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Infection and Immunity, June 2006, p. 3657-3662, Vol. 74, No. 6
0019-9567/06/$08.00+0     doi:10.1128/IAI.02030-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Toll-Like Receptor 2 Is Required for Control of Pulmonary Infection with Francisella tularensis

Meenakshi Malik, Chandra Shekhar Bakshi, Bikash Sahay, Aaloki Shah, Steven A. Lotz, and Timothy J. Sellati*

Center for Immunology and Microbial Disease, Albany Medical College, Albany, New York 12208

Received 16 December 2005/ Returned for modification 18 January 2006/ Accepted 11 March 2006


   ABSTRACT
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Toll-like receptor 2 (TLR2) deficiency enhances murine susceptibility to infection by Francisella tularensis as indicated by accelerated mortality, higher bacterial burden, and greater histopathology. Analysis of pulmonary cytokine levels revealed that TLR2 deficiency results in significantly lower levels of tumor necrosis factor alpha and interleukin-6 but increased amounts of gamma interferon and monocyte chemoattractant protein 1. This pattern of cytokine production may contribute to the exaggerated pathogenesis seen in TLR2–/– mice. Collectively, these findings suggest that TLR2 plays an important role in tempering the host response to pneumonic tularemia.


   TEXT
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Francisella tularensis is capable of causing the lethal disease known as tularemia in several mammalian species. F. tularensis can be transmitted to humans and other mammals via arthropod bites, direct contact with infected tissues, ingestion of contaminated food or water, and, importantly, inhalation (19). F. tularensis has been classified as a category A biological warfare agent by The Working Group on Civilian Biodefense because of its extreme infectivity, substantial capacity to cause illness and death, and ease of artificial dissemination via aerosol. Two clinically relevant forms of F. tularensis exist, the European biovar B (F. tularensis subsp. holarctica) which produces acute though mild self-limiting infections and the more virulent biovar A (F. tularensis subsp. tularensis) found in the United States that is associated with pneumonic tularemia and a more severe clinical course (19).

An attenuated live vaccine strain (LVS) derived from a type B strain of F. tularensis has served as an invaluable tool in the study of tularemia pathogenesis (7, 8, 30). This strain, while not infectious for humans, is virulent in laboratory mice and causes a fulminant and lethal infection with features reminiscent of human tularemia (13, 29, 30). Despite these similarities, immunity to F. tularensis is not completely understood, particularly the early innate responses that contribute to inflammation, pathology, and the subsequent generation of adaptive immunity.

Toll-like receptors (TLRs) represent an ancient family of proteins that have been implicated in host antimicrobial defenses and in coordinating both innate and adaptive immune responses to pathogens (22). TLRs are expressed on a number of immune effector cells including monocytes/macrophages, neutrophils, dendritic cells, and both epithelial and endothelial cells. TLR2 recognizes a variety of pathogen-associated molecular patterns including bacterial lipoproteins (1, 3, 11, 14), peptidoglycan (5, 12), lipoarabinomannan (28), lipoteichoic acid (5), porins (17), and ß-glucan (the cell-stimulatory component of zymosan) (18, 20). It has been demonstrated that TLR2 forms heterodimers in association with TLR1 or TLR6 (20), which could in part explain the ability of TLR2 to recognize such diverse pathogen-associated molecular patterns. To date, the role of this important pattern recognition receptor in innate immunity to F. tularensis has not been investigated.

To explore the role of TLR2 in host resistance to F. tularensis infection, we employed TLR2–/– mice backcrossed onto a C57BL/6 background for 10 generations and used their congenic C57BL/6 wild-type (TLR2+/+) counterparts as controls. F. tularensis LVS (ATCC 29684; American Type Culture Collection, Rockville, MD) was kindly provided by Karen Elkins (U.S. Food and Drug Administration, Bethesda, MD), and aliquots of mid-log-phase growth cultures were stored in liquid nitrogen. For each experiment, the viability of frozen aliquots of bacteria and the inocula dosage after serial dilution in phosphate-buffered saline (PBS) were confirmed by colony counting. Mice were inoculated intranasally with 104, 103, or 102 CFU of F. tularensis LVS in a volume of 20 µl of PBS (10 µl per nare) or with an equal volume of PBS alone. The relative susceptibility of mice to infection was based upon cumulative survival proportions and median time to death (MTD) of the entire group of mice. A log rank test was used to determine the level of significance for the Kaplan-Meir survival analyses. Differences between control and experimental groups were considered significant at a P value of <0.05.

