M A J O R A R T I C L E
Staphylococcus aureus a-Hemolysin Mediates
Virulence in a Murine Model of Severe
Pneumonia Through Activation of the NLRP3
Chahnaz Kebaier,1Robin R. Chamberland,1,aIrving C. Allen,2Xi Gao,1Peter M. Broglie,1Joshua D. Hall,3Corey Jania,4
Claire M. Doerschuk,2,4Stephen L. Tilley,4and Joseph A. Duncan1,2,5
1Department of Medicine, Division of Infectious Diseases,2Lineberger Comprehensive Cancer Center,3Department of Microbiology and Immunology,
4Department of Medicine, Division of Pulmonary and Critical Care Medicine, and5Department of Pharmacology, University of North Carolina, Chapel Hill
Staphylococcus aureus is a dangerous pathogen that can cause necrotizing infections characterized by massive
inflammatory responses and tissue destruction. Staphylococcal a-hemolysin is an essential virulence factor in
severe S. aureus pneumonia. It activates thenucleotide-binding domain and leucine-rich repeat containing gene
family, pyrin domain containing 3 (NLRP3) inflammasome to induce production of interleukin-1b and
programmed necrotic cell death. We sought to determine the role of a-hemolysin–mediated activation of
NLRP3 in the pathogenesis of S. aureus pneumonia. We show that a-hemolysin activates the NLRP3
inflammasome during S. aureus pneumonia, inducing necrotic pulmonary injury. Moreover, Nlrp32/2mice have
less-severe pneumonia. Pulmonary injury induced by isolated a-hemolysin or live S. aureus is independent of
interleukin-1b signaling, implicating NLRP3-induced necrosis in the pathogenesis of severe infection. This work
as a potential target for pharmacologic interventions in severe S. aureus infections.
Community-acquired methicillin-resistant Staphylococcus
aureus (CA-MRSA) is now the primary cause of skin
infections requiring emergency medical attention in the
United States . In addition, CA-MRSA can also cause
severe, life-threatening infections including necrotizing
pneumonias and fasciitis, which are associated with high
mortality rates, even in previously healthy patients [2, 3].
These necrotizing infections are among the most severe
complications of S. aureus infection and are characterized
by localized necrosis and inflammation.
All S. aureus produce secreted exotoxin virulence
factors, including several cytolysins: a-hemolysin,
b-hemolysin, and bicomponent leukocidins .
a-Hemolysin is one of several critical virulence
factors in a murine model of S. aureus necrotizing
pneumonia, including those caused by CA-MRSA
[5, 6]. Purified a-hemolysin induces pulmonary in-
flammation in rats and rabbits [7–9]. Immunization
with inactive a-hemolysin or pharmacologic inhibition
of a-hemolysin can prevent or reduce the severity of
S. aureus pneumonia in mice [10–12]. The mecha-
nisms by which a-hemolysin increases the virulence
of S. aureus in necrotizing pneumonia are not fully
The nucleotide-binding domain and leucine-rich re-
peat containing gene family, pyrin domain containing 3
protein (NLRP3) inflammasome is a signaling complex
that activates procaspase-1, the processing and secretion
of the cytokines interleukin (IL) 1b and IL-18, and the
Received 16 August 2011; accepted 14 October 2011; electronically published
25 January 2012.
Presented in part: 2010 International Conference on Gram-positive Pathogens,
Omaha, Nebraska, 10–13 October 2010 (oral presentation); 2010 Keystone
Symposium: Innate Immunity: Mechanisms Linking with Adaptive Immunity, Dublin,
Ireland, 7–13 June 2010 (D3, poster number 179).
aPresent affiliation: Department of Pathology, Saint Louis University School of
Medicine, St Louis, Missouri.
Correspondence: Joseph A. Duncan, MD, PhD, Department of Medicine,
Division of Infectious Diseases, University of North Carolina School of Medicine,
130 Mason Farm Rd, Chapel Hill, NC 27599-7030 (email@example.com).
The Journal of Infectious Diseases
? The Author 2012. Published by Oxford University Press on behalf of the Infectious
Diseases Society of America. All rights reserved. For Permissions, please e-mail:
S. aureus Co-opts NLRP3 Signaling
d JID 2012:205 (1 March)
initiation of programmed cellular necrosis [13–16]. The NLRP3
inflammasome is activated in response to many pathogen-
derived molecules, sterile inducers of inflammation, and mi-
crobial pore-forming toxins [17–20]. We recently demonstrated
that S. aureus a-hemolysin induces NLRP3-mediated signaling
in cultured cells . S. aureus b-hemolysin and c-hemolysin
also activate the NLRP3 inflammasome, suggesting that nu-
merous S. aureus virulence factors converge on this common
host-signaling pathway . We sought to investigate whether
the activation of the NLRP3 inflammasome was important in
the pathogenesis of these infections.
All protocols were conducted in accordance with National In-
stitutes of Health guidelines for the care and use of laboratory
animals and approved by the Institutional Animal Care and Use
Committee of the University of North Carolina at Chapel Hill.
