Download pdf - Distinct Roles of Reactive Nitrogen and Oxygen Species To Control Infection with the Facultative Intracellular Bacterium Francisella tularensis

INFECTION AND IMMUNITY, Dec. 2004, p. 7172–7182 Vol. 72, No. 120019-9567/04/$08.00�0 DOI: 10.1128/IAI.72.12.7172–7182.2004Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Distinct Roles of Reactive Nitrogen and Oxygen Species To ControlInfection with the Facultative Intracellular Bacterium

Francisella tularensisHelena Lindgren,1,2 Stephan Stenmark,1,2 Wangxue Chen,3

Arne Tarnvik,2 and Anders Sjostedt1*Department of Clinical Microbiology, Clinical Bacteriology,1 and 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 withthe facultative intracellular bacterium Francisella tularensis. When intradermally infected with the live vaccinestrain F. tularensis LVS, mice deficient in production of RNS (iNOS�/� mice) or in production of ROS by thephagocyte oxidase (p47phox�/� mice) showed compromised resistance to infection. The 50% lethal dose (LD50)for iNOS�/� mice was <20 CFU, and the LD50 for p47phox�/� mice was 4,400 CFU, compared to an LD50 of>500,000 CFU for wild-type mice. The iNOS�/� mice survived for 26.4 � 1.8 days, and the p47phox�/� micesurvived for 10.1 � 1.3 days. During the course of infection, the serum levels of gamma interferon (IFN-�) andinterleukin-6 were higher in iNOS�/� and p47phox�/� mice than in wild-type mice. Histological examination oflivers of iNOS�/� mice revealed severe liver pathology. Splenocytes obtained 5 weeks after primary infectionfrom antibiotic-treated iNOS�/� mice showed an in vitro recall response that was similar in magnitude andgreater 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 andROS seemed to be distinct since mice deficient in production of ROS showed dissemination of infection anddied during the early phase of infection, whereas RNS deficiency led to severe liver pathology and a contractedcourse of infection.

Francisella tularensis is a highly virulent bacterium thatcauses tularemia in many mammalian species. In humans, di-rect contact with infected animals or transmission by infectedarthropods, 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 ischaracterized 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 thehost protective mechanisms are similar to those that are activeagainst listeriae and mycobacteria (24, 28). During the first fewdays of infection, T-cell-independent transient host resistanceis evoked. During this phase, both gamma interferon (IFN-�)and tumor necrosis factor alpha (TNF-�) are crucial mediatormolecules, 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 protectiveimmunity to F. tularensis develops. The critical role of thiscell-mediated immunity is demonstrated by the finding thatSCID mice or mice lacking �� T cells succumb to even thesmallest inocula of F. tularensis (11, 13, 32).

Reactive nitrogen species (RNS) and reactive oxygen species(ROS) are intermediates that are involved in the host defenseagainst various intracellular pathogens (4, 7, 8, 21). RNS canbe generated by constitutive nitric oxide synthases, but highlevels of RNS are produced only after the activation of induc-ible nitric oxide synthases (iNOS) (10). Production of ROSmessengers (for example, superoxide) depends on the induc-tion of phagocyte oxidase (phox) (2). Activation of iNOS andphox is induced in macrophages when they are exposed toproinflammatory cytokines, including IFN-� or TNF-� (4, 22).

A role for RNS in the host resistance to tularemia has beensuggested 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 defenseagainst tularemia is, however, not completely understood, andits importance seems to vary with the organ localization ofmacrophages (23). No studies have directly assessed the role ofRNS or ROS in vivo. The availability of mice deficient inexpression of iNOS or phox provides models to directly assessthe roles of these reactive molecular species in killing of F.tularensis.

We demonstrated in this study that mice lacking expressionof RNS or ROS show extreme susceptibility to infection withthe live vaccine strain F. tularensis LVS. However, RNS andROS seem to be predominantly involved at different stages ofthe infection. Mice deficient in production of ROS died within

* Corresponding author. Mailing address: Department of ClinicalMicrobiology, Clinical Bacteriology, Umea University, SE-901 85Umea, Sweden. Phone: 46-90-7851120. Fax: 46-90-7852225. E-mail:[email protected].

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2 weeks, whereas RNS deficiency rapidly resulted in severeliver pathology but a contracted course of infection, and deathoccurred within 4 weeks.

MATERIALS AND METHODSBacterial 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 inmodified Mueller-Hinton broth, harvested, and stored frozen at �70°C.

