Lack of In Vitro and In Vivo Recognition of Francisella tularensis Subspecies Lipopolysaccharide by Toll-Like Receptors

Bacterial strains and growth conditions.F. novicida strain U112, obtained from Francis Nano (University of Victoria, Victoria, Canada) was grown in tryptic soy broth (Gibco BRL, Grand Island, NY) supplemented with 0.1% cysteine (TSB-C) (Sigma-Aldrich, St. Louis, MO) at 37°C with aeration and harvested in stationary phase. F. tularensis subsp. tularensis (type A), F. tularensis subsp. holarctica (type B), and F. tularensis subsp. mediasiatica were from the University of Umeå, Umeå, Sweden. All strains are catalogued in the Francisella strain collection (Defense Research Agency, Umeå, Sweden). These strains were grown on chocolate II agar plates supplemented with hemoglobin and IsoVitaleX (Becton Dickinson Diagnostic Systems, San Jose, CA) at 37°C in a 5% CO₂ incubator for 7 or 5 days, respectively, before harvesting. The Francisella subspecies isolates used are listed in Table 1.

LPS purification and lipid A isolation.Large-scale F. novicida LPS preparations were extracted using a hot phenol-water extraction method (45). Subsequently, LPS was treated with RNase A, DNase I, and proteinase K to ensure purity from contaminating nucleic acids and proteins (14). Individual LPS samples were additionally extracted to remove contaminating phospholipids (15) and TLR2-contaminating proteins (21). The yield of LPS per mg dry cells was 0.91 mg LPS/10 mg dry cells. Small-scale LPS preparations were isolated using the rapid isolation method for mass spectrometry analysis as described previously (46). Lipid A was isolated after hydrolysis in 1% sodium dodecyl sulfate at pH 4.5 as described previously (7). Briefly, 500 μl of 1% sodium dodecyl sulfate in 10 mM Na acetate, pH 4.5, was added to a lyophilized sample. Samples were incubated at 100°C for 1 h, frozen, and lyophilized. The dried pellets were resuspended in 100 μl of water, and 1 ml of acidified ethanol (100 μl 4 N HCl in 20 ml 95% ethanol). Samples were centrifuged at 5,000 rpm for 5 min. The lipid A pellet was further washed (three times) in 1 ml of 95% ethanol. The entire series of washes was repeated twice. Samples were resuspended in 500 μl of water, frozen on dry ice, and lyophilized.

Fatty acid analysis.LPS fatty acids were derivatized to fatty acid methyl esters and analyzed by gas chromatography as described previously (9, 37). Briefly, LPS fatty acids were derivatized to fatty methyl esters with 2 M methanolic HCl at 90°C for 18 h (Alltech, Lexington, KY) and identified and quantified by gas chromatography using an HP 5890 series II with a 7673 autoinjector. Pentadecanioc acid (10 μg; Sigma-Aldrich, St. Louis, MO) was added as an internal standard.

Mass spectrometry procedures.Negative-ion matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) experiments were performed as described for the analysis of LPS or lipid A preparations with the following modifications (13, 18). Lyophilized lipid A was dissolved with 10 μl 5-chloro-2-mercaptobenzothiazole (Sigma-Aldrich, St. Louis, MO) MALDI matrix in chloroform-methanol, 1:1 (vol/vol), and then applied (1 μl) onto the sample plate. All MALDI-TOF experiments were performed using a Bruker Autoflex II MALDI-TOF mass spectrometer (Bruker Daltonics, Inc., Billerica, MA). Each spectrum was an average of 200 shots. ES tuning mix (Agilent, Palo Alto, CA) was used to calibrate the MALDI-TOF MS.

Additional endotoxins.Escherichia coli O111:B4 and Salmonella minnesota Re595 LPS (Sigma-Aldrich, St. Louis, MO) were reconstituted to 5 mg/ml in 20 mM EDTA or H₂O, clarified in an ultrasonic water bath (Cole-Parmer, Vernon Hills, IL), aliquoted, and stored at −80°C. Lipid IV_A and Rhodobacter sphaeroides lipid A (RSLA) were from D. T. Golenbock (University of Massachusetts).

