Seminars in Arthritis and Rheumatism 49 (2019) S43 S48

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Seminars in Arthritis and Rheumatism

journal homepage: www.elsevier.com/locate/semarthrit

Updates on NET formation in health and disease

Moritz Leppkesa, Maximilian Schickb, Bettina Hohbergerc, Aparna Mahajanb, Jasmin Knopfb,
~ ozb,*, Martin Herrmannb
Georg Schettb, Luis E. Mun
a
€ rnberg (FAU), Department of Internal Medicine 1 Gastroenterology, Pneumology and Endocrinology,
Friedrich Alexander University Erlangen-Nu
Universita€tsklinikum Erlangen, 91054 Erlangen, Germany
b
€ rnberg (FAU), Department of Internal Medicine 3 Rheumatology and Immunology, Universita
Friedrich Alexander University Erlangen-Nu €tsklinikum Erlangen,
90154 Erlangen, Germany
c
Friedrich Alexander University Erlangen-Nu €tsklinikum Erlangen, 90154 Erlangen, Germany
€ rnberg (FAU), Department of Ophtalmology, Universita

A R T I C L E I N F O A B S T R A C T

Keywords: Following a recent presentation at ATT Mallorca in May 2019, this paper gives insight into the current
aggNETs research of neutrophil extracellular traps (NETs) and their role in conditions of health and disease. Though
Apoptosis NETs reportedly support disease progression and play a role in the development of autoimmune diseases, we
Clearance argue that NETs are mandatory for the mammalian immune system. They are especially important to patrol
Ductal occlusion
and surveil outer and inner body surfaces and are capable to perform major anti-microbial activities. Neutro-
Innate inflammation
phils are the first cells to be recruited to wounds, where they form NETs and aggregated NETs (aggNETs). The
NETs
Thrombi latter close the wounds and are ever-present in skinfolds, where the integrity of the skin is impaired. On
infected ocular surfaces NETs form an antimicrobial barrier, which prevents bacterial dissemination into the
brain. In the oral cavity, NETs display anti-bacterial properties. Although NETs on internal body surfaces like
ducts and vessels offer superficial surveillance, exaggerated aggNET formation may directly block vessels and
ducts and thus cause thrombi and ductal occlusion, respectively. In the case of biliopancreatic ducts, clogging
by aggNETs may even cause acute pancreatitis. Insufficient clearance of apoptotic remnants and NETs can
lead to autoimmune diseases or unwanted, chronic inflammation. To prevent this, macrophages cloak dead
cells, while apoptotic cells are cleared. We conclude that neutrophils, NETs and aggNETs can be considered
double edged swords that orchestrate the innate immune response but carry the risk to precipitate autoim-
munity and epithelial damage.
© 2019 Elsevier Inc. All rights reserved.