Mice. Breeding stocks of iNOS gene-deficient mice (iNOS�/� mice) andwild-type C57BL/6 mice (designated iNOS�/� mice) were obtained from JacksonLaboratories, 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 asdescribed elsewhere (16) from heterozygous parents (C57BL/6 � 129 back-crossed to F5 in a C57BL/6 lineage). The wild-type mice also represented the F5generation. Breeding stocks of leaky iNOS�/� mice were obtained through thecourtesy 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 werebred and housed at the Animal Facility of the Swedish Defense ResearchAgency, Umea, Sweden, under conventional conditions and were given food andwater ad libitum. The mice were 8 to 14 weeks old and age and sex matched whenthey were used in experiments. They were found to be free of specific pathogens.Permission for the experiments was obtained from the Ethical Committee, UmeaUniversity, Umea, Sweden.

Inoculation and enumeration of bacteria in mice. For primary infection, micewere inoculated intradermally in the thoracic region with F. tularensis LVS cells

suspended in 50 �l of saline. One day before the intradermal inoculation, theregion was shaved, and at various times after injection, animals were killed, anda 1-cm2 sample of the region was excised, briefly washed in 70% ethanol, andadded to a tube with saline. Spleens, livers, and lungs were also collected.Samples from each organ were homogenized, and 10-fold serial dilutions werecultured 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 collectedfrom the retroorbital vein. Serial dilutions of lavage and blood samples wereplated for enumeration of bacteria. Bacterial numbers were expressed as thenumber of CFU per organ (lung, liver, or spleen), per 1.0 ml of peritoneal lavagefluid, or per 100 �l of blood.

The 50% lethal dose (LD50) was determined by the method of Reed andMuench as previously described (25).

Blood chemistry for organ function. On days 3, 6, and 9 of infection, blood wasobtained from three mice per strain. Sera from mice (�300 �l) were storedfrozen until analysis. Alanine aminotransferase, aspartate aminotransferase, to-tal bilirubin, amylase, and creatinine in serum were analyzed by using a Vitros950 multianalyzer (Johnson-Johnson, Clinical Diagnostics).

Histological examination. On days 3, 6 and 9, livers, spleens and lungs werecollected from mice of each strain. The organs were prepared for histologicalstaining by snap freezing them in liquid propane and were placed in OCTcompound (EMS, Hatfield, Pa.). Samples were stored at �70°C until they weresectioned, 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 ofsaline. Debris was removed by centrifugation at 200 � g for 30 s. The cells in theresulting 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 intradermallywith 2 � 101 CFU (�), 2 � 103 CFU (‚), or 2 � 104 CFU (E). p47phox�/� mice that survived for 28 days were sacrificed, and no bacteria werefound in the livers or spleens. (A) iNOS �/� mice. (B) p47 phox�/� mice.

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gation at 200 � g for 10 min, resuspended in 1 ml of 0.8% NH4Cl, and incubatedfor 10 min at room temperature to lyse erythrocytes. The cells were resuspendedin 10 ml of ice-cold saline and centrifuged. This procedure was repeated once.The splenocytes were resuspended in 5 ml of cell culture medium (Dulbeccomodified Eagle medium; Invitrogen, Paisley, United Kingdom) supplemented

with 10% fetal bovine serum (Invitrogen), and enumeration was performed byusing a Burker chamber. Cells were stained by using May Grunwall Giemsa stainfor differential counting of the splenocytes.

Assay of ROS production by splenocytes. At different times, mice infected with5 � 104 CFU 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 � 102 CFU of F. tularensis LVS, and the numbers of bacteria (CFU per organ sample) weredetermined at different times. The means standard errors of the means for five mice per group and time are shown. One asterisk indicates thatthe 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 inoculation 5 Days after inoculation 9 Days after inoculation

p47phox�/� mice p47phox�/� mice p47phox�/� mice p47phox�/� mice p47phox�/� mice p47phox�/� mice

Liver 3.56 0.61 4.79 0.22 Ab 4.76 0.21 7.35 0.29 C 2.89 0.20 4.60 0.41 ALung 3.16 0.58 2.74 0.52 BDL 5.38 0.76 B BDL 3.10 0.73 ABloodc BDLd 2.74 0.52 A BDL 1.77 0.08 A NDc NDPeritoneumf 2.08 1.1 3.22 0.48 BDL 3.77 0.45 B BDL 1.78 0.64 A

a The inoculum was 5 � 104 CFU of F. tularensis LVS per mouse, and the results are expressed as the mean log10 CFU standard error of the mean for threesamples.

b Significance 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.c Log10 CFU per 100 �l of blood.d BDL, below the detection limit.e ND, not determined.f Log10 CFU per milliliter of peritoneal lavage fluid.

