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The Journal of Immunology, 2006, 176: 4923-4930.
Copyright © 2006 by The American Association of Immunologists

Targeted Immunomodulation of the NF-{kappa}B Pathway in Airway Epithelium Impacts Host Defense against Pseudomonas aeruginosa1

Ruxana T. Sadikot2,*,¶, Heng Zeng{dagger}, Myungsoo Joo{dagger}, M. Brett Everhart§, Taylor P. Sherrill{dagger}, Bo Li{dagger}, Dong-sheng Cheng{dagger}, Fiona E. Yull{ddagger}, John W. Christman and Timothy S. Blackwell*,{dagger},{ddagger},§

* Department of Veterans Affairs, Nashville, TN 37232; {dagger} Department of Medicine, Division of Allergy, Pulmonary and Critical Care Medicine, {ddagger} Department of Cancer Biology, and § Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232; and Section of Pulmonary, Critical Care, and Sleep Medicine, University of Illinois, Chicago, Chicago, IL 60612


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
We investigated the impact of inflammatory signaling in airway epithelial cells on host defense against Pseudomonas aeruginosa, a major cause of nosocomial pneumonia. In mice, airway instillation of P. aeruginosa resulted in NF-{kappa}B activation in the lungs that was primarily localized to the bronchial epithelium at 4 h, but was present in a variety of cell types by 24 h. We modulated NF-{kappa}B activity in airway epithelium by intratracheal delivery of adenoviral vectors expressing RelA (AdRelA) or a dominant inhibitor of NF-{kappa}B before P. aeruginosa infection. Bacterial clearance was enhanced by up-regulation of NF-{kappa}B activity following AdRelA administration and was impaired by treatment with a dominant inhibitor of NF-{kappa}B. The TNF-{alpha} concentration in lung lavage was increased by AdRelA treatment and beneficial effects of NF-{kappa}B up-regulation were abrogated in TNF-{alpha}-deficient mice. In contrast, NF-{kappa}B inhibition reduced MIP-2 expression and neutrophil influx following P. aeruginosa infection. Therefore, inflammatory signaling through the NF-{kappa}B pathway in airway epithelial cells critically regulates the innate immune response to P. aeruginosa.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Pseudomonas aeruginosa is an opportunistic pathogen that causes disease in patients with impaired host defenses and is often a cause of life-threatening nosocomial infection in critically ill and immunocompromised patients (1, 2, 3, 4). It is also the leading cause of mortality and morbidity in patients with cystic fibrosis (5, 6, 7). Due to the ubiquitous nature of P. aeruginosa and its ability to develop resistance to antibiotics, it continues to be problematic from a treatment perspective. Although the pathobiology of P. aeruginosa pneumonia involves complex interactions between a variety of bacterial and host factors (8, 9), the pivotal host defense mechanisms responsible for determining outcome of this infection remain incompletely defined. Improved understanding of critical signaling pathways in specific cell populations could lead to the development of targeted immunomodulatory treatments to improve patient outcomes.

Bronchial epithelial cells are capable of sensing infecting organisms and mobilizing an innate immune response through the secretion of effector molecules (10, 11). Infection with P. aeruginosa results in activation of signaling cascades in epithelial cells that lead to expression of cytokines, chemokines, and defensins (12), and may induce apoptosis (13, 14). In vitro studies have shown that interaction of P. aeruginosa with specific receptors on epithelial cells is a potent stimulus for nuclear translocation of the transcription factor, NF-{kappa}B (15, 16). However, the role of airway epithelium in vivo in pulmonary host defense against P. aeruginosa pneumonia has not been well characterized and the impact of specific signal transduction pathways, such as NF-{kappa}B, on generation of an effective innate immune response is unknown. NF-{kappa}B comprises of a family of transcription factors that regulates expression of genes involved in inflammatory/immune responses (17). Usually, the term NF-{kappa}B is collectively used for homo- and heterodimeric complexes formed by Rel/NF-{kappa}B proteins. Signal-induced NF-{kappa}B activation is mainly accomplished by phosphorylation of I{kappa}B proteins at two specific serine residues, followed by polyubiquitination and degradation by the 26S proteasome. Liberated NF-{kappa}B then translocates to the nucleus, where it modulates gene expression by binding to {kappa}B motifs in promoters of target genes (18, 19, 20).

