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Originally published online as doi:10.1189/jlb.0406294 on August 8, 2006

Published online before print August 8, 2006
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(Journal of Leukocyte Biology. 2006;80:766-773.)
© 2006 by Society for Leukocyte Biology

Innate immune response to Francisella tularensis is mediated by TLR2 and caspase-1 activation

Hanfen Li, Suba Nookala, Xiaowen R. Bina, James E. Bina and Fabio Re1

Department Molecular Sciences, University of Tennessee Health Science Center, Memphis, Tennessee

1 Correspondence: Department Molecular Sciences, University of Tennessee Health Science Center, 858 Madison Avenue, MSB 501B, Memphis, TN 38163. E-mail: fre{at}utmem.edu

ABSTRACT

Francisella tularensis, a gram-negative, facultative, intracellular bacterium, is the etiologic agent of tularemia and a category A bioterrorism agent. Little is known about the mechanism of pathogenesis of tularemia. In this paper, we describe the interaction of the live vaccine strain of F. tularensis with the innate immune system. We have found that in human and mouse dendritic cells, F. tularensis elicited a powerful inflammatory response, characterized by production of a number of cytokines and chemokines. Using cells derived from TLR2-deficient mice and in vitro transfection assays, we demonstrated that this response was mediated by TLR2 and did not require the LPS-binding protein. F. tularensis appeared to activate TLR2/TLR1 and TLR2/TLR6 heterodimers. IL-1ß secretion, a reflection of caspase-1 activation, was induced by live but not heat-killed F. tularensis, despite the fact that both forms of the bacterium equally induced the IL-1ß transcript. Our results identified activation of TLR2 and caspase-1 as the two main cellular pathways responsible for the inflammatory response to F. tularensis.

Key Words: Toll-like receptors • inflammation • dendritic cells • bacterial infection

INTRODUCTION

Francisella tularensis is a Gram-negative, facultative, intracellular bacterium, which causes tularemia. F. tularensis is one of the most infectious bacterial pathogens known, requiring less than 10 organisms to cause disease. As F. tularensis is highly infective and easy to propagate and disseminate by aerosol and as infection often requires intensive medical care, it is a prime candidate for use in biological warfare and bioterrorism.

Although F. tularensis can be cultured easily on axenic media, it is believed to replicate intracellularly, mainly within macrophages, during infection of mammalian host. It has been demonstrated that F. tularensis inhibits phagosome/lysosome fusion and the respiratory burst [1 ] and may persist within the acidified endosomal compartment, where the bacteria are able to acquire iron, which is essential for their survival and growth [2 ]. Recent findings suggest that F. tularensis can rapidly escape the phagosome and resides and replicates within the cytoplasm of host cells [3 ], a life-cycle similar to Listeria monocytogenes. Little is known at the molecular level about how F. tularensis causes disease, and only a few potential virulence factors have been identified. The interaction of F. tularensis with the innate immune system likely plays a major role in the pathogenesis of F. tularensis infection, yet it remains an area that has received little attention.

It is well established that infection of monkeys, rabbits, and rodents with F. tularensis results in a pronounced, inflammatory response with high TNF-{alpha} and IFN-{gamma} (reviewed in ref. [4 ]). However, the components of the bacterium that induce inflammation, and the cellular receptors that mediate it remain a mystery. In contrast to the LPS of most Gram-negative bacteria, F. tularensis LPS does not possess proinflammatory activities and is unable to act as a TLR4 agonist or antagonist [5 , 6 ], suggesting that other bacterial products are likely responsible for the induction of the proinflammatory cytokines. In agreement with this result is the discovery that TLR4 does not contribute to resistance of mice to airborne or intradermal F. tularensis infection [7 , 8 ].