THP-1 cell stimulations.THP-1 cells (200 μl at 2 × 10⁵ cells/ml) were plated in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum (HyClone, Logan, UT), 2 mM l-glutamine, and 50 nM vitamin D₃ (Sigma-Aldrich, St. Louis, MO) in 96-well plates (Corning Costar, Acton, MA) and incubated at 37°C in humid air with 5% CO₂ (13). After 72 h, the medium was replaced with fresh medium containing sonically dispersed LPS ligands or no added stimulus. After 6 h or 24 h of incubation, supernatants were harvested and stored at −80°C until assayed. Interleukin-8 (IL-8) and tumor necrosis factor alpha (TNF-α) production by THP-1 cells was measured by enzyme-linked immunosorbent assay (ELISA) 22 h after stimulation per the manufacturer's instructions (Pierce-Endogen, Rockford, IL).

RAW 264.7 cell stimulation.RAW 264.7 cells (200 μl at 2 × 10⁵ cells/ml) were plated in RPMI containing 10% fetal calf serum (HyClone, Logan, UT) in flat-bottom 96-well plates 2 days before stimulation. On the day of stimulation, the medium was replaced with fresh medium containing various ligands in the presence of 5 μg/ml brefeldin A. Cells were stimulated for 3 h at 37°C in a CO₂ incubator followed by staining for intracellular TNF-α as previously described (20). Fc receptors were blocked with 5% goat serum in 50 μl fluorescence-activated cell sorter (FACS) buffer (1% bovine serum albumin in phosphate-buffered saline plus 0.09% sodium azide) on ice for 10 min. Paraformaldehyde (150 μl at 2% [vol/vol] concentration) was then added to each well, and the cells were incubated at room temperature for 15 min. Cells were washed once in phosphate-buffered saline (PBS) and then stained and permeabilized with 0.1% saponin (in PBS with 1% fetal calf serum and 0.1% sodium azide) and 1:100 R-phycoerythrin-anti-TNF-α (PharMingen, San Diego, CA) for 30 min at room temperature. Cells were washed twice in PBS and analyzed on a FACScan (BD Biosciences, San Jose, CA) using CellQuest Pro software.

HEK-293 transfections.HEK-293 cells (200 μl at 2 × 10⁵ cells/ml) were plated in Dulbecco's modified Eagle's medium plus 10% fetal calf serum in flat-bottom 96-well plates the day before transfection (19). The next day, 5 μl of transfection reagent (1:1 mix of 0.25 M CaCl₂ containing DNA and 2× BBS [50 mM BES, 280 mM NaCl, 1.5 mM Na₂HPO₄]) were added to each well. The following amounts of DNA were added per well: ELAM-Luc, 0.01 μg; Renilla-Luc, 0.0002 μg; murine CD14 (mCD14) or human CD14 (hCD14), 0.0025 μg; mMD-2 or hMD-2, 0.0025 μg; mTLR2 or hTLR2, 0.0025 μg (when alone) or 0.00125 μg (when cotransfected with TLR1 or TLR6); mTLR1, 0.0125 μg; mTLR6, 0.00125 μg; hTLR1, 0.00125 μg; hTLR6, 0.0125 μg; mTLR4, 0.00025 μg; hTLR4, 0.002 μg. All TLR constructs were hemagglutinin tagged, and the amount of TLR DNA used was normalized based on relative expression from anti-hemagglutinin Western blots. All transfections were normalized to 0.05 μg total DNA with the addition of empty vector. After 3 h, the medium was replaced with fresh medium. The cells were stimulated the next day for 4 h and then lysed with 50 μl passive lysis buffer (Promega, Madison, WI), and luciferase activity was measured in 10 μl of the lysate using the Dual Luciferase reporter assay system (Promega, Madison, WI).

Stimulation of mouse MH-S alveolar macrophages.Low-passage MH-S cells in RPMI 1640 medium containing 10% heat-inactivated fetal calf serum, 10 mM HEPES, 2 mM l-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin were seeded onto 24- or 48-well plates (Corning Costar, Acton, MA) that had been pretreated with 0.01% poly-l-lysine (Sigma-Aldrich, St. Louis, MO) and incubated at 37°C in humid air with 5% CO₂. After 20 h, the medium was replaced with fresh medium containing sonically dispersed LPS ligands or no added stimulus. After 6 h or 24 h of incubation, supernatants were harvested and stored at −80°C until assayed.

Animals.Male and female C57BL/6 mice, 6 to 8 weeks of age, were purchased from Jackson Laboratories (Bar Harbor, ME). Mice were housed in filtered cages under specific-pathogen-free conditions and permitted unlimited access to sterile food and water. All animal experiments were approved by the Institutional Animal Care and Use Committee of the University of Washington.

