Differential effects of cytokines and corticosteroids on Toll-like receptor 2 expression and activity in human airway epithelia
© Winder et al; licensee BioMed Central Ltd. 2009
Received: 10 February 2009
Accepted: 16 October 2009
Published: 16 October 2009
The recognition of microbial molecular patterns via Toll-like receptors (TLRs) is critical for mucosal defenses.
Using well-differentiated primary cultures of human airway epithelia, we investigated the effects of exposure of the cells to cytokines (TNF-α and IFN-γ) and dexamethasone (dex) on responsiveness to the TLR2/TLR1 ligand Pam3CSK4. Production of IL-8, CCL20, and airway surface liquid antimicrobial activity were used as endpoints.
Microarray expression profiling in human airway epithelia revealed that first response cytokines markedly induced TLR2 expression. Real-time PCR confirmed that cytokines (TNF-α and IFN-γ), dexamethasone (dex), or cytokines + dex increased TLR2 mRNA abundance. A synergistic increase was seen with cytokines + dex. To assess TLR2 function, epithelia pre-treated with cytokines ± dex were exposed to the TLR2/TLR1 ligand Pam3CSK4 for 24 hours. While cells pre-treated with cytokines alone exhibited significantly enhanced IL-8 and CCL20 secretion following Pam3CSK4, mean IL-8 and CCL20 release decreased in Pam3CSK4 stimulated cells following cytokines + dex pre-treatment. This marked increase in inflammatory gene expression seen after treatment with cytokines followed by the TLR2 ligand did not correlate well with NF-κB, Stat1, or p38 MAP kinase pathway activation. Cytokines also enhanced TLR2 agonist-induced beta-defensin 2 mRNA expression and increased the antimicrobial activity of airway surface liquid. Dex blocked these effects.
While dex treatment enhanced TLR2 expression, co-administration of dex with cytokines inhibited airway epithelial cell responsiveness to TLR2/TLR1 ligand over cytokines alone. Enhanced functional TLR2 expression following exposure to TNF-α and IFN-γ may serve as a dynamic means to amplify epithelial innate immune responses during infectious or inflammatory pulmonary diseases.
The airway epithelium plays an important role in orchestrating pulmonary innate and adaptive immune responses. This mucosal surface is a site of first contact with the environment and has evolved many mechanisms to recognize and respond to inhaled or aspirated microorganisms. Epithelial responses to microbes are initiated via pattern recognition receptors including the Toll-like receptors (TLRs) . TLRs are a family of 10 receptors in humans that recognize a variety of microbial molecular patterns and regulate immune responses. Airway epithelial cell responses to a number of TLRs, including TLR2 [2–4], TLR3 , TLR4 , TLR5 [7, 8], and TLRs 6 through 9 [9, 10] have been investigated in human cells and animal models. TLR activation initiates signaling that culminates in a number of host defense responses including the secretion of antimicrobial peptides, cytokines, and chemokines by epithelial cells [11, 12].
TLR expression is regulated in a cell and tissue specific manner [11, 13–15]. Experimental evidence in humans  and animal models [17–20] indicates that the expression and function of several TLRs is developmentally regulated. Here we focus on TLR2 expression and function in well-differentiated human respiratory epithelia. TLR2 forms heterodimers with either TLR6 or TLR1; these heterodimers recognize diacyl and triacyl lipopeptides, respectively [21, 22]. TLR2 signaling occurs via a MyD88-dependent pathway leading to the nuclear translocation of NF-κB , and induction of various inflammatory cytokines, including IL-8, and antimicrobial peptides, such as human beta-defensin 2 (HBD-2) [4, 23].
We hypothesized that first response cytokines would influence both the array of functional TLRs and their responses to stimuli. Further, dexamethasone, when co-administered with pro-inflammatory cytokines, was reported to synergistically enhance TLR2 expression in the human alveolar and bronchial epithelial cell lines A549  and BEAS-2b . We therefore investigated the effects of both cytokine and glucocorticoid exposure on TLR2 expression in primary cultures of human airway epithelia.
