Article

Staphylococcus aureus Panton-Valentine

leukocidin induces an inflammatory
response in human phagocytes via the
NLRP3 inflammasome
Dirk Holzinger,*, Laura Gieldon,* Vijayashree Mysore,* Nadine Nippe,* Debra J. Taxman,,
Joseph A. Duncan,,, Peter M. Broglie, Kristina Marketon,* Judith Austermann,* Thomas Vogl,*
Dirk Foell,* Silke Niemann,# Georg Peters,# Johannes Roth,*
and Bettina Lffler#,1
*Institute of Immunology, Department of General Pediatrics, University Childrens Hospital Mnster, University of Mnster,
Mnster, Germany; Department of Microbiology and Immunology, Lineberger Comprehensive Cancer Center, and Departments of

Medicine, Division of Infectious Diseases, and Pharmacology, University of North Carolina, Chapel Hill, North Carolina, USA; and
#
Institute of Medical Microbiology, University Hospital of Mnster, Mnster, Germany
RECEIVED JANUARY 12, 2012; REVISED JULY 9, 2012; ACCEPTED JULY 23, 2012. DOI: 10.1189/jlb.0112014

ABSTRACT
The Staphylococcus aureus pore-forming toxin PVL is
most likely causative for life-threatening necrotizing infections, which are characterized by massive tissue inflammation and necrosis. Whereas the cytotoxic action of
PVL on human neutrophils is already well established, the
PVL effects on other sensitive cell types, such as monocytes and macrophages, are less clear. In this study, we
used different types of human leukocytes (neutrophils,
monocytes, macrophages, lymphocytes) to investigate
cell-specific binding of PVL subunits and subsequent proinflammatory and cytotoxic effects. In all PVL-sensitive
cells, we identified the binding of the subunit LukS-PV as
the critical factor for PVL-induced cytotoxicity, which was
followed by binding of LukF-PV. LukS-PV binds to
monocytes, macrophages, and neutrophils but not to
lymphocytes. Additionally, we showed that PVL binding to monocytes and macrophages leads to release

of caspase-1-dependent proinflammatory cytokines

IL-1 and IL-18. PVL activates the NLRP3 inflammasome, a signaling complex of myeloid cells that is
involved in caspase-1-dependent IL-1 processing in
response to pathogens and endogenous danger signals. Specific inhibition of this pathway at several
steps significantly reduced inflammasome activation
and subsequent pyronecrosis. Furthermore, we found
that PAMPs and DAMPs derived from dying neutrophils can dramatically enhance this response by upregulating pro-IL-1 in monocytes/macrophages. This
study analyzes a specific host signaling pathway that
mediates PVL-induced inflammation and cytotoxicity,
which has high relevance for CA-MRSA-associated
and PVL-mediated pathogenic processes, such as
necrotizing infections. J. Leukoc. Biol. 92: 1069 1081;
2012.

Introduction
Abbreviations: -toxin-hemolysin, ASCapoptosis-associated speck-like
protein, BMDMbone marrow-derived macrophage, CA-MRSA
community-acquired methicillin-resistant Staphylococcus aureus, CTSB
cathepsin B, DAMPdanger-associated molecular pattern, EVempty
vector, HKSAheat-killed Staphylococcus aureus, HMGB1high-mobility
group protein B1, LTAlipoteichoic acid, LukF-PVsubunit F (F for fast)
of Panton-Valentine leukocidin, LukS-PVsubunit S (S for slow) of
Panton-Valentine leukocidin, MFImean fluorescence intensity, MRP8/
14myeloid-related protein complex 8/14, NLRP3nucleotide-binding domain and leucine-rich repeat-containing gene family, pyrin domain containing 3 protein, PAMPPathogen associated molecular pattern,
PRRpattern recognition receptors, PSMphenol soluble modulins (expressed by Staphylococcus aureus), PVLPanton-Valentine leukocidin
(Staphylococcus aureus pore-forming toxin), shshort hairpin,
shASCmutscrambled sequence with base content equal to short hairpin
apoptosis-associated speck-like protein, shGFPshort hairpin RNA against
GFP, siRNAsmall interfering RNA, zYVAD-fmkz-Tyr-VAD-fmk
The online version of this paper, found at www.jleukbio.org, includes
supplemental information.

S. aureus is an important human pathogen that can cause a wide

variety of infections, ranging from superficial skin infections to
invasive diseases, such as soft tissue infections, pneumonia, osteomyelitis, or sepsis [1]. To establish infections, S. aureus expresses
a multitude of virulence factors, including secreted toxins, enzymes, and peptides (e.g., PSMs), as well as surface proteins (e.g.,
protein A) and wall components (e.g., LTA), that have been described to contribute to disease development [1, 2]. Particularly,
the pore-forming toxins, -toxin [3] and PVL [4], have been implicated in the pathogenesis of severe S. aureus infections. These
proteins are secreted as monomers, which insert into host cell
membranes and assemble into heptameric structures that pene-

1. Correspondence: Institute of Medical Microbiology, University Hospital of

Mnster, Domagkstrasse 10, 48149 Mnster, Germany. E-mail: loeffleb@
uni-muenster.de

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trate cell membranes [5]. In general, -toxin is supposed to be a

crucial toxin of S. aureus, which is expressed by the majority of
clinical isolates, and its contribution to virulence has been demonstrated in various invasive disease models, such as pneumonia,
soft tissue infections, and sepsis [6 8]. By contrast, PVL is a bicomponent leukotoxin composed of two distinct proteins
LukS-PV and LukF-PVwhich target defined cells of the immune
system, mainly neutrophils from humans and rabbits [5, 9, 10].
Only a small percentage of S. aureus isolates (3%) carries the
gene for PVL [11], but PVL-positive S. aureus strains have been
associated with severe necrotizing diseases, such as necrotic cutaneous lesions and necrotizing hemorrhagic pneumonia [12, 13].
In this respect, a rapidly progressive hemorrhagic and necrotizing
pneumonia with severe leukopenia in children and young adults
has been reported. This type of S. aureus pneumonia seems to be
a specific disease entity with a high lethality rate. Because of the
inflammatory and necrotic histopathological appearance of the
lung, the illness was designated S. aureus hemorrhagic necrotizing pneumonia [12, 14].
Since the early 1990s, the number of MRSA strains has risen
dramatically, which emerged as a leading cause of hospital-acquired infections. Additionally, there has been an alarming increase in the number of CA-MRSA infectionsmainly severe skin
infectionsworldwide [15]. In the United States, both MRSA
clones that are most closely associated with community outbreaks,
USA300 and USA400, contain pvl genes, as do other successfully
CA-MRSA clones, e.g., an ST80 clone in Europe and an ST30
clone in Australia and the United States [16 19]. Despite a clear
epidemiological association between the pvl genes and severe,
often recurrent primary skin infections or necrotizing pneumonia
[12, 13], a definite pathogenic role for PVL in disease development could not be determined, as murine models are not adequate to study the pathogenicity of PVL [9]. Whereas recent
work in PVL-sensitive disease models (human cells, rabbits)
clearly supports a pathogenic function of PVL in severe necrotizing diseases [9, 20, 21], the precise mechanisms that underlie the
PVL-induced inflammation and tissue destruction are largely unknown.
During necrotizing diseases, there are massive inflammation
and influx of immune cells to the infection side [12, 22] that
cannot be explained by PVL-induced cytotoxicity on neutrophils
alone [22]. PVL induces the release of proinflammatory mediators from neutrophils such as histamine, leukotriene B4, and IL-8
[2325], but research of proinflammatory processes has been
focused mostly on neutrophils, whereas there is binding to monocytes and macrophages as well [10]. A recent study comparing
the release of proinflammatory mediators from PBMCs by PVL
and other staphylococcal toxins indicated a role of all different
cell types [26]. This is of great importance, as the activation of
monocytic cells can lead to a strong proinflammatory reaction
with the secretion of cytokines, such as IL-1.
IL-1 has multiple biologic effects including the ability to increase the expression of proinflammatory cytokines (IL-1R antagonist, TNF, IL-6, and IL-37), mediators (iNOS, type-2 COX-2 and
phospholipase A2, G-CSF, and GM-CSF), and adhesion molecules
(ICAM-1, VCAM-1, and VEGF). Moreover, IL-1 mediates the
activation of neutrophils to eliminate invading pathogens [27,
28]. The secretion of IL-1 is tightly regulated, and two separate
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signals are necessary to induce the full range of activation [29].

