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The Journal of Immunology, 2007, 178: 3170-3176.
Copyright © 2007 by The American Association of Immunologists, Inc.

A Role of Toll-IL-1 Receptor Domain-Containing Adaptor-Inducing IFN-beta in the Host Response to Pseudomonas aeruginosa Lung Infection in Mice1

Melanie R. Power*, Bo Li*, Masahiro Yamamoto{ddagger}, Shizuo Akira{ddagger} and Tong-Jun Lin2,{dagger}

* Department of Microbiology and Immunology, and {dagger} Department of Pediatrics, Dalhousie University, Halifax, Nova Scotia, Canada; and {ddagger} Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Toll-IL-1R domain-containing adaptor-inducing IFN-beta (TRIF) is an adaptor molecule that mediates a distinct TLR signaling pathway. Roles of TRIF in the host defense have been primarily associated with virus infections owing to the induction of IFN-{alpha}beta. In this study, we investigated a role of TRIF in Pseudomonas aeruginosa infection. In vitro, TRIF-deficient mouse alveolar and peritoneal macrophages showed a complete inhibition of RANTES (CCL5) production, severely impaired TNF and KC (CXCL1) production, and reduced NF-{kappa}B activation in response to P. aeruginosa stimulation. In vivo, TRIF-deficient mice showed a complete inhibition of RANTES production, a severely impaired TNF and KC production, and an efficient MIP-2 and IL-1beta production in the lung following P. aeruginosa infection. This outcome was associated with a delayed recruitment of neutrophils into the airways. These results suggest that TRIF mediates a distinct cytokine/chemokine profile in response to P. aeruginosa infection. P. aeruginosa-induced RANTES production is completely dependent on TRIF pathway in mice. Importantly, TRIF deficiency leads to impaired clearance of P. aeruginosa from the lung during the initial 24–48 h of infection. Thus, TRIF represents a novel mechanism involved in the development of host response to P. aeruginosa infection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The bacterium Pseudomonas aeruginosa is a major opportunistic pathogen in patients with hospital-acquired pneumonia and in individuals with cystic fibrosis. Airway infection with P. aeruginosa triggers both acute and chronic inflammatory responses including elevated production of various cytokines and chemokines (1, 2, 3, 4). It is generally accepted that the host defense against P. aeruginosa lung infection is initiated by the recognition of this bacterium by local resident cells, followed by secretion of inflammatory mediators (cytokines/chemokines) that attract and activate immune effector cells for the clearance of this pathogen (1, 2, 3, 4).

Recently, a number of in vitro and in vivo studies have shown that TLRs are involved in the recognition of P. aeruginosa (5, 6, 7, 8, 9, 10). TLRs are a family of pattern recognition receptors that are critical for cellular responses to microbial products (11). TLRs mediate distinct immune responses depending upon their usage of specific adaptor molecules, which transduce signals from TLRs to transcription factors. Increasing evidence suggests that the adaptor proteins MyD88 and Toll-IL-1R domain-containing adaptor-inducing IFN-beta (TRIF),3 also known as TICAM-1, mediate two distinct TLR signaling pathways (12, 13). MyD88 mediates NF-{kappa}B activation and subsequent expression of NF-{kappa}B-regulated genes, such as TNF and IL-1. TRIF mediates IFN regulatory factor (IRF)3 and IRF7 activation leading to IFN-{alpha}beta expression (14, 15). In addition to activation of IRF3/7 pathway, TRIF also mediates a second pathway leading to NF-{kappa}B activation (16, 17). Global gene expression profiles distinctly regulated by the MyD88-dependent and the MyD88-independent pathways have been characterized (18). In fact, only 21.5% of LPS responsive genes are MyD88-dependent, whereas the majority of these genes (74.5%) are MyD88-independent (18). Thus, activation of the MyD88-dependent pathway and the MyD88-independent TRIF pathway leads to distinct gene expression profiles.