Bacteria, Mice, and Reagents
S. aureus strain Newman and the a-hemolysin–deficient iso-
genic strain (Newman hla::erm) were provided by Dr Juliane
Bubeck Wardenburg (University of Chicago) [5, 6]. Wild-type
mice were from Jackson Laboratory (Bar Harbor, Maine).
Nlrp32/2and Il1r12/2mice, back-crossed onto the C57BL/6J
genetic background for 9 and 6 generations, respectively,
were from Dr John Bertin (Millennium Pharmaceuticals) and
Dr Jacques Peschon (Immunex; Amgen). S. aureus a-hemolysin
(Sigma-Aldrich) was previously determined to be ?50% pure
and to activate the NLRP3 inflammasome without triggering
Toll-like receptor–dependent pro–IL-1b production . The
endotoxin content of the a-hemolysin was determined to be
,0.003 International Endotoxin Units/lg of toxin using a chro-
mogenic limulus amebocyte lysate (ToxinSensor; Genscript).
Lung Cell Preparation
Lung sections were incubated in 2.5 mg/mL collagenase/
0.5 mg/mL DNase-1 (Sigma-Aldrich) at 37?C for 60 minutes.
Red blood cells were lysed with ammonium chloride potassium-
containing lysing buffer (Gibco). Single-cell suspensions were
adjusted to 2 3 106/mL in complete medium and then
stimulated with heat-killed S. aureus (HKSA; 1 particle/cell;
Invivogen), 10 lg/mL S. aureus a-hemolysin (Sigma-Aldrich),
or both for 4 hours. CD11b1cells were purified using anti-
CD11b magnetic microbeads according to the protocol provided
by the manufacturer (Miltenyi Biotec).
Lactate Dehydrogenase Release, Cytokines, and High-Mobility
Group Box 1 Measurements
Lactate dehydrogenase release, enzyme-linked immunosorbent
assay (ELISA) for IL-1b and tumor necrosis factor (TNF) a, and
high-mobility group box 1 (HMGB1) immunoblot analyses
were analyzed using the HMGB1 ELISA kit (IBL International).
IL-6 and macrophage inflammatory protein 1a levels were de-
termined using BioPlex multiplex cytokine analysis (BioRad).
Murine Pneumonia and Pneumonitis
Inoculi were prepared from S. aureus grown for 5 hours at
37?C in tryptic soy broth after 1:100 dilution of an overnight
culture. The bacteria were washed and suspended in phosphate-
buffered saline (PBS) at a concentration of 2 3 109colony-
forming units/mL, confirmed by colony counting of plated
dilutions of the suspension. Age-matched (10–13-week-old) and
sex-matched mice were anesthetized, hung in an upright
position by their incisors, and inoculated with 50 lL of PBS
containing S. aureus, HKSA, or a-hemolysin into the buccal
cavity; the nares were blocked to induce aspiration. Intra-
rectal temperature of infected mice was monitored with
a Thermalert TH-5 (Physitemp). Arterial oxygen saturation
and pulse distention were determined using the MouseOx
pulsoximeter (STARR Life Sciences) in nonanesthetized
mice. For studies of lung mechanics, infected mice were given
vancomycin (3 mg/d) intraperitoneally starting 4 hours after
Bronchoalveolar Lavage Collection, Cell Counts, and Bacterial
Mice were euthanized by CO2asphyxiation, the trachea was
canulated, and the lungs were flushed 3 times with 1 mL of
0.5 mmol/L ethylenediaminetetraacetic acid (EDTA-Dulbecco’s
PBS; Invitrogen). Total cell counts were determined using
a Neubauer hemacytometer. Polymorphonuclear cell counts
were determined by microscopic analysis of cells prepared using
the CytoSpin 4 cytocentrifuge (Thermoscientific) and visualized
using Quik-Dip Stain (Mercedes Medical). Bronchoalveolar
lavage fluid (BALF) and aseptically excised lungs, spleens, and
kidneys were collected from euthanized animals. Organ homo-
genates were prepared in sterile Whirl-Pack bags (Nasco), and
serial dilutions were plated in duplicate on mannitol salt agar
plates (BD). Bacterial burdens were determined by colony
counting after overnight incubation.
Lung Mechanics Studies
Mice were anesthetized and then paralyzed with sequential
intraperitoneal injection of 70–90 mg/kg pentobarbital so-
dium (American Pharmaceutical Partners) and 0.8 mg/kg
pancuronium bromide (Baxter Healthcare). Invasive meas-
urements of lung mechanics, including dynamic resistance
and dynamic and static compliance, were performed using
a computer-controlled small animal ventilator (flexiVent
system; Scireq), as described elsewhere .
Mouse lungs were inflated with 10% buffered formalin
(20-cm pressure), processed, stained with hematoxylin-eosin
d JID 2012:205 (1 March)
d Kebaier et al
stain, and analyzed microscopically. The extent of lung
pathology was scored as described in Supplemental Table 1.
