Male C3H/FeJ and C3H/HeJ mice (8- to 12-week-old) were purchased from Jackson Laboratories (Bar Harbor, ME). Animals were housed in cages with filter tops in specific pathogen-free conditions. Sterile food and water were provided ad lib. All experiments were done in compliance with the Animal Care Committee Rules of the University of California at San Francisco, and all protocols were approved.

P. aeruginosa parental PA103 strain and its isogenic mutant PA103ΔUT missing both ExoU and ExoT toxins were generously provided by Dara W. Frank (Medical College of Wisconsin, Milwaukee, WI) 15. Bacteria from frozen stocks were streaked onto trypticase soy agar plates and grown in a deferrated dialysate of tryptic soy broth supplemented with 10 mM nitrilotriacetic acid (Sigma Chemical), 1% glycerol, and 100 mM monosodium glutamate and grown at 33°C for 13 h in a shaking incubator. Cultures were centrifuged at 8,500 × g for 5 minutes, and the bacterial pellet was washed twice and diluted into the appropriate number of CFU/ml in lactated Ringer's solution, determined by spectrophotometry. The bacterial concentration was confirmed by plating out the known dilutions of the samples on sheep blood agar plates.

Mice were briefly anesthetized with inhaled sevoflurane (Ultane, Abbot Laboratories, Abbot Park, Illinois, USA) and placed supine, at an angle of approximately 30°. Fifty microliters of bacterial solution were instilled slowly in the left lobe of the lungs through a gavage needle (Modified animal feeding needle, 24 G, Popper & Sons, Inc., New Hyde Park, NY) inserted into the trachea via the oropharynx. The proper insertion of the needle in the trachea was confirmed by palpation of the tip of the blunt needle through the skin and the observation of movement of the solution inside the syringe with respiratory efforts 28.

The severity of epithelial lung injury was quantified by measuring the efflux of the ¹³¹I-labeled albumin (the alveolar protein tracer) across the alveolar epithelium into the circulation, as previously described 28. The alveolar instillate was made up of 0.5 μCi of ¹³¹I-labeled human serum albumin (Merck-Frost, Quebec, Canada) and 0.05 mg of anhydrous Evan's blue in 2 ml of lactated Ringer's solution with 5% BSA. An appropriate quantity of the specified P. aeruginosa was added to the 50 μl volume, if the experiment was to include bacteria. The total radioactivity (in counts/minute/g) in the instillate was measured in a γ-ray counter (Auto-Gamma, model 5550, Packard, Downers Grove, IL). Four hours after the instillation, mice were deeply anesthetized with intraperitoneal sodium pentobarbital. Blood was collected by a carotid arterial puncture and median sternotomies were done to harvest and measure the radioactivity of pleural fluids, lungs, tracheas, oropharynxes and stomachs. The percentage of radioactive albumin that left the lung and entered the circulation was calculated by multiplying the counts per gram measured in the terminal blood sample times the blood volume of the animal (body weight × 0.07). The lungs were homogenized in sterile containers with sterile 0.9% saline solution. Lung homogenates were placed in pre-weighed aluminium pans and dried to constant weight in an oven at 80°C for 3 days. The water to dry weight ratios of lung homogenates were used as an index of lung edema.

Blood was plated onto sheep blood agar plates for quantitative assessment of CFU/ml. Lung homogenates were sequentially diluted then plated onto sheep blood agar plates. Colony forming units were calculated per gram of tissue.

Mouse lungs were lavaged by instilling and withdrawing 1 ml of phosphate buffered saline (PBS). Supernatants of centrifuged BAL fluids (BALF) were collected and processed for cytokine measurement.

BALF samples were mixed with the solution including 0.01% crystal violet dye and 2.7% acetic acid for leukocyte staining and erythrocyte hemolysis. The number of leukocytes in BLAF was counted by a hemacytometer under a microscope.

Biological activity of TNFα in plasma and BALF was measured by analyzing the cytotoxicity of TNFα on the fibrosarcoma cell line WEHI-13VAR, as reported previously 1529. Cytokine ELISA was performed for IL-10, as previously described 28. Sample concentrations were calculated by comparison with standard curves using recombinant cytokine.

