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Am J Physiol Lung Cell Mol Physiol 292: L312-L322, 2007. First published August 25, 2006; doi:10.1152/ajplung.00250.2006
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Redundant Toll-like receptor signaling in the pulmonary host response to Pseudomonas aeruginosa

Shawn J. Skerrett,1 Christopher B. Wilson,2 H. Denny Liggitt,3 and Adeline M. Hajjar2

Departments of 1Medicine, 2Immunology, and 3Comparative Medicine, University of Washington School of Medicine, Seattle, Washington

Submitted 30 June 2006 ; accepted in final form 22 August 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Activation of pulmonary defenses against Pseudomonas aeruginosa requires myeloid differentiation factor 88 (MyD88), an adaptor for Toll-like receptor (TLR) signaling. To determine which TLRs mediate recognition of P. aeruginosa, we measured cytokine responses of bone marrow cells from wild-type mice and mice lacking TLR2 (TLR2–/–), TLR4 (TLR4–/–), TLR2 and TLR4 (TLR2/4–/–), or MyD88 (MyD88–/–) to wild-type P. aeruginosa and to fliC P. aeruginosa, which lacks the TLR5 ligand flagellin. Mice also were challenged with aerosolized bacteria to determine cytokine responses, lung inflammation, and bacterial clearance. TNF induction required MyD88 and was absent in TLR2/4–/– cells in response to fliC but not wild-type P. aeruginosa, whereas TLR2–/– cells exhibited augmented responses. In vivo, TLR4–/– mice responded to wild-type P. aeruginosa with reduced cytokine production and inflammation, but intact bacterial clearance, while TLR2–/– mice had partially impaired cytokine responses and delayed bacterial killing despite normal inflammation. When challenged with fliC, MyD88–/– mice failed to mount early cytokine and inflammatory responses or control bacterial replication, resulting in necrotizing lung injury and lethal disseminated infection. TLR4–/– and TLR2/4–/– mice responded to fliC infection with severely limited inflammatory and cytokine responses but intact bacterial clearance. TLR2–/– mice had partially reduced cytokine responses but augmented inflammation and preserved bacterial killing. These data indicate that TLR4- and flagellin-induced signals mediate most of the acute inflammatory response to Pseudomonas and that TLR2 has a counterregulatory role. However, MyD88-dependent pathways, in addition to those downstream of TLR2, TLR4, and TLR5, are required for pulmonary defense against P. aeruginosa.

bacterial pneumonia; lung inflammation

PSEUDOMONAS AERUGINOSA is an important cause of acute pneumonia in critically ill and immunocompromised individuals and of chronic lower respiratory tract infection in the setting of cystic fibrosis (47). Host resistance to P. aeruginosa pneumonia is dependent on the activation of an innate immune response that recruits neutrophils to the lungs (47, 63). The initiation of this response requires the recognition of bacterial ligands by signaling receptors that induce proinflammatory cytokines and chemokines (30). In turn, these mediators stimulate the upregulation of adhesion molecules and create chemotactic gradients that result in the emigration of neutrophils from the bloodstream into the air spaces of the lungs (39).

Toll-like receptors (TLRs) have been found to recognize diverse pathogen-associated molecular patterns and to have prominent roles in the activation of innate immune responses to infection (60). TLRs are type I transmembrane glycoproteins that share structural homology and signaling pathways with the interleukin-1 (IL-1) receptor family (3). More than 10 mammalian TLRs have been described, each with distinct ligand specificities (3, 60). Gram-negative bacteria, such as P. aeruginosa, present multiple structures for specific recognition by TLRs. P. aeruginosa LPS is recognized by TLR4 (22) and has been shown to induce lung inflammation in vivo (54). Cell wall lipopeptides produced by P. aeruginosa activate mammalian cells through TLR2 (4, 18). Both TLR2 and TLR4 are involved in signaling responses to the capsular alginate produced by mucoid strains of P. aeruginosa (19) and to exoenzyme S, a virulence factor secreted by respiratory isolates of P. aeruginosa (11). Flagellin expressed by P. aeruginosa is recognized by TLR5 (2, 23), possibly with some signaling assistance from TLR2 (2), and can induce lung inflammation in vivo (2, 26, 37). Bacterial DNA contains unmethylated cytosine-phosphate-guanosine (CpG) motifs that are known ligands for TLR9 (24) and can elicit neutrophil recruitment to the lungs (51).

TLRs 1–10 are expressed in lung tissue (68), and individual TLRs are differentially regulated in specific lung cell populations in response to microbial stimulation (2, 13, 40, 43, 58). Of the TLRs likely to participate in recognition of gram-negative bacteria, functional TLR2, TLR4, TLR5, and TLR9 are expressed by respiratory epithelial cells (2, 5, 20, 52). Alveolar macrophages express TLR2, TLR4, and TLR5 (10, 13, 43, 45) and are known to respond to ligands for TLR2, TLR4, and TLR9 (16, 45). Thus TLR2, TLR4, TLR5, and TLR9 are the TLRs most likely involved in recognition of bacteria in the lungs.