The total pathologic score for each mouse was calculated as
the sum of scores from each category for that individual. All
hematoxylin-eosin–stained sections were scored by a veterinary
pathologist (Jackson Laboratory), who was blinded to treatment
Statistical analysis was performed using GraphPad software by
Prism. All data are expressed as means 6 standard error of the
mean. In experiments in which only 2 groups were studied,
an unpaired Student t test was used to determine signifi-
cance. Significance in differences between multiple groups
was analyzed by analysis of variance with Bonferroni as
a posttest. Survival curves were created using the Kaplan–
Meier method and compared using the log-rank (Mantel-Cox)
test. In all cases, differences were considered statistically
significant at P , .05.
a-Hemolysin Activation of NLRP3 Inflammasome in Pulmonary
We have reported that human acute monocytic leukemia cell
line, THP1 cells, and murine peritoneal macrophages process
and secrete IL-1b in response to treatment with HKSA and
purified recombinant a-hemolysin. In these studies, HKSA
induced pro–IL-1b production, whereas a-hemolysin trig-
gered NLRP3-dependent caspase-1 activation and pro–IL-1b
processing . Because a-hemolysin is a critical virulence
factor in murine pneumonia models, we sought to determine
whether it could induce NLRP3 signaling in cells from mouse
lungs. Mixed lung cell preparations treated with a-hemolysin
secreted modest levels of IL-1b, regardless of whether they
were pretreated with HKSA (Figure 1A). To examine the
response of pulmonary macrophages to a-hemolysin treat-
ment, CD11b1 cells were isolated from these mixed prepa-
rations. These cells secreted higher levels of IL-1b after
treatment with either a-hemolysin or HKSA followed by
a-hemolysin than preparations depleted of CD11b1 cells
(Figure 1B). a-Hemolysin did not induce IL-1b secretion in
mixed or CD11b1 cells from Nlrp32/2mice (Figure 1A and
1B). a-hemolysin–induced IL-1b secretion did not require
prestimulation (Figure 1B). Supernatants from lung cell
preparations induced pro–IL-1b expression when applied
to THP1 cells (Figure 1C), suggesting that pro–IL-1b is
up-regulated in the lung cells as a result of the preparation
method rather than constitutive high-level expression.
We also sought to determine whether NLRP3-mediated
signaling was activated by a-hemolysin in vivo. HKSA,
a-hemolysin, and HKSA1a-hemolysin were administered
to mice by intratracheal instillation. Six hours after in-
stillation of HKSA (5 3 108particles, a lethal dose of living
S. aureus), BALF contained minimal IL-1b and robust levels
of TNF-a (Figure 1D and 1E). Treatment with a-hemolysin
alone did not induce TNF-a or IL-1b secretion. Instillation
of HKSA1a-hemolysin induced detectable IL-1b, which
was markedly diminished in the Nlrp32/2mice (Figure 1D).
Because instillation of both HKSA and a-hemolysin was
required for pulmonary IL-1b production, it is likely that
a priming step that induces pro–IL1b production is required
for a-hemolysin–induced IL-1b secretion in vivo, as has been
documented in cell culture systems . The addition of
HKSA1a-hemolysin induced less TNF-a in BALF from wild-
type mice compared with HKSA treatment alone, and BALF
TNF-a followed a similar trend in Nlrp32/2mice (Figure 1E).
Thus, HKSA-induced TNF-a secretion may be carried out
by cells that do not express NLRP3, or a-hemolysin may
interfere with signaling pathways for TNF-a production
independent of NLRP3.
NLRP3 Mediation of a-Hemolysin–Induced Pulmonary Injury
We also characterized the pulmonary pathology associated
with HKSA1a-hemolysin treatment. Despite the difference
in IL-1b secretion between wild-type and Nlrp32/2mice
treated with HKSA1a-hemolysin at 6 hours, no animals
survived .12 hours (data not shown). To further investigate
a-hemolysin–induced pulmonary pathology, mice were given
a sublethal dose of HKSA1a-hemolysin (HKSA, 5 3 107
particles; a-hemolysin, 0.5 lg). After 24 hours, the BALF of
HKSA1a-hemolysin–treated animals demonstrated robust
neutrophilia, which was markedly diminished in the Nlrp32/2
animals (Figure 2A). Nlrp32/2mice also exhibited less lung
pathology in a composite histopathologic index (Figure 2B
and 2C) and individual indices of alveolar and vasculitic in-
flammation (data not shown). Thus, NLRP3 activation
plays an important role in a-hemolysin–induced pulmonary
injury and inflammation.