Host cells use several types of pattern recognition receptors (PRR) to detect the presence of microbial products. TLRs are expressed on the host cell surface or in endosomal compartments, where they detect several microbial products derived from bacteria, viruses, yeast, and protozoans (reviewed in ref. [9 ]). For example, TLR4 recognizes the LPS derived from gram-negative bacteria, and TLR2, in combination with TLR1 or TLR6, mediates response to triacylated or diacylated bacterial lipopeptides, respectively. All TLR, with the exception of TLR3, use the cytoplasmic adaptor molecule MyD88 to activate a signaling cascade, which leads to the induction of a large number of cytokines and chemokines. In addition, a different subset of cytokines, which includes IFN-ß, IFN-inducible protein 10 (IP-10), and RANTES, is induced in a MyD88-independent way by TLR4 and TLR3 using the adaptor molecule Toll/IL-1 domain-containing adaptor-reducing IFN-beta [10 , 11 ]. Although TLRs play a predominant role in microbial pattern recognition, other classes of PRR exist, which are able to detect bacterial products in the cytoplasm (a location inaccessible to TLR monitoring). Members of the NACHT-leucine-rich repeat (NLR) family of PRR (reviewed in refs. [12 , 13 ]), which includes the NOD and NALP proteins, can detect in the cytoplasm microbial products such as the muramyl dipeptide (MDP) derived from peptidoglycan (PGN) breakdown [14 15 16 ], bacterial RNA and toxins [17 ], and other endogenous "danger" signals [18 ]. The members of the NLR family are characterized by a carboxy-terminal LRR, probably involved in ligand binding, an intermediate NACHT domain necessary for oligomerization and activation, and an amino-terminal effector domain, which links the receptor to downstream effector molecules. Some NLR family members are part of a multiprotein complex called inflammasome, which also contains the proteases caspase-1 and caspase-5. Activation of caspase-1, which cannot be triggered directly by TLR stimulation alone, occurs in the context of the inflammasome in response to cytoplasmic detection of bacterial products and leads to cleavage of pro-IL-1ß and IL-18, a necessary step for the secretion of these powerful, proinflammatory cytokines.

In this paper, we analyzed the interaction of F. tularensis with cells of the innate immune system. We found that this bacterium elicited a potent inflammatory response in mouse and human dendritic cells (DC). The induction of a number of proinflammatory cytokines and chemokines by live or heat-killed F. tularensis was mediated by TLR2. In addition, live but not heat-killed F. tularensis was able to induce caspase-1 activation and release of mature IL-1ß.

MATERIALS AND METHODS

Reagents
LPS (Escherichia coli K12 LCD25) was from List Biological Laboratories (Campbell, CA). It was purified from contaminant lipoproteins normally found in commercially available LPS preparations by double-phenol extraction, exactly as described in ref. [19 ]. A crude preparation of Staphylococcus aureus PGN was from Fluka (Milwaukee, WI). Triacylated synthetic lipopeptide trispalmitoyl-cysteyl-seryl-lysyl-lysyl-lysyl-lysine (Pam3Cys)-Ala-Gly was purchased from Bachem (Torrance, CA). Diacylated synthetic lipopeptide fibroblast-stimulating lipopeptide-1 (FSL-1) and macrophage-activating lipopeptide-2 (MALP-2) were purchased from Invivogen (San Diego, CA). Caspase-1 inhibitor Z-Tyr-Val-Ala-Asp-fluoromethylketone (Z-YVAD-FMK) was from Alexis (San Diego, CA).

Cell lines and primary cell isolation
A subclone of HeLa, previously isolated in our lab, was grown in DMEM-10% FCS. The stably transfected HeLa-TLR2 and HeLa-TLR4/myeloid differentiation protein 2 (MD-2) cell lines were described previously [20 ]. The mouse peritoneal macrophage immortalized cell lines HeNC2 and GG2EE [21 ], derived from the C3H/HeN and TLR4-deficient C3H/HeJ mouse strains, respectively, were donated by Steven Mizel (Wake Forest University, Winston-Salem, NC) and grown in RPMI 1640-10% FCS.

Human PBMC were isolated from Leukopacks by Ficoll-Hystopaque density gradient centrifugation. Monocytes were purified from human PBMC using MACS CD14 microbeads (Miltenyi Biotec, Auburn, CA) according to the manufacturer’s recommendation. Purity was checked by staining with a FITC-conjugated anti-CD14 antibody (Sigma Chemical Co., St. Louis, MO) and FACScan analysis and routinely found to be greater than 94%. Immature DC were obtained by incubating monocytes at 3 x 106/ml in RPMI 1640-10% FCS supplemented with recombinant human (rh)GM-CSF (10 ng/ml) and rhIL-4 (10 ng/ml; both from R&D Systems, Minneapolis, MN) for 8 days. Fresh complete medium was replaced every 4 days.

