Gene profile changes after Pseudomonas aeruginosa exposure in immortalized airway epithelial cells

Abstract

To test the hypothesis that the excess inflammatory response in cystic fibrosis airway epithelial cells is the result of differential activation of genes in response to a laboratory strain of Pseudomonas aeruginosa (PAO1), a 48-h time course of genes expressed following PAO1 stimulation (10⁹CFU for 1 h) was studied in two pairs of airway epithelial cells: 9/HTEo⁻and 16HBE14o⁻, each with a matched normal and CF phenotype pair. cRNA was hybridized to Affymetrix HG-U95Av2 chips and pairwise comparisons against zero time (no PAO1) were calculated for each time point. PAO1 elicited profound changes in both cell pairs: for 9/HTEo⁻, 144 genes changed significantly in the normal pair, and 116 for the CF pair; for the 16HBE14o⁻pair, 57 genes changed significantly for the normal pair and 53 for the CF pair. Changes were much greater in the 9/HTEo⁻than in the 16HBE14o⁻pair, but basal levels of expression of inflammatory genes are higher in the 16HBE14o⁻pair, so 16HBE14o⁻was used mainly to corroborate the results of 9/HTEo⁻. Clustering analysis indicated that the pattern of gene expression is similar in the CF cells and their normal counterparts. However, there were substantial quantitative differences in gene expression. Thus, the difference between CF and normal resides in the magnitude, not the pattern, of the changes.

abbreviations:

CF – cystic fibrosis; CFTR – cystic fibrosis transmembrane conductance regulator; CFU – colony forming units; PAO1 – laboratory strain of Pseudomonas aeruginosa; BAL – bronchoalveolar lavage; IL – interleukin; NF-kB – nuclear transcription factor kappa B; NOS−2 – nitric oxide synthase; DMEM – Dulbecco’s modified Eagle medium; GM-CSF – granulocyte-macrophage colony-stimulating factor; GROα– melanoma growth stimulating activity, alpha (CXCL1); CXCL2 – chemokine (C-X-C motif) ligand 2, GROβ; TNFAIP3 – tumor necrosis factor, alpha-induced protein 3; NFKBIA – nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha; ELISA – enzyme-linked immunosorbent assay; RT-PCR – reverse transcriptase polymerase chain reaction; GAPDH – glyceraldehyde-3-phosphate dehydrogenase; UPGMA – unweighted pair-group method with arithmetic mean; GO – Gene Ontology.

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