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The Journal of Immunology, 2008, 180, 7574-7581
Copyright © 2008 by The American Association of Immunologists, Inc.
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Administration of a Synthetic TLR4 Agonist Protects Mice from Pneumonic Tularemia1

Annalisa Lembo*, Mark Pelletier*, Ravi Iyer*, Michele Timko*, Jan C. Dudda{ddagger}, T. Eoin West*, Christopher B. Wilson{dagger}, Adeline M. Hajjar{dagger} and Shawn J. Skerrett2,*

* Department of Medicine and {dagger} Department of Immunology, University of Washington, Seattle, WA 98104; and {ddagger} Benaroya Research Institute, Seattle, WA 98101


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Francisella tularensis is a Gram-negative intracellular pathogen that causes the zoonosis tularemia. Because F. tularensis LPS causes weak TLR4 activation, we hypothesized that administration of a synthetic TLR4 agonist, aminoalkyl glucosaminide phosphate (AGP), would boost the innate immune system and compensate for reduced TLR4 stimulation. Intranasal administration of AGPs induced intrapulmonary production of proinflammatory cytokines and chemokines. Mice treated with AGPs before and after inhalation of Francisella novicida exhibited augmented cytokine and inflammatory responses to infection; reduced bacterial replication in lung, liver, and spleen; and increased survival, whereas all PBS-treated control mice died within 4 days of infection, all AGP-treated mice showed prolonged time-to-death, and 30–60% of AGP-treated mice survived. The protective effect of AGP was lost in mice lacking IFN-{gamma}. Long-term survivors developed specific Th1 splenocyte responses and specific Abs dominated by IgG2 isotypes. Survivors were fully protected from rechallenge with aerosolized F. novicida. Thus, preventive administration of AGP successfully modulated innate immune responses to aerosolized F. novicida, leading to protective immunity to pneumonic tularemia. This is the first report of the protective effect of a TLR ligand on resistance to F. novicida-induced pneumonic tularemia.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Francisella tularensis is a Gram-negative, facultative intracellular pathogen that causes the zoonotic infection tularemia. The high infectivity and lethality of airborne infection with F. tularensis have fostered development of this agent as a biological weapon (1). Heightened concern regarding the potential hostile use of aerosolized F. tularensis has intensified the interest of the scientific community in understanding the pathogenesis of this infection and in identifying novel ways to boost resistance to the disease.

There are four closely related subspecies of F. tularensis that differ in geographic distribution and in host range (1, 2, 3, 4). F. tularensis tularensis (type A) is the most virulent for humans and is largely confined to North America (2). F. tularensis holarctica is endemic in the Northern Hemisphere, where it causes a milder (type B) form of human disease (2, 3). The live vaccine strain (LVS)3 derived from Francisella holarctica is attenuated in humans, but virulent in rodents (1). F. tularensis mediasiatica, localized to central Asia, and F. tularensis novicida, found predominantly in North America, are rare human pathogens (3, 5). However, Francisella novicida is highly virulent in mice, inducing a disease that mimics type A human tularemia (5).

The pathogenicity of F. tularensis is linked to its capacity to evade or suppress the activation of mammalian defenses as it gains access to and replicates in its intracellular niche (5, 6). The ability of F. tularensis to escape early detection by the host is facilitated by an unusual LPS structure that is poorly recognized by LPS-binding protein and by TLR4 (7, 8, 9). As a result, F. tularensis LPS is a weak stimulus of innate immune responses in vitro and in vivo (7, 8, 9, 10, 11, 12). Consistent with a lack of TLR4-mediated recognition of F. tularensis, TLR4 has been found to play little or no role in resistance to airborne infection with type A F. tularensis tularensis (13) or intradermal infection with F. tularensis holarctica LVS (14). In contrast, F. novicida mutants that express more stimulatory lipid A structures exhibit attenuated virulence in mice (15, 16).