Because NF-{kappa}B is a key regulator of many important host defense genes and P. aeruginosa is known to activate NF-{kappa}B in epithelial cells, we hypothesized that modulation of NF-{kappa}B in airway epithelium would impact bacterial clearance in the lungs by altering the innate immune response following infection. Although bacterial components are known to generate a host response by activation of NF-{kappa}B via TLR in macrophages and other inflammatory cells, it is becoming increasingly apparent that NF-{kappa}B signaling in structural cells, including epithelial cells, is necessary to mount a robust inflammatory response in the lungs (21, 22). Previously, we have shown that activation of NF-{kappa}B in airway epithelium is sufficient to induce neutrophilic lung inflammation (23). The present studies were designed to determine whether selectively intervening in the NF-{kappa}B pathway in airway lung epithelium in vivo alters the pathogenesis of P. aeruginosa pneumonia.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animal model

Wild-type (WT)3 C57B6/DBA mice or transgenic NF-{kappa}B reporter mice weighing 20–30 g were used. Transgenic mice expressing photinus luciferase cDNA under control of the proximal 5' HIV-LTR mice on a C57B6/DBA background (HLL) were used (24). Another transgenic mouse line (NGL) used in some studies contains a construct in which four copies of the HIV-LTR NF-{kappa}B enhancer element were placed upstream of the herpes virus thymidine kinase minimal promoter to drive expression of enhanced GFP-luciferase fusion protein (25). Additional studies were done using TNF-{alpha}-deficient mice (C57B/6 background) obtained from Dr. M. Marino (Sloan-Kettering Cancer Center, New York, NY), and WT (C57B/6) controls (26, 27).

Mice were treated with intratracheal (IT) administration of adenoviral vectors after sedation with ketamine/xylazine. Mouse tracheas were directly exposed by surgical resection, pierced with a 26-gauge needle, and injected with 100 µl of the adenovirus (Ad) preparation diluted in sterile PBS. The neck wound was closed with sterile sutures under aseptic conditions.

At the conclusion of experiments, mice were euthanized and lungs were removed; one lung was ground in 1 ml of reporter lysis buffer (Promega) and stored at –20°C for luciferase assays, bacterial colony counts, and the other lung was frozen at –70°C. In some experiments, tracheas were cannulated and lungs were lavaged in situ with sterile pyrogen-free physiological saline that was instilled in three 1-ml aliquots and gently withdrawn with a 1-ml tuberculin syringe. The studies were approved by the Vanderbilt Institutional Animal Care and Utilization Committee.

P. aeruginosa (PA103 strain)

Bacteria from frozen stocks were streaked onto trypticase soy agar plates and grown in a deferrated dialysate of trypticase soy broth supplemented with 10 mM nitrilotriacetic acid (Sigma-Aldrich), 1% glycerol, and 100 mM monosodium glutamate at 33°C for 1–3 h in a shaking incubator. Cultures are centrifuged at 8500 x g for 5 min and the bacterial pellet was washed twice in Ringers lactate and diluted into the appropriate number of CFU per milliliter in Ringers lactate solution determined by spectrophotometer. The bacterial concentration was confirmed by diluting all samples and plating out the known dilution on sheep blood agar plates.

Adenoviral vectors

Replication-deficient rAd type 5 was used to construct adenoviral vectors expressing RelA. An expression cassette containing a CMV promoter driving the expression of RelA was inserted into this vector. Adenoviral vectors expressing a dominant inhibitor of NF-{kappa}B (I{kappa}B-{alpha}DN), which represents a S36-40A mutant of the avian I{kappa}B-{alpha} that cannot be phosphorylated or degraded, and beta-gal have been previously reported (23). Adenoviral vectors expressing firefly luciferase under the control of an NF-{kappa}B-dependent promoter (NF-{kappa}Bluc) were a gift from Dr. L. Prince (University of Alabama, Birmingham, AL) (28, 29). Adenoviral vectors expressing luciferase were a gift from Dr. A. Powers (Vanderbilt University, Nashville, TN). Adenoviral vectors were propagated, purified, and stored at –70°C.

Bacterial colony counts

The right middle lobe was removed aseptically and placed in 1 ml of sterile saline. Lungs were homogenized in a tissue homogenizer in a vented hood under sterile conditions. Serial dilutions of the homogenates were made and 10 µl of each dilution were plated in soy base blood agar plates (Difco). Plates were incubated for 18 h at 37°C and then number of colonies was counted.

