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Implications for Cystic Fibrosis DiseasePatterns in Airway Epithelial Cells:

ExpressionAlginate Elicit Very Distinct Gene Flagellin andPseudomonas aeruginosa

and Yolanda S. López-BoadoLaura M. Cobb, Josyf C. Mychaleckyj, Daniel J. Wozniak

http://www.jimmunol.org/content/173/9/5659doi: 10.4049/jimmunol.173.9.5659

2004; 173:5659-5670; ;J Immunol

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Pseudomonas aeruginosa Flagellin and Alginate Elicit VeryDistinct Gene Expression Patterns in Airway Epithelial Cells:Implications for Cystic Fibrosis Disease1

Laura M. Cobb,2* Josyf C. Mychaleckyj,2† Daniel J. Wozniak,‡ and Yolanda S. Lopez-Boado3*‡

Infection with the opportunistic pathogen Pseudomonas aeruginosa remains a major health concern. Two P. aeruginosa pheno-types relevant in human disease include motility and mucoidy. Motility is characterized by the presence of flagella and is essentialin the establishment of acute infections, while mucoidy, defined by the production of the exopolysaccharide alginate, is critical inthe development of chronic infections, such as the infections seen in cystic fibrosis patients. Indeed, chronic infection of the lungby mucoid P. aeruginosa is a major cause of morbidity and mortality in cystic fibrosis patients. We have used Calu-3 human airwayepithelial cells to investigate global responses to infection with motile and mucoid P. aeruginosa. The response of airway epithelialcells to exposure to P. aeruginosa motile strains is characterized by a specific increase in gene expression in pathways controllinginflammation and host defense. By contrast, the response of airway epithelia to the stimuli presented by mucoid P. aeruginosa isnot proinflammatory and, hence, may not be conducive to the effective elimination of the pathogen. The pattern of gene expressiondirected by flagellin, but not alginate, includes innate host defense genes, proinflammatory cytokines, and chemokines. By contrast,infection with alginate-producing P. aeruginosa results in an overall attenuation of host responses and an antiapoptotic effect. TheJournal of Immunology, 2004, 173: 5659–5670.

Pseudomonas aeruginosa, a Gram-negative bacillus commonlypresent in the environment, acts as an opportunistic pathogenin a variety of settings (1). A number of P. aeruginosa viru-

lence factors, including flagella, pili, LPS, quorum-sensing molecules,proteases, toxins, and others, are critical in the establishment of acuteinfections, as well as in chronic lung infections associated with cysticfibrosis (CF)4 (1, 2). This repertoire of virulence factors promotesadherence to host cells, damages host tissues, elicits inflammation,and possibly disrupts host defenses by altering gene expression in hostcells (3–5). P. aeruginosa environmental strains are usually flagel-lated and therefore motile, in contrast to many CF isolates (6). Thus,most P. aeruginosa acute infections are by strains producing flagellin,a virulence factor that directs a proinflammatory program in epithelialand other cell types (7, 8). However, a prominent feature of P. aerugi-nosa strains infecting CF patients is the conversion to a mucoid,exopolysaccharide alginate-overproducing phenotype (9). This phe-nomenon has been associated with the establishment of the chronic P.aeruginosa respiratory infections that plague the CFpatient. The overproduction of alginate by P. aeruginosa may beadvantageous for the bacteria by impeding phagocytosis, and provid-

ing protection against reactive oxygen species and antibiotics (10–12). The subsequent intense neutrophil-dominated airway inflamma-tion and progressive lung disease are major causes of morbidity andmortality in this disease (13, 14). In vivo studies suggest that clearanceof mucoid strains from murine lungs is diminished compared withnonmucoid strains, indicating improved survival of alginate-producing strains in the respiratory tract (15–18). Alginate enhancesmucin secretion by tracheal epithelial cells (19), and may inhibitneutrophil migration to the sites of infection (20). Interestingly, theproduction of flagellin and alginate by P. aeruginosa are inverselyregulated by the alternative sigma factor AlgT, which is a positiveregulator of mucoidy and a negative regulator of flagella-mediatedmotility (21).

During normal growth and infection, many bacteria secreteflagellin, the structural component of the bacterial flagellum (22).In epithelial cells, flagellin from different bacterial species elicits astrong inflammatory program including IL-8 secretion, inducibleNO synthase activity (23–26), and induced expression of innatehost defense genes, such as matrilysin and human �-defensin-2(h-BD-2) (27–29). Furthermore, secretion of flagellin is involvedin the activation of proinflammatory signaling pathways and neu-trophil trans-epithelial migration (30). Other cell types, includingmonocytes, respond to flagellin inducing the production of proin-flammatory cytokines (31). Furthermore, flagellin plays a role intriggering adaptive immune responses by stimulating chemokinesecretion and migration and maturation of dendritic cells (32, 33),and by modulating T cell activation in vivo (34). Flagellin is theligand of TLR-5 (35, 36), although additional receptors may mod-ulate signaling (37, 38). By contrast, alginate signals throughTLR-2 and TLR-4 to induce cytokine expression in monocytes andmacrophages (39), but the molecular mechanisms mediating theeffects of alginate on epithelial cells, which constitute a first line ofdefense against pathogens in the airways, are unknown.

In this work, we have examined genomic responses in airwayepithelial cells exposed to isogenic motile and mucoid P. aerugi-nosa strains, the two phenotypes relevant in acute and chronic

*Department of Internal Medicine (Molecular Medicine), †Center for Human Genom-ics, and ‡Department of Microbiology and Immunology, Wake Forest UniversitySchool of Medicine, Winston-Salem, NC 27157

Received for publication February 5, 2004. Accepted for publication August 18, 2004.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported in part by the American Lung Association and the CysticFibrosis Foundation (to Y.S.L.B.). D.J.W. is supported by Public Health ServiceGrants AI-35177 and HL-58334.2 L.M.C. and J.C.M. contributed equally to this work.3 Address correspondence and reprint requests to Dr. Yolanda S. Lopez-Boado, Depart-ment of Internal Medicine, Section on Molecular Medicine, Wake Forest UniversitySchool of Medicine, Winston-Salem, NC 27157. E-mail address: [email protected] Abbreviations used in this paper: CF, cystic fibrosis; GO, gene ontology; MOI,multiplicity of infection; h-BD, human �-defensin; LDH, lactate dehydrogenase.

The Journal of Immunology

Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00


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respiratory tract infections, respectively. The responses of airwayepithelial cells to these bacterial phenotypes are qualitatively andquantitatively different. We show that infection with flagellatedmotile strains specifically results in the increased expression ofinflammation and host defense genes. By contrast, infection withmucoid alginate-overproducing strains results in an overall atten-uation of host responses to P. aeruginosa and a decrease in apo-ptosis. Our findings show that flagellin is a critical proinflamma-tory determinant of this bacterium and suggest that other factors,independently of P. aeruginosa mucoidy, may contribute to thepersistent inflammation characteristic of CF.