Recently, we (55) reported that mice with targeted deletions of myeloid differentiation factor 88 (MyD88) were profoundly susceptible to P. aeruginosa pneumonia. MyD88 is an adaptor molecule that functions to link receptors of the TLR/IL-1R family with downstream signaling cascades that lead to NF-{kappa}B translocation and transcription of proinflammatory genes (3). We (55) found that MyD88–/– mice (mice lacking MyD88) were unable to mount rapid cytokine and inflammatory responses to pulmonary challenge with P. aeruginosa, failed to contain local or systemic bacterial replication, and succumbed to overwhelming infection. These findings indicated that MyD88-dependent signaling pathways are essential for the activation of innate immunity to P. aeruginosa in the lungs. We hypothesized that TLR2, TLR4, and TLR5 participated in MyD88-dependent recognition of P. aeruginosa in the lungs and that deficient activation of one or more of these TLRs would reproduce the phenotype of MyD88–/– mice. To test this hypothesis, we challenged mice lacking TLR2, TLR4, or both TLR2 and TLR4 with wild-type P. aeruginosa or with bacteria lacking the TLR5 ligand flagellin (fliC). Our results support discrete roles for TLR2, TLR4, and TLR5 in pulmonary innate immunity to P. aeruginosa, but also indicate that MyD88 is an essential component of redundant pathways involved in host defense against bacterial pneumonia.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Mice.

Mice with targeted deletions of MyD88 (MyD88–/–), TLR2 (TLR2–/–), and TLR4 (TLR4–/–) were obtained from Dr. S. Akira (Osaka, Japan) (1, 27, 62) and were backcrossed to C57BL/6 mice (Jackson Laboratories, Bar Harbor, ME) for at least six generations. Heterozygous progeny were intercrossed to generate null and wild-type control mice on similar genetic backgrounds for each strain. Mice lacking both TLR2 and TLR4 (TLR2/4–/–) were created by crossing TLR2–/– and TLR4–/– mice. Genotypes were confirmed by analysis of tail-snip DNA. In some experiments, C57BL/6 mice purchased from Jackson Laboratories were used as wild-type controls. TLR4 mutant C3H/HeJ and genetically similar but TLR4 wild-type C3HeB/FeJ mice were purchased from Jackson Laboratories. Mice were maintained under specific pathogen-free conditions and had unlimited access to sterile food and water. Mice were 6–12 wk of age at the time of experimental infection. All animal experiments were approved by the Institutional Animal Care and Use Committee of the University of Washington (Seattle, WA).

Bacteria.

The wild-type, motile PAK strain of P. aeruginosa was a gift from Dr. S. Lory (Harvard University) and was prepared as described (56). The fliC nonmotile mutant of PAK, in which the gene encoding flagellin protein has been interrupted (15), was a gift from Dr. A. Prince (Columbia University). For each experiment, frozen bacteria were inoculated into Luria-Bertani (LB) broth (Invitrogen, Carlsbad, CA), incubated for 6 h at 37°C on a rotating platform, and then diluted 1:100 in fresh LB broth. After 16–18 h of incubation at 37°C, the stationary phase bacteria were pelleted, washed twice in PBS, and suspended in PBS to a concentration adjusted by optical density at 540 nm. Bacterial suspensions were quantitatively cultured on LB agar using the pour plate method. Media used for culturing fliC P. aeruginosa contained 60 µg/ml gentamicin (Abbott Laboratories, Chicago, IL). Heat-killed bacteria were prepared by incubating washed bacterial suspensions at 65°C for 30 min.

Cytokine induction in vitro.

Bone marrow cells were flushed from the femurs and tibias of mice and washed by centrifugation and resuspension, and red blood cells were lysed in buffered ammonium chloride (0.15 M NH4Cl, 1 mM NaHCO3, 0.1 mM EDTA, pH 7.4). The cells were plated in 96-well microtiter plates (1 x 106 cells/well) in DMEM containing 10% FBS (Hyclone, Logan, UT). For intracellular cytokine induction, the cells were stimulated with heat-killed bacteria [multiplicity of infection (MOI) 0.1 or 1.0] at 37°C in the presence of 0.15 µl/well of monensin (GolgiStop; BD Pharmingen, San Diego, CA) to allow for intracellular accumulation of TNF-{alpha}. Control conditions included the TLR2 agonist Pam3CSK4 (1 µg/ml; EMC Microcollections, Tuebingen, Germany), the TLR4 agonist Salmonella minnesota Re595 LPS (100 ng/ml; Sigma-Aldrich, St. Louis, MO), the TLR5 agonist purified S. typhimurium flagellin (2 µg/ml; a gift from Dr. K. Smith, University of Washington; Ref. 57), the TLR7/8 agonist R848 (20 µM; GLSynthesis, Worcester, MA), or no added stimulus. After 5 h of incubation, the cells were centrifuged in the plate, blocked with anti-Fc receptor antibody (clone 2.4G2; BD Biosciences, San Jose, CA) in PBSA (1% BSA in PBS plus 0.09% sodium azide), and then stained with phycoerythrin-conjugated rat anti-CD11b (BD Pharmingen). The cells then were fixed and permeabilized with 100 µl/well Cytofix/Cytoperm (BD Pharmingen) and stained with FITC-conjugated rat anti-mouse TNF-{alpha} (Caltag, Burlingame, CA). The cells were analyzed on a FACScan flow cytometer using CellQuest Pro software (Becton-Dickinson, Franklin Lakes, NJ).

Murine model of pneumonia and tissue processing.

Mice were exposed to aerosolized P. aeruginosa in a whole animal chamber as described previously (55, 56). In each experiment, mice from one or more TLR-deficient line and an equal number of wild-type controls were infected simultaneously. Immediately, 4 and 24 h after infection, mice were killed with an intraperitoneal injection of pentobarbital then exsanguinated by cardiac puncture. Bacterial deposition was determined by quantitative culture of homogenized lung tissue harvested from four mice immediately after infection. At the 4- and 24-h time points, the left lungs and spleens from 4–6 mice in each group were homogenized in 1 ml PBS for quantitative culture and cytokine measurements. The trachea of each mouse was cannulated, and the right lung lavaged with four 0.5-ml aliquots of 0.9% NaCl supplemented with 0.6 mM EDTA. The right lung was then inflated to 15-cm pressure with 4% paraformaldehyde and immersed in the same fixative.

