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Kineret
H
/IL-1ra Blocks the IL-1/IL-8 Inflammatory Cascade
during Recombinant Panton Valentine Leukocidin-
Triggered Pneumonia but Not during
S. aureus
Infection
Delphine Labrousse
1.
, Magali Perret
2,3,4,5.
, Davy Hayez
1
, Sonia Da Silva
1
,Ce
´dric Badiou
2,3,4,5
,
Florence Couzon
2,3,4,5
, Miche
`le Bes
2,3,4,5
, Pascal Chavanet
6
,Ge
´rard Lina
2,3,4,5
, Franc¸ois Vandenesch
2,3,4,5
,
Delphine Croisier-Bertin
1,6
, Thomas Henry
2,3,4,5
*
1Vivexia, Dijon, France, 2Universite
´de Lyon, Lyon, France, 3INSERM U1111, Lyon, France, 4CNRS, UMR 5308, Lyon, France, 5Ecole Normale Supe
´rieure, Lyon, France,
6Infectious Diseases Department, University Hospital, Dijon, France
Abstract
Objectives:
Community-acquired Staphylococcus aureus necrotizing pneumonia is a life-threatening disease. Panton
Valentine Leukocidin (PVL) has been associated with necrotizing pneumonia. PVL triggers inflammasome activation in
human macrophages leading to IL-1brelease. IL-1bactivates lung epithelial cells to release IL-8. This study aimed to assess
the relevance of this inflammatory cascade in vivo and to test the potential of an IL-1 receptor antagonist (IL-1Ra/Kineret) to
decrease inflammation-mediated lung injury.
Methods:
We used the sequential instillation of Heat-killed S. aureus and PVL or S. aureus infection to trigger necrotizing
pneumonia in rabbits. In these models, we investigated inflammation in the presence or absence of IL-1Ra/Kineret.
Results:
We demonstrated that the presence of PVL was associated with IL-1band IL-8 release in the lung. During PVL-
mediated sterile pneumonia, Kineret/IL-1Ra reduced IL-8 production indicating the relevance of the PVL/IL-1/IL-8 cascade in
vivo and the potential of Kineret/IL-1Ra to reduce lung inflammation. However, Kineret/IL-1Ra was ineffective in blocking IL-
8 production during infection with S. aureus. Furthermore, treatment with Kineret increased the bacterial burden in the
lung.
Conclusions:
Our data demonstrate PVL-dependent inflammasome activation during S.aureus pneumonia, indicate that IL-1
signaling controls bacterial burden in the lung and suggest that therapy aimed at targeting this pathway might be
deleterious during pneumonia.
Citation: Labrousse D, Perret M, Hayez D, Da Silva S, Badiou C, et al. (2014) Kineret
H
/IL-1ra Blocks the IL-1/IL-8 Inflammatory Cascade during Recombinant Panton
Valentine Leukocidin-Triggered Pneumonia but Not during S. aureus Infection. PLoS ONE 9(6): e97546. doi:10.1371/journal.pone.0097546
Editor: Suzan H. M. Rooijakkers, University Medical Center Utrecht, Netherlands
Received November 13, 2013; Accepted April 17, 2014; Published June 6, 2014
Copyright: ß2014 Labrousse et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by a FINOVI young investigator grant to TH. SOBI had no role in study design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing Interests: Kineret H113/IL-1Ra employed in the study was provided by SOBI ß, SOBI had no role in study design, data collection and analysis,
decision to publish or preparation of the manuscript. Authors DL, DH, SDS and DCB are employees of Vivexia, a company specialized in the evaluation of the
efficacy of anti-infectious drug candidates. This does not alter the authors’ adherence to PLOS ONE policies on sharing data and materials.
* E-mail: Thomas.henry@inserm.fr
.These authors contributed equally to this work.
Introduction
The prevalence of community-acquired Staphylococcus aureus
pneumonia is low, but the disease can be very severe, with lethality
higher than 40% in children and young adults [1,2]. Due to the
spread of community-acquired methicillin-resistant S. aureus (CA-
MRSA) and the increased resistance of these strains to antibiotics,
it is crucial to understand the pathophysiological mechanisms at
play during severe CA-S. aureus pneumonia and to find novel
therapeutic options.