Culture of human airway epithelia
Primary cultures of human airway epithelia were grown at an air-liquid interface as described previously . Human donor lungs were obtained from individuals without primary pulmonary diseases whose lungs were determined to be unsuitable for organ transplantation. The use of human tissues was approved by the University of Iowa Institutional Review Board. Well-differentiated (> 2 weeks in culture) tracheal and bronchial epithelia were used in all studies. Epithelia were maintained in DMEM/F-12 with 1% penicillin-streptomycin, 50 μg/ml gentamicin, and 2% Ultroser G. In a microarray experiment (described below), epithelia were incubated for 24 hours in 2% Ultroser G media containing 100 ng/ml each of recombinant interleukin-1-beta (IL-1β; Sigma, St. Louis, MO), tumor necrosis factor-alpha (TNF-α; Sigma), and interferon-gamma (IFN-γ; Sigma). For all other experiments, epithelia were placed in serum free DMEM/F-12 for 48 hours, then incubated overnight (18 hr) in media containing cytokines (100 ng/ml each of TNF-α and IFN-γ), 1 μM water-soluble dexamethasone (D2915; Sigma), cytokines plus dexamethasone, or control serum-free media (100 μl volume applied apically, and 500 μl basolaterally). Cell viability was similar under all experimental conditions.
In TLR2 receptor agonist experiments, epithelia were rinsed with media, then incubated for an additional 24 hours in media containing 25 μg/ml Pam3CSK4 (tlrl-pms; InvivoGen, San Diego, CA), a synthetic bacterial lipoprotein TLR2/TLR1 ligand, or control serum-free media. Where specified, media containing cytokines ± dexamethasone or Pam3CSK4 were applied to only the apical or basolateral surface, with control serum-free media applied contralaterally. Prior to assays of airway surface liquid antimicrobial activity (see Antimicrobial Assays, below), epithelia were incubated for 5 days in antibiotic-free media and washed daily with antibiotic-free media to remove residual antibiotics.
RNA isolation and quantitative reverse transcription-PCR (RT-PCR)
RNA was extracted using an RNeasy Mini Kit (Qiagen Inc., Valencia, CA) according to manufacturer's protocol. For each sample, 1 μg of total RNA was used as a template for first-strand cDNA synthesis. Quantitative PCR (ABI 7900) was used to amplify the TLR or HBD-2 PCR products along with GAPDH transcripts in a single reaction. Forward and reverse primers and TaqMan probes were designed using Primer Express software (P-E Applied Biosystems, Foster City, CA). Primers and probes were: TLR2 forward, 5'-GGCCAGCAAATTACCTGTGTG-3'; TLR2 reverse, 5'-AGGCGGACATCCTGAACCT-3'; TLR2 probe, 5'-TCCATCCCATGTGCGTGGCC-3'; TLR1 forward, 5'-CAGTGTCTGGTACACGCATGGT-3'; TLR1 reverse, 5'-TTTCAAAAACCGTGTCTGTTAAGAGA-3'; TLR1 probe, 5'-TGCCCATCCAAAATTAGCCCGTTC-3'; TLR6 forward, 5'-GAAGAAGAACAACCCTTTAGGATAGC-3'; TLR6 reverse, 5'-AGGCAAACAAAATGGAAGCTT-3'; TLR6 probe, 5'-TGCAACATCATGACCAAAGACAAA-3'; HBD-2 forward, 5'-CCTGTTACCTGCCTTAAGAGTGGA-3'; HBD-2 reverse, 5'-ACCACAGGTGCCAATTTGTTTA-3'; HBD-2 probe: 5'-CCATATGTCATCCAGTCTTTTGCC-3'. The TLR and HBD-2 probes were labeled with the fluorophore FAM, and the GAPDH probe with the fluorophore JOE. C T for the TLR or HBD-2 PCR product was normalized against the C T for GAPDH.