Materials and MethodsCell culture, bacteria and other reagents

The human lung carcinoma cell lines Calu-3 and A549 were obtained fromthe American Type Culture Collection (ATCC, Manassas, VA), and rou-tinely maintained in RPMI 1640 medium supplemented with 10% FBSwithout antibiotics. P. aeruginosa FRD1 (mucA22) is a mucoid strain iso-lated from a CF patient, and produces the exopolysaccharide alginate, butnot flagellin. The isogenic strain FRD440 (mucA22 algT::Tn501) producesflagellin and not alginate. The strains FRD875 (mucA22 algD::xylEaacC1)and FRD1234 (mucA22 algT::Tn501 fliC:: xylEaacC1) are nonmotile anddo not produce alginate (40, 41) (Table I). All the mutations in FRD1-derived strains were generated using nonpolar cassettes to minimize effectson other genes. The mutations in fliC and algD are in gene clusters thataffect the flagellar and alginate pathways only. Motile P. aeruginosa 56173and 10145 were obtained from ATCC. Alginate-producing strains CF91,CF103, CF1025, and CF1028, are part of a collection of mucoid CF iso-lates used in previous studies (42). The anti-flagellin polyclonal Ab (43)was provided by Dr. A. Prince (Columbia University, New York, NY).Bacteria were routinely grown overnight at 37°C in 3% tryptic soy brothwith the appropriate antibiotics. Gentamicin, FBS, and chemicals wereobtained from Sigma-Aldrich (St. Louis, MO). Cathepsin G was obtainedfrom Elastin Products (Owensville, MO).

Infection of epithelial cells for gene expression analysis

Calu-3 human lung epithelial cells were seeded onto 6-well plates andgrown to �90% density. Epithelial monolayers were infected with 108

CFUs per milliliter of each bacterial strain for 60 min and then washedextensively with PBS. The cultures were subsequently incubated in freshRPMI 1640 medium supplemented with 10% FBS, 100 �g/ml gentamicin,and 10 �g/ml chloramphenicol. After 6 h, total RNA from the cells wasprepared with RNAzol B (Tel-Test, Friendswood, TX), and further purifiedwith RNeasy (Qiagen, Valencia, CA) for microarray hybridization.

Adherence, invasion, and cytotoxicity assays

Adherence and invasion assays were performed essentially as described in(44). Briefly, airway epithelial cells were seeded into 6-well plates andgrown to confluency. Following infection at a multiplicity of infection(MOI) of 50 for 1 h, epithelial cells were washed five times in PBS, andlysed in 0.1% Triton X-100 in distilled H2O. Bacteria were plated on tryp-tic soy agar plates, incubated overnight at 37°C, and counted to determinethe number of adhered bacteria. To calculate the total number of bacteriaper well, sets of duplicate wells were lysed by the addition of 20 �l ofTriton X-100. Bacteria in these lysates, representing the total number ofbacteria present, both intra- and extracellularly, were titrated. Adherencefrequencies were calculated as the number of bacteria recovered after PBS

washes divided by the total number of bacteria present in each well. Todetermine invasion frequencies, after the 1-h initial incubation and PBSwashes, cells were incubated for 4 h in the presence of 100 �g/ml genta-micin or amikacin (Sigma-Aldrich) to eliminate extracellular bacteria.Cells were washed five times with PBS, lysed in 1 ml of 0.1% Triton X-100in distilled H2O, and bacteria were plated on tryptic soy agar plates. In-vasion frequencies were calculated as the number of bacteria survivingincubation with antibiotics divided by the total number of bacteria presentjust before the addition of antibiotics. Cytotoxicity was determined using alactate dehydrogenase-based in vitro toxicology kit (Sigma-Aldrich), ac-cording to the manufacturer’s instructions. Data were analyzed by ANOVAand Bonferroni-type multiple t test. A p-value �0.01 was consideredsignificant.

Cytochrome c release analysis

Quantification of cytochrome c release from the mitochondria was per-formed by enzyme-linked immunoabsorbent assay (Oncogene ResearchProducts, San Diego, CA), according to the manufacturer’s instructions.For these experiments, Calu-3 cells were infected for 4 h at a MOI of 50with the different P. aeruginosa strains, and total cell extracts and cytosolicfractions were prepared by differential centrifugation as described in Ref.45. Data were analyzed by ANOVA and Bonferroni-type multiple t test. Ap-value �0.01 was considered significant. In similar experiments, cyto-chrome c distribution was analyzed by Western blotting analysis of cyto-solic and total cell extract samples (46), with a monoclonal anti-cytochrome c Ab (BD Biosciences-Pharmingen).

Annexin V staining

Apoptosis was analyzed by annexin V binding with commercial reagents,according to the manufacturer’s instructions (R&D Systems, Minneapolis,MN). Briefly, Calu-3 cells were infected for 4 h at a MOI of 50, extensivelywashed, and stained with annexin V-FITC. Simultaneous staining withpropidium iodide was used to detect necrosis. Staining was analyzed byusing an inverted microscope (Nikon Eclipse TE2000-E; Nikon, Melville,NY) at a �100 magnification and QCapture software (Quantitative Imag-ing Corporation, Burnaby, British Columbia, Canada).

Microarray hybridization experiments

Total RNA was extracted from Calu-3 cells after exposure to four differentstrains of P. aeruginosa: FRD1, FRD440, FRD875, and FRD1234. Briefly,biotin-labeled RNA was hybridized to Affymetrix Human Genome U133A(HG-U133A; Affymetrix, Santa Clara, CA) probe arrays, stained withstreptavidin-PE conjugate (Molecular Probes, Eugene, OR), and the fluo-rescence intensities were measured with a laser confocal scanner (Af-fymetrix GeneScanner), according to the manufacturer’s instructions. Fourindependent infection-hybridization experiments were performed for eachstrain with the exception of FRD1234, which was subjected to three rep-licate experiments. Four independent control hybridization experimentswere also performed on noninfected Calu-3 cell samples.

Statistical analysis

Raw data from the hybridization experiments was processed using the Af-fymetrix Microarray Suite Version 5.0 (MAS 5.0; Affymetrix) to extracttranscript detection calls and intensities. All arrays were globally scaled tothe same target intensity and scaling factors checked for consistencyaccording to standard Affymetrix protocols. To smooth within-strainbiological and empirical variation, the independent control and strainarray data sets were analyzed as separate groups of replicates. Additionalinformation on statistical methods is available at www.wfubmc.edu/genomics/publicationdata.htm.

We combined the individual array MAS 5.0 detection statistics (genetranscript present/absent call and p-value) into an overall statistic for eachgroup to classify gene transcripts (i.e., probe sets on the array) as presentor absent at the group level. Changes in expression levels of gene tran-scripts were detected through two separate tests. Gene transcripts that weredetected as present in one control/strain group of arrays, but absent inanother, were classified as showing absolute change. For example, a tran-script was identified as significantly up-regulated in the control � FRDstrain group comparison if the transcript (array probe set) was detected asabsent in the control group and present in the FRD strain group (i.e., anabsolute change up). Absolute down-regulation (change down) is the con-verse situation. Gene transcripts that were detected as present in both com-pared groups were classified as showing relative change. Using the pres-ence/absence tests, we filtered the gene transcripts within each strain groupto remove genes that were absent. Because absent genes have low signalintensity, this effectively also removes weakly expressed genes. We applied

Table I. P. aeruginosa strains

Strain Genotype Phenotype Active AlgT

FRD1 mucA22 Alginate� YesFlagellin�

FRD440 mucA22 Alginate� NoalgT::Tn501 Flagellin�

FRD875 mucA22 Alginate� YesalgD::xylEaacC1 Flagellin�

FRD1234 mucA22 Alginate� NoalgT::Tn501 Flagellin�

fliC::xylEaacC1

5660 GENOMIC RESPONSES TO MUCOID AND MOTILE P. aeruginosa


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a two-sample t test using empirical (permutation-derived) p-values to iden-tify gene transcripts showing a significant relative change in expression be-tween compared strain groups. The maximum p-values for significant t testswere set to 0.0011 (control � FRD1), 0.012 (control � FRD440), 0.0055(control � FRD875), and 0.00087 (control � FRD1234). These p-valuethresholds were chosen to ensure a uniform false discovery rate (47) of 20%across all four group comparisons. Genes were mapped to probes sets andclassified by molecular function (ontology classification) using EASE (http://david.niaid.nih.gov/david) and Netaffyx (http://www.affymetrix.com).