Intratracheal HKSA1a-hemolysin induced severe hypo-
thermia and labored respiration, a syndrome similar to pneu-
monia induced by living bacteria. The clinical consequences of
intratracheal HKSA (1 3 108particles), a-hemolysin (1 lg), or
HKSA1a-hemolysin were studied in wild-type mice. Although
mice completely recovered from treatment with HKSA alone,
treatment with a-hemolysin resulted in ?40% mortality and
treatment with HKSA1a-hemolysin induced 80%–100%
mortality (Figure 2D and 2E). Nlrp32/2mice were protected
from death when compared with their wild-type counterparts
(Figure 2E).SurvivingNlrp32/2micewere less hypothermic and
recovered more rapidly than wild-type mice (Figure 2F). These
data suggest that NLRP3-induced inflammation plays a signi-
ficant role in the clinical phenotype of a-hemolysin–induced
S. aureus Co-opts NLRP3 Signaling
d JID 2012:205 (1 March)
Effect of a-Hemolysin–Mediated Activation of NLRP3 on
Severity of S. aureus Pneumonia
Because IL-1b is important in host clearance of S. aureus from
skin infection models, we used a murine model to further
delineate the role of NLRP3 inflammasome activation in
S. aureus pneumonia. Wild-type mice experienced severe
hypothermia and markedly diminished pulse distention
consistent with severe systemic inflammatory response within
24 hours of S. aureus administration. Nlrp32/2mice were less
hypothermic and had increased pulse distention compared
with their wild-type counterparts (Figure 3A and 3B). In mice
surviving to 48 hours, Nlrp32/2animals were less hypoxemic
than wild-type mice (Figure 3C). The overall mortality in
this model of S. aureus pneumonia was lower in Nlrp32/2
mice (14% mortality) than in wild-type mice (37% mor-
tality), but this difference was not statistically significant
(data not shown). Compared with wild-type mice, Nlrp32/2
mice exhibited significantly better survival free of severe
pneumonia, as defined by death, oxygen saturation ,95%,
or a temperature drop .10?C (Figure 3D).
containing 3 protein (NLRP3) inflammasome–mediated interleukin (IL) 1b secretion in pulmonary macrophages and murine lungs. A, Cells were isolated
from the lungs of wild-type (WT) and Nlrp32/2C57L/B6 mice and left untreated (NT) or treated with heat-killed S. aureus (HKSA), alpha-hemolysin (Hla),
or a combination of the two. Secreted IL-1b was measured in the culture supernatant using enzyme-linked immunosorbent assay (ELISA). B, Mouse lung
cells were prepared and separated into CD11b1and CD11b2populations using paramagnetic particle-based separation. The cells were treated as
described for A, and IL-1b secretion was assessed by ELISA. C, THP-1 cells were treated with the indicated dilutions of lung cell preparation supernatant
or untreated preparation medium as a negative control for 4 hours. Cells were also treated with lipopolysaccharide (LPS) (1 lg/mL; 4 hours). Cell pellets
were analyzed by immunoblot with antibodies against pro–IL-1b (3ZD (upper panel) and actin (lower panel). The samples and controls are from the same
immunoblot; however, these samples were not in adjacent lanes. A representative immunoblot from duplicate experiments is shown. D, E, C57BL/6 WT
and Nlrp32/2mice were anesthetized and phosphate-buffered saline (PBS), HKSA (5 3 108), Hla (1 lg), or a combination of HKSA and Hla was instilled
intratracheally. D, E, Bronchoalveolar lavage fluid was collected at 6 hours and analyzed for IL-1b (D) and tumor necrosis factor (TNF) a (E) by ELISA. Bars
represent mean 6 standard error of the mean of 3 experiments. Statistical significance was determined by 1-way analysis of variance with Bonferroni
test. **P 5 .0012.01; ***P ,.001. Abbreviation: ns, not significant.
Staphylococcus aureus a-hemolysin induces nucleotide-binding domain and leucine-rich repeat containing gene family, pyrin domain
d JID 2012:205 (1 March)
d Kebaier et al
The consequences of severe S. aureus pneumonia on lung
function were determined in surviving mice. Wild-type and
Nlrp32/2mice were treated with vancomycin, an antibiotic
used to treat severe S. aureus infections, at the first signs of
illness after inoculation with S. aureus. All mice survived
and normalized their temperature within 3 days (data not
shown), and pulmonary function was assessed on day 4.
Postpneumonia mice had reduced dynamic lung compliance
and increased airway resistance compared with uninfected
mice (Figure 3E and 3F). Along with milder clinical dis-
ease, lung compliance was not reduced in Nlrp32/2mice
after pneumonia. Thus, the NLRP3 inflammasome plays
a key role in mediating pulmonary injury during S. aureus
NLRP3 Inflammasome–Induced Inflammation and Clearance of
S. aureus Pneumonia
Given the unexpected findings that the NLRP3 inflammasome
was not protective in murine S. aureus pneumonia and that the
disease process was worse in Nlrp31/1mice, further studies of
the effects of NLRP3 on the host and bacteria during S. aureus
pneumonia were carried out. Quantitative culture of BALF and
lung homogenates revealed that wild-type and Nlrp32/2mice
had equivalent S. aureus burdens after 24 hours and had almost
completely cleared the bacteria by 48 hours (Figure 4A and 4B).