Introduction hypothesis that the major playground of NET-forming neutrophils

Since the first report of neutrophil extracellular traps in the
defence against microbes [1], their initially positive image was get-
ting continuously worse. Several papers reported favourable disease
outcomes, when NET formation was abrogated or reduced in genetic
or pharmacological models of often chronic diseases [2 20]. NET-
associated enzymes like serine proteases and clotting of vessels and
ducts by NET aggregates (aggNETs) can precipitate tissues damage,
thromboses, and ductal occlusion [21] (Figs. 1 and 2).
The ability to form NETs is widespread in mammalians and argues
for a mainly beneficial, evolution-approved role of NETs [22]. It is
now time to challenge the paradigm of NET as “bad guys”. We instead
consider neutrophils and NETs as often altruistic defenders of our
health that orchestrate the resolution of inflammation and initiate
the healing process. Though some reports describe NET formation in
the connective tissue and the parenchyma of organs we favour the
*Corresponding author.
~ oz).
E-mail address: luis.munoz@uk-erlangen.de (L.E. Mun
are body surfaces, vessels, ducts, preformed holes, cysts, and wounds
(Table 1).
Initially reported as mechanism to sequester and kill bacteria [1]
alternative functional properties of NETs got into the focus of basic
and clinical researchers. In the current view NETs (I) trap and kill bac-
teria and hyphal fungi (II) entrap and degrade soluble pro-inflamma-
tory mediators (III) sequester crystals (IV) orchestrate the resolution
of neutrophil-driven inflammation. However, NETs (I) cause or pre-
cipitate thrombotic events (II) occlude ducts (III) act as immunogens
(IV) cause interferon-driven inflammation and (V) harbour antigens
for interferogenic immune complexes. Many inducers of NET forma-
tion have been reported in the last decades (Table 2).
Among the immune cells, all granulocytes, mononuclear phagocytes
and mast cells reportedly share the feature to externalise chromatin
[18,23,24]. Macrophages externalise the DNA from phagocytosed apo-
ptotic prey; however, the DNA is of relatively small size and does not
form NET-like structures [25]. Reports on decondensed extracellular
chromatin by other cells like epithelial cells are anecdotal and need
further confirmation.

https://doi.org/10.1016/j.semarthrit.2019.09.011
0049-0172/© 2019 Elsevier Inc. All rights reserved.
S44 M. Leppkes et al. / Seminars in Arthritis and Rheumatism 49 (2019) S43 S48

Fig. 1. Schematic display of neutrophils, NETs and aggNETs. (1) Viable neutrophil. When activated, they (a) release proinflammatory mediators, (b) phagocytose bacteria and par-
ticles, such as crystals and (c) release antimicrobial granule contents into extracellular space. (2) Cloudy NET released after stimulation with for instance LPS or crystals (black). These
NETs can, among other things, catch bacteria (pink). (3) Stimulation with C5a forms spikey NETs. (4) At high neutrophil count, aggNETs are formed. (5) Stimulation with bicarbonate
also leads to the formation of notably large aggNETs. This can happen in the pancreas during peripheral leukocytosis. They are specialised to cap large surfaces. (For interpretation of
the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2. Composite of microphotographs of various forms of NETs. Formation of neutrophil extracellular traps (NETs) in vitro or in vivo under fluorescent microscope. Blue: extracellular DNA
stained with propidium iodide (A N); Red: cytoplasmic lysosomes stained with lysotracker (B N). (A) Partial aggNET formation containing neutrophil cell remnants (yellow circles), induced
by 48 mM bicarbonate in vitro. Nuclei are mostly decondensed. (B H) Middle panel: neutrophils were treated with LPS in vitro to induce NET formation. (B) Neutrophils in resting state (C)
activated neutrophils (D) early NET formation (E) spiky NET (yellow arrow) (F) cloudy NET with neutrophil remains (green arrow) (G) extended cloudy NET (H) cloudy NET with spike. (I N)
Right panel: aggNETs isolated from tear fluid. Viable (red asterisk) and dead neutrophils (blue asterisk) are glued together with dead epithelial cells and dust particles by NETs, forming agg-
NETs. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Neutrophils not only respond against invaders but also against
endogenous danger/damage signals like various crystals [26] and
immune complexes [27]. An impaired capability to clear NET rem-
nants contributes to the chronification of inflammation [28]. In this
case, NETs can harbour antigens in pathogenic interferogenic
immune complexes [29]. Both phagocytosis and NET formation com-
pete in fighting fungal infection [30]. Size, number and species of the
pathogen determine the responses of the neutrophils. Inhibition of
NET formation often protects from tissue damage, when infectious
pathogens can be phagocytosed and killed intracellularly.
large aggNET clusters. However, during massive infectious insults
extended areas of solid tissues may perish, and, thus form secondary
infected areas referred to as abscesses with low cellular density. The
formation of abscesses is a consequence of microbial cytotoxicity and
the invasion and action of neutrophils that fight the pathogens with
their full arming, including NET formation. Several bacteria report-
edly harbour nucleases that enable them to digest the DNA scaffold
of the NETs and to produce pus [33].
NETs on outer body surfaces