We hypothesized that administration of an exogenous TLR4 ligand would boost the innate immune response to infection with F. novicida. To pursue this hypothesis, we tested the effects of aminoalkyl glucosaminide phosphates (AGPs), which are synthetic lipid A mimetics that require both TLR4 and MD-2 for recognition (17, 18, 19). AGPs have been shown to induce protection against influenza virus and Listeria monocytogenes infection in wild-type mice, but not in TLR4-deficient mice (20). We found that AGPs are potent activators of alveolar macrophages and dendritic cells in vitro, and that intranasal (i.n.) delivery of AGPs protected 30–60% of mice from lethal airborne infection with F. novicida. This effect required IFN-{gamma} and was associated with augmented innate immune responses. The survivors developed specific cellular and humoral immunity to F. novicida and were fully protected from secondary aerosol challenge with the same bacterium.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Synthetic lipid A mimetics

AGPs CRX 524 and CRX 527 were provided by Corixa (19, 21), which is now GlaxoSmithKline Biologicals North America (22). The synthesis and general structure of AGPs have been described previously (18, 20). AGPs are a class of lipid A mimetics composed of a monosaccharide unit with an N-acylated aminoalkyl aglycon spacer arm (20). AGP 524 and 527 differ in the R4 functional group of the aglycon moiety: a hydrogen atom for 524 and a carboxyl residue in the 527 (20). AGPs were diluted in PBS for all experiments. For in vivo administration, mice were anesthetized with isoflurane, and AGPs (20 or 40 µg diluted in 44 µl PBS/mouse/each administration) or PBS diluent (44 µl PBS/mouse/each administration) were gradually deposited in the nares using a micropipettor.

Bacteria

F. novicida U112 strain was provided by T. Guina (University of Washington, Seattle, WA) (23) and was originally obtained from F. Nano (University of Victoria, British Columbia, Canada) (24). A stock was prepared from overnight growth in trypticase soy broth (Life Technologies) supplemented with 0.1% L-cysteine (Sigma-Aldrich) at 37°C with aeration. Bacteria were harvested in stationary phase, diluted in 20% glycerol, aliquoted, and stored at –80°C.

Mice

Male and female wild-type and IFN-{gamma}–/– on C57BL/6 background mice were obtained from The Jackson Laboratory at 6–8 wk of age. TLR4–/– mice were originally obtained from S. Akira (Osaka University, Suita, Japan) (25) and backcrossed at least eight generations onto a C57BL/6 background. Animals were housed in a high-efficiency particulate air-filtered, laminar flow cage rack and permitted unlimited access to sterile food and water. All experiments were approved by the University of Washington Institutional Animal Care and Use Committee.

Macrophages and dendritic cell cultures

MH-S cells, an SV40-transfected alveolar macrophage cell line derived from BALB/c mice, were purchased from American Type Culture Collection. MH-S cells were seeded in 96-well plates (7 x 104 cells/well) in RPMI 1640 (Life Technologies) supplemented with 10% FCS (HyClone), L-glutamine (2 mM), and HEPES (25 mM) (complete medium). The cells were stimulated with AGP 524 or 527 (from 0.1 to 100 ng/ml). After 24 h, supernatants were harvested and stored at –80°C for measurement of IL-6 and TNF-{alpha}. Bone marrow-derived dendritic cells (BMDC) were prepared, as previously described (26), with some modifications. Briefly, bone marrow cells were flushed from the femurs with a needle and syringe, then washed in complete medium and counted. Six million bone marrow cells were plated in 10-cm petri dishes (Falcon) in 10 ml of complete medium supplemented with murine 40 ng/ml rGM-CSF (PeproTech) plus 10 ng/ml murine rIL-4 (PeproTech). On day 3, 10 ml of fresh medium containing 40 ng/ml GM-CSF was added. On days 5 and 7, 10 ml of medium was replaced with fresh complete medium supplemented with GM-CSF. BMDC were used on day 7, at which point >90% of cells were CD11c+ and MHC class II I-Ab low, measured by FACScan flow cytometer (BD Biosciences), using Abs purchased from BD Pharmingen. BMDC were transferred to 96-well plates (7 x 104 cells in 200 µl medium/well) and stimulated with AGP 524 or 527 (from 0.1 to 100 ng/ml). Supernatants were collected after 24 h and stored at –80°C for measurement of IL-12p40 and IL-12p70.