Depending upon the nature of microbial pathogens, different signaling pathways are used to develop an effective host response to a specific pathogen. We and others have demonstrated that the MyD88 pathway is required for the development of early immune response to P. aeruginosa lung infection (8, 19). However, MyD88 is not essential for innate immunity to Staphylococcus aureus (19) or Mycobacterium tuberculosis (20). In fact, MyD88 deficiency appears to improve resistance against sepsis caused by polymicrobial infection (21). Thus, effective immunity against bacterial infection can be developed through MyD88-independent mechanisms. This notion is supported by a recent study demonstrating that the TRIF pathway but not the MyD88 pathway is needed for P fimbriated Escherichia coli-induced signaling in epithelial cells (22).

TRIF is a major TLR adaptor protein that mediates MyD88-independent pathways (13, 23). A role of TRIF in the host defense has been primarily associated with virus infections due to its significant roles in the induction of IFN-{alpha}beta (24, 25). It is less clear to what extent TRIF contributes to the development of host defense against bacterial infection. In vitro, two recent studies showed that TRIF but not MyD88 is required for Yersinia-induced macrophage apoptosis (26) and E. coli-induced dendritic cell apoptosis (27). In vivo, TRIF appears to be dispensable for the clearance of Gram-negative bacteria Haemophilus influenzae from mouse lung (28). In addition, activation of IRF-IFN-{alpha}beta pathway impairs host resistance to the Gram-positive intracellular bacterium Listeria monocytogenes (29). A role of TRIF in P. aeruginosa infection has not been investigated previously.

In this study, we demonstrated that TRIF deficiency leads to a complete inhibition of RANTES (CCL5) production and a significant reduction of KC (CXCL1) and TNF production by alveolar and peritoneal macrophages in response to P. aeruginosa stimulation in vitro. In vivo, by using TRIF-deficient mice, we demonstrated that TRIF deficiency leads to markedly reduced pulmonary eradication of P. aeruginosa during the initial 48 h of infection. This result was associated with delayed neutrophil recruitment and a distinct pattern of cytokine and chemokine production in the airways. P. aeruginosa-induced production of several NF-{kappa}B-regulated gene products, including RANTES, KC, and TNF was impaired in TRIF-deficient mice. Thus, TRIF plays a positive role in the development of host defense against P. aeruginosa lung infection. This finding is in contrast to the lack of effect or a negative role of TRIF pathway in H. influenzae or L. monocytogenes infection.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

TRIF knockout (TRIF–/–) mice were backcrossed eight times to the C57BL/6 (Osaka University) (14, 30). C57BL/6 mice were purchased from Charles River Breeding Laboratories. TRIF–/– mice were age- and sex-matched with C57BL/6 mice. The protocols were approved by the University Committee on Laboratory Animals (Dalhousie University) in accordance with the guidelines of the Canadian Council on Animal Care.

Bacterial preparation and macrophage activation

P. aeruginosa strain 8821, which was a gift from Dr. A. Chakrabarty (University of Illinois, Chicago, IL), is a mucoid strain isolated from a cystic fibrosis patient (31). P. aeruginosa were cultured in Luria-Bertani broth and harvested when the culture reached an OD at 640 nm of 2 U (early stationary phase). Bacteria were washed in PBS and density adjusted to an OD of 1 U before use. For in vitro macrophage activation experiments, P. aeruginosa were treated with gentamicin (100 µg/ml) for 2 h and exposed directly to UV light illumination for 20 min before experimental use. For in vivo studies, live P. aeruginosa were used.

Alveolar macrophages and peritoneal macrophages were recovered from the lungs or peritoneum of TRIF–/– or TRIF+/+ mice by bronchoalveolar or peritoneal lavage. Cells were treated with killed P. aeruginosa for 3, 6, 24, or 48 h at the multiplicity of infection (MOI) of 1:100. After macrophage and bacteria coincubation, cell-free supernatants were collected for measuring cytokine and chemokine production by ELISA. Cell pellets were used for nuclear protein isolation for examining NF-{kappa}B activation by EMSA.

EMSA analysis

A consensus double-stranded NF-{kappa}B oligonucleotide (5'-AGT TGA GGG GAC TTT CCC AGG C-3'; Promega) was used for EMSA as previously described (8). Briefly, 10 µg of nuclear protein was added to a 10-µl volume of binding reaction with 1 µg of poly(deoxyinosinic-deoxycytidylic acid) (Amersham Biosciences) and incubated at room temperature for 15 min. Double-stranded NF-{kappa}B oligonucleotide was 32P-labeled, then added to each reaction mixture, incubated at room temperature for 30 min, and separated by electrophoresis on a 6% polyacrylamide gel in 0.5x Tris-boric acids-EDTA buffer. Gels were vacuum-dried and subjected to autoradiography.

