous diagnosis can
occur and has a calamitous outcome because patients
become far more susceptible to severe infections and
sepsis than those patients who do not develop the syn-
drome121. Patients with Felty syndrome have abundant
anti-​histone autoantibodies in circulation, the source
of which could be histones released during NETosis. In
Felty syndrome, neutropenia occurs because neutrophils
are sequestered in the spleen or die. The bone marrow
might also fail to produce these cells122. Thus, evidence
suggests that Felty syndrome is also a disease in which
NETs are detrimental.
Gout
Gout is an acute inflammatory arthritic disease induced
by the accumulation of needle shaped monosodium
urate (MSU) crystals. Crystal deposition in joints such
as the big toe or in the kidneys initiates an inflammatory
event that rapidly progresses and then resolves. Gout
occurs in 1–2% of the adult population in developed
countries. Gout has a higher incidence in men than
in women and was described in detail as far back as
ancient Egypt123–126.
The role of neutrophils in the progression and res-
olution of the acute inflammatory flare in gout is not
well characterized127. A marked influx of neutrophils
into the synovium of the affected joint occurs after acti-
vation of local immune cells including macrophages,
dendritic cells and synoviocytes128,129. These cells then
phagocytose the MSU crystals, thus producing ROS
and initiating inflammatory pathways. The activation
of the inflammasome and release of IL-1β recruits neu-
trophils and additional immune cells130. These cells
produce chemokines, which in turn exacerbate neu-
trophil recruitment131. These neutrophils generate ROS
and release of antimicrobial peptides, such as IL-1β and
IL-8 (ref.132).
Neutrophils isolated from patients with gout spon-
taneously release NETs, and serum from patients with
gout induces NET formation in neutrophils of healthy
 Reviews

donors 133. In the synovium, MSU crystals induce
NET production, which can be visualized by detect-
ing DNA, myeloperoxidase and neutrophil elastase
in gouty tophi. Upon reaching a certain threshold,
NETs sequester and form aggregations of NETs or
aggNETs134, which might degrade cytokines. A study
demonstrated that NADPH-​i ndependent NETosis
occurs in response to MSU crystals135. MSU crystals
interact with lysosomes, and chromatin decondensa-
tion mediated by neutrophil elastase occurs upon cell
death. The resulting NETs could potentially sequester
DNA-​coated MSU crystals in tophi135. Thus, neutro-
phils might also have a role in the rapid resolution of
inflammation in gout.
This resolution is not due to the removal of MSU
crystals, as they remain in the affected joint after the
inflammation dissipates. Generation of aggNETs is
thought to provide a backbone for the inflammation-​
silent deposits of MSU crystals that are visualized via
dual-​energy CT. This disease state is referred to as
chronic tophaceous gout136. However, this area of gout
research remains to be clarified.
NET aggregation has been shown to require ROS,
as injecting MSU crystals into the paw of NADPH-​
oxidase-deficient mice results in accumulation of pro-​
inflammatory cytokines and paw swelling that persists
longer than the inflammatory response to MSU crystals
in wild-​type control mice134. Furthermore, proteases in
aggNETs degrade IL-6, TNF and macrophage inflamma-
tory protein 1α (MIP1α; also known as CCL3)134. Hence,
the accumulation of MSU crystals and NETs in the
affected area is central to the resolution of inflammation
by trapping the MSU and degrading pro-​inflammatory
cytokines. Notably, a study from 2016 challenged
the role of neutrophils in gout resolution owing to diffi­
culty in reproducing the data showing that aggNETs
degrade cytokines137.
Although we have some understanding of the role
of neutrophils in gout in humans, the role of neutro-
phils in mouse models of gout remains uncertain.
This uncertainty is yet another example of the differ-
ences in how human and mouse neutrophils respond
to attack and generate NETs. Neutrophils seem to be
detrimental in the inflammatory phase of a gout attack,
but their ability to aid in resolution requires further
investigation (Fig. 2d).
Therapeutic approaches
Treatment of rheumatic diseases is mainly aimed at
reducing the inflammation of the affected area. A com-
mon treatment is steroid-​induced immunosuppression
via cyclophosphamide or glucocorticoids. In SLE, this
treatment is boosted with chemotherapy and rituximab,
an anti-​C D20 monoclonal antibody that depletes
B cells138,139. Patients with vasculitis also utilize steroids
to control inflammation, along with rituximab. Even with
these therapies, the mortality of patients with vasculitis
is high, mostly owing to infection, cardiovascular disease
or cancer140. Patients with RA are treated with steroids
to reduce inflammation but also receive DMARDs such
as methotrexate or infliximab, some of which target
cytokines produced during flares102. Rituximab is also
used to treat RA. Gout is a well-​understood and well-​
managed rheumatic disease. Steroid treatment and the
development of modern drugs along with patient edu-
cation and lifestyle alterations have had a major effect on
patient well-​being and mortality141.
Rituximab treatment in patients with autoimmune
diseases results in reduced NET production and autoan-
tigen generation; hence, this treatment also targets neu-
trophils4. Therapies that target cytokines also reduce
NETosis, and PAD inhibitors have been used with some
success in mouse models of RA142 and SLE78,79. Regulating
neutrophils, particularly NETosis and the effects of NETs,
are approaches that need to be developed and hold great
promise (reviewed elsewhere4).
Much work is now focusing on targeting NETs in the
hope of finding a NET inhibitor to aid in halting the pro-
gression of these diseases. This endeavour is challenging, as
neutrophils are fundamental to the host immune response,
and as such, the NETs must be targeted in only specific
situations while leaving the patient immunologically intact.
Conclusions
A growing body of evidence indicates that neutrophils,
and specifically the process of NETosis, are involved in
the progression of rheumatic diseases. Many studies
have demonstrated the presence of NET material in
the affected tissues of patients, and autoantibodies are
commonly directed against neutrophil proteins that
are known to be released during the process of NETosis.
Evidence also suggests that neutrophils are required for
resolution of inflammation. Questions remain, however,
as to the mechanism of NET release in these diseases; as
such, the role of infection prior to disease onset is of
great interest for the development of therapeutics.
Another factor in the study of rheumatic diseases in
terms of the role of neutrophils is the inconsisten-
cies between patient data and mouse models. These
inconsistencies highlight the stark differences between
neutrophils from humans and mice, which are rarely
discussed. Overall, further studies into the role of NETs
in these diseases will aid in the development of new
therapeutic approaches.
Published online 21 June 2018

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Author contributions
All authors contributed to substantial discussion of content,
researching data, writing and reviewing and/or editing the
manuscript before submission.
Competing interests
The authors declare no competing interests.
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Reviewer information
Nature Reviews Rheumatology thanks M. Herrmann, M. Radic
and the other anonymous reviewer(s) for their contribution to
the peer review of this work.
volume 14 | AUGUST 2018 | 475