S. aureus burdens in the kidneys were low, with no significant
difference between wild-type and Nlrp32/2mice with S. aureus
pneumonia (Figure 4C). No dissemination to the spleen was
observed in either mouse strain (data not shown). Thus, NLRP3
dependent acute pulmonary inflammation and injury. A–C, C57BL/6 wild-type (WT) and Nlrp32/2mice were challenged intratracheally with 5 3 107heat-
killed Staphylococcus aureus (HKSA) and 0.5 lg Hla (HKSA1Hla) or phosphate-buffered saline (PBS). Bronchoalveolar lavage fluid (BALF) was collected at
24 hours and the lungs were subsequently inflated, fixed, and stained with hematoxylin-eosin (HE). A, BALF collected at 24 hours was processed for
neutrophil counts. B, HE-stained lung sections were stained and scored on a pathologic index that includes alveolar neutrophilic inflammation, vasculitis and
vascular extravasation, and bronchial epithelial sloughing or necrosis, as described in the Methods section. C, Representative photomicrographs of
HE-stained lung tissue from WTand Nlrp32/2mice, harvested 24 hours after challenge with PBS or 5 3 107HKSA and 0.5 lg Hla (HKSA1Hla). Scale bar,
50 lm. D, C57BL/6 WT mice were anesthetized and treated with intratracheal PBS, HKSA (108), Hla (1 lg), or a combination of HKSA and Hla. Survival was
monitored over a 60-hour time course, as detailed in the Methods section, and is shown by Kaplan–Meier plot. E, F, WTand Nlrp32/2C57BL/6 mice were
treated with a combination of intratracheal a-hemolysin (1 lg) and HKSA (108), as described for D. E, Survival and intrarectal temperature of survivors was
monitored for 60 hours. Survival of mice from each group is shown by Kaplan–Meier plot. Statistical significance was determined by log-rank test, P ,.05.
F, Intrarectal temperature is plotted for all surviving animals at each time point. Values are expressed as means 6 standard error of the mean. Statistical
significance was determined by 1-way analysis of variance with Bonferroni test. **P 5 .0012.01; ***P ,.001.
a-Hemolysin induces nucleotide-binding domain and leucine-rich repeat containing gene family, pyrin domain containing 3 protein (NLRP3)–
S. aureus Co-opts NLRP3 Signaling
d JID 2012:205 (1 March)
inflammasome activity does not influence clearance of S. aureus
during pulmonary infection.
We also examined the role of NLRP3 in the inflammatory
response to live S. aureus pneumonia. As expected, Nlrp32/2
mice had reduced levels of IL-1b in BALF and lung homo-
genates (Figure 4D and 4E). TNF-a (Figure 4F), IL-6 (not
shown), and macrophage 300 inflammatory protein 1a (not
shown) levels in BALF were similar between wild-type and
Nlrp32/2 mice. Thus, Nlrp32/2mice had reduced IL-1b levels
during S. aureus pneumonia, while production of other
inflammatory cytokines and chemokines was largely intact.
Wild-type mice with S. aureus pneumonia had hemorrhagic
lungs on gross examination, and the Nlrp32/2mice had pink,
healthy-appearing lung tissue (Figure 4G). The BALF samples
from Nlrp32/2mice with S. aureus pneumonia contained fewer
neutrophils than their wild-type counterparts (Figure 4H).
Histologic examination also revealed significantly less pulmo-
nary pathology in the Nlrp32/2mice using a composite index
PBS S. aureus PBS S. aureus
3 protein (NLRP3) controls severity of Staphylococcus aureus pneumonia. A–D, Wild-type (WT) and Nlrp32/2C57BL/6 mice were challenged
intratracheally with phosphate-buffered saline (PBS) or 1–2 3 108colony-forming units of live S. aureus (strain Newman). A, Intrarectal temperature was
monitored daily; the change in temperature between day 0 and day 1 (Temp D1 2 Temp D0) is plotted for each mouse. Pulse distention and blood oxygen
saturation were measured in the mice using a MouseOx small animal pulsoximeter, as described in the Methods section. B, Pulse distention 1 day after
challenge is plotted for each surviving animal. C, Blood oxygen saturation on day 2 is plotted for each surviving animal. Values are expressed as means of
3 independent experiments (n 5 6–22 mice per group). Statistical significance was evaluated by 1-way analysis of variance (ANOVA) with Bonferroni test
(*P ,.05, ***P ,.001). D, Composite end point defined by death, oxygen saturation ,95%, or temperature drop .10?C was used to define severe
pneumonia. Survival free of severe pneumonia is shown using the Kaplan–Meier plot. Statistical significance was determined by log-rank test (*P ,.05).
WT and Nlrp32/2C57BL/6 mice were challenged intratracheally with PBS or live S. aureus (as above) and subsequently treated with vancomycin.
E, F, Dynamic compliance (Cdyn) (E) and pulmonary resistance (R) (F) were measured and control mice were compared with pneumonia
survivors (all mice survived with vancomycin administration); n 5 4–8 mice per group. Statistical significance was evaluated by 1-way ANOVA with
Bonferroni test. *P ,.05; ***P ,.001.
a-Hemolysin–mediated activation of nucleotide-binding domain and leucine-rich repeat containing gene family, pyrin domain containing
d JID 2012:205 (1 March)
d Kebaier et al
(Figure 4I). In total, our data suggest that activation of the
NLRP3 inflammasome during pneumonia leads to excessive
inflammation that is deleterious, rather than protective, to the
host lung tissue.