NETs in tissues NETs in skin wounds

Tissue insults causes imbalance of electrolytes (e.g. high extracel-
lular potassium), low pH, ATP, and other indicators of cell death. If
the dead corpses cannot be “cloaked” by local macrophages [31] the
sentinel cells attract further immune cells from circulation. Neutro-
phils are the dominant cells of the first wave of recruitment and occur
as soon as 40 min after an insult at the site of inflammation. Their
more flexible nuclei compared to mononuclear cells enable a higher
speed of the latter when passing the tissues (»8 as compared to
»4 mm/min) [32]. However, if NET formation is induced in the neu-
trophils in parenchymal tissue, there is usually limited space to form
Most parts of the outer surface of a body are covered by the skin. It
forms a tight barrier that shields the vital parts of a body from the
hostile environment. As long as the skin is fully functional, it is a reli-
able protection against external influences. However, physical of
chemical damage disturbs the integrity and are potential entry points
for pathogens. For that reason, it is not surprising that neutrophils
are immediately recruited to wounds. One may speculate that neu-
trophils form NETs and aggNETs that mechanically form a surrogate
barrier and temporarily close the wound and initiate healing. Dia-
betics are well-known to suffer from impaired wound healing. Wong
 M. Leppkes et al. / Seminars in Arthritis and Rheumatism 49 (2019) S43 S48
Table 1
The physiological functions and pathological aspects of NET and aggNET formation.
Physiology
NETs and aggNETs immobilise pathogenic microorganisms
NETs kill microorganisms
aggNETs form barriers that segregate healthy tissue from necrotic areas
aggNETs form temporary wound closures and initiate wound closure and
healing
aggNETs degrade inflammatory mediators and thus contribute to the resolu-
tion of inflammation
Pathogenesis
Pro-inflammatory compounds released during NET-formation can injure
tissues
NETs can initiate canonical thromboses
aggNETs can clot vessels
aggNETs can clot ducts
Uncleared NETs and aggNETs serve as repository for modified autoantigens
linked to nucleic acids and pathogen-associated molecular patterns
NETs can promote tumour growth
Table 2
The induction of NET formation.
Infectious agents Bacteria, fungi, parasites, and viruses
Bacterial and fungal products LPS, zymosan
Endogenous homeostatic High bicarbonate in bile, pancreatic
microenvironments juice, tears, and saliva
Endogenous molecules from inflam- HMGB1, ATP, histones
matory microenvironments
Agents activating NET formation PMA, ionomycin,
Drugs inhibiting N extravasation and Anti-oxidatives, b-blockers
NET formation
Drugs inhibiting NET expansion and Inhibitors of PAD4
aggNET formation
et al. demonstrated that under high glucose conditions in diabetes
mellitus, neutrophils are prone to undergo NET formation and an
exaggerated formation of NETs may actually disturb the delicate bal-
ance of wound healing under these circumstances [34].
NETs in skinfolds
In elderly and in overweight individuals skin tends to form warm
and humid folds. Between the thighs under breasts and belly, behind
the ears, at the genitalia and the armpits, and between the toes and
the fingers chafing of the warm, moist skin can disturb skin integrity
and destroy their barrier function. If the skinfold is infected, the
resulting rash is usually referred to as intertrigo. The latter appears
inflamed and infected. It tends to itch, ooze, and be sore. It is fre-
quently seen among overweight individuals, those with diabetes, bed
rest or diaper use. This milieu supports the proliferation of microor-
ganisms like Streptococci and Corynebacterium minutissimum [6],
viruses, and fungi (e.g. Candida albicans) [6]. In intertrigo neutrophils
are prone to be important players and it is fair to speculate that agg-
NETs may protect these areas and may limit the inflammatory
response by proteolysis of inflammatory mediators [35].
NETs on ocular surfaces
The ocular surface is a highly specialised tissue. It is warm and con-
