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American Journal of Physiology - Lung Cellular and Molecular Physiology logoLink to American Journal of Physiology - Lung Cellular and Molecular Physiology
. 2012 Jul 20;303(6):L509–L518. doi: 10.1152/ajplung.00226.2011

Does the ΔF508-CFTR mutation induce a proinflammatory response in human airway epithelial cells?

Thomas H Hampton 1, Alicia E Ballok 1, Jennifer M Bomberger 1, Melanie R Rutkowski 1, Roxanna Barnaby 1, Bonita Coutermarsh 1, José R Conejo-Garcia 1, George A O'Toole 1, Bruce A Stanton 1,✉
PMCID: PMC3468482  PMID: 22821996

Abstract

In the clinical setting, mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene enhance the inflammatory response in the lung to Pseudomonas aeruginosa (P. aeruginosa) infection. However, studies on human airway epithelial cells in vitro have produced conflicting results regarding the effect of mutations in CFTR on the inflammatory response to P. aeruginosa, and there are no comprehensive studies evaluating the effect of P. aeruginosa on the inflammatory response in airway epithelial cells with the ΔF508/ΔF508 genotype and their matched CF cell line rescued with wild-type (wt)-CFTR. CFBE41o- cells (ΔF508/ΔF508) and CFBE41o- cells complemented with wt-CFTR (CFBE-wt-CFTR) have been used extensively as an experimental model to study CF. Thus the goal of this study was to examine the effect of P. aeruginosa on gene expression and cytokine/chemokine production in this pair of cells. P. aeruginosa elicited a more robust increase in cytokine and chemokine expression (e.g., IL-8, CXCL1, CXCL2 and TNF-α) in CFBE-wt-CFTR cells compared with CFBE-ΔF508-CFTR cells. These results demonstrate that CFBE41o- cells complemented with wt-CFTR mount a more robust inflammatory response to P. aeruginosa than CFBE41o-ΔF508/ΔF508-CFTR cells. Taken together with other published studies, our data demonstrate that there is no compelling evidence to support the view that mutations in CFTR induce a hyperinflammatory response in human airway epithelial cells in vivo. Although the lungs of patients with CF have abundant levels of proinflammatory cytokines and chemokines, because the lung is populated by immune cells and epithelial cells there is no way to know, a priori, whether airway epithelial cells in the CF lung in vivo are hyperinflammatory in response to P. aeruginosa compared with non-CF lung epithelial cells. Thus studies on human airway epithelial cell lines and primary cells in vitro that propose to examine the effect of mutations in CFTR on the inflammatory response to P. aeruginosa have uncertain clinical significance with regard to CF.

Keywords: cystic fibrosis transmembrane conductance regulator, gene arrays, IL-8, Pseudomonas aeruginosa, mucoviscidosis

cystic fibrosis (cf) is an inherited disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene (5, 13, 32). The most common mutation in CFTR is a three-base-pair deletion resulting in the loss of a phenylalanine at position 508 of the protein (ΔF508). Approximately 90% of patients with CF have at least one ΔF508 CFTR allele, which codes for a protein that misfolds in the endoplasmic reticulum and is subsequently degraded by the proteasome, preventing it from trafficking to the plasma membrane, where it functions as a secretory chloride ion channel in a variety of tissues including the lung, pancreas, and intestine (32). CFTR is also expressed in immune cells including neutrophils and macrophages, and reduction in CFTR expression in zebrafish reduces the ability of neutrophils to phagocytose and kill Pseudomonas aeruginosa (P. aeruginosa) (29). Individuals with the ΔF508 CFTR mutation exhibit defects in pulmonary host defense mechanisms that lead to chronic bacterial infection, primarily P. aeruginosa, and inflammation, resulting in a progressive decline in lung function and death before an average age of 38 (27).

Clinical measures of lung inflammation (e.g., assessment of proinflammatory cytokines in bronchoalveolar lavage fluid) consistently demonstrate more robust inflammation in patients with CF chronically infected with bacteria compared with patients without CF with similar lung infections (7, 25, 33), leaving little doubt that the infected CF airway as a whole is more inflamed than an infected non-CF airway. Although it is generally assumed that CF airway epithelial cells are hyperinflammatory in response to P. aeruginosa compared with non-CF airway epithelial cells, human CF airway cells in culture often fail to show a hyperinflammatory phenotype compared with airway epithelial cells expressing wild-type (wt)-CFTR (16, 21, 35). Indeed, one-third of published studies reveal that CF airway cells elaborate a more robust increase in IL-8 production than non-CF cells in response to P. aeruginosa (28, 37, 39, 41), one-third report no difference (4, 6, 8, 16, 26), and one-third actually report that wt-CFTR cells release more IL-8 than CF cells in response to P. aeruginosa (18, 22, 31). Moreover, because the lungs during infection with P. aeruginosa contain immune cells that, like airway epithelial cells, produce cytokines and chemokines, it is not possible to determine whether the cytokines and chemokines in vivo originate from immune cells and/or airway epithelial cells. (11). Thus it cannot be concluded based on clinical data that mutations in CFTR lead to a more exuberant inflammatory response to P. aeruginosa in CF airway epithelial cells compared with non-CF airway epithelial cells.

The lack of a consensus regarding the effect of P. aeruginosa on the inflammatory response in airway epithelial cells in culture noted above may be due, for example, to experimental differences, the type of cells examined, the analytical tools used to analyze gene array data, and/or the CFTR genotype. It is notable that very few studies have examined the effect of P. aeruginosa on the inflammatory response on matched pairs of cells, a comparison that allows an examination of the effect of mutations in CFTR on the proinflammatory response to P. aeruginosa. However, even studies on matched cell lines do not produce consistent results. For example, studies on IB3–1 (ΔF508/W1282X) and S9 cells (IB3–1 cells complemented with wt-CFTR) demonstrate that P. aeruginosa elicits a more robust increase in IL-8 production in IB3–1 CF cells than in wt-CFTR-corrected S9 cells (1, 10), whereas studies on other matched cell lines reveal that, when challenged with P. aeruginosa, CF cells actually produce less IL-8 than non-CF cells, contrary to the generally accepted view that CF mutations enhance the inflammatory response of airway epithelial cells to P. aeruginosa (16, 22, 31).