To obtain mouse DC, the femur/tibiae were removed and freed of muscles and tendons. Bone ends were cut with scissors, and bone marrow cells were flushed out of the bone cavity with Dulbecco’s PBS (D-PBS) using a syringe with a #25 gauge needle. The cell suspension was filtered through a 70-µm cell strainer, washed once, and resuspended in RPMI 1640-10% FCS supplemented with penicillin (100 U/ml), streptomycin (100 µg/ml), and recombinant mouse (rm)GM-CSF (20 ng/ml; R&D Systems). The cell suspension was plated at 1 x 106 cells/ml in 100 mm petri dishes. The medium was replaced on Day 4, and suspension cells were collected and stimulated on Day 8. This procedure routinely results in >80% CD11c+ cells. The University of Tennessee Animal Care and Use Committee (Memphis) approved the use of animals for these experiments.

Bacterial strains, plasmids, and growth conditions
F. tularensis live vaccine strain (LVS) containing pFNLTP6-groGFP was used for all experiments. F. tularensis LVS was obtained from Mark Miller (University of Tennessee Health Science Center). pFNLTP6-groGFP, an E. coli-F. tularensis shuttle vector, which constitutively produces GFP, was obtained from Tom Zahrt (Wisconsin Medical College, Milwaukee) [22 ]. pFNLTP6-groGFP was transformed into F. tularensis LVS by electroporation, according to the protocol described by Kawula et al. [23 ]. F. tularensis LVS-pFNLTP6-groGFP was grown in modified brain heart infusion (BHI) broth (BHI supplemented with 50 µg/ml hemin and 1% IsoVitalex, BBL, Cockeysville, MD) or on BHI-chocolate agar (BHI agar supplemented with 1% hemoglobin and 1% IsoVitalex). Kanamycin was added to all growth media at 10 µg/ml for plasmid maintenance. F. tularensis LVS-pFNLTP6-groGFP was grown to mid-logarithmic phase (OD600 of ~0.5) in modified BHI broth. Bacteria were collected by centrifugation, washed three times with 1x D-PBS, and resuspended and stored at 4°C in D-PBS. No loss of viability was observed over prolonged storage.

F. tularensis infection of primary DC or macrophage cell lines
Macrophage cell lines or human and mouse DC were resuspended in RPMI 1640-10% FBS, at 1 x 106 cells per tube. Dilutions of the bacteria were used to infect cells at a multiplicity of infection (MOI) of ~100. The MOI for each experiment was verified by serial dilution and plating of an aliquot of the inoculum onto modified Mueller-Hinton (MH) broth. The cells were incubated with bacteria for 2 h (to allow bacteria adhesion and ingestion), after which gentamycin was added (10 µg/ml) to the culture medium to kill extracellular bacteria. This procedure resulted in infection of 40–80% of cultured macrophage or DC cells at 24 h postinfection, as determined by the presence of GFP-positive cellular inclusions by fluorescent microscopy. Heat-killed bacteria were prepared as described above, except the cells were first incubated for 20 min in a heating block at 80°C before use.

RNase protection assay
Total RNA was isolated using TRIzol reagent (Life Technologies, Grand Island, NY). RNase protection assay was performed using 4–6 µg total RNA using the Riboquant kit (BD PharMingen, San Diego, CA) according to the manufacturer’s recommendations. The hCK-2b, hCK-5, and mCK-2 and mCK-5 multiprobe template sets were used.

Luciferase assay
HeLa, HeLa-TLR2, or HeLa-TLR4/MD-2 was transiently transfected in 24-wells plates using Effectene reagent (Qiagen, Valencia, CA) with 0.4 µg endothelial cell-adhesion molecule (ELAM)-luciferase and 0.2 µg pcDNA-CD14, 0.1 µg CMV-ß-galactosidase (ß-Gal), and (for parental HeLa only) 0.6 µg TLR5 or TLR7/8 expression plasmids (recipe for 24 wells). Forty-eight hours after transfection, cells were stimulated for 6 h with different agonists. Luciferase assay was performed using Promega (Madison, WI) reagents according to the manufacturer’s recommendations. Efficiency of transfection was normalized by measuring ß-Gal in cell lysates.

Mammalian expression vectors
The chimeric TLR2/TLR1 (TLR[2-1]) and TLR1-TLR2 (TLR[1-2]) constructs have been described previously [24 ]. The TLR[6-2] was constructed using a similar strategy. A fragment of human TLR6 (aa 1–579) was PCR-amplified using Pfu Turbo polymerase (Stratagene, La Jolla, CA) and thefollowing primers: TLR6Bam-GCGCGGATCCACCATGACCAAAGACAAAGAACCTATTG and TLR6Xho-GCGCCTCGAGTTCAGACATGTGAAAGTCCTTTAG.