Bronchoalveolar lavage cell counts and lung histopathology.

Cell counts in bronchoalveolar lavage (BAL) specimens were measured in a hemocytometer. Differentials were determined from examination of cytocentrifuge slides (Thermo Shandon, Pittsburgh, PA) that were stained with a modified Wright-Giemsa technique (Diff-Quik; Dade Behring, Dudingen, Switzerland). Lung tissue was embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Tissue sections were examined by a veterinary pathologist who was blinded to each specimen's genotype and time after infection. The intensity of peribronchial, perivascular, and alveolar inflammation and intensity of bronchial, vascular, and alveolar necrosis each were scored on a scale of 0–4, as described (54).

Measurements of cytokines and total protein.

Lung homogenates in PBS were diluted 1:1 in lysis buffer containing 2x protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany), incubated on ice for 30 min, and then centrifuged at 1,500 g. Supernatants were harvested and stored at –80°C until assayed. Immunoreactive TNF-{alpha}, IL-1beta, macrophage inflammatory protein-2 (MIP-2), and keratinocyte-derived chemokine (KC) were measured in lung homogenate specimens and in bone marrow cell supernatants by sandwich ELISA, using DuoSet antibody pairs and recombinant standards from R&D Systems (Minneapolis, MN) according to the manufacturer's instructions. Total protein in BAL fluid was measured using the bicinchoninic acid assay (Pierce, Rockford, Il).

Data analysis.

Data are expressed as means ± SE. Statistical comparisons among multiple groups were made by one-way ANOVA, with Tukey's post hoc test for multiple comparisons to identify individual differences. In experiments where only two groups were compared, Student's t-test was used for statistical analysis. A P value of <0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Cytokine response to P. aeruginosa in vitro.

To determine which TLRs mediate recognition of P. aeruginosa in vitro, we incubated primary bone marrow cells harvested from wild-type, MyD88–/–, TLR2–/–, TLR4–/–, and TLR2/4–/– mice with the PAK strain of P. aeruginosa or a flagellin-deficient (fliC) mutant of PAK and then measured intracellular TNF-{alpha} accumulation as an indicator of early cytokine response. Representative histograms from one experiment are shown in Fig. 1A, and the combined results of three separate experiments including results with positive and negative controls are shown in Fig. 1B. Wild-type cells responded to both strains of P. aeruginosa with induction of TNF-{alpha}. MyD88–/– cells did not produce TNF-{alpha} in response to either PAK or fliC. TLR2-deficient cells exhibited normal responses to flagellated and unflagellated bacteria, whereas TLR4-deficient cells showed significantly blunted responses to both PAK and fliC. Bone marrow cells lacking both TLR2 and TLR4 exhibited a reduced response to flagellated P. aeruginosa but no response to fliC. As expected, TLR2-deficient cells failed to respond to Pam3CSK4, TLR4-deficient cells failed to respond to LPS, and TLR2/4-deficient cells did not respond to either stimulus. Cells lacking TLR2 and/or TLR4 responded normally to the TLR7/8 ligand R848 (Fig. 1B) and to flagellin (single experiment, not shown), whereas MyD88-deficient cells did not respond to any of the TLR ligands. Thus the rapid TNF-{alpha} response of bone marrow cells to P. aeruginosa is MyD88-dependent and can be entirely accounted for by signaling mediated by TLR2, TLR4, and flagellin (TLR5).


Figure 1
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  		Fig. 1. Pseudomonas aeruginosa stimulates TNF-{alpha} production via Toll-like receptor 2 (TLR2), TLR4, and TLR5. Primary bone marrow cells from the indicated mice were stimulated with heat-killed P. aeruginosa (HKPA) or the flagellin-deficient fliC mutant of P. aeruginosa (HKfliC). A: intracellular cytokine responses after 5-h stimulation at a multiplicity of infection (MOI) of 1; the % of cells producing TNF-{alpha} is indicated in each density plot. Unstim, unstimulated; WT, wild-type; MyD88–/–, mice with targeted deletions of myeloid differentiation factor 88 (MyD88). DKO, double knockout. B: combined results of 3 separate experiments. *P < 0.05 compared with WT, mice lacking TLR4 (TLR4–/–); {dagger}P < 0.05 compared with WT, TLR2–/–; {ddagger}P < 0.05 compared with all other groups; §P < 0.05 compared with WT; ¶P < 0.05 compared with WT, TLR2–/–, TLR4–/–. ICC, intracellular cytometry; Pam3, Pam3CSK4.

 
Role of TLR4 in response to P. aeruginosa in vivo.

To determine the role of TLR4 in the innate immune response to P. aeruginosa in the lungs, wild-type mice and mice with targeted deletions or spontaneous mutations of TLR4 were exposed to aerosolized bacteria. In TLR4–/– mice that were challenged with a low inoculum of P. aeruginosa [bacterial deposition 5.8 x 104 ± 7.2 x 103 colony-forming units (CFU)/lung], intrapulmonary cytokine and chemokine responses were significantly blunted in the TLR4–/– mice both 4 and 24 h after infection (Fig. 2A). At the 4-h time point, mean concentrations of TNF-{alpha}, IL-1beta, and MIP-2 in lung homogenates from TLR4-deficient mice were 64%, 46%, and 49% lower, respectively, than in samples from wild-type controls (all P < 0.05), whereas the reduction in KC was not significant (P = 0.12). In parallel with reduced cytokine responses to infection in TLR4-deficient mice, the mean number of neutrophils present in BAL specimens was reduced in TLR4–/– mice by 43% at 4 h after infection (P = 0.18) and by 45% at 24 h after infection (P = 0.02) compared with wild-type controls (Fig. 2B). However, bacterial clearance from the lungs did not differ significantly between TLR4–/– and wild-type animals (Fig. 2C). There also were no differences between the two groups of mice in the number of bacteria cultured from spleens 4 or 24 h after infection (not shown).