IL-1 mRNA is induced via activation of PRRs (triggered by
pathogen- and host-derived molecules) and leads to synthesis of
pro-IL-1 protein. A second signal, e.g., a K-ionophore, activates
caspase-1, a protease that cleaves pro-IL-1 into the biologically
active, mature IL-1 [29]. The activation of caspase-1 is mediated
by a cytosolic protein complex, the inflammasome [30]. So far,
different inflammasomes have been described, including the
NLRP3 inflammasome [31]. Compounds that activate the NLRP3
receptor include pore-forming toxins, e.g., nigericin and S. aureus
-toxin [32, 33]. Activation leads to the oligomerization and recruiting of caspase-1 through the adaptor protein ASC, thus
forming the active NLRP3 inflammasome complex [31]. NLRP3
receptors are activated by diverse stimuli with variable structures,
suggesting that the NLRP3 receptor senses them indirectly.
Therefore, cellular stress signals could serve as intermediate steps
in NLRP3 activation [31, 34]. For instance, the K-ionophore
nigericin induces an activation of the inflammasome that depends on the release of the lysosomal cysteine protease CTSB
[35, 36]. CTSB is not only an activator of the NLRP3 inflammasome but also induces a caspase-1-independent programmed
necrotic cell death [36].
It has already been shown that living S. aureus and -toxin
from S. aureus can activate the NLRP3 inflammasome [32, 33],
but the role of PVL in inflammasome activation has not been
elucidated yet. Here, we demonstrate that PVL is a strong inducer of IL-1 secretion via a CTSB-mediated activation of the
NLRP3 inflammasome, which likely contributes to the severe inflammation associated with necrotizing infections.

MATERIALS AND METHODS

Ethics statement
Taking of blood samples from humans and animals and cell isolation were
conducted with approval of the local ethics committee (Ethik-Komission der
rztekammer Westfalen-Lippe und der Medizinischen Fakultt der WestflisA
chen Wilhelms-Universitt Mnster; Az. 2008-034-f-S). Human blood samples
were taken from healthy blood donors, who provided written, informed consent for the collection of samples and subsequent neutrophil isolation and
analysis. All animals were handled in strict accordance with good animal practice and animal keeping, according to European guidelines (86/609/EWG),
and in strict accordance with the German Protection of Animal Act (Deutsches
Tierschutzgesetz). Taking of blood samples was supervised by the veterinary
office of Mnster (Veterinramt der Stadt Mnster; Az. 39.32.7.1).

Preparation and culture of professional phagocytes

Buffy coats were obtained from healthy human donors (Red Cross Blood Service, Mnster, Germany). PBMCs and lymphocytes were isolated from buffy
coats using a Ficoll-Paque density gradient (Amersham Biosciences, GE Healthcare Biosciences, Piscataway, NJ, USA), as described previously [37]. PBMCs
were cultivated from McCoys medium supplemented with 2 mM L-glutamine,
penicillin/streptomycin, and 15% FCS in Teflon bags and allowed to rest for
24 h prior to stimulation. Human MDMs were obtained from PBMCs. Briefly,
PBMCs were cultured in Teflon bags in RPMI 1640 supplemented with 2 mM
L-glutamine, penicillin/streptomycin, and 10% human serum for 7 days with
fresh medium changed every 2 days. After 7 days, cells were plated, and 24 h
later, nonadherent cells were removed by washing with complete medium before the experiment. Human polymorphonuclear cells (neutrophils) were
freshly isolated from Na citrate-treated blood of healthy donors and isolated by
dextran sedimentation and Ficoll-Paque density gradient. Neutrophils were

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Holzinger et al. S. aureus PVL-induced inflammasome activation

resuspended at a final density of 1 106 cells/0.5 ml in RPMI 1640 supplemented with 10% heat-inactivated FCS and used immediately for the experiments. All incubations were performed at 37C in humidified air with 5%
(monocytes) or 7% CO2 (all other cell types).

Stimulation of phagocytes
Primary monocytes, macrophages, or neutrophils were cultured on 24-well or
six-well plates and subjected to PVL for 15 min16 h at 37C under 5%
(monocytes) or 7% CO2 (all other cell types). Where indicated, 100 ng/ml
ultrapure LPS from Escherichia coli (InvivoGen, San Diego, CA, USA; serotype
0111:B4), 25 M caspase-1 inhibitor zYVAD-fmk (Enzo Life Sciences, Germany), 30 M CTSB inhibitor CA-074Me (Enzo Life Sciences), or 130 mM
KCl was added to the incubation medium. For the costimulation experiments,
HKSA from strain 6850 (80C for 30 min), LTA (Sigma-Aldrich, St. Louis,
MO, USA), MRP8, or S100A12, manufactured at our institute [38, 39], was
used. After the incubations, culture media were collected for analysis of their
cytokine contents, and cells were washed and lysed for RNA analysis or Western blots.

