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* Department of Microbiology and Immunology, University of Maryland, Baltimore, School of Medicine, Baltimore, MD 21201;
Laboratory of Mycobacterial Diseases and Cellular Immunology, Division of Bacterial, Allergenic, and Parasitic Products, Center for Biologics Evaluation and Research/Food and Drug Administration, Bethesda, MD 20852;
Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294;
Department of Basic Medical Science and/or Surgery, Shock/Trauma Research Center, University of Missouri-Kansas City, School of Medicine, Kansas City, MO 64108; and
¶ List Biological Laboratories, Campbell, CA 95008
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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108 Ft is required to cause glandular infection (8, 9). The severity of illness is also dependent upon the route of exposure; the pneumonic form of tularemia is most severe and has the highest mortality rate, i.e., >30%, when untreated (1, 2, 3, 5). As with tularemia in humans, the outcome of Ft LVS exposure in mice is dependent on both the size of the inoculum and the route of inoculation. The LD50 for an i.p. injection is <10 organisms; however, the outcome is quite different when Ft LVS are introduced s.c. or intradermally (i.d.), where the LD50 can range from 105 to 108 bacteria (6, 10, 11). Infection by the i.d. route protects some strains of mice against a subsequent lethal i.p. LVS infection (6, 10, 12), suggesting that a protective immune response is achievable. Interestingly, part of this protective response is generated quite rapidly. Two days after an i.d. inoculation, mice are able to survive an otherwise lethal i.p. or i.v. challenge (13). Infection with Ft also results in a pronounced inflammatory response (14, 15, 16, 17) that has been suggested to be responsible for most of the tissue damage associated with human tularemia (1, 18). However, little is known about how Ft elicits this profound response. Because the route of entry for Ft has such a direct impact on the outcome of infection in both humans and mice, we proposed that analysis of the cytokines and chemokines expressed after either i.p. or i.d. infection might provide insights into how inoculation by these two routes could lead to such disparate outcomes.
LPS is a primary mediator of inflammatory damage induced by Gram-negative infection. However, several previous studies indicated that Ft LVS LPS did not possess strong endotoxic activity (19, 20, 21). Recent reports that the lipid A structure of Ft LPS is tetra-acylated (22, 23) may account, in part, for its reduced endotoxicity (24). Despite its apparent lack of endotoxic properties, mice immunized with Ft LVS LPS are protected against subsequent i.p. challenge with Ft LVS (21, 25, 26). Passive transfer of sera from Ft LVS LPS-immunized mice provides some protection to naive animals against subsequent Ft LVS challenge, suggesting that anti-LPS (e.g., O-polysaccharide-specific) Abs mediate this protection (10, 27). However, mice immunized with 100 ng of Ft LVS LPS 2 days before i.p. challenge with Ft LVS LPS are fully protected, even though no anti-LPS Abs were detected at the time of challenge (21). Therefore, we theorized that Ft LVS LPS might be at least partially responsible for the proinflammatory gene expression seen after i.p. and i.d. inoculation.
In this study, we compared the abilities of Ft LVS LPS and live Ft LVS to activate genes that encode a broad spectrum of inflammatory mediators in both macrophage culture and whole animal experiments. Moreover, we sought to determine how mice could be protected against an otherwise lethal Ft LVS challenge, even when anti-LPS Abs are not present. We found that immunization with Ft LVS LPS as few as 2 days before a lethal i.p. Ft LVS challenge markedly curtailed both the bacterial burden in the liver as well as the inflammatory response. Although Abs to Ft LVS or Ft LVS LPS were not detected on the day of infection, high titers of Ab were achieved in Ft LVS LPS-pretreated mice following infection. This suggests that whereas Ft LVS LPS is not endotoxic, it is not inert and modifies the host response such that mice are better able to clear Ft LVS and, thus, prevent the tissue damage that occurs as the consequence of a robust inflammatory response. In addition, Ft LVS LPS primes the adaptive immune system such that a stronger Ab response is elicited upon infection.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Five to 6-wk-old C57BL/6J mice were purchased from The Jackson Laboratory. Mice were injected i.p. with purified Ft LVS LPS or inoculated i.p. or i.d. with Ft LVS bacteria. At the indicated times after inoculation, mice were sacrificed, and livers, lungs, spleen, and blood were collected. Organs were snap frozen in an ethanol/dry ice bath and then stored at –80°C for subsequent RNA extraction. Serum samples were collected and stored at –80°C. Serum cytokine concentrations were determined via ELISA or the Luminex 100 Total System (Luminex) by the Cytokine Core Laboratory (University of Maryland, Baltimore, MD).