Northern blotting and RT-PCR analysis

Total RNA samples were separated by electrophoresis in 1.2% agarose-formaldehyde gels, and blotted onto Hybond nylon filters (AmershamPharmacia Biotech, Buckinghamshire, U.K.). The integrity of the RNA inthe different samples was ascertained by direct visualization of the gelsunder UV light. Northern hybridization for matrilysin and GAPDH mR-NAs was done as described before (27). For the analysis of the expressionof h-BD, total RNA samples were reverse transcribed using random hex-amer primers (PerkinElmer, Branchburg, NJ), and cDNAs were then am-plified by PCR as described before (29). The sizes of the amplified productsfor h-BD-2 and h-BD-1 are 241 and 258 bp, respectively. For the analysisof Nckap1 (Nap1), cDNAs were amplified for 21 cycles using the primersand conditions described in (48). The size of the amplified product was 184bp. Reactions were analyzed on 3% agarose gels or 6% acrylamide gels.

Infection of epithelial cells for collection of conditioned mediumsamples

Calu-3 and A549 human lung epithelial cells were seeded onto 6-wellplates and grown to �90% density. Monolayers were infected with 108

CFUs of each bacterial strain (corresponding to a MOI of 50) in 1 ml ofRPMI 1640 medium without serum or antibiotics, and incubated at 37°Cfor a period of 60 min. Conditioned medium samples from the 60-mininfection period were collected, centrifuged at 10,000 � g for 10 min toremove debris, concentrated 10-fold by lyophilization, and analyzed byWestern blotting with flagellin specific Abs (43), as described below. Inother experiments, cells were washed extensively after infection and fur-ther incubated in the presence of antibiotics for 24 h postinfection. Con-ditioned media were then collected for the analysis of cytokine and che-mokine secretion by enzyme-linked immunoabsorbent assay withQuantikine reagents (R&D Systems), according to the manufacturer’s in-structions. Data are reported as the means � the SD. All determinations ofcytokine and chemokine secretion were done in duplicate and repeated inat least three independent experiments. Data were analyzed by ANOVAand Bonferroni-type multiple t test. A p-value �0.01 was consideredsignificant.

Purification of flagellin

P. aeruginosa flagellin was purified from overnight culture supernatants asdescribed before (27). For some experiments, flagellin was further purifiedby using polymyxin B beads (Sigma-Aldrich), according to the manufac-turer’s instructions. Removal of endotoxin to �0.06 endotoxin U/ml wasverified by using the Limulus amebocyte lysate detection kit (BioWhit-taker, Walkersville, MD).

Purification of alginate

Alginate was purified from the strain FRD1 as described in Ref. 49, withsome modifications. Briefly, P. aeruginosa FRD1 was grown overnight at37°C in 3% tryptic soy broth medium. Following the addition of 1 vol of

Table II. Gene ontology (GO) processes showing significant regulatory changesa

GO Biological ProcessFRD440 (motile)

vs UninfectedFRD1234 (nonmotile) vs

UninfectedFRD1 (mucoid) vs

UninfectedFRD875 (nonmucoid) vs

Uninfected

Inflammatory/innate immune response 25 (8.2E � 001) 0 1 (2.5E � 003) 0Chemoattractant activity 16 (9.2E � 003) 0 1 (2.5E � 003) 0Viral infectious cycle 3 (2.3E � 002) 0 0 0Plasminogen activator activity 3 (1.4E � 002) 0 0 0Small GTPase mediated signal transduction 49 (5.0E � 004) 4 (1.3E � 002) 5 (2.01E � 002) 12 (2.0E � 001)Apoptosis/cell death 32 (2.8E � 003) 10 (1.97E � 001) 4 (1.00E � 000) 7 (6.4E � 001)Cell cycle 24 (3.8E � 003) 4 (9.8E � 002) 10 (1.5E � 003) 3 (5.6E � 002)Actin cytoskeleton 45 (3.6E � 002) 2 (1.0E � 000) 5 (3.8E � 002) 27 (8.0E � 001)Detoxification/cellular stress/protein

phosphatase activity20 (4.4E � 001) 0 4 (3.8E � 002) 1 (1.00E � 000)

Response to external stimulus 53 (8.2E � 001) 4 (8.1E � 001) 4 (9.5E � 001) 15 (9.6E � 001)Protein metabolism 114 (2.2E � 001) 5 (7.1E � 002) 17 (9.2E � 002) 44 (2.1E � 002)Intracellular 393 (2.2E � 008) 33 (5.5E � 003) 44 (3.3E � 002) 142 (2.5E � 005)Physiological processes 454 (2.0E � 002) 35 (2.8E � 002) 52 (1.3E � 001) 160 (4.9E � 004)

a Values represent the number of significant changes (up- and down-regulated genes) in the selected pathways for each condition (Calu-3 cells exposed to the different P.aeruginosa strains). EASE scores for each pathway are given in parenthesis. The EASE score measures the statistical significance of changes in biological process regulationby comparing the count of genes that were seen to change experimentally, to the total number of genes spotted on the HG133A array that are annotated for that process (a lowscore meaning a more significant, nonrandom event). Technical details on the program EASE are available at http://david.niaid.nih.gov/david/ease/help1.htm.

FIGURE 1. P. aeruginosa-infected airway epithelial cells transcriptomeexpression summary. Control (uninfected) and P. aeruginosa strain-ex-posed transcriptome counts are the number of unique annotated genes (N)statistically detected in each treatment group of arrays. The area of eachcircle is also proportional to these counts. Gene transcriptional changes areindicated by black and patterned arrows, and � indicates counts of uniquegenes that show either a relative change in expression (genes statisticallydetected both in control uninfected and in infected airway epithelial cells),or absolute changes in gene expression (genes detected in only one of thepair) for each infected vs uninfected pairwise comparison groups. Blackarrow lengths are proportional to unique up-regulated gene changes in thecomparison of uninfected cells to cells exposed to each P. aeruginosastrain. Patterned arrows represent the same for down-regulated uniquegenes. FRD1 is a mucoid nonmotile CF isolate. The strains FRD440 (non-mucoid, motile), FRD875 (nonmucoid, nonmotile), and FRD1234 (non-mucoid, nonmotile, algT mutant) were derived from FRD1 (Table I).

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saline, alginate was precipitated from the supernatant of this culture (whichhad an obvious mucoid appearance) by the addition of an equal volume of2% cetylpyridinium chloride (Sigma-Aldrich). After a centrifugation stepat 25,000 � g for 30 min, the pellet was resuspended in the initial volumeof 1 M NaCl. Finally, alginate was precipitated by the addition of 1 vol ofchilled isopropanol, resuspended in PBS, and quantified in a colorimetricassay using alginic acid (Sigma-Aldrich) to plot a standard curve (50).

