Distinct roles of reactive nitrogen and oxygen species to control infection with the facultative intracellular bacterium Francisella tularensis.

Abstract

Reactive nitrogen species (RNS) and reactive oxygen species (ROS) are important mediators of the bactericidal host response. We investigated the contribution of these two mediators to the control of infection with the facultative intracellular bacterium Francisella tularensis. When intradermally infected with the live vaccine strain F. tularensis LVS, mice deficient in production of RNS (iNOS(-/-) mice) or in production of ROS by the phagocyte oxidase (p47(phox-/-) mice) showed compromised resistance to infection. The 50% lethal dose (LD(50)) for iNOS(-/-) mice was <20 CFU, and the LD(50) for p47(phox-/-) mice was 4,400 CFU, compared to an LD(50) of >500,000 CFU for wild-type mice. The iNOS(-/-) mice survived for 26.4 +/- 1.8 days, and the p47(phox-/-) mice survived for 10.1 +/- 1.3 days. During the course of infection, the serum levels of gamma interferon (IFN-gamma) and interleukin-6 were higher in iNOS(-/-) and p47(phox-/-) mice than in wild-type mice. Histological examination of livers of iNOS(-/-) mice revealed severe liver pathology. Splenocytes obtained 5 weeks after primary infection from antibiotic-treated iNOS(-/-) mice showed an in vitro recall response that was similar in magnitude and greater secretion of IFN-gamma compared to cells obtained from wild-type mice. In summary, mice lacking expression of RNS or ROS showed extreme susceptibility to infection with F. tularensis LVS. The roles of RNS and ROS seemed to be distinct since mice deficient in production of ROS showed dissemination of infection and died during the early phase of infection, whereas RNS deficiency led to severe liver pathology and a contracted course of infection.

Full text

Page 1

INFECTION AND IMMUNITY, Dec. 2004, p. 7172–7182
0019-9567/04/$08.00?0DOI: 10.1128/IAI.72.12.7172–7182.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Vol. 72, No. 12
Distinct Roles of Reactive Nitrogen and Oxygen Species To Control
Infection with the Facultative Intracellular Bacterium
Francisella tularensis
Helena Lindgren,1,2Stephan Stenmark,1,2Wangxue Chen,3
Arne Ta ¨rnvik,2and Anders Sjo ¨stedt1*
Department of Clinical Microbiology, Clinical Bacteriology,1and Infectious Diseases,2
Umea ˚ University, Umea ˚, Sweden, and National Research Council Canada,
Institute for Biological Sciences, Ottawa, Canada3
Received 24 March 2004/Returned for modification 14 June 2004/Accepted 2 September 2004
Reactive nitrogen species (RNS) and reactive oxygen species (ROS) are important mediators of the bacte-
ricidal host response. We investigated the contribution of these two mediators to the control of infection with
the facultative intracellular bacterium Francisella tularensis. When intradermally infected with the live vaccine
strain F. tularensis LVS, mice deficient in production of RNS (iNOS?/?mice) or in production of ROS by the
phagocyte oxidase (p47phox?/?mice) showed compromised resistance to infection. The 50% lethal dose (LD50)
for iNOS?/?mice was <20 CFU, and the LD50for p47phox?/?mice was 4,400 CFU, compared to an LD50of
>500,000 CFU for wild-type mice. The iNOS?/?mice survived for 26.4 ? 1.8 days, and the p47phox?/?mice
survived for 10.1 ? 1.3 days. During the course of infection, the serum levels of gamma interferon (IFN-?) and
interleukin-6 were higher in iNOS?/?and p47phox?/?mice than in wild-type mice. Histological examination of
livers of iNOS?/?mice revealed severe liver pathology. Splenocytes obtained 5 weeks after primary infection
from antibiotic-treated iNOS?/?mice showed an in vitro recall response that was similar in magnitude and
greater secretion of IFN-? compared to cells obtained from wild-type mice. In summary, mice lacking expres-
sion of RNS or ROS showed extreme susceptibility to infection with F. tularensis LVS. The roles of RNS and
ROS seemed to be distinct since mice deficient in production of ROS showed dissemination of infection and
died during the early phase of infection, whereas RNS deficiency led to severe liver pathology and a contracted
course of infection.
Francisella tularensis is a highly virulent bacterium that
causes tularemia in many mammalian species. In humans, di-
rect contact with infected animals or transmission by infected
arthropods, such as ticks or mosquitoes, leads to the ulcer-
oglandular form of the disease, whereas inhalation of F. tula-
rensis results in the more rare respiratory form. The disease is
characterized by flu-like symptoms and prominent enlarge-
ment of draining lymph nodes. Life-threatening manifesta-
tions, such as sepsis and rhabdomyolysis, may occur (29).
F. tularensis is a facultative intracellular bacterium, and the
host protective mechanisms are similar to those that are active
against listeriae and mycobacteria (24, 28). During the first few
days of infection, T-cell-independent transient host resistance
is evoked. During this phase, both gamma interferon (IFN-?)
and tumor necrosis factor alpha (TNF-?) are crucial mediator
molecules, primarily due to their ability to activate the antimi-
crobial mechanisms of mononuclear phagocytes (14, 18, 27).
After the initial phase, T-cell-dependent long-term protective
immunity to F. tularensis develops. The critical role of this
cell-mediated immunity is demonstrated by the finding that
SCID mice or mice lacking ?? T cells succumb to even the
smallest inocula of F. tularensis (11, 13, 32).
Reactive nitrogen species (RNS) and reactive oxygen species
(ROS) are intermediates that are involved in the host defense
against various intracellular pathogens (4, 7, 8, 21). RNS can
be generated by constitutive nitric oxide synthases, but high
levels of RNS are produced only after the activation of induc-
ible nitric oxide synthases (iNOS) (10). Production of ROS
messengers (for example, superoxide) depends on the induc-
tion of phagocyte oxidase (phox) (2). Activation of iNOS and
phox is induced in macrophages when they are exposed to
proinflammatory cytokines, including IFN-? or TNF-? (4, 22).
A role for RNS in the host resistance to tularemia has been
suggested by in vitro studies of F. tularensis (1, 15) After acti-
vation with IFN-?, macrophages are capable of arresting bac-
terial replication, an effect prevented by an inhibitor of iNOS.
The role of macrophage-derived NO in the host defense
against tularemia is, however, not completely understood, and
its importance seems to vary with the organ localization of
macrophages (23). No studies have directly assessed the role of
RNS or ROS in vivo. The availability of mice deficient in
expression of iNOS or phox provides models to directly assess
the roles of these reactive molecular species in killing of F.
tularensis.
We demonstrated in this study that mice lacking expression
of RNS or ROS show extreme susceptibility to infection with
the live vaccine strain F. tularensis LVS. However, RNS and
ROS seem to be predominantly involved at different stages of
the infection. Mice deficient in production of ROS died within
* Corresponding author. Mailing address: Department of Clinical
Microbiology, Clinical Bacteriology, Umea ˚ University, SE-901 85
Umea ˚, Sweden. Phone: 46-90-7851120. Fax: 46-90-7852225. E-mail:
Anders.Sjostedt@climi.umu.se.
7172

