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The NLRP12 Inflammasome Recognizes Yersinia
Gregory I. Vladimer
University of Massachusetts Medical School, firstname.lastname@example.org
University of Massachusetts Medical School, Dan.Weng@umassmed.edu
Sara W. Montminy Paquette
University of Massachusetts Medical School, Sara.PaquetteMontminy@umassmed.edu
Sivapriya Kailasan Vanaja
University of Massachusetts Medical School
Vijay A.K. Rathinam
University of Massachusetts Medical School, email@example.com
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Vladimer, Gregory I.; Weng, Dan; Paquette, Sara W. Montminy; Vanaja, Sivapriya Kailasan; Rathinam, Vijay A.K.; Aune, Marie
Hjelmseth; Conlon, Joseph E.; Burbage, Joseph J.; Proulx, Megan K.; Liu, Qin; Reed, George W.; Mecsas, Joan C.; Iwakura, Yoichiro;
Bertin, John; Goguen, Jon D.; Fitzgerald, Katherine A.; and Lien, Egil, "The NLRP12 Inflammasome Recognizes Yersinia pestis"
(2012).GSBS Student Publications.Paper 1782.
Gregory I. Vladimer, Dan Weng, Sara W. Montminy Paquette, Sivapriya Kailasan Vanaja, Vijay A.K. Rathinam,
Marie Hjelmseth Aune, Joseph E. Conlon, Joseph J. Burbage, Megan K. Proulx, Qin Liu, George W. Reed,
Joan C. Mecsas, Yoichiro Iwakura, John Bertin, Jon D. Goguen, Katherine A. Fitzgerald, and Egil Lien
This article is available at eScholarship@UMMS:http://escholarship.umassmed.edu/gsbs_sp/1782
Gregory I. Vladimer,1Dan Weng,1,8Sara W. Montminy Paquette,1,8Sivapriya Kailasan Vanaja,1Vijay A.K. Rathinam,1
Marie Hjelmseth Aune,2Joseph E. Conlon,1Joseph J. Burbage,1Megan K. Proulx,3Qin Liu,4George Reed,4
Joan C. Mecsas,5Yoichiro Iwakura,6John Bertin,7Jon D. Goguen,3Katherine A. Fitzgerald,1and Egil Lien1,2,*
1Division of Infectious Diseases and Immunology, UMass Medical School, Worcester, MA 01605, USA
2Department of Cancer Research and Molecular Medicine, NTNU, 7489 Trondheim, Norway
3Dept of Molecular Genetics and Microbiology
4Division of Preventive and Behavioral Medicine
UMass Medical School, Worcester, MA 01655, USA
5Department of Molecular Biology and Microbiology, Tufts University, Boston, MA 02111, USA
6Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan
7Pattern Recognition Receptor DPU, GlaxoSmithKline, Collegeville, PA 19426, USA
8These authors contributed equally to this work
Yersinia pestis, the causative agent of plague, is able
to suppress production of inflammatory cytokines
IL-18 and IL-1b, which are generated through cas-
pase-1-activating nucleotide-binding domain and
somes. Here, we sought to elucidate the role of NLRs
and IL-18 during plague. Lack of IL-18 signaling led
to increased susceptibility to Y. pestis, producing
tetra-acylated lipid A, and an attenuated strain
producing a Y. pseudotuberculosis-like hexa-acyl-
ated lipid A. We found that the NLRP12 inflamma-
some was an important regulator controlling IL-18
and IL-1b production after Y. pestis infection, and
NLRP12-deficient mice were more susceptible to
bacterial challenge. NLRP12 also directed inter-
feron-g production via induction of IL-18, but had
minimal effect on signaling to the transcription factor
NF-kB. These studies reveal a role for NLRP12 in
host resistance against
NLRP12 inflammasome activation may have been
a central factor in evolution of the high virulence of
Inflammasomes are multimolecular complexes consisting of
inactive pro-caspase-1 and members of the nucleotide-binding
domain-leucine-rich repeat (NLR) family of immune system
proteins (Latz, 2010). The assembly of an inflammasome leads
to proteolytic activation of caspase-1, which in turn cleaves
pro-interleukin (IL)-1b and pro-IL-18 into mature forms (Latz,
2010). Active IL-1b and IL-18 are essential members of host
defenses toward various pathogens and may also participate
in sterile inflammatory processes. The NLR family has more
than 20 members; however, many of these proteins have
unknown functions (Martinon et al., 2009), and their relative roles
in promoting resistance to infection are in many instances
unclear. There is evidence supporting a function in bacterial
recognition for several NLRs. These include NOD1/2 (recog-
nizing peptidoglycan fragments) (Martinon et al., 2009), NLRP1
(sensing anthrax lethal toxin) (Averette et al., 2009), NLRP3
(activated by exposure to many pathogens, bacterial RNA,
toxins, and crystal structures) (Davis et al., 2011; Duewell
et al., 2010; Halle et al., 2008; Hornung et al., 2008; Kanneganti
et al., 2006; Sander et al., 2011), NLRC4 (sensing of Salmonella,
intracellular flagellin and bacterial type III secretion rod proteins)
(Franchi et al., 2006; Miao et al., 2010), and Naip5 (promoting
resistance to Legionella) (Kofoed and Vance, 2011; Molofsky
et al., 2006; Ren et al., 2006). Recent results also suggested
a role for NLRP6 in maintenance of bacterial homeostasis in
the colon and for NLRP7 in the recognition of lipoproteins (Khare
et al., 2012). NLRP12 (also called Nalp12, Monarch-1, and
Pypaf-7) was the first NLR shown in biochemical assays to
interact with the adaptor protein Asc to form an active IL-
1b-maturing inflammasome (Wang et al., 2002). The role of
NLRP12 in innate immunity has remained unclear. Both inflam-
matory and inhibitory functions have been suggested, as has
a role in hypersensitivity (Allen et al., 2012; Arthur et al., 2010;
Lich and Ting, 2007; Lich et al., 2007; Wang et al., 2002; Zaki
et al., 2011). Interestingly, like for NLRP3, mutations in NLRP12
are linked to hereditary inflammatory disease (Je ´ru et al.,
2008), and mutations may lead to increased Asc speckle forma-
tion and caspase-1 activity (Je ´ru et al., 2011b). It has been re-
ported that patients carrying NLRP12 mutations associated
with increased inflammasome activation have been successfully
treated with anti-IL-1 therapy, similar to patients containing
mutations in NLRP3 (Hawkins et al., 2003; Je ´ru et al., 2011a;
Lachmann et al., 2009). No previous studies have addressed
the role of NLRP12 in host resistance to infectious agents.
