Restoration of anti-Aspergillus defense by neutrophil
extracellular traps in human chronic granulomatous disease
after gene therapy is calprotectin-dependent
Matteo Bianchi, MSc,a* Maria J. Niemiec, MSc,b* Ulrich Siler, PhD,aConstantin F. Urban, PhD,b?
and Janine Reichenbach, MDa?
Zurich, Switzerland, and Ume? a, Sweden
Background: Aspergillus spp infection is a potentially lethal
disease in patients with neutropenia or impaired neutrophil
function. We showed previously that Aspergillus hyphae, too
large for neutrophil phagocytosis, are inhibited by reactive
oxygen species-dependent neutrophil extracellular trap (NET)
formation. This process is defective in chronic granulomatous
disease (CGD) because of impaired phagocyte nicotinamide
adenine dinucleotide phosphate (NADPH) oxidase function.
Objective: To determine the antifungal agent and mechanism
responsible for reconstitution of Aspergillus growth inhibition
within NETs after complementation of NADPH oxidase function
by gene therapy (GT) for CGD.
Methods: Antifungal activity of free and NET-released
calprotectin was assessed by incubation of Aspergillus nidulans
with purified calprotectin, induced NETs from human controls,
and CGD neutrophils after GT in the presence or absence of
Zn21or a-S100A9 antibody, and with induced NETs from wild-
type or S100A9–/–mouse neutrophils.
Results: We identified the host Zn21chelator calprotectin as a
neutrophil-associated antifungal agent expressed within NETs,
reversibly preventing A nidulans growth at low concentrations,
and leading to irreversible fungal starvation at higher
concentrations. Specific antibody-blocking and Zn21addition
abolished calprotectin-mediated inhibition of A nidulans
proliferation in vitro. The role of calprotectin in anti-Aspergillus
defense was confirmed in calprotectin knockout mice.
Conclusion: Reconstituted NET formation by GT for human
CGD was associated with rapid cure of pre-existing therapy-
refractory invasive pulmonary aspergillosis in vivo, underlining
the role of functional NADPH oxidase in NET formation and
calprotectin release for antifungal activity. These results
demonstrate the critical role of calprotectin in human innate
immune defense against Aspergillus infection. (J Allergy Clin
Key words: Chronic granulomatous disease, gene therapy, neutro-
phil extracellular trap, calprotectin, Aspergillus infection
Neutrophils kill microbes by distinct reactive oxygen species
(ROS)–dependent processes: intracellularly after phagocytosis
and by extracellular mechanisms including neutrophil extracellu-
lar traps (NETs), which capture and kill bacteria,1parasites,2and
fungi.3,4NETs are composed of chromatin (histones and DNA),
decorated with at least 20 other proteins.1,4,5In naive neutrophils,
8 of these proteins localize to granules, such as neutrophil elas-
tase, myeloperoxidase, or azurocidin, and 11 to the cytoplasm,
such as catalase and the calprotectin complex.4This heteroduplex
formed by subunits S100A8 and S100A9 belongs to the calcium-
binding protein family of S100 proteins and exerts strong micro-
bistatic activity against a variety of microorganisms in vitro.6
(NADPH) oxidase function accounts for impaired phagocyte
ROS production and poor microbial killing in chronic granulom-
atous disease (CGD), leading to recurrent life-threatening bacte-
rial and fungal infections. Aspergillus spp infections cause
pneumonia and disseminated disease and are the leading cause
of death in these patients.7-10ROS production by the NADPH
oxidase is indispensable for microbe-triggered NET forma-
tion,5,11,12whereas their mechanism of action in this process is
unknown. After release from granules, neutrophil elastase (NE)
and myeloperoxidase synergize to drive decondensation of chro-
matin, an early event preceding the release of NETs.13,14ROS
might contribute to the release of NE and myeloperoxidase
from granules. We previously reported impaired NET formation
fection in vivo. NADPH oxidase–dependent ROS production and
NET inhibition of Aspergillus conidia and hyphae were restored
early after gene therapy (GT) in a patient with X-linked
Fromathe Division of Immunology/Hematology/BMT, University Children’s Hospital
Zurich; andbthe Antifungal Immunity Group, Molecular Biology Department, Labo-
ratory for Molecular Infection Medicine Sweden, Ume? a University.
*These authors contributed equally to this work.
?These authors contributed equally to this work.
Supported by a grant of the Chronic Granulomatous Disorder Research Trust, United
Kingdom (grant no. J4G/08/01 to J.R. and M.B.), a grant of GEBERT R€UF STIF-
TUNG, Switzerland (grant number GRS-046/10 to J.R.), a grant of the EU FP7
CELL-PID programme (J.R. and U.S.), and a grant of the Swiss National Science
Foundation (grant no. 320000-121983 to U.S.). M.J.N. and C.F.U. were supported
by a starting grant from MIMS, a stipend from Stiftelsen J. C. Kempes Minnes Stipen-
diefond, and a travel financing grant from Medicinska Fakulteten Ume? a Universitet.
The funders had no role in study design, data collection and analysis, decision to pub-
lish, or preparation of the article.