The PCR product was digested with BamHI-XhoI and cloned into a modified pEF1{alpha} vector (Invitrogen, Carlsbad, CA) containing the transmembrane and cytoplasmic region of human TLR2 (aa 585–784).

The pEF-LPS-binding protein (LBP)-Flag-His vector for expression of LBP was constructed as follows: The cDNA of human LBP was obtained by RT-PCR of PBMC total RNA and using the following primers: LBP5-GCGAGATCTACCATGGGGGCCTTGGCAAGAGCC and LBP3-GCGAGATCTAACCTCATGTATTGGACATTGG. The PCR product was digested with BglII and cloned into the BamHI site of a modified pEF1{alpha} vector containing the FLAG and 6HIS tag. To obtain the LBP-containing media, this vector was transfected into human embryo kidney (HEK) 293T by the calcium-phosphate method, and the medium was collected 48 h later and used for the experiments seen in Figure 5 .


Figure 5
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  		Figure 5. Contribution of serum and LBP to TLR2 activation by F. tularensis. HeLa-TLR2 cells, transiently transfected with ELAM-luciferase reporter construct, CMV-CD14, and CMV-ß-Gal (for normalization), were stimulated for 6 h in SFM, in SFM supplemented with 10% FCS, and in SFM containing LBP (~2 µg/ml). NF-{kappa}B activation was measured by luciferase assay. Agonists were used at the following concentrations: crude PGN (10 µg/ml), FSL-1 (10 ng/ml), Pam3Cys (2 µg/ml), and F. tularensis at a MOI of 500.

 
Cytokines measurements
Cytokines levels in conditioned media were measured by ELISA using the following paired antibody kits: mIL-1ß, mIL-10 (eBioscience, San Diego, CA), hIL-1ß, hIL-8, hIL-10, and mTNF-{alpha} (R&D Systems).

RESULTS

Characterization of the inflammatory response to F. tularensis
To characterize the inflammatory response to F. tularensis LVS, human immature DC and PBMC were stimulated for 5 h with live or heat-killed F. tularensis, and the induction of cytokines and chemokines was analyzed by RNase protection assay. For comparison, cells were also stimulated with purified agonists of different TLR {LPS, TLR4; crude PGN, TLR2; polyinosinic:polycytidylic acid [poly(I:C)], TLR3; flagellin, TLR5; R848, TLR7 and TLR8}. As shown in Figure 1 , F. tularensis was a potent inducer of several cytokines and chemokines. The pattern of cytokine induced by F. tularensis was different from that of the TLR4 agonist LPS and resembled more closely that of a crude preparation of PGN, capable of stimulating TLR2. In particular, the inability of F. tularensis to induce IP-10, a chemokine induced only through the MyD88-independent pathway [10 ], would suggest that F. tularensis may only activate the MyD88-dependent pathway. It is interesting that heat-killed F. tularensis was equally capable of inducing cytokines as the live bacteria, suggesting that the ability to infect DC did not play a role in the early phase of cellular stimulation.


Figure 1
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  		Figure 1. Comparison of cytokine and chemokine induction by different TLR agonists and F. tularensis in human DC and PBMC. Human immature DC (A) and PBMC (B) were stimulated with the indicated agonists for 5 h, and RNase protection assay was used to analyze cytokine and chemokine induction. Agonists were used at the following concentration: LPS (1 ng/ml), crude PGN (10 µg/ml), poly(I:C) (PIC; 50 µg/ml), flagellin (Flag; 10 µg/ml), R848 (1 µg/ml), or live or heat-killed F. tularensis (FT-HK) at a MOI of 100. One experiment representative of several donors is shown. Ltn, Lymphotactin.

 
Similar experiments were repeated using mouse macrophage cell lines (Fig. 2 ). Even in these cell types, the pattern of cytokines induced by F. tularensis mirrored that induced by TLR2 agonists, lacking IP-10, TCA-3, and RANTES messages, all of which rely on the activation of the MyD88-independent pathway and were induced by the TLR4 agonist. Cytokine induction by F. tularensis was observed in the GG2EE macrophage cell line (derived from TLR4-deficient C3H/HeJ mice), indicating that TLR4 is not involved in the cellular stimulation by F. tularensis. This result is consistent with the reported inability of F. tularensis LPS to act as a TLR4 agonist [5 , 6 ] and with other reports that rule out TLR4 involvement in protection of mice from F. tularensis infection [7 , 8 ]. Taken together, these results demonstrate that F. tularensis elicits a powerful, inflammatory response, indistinguishable from that induced by TLR2 agonists, suggesting that TLR2 may mediate the response to F. tularensis.