NLRP3 Inflammasome Activation and S. aureus Pneumonia
Pathogenesis in the Absence of a-Hemolysin
To determine whether a-hemolysin from live S. aureus was
driving the NLRP3-induced lung pathology, we used an isogenic
strain of S. aureus with a deletion of the hla gene. As reported
elsewhere, the hla-deficient S. aureus was less virulent than
a-hemolysin-expressing S. aureus (data not shown) . To
observe a clinical phenotype, we induced S. aureus pneumonia
with hla::erm S. aureus with a larger dose than was used with
wild-type S. aureus. There was no difference between wild-type
and Nlrp32/2mice in the recovered bacteria in BALF or lung
homogenates after challenge with hla-deficient S. aureus
(Figure 5A and 5B). In contrast to findings with wild-type
S. aureus, the mouse strains challenged with hla::erm S. aureus
were equally hypothermic, with no difference in BALF neutrophil
controls inflammation but not bacterial growth during Staphylococcus aureus pneumonia. A–H, Wild-type (WT) and Nlrp32/2mice were instilled
intratracheally with 1–2 3 108colony-forming units (CFU) S. aureus (strain Newman). A–C, Bronchoalveolar lavage fluid (BALF) samples (A), lung
homogenates(B),and kidney homogenates (C) were harvested at 24and 48hours then assessedfor the presenceof bacteria by plating of serial dilutions on
mannitol salt agar plates. Dashed lines represent limit of detection (100 total CFU). Statistical significance was determined by 1-way analysis of variance
(ANOVA)withBonferronitestandnosignificantdifferenceswerepresent(n5 5–12micepergroup).D–F,LevelsofIL-1b inBALFsamples(D),interleukin(IL)
1b in lung homogenates (E), and tumor necrosis factor (TNF) a in BALF samples (F) were determined by enzyme-linked immunosorbent assay. G, Lungs from
WTand Nlrp32/2C57BL/6 mice infected with live S. aureus Newman were exposed and photographed to show gross pathology in situ at day 4. Control
micetreated with phosphate-bufferedsaline(PBS)are shownforcomparison. H,TotalnumberofneutrophilsinBALF sampleswas determined24hours after
challenge and represented as means for 5–10 mice per group. I, HE-stained lung sections prepared 24 hours after challenge were examined and scored for
pathologic findings, as described in Methods. Levels of pulmonary pathology determined using a combined index that includes alveolar inflammation,
vascular inflammation, and epithelial sloughing and necrosis are plotted for each animal. Statistical significance was evaluated by 1-way ANOVA with
Bonferroni test. *P ,.5; **P 5 .001–.01; ***P ,.001.
Nucleotide-binding domain and leucine-rich repeat containing gene family, pyrin domain containing 3 protein (NLRP3) inflammasome activation
S. aureus Co-opts NLRP3 Signaling
d JID 2012:205 (1 March)
counts (Figure 5C and 5D). These data support the hypothesis
that activation of NLRP3 is a major mechanism by which
a-hemolysin mediates induction of severe S. aureus pneumonia.
Role of IL-1b in a-Hemolysin Induced Pulmonary Injury and
Promotion of S. aureus Virulence
Anti-IL-1 therapy is highly efficacious in the treatment of peri-
odic fever syndromes in patients with mutations in the Nlrp3
gene, suggesting that processing and secretion of IL-1b is the
primary downstream signaling event of NLRP3 inflamma-
some activation [24–26]. We sought to determine whether
excessive IL-1b signaling induced by a-hemolysin was in-
volved in S. aureus virulence. First, we studied the effects of
intratracheal HKSA1a-hemolysin intoxication in Il1r12/2
mice. Unlike Nlrp32/2mice, HKSA1a-hemolysin–treated
Il1r12/2mice had mortality and hypothermia equivalent to
that in wild-type mice (Figure 6A and 6B). We also studied
pneumonia with live S. aureus in wild-type and Il1r12/2mice.
Survival free from severe pneumonia (Figure 6C) and
absolute mortality (not shown) was also indistinguishable
between strains. Il1r12/2mice with S. aureus pneumonia
also had worse blood oxygen saturation than wild-type
mice (Figure 6D). Combined, these data suggest that the
pathogenic mechanism of a-hemolysin–mediated NLRP3
activation during S. aureus pneumonia does not require
production of IL-1b.
Because a-hemolysin enhanced S. aureus virulence through
a mechanism that was NLRP3 dependent and IL-1 signaling
independent, we sought to examine additional NLRP3-
dependent signaling pathways in CD11b1 cells and in mice.