tinuously wetted by the eye’s tear system consisting of a delicate array
of glands that deliver aqueous, proteinaceous and lipidic fluids forming
the three layered tear film. In the open eye situation, the ocular surface
is exposed to a plethora of pathogenic microorganisms and viruses. The
protection of the ocular surface is based on antibodies, complement and
on the cells of the innate immune system e.g. neutrophils and mononu-
clear phagocytes [36]. Failure of innate immune mechanisms and the
resulting activity of T cells may irreversibly scar the translucent cornea,
calling for robust innate immune mechanisms [37].
S45
Bacterial keratitis by ocular biofilm formation of Pseudomonas
aeruginosa is fostered by the exopolysaccharide Psl. The timely
recruitment to the outer eye surface of neutrophils prevented the
spread of the bacteria into the murine brain. In response to the bacte-
rial type-3 secretion system (T3SS) the neutrophils reportedly form
NETs at the base of the developing biofilm and formed a barrier
referred to as “dead zone” [38]. The latter confined the bacteria to the
outside of the eye and thus inhibited bacterial dissemination into the
brain. Importantly, the clearance of established ocular biofilms
required synchronous blocking of Psl and T3SS as well as appropriate
antibiotic therapy [38].
NETs in the oral cavity
A location where NETs can regularly be found in healthy individuals is
the oral cavity. There are three major sources (I) the bronchi (II) direct
access via mucosal exit of neutrophils and (III) the plethora of oral glands
via their ductal systems. This is not surprising as we are exposed to
microorganisms, whenever we breathe or eat. Mucosal surfaces are
mostly colonised with microorganisms and are thus constantly chal-
lenged by residing microorganisms. Besides colon and vagina, the oral
cavity is the most heavily colonised part of the human body. The saliva
and its antimicrobial components, like secretory IgA (sIgA), IgG lysozyme,
and mucins comprise the humoral defense. IgA is produced by plasma
cells in the salivary glands and is exported in the polymeric Ig receptor
(pIgR) pathway. IgG is mainly derived from serum (via gingival crevices),
with little local production. Neutrophils are part of the host defense at
the oral mucosa. One way that they are driven to undergo NET formation
is driven by the interaction of sialyl Lewis(X) of salivary mucins with
L-selectin of neutrophils. This pathway of NET induction is not depending
on elastase and less on NAD phosphate-oxidase activity. Saliva-induced
NETs displayed high extra- and intracellular bactericidal activities [39].
Interestingly, saliva from patients with aphthous ulcers or Behçet disease
prone to oral ulcers showed impaired NET formation [39].
Neutrophils continuously migrate to oral mucosa and neutropenia
or functional neutrophil deficiencies can alter the local microflora
causing gingivitis, ulcerations, periodontitis and possibly Behçet’s
vre-syndrome, deficiency of functional neu-
disease. In Papillon Lefe
trophil serine proteases by a cathepsin c-mediated maturation defi-
ciency typically features debilitating periodontitis [40]. The
formation of NETs in saliva is critical to maintain a healthy oral
microbiota. Pharmacologic targeting NET formation could be a novel
approach to treat oral manifestations of inflammatory diseases.
NETs on internal body surfaces
After injection of inflammatory stimuli like MSU or zymosan into
murine peritoneal cavities usually a huge number of neutrophils are
recruited that, at least partially, form NETs that tend to aggregate. As
crystals tend to bind to the epithelia lining the inner surfaces of the
abdominal cavity, aggNETs are preferentially found in this region.
The binding of the aggNETs to the peritoneal surface is extremely sta-
ble; aggNETs are difficult to remove mechanically [28]. While NETs
on internal body surfaces beneficially monitor potential hazards,
excessive NET-formation may lead to epithelial damage as well as
vascular and ductal occlusions.
NETs in vessels
It is commonly accepted that in a plethora of infections and non-