CFBE41o- cells, human airway epithelial cells homozygous for ΔF508 (CFBE-ΔF508-CFTR), and their matched pair complemented with wt-CFTR (CFBE-wt-CFTR) have been used extensively as an experimental model to study CF (18, 20, 23, 40); however, a comprehensive study examining the effect of P. aeruginosa on gene expression combined with cytokine/chemokine profiles in these cells has not been undertaken. Thus the goal of this study was to examine the effect of P. aeruginosa on gene expression and cytokine/chemokine production in this matched pair of cells, which express the most common genotype in CF (ΔF508/ΔF508) with the ultimate goal of determining the utility of these cell lines as models to better understand the effects of the ΔF508 mutation on the immune response to P. aeruginosa. Analysis of gene array, real-time RT-qPCR, and ELISA data revealed that P. aeruginosa elicited a more robust increase in IL-8, CXCL1, CXCL2, and TNF-α production in CFBE-wt-CFTR cells compared with CFBE-ΔF508-CFTR cells. Taken together with other published studies, our data demonstrate that there is no compelling evidence to support the view that mutations in CFTR induce a hyperinflammatory response to P. aeruginosa in human airway epithelial cells in vitro. Although the lungs of patients with CF have abundant levels of proinflammatory cytokines and chemokines, because the lung is populated by immune cells and epithelial cells, there is no way to know, a priori, whether airway epithelial cells in vivo are hyperinflammatory in response to P. aeruginosa. Thus studies on human airway epithelial cell lines and primary cells in vitro that propose to examine the effect of mutations in CFTR on the inflammatory response to P. aeruginosa have uncertain clinical significance.

MATERIALS AND METHODS

Cell culture and exposure to P. aeruginosa.

CFBE41o- cells, a human bronchial epithelial cell line homozygous for the ΔF508-CFTR mutation, and CFBE41o- cells complemented with either ΔF508-CFTR (CFBE-ΔF508-CFTR) or wt-CFTR (CFBE-wt-CFTR) were generously provided by Dr. J. P. Clancy (Cincinnati Children's Hospital Medical Center). In all studies, we examined the effect of P. aeruginosa strain PAO1 on CFBE-ΔF508-CFTR cells compared with CFBE-wt-CFTR cells because both lines contain three copies of CFTR (either ΔF508/ΔF508/ΔF508 or ΔF508/ΔF508/wt-CFTR). Cells were grown as polarized monolayers in air-liquid interface culture on Transwell filters as described previously (2). Briefly, epithelial cells were seeded at 1 × 106 on 24-mm Transwell filters and grown for 8 days (to develop polarized monolayers) in MEM (Mediatech, Herndon, VA) with 10% fetal bovine serum, 2 mM l-glutamine, 50 U/ml penicillin, and 50 g/ml streptomycin at 37°C and 5% CO2, balance air. In all experiments, the medium was removed from the apical side of cultures and replaced with an equivalent volume of medium containing vehicle (medium with no P. aeruginosa) or medium with P. aeruginosa (washed twice in medium) to maintain air-liquid-interface culture. None of the experimental treatments changed LDH release or MTT production (n = 3/group), indicating that neither the ΔF508 mutation nor the experimental treatment with P. aeruginosa altered cell viability or metabolism, as determined by these assays.

Cytokine analysis for IL-8, CXCL1, CXCL2, and TNF-α secretion.

IL-8, CXCL1, and TNF-α produced by polarized monolayers of CFBE-ΔF508-CFTR and CFBE-wt-CFTR cells were measured using the Bio-Rad Bio-Plex cytokine arrays (Hercules, CA) according to the manufacturer's instructions. CXCL2 produced by polarized monolayers of CFBE-ΔF508-CFTR and CFBE-wt-CFTR cells was measured using a PromoKine ELISA kit (Heidelberg, Germany) according to the manufacturer's instructions. CFBE-ΔF508-CFTR and CFBE-wt-CFTR cells were grown on Transwell filters as described above, and PAO1 was added to the apical side of monolayers at a multiplicity of infection (MOI) of 30:1 for 1 h in the absence of antibiotics, and then planktonic PAO1 was removed by replacing the apical medium with MEM supplemented with 0.4% arginine (2). Control monolayers were treated identically except that vehicle only (MEM supplemented with 0.4% arginine) was added to the apical side of cells. Five hours after planktonic P. aeruginosa was washed from monolayers (visual inspection confirmed that P. aeruginosa had attached to the surface of the cells) (23), the apical and basolateral media were removed for analysis of cytokines/chemokines. Data are reported as total cytokine secretion because the results were similar if apical and basolateral cytokine/chemokine secretion were evaluated separately.

Quantitative, real-time RT-PCR.

Cells were grown and treated with P. aeruginosa as described above for the cytokine assay. Quantitative-RT-PCR (qPCR) experiments were conducted to examine the effects of P. aeruginosa on IL-8, CXCL1, CXCL2, and TNF-α mRNA expression by CFBE-wt-CFTR and CFBE-ΔF508 cells as described in detail previously (36). Airway cells were treated with P. aeruginosa, as described above, and stored in RNAlater (Ambion, Austin, TX). Total RNA was isolated using the RNAeasy Mini Kit (Qiagen, Valencia, CA), and tRNA concentration was measured using spectrophotometry (NanoDrop; NanoDrop Technologies, Rockland, DE). RNA quality was assessed with an Agilent 2100 Bioanalyzer (Agilent Technologies, Wilmington, DE). cDNA was synthesized from 1 μg of total RNA and random decamers using Retroscript Reverse Transcriptase (Ambion, Austin, TX). TaqMan Gene Expression Assays were purchased for human IL8 (TaqMan Gene Expression Assays, Inventoried assay ID Hs00174103_m1), human CXCL1 (TaqMan Gene Expression Assays, Inventoried assay ID Hs00236937_m1), human CXCL2 (TaqMan Gene Expression Assays, Inventoried assay ID Hs00601975_m1), and human TNF-α (TaqMan Gene Expression Assays, Inventoried assay ID Hs01113624_g1) from Applied Biosystems (ABI, Foster City, CA). From each experiment, triplicate reactions of each sample were incubated at 95°C for 10 min, followed by 40 cycles of 15 s at 95°C and 1 min at 60°C in a 96-well ABI Prism 7500 Sequence Detection System (Foster City, CA). In preliminary studies, qPCR products were analyzed on LMP agarose gels to confirm product size, subcloned into pCR4-TOPO (Invitrogen, Carlsbad, CA) and submitted for sequence analysis to confirm identity of the products. Dilutions of plasmid DNA prepared from the qPCR products for each gene were used to construct a standard curve. The standard curves showed a correlation coefficient close to 1 (R2 > 0.99) and were linear over a 6-log range. Raw data were analyzed, baseline and threshold values set, and gene expression interpolated using the external standard curves.