Evading innate immunity early in infection plays a key role in
virulence of many microorganisms including the plague bacillus
Yersinia pestis (Cornelis, 2000; Perry and Fetherston, 1997;
Stenseth et al., 2008). This pathogen has several means of mini-
mizing immune activation (Lathem et al., 2007; Monack et al.,
1998; Mukherjee et al., 2006; Sodeinde et al., 1992; Zhou
etal.,2005),with the effect thatbacterialreplication can proceed
with minimal interference by the immune system. As a result,
96 Immunity 37, 96–107, July 27, 2012 ª2012 Elsevier Inc.
plague is often characterized by very high bacterial numbers in
patient sera and organs (Perry and Fetherston, 1997). Major
factors neutralizing host defenses by active means include
a complex type III secretion system (T3SS) (Cornelis, 2002; Perry
and Fetherston, 1997), the plasminogen activator Pla (Lathem
et al., 2007; Sodeinde et al., 1992), and a high-affinity iron acqui-
sition system (Perry and Fetherston, 1997). The Yersinia T3SS
delivers effector proteins, which disrupt signaling within the
host cell to prevent phagocytosis, induce apoptosis, and evade
the immune response (Cornelis, 2002). Many Gram-negative
bacteria, including Y. pseudotuberculosis, a very close ancestor
of Y. pestis, produce a hexa-acylated lipid A and LPS, which has
the potential of strongly triggering innate immunity via Toll-like
receptor 4 (TLR4)-MD-2 signaling (Munford, 2008; Raetz et al.,
2007; Rebeil et al., 2004; Therisod et al., 2002). In contrast,
Y. pestis generates a tetra-acylated lipid A-LPS that poorly
induces TLR4-mediated cellular activation (Kawahara et al.,
2002; Knirel et al., 2005; Montminy et al., 2006; Rebeil et al.,
2006). We have reported that expression of E. coli lpxL in
Y. pestis, which lacks a homolog of this gene, forces the bio-
synthesis of a hexa-acylated LPS (Montminy et al., 2006) and
that this single modification dramatically reduces virulence in
wild-type mice, but not in mice lacking a functional TLR4. This
emphasizes that avoiding activation of innate immunity is
important for Y. pestis virulence. It also provides a model in
which survival is strongly dependent on innate immune
defenses, presenting a unique opportunity for evaluating relative
importance of innate immunity signals in protection against
One implication of TLR4 engagement is the induction of the
immature forms of the central proinflammatory cytokines IL-1b
and IL-18. TLR4 signaling can also promote expression of
inflammasome components such as Nlrp3 (Bauernfeind et al.,
2009). This establishes links between TLR4 activation and the
inflammasome pathways. In this study, we have used wild-
type Y. pestis and attenuated strains expressing a strong
TLR4-activating hexa-acylated LPS as a model system to inves-
tigate the involvement of NLRP12 in pathogen recognition and
IL-18 - IL-1b release.
Here, we show that NLRP12 is an inflammasome component
that is central in the recognition of Y. pestis and that IL-18
signaling substantially contributes to resistance against bac-
teria. Compared to wild-type mice, NLRP12-deficient animals
had higher mortality and increased bacterial loads after infec-
tion, correlated with lower amounts of IL-18, IL-1b, and IFN-g.
We propose a role for NLRP12 in the sensing of microbial
We have found that all members of the genus Yersinia other
thanY. pestis, and including
Y. pseudotuberculosis, contain the lpxL gene (S. Paquette
et al., unpublished data). Absence of lpxL and the resulting
production of a tetra-acylated LPS was proposed to be essential
for Y. pestis virulence (Montminy et al., 2006). To study the
evasion of TLR4 signaling in an evolutionary perspective, we
the very closely related
cloned lpxL from the closely related Y. pseudotuberculosis and
expressed it in Y. pestis, generating Y. pestis-pYtbLpxL, to
determine its effects on virulence. Y. pestis grown at 37?C has
a tetra-acylated lipid A (Figure S1A available online) (Montminy
et al., 2006), whereas Y. pseudotuberculosis and Y. pestis-
pYtbLpxL have a hexa-acylated lipid A (Figure S1B). Mice in-
fected subcutaneously (s.c.) with 500 colony forming units
(CFUs) of highly virulent Y. pestis KIM1001 rapidly succumb to
infection (Figure 1A). All wild-type mice infected with KIM1001-
pYtbLpxL expressing a hexa-acylated Y. pseudotuberculosis-
like lipid A survived (Figure 1A), and the animals were protected
toward challenge with virulent KIM1001 (Table S1). Survival of
mice was strongly TLR4 dependent (Figure 1A). To determine
the pathways responsible for in vivo clearance, we infected
mice from several strains deficient in inflammatory cytokines or
cytokine receptors s.c. with 500 CFUs of KIM1001-pYtbLpxL
(Figure 1B). Interestingly, 100% of the animals lacking IL-18
and IL-18R died, as did the TLR4-deficient mice and 70% of
the IL-1R1-deficient mice. Weaker effects were observed in
animals lacking IFN-abR, TNFR1, or IL-12p40 (Figure 1B). Resis-
tance to infection in IL-1b- and IL-1R1-deficient animals was
reduced to a similar degree, with ?30% of animals surviving
(Figure 1C). However, IL-18 was critically important for resis-
tance to infection in this model, given that IL-18 and IL-18R-
deficient mice developed symptoms of bubonic plague and
rapidly succumbed to disease when infected with KIM1001-
pYtbLpxL (Figures 1B and 1D). Because inflammasomes are
responsible for processing of IL-18 and IL-1b into mature forms,
this result indicates that this infection model is well-suited for the
study of inflammasome mechanisms and implications of IL-18
release. Mice deficient in MyD88, an adaptor molecule common
to TLR, IL-1R, and IL-18R signaling pathways, were more
susceptible to wild-type Y. pestis KIM1001 than wild-type
C57Bl/6 mice (Figure S1C) and are also highly susceptible to
strains expressing lpxL (Montminy et al., 2006). Intravenous
(i.v.) infection causes systemic infection even when attenuated
bacterial strains are used; hence the inflammatory capacity in
tissues for various bacterial strains can better be compared
with this route of delivery. We found elevated levels of spleen
IL-1b and IL-18 after i.v. infection with Y. pestis and fully virulent
KIM1001-induced lower cytokine levels as compared to
KIM1001-pYtbLpxL producing the potent LPS (Figures 1E and
1F). A similar release pattern could also be seen in vitro with
bone marrow-derived macrophages (BMDMs) (Figure 1G) after
stimulation with KIM5 (a pgm mutant attenuated strain used for
in vitro experiments) or KIM5-pYtbLpxL. Immunoblot analysis
(Figure 1H) indicated that pro-IL-1b was indeed cleaved
into mature IL-1b after infection with Y. pestis strains, a sign of
inflammasome action. Infection with the Y. pestis-YtbLpxL strain
markedly increased levels of pro- and cleaved IL-1b. These
results indicate that minimizing inflammasome priming may
have been an important implication of lpxL loss during evolution
of Y. pestis from Y. pseudotuberculosis.