Transient transfection and NF-�B activity determination

A549 cells were seeded in 96-well plates at �90% confluency in RPMI1640 medium containing 10% FBS 24 h before the transfection. The re-porter plasmid pNF-�B-Luciferase (Stratagene, La Jolla, CA) was used toanalyze the effect of external stimuli on NF-�B activity in A549 cells.Transfections were conducted using 200 ng of DNA and 1 �l of Lipo-fectamine 2000 (Invitrogen Life Technologies, Carlsbad, CA) per well.After an incubation of 48 h to allow maximal expression of the transgene,cells were stimulated with flagellin (10�7-10�9 M) or alginate (20–40�g/ml) for different periods of time. Cells were lysed in Glo-Lysis buffer(Promega, Madison, WI) for the analysis of luciferase expression withBright-Glo reagents (Promega), according to the manufacturer’s instruc-tions. Luciferase activity in each sample was measured with a ReporterMicroplate Luminometer (Turner Designs, Sunnyvale, CA). Data are re-ported as the means � the SD. All transfection experiments were done intriplicate and repeated at least three times. Data were analyzed by ANOVAand Bonferroni-type multiple t test. A p-value �0.01 was considered sig-nificant. Transfection efficiency was assessed by cotransfection with a plas-mid containing Renilla luciferase under the control of the SV40 viral pro-moter (phRL; Stratagene), and did not vary �25% among the differentsamples. However, because the expression of this internal standard was

modified by the ligands used in our experiments, Renilla expression datawere not used to correct the firefly luciferase expression data (51).

Immunoblotting

Conditioned medium samples were separated on 12% SDS-polyacrylamidegels and transferred by semidry electrophoretic transfer to nitrocellulosemembranes (Hybond ECL; Amersham Pharmacia Biotech), and probedwith anti-flagellin Abs as described before (27, 29).

ResultsMicroarray analysis of airway epithelial cells exposed to motileand mucoid P. aeruginosa reveals very distinct patterns of geneexpression

To gain insight into the molecular processes underlying the inter-action of airway epithelial cells with P. aeruginosa phenotypesrelevant in lung disease, we conducted a genechip analysis of air-way epithelial cell responses to P. aeruginosa mucoid and motilestrains. For this, we used an in vitro model of Calu-3 human lungepithelial cells infected with the alginate-producing CF isolateFRD1 and a series of isogenic mutant strains, including a motilestrain that does not express alginate (FRD440), and mutants thatlack the expression of alginate and flagellin (FRD875), and algi-nate, flagellin, and the alternative sigma factor AlgT (FRD1234)(Table I). Calu-3 cells were exposed to the different strains at aMOI of 50, and gene expression examined at 6 h postinfection.

Table III. Genes up-regulated in P. aeruginosa-infected cellsa

GO Biological Process Gene Symbol Gene Name Fold Induction

FRD440-induced genesInflammation/chemoattractant ICAM-1 Intercellular adhesion molecule 1 Absolute change

activity/acute phase response CCL20 Chemokine (CC motif) ligand 20 (MIP3�) Absolute changePTX3 Pentraxin 3 Absolute changeCXCL6 Chemokine (CXC motif) ligand 6 5.5CXCL1 Chemokine (CXC motif) ligand 1 5.0CXCL2 Chemokine (CXC motif) ligand 1 4.7CXCL3 Chemokine (CXC motif) ligand 3 3.4CXCL5 Chemokine (CXC motif) ligand 5 1.2IL-8 Interleukin-8 4.0NFKBIA NF-�B inhibitor, � 2.8

Plasminogen activator activity PLAU Plasminogen activator, urokinase 3.4PLAT Plasminogen activator, tissue 5.0

Detoxification/cellular stress/proteinphosphatase activity

CYP1A1 Cytochrome P450, family 1, subfamily A, polypeptide 1 11.4

ALDH1A3 Aldehyde dehydrogenase 1 family, member A3 2.0PPP2R1B Protein phosphatase 2, regulatory subunit A, � isoform 1.3

Microtubules/cytoskeleton TUBB Tubulin, � polypeptide Absolute changeFRD1234-induced genes

Small GTPase mediated signaltransduction

RAB5C Member RAS oncogene family, RAB5C 3.0

Physiological processes LRP-8 Low density lipoprotein receptor-related protein 8 1.8ILF2 Interleukin enhancer binding factor 2 1.4

FRD1-induced genesApoptosis MCL1 Myeloid cell leukemia sequence 1 (Bcl-2-related) Absolute change

NCKAP1 NCK-associated protein 1 1.9Inflammation/chemoattractant

activityCXCL1 Chemokine (CXC motif) ligand 1 1.8

IL-8 Interleukin 8 1.6Detoxification/cellular stress/protein

phosphatase activitySLC26A2 Solute carrier family 26 (sulfate transporter), member 2 1.8

PTPRE Protein tyrosine phosphatase, receptor type, E 1.6PTP4A1 Protein tyrosine phosphatase type IV A, member 1 1.4

FRD875-induced genesMicrotubules/cytoskeleton KRT17 Keratin17 1.9Detoxification/cellular stress/protein

phosphatase activityALDH1A3 Aldehyde dehydrogenase 1 family, member A3 1.9

PTPRE Protein tyrosine phosphatase receptor type E 1.6PPP2R3A Protein phosphatase 2 1.4

a Genes up-regulated in Calu-3 cells by exposure to each of the strains were sorted by fold induction and GO biological process. Genes showing the highest level of inductionare listed here (relative changes). Also listed are the genes that show an absolute change (i.e., signal detection was not significant in uninfected cells).



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Fig. 1 summarizes the number of unique annotated genes statisti-cally detected in each treatment group of arrays. There were a totalof 612 statistically significant changes (313 up-regulated, 299down-regulated) in gene expression in response to the motilestrain, FRD440, while only 48 statistically significant changeswere observed in response to the isogenic fliC mutant, FRD1234.Therefore, the presence of flagellin in the bacteria correlates with�500 changes in gene expression in infected airway epithelialcells. Furthermore, the conversion of P. aeruginosa to a mucoidphenotype, such as what takes place in CF (strain FRD1), resultedin only 67 statistically significant changes (39 up-regulated, 28down-regulated) in gene expression. Remarkably, our transcrip-tional profile analysis shows that a nonmucoid and nonmotilestrain (FRD875) regulates the expression of 231 genes in airwayepithelial cells. Interestingly, strain FRD875 is a nonalginate-pro-ducing mutant derived from the mucoid isolate FRD1. The alter-native sigma factor AlgT, which regulates the expression of manyvirulence factors by P. aeruginosa (52), is active in both strainsFRD1 and FRD875 (Table I). Therefore, our results show that theproduction of alginate by P. aeruginosa actually attenuates themagnitude of the host response to this bacterium. Taken together,these results demonstrate that a P. aeruginosa motile phenotypehas a much more extensive effect on host gene transcription thana mucoid phenotype, and suggest that flagellin and alginate directsubstantially different patterns of gene expression in airway epi-thelial cells. Consistent with this hypothesis, the lowest number ofhost gene expression changes (only 48 statistically significantchanges) was observed in response to the strain FRD1234, a non-mucoid, nonmotile, algT mutant strain (Fig. 1). Full data sets forall microarrays analyzed in this study are available in the NationalCenter for Biotechnology Information Gene Expression Omnibus(NCBI GEO) database (www.ncbi.nlm.nih.gov/geo). The genomicdata have the following GEO accession numbers: GSM14498-GSM14516 (GSE923, NCBI tracking system no. 15031016).