Disclosure of potential conflict of interest: M. Bianchi and J. Reichenbach have received
research support from the CGD Research Trust. M. J. Niemiec has received research
support from the Stiftelsen J. C. Kempes Minnes. U. Siler has received research sup-
port from the Swiss National Science Foundation. C. F. Urban has received research
support fromMolecular InfectionMedicinesSweden, Stiftelsen J.C.Kempes Minnes,
and Med/Cinska Fakuleten Umea University.
Received for publication October 28, 2010; revised December 26, 2010; accepted for
publication January 10, 2011.
Reprint requests: JanineReichenbach,MD, Division ofImmunology/Hematology/BMT,
E-mail: firstname.lastname@example.org. Constantin F. Urban, PhD, Antifungal Im-
munity Group, Molecular Biology Department, Laboratory for Molecular Infection
Medicine Sweden, Ume? a University, 90187 Ume? a, Sweden. E-mail: constantin.
? 2011 American Academy of Allergy, Asthma & Immunology
CGD: Chronic granulomatous disease
DAPI: 4’-6-diamidino-2-phenylindole, dihydrochloride
GT: Gene therapy
LTR: Long terminal repeat
NADPH: Nicotinamide adenine dinucleotide phosphate
NE: Neutrophil elastase
NET: Neutrophil extracellular trap
phox: Phagocyte oxidase
PMA: Phorbol 12-myristate 13-acetate
ROS: Reactive oxygen species
gp91phox-deficient CGD, paralleled by clearance of therapy-
refractory Aspergillus nidulans lung infection.11
The antifungal agent responsible for A nidulans growth inhibi-
tion within NETs has not been characterized. Here we show that
among the 24 NET-associated proteins, the major antifungal
agent inhibiting Aspergillus growth within NETs is the cytoplas-
mic calprotectin protein complex (S100A8/A9 heteroduplex).
This is in good agreement with our previous report showing
that calprotectin is the major NET component against Candida
ocyte oxidase)-deficient CGD and multifocal therapy–refractory A nidulans
lung infection with a monocistronic long terminal repeat–driven g-retroviral
SF71gp91phoxvector (see reference11for details; additional information in
this article’s Methods section in the Online Repository at www.jacionline.
org) under a protocol approved by the ethics review board of the University
Children’s Hospital Zurich and the Swiss Expert Committee for Bio-Safety
after written informed consent of the parents.
NET induction and quantification
Neutrophil extracellular trap formation was quantified as described11after
stimulation of 5 3 104neutrophils (control, CGD unsorted, CGD gp91phox-
negative [gp91phox2], and gp91phox-positive [gp91phox1] sorted neutrophils)
for 4 hours with 40 nmol/L phorbol 12-myristate 13-acetate (PMA, Sigma-
tox green (Invitrogen, Molecular Probes, Leiden, The Netherlands) in a black
96-well plate. Nonstimulated neutrophils were used as negative control. The
plates were read in a fluorescence microplate reader (Victor 3, PerkinElmer,
Waltham, Mass) with a filter setting of 485/535 nm (excitation/emission).
A nidulans growth inhibition
Antifungal activity of purified calprotectin was assessed by incubating
S100A9 (ProtEra, Florence, Italy) 6 1 mmol/L ZnSO4(Sigma-Aldrich) or 15
mg/mL rabbit polyclonal a-S100A9 antibody (H00006280-D01P, Abnova,
Taipei, Taiwan). Antifungal activity of NETs was determined by incubating
olar lavage fluid of the patient) with human and murine NETs or human NET
extracts6 0to2mmol/LZnSO4or15mg/mL rabbitpolyclonala-S100A9 an-
tibody, respectively (additional information in the Methods section in the On-
NET induction with A nidulans
In a 24-well plate, 2 3 105control, CGD gp91phox2and gp91phox1neutro-
phils were incubated with A nidulans hyphae obtained from culture of 6 3 104
A nidulans conidia on polylysine-coated cover slips for 5 hours at 378C, then
fixed in 4% paraformaldehyde for bright field microscopy and immunostain-
copy. For confocal microscopy (SP5; Leica, Wetzlar, Germany), specimens
were blocked with 5% donkey serum (Jackson ImmunoResearch, Suffolk,
United Kingdom), 1% BSA, 3% cold water fish gelatin (Sigma-Aldrich), and
0.25% Tween 20 in PBS, incubated with primary antibodies directed against
S100A8/A9 (BM4029 and BM4027; Acris, Herford, Germany), common As-
Cambridge, United Kingdom), and species-specific secondary antibodies cou-
pled to Alexa Fluor 488 and 568 (Invitrogen, Molecular Probes). DNA was
stained with 4’-6-diamidino-2-phenylindole, dihydrochloride (DAPI; Sigma-
Aldrich). For scanning electron microscopy (CM208; Philips, Eindhoven,
TheNetherlands), specimenswerepostfixedwith 2%osmiumtetroxide,dehy-
drated with graded ethanol series (50% to 100%), critical point–dried, and
coated with 10 nm platinum.