Staphylococcal virulence factors

The six-His-tagged LukS-PVL and LukF-PVL proteins from E. coli were purified
by nickel-nitrilotriacetic acid affinity resin (Qiagen, Germany), as described
previously [9]; -toxin and Protein A (P3838) were obtained from Sigma-Aldrich Chemie GmbH (Germany). PSM1 and PSM2 were synthesized by
Genosphere Biotechnology (France).

out FCS) were harvested and precipitated with TCA, protein pellets were resuspended in SDS-PAGE buffer. Cell lysates were prepared for Western blot analysis as described earlier [42]. Western blot analysis was performed by standard
procedures with the following antibodies: human anticleaved-IL-1 antibody
(2021S; Cell Signaling Technology, Danvers, MA, USA) was used at 1/1000
dilution, anti-IL-1 antibody (R&D Systems) was used at 1/500 dilution, and
-actin antibody (Sigma-Aldrich Chemie GmbH) was used at 1/500 dilution.
Cell lysates of THP1-knockdown cells were analyzed with human anti-ASC
(AL177; Enzo Life Sciences), dilution 1/1000; anti-NLRP3 (Cryo-2; Enzo Life
Sciences), dilution 1/1000; anti-CTSB (Enzo Life Sciences), dilution 1/1000;
and anti-HMGB1 (4C3; Abcam, Cambridge, MA, USA), dilution 1/1000.

Quantitative RT-PCR
After stimulation, RNA was isolated from the cells, and expression of IL-1
and TNF- RNA was analyzed by real-time RT-PCR, as described previously
[42]. Each measurement was set up in duplicate, and three independent experiments were performed. After normalization to the endogenous housekeeping control gene GAPDH, the relative expression was calculated. The primers
used for PCR analysis were: IL-1 forward, 5=-GCGGCCAGGATATAACTGACTTC-3=, and IL-1 reverse, 5=-TCCACATTCAGCACAGGACTCTC-3=; TNF
forward, 5=-CTTCTCGAACCCCGAGTGAC-3=, and TNF reverse, 5=-TGAGGTACAGGCCCTCTGATG-3=; GAPDH forward, 5=-TGCACCACCAACTGCTTAGC3=, and GAPDH reverse, 5=-GGCATGGACTGTGGTCATGAG-3=.

Stable, transfected THP-1 cells

Labeling of PVL
LukS-PV and LukF-PV were labeled with fluorescein (LukS-FITC and LukFFITC) as follows. The proteins were dialyzed against PBS and dialyzed further
against 0.1 M NaCO3, pH 9, at 4C. Protein concentration was measured, and
proteins were incubated further with a tenfold excess of FITC (Sigma-Aldrich
Chemie GmbH) for 2 h at room temperature. NH4Cl was added to a final
concentration of 50 mM and incubated for 2 h at room temperature. The
FITC-coupled PVL subunits were separated from free FITC by a G25 column.
The solution was again dialyzed against PBS for 3 h at 4C. Absorption was
measured at 280 nm and 495 nm. The coupling rate had to be between 0.3
and 1.0 and was calculated as follows: molar FITC/PVL MWPVL/MWFITC
[A495/195/(A2800.35A495/PVL)].

Analysis of cytokine secretion

Human IL-1, IL-18, TNF-, and p20 subunit of caspase-1 were quantified
in the culture media samples using commercial ELISA according to the
manufacturers protocols (IL-1 and TNF- ELISAs were from BD Biosciences, Heidelberg, Germany; IL-18 and caspase-1 from R&D Systems, Minneapolis, MN, USA). Concentrations of MRP8/14 and S100A12 were determined by an in-house ELISA, as described previously [37].

Analysis of cell death

Cells were stained with PI (Sigma-Aldrich Chemie GmbH) to detect necrosislike membrane damage by flow cytometry. Cell death was quantified further by
determination of lactate dehydrogenase activity in the medium at the end of
exposure periods as described earlier [40].

Analysis of CTSB activation

Monocytes were incubated with or without PVL as described above. All cells
were then incubated with Magic Red CTSB substrate (ImmunoChemistry
Technologies, Bloomington, MN, USA) for 15 min. The levels of fluorescent
Magic Red CTSB substrate present in the cells were quantified using flow cytometry as described previously [41].

Western blotting
IL-1 processing was analyzed in a serum-free culture medium sample corresponding to 2 106 cells. After 3 h of treatment supernatants (McCoys with-

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THP1 cells (American Type Culture Collection, Manassas, VA, USA) were cultured in RPMI, 10% FCS. shRNAs for knockdown of ASC (shASC), NLRP3
(shNLRP3), CTSB, or an EV and shASCmut and shGFP as negative controls
were stably introduced using retrovirus as described previously [4345]. Efficient knockdown of targeted proteins was confirmed by Western blotting. To
assess effects of knockdown, cells were plated at 106 cells/ml. Equal amounts
of LukS-PV and LukF-PV were added to yield a final concentration of 210
nM, and cells were incubated for 24 h. Cell supernatants were collected and
assayed for IL-1 levels. Results represent the average plus sd of triplicate wells
and are representative of three independent experiments.

Plasmid construction and transformation

To create Staphylococcus carnosus strains that express virulence factors of S. aureus, we used two basic vectors: the xylose-inducible pXR100 and the
pNXR100, which is a noninducible derivate of the pXR100. For the expression
of LukS-PV and LukF-PV in E. coli TG1, the commercial IPTG-inducible
pQE30UA was used. For creation of the expression vectors, the respective
genes were amplified by PCR, purified, and digested. The basic vectors were
also digested corresponding to the genes. After ligation, S. carnosus TM300 and
E. coli were transformed by protoplast transformation or the CaCl method.

Bacterial strains and cultures

TM300 strains were characterized for the presence of genes encoding PVL
by PCR and by Western blots, as described recently [9, 46]. For cell culture
experiments with live staphylococci, bacteria were grown overnight at 37C
in Mller-Hinton medium (containing antibiotics/xylose, if mutants are
used) without shaking. Bacteria were washed in PBS and resuspended in
PBS with 1% HSA. Monocytes were incubated with bacterial suspensions,
resulting in a MOI as indicated. Bacterial supernatants were prepared by
growing bacteria in 5 ml brain-heart infusion broth (Merck, Rahway, NJ,
USA) in a rotary shaker (160 rpm) at 37C for 1214 h and pelleted for 10
min at 3350 g. Supernatants were sterile-filtered through a Millex-GP filter
unit (0.22 m; Millipore, Billerica, MA, USA) and used for the experiments. For PVL isolation, E. coli TG1 strains containing expression vectors
for LukS-PV and LukF-PV were grown in LB media with IPTG (1 mM) and
ampicillin (100 mg/ml), and cell lysates were used to purify PVL as described recently [9].

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Mice
Eight- to 12-week-old C57BL/6 mice were obtained from Harlan Winkelmann (Borchen, Germany). Animals were maintained under specific pathogen-free conditions and according to the institutional guidelines in the animal facility of the University of Mnster, Institute of Immunology.