NLRP12 Is Involved in Recognition of Y. pestis
We next wanted to determine which NLRs were involved in
resistance to Y. pestis strains and in IL-18 and IL-1b release.
NLRP12 and NLRP3 have both been shown to interact with
Asc in generating an IL-1b-processing inflamammasome
The NLRP12 Inflammasome Recognizes Yersinia pestis
Immunity 37, 96–107, July 27, 2012 ª2012 Elsevier Inc. 97
(Agostini et al., 2004; Manji et al., 2002; Wang et al., 2002), but
little is known of the role of NLRP12 during infection. We infected
both NLRP3-deficient and NLRP12-deficient mice (Figures 2A
and 2B) s.c. with 500 CFUs of KIM1001-pYtbLpxL and found
that only 20% of NLRP12-deficient mice survived the infection,
whereas ?50% of mice lacking NLRP3 survived. This suggests
that NLRP12 plays an important role in host defense against
some bacterial pathogens. In contrast, NLRP12-deficient
mice were resistant to infection with Salmonella typhimurium,
whereas TLR4-deficient mice all succumbed to the infectious
challenge (Figure 2C). This indicates that NLRP12 deficient
several organs and immune cells (Figures S2A and S2B),
including macrophages, although
maturation led to a decrease in expression (Figure S2C).
NLRP12-deficient mice (Figure S2D) had a normal composition
of cell populations in spleen and bone marrow (Figure S2E).
Figure 1. Infection of Mice with Y. pestis-pYtbLpxL Is Controlled by IL-18
(A) Survival of mice infected s.c. with 500 CFUs of Yersinia pestis KIM1001 (C57Bl/6: n = 8) or KIM1001-pYtbLpxL (C57Bl/6 and Tlr4?/?[TLR4 KO]: n = 10)
(B)Mortalityof animals infecteds.c. with 500CFUsof KIM1001-pYtbLpxL (n=7 forIfnar?/?[IFNabRKO],8 forIl12b?/?[IL-12p40 KO],10 forC57Bl/6, Il1r1?/?[IL-
1R1 KO], Il18r1?/?[IL-18R KO], Il18?/?[IL-18 KO], and TLR4 KO). Statistical differences in TLR4, IL-18, or IL-18R versus IL-1R and other strains: p < 0.002.
Statistical differences in IL-12p40, IL-18, IL-18R, and TLR4 versus C57Bl/6: p < 0.001.
(C and D) Survival of mice deficient in (C) IL-1b (Il1b?/?, IL1b KO) and IL-1R and (D) IL-18 infected s.c. with 500 CFUs of KIM1001-pYtbLpxL (n = 10 of each
(E and F) Concentrations of spleen IL-1b and IL-18 from C57BL/6 mice infected i.v. with 500 CFU of KIM1001 or KIM1001-pYtbLpxL for 44 hr.
(G) IL-1b in supernatants from BMDM stimulated with 10 multiplicity of infection (m.o.i.) of KIM5 or KIM5-pYtbLpxL for 6 hr, 50 mg/ml of gentamicin was added to
wells after 3 hr.p.i.; error bars represent the SD.
(H) Immunoblot of IL-1b in the combined lysates and supernatants of BMDMs stimulated with 10 m.o.i. of Y. pestis KIM5, KIM5-pEcLpxL, and KIM5-pYtbLpxL
and 1m.o.i. of Salmonella typhimurium.Both pro-IL-1b (upper band) and mature IL-1b (lower band) are shown. Shown is representativeof threeto five performed
experiments. *p < 0.05; **p < 0.01; ***p < 0.001. Also see Figure S1 and Table S1.
The NLRP12 Inflammasome Recognizes Yersinia pestis
98 Immunity 37, 96–107, July 27, 2012 ª2012 Elsevier Inc.
The possible involvement of NLRP12 in maturation of IL-1b and
IL-18 led us to perform in vitro experiments with mouse cells to
study inflammasome components that promote caspase-1
cleavage and IL-1b-IL-18 release after infection with Y. pestis
and modified strains. Neutrophils express more Nlrp12 than
macrophages (Figure S2B), but the role of inflammasomes in
pathogen-induced neutrophil release of IL-1b and IL-18 is not
late-elicited neutrophil-enriched peritoneal cells released IL-1b
after Y. pestis infection (Figure 3A). When compared to cells
from wild-type mice, the amounts of IL-1b, but not TNF (Fig-
ure S3A) released from the neutrophils lacking NLRP12, were
markedly reduced after stimulation with Y. pestis strains. More-
over, infected neutrophils from the caspase-1-deficient mice
lack IL-1b in the supernatant, suggesting that Y. pestis-induced
neutrophil IL-1b release involves caspase-1 inflammasomes,
although we cannot rule out a role for other neutrophil proteases
relative to caspase-1 in Y. pestis-induced inflammasome activa-
tion, giventhatthe caspase-1-deficient mice utilizedin thisstudy
contain the same truncated and apparently nonfunctional cas-
pase-11 as previously published (Kayagaki et al., 2011). Macro-
phages deficient in NLRP12 or NLRP3 also had a reduced ability
to release both IL-18 and IL-1b after infection with parental
Y. pestis and Y. pestis-pYtbLpxL (Figures 3B and 3C). These
observations are consistent with the survival data (Figure 2),
which indicated that host recognition of Y. pestis involves
NLRP12. Cells deficient in Asc and caspase-1 also had
decreased IL-18 and IL-1b release (Figures 3B and 3C). Thus,
NLRP12 signaling may occur parallel to or in cooperation with
additional inflammasome components because NLRP12 defi-
ciency did not completely block cytokine release. NLRP12
KO macrophages responded normally to alum, S. typhimurium
(Figure 3C), nigericin, and poly(dA:dT) (Figures S3B and S3C),
suggesting that NLRP12 may not participate in NLRP3, AIM2,
or NLRC4 inflammasomes formed in response to those stimuli.
