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The Journal of Immunology, 2006, 176: 3890-3894.
Copyright © 2006 by The American Association of Immunologists

CUTTING EDGE

Cutting Edge: Enhanced Pulmonary Clearance of Pseudomonas aeruginosa by Muc1 Knockout Mice1

Wenju Lu*, Akinori Hisatsune*, Takeshi Koga*,{dagger}, Kosuke Kato*,{dagger}, Ippei Kuwahara*, Erik P. Lillehoj{ddagger}, Wilbur Chen{ddagger}, Alan S. Cross{ddagger}, Sandra J. Gendler§, Andrew T. Gewirtz and K. Chul Kim2,*,{dagger},{ddagger}

* Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, MD 21201; {dagger} Lovelace Respiratory Research Institute, Albuquerque, NM 87108; {ddagger} Department of Medicine, School of Medicine, University of Maryland, Baltimore, MD 21201; § Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Scottsdale, AZ 85259; and Department of Pathology and Laboratory Medicine, School of Medicine, Emory University, Atlanta, GA 30322


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 Disclosures
 References
 
MUC1 (MUC1 in human and Muc1 in nonhumans) is a membrane-tethered mucin that interacts with Pseudomonas aeruginosa (PA) through flagellin. In this study, we compared PA pulmonary clearance and proinflammatory responses by Muc1–/– mice with Muc1+/+ littermates following intranasal instillation of PA or flagellin. Compared with Muc1+/+ mice, Muc1–/– mice showed increased PA clearance, greater airway recruitment of neutrophils, higher levels of TNF-{alpha} and KC in bronchoalveolar lavage fluid, higher levels of TNF-{alpha} in media of flagellin-stimulated alveolar macrophages, and higher levels of KC in media of tracheal epithelial cells. Knockdown of MUC1 enhanced flagellin-induced IL-8 production by primary human bronchial epithelial cells. Expression of MUC1 in HEK293T cells attenuated TLR5-dependent IL-8 release in response to flagellin, which was completely ablated when its cytoplasmic tail was deleted. We conclude that MUC1/Muc1 suppresses pulmonary innate immunity and speculate its anti-inflammatory activity may play an important modulatory role during microbial infection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 Disclosures
 References
 
Pseudomonas aeruginosa (PA)3 is an opportunistic pathogen responsible for pneumonia in immunocompromised patients and chronic lung diseases such as cystic fibrosis. Innate immune responses by alveolar macrophages and epithelial cells are a major first line of defense following PA lung infection (1, 2, 3). Interactions between pathogen-associated molecular patterns and pattern recognition receptors, e.g., TLRs on resident airway macrophages and epithelial cells, result in the release of proinflammatory cytokines and chemokines, including TNF-{alpha} and IL-8 (4, 5). Flagellin, the major immunogenic protein of the bacterial flagellum, signals through TLR5 (6). Proinflammatory molecules released by airway cells recruit leukocytes and enhance the bacteriocidal activity of neutrophils and macrophages. However, the detailed molecular and cellular events following PA exposure that are responsible for bacterial clearance from the airways remain to be established.

MUC1 (MUC1 in human, Muc1 in nonhumans) is a membrane-tethered mucin expressed on the apical surface of glandular epithelial cells and hemopoietic cells (7, 8). The MUC1 cytoplasmic tail (CT) is highly conserved among species (9) and is phosphorylated on tyrosine and serine residues located within docking sites for Src homology-2 and non-Src homology-2-signaling molecules (10, 11, 12). It has been suggested that MUC1 functions during signal transduction in a manner similar to cytokine receptors (10), but the cellular function of MUC1 remains to be determined. Our previous studies demonstrated that Muc1 was a receptor for PA, and PA binding was mediated through flagellin leading to phosphorylation of the Muc1 CT (13, 14, 15). The current study was undertaken to elucidate the role of MUC1/Muc1 during PA- and flagellin-induced airway inflammation. Our results demonstrated that Muc1 null mice cleared inhaled PA better than their wild-type littermates and exhibited enhanced inflammatory responses to PA and flagellin. Mechanistic studies using a human epithelial cell line indicated that MUC1 suppressed flagellin-driven production of inflammatory cytokines via crosstalk with TLR5.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 Disclosures
 References
 