Generation of BMDMs
BMDMs were differentiated in vitro from BM cells, as described previously
[47]. Briefly, to generate macrophages, BM cells were cultured in DMEM
(Invitrogen, Karlsruhe, Germany) containing 2 mM L-glutamine (Invitrogen), 1 mM nonessential amino acids (Invitrogen), 100 mg/ml penicillin/
streptomycin (Biochrom, Berlin, Germany), 10% heat-inactivated FCS (Biochrom), and 50 ng/ml M-CSF for 6 days.

Stimulation of BMDMs
Macrophages (1106/ml) were seeded into 96-well tissue-culture plates
(Greiner Bio One, Frickenhausen, Germany) and treated with different
stimuli: 1 g/ml ultrapure LPS from E. coli (InvivoGen; serotype 0111:B4),
4 g/ml PVL, and 20 M nigericin. After 6 h, supernatants were harvested,
and cytokine production was determined using CBA technology (BD Biosciences), according to the manufacturers instructions. CBA FlexSets were
used for IL-1 and TNF.

Statistical analysis
Statistical analyses were performed by using Prism 5.0 software (GraphPad
Software, La Jolla, CA, USA). Analyses between two groups were performed
by using an unpaired two-tailed Students t test. Comparisons among three
or more groups were performed by using one-way ANOVA, followed by
Dunnetts multiple means test for comparing all columns versus control or
by Bonferronis multiple means test for comparing all pairs of columns.
Differences were considered statistically significant at a P value of 0.05.
All experiments were done three times or more.

RESULTS

Binding of LukS-PV and LukF-PV to human

leukocytes and subsequent cytotoxic effects
To investigate the differential sensitivity of human leukocytes toward PVL, we challenged primary isolated human monocytes,
macrophages, neutrophils, and lymphocytes with increasing doses
of PVL. Doses 0.04 g/ml were sufficient to induce cell damage in professional phagocytes (Fig. 1AC). Cell death occurred
rapidly within 1 h and increased further within 16 h. In all sensitive cell types, measured cell death was a result of necrosis, as we
could not detect Annexin V-positive cells at any time or dose
tested (data not shown). Whereas monocytes and neutrophils
showed a comparable rate of cell death, macrophages were less
sensitive to PVL-mediated cytotoxicity. No significant cell death
was observed in cells incubated with one or the other single subunitLukS-PV or LukF-PV even after 16 h incubation. In contrast, PVL did not cause cell death in lymphocytes (Fig. 1D). To
detect the reason for this differential sensitivity toward PVL, we
incubated leukocytes with FITC-labeled and unlabeled LukS-PV
and/or LukF-PV components as indicated and analyzed binding
of the PVL components to the cell surface by flow cytometry (Fig.
1EG). In general, we detected significant binding of LukS-PV to
cells that were tested sensitive to PVL, namely, neutrophils,
monocytes, and macrophages, whereas there was no binding of
LukS-PV to lymphocytes. In lymphocytes, we only found binding
of the LukF-PV component, which did not result in cell death
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(Fig. 1D and H). In neutrophils, monocytes, and macrophages

there was also some binding of the LukF-PV component, but this
binding was enhanced significantly by pretreatment with the
LukS-PV component (Fig. 1EG). Only binding of both components to the cell surface induced cell death. These findings indicate that binding of the LukS-PV is the crucial factor for cell
specificity, which is followed by binding of LukF-PV and subsequent death of the host cell.

PVL induces a proinflammatory response in

professional phagocytes
To analyze the proinflammatory IL-1 response induced by PVL
binding to human leukocytes, we incubated the different cell
types with increasing PVL doses (0.04 0.4 g/ml) for 3 h. Ultrapure LPS (100 ng/ml) was used as a costimulant and was added
30 min before PVL treatment. PVL induced a strong increase of
IL-1 release in monocytes (Fig. 2A) and macrophages (Fig. 2B)
that was even more pronounced after LPS prestimulation. Western blot analysis of cell supernatants revealed that mature IL-1
was secreted by activated monocytes in the absence of pro-IL-1,
confirming the processing of IL-1 induced by PVL. Furthermore,
in nonprimed cells treated with low doses of PVL, pro-IL-1 was
induced. In monocytes, increasing concentrations of PVL did not
further enhance IL-1 secretion (Fig. 2A), whereas in macrophages, the IL-1 production was augmented dose-dependently,
which can be explained by lower sensitivity of macrophages to
PVL. In contrast to IL-1 secretion, TNF- levels were only marginally/not increased after PVL treatment (Fig. 2D and E). In
general, both PVL-induced proinflammatory effects (IL-1 and
TNF release) were much less pronounced in neutrophils, and we
could not detect an enhancing effect of prestimulation with LPS
(Fig. 2C and F). Yet, after PVL treatment of neutrophils, we
found a massive release of the DAMPs MRP8/14 (Fig. 2G) and
S100A12 (Fig. 2H), which are known to act as endogenous amplifiers of innate immunity [48]. Based on these findings, we performed a kinetic of IL-1 and IL-18 release (Fig. 3). Both inflammasome-regulated proteins showed a quick release with a climax
after 1 h, indicating that the secretion of these proteins is a fast
mechanism.

PVL-induced IL-1 secretion is mediated by the

NLRP3 inflammasome
Next, we studied the involved mechanism of PVL-induced IL-1
secretion in human monocytes (Fig. 4A). Firstly, we found that
the caspase-1 inhibitor zYVAD-fmk abrogated PVL-induced IL-1
secretion, confirming that IL-1 maturation is indeed caspase-1dependent. As PVL is a known K-ionophore, and K-efflux has
been linked to activation of NLRP3 and NLRP1 inflammasomes
[49], we incubated cells with high extracellular K-concentrations
that prevented the IL-1 response to PVL as well. Furthermore
activation of the lysosomal cysteine protease cathepsin has been
associated with activation of the NLRP3 inflammasome. The
CTSB inhibitor CA-074Me completely abolished the IL-1 response to PVL. All of our results could be confirmed by Western
blots of cell pellets and supernatants showing the inhibition of
secreted, mature IL-1 in the presence of high intracellular proIL-1 levels, suggesting a major role for CTSB and NLRP3 in

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Holzinger et al. S. aureus PVL-induced inflammasome activation

Figure 1. Cytolytic effects and binding of purified PVL to human leukocytes. Primary bloodderived monocytes (A), macrophages (B), neutrophils (C), and lymphocytes (D; 11060.5
ml1 cells) were incubated with increasing doses
of PVL [0.04 0.4 g/ml (110 nM)]. Cells were
stimulated for 1, 3, or 16 h, respectively, or with
LukS-PV and LukF-PV for 16 h and then were
washed and stained with PI, and cell death was
measured by flow cytometry. Statistical differences between control and stimulated cells were
determined by Students t test. Leukocytes
[monocytes (E), macrophages (F), neutrophils
(G) and lymphocytes (H)] (11060.5 ml1
cells) were incubated with untreated and FITCconjugated LukS and/or LukF (1.0 g/ml) for 1 h and washed, and binding of toxin was quantified by flow cytometry. Rate of binding was calculated by the MFI shift between untreated control cells and toxin-treated cells. The values represent the mean sd of at least three independent experiments. Statistical differences were determined by ANOVA comparing the MFI between control and treated cells (*P0.05; **P0.01; ***P0.001).