None of the inflammasome proteins had an impact on TNF
release (Figure 3D; Figure S3). Furthermore, NLRP12 deficiency
had little impact on the expression of 31 selected macrophage
genes, including Il1b, in the absence or presence of bacteria
(Figure S3C). Many of those genes are controlled by NF-kB
and/or MAP kinases. In a more detailed study, NF-kB signaling
measured by IKK kinase assay and I-kB degradation was also
largely preserved in NLRP12-deficient cells (Figures S3D and
S3E). Y. pestis pregrown at 26?C naturally expresses a hexa-
acylated LPS (Montminy et al., 2006), and release of IL-1b
in response to infection by 26?C grown bacteria was also
influenced by NLRP12 (Figure S3B). Upon infection of wild-
type and NLRP12 KO BMDMs with the human pathogens
Y. pseudotuberculosis and Y. enterocolitica, ancestors of
IL-1b from the cells lacking NLRP12 (Figure 3E), although TNF
release was normal (Figure S3F). By using KIM6, a derivative of
KIM5 that lacks the pCD1 virulence plasmid containing genes
for the T3SS (Perry and Fetherston, 1997), we found that the
secretion system was necessary for stimulating IL-1b release,
even in the presence of a highly stimulatory LPS as found in
KIM6-pYtbLpxL (Figure 3F). YopJ may participate in inflamma-
some activation (Zheng et al., 2011), and the deletion of YopJ
or the T3SS translocon protein YopB reduced IL-1b release (Fig-
ure 3G). Experiments performed using a strain with the expres-
sion of lpxL on a YopJ mutant background suggested that
YopJ is a key player controlling IL-1b release, even in the
presence of a stimulatory LPS (Figure 3H), although other
T3SS-dependent factors may also regulate IL-1b (Brodsky
et al., 2010). The data suggest that the ligand(s) responsible for
NLRP12 activation are dependent on the Yersinia T3SS. TLR4
plays a critical role in the IL-1b and IL-18 production after infec-
tion of the mouse macrophages (Figure 3I; Figure S3G), although
the relative importance of mouse versus human TLR4-MD-2 in
inducing Y. pestis responses may differ. Rodent cells have
higher ability to recognize hypoacylated lipid A (Lien et al.,
2000; Montminy et al., 2006). This might be influenced by a
shallow positioning of the hypoacylated lipid A in mouse MD-2
compared to human MD-2, and the enabling of enhanced ionic
interactions between hypoacylated lipid A and mouse TLR4,
facilitating receptor cluster dimerization and signaling (Meng
et al., 2010). Our results indicate a role for both TLR4 and
NLRP12 in the proinflammatory macrophage response against
Y. pestis strains.
NLRP12 Is an Inflammasome Component
Upregulation of NLRP3 has been suggested to positively affect
the activity of the NLRP3 inflammasome (Bauernfeind et al.,
2009). We therefore studied expression of Nlrp12 and Nlrp3
(Figures 4A and 4B) after infection of macrophages with KIM5
or KIM5-pYtbLpxL. Expression of Nlrp12 in BMDM was mark-
edly increased after infection with Y. pestis strains and this
may boost host responses to an infection. Treatment with LPS
alone induced upregulation of Nlrp12 gene expression (Fig-
ure S4A). Furthermore, Y. pestis-induced formation of cleaved
and active caspase-1, as measured by an assay showing
binding of active caspase-1 to a fluorescent substrate, was
also impaired in NLRP12-deficient cells, providing evidence for
NLRP12-dependent inflammasome function (Figures 4C and
4D). Caspase-1 cleavage measured by this assay is also
Figure 2. NLRP12 Is Involved in Host Resis-
tance to Attenuated Y. pestis
(A and B) Survival of C57Bl/6 (circles), (A) Nlrp3?/?
(NLRP3 KO, squares), and (B) Nlrp12?/?(NLRP12
KO, squares) mice infected s.c. with 500s CFU of
(C) Survival of C57BL/6 (circles), NLRP12 KO
(squares), or TLR4 KO (triangle) mice infected i.p.
with 500 CFUs of S. typhimurium; p < 0.003
(NLRP12 KO or WT versus TLR4 KO). Also see
The NLRP12 Inflammasome Recognizes Yersinia pestis
Immunity 37, 96–107, July 27, 2012 ª2012 Elsevier Inc. 99
decreased in spleen macrophages or neutrophils from NLRP12
KO mice 24 hr after infection with KIM1001 or KIM1001-
pYtbLpxL (Figures 4E and 4F). Il1b gene expression was similar
in infected wild-type cells and NLRP12 KO cells infected with
Y. pestis (Figure 4G; Figure S3D). The macrophages infected
in vitro showed a reduction in caspase-1 and IL-1b processing
by immunoblotting (Figure 4H), also cells infected at a higher
m.o.i. (Figure S4B). Thus, several lines of evidence support the
hypothesis that NLRP12 is a component of inflammasomes
formed after Y. pestis infection. Macrophage cell death induced
et al., 2008). We confirmed those data (not shown), and in line
with this observation, NLRP12-deficient cells did not show an
altered cell death in response to Y. pestis infection (Figure S4C).
in macrophages infected with Y. pestis.
NLRP12 and IL-18 Mediate Host Resistance
to Y. pestis Infection
As shown in Figures 1 and 2, NLRP12 knockout (KO) and IL-18
KO mice are more susceptible than wild-type mice to infection
with Y. pestis-pYtbLpxL. To monitor changes in IL-18 and IL-
1b in tissues during systemic disease, we subjected WT and
NLRP12 mice to intravenous (i.v.) infection with fully virulent or
Figure 3. NLRP12 Mediates Y. pestis-Induced Release IL-1b and IL-18
(A) IL-1b released from neutrophil-enriched peritoneal cells from C57BL/6 (black bars), NLRP12 KO (gray bars), and Casp1?/?(Caspase-1 KO, white bars) mice.