Experimental PA lung infection

Muc1–/– mice and their FVB background Muc1+/+ littermates have been described previously (16). PA strain K (PAK) was cultured in Luria broth and resuspended in PBS. Mice were anesthetized by i.p. injection of ketamine-xylazine (Sigma-Aldrich) and 1 x 105 or 7.0 x 105 CFU applied intranasally (i.n.) in a 40-µl suspension. Lungs were excised at 4 or 16 h postinfection, homogenized in 10 ml of PBS, and CFU were enumerated on Luria agar plates. Bronchoalveolar lavage fluid (BALF) was collected by 3 x 1.0-ml instillation ofnormal saline. All animal procedures were approved by the Institutional Animal Care and Use Committee of the University of Maryland (Baltimore, MD).

Isolation and culture of mouse macrophages

Macrophages were harvested from BALF or the peritoneal space, and erythrocytes lysed with 10 mM Tris-HCl (pH 7.2) containing 150 mM NH4Cl. Peritoneal macrophages (PM) were harvested after treating mice i.p. with 3% thioglycollate for 3 days. The recovered cells consisted of >95% macrophages as determined by immunofluorescence using the F4/80 macrophage marker (eBioscience). Cells were seeded in 24-well tissue culture plates at 1.0 x 105 cells/well in DMEM containing 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin, and allowed to adhere for 24 h before flagellin or LPS (PA10; Sigma-Aldrich) treatment.

Nucleofection of normal human bronchial epithelial (NHBE) cells with MUC1 small interfering RNA (siRNA)

Primary NHBE cells (Cambrex) were propagated in antibiotics-free BEGM medium (BulletKit; Cambrex) and transfected with 1.5 µg of a 21-bp siRNA with a sequence derived from the MUC1 gene (17) (Dharmacon) or a nontargeting control RNA (Dharmacon) using the NHBE Nucleofector kit (Amaxa) according to the manufacturer’s instructions. Following nucleofection, the cells were cultured in 12-mm Millicell inserts coated with human placenta collagen type IV (7.5 µg/insert) (Sigma-Aldrich) at 2.5 x 105 cells/insert in 24-well plates using air-liquid interface medium (18).

Plasmids and transfections

The MUC1-{Delta}CT plasmid containing a deletion of the CT was constructed by BamHI/KpnI digestion of the full-length MUC1 encoding plasmid (19), purification of the 3.3-kb fragment containing the MUC1 extracellular and transmembrane regions, and in-frame ligation with an oligonucleotide linker (sense, 5'-CATTGCCTTGGCTGTCTAG-3'; antisense, 5'-AATTCTAGACAGCCAAGGCAATGAG-3'). HEK293T cells were stably transfected with plasmids encoding the full-length MUC1 or MUC1-{Delta}CT molecules (12) and transiently transfected with a pEF6/V5-His (Invitrogen Life Technologies)-based expression plasmid encoding TLR5 (20) or empty vector using Lipofectamine 2000 (Invitrogen Life Technologies) (21). Transient transfection efficiencies were >95% in both cases.

ELISA

Mouse KC and TNF-{alpha} and human IL-8 were quantified by ELISA using commercially available Abs (eBioscience; R&D Systems). All samples were analyzed in triplicate, and standard curves were performed on each plate.