Lung infection with P. aeruginosa and collection of lung and bronchoalveolar lavage fluid (BALF)

Mice were intranasally infected with 1 x 107 or 1 x 109 CFU of P. aeruginosa. After 4, 8, 12, 24, 48 h, or 6 days, BALF was obtained by lavaging the lung with 3x 1 ml of phosphate buffer solution containing soybean trypsin inhibitor (100 µg/ml). The lung tissues were obtained for detection of cytokines, myeloperoxidase (MPO), and bacterial CFU counting.

Lung tissues were homogenized (maximum speed, 20 s, PowerGen 125; Fisher Scientific) in 50 mM HEPES buffer (4 µl/mg lung) containing soybean trypsin inhibitor (100 µg/ml). For counting bacterial CFU, 10 µl of the homogenate was plated on an agar dish and incubated for 24–48 h at 37°C. The homogenate was centrifuged at 4°C for 30 min at 18,000 x g. The supernatant was stored at –80°C for later cytokine analysis. The pellet was resuspended and homogenized in 0.5% cetyltrimethylammonium chloride (4 µl/mg lung) and centrifuged as described. The clear extract was used for MPO assay.

BALF (10 µl) was plated on an agar dish and incubated for 24–48 for CFU counting. For detection of cytokines and MPO activity, BALF was centrifuged at 300 x g for 5 min at 4°C. The supernatants were used for cytokine analysis. The pellets (from both infected and noninfected mice) were resuspended in 1 ml of NH4Cl (0.15 M) and spun as before to lyse RBC. The supernatants were discarded. The pellets were resuspended in 0.5% cetyltrimethylammonium chloride (250 µl/mouse) and centrifuged. The clear extracts were used for MPO assay.

MPO assay

The MPO assay was used to determine the infiltration of neutrophils into the lungs of the mice as previous described (32). Briefly, samples in duplicate (75 µl) were mixed with equal volume of the substrate (3 mM 3,3',5,5'-tetramethylbenzidine dihydrochloride, 120 µM resorcinol, and 2.2 mM H2O2) for 2 min. The reaction was stopped by adding 150 µl of 2 M H2SO4. The OD was measured at 450 nm.

Histology

Mice lungs were fixed in 10% formalin overnight then in 100% ethanol for paraffin embedding and sectioning. Slides were deparaffinized with CitriSolv (Fisher Scientific), and rehydrated through decreasing concentrations of ethanol. Slides were stained with Harris H&E to illustrate lung histology.

Cytokine production

The concentrations of KC, IL-1beta, TNF, MIP-2, RANTES (CCL5), or IFN-{alpha}beta in the lung and BALF or cell-free supernatants were determined by ELISA as previously described using Ab pairs from R&D Systems (33).

Statistics

Data are presented as the mean ± SEM of the indicated number of experiments. Statistical significance was determined by assessing means with ANOVA and the Tukey-Kramer multiple comparison tests, or by using an unpaired t test. Differences were considered significant at p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Impaired cytokine/chemokine expression and NF-{kappa}B activation in TRIF-deficient macrophages in response to P. aeruginosa stimulation in vitro

To examine whether TRIF plays a role in P. aeruginosa-induced macrophage activation, alveolar and peritoneal macrophages from TRIF-deficient mice and wild-type mice were treated with P. aeruginosa strain 8821 (MOI = 100) for 3, 6, 24, or 48 h. Cell-free supernatants were collected for measuring cytokines and chemokines by ELISA. P. aeruginosa stimulation induced a significant production of RANTES by macrophages from wild-type mice. In contrast, P. aeruginosa induced little to none RANTES production in alveolar macrophages or peritoneal macrophages from TRIF-deficient mice (Fig. 1, A and B).