(B–I) IL-18 (B), IL-1b (C and E–I) and TNF (D and G) released from C57BL/6, NLRP12 KO, NLRP3 KO, ASC KO, and caspase-1 KO BMDM (B–D); C57BL/6
and NLRP12 KO BMDM (E); C57BL/6 BMDM (F and G); or C57BL/6 and TLR4 KO BMDMs (I). Infection with Yersinia strains occurred for 6 hr, with an addition of
50 mg/ml gentamicin to limit bacterial growth after 3 hr. Yersinia strains were added at 10 m.o.i., S. typhimurium at 1 m.o.i. Alum (130 mg/ml) stimulations (C and I)
lasted 6 hr after priming for 3 hr with 10 ng/ml KIM5-YtbLpxL LPS. Shown are mean for triplicate cultures (with SD) in representative experiments out of three
to ten performed. *p < 0.05; **p < 0.01; ***p < 0.001. Statistical comparisons are between wild-type cells and multiple mutant cells (A–E and I) or between
unstimulated and multiple bacterial strains (F). Also see Figure S3.
The NLRP12 Inflammasome Recognizes Yersinia pestis
100 Immunity 37, 96–107, July 27, 2012 ª2012 Elsevier Inc.
attenuated Y. pestis (KIM1001 or KIM1001-pYtbLpxL). At 44 hr
after infection with KIM1001, IL-18 cytokine amounts were
considerably lower in the NLRP12 KO mice, expressed as both
cytokine normalized to the spleen bacterial load in each partic-
ular animal (Figure 5A) or simply as cytokine concentration in
homogenate (Figure S5A). A decrease of IL-18 and IL-1b in the
spleen (Figures 5B and 5C) and serum (Figures 5D and 5E)
was also observed after KIM1001-pYtbLpxL infection in the
Figure 4. NLRP12 Is Necessary for Optimal Maturation of IL-1b and Caspase-1 after Infection with Y. pestis
(A, B, and G) Q-PCR of (A) Nlrp12, (B) Nlrp3, or (G) Il1b from BMDMs infected with 10 m.o.i. of Y. pestis KIM5 or KIM5-pYtbLpxL for (G) 4 hr or (A and B) 6 hr, with
gentamicin addition after 3 hr. Error bars represent the SD.
(C–F) FACS histograms (C and E) showing active caspase-1 after FLICA reagent staining with corresponding (D and F) percent positive cells of (C and D) bone
marrow cells after 6 hr of challenge with 10 m.o.i. of Y. pestis strains (gentamicin added after 3 hr) or (E and F) Ly6G- or F4/80-positive splenocytes from mice
infected with 500 CFUs of KIM1001 or KIM1001-pYtbLpxL for 24 hr. Values from unstimulated cells are subtracted in (D). LPS primed cells treated with nigericin
(10 mM) served as a control (C and D). Error bars in (D) and (F) represent the SD.
(H) Immunoblot for caspase-1 p10 or IL-1b p17 in supernatant or cell lysate from BMDMs exposed for 10 hr to poly(dA:dT) (LPS primed as in Figure 3), KIM5, or
KIM5-pYtbLpxL. Shown is a representative of two (E–G) or three to five (A–D, H, and I) experiments. *p < 0.05; **p < 0.01; ***p < 0.001. Also see Figure S4.
The NLRP12 Inflammasome Recognizes Yersinia pestis
Immunity 37, 96–107, July 27, 2012 ª2012 Elsevier Inc. 101
Figure 5. NLRP12 and IL-18 Control Infection with Y. pestis In Vivo
(B–D) IL-18 or IL-1b in (B and C) spleen or (D) serum of WT or NLRP12 KO mice infected i.v. with 500 CFU KIM1001-pYtbLpxL for 44 hr. In (B) and (C), uninfected
mice: n = 3; infected animals: n = 8. In (D), uninfected mice: n = 4; infected mice: n = 5.
(E) Spleen CFUs of mice infected i.v. with either KIM1001 or KIM1001-pYtbLpxL (n = 5). Horizontal lines indicate mean values.
(F) Histology of fixed H&E stained liver sections from (top) WT, (middle) NLRP12 KO, or (bottom) IL-18R KO mice infected i.v. with (left) KIM1001 or (right)
KIM1001-pYtbLpxL for 44 hr. Asterisks represent bacterial clusters; arrows represent foci of inflammatory cells, primarily neutrophils.
(G) Survival of C57Bl/6 (n = 10), IL-18 KO, and NLRP12 KO (n = 8) mice with 10 CFU s.c. of KIM1001.
(H and I) Spleen CFU (H) or spleen IL-1b (I) of C57Bl/6 and NLRP3 KO mice infected for 44 hr with 500 CFU i.v. of KIM1001-pYtbLpxL. Shown is a representative
of three performed experiments. *p < 0.05; **p < 0.001. Also see Figure S5.