Release of calprotectin
In a 96-well plate, 5 3 104control, CGD gp91phox2and gp91phox1neutro-
phils were stimulated in duplicate with 40 nmol/L PMA at 378C and 5% CO2
for 4 hours to form NETs. After 30 minutes NET digestion with 5 U/mL Mi-
crococcal nuclease (Worthington Biochemicals, Lakewood, NJ) 1 1 U/mL
Deoxyribonuclease-1 (Sigma-Aldrich), samples was centrifuged for 10 min-
utes at 10,000g to remove debris. Heterodimeric calprotectin (S100A8/A9)
concentration was measured by ELISA (Hycult, Uden, The Netherlands; de-
tection limit 1.6 ng/mL) with 1/10, 1/40, and 1/80 dilutions. As control, total
cytoplasmic calprotectin was quantified after neutrophil lysis.
NET quantification in CGD neutrophils after GT
We aimed to identify factors that limit A nidulans growth in
NADPH oxidase–dependent NETs. Therefore, we first analyzed
whether NET formation was restored 2.6 years after GT in
gp91phox1(transduced) compared with gp91phox2(nontrans-
with a-gp91phoxantibody showed 22.8% gp91phox1neutrophils.
Sorting of gp91phox2and gp91phox1neutrophils by autoMACS
CGD neutrophils after GT correlated well with the percentage of
gp91phox1cells, 20.5% of unsorted, 3.2% of gp91phox2, and
87.6% of gp91phox1neutrophils releasing NETs (Fig 1, B). The
O2-production of individual gp91phox1neutrophils was 27.8%
(compared with control), measured by reduction of cytochrome
c (data not shown),16indicating this threshold as enough for sub-
stantial, albeit not 100%, NET formation.
Calprotectin inhibits A nidulans growth efficiently
We previously identified the cytoplasmic protein complex
calprotectin as major antifungal effector in NETs preventing
growth of C albicans4and therefore reasoned that calprotectin
might also be responsible for NET-mediated growth inhibition
purified calprotectin isabletoinhibitAspergillusspp.Toevaluate
the inhibitory activity of each calprotectin subunit on A nidulans
growth, we incubated conidia and hyphae with purified subunits
S100A8 and S100A9.17Addition of S100A8 did not inhibit fun-
gal growth, whereas S100A9 had a small effect on conidia germi-
nation but not on hyphae growth (Fig 2, A and B; see this article’s
Fig E1 in the Online Repository at www.jacionline.org). Only
J ALLERGY CLIN IMMUNOL
2 BIANCHI ET AL
both subunitstogether strongly inhibited A nidulans growth, indi-
cating that the heteroduplex is required for full inhibition of
Aspergillus spp, consistent with a report on its anticandidal activ-
ity.18In all cases, addition of 1 mmol/L zinc (Zn21) restored fun-
gal growth, confirming the putative role of Zn21chelation by
calprotectin in suppressing fungal growth.18-20In addition, we
preincubated combined S100A9 and S100A8 with a polyclonal
a-S100A9 antibody. A nidulans growth of conidia and hyphae
was restored in the presence of specific antibody (Fig 2, C and
D; see this article’s Fig E2 in the Online Repository at www.
jacionline.org), confirming the requirement for interaction of
both calprotectin subunits for antifungal activity. Unspecific con-
trol antibody did not have any effect. Here we used an excess
amount of 5 mg/mL purified S100A8 and S100A9, but similar in-
tectin amount found in NETs from about 4 million neutrophils.4
FIG 1. NET quantification in CGD gp91phox1and gp91phox2sorted neutrophils. A, Neutrophils were sorted
by magnetic beads and autoMACS. B, NET formation was quantified after PMA activation of neutrophils by
staining of released NET-DNA with Sytox green. Data are means 6 SDs of representative triplicate experi-
ments. The difference between control and gp91phox1neutrophils was nonsignificant (P > .05).
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BIANCHI ET AL 3
Antifungal activity of CGD gp91phox1cells is
To study whether restored antifungal activity of CGD
gp91phox1neutrophils after GT was attributable to calprotectin,
we PMA-stimulated gp91phox1and gp91phox2neutrophils to in-
duce NETs, and coincubated with A nidulans conidia and hyphae
6 Zn21. Only gp91phox1neutrophils showed strong antifungal
activity, equivalent to control (Fig 3, A and B; see this article’s
Fig E3 in the Online Repository at www.jacionline.org).
gp91phox2neutrophils did not form NETs (Fig 1, B), yet partially
inhibited conidia germination, probably by phagocytosis,21but
were inefficient against hyphae. Full restoration of A nidulans
growth by addition of Zn21supports the assumption that NET
antifungal activity was calprotectin-dependent (at calprotectin
concentrations of 450-500 ng/mL, measured by ELISA).
Restoration of A nidulans conidia and hyphae growth by Zn21
addition was dose-dependent (see this article’s Fig E4 in the On-
line Repository at www.jacionline.org). Addition of Mn21, Fe21,
and Cu21(0-2.5 mmol/L), however, did not restore fungal growth
(not shown), indicating that Zn21binding by calprotectin has a
major NET anti-Aspergillus activity. Because addition of metal
ions can have pleiotropic effects, we applied an a-S100A9
FIG 2. Inhibition of A nidulans growth by purified calprotectin. A and B, Conidia and hyphae incubated with
5 mg/mL S100A8 and/or S100A9 6 1 mmol/L Zn21. C and D, Conidia and hyphae incubated with 5 mg/mL
S100A8 and S100A9 6 15 mg/mL polyclonal a-S100A9 antibody or unspecific control antibody. Data are
means 6 SDs of representative triplicate experiments.