Figure 1
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  		FIGURE 1. TRIF deficiency selectively impairs a distinct set of cytokine and chemokine production by macrophages in response to P. aeruginosa stimulation in vitro. Macrophages were collected from BALF (B, D, F, and H) or peritoneum (A, C, E, and G) of TRIF+/+ ({square}) and TRIF–/– mice ({blacksquare}). Cells were treated with P. aeruginosa (mucoid strain 8821, MOI = 1:100) or treated with medium as a control (NT) for 3, 6, 24, or 48 h. Cell-free supernatant were collected for the determination of RANTES, TNF, KC, and MIP-2 by ELISA. Data are the mean ± SE of three independent experiments. Macrophages were pooled from three mice in each experiment.

 
Similarly, P. aeruginosa-induced TNF and KC production was significantly impaired in TRIF-deficient macrophages compared with wild-type macrophages (Fig. 1, C–F). However, a significant MIP-2 production by TRIF-deficient macrophages was observed (Fig. 1, G and H), suggesting a selective effect of TRIF on P. aeruginosa-induced cytokine/chemokine production. TNF and KC are primarily regulated by NF-{kappa}B (34). Consistent with this finding, P. aeruginosa-induced NF-{kappa}B activation was severely impaired in peritoneal macrophages from TRIF-deficient mice as examined by EMSA (Fig. 2).


Figure 2
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  		FIGURE 2. TRIF deficiency leads to a significant inhibition of P. aeruginosa-induced NF-{kappa}B activation in macrophages in vitro. Peritoneal macrophages were collected from TRIF+/+ and TRIF–/– mice. Macrophages were treated with medium as a control (NT) or treated with P. aeruginosa (strain 8821, MOI = 1:100) for 0.5, 2, 6, 12, or 24 h. Nuclear proteins were extracted and subject to EMSA using 32P-labeled NF-{kappa}B probes. Representative results of two independent experiments are shown.

 
Impaired cytokine and chemokine production in the airways of TRIF-deficient mice following P. aeruginosa lung infection

To determine a role for TRIF in the development of immune response to P. aeruginosa lung infection in vivo, TRIF–/– mice were infected with P. aeruginosa strain 8821 at the concentration of 1 x 107 or 1 x 109 CFU/mouse for 4 or 24 h. BALF and lung tissues were collected for the determination of RANTES, KC, MIP-2, TNF, and IL-1beta. P. aeruginosa-induced RANTES production was completely inhibited in TRIF–/– mice (Fig. 3, A and B). The reaction appeared to be different from MyD88–/– mice, which showed significant RANTES production after P. aeruginosa (1 x 109 CFU/mouse) infection for 24 or 48 h (data not shown). Similarly, neutrophil chemoattractant KC was also markedly reduced in TRIF–/– mice (Fig. 3, C and D). However, TRIF–/– mice had little or no defects in the production of MIP-2 or IL-1beta in response to P. aeruginosa lung infection (Fig. 4, A–C). This finding is in contrast to MyD88 deficiency, which led to significant impairment of MIP-2 and IL-1beta production in the lung (8).


Figure 3
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  		FIGURE 3. P. aeruginosa-induced RANTES (CCL5) and KC production in the airways is dependent on TRIF. TRIF+/+ and TRIF–/– mice were inoculated intranasally with P. aeruginosa (strain 8821, 1 x 107 CFU/mouse or 1 x 109 CFU/mouse) (Psa) for 4 or 24 h. Mice that were not treated with bacteria were used as controls (NT). BALF and lung tissues were collected for the determination of RANTES (A, B) and KC (C, D) protein by ELISA. Data are the mean ± SE of 9–11 mice per group.

 

Figure 4
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  		FIGURE 4. TRIF deficiency has limited effects on MIP-2, IL-1beta, and TNF production following P. aeruginosa lung infection. TRIF+/+ and TRIF–/– mice were inoculated intranasally with P. aeruginosa (mucoid strain 8821, 1 x 107 or 1 x 109 CFU/mouse) (Psa) for 4 or 24 h. Mice that were not treated with bacteria were used as controls (NT). BALF and lung tissues were collected for the determination of MIP-2 (A, B), IL-1beta (C), and TNF (D) protein by ELISA. There are low levels of IL-1beta in the BALF and TNF in the lung (data not shown). Data are the mean ± SE of 8–11 mice per group.