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described above. ROS production by fresh splenocytes was measured by a lumi-nol-based chemiluminescence method (12). In preliminary experiments, themethod showed a good correlation with a colorimetric assay based on ROS-mediated reduction of cytochrome c. The kinetics of the chemiluminescenceresponse were similar for all samples tested and were maximal within the 5-minlength of the assay. Splenocytes (1 � 106 cells) were incubated at 37°C for 5 minin 1 ml of KRG buffer (Krebs-Ringer phosphate buffer containing 10 mMglucose, 1 mM Ca2�, and 1.5 mM Mg2�; pH 7.3) supplemented with 100 �Mluminol (Sigma, Madison, Wis.). Immediately before measurement of the chemi-luminescence response, 0.5 �l of phorbol myristate acetate (2 �g/ml) was addedto the tube. In preliminary experiments, this concentration was found to beoptimal. The chemiluminescence was measured with a luminometer (Luminom-eter 1250; LKB Wallac, Turku, Finland) for 5 min. The results were expressed inpeak relative light units.

Assay of RNS production by splenocytes. At different times, infected micewere killed, and spleen cells were prepared as described above. Cells were seeded

in a 96-well cell culture plate at a density of 3 � 105 cells/well in 200 �l andincubated at 37°C in 5% CO2. Preliminary experiments indicated that measur-able levels of the end metabolite NO2

� were found only after incubation for atleast 4 days. The concentration of NO2

� was measured by the Griess reaction.Fifty microliters of the culture supernatant was mixed with 50 �l each of theGriess 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 limitof detection of the assay was 2.5 �M.

Assay of serum cytokine levels. All procedures for the cytokine enzyme-linkedimmunosorbent assay (OptEIA; BD Biosciences, San Diego, Calif.) were per-formed by following the instructions of the manufacturer. Dilutions of serumsamples 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 andIFN-� was 30 pg/ml, and the level of detection for IL-4, IL-6, and TNF-� was 15pg/ml.

Assay of the T-cell response. Mice were inoculated with 2 � 102 CFU of F.tularensis LVS, and 15 days later moxifloxacin was administered intraperitoneallyat a dose of 0.1 mg/g of body weight once daily for 7 days. Mice were killed 5weeks after inoculation, and spleen cell cultures were established. Each culture(200 �l) contained 6 � 105 splenocytes in RPMI (GIBCO BRL) supplementedwith 15% inactivated fetal calf serum, 10 �g of gentamicin per ml, 5 � 10�5 M�-mercaptoethanol, and 2 mM L-glutamine (GIBCO BRL). Heat-killed F. tula-rensis LVS (106 cells/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 hwith 0.5 �Ci of [3H]thymidine (18 Ci/mmol), and harvested with an automatedcell harvester (Inotech, Basel, Switzerland). For determination of secreted cyto-kines, parallel cultures were established, and after 2 days of incubation withantigen, 100 �l of supernatant was collected. The proliferative responses andcytokine levels have been found to be optimal at these times (26).

Statistical analysis. Student’s t test and Pearson’s correlation coefficient testwere used.

RESULTS

Numbers of bacteria in iNOS�/�, iNOS�/�, p47phox�/�, andp47phox�/� mice after primary intradermal infection with F.tularensis. The LD50 for intradermal infection was found to be4.4 � 103 CFU of F. tularensis LVS for p47phox�/� mice andless than 20 CFU for iNOS�/� mice (Fig. 1). The mean time todeath was determined for all of the mice in the experiment,irrespective of the inoculum. In spite of a lower LD50, themean time to death was significantly longer for the iNOS�/�

mice (26.4 1.8 days, compared with 10.1 1.3 days for thep47phox�/� mice). For the iNOS�/� mice the LD50 was 5.0 �105 to 1.2 � 106 CFU and the mean time to death was 7.2 1.2 days, and for the p47phox�/� mice the LD50 was �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 inoculation 6 Days after inoculation 9 Days after inoculation

iNOS�/� mice iNOS�/� mice iNOS�/� mice iNOS�/� mice iNOS�/� mice iNOS�/� mice

Skin 6.91 0.17 7.82 0.26 Ab 5.04 0.01 7.27 0.11 C 1.31 1.31 6.72 0.24 CLiver 3.99 0.33 3.91 0.19 4.61 0.21 5.36 0.22 A 2.72 0.01 5.80 1.13 ALung BDLc BDL BDL 2.34 0.30 A BDL 2.00 0.34Peritoneumd 2.07 0.47 2.93 0.62 BDL 2.80 0.48 B BDL 2.5 0.18 CBlood BDL BDL BDL BDL BDL BDL

a The inoculum was 5 � 104 CFU of F. tularensis LVS per mouse, and the results are expressed as the mean log10 CFU standard error of the mean for threesamples.

b Significance 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.c BDL, below the detection limit.d Log10 CFU per milliliter of peritoneal lavage fluid.