Exposure to aerosolized F. novicida

Mice were exposed to aerosolized bacteria in a nose-only exposure chamber (In-Tox). Bacterial suspensions of 5 x 108 CFU/ml from glycerol stock were aerosolized using Uni-Heart nebulizers (Westmed) driven at a pressure head of 40 psi to a flow rate of nebulizer of 1 L/min. Airflow through the chamber was maintained at 5 L/min. After a 10-min exposure to aerosolized bacteria, the chamber was purged with air for 5 min. To determine bacterial deposition in each experiment, three mice were anesthetized with pentobarbital sodium and exanguinated by cardiac puncture immediately after exposure to aerosolized bacteria. The left lungs were harvested, homogenized in PBS, serially diluted in the same buffer, and quantitatively cultured in duplicate by spreading 0.1-ml aliquots on trypticase soy agar supplemented with 0.1% L-cysteine. Colonies were counted after 72-h incubation at 37°C.

Bronchoalveolar lavage and organ tissue harvest

At indicated time points after administration of AGPs and/or exposure to aerosolized bacteria, mice were euthanized for harvest of bronchoalveolar lavage fluid and organs, as described (27). The trachea of each mouse was exposed and cannulated with a 20-gauge polyethylene catheter, the left lung was harvested, and the right lung was lavaged four times with 0.5-ml vol of 0.9% sodium chloride containing 0.9% mM EDTA and prewarmed to 37°C. Bronchoalveolar lavage specimens were centrifuged at 300 x g, and the cell pellets were resuspended in RPMI 1640 medium containing 10% heat-inactivated FCS. Cell counts were performed with a hemacytometer, and differential counts were determined from cytocentrifuged specimens prepared with a modified Wright-Giemsa stain (Diff-Quik; Dade Behring). Left lungs, livers, and spleens were homogenized and serially diluted in PBS, then quantitatively cultured on trypticase soy agar supplemented with 0.1% L-cysteine. For cytokine measurements, lung homogenates were diluted 1/1 in lysis buffer (150 mM NaCl/15 mM Tris/1 mM MgCl2/1 mM CaCl2/1% Triton X-100), then incubated for 30 min on ice. The lysates were centrifuged at 1500 x g for 15 min at 4°C, and the supernatants were stored at –80°C.

Splenocyte stimulation

Mice that survived F. novicida infection were euthanized 20 days after infection. Spleens were collected and pressed through a strainer (0.7 µm FALCON) to prepare single-cell suspensions. RBC were lysed with lysing buffer (BD PharM Lyse). Splenocytes were washed and plated in 96-well U-bottom plates at concentration of 6.6 x 106 cells/ml in DMEM medium/10% FCS/1% HEPES/1% L-glutamine. Cells were stimulated with either control medium or PMA (50 ng/ml) plus ionomycin (2 µM) or heat-killed F. novicida (multiplicity of infection 100). After 48 h, plates were centrifuged and supernatants were collected and stored at –80°C for IFN-{gamma} measurement by ELISA.

Measurement of cytokines

MIP-2, TNF-{alpha}, IFN-{gamma}, IL-6, IL-12p40, and IL-12p70 in culture supernatants or lung homogenates were measured by ELISA using DuoSet reagents purchased from R&D Systems, according to the manufacturer’s instructions.

Measurement of Ab to F. novicida

Sera were harvested from surviving mice 20 days after infection with F. novicida and from uninfected controls. Specific Abs to F. novicida were detected by ELISA (56). To prepare Ag, 120 ml of 1 x 1011 CFU/ml F. novicida U112 in sterile PBS was lysed with 3 ml of QIAamp ATL lysis buffer (Qiagen) and diluted 10-fold, and 100 µl was applied to 96-well Nunc-Immuno Maxisorp plates (Nalge Nunc International) at 4°C overnight. Plates were washed three times with wash buffer (0.05% Tween 20 (Fisher Scientific) in PBS) and blocked with 1% BSA in PBS. After repeat washing, serially diluted sera from infected and uninfected animals were added to the plates for 2 h. After repeat washing, goat anti-mouse Igs conjugated to biotin (IgG2a, IgG2b, IgG1, IgA; Southern Biotechnology Associates) or streptavidin (IgG2c; Bethyl Laboratories) were added for 1 h. Streptavidin-HRP was added for 20 min on the biotin-conjugated Abs. Color development was obtained by adding substrate solution from a DuoSet ELISA kit (R&D Systems), and the reaction was quenched with 1 M phosphoric acid. Plates were read at 405 nm using 570 nm for correction. A positive Ab titer was defined as an OD of infected serum that was greater than 0.05 and twice the mean OD of uninfected serum from three mice at a comparable dilution. The endpoint titer was defined as the lowest dilution of serum that gave an OD at 405 nm that was 2-fold greater than the value of the matched dilution of uninfected mouse serum (28).