The NLRP12 Inflammasome Recognizes Yersinia pestis
102 Immunity 37, 96–107, July 27, 2012 ª2012 Elsevier Inc.
NLRP12 KO mice as compared to wild-type mice. Experiments
with IL-1R KO, IL-1b KO, and IL-18R KO mice suggested that
IL-18 signaling had the greatest impact on resistance to
Y. pestis-pYtbLpxL, given that 100% of IL-18 and IL-18R KO
animals died after infection (Figures 1B and 1D). IL-18R KO
mice had reduced IL-18 and IL-1b in the spleens compared to
WT mice after infection with KIM1001-pYtbLpxL (Figure S5B),
suggesting a positive feedback loop via IL-18R for IL-1b and
A reduction of several orders of magnitude in spleen bacterial
load was seen when mice were infected i.v. with KIM1001-
pYtbLpxL compared to wild-type KIM1001 (Figure 5E), indi-
cating beneficial host responses induced by the presence of
the hexa-acylated LPS. These differences in systemic bacterial
load between the two bacterial strains were absent in mice lack-
ing NLRP12 or IL-18R. NLRP12-deficient and IL-18R-deficient
mice also had increased bacterial loads compared to wild-type
mice when infected with the virulent Y. pestis KIM1001 (Fig-
ure 5E, p = 0.01, WT versus Nlrp12?/?; p < 0.001, WT versus
Il18r1?/?). This is important in that it shows that NLRP12 and
IL-18R participate in host resistance in vivo toward both virulent
and attenuated strains of Y. pestis. Thus, it appears that Y. pestis
has an inherent ability to activate NLRP12-dependent recogni-
tion and that the potent LPS found in strains expressing LpxL
increases the formation of proforms and subsequently mature
forms of inflammasome-controlled cytokines such as IL-1b
(Figures 1H and 4C). Livers from animals infected with wild-
type Y. pestis have large extracellular clusters of bacteria
(Figure 5F, left panels, marked with an asterisk) and remarkably
few signs of inflammation, probably reflecting active suppres-
sion of immunity combined with stealth via limited initiation of
TLR4 signaling. Livers from animals infected with Y. pestis-
upper right, indicated byarrows) and absence of visible bacterial
masses, suggesting that recruitment of phagocytes limits
bacterial growth (Montminy et al., 2006). Livers from NLRP12
KO mice infected with KIM1001-pYtbLpxL had recruitment of
inflammatory cells (Figure 5F, arrows). Such masses of inflam-
matory cells typically contain large number of neutrophils
and some mononuclear cells (Montminy et al., 2006), and a
calculation of number of recruited cells showed no significant
difference between infected wild-type versus NLRP12-deficient
livers (Figure S5C). However, this cell recruitment did not corre-
late with suppression of bacterial growth, given that bacterial
masses were visible (Figure 5F). These results suggest that
to infected sites in the liver, but is central to the effective antibac-
terial actions they perform. Few if any inflammatory cells were
visible in livers of IL-18R-deficient mice (Figure 5F), indicating
failures of both cell recruitment and antibacterial defenses.
Taken together, the results suggest that NLRP12 and IL-18
contribute to host resistance against Y. pestis and Y. pestis-
pYtbLpxL. We also found that NLRP12 KO mice infected with
KIM1001-pYtbLpxL had reduced amounts of TNF and the
chemokine CXCL12 compared to C57Bl/6 mice (Figures S5D–
S5F), possibly secondary effects of reduced IL-1b and IL-18
release, given that primary cells lacking NLRP12 did not
display decreased TNF release in culture (Figure 2). In contrast,
NLRP12-deficient mice injected with an alum-LPS mixture did
not show decreased serum IL-1b, IL-18, TNF-a, and CXCL12
(Figure S5G). Furthermore, we found similar recruitment of
neutrophils to the peritoneum of wild-type mice or NLRP12-
deficient mice injected intraperitoneally (i.p.) with sterile
thioglycollate (Figure S5H). Movement of neutrophils (Figure 5F;
Figure S5C) and DCs (Figure S5I) during infection of NLRP12-
deficient mice appears to be preserved. Differences in survival
between NLRP12-deficient or IL-18-deficient mice and wild-
type mice after s.c. infection with only 10 CFUs of fully virulent
KIM1001 were not significant (Figure 5G). This result is of uncer-
tain importance because the very low LD50 of Y. pestis by s.c.
infection (less than 10 CFUs) makes it difficult to demonstrate
reductions in host resistance impacting survival without the
use of very large numbers of animals. Tissue bacterial loads
(Figure 5E) appear to be more sensitive assays for analyzing
host resistance to Y. pestis.
NLRP3 has also been proposed as an inflammasome com-
ponent recognizing Y. pestis (Zheng et al., 2011) (Figures 2 and
3). NLRP3-deficient animals also were less resistant to infection
by KIM1001-pYtbLpxL, in that they displayed increased bacte-
rial loads in the spleen (Figure 5H) that correlated with reduced
spleen cytokines (Figure 5I). In summation, NLRP12 and
NLRP3 both contribute to the host resistance toward Y. pestis
NLRP12 and IL-18 Signaling Induce IFN-g that
IL-18 is a known inducer of IFN-g (Okamura et al., 1995), a key
protein in many host responses to pathogens. This suggests
that signaling via NLRP12 and the IL-18R, resulting in the release
of IFN-g, could mediate resistance to Y. pestis-pYtbLpxL. Mice
lacking both IFN-abR and IFN-gR (dKO) were infected with
KIM1001-pYtbLpxL s.c., and we found that all the dKO animals
succumbed to the infection (Figure 6A). This phenomenon was
largely attributed to IFN-gR signaling, given that only a few
mice lacking IFN-abR died upon infection, whereas almost all
mice lacking IFN-gR succumbed (Figure 6B). No differences in
IFN-g concentrations were observed between spleens of unin-
fected WT, NLRP12-deficient, and IL18R-deficient mice (Figures
6C and 6D). However, the IFN-g concentrations in spleens from
KIM1001-pYtbLpxL-infected NLRP12-deficient mice compared
to wild-type mice were drastically reduced (Figure 6C), as was
also true for the mice lacking IL-18R (Figure 6D). Thus, we
propose a cascade of signals from NLRP12 to IL-18 maturation
that in turn mediates IFN-g release after infection with Y. pestis
LPS by TLR4 leads to upregulation of NLRP12 and proinflam-
matory cytokines such as IL-18 and IL-1b. NLRP12 then recog-
nizes a ligand produced upon Y. pestis infection and assembles
into an inflammasome that processes IL-18 and IL-1b. Although
the precise nature of the true NLRP12 ligand is unknown,
and it may be a host or bacterial protein, the generation of
the ligand appears to require the virulence-associated T3SS
of Yersinia. Models for activation may include possibilities that
cells sense membrane damage associated with the T3SS,
The NLRP12 Inflammasome Recognizes Yersinia pestis
Immunity 37, 96–107, July 27, 2012 ª2012 Elsevier Inc. 103
secreted effectors or other molecules channeled by the T3SS,
and modified host proteins. NLRP3 also contributes to IL-18-
IL-1b release. IL-18 seems to be more critical than IL-1b and
plays a key role in induction of IFN-g.
We show that NLRP12 is an inflammasome component
recognizing Y. pestis and contributes to in vivo resistance to
infection with Y. pestis strains. To our knowledge, this is the
first demonstration of a clear role for NLRP12 in resistance to
infection. Our data suggest an inflammasome role for NLRP12
in pathogen recognition and that the NLRP12-IL-18-IFN-g axis
is effective in limiting infection with Y. pestis-pYtbLpxL. We
also show that the expression of Y. pseudotuberculosis LpxL
in Y. pestis increases TLR4-dependent release of IL-18 and
IL-1b. This increase correlates with increased resistance to the
modified pathogen. In fact, the results indicate that a major
consequence of producing LPS with low TLR4-activating poten-
tial could be lack of priming necessary for effective synthesis
of active IL-1b and IL-18. Therefore, Y. pestis is able to utilize
neutralize the immune response without an effective activation
of an inflammatory response. This phenomenon may have
played a role in evolution of high virulence in Y. pestis.