Panton Valentine Leukocidin (PVL) is a bi-component leuko-
toxin composed of LukS-PV and LukF-PV. PVL is very cytotoxic
to human neutrophils, monocytes and macrophages. Furthermore,
PVL triggers the production of IL-8 [3] by neutrophils and of IL-
1bby monocytes and macrophages [4,5]. We have recently shown
that IL-1breleased by rPVL-intoxicated macrophages activates
lung epithelial cells to release large amounts of IL-8. IL-1band IL-
8 are key cytokines to recruit neutrophils [6]. This inflammatory
cascade could thus contribute to acute lung inflammation observed
during infection. While inflammation is important to clear
bacteria, it can be detrimental to the host by triggering tissue
damage. Indeed, Diep et al. demonstrated that PVL was
associated with increased inflammation and neutrophil recruit-
ment, both of which trigger lung injury [7].
Kineret, also known as Anakinra, is a drug used to treat
rheumatoid arthritis and several inflammasome-related diseases
[8]. Kineret is a recombinant form of the naturally occurring IL-1
receptor antagonist (IL-1Ra). Kineret competes with the IL-1
PLOS ONE | www.plosone.org 1 June 2014 | Volume 9 | Issue 6 | e97546
receptor for the binding of IL-1aand IL-1b. The safety of Kineret
is well-characterized, thus allowing the drug to be used to treat
other diseases [9].
In this work, we first characterized IL-8 secretion by human
neutrophils, macrophages and lung epithelial cells in response to
PVL and toxin-containing bacterial supernatant in vitro. We then
performed an in vivo study with two specific aims: i) To assess
whether inflammasome activation and the rPVL/IL-1/IL-8
inflammatory cascade were relevant during pneumonia. ii) To
test whether Kineret/IL-1Ra could block this cascade and
alleviate lung inflammation and injury.
Our results confirmed that PVL is a virulence factor that
contributes to lung inflammation. Furthermore, we demonstrated
that the instillation of heat-killed S. aureus (HKS) and rPVL,
Kineret/IL-1ra reduced PVL-mediated IL-8 secretion, thus
indicating the functionality of the rPVL/IL-1/IL-8 cascade in
vivo. However, during infection with PVL
+
S. aureus, we found that
Kineret/IL-1ra had no effect on IL-8 levels, suggesting that other
inflammatory mechanisms were at play. Finally, treatment with
Kineret/IL-1ra increased bacterial replication in the lung,
indicating that the IL-1 inflammatory pathway contributed to
bacterial clearance. This latter result highlights the possible caveat
of targeting inflammatory pathways and indicates that if such a
therapeutic option were chosen, it could only be applied in the
presence of a potent adjunctive antibiotic therapy.
Materials and Methods
Ethics Statement
The experimental protocol was approved by the local ethics
committee for animal experimentation (Burgundy, Dijon, France-
protocol number CE 1311). Blood packs from healthy donors were
obtained anonymously through an agreement (InsermU1111/
11_02_2013) with the Etablissement Franc¸ais du Sang (EFS,
Lyon, France) in accordance with French laws #98-535 and 99-
1143 and with the principles of the Declaration of Helsinki. Each
donor provided written informed consent, which is available upon
request. The human cell lines (THP-1 and A549) used in this study
were previously described [4].
Bacterial strains, culture conditions and reagents
The MRSA Staphylococcus aureus USA 300 PVL
+
Los Angeles
Clone 0114 (hereafter termed USA300 LAC), SF8300 (BD425)
and their isogenic PVL
2
derivative strains (HT20060753 and
BD0452) kindly provided by Franck DeLeo [10] and Binh Diep
[7] were grown in CCY broth, which promotes the production of
leukocidins. The clinical MRSA strain ST20120376 (a clonal
complex CC121 PVL
+
strain) was isolated from a patient suffering
from necrotizing pneumonia. Supernatant was prepared from a
16 h culture and diluted 625 times before addition to cells. rPVL
was produced and purified as previously described [4].
Cells, culture conditions and in vitro tests
Human neutrophils were purified as previously described [11].
When applicable, Heat-killed S. aureus (HKS MOI 100:1) and
Kineret were added to the neutrophils 3 h before rPVL
intoxication.
THP-1 cells and A549 cells were cultured as previously
described [4]. For the mixed culture experiment, 10
5
A549 cells
were added to 10
3
PMA-differentiated THP-1 cells 24 h before
the addition of PVL. HKS was added for 16 h before cell
intoxication.
Production and treatment of necrotizing pneumonia in
rabbits.