TABLE 3. Histological examination of livers from mice infectedwith F. tularensis LVSa

Dayspostinfection Mouse strain Liver

damageb

Inflammatory response

Extentc Intensityd

3 iNOS�/� 1, 1, 1c 1, 1, 1 1, 1, 1iNOS�/� 1, 2, 2 1, 2, 2 1, 2, 2p47phox�/� 1, 1, 1 1, 1, 1 1, 1, 1p47phox�/� 1, 1, 1 1, 1, 1 1, 1, 1

6 iNOS�/� 1, 1, 1 2, 2, 2 2, 2, 2iNOS�/� 4, 4, 4 3, 4, 4 4, 4, 4p47phox�/� 2, 2, 2 2, 2, 2 2, 2, 2p47phox�/� 2, 2, 2 2, 2, 2 2, 2, 2

9 iNOS�/� 1, 2, 3 1, 2, 3 1, 2, 3iNOS�/� 4, 4, 4 4, 4, 4 4, 4, 4p47phox�/� 2, 3, 3 2, 3, 3 2, 3, 3p47phox�/� 3, 3, 3 3, 3, 3 3, 3, 3

a The intradermal inoculum was 5 � 104 CFU of F. tularensis LVS per mouse.b Liver damage: 1, degeneration and necrosis of individual hepatocytes occa-

sionally seen; 2, clusters or small aggregates of hepatocyte degeneration andnecrosis; 3, medium to large aggregates of hepatocyte necrosis; 4, large areas ofhepatocyte necrosis with loss of normal liver anatomic architecture.

c Extent 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/�100field); 4, large numbers of inflammatory infiltrates throughout the section.

d Intensity of the inflammatory response: 1, small inflammatory infiltrates witha few inflammatory cells; 2, medium-size inflammatory infiltrates with small tomoderate numbers of inflammatory cells; 3, large inflammatory infiltrates withmoderate to large numbers of inflammatory cells; 4, extensive infiltration withlarge numbers of inflammatory cells. The values are averages for three individualmice.

e Individual scores for liver samples for three mice for each day and group.



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Bacterial counts in the skin, livers, and spleens of p47phox�/�

and iNOS�/� mice and the corresponding wild-type mice weredetermined at various intervals after intradermal inoculationof 5 � 102 CFU of F. tularensis LVS, a dose at which allanimals survived for �10 days. The data are summarized inFig. 2. Compared to the wild-type mice, the iNOS�/� miceshowed no significant differences in the numbers of bacteria inthe liver and spleen during the first 6 days of infection (Fig.2A). After this, the numbers of bacteria decreased in theiNOS�/� mice but remained stationary in the iNOS�/� mice.In the skin, starting at day 4, the iNOS�/� mice showed highernumbers of bacteria than the wild-type mice. By day 35, thewild-type mice had cleared the infection, while the iNOS�/�

mice still had more than 5 log10 CFU of bacteria in the liver,spleen, and skin (Fig. 2A).

Compared to the wild-type mice, the p47phox�/� mice hadhigher numbers of bacteria in the liver and spleen at all timesexamined (Fig. 2B). In contrast, the numbers of bacteria in theskin of the p47phox�/� mice did not differ from the numbers ofbacteria in the skin of the p47phox�/� mice during the first 7days of infection, but the numbers of bacteria were significantlyhigher on day 10 after inoculation.

We extended these experiments by monitoring the course ofinfection after inoculation of 5 � 104 CFU of F. tularensis LVS,a dose 10 times higher than the LD50 for p47phox�/� mice.Compared to liver samples of p47phox�/� mice, liver samples ofp47phox�/� 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 ofp47phox�/� mice, bacteria were present at detectable levelsduring the whole period, whereas in wild-type mice, bacteria

were found only in samples from the lungs and peritoneum andonly on day 3 (Table 1).