Gene array studies.

CFBE-ΔF508 and CFBE-wt-CFTR cells were grown on Transwell filters as described above, and PAO1 was added to the apical side of monolayers at an MOI of 30:1 for 1 h in the absence of antibiotics; then planktonic PAO1 was removed by replacing the apical medium with MEM supplemented with 0.4% arginine, as described above (2). Control monolayers were treated identically except that vehicle only (medium used to grow PAO1, MEM supplemented with 0.4% arginine) was added to the apical side of cells. Five hours after planktonic P. aeruginosa was washed from the cell monolayers, mRNA was isolated as described in detail (38). First-strand cDNA synthesis was conducted by priming the mRNA with a T7-(dT24) primer. Double-stranded cDNA was prepared using Life Technology Superscript cDNA Synthesis System (Invitrogen, Carlsbad, CA). From cDNA, cRNA was synthesized using the T7 MegaScript In Vitro Transcription kit (Ambion, Austin, TX). Synthesized cRNA was labeled during transcription (Megascript system; Ambion) with biotin-11-cytidine triphosphate and biotin-16-uridine triphosphate (Enzo Diagnostics, Farmingdale, NY). Biotinylated cRNA products were hybridized to Affymetrix genechip HGU 133Plus2 arrays for 16 h at 40°C in a Gene Chip Hybridization oven 640 using the manufacturer's hybridization buffer (Affymetrix, Santa Clara, CA) in the Dartmouth Genomics and Microarray Laboratory (http://dms.dartmouth.edu/dgml/background/). After hybridization, the arrays were washed and stained using the GeneChip Fluidics Station 450 (Affymetrix). The chips were scanned with the 7G Affymetrix Gene Scanner (Affymetrix) according to manufacturer's procedures.

Gene array analysis and meta-analysis.

As described in detail previously (12, 15), we normalized and summarized raw gene array data using RMA as implemented in Bioconductor (17). We assed group differences in two complementary ways. First, we used simple t-tests to identify genes whose mean expression value was different in a given cell line following exposure to P. aeruginosa. In accordance with suggestions from the Microarray Quality Control project (MAQC) (14), we chose to define significantly regulated genes as those with a P value <0.05 as well as a substantial fold change >2 as significantly regulated. Using this approach, the MAQC project and our laboratory obtain results that are more reproducible between replicate experiments compared with approaches that rank genes on the basis of adjusted P values, such as false discovery rates (12, 14, 15). To determine whether the strength of the response to P. aeruginosa differed between cell lines, we ran linear models of CFTR genotype, P. aeruginosa exposure, and the interaction of these two factors to establish P values and estimate effect size in R. We identified genes whose interaction estimate exceeded a fold of 2 and a P value <0.05 as significant. Significant genes identified by either method were used as input for Ingenuity (www.ingenuity.com) pathway analysis. Pathways selected by Ingenuity as significantly enriched in differentially expressed genes were further explored as previously reported (15). Hierarchical clustering of genes was performed using Euclidian distance and complete clustering using the heatmap.2 function from the R gplots library. In addition to analyzing the data from gene array studies on CFBE-ΔF508-CFTR and CFBE-wt-CFTR cells, we queried the Gene Expression Omnibus at NCBI, (3) searching for data on other sets of matched human airway epithelial cells exposed to P. aeruginosa, and found none available for download. However, microarray data from a comparison of IB3–1 and S9 cells previously published by Virella-Lowell et al. (41) was kindly supplied by the authors. These data were analyzed as described above to allow a comparison of the results between the two matched sets of cells. The gene array data from CFBE-ΔF508-CFTR and CFBE-wt-CFTR cells have been deposited in the Gene Expression Omnibus (GEO-accession number GSE30439) (http://www.ncbi.nlm.nih.gov/geo/).

General statistics.

Statistics on gene arrays were performed in R from the Foundation for Statistical Computing (http://www.R-project.org), Vienna, Austria as described previously (15). Other statistical analysis was performed using GraphPad Prism version 4.0a for Macintosh (GraphPad, San Diego, CA). When appropriate, experimental triplicates were performed and averaged. Means were compared using a two-sample t-test assuming unequal variance or linear model, as appropriate. P < 0.05 was considered significant for qPCR and ELISA assays.

RESULTS

Microarray gene expression analysis identifies a proinflammatory phenotype in CFBE-wt-CFTR cells.

Gene microarray studies were conducted to characterize the inflammatory response to P. aeruginosa in CFBE-ΔF508-CFTR cells and CFBE-wt-CFTR cells. The rationale for these experiments was twofold. First, there is only one published gene array study examining the effect of P. aeruginosa on gene expression in a CF cell line and its matched pair complemented with wt-CFTR. This experiment involved IB3–1 cells, which have a relatively uncommon CF genotype, ΔF508/W1282X (41). Second, there are no gene array studies examining the effect of P. aeruginosa on gene expression in matched CFBE-ΔF508-CFTR cells and wt-CFTR complemented CFBE cells, which have the most common CF genotype (homozygous ΔF508). Accordingly, CFBE-ΔF508-CFTR and CFBE-wt-CFTR cells were grown on Transwell filter supports to form polarized monolayers, and P. aeruginosa (PAO1) was added to the apical side of the monolayers at a multiplicity of infection of 30:1 as described in materials and methods. Previous studies by our laboratory have shown that the addition of PAO1 to the apical side of CFBE-ΔF508-CFTR and CFBE-wt-CFTR cells in the time frame of these studies does not affect the integrity of the epithelium, that P. aeruginosa remains in the apical compartment, and that this strain does not cross or disrupt cell monolayers (23, 24 and unpublished observations). This experimental approach was designed to limit the access of P. aeruginosa to the basolateral side of airway cells, which would otherwise result in rapid destruction of epithelial cells. Control monolayers were treated identically except that vehicle only (medium used to grow PAO1) was added to the apical side of cells.