Figure 2
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  		Figure 2. Cytokine and chemokine induction by F. tularensis is not mediated by TLR4. The mouse macrophage cell lines HeNC2 (wild-type) and GG2EE (TLR4-deficient) were stimulated with the indicated agonists for 5 h, and RNase protection assay was used to analyze cytokine and chemokine induction. MIF, Migration inhibitory factor.

 
TLR2 mediates the innate immune response to F. tularensis
To test TLR2 involvement in the inflammatory response to F. tularensis, DC derived from the bone marrow of wild-type or TLR2-deficient mice were infected with F. tularensis, and cytokine and chemokine induction was analyzed. As shown in Figure 3 , induction of these factors by F. tularensis was potent in wild-type DC but was completely abrogated in cells derived from TLR2-deficient mice, demonstrating that the innate immune response to F. tularensis is mediated by activation of TLR2. The absolute lack of induction of cytokine transcripts in TLR2-deficient cells is remarkable and suggests that in these cell types, no other TLR is activated by F. tularensis, a surprising finding considering the theoretical potential for activation of multiple TLR by whole bacteria (CpG DNA, heat shock proteins, flagellin, and others). It is surprising that although the cytokine induction was abrogated completely in TLR2-deficient cells stimulated with synthetic agonists, residual cytokine induction was still observed in cells stimulated with crude preparation of PGN. It has been reported that the ability of commercial PGN preparations to stimulate TLR2 is in fact a result of contaminating bacterial lipoproteins and lipoteichoic acid [25 ]. It is therefore conceivable that bacterial contaminant or PGN-breakdown products present in our crude PGN are responsible for the residual cytokine induction observed in PGN-stimulated TLR2–/– cells.


Figure 3
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  		Figure 3. Cytokine and chemokine induction by F. tularensis is mediated by TLR2. DC derived from the bone marrow of TLR2-deficient mice or wild-type (WT) littermates were stimulated with the indicated agonists for 5 h, and RNase protection assay was used to analyze cytokine and chemokine induction.

 
To confirm and characterize in more detail the ability of F. tularensis to stimulate TLR2 signaling, we measured activation of NF-{kappa}B in HeLa cell lines transfected with different TLRs. The results shown in Figure 4A indicated that F. tularensis was capable of stimulating only cells expressing TLR2.


Figure 4
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  		Figure 4. TLR2 activation by F. tularensis does not require LBP and occurs through TLR2/TLR1 and TLR2/TLR6 heterodimers. HeLa cell lines expressing the indicated TLR constructs were transiently transfected with ELAM-luciferase reporter construct, CMV-CD14, and CMV-ß-Gal (for normalization) and stimulated for 6 h with the indicated agonists. NF-{kappa}B activation was measured by luciferase assay. (A) HeLa-TLR2 and HeLa-TLR4/MD-2 are stable cell lines, and expression of TLR5 and TLR7/TLR8 was established via transient transfections. (B) Parental HeLa cells were transiently transfected with the indicated TLR chimeric constructs and stimulated with the indicated agonists, which were used at the following concentrations: LPS (1 ng/ml), crude PGN (10 µg/ml), FSL-1 (10 ng/ml), MALP-2 (10 ng/ml), Pam3Cys (2 µg/ml), flagellin (10 µg/ml), R848 (1 µg/ml), live or heat-killed F. tularensis at MOIs of 500 and 50, and PMA (10 ng/ml). (C) Schematic illustration of the TLR chimeric constructs and their modality of signaling. On the left, transfected TLR2 heterodimerizes with endogenous TLR1 or TLR6 and signals in response to triacylated or diacylated lipopeptides. Center, Chimeric constructs transfected in isolation cannot signal as a result of absence of heterodimerization of extracellular or cytoplasmic regions. On the right, complementing chimeric construct pairs restore signaling by providing simultaneously extracellular and cytoplasmic regions that can heterodimerize.