FIG 3. Calprotectin-dependent antifungal activity of CGD gp91phox1neutrophils. A nidulans conidia or hy-
phae incubated with NETs (A and B) or NET supernatant (C and D) from control and CGD-sorted neutrophils
6 1 mmol/L Zn21or 15 mg/mL a-S100A9, respectively. E, Wild-type (WT) and S100A9–/–mouse NETs infected
with A nidulans conidia or hyphae. Data are means 6 SDs of representative triplicate experiments.
J ALLERGY CLIN IMMUNOL
4 BIANCHI ET AL
antibody to block calprotectin-mediated inhibition in concen-
trated NET extracts, subsequently incubated with A nidulans co-
nidia or hyphae. NETextracts from gp91phox1neutrophils in the
presence of unspecific control antibody showed strong antifungal
in the Online Repository at www.jacionline.org). Supernatants
from non–NET-forming gp91phox2neutrophils in the presence
of unspecific control antibody did not have antifungal activity,
even against conidia germination, supporting the hypothesis
that their low antifungal activity observed against conidia (Fig
3, A) was a result of phagocytosis.
We then tested whether NET-mediated A nidulans inhibition
calprotectin-deficient mouse neutrophils. NETs were PMA-
induced in mature neutrophils from both mouse strains and incu-
bated with A nidulans conidia and hyphae. Indeed, NETs from
calprotectin-deficient neutrophils did not prevent A nidulans
growth, whereas NETs from wild-type neutrophils did (Fig 3,
E), although the amount of NET formation was similar in
calprotectin-deficient and wild-type neutrophils.4Complete res-
toration of fungal growth in the presence of a-S100A9 antibody
in human gp91phox1and control NET extracts and the inability
of calprotectin-deficient murine NETs to block fungal growth
confirmed the essential antifungal role of NET-associated calpro-
tectin against A nidulans.
Antifungal effect of calprotectin
To determine whether calprotectin-mediated inhibition of A
nidulans growth within NETs was reversible or irreversible, we
incubated conidia and hyphae on NETs for 4 days to 4 weeks at
neutral pH, then added Zn21to restore fungal growth. NETs
strongly inhibited A nidulans conidia germination and hyphae
growth even after 4 weeks (Fig 4, A-P), indicating strong stability
stability against proteases.22Surprisingly, addition of Zn21re-
stored fungal growth even after 4 weeks, demonstrating that cal-
protectin has fungistatic but not fungicidal activity on A nidulans
at low concentrations (450-500 ng/mL, measured by ELISA).
We next tested whether antifungal activity was still reversible
at higher calprotectin concentrations that might occur invivo. We
incubated conidia and hyphae for 4 days with increasing concen-
trations of purified calprotectin S100A8 and S100A9, then added
at any concentration (Fig 4, Q, white bars), whereas hyphae were
slightly more resistant, showing a dose-dependent increase of
growth arrest between 0.5 and 4 mg/mL S100A8/A9 (Fig 4, R,
white bars). Fungal growth was not restored by Zn21when the
S100A8/A9 concentration was higher than 16 mg/mL (Fig 4,
Q-R, black bars). Light microscopy showed intact hyphae at
low S100A8/A9 concentrations (Fig 4, S), whereas in the pres-
ence of >16 mg/mL S100A8/A9, hyphae had a translucent cell
wall and a fragmented internal structure (Fig 4, T). Thus, we
demonstrate a previously unknown microbicidal effect against
A nidulans at high calprotectin concentrations.
A nidulans–induced NET formation is strictly
dependent on NADPH oxidase
We investigated whether the induction of NETs by A nidulans
is dependent on functional NADPH oxidase and is restored after
CGD GT. CGD gp91phox1and gp91phox2neutrophils were incu-
light microscopy, scanning electron microscopy, and immuno-
trol and gp91phox1neutrophils but, as expected, less strongly
compared with PMA stimulation. We observed that NET forma-
hyphae (Fig 5, A and B, black arrows). This might suggest that
close pathogen-neutrophil interaction promotes induction of
NETs. The molecular details behind this trigger mechanism
will be subject to further investigation. In contrast, gp91phox2
neutrophils did not form NETs when in contact with hyphae
(Fig 5, C, white arrowheads), underlining the role of functional
NADPH oxidase in NET formation and antifungal defense.
Analysis by scanning electron microscopy showed that A
nidulans hyphae were entangled by NETs from control and
neutrophils surrounded and wrapped around hyphae but did not
make NETs (Fig 5, F, I).