Animal exposure to aerosolized LPS and bacteria.For each LPS exposure, an aliquot of F. novicida or E. coli LPS was thawed, sonically dispersed, and diluted in sterile, endotoxin-free PBS (Mediatech, Herndon, VA). For challenges with live bacteria, F. novicida was grown to stationary phase in TSB-C, washed twice, and resuspended in endotoxin-free PBS. Mice were exposed to aerosolized LPS or live bacteria in a 36-port nose-only chamber (In-Tox Products, Moriarty, NM). Aerosols were generated from UniHeart jet nebulizers (Westmed, Tucson, AZ) containing LPS suspended in 5 ml PBS at concentrations of 10, 100, or 1,000 μg/ml or live F. novicida suspended in 5 ml PBS at 10⁸ (low dose) or 10⁹ (high dose) CFU/ml (estimated by optical density and confirmed by quantitative culture). Control animals were exposed to aerosolized PBS. The nebulizers were driven at 40 lb/in², and airflow through the chamber was maintained at 5 liter/min by negative pressure for the 10-min exposure period. Immediately after exposure to each concentration of live bacteria, 3 animals were killed with an overdose of intraperitoneal pentobarbital to determine bacterial deposition by quantitative culture of homogenized lung tissue. Four hours and 24 h after aerosol exposure, mice were killed with pentobarbital and exsanguinated by cardiac puncture before undergoing bronchoalveolar lavage (BAL), as described previously (36). Normal mice unexposed to aerosols also were lavaged for additional control specimens. BAL cells were pelleted by centrifugation and counted in a hemacytometer, and differentials were determined by examination of cytocentrifuge slides stained with Diff-Quik (Dade Behring, Dudingen, Switzerland). BAL fluid supernatants were stored at −80°C.

Measurement of cytokines.Murine TNF-α, macrophage inflammatory protein-2 (MIP-2, CXCL2), keratinocyte-derived chemokine (KC, CXCL1), IL-10, and human IL-8 (CXCL8) were measured by sandwich ELISA, using antibody pairs and recombinant standards purchased from R&D Systems (Minneapolis, MN).

Data analysis.Data are expressed as means ± standard errors. Statistical comparisons among groups for continuous variables measured at multiple time points were made by one-way analysis of variance with Tukey's post hoc test. A P value of ≤0.05 was considered significant.

Characterization of lipid A isolated from laboratory and clinical isolates of Francisella tularensis subspecies.The structure of the lipid A component of LPS was determined from clinical and environmental isolates from F. tularensis subspecies. Lipid A was isolated from 7 F. tularensis subsp. tularensis (type A) isolates, 10 F. tularensis subsp. holarctica (type B) isolates, 3 F. tularensis subsp. mediasiatica isolates, and 1 F. novicida isolate grown at 37°C (Table 1) in TSB-C. Individual LPS preparations were hydrolyzed to lipid A using mild acid hydrolysis conditions and analyzed by MALDI-TOF MS (Fig. 1; representative examples for each F. tularensis subspecies are shown).

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FIG. 1.

MALDI-TOF MS of lipid A of Francisella tularensis subspecies. Lipid A was isolated from clinical and environmental isolates of F. tularensis subspecies (Table 1) after growth at 37°C and analyzed by MALDI-TOF MS in the negative-reflection ion mode. Representative examples for each F. tularensis subspecies are shown. (A to C) F. tularensis subsp. Tularensis (type A); (D to F) F. tularensis subsp. holarctica (type B); (G and H) F. tularensis subsp. mediasiatica; (I) F. novicida.

MALDI-TOF analysis of lipid A isolated from all clinical and environmental isolates of F. tularensis subspecies grown at 37°C (Fig. 1; Table 1) contained two tetra-acylated molecular ion species, m/z 1,665 and m/z 1,637. Higher-order MS (IRC-FT-MS) structural analysis of only these molecular ion species for F. novicida lipid A showed that the species at m/z 1,665 represented a single lipid A structure that contained 2 3-OH C₁₈, 1 3-OH C₁₆, and 1 C₁₈ acyl group, as previously described for two type B isolates (30, 44). However, the molecular ion species at m/z 1,637 represented a mixture of four different lipid A structures (three structures that contained 2 3-OH C₁₈, 1 3-OH C₁₆, and 1 C₁₆ acyl groups at different locations on the lipid A backbone and one structure that contained 3 3-OH C₁₈ and 1 C₁₄ acyl group) (data not shown). In addition, lipid A structures (m/z 1,637 and 1,665) contained a phosphogalactosamine group located at the 1 position on the diglucosamine backbone as previously described (30). Finally, the two smaller molecular ion species at m/z 1,476 (from m/z 1,637) and 1,504 (from m/z 1,665) represent tetra-acylated lipid A structures corresponding to the loss of the 1 position phosphogalactosamine (m/z 161) and may represent precursors in the biosynthetic pathway of F. novicida lipid A or possibly artifacts of the samples preparation or MS analysis. As shown previously, the m/z 1,504 structure is similar to the lipid A structure isolated from F. tularensis subsp. holarctica strain LVS (44). Fatty acid quantitation by gas chromatography corroborated the interpretation of the MALDI-TOF spectra (data not shown).