Five micrograms of total RNA was processed using the Affymetrix GeneChip one-cycle target labeling kit (Affymetrix, Inc., Santa Clara, CA) following the manufacturer's protocols. The resultant biotinylated cRNA was hybridized to a custom GeneChip Human Airway Array (HsAirway, Affymetrix, Inc.). Tracheal and bronchial epithelial cells from seven donors were used in this study. The custom Affymetrix array (HsAirway) was comprised of approximately 23,000 probe sets derived from sequencing of cDNA libraries prepared from human lung, primary airway epithelial cells, and human alveolar macrophages . The arrays were washed, stained, and scanned using the Affymetrix Model 450 Fluidics Station and Affymetrix Model 3000 scanner. Each sample and hybridization underwent quality control evaluation, including cRNA amplification of more than 4-fold, percentage of probe sets reliably detecting between 40 and 60 percent present call, and 3'-5' ratio of GAPDH gene less than 3.
The hybridizations were normalized using the RMA (robust multi-chip averaging)  method from Bioconductor  to obtain summary expression values for each probe set. Gene expression levels were analyzed on a logarithmic scale. Differentially expressed genes were identified using a 1-factor ANOVA test. Heat-map visualizations were generated with GenePattern . Global scaling of the expression levels was used to allow experiment-wide comparison of gene expression.
Protein quantification by ELISA
IL-8 and CCL20 protein abundance in the basolateral media was measured by ELISA (Duoset DY208 (IL-8) and DY360 (CCL20); R&D Systems, Minneapolis, MN).
Apical washings from epithelia were obtained after a 24-hour incubation with the TLR2 agonist Pam3CSK4 or control serum-free, antibiotic-free media. Airway surface liquid (ASL) was obtained by adding 100 μl of sterile 1× PBS to the apical surface and immediately pipetting off the fluid. We used a modified radial diffusion assay to quantify ASL antimicrobial activity as described previously . Briefly, 4 × 106 bacteria in mid-log phase were suspended in an underlay gel. 2.5 mm diameter wells were punched into the gel and filled with 0.04-5 μl of ASL, with 0.02% acetic acid with 0.1% human serum albumin (Sigma) to equal 5 μl, or control antibiotic (gentamicin, 0.4-50 μg/ml). The plates were then incubated for 3 hours at 37°C. Nutrient rich gels were then overlaid, and the plates incubated at 37°C overnight. Zones of clearance were manually measured and plotted on a semi-log graph where the X-intercept represents the minimal inhibitory concentration (MIC) . Test organisms included Escherichia coli DH5α, Pseudomonas aeruginosa PA01, and a clinical strain of Listeria monocytogenes.
Whole cell protein extract preparation and immunoblot analysis were performed as described previously [32, 33]. Primary antibodies used to detect specific cellular and nuclear proteins were: mouse IgG1 mAb clone L35A5 against human IκBα, rabbit polyclonal IgG 9171 against human Stat1 phosphorylated on tyrosine-701, rabbit polyclonal IgG 9172 against human total Stat1, rabbit IgG mAb clone 3D7 against human p38 MAP kinase phosphorylated on threonine-180 and tyrosine-182, rabbit IgG mAb clone 7D6 against human total p38 MAP kinase from Cell Signaling Technology (Beverly, MA); mouse IgG2α mAb clone AC-74 against human β-actin from Sigma-Aldrich (St. Louis, MO); rabbit polyclonal antiserum against human heat shock protein (HSP)-90 from Assay Designs (Ann Arbor, MI). Primary antibody binding was detected using goat antirabbit or antimouse IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA or Cell Signaling Technologies) and an enhanced chemiluminescence detection system (Amersham Biosciences, Uppsala, Sweden). Reprobing of membranes was done after washing twice in Restore™ buffer (Pierce, Rockford, IL) for 15 min at 37°C. In some experiments, radiographic film images were analyzed using ImageJ software . To generate an integrated density level, band area was multiplied by the band mean gray value, and the integrated density for IκBα or phosphorylated p38 was divided by the corresponding β-actin or HSP-90 level creating a ratio for each sample.
NF-κB Activation Assay
NF-κB-dependent gene activation was determined using a recombinant adenoviral vector that expresses a luciferase gene driven by four tandem NF-kB enhancer sequences as described previously [32, 35]. Photinus pyralis luciferase activity was determined using a commercial luciferase reporter assay kit (Promega) and a Lumat LB 9501 luminometer (Berthold, Bad Wildbad, Germany).