Page 2

2 weeks, whereas RNS deficiency rapidly resulted in severe
liver pathology but a contracted course of infection, and death
occurred within 4 weeks.
MATERIALS AND METHODS
Bacterial strain. The live vaccine strain F. tularensis LVS (? ATCC 29684)
was supplied by the U.S. Army Medical Research Institute of Infectious Dis-
eases, Fort Detrick, Frederick, Md. It was grown to the logarithmic phase in
modified Mueller-Hinton broth, harvested, and stored frozen at ?70°C.
Mice. Breeding stocks of iNOS gene-deficient mice (iNOS?/?mice) and
wild-type C57BL/6 mice (designated iNOS?/?mice) were obtained from Jackson
Laboratories, Bar Harbor, Maine. p47phox?/?mice and wild-type mice (desig-
nated p47phox?/?mice) were obtained through the courtesy of Steven Holland,
National Institutes of Health,, Bethesda, Md. p47phox?/?mice were derived as
described elsewhere (16) from heterozygous parents (C57BL/6 ? 129 back-
crossed to F5 in a C57BL/6 lineage). The wild-type mice also represented the F5
generation. Breeding stocks of leaky iNOS?/?mice were obtained through the
courtesy of F. Y. Liew, Department of Immunology, University of Glasgow,
Glasgow, United Kingdom (31). Strain 129 mice (designated iNOS?/?129 mice)
were obtained from Harlan, Austerlitz, The Netherlands. All strains of mice were
bred and housed at the Animal Facility of the Swedish Defense Research
Agency, Umea ˚, Sweden, under conventional conditions and were given food and
water ad libitum. The mice were 8 to 14 weeks old and age and sex matched when
they were used in experiments. They were found to be free of specific pathogens.
Permission for the experiments was obtained from the Ethical Committee, Umea ˚
University, Umea ˚, Sweden.
Inoculation and enumeration of bacteria in mice. For primary infection, mice
were inoculated intradermally in the thoracic region with F. tularensis LVS cells
suspended in 50 ?l of saline. One day before the intradermal inoculation, the
region was shaved, and at various times after injection, animals were killed, and
a 1-cm2sample of the region was excised, briefly washed in 70% ethanol, and
added to a tube with saline. Spleens, livers, and lungs were also collected.
Samples from each organ were homogenized, and 10-fold serial dilutions were
cultured on agar plates. Peritoneal lavage was performed by injection and aspi-
ration of 1.0 ml of saline with a Pasteur pipette. Blood (100 ?l) was collected
from the retroorbital vein. Serial dilutions of lavage and blood samples were
plated for enumeration of bacteria. Bacterial numbers were expressed as the
number of CFU per organ (lung, liver, or spleen), per 1.0 ml of peritoneal lavage
fluid, or per 100 ?l of blood.
The 50% lethal dose (LD50) was determined by the method of Reed and
Muench as previously described (25).
Blood chemistry for organ function. On days 3, 6, and 9 of infection, blood was
obtained from three mice per strain. Sera from mice (?300 ?l) were stored
frozen until analysis. Alanine aminotransferase, aspartate aminotransferase, to-
tal bilirubin, amylase, and creatinine in serum were analyzed by using a Vitros
950 multianalyzer (Johnson-Johnson, Clinical Diagnostics).
Histological examination. On days 3, 6 and 9, livers, spleens and lungs were
collected from mice of each strain. The organs were prepared for histological
staining by snap freezing them in liquid propane and were placed in OCT
compound (EMS, Hatfield, Pa.). Samples were stored at ?70°C until they were
sectioned, and they were stained with Mayer’s hematoxylin and eosin. Micros-
copy and photography were performed by using a Leica DMLB 100T micro-
scope. The sections were assessed in a blind fashion.
Preparation of splenocytes. Spleens from mice were homogenized in 10 ml of
saline. Debris was removed by centrifugation at 200 ? g for 30 s. The cells in the
resulting supernatant were resuspended in 10 ml of saline, pelleted by centrifu-
FIG. 1. Percentage of surviving mice after intradermal injection of F. tularensis LVS. Mice (10 mice per group) were inoculated intradermally
with 2 ? 101CFU (?), 2 ? 103CFU (‚), or 2 ? 104CFU (E). p47phox?/?mice that survived for 28 days were sacrificed, and no bacteria were
found in the livers or spleens. (A) iNOS?/?mice. (B) p47phox?/?mice.
VOL. 72, 2004 RNS AND ROS AND CONTROL OF F. TULARENSIS INFECTION7173

Page 3

gation at 200 ? g for 10 min, resuspended in 1 ml of 0.8% NH4Cl, and incubated
for 10 min at room temperature to lyse erythrocytes. The cells were resuspended
in 10 ml of ice-cold saline and centrifuged. This procedure was repeated once.
The splenocytes were resuspended in 5 ml of cell culture medium (Dulbecco
modified Eagle medium; Invitrogen, Paisley, United Kingdom) supplemented
with 10% fetal bovine serum (Invitrogen), and enumeration was performed by
using a Bu ¨rker chamber. Cells were stained by using May Gru ¨nwall Giemsa stain
for differential counting of the splenocytes.
Assay of ROS production by splenocytes. At different times, mice infected with
5 ? 104CFU of F. tularensis LVS were killed, and spleen cells were prepared as
FIG. 2. Growth curves for F. tularensis LVS in the skin, spleens, and livers of iNOS?/?and iNOS?/?mice (A) and p47phox?/?and p47phox?/?
mice (B). Mice were inoculated intradermally with 5 ? 102CFU of F. tularensis LVS, and the numbers of bacteria (CFU per organ sample) were
determined at different times. The means ? standard errors of the means for five mice per group and time are shown. One asterisk indicates that
the P value is ?0.05, two asterisks indicate that the P value is ?0.01, and three asterisks indicate that the P value is ?0.001.
TABLE 1. Viable counts in organs and tissues after intradermal inoculation of p47phox?/?or p47phox?/?mice with F. tularensis LVSa
Organ or tissue
Viable counts
3 Days after inoculation5 Days after inoculation 9 Days after inoculation
p47phox?/?mice
p47phox?/?mice
p47phox?/?mice
p47phox?/?mice
p47phox?/?mice
p47phox?/?mice
Liver
Lung
Bloodc
Peritoneumf
3.56 ? 0.61
3.16 ? 0.58
BDLd
2.08 ? 1.1
4.79 ? 0.22 Ab
2.74 ? 0.52
2.74 ? 0.52 A
3.22 ? 0.48
4.76 ? 0.21
BDL
BDL
BDL
7.35 ? 0.29 C
5.38 ? 0.76 B
1.77 ? 0.08 A
3.77 ? 0.45 B
2.89 ? 0.20
BDL
NDc
BDL
4.60 ? 0.41 A
3.10 ? 0.73 A
ND
1.78 ? 0.64 A
aThe inoculum was 5 ? 104CFU of F. tularensis LVS per mouse, and the results are expressed as the mean log10CFU ? standard error of the mean for three
samples.
bSignificance is indicated as follows: A, P ? 0.05 compared to p47phox?/?mice; B, P ? 0.01 compared to p47phox?/?mice; C, P ? 0.001 compared to p47phox?/?
mice.
cLog10CFU per 100 ?l of blood.
dBDL, below the detection limit.
eND, not determined.
fLog10CFU per milliliter of peritoneal lavage fluid.
7174 LINDGREN ET AL.INFECT. IMMUN.