When in similar experiments iNOS�/� mice were intrader-mally inoculated with 5 � 104 CFU, numbers of bacteria thatwere significantly higher than the numbers in wild-type micewere found in samples from livers on days 6 and 9 and in skinsamples on day 3 and beyond (Table 2). In samples from theperitoneum, bacteria were detectable in iNOS�/� mice duringthe whole period, and in wild-type mice bacteria were detect-able only on day 3. In lung samples, bacteria were isolated fromiNOS�/� 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 fromiNOS�/� 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 ofinfection occurred, as shown by bacteremia and peritonitis.The iNOS�/� mice had increased numbers of bacteria in theskin even during the early phase and exhibited a loss of controlalso in the liver and spleen after the first week of infection. TheiNOS�/� mice were unable to eradicate the infection, and aftera protracted course of infection, they eventually succumbed toeven the lowest challenge dose.

Histological examination of livers and assay of s-amino-transferase levels of infected mice. Histological examination ofspleens, livers, and lungs from infected mice showed that themost pronounced differences were in livers, which became thefocus of the comparative analysis. Examinations were per-formed on days 3, 6, and 9 after inoculation of 5 � 104 F.tularensis LVS cells, a dose found to be high enough to induce

FIG. 3. After inoculation of 5 � 104 CFU of F. tularensis LVS, the serum liver enzymes s-ALT and s-AST were monitored in iNOS�/� andiNOS�/� mice (A) and in p47phox�/� and p47phox�/� mice (B). The means standard errors of the means for five mice per group and time areshown. An asterisk indicates that the P value is 0.05.



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pathological changes. During the 9-day period, histologicallesions developed in p47phox�/� and p47phox�/� mice (Table 3).Increased serum levels of s-ALT and s-AST, sensitive markersfor liver damage, were found in p47phox�/� mice on day 9 (Fig.3). The iNOS�/� mice showed even more severe histologicalchanges at all times (Table 3), and the enzyme levels on day 9were higher than those in any other group of mice (Fig. 3).Representative histological sections from day 6 are shown inFig. 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 leakyiNOS�/� and iNOS�/� mice after primary infection with F.tularensis. To obtain more information about the role of RNSin 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 beenshown 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 � 102 CFUof 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, thenumbers of bacteria were consistently 1 to 2 log10 higher iniNOS�/� mice than in iNOS�/� 129 mice (Fig. 5). In each offive separate experiments, the numbers of bacteria in the skinwere higher in iNOS�/� mice on days 4 to 7 of infection, andthe bacteria were eradicated within 3 weeks. In livers andspleens, there were no significant differences between the twostrains of mice (Fig. 5). After peak numbers of bacteria werereached on day 4, complete eradication occurred within 3weeks postinfection. The data indicate that low levels of NOare not sufficient for control of LVS infection in the skin,although they are sufficient for control of LVS infection in theliver and spleen.

Production of RNS and superoxide ex vivo by splenocytes ofmice infected with F. tularensis LVS. After intradermal injec-tion of 5 � 104 CFU of F. tularensis LVS (the same dose thatwas used in the histological examinations and liver enzyme andcytokine assays), mice were killed on days 3 to 9 in order todetermine the capacity of splenocytes to produce RNS andROS. The ROS assay was highly sensitive and allowed detec-tion of superoxide-dependent chemoluminescence after onlyminutes of incubation, whereas RNS had to be accumulated byin 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 � 104 CFU 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-sizeinflammatory 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 someseverity (in both cases the scores were 2, 2, and 2). Hematoxylin and eosin staining was used. Bars � 20 �m.



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tifiable amounts were present. Although these differences inthe assay time cannot be reliably translated to time of occur-rence in vivo, both assays were nonetheless thought to reflectthe in vivo ability of the splenocytes to produce the corre-sponding mediators.

Splenocytes harvested from infected iNOS�/� mice pro-duced significant levels of NO2

� only when they were obtainedon day 6 of infection or later (Fig. 6A). Similarly, splenocytesobtained from p47phox�/� and p47phox�/� mice started to pro-duce significant levels of NO2

� only when they were obtained�7 days after inoculation. On days 7 and 9, the levels weresignificantly higher for p47phox�/� mice than for p47phox�/�

mice. Splenocytes derived from iNOS�/� mice showed no pro-duction of NO2

� at any time.Splenocytes from wild-type (iNOS�/� and p47phox�/�) and

iNOS�/� mice harvested on day 3 produced ROS at levels thatwere significantly higher than the levels produced by spleno-cytes from noninfected mice (Fig. 6B). At this time, the level ofROS produced by cells from iNOS�/� mice was similar to thelevel 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 asthe levels produced by cells from iNOS�/� mice, and on day 9the levels were more than three times as high as the levelsproduced by cells from iNOS�/� mice (Fig. 6B). Splenocytesfrom p47phox�/� mice did not produce ROS at any time.

