Published Ahead of Print 17 June 2013.
2013, 81(9):3099. DOI: 10.1128/IAI.00203-13.
Marjorie D. Sutherland, John T. Belisle and David W.
Jerod A. Skyberg, MaryClare F. Rollins, Joshua W. Samuel,
but Not against a Virulent F. tularensis Type
Francisella tularensis Live Vaccine Strain
Interleukin-17 Protects against the
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Interleukin-17 Protects against the Francisella tularensis Live Vaccine
Strain but Not against a Virulent F. tularensis Type A Strain
Jerod A. Skyberg,a,b,cMaryClare F. Rollins,aJoshua W. Samuel,b,cMarjorie D. Sutherland,dJohn T. Belisle,dDavid W. Pascuala,e
Department of Immunology and Infectious Diseases, Montana State University, Bozeman, Montana, USAa; Department of Veterinary Pathobiology, College of Veterinary
Medicine, University of Missouri, Columbia, Missouri, USAb; Laboratory for Infectious Disease Research, University of Missouri, Columbia, Missouri, USAc; Rocky Mountain
Regional Center for Excellence in Bioterrorism and Emerging Infectious Diseases and Department of Microbiology, Immunology and Pathology, Colorado State University,
Fort Collins, Colorado, USAd; Department of Infectious Diseases and Pathology, University of Florida, Gainesville, Florida, USAe
Francisella tularensis is a highly infectious intracellular bacterium that causes the zoonotic infection tularemia. While much
literature exists on the host response to F. tularensis infection, the vast majority of work has been conducted using attenuated
against attenuated F. tularensis versus F. tularensis type A differs. Several groups have recently reported that interleukin-17 (IL-
more susceptible to F. tularensis LVS infection, our studies, using a virulent type A strain of F. tularensis (SchuS4), indicate that
model. IL-17 protein levels were also higher in the lungs of mice infected with the LVS rather than F. tularensis type A, while IL-
these results demonstrate that IL-17 is dispensable for host immunity to type A F. tularensis infection, and that induced and pro-
tective immunity differs between attenuated and virulent strains of F. tularensis.
tion tularemia. The type and severity of tularemia depends on the
strain, dose, and route of infection (1). F. tularensis subspecies
tularensis (type A) and holarctica (type B) cause the majority of
human cases, with subspecies tularensis being more virulent (1).
Inhalation of F. tularensis results in respiratory or pneumonic tu-
laremia and can present anything from a mild pneumonia to an
acute infection with high fever, malaise, chills, cough, delirium,
and pulse-temperature dissociation (1). Untreated respiratory
forms of tularemia due to type A strains have mortality rates of
isms) (4) and mortality of F. tularensis infections have led to the
tibiotic resistance, by several nations (3). In addition, no vaccines
agent by the CDC.
Although a live vaccine strain (LVS) derived from F. tularensis
subspecies holarctica was created over 50 years ago, questions re-
it is not licensed for human use (1). LVS is attenuated in humans
but retains dose- and route-dependent virulence for mice (5), al-
though it is not as virulent as wild-type A and B strains. As LVS
causes disease in mice, it has been studied extensively as a model
intracellular pathogen (6). However, emerging evidence suggests
that in vitro and in vivo immune responses differ between type A
Francisella and the LVS (7–10). In addition, immunotherapeutic
strategies that confer potent protection against pulmonary LVS
infection only confer partial or negligible protection against pul-
rancisella tularensis is a highly infectious, Gram-negative fac-
ultative intracellular bacterium that causes the zoonotic infec-
monary infection with type A Francisella (6, 11, 12). Due to the
differences in induced and protective immune responses between
attenuated and virulent F. tularensis subspecies, it is important to
study the immune response to virulent strains of F. tularensis that
cause disease in humans in order to determine truly protective
correlates of immunity with relevance to human disease.
Interleukin-17 (IL-17) is a proinflammatory cytokine that can
confer protection against a variety of extracellular and intracellu-
lar bacterial pathogens (13–15). While no literature exists on the
role of IL-17 during infection with virulent F. tularensis, several
One study showed that IL-12p35-deficient mice, as well as wild-
type mice, are capable of resolving infection; however, IL-12p40-
deficient mice are much more susceptible to disease, suggesting
that IL-23 (a heterodimer of IL-23p19 and IL-12p40), which can
induce IL-17 production, is important in resolving F. tularensis
infections (39). It was also shown that intranasal LVS infection
leads to increased IL-17 expression in the bronchoalveolar lavage
blood mononuclear cells (PBMCs) with LVS in vitro also induces
IL-17 responses (17). In addition, the infection of human PBMCs
Received 12 February 2013 Returned for modification 4 March 2013
Accepted 2 June 2013
Published ahead of print 17 June 2013
Editor: R. P. Morrison
Address correspondence to Jerod A. Skyberg, email@example.com.