Similar to our findings with IL-1b secretion, a-hemolysin
induced NLRP3-dependent death that was accompanied by
release of HMGB1, a marker of programmed necrosis in
CD11b1 cells (Figure 6EdG). Cell death was not significantly
induced in the CD11b-negative cells by this concentration
of a-hemolysin (data not shown). HMGB1 levels were also
increased in BALF from HKSA1a-hemolysin–treated wild-
type mice but not Nlrp32/2mice, confirming that NLRP3
mediates a-hemolysin–induced necrotic cell death in vivo
(Figure 6H). a-Hemolysin also induced IL-18 secretion from
wild-type but not Nlrp32/2CD11b1 cells (Figure 6I). IL-18
levels were increased in BALF from wild-type mice with
S. aureus pneumonia but not Nlrp32/2mice (Figure 6J).
S. aureus produces many virulence factors, including both
surface proteins and exotoxins that have been shown to be
Staphylococcus aureus. WTand Nlrp32/2animals were intratracheally challenged with 5 3 108colony-forming units (CFU) of S. aureus strain Newman
hla::erm (a transposon insertion Hla mutant), as described in Methods. A, B, Bacterial burden in bronchoalveolar lavage fluid (BALF) samples (A) and lung
homogenates (B) was determined 24 hours after challenge by plating serial dilutions on mannitol salt agar. C, Intrarectal temperature was monitored
daily; the change in temperature from day 0 to day 1 (Temp D1 2 Temp D0) is plotted for each animal. PBS, phosphate-buffered saline. D, Total number of
neutrophils was measured in BALF samples collected 24 hoursafter challenge. Statistical significance was determined by 1-way analysis of variance with
Bonferroni test (bars represent means. **P 5 .0012.01, ***P ,.001. Abbreviation: ns, not significant.
There is no difference in pneumonia severity between wild-type (WT) and Nlrp32/2mice infected with a-hemolysin–deficient
d JID 2012:205 (1 March)
d Kebaier et al
important in murine pneumonia models. Recent studies
have focused on the role of a-hemolysin in this model .
Numerous host cell types are susceptible to this cytolytic
toxin. Although lysis of red blood cells to provide heme as an
iron source may play a role in a-hemolysin–mediated viru-
lence in humans, this is not the case in mice, because murine
hemoglobin is not recognized by the S. aureus hemoglobin
receptor . Cytolysis of host epithelial cells and leukocytes
must be the primary mechanism by which this toxin medi-
ates virulence in the murine pneumonia. Several studies
indicated that the NLRP3 inflammasome may be an im-
portant target pathway for a-hemolysin; however, in vivo
evidence was lacking [21, 22]. In this study we show that
pulmonary macrophages are a target of a-hemolysin activity
among cells isolated from murine lungs. S. aureus a-hemolysin
activates the NLRP3 inflammasome both in cultured cells in
leucine-rich repeat containing gene family, pyrin domain containing 3 protein (NLRP3). A, B, C57BL/6 wild-type (WT) and Il1r12/2mice were challenged
intratracheally with 1 3 108heat-killed Staphylococcus aureus (HKSA) and 1 lg of Hla (HKSA1Hla) or phosphate-buffered saline (PBS), as described for
Figure 2. A, Survival and intrarectal temperature of survivors was monitored for 60 hours. Survival of mice from each group is shown by Kaplan–Meier
plot. B, Statistical significance was determined by log-rank test (P ,.05). Intrarectal temperature is plotted for all surviving animals at each time point.
C, D, WTand Il1r12/2C57BL/6 mice were challenged intratracheally with PBS or 1–2 3 108colony-forming units (CFU) of live S. aureus (strain Newman)
and monitored as described for Figure 3. C, A composite end point defined by death, oxygen saturation ,95%, or temperature drop .10?C was used to
define severe pneumonia. Survival free of severe pneumonia is shown by Kaplan–Meier plot. No statistically significant difference was observed
between WT and Il1r12/2mice, determined by log-rank test. D, Blood oxygen saturation on day 2 is plotted for each surviving animal. Values are
expressed as means of 3 independent experiments (n 5 6–22 mice per group). CD11b1pulmonary cells were isolated from WT C57BL/6 and Nlrp32/2
mice, as described for Figure 1. CD11b1cells were prepared and left untreated (NT) or stimulated with a-hemolysin. E, Cell death was assessed by
measurement of the release of cytoplasmic lactate dehydrogenase (LDH) into the culture medium of CD11b1cells and is plotted as a percentage of
maximum (max) release achieved by treatment with detergent. F, G, Necrotic cell death, defined by release of high-mobility group box 1 (HMGB1), was
assessed using immunoblot analysis of the cell culture supernatants. F, Representative immunoblot showing media from untreated cells (NT) and from
a-hemolysin–treated (Hla) CD11b1cells. G, Quantities of HMGB1 were determined using the FluorChemE imaging system and analysis software (Cell
Biosystems); the level of HMGB1 in the culture supernatant expressed as a percentage of the total is plotted. H, C57BL/6 WTand Nlrp32/2mice were
challenged intratracheally with 5 3 107HKSA and 0.5 lg of Hla (HKSA1Hla) or PBS. Bronchoalveolar lavage fluid (BALF) was collected at 24 hours, as
described for Figure 2. The levels of HMGB1 (a marker of necrotic cell death) in BALF samples were assessed by enzyme-linked immunosorbent assay
(ELISA). I, IL-18 secretion was measured using ELISA. J, WTand Nlrp32/2mice were challenged intratracheally with PBS or 1–2 3 108CFU of live S.
aureus and IL-18 secretion in BALF was determined after 24 hours. Bars represent mean 6 standard error of the mean of 3 (E, F, J) or 4 (G, H, I)
experiments. Statistical significance was evaluated by 1-way analysis of variance with Bonferroni test. *P ,.5; **P 5 .0012.01; ***P ,.001.