Peritoneal macrophages were isolated from mice 4 days after i.p. injection of sterile 3% thioglycollate as described previously (28). Briefly, cells were washed in sterile PBS and resuspended in RPMI 1640 (Invitrogen Life Technologies) containing 2% FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. Macrophages were plated in 6-well (4 x 106 cells/well) or 24-well (1 x 106 cells/well) tissue culture plates (Corning). After overnight incubation, cells were washed with PBS to remove nonadherent cells. In experiments in which Ft LVS organisms were used in vitro, cells were cultured in antibiotic-free medium both 24 h before and during the experiment. Treatments were conducted in duplicate or triplicate. All animal experiments were conducted with Institutional Animal Care and Use Committee approval.
Reagents
The synthetic lipopeptide S-[2,3-Bis(palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-(R)-Cys-(S)-Ser-Lys4-OH, trihydrochloride (Pam3Cys) was purchased from EMC Microcollections. Escherichia coli K235 LPS was prepared by a modification of the method of McIntire et al. (29) using two rounds of hot phenol-water extraction. Ft LVS LPS was purified by List Biological Laboratories. Briefly, the LPS was extracted from a wet cell pellet of Ft LVS using a modified Westphal/Jann protocol (30). The aqueous fraction, which contained >75% of the LPS as determined by a KDO assay, was treated with RNase, DNase, and proteinase K. After treatment, the LPS mixture was centrifuged at 3076 x g to remove solids and diafiltered against 0.85% NaCl and distilled water using a 100-kDa AGT hollow fiber cartridge (Amersham Pharmacia/GE Healthcare). Lyophilized LPS was then reconstituted in water and re-extracted using a modified deoxycholate-phenol extraction protocol as described by Manthey and Vogel (31) and again subjected to treatment with DNase, RNase, and proteinase K. Finally, the LPS was subjected to a chloroform:methanol (2:1) solvent extraction and dialyzed against water. Lyophilized LPS was resuspended in pyrogen-free saline at a concentration of 1 mg/ml for use in all assays.
Bacterial infections
Frozen aliquots of Ft LVS (ATCC 29684; American Type Culture Collection) were prepared as described previously (12). Briefly, bacterial cultures were grown to mid-log phase in Mueller-Hinton (MH) broth (Difco Laboratories) supplemented with ferric pyrophosphate and IsoVitaleX (BD Biosciences), aliquot, and frozen at –70°C. Viable bacteria were quantified by plating serial dilutions on MH agar plates. For inoculations, bacteria were diluted in pyrogen-free saline, and mice were injected i.p. or i.d. with 4–5 x 104 Ft LVS as indicated in the text. Control animals were injected with saline only. Groups of five animals per treatment were sacrificed at the indicated times after inoculation. For experiments in which mice were pretreated with Ft LVS LPS before infection, groups of five animals were injected i.p. with saline or 100 ng of Ft LVS LPS 2 or 7 days before i.p. challenge with 4 x 104 Ft LVS. Forty-eight hours after Ft LVS challenge, all animals were sacrificed. All work involving Ft LVS was conducted under biosafety level 2 conditions.
Detection of anti-Ft LVS or anti-Ft LVS LPS Abs
Abs (IgM and IgG) against Ft LVS organisms or Ft LVS LPS were measured in sera as described previously (21, 27).
Real-time PCR
Total RNA was isolated from macrophage cultures and homogenized organs using RNA Stat60 (Tel-Test). Real-time PCR was performed in a sequence detector system (ABI Prism 7900 Sequence Detection System and software; Applied Biosystems) as described previously (32). Levels of mRNA for specific murine genes are reported as fold induction (relative gene expression) over background levels detected in control samples unless otherwise noted. For some genes (e.g., IFN-
), no baseline level could be detected in macrophage cultures; therefore, fold induction was calculated based on extrapolation of real-time PCR data from a standard curve of an RNA sample with detectable levels of IFN--specific mRNA. Primers were designed using the Primer ExpressÔ Program (Applied Biosystems) in conjunction with GenBank sequences or were from the literature. The following primer sets were used in these studies: IFN-