The effect of P. aeruginosa exposure on gene expression by the CFBE cells was evaluated using Affymetrix genechip HGU 133Plus2 arrays, which contain 50,000 probe sets and 500,000 distinct oligonucleotide features that represent 14,500 well-characterized human genes. We considered gene expression significantly affected if the change in expression was greater than twofold and P < 0.05 in a t-test comparing exposed cells to control cells of the same genotype, or a linear model of gene expression values estimating the effect of P. aeruginosa exposure, CFBE genotype, and their interaction. Ingenuity pathway analysis of significant probes identified five pathways (TNFR1, TNFR2, HMGB1, MIF regulation of innate immunity, and IL-10) as significantly associated with genes induced by P. aeruginosa exposure (P < 10−6 in Fisher's Exact Test). An inspection of the 105 Ingenuity canonical paths that reached ordinary statistical significance (P < 0.05) revealed that the response involved genes associated with the Toll-like receptor signaling (99 genes) and chemokine signaling (170 genes) paths as defined by KEGG (19). The Toll-like receptor signaling path includes genes expressed in epithelial cells that might sense the presence of a specific pathogen-associated molecular pattern (e.g., lipopolysaccharide, which binds to Toll-like receptor 4), as well as many signal transduction elements such as the adaptor protein MyD88, transcription factors like NF-κB, and genes regulated by these transcription factors such as IL-8, a chemokine. Release of chemokines like IL-8 by epithelial cells in vivo recruits neutrophils from the bloodstream but also signals to adjacent epithelial cells through the chemokine signaling pathway.

P. aeruginosa induced 14 genes in the KEGG Toll-like receptor and cytokine signaling pathways (14/278 or 6%). The Venn diagram in Fig. 1 shows that the two cell lines responded similarly. Twelve genes were significantly induced by P. aeruginosa in both CFBE-ΔF508-CFTR and CFBE-wt-CFTR cells, two were induced only in CFBE-wt-CFTR cells, and there were no genes that were only induced in CFBE-ΔF508-CFTR cells (Fig. 1). By contrast, only two genes in the Toll-like receptor and cytokine signaling pathways were repressed by P. aeruginosa in CFBE-wt-CFTR cells (FADD and VAV3), and no genes in the Toll-like receptor or cytokine signaling pathways were repressed in CFBE-ΔF508-CFTR cells.

Fig. 1.

Fig. 1.

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Venn diagram of gene array data from CFBE-ΔF508-CFTR and CFBE41o- cells complemented with wild-type cystic fibrosis transmembrane conductance regulator (wt-CFTR) (CFBE-wt-CFTR) cells exposed to Pseudomonas aeruginosa (P. aeruginosa) showing induced genes belonging to either the KEGG Toll-like receptor or the KEGG chemokine signaling pathways (n = 4 biological replicates for each condition). 12 genes were induced (fold change >2, P < 0.05) by P. aeruginosa in both CFBE-ΔF508-CFTR and in CFBE-wt-CFTR cells (CXCL1, CXCL2, CXCL3, CXCR4, FOS, IL-6, IL-8, JUN, LYN, MAP3K8, NF-κBI, and TICAM1). NF-κBI and TNF-α were significantly induced (fold change >2, P < 0.05) by P. aeruginosa in CFBE-wt-CFTR cells but not in CFBE-ΔF508-CFTR cells. Please note that in this figure the relative change in gene expression between CFBE-wt-CFTR cells and CFBE-ΔF508-CFTR cells is not described. In this Venn diagram the number of genes that change in both or one group are denoted. Table 1 reports the relative changes in gene expression between CFBE-wt-CFTR cells and CFBE-ΔF508-CFTR cells.

Venn diagrams like the one shown in Fig. 1 may overstate differences between gene subsets because Venn diagrams have no mechanism to communicate similarity between genes that fall into different groups: a gene either falls into a group or it does not. Looking at Fig. 1, one might imagine that the two genes “not significantly induced by P. aeruginosa” in CFBE-ΔF508-CFTR were either repressed by P. aeruginosa or not induced at all, when in fact all 14 genes induced by P. aeruginosa in CFBE-wt-CFTR behaved quite similarly in CFBE-ΔF508-CFTR cells, as shown in Fig. 2. This figure shows the average response in log base 2 units for each of the 14 genes identified in Fig. 1 (blue and orange triangles), as well as uninduced genes belonging to the Toll-like receptor and cytokine signaling pathways (red triangles) and the other 50,000 Affymetrix probes (black dots) measured in CFBE-ΔF508-CFTR cells (x-axis) and CFBE-wt-CFTR cells (y-axis). In many ways, the response to P. aeruginosa shown in Fig. 2 agrees well with what one would expect. P. aeruginosa strongly induces IL-8 and several other inflammatory genes, whereas the vast majority of genes were unaffected by P. aeruginosa exposure and therefore cluster near (0,0). Figure 2 does not show the hyperinflammatory phenotype in CFBE-ΔF508-CFTR cells compared with CFBE-wt-CFTR cells that some would predict. Most inflammatory genes were similarly induced regardless of CFTR genotype (blue triangles, 11 probes corresponding to genes for CXCL1, CXCR4, FOS, IL6, IL8, JUN, LYN, MAP3K8, NF-κB1, NF-κBIA, and TICAM1), and where a linear model was able to detect a significant interaction (P < 0.05, fold change >2) between genotype and P. aeruginosa exposure (orange triangles corresponding to CXCL2, CXCL3, and TNF-α), the hyperinflammatory phenotype was evident in the CFBE-wt-CFTR cells, not the CFBE-ΔF508-CFTR cells. There were no genes in the Toll-like receptor and cytokine signaling pathways that were significantly more induced in CFBE-ΔF508-CFTR cells vs. CFBE-wt-CFTR cells. Thus P. aeruginosa induced a more robust inflammatory response in polarized CFBE-wt-CFTR cells than in CFBE-ΔF508-CFTR cells.

Fig. 2.

Fig. 2.

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Gene array data from CFBE-wt-CFTR and CFBE-ΔF508-CFTR cells exposed to P. aeruginosa. CFBE-wt-CFTR cell fold change (log base 2) in response to P. aeruginosa (y-axis) is plotted against the CFBE-ΔF508-CFTR cell response to P. aeruginosa (x-axis). Values near the 0,0 intercept indicate no response. Genes lying near the solid diagonal line responded similarly in both cell lines. Colored triangles highlight genes in the chemokine or Toll-like receptor signaling paths as defined by KEGG (19). Red triangles indicate genes in these paths that were not significantly induced, where significant induction is defined as a fold change greater than 2, and P < 0.05. Blue triangles correspond to 15 probes for 11 unique genes on the Toll-like receptor or chemokine signaling pathways: CXCL1, CXCR4, FOS, IL6, IL8, JUN, LYN, MAP3K8, NF-κB1, NF-κBIA, and TICAM1. Orange triangles correspond to an alternate probe for IL8 as well as probes for CXCL2, CXCL3, and TNF-α, all of which were significantly more induced in CFBE-wt-CFTR cells than in CFBE-ΔF508-CFTR cells, based on a P value of <0.05 in the interaction term of a linear model of CFTR genotype and P. aeruginosa exposure. Black dots signify genes not associated with chemokine or Toll-like receptor signaling pathways (n = 4 biological replicates for each condition).