Page 4

described above. ROS production by fresh splenocytes was measured by a lumi-
nol-based chemiluminescence method (12). In preliminary experiments, the
method showed a good correlation with a colorimetric assay based on ROS-
mediated reduction of cytochrome c. The kinetics of the chemiluminescence
response were similar for all samples tested and were maximal within the 5-min
length of the assay. Splenocytes (1 ? 106cells) were incubated at 37°C for 5 min
in 1 ml of KRG buffer (Krebs-Ringer phosphate buffer containing 10 mM
glucose, 1 mM Ca2?, and 1.5 mM Mg2?; pH 7.3) supplemented with 100 ?M
luminol (Sigma, Madison, Wis.). Immediately before measurement of the chemi-
luminescence response, 0.5 ?l of phorbol myristate acetate (2 ?g/ml) was added
to the tube. In preliminary experiments, this concentration was found to be
optimal. The chemiluminescence was measured with a luminometer (Luminom-
eter 1250; LKB Wallac, Turku, Finland) for 5 min. The results were expressed in
peak relative light units.
Assay of RNS production by splenocytes. At different times, infected mice
were killed, and spleen cells were prepared as described above. Cells were seeded
in a 96-well cell culture plate at a density of 3 ? 105cells/well in 200 ?l and
incubated at 37°C in 5% CO2. Preliminary experiments indicated that measur-
able levels of the end metabolite NO2
least 4 days. The concentration of NO2
Fifty microliters of the culture supernatant was mixed with 50 ?l each of the
Griess reagents p-aminobenzenesulfonamide (58 mM in 5% H3PO4) and 2,6,8-
trihydroxypurine (3.9 mM) (Sigma). After 10 min of incubation at room tem-
perature, the absorbance at 540 nm was recorded. The concentration of NO2
was determined by preparing a standard curve for sodium nitrite. The lower limit
of detection of the assay was 2.5 ?M.
Assay of serum cytokine levels. All procedures for the cytokine enzyme-linked
immunosorbent assay (OptEIA; BD Biosciences, San Diego, Calif.) were per-
formed by following the instructions of the manufacturer. Dilutions of serum
samples from LVS-infected mice were analyzed for the presence of IFN-?,
TNF-?, interleukin-6 (IL-6), IL-4, and IL-12. The level of detection for IL-12 and
IFN-? was 30 pg/ml, and the level of detection for IL-4, IL-6, and TNF-? was 15
pg/ml.
Assay of the T-cell response. Mice were inoculated with 2 ? 102CFU of F.
tularensis LVS, and 15 days later moxifloxacin was administered intraperitoneally
at a dose of 0.1 mg/g of body weight once daily for 7 days. Mice were killed 5
weeks after inoculation, and spleen cell cultures were established. Each culture
(200 ?l) contained 6 ? 105splenocytes in RPMI (GIBCO BRL) supplemented
with 15% inactivated fetal calf serum, 10 ?g of gentamicin per ml, 5 ? 10?5M
?-mercaptoethanol, and 2 mM L-glutamine (GIBCO BRL). Heat-killed F. tula-
rensis LVS (106cells/ml) or concanavalin A (10 ?g/ml; Pharmacia, Uppsala,
Sweden) was used as the stimulating agent. To estimate the proliferative re-
sponse, splenocyte cultures were incubated at 37°C for 4 days, pulsed for 18 h
with 0.5 ?Ci of [3H]thymidine (18 Ci/mmol), and harvested with an automated
cell harvester (Inotech, Basel, Switzerland). For determination of secreted cyto-
kines, parallel cultures were established, and after 2 days of incubation with
antigen, 100 ?l of supernatant was collected. The proliferative responses and
cytokine levels have been found to be optimal at these times (26).
Statistical analysis. Student’s t test and Pearson’s correlation coefficient test
were used.
?were found only after incubation for at
?was measured by the Griess reaction.
?
RESULTS
Numbers of bacteria in iNOS?/?, iNOS?/?, p47phox?/?, and
p47phox?/?mice after primary intradermal infection with F.
tularensis. The LD50for intradermal infection was found to be
4.4 ? 103CFU of F. tularensis LVS for p47phox?/?mice and
less than 20 CFU for iNOS?/?mice (Fig. 1). The mean time to
death was determined for all of the mice in the experiment,
irrespective of the inoculum. In spite of a lower LD50, the
mean time to death was significantly longer for the iNOS?/?
mice (26.4 ? 1.8 days, compared with 10.1 ? 1.3 days for the
p47phox?/?mice). For the iNOS?/?mice the LD50was 5.0 ?
105to 1.2 ? 106CFU and the mean time to death was 7.2 ?
1.2 days, and for the p47phox?/?mice the LD50was ?8 ? 105
CFU and the mean time to death was 6.9 ? 1.4 days.
TABLE 2. Viable counts in organs and tissues after intradermal inoculation of iNOS?/?or iNOS?/?mice with F. tularensis LVSa
Organ or tissue
Viable counts
3 Days after inoculation6 Days after inoculation9 Days after inoculation
iNOS?/?mice
iNOS?/?mice
iNOS?/?mice
iNOS?/?mice
iNOS?/?mice
iNOS?/?mice
Skin
Liver
Lung
Peritoneumd
Blood
6.91 ? 0.17
3.99 ? 0.33
BDLc
2.07 ? 0.47
BDL
7.82 ? 0.26 Ab
3.91 ? 0.19
BDL
2.93 ? 0.62
BDL
5.04 ? 0.01
4.61 ? 0.21
BDL
BDL
BDL
7.27 ? 0.11 C
5.36 ? 0.22 A
2.34 ? 0.30 A
2.80 ? 0.48 B
BDL
1.31 ? 1.31
2.72 ? 0.01
BDL
BDL
BDL
6.72 ? 0.24 C
5.80 ? 1.13 A
2.00 ? 0.34
2.5 ? 0.18 C
BDL
aThe inoculum was 5 ? 104CFU of F. tularensis LVS per mouse, and the results are expressed as the mean log10CFU ? standard error of the mean for three
samples.
bSignificance is indicated as follows: A, P ? 0.05 compared to iNOS?/?mice; B, P ? 0.01 compared to iNOS?/?mice; C, P ? 0.001 compared to iNOS?/?mice.
cBDL, below the detection limit.
dLog10CFU per milliliter of peritoneal lavage fluid.
TABLE 3. Histological examination of livers from mice infected
with F. tularensis LVSa
Days
postinfection
Mouse strain
Liver
damageb
Inflammatory response
Extentc
Intensityd
3 iNOS?/?
iNOS?/?
p47phox?/?
p47phox?/?
1, 1, 1c
1, 2, 2
1, 1, 1
1, 1, 1
1, 1, 1
1, 2, 2
1, 1, 1
1, 1, 1
1, 1, 1
1, 2, 2
1, 1, 1
1, 1, 1
6iNOS?/?
iNOS?/?
p47phox?/?
p47phox?/?
1, 1, 1
4, 4, 4
2, 2, 2
2, 2, 2
2, 2, 2
3, 4, 4
2, 2, 2
2, 2, 2
2, 2, 2
4, 4, 4
2, 2, 2
2, 2, 2
9iNOS?/?
iNOS?/?
p47phox?/?
p47phox?/?
1, 2, 3
4, 4, 4
2, 3, 3
3, 3, 3
1, 2, 3
4, 4, 4
2, 3, 3
3, 3, 3
1, 2, 3
4, 4, 4
2, 3, 3
3, 3, 3
aThe intradermal inoculum was 5 ? 104CFU of F. tularensis LVS per mouse.
bLiver damage: 1, degeneration and necrosis of individual hepatocytes occa-
sionally seen; 2, clusters or small aggregates of hepatocyte degeneration and
necrosis; 3, medium to large aggregates of hepatocyte necrosis; 4, large areas of
hepatocyte necrosis with loss of normal liver anatomic architecture.
cExtent of the inflammatory response: 1, occasional inflammatory infiltrates
(?1 foci/?100 field); 2, small numbers of inflammatory infiltrates (1 to 5 foci/
?100 field); 3, moderate numbers of inflammatory infiltrates (?5 foci/?100
field); 4, large numbers of inflammatory infiltrates throughout the section.
dIntensity of the inflammatory response: 1, small inflammatory infiltrates with
a few inflammatory cells; 2, medium-size inflammatory infiltrates with small to
moderate numbers of inflammatory cells; 3, large inflammatory infiltrates with
moderate to large numbers of inflammatory cells; 4, extensive infiltration with
large numbers of inflammatory cells. The values are averages for three individual
mice.
eIndividual scores for liver samples for three mice for each day and group.
VOL. 72, 2004RNS AND ROS AND CONTROL OF F. TULARENSIS INFECTION7175