, sense (5'-CACTTGAAGAGCTATTACTGGAGGG-3') and antisense (5'-CTCGGACCACCATCCAGG-3'); IFN-, sense (5'-CTGCCACGGCACAGTCATTG-3') and antisense (5'-TGCATCCTTTTTCGCCTTGC-3') (33); IL-10, sense (5'-ATTTGAATTCCCTGGGTGAGAAG-3') and antisense (5'-CACAGGGGAGAAATCGATGACA-3'); IL-1, sense (5'-ACAGAATATCAACCAACAAGTGATATTCTC-3') and antisense (5'-GATTCTTTCCTTTGAGGCCCA-3'); IL-12p35, sense (5'-GACGTCTTTGATGATGACCCTG-3') and antisense (5'-TGTGATTCTGAAGTGCTGCGTT-3'); IL-12p40, sense (5'-TCTTTGTTCGAATCCAGCGC-3') and antisense (5'-GGAACGCACCTTTCTGGTTACA-3'); IL-4, sense (5'-GCATTTTGAACGAGGTCACAGG-3') and antisense (5'-TATGCGAAGCACCTTGGAAGC-3'); IL-6, sense (5'-TCAGGAAATTTGCCTATTGAAAATTT-3') and antisense (5'-GCTTTGTCTTTCTTGTTATCTTTTAAGTTGT-3'); inducible NO synthase (iNOS), sense (5'-GTCTTTGACGCTCGGAACTGT-3') and antisense (5'-GATGGCCGACCTGATGTTG-3'); IFN--inducible protein 10 (IP-10), sense (5'-CTTGGGATCCACACTCTCCAG-3') and antisense (5'-TTTTTGGCTAAACGCTTTCATTAA-3'); hypoxanthine phosphoribosyltransferase (HPRT), sense (5'-GCTGACCTGCTGGATTACATTAA-3') and antisense (5'-TGATCATTACAGTAGCTCTTCAGTCTGA-3'); KC, sense (5'-TGTCAGTGCCTGCAGACCAT-3') and antisense (5'-GCTATGACTTCGGTTTGGGTG-3'); MCP-1, sense (5'-TGGCTCAGCCAGATGCAG-3') and antisense (5'-GGTGATCCTCTTGTAGCTCTCCAG-3'); RANTES, sense (5'-GAGTGACAAACACGACTGCAAGAT-3') and antisense (5'-CTGCTTTGCCTACCTCTCCCT-3'); and TNF-
, sense (5'-GACCCTCACACTCAGATCATCTTCT-3') and antisense (5'-CCACTTGGTGGTTTGCTACGA-3').
To quantify bacterial replication in vivo and in vitro, primers specific for Ft LVS 16S rRNA were also designed based on the bacterial DNA sequence reported in GenBank (gi:46911542), and quantitative real-time PCR was conducted as described for the detection of murine mRNA species. The following primers were used: Ft LVS 16S rRNA, sense (5'-CAGCCACATTGGGACTGAGA-3') and antisense (5'-CACACATGGCATTGCTGGAT-3').
To ensure that detection of Ft 16S rRNA was a reliable correlate of bacterial burden, a side-by-side comparison of bacterial plate counts and levels of Ft-specific 16S rRNA was initially conducted using macrophages. Set amounts of Ft LVS (2–200,000 CFU as determined by plate counts) were added to macrophage cultures. Total RNA was isolated immediately, and Ft-specific 16S rRNA was quantified by real-time PCR. There was a direct correlation between the number of bacteria added and the level of Ft 16S rRNA in the total RNA samples derived from the cultures (r2 = 0.9989) (data not shown), indicating that eukaryotic mRNA did not interfere with the detection of Ft-specific 16S rRNA. We also compared bacterial recovery from liver with the detection of Ft 16S rRNA. Mice were injected i.p. with 4 x 104 Ft LVS, and 24, 48, and 72 h after inoculation the animals were sacrificed and the livers removed. Half of each liver sample was homogenized in 10 ml of PBS and plated for colony counts, whereas total RNA isolated from the other half was subjected to real-time PCR for detection of Ft 16S rRNA.
All primers were synthesized at the Biopolymer Core Facility (University of Maryland, Baltimore, MD).
iNOS staining of liver sections
Livers were removed and fixed in 10% buffered formalin (Sigma-Aldrich) and embedded in paraffin. After deparaffinization, endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 30 min at room temperature. For Ag unmasking, the slides were submerged in citrate buffer (pH 6) and heated for 10 min in a microwave oven. The sections were cooled in PBS and subsequently incubated with 3% nonfat dried milk for 30 min at room temperature to block nonspecific binding. Slides were treated with rabbit polyclonal Ab against mouse iNOS (Santa Cruz Biotechnology) at a 1/750 dilution at 4°C overnight. Sections were incubated with biotinylated goat anti-rabbit IgG (Vector Laboratories; 1:200) and with avidin-biotin peroxidase complexes (Vector Laboratories; 1:100) for 30 min at room temperature. Peroxidase activity was visualized with diaminobenzoate (Vector Laboratories), and nuclear counterstaining was performed with Harris hematoxylin. The slides were dehydrated and mounted with Permount (Fisher Scientific).