PVL-induced inflammasome activation. Cleavage of caspase-1 after PVL treatment was confirmed by measuring the p20 subunit
in the supernatant of PVL- or nigericin-treated cells. Caspase-1
levels increased fast in a time-dependent manner (Fig. 4B). Our
hypothesis was strengthened further by using stable THP-1-derived cell lines with expression of the inflammasome components
NLRP3 or ASC knocked down by expression of shRNAs targeting the mRNA encoding those proteins. Western blot analysis
indicates that levels of NLRP3 and ASC proteins are reduced by
90% in these cells (Fig. 4C). Testing the effect of PVL on both
cell lines revealed a strong reduction of IL-1 secretion when
compared with control cell lines with intact expression of NLRP3
and ASC (Fig. 4C). The functionality of the model was confirmed
by treatment with LPS and ATP (Supplemental Fig. 1A). Interest-

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ingly, THP-1 cells show a high resistance against PVL-induced

cytoxicity, demonstrated by the inability of the toxin to induce PI
permeability or release of HMGB1 into the culture supernatant
in PVL-treated cells (Supplemental Fig. 1B and C). In the absence of cell death, we could rule out passive release of pro-IL-1
to the supernatant but also, could not study cell death mechanisms in this model.
Furthermore, we tested whether inflammasome activation depends on pore-formation of PVL or whether it is mediated by
LukS-PV or LukF-PV components alone. Only the combination of
LukS-PV and LukF-PV was able to induce a strong IL-1 release,
whereas the single components did not show any effect (Fig. 4D).
On the transcriptional level, LukS-PV or LukF-PV alone did not
cause any IL-1 gene expression as well. Treatment with the com-

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Figure 2. Human phagocytes respond to PVL by IL-1 secretion and release of DAMPs. Primary monocytes (A and D),
macrophages (B and E), and neutrophils (C and FH; 11060.5 ml1 cells) were incubated with PVL (0.04 0.4 g/
ml) for 3 h. Ultrapure LPS (100 ng/ml) was used as a costimulant for the primary cells and was added 30 min before
PVL treatment. Concentrations of IL-1, TNF-, MRP8/14, and S100A12 were subsequently determined from cell culture supernatants. The inset in A verifies the presence of mature 17 kDa IL-1 and absence of pro-IL-1 in cell culture
supernatants (SN) of primary monocytes, as well as the induction of intracellular pro-IL-1 (IC) by Western blotting.
The values represent the means sd from at least three experiments. Statistical differences were determined by Students t test (*P0.05; **P0.01; ***P0.001 compared with untreated cells).

bination of LukS-PV and LukF-PV induced expression of the

IL-1 gene (Fig. 4E) that correlated with increased pro-IL-1 protein found after PVL stimulation, as measured by Western blot
and ELISA (Fig. 2A). As IL-1 expression can be activated by NFB, we analyzed NF-B activation by PVL. In comparison with
LPS, PVL was not able to activate NF-B (Supplemental Fig. 2A
and B).

PVL-mediated K-efflux leads to CTSB activation and

pyronecrosis
In further experiments, we aimed to analyze the inflammasome
activation in more detail. For this, we used several inhibitors/
compounds interfering at different stages of the inflammasome
pathway. IL-1 secretion and cell death induced by the K-ionophore nigericin are blocked by inhibitors of the cysteine proteinase CTSB. Additionally, NLRP3-dependent pyronecrosis, which
does not require caspase-1 activity, can be abrogated by CTSB
inhibitors [36]. As we could block IL-1 secretion by the CTSB
inhibitor CA-074Me, we investigated the impact of CTSB activation on IL-1 secretion by PVL and its role in cell death. Firstly,
we used stable THP-1-derived cell lines with CTSB knocked down
by expression of shRNAs targeting the mRNA encoding those
proteins. Western blots demonstrate that the levels of CTSB protein are reduced by 90% in these cells without affecting expression of NLRP3 and ASC. These cell lines exhibited a large reduction in IL-1 secretion after stimulation with PVL when compared with control cell lines with intact expression of CTSB
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(Fig. 5A). Furthermore, we found that 15 min stimulation of

monocytes with PVL was followed by cleavage of the fluorescent
CTSB substrate, Magic Red-(RR)2, and this could be reduced significantly by high extracellular K-concentrations, indicating a
direct link between K-efflux and CTSB activation. CA-074Me was
used as a positive control for this assay, whereas inhibition of
caspase-1 downstream of CTSB activation did not influence the assay. These findings could be reproduced with the inflammasome
activators -toxin and nigericin (Fig. 5B). Finally, we quantified PVLinduced necrotic cell death by PI staining and LDH activity in the
presence of increasing doses of CA-074Me, KCl, and zYVAD-fmk.
With high doses of CA-074Me and KCl, we were able to inhibit cell
death by 40%, whereas zYVAD-fmk did not show any effect (Fig.
5B). These results confirm that CTSB-mediated pyronecrosis contributes partially to PVL-induced cytotoxicity.

PVL-expressing bacteria cause cytotoxicity and IL-1

secretion
To investigate the impact of PVL expression, we used the strain
S. carnosus TM300 (lacking most S. aureus virulence factors) that
has been transformed with a plasmid encoding the genes for
PVL. Infecting monocytes with live bacteria of TM300 PVL or
with live bacteria of the parent strain TM300 revealed that the
expression of PVL is clearly responsible for cell death induction
(Fig. 6A) and IL-1 expression (Fig. 6B) in monocytes. As PVL is
a bacterial exotoxin, which is rapidly released and acts on cells at
the site of infection, we additionally challenged monocytes with

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Holzinger et al. S. aureus PVL-induced inflammasome activation

used the DAMPs MRP8, the active subunit of the MRP8/14 complex, and S100A12, which are released by PVL-damaged neutrophils (Fig. 2G and H), to preactivate monocytes. The stimulation
with DAMPs and PAMPs alone was not sufficient to induce IL-1
secretion, whereas the addition of PVL leads to massive release of
IL- (Fig. 7A). By contrast, the activation with PAMPs and
DAMPs induced TNF- secretion in monocytes, which was not
further increased by PVL (Fig. 7B). All costimulants were capable
of inducing IL-1 gene expression (Fig. 7C), leading to increased
production of pro-IL-1, which resulted in enhanced processing
and a strong IL-1 release after PVL stimulation. Therefore,
DAMPs and PAMPs are capable of amplifying IL-1 release by
priming monocytes before activation by PVL.