Calprotectin is NET-associated after A nidulans
The association of calprotectin and A nidulans–induced NETs
was studied by immunofluorescence and confocal microscopy,
staining DNA with DAPI and calprotectin and hyphae with pri-
mary antibodies. Colocalization of DNA (blue) and calprotectin
(red) demonstrates that calprotectin was released and associated
with NETs when control (Fig 5, J, M, P) and gp91phox1(Fig 5,
K, N, Q) neutrophils were incubated with A nidulans hyphae
(green). Moreover, we demonstrate that gp91phox2neutrophils
showed neither NET formation nor extracellular calprotectin on
A nidulans incubation (Fig 5, L, O, R), although they were able
ping around hyphae (Fig 5, R). DNAwas confined to the nucleus
(Fig 5, L) and calprotectin to the cytoplasmic compartment of
these cells (Fig 5, O).
our microscopic findings. Control, gp91phox1and gp91phox2neu-
trophils were activated with PMA for 4 hours, and calprotectin
release was determined by ELISA after NET formation and sub-
sequent digestion of NETs. Control and gp91phox1neutrophils
released approximately 4 times the calprotectin concentration
compared with gp91phox2neutrophils (Fig 6), supporting the
previous results. Differences in total cytoplasmic calprotectin
concentration among control, gp91phox1, and gp91phox2neutro-
phils were not statistically significant (data not shown). Thus,
we conclude that NADPH oxidase is required not only for the re-
lease of NETs but also for the release of calprotectin, the crucial
component for the inhibition of A nidulans hyphae.
Recent work suggests NET contribution to elimination of
fungal infections in healthy subjects because hyphae are too large
to be phagocytosed.23,24Patients with CGD are unable to make
NETs5,11and are susceptible to therapy-refractory infection
with Aspergillus spp.10Especially infections with A nidulans
are a major threat to these patients because of higher virulence
and frequent resistance to antifungal treatment compared to As-
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BIANCHI ET AL 5
FIG 4. Antifungal effect of calprotectin. A-P, Fungistatic activity: A nidulans conidia or hyphae incubated
with NETs for 4 days to 4 weeks, after which 1 mmol/L Zn21was added overnight. Q and R, Fungicidal ac-
tivity: A nidulans conidia or hyphae incubated with 0.5 (S) to 128 (T) mg/mL purified S100A8/A9 for 4
days, after which 50 mmol/L Zn21was added overnight.
J ALLERGY CLIN IMMUNOL
6 BIANCHI ET AL
FIG 5. A nidulans-induced NETs contain calprotectin. Control and CGD sorted neutrophils were infected
with hyphae, and NET formation was determined by bright field microscopy (A-C), scanning electron mi-
croscopy (D-I), and immunofluorescence and confocal microscopy (J-R). NET formation was determined
by DAPI (blue), calprotectin by staining with antibodies against S100A8/A9 (red), and hyphae with anti-
bodies against Aspergillus soluble proteins (green).
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BIANCHI ET AL 7
of NADPH oxidase by human CGD GT restored the ability of
neutrophils to release NETs and NET-associated calprotectin,
which is responsible for growth inhibition of A nidulans by
Zn21sequestration. A threshold of 27.8% of O2-production per
individual gp91phox1neutrophil and a chimerism of 22.8% of
oxidase-positive cells was enough for sufficient NET formation
in vitro and clinical clearance of multifocal therapy–refractory
A nidulans lung infection in a patient with CGD in vivo.
The S100 Ca21-binding protein calprotectin is a heteroduplex
of subunits S100A8 and S100A9 that is expressed by granulo-
cytes, monocytes, and early differentiation stages of macro-
phages.25It represents about 40 % of neutrophil cytoplasm,26
supernatants,27and has been shown to bind Zn21in vitro.28Its
chelation and is reversible by micromolar quantities of
Zn21.18,27,28We set out to study the role of calprotectin in innate
defense against CGD-relevant A nidulans. Growth of A nidulans
was strongly inhibited when coincubated with both purified
calprotectin subunits, which is in accordance with a report by
Sohnle et al17and with crystal structure29as well as mass
spectrometric analyses30suggesting that theCa21-dependent for-
mation of S100A8/A9 heterotetramers would lead to enhanced
Zn21-binding capacity, whereas none of the homodimers should
display significant affinity for Zn21.29
Our study demonstrates the major role of NET-associated
calprotectin in suppressing growth of A nidulans conidia and hy-
phae and shows the presence of calprotectin in NETs induced by
that could be prevented by Zn21.32However, the mechanism of
inhibition was not clearly shown in these reports. Here we show
that blocking of calprotectin by specific antibodies and the ab-
sence of calprotectin in NETs from calprotectin-deficient mouse
neutrophils completely abrogated the inhibition of A nidulans
conidia and hyphae growth. This is a more precise analysis
because NETs also contain other Zn21-binding proteins, such
as S100A12,33which is involved in antiparasite responses.
According to our findings, calprotectin has to be presented
extracellularly for antifungal activity against A nidulans. To the
best of our knowledge, leakage of a cytoplasmic protein into
phagolysosomes has not been described. We previously showed
that neutrophil degranulation stimulated by the bacterial peptide
formyl-Methionyl-Leucyl-Phenylalanine does not induce secre-
tion of calprotectin, indicating nonvesicular localization.4How-
ever, we cannot entirely exclude that minor amounts of
calprotectin are found in phagolysosomes and contribute to the
slight inhibitory effect we reported on CGD neutrophils infected
with A nidulans conidia.