Statistical analysis

Differences between treatment groups were assessed using the Student’s t test, and considered significant at p < 0.05.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
Disclosures
 References
 
Muc1–/– mice exhibit increased pulmonary clearance of PA and proinflammatory responses

Because our previous studies showed that Muc1 is a receptor for PA (13, 14, 15), we investigated whether or not Muc1 influences airway clearance of the bacteria using Muc1+/+ and Muc1–/– mice. As shown in Fig. 1A, 4 h after i.n. instillation of 1 x 105 CFU/mouse, Muc1+/+ animals displayed ~2-fold greater numbers of PA in the lungs compared with Muc1–/– mice. The difference in lung bacteria between Muc1+/+ and Muc1–/– mice increased with both time and PA inoculum; a 5-fold increase with 1.0 x 105 CFU at 16 h postinfection and >10-fold increase with 7.0 x 105 CFU at 16 h (data not shown). This difference could have resulted from either Muc1 improving PA adherence/colonization or improved PA clearance in Muc1–/– mice. The report by Danjo et al. (22) showing no difference in the number of PA binding to corneal epithelium between Muc1+/+ and Muc1–/– mice and our similar observations in mouse primary tracheal surface epithelial (TSE) cells (our unpublished data) suggested greater PA clearance in Muc1–/– vs Muc1+/+ mice. Bacterial clearance from the airways is mediated by mucociliary processes and phagocytosis by neutrophils and macrophages (3). The former depends on the efficiency of ciliary beating, but, to the best of our knowledge, there are no published studies showing that Muc1 modifies cilia beating. Therefore, we investigated whether the reduced number of PA in lungs of Muc1–/– mice was due to greater leukocyte infiltration. Muc1–/– mice had 73% more neutrophils and 62% more macrophages in BALF compared with Muc1+/+ mice at 4 h postinfection (Fig. 1B). Leukocyte infiltration into airways in response to bacterial infection is mediated by chemokines, particularly IL-8, the expression of which is enhanced by cytokines such as TNF-{alpha}. Therefore, we next examined KC and TNF-{alpha} levels in BALF of Muc1+/+ and Muc1–/– mice following i.n. instillation of PA. KC and TNF-{alpha} levels were significantly greater in Muc1–/– mice compared with Muc1+/+ mice (Fig. 1, C and D). Collectively, these results suggested that the increased PA clearance in Muc1–/– mice was due to increased proinflammatory cytokine production and leukocyte influx into the airways.


Figure 1
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  		FIGURE 1. Muc1+/+ and Muc1–/– mice were treated i.n. with PBS or PAK (1.0 x 105 CFU/mouse). At 4 h postinfection, lung CFU were enumerated (A), BALF leukocytes, neutrophils, and macrophages were counted (B), and KC (C) and TNF-{alpha} (D) levels in BALF were quantified by ELISA. Each bar represents the mean ± SEM (n = 5). *, p < 0.05 comparing PA-treated Muc1–/– mice with Muc1+/+ mice.

 
Muc1–/– mice exhibit increased proinflammatory cytokine production in response to flagellin

Flagellin is the major PA factor inducing proinflammatory cytokines during acute airway infection (23, 24, 25, 26). Accordingly, we asked whether or not the differential responses of Muc1–/– and Muc1+/+ mice to PA could be reproduced by flagellin. PAK flagellin was purified as described previously (27) and determined to have undetectable pilin by Western blotting and LPS levels <0.1 E.U./µg by the Limulus amebocyte lysate test. KC and TNF-{alpha} levels in BALF following i.n. application of flagellin were significantly greater in Muc1–/– mice compared with Muc1+/+ mice (Fig. 2). This amount of flagellin corresponds to the amount recovered from 2.0 x 106 CFU of PA. The increase in flagellin-induced TNF-{alpha} production was greater than that recently reported by Honko et al. (26), possibly due to differences in bacterial strain (PAK vs PAO1), flagellin preparation (native vs recombinant protein), and/or mouse strain (FVB vs BALB/c). Nevertheless, the relative increase in cytokines was virtually identical with the results with PA, suggesting that the proinflammatory response to whole bacteria was mediated by flagellin.


Figure 2
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  		FIGURE 2. Muc1+/+ and Muc1–/– mice were treated i.n. with 20 ng/mouse of flagellin in 40 µl of pyrogen-free PBS or an equal volume of PBS, and KC (A) and TNF-{alpha} (B) levels in BALF were quantified at 4 h. Each bar represents the mean ± SEM (n = 5).