Induction of IL-1 secretion by different

staphylococcal virulence factors

Figure 3. PVL induces a quick secretion of inflammasome-dependent

proteins IL-1 and IL-18. Primary monocytes (11060.5 ml1 cells)
were incubated with 0.04 g/ml PVL for the indicated time-points; the
K-ionophore nigericin (20 M) served as a positive control. Release of
IL-1 (A) and IL-18 (B) was quantified by ELISA in the supernatants.
The values represent the means sd from at least three experiments.
Statistical differences were determined by Students t test. (*P0.05;
**P0.01; ***P0.001 compared with untreated cells).

sterile-filtered bacterial supernatants from overnight cultures. Culture media from strain TM300 PVL induced rapid cell death
within 1 h (Fig. 6C) and secretion of IL-1 (Fig. 6D), whereas
supernatants from the control strain TM300 did not show any
effects.

Costimulation with PAMPs and DAMPs increases

PVL-induced IL-1 secretion
LPS is a common experimental costimulus to increase IL- secretion by induction of pro-IL-1 [50]. However, LPS is restricted to
Gram-negative bacteria and is not present during an infection
with Gram-positive S. aureus. Therefore, we analyzed other compounds that are supposed to accumulate at the S. aureus infection
side for their potential to prime monocytes to generate pro-IL-1.
In this respect, we analyzed costimulatory effects of PAMPs, which
are associated with groups of pathogens and are recognized by
cells of the innate immune system, mostly via TLRs, and DAMPs,
which are highly released from neutrophils after PVL challenge
(see Fig. 2). In our experiments, we costimulated monocytes with
the PAMPs HKSA or the cell wall component LTA, a known
TLR2 agonist, followed by incubation with PVL. Furthermore, we

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As PVL is not the only important S. aureus virulence factor that

can induce a proinflammatory reaction, we compared PVL with
-toxin (which is known to be an inflammasome activator) [32],
with protein A (which has been demonstrated to activate TNFR1)
[51], and with the PSM1 and -2 (which have chemoattractive
activity for immune cells) [52]. All agents, despite protein A,
caused a strong cytotoxic effect in monocytes (Fig. 8A), which is
in accordance with previously published work. Interestingly, only
the pore-forming toxins PVL, -toxin, and nigericin, which we
used as a positive control, were able to induce a strong IL-1 release (Fig. 8B). By contrast, PSMs only induced weak IL-1 secretion at high concentrations, indicating that cytotoxicity alone is
not the responsible factor for IL-1 release but rather, the specific formation of a K-ionophore.

PVL has no effect on murine BMDMs

Recently, we could show the species specificity of PVL and excluded its cytotoxic and proinflammatory effects on murine neutrophils [9]. To analyze a potential proinflammatory action of
PVL on other murine cell types, we challenged BMDMs with high
concentrations of PVL that have been reported to be effective in
murine cells before [53]. Despite costimulation with LPS and
different time periods of incubation, we could not find any effect
of PVL with respect to cytotoxicity (Fig. 9A), IL-1 secretion
(Fig. 9B), or TNF- release (Fig. 9C), whereas nigericin was
highly effective to cause cytotoxicity and IL-1 secretion.
LukS-PV and LukF-PV are not able to bind to murine BMDMs,
which could be demonstrated by FACS analysis with FITC-labeled components (Fig. 9D). These experiments underlined
the strict species specificity of PVL.

DISCUSSION
The pathogenic mechanisms of S. aureus necrotizing infections
that lead to massive inflammation and tissue destruction are
largely unknown. According to epidemiological data [12] and to
results from an in vivo model in rabbits [22], PVL could be identified as a crucial, responsible factor. Yet, the exact molecular
mechanisms that are induced by PVL at the cellular level are not
fully elucidated. Previous work largely focused on PVL-induced
cytotoxicity of human neutrophils, whereas there is binding to

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1075

Figure 4. Mechanism of PVL-induced IL-1 secretion. Primary monocytes (11060.5 ml1 cells; A) were
incubated with 0.04 g/ml PVL in the absence or presence of KCl (130 mM), CTSB inhibitor CA-074Me
(30 M), or caspase-1 inhibitor zYVAD-fmk (25 M). After the incubation, IL-1 (average response to PVL,
690 pg/ml) was analyzed by ELISA. The values represent the means sd from at least three experiments.
The inset in A verifies the inhibition of processing and secretion of mature 17 kDa IL-1 and the absence
of pro-IL-1 in cell culture supernatants of LPS-primed primary monocytes (51062.5 ml1 cells) and
the presence of intracellular pro-IL-1 by Western blotting. Statistical differences were determined by
ANOVA comparing PVL-treated cells with PVL-treated cells inhibitors (***P0.001). Further, cells were
treated with PVL (0.04 g/ml), and p20 caspase-1 subunit was analyzed by ELISA in the supernatant after
different time-points; the K-ionophore nigericin (20 M) served as a positive control. Statistical differences were determined by Students t test (*P0.05; **P0.01; ***P0.001 compared with untreated cells; B). THP-1-derived cell lines (C) stably transduced with shRNA expressing retrovirus were treated with 0.08 or 0.4 g/ml PVL, and cell culture supernatants were analyzed for IL-1 after 24 h. The
shRNAs are directed to knock down expression as follows: EV, negative control; shASCmut, negative control; shASC, shRNA directed against ASC protein; shNLRP3, shRNA directed against NLRP3. Knockdown of ASC and NLRP3 and expression of pro-IL-1 were confirmed by Western blot. Results
represent the means sd of triplicate wells and are representative of three independent experiments. Primary monocytes (51062.5 ml1 cells) were
treated with LukS or LukF or both for 3 h, and cell culture supernatants were analyzed for IL-1 or TNF- (D). Induction of IL-1 and TNF- RNA was
determined by RT-PCR (E) and expressed as n-fold compared with untreated control. The values represent the means sd from at least three experiments. Statistical differences were determined by ANOVA comparing PVL-treated cells to control (*P0.05; **P0.01; ***P0.001).

monocytes and macrophages as well [10]. It has already been

shown that binding of LukS-PV is a critical factor for LukF-PV
binding [54 56], but to our knowledge, no previous study has
given a comprehensive overview of PVL components binding to
all different types of human leukocytes and the subsequent impact on cell viability. In this work, we could confirm the binding
of LukS-PV as the critical factor for PVL-induced cytotoxicity, and
we performed the first binding studies with human macrophages,
demonstrating that LukS-PV binds to monocytes, macrophages,
and neutrophils but not to lymphocytes.
Furthermore, we present a novel, specific mechanism of how S.
aureus PVL induces a proinflammatory response in primary human monocytic cells. Monocytes are mobile cells that can move
quickly to sites of infection to elicit an early immune response.
1076 Journal of Leukocyte Biology