Copyright © 2013, American Society for Microbiology. All Rights Reserved.
September 2013 Volume 81 Number 9 Infection and Immunityp. 3099–3105iai.asm.org
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in vitro with either Francisella novicida or type A F. tularensis
SchuS4 was shown to induce IL-23p19 mRNA (18). Taken to-
in C57BL/6 mice during pulmonary infection with F. tularensis
LVS; however, no investigation was made on the role of IL-17
during infection with type A F. tularensis (19–21). Here, we show
that IL-17 receptor-deficient mice are impaired in their ability to
dispensable for immunity to type A F. tularensis SchuS4.
MATERIALS AND METHODS
Bacterial strains, culture conditions, and mice. F. tularensis SchuS4 or
LVS was cultured in modified Mueller-Hinton (MMH) broth (0.025%
ferric pyrophosphate, 2% IsoVitaleX, and 0.1% glucose) at 37°C with
constant shaking overnight, aliquoted into 1-ml samples, frozen at
?80°C, and thawed just before use as previously described (22). Titers of
dilutions plated on MMH agar (0.025% ferric pyrophosphate, 2%
period. These stocks were used to generate cultures for F. tularensis
were performed in biosafety level 3 (BSL-3) facilities at Montana State
University or the University of Missouri.
Mice. Six-week-old female C57BL/6 or BALB/c mice were purchased
from Charles River Laboratories. A breeding colony of gamma interfer-
on?/?(IFN-??/?) mice (The Jackson Laboratory, Bar Harbor, ME) on a
BALB/c background was maintained at Montana State University. IL-
animal resources facility at Montana State University or the University of
Missouri and were provided with sterile water and food ad libitum. All
animal care and experimental procedures were in accordance with insti-
tutional and federal policies for animal health and well-being.
Mouse infection. For intratracheal (i.t.) infections, mice were anes-
thetized with isoflurane gas, and a 22-gauge blunted needle was passed
through the oral pharynx into the trachea. A 50-?l volume of phosphate-
with F. tularensis LVS, mice were orally gavaged with 1 ? 108CFU of F.
tularensis LVS in 200 ?l PBS (after neutralizing stomach acidity by oral
titer into PBS. In some experiments, mice were treated intraperitoneally
(i.p.) on days 1 to 9 postinfection with gentamicin (10 mg/kg of body
weight; Sigma-Aldrich, St. Louis, MO) in order to extend the time to
mortality (24). For survival experiments, mice were monitored for mor-
bidity and mortality twice daily for up to 28 days, at which time survivors
were euthanized. In some experiments, IL-17 was depleted in vivo via the
intraperitoneal (i.p.) administration of 0.25 mg of anti-IL-17A monoclo-
and 7 in relation to infection, while control mice received rat IgG, as we
to enhance the susceptibility of wild-type mice to infection with F. tular-
ensis LVS (unpublished observation).
in lung and/or spleen homogenates. For CFU determination, mouse or-
gans were homogenized in sterile PBS, and homogenates were serially
diluted and plated on MMH plates. The plates then were incubated at
37°C for 48 h, at which time CFU were enumerated. For enzyme-linked
immunosorbent assay (ELISA), mouse lungs were homogenized, centri-
IL-17A concentrations in these lung filtrates were determined using the
Ready-Set-Go IL-17A ELISA kit from eBioscience (San Diego, CA) ac-
cording to the manufacturer’s instructions.
Isolation and infection of murine peritoneal macrophages. Perito-
were given a single intraperitoneal injection of 1.0 ml of expired thiogly-
colate medium (Difco, Detroit, MI), and 3 days later, the peritoneum of
each mouse was washed with RPMI 1640 (Gibco BRL-Life Technologies
[Life Technologies], Grand Island, NY) containing 2% fetal calf serum
(Life Technologies) without antibiotics. Peritoneal cells were washed
twice in the same medium without antibiotics and allowed to adhere
overnight to 24-well microtiter plates. Macrophages (1 ?106/ml) were
RPMI 1640 complete medium (CM) containing 10% fetal bovine serum,
and 50 ?g/ml gentamicin (Sigma) was added and cells incubated for 30
min at 37°C and 5% CO2to kill extracellular bacteria. Cells were then
time point). For time points of ?8 h, gentamicin was added to the wells
cells were washed three times with PBS and then lysed with sterile 1%
saponin in PBS (27). Serial logarithmic dilutions of macrophage lysates
were then performed and plated in triplicate onto MMH agar for incuba-
treated overnight prior to infection with 50 ?M caspase-1 inhibitor
YVAD-cmk (Cayman Chemical, Ann Arbor, MI) or dimethylsulfoxide
in the wells throughout the experiments, other than during washes.