Neither human nor murine monocyte/macrophage cell lines respond to F. novicida LPS.Since the structure of F. novicida lipid A was identical to that of all of the clinical and environmental isolates of F. tularensis subspecies (Fig. 1), we further characterized the biological activity of F. novicida LPS (Fig. 2C). The human monocytic THP-1 cell line and the murine macrophage RAW 264.7 cell line were each stimulated with increasing concentrations of F. novicida LPS (1 to 1,000 ng/ml). Salmonella minnesota Re595 LPS was used as a positive control (Fig. 2D). F. novicida LPS did not stimulate IL-8 production by THP-1 cells or TNF-α by RAW 264.7 cells at all concentrations tested (Fig. 2A and B). S. minnesota Re595 LPS, a prototypic highly acylated LPS, was a potent stimulator of both human and murine cells, resulting in production of high levels of IL-8 and TNF-α at all concentrations tested (Fig. 2A and B). These results demonstrate that human and murine cells that can respond to prototypical enteric LPS do not recognize tetra-acylated F. novicida LPS.

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FIG. 2.

Lack of responsiveness to F. novicida LPS by monocytic cells. Human THP-1 cells (A) or murine RAW 264.7 cells (B) were stimulated with increasing amounts of F. novicida LPS or S. minnesota Re595 LPS. IL-8 secretion was measured in supernatants by ELISA 18 h after stimulation of THP-1 cells, and intracellular TNF-α production was measured by FACS in RAW cells 3 h after stimulation. Values are the means of results from triplicate wells ± standard deviations. One experiment representative of three similar experiments is shown. Chemical structures of the tetra-acylated lipid A from F. novicida (m/z 1,665) (C) and the hexa-acylated lipid A from S. minnesota Re595 (m/z 1797) (D) are shown.

Neither human nor murine TLR4 or TLR2 recognize F. novicida LPS structures.To test whether F. novicida LPS was recognized by TLR4 or TLR2, a human embryonic kidney cell line (HEK-293) that does not respond to LPS was transiently transfected with either mTLR4 or hTLR4, MD-2, and hCD14. Using ELAM-luciferase as a readout of NF-κB activity and β-actin-Renilla luciferase as a transfection control, cells transfected with hTLR4-MD-2 or mTLR4-MD-2 were found to respond only to the highest concentration (1,000 ng/ml) of F. novicida LPS (Fig. 3A and B). In contrast, S. minnesota Re595 LPS was a potent stimulator of both hTLR4 and mTLR4 even at 1 ng/ml (Fig. 3A and B). These responses required expression of all three components of the LPS receptor complex (CD14, MD-2, and TLR4), and similar responses were observed when mCD14 was substituted for hCD14 (data not shown). Identical responses were also observed when LPS preparations were repurified by phenol extraction to eliminate trace amounts of lipoprotein and when lipid A rather than LPS was used (data not shown). As controls, cells were also stimulated with 1,000 ng/ml of the disaccharide precursor of lipid A from enteric bacteria termed lipid IV_A, a hTLR4 antagonist but mTLR4 agonist structure, and RSLA, an hTLR4 and mTLR4 antagonist structure (17). As expected, RSLA did not induce the reporter in either transfection, whereas lipid IV_A did so only in mTLR4-MD-2-transfected cells (Fig. 3A and B).

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FIG. 3.

Neither TLR4 nor TLR2 mediates recognition of F. novicida LPS. HEK-293 cells were transiently transfected with hTLR4-MD-2 (A), mTLR4-MD-2 (B), hTLR2 ± hTLR1 or TLR6 (C), or mTLR2 ± mTLR1 or TLR6 (D). All cells were also transfected with hCD14. Cells were stimulated for 4 h with indicated ligands, and ratios of the ELAM-luciferase reading to the β-actin-Renilla luciferase reading (relative light units) are plotted. IL-1β (10 ng/ml), Pam₃CSK₄ (1 μg/ml), or F. novicida LPS (1 μg/ml) was added to each well in experiments for which results are shown in panels C and D. Values are the means of results from triplicate wells ± standard deviations. One experiment representative of three similar experiments is shown. Unstim, unstimulated.