With an inoculum of 104 CFU of F. tularensis, TLR2+/+ mice had an MTD of 10 days, while TLR2–/– mice died more rapidly, with an MTD of only 6.5 days (Fig. 1 and Table 1). Although reducing the inoculum size lengthened the MTD, TLR2 deficiency hastened death in all cases. These results suggest that signaling through TLR2 enhances host resistance to F. tularensis infection. To determine whether the increased mortality observed in TLR2–/– mice reflects an impaired ability to control bacterial replication, we quantified the bacterial burden in various tissues. Mice were infected with 103 CFU of F. tularensis, and lungs, liver, and spleen were removed at various times postinfection. Portions of lung (20 mg), liver (30 mg), and spleen (5 mg) were homogenized in 0.5 ml of sterile PBS containing a protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN), and 10-fold serial dilutions of clarified supernatant were spotted onto Mueller Hinton II chocolate agar plates to enumerate the bacterial colonies. At days 5 and 7 postinfection, the number of bacteria was consistently higher in the tissues of TLR2–/– mice than in TLR2+/+ mice. Bacterial burden in the lungs of TLR2–/– mice was nearly 3 logs higher than in wild-type animals. Under these experimental conditions, the earliest point at which bacteria were detected in the liver and spleen of either genotype was 2 days postinfection (unpublished data). Compared with TLR2+/+ mice, bacterial burden in the TLR2–/– mice was significantly higher by day 5 for the liver and by day 3 for the spleen (Fig. 2). Comparisons between the groups were made using one-way analysis of variance (ANOVA), followed by Bonferroni's correction or nonparametric Mann-Whitney test. These findings demonstrate that the increased susceptibility to infection observed in TLR2–/– mice is associated with either an impaired ability to control bacterial growth and/or eliminate organisms from infected tissues.


Figure 1
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  		FIG. 1. TLR2 deficiency influences host susceptibility to F. tularensis infection. TLR2+/+ and TLR2–/– mice were inoculated intranasally with different doses of F. tularensis LVS and monitored for survival. Results are expressed as Kaplan-Meier curves and P values were determined using a log rank test. The results show pooled data of two independent experiments (4 to 6 mice per group, for a total of 10 mice).

 

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  		TABLE 1. MTD of F. tularensis-infected TLR2+/+ and TLR2–/– mice

 

Figure 2
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  		FIG. 2. TLR2–/– mice fail to limit F. tularensis replication in the lungs. TLR2+/+ and TLR2–/– mice were inoculated intranasally with 103 CFU of F. tularensis LVS. At the times indicated, mice were sacrificed, and homogenates of the lungs, liver, and spleen were plated for determination of bacterial burden. Results are shown as the means ± standard errors of the means of CFU counts and are representative of two independent experiments (n = 6 mice per group, for a total of 12 mice). *, P < 0.05; **, P < 0.01; and ***, P < 0.001 (by a nonparametric Mann-Whitney test).

 
Given the greater bacterial burden found in TLR2–/– mice, we next evaluated the lungs, liver, and spleen for histopathological changes. Tissues were excised from mice infected with 103 CFU of F. tularensis LVS at day 7 postinfection. Lungs were perfused with PBS prior to removal, and sections stained with hematoxylin and eosin were assessed using a histopathologic scoring system (Table 2). A numerical score for each animal was assigned by adding the subscores from nine parameters, each of which was graded from 0 to 3 and then combined for a maximum cumulative score of 27. TLR2–/– mice presented with severe lesions consisting of extensive peribronchial and perivascular infiltrates composed of neutrophils, macrophages, and lymphocytes. They also exhibited severe necrotizing bronchopneumonia, which typically involved multiple lobes of the lung. In contrast, TLR2+/+ mice showed only mild to moderate peribronchial and perivascular inflammation and mild focal pneumonia (Fig. 3). Based upon the extent and severity of these lesions as observed in a total of 12 mice per group from two independent experiments, the mean cumulative histological score for TLR2–/– mice (21.6 ± 1.56) was significantly higher (P < 0.01) than that of TLR2+/+ mice (13.2 ± 1.15). Owing to the greater inflammation, considerably more macrophages were found in the pulmonary lesions of TLR2–/– mice than in their TLR2+/+ counterparts. The livers of TLR2–/– mice showed nondiscrete, granulomatous lesions and focal necrosis of hepatocytes while those of the TLR2+/+ mice showed discrete lesions with no indication of necrosis. Nearly 20 to 25 granulomas per field were seen in the livers of TLR2–/– mice compared with only 5 to 10 per field in TLR2+/+ mice, suggestive of more extensive liver damage as a result of TLR2 deficiency. Spleens from infected TLR2–/– mice also showed more severe inflammation with greater numbers of infiltrating cells and significant disruption of the normal splenic architecture. In contrast, spleens from infected wild-type mice were less inflamed, contained fewer infiltrating cells, and retained a normal splenic architecture (Fig. 3).