Animals. Male New Zealand rabbits were bred and housed
at the Zootechnical Center (University of Burgundy, Dijon,
France) in accordance with current European Institute of Health
guidelines.
Inoculum. USA 300 LAC and its isogenic PVL mutant were
grown in CCY medium supplemented with pyruvic acid for
10 hours. The inoculum was adjusted to 3610
9
CFU/mL.
Rabbit Model of Necrotizing Pneumonia. The central
venous catheters were installed and the pneumonia model was
established as previously described [12,13]. Bacterial pneumonia
was induced by endobronchial challenge with 0.5 mL of saline
containing 3610
9
CFU/mL. rPVL-induced pneumonia was
achieved by endotracheal instillation of 12 mg each of LukS-PV
and LukF-PV 3 hours after the delivery of 0.5 mL of HKS.
Kineret/IL-1Ra was kindly provided EXW by SOBI
ß
and
administered at doses of 10 mg/kg (Fig. 1).
Evaluation of necrotizing pneumonia
Bacterial counts. The rabbits were euthanized 6 hours after
challenge (Fig. 1). Each pulmonary lobe was weighed and
homogenized in 5 mL of sterile saline for bacterial counts.
Bacterial concentrations in each lobe were determined after
adjusting for weight. The threshold value was 1 log
10
CFU/g.
Macroscopic scoring. Macroscopic pulmonary injury scores
were calculated according to a macroscopic scoring grid as
previously described [14].
Bronchoalveolar Lavage Fluid. At the time of euthanasia
(6 h after challenge), the lungs were excised and bronchoalveolar
lavage (BAL) was performed on both inferior lobes by the
instillation of 4 ml physiological saline. Total protein concentra-
tion in the BAL fluid (BALF) was determined by the Bradford
protein assay according to the manufacturer’s instructions (Pierce).
Measurements of IL-1band IL-8. Concentrations of rabbit
IL-1band IL-8 in the BALF and in the crude homogenate of each
pulmonary lobe were assessed by ELISA (Euromedex). The release
of human IL-1band IL-8 was quantified by ELISA using DuoSet
ELISA kits (R&D systems, Lille, France).
Statistical analysis. In vitro and in vivo data were analyzed
by an unpaired t-test and Mann-Whitney analysis, respectively,
using Prism software (GraphPad, San Diego, USA). For in vitro
data, means and standard deviations are shown. Two tailed p
values are shown: n.s.: not significant, * p,0.05; ** p,0.01; ***
p,0.0001.
Results
Kineret/IL-1Ra blocks the IL-1b/IL-8 inflammatory cascade
observed in a co-culture of human macrophages and
lung epithelial cells exposed to S. aureus supernatant,
but has no effect on PVL-mediated neutrophil response
Using a co-culture system of macrophages (1%) and lung
epithelial cells (99%), we previously showed that very low
concentrations of IL-1breleased by rPVL-intoxicated macrophag-
es triggered the release of large amounts of IL-8 by lung epithelial
cells [4]. In line with this result, the addition of Kineret/IL-1Ra to
a co-culture of macrophages (1%) and lung epithelial cells (99%)
exposed to supernatant from a clinical PVL
+
S. aureus strain
isolated from a patient suffering from necrotizing pneumonia
reduced IL-8 secretion in a dose-dependent manner (Fig. 2A).
Though Kineret/IL-1Ra also reduced IL-8 levels in the same
cellular model in response to supernatant from USA300 SF8300
Kineret H/IL-1Ra in CA-MRSA-Pneumonia
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[7] or LAC strains, we found no difference in IL-8 levels between
WT USA300 strains and their isogenic DPVL mutants (Fig. 2B
and data not shown). This result indicates that other staphylococ-
cal secreted factors besides PVL can trigger the IL-1/IL-8 cascade.
Altogether, these results suggested that the majority of IL-8
produced early on during infection with S. aureus could be
secondary to IL-1 production triggered by the exposure of
macrophages to PVL or other staphylococcal secreted factors.