LPS from some bacterial species have been demonstrated to signal through TLR2 (reviewed in (23). Therefore, we also transfected HEK-293 cells with hTLR2 or mTLR2 either alone or in combination with TLR1 or TLR6. The cells were then stimulated with hIL-1 as a positive control (HEK-293 cells express IL-1 receptors), the TLR2 ligand Pam₃CSK₄, or F. novicida LPS (Fig. 3C and D). Although good induction of the reporter construct was observed for IL-1 and Pam₃CSK₄, the F. novicida LPS preparation did not demonstrate any activity in this assay.

F. novicida LPS is not a TLR4 antagonist.Since F. novicida LPS did not mediate activation through TLR4, we next determined whether it could act as an antagonist. RSLA, which does not signal via hTLR4 or mTLR4 (Fig. 3A and B) but rather inhibits responses to stimulatory LPS, was used as a positive control of antagonism (Fig. 4). RAW 264.7 or MH-S cells, mouse alveolar macrophages, were stimulated with a constant amount of stimulatory S. minnesota Re595 LPS in the presence of increasing amounts of RSLA or F. novicida LPS. While the response to S. minnesota Re595 LPS was completely inhibited in the presence of 100 ng/ml RSLA in both cell lines, this response was unaffected even in the presence of 100 ng/ml of F. novicida LPS. A similar lack of antagonism by F. novicida LPS was observed in HEK-293 cells transiently transfected with hTLR4 or mTLR4 (data not shown).

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FIG. 4.

F. novicida LPS does not inhibit responsiveness to Re595 LPS. RAW cells (A) or MH-S cells (B) were stimulated with S. minnesota Re595 LPS (0.1 ng/ml [A] or 1 ng/ml [B]) alone or in the presence of R. sphaeroides lipid A (1, 10, or 100 ng/ml) or F. novicida LPS (1, 10, or 100 ng/ml). Intracellular TNF-α production after 3 h is shown in panel A, and TNF-α in supernatants after 6 h is shown in panel B. Data are means ± standard deviations (A) or means ± standard errors of the means (B) of results from triplicate wells. Data are representative of two independent experiments.

Aerosolized F. novicida LPS does not stimulate TLR4-mediated responses.To determine whether F. novicida LPS was stimulatory in vivo, we exposed mice to aerosolized F. novicida or E. coli LPS and compared neutrophil (PMN) recruitment and chemokine production in BAL fluid (Fig. 5). E. coli LPS at concentrations of 100 or 1,000 μg/ml induced intrapulmonary secretion of the CXCR2 ligands MIP-2 and KC and stimulated an influx of neutrophils into the bronchoalveolar airspaces by 4 h after aerosol exposure. In contrast, F. novicida LPS did not induce chemokine production or neutrophil recruitment either 4 h after exposure (Fig. 5A to C) or 24 h after exposure (not shown).

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FIG. 5.

Aerosolized F. novicida LPS or whole bacteria do not induce lung inflammation. Mice were exposed to aerosols containing F. novicida LPS or E. coli 0111:B4 LPS at concentrations of 100 or 1,000 μg/ml of LPS. Aerosols of live bacteria resulted in bacterial depositions of 52 ± 16 CFU (low, mean ± standard error of the mean) or 3,042 ± 1,048 CFU (high) in the lungs. After 4 h and 24 h, mice underwent bronchoalveolar lavage to enumerate cell counts (% PMN in panel A) and assay MIP-2 (B) and KC (C) by ELISA. Control animals were exposed to aerosolized PBS or were not exposed (none). Data are means ± standard errors of the means (n = 3 to 8 mice). ***, P < 0.001 compared to all other groups.

Aerosolized live F. novicida does not stimulate airway inflammation, consistent with lack of recognition of LPS.To determine whether whole F. novicida was stimulatory in vivo, we exposed mice to two concentrations of aerosolized F. novicida and measured PMN recruitment and chemokine production in BAL (Fig. 5). As a control for airway immune responses, aerosolization of the gram-negative opportunistic pathogen Pseudomonas aeruginosa resulted in recruitment of large numbers of PMN (>10⁵) and production of significant amounts of cytokines and chemokines by 4 h postinfection (34) (data not shown). In contrast, F. novicida did not induce chemokine, TNF, or IL-10 production or neutrophil recruitment 4 h postexposure (Fig. 5A to C and data not shown).