P. aeruginosa elicits a more robust increase in IL-8 gene and cytokine expression in CFBE-wt-CFTR cells compared with CFBE-ΔF508-CFTR cells.

To confirm the gene microarray data that showed P. aeruginosa elicits a more robust increase in IL-8 gene expression in CFBE-wt-CFTR cells compared with CFBE-ΔF508 cells (Fig. 3A), we measured IL-8 mRNA by qPCR as described in materials and methods (Fig. 3B). We focused first on IL-8 because it is a standard marker of inflammation and is a major chemotactic factor for neutrophils, a cell type known to respond to infections in the CF lung (9, 27). qPCR studies confirmed the microarray studies that IL-8 mRNA levels were higher in CFBE-wt-CFTR cells compared with CFBE-ΔF508-CFTR cells treated with P. aeruginosa (Fig. 3B). Moreover, P. aeruginosa induced a significantly more robust increase in IL-8 cytokine production in CFBE-wt-CFTR cells compared with CFBE-ΔF508-CFTR cells, as determined using the Bio-Rad Bio-Plex cytokine arrays (Fig. 3C).

Fig. 3.

Fig. 3.

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IL-8 gene expression and cytokine secretion in response to P. aeruginosa (PA) in CFBE-wt-CFTR (WT) and CFBE-ΔF508-CFTR (ΔF508) cells as determined in gene microarray studies, n = 4/group (A), quantitative RT-PCR (qPCR), n = 3/group (B), and IL-8 protein secretion n = 3/group (C). Asterisks indicate significantly larger increase in expression in CFBE-wt-CFTR cells compared with CFBE-ΔF508-CFTR cells (P < 0.05).

P. aeruginosa elicits a more robust increase in CXCL1 gene and cytokine expression in CFBE-wt-CFTR cells compared with CFBE-ΔF508-CFTR cells.

The gene microarray data revealed that P. aeruginosa elicits a more robust increase in CXCL1 gene expression in CFBE-wt-CFTR cells compared with CFBE-ΔF508 cells although the difference did not achieve statistical significance (Fig. 4A). However, qPCR studies revealed that P. aeruginosa elicited a more robust, and statistically significant, increase in CXCL1 mRNA in CFBE-wt-CFTR cells compared with CFBE-ΔF508-CFTR cells (Fig. 4B). Consistent with the qPCR studies, P. aeruginosa induced a significantly more robust increase in CXCL1 cytokine production in CFBE-wt-CFTR cells compared with CFBE-ΔF508-CFTR cells, as determined using the Bio-Rad Bio-Plex cytokine arrays (Fig. 4C).

Fig. 4.

Fig. 4.

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CXCL1 gene expression and cytokine secretion in response to P. aeruginosa (PA) in CFBE-wt-CFTR (WT) and CFBE-ΔF508-CFTR (ΔF508) cells as determined in gene microarray studies, n = 4/group (A), quantitative RT-PCR (qPCR) studies (B) n = 3/group, and CXCL1 cytokine secretion, n = 3/group (C). Asterisks indicate significantly larger increase in expression in CFBE-wt-CFTR cells compared with CFBE-ΔF508-CFTR cells (P < 0.05).

P. aeruginosa elicits a more robust increase in CXCL2 gene and cytokine expression in CFBE-wt-CFTR cells compared with CFBE-ΔF508-CFTR cells.

The gene microarray data revealed that P. aeruginosa elicits a more robust increase in CXCL2 gene expression in CFBE-wt-CFTR cells compared with CFBE-ΔF508 cells although the difference did not achieve statistical significant (Fig. 5A). However, qPCR studies revealed that P. aeruginosa elicited a significantly more robust increase in CXCL2 mRNA in CFBE-wt-CFTR cells compared with CFBE-ΔF508-CFTR cells (Fig. 5B). Consistent with the qPCR studies, P. aeruginosa induced a significantly more robust increase in CXCL2 cytokine production in CFBE-wt-CFTR cells compared with CFBE-ΔF508-CFTR cells, as determined using PromoKine ELISA kit (Fig. 5C).

Fig. 5.

Fig. 5.

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CXCL2 gene expression and cytokine secretion in response to P. aeruginosa (PA) in CFBE-wt-CFTR (WT) and CFBE-ΔF508-CFTR (ΔF508) cells as determined in gene microarray studies, n = 4/group (A), quantitative RT-PCR (qPCR) studies, n = 3/group (B), and CXCL2 cytokine secretion, n = 3/group (C). Asterisks indicate significantly larger increase in expression in CFBE-wt-CFTR cells compared with CFBE-ΔF508-CFTR cells (P < 0.05).

P. aeruginosa elicits a more robust increase in TNF-α gene and cytokine expression in CFBE-wt-CFTR cells compared with CFBE-ΔF508-CFTR cells.

Gene array, qRT-PCR, and Bio-Plex studies were also conducted to examine the effect of P. aeruginosa on gene and protein expression of TNF-α, which is a proinflammatory cytokine. The gene microarray data revealed that P. aeruginosa elicits a more robust increase in TNF-α gene expression in CFBE-wt-CFTR cells compared with CFBE-ΔF508 cells (Fig. 6A). qRT-PCR studies confirmed the gene microarray studies demonstrating that P. aeruginosa elicited a more robust increase in TNF-α mRNA in CFBE-wt-CFTR cells compared with CFBE-ΔF508-CFTR cells (Fig. 6B). Consistent with the gene array and qPCR studies, P. aeruginosa induced a significantly more robust increase in TNF-α protein production in CFBE-wt-CFTR cells compared with CFBE-ΔF508-CFTR cells (Fig. 6C).

Fig. 6.

Fig. 6.

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Relative TNF-α gene expression in response to P. aeruginosa (PA) in CFBE-wt-CFTR (WT) and CFBE-ΔF508-CFTR (ΔF508) cells as determined in gene array studies, n = 4/group (A), by quantitative RT-PCR (qPCR), n = 3/group (B), and TNF-α cytokine production, n = 3/group (C). Asterisks indicate significantly larger increase in expression in CFBE-wt-CFTR cells compared with CFBE-ΔF508-CFTR cells (P < 0.05).