 
P. aeruginosa-induced TNF production was partially reduced in TRIF–/– mice (Fig. 4D). Taken together, TRIF–/– mice have a specific pattern of cytokine and chemokine production in response to P. aeruginosa lung infection, which is distinct from MyD88–/– mice (7, 8, 19).

IFN-{alpha} or IFN-beta in the BALF or lung were also tested by ELISA. P. aeruginosa infection had little effect on IFN-{alpha} or IFN-beta production in wild-type or TRIF–/– mice (data not shown).

To examine P. aeruginosa-induced NF activation in the lung, wild-type and TRIF–/– mice were challenged with P. aeruginosa intranasally for 4 h. Lung tissue was harvested from these mice. Nuclear proteins were extracted and were subjected to EMSA analysis for NF-{kappa}B or IRF binding. P. aeruginosa infection induced NF-{kappa}B activation in wild-type mice. Interestingly, TRIF deficiency only induced a mild reduction of P. aeruginosa-induced NF-{kappa}B activation in the lung (Fig. 5), an effect which is different from that in macrophages. However, no IRF binding activity can be observed in P. aeruginosa-treated or untreated lung tissues (data not shown).


Figure 5
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  		FIGURE 5. P. aeruginosa induces a reduced NF-{kappa}B activation in the lung of TRIF-deficient mice. TRIF+/+ and TRIF–/– mice were inoculated intranasally with P. aeruginosa (mucoid strain 8821, 1 x 107 CFU/mouse) (Psa) or saline (NT) for 4 h. Nuclear proteins were extracted from lung tissues obtained from individual mouse and were subjected to EMSA by incubation with 32P-labeled NF-{kappa}B DNA probe. Unlabeled NF-{kappa}B or AP-1 DNA probes (50 times) were used for competitive binding assay using nuclear proteins from the lung of P. aeruginosa-treated wild-type mice. Each lane represents sample from individual mouse. Representative results of three independent experiments are shown.

 
Delayed neutrophil recruitment into the airways in TRIF–/– mice following P. aeruginosa lung infection

Neutrophils are involved in the clearance of P. aeruginosa and are recruited by the neutrophil chemoattractants produced in the airways (4). The distinct pattern of cytokine and chemokine production in TRIF–/– mice led us to examine whether TRIF deficiency affects neutrophil recruitment into the airways. TRIF–/– mice were infected intranasally with 1 x 107 or 1 x 109 CFU of P. aeruginosa strain 8821. BALF and lung tissues were collected 4, 8, or 24 h later for the detection of neutrophil infiltration by MPO assay. MPO levels in the earlier time points (8 h in the BALF, 4 and 8 h in the lung tissue) in TRIF–/– mice were lower than levels in TRIF+/+ mice (Fig. 6, A and B). Similarly, lung histology showed that there was limited number of neutrophils reached the alveolar airspace in the earlier stages (8 h) of infection in TRIF-deficient mice. However, at the later stages of infection, a similar level of neutrophil infiltration into the alveolar airspace was observed in wild-type and TRIF-deficient mice (Fig. 6, C and D). This result suggests that TRIF deficiency caused a delayed neutrophil recruitment into the airways.


Figure 6
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  		FIGURE 6. TRIF deficiency leads to delayed neutrophil recruitment into the airways following P. aeruginosa lung infection. TRIF+/+ and TRIF–/– mice were inoculated intranasally with P. aeruginosa (mucoid strain 8821, 1 x 107 or 1 x 109 CFU/mouse) (Psa) for 4, 8, or 24 h. Mice that were not treated with bacteria were used as controls (NT). BALF (A) and lung tissues (B) were collected for the determination of MPO activities. Data are the mean ± SE of 5–11 mice per group. Wild-type mice (C) or TRIF-deficient mice (D) were treated with P. aeruginosa (1 x 109 CFU/mouse) (Psa) for 8 or 48 h. The upper lobe of the left lung was collected for H & E staining (original magnification, x100). Arrow indicates neutrophil in the alveolar airspace.