Figure 2
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  		Fig. 2. Role of TLR4 and flagellin in host response to P. aeruginosa pneumonia. TLR4–/– and WT control mice were exposed to aerosolized wild-type P. aeruginosa (PAK; A--C) or to the fliC mutant of P. aeruginosa (D–F). Immediately, 4 h, and 24 h after infection, left lungs were homogenized for cytokine measurements by ELISA (A and D) and for quantitative culture (C and F), and right lungs were lavaged for enumeration of neutrophils (PMN) (B and E). Data are means ± SE, n = 4–5 mice per group per time point. *P < 0.05 compared with control. {dagger}P < 0.001 compared with control. Representative of 2 separate experiments with PAK and 2 separate experiments with fliC. MIP-2, macrophage inflammatory protein-2; KC, keratinocyte-derived chemokine; CFU, colony-forming units.

 
Similar observations were made in experiments comparing the responses of TLR4 mutant C3H/HeJ mice with those of wild-type C3HeB/FeJ controls. After low-dose infection (bacterial deposition 3.4 x 104 ± 1.0 x 104 CFU/lung), mean concentrations of TNF-{alpha}, IL-1beta, MIP-2, and KC in BAL samples of TLR4 mutant mice were reduced by 86%, 53%, 78%, and 75%, respectively, compared with controls 4 h after infection (all P < 0.05). The number of neutrophils present in BAL samples 4 h after infection was more than 10-fold lower in TLR4 mutant mice compared with wild-type controls [2.1 x 106 ± 2.3 x 105 vs. 1.7 x 105 ± 4.4 x 104 polymorphonuclear leukocytes (PMN)/lung, n = 4, P = 0.003]. However, the number of viable bacteria present in lung tissue 4, 24, and 48 h after infection did not differ significantly between the two groups of mice. After aerosol challenge with a higher inoculum of P. aeruginosa (bacterial deposition 5.3 x 105 ± 1.6 x 105 CFU/lung), intrapulmonary cytokine levels and BAL neutrophil responses again were significantly lower in C3H/HeJ vs. C3HeB/FeJ mice without affecting bacterial clearance (not shown). Thus TLR4-mediated signaling is involved in the cytokine and inflammatory responses to P. aeruginosa in the lungs but is not required for effective bacterial clearance.

Role of flagellin/TLR5-mediated signaling in response to P. aeruginosa in vivo.

To determine the role of flagellin and, by inference, TLR5 in mediating the innate immune response to P. aeruginosa, wild-type and TLR4–/– mice were infected with the flagellin-deficient fliC mutant of PAK using an inoculum nearly identical to that used for PAK (bacterial deposition 6 x 104 CFU/lung). Wild-type mice responded to this infection with cytokine and PMN responses that were indistinguishable from those observed when wild-type mice were challenged with the same inoculum of PAK (Fig. 2, D and E compared with A and B). However, intrapulmonary cytokine levels 4 h after infection with fliC were >80% reduced in TLR4–/– mice compared with wild-type controls, and early neutrophil recruitment was blunted by >99% in TLR4-deficient mice (Fig. 2, D and E). The impairment in cytokine and inflammatory responses of TLR4-deficient mice to fliC was greater than the reduction in the responses of TLR4-deficient mice to PAK (Fig. 2, D and E compared with A and B). The clearance of flagellin-deficient P. aeruginosa from the lungs was not affected by the absence of TLR4 and followed a similar course to that observed for PAK. These observations suggest that in the presence of TLR4, flagellin-mediated signaling is not required to initiate cytokine and inflammatory responses to P. aeruginosa in the lungs, but in the absence of TLR4, flagellin stimulation contributes importantly to these responses. Furthermore, TLR4- and flagellin-mediated signaling accounts for most of the early cytokine and inflammatory responses to P. aeruginosa but is not required for successful bacterial clearance.

Role of TLR2 in response to P. aeruginosa in vivo.

To determine the role of TLR2 in the innate immune response to wild-type P. aeruginosa, mice with targeted deletions of TLR2 and wild-type control mice were exposed to aerosolized PAK. As shown in Fig. 3, the levels of KC in lung homogenates 4 h after infection were significantly reduced in TLR2–/– mice compared with wild-type controls. Intrapulmonary concentrations of TNF-{alpha}, MIP-2, and IL-1beta did not differ between the two strains of mice at either time point. No significant differences in PMN recruitment or bacterial clearance were observed. Thus TLR2 has a more limited role than TLR4 in triggering cytokine responses to P. aeruginosa in the lungs and is not required either for neutrophil recruitment or bacterial clearance.


Figure 3
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  		Fig. 3. Role of TLR2 in host response to P. aeruginosa pneumonia. TLR2–/– and WT control mice were exposed to PAK and processed as described in Fig. 2. A: levels of immunoreactive cytokines measured by ELISA. B: PMN enumerated in bronchoalveolar lavage (BAL) specimens. C: bacterial CFU in lung homogenates. Data are means ± SE, n = 10 mice per group per time point; combined results of 2 separate experiments. *P < 0.05 compared with control.