Page 5

Bacterial counts in the skin, livers, and spleens of p47phox?/?
and iNOS?/?mice and the corresponding wild-type mice were
determined at various intervals after intradermal inoculation
of 5 ? 102CFU of F. tularensis LVS, a dose at which all
animals survived for ?10 days. The data are summarized in
Fig. 2. Compared to the wild-type mice, the iNOS?/?mice
showed no significant differences in the numbers of bacteria in
the liver and spleen during the first 6 days of infection (Fig.
2A). After this, the numbers of bacteria decreased in the
iNOS?/?mice but remained stationary in the iNOS?/?mice.
In the skin, starting at day 4, the iNOS?/?mice showed higher
numbers of bacteria than the wild-type mice. By day 35, the
wild-type mice had cleared the infection, while the iNOS?/?
mice still had more than 5 log10CFU of bacteria in the liver,
spleen, and skin (Fig. 2A).
Compared to the wild-type mice, the p47phox?/?mice had
higher numbers of bacteria in the liver and spleen at all times
examined (Fig. 2B). In contrast, the numbers of bacteria in the
skin of the p47phox?/?mice did not differ from the numbers of
bacteria in the skin of the p47phox?/?mice during the first 7
days of infection, but the numbers of bacteria were significantly
higher on day 10 after inoculation.
We extended these experiments by monitoring the course of
infection after inoculation of 5 ? 104CFU of F. tularensis LVS,
a dose 10 times higher than the LD50for p47phox?/?mice.
Compared to liver samples of p47phox?/?mice, liver samples of
p47phox?/?mice contained significantly higher numbers of bac-
teria during the whole 9-day period of observation (Table 1).
In lung, blood, and peritoneal lavage fluid samples of
p47phox?/?mice, bacteria were present at detectable levels
during the whole period, whereas in wild-type mice, bacteria
were found only in samples from the lungs and peritoneum and
only on day 3 (Table 1).
When in similar experiments iNOS?/?mice were intrader-
mally inoculated with 5 ? 104CFU, numbers of bacteria that
were significantly higher than the numbers in wild-type mice
were found in samples from livers on days 6 and 9 and in skin
samples on day 3 and beyond (Table 2). In samples from the
peritoneum, bacteria were detectable in iNOS?/?mice during
the whole period, and in wild-type mice bacteria were detect-
able only on day 3. In lung samples, bacteria were isolated from
iNOS?/?mice on days 6 and 9 and not at all from iNOS?/?
mice (Table 2). In blood samples, no bacteria were detectable,
either in samples from iNOS?/?mice or in samples from
iNOS?/?mice.
In summary, the control of infection was found to be com-
promised in p47phox?/?mice throughout the course of infec-
tion, and when a large inoculum was used, dissemination of
infection occurred, as shown by bacteremia and peritonitis.
The iNOS?/?mice had increased numbers of bacteria in the
skin even during the early phase and exhibited a loss of control
also in the liver and spleen after the first week of infection. The
iNOS?/?mice were unable to eradicate the infection, and after
a protracted course of infection, they eventually succumbed to
even the lowest challenge dose.
Histological examination of livers and assay of s-amino-
transferase levels of infected mice. Histological examination of
spleens, livers, and lungs from infected mice showed that the
most pronounced differences were in livers, which became the
focus of the comparative analysis. Examinations were per-
formed on days 3, 6, and 9 after inoculation of 5 ? 104F.
tularensis LVS cells, a dose found to be high enough to induce
FIG. 3. After inoculation of 5 ? 104CFU of F. tularensis LVS, the serum liver enzymes s-ALT and s-AST were monitored in iNOS?/?and
iNOS?/?mice (A) and in p47phox?/?and p47phox?/?mice (B). The means ? standard errors of the means for five mice per group and time are
shown. An asterisk indicates that the P value is ?0.05.
7176LINDGREN ET AL.INFECT. IMMUN.