The changes in biochemical and cellular pathways in responseto P. aeruginosa exposure are summarized in Table II. The mostsignificant changes in gene expression, both qualitative and quan-

titative, were elicited by the motile strain FRD440. The isogenicfliC mutant, FRD1234, had no significant effect on many of thesebioprocesses. Thus, flagellin specifically directs gene expressionchanges in pathways related to the inflammatory and innate im-mune responses, chemoattractant activity, cell cycle control, andresponse to external stimuli. By contrast, the conversion of P.aeruginosa to mucoidy results in a very limited effect on pathwayscoordinating the immune and inflammatory responses, as seen inthe response to the strain FRD1. Furthermore, the number of genesthat showed differential expression in response to the strains FRD1and FRD875 (nonalginate producing) in the broad functional cat-egories of protein metabolism, intracellular localization, and phys-iological processes illustrates the fact that the production of algi-nate by the mucoid strain FRD1 modulates, and in fact stronglyattenuates, the extent of the host response to P. aeruginosa. TablesIII and IV present a summary of changes in expression for selectedgenes in response to each bacterial strain. Confirming our microar-ray analysis, up-regulation of the chemokine CCL20, ICAM-1, andthe acute phase reaction protein pentraxin 3 by flagellin has beenrecently described (32, 53, 54). Interestingly, the most significantup-regulating changes in response to FRD1 exposure were ingenes related to apoptosis (Table III). Genes down-regulated inresponse to bacterial exposure were included in various cell sig-naling categories (Table IV).

P. aeruginosa attaches to Calu-3 human airway epithelial cells

To further characterize the interaction between P. aeruginosa andCalu-3 airway cells, we determined the adherence and invasionfrequencies of the bacterial strains listed in Table I. As shown inTable V, FRD440 had a higher rate of attachment and invasionthan the other strains, suggesting that the presence of flagellinand/or motility may favor these interactions. However, and moreimportantly, there were no significant differences in adherence andinvasion between the mucoid strain FRD1 and the isogenic non-alginate-producing strain FRD875. Therefore, the data suggest thatalginate does not significantly modulate these interactions of P.aeruginosa with Calu-3 cells, while very significantly affecting

Table IV. Genes down-regulated in P. aeruginosa-infected cellsa

GO Biological Process Gene Symbol Gene Name Fold Repression

FRD440-repressed genesProtein synthesis RPS11 Ribosomal protein S11 0.31Unknown MRF2 Modulator recognition factor 2 0.32Cholesterol synthesis HMGCS1 3-hydroxi-3-methylglutaryl-coenzyme A synthase 1 0.33

FRD1234-repressed genesUnknown MGC14799 Hypothetical protein MGC14799 Absolute change

DKFZp566C0424 Putative MAPK activating protein PM20, PM21 Absolute changeGTF2H3 General transcription factor IIH, polypeptide 3 Absolute change

Small GTPase mediated signal transduction RALBP1 ralA binding protein 1 0.42Ubiquitin-proteasome pathway UBXD2 UBX domain containing 2 0.43Metabolism DLST Dyhidrolipoamide S-succinyl-transferase 0.44

FRD1-repressed genesSmall GTPase mediated signal transduction IQGAP1 IQ motif-containing GTPase-activating protein 1 0.48Phosphoinositide binding proteins PLEKHA1 Pleckstrin homology domain-containing family A member 1 0.65Nuclear receptor activators BRD8 Bromodomain-containing protein 8 0.66

FRD875-repressed genesApoptosis TRAF4 TNF receptor-associated factor 4 Absolute changeUnknown C14orf106 Chromosome 14 open reading frame 106 Absolute change

CYR61 Cysteine-rich angiogenic inducer 61 Absolute changePhosphoinositide binding proteins PICALM Phosphatidylinositol-binding clathrin assembly protein Absolute changeMetabolism/physiological processes APP Amyloid � (A4) precursor protein 0.20

RAD23B RAD23B homolog B (yeast) 0.22CHC1 Chromosome condensation 1 0.26

a Genes down-regulated in Calu-3 cells by exposure to each of the strains were sorted by fold repression and GO biological process. Genes showing the highest level ofrepression are listed here (relative changes). Also listed are the genes that show an absolute change (i.e., signal detection was not significant in infected cells compared touninfected cells).



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gene expression (Fig. 1, Table II). Furthermore, in our experimen-tal design of 1-h infection followed by a 5-h or 23-h postinfectionincubation in the presence of antibiotics, no significant cytotoxicitywas caused by the different P. aeruginosa strains, as determined bylactate dehydrogenase (LDH) release (Table VI).

Gene regulation by different P. aeruginosa phenotypes

To further examine the regulation of gene expression by P. aerugi-nosa phenotypes relevant in airway infection, we exposed Calu-3human lung epithelial cells to the strains listed in Table I. We havepreviously shown that bacterial exposure, and specifically Gram-negative flagellin, up-regulates the expression of matrilysin, a ma-trix metalloprotease involved in host defense (27, 29, 55). Expo-sure to the flagellated strain FRD440 resulted in a 5-fold inductionin the expression of matrilysin (Fig. 2A). By contrast, neither thealginate-producing P. aeruginosa strain FRD1 or the double mu-tants lacking alginate and flagellin production (FRD875 andFRD1234) had any effect on matrilysin expression in human lungcarcinoma cells (Fig. 2A, data not shown). To further explore theregulation of host defense gene expression, we examined the ex-pression of the h-BDs, h-BD-1 and h-BD-2, in infected Calu-3cells. As shown in Fig. 2B, the expression of h-BD-2 is specificallyup-regulated by infection with flagellated P. aeruginosa strains(ATCC 51673 and FRD440), but not by the CF isolate, alginate-producing strain FRD1, or the nonmucoid/nonmotile strains,FRD875 and FRD1234 (data not shown). The expression of h-BD-1, which is constitutive (56), did not change in response toexposure to any of these strains and thus served as an internalcontrol. The expression of Nckap1 was induced by FRD1, but notthe other strains (Fig. 2C), in agreement with the microarray data.We examined the response of additional markers of inflammationto P. aeruginosa virulence factors in airway epithelial cells. Forthis, we determined the levels of secretion of IL-8 and GM-CSF,which are involved in neutrophil recruitment and survival in theairways (57) (Fig. 2D). Infection with the flagellated strains ATCC51673 and FRD440 resulted in a 5- to 10-fold induction in IL-8and GM-CSF secretion at 24 h (Fig. 2D). By contrast, infectionwith the alginate-producing strain FRD1, as well as the nonmotile,nonmucoid strains FRD875 and FRD1234, had no significant ef-fect on chemokine secretion (Fig. 2D, data not shown). Further-more, the increased expression of host defense genes correlatedwith the presence of soluble flagellin released by P. aeruginosamotile strains to the supernatant of infected epithelial cells (Fig.

2E) (27). In fact, the degree of gene expression increase in theseexperiments correlates with the amount of soluble flagellin de-tected in the supernatant of infected cells, which varies among P.aeruginosa strains (Fig. 2F). Alginate, at a concentration between0.1–0.5 �g/ml, was detected in the 1-h-conditioned medium ofcells exposed to FRD1 (mucoid strain).

P. aeruginosa mucoid strains do not induce the expression of asubset of host defense genes

The differential regulation of gene expression by mucoid and mo-tile phenotypes was not restricted to the CF isolate FRD1 andFRD1-derived strains. Thus, the specificity of the increase in hostdefense gene expression and chemokine secretion by flagellated P.aeruginosa was further confirmed by exposure of Calu-3 airwayepithelial cells to a panel of mucoid and motile P. aeruginosastrains, i.e., only the flagellated strains were able to induce signif-icantly the expression of the examined host defense genes (Fig. 3).