Assessment of statistical significance for quantitive PCR and ELISA data was performed using one-tailed Student's t tests with Microsoft Excel. P values < 0.05 were considered significant. Luciferase assays and densitometry analysis were analyzed for statistical significance using ANOVA for a factorial experimental design. The multicomparison significance level for the ANOVA was 0.05. If significance was achieved by one-way analysis, post-ANOVA comparison of means was performed using Tukey's test .
Results and Discussion
Microarray profiling of TLR expression in airway epithelia
Induction of TLR2 mRNA expression in airway epithelia
TLR2 expression was previously documented in human airway cell lines, passaged cells in submersion culture, or in lung tissues [2, 4, 9, 10, 37]. Although these studies noted TLR2 expression in several model systems, the functional consequences of combined cytokine and glucocorticoid treatment were not investigated, nor were the polarity of TLR2 responses. We focused on functional TLR2 responses in well-differentiated primary airway epithelia. With the exception of studies by Becker et al  and Hertz and colleagues , TLR2 expression and function has been little studied in this model that closely mimics the in vivo airways.
As cell-surface TLR2 exists as a heterodimer with either TLR1 or TLR6 [21, 22], we next investigated the effects of cytokine and dexamethasone exposure on TLR1 and TLR6 mRNA expression. As shown in Figure 2B, TLR1 mRNA abundance remained unchanged following cytokine ± dexamethasone treatment. Unlike TLR2, TLR6 mRNA decreased modestly following cytokine treatment, both in the presence, and absence, of dexamethasone (P < 0.05).
Treatment with cytokines ± dexamethasone alters effects of TLR2 receptor ligation in airway epithelia
To determine the functional effects of increased TLR2 expression, epithelia pre-treated with cytokines ± dexamethasone were stimulated with the TLR1/2 ligand Pam3CSK4 for 24 hours. Two different concentrations of Pam3CSK4, 10 and 25 μg/ml, were studied initially. As greater changes in IL-8 abundance were seen with 25 μg/ml (data not shown), this concentration was used for further studies.
Since epithelia may preferentially express proteins on either their apical or basolateral membrane domains, we asked whether cells exhibited polarized responses to Pam3CSK4. As shown in Figure 3B, IL-8 abundance increased following either apical or basolateral Pam3CSK4 application. In control cells, mean IL-8 production was over 3-fold higher following apical, compared to basolateral application (P < 0.05). A similar trend was seen in cytokine pre-treated cells, but did not reach statistical significance. These functional data suggest that TLR2 protein abundance is greater on the apical surface of polarized airway epithelia; however airway epithelia can respond to a TLR2 ligand from either surface.
The changes in TLR2 mRNA abundance were similar in pattern, though less pronounced, than those reported in epithelial cell lines. Prior studies in BEAS-2b  and A549  cells demonstrated similar increases in TLR2-mediated IL-8 production following pre-treatment with cytokines plus dexamethasone, but did not compare findings to pre-treatment with cytokines alone. While we detected TLR2 protein in cell lines overexpressing the protein, we were unable to conclusively localize TLR2 protein expression in primary cells using commercially available antibodies and methods including immunohistochemistry and surface biotinylation (data not shown). We conclude that TLR2 protein is functionally present in this model but below the limits of immunodetection.
Effects of Cytokines on Pam3CSK4 Signaling Responses
Human airway epithelial cells respond to cytokines and bacterial products through induction of specific signaling pathways. The functional expression of TLR2 may be regulated by more than one receptor-mediated signaling pathway prominent in the early responses to bacterial and viral pathogens . Several groups have studied the mechanisms by which dexamethasone enhances TLR2 expression. Glucocorticoids synergistically enhanced nontypeable Haemophilus influenzae-induced TLR2 expression via induction of MAPK phosphatase-1, leading to inhibition of p38 MAPK . The results of Hermoso et al  suggest that TNF-α and dexamethasone cooperatively regulate the TLR2 promoter, through the involvement of NF-kB and STAT transcription factors, as well as a 3'-glucocorticoid response element.