However, neutrophils are rapidly recruited to the lung and rapidly
out-compete alveolar macrophages. We thus decided to quantify
the production of IL-1band IL-8 by human neutrophils treated
with rPVL. In agreement with the low ability of human
neutrophils to produce IL-1b[15,16,17], IL-1bproduction by
rPVL-intoxicated primary human neutrophils was very low even
when they were primed with HKS (Figure 2C [4]). As previously
described [3,18], human neutrophils intoxicated with rPVL at
10 mg/ml produced high levels of IL-8 (Fig. 2D). In contrast, rPVL
at 100 mg/ml did not lead to consistent IL-8 production. This was
probably due to the rapid death of neutrophils at this concentra-
tion [7,11,19,20]. We then checked whether IL-8 production by
neutrophils or by lung epithelial cells exposed to PVL-intoxicated
neutrophils could be due to IL-1 signaling. Kineret/IL-1Ra had
no impact on IL-8 production by neutrophils (Fig. 2E) or by a co-
culture of neutrophils and lung epithelial cells (not shown), thus
indicating that IL-8 production in neutrophils is independent of
the IL-1/IL-8 cascade observed in macrophages ([4] and Fig. 2A).
Altogether, these results indicated that PVL triggers different
signaling pathways in human macrophages and neutrophils.
Furthermore, these in vitro results suggested that Kineret/IL-
1Ra could block IL-8 in vivo if the inflammatory response is driven
by macrophages, with no major contribution from neutrophils.
Infection with PVL
+
S. aureus triggers IL-1band IL-8
release in a rabbit model of necrotizing pneumonia
We and others have recently identified PVL as a major
inflammasome activator in human monocytes and macrophages
[4,5]. However, whether PVL activates the inflammasome
pathway in vivo remains to be determined. The rabbit model is
a well established model to study PVL
+
S. aureus diseases
[7,18,20,21,22] and to discriminate between active and inactive
anti-infective treatments [23][13]. We thus investigated the role of
PVL and its ability to activate the inflammasome pathway in a
rabbit model of pneumonia [7]. Rabbits were inoculated
intratracheally with 1.5610
9
CFU of the MRSA USA300 LAC
strain or its isogenic DPVL mutant strain (Fig. 1). This infectious
dose led to the rapid PVL-dependent death of infected rabbits
(Figure S1), with macroscopic lesions of the lung at 15 h PI
indicating necrosis (Figure S2). Necrotizing pneumonia is a
fulminant disease. We therefore focused on early events. Infected
animals were euthanized at 6 h PI. Bronchoalveolar lavage fluid
(BALF) and lung lysates were collected to quantify cytokines.
Infection with the PVL
+
strain was associated with greater IL-1b
release in the BALF than was the case with a DPVL mutant strain
(Fig. 3A). This cytokine is notoriously difficult to detect in
biological fluids [24] suggesting that the low levels of IL-1b
Figure 1. Study protocol.
doi:10.1371/journal.pone.0097546.g001
Kineret H/IL-1Ra in CA-MRSA-Pneumonia
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detected in the BALF of PVL
+
S. aureus infected rabbits were
probably biologically relevant. As previously described [7], PVL
+
S. aureus-mediated pneumonia was associated with a greater
increase in IL-8 levels in the BALF (Fig. 3B) and the lung lysates
than that observed during infection with the DPVL strain (Fig. 3C).
This increase in inflammatory cytokines was associated with an
increase i) in the permeability of the alveolar-capillary barrier as
measured by the protein content of the BALF (Fig. 3D), ii) in the
overall lung histopathology as determined by macroscopic scoring
(Fig. 3E), iii) in the lung weight/body weight ratio (a measurement
of pulmonary edema-Fig. 3F) and iv) in the lung bacterial burden
(Fig. 3G).
Figure 2. IL-8 produced in response to
S. aureus
supernatant by a co-culture of macrophages and lung epithelial cells is dependent
on IL-1 signaling, while IL-8 produced by neutrophils is independent of IL-1. (A, B) THP-1 macrophages (10
3
cells), A549 lung epithelial cells
(10
5
) were cultured alone or mixed and treated as indicated with HKS, S. aureus CCY broth culture supernatant and Kineret/IL-1Ra. IL-8 was quantified
by ELISA at 6 h post-intoxication. (C–E) As indicated, primary human neutrophils were treated with HKS, Kineret/IL-1Ra (E) and rPVL. IL-1b(C) and IL-8
levels were quantified at 6 h PI (E) or at the indicated time post-intoxication (C, D). One experiment representative of three independent experiments
with three independent donors is shown.
doi:10.1371/journal.pone.0097546.g002
Kineret H/IL-1Ra in CA-MRSA-Pneumonia
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Sequential instillation of Heat-killed S. aureus and rPVL
triggers pneumonia-like symptoms associated with IL-1b
and IL-8 production.