Reanalysis of microarray gene expression identifies a proinflammatory phenotype in IB3–1 cells (ΔF508/W1282X) compared with S9 cells (ΔF508/W1282X/wt-CFTR).

In a previous study, Virella-Lowell et al. (41) examined the effect of P. aeruginosa on gene expression in IB3–1 and S9 cells. They reported that P. aeruginosa elicited an exaggerated increase in the gene expression of IL-8 in CF cells (IB3–1 cells) compared with CF cells complemented with wt-CFTR (S9 cells). Although some genes reported to be induced by P. aeruginosa were similar in the Virella-Lowell study and the present study (i.e., IL-8 was induced by P. aeruginosa), several genes in the cytokine signaling pathway were induced in only one of the two studies. Thus, to determine whether the differences in results could be due in part to the statistical approach used to analyze the data in the two studies, we reanalyzed the Virella-Lowell published gene array data using the same statistical approach described in materials and methods that we used for the analysis of the CFBE-ΔF508-CFTR and CFBE-wt-CFTR cells.

Analysis of the gene array data in IB3–1 and S9 cells exposed to P. aeruginosa revealed that P. aeruginosa induced the expression of several genes in the Toll-like receptor and cytokine signaling pathways (Fig. 7). Only 13 of 278 genes (6%) in the two pathways analyzed were induced by P. aeruginosa. Three genes (IL-8, JUN, and RAP1A) were significantly induced (fold change >2, P < 0.05) by P. aeruginosa in both IB3–1 and S9 cells. Four genes were induced only in IB3–1 cells (CCL20, CXCL1, CXCL2, IL-6), and six genes (GNG11, GNG12, KRAS, PLCB4, RAC1, RAP1B) were induced only in S9 cells (Fig. 7). By contrast, only 5 of 278 genes were repressed by P. aeruginosa. One gene (PIK3CD) was significantly repressed (fold change >2, P < 0.05) by P. aeruginosa in both IB3–1 and S9 cells, three genes were repressed only in IB3–1 cells (GNAI2, RAC2, STAT3), and one gene (RASGRP2) was repressed only in S9 cells (Fig. 8).

Fig. 7.

Fig. 7.

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Venn diagram of gene array data from IB3–1 and S9 cells exposed to P. aeruginosa showing significantly induced genes belonging to either the KEGG Toll-like receptor or the KEGG chemokine signaling pathways. 3 genes (IL8, JUN, and RAP1A) were induced (fold change >2, P < 0.05) by P. aeruginosa in both IB3–1 and S9 cells. 4 genes were induced only in IB3–1 cells (CCL20, CXCL1, CXCL2, IL-6), and 6 genes (GNG11, GNG12, KRAS, PLCB4, RAC1, RAP1B) were induced only in S9 cells. Data from Virella-Lowell (41) re-analyzed as described in materials and methods.

Fig. 8.

Fig. 8.

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Venn diagram of gene array data from IB3–1 and S9 cells exposed to P. aeruginosa showing significantly repressed genes belonging to either the KEGG Toll-like receptor or the KEGG chemokine signaling pathways. 1 gene (PIK3CD) was significantly repressed (fold change >2, P < 0.05) by P. aeruginosa in both IB3–1 and S9 cells. 3 genes were repressed only in IB3–1 cells (GNAI2, RAC2, STAT3), and 1 gene (RASGRP2) was repressed only in S9 cells. Data from Virella-Lowell (41) were re-analyzed as described in materials and methods.

Figure 9 presents all of the genes in the Affymetrix chips from IB3–1 and S9 cells exposed to P. aeruginosa. The Affymetrix genechip U95Av2 used in the Virella-Lowell study contains 12,625 probe sets that represent 8,799 well-characterized human genes. Plotting the change in gene expression in log base 2 units for S9 cells on the y-axis vs. the change in gene expression for IB3–1 cells on the x-axis reveals that the vast majority of genes was not affected by P. aeruginosa (i.e., most genes, including those plotted as black dots or colored triangles, clustered around the 0,0 intersection). Genes in the Toll-like receptor and cytokine signaling pathways are presented in color. Very few genes in the Toll-like receptor and cytokine signaling pathways were changed by P. aeruginosa in either cell line (i.e., most genes in this pathway, shown in red, clustered about the 0,0 intersection). Thirteen genes in the KEGG Toll-like pathway and the KEGG cytokine signaling pathway were significantly induced (fold change >2 and P < 0.05) by P. aeruginosa in either IB3–1 or S9 cells: CCL20, CCL23, GNG11, IL6, IL8, JUN, KRAS, PLCB4, RAC1 RAP1A, and RAP1B (orange and blue triangles in Fig. 9). Of these, 10 showed significantly different levels of induction (orange triangles) and had P values of <0.05 in the interaction term of the linear model that included CFTR genotype and P. aeruginosa exposure. Three genes (blue triangles) were similarly induced in IB3–1 and S9 cells and included CCL23, CXCL1, and JUN. Genes responding significantly less in S9 (wild-type CFTR) cells included IL8 and two other genes, IL6 and CCL20 (orange triangles below the diagonal line), whereas GNG11, GNG12, KRAS, PLCB4, RAC1, RAP1A, and RAP1B responded more strongly in S9 cells (orange triangles above the diagonal line). This re-analysis of the Virella-Lowell data reveals that the differences in results between their study and the data in this study are not due to differences in the statistical approach used to analyze the data.

Fig. 9.

Fig. 9.

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Gene array data from S9 (wt-CFTR) and IB3–1 (ΔF508/W1282X) cells exposed to P. aeruginosa. S9 wt-CFTR cell fold change response (log base 2) to P. aeruginosa (y-axis) is plotted against the IB3–1 ΔF508-CFTR response to P. aeruginosa (x-axis). Values near the 0,0 intercept indicate no response. Genes lying near the solid diagonal line responded similarly in both cell lines. Colored triangles highlight genes in the chemokine or Toll-like receptor signaling pathways as defined by KEGG (19). Red triangles indicate genes in these pathways that were not significantly induced in either cell line, where significant induction is defined as a fold change greater than 2, and P < 0.05. Blue triangles denote genes similarly induced in IB3–1 and S9 cells and included CCL23, CXCL1, and JUN. Orange triangles indicate genes responding either significantly less in S9 cells (below the diagonal line) included IL8 and two other genes, IL6 and CCL20, as well as genes responding significantly more in S9 cells (above the diagonal line) GNG11, GNG12, KRAS, PLCB4, RAC1, RAP1A and RAP1B. Black dots signify probes not associated with chemokine or Toll-like receptor signaling paths.