 
Roles for TLR4, TLR2, and MyD88 in cytokine and inflammatory responses to flagellin-deficient P. aeruginosa.

To determine whether combined deficiencies of TLR2-, TLR4-, and flagellin-mediated signaling could reproduce the phenotype of mice lacking MyD88, TLR2–/–, TLR4–/–, TLR2/4–/–, MyD88–/–, and wild-type C57BL/6 mice were simultaneously exposed to aerosolized fliC P. aeruginosa. In mice lacking MyD88, the lung homogenate levels of TNF-{alpha}, IL-1beta, MIP-2, and KC were nearly absent 4 h after infection (Fig. 4), similar to what was observed after infection with PAK (55), and were significantly different from all other groups of mice with the exception of TNF-{alpha}, which differed from all groups except TLR2/4–/– mice. In TLR4–/– mice, the local expression of TNF-{alpha}, IL-1beta, MIP-2, and KC after infection with P. aeruginosa was significantly reduced compared with wild-type animals but not to the degree observed in MyD88–/– mice. In mice lacking TLR2, the TNF-{alpha} and KC responses were blunted 4 h after infection but the intrapulmonary levels of MIP-2 and IL-1beta were not distinguishable from wild-type controls. At the 24-h time point, IL-1beta levels in TLR2–/– were significantly higher than all other groups. The cytokine response pattern among mice lacking both TLR2 and TLR4 was the same as that of TLR4–/– mice with the exception that the early KC response in these mice was not significantly reduced compared with wild-type mice. Thus the cytokine response to P. aeruginosa is largely mediated by TLR2, TLR4, and flagellin/TLR5, but MyD88-dependent signals independent of these receptors contribute to the recognition response.


Figure 4
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  		Fig. 4. Role of MyD88, TLR2, and TLR4 in mediating cytokine responses to flagellin-deficient P. aeruginosa. MyD88–/–, TLR2–/–, TLR4–/–, TLR2/4–/–, and WT mice were exposed simultaneously to aerosolized fliC P. aeruginosa, and left lungs were homogenized for cytokine measurements by ELISA. Levels in uninfected C57BL/6 mice in picograms per milliliter: TNF-{alpha}, 85.0 ± 1.3; IL-1beta, 61.0 ± 8.6; MIP-2, 95.1 ± 8.3; KC, 150.1 ± 9.8. Data are means ± SE, n = 5–9 mice; combined results of 2 separate experiments. *P < 0.05 compared with all other groups; {dagger}P < 0.05 compared with WT, TLR2–/–, MyD88–/–; {ddagger}P < 0.05 compared with WT, TLR2–/–; §P < 0.05 compared with WT, TLR2–/–, TLR4–/–.

 
Wild-type mice responded to inhalation of fliC P. aeruginosa with an influx of neutrophils, indicating that flagellin expression was not required for the acute inflammatory response to P. aeruginosa (Fig. 5A). BAL neutrophils were dramatically reduced in the MyD88–/– mice and were significantly reduced in TLR4–/– and TLR2/4–/– mice compared with wild-type animals. In contrast, TLR2–/– mice exhibited a significantly augmented early inflammatory response to flagellin-deficient P. aeruginosa. After 24 h, bronchoalveolar neutrophilic inflammation was more evident in MyD88–/– mice than after 4 h but remained significantly blunted compared with other groups. Thus the acute neutrophilic inflammatory response to P. aeruginosa infection of the lungs does not require flagellin-mediated signaling, is almost entirely dependent on MyD88, and is partially dependent on TLR4. TLR2 is not required for this response and plays a counterregulatory role, serving to limit neutrophil emigration into the air spaces. Examination of tissue sections in these mice confirmed that early peribronchial, perivascular, and alveolar inflammation was absent in the MyD88–/– mice and markedly reduced in TLR4–/– and TLR2/4–/– mice (Fig. 6). After 24 h, foci of bronchial, vascular, and alveolar necrosis were evident in the MyD88–/– mice, with inflammatory cells trapped within fibrin matrices associated with these necrotic foci. Areas outside necrotic foci remained relatively free of inflammation in MyD88–/– mice. Lesser degrees of tissue necrosis were present in the TLR4–/– and TLR2/4–/– mice but not in TLR2–/– or wild-type animals. The lung injury scores, composite measures of tissue inflammation and necrosis, were significantly lower in the MyD88–/–, TLR2/4–/–, and TLR4–/– mice 4 h after infection compared with wild-type mice, reflecting the absent or reduced early inflammatory responses in these animals (Fig. 5B). In contrast, lung injury scores were highest in MyD88–/– mice 24 h after infection because of the appearance of tissue necrosis in this group. BAL protein levels were not significantly different among the five groups 4 h after infection. However, 24 h after infection the BAL protein levels were significantly higher in the MyD88–/– mice than in all other groups except TLR2/4–/–, consistent with the greater degree of lung injury in these animals (Fig. 5C). BAL protein concentrations in TLR2/4–/– and TLR4–/– mice also were significantly higher than in wild-type mice.


Figure 5
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  		Fig. 5. Role of MyD88, TLR2, and TLR4 in mediating inflammatory responses to flagellin-deficient P. aeruginosa. Animals were infected as in Fig. 4, and right lungs were lavaged for cell counts and protein measurements and then fixed for histological analysis. A: BAL PMN. B: lung injury score determined from light microscopic analysis of fixed tissue, as described in MATERIALS AND METHODS. C: BAL total protein. Data are means ± SE, n = 5–9 mice, except lung injury scores in TLR2–/– mice, n = 2. Combined results of 2 separate experiments. *P < 0.05 compared with all other groups; {dagger}P < 0.05 compared with WT, TLR2–/–, MyD88–/–; {ddagger}P < 0.05 compared with WT, TLR2–/–; §P < 0.05 compared with WT, TLR2–/–, TLR4–/–; ¶P < 0.05 compared with WT, MyD88–/–; P < 0.05 compared with TLR4–/–, MyD88–/–; #P < 0.05 compared with WT, TLR2–/–, TLR2/4–/–; **P < 0.05 compared with MyD88–/–.