Plasmid constructs and reporter assay
HEK293T cells were cultured in DMEM (BioWhittaker) supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. All cells were maintained in a 37°C humidified atmosphere with 5% CO2. HEK293T cells were transfected with plasmids encoding various TLRs, coreceptor, and reporter constructs as described previously (34). Briefly, 2 x 105 HEK293T cells were seeded into each well of 12-well Costar plates (Corning). After an overnight incubation, cells in each well were transfected for 3 h with 0.5 µg of pELAM_Luc and 0.1 µg of pCMV1--gal, along with either 0.3 µg of pCDNA3-TLR4, 30 ng of pCDNA3-huCD14 (provided by Dr. D. T. Golenbock, University of Massachusetts Medical School, Worcester, MA), and 3 ng of pEFBOS-HA-huMD-2 (provided by Dr. K. Miyake, Saga Medical School, Saga, Japan), or 0.3 µg of pFLAG_TLR2 (provided by Dr. D. T. Golenbock, University of Massachusetts Medical School, Worcester, MA). pCDNA3 (Invitrogen Life Technologies) was added such that the total amount of DNA was 1.5 µg/well. All plasmid DNA was isolated using Endo-Free plasmid prep kits (Qiagen). Transfection was conducted with SuperFect transfection reagent (Qiagen). After an overnight recovery, cells were stimulated with LPS (Ft LVS or E. coli), Pam3Cys, or Ft LVS bacteria for either 5 or 24 h. Cells were lysed in reporter assay lysis buffer (Promega), and -galactosidase (Galacto-Light system; Tropix) and luciferase (Luciferase assay system; Promega) activities were analyzed using a Berthold LB 9507 Luminometer (Berthhold Technologies). Relative luciferase activity was calculated by normalizing each sample’s luciferase activity with the -galactosidase activity measured within the same sample.
Statistics
Results were analyzed using a one-way ANOVA with repeated measures, followed by a Tukey’s post hoc test for multiple paired comparisons. Data analysis was performed using GraphPad PRISM 4 program for Windows (GraphPad).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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In mice, the outcome of Ft LVS infection is dependent on the size of the inoculum as well as the route of inoculation. When mice are injected i.p., the LD50 has been estimated to be <10 organisms, whereas the LD50 following i.d. inoculation (i.e., into the base of the tail) ranges from 1 x 105 to 1 x 108 CFU (10, 11, 12). Because lethal infection with Ft in man is associated with a profound inflammatory response, we sought to measure bacterial load and cytokine expression to gain insights into how inoculation by these two routes could lead to such disparate outcomes.
Using real-time PCR, we compared the expression of Ft LVS-specific 16S ribosomal RNA (as an indirect measure of bacterial burden) in the total RNA derived from the organs of mice inoculated with 5 x 104 CFU mouse i.p. or i.d. To insure that detection of Ft 16S rRNA was a reliable correlate of bacterial burden, we compared bacterial recovery from mouse livers to levels of Ft-specific 16S rRNA in the same livers. Fig. 1A illustrates that recovery of viable bacteria from livers over time correlates directly with the detection Ft 16S rRNA (r2 = 0.9213). Therefore, using Ft 16S rRNA as a surrogate marker for Ft replication in mice challenged i.p. or i.d., we found that the bacterial burden was greatest in mice infected with Ft LVS i.p. and that the liver contained the greatest amount of specific transcript regardless of route of infection. Even at 72 h postinfection, the amount of Ft LVS 16S rRNA was considerably lower in mice infected i.d. than in the 48-h samples from mice infected i.p. These data demonstrate that i.p. infection leads to higher bacterial burden in the liver, followed by spleen and lung, than i.d. infection.
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Regardless of the route of inoculation, the liver always displayed the largest relative increases in proinflammatory gene expression (summarized in Table I) and the highest bacterial burden (Fig. 1). TNF-, KC, IFN-, and iNOS mRNA species, in particular, were most strongly up-regulated in the livers of mice inoculated by either route, whereas there was far less expression of these specific genes elicited in lungs and spleens (Table I). Histological examination of the livers of mice sacrificed at 48 h also revealed that mice inoculated i.p. had larger and more numerous granulomas than either the control mice injected with saline only or mice inoculated with Ft LVS i.d. (Fig. 3). In addition, only mice infected i.p. developed liver granulomas that expressed intracellular iNOS by this time point.
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Recognition of LPS by the innate immune system is a critical first step in controlling Gram-negative infection. Although enterobacterial LPS signals through TLR4 (reviewed in Refs. 35, 36, 37), several nonenterobacterial species of LPS are able to signal through TLR2 (e.g., Porphyromonas gingivalis (38), Leptospira interrogans (39), Bacteroides fragilis (40), Chlamydia trachomatis (40), and Pseudomonas aeruginosa (40)). It has been proposed that the three-dimensional shape of the LPS dictates which TLR will be used, with conical-shaped LPS (e.g., E. coli) signaling through TLR4 and more cylindrically shaped LPS (e.g., P. gingivalis) signaling through TLR2 (41). Because the lipid A of Ft LVS LPS has been reported to have an unusual tetra-acylated structure (rather than penta- or hexa-acylated) and other chemical modifications (22, 23), we initially sought to determine whether Ft LVS LPS signaled through TLR4 or TLR2.