TLR2 receptor ligation enhances expression of inducible host defense proteins and increases antimicrobial activity in airway surface liquid
We also assayed expression of CCL20, a peptide with both innate and adaptive immune functions produced by airway epithelia. CCL20 stimulates B-cell migration and has antimicrobial activity comparable to the β-defensins . Following Pam3CSK4 stimulation, CCL20 increased 25- to 30-fold over baseline in cells pre-treated with cytokines (Figure 7B). Dexamethasone significantly blunted this response, as CCL20 abundance in epithelia pre-treated with cytokines plus dexamethasone was approximately one-third that of cells treated with cytokines alone (P < 0.05).
We conclude that TLR2 is an inducible component of airway epithelial defenses. Enhanced functional TLR2 expression following TNF-α and IFN-γ exposure may serve as a dynamic means to amplify innate immune responses during infectious or inflammatory pulmonary diseases. Under conditions where first response cytokines are present, enhanced TLR2 signaling allows for further amplification of mucosal immunity. This increase in signalling does not correlate well with NF-κB, Stat1, or p38 MAP kinase pathway activation. The importance of lipopeptide recognition in airway defense is further demonstrated by the upregulation of several host defense proteins/peptides following receptor engagement. Our finding that glucocorticoids can act as a negative regulator of functional TLR2 expression in well-differentiated human airway epithelia has clinical implications in settings of systemic or inhaled corticosteroid use. Since TLR2-mediated responses may occur early in the host response to infection, any factors that negatively impact TLR2 expression or signaling might influence disease outcomes.
We thank Tim Starner and Ariadna Arias for their guidance and technical assistance with the antimicrobial assays. We acknowledge the support of NIH T32 AI007343 (A.W.), P50 HL-61234 (P.B.M.), N01 AI-30040 (P.B.M.), the Roy J. Carver Charitable Trust (P.B.M.), a Career Development Award from Research to Prevent Blindness (T.E.S.), HL-082505 (D.C.L.), and HL-075559 (D.C.L.). We also acknowledge the support of the Cell and Tissues and Cell Morphology Cores, partially supported by the Center for Gene Therapy for Cystic Fibrosis (NIH P30 DK-54759) and the Cystic Fibrosis Foundation.
- Akira S, Takeda K, Kaisho T: Toll-like receptors: critical proteins linking innate and acquired immunity. Nat Immunol. 2001, 2 (8): 675-680. 10.1038/90609.PubMedView ArticleGoogle Scholar
- Soong G, Reddy B, Sokol S, Adamo R, Prince A: TLR2 is mobilized into an apical lipid raft receptor complex to signal infection in airway epithelial cells. J Clin Invest. 2004, 113 (10): 1482-1489.PubMedPubMed CentralView ArticleGoogle Scholar
- Homma T, Kato A, Hashimoto N, Batchelor J, Yoshikawa M, Imai S, Wakiguchi H, Saito H, Matsumoto K: Corticosteroid and cytokines synergistically enhance toll-like receptor 2 expression in respiratory epithelial cells. Am J Respir Cell Mol Biol. 2004, 31 (4): 463-469. 10.1165/rcmb.2004-0161OC.PubMedView ArticleGoogle Scholar
- Hertz CJ, Wu Q, Porter EM, Zhang YJ, Weismuller KH, Godowski PJ, Ganz T, Randell SH, Modlin RL: Activation of Toll-like receptor 2 on human tracheobronchial epithelial cells induces the antimicrobial peptide human beta defensin-2. J Immunol. 2003, 171 (12): 6820-6826.PubMedView ArticleGoogle Scholar