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  		TABLE 2. Histopathologic scoring system for lungs

 

Figure 3
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  		FIG. 3. TLR2–/– mice show more extensive histopathological changes than their wild-type counterparts. Histopathological changes in the lungs, liver, and spleen of TLR2+/+ and TLR2–/– mice were evaluated at day 7 after intranasal inoculation with 103 CFU of F. tularensis LVS. Lung, liver, and spleen sections of sham-inoculated TLR2+/+ mice served as a control. Magnification, x40 (lung, liver, and spleen). Arrows indicate neutrophil-rich granulomas with central areas of necrosis.

 
Next we sought to determine whether the more severe histopathological lesions observed in TLR2–/– mice were associated with an altered profile of pro- and anti-inflammatory cytokines and chemokines. Levels of proinflammatory cytokines in the lung homogenates of the infected TLR2–/– and TLR2+/+ mice were determined using a cytometric bead array (BD Pharmingen, San Diego, CA), followed by four-parameter flow cytometric analysis using a FACSArray flow cytometer (BD Immunocytometry Systems, San Jose, CA). At 5 days postinfection, the levels of tumor necrosis factor alpha (TNF-{alpha}) and interleukin-6 (IL-6), two proinflammatory cytokines required for resistance to F. tularensis (9, 26), were significantly lower in the lungs of infected TLR2–/– mice than in their wild-type counterparts. However, by day 7 postinfection levels of interferon gamma (IFN-{gamma}) and monocyte chemoattractant protein 1 (MCP-1) were significantly elevated in the lungs of TLR2–/– mice (Fig. 4). With the exception of IL-6, there was a progressive increase in immunomodulator levels over time. In contrast, it was consistently observed that IL-6 production began after 3 days of infection, peaked by 5 days, and returned to baseline levels by day 7. Although levels of active IL-12 (i.e., IL-12p70) were below detectable limits, an elevation in the level of the IL-12p40 subunit was observed (unpublished data). For a brief period early in the disease process, IL-12p40 levels were higher in TLR2+/+ than in TLR2–/– mice; however, by day 5 postinfection, the reverse was observed, with higher levels seen in mice deficient for TLR2. In contrast, IL-10 levels never rose above detectable limits for the duration of the experiment (unpublished data).


Figure 4
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  		FIG. 4. Expression of proinflammatory cytokines TNF-{alpha} and IL-6, but not IFN-{gamma} and MCP-1, is impaired in TLR2–/– mice. Following intranasal inoculation with 103 CFU of F. tularensis, lung homogenates were prepared, and cytokine levels were measured using sham-inoculated mice as a control. Results represent the means ± standard errors of the means of the cytokine concentrations from two independent experiments (n = 6 mice per group, for a total of 12 mice). *, P < 0.05; **, P < 0.01; ***, P < 0.001 (by one-way ANOVA followed by Bonferroni's correction).