DISCUSSION

This study provides the first comprehensive analysis of the effects of P. aeruginosa on gene expression and cytokine/chemokine production in CFBE-wt-CFTR and CFBE-ΔF508-CFTR cells, a pair of matched cell lines utilized extensively to study CF. We report that P. aeruginosa elicited a more robust increase in cytokine and chemokine expression (e.g., IL-8, CXCL1, CXCL2, and TNF-α) in CFBE-wt-CFTR cells compared with CFBE-ΔF508-CFTR cells. These data add to a growing body of evidence revealing that the inflammatory response of human airway epithelial cells expressing wt-CFTR and ΔF508-CFTR to P. aeruginosa is model dependent, even when considering matched cell lines and human airway epithelial cells in primary culture (4, 6, 8, 16, 18, 22, 26, 28, 31, 37, 39, 41). Taken together with other published studies, our data demonstrate that there is no compelling evidence to support the view that mutations in CFTR induce a hyperinflammatory response in human airway epithelial cells in vivo. Although the lungs of patients with CF have abundant levels of proinflammatory cytokines and chemokines compared with non-CF lungs, because the lung is populated by immune cells and epithelial cells, there is no way to know, a priori, whether airway epithelial cells in the CF lung in vivo are hyperinflammatory in response to P. aeruginosa compared with non-CF lung epithelial cells. Thus studies on human airway epithelial cell lines and primary cells in vitro that propose to examine the effect of mutations in CFTR on the inflammatory response to P. aeruginosa have uncertain clinical significance with regard to CF.

The goal of this study was to provide insight into the response of CFBE-ΔF508-CFTR cells and CFBE-wt-CFTR cells to P. aeruginosa using Affymetrix gene arrays, qPCR, and direct measurement of cytokines/chemokines protein production. P. aeruginosa induced a relatively small number of genes in the KEGG Toll-like receptor and cytokine signaling pathways (∼6% of 278 unique genes), including CXCL1, CXCL2, IL-8, and TNF-α in both cell lines. We selected these KEGG pathways because they include the major factors involved in inflammatory response in epithelial cells. Interestingly, there were no genes in the Toll-like receptor and cytokine signaling pathways that were significantly more induced in CFBE-ΔF508-CFTR cells vs. CFBE-wt-CFTR cells. Indeed, P. aeruginosa induced a more robust increase in CXCL1, CXCL2, IL-8, and TNF-α in CFBE-wt-CFTR cells vs. CFBE-ΔF508-CFTR cells. Remarkably, only two genes in these KEGG pathways were repressed by P. aeruginosa, FADD (a gene that codes for an adaptor protein that interacts with membrane receptors involved in apoptosis) and VAV3 (a gene that codes for a guanine nucleotide exchange factor for the Rho family of GTPases that activates actin cytoskeletal rearrangement).

qPCR and ELISA assays confirmed our gene array studies and demonstrated that P. aeruginosa elicited a more robust increase in CXCL1, CXCl2, IL-8, and TNF-α protein expression in CFBE-WT-CFTR cells compared with CFBE-ΔF508-CFTR cells. Moreover, P. aeruginosa induced a larger increase in MAP3K8 and NF-κBI gene expression in CFBE-wt-CFTR cells compared with CFBE-ΔF508-CFTR cells, demonstrating that P. aeruginosa activates the inflammatory response in both cell lines, but the effect is more robust in CFBE-wt-CFTR cells compared with CFBE-ΔF508-CFTR cells. These data suggest that the ΔF508/ΔF508 genotype in CFTR reduces the ability of CFBE cells to elaborate an inflammatory response to P. aeruginosa.

The present study reveals similarities and a key difference between the present study and that of Virella-Lowell et al. (41). First the similarities include the following: very few genes are significantly induced or repressed by P. aeruginosa in CF cells and CF cells expressing wt-CFTR in the pathways examined (∼6% of the 278 genes in the Toll-like receptor and cytokine pathways), regardless of the matched pair of cells. The major difference between the two studies was that, in the IB3–1/S9 set of cells, P. aeruginosa induced a more dramatic induction of IL-8 in IB3–1 cells compared with S9 cells, whereas, in the present study, P. aeruginosa elicited a more robust response in CXCL1, CXCL2, IL-8, and TNF-α in CFBE-wt-CFTR cells compared with CFBE-ΔF508-CFTR cells. It is notable that, in S9 cells, P. aeruginosa did not significantly induce IL-6, CXCL2, CXCL3, CXCR4, TNF-α, MAP3K8, or NF-κBI (See Table 1), an observation suggesting that the S9 cell line has lost the ability to elaborate an inflammatory response to P. aeruginosa and one that may explain the observation that IB3–1 cells are more inflammatory than S9 cells. Although it is not entirely clear why the proinflammatory response in the IB3–1/S9 pair is opposite to the response observed in the CFBE-ΔF508-CFTR/CFBE-wt-CFTR pair, several possibilities should be considered. First, as noted above, it appears that the S9 cells have lost the ability to respond to P. aeruginosa. Second, the genotype of the cells is different (i.e., IB3–1 cells have the ΔF508/W1282X genotype and CFBE cells are ΔF508/ΔF508). Third, IB3–1 cells do not form polarized monolayers as determined by their inability to generate a significant transepithelial resistance when grown on permeable filters, thus it is possible that P. aeruginosa and/or its products may have had access to basolateral membrane receptors, which would elicit a different response than activation of apical membrane receptors in polarized CFBE-ΔF508-CFTR cells and CFBE-wt-CFTR cells. Finally, other possible factors contributing to differential responses are clonal selection and epithelial phenotype drift.

Table 1.