 
TLR2 is known to recognize its ligands by forming heterodimers with TLR1 or TLR6. The TLR1/TLR2 heterodimer mediates response to triacylated bacterial lipopeptides, and cell activation by diacylated lipopeptides occurs through the TLR6/TLR2 heterodimer [26 , 27 ] (although activation of the TLR1/TLR2 has also been reported in response to some diacylated lipopeptides [28 ]). As our HeLa or HEK 293 cell lines express endogenous TLR1 and TLR6, we used chimeric constructs, which swap the extracellular and intracellular domains among TLR1, TLR2, and TLR6, to determine which heterodimer is activated by F. tularensis. As we previously demonstrated [24 ] and illustrated in Figure 4C , these chimeric receptors can only function when used in complementing pairs that simultaneously provide the cytoplasmic and the extracellular domain of TLR1 and TLR2 (or TLR6 and TLR2). As expected (Fig. 4B) , the chimeric pair TLR[1-2] plus TLR[2-1] was activated by the triacylated lipopeptide Pam3Cys but not by the diacylated lipopeptide MALP-2. Conversely, the chimeric pair TLR[6-2] plus TLR[2-1] was activated exclusively by diacylated synthetic lipopeptides FSL-1 and MALP-2. It is interesting that FSL-1, which differs in structure from MALP-2, was also capable of stimulating the TLR[1-2] plus TLR[2-1] pair, in agreement with a published study [28 ]. In this assay, F. tularensis was able to activate cells transfected with TLR1 or TLR6 extracellular domain-containing chimeric constructs, suggesting that F. tularensis can agonize both TLR2 heterodimers. This conclusion seems to contrast with a study that was published during the reviewing process of our manuscript. In that study, Katz et al. [29] demonstrated that F. tularensis induction of TNF-{alpha} was mediated by TLR2 and was impaired in TLR6-deficient cells but unaffected in TLR1-deficient cells. The reason for the discrepancy between our study and the Katz et al. study is unclear. One likely explanation is the fact that the bacteria stocks used in the respective studies were grown using considerably different culture media—BHI broth in our study and MH broth in Katz et al. An obvious difference between our study and that of Katz et al. is the experimental approach used to determine the heterodimer requirements. Although this remains a possible source of the results discrepancy, it should be noted that our model, although clearly artificial, faithfully recapitulates the heterodimer specificities of different lipopeptides, as determined previously by other investigators using different approaches. Finally, it is interesting to note that the ability to stimulate the TLR[1-2] plus TLR[2-1] pair was more pronounced using the heat-killed F. tularensis than the live bacterium. It is possible that the heat treatment (a condition not tested by Katz et al. [29]) may unmask a ligand for the TLR2-TLR1 heterodimer.

It is interesting that a supernatant obtained from heat-killed F. tularensis was still able to activate the TLR[6-2] plus TLR[2-1] pair (not shown), suggesting that the F. tularensis-derived TLR2 agonist is a soluble product.

The recognition of several microbial products by TLR2 is known to be enhanced by serum factors such as LBP and CD14. To determine the contribution of serum and LBP to TLR2 activation by F. tularensis, the HeLa-TLR2 cell line, transiently transfected with CD14, was stimulated with three different TLR2 agonists (crude PGN, diacylated lipopeptide FSL-1, and triacylated lipopeptide Pam3Cys) and with F. tularensis in serum-free medium (SFM), FCS-containing SFM, and SFM containing recombinant LBP (Fig. 5 ). NF-{kappa}B activation was measured by luciferase assay. Stimulation with crude PGN, FSL-1, and F. tularensis was enhanced greatly in SFM, suggesting that an unidentified serum factor antagonized TLR2 stimulation by these agonists. Such effect has been reported previously for PGN stimulation of TLR2 [30 ]. In contrast, stimulation with Pam3Cys was completely abolished in SFM. Stimulation in LBP-containing medium totally restored the response to Pam3Cys, only marginally increasing the response to crude PGN, FSL-1, and F. tularensis. Thus, TLR2 stimulation by F. tularensis, being decreased by serum and unaffected by LBP presence, appears to occur with modalities that closely resemble PGN and diacylated lipopeptides, a possible indication of the nature of the F. tularensis-derived TLR2 agonists. The simplest (although not sole) explanation for this set of results (Figs. 4 and 5) is that F. tularensis contains a diacylated lipopeptide capable of stimulating TLR2/TLR1 and TLR2/TLR6 heterodimers.