- Guillot L, Le Goffic R, Bloch S, Escriou N, Akira S, Chignard M, Si-Tahar M: Involvement of toll-like receptor 3 in the immune response of lung epithelial cells to double-stranded RNA and influenza A virus. J Biol Chem. 2005, 280 (7): 5571-5580. 10.1074/jbc.M410592200.PubMedView ArticleGoogle Scholar
- Jia HP, Kline JN, Penisten A, Apicella MA, Gioannini TL, Weiss J, McCray PB: Endotoxin responsiveness of human airway epithelia is limited by low expression of MD-2. Am J Physiol Lung Cell Mol Physiol. 2004, 287 (2): L428-437. 10.1152/ajplung.00377.2003.PubMedView ArticleGoogle Scholar
- Tseng J, Do J, Widdicombe JH, Machen TE: Innate immune responses of human tracheal epithelium to Pseudomonas aeruginosa flagellin, TNF-alpha, and IL-1beta. Am J Physiol Cell Physiol. 2006, 290 (3): C678-690. 10.1152/ajpcell.00166.2005.PubMedView ArticleGoogle Scholar
- Adamo R, Sokol S, Soong G, Gomez MI, Prince A: Pseudomonas aeruginosa flagella activate airway epithelial cells through asialoGM1 and toll-like receptor 2 as well as toll-like receptor 5. Am J Respir Cell Mol Biol. 2004, 30 (5): 627-634. 10.1165/rcmb.2003-0260OC.PubMedView ArticleGoogle Scholar
- Ritter M, Mennerich D, Weith A, Seither P: Characterization of Toll-like receptors in primary lung epithelial cells: strong impact of the TLR3 ligand poly(I:C) on the regulation of Toll-like receptors, adaptor proteins and inflammatory response. J Inflamm (Lond). 2005, 2 (1): 16-10.1186/1476-9255-2-16.View ArticleGoogle Scholar
- Greene CM, Carroll TP, Smith SG, Taggart CC, Devaney J, Griffin S, O'Neill SJ, McElvaney NG: TLR-induced inflammation in cystic fibrosis and non-cystic fibrosis airway epithelial cells. J Immunol. 2005, 174 (3): 1638-1646.PubMedView ArticleGoogle Scholar
- Greene CM, McElvaney NG: Toll-like receptor expression and function in airway epithelial cells. Arch Immunol Ther Exp (Warsz). 2005, 53 (5): 418-427.Google Scholar
- Hornef MW, Bogdan C: The role of epithelial Toll-like receptor expression in host defense and microbial tolerance. J Endotoxin Res. 2005, 11 (2): 124-128.PubMedView ArticleGoogle Scholar
- Abreu MT, Fukata M, Arditi M: TLR signaling in the gut in health and disease. J Immunol. 2005, 174 (8): 4453-4460.PubMedView ArticleGoogle Scholar
- Kaisho T, Akira S: Toll-like receptor function and signaling. J Allergy Clin Immunol. 2006, 117 (5): 979-987. 10.1016/j.jaci.2006.02.023.PubMedView ArticleGoogle Scholar
- Wira CR, Fahey JV, Sentman CL, Pioli PA, Shen L: Innate and adaptive immunity in female genital tract: cellular responses and interactions. Immunol Rev. 2005, 206: 306-335. 10.1111/j.0105-2896.2005.00287.x.PubMedView ArticleGoogle Scholar
- Levy O, Coughlin M, Cronstein BN, Roy RM, Desai A, Wessels MR: The adenosine system selectively inhibits TLR-mediated TNF-alpha production in the human newborn. J Immunol. 2006, 177 (3): 1956-1966.PubMedPubMed CentralView ArticleGoogle Scholar
- Harju K, Ojaniemi M, Rounioja S, Glumoff V, Paananen R, Vuolteenaho R, Hallman M: Expression of toll-like receptor 4 and endotoxin responsiveness in mice during perinatal period. Pediatr Res. 2005, 57 (5 Pt 1): 644-648. 10.1203/01.PDR.0000156212.03459.A9.PubMedView ArticleGoogle Scholar
- Schaub B, Bellou A, Gibbons FK, Velasco G, Campo M, He H, Liang Y, Gillman MW, Gold D, Weiss ST, et al: TLR2 and TLR4 stimulation differentially induce cytokine secretion in human neonatal, adult, and murine mononuclear cells. J Interferon Cytokine Res. 2004, 24 (9): 543-552.PubMedPubMed CentralView ArticleGoogle Scholar