Purified flagellin and alginate recapitulate the effects ofexposure to whole bacteria

Taken together, our studies demonstrate that motility and mucoidy,two critical P. aeruginosa virulence phenotypes, have very distinctand specific effects on host gene expression. Our data also showthat motility, but not mucoidy, is the bacterial phenotype specifi-cally up-regulating host defense gene expression (Table II, Figs. 2and 3). We next investigated whether the effects of exposure to themucoid and motile strains could be reproduced by challenging thecells with purified flagellin and alginate. In these experiments,Calu-3 cells were challenged for 1 h with purified components, andgene expression examined at 6 h posttreatment. As shown in Fig.4 (A and B), matrilysin and h-BD-2 were induced by flagellin andnot alginate. Therefore, these data correlate with the expressionpattern seen in cells directly exposed to motile and mucoid strains(Figs. 2 and 3). Furthermore, treatment of Calu-3 cells with puri-fied flagellin, but not alginate, resulted in a dose-dependent in-crease in chemokine and cytokine secretion (Fig. 4, C and D). Infact, treatment with 10�8 M purified flagellin resulted in levels ofIL-8, IL-6, CCL20 (MIP-3�), and GM-CSF secretion very similarto those observed with infection (Figs. 2, 3, and 4, data not shown).Based on the yield of our flagellin purification procedure and theamount of flagellin detected in infected cell supernatants (27) (Fig.2, D and E), challenge of Calu-3 epithelial cells with 10�8 Mflagellin is roughly equivalent to a direct infection at a MOI of

Table V. Attachment and invasion of P. aeruginosa strainsa

FRD1 FRD440 FRD875 FRD1234

Attachment (% of total bacteria) 0.33 � 0.11 1.1 � 0.09� 0.26 � 0.08 0.15 � 0.07Invasion (% of adhered bacteria) 0.008 � 0.0005 0.01 � 0.007� 0.005 � 0.0015 0.002 � 0.001

a Attachment is expressed as the number of bacteria adhered to Calu-3 cells with respect to the total number of bacteria present in the well for each strain. Invasion frequencieswere calculated as the number of bacteria surviving incubation with antibiotics divided by the total number of bacteria present just before the addition of antibiotics.

�, Statistically significant difference by ANOVA and Bonferroni-type multiple t test ( p � 0.01).

Table VI. Cytotoxicity of P. aeruginosa strainsa

Uninfected FRD1 FRD440 FRD875 FRD1234

LDH (6 h post 1-h infection) ND ND ND ND NDLDH (24 h post 1-h infection) ND ND ND ND NDLDH (8 h continuous infection) ND 0.014 � 0.004 0.022 � 0.009 0.017 � 0.005 0.025 � 0.007

a LDH activity was determined in the conditioned media of Calu-3 cells infected for 1 h and incubated for an additional 5 or 23 h in the presence of antibiotics, and in theconditioned media of cells continuously infected for 8 h. ND, No absorbance at 490 nm was detected. Data from continuous infection experiments are shown as positive control.LDH is expressed as millimoles of formazan per 106 cells, using a molar extinction coefficient of 19.9 mmol�1 cm�1.



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10–50. By contrast, treatment of Calu-3 cells with purified alginateat concentrations 20–80 �g/ml had no effect on host defense geneexpression and chemokine and proinflammatory cytokine secre-tion, even for periods of treatment up to 24 h (Fig. 4). It is worthmentioning that the concentration of alginate in CF sputum variesbetween 4 and 100 �g/ml (58). Furthermore, treatment of flagellinwith polymyxin B did not affect chemokine secretion by airwayepithelial cells, suggesting that the effect is LPS-independent (datanot shown). This finding confirmed our previous observation thatmatrilysin induction by flagellin was not inhibited by polymyxinB, and was in fact LPS-unrelated (27). Furthermore, the effect offlagellin is completely dependent on the integrity of the protein,and flagellin bioactivity is lost when the protein is specificallycleaved by neutrophil serine proteases, including cathepsin G (Fig.4E) (29).

Similar responses of other human airway epithelial cells tomucoid and motile P. aeruginosa

We further investigated the effects of exposure to P. aeruginosa inepithelial cells from distal lung by infecting type II pneumocyte-like A549 human cells with FRD1 and the FRD1-derived strains(Fig. 5). A549 cells do not express matrilysin or defensins (Y. S.Lopez-Boado, unpublished observations), but responded to in-fection with increased secretion of IL-8 upon exposure to all thestrains (Fig. 5A). However, the presence of flagellin resulted ina further 5-fold increase in the amount of IL-8 detected in theconditioned medium of infected cells (compare the levels ob-tained in response to the motile strain FRD440 and the corre-sponding isogenic fliC mutant, FRD1234), while the presence ofalginate did not augment IL-8 secretion by these cells (comparethe levels in response to the mucoid strain FDR1 and the

FIGURE 2. Host defense gene expression is not up-regulated by exposure to FRD1, a P. aeruginosa mucoid CF isolate, in airway epithelial cells. A,Calu-3 cells were infected for 1 h at a ratio of 50 bacteria per epithelial cell with the strains FRD1 (mucoid), FRD440 (motile), and FRD875 (nonmucoid,nonmotile), and the expression of matrilysin and GAPDH examined by Northern blotting with specific probes at 6 h postinfection. Only the motile strainup-regulates matrilysin expression. Cntl, uninfected cells. B, In a similar experiment, the expression of h-BD-2 and h-BD-1 was examined by RT-PCR.h-BD-2 expression was exclusively induced by the motile strains ATCC 51673 and FRD440, while the expression of h-BD-1 is constitutive. Amplifiedproducts for h-BD-2 and -1 (241 and 258 bp, respectively) were resolved on a 3% agarose gel. C, In a similar experiment, the expression of Nckap1 ininfected cells was examined by RT-PCR. Only FRD1 induced Nckap1 expression. Amplified products (184 bp) were resolved on an 8% acrylamide gel.D, Secretion of chemokines is increased by exposure to motile, but not mucoid, P. aeruginosa. Calu-3 cells were exposed to FRD1 (mucoid), FRD440(motile), and FRD875 (nonmucoid, nonmotile) for 1 h at a MOI of 50. After extensive washing and the addition of fresh medium containing antibiotics,conditioned medium were collected at 24 h postinfection and the expression of IL-8 and GM-CSF was examined by ELISA. P. aeruginosa ATCC 51673(a flagellated strain) and TNF-� (100 ng/ml) were used as positive controls. �, Significantly different from control (p � 0.01). E, Soluble flagellin wassecreted to the conditioned medium (of the 1-h period of infection) of Calu-3 cells infected with the motile strain FRD440, where it was detected by Westernblotting with anti-flagellin Abs. F, Different amounts of soluble flagellin are secreted by motile P. aeruginosa strains as detected by Western blotting inconditioned medium (1 h infection at a MOI of 50). PAO1-NP is a pilin (pilA) mutant isogenic to PAO1. The molecular mass of flagellin varies between45 and 53 kDa depending on the strain.



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corresponding isogenic nonalginate-producing strain FRD875).These results were further confirmed by using purified flagellinand alginate (Fig. 5B). A549 cells challenged with flagellin re-sponded with a 20-fold increase in IL-8 secretion, while theexopolysaccharide alginate had no significant effect. Indeed, theregulation of IL-8 and other markers of inflammation (data notshown) in the alveolar-like A549 cells in response to P. aeruginosaexposure is similar to the response observed in Calu-3 cells.