 
TNF-{alpha} is produced by alveolar macrophages and KC by TSE cells (1, 2, 28). Therefore, we hypothesized that these cells in Muc1–/– mice produce more proinflammatory cytokines than Muc1+/+ cells in response to flagellin. Flagellin stimulated greater TNF-{alpha} levels in media of alveolar macrophages from Muc1–/– mice compared with Muc1+/+ mice (Fig. 3A). A similar biphasic response to flagellin was previously reported (29). Additionally, mouse primary TSE cells from Muc1–/– mice produced significantly higher levels of KC compared with Muc1+/+ cells (Fig. 3B). Collectively, the results presented in Figs. 1–3 indicated that Muc1–/– mice exhibited a greater innate immune response both in vitro and in vivo following treatment with PA or flagellin compared with Muc1+/+ mice.


Figure 3
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  		FIGURE 3. A, Alveolar macrophages from Muc1+/+ and Muc1–/– mice were treated for 4 h with flagellin or PBS, and TNF-{alpha} levels in culture media were quantified. B, TSE cells from Muc1+/+ and Muc1–/– mice were harvested and cultured at an air-liquid interface as described previously (31 ), treated with flagellin (10 ng/ml) or PBS for 4 h, and KC levels in basolateral media were measured. Each bar represents the mean ± SEM (n = 3). *, p < 0.05.

 
Knockdown of MUC1 expression increases flagellin-stimulated IL-8 release

Although the above results suggested a role for Muc1 as an anti-inflammatory protein, we could not eliminate the possibility that Muc1–/– mice developed a compensatory Muc1-independent mechanism accounting for the differences with wild-type mice. To test this possibility, primary NHBE cells were treated with a MUC1 siRNA, and flagellin-induced IL-8 levels were determined. Nucleofection of MUC1 siRNA resulted in ~95% knockdown of total cellular MUC1 expression (Fig. 4A), 67% decrease in the number of cells with surface MUC1 expression (Fig. 4B), and 50% greater IL-8 levels (Fig. 4C) compared with cells treated with control RNA.


Figure 4
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  		FIGURE 4. Primary NHBE cells were nucleofected with 1.5 µg of a MUC1 siRNA or a nontargeting control RNA. At 48 h postnucleofection, total MUC1 protein was determined by immunoblotting as described previously (19 ) (A), and cell surface MUC1 was determined by FACS analysis using anti-MUC1 (GP1.4, mouse IgG1, {kappa}; Biomeda) as primary Ab and R-PE-conjugated goat anti-mouse IgG (Santa Cruz Biotechnology) as secondary Ab. Mouse IgG1 ({kappa} isotype; eBioscience) was used as primary Ab to determine threshold fluorescence signal (B). C, Cells were stimulated with 10 ng/ml flagellin for 6 h, and IL-8 levels in basolateral media were measured. Each bar represents the mean ± SEM (n = 3).

 
MUC1 expression inhibits flagellin-stimulated TLR5-dependent IL-8 release

TLR5 mediates flagellin-stimulated innate immune responses (6). Because TLR5 and MUC1/Muc1 are flagellin receptors and both are expressed by airway macrophages and epithelial cells (data not shown), we hypothesized that these two signaling molecules may crosstalk through flagellin. To explore the relationship between MUC1 and TLR5 in response to flagellin, HEK293T cells were stably transfected with a MUC1-expressing plasmid, and flagellin-driven IL-8 release was determined following transient transfection with a TLR5-expressing plasmid. HEK293T cells express TLR5 but not MUC1 (data not shown). Cells transfected with empty vector increased IL-8 release by 55% compared with PBS treatment, whereas cells transfected with TLR5 displayed 164% increase following flagellin treatment (Fig. 5). Flagellin-induced increase in IL-8 release was completely abolished in cells cotransfected with MUC1 and TLR5. In contrast, cells cotransfected with TLR5 and MUC1 containing a deletion of the CT (MUC1-{Delta}CT) exhibited IL-8 secretion that was undistinguishable from cells expressing TLR5 alone. These results indicated that overexpression of MUC1 in HEK293T cells inhibited flagellin-induced TLR5 signaling.