Histology and GFP immunohistochemistry

To collect lung tissue, mice were perfused with saline and lungs were inflated with 1 ml of 10% neutral-buffered formalin. After paraffin embedding, 5-µm sections were cut and placed on charged slides. Following paraffin removal, sections were rehydrated and placed in heated Target Retrieval Solution, High pH (DakoCytomation) for 20 min. Tissues were incubated with anti-GFP Ab (BD Clontech) for 2 h at room temperature. Sections without primary Ab served as negative controls. The Vectastain ABC Elite (Vector Laboratories) System and DAB+ (DakoCytomation) were used to produce localized, visible staining. Slides then were lightly counterstained with Mayer’s hematoxylin, dehydrated, and coverslipped.

Bioluminescence imaging

Mice were anesthetized and shaved over the chest and abdomen before imaging. Luciferin (1 mg/mouse in 200 µl of isotonic saline) was administered by i.p. injection and mice were imaged with an intensified charge couple device camera (model no. C2400-32; Hamamatsu). For the duration of photon counting, mice were placed inside a light tight box. Light emission from the mouse was detected as photon counts using the intensified charge couple device camera and customized image processing hardware and software (Hamamatsu). A digital false-color photon emission image of the mouse was generated and photons were counted over a standard area corresponding to the region of the chest overlying the mid lung zone. Photon counts were obtained before and following treatment with adenoviruses so that each mouse could be used as its own control.

Measurement of luciferase activity

Luciferase activity was measured in cells or postmortem tissue samples by adding 100 µl of freshly reconstituted luciferase assay buffer to 20 µl of cell lysate or homogenated lung tissue that was ground in reporter lysis buffer (Promega). Luciferase activity was expressed as relative light units normalized for protein content, which was measured by Bradford assay.

Total and differential cell counts in bronchoalveolar lavage (BAL)

BAL fluid was centrifuged at 400 x g for 10 min to separate cells from supernatant. Supernatant was saved separately and frozen at –70°C. The cell pellet was suspended in serum-free RPMI 1640 culture medium and total cell counts were determined on a grid hemocytometer. Differential cell counts were determined by staining cytocentrifuge slides with a modified Wright stain (Diff-Quick; Baxter) and counting 400–600 cells in complete cross-sections.

Chemokine and cytokine ELISAs

MIP-2, IL-beta, IL-12 (p70 and p40), and TNF-{alpha} levels in BAL were measured using specific ELISA kits according to the manufacturer’s instructions (R&D Systems).

Extraction of nuclear proteins and Western blot for RelA

Nuclear proteins were extracted from whole-lung tissue. A total of 50–100 mg of tissue was mechanically homogenized in liquid nitrogen, to which 4 ml of buffer A (150 mM NaCl, 10 mM HEPES (pH 7.9), 0.6% (v/v) Nonidet P-40, 0.2 M EDTA, 0.1 M PMSF) was added. The homogenate was transferred to a 15-ml Falcon tube and centrifuged at 850 x g in a tabletop centrifuge for 30 s to remove cellular debris. The supernatant was then transferred to a 50-ml Falcon tube and incubated on ice for 5 min before being centrifuged for 10 min at 3500 x g. Supernatant was collected as a cytoplasmic extract. The pellet was resuspended in 300 µl of buffer B (sterile water, 25% (v/v) glycerol, 20 mM HEPES (pH 7.9), 5 min NaCl, 1 M MgCl2, 0.2 min EDTA, 0.1 M phenylsulfonyl fluoride, 1 mg of mindithiothreitol, 10 mg of benzamidine/ml, 1 mg of pepstatin/ml, 1 mg of leupeptin/ml, 1 mg of aprotinin/ml) and incubated on ice for 30 min. Following centrifugation at 14,000 rpm in an Eppendorf microcentrifuge for 2 min, the supernatant was collected as the nuclear extract and was frozen at –70°C. Protein concentrations in nuclear extracts were determined by using the Bradford assay. For Western blot, 25 µg of nuclear protein was separated on a 10% acrylamide gel, transblotted and immunodetected. Abs to RelA were obtained from Santa Cruz Biotechnology.

Statistical analysis

Our statistical analyses were performed with GraphPad InStat software, version 3.01 for Windows NT (GraphPad) using an unpaired t test and unpaired ANOVA. Values of p <0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
To evaluate induction of NF-{kappa}B-dependent transcriptional activity in lung epithelial cells by P. aeruginosa (strain PA103), we treated A549 cells with adenoviral vectors expressing luciferase under the control of an NF-{kappa}B dependent promoter (AdNF-{kappa}Bluc). Forty-eight hours after adenoviral treatment, PA103 was added to cell cultures and incubated for a total of 4 or 8 h. Cells treated with P. aeruginosa showed a significant increase in luciferase activity at both 4 and 8 h compared with uninfected controls (data not shown), indicating that P. aeruginosa directly activates NF-{kappa}B transcriptional activity in lung epithelial cells.