Therefore, the PVL-induced proinflammatory effect on monocytes could play an important role to initiate an infection and to
attract further immune cells, such as neutrophils and lymphocytes, which are usually present in large numbers during necrotizing infections. In our experiments, we demonstrate that PVL is
capable of inducing activation of caspase-1 through the NLRP3
inflammasome. Pore formation of PVL leads to K-efflux and the
consecutive activation of CTSB, which mediates programmed necrosis and activation of NLRP3. By performing siRNA silencing
experiments, we confirmed the NLRP3 receptor as the PVL-responsive element in monocytes. Previous work revealed that S.
aureus, lacking -toxin or - or -hemolysin, was still capable of
activating IL-1 secretion, indicating that other staphylococcal
factors cause inflammasome activation as well [33]. Our findings

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Holzinger et al. S. aureus PVL-induced inflammasome activation

Figure 5. PVL-mediated K-efflux leads to CTSB activation and pyronecrosis. THP-1-derived cell
lines (A), stably transduced with shRNA expressing retrovirus, were treated with 0.4 g/ml PVL,
and cell culture supernatants were analyzed for IL-1 after 24 h. The shRNAs are directed to
knockdown expression as follows: EV, negative control; shGFP, negative control; shCTSB#1 and
#2, two shRNAs directed against CTSB. Knockdown of CTSB and the presence of ASC and NLRP3 were confirmed by Western blot. Results represent
the means sd of triplicate wells and are representative of three independent experiments. Statistical differences were determined by ANOVA comparing knockdown cells with control (***P0.001). CTSB activity was measured via degradation of the fluorescent Magic Red CTSB substrate (ImmunoChemistry Technology). Magic Red CTSB substrate was added to primary monocytes (11060.5 ml1 cells) treated with PVL (0.4 g/ml), -toxin (10
g/ml), or nigericin (20 M) for 15 min, with or without pretreatment with KCl (130 mM), CA-074Me (30 M), or zYVAD-fmk (25 M) (B). Degradation of the Magic Red substrate was measured via flow cytometry on a BD FACSCalibur. Analysis of the data was accomplished with detection of the optimal Magic Red substrate fluorescence emission in fluorescence-2 on a logarithmic scale. The values represent the means sd from three experiments. Statistical differences were determined by ANOVA comparing the MFI between control and treated cells (*P0.05; **P0.01). Primary monocytes (11060.5 ml1 cells; C) were incubated with 0.04 g/ml PVL for 3 h in the absence or presence of CTSB inhibitor CA-074Me (12.5100 M),
KCl (32.5130 mM), or caspase-1 inhibitor zYVAD-fmk (25 or 100 M). LDH was measured in the supernatants, and cells were washed and stained with
PI. The values represent the means sd from at least three experiments. Statistical differences were determined by ANOVA comparing PVL-treated
cells with PVL-treated cells inhibitors (**P0.01; ***P0.001).

demonstrate that PVL is a strong inducer of NLRP3 activation.

This effect was independent of other S. aureus virulence factors,
as the strain S. carnosus TM300 dramatically increased cytotoxicity
and IL-1 secretion in monocytes upon complementation with a

plasmid containing the genes for PVL. Additionally, we could

show that this ability is not shared by all S. aureus toxins. We confirmed the -toxin-mediated IL-1 secretion that was reported
previously [32], but we also provide evidence that PSMs fail to

Figure 6. The cytotoxic effect and induction of IL-1 secretion of TM300 and its supernatant are dependent on PVL expression. Primary monocytes (51062.5 ml1 cells) were incubated with 10 MOI of bacteria (A and B) for 3 h or for 1 h with bacterial supernatants (C and D), which
were prepared from overnight cultures and used for stimulating cells (10% vol/vol) or were left untreated. In this experiment, the heterologous
expression strain TM300 PVL and its WT strain were used. After incubation of the cells with bacterial supernatants or whole bacteria, cells were
washed and stained with PI for analysis by flow cytometry. IL-1 was measured in the cell supernatants by ELISA. The values represent the means sd
of at least three independent experiments. Statistical differences were determined by Students t test (***P0.001 compared with TM300).

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1077

masome-dependent protein IL-18 [57] is released by PVL stimulation. In contrast to pro-IL-1, pro-IL-18 is constitutively expressed
in cells, independently of a primary stimulus [58], but it shares
the same effects of IL-1 and is especially involved in T cell activation [59].
With respect to IL-1 secretion, primary stimuli, such as LPS,
induce synthesis of pro-IL-1 and may provide signals that enhance the effect of the secondary stimulus [50]. In this work, we
demonstrate that PVL does not require a LPS-priming step, as
PVL alone was sufficient to induce pro-IL-1 and activate
caspase-1 and mature IL-1 release. PVL treatment is capable of
inducing IL-1 gene expression, independent from an early
NF-B activation, and might be a result of an intermediate step
by released DAMPs (e.g., MRPs) or cytokines. Nevertheless, priming with LPS enhanced IL-1 release significantly. As LPS is restricted to Gram-negative bacteria, we tested other factors that are

Figure 7. Costimulation with PAMPs and DAMPs increases PVL-induced

IL-1 secretion. Primary monocytes (11060.5 ml1 cells) were incubated with 0.04 g/ml PVL for 3 h or were left untreated. Ultrapure LPS
(100 ng/ml), HKSA (100 MOI), and LTA (10 g/ml) were used as costimulatory PAMPs, and MRP8 and S100A12 (each 5 g/ml) served as
DAMPs and were added 30 min before PVL treatment, or cells were left
untreated as controls. After incubation, cell culture supernatants were
analyzed for IL-1 (A) or TNF- (B). Statistical differences were determined by ANOVA (*P0.05; **P0.01; ***P 0.001 compared with controls). Furthermore, primary monocytes (51062.5 ml1 cells) were
treated with the above-mentioned PAMPs and DAMPS or PVL (0.04 g/
ml), and induction of IL-1 and TNF- RNA was determined by RT-PCR
(C) and expressed as n-fold compared with untreated controls.

activate the inflammasome. Accordingly, the ability to activate the

inflammasome is restricted to pore-forming toxins and is not
shared by other S. aureus components, such as PSMs, protein A,
or LTA. Furthermore, we could show that another inflam1078 Journal of Leukocyte Biology

Figure 8. Induction of IL-1 and TNF- secretion by staphylococcal components. Primary monocytes (11060.5 ml1 cells) were incubated with
PVL (0.04 g/ml), -toxin (10 g/ml), Protein A (10 and 100 g/ml),
and PSM1 and -2 (25100 g/ml) for 3 h or were left untreated; the
K-ionophore nigericin (20 M) served as positive control. After incubation, cells were washed and stained with PI for flow cytometry (A), and
cell culture supernatants were analyzed for IL-1 or TNF- by ELISA (B).
The values represent the means sd from at least three experiments.
Statistical differences were determined by ANOVA (*P0.05; **P0.01;
***P0.001 compared with control).