To investigate whether the increase in the capacity of

splenocytes from iNOS�/� mice to produce ROS was associ-ated with changes in the proportion of phagocytes in thespleen, differential counts were obtained on days 3, 6, and 9 ofinfection. Infection resulted in increased proportions of neu-trophils and monocytes in the spleens of all four strains of miceat all times compared to noninfected mice. However, nomarked differences were found between the gene-deficientmouse strains and the corresponding wild-type strains (Table4). Thus, the enhanced production in cultures from infectedmice compared with cultures from noninfected mice may havebeen related to the increased numbers of ROS-producing neu-trophils and monocytes in the former mice.

In conclusion, p47phox�/� mice seemed to compensate forthe lack of the phagocyte oxidase by increased expression ofNO2

�, and in contrast, iNOS�/� mice seemed to compensatefor the lack of iNOS by an increased ability to produce ROS.The increased ROS levels observed in the latter mice was likelyrelated to an enhanced cellular capability for ROS production,since there were no significant differences in the proportion ofphagocytes 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 � 104 CFU of F.tularensis LVS, blood samples were collected for an assay ofcytokines. Both p47phox�/� mice and iNOS�/� mice had muchhigher serum levels of IFN-� than the corresponding wild-typemice (Table 5). On day 10, the levels in the gene-deficient micewere �2,000 pg/ml, compared to 500 pg/ml in the wild-typestrains. Leaky iNOS�/� mice showed down-regulation ofIFN-� levels in the same manner as wild-type mice, and thelevel on day 10 was 200 pg/ml. In serum from noninfectedanimals, IFN-� was not detectable (concentration, 30 pg/ml).Based on the values for individual animals on days 3, 6, 9, and10, the levels of IFN-� showed a correlation coefficient of 0.5or higher with the numbers of bacteria in spleens and liversirrespective of the mouse strain.

Also, the IL-6 levels were generally higher in sera from theiNOS�/� and p47phox�/� mice than in the sera from the cor-responding wild-type mice (Table 6). The IL-12p40 levels were2,000 pg/ml in all serum samples and were not different ingene-deficient and wild-type mice. TNF-� and IL-4 were notdetected in any serum sample from any strain of mice.

In vitro recall response of splenocytes from iNOS�/� andiNOS�/� mice. To determine whether the high level of sus-ceptibility of the iNOS�/� mice was related to an impairedability to respond to F. tularensis antigen with T-cell prolifer-ation and IFN-� production, mice were primed by intradermalinoculation of a low dose of F. tularensis LVS, treated withmoxifloxacin on days 15 to 22, and killed on day 35 for prep-aration of splenocyte cultures. Naıve iNOS�/� and iNOS�/�

mice showed low or nonsignificant proliferative responses invitro to heat-killed F. tularensis (stimulatory indices, �2.4),whereas primed mice from both groups displayed a strongresponse, with stimulatory indices of �10.5 (Table 7). In re-sponse to heat-killed F. tularensis, splenocytes from immuneiNOS�/� mice secreted much higher levels of IFN-� than cellsfrom primed iNOS�/� mice secreted (Table 7). Irrespective ofimmunization, the spleen cells showed vigorous proliferationand a high level of secretion of IFN-� in response to the T-cellmitogen concanavalin A. Thus, the immune iNOS�/� mice

FIG. 5. Growth curves for F. tularensis LVS in the skin, livers, andspleens of leaky iNOS�/� mice and iNOS�/� 129 mice after primaryintradermal infection with 5 � 102 F. tularensis LVS cells. The means standard errors of the means for five mice per group and time areshown. One asterisk indicates that the P value is 0.05, and threeasterisks indicate that the P value is 0.001.



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showed recall responses that were as strong as those of im-mune iNOS�/� mice and produced much higher levels of theTh1 cytokine IFN-� than immune iNOS�/� mice produced.

DISCUSSION

F. tularensis is a potent pathogen, and the principal virulencemechanism seems to be intracellular survival. As observed forother facultative intracellular bacteria, the mechanisms of hostprotection are critically dependent on cell-mediated immunity(30) (i.e., the activation of macrophages by T cells to killintracellularly located bacteria). The T-cell-dependent activa-tion requires the involvement of IFN-�, and previous in vitrostudies demonstrated that a release of nitric oxide by the mac-rophages might contribute to killing (1, 15). By usingp47phox�/� and iNOS�/� mice, we demonstrated here thatboth ROS and RNS play an essential role in vivo for thecontrol of murine tularemia. Mutant mice were significantly

more susceptible to death caused by F. tularensis LVS. TheLD50 for the iNOS�/� mice was 20 CFU, the LD50 for thep47phox�/� mice was 4,400 CFU, and the LD50 for the wild-type mice was �500,000 CFU. In p47phox�/� mice the infectionwas 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 after26.4 days of infection (mean value) and there was severe liverdamage, whereas in p47phox�/� mice the disease was moreacute, the mean time to death was 10.1 days, and there was lesspronounced liver damage.