After demonstrating direct activation of NF-{kappa}B in lung epithelial cells in vitro, we examined the distribution of NF-{kappa}B activity in the lungs of mice following IT infection with P. aeruginosa. To assess NF-{kappa}B-dependent transcriptional activity in individual cells in vivo, transgenic reporter mice that express GFP-luciferase fusion protein under the control of an NF-{kappa}B-dependent promoter were infected with PA103 (106 CFU) and lungs were harvested at 4 and 24 h. By immunohistochemical detection, GFP was expressed predominantly in bronchial epithelial cells at 4 h; however, by 24 h, NF-{kappa}B-dependent GFP expression was identified in epithelium and a variety of other cell types, particularly macrophages and infiltrating neutrophils (Fig. 1a). These data indicate that initial NF-{kappa}B activation occurs in epithelial cells following airway challenge with P. aeruginosa and suggests that the NF-{kappa}B pathway in epithelial cells is positioned to play a central role in coordinating the host response to P. aeruginosa.


Figure 1
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  		FIGURE 1. Activation of NF-{kappa}B in epithelial cells by P. aeruginosa. a, Immunohistochemistry for GFP in the lungs of NF-{kappa}B reporter mice (NGL) to demonstrate cellular localization of NF-{kappa}B activity. Mice were harvested at 0, 4, and 24 h after IT administration of P. aeruginosa (PA103) (106 CFU). GFP staining (brown) is identified predominantly in airway epithelial cells at 4 h, whereas staining is also seen in other cell types at 24 h. b, Bioluminescent detection of luciferase activity in vivo in WT mice treated with AdNF-{kappa}Bluc (109 PFU) by IT injection 48 h before administration of PA103 (106 CFU). Following i.p. injection of luciferin, images of representative mice show that photon emission from the lungs was increased after treatment with PA103 but not PBS. c, Photon counts measured over the chest show a significant increase in lung luciferase activity as compared with mice that were treated with PBS, p < 0.001 (n = 5).

 
After identifying prominent NF-{kappa}B activation in airway epithelial cells following P. aeruginosa infection, we used adenoviral vectors to target the NF-{kappa}B pathway in these cells. First, we used a construct that contains an NF-{kappa}B reporter to determine whether P. aeruginosa induces NF-{kappa}B activation in cells that express transgenes delivered by adenoviral vectors. AdNF-{kappa}Bluc (109 PFU) was administered by IT injection into WT mice 48 h before challenge with P. aeruginosa. Following i.p. injection of 1 mg of luciferin, in vivo luciferase activity was detected by bioluminescence imaging. No increase in chest photon emission over baseline was detected at 48 h after AdNF-{kappa}Bluc injection (not shown). P. aeruginosa or control (PBS) was then administered intratracheally and bioluminescence was re-evaluated 24 h later. A marked increase in photon emission from the lungs was detected 24 h after P. aeruginosa infection compared with the same mice before P. aeruginosa infection or controls treated with IT administration of PBS (Fig. 1b). These findings were supported by postmortem luciferase activity measurements in lung homogenates, which revealed luciferase activity of 42 ± 7 relative light units (RLU)/µg of protein in mice treated with PBS compared with 285 ± 38 RLU/µg protein in mice treated with P. aeruginosa, p < 0.001 (Fig. 1c). Together, these experiments indicate that intratracheally delivered adenoviral vectors target a population of airway epithelial cells that activate NF-{kappa}B as a consequence of P. aeruginosa infection.