infectious diseases, NETs are formed inside the vasculature. There
they form both scaffold and stimulus for canonical thrombus forma-
tion [41]. In vitro, NETs bound and activated platelets, initiated their
aggregation and induced the formation of thrombi. DNase or heparin
inhibited the formation of the latter. Purified histones were also suffi-
cient to initiate platelet aggregation. NETs bound erythrocytes and
S46 M. Leppkes et al. / Seminars in Arthritis and Rheumatism 49 (2019) S43 S48
fibrin and induced a red venous thrombus [41]. In a more recent work
it was described that NETs can directly occlude blood vessels and
form non-canonical thrombi that do not require platelet activation.
The presence of functional deoxyribonucleases (DNases), DNase1 and
DNase1-like 3 controlled the pro-thrombotic effects of NETs in the
circulation. In the absence of both DNases, intravascular NETs
obstructed the blood vessels and caused dramatic mortality during
sterile leukocytosis as well as in bacterial sepsis [42]
NETs in ducts
In addition to the vessels, the mammalian body is also traversed
by ducts and channels of a variety of sizes and functions. The immu-
nological surveillance of these tubular structures is executed by the
humoral and cellular branches of the immune system. The dominant
cells are neutrophils. In the case of the pancreas the neutrophils tend
to externalise their chromatin shortly after entering the hostile vicin-
ity of the ductal fluids characterised by alkaline pH, super threshold
bicarbonate and calcium concentrations, lysosomal stress, and leak-
age. In the case of the ductal system of the pancreas, peripheral leu-
kocytosis causes increased invasion of the ducts via the acini.
Extreme conditions induce neutrophil chromatin decondensation
independently of PAD activity or ROS, however, mild disturbances of
the pH pCO2-balance favours calcium influx and PAD-mediated
chromatin decondensation [43]. In analogy to canonical NET forma-
tion the extracellular chromatin tends to aggregate and occlude the
pancreatic ducts. As consequence, the restriction of the flow of the
pancreatic fluid may propagade acute pancreatitis [21].
Clearance of apoptotic remnants and NETs
After the NETs have immobilised and eventually killed the patho-
gens [1], they form a highly interferogenic potential pathogenic load. In
patients with systemic lupus erythematodes (SLE) a defective clearance
of NETs was associated with the development of nephritis [44]. NETs
may harbour autoantigens with autoantibodies immune-complexes
and promote type I IFN production further augmented by cathelicidin
(LL37)-mediated activation of pDC [29]. In pediatric forms of SLE, NETs
are the cardinal inducers of type I IFN. In this case LL37, HMGB1, and
DNA synergistically activate an interferogenic response depending on
the Toll-like receptors (TLR) 4 and 9, respectively [45]. Autoinflamma-
tion and necrosis of blood vessel walls are hallmarks of antineutrophil
cytoplasmic antibody (ANCA)-associated vasculitis (AAV). The renal
involvement is characterised by a pauci-immune crescentic glomerulo-
nephritis (PiCGN) followed by a rapid decline in renal function [46]. To
avoid these pathological interferon-driven responses, the body has
developed several systems to handle remnants from dying and dead
cells. In the case of apoptosis the macrophage clear by an anti-inflam-
matory macropinocytotic pathway referred to as efferocytosis [47]. The
process involves phospholipid and glucan moieties for recognition [48].
If the clearance is impaired, (secondary) necrosis may ensue and the
uptake of the cellular remnants shifts to pro-inflammation, especially in
the presence of anti-nuclear autoantibodies [49]. Since DNA is rather
stodgy for an individual phagocyte, DNA is cleaved into oligonucleotides
that are released into the phagocyte’s vicinity [25].
The role of phagocytic cells
Neutrophils that had been recruited to sites of cell damage often
form dense swarms that tend to prolong and enhance the damage
induced by the insult. Resident macrophages immediately sense
death or NET formation of individual cells, cloak their dead corpses