Page 6

pathological changes. During the 9-day period, histological
lesions developed in p47phox?/?and p47phox?/?mice (Table 3).
Increased serum levels of s-ALT and s-AST, sensitive markers
for liver damage, were found in p47phox?/?mice on day 9 (Fig.
3). The iNOS?/?mice showed even more severe histological
changes at all times (Table 3), and the enzyme levels on day 9
were higher than those in any other group of mice (Fig. 3).
Representative histological sections from day 6 are shown in
Fig. 4. Two indicators of kidney and pancreas function, s-
creatinine and s-amylase, respectively, were also monitored,
but no significant differences were observed between the gene-
deficient and wild-type mice.
Numbers of bacteria in skin, spleens, and livers of leaky
iNOS?/?and iNOS?/?mice after primary infection with F.
tularensis. To obtain more information about the role of RNS
in control of F. tularensis, we monitored the growth of F.
tularensis LVS in mice with a partially defective iNOS gene,
which were designated leaky mice. These mice have been
shown to exhibit NO-dependent killing of Leishmania, al-
though they display no measurable serum levels of NO (22).
At various times after intradermal injection of 5 ? 102CFU
of F. tularensis LVS, a dose that was sublethal for the mice,
bacterial counts in the skin, livers, and spleens were deter-
mined. In samples from skin obtained on day 4, 6, or 10, the
numbers of bacteria were consistently 1 to 2 log10higher in
iNOS?/?mice than in iNOS?/?129 mice (Fig. 5). In each of
five separate experiments, the numbers of bacteria in the skin
were higher in iNOS?/?mice on days 4 to 7 of infection, and
the bacteria were eradicated within 3 weeks. In livers and
spleens, there were no significant differences between the two
strains of mice (Fig. 5). After peak numbers of bacteria were
reached on day 4, complete eradication occurred within 3
weeks postinfection. The data indicate that low levels of NO
are not sufficient for control of LVS infection in the skin,
although they are sufficient for control of LVS infection in the
liver and spleen.
Production of RNS and superoxide ex vivo by splenocytes of
mice infected with F. tularensis LVS. After intradermal injec-
tion of 5 ? 104CFU of F. tularensis LVS (the same dose that
was used in the histological examinations and liver enzyme and
cytokine assays), mice were killed on days 3 to 9 in order to
determine the capacity of splenocytes to produce RNS and
ROS. The ROS assay was highly sensitive and allowed detec-
tion of superoxide-dependent chemoluminescence after only
minutes of incubation, whereas RNS had to be accumulated by
in vitro incubation of the splenocytes for 4 days before quan-
FIG. 4. Representative liver histopathology for different strains of mice killed on day 6 after intradermal infection with 5 ? 104CFU of F.
tularensis LVS. (A) Liver from an iNOS?/?mouse, showing a medium-size focal accumulation of mixed inflammatory cells and hepatic necrosis
(the scores were 1, 1, and 1, as shown in Table 3). (B) Liver from an iNOS?/?mouse, showing the presence of numerous small to medium-size
inflammatory infiltrates throughout the entire liver section (the scores were 4, 4, and 4). (C and D) Livers from p47phox?/?(C) and p47phox?/?
(D) mice, showing the presence of small to medium-size focal accumulations of mixed inflammatory cells and occasional hepatic necrosis of some
severity (in both cases the scores were 2, 2, and 2). Hematoxylin and eosin staining was used. Bars ? 20 ?m.
VOL. 72, 2004RNS AND ROS AND CONTROL OF F. TULARENSIS INFECTION 7177

Page 7

tifiable amounts were present. Although these differences in
the assay time cannot be reliably translated to time of occur-
rence in vivo, both assays were nonetheless thought to reflect
the in vivo ability of the splenocytes to produce the corre-
sponding mediators.
Splenocytes harvested from infected iNOS?/?mice pro-
duced significant levels of NO2
on day 6 of infection or later (Fig. 6A). Similarly, splenocytes
obtained from p47phox?/?and p47phox?/?mice started to pro-
duce significant levels of NO2
?7 days after inoculation. On days 7 and 9, the levels were
significantly higher for p47phox?/?mice than for p47phox?/?
mice. Splenocytes derived from iNOS?/?mice showed no pro-
duction of NO2
Splenocytes from wild-type (iNOS?/?and p47phox?/?) and
iNOS?/?mice harvested on day 3 produced ROS at levels that
were significantly higher than the levels produced by spleno-
cytes from noninfected mice (Fig. 6B). At this time, the level of
ROS produced by cells from iNOS?/?mice was similar to the
level of ROS produced by cells from iNOS?/?mice. After this,
the capacity of cells from iNOS?/?mice increased signifi-
cantly; on days 6 and 7 the levels were almost twice as high as
the levels produced by cells from iNOS?/?mice, and on day 9
the levels were more than three times as high as the levels
produced by cells from iNOS?/?mice (Fig. 6B). Splenocytes
from p47phox?/?mice did not produce ROS at any time.
To investigate whether the increase in the capacity of
?only when they were obtained
?only when they were obtained
?at any time.
splenocytes from iNOS?/?mice to produce ROS was associ-
ated with changes in the proportion of phagocytes in the
spleen, differential counts were obtained on days 3, 6, and 9 of
infection. Infection resulted in increased proportions of neu-
trophils and monocytes in the spleens of all four strains of mice
at all times compared to noninfected mice. However, no
marked differences were found between the gene-deficient
mouse strains and the corresponding wild-type strains (Table
4). Thus, the enhanced production in cultures from infected
mice compared with cultures from noninfected mice may have
been related to the increased numbers of ROS-producing neu-
trophils and monocytes in the former mice.
In conclusion, p47phox?/?mice seemed to compensate for
the lack of the phagocyte oxidase by increased expression of
NO2
for the lack of iNOS by an increased ability to produce ROS.
The increased ROS levels observed in the latter mice was likely
related to an enhanced cellular capability for ROS production,
since there were no significant differences in the proportion of
phagocytes between the iNOS?/?mice and the iNOS?/?mice.
Cytokine levels in sera from iNOS?/?, iNOS?/?, p47phox?/?,
and p47phox?/?mice after infection with F. tularensis. At var-
ious times after intradermal inoculation of 5 ? 104CFU of F.
tularensis LVS, blood samples were collected for an assay of
cytokines. Both p47phox?/?mice and iNOS?/?mice had much
higher serum levels of IFN-? than the corresponding wild-type
mice (Table 5). On day 10, the levels in the gene-deficient mice
were ?2,000 pg/ml, compared to ?500 pg/ml in the wild-type
strains. Leaky iNOS?/?mice showed down-regulation of
IFN-? levels in the same manner as wild-type mice, and the
level on day 10 was ?200 pg/ml. In serum from noninfected
animals, IFN-? was not detectable (concentration, ?30 pg/ml).
Based on the values for individual animals on days 3, 6, 9, and
10, the levels of IFN-? showed a correlation coefficient of 0.5
or higher with the numbers of bacteria in spleens and livers
irrespective of the mouse strain.
Also, the IL-6 levels were generally higher in sera from the
iNOS?/?and p47phox?/?mice than in the sera from the cor-
responding wild-type mice (Table 6). The IL-12p40 levels were
?2,000 pg/ml in all serum samples and were not different in
gene-deficient and wild-type mice. TNF-? and IL-4 were not
detected in any serum sample from any strain of mice.
In vitro recall response of splenocytes from iNOS?/?and
iNOS?/?mice. To determine whether the high level of sus-
ceptibility of the iNOS?/?mice was related to an impaired
ability to respond to F. tularensis antigen with T-cell prolifer-
ation and IFN-? production, mice were primed by intradermal
inoculation of a low dose of F. tularensis LVS, treated with
moxifloxacin on days 15 to 22, and killed on day 35 for prep-
aration of splenocyte cultures. Naı ¨ve iNOS?/?and iNOS?/?
mice showed low or nonsignificant proliferative responses in
vitro to heat-killed F. tularensis (stimulatory indices, ?2.4),
whereas primed mice from both groups displayed a strong
response, with stimulatory indices of ?10.5 (Table 7). In re-
sponse to heat-killed F. tularensis, splenocytes from immune
iNOS?/?mice secreted much higher levels of IFN-? than cells
from primed iNOS?/?mice secreted (Table 7). Irrespective of
immunization, the spleen cells showed vigorous proliferation
and a high level of secretion of IFN-? in response to the T-cell
mitogen concanavalin A. Thus, the immune iNOS?/?mice
?, and in contrast, iNOS?/?mice seemed to compensate
FIG. 5. Growth curves for F. tularensis LVS in the skin, livers, and
spleens of leaky iNOS?/?mice and iNOS?/?129 mice after primary
intradermal infection with 5 ? 102F. tularensis LVS cells. The means
? standard errors of the means for five mice per group and time are
shown. One asterisk indicates that the P value is ?0.05, and three
asterisks indicate that the P value is ?0.001.
7178 LINDGREN ET AL.INFECT. IMMUN.