To modulate NF-{kappa}B activation in airway epithelial cells, we used adenoviral vectors that express RelA, which is the transactivating component of the NF-{kappa}B heterodimer, or adenoviral vectors expressing a dominant inhibitor of NF-{kappa}B (AdI{kappa}Bdn) (23). Transgenic NF-{kappa}B reporter mice that express luciferase under the control of an NF-{kappa}B promoter (HLL) (24, 30) were treated by IT injection of PBS or adenoviral vectors (109 PFU) expressing RelA, I{kappa}Bdn, or a control (beta-gal). In previous studies using this method of IT adenoviral injection, we showed that transgene expression occurs selectively in airway epithelium and not other cell types, including alveolar macrophages (23). To rule out a direct effect of adenoviral vectors on alveolar macrophages in the current studies, we evaluated NF-{kappa}B activation in this cell population after IT Ad injection. HLL reporter mice were treated with Adbeta-gal, AdRelA, AdI{kappa}Bdn, or PBS and BAL was performed 48 h later. Adherent cells (2 x 105) pooled from three mice per group were treated with TNF-{alpha} (10 ng/ml) or vehicle (PBS) for 4 h in DMEM medium. Luciferase activity was measured from the cell lysates at 4 h as a surrogate marker of NF-{kappa}B activation. The luciferase activity in lavage macrophages from mice treated with PBS, Adbeta-gal, AdRelA, or AdI{kappa}Bdn followed by treatment with vehicle (PBS) was 85, 72, 92, and 71 RLU, respectively. After treatment with TNF-{alpha}, luciferase activity in cells from the four groups (PBS, Adbeta-gal, AdRelA, or AdI{kappa}Bdn) increased to 172, 153, 145, and 156 RLU, respectively. These findings indicate that basal and inducible NF-{kappa}B activation in alveolar macrophages are unaffected after delivery of adenoviral vectors to the airways.

Forty-eight hours after IT injection of adenoviral vectors or PBS, NF-{kappa}B activation was determined by bioluminescence imaging of luciferase expression in HLL mice (following i.p. injection of luciferin) (Fig. 2a). Mice treated with AdRelA showed increased photon emission from the lungs compared with PBS treated controls, mice treated with control adenoviral vectors (Adbeta-gal), or mice treated with AdI{kappa}Bdn (Fig. 2b). Mice were then treated with P. aeruginosa (106 CFU) and bioluminescent imaging was repeated at 24 h after infection. Mice treated with AdRelA before P. aeruginosa showed a significant increase in chest bioluminescence compared with the other groups (Fig. 2, a and b). In addition, mice treated with AdI{kappa}Bdn exhibited significantly less photon emission from the lungs than did mice treated with PBS or Ad-beta-gal at 24 h after P. aeruginosa infection. These data indicate: 1) total NF-{kappa}B activity in the lungs is up-regulated after treatment with AdRelA, and 2) P. aeruginosa induced NF-{kappa}B activity is significantly suppressed by administering viruses that express the dominant inhibitor of NF-{kappa}B.


Figure 2
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  		FIGURE 2. Treatment with adenoviral vectors alters NF-{kappa}B activity in lungs following P. aeruginosa infection. a, Representative bioluminescent images from HLL NF-{kappa}B reporter mice after treatment with PBS, Adbeta-gal, RelA, or AdI{kappa}Bdn are shown. Mice were treated with PBS or adenoviral vectors (109 PFU) for 48 h and luciferase activity was measured by bioluminescence following i.p. luciferin injection. Mice were then infected with P. aeruginosa (PA103) and reimaged 24 h later following luciferin injection. b, Quantification of photon emission over the chest in mice described in a. P. aeruginosa induced NF-{kappa}B activity in all groups of mice; however, photon emission was increased in mice treated with AdRelA and inhibited in mice treated with AdI{kappa}Bdn compared with controls treated with PBS or Adbeta-gal (*, p < 0.05 compared with PA103-treated control groups; **, p < 0.05 compared with other groups before PA103 treatment, n = 6–8/group). c, Western blot for detection of nuclear RelA in lung nuclear protein extracts. d, Densitometry of Western blots identified increased nuclear RelA in lungs of mice treated with AdRelA before PA103 and reduced RelA in the group treated with AdI{kappa}Bdn compared with control (Adbeta-gal) (*, p < 0.05 compared with Adbeta-gal control, n = 6/group).

 
To corroborate bioluminescent imaging for evaluation of NF-{kappa}B activity in these mice, luciferase activity was measured in lung homogenates and nuclear translocation of RelA was measured in lung nuclear protein extracts. At the time of harvest (24 h after P. aeruginosa infection), lung luciferase activity was 520 ± 75 RLU/µg protein for the AdRelA-treated group, 260 ± 51 RLU/µg protein for the Adbeta-gal group, and 112 ± 35 RLU/µg protein for the AdI{kappa}Bdn group (p < 0.05 for both AdRelA and AdI{kappa}Bdn groups compared with Adbeta-gal). Although NF-{kappa}B-dependent lung luciferase activity was significantly higher in mice treated with AdRelA compared with mice that were treated with control adenoviral vectors (Adbeta-gal) or AdI{kappa}Bdn, mice treated with AdI{kappa}Bdn contained less luciferase activity in the lungs than mice treated with Adbeta-gal. By Western blot analysis for nuclear RelA, lungs from AdRelA-treated mice showed greater amounts of nuclear RelA than the other groups, and lungs from AdI{kappa}Bdn-treated mice showed reduced nuclear RelA (Fig. 2, c and d). Together, these experiments indicate that IT administration of adenoviral vectors targeting the NF-{kappa}B pathway can substantially modulate P. aeruginosa-induced NF-{kappa}B activation in the lungs.