Extraction of RNA and qRT-PCR analysis of F. tularensis-infected
murine macrophages and lungs. F. tularensis-infected murine macro-
phages or lungs were homogenized and lysed in Tri reagent (Qiagen,
Valencia, CA). RNA was then isolated according to the manufacturer’s
instructions prior to being further purified via extraction with an RNeasy
Mini kit (Qiagen). cDNA was generated using the Superscript III first-
strand synthesis system (Life Technologies, Grand Island, NY). Primers
PrimerQuest application from Integrated DNA Technologies. Relative
specific mRNA was quantified by measuring SYBR green incorporation
during real-time quantitative reverse transcription-PCR (qRT-PCR)
Statistical analysis. Statistical differences between two groups were
analysis of variance (ANOVA) followed by Tukey’s multiple-comparison
test with significance determined at P ? 0.05. For in vivo studies, signifi-
at P ? 0.05.
IL-17 receptor-deficient mice are impaired in innate immunity
to F. tularensis LVS but not F. tularensis SchuS4. To investigate
a C57BL/6 background) were infected i.t. with 5,000 CFU of F.
tularensis LVS or 50 CFU of F. tularensis SchuS4. These doses of
Francisella were chosen because we have found them to induce
100% lethality. We found that at 96 h postinfection, IL-17R??/?
mice were significantly more susceptible to colonization of the
spleen by F. tularensis LVS than were wild-type mice (Fig. 1A). In
addition, we found that at time points ?144 h postinfection, IL-
17R??/?mice had higher splenic and pulmonary levels of LVS
Skyberg et al.
iai.asm.org Infection and Immunity
on June 12, 2014 by guest
than did C57BL/6 mice, and they succumbed to infection more
quickly (data not shown). However, IL-17R??/?mice infected
with SchuS4 were unimpaired in their ability to control bacterial
relation to C57BL/6 mice. IL-17R??/?mice actually displayed a
slight but statistically significant reduction in pulmonary coloni-
zation by SchuS4 compared to wild-type animals; however, this
was only observed at 96 h postinfection (Fig. 1B).
IL-17R??/?mice display an unimpaired IFN-? response
others showed that IL-17 was required for the induction of an
(21), whereas in a parasite infection model, IL-17R??/?mice
were found to have an exaggerated IFN-? response (30). There-
fore, to investigate the effect of impaired IL-17 signaling on the
induction of IFN-? during infection with virulent F. tularensis,
wild-type C57BL/6 or IL-17R??/?mice were infected i.t. with 50
CFU of F. tularensis SchuS4. At 48 and 96 h after infection, RNA
was extracted from the lungs of infected animals, and qRT-PCR
was performed to assess the induction of IL-17 and IFN-? mRNA
relative to that of mock-infected animals. We found that IL-
At 96 h postinfection, it was also observed that IFN-? mRNA was
more potently induced than IL-17 in the lungs of both wild-type
and IL-17R??/?mice (Fig. 2).
It has been suggested that during infection with F. tularensis LVS,
the role of IL-17 is more important in convalescence (19). In ad-
dition, the protective role of IL-17 in bacterial infection models
has been proposed to be either dependent on IFN-? (21) or en-
hanced in the absence of IFN-? (15). Therefore, we neutralized
IL-17 in both wild-type and IFN-??/?mice in a convalescent
model of tularemia. BALB/c or IFN-??/?mice were depleted of
IL-17 via neutralizing MAb (or treated with rat IgG as a control)
and treated i.p. with 10 mg/kg gentamicin on days 1 to 9 postin-
fection with 50 CFU of F. tularensis SchuS4 (24). Morbidity and
mortality were recorded over time. IL-17 neutralization had no
effect on morbidity in wild-type animals, as mice lost weight at a
tality was also unaffected in wild-type mice by IL-17 neutraliza-
IFN-? had no effect on weight loss (Fig. 3A), it was critically im-
portant for survival in this model (Fig. 3B). Similar to what we
mice did not affect weight loss or survival (Fig. 3A and B).
background (IL-17R??/?) were infected intratracheally (i.t.) with 5,000 CFU of F. tularensis LVS (A) or 50 CFU of F. tularensis SchuS4 (B). At 48 or 96 h
postinfection, lung and splenic bacterial burdens were determined. Error bars represent standard deviations (SD). *, P ? 0.05 compared to C57BL/6 mice
infected with the same strain of Francisella at the same time point. Data shown in panel A and at 48 h in panel B were calculated by averaging the values from
mice pooled from 5 independent experiments (20 mice/group).