P. aeruginosa flagellin, but not alginate, induces NF-�B activityin airway epithelial cells

We examined the effects of flagellin and alginate on NF-�B ac-tivity by using cells transiently transfected with an NF-�B reporterplasmid. As shown in Fig. 6A, flagellin, but not alginate, was able

to induce NF-�B activity in A549 airway epithelial cells. The ef-fect was dose- and time-dependent, with maximum induction ofNF-�B activity observed after 3 h of challenge with 10�7 M flagel-lin (Fig. 6, data not shown). By contrast, alginate (at concentra-tions between 20 and 40 �g/ml) was unable to induce NF-�Bactivity in transfected cells (Fig. 6A). Altogether, these results in-dicate that flagellin, but not alginate, activates NF-�B-dependentpathways in airway epithelial cells. Consistent with these data, theinduction of matrilysin and other host defense gene expression wasspecifically inhibited by the proteasome inhibitor MG132 (whichblocks NF-�B activity), but not the p38-MAPK inhibitorSB203580 or the MEK1 inhibitor PD98059 (Fig. 6B, data notshown).

Exposure to mucoid P. aeruginosa has an antiapoptotic effect onairway epithelial cells

Infection with P. aeruginosa causes apoptosis of airway epithelialcells, a mechanism involved in bacterial clearance by the host (59),which is accompanied of release of cytochrome c from the mito-chondria (46). Previous studies have examined the effect of motileP. aeruginosa on apoptosis (46, 60, 61). To compare the effects ofexposure to the mucoid and motile P. aeruginosa phenotypes onCalu-3 cell apoptosis, we determined the degree of cytochrome crelease from the mitochondria in infected cells. As shown in Fig.7A, cytochrome c was released in response to P. aeruginosa in-fection. However, there was significantly less cytochrome c re-leased to the cytosol in cells exposed to the mucoid strain FRD1,compared with the other strains. Similar results were observedwhen cytochrome c was detected by Western blotting in cytosolicand total extracts from infected cells (Fig. 7B). Finally, we exam-ined P. aeruginosa-induced apoptosis by performing an annexin Vstaining analysis of infected Calu-3 cells. Annexin V staining de-tects phosphatidyl serine flipped to the outer leaflet of the plasmamembrane, an early apoptotic event during infection (62). Asshown in Fig. 8, exposure to the motile strain FRD440 resulted inthe staining of virtually all cells. However, infection with the mu-coid strain FRD1 resulted in markedly reduced apoptosis com-pared with cells exposed to the other strains. No staining withpropidium iodide was observed in infected cells (data not shown).Thus, these data strongly suggest that the production of alginate byP. aeruginosa results in less apparent apoptosis in infected cells.

DiscussionIn this work, we have analyzed the transcriptome of human airwayepithelial cells exposed to P. aeruginosa phenotypes relevant inacute and chronic infections. Our model of coculture of humanlung cells with isogenic strains of this bacterium has allowed usto identify changes in expression patterns that can be ascribed to spe-cific P. aeruginosa virulence determinants. Thus, exposure to motilestrains directs a response characterized by the increased expression inpathways related to inflammation and host defense. Furthermore,this effect is specifically orchestrated by flagellin, as demonstratedby the lack of effect of isogenic fliC mutants, and many features ofthese responses can be reproduced by challenging airway epithelialcells with purified flagellin. By contrast, the response of Calu-3cells exposed to mucoid P. aeruginosa strains and purified alginateis not proinflammatory and is much more restricted in the numberof genes whose expression was significantly changed, comparedwith motile strains. Therefore, our data show that P. aeruginosamucoid strains, which chronically infect CF patients, do not elicitthe expression of proinflammatory pathways in this model of air-way epithelial cells. Interestingly, our microarray analysis showedFRD1-dependent up-regulation of genes with antiapoptotic effecton epithelial and other cell types (63–65), and exposure to an

FIGURE 3. P. aeruginosa mucoid CF isolates do not induce host de-fense gene expression in airway epithelial cells. A, Calu-3 cells were in-fected for 1 h at a ratio of 50 bacteria per epithelial cell with a series ofmucoid CF isolates (CF91, CF1025, and CF1028), and the expression ofmatrilysin and GAPDH examined by Northern blotting with specificprobes at 6 h postinfection. P. aeruginosa ATCC 10145 (motile strain) wasused as a positive control of the induction of matrilysin expression. Cntl,uninfected cells. B, In a similar experiment, the expression of h-BD-2 andh-BD-1 was examined by RT-PCR at 6 h postinfection. h-BD-2 expressionwas exclusively induced by the flagellated strain ATCC 10145, but not themucoid isolates CF91, CF103, CF1025, and CF1028, while the expressionof h-BD-1 is constitutive. Amplified products were resolved on a 6% acryl-amide gel. C, Calu-3 cells were exposed to mucoid and motile strains for1 h as indicated above. After extensive washing and the addition of freshmedium containing antibiotics, conditioned medium were collected at 24 hpostinfection and the secretion of IL-8 was examined by ELISA. Motilestrains stimulated IL-8 secretion by 15- to 20-fold, while mucoid strainsstimulated IL-8 secretion by 2- to 3-fold. Representative results obtainedwith the strains ATCC 51673 (motile) and CF1025 (mucoid) are shown. �,Significantly different from control (p � 0.01).



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alginate-producing strain results in diminished apoptosis in Calu-3airway epithelial cells. Thus, the lack of the appropriate host de-fense and inflammatory milieu in the airways, and impaired bac-terial clearance because of reduced epithelial cell apoptosis (59,66), may explain the increased persistence of these strains in an-imal models of acute infection (15–18).

Our model of coculture of epithelial cells and genetically de-fined P. aeruginosa has allowed us to explore the complex bacte-ria-cell interactions shaping the response of Calu-3 human airwayepithelial cells to phenotypes of this bacterium relevant in lunginfections. Bacteria-epithelial cells interactions are determined bythe virulence factors expressed by bacteria as well as the effect ofthese virulence factors on mammalian signaling pathways. Al-though differences in attachment or invasion may partially con-tribute to the very distinct cellular response of Calu-3 cells to thestrains used in this study, our work demonstrates that P. aerugi-nosa mucoid and motile phenotypes direct fundamentally differentresponses in host cells, both in the number of genes and the typeof cellular bioprocesses affected. Although our microarray analysiswas limited to one time point after bacterial infection and we haveused an immortalized lung epithelial cell line, our data show forthe first time that the conversion to mucoidy by the bacteriumcorrelates in this model with a fundamental switch in host geneexpression patterns. Our work also underscores the extent to whichproinflammatory and host defense responses to P. aeruginosa inthe airways are dependent on the presence of flagellin, and notalginate. Our previous work identified flagellin as a P. aeruginosavirulence factor that specifically up-regulates host defense gene

FIGURE 4. Host defense gene expression is up-regulated by challenge with P. aeruginosa flagellin,but not alginate, in airway epithelial cells. A, Calu-3cells were treated for 6 h with 10�8 M LPS-free pu-rified flagellin and 200 �g/ml alginate, and the ex-pression of matrilysin and GAPDH was examined byNorthern blotting as described above. B, Calu-3 cellswere treated with flagellin and alginate as describedabove and the expression of h-BD examined by RT-PCR. C and D, Secretion of chemokines and proin-flammatory cytokines is increased by challenge withP. aeruginosa flagellin, but not alginate, in airwayepithelial cells. Calu-3 cells were challenged for 24 hwith different concentrations of purified flagellin andalginate, and the secretion of IL-8 (C) and IL-6 (D)was examined by ELISA. E, Flagellin bioactivity de-pends on protein integrity. Purified flagellin was in-cubated with cathepsin G at 37°C for 15 min at theindicated molar ratios, and added to Calu-3 epithelialcells. Secretion of IL-8 was determined by ELISA in24 h-conditioned medium as described above. �, Sig-nificantly different from control (p � 0.01).