- Sadeghi K, Berger A, Langgartner M, Prusa AR, Hayde M, Herkner K, Pollak A, Spittler A, Forster-Waldl E: Immaturity of infection control in preterm and term newborns is associated with impaired toll-like receptor signaling. J Infect Dis. 2007, 195 (2): 296-302. 10.1086/509892.PubMedView ArticleGoogle Scholar
- Hillman NH, Moss TJ, Nitsos I, Kramer BW, Bachurski CJ, Ikegami M, Jobe AH, Kallapur SG: Toll-Like Receptors and Agonist Responses in the Developing Fetal Sheep Lung. Pediatr Res. 2008, 63 (4): 388-393. 10.1203/PDR.0b013e3181647b3a.PubMedView ArticleGoogle Scholar
- Takeuchi O, Kawai T, Muhlradt PF, Morr M, Radolf JD, Zychlinsky A, Takeda K, Akira S: Discrimination of bacterial lipoproteins by Toll-like receptor 6. Int Immunol. 2001, 13 (7): 933-940. 10.1093/intimm/13.7.933.PubMedView ArticleGoogle Scholar
- Takeuchi O, Sato S, Horiuchi T, Hoshino K, Takeda K, Dong Z, Modlin RL, Akira S: Cutting edge: role of Toll-like receptor 1 in mediating immune response to microbial lipoproteins. J Immunol. 2002, 169 (1): 10-14.PubMedView ArticleGoogle Scholar
- Singh PK, Jia HP, Wiles K, Hesselberth J, Liu L, Conway BA, Greenberg EP, Valore EV, Welsh MJ, Ganz T, et al: Production of beta-defensins by human airway epithelia. Proc Natl Acad Sci USA. 1998, 95 (25): 14961-14966. 10.1073/pnas.95.25.14961.PubMedPubMed CentralView ArticleGoogle Scholar
- Hermoso MA, Matsuguchi T, Smoak K, Cidlowski JA: Glucocorticoids and tumor necrosis factor alpha cooperatively regulate toll-like receptor 2 gene expression. Mol Cell Biol. 2004, 24 (11): 4743-4756. 10.1128/MCB.24.11.4743-4756.2004.PubMedPubMed CentralView ArticleGoogle Scholar
- Karp PH, Moninger TO, Weber SP, Nesselhauf TS, Launspach JL, Zabner J, Welsh MJ: An in vitro model of differentiated human airway epithelia. Methods for establishing primary cultures. Methods Mol Biol. 2002, 188: 115-137.PubMedGoogle Scholar
- Scheetz TE, Zabner J, Welsh MJ, Coco J, Eyestone Mde F, Bonaldo M, Kucaba T, Casavant TL, Soares MB, McCray PB: Large-scale gene discovery in human airway epithelia reveals novel transcripts. Physiol Genomics. 2004, 17 (1): 69-77. 10.1152/physiolgenomics.00188.2003.PubMedView ArticleGoogle Scholar
- Irizarry RA, Hobbs B, Collin F, Beazer-Barclay YD, Antonellis KJ, Scherf U, Speed TP: Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics. 2003, 4 (2): 249-264. 10.1093/biostatistics/4.2.249.PubMedView ArticleGoogle Scholar
- Gentleman RC, Carey VJ, Bates DM, Bolstad B, Dettling M, Dudoit S, Ellis B, Gautier L, Ge Y, Gentry J, et al: Bioconductor: open software development for computational biology and bioinformatics. Genome Biol. 2004, 5 (10): R80-10.1186/gb-2004-5-10-r80.PubMedPubMed CentralView ArticleGoogle Scholar
- Reich M, Liefeld T, Gould J, Lerner J, Tamayo P, Mesirov JP: GenePattern 2.0. Nat Genet. 2006, 38 (5): 500-501. 10.1038/ng0506-500.PubMedView ArticleGoogle Scholar
- Starner TD, Swords WE, Apicella MA, McCray PB: Susceptibility of nontypeable Haemophilus influenzae to human beta-defensins is influenced by lipooligosaccharide acylation. Infect Immun. 2002, 70 (9): 5287-5289. 10.1128/IAI.70.9.5287-5289.2002.PubMedPubMed CentralView ArticleGoogle Scholar
- Lehrer RI, Barton A, Daher KA, Harwig SS, Ganz T, Selsted ME: Interaction of human defensins with Escherichia coli: mechanism of bactericidal activity. J Clin Invest. 1989, 84: 553-561. 10.1172/JCI114198.PubMedPubMed CentralView ArticleGoogle Scholar