 
Impaired clearance of P. aeruginosa from the lungs of TRIF–/–mice during the initial 24- to 48-h infection

To determine whether TRIF-dependent immune responses in the airways have an effect on the clearance of P. aeruginosa from the lung, the BALF and lung tissues from TRIF+/+ and TRIF–/– mice were collected for the detection of viable bacteria by CFU counting after intranasal administration of P. aeruginosa strain 8821 (1 x 109 CFU/mouse) for 24 or 48 h. After infection for 24 h, CFU counts in the lung tissues from TRIF–/– mice were 7-fold higher compared with TRIF+/+ mice. This difference between TRIF+/+ and TRIF–/– mice in the bacterial clearance appeared to last to 48 h (Fig. 7). Similarly, CFU counts in the BALF in TRIF–/– mice (24 or 48 h) were consistently higher than that in TRIF+/+ mice (data not shown). These results suggest that TRIF is required for the clearance of P. aeruginosa from the lung during the initial 24- to 48-h infection period.


Figure 7
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  		FIGURE 7. TRIF deficiency leads to impaired clearance of P. aeruginosa from the lung during the early stage of infection (24–48 h). TRIF+/+ and TRIF–/– mice were challenged intranasally with P. aeruginosa (mucoid strain 8821, 1 x 109 CFU/mouse for 24 or 48 h). After 24 or 48 h, the right lobe of the lung was collected and homogenized for colony counting. Data are the mean ± SE of 12 mice per group.

 
To examine whether P. aeruginosa were cleared from the lung at the later time points of infection, wild-type mice and TRIF–/– mice were infected with P. aeruginosa (1 x 109 CFU/mouse, n = 6 mice per group) for 6 days. For comparison, MyD88–/– mice were also infected for 6 days (1 x 109 CFU/mouse, n = 6 mice). Lung tissues and BALF were collected for CFU counting. No bacterial colonies were detected in either lung homogenates or BALF in wild-type, TRIF–/–, or MyD88–/– mice. Thus, in this acute infection model, this bacterium at the sublethal dose was cleared by all of these animals at the later time points of infection.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Despite advances in antibiotic therapy, P. aeruginosa lung infection remains a major cause of death in cystic fibrosis patients and in immunocompromised individuals. Molecular mechanisms involved in the immune response to this clinically important bacterium are still under investigation. In this study, we demonstrated for the first time that TRIF is involved in the development of host defense against P. aeruginosa infection.

P. aeruginosa infection is associated with altered production of a plethora of cytokines and chemokines in the lung (1, 2, 3, 4). Because cytokines and chemokines play a critical regulatory role in the host defense against P. aeruginosa infection, it is important to define the molecular pathways responsible for a specific pattern of cytokine and chemokine production during P. aeruginosa infection. In this study, we demonstrated that TRIF is essential for RANTES, KC, and TNF, but not MIP-2 or IL-1beta production in response to P. aeruginosa infection, a pattern that appears to be distinct from that in MyD88-deficient mice (8).

Previous studies using gene knockout and dominant-negative mutants suggest that TRIF is involved in TLR2- and TLR4-mediated cellular activation (14, 35, 36). Various TLRs, including TLR2, TLR4, and TLR5, have been implicated in P. aeruginosa infection in vivo and in vitro (5, 6, 8, 10). Two major TLR signaling pathways, including the MyD88 pathway and the TRIF pathway, have been described (12). Activation of the MyD88 pathway leads to NF-{kappa}B activation and subsequent production of various inflammatory cytokines and chemokines such as TNF and IL-1beta. An essential role of MyD88 pathway in the host defense against bacterial infection has been recognized (7, 8, 19). We and others showed that MyD88 deficiency leads to impaired clearance of P. aeruginosa from the lung (7, 8, 19). In contrast, the TRIF pathway is responsible for the activation of IRF3/7 leading to type I IFN (IFN-{alpha}beta) production. Accordingly, TRIF has been primarily associated with the host defense against virus infections. In this study, we found that TRIF is involved in the development of an effective host response to a Gram-negative bacterium P. aeruginosa infection in vitro and in vivo. A similar order of magnitude of bacterial clearance defect was observed in the MyD88- and TRIF-deficient mice. After 24-h infection, TRIF deficiency leads to a 7-fold increase of CFU in the lung, whereas MyD88 deficiency leads to a 9-fold increase of lung CFU when compared with their control mice. Thus, the full development of host defense against P. aeruginosa lung infection likely requires a coordinated effect of both the MyD88 pathway and the TRIF pathway.