Statistical analysis

The Mann-Whitney U test was performed on differences between cytokine levels, numbers of cells, and CFU/organ between treatment groups. Survival analyses were performed using a log rank test. A p value of <0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
AGPs induced proinflammatory cytokine release by alveolar macrophages and dendritic cells

To test the effects of AGP stimulation on alveolar macrophages and dendritic cells in vitro, we incubated MH-S cells and BMDC with AGP 527 or AGP 524, then harvested supernatants for cytokine analysis (Fig. 1). Alveolar macrophages produced proinflammatory cytokines IL-6 and TNF-{alpha}, and BMDC generated IL-12p40 when stimulated with either AGP 527 or AGP 524. AGP 527 was a more potent stimulus than AGP 524, inducing greater cytokine secretion at each concentration tested. Neither MH-S cells nor BMDC produced IL-12p70 after stimulation with AGPs (data not shown).


Figure 1
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  		FIGURE 1. AGP 527 and AGP 524 induced IL-6 and TNF-{alpha} production by MH-S cells and IL-12p40 production by BMDC. MH-S cells (A and B) were stimulated with AGP 527 or AGP 524 (from 0.1 to 100 ng/ml). After 24 h, supernatants were collected and assayed by ELISA for IL-6 (A) and TNF-{alpha} (B). The threshold of detection was 15 pg/ml for both cytokines. Data are mean ± SD of triplicate wells and are representative of three separate experiments. Supernatants of control cells stimulated with medium only contained 15 pg/ml IL-6 and 22 pg/ml TNF-{alpha}. BMDC (C) were stimulated with AGP 527 or AGP 524 (from 0.1 to 100 ng/ml). After 24 h, supernatants were collected and IL-12p40 was measured by ELISA. Supernatants of control BMDC stimulated with medium only contained 388 ± 68.7 pg/ml IL-12p40. The lower limit of detection level was 30 pg/ml. Data are mean ± SD of triplicate wells. Results are representative of two separate experiments.

 
Intranasal administration of AGP 527 induced lung inflammation

To test the effects of AGPs in vivo, we measured intrapulmonary chemokine, cytokine, and inflammatory responses after i.n. administration of AGP 527 or PBS (Fig. 2). AGP 527 induced high lung levels of MIP-2, TNF-{alpha}, and IFN-{gamma} within 24 h that persisted for at least 48 h after AGP administration. IL-12p70 also was induced by AGP 527, but with delayed kinetics; IL-12p70 concentrations in lung homogenates were significantly increased at 48 h, but not 24 h, after AGP treatment, whereas IL-12p40 was increased at 24 h (data not shown). In concert with the chemokine and cytokine responses, AGP 527 elicited neutrophilic lung inflammation that persisted for at least 48 h (Fig. 3). There was no significant change in the number of bronchoalveolar mononuclear cells after AGP administration. To confirm that the proinflammatory effects of AGP were mediated by TLR4, we compared the responses of wild-type and TLR4–/– mice to i.n. administration of AGP 527 (20 µg). In contrast to wild-type animals, no recruitment of PMN was observed 24 h after administration of AGP 527 in TLR4–/– mice (data not shown).


Figure 2
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  		FIGURE 2. Intranasal administration of AGP 527 induced intrapulmonary production of cytokines. Mice were administered 44 µl of PBS or AGP 527 (20 µg in 44 µl of PBS) i.n. After 24 and 48 h, mice were euthanized, and the left lung of each mouse was homogenized for measurement of MIP-2 (A), TNF-{alpha} (B), IFN-{gamma} (C), and IL-12p70 (D) by ELISA. The lower detection limit was 15 pg/ml for MIP-2, 23 pg/ml for IL-12p70, and 30 pg/ml for the other cytokines. The results are from a representative experiment of two performed. Each symbol represents the cytokine induced by one mouse. Bars indicate median values. *, p < 0.05.