To this end, HEK293T cells were transfected with plasmids encoding either human TLR2 or TLR4/MD-2/CD14 to reconstitute TLR2 and TLR4 signaling complexes, respectively. TLR4/MD-2/CD14-transfected cells were activated only at the highest concentration of Ft LVS LPS tested, i.e., 10,000 ng/ml. Although statistically significant (p < 0.05), this level of activation is minimal when compared with the positive control for TLR4 signaling, E. coli LPS, which induced 10-fold greater activation of the NF-
B luciferase reporter at a concentration that was 1,000-fold less. Cells transfected with TLR2 did not respond to Ft LVS LPS at any concentration tested (Fig. 4A). We also considered that Ft LVS LPS might signal through another TLR; however, no signaling through TLR3, TLR5, TLR7, TLR8, or TLR9 was observed when tested in the same system (data not shown).
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We also used the HEK293T transient transfection system to study whether live Ft LVS bacteria, rather than the purified Ft LVS LPS, signaled though either TLR2 or TLR4. Live Ft LVS organisms failed to elicit signaling through TLR4 (Fig. 4C, top graph), but organisms induced significant, dose-dependent NF-B reporter activity through TLR2 (Fig. 4C, bottom graph) (p < 0.0001). Equivalent concentrations of MH broth alone did not induce NF-B reporter activity through TLR2 (data not shown).
Effect of Ft LVS LPS and Ft LVS infection of murine macrophages
Although Ft LVS LPS induced essentially no activation through TLR4 or TLR2, we considered the possibility that the lack of activation was not due to lack of engagement of the receptor per se, but rather, lack of an appropriate coreceptor or adapter protein in the HEK293T cells. To investigate this possibility, we exposed primary murine macrophages to Ft LVS LPS for 3 h at concentrations ranging from 0.1–10,000 ng/ml. Table III illustrates that only at Ft LVS LPS concentrations of 
1,000 ng/ml was there minimal activation of some of the proinflammatory genes, with the level of gene expression being much lower than that induced by 0.1 ng/ml E. coli LPS for most genes examined. Measurement of cytokine concentrations in the macrophage culture supernatants confirmed the real-time PCR data (data not shown). As was observed in the HEK239T cells transfected with TLR2 or TLR4/MD-2/CD14, concurrent or pretreatment of macrophages with 10,000 ng/ml Ft LVS LPS failed to enhance or inhibit inflammatory gene expression induced by E. coli LPS (0.1–10 ng/ml) at either the mRNA or protein level (data not shown).
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, IL-6, IL-12 p40, IL-12 p35, and KC (data not shown). We next sought to determine whether live Ft LVS could modulate macrophage gene expression. Peritoneal macrophages were exposed to Ft LVS for 24 h at a multiplicity of infection (MOI) of 1, 5, or 20. These results sharply contrast with that seen in response to Ft LVS LPS. Analysis by real-time PCR showed that proinflammatory genes were strongly up-regulated after exposure to Ft LVS, even at a MOI = 1 (Table IV).
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Previous studies have shown that when mice are pretreated with as little as 1 ng of Ft LVS LPS 3 days before i.p. infection or with 100 ng of Ft LVS LPS 2 days before i.p. infection, they survived an otherwise lethal challenge with Ft LVS. However, no anti-LPS Abs were detected in the sera of these mice on the day of challenge (21). Because Ft LVS LPS apparently lacks endotoxic properties, we sought to determine how such a small amount of a seemingly inert material could afford protection against an otherwise lethal challenge.