To determine whether modulation of epithelial cell NF-{kappa}B alters bacterial clearance after P. aeruginosa infection, we measured bacterial colony counts from the right middle lobe at 24 and 48 h after bacterial inoculation (Fig. 3). Although mice treated with Adbeta-gal, AdRelA, AdI{kappa}Bdn, or PBS received an equal initial dose of bacteria, AdRelA-treated mice showed significantly lower bacterial colony counts at 24 and 48 h compared with the other groups. At 48 h, mice treated with AdI{kappa}Bdn had increased bacterial colony counts compared with controls treated with Adbeta-gal or PBS before P. aeruginosa. No differences in colony counts were observed at 24 or 48 h between Adbeta-gal- and PBS-treated mice. Because no differences in bacterial clearance (or measurements of NF-{kappa}B activation) were observed between mice treated with PBS or control adenoviral vectors (Ad-beta-gal) before P. aeruginosa infection, Adbeta-gal-treated mice were used as a single control group for subsequent experiments. These data show that accentuation of NF-{kappa}B activity in airway epithelium enhances bacterial clearance whereas NF-{kappa}B inhibition impairs host defense in this model of P. aeruginosa infection.


Figure 3
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  		FIGURE 3. Modulation of NF-{kappa}B activity in airway epithelial cells alters the pathogenesis of P. aeruginosa pneumonia. Bacterial colony counts from the right middle lobe 24 and 48 h after IT administration of P. aeruginosa (PA103) into mice that were pretreated with Adbeta-gal, AdRelA, AdI{kappa}Bdn, or PBS. Initial colony counts were obtained by harvesting a group of mice 30 min after infection (*, p < 0.05 compared with Adbeta-gal and PBS controls, n = 6/group).

 
We performed additional studies to evaluate mechanisms by which NF-{kappa}B modulation could impact clearance of P. aeruginosa from the lungs. BAL was performed at the time of harvest (24 h after infection) (Fig. 4a). At this time point, neutrophils account for the majority of BAL cells in all three treatment groups (Adbeta-gal, AdRelA, or AdI{kappa}Bdn). Although there was a trend toward increased total cells and neutrophils from BAL in mice that were treated with AdRelA compared with Adbeta-gal before P. aeruginosa infection, mice treated with AdI{kappa}Bdn showed significantly lower total cells and neutrophils in BAL compared with the other groups. We also measured concentrations of NF-{kappa}B-dependent mediators in BAL, including TNF-{alpha}, IL-1beta, MIP-2, and IL-12. IL-1beta and IL-12 levels were not different among the three groups. Interestingly, MIP-2 levels paralleled neutrophil counts with no significant differences between MIP-2 levels from mice treated with AdRelA and Adbeta-gal, but decreased levels of MIP-2 in BAL from mice treated with AdI{kappa}Bdn (Fig. 4b). For TNF-{alpha}, mice treated with AdRelA before P. aeruginosa infection developed significantly higher TNF-{alpha} levels in BAL compared with the other groups of mice (Fig. 4c).


Figure 4
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  		FIGURE 4. Total and differential cell counts, MIP-2, and TNF-{alpha} in the BAL after IT administration of P. aeruginosa (PA103) in mice that were pretreated with Adbeta-gal, AdRelA, or AdI{kappa}Bdn. a, Total cells and neutrophils in BAL from mice harvested 24 h after P. aeruginosa infection (*, p < 0.05 compared with Adbeta-gal control). b and c, MIP-2 and TNF-{alpha} levels in BAL from mice harvested 24 h after P. aeruginosa infection (*, p < 0.05 compared with Adbeta-gal control, n = 6/group).