Page 8

showed recall responses that were as strong as those of im-
mune iNOS?/?mice and produced much higher levels of the
Th1 cytokine IFN-? than immune iNOS?/?mice produced.
DISCUSSION
F. tularensis is a potent pathogen, and the principal virulence
mechanism seems to be intracellular survival. As observed for
other facultative intracellular bacteria, the mechanisms of host
protection are critically dependent on cell-mediated immunity
(30) (i.e., the activation of macrophages by T cells to kill
intracellularly located bacteria). The T-cell-dependent activa-
tion requires the involvement of IFN-?, and previous in vitro
studies demonstrated that a release of nitric oxide by the mac-
rophages might contribute to killing (1, 15). By using
p47phox?/?and iNOS?/?mice, we demonstrated here that
both ROS and RNS play an essential role in vivo for the
control of murine tularemia. Mutant mice were significantly
more susceptible to death caused by F. tularensis LVS. The
LD50for the iNOS?/?mice was ?20 CFU, the LD50for the
p47phox?/?mice was 4,400 CFU, and the LD50for the wild-
type mice was ?500,000 CFU. In p47phox?/?mice the infection
was exacerbated on day 4, whereas in iNOS?/?mice exacer-
bation occurred in the second week of infection. In iNOS?/?
mice there was a prolonged course of disease until death after
26.4 days of infection (mean value) and there was severe liver
damage, whereas in p47phox?/?mice the disease was more
acute, the mean time to death was 10.1 days, and there was less
pronounced liver damage.
Thus, our results indicate that ROS have a critical role
during the first few days of infection, whereas RNS seemed to
be critical at a subsequent phase. Few previous studies have
assessed the role of ROS in the control of tularemia. One study
demonstrated that superoxide and hydrogen peroxide are pro-
duced during the course of experimental tularemia but did not
FIG. 6. iNOS?/?mice (solid bars), iNOS?/?mice (striped bars), p47phox?/?mice (gray bars), and p47phox?/?mice (open bars) were inoculated
with 5 ? 104CFU of F. tularensis LVS. On different days after inoculation mice were sacrificed, and splenocytes were prepared and analyzed for
NO production (A) and ROS production (B). The values are the means ? standard errors of the means for three mice per group and time. Samples
were analyzed in triplicate. No values are given for iNOS?/?and iNOS?/?mice on day 4. One asterisk indicates that the P value is ?0.05, two
asterisks indicate that the P value is ?0.01, and three asterisks indicate that the P value is ?0.001. RLU, relative light units.
VOL. 72, 2004 RNS AND ROS AND CONTROL OF F. TULARENSIS INFECTION 7179

Page 9

analyze if this production correlated with a bactericidal effect
(17). The high level of susceptibility of p47phox?/?mice may be
related to a lack of macrophage- and neutrophil-mediated
bactericidal mechanisms. Neutrophils have been shown to play
a crucial role in the control of murine tularemia (26), and
human neutrophils display bactericidal activity against F. tula-
rensis by formation of hypochloric acid (19). This acid is pro-
duced as a result of the reaction between hydrogen peroxide
and chloride anions and is thus not formed in neutrophils from
p47phox?/?mice. Although killing of F. tularensis by murine
neutrophils has not been demonstrated, the important role of
these cells in the control of infection may be one explanation
for why in the present experiments p47phox?/?mice showed
such a high level of susceptibility to tularemia. During the
interval from day 4 to 7, when exacerbation was observed in
p47phox?/?mice, splenocytes derived from wild-type mice pro-
duced high levels of superoxide, indirectly supporting the hy-
pothesis that ROS production plays an important role during
this stage. Collectively, our data indicate that ROS have an
important role in control of the early phase of the F. tularensis
infection when innate immune mechanisms are operative.
However, since small challenge inocula were controlled in
p47phox?/?mice, ROS-independent mechanisms are appar-
ently operative as well.
In contrast to the role of ROS, the absence of RNS did not
affect the numbers of bacteria in livers and spleens during the
first week of infection. After this, the numbers of bacteria
increased in the iNOS?/?mice, which developed severe liver
pathology and showed high serum levels of liver-specific en-
zymes and IFN-?. Together, these results indicate that RNS
have both immunoregulatory and bactericidal effects. Besides
their lack of possible NO-mediated antimicrobial mechanisms,
the iNOS?/?mice may be afflicted with dysregulation of the
immune response, leading to liver damage and persistent, high
serum levels of IFN-?. This is in line with extensive evidence
concerning the important immunoregulatory role of NO (5, 6,
9). Since iNOS?/?mice with a leaky phenotype displayed no
measurable serum levels of NO but showed no signs of exac-
erbation of systemic infection, no liver damage, and normal
serum levels of IFN-? on day 10, such regulatory mechanisms
might depend on the presence of very low serum levels of NO.
Previous studies have demonstrated that immune mice are
effectively protected by a number of overlapping, compensa-
tory mechanisms (11, 14, 26).
The role of RNS in killing of F. tularensis in skin was evident
even during the early phase of infection in both iNOS?/?mice
and leaky iNOS?/?mice and persisted throughout the course
of infection. Thus, in skin, low levels of NO did not appear to
be sufficient for host control of an F. tularensis infection. The
skin is an important barrier for controlling both murine and
human tularemia. The data suggest that NO-dependent, der-
mal killing mechanisms may be important for control of tula-
remia.
In mice, the lack of IFN-? leads to lethal exacerbation of
TABLE 4. Spleen cell composition in spleens of mice infected with F. tularensis LVSa
Cell typeMouse strain
No. of cells (?106) in spleens on:
Day 0a,b
Day 3c
Day 6c
Day 9c
Neutrophilsp47phox?/?
p47phox?/?
iNOS?/?
iNOS?/?
0.3 (1.1)
0.3 (1.1)
0.6 (2.0)
0.6 (2.0)
7.8 ? 0.6 (14)
4.9 ? 2.7 (9.8)
13.5 ? 2.3 (17)
14.9 ? 2.9 (21)
14.9 ? 2.9 (18)
12.0 ? 2.7 (16)
12.6 ? 4.6 (15)
5.9 ? 2.9 (9.3)
6.4 ? 2.2 (7.3)
10.3 ? 1.9 (14)
6.7 ? 4.3 (6.6)
11.0 ? 0.9 (15)
Monocytesp47phox?/?
p47phox?/?
iNOS?/?
iNOS?/?
0
0
0
0
1.7 ? 0.3 (3.0)
0.5 ? 0.3 (1.0)
1.9 ? 0.6 (2.4)
2.1 ? 1.1 (2.9)
4.8 ? 1.6 (5.8)
4.8 ? 1.3 (6.5)
4.2 ? 0 (5.0)
2.6 ? 1.6 (4.1)
1.0 ? 0.6 (1.1)
1.0 ? 0.8 (1.4)
3.3 ? 1.5 (3.2)
3.7 ? 1.0 (5.0)
aThe intradermal inoculum was 5 ? 104CFU of F. tularensis LVS per mouse.
bThe values are the numbers of cells before inoculation of F. tularensis LVS. The numbers in parentheses are the percentages of the total numbers of cells in the
spleens.
cThe values are means ? standard errors of the means for three samples. The numbers in parentheses are the percentages of the total numbers of cells in the spleens.
TABLE 5. IFN-? in sera of mice infected with F. tularensis LVSa
Mouse strain
IFN-? concn in serum (pg/ml)
Day 0b
Day 3c
Day 6c
Day 9c
Day 10c
p47phox?/?
p47phox?/?
iNOS?/?
iNOS?/?
iNOS?/?129
Leaky iNOS?/?
?30
?30
?30
?30
?30
?30
1,070 ? 390
2,088 ? 418d
1,530 ? 620
2,790 ? 1,500
1,190 ? 440
1,530 ? 525
4,340 ? 2,590
6,190 ? 2,510
3,243 ? 1,641
12,080 ? 575 A
1,650 ? 240
4,000 B
220 ? 25
3,600 ? 640 C
NDe
6,490 ? 1,220
ND
ND
280 ? 15
2,010 ? 75 B
?30
2,020 ? 710 A
220 ? 120
150 ? 45
aThe intradermal inoculum was 5 ? 104CFU of F. tularensis LVS per mouse.
bIFN-? serum levels before inoculation of F. tularensis LVS.
cMeans ? standard errors of the means for three samples.
dSignificance is indicated as follows: A, P ? 0.05 compared to wild-type mice; B, P ? 0.01 compared to wild-type mice; C, P ? 0.001 compared to wild-type mice.
eND, not determined.
7180 LINDGREN ET AL.INFECT. IMMUN.