Groups of five mice were injected i.p. with either saline or 100 ng of Ft LVS LPS, 2 or 7 days before i.p. challenge (on day 0) with 4 x 104 CFU Ft LVS. Forty-eight hours after challenge, all animals were sacrificed. Hepatic mRNA was analyzed for both bacterial burden and gene expression. Fig. 5A illustrates that pretreatment with Ft LVS LPS strongly blunts the bacterial burden in mice subsequently infected with Ft LVS i.p. Specifically, Ft-specific 16S rRNA was nearly undetectable in mice pretreated with Ft LVS LPS 7 days before infection (day –7), and even a 2-day pretreatment (day –2) greatly decreased the amount of detectable 16S rRNA compared with that seen in mice pretreated with saline. In parallel with reduced bacterial burden, induction of all proinflammatory genes measured was markedly inhibited (Fig. 5B; Table V provides a summary of all genes examined). IFN- and iNOS mRNA levels were particularly sensitive to Ft LVS LPS pretreatment. These data were confirmed at the level of protein for TNF-, IL-12 p70, IL-6, IFN-, KC, MCP-1, RANTES, and IL-10 (Table VI). In addition, the number of granulomas detected in the liver 2 days after Ft LVS infection was reduced in mice pretreated with Ft LVS LPS when compared with mice pretreated with saline (7.5 ± 2.9 vs 21.4 ± 4.3 granulomas/field).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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B and MAPK activation and TNF- secretion in vitro (47, 48, 49, 50), infection with Ft in vivo invokes a profound inflammatory response (14, 15, 16, 17) that is thought to underlie the tissue damage associated with tularemia (18). Although attenuated for humans, the Ft LVS strain is lethal for mice when administered i.p. and results in a disease that closely resembles human tularemia (6, 10, 51). In contrast, administration of Ft LVS to mice i.d. is not lethal unless the innate or adaptive immune response is compromised. Specifically, mice deficient in MyD88 (52) or IFN- (53) are susceptible to i.d. infection, as are mice that lack cellular components of the adaptive immune response (e.g., SCID (53) and nude mice (13)). Previous studies have also shown that mice inoculated s.c. with live Ft LVS, but not with killed bacteria, exhibit increased levels of hepatic TNF-, IFN-, IL-12, and IL-10 mRNA within 48-h postinfection (16, 17). These data suggest that Ft viability is an important component of the proinflammatory response to infection in vivo.
The data presented in this study represent the first comprehensive analysis of the early cytokine response to Ft LVS infection in mice and have revealed unexpected results. Using quantitative real-time PCR, we found that whether mice were injected i.p. or i.d., the largest bacterial burdens and strongest cytokine responses were consistently detected in the liver. A notable exception was IL-10 mRNA, which was greatly enhanced only in the lungs at 48 and 72 h after i.p. and i.d. infection, respectively (Fig. 3 and Table I). Although qualitatively similar patterns of proinflammatory gene expression were observed after i.p. or i.d. administration, i.p. infection consistently led to an earlier and more robust proinflammatory response and higher bacterial burdens. The exception to this was IFN- mRNA, which was increased in the liver only after i.d. inoculation.
TNF- and IFN- were expressed almost exclusively in the liver after both i.p. and i.d. challenge. These cytokines have been shown previously to be crucially important in the control of Ft LVS infection. IFN- knockout mice die after i.d. challenge (53), and, conversely, IFN- treatment of mice enhances resistance to infection (54). IFN- treatment of peritoneal or bone marrow-derived macrophages also severely limits Ft LVS replication (55). Ab neutralization of TNF- or IFN- converts an otherwise nonlethal i.d. infection into one that is lethal (56), but anticytokine treatment is only effective when given within 2 days of challenge. SCID (53, 57) and nude (13) mice are able to survive for several weeks after i.d. Ft LVS challenge; however, the mean time to death can be shortened if mice are treated with Abs to TNF- or IFN-. Collectively, these findings suggest that cytokines play an important role in the initial control of infection (56). Our data indicate that up-regulation of iNOS, KC, IL-12 p35, and IL-12 p40 mRNA was also primarily confined to the liver, and iNOS was the most strongly up-regulated gene after i.p. infection. Both reactive oxygen and nitrogen species have been implicated in control of Ft LVS infection; iNOS-deficient mice exhibit severe liver pathology after i.d. Ft LVS infection, with an accompanying decrease in the LD50 (58). Interestingly, we observed that mice infected i.p. had numerous granulomas in the liver that expressed iNOS by 48 h (Fig. 3), whereas mice infected i.d. have far fewer granulomas that were smaller and expressed little or no iNOS. The up-regulation of KC is interesting because KC is a neutrophil chemoattractant, and neutrophils have been shown to be critical for the control of Ft infection: mice depleted of neutrophils die after low-dose i.v. or i.d. challenge (59, 60). In summary, our data support the role of these cytokines in the pathology associated with lethal i.p. infection and strengthen the hypothesis that the liver is the primary site for both bacterial replication and the proinflammatory storm that underlies the tissue damage associated with tularemia.