Interleukin (IL) 1b secretion is not responsible for lung injury induced by a-hemolysin–mediated activation of nucleotide-binding domain and
S. aureus Co-opts NLRP3 Signaling
d JID 2012:205 (1 March)
vitro and in the lungs in vivo. Interestingly, a-hemolysin’s ac-
tivity toward other cell types in the respiratory system may be
mediated through signaling systems independent of NLRP3,
such as a disintegrin and metalloprotease 10 (ADAM10), which
is a cellular receptor for a-hemolysin and was recently shown
to play an important role in murine S. aureus pneumonia
[28, 29]. Mice lacking NLRP3 are not completely protected from
a-hemolysin-expressing S. aureus, suggesting that a-hemolysin
promotes virulence through NLRP3-dependent and NLRP3-
Given previous reports showing that a-hemolysin acti-
vates NLRP3, it was not unanticipated that IL-1b secretion is
diminished in Nlrp32/2mice after intratracheal a-hemolysin
administration or S. aureus pneumonia. However, because
IL-1b and other inflammasome components are important
in the clearance of S. aureus in skin infection models, it is
surprising that Nlrp32/2mice are not more susceptible to
S. aureus pneumonia . S. aureus lacking O-acetylated
peptidoglycan induces increased inflammasome activation
and has decreased virulence in a skin infection model com-
pared with S. aureus with O-acetylated peptidoglycan .
However, O-acetylation of peptidoglycan also protects the
bacteria from lysozyme, making it unclear whether the
mechanism leading to the diminished virulence is related to
increased inflammasome activation. We report that S. aureus
pneumonia is slightly more severe in Il1r12/2mice, paral-
leling previous reports that IL-1b signaling is involved in
clearance of S. aureus in other models of infection. Mice
lacking NOD2, an NLR protein involved in recognition of
muramyl dipeptide, have decreased sensitivity to S. aureus
pneumonia and increased sensitivity to intraperitoneal and
cutaneous S. aureus infections [32–34]. NOD2 signaling
up-regulates production of pro–IL-1b, antimicrobial pep-
tides, and other host defense mechanisms, leaving the mecha-
nism underlying the site-specific phenotypes of S. aureus
infection in Nod22/2mice still to be determined . It also
remains to be determined whether NLRP3 deficiency will carry
a consistent phenotype with varying sites of infection.
Although S. aureus pneumonia in mice may differ from
human disease, a-hemolysin does activate the NLRP3 in-
flammasome in both human and murine cells. Thus, in-
hibition of the NLRP3 inflammasome may someday prove to
be useful adjunctive therapy to antibiotics in severe S. aureus
pneumonia. At this time, specific NLRP3 inflammasome
inhibitors are not available; however, glyburide, an anti-
hyperglycemic medication, has been shown to have NLRP3-
inhibitory effects . Interestingly, a recent analysis of patient
survival during infection with Burkholderia pseudomallei dem-
onstrated that diabetics taking glyburide had better survival
rates than nondiabetics, hinting that inhibition of the NLRP3
inflammasome may indeed be beneficial in some acute
human infections . Because the protection from S. aureus
pneumonia observed in Nlrp32/2mice was independent of
IL-1b signaling, IL-1 antagonists, which are efficacious in
ameliorating fever syndromes associated with NLRP3 signal-
ing hyperactivity, are unlikely to be beneficial and may be
harmful in the setting of S. aureus infection .
S. aureus produces multiple pore-forming toxins that are
implicated in the pathogenesis of severe infections and are
capable of activating the NLRP3 inflammasome [38, 39].
The presence of NLRP3-activating, pore-forming toxins in
other respiratory pathogens, like pneumolysin in Streptococcus
pneumoniae, suggests that the NLRP3-induced inflammation
may be an important factor in many bacterial pneumonias
[40–42]. The ability to activate programmed necrosis at
a distance may protect these bacteria from host phagocytes,
and inflammatory damage to the pulmonary architecture may
facilitate bacterial penetration of the epithelial barrier [7, 43].
Further studies are needed to clarify the role of toxin-mediated
activation of NLRP3 in other bacterial pneumonias.
Supplementary materials are available at the Journal of Infectious Diseases
online (http://www.oxfordjournals.org/our_journals/jid/). Supplementary
materials consist of data provided by the author that are published to benefit
the reader. The posted materials are not copyedited. The contents of all
supplementary data are the sole responsibility of the authors. Questions or
messages regarding errors should be addressed to the author.
Sparling for their insights into these studies. The Imaging and Biomarker
Analysis Core of the Southeastern Regional Center of Excellence for
Emerging Infections and Biodefense (SERCEB) carried out multiplex
Financial support.Studies were supported by the Burroughs Wellcome
Fund: Career Award for Medical Scientists (to J. A. D.) and the National
Institutes of Health (grants AI057157 [SERCEB Career Development Award]
and AI088255 to J. A. D., AI077437 [Lung Disease Model Core] and
HL071802 to S. L. T.; and HL048160 to C.M.D.). P. M. B. is a trainee of
the University of North Carolina STD and HIV Training Program
Potential conflicts of interest.All authors: No reported conflicts.
All authors have submitted the ICMJE Form for Disclosure of Potential
Conflicts of Interest. Conflicts that the editors consider relevant to the
content of the manuscript have been disclosed.
We thank Jenny P. Y. Ting and P. Fredrick
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