Page 10

tularemia even with the smallest challenge inocula (18, 27). In
the present study, a paradoxical relationship between the se-
rum levels of IFN-? and the numbers of bacteria in the liver
and spleen was demonstrated and was evident especially dur-
ing protracted infection in iNOS?/?mice. The simplest expla-
nation for the paradox is the occurrence of frustrated overpro-
duction of IFN-?, which is not sufficient to control infection in
the absence of complementary effector molecules generated
directly or indirectly by iNOS and phox. The present data and
previous studies (14, 18, 27) together show that although
IFN-? is absolutely required in vivo for control of tularemia,
high, excessive levels of the cytokine do not necessarily lead to
more effective killing of F. tularensis.
The host protective mechanisms for murine tularemia seem
to resemble those that are operative in other experimental
models of intracellular infections. For example, in cutaneous
leishmaniasis, RNS were needed for killing of the parasite in
the skin and draining lymph nodes, whereas the activity of phox
was dispensable for resolution of the acute skin infection but
essential for clearance of the parasites in the spleen (3). Re-
sistance to virulent Salmonella enterica biovar Typhimurium in
the mouse model was dependent on ROS, because phox-defi-
cient mice showed dramatic exacerbation and succumbed to
infection within 5 days. In contrast, iNOS?/?mice contained
increased numbers of bacteria only after the first week of
infection (20). Thus, in these two infection models there is
RNS- and ROS-dependent organ- and stage-specific control,
and the roles of the two classes of effector molecules appear to
be quite distinct.
Our results demonstrate that both RNS and ROS play im-
portant roles in the control of experimental tularemia. Exac-
erbation was observed in p47phox?/?mice during the early
phase of infection, at a stage when a lack of NO resulted in no
systemic exacerbation. However, iNOS?/?mice subsequently
succumbed to the infection, and they succumbed to even the
lowest dose. The extreme susceptibility of iNOS?/?mice might
be in line with a complex regulatory role for NO in intracellular
infections, and death may be an effect of dysregulation of the
cytokine response and resulting severe liver damage.
ACKNOWLEDGMENTS
Grant support was obtained from the Swedish Medical Research
Council, Samverkansna ¨mnden, Norra Sjukva ˚rdsregionen, Umea ˚, Swe-
den, and the Medical Faculty, Umea ˚ University, Umea ˚, Sweden.
We thank Steven Holland for supplying the p47phox?/?mice and
F. Y. Liew for supplying leaky iNOS?/?mice.
REFERENCES
1. Anthony, L. S., P. J. Morrissey, and F. E. Nano. 1992. Growth inhibition of
Francisella tularensis live vaccine strain by IFN-gamma-activated macro-
phages is mediated by reactive nitrogen intermediates derived from L-argi-
nine metabolism. J. Immunol. 148:1829–1834.
2. Babior, B. M., J. D. Lambeth, and W. Nauseef. 2002. The neutrophil
NADPH oxidase. Arch. Biochem. Biophys. 397:342–344.
3. Blos, M., U. Schleicher, F. J. Soares Rocha, U. Meissner, M. Rollinghoff, and
C. Bogdan. 2003. Organ-specific and stage-dependent control of Leishmania
major infection by inducible nitric oxide synthase and phagocyte NADPH
oxidase. Eur. J. Immunol. 33:1224–1234.
4. Bogdan, C. 2001. Nitric oxide and the immune response. Nat. Immunol.
2:907–916.
5. Bogdan, C., M. Rollinghoff, and A. Diefenbach. 2000. Reactive oxygen and
reactive nitrogen intermediates in innate and specific immunity. Curr. Opin.
Immunol. 12:64–76.
6. Bogdan, C., M. Rollinghoff, and A. Diefenbach. 2000. The role of nitric oxide
in innate immunity. Immunol. Rev. 173:17–26.
7. Brunet, L. R. 2001. Nitric oxide in parasitic infections. Int. Immunopharma-
col. 1:1457–1467.
8. Chakravortty, D., and M. Hensel. 2003. Inducible nitric oxide synthase and
control of intracellular bacterial pathogens. Microbes Infect. 5:621–627.
9. Cifone, M. G., S. Ulisse, and A. Santoni. 2001. Natural killer cells and nitric
oxide. Int. Immunopharmacol. 1:1513–1524.
10. Coleman, J. W. 2001. Nitric oxide in immunity and inflammation. Int. Im-
munopharmacol. 1:1397–1406.
11. Conlan, J. W., A. Sjo ¨stedt, and R. J. North. 1994. CD4?and CD8?T-cell-
dependent and -independent host defense mechanisms can operate to con-
trol and resolve primary and secondary Francisella tularensis LVS infection in
mice. Infect. Immun. 62:5603–5607.
12. Dahlgren, C., and A. Karlsson. 1999. Respiratory burst in human neutro-
phils. J. Immunol. Methods 232:3–14.
13. Elkins, K. L., T. Rhinehart-Jones, C. A. Nacy, R. K. Winegar, and A. H.
Fortier. 1993. T-cell-independent resistance to infection and generation of
immunity to Francisella tularensis. Infect. Immun. 61:823–829.
TABLE 6. IL-6 levels in sera of mice infected with F. tularensis LVSa
Mouse strain
IL-6 concn in serum (pg/ml)
Day 0b
Day 3c
Day 6c
Day 9c
Day 10c
p47phox?/?
p47phox?/?
iNOS?/?
iNOS?/?
?15
?15
?15
?15
419 ? 137
1,260 ? 588 Ae
522 ? 145
625 ? 43
115 ? 50
1,007 ? 891
140 ? 81
1450 ? 86 B
52 ? 10
203 ? 25 A
57 ? 11
1,480 ? 420 B
NDd
ND
102 ? 9
684 ? 5 C
aThe intradermal inoculum was 5 ? 104CFU of F. tularensis LVS per mouse.
bIL-6 serum levels before inoculation of F. tularensis LVS.
cMeans ? standard errors of the means for three samples.
dND, not determined.
eSignificance is indicated as follows: A, P ? 0.05 compared to wild-type mice; B, P ? 0.01 compared to wild-type mice; C, P ? 0.001 compared to wild-type mice.
TABLE 7. In vitro proliferation and IFN-? production in
splenocytes from naı ¨ve or F. tularensis-primed iNOS?/?or iNOS?/?
mice after stimulation with heat-killed F. tularensisa
Assay
Mice
F. tularensis immuneb
Nonimmune
iNOS?/?
iNOS?/?
iNOS?/?
iNOS?/?
Proliferative
response (SI)c
IFN-? (ng/ml)
10.5 ? 2.7 12.3 ? 1.5 2.4 ? 0.51.9 ? 0.2
7.5 ? 0.4 24.5 ? 2.1
?0.03
?0.03
aTo estimate the proliferative response, spleen cell cultures were stimulated
for 4 days with 106heat-killed cells of F. tularensis LVS and pulsed with [3H]thy-
midine. Cytokine levels were determined in cell supernatants after 2 days of
stimulation. The values are means ? standard errors of the means for five
cultures.
bMice were subjected 5 weeks previously to intradermal infection with 5 ? 103
CFU of F. tularensis LVS and given moxifloxacin on days 15 to 22.
cStimulation index (SI) ? mean counts per minute for five cultures containing
antigen/mean counts per minute for five cultures lacking antigen. Stimulation
with concanavalin A resulted in a stimulation index of ?28.7. Cultures without
antigen gave 1,400 to 1,800 cpm.
VOL. 72, 2004 RNS AND ROS AND CONTROL OF F. TULARENSIS INFECTION7181