FIGURE 5. IL-8 secretion is induced by flagellin, but not alginate, inA549 airway epithelial cells. A, A549 airway cells were exposed to the P.aeruginosa strains FRD1 (mucoid), FRD440 (motile), FRD875 (nonmu-coid, nonmotile), and FRD1234 (nonmucoid, nonmotile, algT mutant), for1 h at a MOI of 50. After extensive washing and the addition of freshmedium containing antibiotics, conditioned medium were collected at 24 hpostinfection and the expression of IL-8 was examined by ELISA. Motilitysignificantly increased IL-8 secretion (FRD440 vs FRD1234), while mu-coidy had no effect (FRD1 vs FRD875). B, A549 cells were challengedwith 10�7 M purified flagellin and 40 �g/ml alginate for 24 h, and thesecretion of IL-8 was examined by ELISA. �, Significantly different fromcontrol (p � 0.01).



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expression in airway epithelial cells (27, 29). Flagellin mutantswere unable to induce the expression of the matrix metalloprotein-ase matrilysin both in vivo and in vitro (27). Furthermore, flagellinis a substrate for host proteases, and the outcome of the interactionwith the pathogen is further modulated by the host via the specificcleavage of flagellin by neutrophil serine proteases and the subse-quent inactivation of flagellin signaling (29).

In Pseudomonas spp., the alternative sigma factor AlgT is aglobal regulator of gene expression, which specifically modulatesthe expression of virulence factors (52), and inversely regulatesmucoidy and flagellin expression (21). A remarkable finding of ourstudy is the difference in the magnitude of host responses to themucoid strain FRD1 (67 gene expression changes) and the non-mucoid isogenic strain FRD875 (231 gene expression changes).Both strains have an active AlgT, suggesting that the presence ofalginate itself in the mucoid strain attenuates host responses andhelps the bacterium to evade host detection. Finally, FRD1234, anonmotile, nonmucoid, and algT mutant strain derived fromFRD1, had a very limited effect on host gene expression in ourexperimental system. It is tempting to speculate that factors not yetdetermined in the CF airway milieu promote and select the con-version of P. aeruginosa to a mucoid phenotype. Thus, the AlgT-mediated P. aeruginosa conversion to a mucoid phenotype in CFserves a double purpose for the bacterium, and by simultaneouslyrepressing flagellin synthesis and derepressing alginate production,

the bacterium further favors chronic colonization of the airways.Interestingly, a recent study shows that flagellin expression in P.aeruginosa is regulated by factors present in CF airway fluid (67).Our future studies will examine the possibility that mutations inCF transmembrane conductance regulator (68) modulate theinflammatory responses of airway epithelial cells to mucoid andmotile P. aeruginosa strains.

A recent analysis of the transcriptional response of airway ep-ithelial cells exposed to P. aeruginosa suggests a role for specifictype III-secreted factors in the regulation of host gene expression(4). However, because the nonmotile strain PA103 (69) was thegenetic background for the generation of the type III-secretion mu-tations, this study did not detect a significant increase in proin-flammatory and innate host defense pathways (4). A previous anal-ysis of the interaction of P. aeruginosa with type II pneumocyte-like human airway epithelial cells (3) exposed A549 cells to themotile wild-type strain PAK (70) and the isogenic type IV pilimutant PAK-NP (a pilA mutant, defective in adherence to epithe-lial cells) (71). In this study, which was more limited than ours inthe scope of genes interrogated, the increased expression of a lim-ited number of these proinflammatory genes was dependent tosome degree on adherence (3). Furthermore, a recent study of CFairway epithelial cells exposed to P. aeruginosa suggests thatflagellin, and not pilin, is the factor responsible for cytokine geneup-regulation (5). Remarkably, our and other studies also point tothe general unresponsiveness of lung and intestinal epithelial cellsto LPS (3, 5, 8, 27). Altogether, the data indicate that epithelialcells, which constitute a first line of host defense and the initial

FIGURE 6. Flagellin, but not alginate, up-regulates NF-�B activity in atime- and dose-dependent manner. A, A549 cells were transfected with aNF-�B reporter plasmid, treated with purified flagellin or alginate for 3 h,and luciferase activity was determined in cell extracts. pFC-MEKK-trans-fected cells were used as a negative control. Untreated, A549 cells trans-fected with the reporter and treated with PBS. �, Significantly differentfrom control (p � 0.01). B, Calu-3 cells were infected with motile P.aeruginosa as indicated above or treated with 10�7 M purified flagellin,with and without the inhibitors MG132 (5 nM), PD98059 (2 �M), andSB203580 (60 nM). The expression of matrilysin and GAPDH was exam-ined by Northern blotting as described above.

FIGURE 7. Release of cytochrome c from mitochondria in response to P.aeruginosa. A, Calu-3 cells were infected at a MOI of 50, and cytosolic frac-tions and total cell extracts were prepared by differential centrifugation. Sam-ples were analyzed by ELISA, and the data are expressed as percentage ofcytochrome c detected in the cytosolic extracts with respect to total cyto-chrome c determined per each condition. Data were analyzed by ANOVA andBonferroni-type multiple t test. A p-value �0.01 was considered significant. �,Significantly different with respect to uninfected cells (p � 0.01). ��, FRD1-induced cytochrome c release was significantly different with respect to theother strains (p � 0.01). B, Samples of cytosolic fractions and total cell extractsfrom uninfected and infected Calu-3 cells were resolved by SDS-PAGE. Cy-tochrome c was detected by Western blotting.



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barrier encountered by a pathogen, are geared to readily respond toflagellin with a program of “cell activation,” mediated by TLR-5(35, 36). Indeed, TLR-5 is constitutively expressed by Calu-3 cellsand mediates the responses to flagellin (Y. S. Lopez-Boado, un-published observations). Alginate can signal through TLR-2 andTLR-4 to activate monocytes and macrophages (39), and it islikely that alginate can act on other cell types relevant in CF air-way disease. However, alginate seems unable to activate NF-�B inairway epithelial cells, although the expression of TLR-2, -4, and-5 in these cells has been reported (72). Our future studies willaddress the mechanism by which alginate affects gene expressionin airway epithelial cells.

Finally, a recent study suggests that interspecies communicationbetween the host microflora and P. aeruginosa modulates the ex-pression of virulence factors in the latter organism (73). Remark-ably, this study shows that the interaction between oropharyngealflora and P. aeruginosa in CF results specifically in the up-regu-lation of fliC expression. In this context, our study suggests that theunderlying cause of the exacerbations frequently observed in adultCF patients, which are not the result of acquisition of new strains(74), may be the potent proinflammatory activity of flagellin.

AcknowledgmentsWe thank Amanda Kuber (Wake Forest University Microarray Core),Rebecca Keyser, and Haiping Lu for technical help. We also thankDr. Alice Prince for the anti-flagellin Ab.

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FIGURE 8. Infection with mucoid P. aeruginosa results in less airwayepithelial cell apoptosis. Calu-3 cells were infected at a MOI of 50 with thedifferent P. aeruginosa strains, washed extensively, and stained for annexinV as an indicator of apoptosis. Left panels show annexin V staining; rightpanels show phase contrast views of the same fields. Representative resultsof four independent experiments are shown.



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