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Holzinger et al. S. aureus PVL-induced inflammasome activation

Figure 9. PVL has no effect on murine BMDMs. BMDMs were prestimulated with ultrapure LPS (1 g/ml) for 6 h and then incubated further
with PVL (4 g/ml) for 30 min or costimulated for 6 h with LPS and PVL. Thirty minutes of treatment with nigericin served as a positive control.
After incubation, cells were washed and stained with PI (A), and cell culture supernatants were analyzed for IL-1 (B) or TNF- (C) by CBA. The
values represent the means sd from at least three experiments. Statistical differences were determined by ANOVA (**P0.01 compared with
control). BMDMs were incubated with unlabeled and FITC-conjugated LukS and/or LukF (1.0 or 4.0 g/ml) for 1 h and washed, and binding of
toxin was quantified by flow cytometry. Rate of binding was calculated by the MFI shift between untreated control cells and toxin-treated cells. The
values represent the mean sd of at least three independent experiments (D).

usually present during a S. aureus infection. Firstly, we used HKSA

and the bacterial wall component LTA as primary stimuli provided by bacteria as PAMPs. For both components, we could
demonstrate a priming effect for IL-1 secretion, which is in line
with previously published work [32, 60]. In addition, our data
point to a novel, positive feedback mechanism by which PVL may
induce overwhelming inflammation. We demonstrate for the first
time that PVL induces release of the endogenous TLR4 agonists
MRP8/14 [39] and the receptor for advanced glycation endproducts agonist S100A12 [38] by neutrophils, which we have previously shown to drive inflammation via activation of endothelial
cells and phagocytes [38, 39, 61]. Endogenous DAMPs as well as
the PAMPs of S. aureus were all capable of inducing IL-1 mRNA
and consecutively enhancing PVL-mediated IL-1 release, indicating that IL-1 secretion in vivo can be enhanced strongly by host
and bacterial factors.
A common feature of secondary stimuli is their ability to induce cell death concomitant with the release of IL-1 [62, 63].
The ability of PVL to activate programmed cell death in host cells
has been the subject of different studies [9, 64]. Recently, we
demonstrated that PVL induces rapid necrosis in neutrophils
without any signs of apoptosis [9]. In this study, we were not able
to detect apoptosis in all investigated cell types as well. Pretreatment with CA-074Me and elevated extracellular K-concentrations significantly reduced PVL-induced cell death. Moreover, we
could reveal that although the caspase-1-specific inhibitor zYVADfmk effectively blocks the secretion of mature IL-1 from monocytes, it does not protect cells from PVL-induced necrosis. This
indicates that PVL-induced necrosis is independent from
caspase-1 activation. Nevertheless, CTSB inhibition only partially
reduced cell death, and therefore, programmed necrosis, socalled pyronecrosis [65], is not the only pathway leading to PVLmediated necrosis. Our study was limited by the resistance of
THP-1 cells against PVL-induced cytotoxicity, and therefore, our
findings are based on experiments with specific inhibitors. Our
findings support the hypothesis that PVL leads to K-efflux and
activation of CTSB, which induces necrosis and activation of

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caspase-1 (Fig. 10). This CTSB-dependent activation of caspase-1

was already shown for different inflammasome activators, such as
the pore-forming toxin nigericin, serum amyloid A, and cholesterol crystals [36, 66, 67]. By performing siRNA silencing experiments, we confirmed the crucial role of CTSB for PVL-induced
inflammasome activation. K-efflux [49] and activation of CTSB
[68] seem to be essential intermediate steps in activation of the
NLRP3 receptor, but the connection between these processes is
poorly understood [34]. Our findings suggest that both K-efflux
and CTSB activity contribute to PVL-mediated activation of the
NLRP3 inflammasome. Nevertheless, further studies are necessary
to identify a possible interaction of these cellular factors in PVLinduced NLRP3 activation, but our findings indicate that the Kefflux is upstream of CTSB activation.
In conclusion, our study provides the first evidence that PVL
activates the NLRP3-inflammasome in a CTSB-dependent manner. This finding is of high biological relevance with regard to
severe infections caused by PVL-expressing S. aureus strains.

Figure 10. Proposed mechanism of PVL-induced inflammasome activation. PVL leads to K-efflux and CTSB activation, which consecutively
activates the inflammasome and induces pyronecrosis. Activated
caspase-1 processes IL-1 and IL-18. All steps can be blocked by specific inhibitors.

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PVL induces cell death in monocytes, macrophages, and neutrophils, which is most likely a strategy of the pathogen to
overcome the cellular innate immune system. In addition, our
findings regarding priming of phagocytes for PVL responses by
endogenous DAMPs may be of relevance for the tissue damage
associated with overwhelming disease activity during PVLdriven inflammation and may thus represent a novel molecular target for future adjuvant therapy to appropriate antibiotic
use in severe S. aureus infections.

10.

11.

12.

13.

AUTHORSHIP
D.H., J.R., G.P., and B.L. conceived of and designed the experiments. D.H., L.G., V.M., N.N., D.J.T., K.M., T.V., D.F., S.N.,
J.A., J.A.D., and P.M.B. performed the experiments. D.H. and
B.L. analyzed the data and wrote the manuscript. J.R. and G.P.
initiated the research, supervised the program, and analyzed
and discussed data.

14.

15.
16.

17.

ACKNOWLEDGMENTS
This work was supported by Deutsche Forschungsgemeinschaft
of Germany (DFG) grants (HA3177/2-1 and SFB1009) to B.L.,
a German Federal Ministry of Education and Research
(BMBF) grant (Flu-Bak Signaling) to B.L., and grants from the
Interdisciplinary Centre for Clinical Research (Lf2/030/10
and Ro2/004/10) to B.L. and J.R. at the University of Mnster. D.H. was supported by an Innovative Medizinische Forschung (IMF) grant of the University of Mnster (HO220912).
P.M.B. is an awardee of the University of North Carolina STD
and HIV Training Program (U.S. National Institutes of Health
T32AI007001). J.A.D. is supported by the Burroughs Wellcome
Fund through the Career Award for Medical Scientists and
U.S. National Institutes of Health (AI088255). We thank Melanie Saers, Heike Berheide, Brigitte Schuhen, and Michaela
Brck for excellent technical assistance.

18.
19.

20.
21.

22.

23.
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KEY WORDS:
Panton-Valentine leukocidine monocytes macrophages IL-1 and
IL-18 expression inflammasome

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1081

Staphylococcus aureus Panton-Valentine leukocidin induces an

inflammatory response in human phagocytes via the NLRP3
inflammasome
Dirk Holzinger, Laura Gieldon, Vijayashree Mysore, et al.
J Leukoc Biol 2012 92: 1069-1081 originally published online August 14, 2012
Access the most recent version at doi:10.1189/jlb.0112014

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