Page 11

14. Elkins, K. L., T. R. Rhinehart-Jones, S. J. Culkin, D. Yee, and R. K. Win-
egar. 1996. Minimal requirements for murine resistance to infection with
Francisella tularensis LVS. Infect. Immun. 64:3288–3293.
15. Fortier, A. H., M. V. Slayter, R. Ziemba, M. S. Meltzer, and C. A. Nacy. 1991.
Live vaccine strain of Francisella tularensis: infection and immunity in mice.
Infect. Immun. 59:2922–2928.
16. Jackson, S. H., J. I. Gallin, and S. M. Holland. 1995. The p47phox mouse
knock-out model of chronic granulomatous disease. J. Exp. Med. 182:751–
758.
17. Kovarova, H., A. Macela, and J. Stulik. 1990. The production of oxygen
metabolites and their possible regulatory role in the course of tularemia
infection. Folia Microbiol. 35:413–422.
18. Leiby, D. A., A. H. Fortier, R. M. Crawford, R. D. Schreiber, and C. A. Nacy.
1992. In vivo modulation of the murine immune response to Francisella
tularensis LVS by administration of anticytokine antibodies. Infect. Immun.
60:84–89.
19. Lo ¨fgren, S., A. Ta ¨rnvik, M. Thore, and J. Carlsson. 1984. A wild and an
attenuated strain of Francisella tularensis differ in susceptibility to hypochlo-
rous acid: a possible explanation of their different handling by polymorpho-
nuclear leukocytes. Infect. Immun. 43:730–734.
20. Mastroeni, P., A. Vazquez-Torres, F. C. Fang, Y. Xu, S. Khan, C. E. Hor-
maeche, and G. Dougan. 2000. Antimicrobial actions of the NADPH phago-
cyte oxidase and inducible nitric oxide synthase in experimental salmonello-
sis. II. Effects on microbial proliferation and host survival in vivo. J. Exp.
Med. 192:237–248.
21. Nathan, C., and M. U. Shiloh. 2000. Reactive oxygen and nitrogen interme-
diates in the relationship between mammalian hosts and microbial patho-
gens. Proc. Natl. Acad. Sci. USA 97:8841–8848.
22. Niedbala, W., X. Q. Wei, D. Piedrafita, D. Xu, and F. Y. Liew. 1999. Effects
of nitric oxide on the induction and differentiation of Th1 cells. Eur. J. Im-
munol. 29:2498–2505.
23. Polsinelli, T., M. S. Meltzer, and A. H. Fortier. 1994. Nitric oxide-indepen-
dent killing of Francisella tularensis by IFN-gamma-stimulated murine alve-
olar macrophages. J. Immunol. 153:1238–1245.
24. Portnoy, D. A., V. Auerbuch, and I. J. Glomski. 2002. The cell biology of
Listeria monocytogenes infection: the intersection of bacterial pathogenesis
and cell-mediated immunity. J. Cell Biol. 158:409–414.
25. Sjo ¨stedt, A., G. Sandstro ¨m, and A. Ta ¨rnvik. 1992. Humoral and cell-medi-
ated immunity in mice to a 17-kilodalton lipoprotein of Francisella tularensis
expressed by Salmonella typhimurium. Infect. Immun. 60:2855–2862.
26. Sjo ¨stedt, A., J. W. Conlan, and R. J. North. 1994. Neutrophils are critical for
host defense against primary infection with the facultative intracellular bac-
terium Francisella tularensis in mice and participate in defense against rein-
fection. Infect. Immun. 62:2779–2783.
27. Sjo ¨stedt, A., R. J. North, and J. W. Conlan. 1996. The requirement of tumour
necrosis factor-alpha and interferon-gamma for the expression of protective
immunity to secondary murine tularaemia depends on the size of the chal-
lenge inoculum. Microbiology 142:1369–1374.
28. Stenger, S., and R. L. Modlin. 1999. T cell mediated immunity to Mycobac-
terium tuberculosis. Curr. Opin. Microbiol. 2:89–93.
29. Ta ¨rnvik, A., and L. Berglund. 2003. Tularaemia. Eur. Respir. J. 21:361–373.
30. Ta ¨rnvik, A., M. Ericsson, I. Golovliov, G. Sandstro ¨m, and A. Sjo ¨stedt. 1996.
Orchestration of the protective immune response to intracellular bacteria:
Francisella tularensis as a model organism. FEMS Immunol. Med. Microbiol.
13:221–225.
31. Wei, X. Q., I. G. Charles, A. Smith, J. Ure, G. J. Feng, F. P. Huang, D. Xu,
W. Muller, S. Moncada, and F. Y. Liew. 1995. Altered immune responses in
mice lacking inducible nitric oxide synthase. Nature 375:408–411.
32. Yee, D., T. R. Rhinehart-Jones, and K. L. Elkins. 1996. Loss of either CD4?
or CD8?T cells does not affect the magnitude of protective immunity to an
intracellular pathogen, Francisella tularensis strain LVS. J. Immunol. 157:
5042–5048.
Editor: A. D. O’Brien
7182 LINDGREN ET AL.INFECT. IMMUN.