- Humlicek AL, Manzel LJ, Chin CL, Shi L, Excoffon KJ, Winter MC, Shasby DM, Look DC: Paracellular permeability restricts airway epithelial responses to selectively allow activation by mediators at the basolateral surface. J Immunol. 2007, 178 (10): 6395-6403.PubMedView ArticleGoogle Scholar
- Manzel LJ, Chin CL, Behlke MA, Look DC: Regulation of bacteria-induced intercellular adhesion molecule-1 by CCAAT/enhancer binding proteins. Am J Respir Cell Mol Biol. 2009, 40 (2): 200-210. 10.1165/rcmb.2008-0104OC.PubMedPubMed CentralView ArticleGoogle Scholar
- Rasband WS: National Institutes of Health, Bethesda, Maryland, USA. Image J. 1997Google Scholar
- Chin CL, Manzel LJ, Lehman EE, Humlicek AL, Shi L, Starner TD, Denning GM, Murphy TF, Sethi S, Look DC: Haemophilus influenzae from patients with chronic obstructive pulmonary disease exacerbation induce more inflammation than colonizers. Am J Respir Crit Care Med. 2005, 172 (1): 85-91. 10.1164/rccm.200412-1687OC.PubMedPubMed CentralView ArticleGoogle Scholar
- Hassard TH: Understanding Biostatistics. Mosby-Year Book. 1991Google Scholar
- Becker S, Dailey L, Soukup JM, Silbajoris R, Devlin RB: TLR-2 is involved in airway epithelial cell response to air pollution particles. Toxicol Appl Pharmacol. 2005, 203 (1): 45-52. 10.1016/j.taap.2004.07.007.PubMedView ArticleGoogle Scholar
- Imasato A, Desbois-Mouthon C, Han J, Kai H, Cato AC, Akira S, Li JD: Inhibition of p38 MAPK by glucocorticoids via induction of MAPK phosphatase-1 enhances nontypeable Haemophilus influenzae-induced expression of toll-like receptor 2. J Biol Chem. 2002, 277 (49): 47444-47450. 10.1074/jbc.M208140200.PubMedView ArticleGoogle Scholar
- Shuto T, Xu H, Wang B, Han J, Kai H, Gu XX, Murphy TF, Lim DJ, Li JD: Activation of NF-kappa B by nontypeable Hemophilus influenzae is mediated by toll-like receptor 2-TAK1-dependent NIK-IKK alpha/beta-I kappa B alpha and MKK3/6-p38 MAP kinase signaling pathways in epithelial cells. Proc Natl Acad Sci USA. 2001, 98 (15): 8774-8779. 10.1073/pnas.151236098.PubMedPubMed CentralView ArticleGoogle Scholar
- Janssen-Heininger YM, Poynter ME, Aesif SW, Pantano C, Ather JL, Reynaert NL, Ckless K, Anathy V, Velden van der J, Irvin CG, et al: Nuclear factor kappaB, airway epithelium, and asthma: avenues for redox control. Proc Am Thorac Soc. 2009, 6 (3): 249-255. 10.1513/pats.200806-054RM.PubMedPubMed CentralView ArticleGoogle Scholar
- Tak PP, Firestein GS: NF-kappaB: a key role in inflammatory diseases. J Clin Invest. 2001, 107 (1): 7-11. 10.1172/JCI11830.PubMedPubMed CentralView ArticleGoogle Scholar
- Look DC, Pelletier MR, Holtzman MJ: Selective interaction of a subset of interferon-gamma response element-binding proteins with the intercellular adhesion molecule-1 (ICAM-1) gene promoter controls the pattern of expression on epithelial cells. J Biol Chem. 1994, 269 (12): 8952-8958.PubMedGoogle Scholar
- Horvath CM, Darnell JE: The antiviral state induced by alpha interferon and gamma interferon requires transcriptionally active Stat1 protein. J Virol. 1996, 70 (1): 647-650.PubMedPubMed CentralGoogle Scholar
- Starner TD, Barker CK, Jia HP, Kang Y, McCray PB: CCL20 is an inducible product of human airway epithelia with innate immune properties. Am J Respir Cell Mol Biol. 2003, 29 (5): 627-633. 10.1165/rcmb.2002-0272OC.PubMedView ArticleGoogle Scholar
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