We show that the presence of functional NADPH oxidase in
human neutrophils is an absolute requirement for A nidulans
growth inhibition by NET-associated calprotectin, because only
gp91phox1and not gp91phox2neutrophils made NETs containing
man CGD restored the amount of released calprotectin during
NET formation to wild-type levels, and NET-associated calpro-
tectin colocalized with A nidulans hyphae. Analysis of gp91phox2
the release of calprotectin during NET formation, which is an ad-
nylene iodonium chloride. We propose that NET formation is an
crete calprotectin into the extracellular compartment on infection
with Aspergillus hyphae, resulting in both NET-bound and free
calprotectin. NET-associated calprotectin is concentrated with
may in addition have distinct functions, such as chemotaxis or
The fact that gp91phox2neutrophils also partially inhibited
conidia germination suggests that NADPH oxidase–independent
mechanisms linked to phagocytosis, such as activity of the gran-
ule serine proteases NE and cathepsin G34-36(although the mech-
anism has been recently object of controversy37-39), might
contribute to early defense against Aspergillus. The need of NE
for NET formation14could explain the susceptibility to infection
of NE-deficient mice described by Reeves et al35as an indirect
action. Other mechanisms such as the tryptophan-catabolizing
enzyme indoleamine-2,3-dioxygenase, considered critical for
regulating immune responses and suppression of inflammation
in mice,40do not play a role in human CGD as we previously
Fungi have a high Zn21requirement (1027-1025mol/L) for
growth43with a low minimum inhibitory concentration of calpro-
tectin.44Because of the lack of a well developed extracellular
FIG 6. Calprotectin release by gp91phox1and gp91phox2neutrophils. Cal-
protectin release was quantified by ELISA after neutrophil activation by
PMA inducing NET formation. NETs were digested by MNase/DNase-1 to
quantify NET-associated calprotectin. Significance was assessed by the
Student t test: NS, not significant (P > .05); ***P < .001.
J ALLERGY CLIN IMMUNOL
8 BIANCHI ET AL
Zn21-scavenging system, A fumigatus is extremely sensitive to
Zn21deprivation.45The Zn21requirement for A nidulans in
our study was in the same range as described for other fungi. In
contrast with Staphylococcus aureus, which is more sensitive to
calprotectin-mediated Mn21deprivation,19we found that A
nidulans could resume growth only after Zn21supplementation,
but not after Mn21, Fe21, or Cu21addition. This supports the
central role of Zn21for A nidulans growth.
Interestingly, A nidulans hyphae were not killed by NETs at
low calprotectin concentrations in vitro, although hyphal struc-
tures seemed to be severely deranged by NETs. This corre-
sponded to the range of fungistatic calprotectin concentrations
much higher calprotectin concentrations have been observed in
human abscesses,27Aspergillus hyphae might still be killed by
NET-associated calprotectin in vivo. Killing of Candida spp or
Cryptococcus neoformans was demonstrated at calprotectin con-
centrations of 3 to 5 mg/mL.44We show for the first time that cal-
protectin concentrations > _16 mg/mL lead to irreversible growth
vation. In contrast with our findings, a previous publication
reported only minor antifungal activity of NETs against A
fumigatus,31probably because of a shorter incubation period. Al-
ternatively, complete in vivo killing might also be achieved by
other antimicrobial peptides46orby sofar unknown mechanisms:
in addition to their antimicrobial properties, S100A8, S100A9,
and S100A8/A9 are chemotactic for neutrophils and mono-
cytes.47-49Notably, for this function, calprotectin needs to be
extracellular, and thus Aspergillus-induced NET formation
supports it as well.
Here we establish that calprotectin released in NETs displays
concentration-dependent reversible and irreversible growth in-
hibitory activity against virulent A nidulans. NETs may be in-
volved in disarming A nidulans and may prevent further
spreading by providing high local concentrations of calprotectin.
Definitive in vivo proof in human beings is obviously ethically
impossible. In conclusion, we show that calprotectin is a critical
factor in the innate immune defense of human neutrophils to
Aspergillus infection and adds to the concept of metal chelation
as a strategy for inhibiting microbial growth at the sites of infec-
tibility to Aspergillus infection of patients with CGD might be
linked to absence of NETs, and restoration of NADPH oxidase
function and NET formation by GT leads to rapid cure of refrac-
tory invasive aspergillosis in X-linked CGD.
We thank the patient and his family for their trust. We are indebted to the
medical and nursing staff of the bone marrow transplantation unit of
University Children’s Hospital Zurich. We thank Reinhard Seger for his sup-
port and helpful discussions,and Manuel Grez and Klaus K€ uhlke fordevelop-
ing and providing the SF71gp91phoxvector, respectively. We are grateful to
Klaus Marquardt for electron microscopy, to Andres Kaech for help with con-
more, we acknowledge Thomas Vogl and Johannes Roth for supplying
S100A9 knockout mice.
Clinical implications: Gene therapy for CGD restores NET for-
mation and calprotectin release by neutrophils, leading to effi-
cient A nidulans inhibition. Calprotectin is a critical factor in
human innate immune defense against Aspergillus infection.