 
Finally, experiments were undertaken to determine whether the in vivo phenotypic differences observed between TLR2+/+ and TLR2–/– mice were reflected in ex vivo macrophage responses. Bone marrow-derived macrophages (BMDM) were incubated with F. tularensis at a multiplicity of infection of 102 for 4 h (unpublished data), 24 h, and 48 h, followed by quantification of bacterial numbers at each time point. As seen in Fig. 5A, the number of total bacteria (both intra- and extracellular) recovered from TLR2–/– BMDM was a log higher than that obtained following coculture with TLR2+/+ cells. Consistent with this finding was the observation that TLR2–/– BMDM were more sensitive to F. tularensis-induced cell death than their wild-type counterparts as measured by lactate dehydrogenase (LDH) release using the CytoTox96 LDH-release kit (Promega, Madison, WI) (Fig. 5B). This increased cell death likely results from the more permissive environment TLR2–/– macrophages provide for intracellular replication. Importantly, at 4 h (a point before significant replication occurs) nearly identical numbers of bacteria were recovered from TLR2+/+ and TLR2–/– cells, suggesting that no difference exists in the kinetics of francisellae uptake by these two cell populations (unpublished data). Lastly, as was found in lung homogenates from TLR2–/– mice, significantly lower amounts of TNF-{alpha} were released by F. tularensis-stimulated TLR2–/– BMDM (Fig. 5C).


Figure 5
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  		FIG. 5. Ex vivo exposure of TLR2–/– macrophages to F. tularensis elicits responses reminiscent of those observed in vivo. BMDM (2.5 x 105/well) from TLR2+/+ and TLR2–/– mice were seeded into 24-well tissue culture plates and infected with F. tularensis LVS (multiplicity of infection of 100) for 2 h followed by gentamicin (50 µg/ml) treatment for 1 h to kill extracellular bacteria. Cells were washed, incubated for the indicated periods of time, and then lysed with 0.1% sodium deoxycholate. Bacteria were enumerated by colony counting (A). Cells (5 x 104/well) seeded in 96-well tissue culture plates were treated as described above, and cell death was quantified at the indicated times using a CytoTox96 LDH-release kit (Promega) according to the manufacturer's instructions (B). Levels of TNF-{alpha} in the tissue culture supernatants derived from experiments outlined in panel A were assayed using the cytometric bead array system (BD Pharmingen) (C). **, P < 0.01; ***, P < 0.001 (by one-way ANOVA followed by Bonferroni's correction).

 
TLR2 signal transduction and the subsequent release of proinflammatory cytokines and chemokines underlie the pathogenesis resulting from infection with a variety of intracellular gram-negative bacteria. F. tularensis-induced TNF-{alpha} and IL-6 production was significantly lower in TLR2–/– mice than in their wild-type counterparts. Further, TLR2–/– mice infected with F. tularensis harbored greater numbers of organisms and developed more severe and extensive pathology. Our finding is reminiscent of the failure of mice to clear Legionella pneumophila and to exhibit persistent pneumonitis following antibody-mediated depletion of TNF-{alpha} (24). TNF-{alpha} is known to enhance the L. pneumophila-killing activity of rat alveolar macrophages via its ability to potentiate the activating effects of IFN-{gamma} through induction of nitric oxide synthesis (25). IFN-{gamma}, nitric oxide, and IL-12 are known to be critical elements of innate immunity to F. tularensis (4, 6, 15). Importantly, the full microbicidal action of IFN-{gamma} is not realized in the absence of the synergistic influence of TNF-{alpha}. Thus, despite the higher IFN-{gamma} levels observed in TLR2–/– mice, diminished production of TNF-{alpha} results in less effective control of F. tularensis growth. This interpretation is further supported by results presented in Fig. 5A, wherein TLR2–/– macrophages harbor significantly greater numbers of bacteria than their wild-type counterparts.

Despite the fact that elevated levels of IL-6 are associated with many pathophysiological processes including the development of chronic inflammation (10, 27), this multifunctional cytokine also plays an important "positive" role as a differentiation and growth factor for B and T cells (27) and as a modulator of the suppressor activity of CD4+ CD25+ T cells (21). In the Chlamydia trachomatis model, IL-6–/– mice present with increased mortality, higher bacterial burdens, and altered histological findings (32). The phenotypic similarity between C. trachomatis-infected IL-6–/– mice and F. tularensis-infected TLR2–/– mice suggests that lower IL-6 levels in the latter may contribute to, rather than result from, greater pathology. TLR-mediated release of IL-6 is known to "unblock" the suppressive effect that CD4+ CD25+ T cells have on pathogen-specific effector T cells (21). In our model where IL-6 levels are lower in TLR2–/– mice, the actions of F. tularensis-specific effector T cells may remain partially suppressed by CD4+ CD25+ T cells, thus resulting in less effective adaptive immune responses. Suboptimal adaptive immunity may, in part, contribute to the larger numbers of TLR2–/– mice that succumb to infection between days 7 and 14.