To ensure that the differences in IL-1band IL-8 production and
lung injury was due to the presence of PVL, we investigated
whether we could reproduce this inflammation using rPVL. In
vitro, inflammasome activation in response to pore-forming toxins
requires prestimulation with TLR agonists [25,26]. We thus
adapted the protocol established by Diep and collaborators [7]
and instilled HKS followed 3 h later by rPVL intratracheally.
Rabbits were euthanized 3 h post-rPVL instillation (Fig. 1). As
described above for infection, in this sterile model, the presence of
rPVL was associated with an increase in the levels of IL-1b
(Fig. 4A) and IL-8 (Fig. 4B) in the BALF, and IL-8 (Fig. 4C) in lung
lysates. Furthermore, the increase in inflammation mirrored an
increase in the lung histopathology as determined by macroscopic
scoring (Fig. 4D) and lung edema (Fig. 4E). Interestingly, at this
time point, HKS alone had no effect on the secretion of cytokines,
but worked in synergy with rPVL to induce IL-8 and pulmonary
pathological features.
Altogether, these two models of HKS-rPVL-mediated and
bacterial pneumonia indicated that the presence of PVL was
associated in vivo with inflammasome activation and confirmed
that PVL contributed to severe inflammation and lung injury in a
rabbit model of necrotizing pneumonia [7,18].
Kineret/IL-1Ra blocks the PVL/IL-1/IL-8 inflammatory
cascade observed in a HKS-rPVL-mediated pneumonia
model
IL-1band IL-8 are two key cytokines involved in the
recruitment of neutrophils [6]. Neutrophils contribute to the
pathology of necrotizing pneumonia [7]. We thus investigated
Figure 3. PVL is associated with an increase in IL-1band IL-8 release in a rabbit model of
S. aureus
-mediated pneumonia. (A–G)
Rabbits were inoculated intratracheally with 1.5610
9
cfu of USA300 LAC WT or DPVL strains and euthanized 6 h post-infection. IL-1b(A), IL-8 levels in
BALF (B) or in lung lysates (C) were quantified by ELISA. (D) Total protein levels in BALF were quantified by a Bradford assay. (E) Gross pathological
scoring was performed. (F) The ratio of lung weight to body weight is shown. (G) Bacterial burden as determined by CFU in each rabbit lung is shown.
(A–D) Each point represents the value obtained in the BALF from one lobe or (E–G) the value obtained for one rabbit. (A–G) The geometric mean is
shown. One experiment (n = 6) representative of two experiments (n = 12) is shown.
doi:10.1371/journal.pone.0097546.g003
Kineret H/IL-1Ra in CA-MRSA-Pneumonia
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whether Kineret/IL-1Ra was effective in vivo to decrease the IL-
1/IL-8 inflammatory cascade and the inflammation-associated
lung damages. We first tested Kineret/IL-1Ra on pneumonia
triggered by sequential instillation of HKS and rPVL. To ensure
the highest potency of this drug, we administered Kineret IV 2 h
post-HKS treatment as well as in co-instillation with rPVL (Fig. 1).
Kineret was used at 10 mg/kg [27]. Interestingly and as observed
in vitro using human macrophages, treatment with Kineret
reduced IL-8 levels in both BALF (Fig. 5A) and lung lysates
(Fig. 5B). The reduction in IL-8 levels in lung lysate was much
greater than in BALF possibly due to the poor bioavailability of
IL-1Ra in the lung lumen. The inhibition of IL-1 signaling and IL-
8 production did not decrease the macroscopic pathological score
(Fig. 5C), edema formation (Fig. 5D) or the permeability of the
alveolar-capillary barrier (Fig. 5E). Overall, these results demon-
strated the presence of this PVL/IL-1/IL-8 cascade in vivo in the
lung and showed that Kineret/IL-1Ra targets this pathway but
has no detectable effect on lung pathophysiology.