 

Figure 6
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  		Fig. 6. Representative histological sections of lungs from mice infected 4 or 24 h earlier with P. aeruginosa. Changes are most noticeable in MyD88–/– (M88–/–) mice where at 4 h there is essentially no evidence of inflammation but 20 h later there is severe necrosis and accumulation of inflammatory cells within and surrounding most bronchioles and associated blood vessels (arrowheads). At these sites the necrotic debris and many inflammatory cells are entrapped within a fibrin-based coagulum. Histologically the other mutant mice do not differ markedly from WT at either 4 or 24 h postinfection in regard to the inflammatory cell coefficient although TLR4–/– (T4–/–) and TLR2/4–/– (T2/4–/–) mice had mild, focal necrosis. Original magnification was either x20 or x40.

 
Resistance to flagellin-deficient P. aeruginosa requires MyD88 but not TLR2 or TLR4.

As shown in Fig. 7, flagellin-deficient P. aeruginosa replicated exponentially (>500-fold over 24 h) in the lungs of MyD88-deficient mice, and the number of bacteria cultured from the spleens of MyD88–/– mice increased >500-fold between 4 and 24 h after infection, similar to what we (55) observed with PAK infection of these mice. These observations demonstrate the invasiveness of the fliC strain, despite the absence of flagella, in mice lacking MyD88. Infection with fliC P. aeruginosa was lethal for these mice, as seven of nine animals held beyond the 4-h time point were dead by 24 h (6 of these animals died 22–23 h after infection and were processed immediately postmortem for the 24-h data shown in Figs. 4, 5, and 7). The uncontrolled bacterial growth in the lungs, systemic dissemination, and mortality after infection of MyD88–/– mice with fliC were very similar to what we observed when these mice were challenged with PAK, indicating that flagellin expression is not required for virulence of P. aeruginosa in animals deficient in MyD88. In contrast, there were no significant differences between TLR2–/–, TLR4–/–, TLR2/4–/–, and wild-type C57BL/6 mice in CFU per lung or CFU per spleen at either time point, and only one death (in the TLR2/4–/– group) was observed. Thus the absence of TLR2-, TLR4-, and flagellin-mediated signaling did not reproduce the highly susceptible phenotype of MyD88-deficient mice to P. aeruginosa pneumonia, indicating that MyD88-dependent signals other than those mediated by TLR2, TLR4, and flagellin/TLR5 are sufficient for control of P. aeruginosa infection in vivo.


Figure 7
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  		Fig. 7. Roles of MyD88, TLR2, and TLR4 in clearance of flagellin-deficient P. aeruginosa. MyD88–/–, TLR2–/–, TLR4–/–, TLR2/4–/–, and WT mice were exposed simultaneously to aerosolized fliC P. aeruginosa. CFUs were determined in homogenized lung and spleen tissue. Combined results of 2 experiments are shown. Bars indicate mean values, n = 5–9 mice. *P < 0.05 compared with TLR4–/–; {dagger}P < 0.001 compared with all other groups.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
This study explored the roles of TLR2, TLR4, and TLR5 in mediating MyD88-dependent defenses against P. aeruginosa. We found that bone marrow cells lacking TLR4 or both TLR2 and TLR4 exhibited blunted TNF-{alpha} responses to P. aeruginosa, whereas the responses of TLR2-deficient cells were unimpaired compared with wild-type cells. Cells lacking both TLR2 and TLR4 failed to respond to flagellin-deficient P. aeruginosa. In vivo, mice lacking functional TLR4 demonstrated impaired pulmonary cytokine and inflammatory responses to P. aeruginosa, whereas mice lacking TLR2 exhibited a selective impairment in KC production with no defect in neutrophil recruitment. When challenged with flagellin-deficient P. aeruginosa, the responses of mice lacking TLR4 or both TLR2 and TLR4 were markedly blunted but not absent. In contrast, TLR2–/– mice had selectively reduced cytokine production but increased airway inflammation. Infection of MyD88–/– mice with flagellin-deficient P. aeruginosa resulted in exponential bacterial replication, systemic dissemination, and death within 24 h. However, mice lacking TLR4 or both TLR2 and TLR4 effectively cleared the organism from the lungs, despite severely reduced inflammatory responses. These observations suggest that TLR2, TLR4, and TLR5 have specific roles in the recognition of P. aeruginosa in the lungs, and that signaling via these receptors accounts for most of the acute cytokine and inflammatory responses to infection. However, TLR2, TLR4, and flagellin-mediated signal transduction were not required for effective bacterial clearance, indicating that MyD88-dependent signals from receptors other than TLR2, TLR4, and TLR5 are sufficient for host resistance to P. aeruginosa pneumonia.