 

Figure 3
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  		FIGURE 3. Intranasal administration of AGP 527 induced recruitment of inflammatory cells in bronchoalveolar lavage fluid (BAL). Mice were administered 44 µl of PBS or AGP 527 (20 µg in 44 µl of PBS) i.n. After 24 and 48 h, mice were euthanized and bronchoalveolar lavage (BAL) was performed on the right lung for enumeration of total cells (A), mononuclear cells (B), and neutrophils (PMN, C). Each symbol represents one mouse. Bars indicate median values. *, p < 0.05.

 
AGP administration augmented early cytokine responses to inhaled F. novicida

To determine whether AGP treatment could augment the innate immune response to F. novicida in vivo, we administered i.n. AGP 527 or PBS 48 h before exposure to aerosolized bacteria. Four hours after infection (Fig. 4), high levels of MIP-2, TNF-{alpha}, and IFN-{gamma} were detected in the lungs of AGP 527-treated mice, whereas in PBS-treated controls the levels of these cytokines were barely detectable (Fig. 4). By 24 h after infection, the intrapulmonary concentrations of MIP-2, TNF-{alpha}, and IFN-{gamma} did not differ significantly between the two treatment groups. To determine whether the effects of AGP administration on cytokine induction were additive or synergistic with responses elicited by infection, we treated mice i.n. with AGP 527 (20 µg) or PBS and 48 h later exposed these animals to aerosolized PBS or F. novicida. Four hours and 24 h after aerosol exposure, intrapulmonary levels of MIP-2, TNF-{alpha}, IL-12p70, and IFN-{gamma} were significantly higher in mice treated with AGP than in PBS controls, but the levels of these cytokines did not differ between AGP-treated mice that were exposed to aerosolized F. novicida vs aerosolized PBS (data not shown). Thus, AGP administration induced cytokine responses independently of signals activated by F. novicida.


Figure 4
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  		FIGURE 4. Intranasal administration of AGP 527 augmented intrapulmonary cytokine responses after inhalation of F. novicida. A total of 44 µl of PBS or AGP 527 (20 µg/44 µl PBS) was administered i.n. to each mouse 48 h before exposure to aerosolized F. novicida (deposition = 88 ± 39 CFU/lung). Left lungs were harvested 4 and 24 h after infection for measurement of MIP-2 (A), TNF-{alpha} (B), and IFN-{gamma} (C) by ELISA. Each symbol represents the cytokine level of one mouse. Bars indicate median values. Data are representative of two experiments. *, p < 0.05.

 
AGP administration reduced organ burden of bacteria after inhalation of F. novicida

To determine whether AGP administration affected bacterial clearance after exposure to aerosolized F. novicida, we administered AGP 527 48 h before and 24 h after infection, then measured the CFU in lung, liver, and spleen 72 h after infection. AGP treatment resulted in significantly reduced bacterial burdens in each organ compared with PBS-treated controls (Fig. 5).


Figure 5
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  		FIGURE 5. AGP 527 administration prior and after inhalation of F. novicida reduced organ burden of bacteria. Mice were treated with PBS or AGP 527 (20 µg/mouse) i.n. 2 days before and 1 day after exposure to aerosolized F. novicida (deposition = 98 ± 43 CFU/lung). Mice were euthanized 72 h after infection. Lungs (A), livers (B), and spleens (C) were harvested and homogenized in PBS. Serial 10-fold dilutions of these homogenates were quantitatively cultured by spreading aliquots onto trypticase soy agar supplemented with 0.1% L-cysteine. Colonies were counted after 3 days of incubation at 37°C, and total CFU/organ was calculated. Each symbol represents one mouse. Bars indicate medians. {dagger}, p < 0.001; *, p < 0.02. The results combine data from two independent experiments.

 
AGP treatment increased survival from pneumonic tularemia

To determine the effect of AGP treatment on survival from pneumonic tularemia, groups of mice were treated i.n. with PBS, AGP 527 (20 or 40 µg), or AGP 524 (20 or 40 µg) 48 h before and 24 h after exposure to aerosolized F. novicida. All PBS-treated mice died within 4 days after infection. In contrast, survival was prolonged significantly in AGP-treated mice (Fig. 6). AGP 527 had similar efficacy at both doses tested (20 and 40 µg), with 30–40% of treated mice showing no signs of illness when the experiments were censored at 20 days after infection. However, long-term survival after administration of AGP 524 was observed only at the 20 µg dose.