 
Because higher levels of TNF-{alpha} were found in BAL from mice treated with AdRelA before P. aeruginosa infection, we performed experiments in TNF-{alpha}-deficient mice. Adenoviral vectors (AdRelA and Adbeta-gal) were administered 48 h before IT administration of P. aeruginosa (105 CFU) and mice were harvested 24 h later. In these studies, we found no significant differences in bacterial colony counts between the two groups (4.1 ± 1.2 x 104 colonies in AdRelA-treated mice and 3.8 ± 0.7 x 104 colonies in Adbeta-gal-treated mice, n = 4/group) (Fig. 5a). In TNF-{alpha}-deficient mice, treatment with AdRelA induced a significant increase in total BAL cells (both macrophages and neutrophils) at 72 h compared with Adbeta-gal treatment (Fig. 5b). Following P. aeruginosa infection, BAL cells from AdRelA-treated mice remained increased in comparison to Adbeta-gal treated mice (Fig. 5b). Thus, treatment with AdRelA did not affect bacterial clearance at 24 h in TNF-{alpha}-deficient mice. These findings imply that overexpression of RelA affects host defense against P. aeruginosa through a mechanism involving downstream production of TNF-{alpha}.


Figure 5
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  		FIGURE 5. Beneficial effects of AdRelA treatment on bacterial clearance are lost in TNF-{alpha}-deficient mice. TNF-{alpha} knockout mice were treated with AdRelA or Adbeta-gal (109 PFU). Mice were harvested at 72 h after adenoviral injection (n = 3/group) or challenged with P. aeruginosa (PA103) (105 CFU) at 48 h after adenoviral injection and harvested 24 h later (n = 4/group). a, Bacterial colony counts in mice treated with P. aeruginosa. b, Total BAL cell counts (*, p < 0.05 compared with corresponding Adbeta-gal control group).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In these studies, we found that infection with P. aeruginosa induced NF-{kappa}B activation in airway epithelial cells in vivo. Early epithelial NF-{kappa}B activity was followed by recruitment of inflammatory cells and further NF-{kappa}B activation in a variety of cell types. By modulating epithelial NF-{kappa}B activity in response to bacterial challenge, we identified NF-{kappa}B signaling in these cells as a major determinant of the outcome of P. aeruginosa pneumonia. Up-regulation of NF-{kappa}B activity improved bacterial clearance, while blockade of NF-{kappa}B activation impaired lung host defense. The beneficial effects of accentuated NF-{kappa}B activation appeared to be related to increased production of TNF-{alpha}, because BAL levels of TNF-{alpha} were increased in mice treated with AdRelA and beneficial effects of AdRelA treatment were abrogated in TNF-{alpha}-deficient mice. In contrast, impaired clearance of bacteria following epithelial NF-{kappa}B blockade may be related to decreased neutrophil recruitment because lower MIP-2 levels and reduced neutrophil counts were found in BAL following treatment with AdI{kappa}Bdn.

Airway epithelium is involved in innate immunity in the lungs in multiple ways: establishing a physical barrier to microbial invasion, providing a structural basis for mucociliary clearance, recognizing microbial products by pattern recognition receptors, secreting antimicrobial substances including defensins, and producing a variety of proinflammatory mediators (11). Recent studies have highlighted specific epithelial processes, such as internalization of bacteria and apoptosis mediated through the CD95 pathway, in effective host defense against P. aeruginosa (13, 14). Our studies, however, focus on the role of the airway epithelium in initiating and regulating inflammatory signaling in the lungs, functions whose importance is being increasingly recognized (21, 22). The expression of TLRs 1–10 by airway epithelial cells (31, 32) indicates that these cells are likely participants in inflammatory signaling in infected lungs. P. aeruginosa products, including pili and flagella, can bind to TLRs on bronchial epithelial cells through an interaction with asialyted glycolipids such as asialoGM1 (33, 34). Interaction with these receptor complexes, present in lipid rafts, leads to downstream activation of NF-{kappa}B and production of inflammatory cytokines (33, 35). Our studies indicate that signaling through the NF-{kappa}B pathway in epithelial cells plays an important host protective role against P. aeruginosa infection in intact animals.