For most Gram-negative bacteria, LPS is a powerful proinflammatory stimulus. Although Ft LVS LPS can activate complement, it is poorly endotoxic (19, 20). Although enterobacterial species of LPS engage TLR4, and some unusual LPS species engage TLR2 (38, 39), Ft LVS LPS failed to activate NF-B in HEK293T cells that expressed TLR2, TLR3, TLR5, TLR7, TLR8, or TLR9, or in wild-type murine macrophages that respond to soluble Toxoplasma gondii tachyzoite Ag through TLR11. Minimal activation of HEK293T cells that express TLR4/MD-2/CD14 heterologously, or murine macrophages that express endogenous TLR4/MD-2/CD14, was detected only at the highest concentration of Ft LVS LPS tested (10,000 ng/ml; Fig. 4A). These findings were confirmed in the RAW 264.7 macrophage cell line stimulated with lipid A derived from this same Ft LVS LPS (N. Qureshi, unpublished observation), indicating that, at best, Ft LVS LPS is a weak TLR4 agonist. Because Ft LVS LPS has been reported to be tetra-acylated (22, 23), we also considered the possibility that it might exert an inhibitory effect on human TLR4 as is seen with tetra-acylated lipid IVA or deacylated LPS (61), or the penta-acylated LPS antagonist, R. sphaeroides LPS or its diphosphoryl lipid A in murine or human cells (42). Ft LVS LPS failed to antagonize E. coli LPS-induced activation of proinflammatory genes in murine macrophages (data not shown) or signaling through either human TLR2 or TLR4 in the HEK293T transfectants (Fig. 4B). Ft LVS LPS also failed to induce a state of endotoxin tolerance in macrophages restimulated with E. coli K235 LPS (data not shown). Perhaps the low toxicity of Ft LVS LPS conveys an infectious advantage, because this may enable bacteria to evade the host innate immune response.
Previous studies have shown that TLR4 expression does not play a role in controlling infection because there was no difference in responses of TLR4-defective C3H/HeJ and wild-type C3H/HeOuJ mice after aerosol challenge with a type A Ft (62), and survival of TLR4 knockout mice was equivalent to wild-type mice after i.d. Ft LVS infection (52). Although Ft LVS LPS did not signal through any of the known TLRs, we found that live Ft LVS activated NF-B in TLR2-expressing HEK239T cells. TLR2 typically recognizes bacterial lipopeptides derived from Gram-positive bacteria (63, 64, 65). Recent studies by Michalek and colleagues (72) have confirmed our findings of a role for TLR2 in responses to Ft LVS using bone marrow-derived dendritic cells from TLR2 and TLR4 knockout mice. Moreover, we have recently found that in contrast to wild-type macrophages, TLR2-null macrophages respond minimally or not at all to Ft LVS infection with the induction of cytokine and chemokine gene expression and protein synthesis (data not shown). Because Ft has been shown to inhibit phagosome/lysosome fusion, ingested organisms would be expected to be retained in the phagosome longer, thereby prolonging the period over which TLR2 signaling could occur. Finally, our data do not preclude the possibility of a synergistic interaction between TLR2 and TLR4. To the best of our knowledge, no such interaction has been described previously, although synergy between TLR4 and TLR5 has been described in response to bacterial flagellin (66).
When injected i.d., MyD88-deficient mice are extremely susceptible to Ft LVS, whereas TLR2 knockout mice are only slightly more susceptible than wild-type controls (52). The differential susceptibility of MyD88-deficient and TLR2 knockout is much less pronounced after intranasal challenge. After intranasal inoculation with Ft LVS, 100% of wild-type mice survived, whereas only 20% of TLR2 knockout and 0% of the MyD88-deficient mice survived with mean times to death of 8 and 6 days, respectively (67). The differential susceptibility of the TLR2 and MyD88 knockout mice suggests that whereas TLR2 recognition of Ft is important for pathogen recognition, an additional MyD88-dependent pathway(s) may contribute to the control of Ft LVS infection. IL-1 and IL-18 also use MyD88 for signaling; however, studies with IL-1 and IL-18 knockout mice indicate that these cytokines do not play a major role in resistance to Ft LVS infection (52).
Although Ft LVS LPS is seemingly inert with respect to its endotoxic activities, it appears to protect against infection. Passive transfer of Abs against Ft LVS LPS provides some protection against low-dose i.p. challenge with Ft LVS (25, 26). However, anti-Ft LVS LPS immunization did not protect mice challenged with the highly virulent type A strain, Schu4, although an increased mean time to death was reported (26). Ft LVS LPS-immunized mice subsequently depleted of T cells survive i.p. challenge with Ft LVS, suggesting that anti-LPS Abs or other B cell functions are sufficient to limit infection, even in the absence of T cell-mediated immunity (26). Abs appear to be directed against the O-polysaccharide of the LPS, because mice vaccinated with protein-conjugated O-polysaccharide of Ft LVS were completely protected against i.d. challenge and partially protected against aerosol challenge with a highly virulent type B strain (68). Analysis of the O-polysaccharides from Ft LVS and Schu S4 strains indicates that the two strains have identical O Ag repeats (69, 70) and similar immunological properties (70). Taken together, such data suggest that Ab alone is not sufficient to confer complete protection against all strains.