Figure 6
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  		FIGURE 6. AGP administration before and after infection augmented survival from pneumonic tularemia. Mice were treated i.n. with AGP 527 (A, 20 and 40 µg), AGP 524 (B, 20 or 40 µg), or PBS (A and B), 2 days before and 1 day after aerosol infection with F. novicida (deposition = 89 CFU/lung). Data from a single experiment shown in divided panels for clarity. Survival was recorded daily over a period of 20 days. Survival analysis was performed using a log rank test. n = 9 for 20 µg of AGP 524; n = 8 for 40 µg of AGP 524; n = 10 in all the remaining groups. *, p < 0.0001 vs PBS and p < 0.05 vs 40 µg of AGP 524.

 
Pretreatment with AGP was required for protection against pneumonic tularemia

To determine whether pretreatment with AGP was required for a protective effect against pneumonia tularemia, groups of mice were treated with AGP 527 or PBS, exclusively before or exclusively after exposure to aerosolized F. novicida. Mice pretreated with a single preinfection dose of AGP exhibited 40% survival. In contrast, survival of mice treated with a single postinfection dose of AGP did not differ from that of PBS-treated controls (Fig. 7). Thus, treatment with AGP before infection is required to elicit a protective response to aerosolized F. novicida.


Figure 7
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  		FIGURE 7. Pretreatment is required for the protective effect of AGP. Mice were treated i.n. with 20 µg of AGP 527 2 days before or 1 day after aerosol infection with F. novicida (depsosition = 144 CFU/lung). Mice treated with PBS 2 days before infection were used as control. Survival was recorded daily over a period of 20 days. PBS-treated mice (n = 4) and mice treated with AGP only after the infection (n = 8) died at day 4 after infection, whereas 40% of mice treated with AGP 2 days before the infection (n = 8) survived. Survival analysis was performed using a log rank test. *, p < 0.0001 vs PBS or postinf. AGP.

 
AGP requires IFN-{gamma} to induce protection against pneumonic tularemia

To determine whether IFN-{gamma} production was necessary for AGP-induced protection against F. novicida infection, wild-type mice and IFN-{gamma}–/– mice were treated with i.n. PBS or AGP 527 (20 µg) before and after infection. PBS-treated IFN-{gamma}–/– mice died within 48 h after infection, whereas PBS-treated wild-type mice died 4 days after infection (Fig. 8). AGP-treated IFN-{gamma}–/– mice died 3 days after infection, and 50% survival was observed in AGP-treated wild-type mice. Thus, IFN-{gamma} contributes to the limited innate resistance of mice to pneumonic tularemia, and is required to express the protective effect induced by treatment with AGP.


Figure 8
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  		FIGURE 8. IFN-{gamma} required for AGP-induced survival of pneumonic tularemia. Wild-type and IFN-{gamma}–/– mice were treated i.n. with 20 µg of AGP 527 or PBS 2 days before and 1 day after aerosol infection with F. novicida (deposition = 177 CFU/lung; n = 8 mice/group). Survival was documented daily for 60 days after infection. IFN-{gamma}–/– mice died more rapidly than comparably treated wild-type mice, and AGP treatment extended survival of IFN-{gamma}–/– mice by only 1 day. Survival analysis was performed using a log rank test. All groups are significantly different from one another, with a p ≤ 0.0001.

 
AGP-treated survivors of pneumonic tularemia developed adaptive immune responses

AGP-treated mice that survived primary infection with F. novicida were sacrificed 20 days after infection to measure specific immune responses. To determine humoral responses, the titer of F. novicida-specific Ab in the serum was tested by ELISA, using lysed F. novicida as Ag. All surviving mice treated with AGP 527 (20 µg) showed high levels of F. novicida-specific IgG and small levels of IgA (Fig. 9). Similar results were obtained with sera from infected mice treated with AGP 524 (20 µg) (data not shown). We investigated which subclasses of IgG were present in the sera of AGP 527-treated mice and found a marked prevalence of IgG2 subsets over IgG1. To measure cell-mediated immunity, spleen cells from surviving and uninfected control mice were tested for IFN-{gamma} production in response to heat-killed F. novicida (Fig. 10). Splenocytes from all AGP-treated mice that survived infection produced high levels of IFN-{gamma} in response to F. novicida Ag, in contrast to cells from naive mice. Low detectable levels of IL-4 were measured in the splenocytes of AGP-treated and infected mice compared with uninfected mice (data not shown).