Thus, our results indicate that ROS have a critical roleduring the first few days of infection, whereas RNS seemed tobe critical at a subsequent phase. Few previous studies haveassessed the role of ROS in the control of tularemia. One studydemonstrated 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 inoculatedwith 5 � 104 CFU of F. tularensis LVS. On different days after inoculation mice were sacrificed, and splenocytes were prepared and analyzed forNO production (A) and ROS production (B). The values are the means standard errors of the means for three mice per group and time. Sampleswere 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, twoasterisks indicate that the P value is 0.01, and three asterisks indicate that the P value is 0.001. RLU, relative light units.



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analyze if this production correlated with a bactericidal effect(17). The high level of susceptibility of p47phox�/� mice may berelated to a lack of macrophage- and neutrophil-mediatedbactericidal mechanisms. Neutrophils have been shown to playa crucial role in the control of murine tularemia (26), andhuman 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 peroxideand chloride anions and is thus not formed in neutrophils fromp47phox�/� mice. Although killing of F. tularensis by murineneutrophils has not been demonstrated, the important role ofthese cells in the control of infection may be one explanationfor why in the present experiments p47phox�/� mice showedsuch a high level of susceptibility to tularemia. During theinterval from day 4 to 7, when exacerbation was observed inp47phox�/� 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 duringthis stage. Collectively, our data indicate that ROS have animportant role in control of the early phase of the F. tularensisinfection when innate immune mechanisms are operative.However, since small challenge inocula were controlled inp47phox�/� mice, ROS-independent mechanisms are appar-ently operative as well.

In contrast to the role of ROS, the absence of RNS did notaffect the numbers of bacteria in livers and spleens during thefirst week of infection. After this, the numbers of bacteria

increased in the iNOS�/� mice, which developed severe liverpathology and showed high serum levels of liver-specific en-zymes and IFN-�. Together, these results indicate that RNShave both immunoregulatory and bactericidal effects. Besidestheir lack of possible NO-mediated antimicrobial mechanisms,the iNOS�/� mice may be afflicted with dysregulation of theimmune response, leading to liver damage and persistent, highserum levels of IFN-�. This is in line with extensive evidenceconcerning the important immunoregulatory role of NO (5, 6,9). Since iNOS�/� mice with a leaky phenotype displayed nomeasurable serum levels of NO but showed no signs of exac-erbation of systemic infection, no liver damage, and normalserum levels of IFN-� on day 10, such regulatory mechanismsmight depend on the presence of very low serum levels of NO.Previous studies have demonstrated that immune mice areeffectively protected by a number of overlapping, compensa-tory mechanisms (11, 14, 26).

The role of RNS in killing of F. tularensis in skin was evidenteven during the early phase of infection in both iNOS�/� miceand leaky iNOS�/� mice and persisted throughout the courseof infection. Thus, in skin, low levels of NO did not appear tobe sufficient for host control of an F. tularensis infection. Theskin is an important barrier for controlling both murine andhuman 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 type Mouse strainNo. of cells (�106) in spleens on:

Day 0a,b Day 3c Day 6c Day 9c

Neutrophils p47phox�/� 0.3 (1.1) 7.8 0.6 (14) 14.9 2.9 (18) 6.4 2.2 (7.3)p47phox�/� 0.3 (1.1) 4.9 2.7 (9.8) 12.0 2.7 (16) 10.3 1.9 (14)iNOS�/� 0.6 (2.0) 13.5 2.3 (17) 12.6 4.6 (15) 6.7 4.3 (6.6)iNOS�/� 0.6 (2.0) 14.9 2.9 (21) 5.9 2.9 (9.3) 11.0 0.9 (15)

Monocytes p47phox�/� 0 1.7 0.3 (3.0) 4.8 1.6 (5.8) 1.0 0.6 (1.1)p47phox�/� 0 0.5 0.3 (1.0) 4.8 1.3 (6.5) 1.0 0.8 (1.4)iNOS�/� 0 1.9 0.6 (2.4) 4.2 0 (5.0) 3.3 1.5 (3.2)iNOS�/� 0 2.1 1.1 (2.9) 2.6 1.6 (4.1) 3.7 1.0 (5.0)

a The intradermal inoculum was 5 � 104 CFU of F. tularensis LVS per mouse.b The 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.c The 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 strainIFN-� concn in serum (pg/ml)