Kineret/IL-1Ra does not block the PVL/IL-1/IL-8
inflammatory cascade observed during lung infection
with PVL
+
S. aureus
Since treatment with Kineret/IL-1Ra led to encouraging results
on the possibility to target the IL-1/IL-8 cascade, we decided to
investigate its potency during PVL
+
S. aureus-mediated pneumonia
(Fig. 1). In contrast to what we observed in the HKS-rPVL model,
treatment with Kineret/IL-1Ra did not reduce IL-8 levels in
either BALF (Fig. 6A) or lung lysates (Fig. 6B). As previously
described in the HKS-rPVL model, treatment with Kineret/IL-
1Ra was ineffective in reducing the pathological score (Fig. 6C),
lung edema (Fig. 6D) or the permeability of the alveolar-capillary
barrier (Fig. 6E). However, treatment with Kineret/IL-1Ra did
result in a significant increase in the bacterial burden per lung
(Fig. 6F) indicating that IL-1 signaling contributes to antibacterial
defenses during necrotizing pneumonia.
Figure 4. Sequential intratracheal instillation of HKS and rPVL triggers IL-1band IL-8 release and reproduces some key aspects of
S.
aureus
-mediated pneumonia. (A–E) Rabbits were inoculated intratracheally with HKS, 3 h later with PBS or with rPVL and euthanized 6 h post-HKS
instillation. IL-1b(A) and IL-8 levels in BALF (B) or in lung lysates (C) were quantified by ELISA. (D) Gross pathological scoring was performed. (E) The
ratio of lung weight on body weight is shown. (A–C) Each point represents the value obtained in the BALF from one lobe or (D, E) the value obtained
for one rabbit. (A–F) The geometric mean is shown. Results from three independent experiments (n = 12) are shown.
doi:10.1371/journal.pone.0097546.g004
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Discussion
CA-S. aureus necrotizing pneumonia is a severe disease with a
high percentage of fatal outcomes. The role of PVL in
experimental infections, in triggering specific human diseases or
in affecting disease outcome is still debated [28,29,30]. Here, using
infection or sequential instillations of HKS and rPVL, we
confirmed the role of PVL in triggering inflammation and lung
injury in a rabbit model of necrotizing pneumonia. Two recent
studies have described the ability of PVL to activate the
inflammasome in primary human monocytes and macrophages
[4,5]. We demonstrated in vivo that the presence of PVL was
associated with an increase in IL-1blevels in the BALF of infected
rabbits, thus highlighting the relevance of this pathway during
pneumonia. Furthermore, we observed that the blockage of IL-1
signaling using Kineret/IL-1Ra led to a ten-fold increase in the
bacterial burden in the lung of infected animals, indicating that IL-
1 is critical for the antibacterial activity during acute pneumonia.
The current guidelines for the treatment of necrotizing
pneumonia are the prompt and aggressive administration of
toxin-suppressing antibiotics such as linezolid [23,31,32,33].
Appropriate antibiotic therapy might not be sufficient in such
fulminant diseases in which inflammation is intense and at least
partly responsible for lung injury [7]. Indeed, numerous deaths
have been observed in patients treated in a timely manner with
effective antibiotic therapy [32,34]. Adjunctive anti-inflammatory
treatment is widely used in bacterial meningitis even though its
efficacy might be limited [35]. The use of non-steroidal
antiinflammatory drugs in CA pneumonia is associated with a
more complicated course [36] and the use of dexamethasone in
treating CA pneumonia is still debated [37,38]. Furthermore, CA-
pneumonia covers a wide range of diseases in terms of both the
causative pathogenic agent and severity, thus highlighting the need
to test adjunctive therapy in specific animal models of diseases.
Here, we used a rabbit model of pneumonia to test the therapeutic
potential of anti-inflammatory drugs. Due to the high sensitivity of
rabbit neutrophils to PVL [20] and as demonstrated by others
Figure 5. Kineret/IL-1Ra inhibits the IL-1/IL-8 cascade triggered by sequential intratracheal instillation of HKS and rPVL. (A–E) Rabbits
were inoculated intratracheally with HKS, 3 h later with rPVL and euthanized 6 h post-HKS instillation. When applicable, Kineret/IL-1Ra was injectedIV
at 2 h post-HKS and was co-instillated with rPVL. IL-8 levels in BALF (A) or in lung lysates (B) were quantified by ELISA. (C) Gross pathological scoring
was performed. (D) The ratio of lung weight to body weight is shown. (E) Total protein levels in BALF were quantified by Bradford assay. (A–B, E) Each
point represents the value obtained in the BALF from one lobe or (C, D) the value obtained for one rabbit. (A–E) The geometric mean is shown.