before these initiate swarming and consequently neutrophil-
mediated inflammatory damage. This mechanism stops excessive
inflammatory responses to injury of single cells that occurs during
normal function [31].
Swift clearance of apoptotic cells, especially that of granulocytes
protects tissues from the toxic contents of dying cells; therefore, it is
cardinal for the resolution of inflammation [8,48,50,51]. Monocytes
reportedly display impaired phagocytosis of autologous apoptotic
PMNs. However, they efficiently clear apoptotic PMNs from healthy
individuals. CD16POS monocytes more efficiently interacted with apo-
ptotic neutrophils than CD16NEG cells. The molecular basis of this
defect is still elusive. The apoptotic cell-dependent LPS-induced
induction of IL-10 and TNF-a were blunted and unchanged in mono-
cytes from SLE, respectively [52].
SLE can be complicated by the development of nephritis for which
current therapeutic regimens work insufficiently. Comparing molec-
ular data from murine and human cases it turned out that inflamma-
tory events peak around the onset of proteinuria, are followed by
stress, and endothelial dysfunction of the tubular system. Renal mac-
rophages and dendritic cells orchestrate the inflammatory responses
during nephritis. In these conditions macrophages tend to switch
from resolution of inflammation to tissue remodelling [53].
The role of humoral factors
In the blood extracellular DNA is degraded by DNase1 and
DNase1-like 3. The activity of either of these nucleases is required to
maintain vascular homeostasis and to preclude unwanted thrombotic
events. Indeed, the genetic absence of both DNase1 and DNase1-like
3 non-canonical vascular clots emerge that were based on neutrophil
extracellular traps. In mice even sterile leucocytosis driven by hepatic
expression of G-CSF resulted in intravascular NETs that obstructed
blood vessels and caused organ damage and death [42]. In humans
with septic conditions vascular occlusions were associated with a
defect to degrade NETs ex vivo and with the vascular occlusions by
NET clots [42]. The clearance of NETs was impaired in a subgroup of
patients with SLE involving two mechanisms (I) the presence of
DNase1 inhibitors and (II) anti-NET antibodies that prevented access
of DNase1 to NETs [44].
SLE is characterised by autoantibodies to DNA, histones, and neutro-
phil proteins. In the presence of cellular debris the autoantibodies form
pathogenic, nephritogenic immune-complexes. Since anti-microbial
NETs are composed of DNA, histones, and neutrophil proteins they may
serve as autoantigen repository for the hallmark autoantibodies of SLE.
The timely and swift removal of NETs independent of (auto)-antibodies
is therefore crucial for tissue homeostasis and to prevent exposure of
self-antigens.
Murine annexinA1 is involved in the regulation of inflammatory
cells and the resolution of inflammation. Studies on human Annex-
inA1 are scarce [54]. Patients with SLE often harbour anti-annexinA1
autoantibodies, especially those with renal complications. It is dis-
cussed that annexin A1 (I) is involved in SLE-related autoimmunity
(II) is post-translational modified when associated with NETs and
that (III) the immunogenic form is citrullinated [54]. The role of the
clearance-related complement components C3 and C4 in SLE and
murine lupus is established since decades [55 57]. The complement
serum levels are useful to monitor the activity of SLE and to predict
flares of membrano-proliferative glomerulonephritides [58].
Conclusion
We consider neutrophils, NETs and aggNETs double edged swords
that orchestrate the innate immune response but carry the risk to
precipitate autoimmunity and epithelial damage.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
 M. Leppkes et al. / Seminars in Arthritis and Rheumatism 49 (2019) S43 S48
Acknowledgement
This work was partially supported by the German Research Foun-
dation (DFG) (SCHE1583714-1; CRC1181 project C03; TRR241 project
B04) and the Volkswagen-Stiftung (90361).
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