Day 0b Day 3c Day 6c Day 9c Day 10c

p47phox�/� 30 1,070 390 4,340 2,590 220 25 280 15p47phox�/� 30 2,088 418d 6,190 2,510 3,600 640 C 2,010 75 BiNOS�/� 30 1,530 620 3,243 1,641 NDe 30iNOS�/� 30 2,790 1,500 12,080 575 A 6,490 1,220 2,020 710 AiNOS�/� 129 30 1,190 440 1,650 240 ND 220 120Leaky iNOS�/� 30 1,530 525 4,000 B ND 150 45

a The intradermal inoculum was 5 � 104 CFU of F. tularensis LVS per mouse.b IFN-� serum levels before inoculation of F. tularensis LVS.c Means standard errors of the means for three samples.d Significance 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.e ND, not determined.



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tularemia even with the smallest challenge inocula (18, 27). Inthe present study, a paradoxical relationship between the se-rum levels of IFN-� and the numbers of bacteria in the liverand 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 inthe absence of complementary effector molecules generateddirectly or indirectly by iNOS and phox. The present data andprevious studies (14, 18, 27) together show that althoughIFN-� is absolutely required in vivo for control of tularemia,high, excessive levels of the cytokine do not necessarily lead tomore effective killing of F. tularensis.

The host protective mechanisms for murine tularemia seemto resemble those that are operative in other experimentalmodels of intracellular infections. For example, in cutaneousleishmaniasis, RNS were needed for killing of the parasite inthe skin and draining lymph nodes, whereas the activity of phoxwas dispensable for resolution of the acute skin infection butessential for clearance of the parasites in the spleen (3). Re-sistance to virulent Salmonella enterica biovar Typhimurium inthe mouse model was dependent on ROS, because phox-defi-cient mice showed dramatic exacerbation and succumbed toinfection within 5 days. In contrast, iNOS�/� mice containedincreased numbers of bacteria only after the first week ofinfection (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 tobe 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 earlyphase of infection, at a stage when a lack of NO resulted in nosystemic exacerbation. However, iNOS�/� mice subsequentlysuccumbed to the infection, and they succumbed to even thelowest dose. The extreme susceptibility of iNOS�/� mice mightbe in line with a complex regulatory role for NO in intracellularinfections, and death may be an effect of dysregulation of thecytokine response and resulting severe liver damage.

ACKNOWLEDGMENTS

Grant support was obtained from the Swedish Medical ResearchCouncil, Samverkansnamnden, Norra Sjukvardsregionen, Umea, Swe-den, and the Medical Faculty, Umea University, Umea, Sweden.

We thank Steven Holland for supplying the p47phox�/� mice andF. Y. Liew for supplying leaky iNOS�/� mice.

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TABLE 6. IL-6 levels in sera of mice infected with F. tularensis LVSa

Mouse strainIL-6 concn in serum (pg/ml)

Day 0b Day 3c Day 6c Day 9c Day 10c

p47phox�/� 15 419 137 115 50 52 10 NDd

p47phox�/� 15 1,260 588 Ae 1,007 891 203 25 A NDiNOS�/� 15 522 145 140 81 57 11 102 9iNOS�/� 15 625 43 1450 86 B 1,480 420 B 684 5 C

a The intradermal inoculum was 5 � 104 CFU of F. tularensis LVS per mouse.b IL-6 serum levels before inoculation of F. tularensis LVS.c Means standard errors of the means for three samples.d ND, not determined.e Significance 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 insplenocytes from naıve 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�/�

Proliferativeresponse (SI)c

10.5 2.7 12.3 1.5 2.4 0.5 1.9 0.2

IFN-� (ng/ml) 7.5 0.4 24.5 2.1 0.03 0.03

a To estimate the proliferative response, spleen cell cultures were stimulatedfor 4 days with 106 heat-killed cells of F. tularensis LVS and pulsed with [3H]thy-midine. Cytokine levels were determined in cell supernatants after 2 days ofstimulation. The values are means standard errors of the means for fivecultures.

b Mice were subjected 5 weeks previously to intradermal infection with 5 � 103

CFU of F. tularensis LVS and given moxifloxacin on days 15 to 22.c Stimulation index (SI) � mean counts per minute for five cultures containing

antigen/mean counts per minute for five cultures lacking antigen. Stimulationwith concanavalin A resulted in a stimulation index of �28.7. Cultures withoutantigen gave 1,400 to 1,800 cpm.



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Editor: A. D. O’Brien



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