Several recent investigations have implicated the NF-{kappa}B pathway in host defense against lung pathogens, including P. aeruginosa (36, 37, 38, 39, 40, 41). Skerrett et al. (36) reported that MyD88, an adaptor protein that facilitates TLR-dependent NF-{kappa}B activation, was essential for clearance of P. aeruginosa from the lungs. Using MyD88-deficient mice, this group showed that MyD88-dependent signaling was integral to cytokine production and neutrophil recruitment in response to respiratory tract infections with P. aeruginosa and Staphylococcus aureus; however, MyD88 was required for clearance of P. aeruginosa but not S. aureus (36). Using a similar study design, Power et al. (37) confirmed that MyD88-deficient mice have impaired bacterial clearance of P. aeruginosa from the lungs compared with the WT controls. This defect in host defense was found to be related to globally deficient activation of NF-{kappa}B in the lungs and reduced production of MIP-2, TNF-{alpha} and IL-1beta. In humans, mutations in genes encoding IRAK-4, an upstream kinase that leads to activation of NF-{kappa}B and the NF-{kappa}B essential modulator, have been linked with impaired NF-{kappa}B activation and recurrent infections with Gram-positive and -negative bacteria (including P. aeruginosa) (38, 39, 40, 41). Together, these studies imply that NF-{kappa}B signaling is important for generating a protective innate immune response to bacterial invasion. Our studies further define this connection between NF-{kappa}B and host defense by providing direct evidence of a host protective function of NF-{kappa}B activation in a defined cell population.

Although the NF-{kappa}B pathway impacts a variety of cellular processes and influences transcription of many genes, our data suggest that the beneficial effects of up-regulating NF-{kappa}B activity in airway epithelium are related to increased TNF-{alpha} production. In our model, it is unclear whether augmented TNF-{alpha} production following AdRelA treatment results from direct up-regulation of NF-{kappa}B-dependent transcription in epithelial cells or from downstream stimulation of alveolar macrophages or other immune cells by mediators released from the epithelium. In A549 epithelial cells, neither infection with AdRelA nor addition of P. aeruginosa were found to induce TNF-{alpha} expression (data not shown). Although the source of TNF-{alpha} is unclear, this mediator has been previously implicated in host defense against P. aeruginosa. The administration of adenoviral vectors expressing TNF-{alpha} to mice before IT challenge with P. aeruginosa enhances bacterial clearance (42), whereas administration of TNF-{alpha}-neutralizing Ab is associated with impaired bacterial clearance (43). Interestingly, TNF-{alpha} knockout mice exhibit a major defect in P. aeruginosa clearance (44) but mice deficient in TNFRs are not more susceptible than controls to P. aeruginosa infection (45, 46). In a recent study, patients with cystic fibrosis who had additional polymorphisms in the TNF-{alpha} gene had an increased susceptibility to P. aeruginosa with severe lung disease compared with the patients who had normal expression of TNF-{alpha} (47). Together, these studies implicate TNF-{alpha} as an important effector molecule in host defense against P. aeruginosa.

In mice treated with the NF-{kappa}B inhibitor (AdI{kappa}Bdn), TNF-{alpha} levels were similar to controls following P. aeruginosa infection; however, decreased expression of the neutrophil chemotactic chemokine MIP-2 and impaired neutrophil recruitment were observed. Recruitment of neutrophils by CXC chemokines is a major component of the protective innate immune response to P. aeruginosa (48, 49). Thus, impaired host defense in AdI{kappa}Bdn-treated mice is likely related to deficient neutrophil recruitment. Although NF-{kappa}B-dependent signaling in the airway epithelium is required for maximal MIP-2 production following P. aeruginosa infection, further up-regulation of NF-{kappa}B by AdRelA treatment did not result in additional chemokine expression and neutrophil recruitment.

In these studies, we have defined the NF-{kappa}B pathway in airway epithelial cells as a specific target for immunomodulatory therapy in P. aeruginosa pneumonia. However, further studies are needed to fully identify the mechanisms by which NF-{kappa}B contributes to host defense and bacterial clearance. By carefully defining targets for intervention, immunomodulatory therapies may prove beneficial as adjuvant therapies to treat patients with severe or recurrent P. aeruginosa infections.


   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 the U.S. Department of Veterans Affairs and National Institutes of Health Grants HL61419 and HL66196 from the National Heart Lung and Blood Institute. Back

2 Address correspondence and reprint requests to Dr. Ruxana T. Sadikot, Section of Pulmonary, Critical Care, and Sleep Medicine, University of Illinois, 840 South Wood Street, Chicago, IL 60612. E-mail address: sadikot{at}uic.edu Back

3 Abbreviations used in this paper: WT, wild type; IT, intratracheal; Ad, adenovirus; BAL, bronchoalveolar lavage; RLU, relative light unit. Back

Received for publication June 13, 2005. Accepted for publication January 31, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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