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10 BIANCHI ET AL
Patient description and GT
We treated a boy 8 years 7 months old with X-linked gp91phox-deficient
ocistronic long terminal repeat–driven g-retroviral SF71gp91phoxvector (see
referenceE1for details) under a protocol approved by the ethics review board
of the University Children’s Hospital Zurich and the Swiss Expert Committee
for Bio-Safety after written informed consent of the parents. Collection of
CD341cells, transduction, conditioning with low-dose intravenous busulfan
(8.8 mg/kg), and clinical follow-up were performed as described.E2The A
nidulans lung infection was completely cleared 6 weeks after GT. Therapy/
prophylaxis with oral voriconazole was continued throughout and has not
yet been tapered 2.6 years after GT. To avoid interactions, voriconazole was
stopped 4 days before Aspergillus experiments and continued thereafter. At
the time of analysis, the patient was free of any infection, and 22.8% of
neutrophils expressed gp91phox, with an O22production of 27.8% per
gp91phox-expressing cell (relative to healthy controls).E3Clinical and molec-
ular follow-up of GT in this patient will be described elsewhere.
A nidulans strain
The A nidulans strain used was isolated from bronchoalveolar lavage fluid
of the patient; conidiaweregrown and collected as described.E4For all exper-
iments, conidia and hyphae were grown in serum-free RPMI medium (phenol
red–free) supplemented with 10 mmol/L HEPES. If not stated otherwise,
fungal growth was quantified in all assays with the tetrazolium dye 2,3-bis
Invitrogen, Molecular Probes, Leiden, The Netherlands) as described.E5
This method, unlike colony-forming unit enumeration, does not require
dispersion of the hyphae.
Isolation of human and murine neutrophils
Human neutrophils were isolated from peripheral blood of healthy donors
or the patient with CGD by using dextran-Ficoll (GE Healthcare, Munich,
Germany)E6for all NET assays with A nidulans and for autoMACS sorting,
whereas Percoll (GE Healthcare) density gradient separationE7was used for
NET quantificationassays. Forall experiments,neutrophils wereresuspended
HEPES and used within 1 hour after isolation.
Murine neutrophils were isolated from S100A92/2mice backcrossed 10
times into C57 BL/6. These mice are deficient in both calprotectin subunits
S100A8 and S100A9 protein.E8Micewere bred in our animal facility accord-
ing to regulations of the Jordbruksverket Sweden (Dnr A29-69). Mature mu-
rine neutrophils were isolated from bone marrow as previously described.E9
Briefly, bone marrow cells from tibia and femur were singularized by using
a 70-mm cell strainer and separated by centrifugation for 30 minutes at
1500g on a discontinuous Percoll gradient with 52% (vol/vol), 69% (vol/
69% and 78% were resuspended in HBSS without Ca21and Mg21.
Sorting of CGD gp91phox1and gp91phox2
Control and patient neutrophils were first stained for 30 minutes at room
temperature with 10 mg/mL mouse a-gp91phox–fluorescein isothiocyanate
antibody (clone 7D5; MBL, Woburn, Md), then 30 minutes at 48C with
anti–fluorescein isothiocyanate MicroBeads (Miltenyi Biotec, Bergisch
Gladbach, Germany) for magnetic separation by autoMACS (Miltenyi
Biotec), according to the manufacturer’s instructions. Purity of the resulting
gp91phox2and gp91phox1populations was directly measured by the
fluorescent-activated cell sorter.
A nidulans growth inhibition by purified
In a 96-well plate, 104A nidulans conidia or hyphae obtained from culture
of 104conidia were incubated 24 hours at 378C with 5 mg/mL recombinant
S100A8 and/or 5 mg/mL recombinant S100A9 (ProtEra, Florence, Italy) 6
polyclonal a-S100A9 antibody (H00006280-D01P; Abnova, Taipei, Taiwan)
versus unspecific control antibody (Sigma-Aldrich).E10This a-S100A9 anti-
plexed with S100A8. Fungal growth is expressed as percentage of control
values (A nidulans conidia or hyphae incubated in media without neutrophils
6 ZnSO4, S100A8/A9, or antibodies).
A nidulans growth inhibition by NETs
In a 96-well plate, 105control, CGD gp91phox2, and gp91phox1sorted neu-
CO2for 4 hours until NET formation was complete. Zero to 2 mmol/L ZnSO4
(Sigma-Aldrich) and A nidulans conidia or hyphae (hyphae previously grown
a cell scraper) with a multiplicity of infection A nidulans/neutrophils of 0.1
were then added, in a final volume of 160 mL. Afterward, the plates were
conidia germination and hyphal outgrowth. Fungal growth is expressed as
percentage of control values (A nidulans conidia or hyphae incubated in
media 6 ZnSO4).