Figure 9
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  		FIGURE 9. Survived mice developed specific anti-F. novicida Abs in serum. Sera were harvested at day 20 from mice treated with AGP 527 (20 µg) before and after aerosol infection and that survived from F. novicida infection. Sera from uninfected mice were used as control. Each symbol represents the titer of one mouse. A positive titer was defined as the dilution of which the OD405 was >0.05 and was twice that of uninfected mice at the same dilution (28 ).

 

Figure 10
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  		FIGURE 10. AGP-treated mice developed specific anti-F. novicida IFN-{gamma}-producing cells. Splenocytes were harvested from mice that survived airborne F. novicida infection 20 days previously and were stimulated in vitro for 48 h with medium, PMA/ionomycin, or heat-killed F. novicida. IFN-{gamma} was measured in the supernatants by ELISA. Lower detection limit was 30 pg/ml. Each symbol represents one mouse. The data of each mouse were the mean cytokine levels of three wells. Bars represent median values. *, p < 0.05

 
AGP-treated survivors of pneumonic tularemia were protected against airborne rechallenge with F. novicida

To determine whether AGP-facilitated survival from F. novicida pneumonia resulted in lasting immunity, surviving mice were rechallenged with aerosolized F. novicida 60 days after the primary infection. All six mice treated with AGP both before and after primary infection and three of four mice treated with AGP only before primary infection survived the secondary challenge, in comparison with zero of four naive mice (Fig. 11).


Figure 11
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  		FIGURE 11. AGP-treated mice surviving pneumonic tularemia are protected against rechallenge with F. novicida. Mice that survived airborne infections with F. novicida (deposition = 177 ± 118 or 144 ± 20 CFU/lung) after i.n. treatment with 20 µg of AGP 527 either before and after infection (n = 6) or only before infection (n = 4) were rechallenged 60 days later with aerosolized F. novicida (deposition = 87.6 ± 40 CFU/lung). Naive mice were used as controls (n = 4). Survival analysis was performed using a log rank test. *, p < 0.05 vs naive.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
These studies yielded three major findings. First, i.n. administration of synthetic TLR4 agonists protected mice against lethal challenge with aerosolized F. novicida, in association with augmented cytokine and inflammatory responses, improved bacterial clearance, and the development of adaptive immunity. Second, this protective effect required IFN-{gamma}. Third, AGP-treated mice that survived primary infection with airborne F. novicida were resistant to rechallenge with the same organism. This is the first report of the protective effect of a pure TLR ligand on resistance to pneumonic tularemia.

The recognition of LPS by TLR4 has a major role in activating innate immunity against Gram-negative bacteria (29). However, the LPS of F. tularensis species have unusual structures (9, 16) that are poorly recognized by LPS-binding protein and TLR4 (7, 8, 9) and fail to stimulate inflammatory responses in vitro and in vivo (7, 8, 9, 10, 11, 12). F. novicida LPS is a very weak stimulus for cytokine production by monocyte/macrophage cell lines and does not elicit an inflammatory response in the lung after inhalation by mice (9). Administration of the TLR4 ligand AGP before infection activated the TLR4 pathway and compensated for the lack of TLR4 triggering by F. novicida LPS. Our data suggest that avoidance of TLR4 activation is an important virulence factor of F. novicida. A similar strategy has been adopted by Yersinia pestis (30).

AGP administration induced production of proinflammatory chemokine and cytokines in the lungs and potentiated their early production in response to inhalation of F. novicida. The induction of CXC chemokines such as MIP-2 is essential for the recruitment and activation of neutrophils in response to infection (31, 32). Neutrophils are required for resistance to systemic challenge with F. holarctica LVS (33, 34), but a protective role for neutrophils in host defense against pneumonic tularemia has not been established. It has been shown that F. holarctica LVS resists killing by human neutrophils (35) and that neutrophil depletion does not impair resistance to LVS pneumonia (34). Indeed, neutrophilic inflammation may exacerbate lung injury and worsen outcome in LVS pneumonia (36).

We found that administration of AGP also augmented the early TNF-{alpha} response after inhalation of F. novicida. TNF-{alpha} is a pluripotent cytokine that promotes inflammation, stimulates phagocyte antimicrobial activities, and contributes to the initiation of adaptive immune responses. TNF-{alpha} has been shown to have a role in innate resistance to intradermal challenge with LVS (37, 38), and to be required for suppression of intracellular replication of LVS by IFN-{gamma}-activated macrophages (39).