Although paradoxical given the typical association of TNF-{alpha} and IL-6 with tissue damage and establishment of chronic inflammation, respectively, diminished levels of these cytokines in TLR2–/– mice may undermine clearance mechanisms, thus allowing greater replication of F. tularensis and exacerbation of disease via production of other proinflammatory molecules. IFN-{gamma} and MCP-1 are modulators of inflammation that variably contribute both to bacterial clearance and tissue damage. Recently, IL-12-induced IFN-{gamma} was shown to be necessary for the protection of mice against lethal respiratory tularemia (4). Nevertheless, production of IFN-{gamma} appears insufficient to prevent death, as evidenced by greater levels of this potent stimulator of macrophage antimicrobial action in highly susceptible TLR2–/– mice. Despite higher IFN-{gamma} levels, bacterial burdens in the lungs, liver, and spleen of TLR2–/– mice exceeded by several logs the number of francisellae in wild-type mice. Consistent with this observation is the finding that although IFN-{gamma} is required for control of tularemia, its excessive production does not result in more effective killing of F. tularensis (15). One potential explanation for this paradoxical observation is that MCP-1 levels are significantly elevated in TLR2–/– mice as well. A potent stimulus for macrophage recruitment to sites of bacterial insult, MCP-1 may facilitate the accumulation of macrophages in the lungs of TLR2–/– mice, thus providing a larger pool of cells in which F. tularensis can replicate.

The more severe course of disease in TLR2–/– mice raised an intriguing question. Does the TLR2 deficiency phenotype reflect a primary TLR2-mediated response or secondary responses resulting from bacterial replication in the lungs? To answer this question UV-killed francisellae were instilled in the lungs of mice, and it was observed that this inoculum failed to elicit any pulmonary histopathological changes or stimulate the production of cytokines at a point in time when fulminant inflammatory responses were elicited by viable organisms in both TLR2+/+ and TLR2–/– mice (unpublished data). A lymphoproliferative response, however, was observed in spleens of mice receiving UV-killed F. tularensis. These findings lead us to propose that both the primary (a direct result of bacterial recognition) and secondary (inflammation) responses to F. tularensis are mediated by and are a consequence of TLR2 signaling events.

Finally, Mariathasan and coworkers recently reported that activation of the ASC/caspase-1 axis is essential for innate immunity against F. tularensis (16). F. tularensis infection of mice deficient for ASC or caspase-1 was associated with markedly increased mortality, bacterial burdens, and pathology compared with wild-type mice, a phenotype shared by TLR2–/– mice. This intriguing similarity is perhaps not surprising given that binding of bacterial lipoproteins to TLR2 on macrophages induces caspase-1 and triggers subsequent apoptosis (2). Although no component of F. tularensis has yet been definitively identified as a TLR2 ligand, possible candidates include lipoproteins such as Tul4 (23) and ß-glucan (31). Whether either or both of these putative ligands act directly through TLR2 to engage the ASC/caspase-1 axis and trigger antibacterial defense mechanisms such as apoptosis requires further investigation. Regardless, our results demonstrate a central role for TLR2 in tularemia pathogenesis and suggest that failure to engage this signaling pathway drastically impairs host immunity to F. tularensis.


   ACKNOWLEDGMENTS
 
This work was supported by U.S. Public Health Service grant PO1 AI-056320.

We are indebted to Paul Feustal for discussions on biostatistics, James Drake and Karsten Hazlett for critical review of the manuscript, and the Center for Immunology and Microbial Disease Immunology Core Facility.


   FOOTNOTES
 
* Corresponding author. Mailing address: Center for Immunology and Microbial Disease, Albany Medical College, 47 New Scotland Avenue, MC151, ME205B, Albany, NY 12208-3479. Phone: (518) 262-8140. Fax: (518) 262-2885. E-mail: sellatt{at}mail.amc.edu. Back

Editor: J. B. Bliska


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Infection and Immunity, June 2006, p. 3657-3662, Vol. 74, No. 6
0019-9567/06/$08.00+0     doi:10.1128/IAI.02030-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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