Mantel-Cox log rank test was used for survival analysis. One-way analysis of variance (ANOVA) followed by the Bonferroni multiple comparison test or the Dunn's Multiple Comparisons test were used for comparisons among 3 groups. Student's t-test or Mann-Whitney test were used for comparisons between 2 groups. The Kruskal-Wallis non-parametric test with Dunn's test was used for the comparisons of leukocyte counts in BALF. Significance was accepted at p < 0.05.

To examine whether a lack of normal TLR4 signaling affects the mortality induced by P. aeruginosa pneumonia, we performed survival studies using LPS-resistant C3H/HeJ mice. LPS-sensitive C3H/FeJ mice (the background strain of C3H/HeJ) were used as control groups. Mice were intratracheally infected with wild type P. aeruginosa PA103 at doses of either 5 × 10³ CFU (low dose), 6 × 10⁴ CFU (medium dose), or 5 × 10⁵ CFU (high dose), or with an isogenic mutant PA103ΔUT, which is missing the expression of TTS toxins (ExoU and ExoT), at a dose of 5 × 10⁵ CFU (high dose).

After the instillation of a low dose (5 × 10³ CFU) wild type PA103, 30% of LPS-resistant C3H/HeJ mice died in 3 days, while all C3H/FeJ survived for a week (Fig. 1a). However, statistically significant difference was not detected between the two strains of mice. After the instillation of a medium dose (6 × 10⁴ CFU) of wild type PA103, 65% of LPS-resistant C3H/HeJ mice died in 4 days, while all C3H/FeJ survived for a week (Fig. 1b). There was statistically significant difference between C3H/HeJ and C3H/FeJ mice. After the instillation of high dose (5 × 10⁵ CFU) wild type PA103, only 30% of C3H/FeJ and only 15% C3H/HeJ mice, respectively, survived for more than 3 days (Fig. 1c). The results suggest that, in the comparison between C3H/HeJ and C3H/FeJ mice, LPS-resistant C3H/HeJ mice were more susceptible to wild type PA103 at three different doses. The result implies that defective TLR4 signaling was not beneficial for the survival of infected mice with cytotoxic PA103.

Next, we examined whether the mortality of infected mice depended on the phenotypes of the TTS toxins of P. aeruginosa. An isogenic mutant PA103ΔUT missing ExoU and ExoT was intratracheally administered at a high dose (5 × 10⁵ CFU) to the airspaces of C3H/HeJ and C3H/FeJ mice. Although instilled at a high dose, none of the mice died for a week (Fig. 1d). Therefore, the mortality we observed in the mice infected with a high dose of P. aeruginosa PA103 was totally dependent upon the TTS virulence of P. aeruginosa rather than the genotype of TLR4 in mice.

We quantified acute lung injury in the mice infected with P. aeruginosa. Acute lung epithelial damage was quantified by measuring the efflux of alveolar protein tracers from the lungs into the bloodstream (Fig. 2a). Lung edema was quantified by measuring the water to dry weight ratios of the lung (Fig. 2b). The instillation of a high dose (5 × 10⁵ CFU) of PA103 led to a significant increase of lung epithelial injury (Fig. 2a) and severe increases of lung edema (Fig. 2b) in both groups of mice. When the mice were infected with a medium dose (6 × 10⁴ CFU) of PA103, both severe lung epithelial injury and lung edema occurred in the both mouse strains (Fig. 2a). Although the injury levels in C3H/HeJ were slightly higher, these were not statistically significant. In contrast, the intratracheal instillation of PA103ΔUT even at a high dose (5 × 10⁵ CFU) did not cause lung injury in either of the mice (Fig. 2a and 2b). This implies that, in our mouse model, the bacterial dose and virulence of the bacteria, but not the TLR4 phenotype of mice, determines the initial severity of acute lung injury. Because more C3H/HeJ mice infected with a medium dose (6 × 10⁴ CFU) of PA103 died 3 days after infection in the survival studies (Fig. 1a), we analyzed lung edema 3 days after a medium dose instillation (6 × 10⁴ CFU) of PA103 in the strains of mice. In contrast to C3H/FeJ mice, C3H/HeJ mice showed a significantly higher level of lung edema (Fig. 2c).