AGPs stimulated IL-12p40 release by dendritic cells and induced IL-12 production in the lungs. An important role for IL-12 in host defense against tularemia is supported by the reduction of bacterial loads when i.n. IL-12 is administered before LVS infection (40). Furthermore, it is known that both chains of IL-12 (p35 and p40) play a critical role in immune protection against LVS i.n. infection (40) and that administration of IL-12 before F. novicida infection promotes bacterial clearance, but not survival, unless the mice are also treated with gentamicin (41).

We found that AGP administration also induced IFN-{gamma} and augmented the early IFN-{gamma} response to inhalation of F. novicida. Indeed, the beneficial effect of AGP on survival from pneumonic tularemia was dependent on IFN-{gamma}. We observed that IFN-{gamma}–/– mice treated with PBS and then infected died 48 h before wild-type mice treated with PBS, indicating that endogenous production of IFN-{gamma} contributes measurably to the limited resistance of mice to airborne challenge with F. novicida. AGP-treated IFN-{gamma}–/– mice also died 1 day earlier than wild-type mice treated with PBS, demonstrating that AGP protection is mediated through IFN-{gamma}. IFN-{gamma} has a variety of immune regulatory functions, including macrophage and neutrophil activation (42). IFN-{gamma} has been shown to suppress the intracellular replication of F. novicida in murine macrophages (41) and to have a role in primary (43) and secondary LVS infection (44). We have not identified the cellular sources of IFN-{gamma} in this model, but NK cells have been shown to produce IFN-{gamma} in the lungs of mice with sublethal LVS infection (45). NKT and CD8+ T cells also have been shown to produce IFN-{gamma} during the innate immune response to intracellular bacteria (46, 47).

AGP-treated survivors of pneumonic tularemia developed specific immunity to F. novicida that was protective against rechallenge with the same organism. This protective immunity was associated with a strikingly IgG2-dominated Ab response typical of a Th1 cell-mediated immune response (48, 49) or of direct stimulation of B cells through TLR activation (50, 51). Splenocytes of AGP-treated survivors also demonstrated Ag-specific IFN-{gamma} responses, further supportive of a Th1 T cell response. It remains to be investigated whether the survivors are protected against a heterologous challenge with type A F. tularensis. Previous reports highlighted the limited protective immunity against aerosol challenge with type A F. tularensis in BALB/c mice previously intradermally immunized with F. novicida (52).

We found that AGP 527 was a more potent inducer of cytokine responses than AGP 524 by macrophages and dendritic cells, and engendered greater protection against F. novicida infection. Similarly, AGP 527 has been previously described as stronger inducer of protection against L. monocytogenes infection compared with other AGPs, such as 524 (20).

In summary, the TLR4 ligand AGP 527 is an immunomodulator with protective effects against pneumonic tularemia. We surmise that it also may provide protection against other subspecies of F. tularensis, all of which share the same lipid A structure with F. novicida (9). AGPs may augment host responses to other Gram-negative bacteria with weakly stimulatory lipopolysaccharides that are poorly recognized by TLR4, such as Legionella pneumophila, Bacterioides fragilis, and some strains of Pseudomonas aeruginosa (53, 54, 55). Similarly, AGPs may be beneficial in augmenting resistance to bacteria with temperature-dependent alterations in LPS phenotype that reduce TLR4 activation, such as Y. pestis (30).


   Acknowledgments
 
We thank Sing Sing Way, Tina Guina, Carolyn Hovde Bohach, and David Persing for valuable advice, and Destry Taylor and Malinka-Jansson-Hutson for technical assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grants AI057141 and AI-50023. Back

2 Address correspondence and reprint requests to Dr. Shawn J. Skerrett, Harborview Medical Center Box 359640, 325 Ninth Avenue, Seattle, WA 98104. E-mail address: shawn{at}u.washington.edu Back

3 Abbreviations used in this paper: LVS, live vaccine strain; AGP, aminoalkyl glucosaminide phosphate; BMDC, bone marrow-derived dendritic cell; i.n., intranasal. Back

Received for publication January 7, 2008. Accepted for publication March 28, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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