TRIF-dependent IRF-regulated IFN-{alpha}beta production accounts for a major role in the host defense against various virus infections. A role of TRIF in bacterium infection is less clear. A recent study demonstrated that IRF3/7-IFN-{alpha}beta pathway is activated by a Gram-positive intracellular bacterium L. monocytogenes (29). Contrary to a protective role of IRF3/7-IFN-{alpha}beta pathway in virus infection, activation of IRF3/7-IFN-{alpha}beta pathway by L. monocytogenes leads to impaired host defense against this bacterium (29). Considering the significant role of TRIF in IRF3/7-IFN-{alpha}beta pathway activation, we examined whether IRF3/7-IFN-{alpha}beta pathway is activated during P. aeruginosa lung infection. Interestingly, no IRF3/7 activation can be observed by EMSA using nuclear extracts from lung tissues after P. aeruginosa infection for 4 h (data not shown). In addition, there were no changes of IFN-{alpha} and IFN-beta production in the BALF or lung tissues from P. aeruginosa-infected mice (4, 8, or 24 h) (data not shown). These results suggest that IRF3/7-IFN-{alpha}beta pathway may not be involved in P. aeruginosa infection. Thus, the usage of a specific signaling pathway is dependent on the nature of a specific pathogen.

In addition to IRF-IFN-{alpha}beta activation, recent studies suggest that TRIF also activates NF-{kappa}B through Rip1 (16, 17). Consistent with this model, TRIF-deficient macrophages demonstrated impaired NF-{kappa}B activation in response to P. aeruginosa infection. In fact, it is surprising that TRIF deficiency leads to a near complete inhibition of NF-{kappa}B activation in macrophages because MyD88-dependent pathway is expected to be functional. Interaction between MyD88 and TRIF has been reported (37). The importance of a coordinated effect for MyD88 and TRIF in resistance to infection has been suggested in a recent review (13). When both MyD88 and TRIF are absent, mice are more susceptible to infection when compared with MyD88 single knockout mice (13). The MyD88 and TRIF double-deficient animals are drastically immunocompromised and showed little or no response to most microbial ligands (13). Thus, it is likely that a coordinated effect of the TRIF pathway and the MyD88 pathway is needed for P. aeruginosa-induced NF-{kappa}B activation.

In vivo, P. aeruginosa-induced NF-{kappa}B activation in the lung was mildly reduced in TRIF-deficient mice. This result is in contrast to the severe defect of NF-{kappa}B activation in macrophages in vitro. It is likely that different cell types may have differential requirement for MyD88 and TRIF. Thus, additional cells other than macrophages contribute to P. aeruginosa-induced NF-{kappa}B activation in the lung. Alternatively, secondary effects of cytokines/chemokines such as IL-1beta or MIP-2 produced in the lung during P. aeruginosa infection may contribute to NF-{kappa}B activation in TRIF-deficient mice.

In contrast to the mild defect of NF-{kappa}B activation in the lung, P. aeruginosa-induced production of several NF-{kappa}B-regulated genes, including TNF, was severely impaired in the lung in TRIF-deficient mice following P. aeruginosa infection. This defect was observed in both shorter (4 h) and longer (24 h) time points after P. aeruginosa infection, suggesting a nonredundant role of TRIF in the regulation of these genes in vivo during P. aeruginosa lung infection. It is possible that a full and complete activation of NF-{kappa}B in the lung is required for P. aeruginosa-induced TNF production. Alternatively, because TNF production is also regulated by additional transcription factors such as CREB, C/EBP-beta, NFATp, ATF-2/Jun, Egr-1, Nrf1, Ets, and others (38), TRIF deficiency may directly or indirectly affect activation of additional transcriptional factor other than NF-{kappa}B. In addition, posttranscriptional regulation such as RNA stability and translational regulation also contributes to the expression level of cytokines and chemokines. It is unclear whether TRIF deficiency affects gene expression at the posttranscription level through direct or indirect mechanisms in the lung in vivo.