List of genes in the KEGG Toll-like receptor and cytokine signaling pathways induced by P. aeruginosa in CFBE-wt-CFTR and CFBE-ΔF508-CFTR cells as discovered by microarray studies and in IB3-1 and S9 cells

Gene CFBE ΔF508/ΔF508-CFTR CFBE wt-CFTR IB3-1 ΔF508/W1282X CFTR S9 wt-CFTR Gene Function
CXCL1 + + + NI Chemotactic for neutrophils
CXCL2 + + + NI Chemotactic for neutrophils
CXCL3 + ++ ++ + Chemotactic for neutrophils
CXCR4 + + ++ + Chemokine receptor to CXCL12, which induces epithelial chemotaxis
TNF-α NI ++ NI NI Multifunctional proinflammatory cytokine
IL-8 + ++ ++ + Involved in the proinflammatory response. A cellular protooncogene and early transcription factor
IL-6 + + ++ NI Induces transcriptional proinflammatory response
MAP3K8 + + NI NI Activates MAP and JNK kinase pathways and IkB kinase (nuclear translocation of NF-κB)
NF B1 NI + NI NI Transcription factor. Regulates the immune response to infection.
NF BIA + + NI NI Inhibitor of NF-κB1 activity
TICAM1 + + NI NI Toll like receptor signaling adapter molecule
FOS + + NI NI Regulator of cell proliferation, differentiation, and transformation
JUN + + + + Regulates gene expression
LYN + + NI NI Mediator that relays suppressing signals from CXCR4
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Data are analyzed as described in materials and methods. + Significant induction compared to control; ++significantly more induction compared to the other cell line in the matched pair. Genes that are not induced by Pseudomonas aeruginosa (P. aeruginosa) as determined by the analysis in this study are depicted as NI. The effects of P. aeruginosa on the expression of all genes that were significantly induced or repressed (i.e., 2-fold change and P < 0.05) are presented in the Supplemental Tables; which are available online at the American Journal of Physiology Lung Cellular and Molecular Physiologywebsite. CFTR, cystic fibrosis transmembrane conductance regulator.

Several other laboratories have examined the effect of the ΔF508 mutation on the production of IL-8 by matched cell lines in response to P. aeruginosa. These studies have produced mixed results and have demonstrated that the ΔF508 mutation either enhances (28, 37, 39, 41), suppresses (18, 22, 31), or has no effect on IL-8 secretion (8, 16, 30) compared with the matched cells complemented with wt-CFTR. For example, one study on IB3–1 and S9 cells demonstrated that P. aeruginosa elicits a more robust increase in IL-8 production in IB3–1 cells compared with the CFTR-corrected S9 cells (10). However, others report that there is no difference in the response to P. aeruginosa in IB3–1 and S9 cells. For example, Cigana et al. (8) found that P. aeruginosa exploits lipid A and muropeptides modification as a strategy to lower innate immunity during cystic fibrosis lung infection (8). They measured IL8 in IB3–1 and C38 cells (IB3–1 cells also complemented with wt-CFTR) and saw no difference in IL8. These observations suggest that clonal selection and/or epithelial phenotype drift of IB3–1/S9 (C38) cells in culture and/or subtle differences in experimental design may be responsible for the variable results. Consistent with this view are the observations that removal of FBS from the cell culture medium eliminates the stimulatory effect of the ΔF508 mutation on the inflammatory response in primary human airway epithelial cells and that with passage primary CF human airway epithelial cells lose the hyperinflammatory response to P. aeruginosa (42).

Studies on humans, mice, and zebrafish have also examined the effect of mutations in the CFTR gene on the inflammatory response to P. aeruginosa. For example, Muhlebach (25) reported that, in bronchoalveolar lavage fluid, IL-8 levels were higher in patients with CF than in patients without CF, at least when P. aeruginosa CFU were less than 107 (25). Interestingly, Muhlebach reported that, at higher CFU of P. aeruginosa, there was no difference in IL-8 between CF and non-CF, and that the slope of the relationship between IL-8 and P. aeruginosa CFU was less in CF than non-CF. In other words, it takes a larger increase in CFU to produce a given increase in IL-8 in patients with CF than it does in patients without CF. An indolent response to P. aeruginosa associated with the absence of CFTR has also been observed in mice and zebrafish. Murine models of CF reveal reduced bacterial clearance of P. aeruginosa in CFTR (−/−) mice (34), whereas, in zebrafish morpholino, knockdown of CFTR suppressed the innate immune response to P. aeruginosa, including decreasing the production of reactive oxygen species and reducing neutrophil migration, resulting in an increase in the load of P. aeruginosa (29). Thus both in vivo models recapitulate the hyperinflammatory response to P. aeruginosa observed in the human CF lung compared with the non-CF lung.

In conclusion, the data in this manuscript taken together with previous studies demonstrate that there is no consistent effect of mutations in CFTR on the proinflammatory response of isolated human airway epithelial cells to P. aeruginosa, even when comparing matched cell lines and human airway epithelial cells in primary culture. Thus we conclude that, although it is clear that the inflammatory response to P. aeruginosa in the CF lung is more robust than in the non-CF lung, the literature, including the present study, does not allow a conclusion regarding the effect of mutations in CFTR on the inflammatory response to P. aeruginosa by isolated airway epithelial cells. Therefore, it is not clear whether the enhanced inflammatory response to P. aeruginosa in the human CF airway compared with the non-CF airway reflects differences in airway epithelial cell function per se, differences in the response of immune cells to P. aeruginosa, differences in P. aeruginosa-induced interactions between immune cells and airway epithelial cells, reduced ability of the mucociliary ladder to clear P. aeruginosa, or a combination of these factors.

GRANTS

This work was supported by NIH grants R01-HL074175, R01-DK045881 (B. Stanton), and K99-HL098342 (J. Bomberger), and grants from the Cystic Fibrosis Foundation (STANTO07R0 and STANTO08GO to B. Stanton).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

Author contributions: T.H.H., G.A.O., and B.A.S. conception and design of research; T.H.H. and B.A.S. analyzed data; T.H.H. and G.A.O. interpreted results of experiments; T.H.H. prepared figures; T.H.H., A.E.B., and B.A.S. drafted manuscript; T.H.H., B.C., J.R.C.-G., G.A.O., and B.A.S. edited and revised manuscript; T.H.H., G.A.O., and B.A.S. approved final version of manuscript; A.E.B., J.M.B., M.R.R., R.B., and B.C. performed experiments.

Supplementary Material

Supplemental Tables
supp_303_6_L509__index.html (1.4KB, html)

ACKNOWLEDGMENTS

The authors thank Dr. Terence Flotte for sharing his gene array data (41).

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental Tables
supp_303_6_L509__index.html (1.4KB, html)
supp_00226.2011_tableS1.xlsx (67.9KB, xlsx)
supp_00226.2011_tableS2.xlsx (88.1KB, xlsx)

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