These findings support the view that inflammasomes, the
cellular protein complexes cleaving IL-18 and IL-1b into mature
forms, are fundamental components of the host response to
many pathogens. Indeed, several viral, bacterial and fungal
microbes have strongly increased ability to induce disease in
the absence of IL-1b, IL-18 and inflammasome components
(Broz et al., 2010; Davis et al., 2011; Hise et al., 2009; Lamkanfi
and Dixit, 2009; Rathinam et al., 2010). In spite of this, only
like theT3SS to
a few mammalian NLRs out of a family of more than 20 members
have currently been shown to directly participate in host
defenses. Here, we show that NLRP12 participates in host
responses to wild-type Y. pestis and modified Y. pestis strains
expressing a potent LPS, although the factor(s) in Y. pestis
responsible for directly activating the NLRP12 inflammasome
are still unknown.
NLRP12 may also be involved in resisting infections caused
by other human pathogens. It is unclear how NLRP12 may
interact with other inflammasome components. NLRP12 defi-
ciency did not cause a complete reduction in ability to release
IL-18 and IL-1b after exposure to Y. pestis and Y. pestis-
pYtbLpxL infection. Also, the increased mortality observed in
NLRP12-deficient mice did not appear as great as observed in
IL-18-deficient animals, and NLRP3 also plays a role in host
defenses. Redundancy between NLRs may occur, and other
NLRs may also participate in optimal responses to infection.
This may support the idea that NLRs work together for optimal
protection of the host (Broz et al., 2010). The generation of
animals with combined deficiencies in NLRP12 and other NLRs
may clarify how NLRP12 functions in cooperation with other
signaling components. NF-kB signaling after bacterial challenge
appeared normal in NLRP12-deficient cells.
IL-18, IL-1b, and IFN-g are all cytokines active at the interface
between innate and adaptive immunity. We have found that
Y. pestis strains generating a hexa-acylated LPS could function
as effective live vaccines (Montminy et al., 2006). It would be of
interest to investigate the role of NLRP12 in promoting the devel-
opment of adaptive immunity and protection after vaccination
with both live and subunit + adjuvant vaccines.
The emerging role of inflammasomes as key players in host
defenses during many infections makes them desirable targets
for therapeutic intervention and drug development. We note
that alum, one of the first components known to activate
specific inflammasomes, already is in widespread use as one
of the few vaccine adjuvants licensed for human use. However,
a delicate balance between pathological effects and enhanced
host defenses arising from inflammasome-stimulating treat-
ments will be necessary. Mutations in NLRs are linked to
inflammatory diseases (Hawkins et al., 2003; Je ´ru et al.,
2011a), and anti-IL-1 treatment does in fact reduce symptoms
in many such patients. More knowledge on the role of NLRs in
inflammation and homeostasis is needed in order to fine-tune
future NLR-based therapies.
Bacterial Strains and Growth Conditions
Y. pestis KIMis originally aclinical isolate from aKurdistan Iran man (Brubaker,
1970; Perry and Fetherston, 1997). Y. pestis strains KIM5, KIM5-pEcLpxL
(containing E. coli lpxL, earlier called pLpxL) and KIM1001 were as reported
(Montminy et al., 2006). Y. pseudotuberculosis IP2666 (containing a comple-
mentation of PhoP/PhoQ deficiency) and Y. enterocolitica 8081 were provided
by J. Mecsas. Strains were grown in tryptose-beef extract (TB) broth with
2.5 mM CaCl2all by shaking at 37?C. lpxL of Y. pseudotuberculosis IP2666
including 480 basepairs upstream and 266 basepairs downstream from
coding region was cloned with Pfu Ultra (Stratagene) and was ligated into
the BamHI and SalI sites of pBR322, creating pSP::YtbLpxL (or ‘‘pYtbLpxL’’).
The resulting plasmid was electroproated into Y. pestis KIM5 (Goguen et al.,
1984) or Y. pestis KIM1001 (Sodeinde et al., 1992), and bacteria were selected
by growth on TB agar supplemented with 2.5 mM CaCl2in the presence of
Figure 6. NLRP12 Induces IFN-g via IL-18 Signaling
(A and B) Survival of mice: in (A), C57Bl/6: n = 10 and Ifnar1?/?3 Ifngr1?/?
(IFNabR 3 IFN-gR DKO; triangles): n = 8, and in (B), C57Bl/6 (squares): n = 10,
IFNabR KO: n = 8, and IFN-gR KO: n = 7, infected s.c. with 500 CFUs of
(C and D) IFN-g in spleen homogenates from C57BL/6 and NLRP12 KO mice
infected i.v. with 500 CFU of KIM1001-pYtbLpxL. Uninfected mice: n = 3; in-
fected mice: n = 8 (C) and n = 5 (D). Samples were harvested 46 hr after
infection. Horizontal lines indicate median values. Experiments shown are
representative out of three performed. *p < 0.05; **p < 0.001.
The NLRP12 Inflammasome Recognizes Yersinia pestis
104 Immunity 37, 96–107, July 27, 2012 ª2012 Elsevier Inc.
100 mg/ml of ampicillin. All strains containing plasmids above remained tetra-
cycline sensitive. KIM1001 (pPCP1+, pCD1+, and pMT1+) is highly virulent
(Perry and Fetherston, 1997), whereas KIM5 bears the chromosomal deletion
‘‘Dpgm,’’ which substantially attenuates virulence. The pgm locus contains no
T3SS-containing pCD1 virulence plasmid. KIM5-DYopB was provided by
G. Plano (Torruellas et al., 2005). For the generation of KIM5-DYopJ, the
following method was used: An in-frame deletion removing codons 4–287
was created via allelic exchange. PCR products made with primer sets A
(50-ATAGAGCTCCACTACTGATTCAACTTGGACG-30), B (50-50-TCCGATCATT
TATTTATCCTTATTCA-30) and C (50-TGAATAAGGATAAATAAATGATCGGAT
AATGTATTTTGGAAATCTTGCT-30), D (50-GGGTCTAGACTGATGTCGTTTATT
TCTGGGTAT-30), respectfully, were used to make a fused product by overlap
allelic exchange vector pRE107 (Edwards et al., 1998) in E.coli K12 strain
B2155 and transferred to Y. pestis by conjugation; recombinants were
selected on TB medium containing 100 mg/ml ampicillin but no diaminopimelic
by PCR. For in vitro infections, bacteria were grown overnight at 37?C in TB
broth with or without ampicillin, diluted 1:4 in fresh media, and cultured for
in DMEM or RPMI. S. enterica serovar typhimurium strain SL1344 was
provided by M. O’Riordan and strain M525P by C. Bryant.