A nidulans growth inhibition by NET extracts
Neutrophil extracellular trap formation was induced in 8 3 105control,
CGD gp91phox2, and gp91phox1neutrophils by activation with 40 nmol/L
PMAat 378C and 5%CO2for5 hours in a 24-wellplate(500mL/well;4wells
digested for 30 minutes with 5 U/mL MNase (Worthington Biochemical,
Lakewood, NJ) 1 1 U/mL DNase-1 (Sigma-Aldrich) and pooled supernatant
concentrated ;10-fold by centrifugation on filter columns with a 3-kd cutoff
(Amicon;Millipore, Zug, Switzerland), following the manufacturer’sinstruc-
tions. In a 96-well plate, 104A nidulans conidia or hyphae obtained from cul-
ture of 6 3 104A nidulans conidia were then incubated for 24 hours at 378C
withthe concentrated NETextracts (diluted 1/3)with or without 15 mg/mLa-
is expressed as percentage of control values (A nidulans conidia or hyphae
incubated in media 6 antibodies).
A nidulans growth inhibition by murine S100A92/2
In a 24-well plate, 5 3 105mouse neutrophils in 500 mL RPMI with 1%
(vol/vol) mouse serum were stimulated with 100 nmol/L PMA for 20 hours
at 378C with 5% CO2to induce NET formation. The supernatant was dis-
carded; NETs were washed once with RPMI and incubated with 500 mL
RPMI containing A nidulans conidia or hyphae at multiplicities of infection
0.1 and 0.01, respectively. Afterward, plates were centrifuged for 5 minutes
centage of control values (A nidulans conidia or hyphae incubated in media).
Fungistatic and fungicidal effect of calprotectin
In a 96-well plate, 104A nidulans conidia or hyphae obtained from culture
of 104conidia were incubated on previously formed NETs (from 105neutro-
phils activated with 40 nmol/L PMA at 378C and 5% CO2for 4 hours) or with
0.5 to 128 mg/mL purified S100A8/A9. After 4 days to 4 weeks, RPMI or
ZnSO4(final concentration, 1 mmol/L for NETs and 50 mmol/L for purified
S100A8/A9) were added to the wells and incubated overnight. Growth of A
(Leica DM IL HC Fluo, Wetzlar, Germany) coupled to a CCD camera (Leica
DFC 480 R2).
considered statistically significant when P < .05. All statistical tests were
performed by using GraphPad Prism.
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BIANCHI ET AL 10.e1
E1. Bianchi M, Hakkim A, Brinkmann V, Siler U, Seger RA, Zychlinsky A, et al.
Restoration of NET formation by gene therapy in CGD controls aspergillosis.
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and aggregation in the murine lung inhibit germination of Aspergillus fumigatus
conidia. Infect Immun 2006;74:6528-39.
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assessment of fungal cell damage with the tetrazolium dye, (2,3)-bis-(2-me-
thoxy-4-nitro-5-sulphenyl)-(2H)-tetrazolium-5-carboxanil ide (XTT). J Infect
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oxygen-dependent extracellular killing of Escherichia coli S15 by human
polymorphonuclear leukocytes. J Clin Invest 1985;76:206-12.
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by the intracellular parasite Leishmania major. J Immunol 2002;169:
E8. Manitz MP, Horst B, Seeliger S, Strey A, Skryabin BV, Gunzer M, et al. Loss of
S100A9 (MRP14) results in reduced interleukin-8-induced CD11b surface
expression, a polarized microfilament system, and diminished responsiveness to
chemoattractants in vitro. Mol Cell Biol 2003;23:1034-43.
E9. Ermert D, Urban CF, Laube B, Goosmann C, Zychlinsky A, Brinkmann V. Mouse
neutrophil extracellular traps in microbial infections. J Innate Immun 2009;1:
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ity of recombinant calprotectin (migration inhibitory factor-related proteins 8 and
14). J Infect Dis 2000;182:1272-5.
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10.e2 BIANCHI ET AL
FIG E1. Inhibition of A nidulans growth by purified calprotectin is abolished by Zn21addition. Incubation
with the S100A8 (5 mg/mL) subunit did not prevent A nidulans conidia or hyphae growth, whereas incuba-
tion with S100A9 (5 mg/mL) partially inhibited conidia germination. Coincubation with both subunits
strongly inhibited conidia germination and hyphae growth. In all cases, antifungal activity was completely
abolished in presence of 1 mmol/L Zn21.
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FIG E2. Inhibition of A nidulans growth by purified calprotectin is abolished by a-S100A9 antibody. A total
of 15 mg/mL polyclonal a-S100A9 antibody fully inhibited antifungal activity of the S100A8/A9 calprotectin
complex on conidia or hyphae. As control, S100A8/A9 was incubated with an unspecific control antibody,
showing no effect on antifungal activity of the S100A8/A9 calprotectin complex. w/o, Without.
J ALLERGY CLIN IMMUNOL
10.e4 BIANCHI ET AL
FIG E3. Calprotectin-dependent antifungal activity of CGD gp91phox1neutrophils is abolished by Zn21 Download full-text
addition. Control and CGD sorted neutrophils were stimulated with PMA for NET formation and incubated
overnight with A nidulans conidia or hyphae in presence or absence of 1 mmol/L Zn21. gp91phox1neutro-
phils showed strong antifungal activity against conidia or hyphae, comparable to control. gp91phox2neutro-
phils were inefficient in controlling fungal growth, with only partial inhibition of conidia germination. In all
cases, antifungal activity was completely abolished in the presence of 1 mmol/L Zn21.
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