FIG 2 IL-17R??/?mice display an unimpaired IFN-? response during pul-
monary infection with F. tularensis SchuS4. C57BL/6 or IL-17R??/?mice
(n ? 4 to 5/group) were infected intratracheally (i.t.) with 50 CFU of F. tula-
rensis SchuS4. At 48 or 96 h postinfection, RNA was extracted from the lungs
and qRT-PCR was performed to determine the log2induction of IFN-? and
IL-17 mRNA in infected mice compared to that of mock-infected mice (nor-
malized to ?-actin). Error bars represent SD. *, P ? 0.05 compared to IL-17
induction by the same strain of mouse at the same time point.
FIG 3 IFN-?, but not IL-17, is required for protection against virulent F.
tularensis infection in a convalescent model of tularemia. BALB/c or IFN-
??/?mice (BALB/c background) (n ? 10/group) were treated i.p. with
0.25 mg of IgG or anti-IL-17 MAb on days ?1, 0, and 7 relative to i.t.
infection with 50 CFU of F. tularensis SchuS4. Mice were also treated i.p.
with 10 mg/kg gentamicin on days 1 to 9. Surviving mice were weighed (A)
and monitored for morbidity and mortality (B). Error bars depict standard
errors of the means (SEM). *^, P ? 0.05 compared to mice of the same
genotype treated with IgG.
IL-17 in Immunity to Virulent F. tularensis
September 2013 Volume 81 Number 9iai.asm.org 3101
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assess the role of IL-17 during a protective, vaccine-induced im-
mune response against virulent F. tularensis, C57BL/6 or IL-
17R??/?mice were immunized twice orally with 1 ? 108CFU of
route of vaccination, as both strains of mice survived oral vacci-
nation with LVS, whereas IL-17R??/?mice were found to suc-
able to provide partial protection against a low-dose pulmonary
loss (Fig. 4A) and delayed mortality/increased survival (Fig. 4B).
However, the absence of IL-17R??/?did not render LVS-vacci-
nated animals (or vehicle-treated animals) more susceptible to
B). In fact, LVS-vaccinated IL-17R??/?mice lost slightly less
weight early after challenge than wild-type animals (Fig. 4A);
however, this modest effect was not pursued further.
LVS-infected mice have higher pulmonary levels of IL-17
i.t. with 5,000 CFU of LVS or 50 CFU of SchuS4. At 96 h after
infection, we found that IL-17 mRNA levels were ?3-fold higher
in animals infected with LVS than in SchuS4-infected animals.
However, using these infectious doses, we found that LVS CFU
SchuS4-infected animals at this time point (data not shown). To
achieve similar levels of SchuS4 and LVS in the lung, we used a
higher dose of SchuS4 and compared bacterial colonization and
and SchuS4 (150 CFU i.t.) at 48 and 110 h. At 48 h postinfection,
mice infected with LVS or SchuS4 displayed similar pulmonary
bacterial burdens and IL-17 protein levels (Fig. 5A and B). How-
ever, at 110 h after infection, LVS-infected mice had higher pul-
monary levels of IL-17 than did SchuS4-infected mice, despite
lower levels of LVS in the lung (Fig. 5A and B), indicating that
enhanced IL-17 mRNA or protein levels in LVS-infected mice are
not dependent on bacterial burden.
pendent in vitro. To investigate the mechanism by which LVS
induces a stronger IL-17 response than SchuS4, we queried the
role of caspase-1 in the induction of IL-23p19 mRNA. Caspase-1
is an inflammatory caspase that mediates the release of bioactive
induce IL-23 (31) and caspase-1-dependent IL-1 can synergize
with IL-23 to enhance IL-17 responses in vivo (32). Therefore, to
study the role of caspase-1 in the induction of IL-23p19 mRNA,
peritoneal macrophages were infected with LVS or SchuS4 and
treated with the caspase-1 inhibitor YVAD-cmk or DMSO as a
vehicle control. Twenty h after infection, some wells containing
macrophages were lysed and intracellular bacteria enumerated,
whereas RNA was extracted from other macrophage wells to de-
FIG 4 IL-17R? is dispensable for oral LVS vaccine-induced immunity against pulmonary challenge with virulent F. tularensis. C57BL/6 or IL-17R??/?mice
Body weights (A) and survival (B) were recorded. Mice that succumbed to infection were assigned a body weight of 75%. Error bars depict SEM. *, P ? 0.05
compared to mice of the same genotype that were vaccinated with LVS. ^, P ? 0.05 compared to LVS-vaccinated C57BL/6 mice.
were infected i.t. with 5,000 CFU of LVS or 150 CFU of SchuS4. At 48 and 110 h postinfection, lungs were homogenized and bacterial burdens were determined
by colony counts (A), and IL-17 levels were determined by ELISA (B). Error bars depict SD. *, P ? 0.05 compared to LVS-infected C57BL/6 mice.
Skyberg et al.
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