We quantified the ability of the different strains of mice to clear the bacteria from their infected lungs and the severity of bacteremia during infection. Four hours after a high dose instillation (5 × 10⁵ CFU) of wild type PA103, both strains of mice had similar quantities of bacteria in their lungs (Fig. 3a). However, 4 h after the instillation of a medium dose (6 × 10⁴ CFU) of the wild type PA103, there was a significantly larger quantity of bacteria in the lungs of the C3H/HeJ mice. The quantity of bacteria in the lungs of the C3H/HeJ mice was significantly increased after 3 days of infection (6 × 10⁴ CFU) compared to the quantity of bacteria in the C3H/FeJ mice (Fig. 3b). The lungs of C3H/HeJ infected with high dose (5 × 10⁵ CFU) PA103ΔUT also showed a significantly higher level of bacterial burden in their lungs in comparison to the lungs of the C3H/FeJ mice (Fig. 3a) although all the mice survived for a week after instillation of the same dose of PA103ΔUT in the survival study (Fig. 1c). This result implies that C3H/HeJ mice could not clear the bacteria efficiently from the infected lungs even 3 days after the infection (Fig. 3b). Persistent lung infection observed in C3H/HeJ mice explained higher edema (Fig. 2c) and mortality (Fig. 1a) of the mice infected with a medium dose of PA103.

We evaluated the severity of bacteremia in C3H/HeJ and C3H/FeJ mice. Only a high dose instillation of PA103 caused detectable bacteremia 4 h after the infection in either strain of mice (Fig. 3c). Among the two strains, C3H/HeJ mice showed a significantly higher level of bacteremia 4 h after infection (Fig. 3c), although more than 70% of mice in the both strains eventually died within a week after the instillation of bacteria (Fig. 1b). Neither the instillation of a high dose of PA103ΔUT nor the instillation of a medium dose of PA103 resulted in bacteremia 4 h after the infection (Fig. 3c). However, 3 days after the instillation of the medium dose PA103, C3H/HeJ mice showed a significantly greater level of bacteremia while C3H/FeJ mice had either minimal bacteremia or none, respectively (Fig. 3d). Therefore, the results imply that decreased bacterial clearance probably lead to a delay in the onset of bacteremia in C3H/HeJ mice.

We measured the number of leukocytes in BALFs which were collected 16 h after infection from the mice infected with either PA103 or PA103ΔUT at a medium dose (Fig. 4). More leukocytes were found in the BALF from the mice infected with PA103ΔUT than in the BALF from the mice infected with PA103. More leukocytes were found in the BALF of C3H/FeJ mice than in the C3H/FeJ mice. The number of leukocytes in the BALF of C3H/FeJ mice infected with PA103 was the lowest among the four groups, and significantly lower than that of C3H/HeJ mice infected with PA103ΔUT. The results implied that C3H/HeJ mice failed to recruit inflammatory cells efficiently into the site of infection. In addition, when infection was by virulent P. aeruginosa secreting TTS toxins, few leukocytes were detected in BALF, implying that virulent P. aeruginosa suppressed the proper inflammatory cell recruitment or eliminated the recruited leukocytes by its cytotoxicity.

We measured concentrations of TNFα in BALF and in the serum of infected animals. TNFα in BALF was almost undetectable in C3H/HeJ mice 4 h after the instillation of a high dose (5 × 10⁵ CFU) of either wild type PA103 or of PA103ΔUT (Fig. 5a). Only in the C3H/FeJ mice there was a significant difference in the TNFα in BALFs between PA103 and PA103ΔUT infections (Fig. 5a). Only C3H/FeJ mice infected with a high dose of PA103 showed a significantly increased serum concentration of TNFα compared to those in the other strains of mice infected with either PA103 or PA103ΔUT (Fig. 5b).

We also measured the serum concentration of the anti-inflammatory cytokine IL-10 in the mice infected with a high dose of PA103. The concentration of IL-10 4 h after infection was higher in the C3H/HeJ mice, but the difference not statistically significant (Fig. 5c).