Results from two independent experiments (n = 12) are shown.
doi:10.1371/journal.pone.0097546.g005
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[7,18], the rabbit is a good animal model to study PVL
+
-S. aureus
pneumonia and to assess treatment efficacy. However, the
limitations of our model include the high inoculum required to
trigger robust pneumonia and the absence of an influenza-like
viral infection preceding S.aureus infection, which is observed in
most severe human clinical cases [1,2]. We found that dexameth-
asone had no effect on IL-8 or any of the other parameters
considered in our model of pneumonia (not shown). In contrast,
we demonstrated that Kineret/IL-1Ra was effective in inhibiting
the rPVL/IL-1/IL-8 cascade after sequential instillation of HKS
and rPVL. Surprisingly, this effect was not observed during
infection despite the detection of PVL-dependent IL-1band IL-8
release. Several hypotheses can explain this difference: i) while the
macrophage response may dominate in the sterile model (HKS+
rPVL), a neutrophil-based response may be predominant during
infection. Indeed, human neutrophils produced IL-8 in response to
rPVL independently of IL-1 signaling (Fig. 2E). Furthermore, IL-8
levels were strongly reduced in neutropenic rabbits [7,18]. ii) S.
aureus and leukocidins/PSMs-intoxicated neutrophils release nu-
merous proteases[39,40][41], which might degrade/inactivate
Kineret/IL-1Ra. iii) Endogenous IL-1Ra, which is up-regulated
during bacterial pneumonia [42], might mask the action of
exogenously added IL-1Ra/Kineret.
In our sterile pneumonia model, despite the efficient block of
IL-1 signaling and a reduction of 50% in IL-8 level, we found no
reduction in neutrophil infiltration into the lung (not shown). This
result suggests that neutrophil recruitment in the lung is based on
several redundant pathways [6] and would require the targeting of
numerous signaling pathways to be significantly reduced. Similar-
ly, the failure of Kineret/IL-1ra clinical trials in severe sepsis led to
the conclusion that strategies aimed at targeting a single
inflammatory mediator were unlikely to work in such complex
diseases. Importantly, such a lesson can be learned from S. aureus,
which has evolved a large number of virulence factors targeting
neutrophils [43,44].
Figure 6. Kineret/IL-1Ra does not reduce IL-8 levels during PVL
+
S. aureus
-mediated pneumonia but increases lung bacterial load.
(A–F) Rabbits were inoculated intratracheally with USA300 LAC strain and euthanized 6 h post-infection. When applicable, Kineret/IL-1Ra was injected
IV 1 h before and 3 h after infection and was co-instilled with S. aureus. IL-8 levels in BALF (A) or in lung lysates (B) were quantified by ELISA. (C) Gross
pathological scoring was performed. (D) The ratio of lung weight to body weight is shown. (E) Total protein levels in BALF were quantified by
Bradford assay. (F) Bacterial burden as determined by CFU assay is shown. (A, B, E) Each point represents the value obtained in the BALF from one
lobe or (C, D, F) the value obtained for one rabbit. (A–F) The geometric mean is shown. Results from two independent experiments (n = 12) are
shown.
doi:10.1371/journal.pone.0097546.g006
Kineret H/IL-1Ra in CA-MRSA-Pneumonia
PLOS ONE | www.plosone.org 8 June 2014 | Volume 9 | Issue 6 | e97546
Supporting Information
Figure S1 Survival curves of LAC PVL and LAC DPVL-
infected rabbits.
(PPTX)
Figure S2 Macroscopic Pulmonary Injury Scores of LAC
PVL and LAC A
¨PVL-infected rabbits.
(PPTX)
Acknowledgments
We thank Frank deLeo and Binh Diep for providing strains, Y. Jamilloux
and F. Ader for stimulating discussions. This work was performed within
the framework of the LABEX ECOFECT (ANR-11-LABX-0042) of
Universite´ de Lyon, within the program ‘‘Investissements d’Avenir’’ (ANR-
11-IDEX-0007) operated by the French National Research Agency (ANR).
We thank Philip Bastable for helpful comments on the manuscript.
Author Contributions
Conceived and designed the experiments: DL PC GL FV DCB TH.
Performed the experiments: DL MP DH SDS CB FC DCB. Analyzed the
data: DL MP CB DCB TH. Contributed reagents/materials/analysis tools:
MP MB CB DCB. Wrote the paper: TH DL DCB PC FV GL.
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