We found that TLR4 had a major role in the activation of innate immune responses to P. aeruginosa. In vitro, bone marrow cells lacking TLR4 exhibited blunted TNF-{alpha} expression when stimulated with P. aeruginosa. In vivo, the cytokine and inflammatory responses to pulmonary infection with P. aeruginosa were significantly impaired in TLR4–/– mice compared with wild-type controls, consistent with recent observations in mice with nonfunctional mutations in TLR4 (14, 44). Ramphal et al. (46) similarly found reduced cytokine responses in TLR4–/– mice after intranasal challenge with PAK but without an associated decrement in neutrophilic inflammation compared with wild-type animals. Multiple bacterial products and host cells may participate in the TLR4-mediated response to P. aeruginosa in the lungs. LPS is the major bacterial ligand for TLR4 (3), which recognizes LPS in conjunction with two coreceptors, MD2 and CD14 (60). LPS derived from the PAK strain of P. aeruginosa contains a penta-acylated lipid A moiety and stimulates the murine TLR4/MD2/CD14 signaling complex (22). In contrast, PAK LPS is poorly recognized by human TLR4, which is highly responsive to hexa-acylated forms of LPS that are found in cystic fibrosis isolates of P. aeruginosa (12, 22). Inhalation of P. aeruginosa LPS has been shown to induce neutrophilic lung inflammation (54, 67). Exoenzyme S is a toxin secreted by P. aeruginosa that also has been found to stimulate cellular activation via a MyD88-dependent pathway that involves recognition by TLR4/MD2/CD14 (11). It is possible that additional bacterial components or host factors released in the context of acute lung injury contribute to TLR4-mediated activation (3). Despite the importance of TLR4-mediated signaling in the initiation of cytokine and inflammatory responses to P. aeruginosa, we found that TLR4 was not required for successful host defense against P. aeruginosa pneumonia: bacterial clearance from the lungs of TLR4-deficient mice was unimpaired compared with wild-type animals under the conditions tested. These results are similar to those of Ramphal and colleagues (46) but differ somewhat from those of Faure et al. (14), who reported inoculum-dependent impairments in bacterial clearance and survival after endobronchial instillation of the PA103 strain of P. aeruginosa. TLR4-deficient mice also have been shown to exhibit reduced resistance to pulmonary infection with other gram-negative bacteria, including Pasteurella pneumotropica (8), Haemophilus influenzae (65, 66), Klebsiella pneumoniae (7, 50), Bordetella bronchiseptica (38), and Acinetobacter baumanii (32), but not Legionella pneumophila (34), Francisella tularensis (9), or Escherichia coli (33).

TLR2 had a subtler role than TLR4 in the host response to P. aeruginosa. TLR2 has been reported to participate in recognition of multiple P. aeruginosa ligands, including lipoproteins (4, 18), alginate (19), flagellin (2), and exoenzyme S (11). In vitro, we found that early TNF-{alpha} responses of TLR2-deficient bone marrow cells to P. aeruginosa were unimpaired compared with wild-type cells. The role of TLR2 in mediating host responses to P. aeruginosa may be time- and cell-dependent: Soong et al. (58) found that transfection of airway epithelial cells with a dominant-negative TLR2 construct blunted rapid NF-{kappa}B activation and overnight secretion of IL-8 in response to P. aeruginosa in vitro. In vivo, however, we found that early cytokine responses to P. aeruginosa pneumonia in TLR2-deficient mice were indistinguishable from those of wild-type mice but for the reduced levels of KC. Others also have observed unimpaired IL-1beta, MIP-2, and TNF-{alpha} responses to P. aeruginosa pneumonia in TLR2–/– mice (36, 44). Despite the lower levels of KC, the inflammatory response to P. aeruginosa infection was not impaired in TLR2–/– mice; indeed, neutrophil recruitment was enhanced after infection with flagellin-deficient P. aeruginosa and tended toward supernormal after infection with wild-type P. aeruginosa. Similarly, Lorenz and colleagues (36) found that TLR2-deficient mice exhibited normal pulmonary inflammatory responses to wild-type P. aeruginosa but increased responses to pilA-deficient P. aeruginosa. Knapp et al. (32) observed augmented lung cytokine and inflammatory responses to A. baumanii in TLR2–/– mice. These observations suggest that signaling through TLR2 may have a suppressive effect on inflammatory responses, perhaps by downregulating TLR4 expression (36) or by selectively inducing IL-10 (31, 42, 53). TLR2-deficient mice have been reported to have increased resistance to pulmonary challenge with pilA-deficient P. aeruginosa (36) or with A. baumanii (32), and to systemic challenge with either Candida albicans (42) or Yersinia enterocolitica (53), but diminished resistance to systemic challenge with Staphylococcus aureus (61). We found no difference between TLR2–/– and wild-type mice in the clearance of either wild-type or flagellin-deficient P. aeruginosa from the lungs. Interestingly, the role of TLR2 was subordinate to that of TLR4 in this model, as the phenotype of mice lacking both TLR2 and TLR4 was indistinguishable from that of TLR4–/– mice. This finding is consistent with the recent observation that the upregulation of TLR2 in the lungs following P. aeruginosa infection requires TLR4 (44).