Dreisbach et al. (21) provided the first evidence of a possible Ft LVS LPS-mediated protection that is possibly Ab independent. Mice injected with as little as 0.1 ng of Ft LVS LPS 3 days before Ft LVS challenge were completely protected against subsequent i.p. challenge with 1 x 103 LVS, yet no anti-LPS Abs were detected on the day of infection (21). In our hands, as little as 0.1 ng of Ft LVS LPS administered 2 days before challenge with 1 x 103 CFU protected four of five mice (data not shown). T cell-deficient, nude mice pretreated with 100 ng of Ft LVS LPS 3 days before challenge survive 2–3 wk longer than euthymic control mice that were not Ft LVS LPS-pretreated; however, the nude mice eventually succumbed (21). This demonstrated that whereas the low-dose Ft LVS LPS pretreatment allows mice to survive the initial innate immune phase of infection, T cells are required for long-term survival. B cells appear to play an important role in mediating the protection afforded by low-dose Ft LVS LPS pretreatment, because B cell-deficient mice pretreated with Ft LVS LPS died in the same time frame as wild-type control mice pretreated with saline (21). Our results show that protection of mice with injection of as little as 100 ng/mouse Ft LVS LPS 2 days before i.p. infection was correlated with a decreased bacterial burden and mitigated proinflammatory response (Fig. 5 and Tables V and VI). Perhaps a dampening effect on bacterial growth, combined with a blunting of the usually strong proinflammatory response that is normally elicited by i.p. infection, allows the mice to survive long enough to develop an Ag-specific, adaptive immune response that is protective. Supportive of this hypothesis is our confirmation that a low dose of Ft LVS LPS given 2 days before challenge fails to elicit a detectable Ab response at the time of challenge with an otherwise lethal dose of bacteria. Yet, 2 days later, when the inflammatory response and bacterial burden are significantly diminished, high Ab titers are detectable. Because it has been previously reported that Ft LVS LPS failed to activate murine B cells for proliferation or polyclonal Ab secretion (21), or induce inflammatory responses (this report), our findings suggest that part of the protective mechanism may be due to priming for subsequent Ab production. This low-dose Ft LVS LPS-induced protection appears to require intact Ft LVS LPS, because neither the free lipid A moiety nor O-polysaccharide alone were protective (71).
Based on these findings, we propose a model of Ft LVS infection in mice that occurs in two phases: 1) an initial, innate immune phase in which cytokine expression is crucial for survival; and 2) a later, adaptive immune phase, where protection is both T cell-dependent and mediated, at least in part, by anti-Ft LVS LPS Abs. Proinflammatory gene expression (as seen in i.d. infection) is key to surviving the innate immune phase of infection, although an overexuberant response (as seen in i.p. infection) may ultimately cause inflammatory damage to organs.
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1 This work was supported by National Institutes of Health (NIH) Grants AI-57168 and AI-44936 (to S.N.V.), AI-56460 and DE-09081 (to S.M.M.), as well as a National Institutes of Allergy and Infectious Diseases/NIH Intramural Biodefense Award (to K.L.E.). 
2 Address correspondence and reprint requests to Dr. Stefanie N. Vogel, Department of Microbiology and Immunology, University of Maryland, Baltimore, 660 West Redwood Street, Room 324, Baltimore, MD 21201. E-mail address: svogel{at}som.umaryland.edu
3 Abbreviations used in this paper: Ft, Francisella tularensis; LVS, live vaccine strain; i.d., intradermal; Pam3Cys, S-[2,3-Bis(palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-(R)-Cys-(S)-Ser-Lys4-OH, trihydrochloride; MH, Mueller-Hinton; iNOS, inducible NO synthase; IP-10, IFN--inducible protein 10; HPRT, hypoxanthine phosphoribosyltransferase; MOI, multiplicity of infection.
Received for publication October 31, 2005. Accepted for publication March 2, 2006.
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, and interferon after cutaneous Francisella tularensis infection in tularemia-immune mice. Infect. Immun. 67: 1789-1797. interferon. Infect. Immun. 68: 1988-1996. interferon and T cells. Infect. Immun. 62: 3129-3137. B signaling pathway components. J. Immunol. 170: 508-519. interferon in resistance of mice to experimental tularemia. Microb. Pathog. 7: 421-428. [Medline]This article has been cited by other articles:
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), 48 (•), or 72 (
) h after i.p. inoculation with 4 x 104 Ft LVS. The calculated number of Ft LVS organisms per liver, determined by plate counts, was plotted against the relative amount of Ft 16S rRNA, as determined by real-time PCR. B, Ft LVS bacterial burden in the liver, lung, and spleen of mice infected i.p. vs i.d. was determined by measuring the relative amount of Ft 16S rRNA present in total RNA derived from the liver, lung, and spleen using real-time PCR. Groups of five animals were injected with saline or 