Figure 5
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  		FIGURE 5. HEK293T cells stably transfected with the pcDNA3.1 empty vector (A) or plasmids encoding the full-length MUC1 or MUC1-{Delta}CT (B) were transiently transfected with a TLR5 expression plasmid or empty vector. At 24 h posttransfection, the cells were treated for 6 h with 10 ng/ml flagellin or PBS, and IL-8 levels in media were measured. Each bar represents the mean ± SEM (n = 3). *, p < 0.05 comparing flagellin with PBS treatment.

 
Although TLR5 is the only TLR specific for flagellin, its signaling pathway is shared by TLR2 and TLR4 (30). Therefore, to address the specificity of the MUC1/Muc1 effect, we investigated whether LPS-induced TLR4 signaling is also affected by the presence of Muc1 by using PM prepared from wild-type and Muc1 null mice. The amounts (mean ± SEM; n = 4) of TNF-{alpha} released into spent media of cultured PM during the 4-h treatment with 1, 10, 100, and 1000 ng/ml LPS were 0.011 ± 0.001, 14.43 ± 0.766, 51.69 ± 1.74, and 85.16 ± 5.91 ng/ml in Muc1+/+ and 0.011 ± 0.001, 26.19 ± 1.274, 120.09 ± 7.33, and 149.82 ± 1.85 ng/ml in Muc1–/–. Thus, Muc1–/– PM produced significantly higher levels of TNF-{alpha} compared with Muc1+/+ PM (p < 0.05) to 10, 100, and 1000 ng/ml concentrations of LPS, suggesting that the suppressive effect of MUC1/Muc1 was downstream of TLR4/5.

In conclusion, our results are the first to demonstrate an anti-inflammatory role for MUC1/Muc1 in response to bacterial infection or exposure to bacterial products. Because MUC1 expression is up-regulated by neutrophil elastase (21), a major product of airway inflammation, we speculate the anti-inflammatory activity of MUC1/Muc1 may play an important role in protecting lungs from excessive inflammation during bacterial infection.


   Acknowledgments
 
We thank Dr. Matthew Fenton (University of Maryland, Baltimore, MD) for critical review.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results and 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 National Institutes of Health (RO1 HL-47125) and the Cystic Fibrosis Foundation. Back

2 Address correspondence and reprint requests to Dr. K. Chul Kim, Asthma and Immunology Program, Lovelace Respiratory Research Institute, Albuquerque, NM 87108. E-mail address: kckim{at}lrri.org Back

3 Abbreviations used in this paper: PA, Pseudomonas aeruginosa; CT, cytoplasmic tail; PAK, PA strain K; i.n., intranasal; BALF, bronchoalveolar lavage fluid; PM, peritoneal macrophage; NHBE, normal human bronchial epithelial; siRNA, small interfering RNA; TSE, tracheal surface epithelial. Back

Received for publication September 13, 2005. Accepted for publication January 23, 2006.


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

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Phosphoinositide 3-kinase is activated by MUC1 but not responsible for MUC1-induced suppression of Toll-like receptor 5 signaling
Am J Physiol Lung Cell Mol Physiol, September 1, 2007; 293(3): L686 - L692.
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 Am. J. Respir. Crit. Care Med.Home page
I. Sabroe, L. C. Parker, D. H. Dockrell, D. E. Davies, S. K. Dower, and M. K. B. Whyte
Targeting the Networks that Underpin Contiguous Immunity in Asthma and Chronic Obstructive Pulmonary Disease
Am. J. Respir. Crit. Care Med., February 15, 2007; 175(4): 306 - 311.
[Abstract] [Full Text] [PDF]

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 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
S. J. Skerrett, C. B. Wilson, H. D. Liggitt, and A. M. Hajjar
Redundant Toll-like receptor signaling in the pulmonary host response to Pseudomonas aeruginosa
Am J Physiol Lung Cell Mol Physiol, January 1, 2007; 292(1): L312 - L322.
[Abstract] [Full Text] [PDF]

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