We used flagellin-deficient bacteria to explore the role of TLR5 in the MyD88-dependent response to P. aeruginosa pneumonia. Flagellin is the only known ligand for TLR5 and the major structural protein of bacterial flagella (3, 23, 57). P. aeruginosa flagellin is recognized by TLR5 (2, 23) and stimulates airway epithelial cell activation via direct interaction with asialoGM1 as well as TLR5 (2). TLR2 also may have a role in modulating signaling responses of respiratory epithelial cells to flagellin but is not required for lung inflammation in response to airway challenge with flagellin in vivo (2). Expression of TLR5 by respiratory epithelial cells is upregulated and mobilized to the apical surface on exposure to P. aeruginosa flagella (2). Alveolar macrophages express mRNA for TLR5 (43) and also respond to flagellin (37). We found that cytokine and inflammatory responses to fliC mutant P. aeruginosa were diminished compared with responses to wild-type P. aeruginosa only in mice lacking TLR4, supporting a limited role for TLR5 in the host response to pulmonary infection with P. aeruginosa. These data are consistent with the recent observation (17) that mice lacking TLR4 and TLR5, but not TLR5 alone, exhibited increased susceptibility to P. aeruginosa pneumonia. We found that flagellin-mediated signaling was not required to elicit intrapulmonary cytokine induction or neutrophilic inflammation in response to P. aeruginosa infection. Furthermore, flagellin expression was unnecessary for P. aeruginosa to establish an invasive, lethal pulmonary infection in mice lacking MyD88. Flagellin-deficient P. aeruginosa replicated exponentially in the lungs and spleens of MyD88–/– mice at the same rate that we (55) observed previously with the parental strain of wild-type P. aeruginosa, and the infection was fatal within 24 h in nearly all mice. In contrast, nasal challenge with fliC mutant P. aeruginosa was less likely than PAK to induce pneumonia, invade the bloodstream, or cause mortality in newborn mice (15). Our observations indicate that in a host unable to initiate rapid inflammatory responses to bacteria, nonmotile P. aeruginosa is fully capable of invasive infection.

The most striking finding in these studies is that the absence TLR2-, TLR4-, and TLR5 (flagellin)-mediated signaling did not reproduce the dramatically susceptible phenotype of the MyD88–/– mice. When TLR2/4–/– mice were challenged with flagellin-deficient P. aeruginosa, cytokine and inflammatory responses were measurable, albeit markedly attenuated, and bacterial clearance was unimpaired. In contrast, MyD88–/– mice failed to mount an inflammatory response or contain the infection. These observations indicate that MyD88-dependent signaling pathways that are independent of TLR2, TLR4, and TLR5 are important in pulmonary resistance to P. aeruginosa. These pathways may stimulate the recruitment of a sufficient number of neutrophils to control the infection or activate other innate defenses against P. aeruginosa, such as the release of antimicrobial peptides (25).

It is possible that additional TLRs contribute to MyD88-dependent defenses. One candidate is TLR9, which recognizes bacterial CpG motifs and signals via MyD88 (21, 24). Alveolar macrophages (16), respiratory epithelial cells (52), and pulmonary endothelial cells (35) all respond to CpG DNA, and endotracheal instillation of bacterial DNA or CpG elicits a rapid neutrophil response (51). TLR9-deficient mice recently have been shown to exhibit increased susceptibility to pulmonary tuberculosis (6) and to P. aeruginosa keratitis (28). However, we have found in preliminary experiments that triple knockout mice lacking TLR2, TLR4, and TLR9 were not more handicapped in their response to aerosolized flagellin-deficient P. aeruginosa than mice lacking TLR2 and TLR4 (unpublished observations), suggesting that TLR9 does not have an essential role in MyD88-dependent resistance to P. aeruginosa pneumonia. Mice also express three recently described TLRs, TLR11–13 (59). TLR11 recognizes uropathogenic strains of E. coli and signals via MyD88 but is not expressed in the lungs (69); whether or not TLR11 recognizes P. aeruginosa is unknown. The functions of TLR12 and TLR13 have not been described (59). Other TLRs are not known to independently recognize bacterial ligands (3).

In addition to TLRs, MyD88 mediates signaling via the IL-1 and IL-18 receptors (1) and other members of the Toll/IL-1 receptor superfamily that possess a Toll/IL-1R (TIR) domain (3). Mice deficient in either IL-1R or IL-18 have paradoxically augmented resistance to pulmonary challenge with P. aeruginosa (41, 48, 49). Furthermore, we found that the IL-1beta response to infection was markedly blunted in MyD88–/– mice. Thus it seems unlikely that aborted IL-1- or IL-18-mediated signaling accounts for the observed defect in MyD88–/– animals, although the host defense functions of IL-1 and IL-18 in TLR-deficient mice have not been studied. Some other members of the IL-1 receptor family with TIR domains, such as single immunoglobulin IL-1R-related molecule (SIGIRR) and ST2, serve as negative regulators of IL-1/TLR signaling (3). Defective signaling via these molecules can influence resistance to bacterial infection (3, 29, 64) but would not explain the lack of early inflammation in MyD88–/– mice challenged with P. aeruginosa. However, it is possible that other members of this family contribute to MyD88-dependent resistance to bacterial pneumonia.

Our studies support distinct and complementary roles for TLR2, TLR4, and TLR5 in the recognition of P. aeruginosa in the lungs. Signaling via these receptors accounts for most of the early cytokine and inflammatory responses to infection. However, it is clear that MyD88-dependent recognition or amplification pathways other than those triggered by TLR2, TLR4, and TLR5 are involved in pulmonary host defense against P. aeruginosa pneumonia. Redundant mechanisms for microbial recognition and activation of innate immune responses serve to protect the host from diverse bacterial challenges.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
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 GRANTS
REFERENCES
 
This work was supported by National Institutes of Health Grants HL-54972 and HL-73996 (S. J. Skerrett), HL-065898 and DK-047754 (C. B. Wilson), and HL-069503 (A. M. Hajjar); and by the Cystic Fibrosis Foundation Research Development Program (C. B. Wilson).


   ACKNOWLEDGMENTS
 
We thank Deanna Penney, Michele Timko, and Brooke Nakhuda for expert technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. J. Skerrett, Division of Pulmonary and Critical Care Medicine, Harborview Medical Center, 325 Ninth Ave., Box 359640, Seattle, WA 98104 (e-mail: shawn{at}u.washington.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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