Live but not heat-killed F. tularensis induces activation of caspase-1 and IL-1ß release
The fact that F. tularensis is able to escape the phagosome and reside in the cytoplasm suggests that this bacterium may be detected by cytoplasmic PRR, leading to the activation of the inflammasome and caspase-1 and resulting in release of mature IL-1ß, one of the most powerful proinflammatory cytokines. To test this, human and mouse DC were stimulated for 20 h with different TLR agonists and with live or heat-killed F. tularensis. Mature IL-1ß was measured in the cell culture supernatants. As shown in Figure 6 , pure TLR2 and TLR4 agonists induced release of negligible amounts of mature Il-1ß, whose levels were increased by costimulation with MDP, a NOD-2 and NALP3 agonist, in agreement with published results by other groups [31 ]. In contrast, cells stimulated with live F. tularensis released large amounts of IL-1ß, which by human DC infected with live F. tularensis, reflected caspase-1 activation, as addition of the caspase-1 inhibitor Z-YVAD-FMK abolished IL-1ß production (Fig. 6A) . In agreement with the results of Figure 3 , IL-1ß production by mouse DC was TLR2-dependent (Fig. 6B) . It is remarkable that heat-killed F. tularensis was unable to induce the release of IL-1ß, despite being an equally powerful inducer of the IL-1ß transcript as the live organism or all the other TLR agonists tested (see Figs. 1 2 3 ). It should be noted that the differences observed between stimulation with live and heat-killed F. tularensis were not a result of difference in the amount of F. tularensis (dead or alive) used, as heat-killed F. tularensis was still unable to induce appreciable IL-1ß release, even when used at a concentration 100-fold higher than the live organism, and was not a result of induction of apoptosis, which was not observed at the MOI and incubation time used in our experiments (not shown). Live and heat-killed F. tularensis were equally capable of inducing the release of other cytokines such as TNF-{alpha} and IL-8. It is interesting that in human and mouse DC, F. tularensis was a much weaker IL-10 inducer than the other TLR2 agonist tested.


Figure 6
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  		Figure 6. IL-1ß secretion is induced by live but not heat-killed F. tularensis. Human (A) and mouse (B) DC were stimulated with the indicated agonists for 20 h, and cytokine levels were measured in conditioned supernatants by ELISA. One experiment representative of three is shown.

 
DISCUSSION

As caspase-1 activation by the inflammasome is a cytoplasmic event, the lack of IL-1ß secretion during stimulation with heat-killed F. tularensis suggests that infection and escape into the cytoplasm from the phagosome are necessary for caspase-1 activation by F. tularensis and are consistent with results recently published by other groups [31 32 33 ]. It is also possible that a bacterial toxin, which may activate the inflammasome, is produced and released into the cytoplasm by live bacteria. However, no type III secretion apparatus has yet been identified in F. tularensis. Thus, our results demonstrate that production of mature IL-1ß in response to F. tularensis is regulated at two separate levels, the induction of the mRNA, which is dependent on TLR2, and the activation of caspase-1, which can be triggered only by live bacteria, which are likely situated in the cytosol.

Our results show that the interaction of F. tularensis with DC results in a powerful inflammatory response, which is primarily mediated by TLR2 and caspase-1 activation. Although TLR2 activation is triggered by live and heat-killed F. tularensis, caspase-1 activation requires live bacteria. The observed differences between the response elicited by live and heat-killed F. tularensis should be taken into consideration during the design and development of a protective vaccine for tularemia. It is tempting to speculate that the ability to detect bacterial products in the cytoplasm through the inflammasome may provide factors such as IL-1ß and IL-18, which are important for the proper development of protective T cell-mediated immunity. This may suggest that similarly to what has been observed for other intracellular bacteria such as L. monocytogenes [34 ], vaccines that rely on dead F. tularensis may not confer complete protection. The identification of the components of F. tularensis that activate TLR2 and caspase-1 should be a priority and may lead to the development of subunit vaccines, which mimic more closely infection with live F. tularensis.

ACKNOWLEDGEMENTS

This work was supported in part by the National Institutes of Health Grant AI-05466501 to F. R. We are grateful to Peter Murray (St. Jude Children’s Research Hospital) and David Hasty (U.S. Department of Veterans Affairs) for the gift of TLR2-deficient mice and to Mark Miller for the gift of F. tularensis LVS and for critically reading the manuscript.

Received April 30, 2006; revised May 25, 2006; accepted June 10, 2006.

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