Mouse BMDMs were prepared by maturing fresh bone marrow cells for 5–
neutrophils were enriched by injecting 1 ml of thioglycolate i.p., peritoneal cells
after flushing with RPMI. Mouse BMDMs were plated at 2 3 105per well in 96-
well plates for ELISA or 2 3 106per well in 12-well plates for immunoblotting.
Stimulation was for 6 hr and supernatants were collected for cytokine analysis.
Three hours after bacterial infections, 50 mg/ml of gentamycin was added.
Alum was from Pierce; nigericin and poly(dA:dT) was from Sigma. IL-1b p17
and Caspase-1 p10 immunoblots were conducted mainly as described (Hor-
nung et al., 2008) with antibodies from Santa Cruz Biotechnology (caspase-1
p10) and R&D (IL-1 b). The antibody against b-actin was from Sigma. Q-PCR
for Nlrp12 and Nlrp3 in resting or infected BMDMs or magnetic bead (StemCell
Technologies)-isolated neutrophils was performed with the RNeasy Mini Kit
(QIAGEN) and the iScript cDNA Synthesis Kit (BioRad). PCR was performed
on transcribed cDNA or mouse tissue cDNA (Clontech) with primers for
detection of mouse Nlpr12 (50-TGCAAGCTTCGAGTCCTGT-30, 50-CCTGG
TCGGCTTCATTCTG-30), Nlrp3 (50-AACCAATGCGAGATCCTGAC-30, 50-AT
GCTGCTTCGACATCTCCT-30), or Il1b (50-GCCCATCCTCTGTGACTCAT-30,
50-AGGCCACAGGTATTTTGTCG-30) with SYBR green (BioRad) in accordance
IFN-g (R&D), and IL-18 (MBL) were used for cytokine detection. Reagents for
FACS detection of active and cleaved caspase-1 by FLICA-FITC substrate
were from Immunochemistry Technologies.
All experiments involving animals were approved by the Institutional Animal
Care and Use Committee. ASC (Pycard?/?), NLRP3 (Nlrp3?/?), and NLRP12-
deficient (Nlrp12?/?) mice were generated by Millennium Pharmaceuticals
and were backcrossed 8–11 generations to C57BL/6 background. Mice
deficient in TLR4 (TLR4?/?) and MyD88 (Myd88?/?) were from S. Akira, and
mice lacking caspase-1 (Casp1?/?) were from M. Starnbach. C57BL/6 mice
and mice deficient in IL-1R1 (Il1r1?/?), IL-18R (Il18r1?/?), IL-18 (Il18?/?),
TNFR1 (Tnfr1?/?), IL-12p40 (Il12b?/?), and IFN-gR (Ifngr1?/?) were all from
Jackson Laboratories. J. Sprent (The Scripps Research Institute) provided
the IFN-abR1 (Ifnar1?/?) and IFN-gR1 3 IFN-abR1 doubly deficient mice. IL-
1b (Il1b?/?)-deficient mice (Horai et al., 1998) were provided by Y. Iwakura.
Wild-type (from Jackson Laboratories or bred at UMass) and knockout mice
were infected s.c. in the nape of the neck with Y. pestis and their survival
was monitored twice a day for 30 days. Mice were infected with 1000 CFUs
of S. typhimurium M525P i.p. and survival was monitored as described above.
For cytokine and CFU analysis, mice were infected either s.c. or i.v. and sacri-
ficed at the indicated time points. Serum was generated by centrifugation in
microtainer tubes (BD), and spleens were homogenized in 0.5 ml PBS with
a closed system Miltenyi gentleMACS dissociator and c-tubes to preserve
intact cells; subsequently cells/debris were removed by centrifugation.
Samples for cytokine analysis were subjected to protease inhibitor (Roche)
treatment. Cytokine amounts normalized by bacterial loads were calculated
by dividing IL-18 concentrations (ng/ml) by the bacterial load (CFUs 3 108)
for each animal. Hematoxylin and eosin (H&E) staining and microscopy were
performed as published (Montminy et al., 2006).
In Vivo Caspase-1 Cleavage Analysis
Mice were infected with 500 CFUs of Y. pestis i.p. After 24 hr, spleens were
harvested and homogenized and cell suspensions were stained with cas-
pase-1 FLICA reagent.
In vitro cytokine release was analyzed by two-way ANOVA with a Bonferroni
post-test. Differences in spleen and serum cytokine concentrations were
analyzed by the unpaired t test. Differences in survival were studied with
Kaplan-Meyer analysis and the logrank test. Differences in spleen CFUs or
cytokine/CFU ratio values between genotypes of mice were evaluated with
the Mann-Whitney test or in more complex comparisons involving multiple
mouse genotypes, with a generalized linear regression model of cubic trans-
formed log CFU values (95% confidence interval), to meet normality assump-
tions. Values of p < 0.05 were considered significant.
Supplemental Information includes five figures, one table, and Supplemental
Experimental Procedures and can be found with this article online at http://
We thank A. Cerny, H. Ducharme, M. Whalen, A. Zacharia, and C. Raskett for
animal husbandry and X. He and members of the Lien, Fitzgerald, and Goguen
labs for help and discussions. Work was supported by the NIH (grants
AI057588-American Recovery and Reinvestment Act and AI075318 to E.L.,
AI64349 and AI083713 to K.F., AI095213 to G.I.V., and NERCE fellowships
AI057159 to S.K.V. and V.A.K.R.), the Research Council of Norway, and the
Norwegian Cancer Society. The study also utilized core services supported
by DERC grant NIH DK32520. We thank those who provided reagents, B.
Monks for help with cloning, and R. Ingalls for critical reading of the manu-
script. J.B. is an employee and shareholder of GSK. J.C. is an employee of
Received: June 22, 2011
Revised: March 10, 2012
Accepted: April 19, 2012
Published online: July 26, 2012
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