The promoter of RANTES contains both NF-{kappa}B binding sites and a IFN-stimulated response element that is sensitive to IRF3/7 (39). Expression of RANTES requires cooperative effect between NF-{kappa}B and IRF3/7 (40). The near complete inhibition of RANTES in TRIF-deficient macrophages and mice lung is likely due to an impaired NF-{kappa}B activation because IRF activation was not observed after P. aeruginosa infection in this study. P. aeruginosa-induced RANTES expression was also observed in human cells because cord blood-derived mast cells and PBL produce a substantial amount of RANTES in response to P. aeruginosa stimulation (data not shown). RANTES is a potent leukocyte chemoattractant. It promotes leukocyte recruitment and activation (41). RANTES activates human neutrophil to release antimicrobial peptide {alpha}-defensins and promote neutrophil phagocytic capacity (42). These effects likely contribute to its host defense against P. aeruginosa infection.

Interestingly, TRIF deficiency had minimal effects on other NF-{kappa}B regulated genes such as IL-1beta and MIP-2. This finding is in contrast to our previous finding that P. aeruginosa-induced IL-1beta and MIP-2 production is dependent on MyD88, which functions through NF-{kappa}B (8). It is likely that different additional transcription factors other than NF-{kappa}B are also involved in TRIF- and MyD88-dependent gene activation. Thus, TRIF deficiency induces a distinct pattern of cytokine and chemokine production that is different from MyD88 deficiency. This is consistent with a recent finding that of the 1055 genes found to be LPS responsive, only 21.5% were dependent on MyD88 expression, with MyD88-independent genes constituting 74.7% of the genetic response (18).

The impaired production of neutrophil chemoattractants such as KC, TNF, and RANTES in TRIF-deficient mice may contribute to the delayed neutrophil recruitment into the airways. It is noticeable that the neutrophil recruitment was only partially impaired in the early time points (4 or 8 h) of infection. After 24-h infection, TRIF-deficient mice recruited comparable levels of neutrophils into the airways as wild-type mice. However, the impairment of bacterial clearance from the lung in TRIF-deficient mice can be seen even after 24- to 48-h infection, suggesting that additional mechanisms other than neutrophils may be involved in TRIF-dependent host defense. These data suggest a nonredundant and indispensable role of TRIF in the clearance of P. aeruginosa from the lung during the early stage (24–48 h) of infection. Further studies on the roles of TRIF in the bactericidal activity of neutrophils and other immune effector cells such as epithelial cells, NK cells, and NKT cells may provide new insights into the mechanism of TRIF in contributing to the host defense against this clinically important bacterium. It is also noteworthy that a sublethal dose of bacterium was used in this acute lung infection model and no bacterial colony was detected in the lungs of wild-type, TRIF–/–, or MyD88–/– mice after 6 days of P. aeruginosa lung infection. Thus, other mechanisms in the absence of TRIF or MyD88 contribute to the clearance of this bacterium. Further studies are needed to determine whether TRIF-dependent host responses, including altered cytokine/chemokine production pattern, impaired NF-{kappa}B activation, and delayed bacterial clearance and neutrophil recruitment, ultimately contribute to the survival of the host when the host encounters a more virulent strain or a lethal concentration of P. aeruginosa.


   Acknowledgments
 
We thank Elana Maydanski, Sandy Edgar, and Fang Liu for their excellent technical help.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


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

1 This work was supported by grants from the Canadian Institutes of Health Research, Canadian Cystic Fibrosis Foundation, and Izaak Walton Killam Health Center. T.J.L. is supported by a New Investigator Award from the Canadian Institutes of Health Research and an Investigatorship from Izaak Walton Killam Health Center. Back

2 Address correspondence and reprint requests to Dr. Tong-Jun Lin, Izaak Walton Killam Health Center, 5850 University Avenue, Halifax, Nova Scotia B3K 6R8, Canada. E-mail address: tong-jun.lin{at}dal.ca Back

3 Abbreviations used in this paper: TRIF, Toll-IL-1R domain-containing adaptor-inducing IFN-beta; IRF, IFN regulatory factor; BALF, bronchoalveolar lavage fluid; MOI, multiplicity of infection; MPO, myeloperoxidase. Back

Received for publication May 30, 2006. Accepted for publication December 18, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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