Formoterol- and budesonide-induced gene expression and effects of combination. (A) The 212 genes induced (≥2-fold, P ≤ 0.05) at any time by formoterol in BEAS-2B cells were grouped according to the time of peak expression (log₂ fold) and are presented as heat maps. Red = induced, blue = repressed, white = log₂(1), and color intensity reflects increasing change in log₂ fold. (B) Expression of the genes showing peak formoterol-induced expression at each time was summarized using local polynomial regression (solid line) ± 95% confidence interval (CI; gray shading) of log₂ fold over time (hours). For each time of peak formoterol-induced expression, the genes were stratified according to the greatest effect (+1, 0, or −1) of budesonide occurring at any time: +1 = ≥2-fold, 0 = >0.5/<2-fold, and −1 = ≤0.5-fold produced by budesonide addition when compared with formoterol-induced expression. Red line = formoterol, blue line = budesonide, and purple line = budesonide + formoterol. (C) Graphical representation showing the gene count for formoterol-induced genes meeting the criteria (+1, 0, or −1) in (B) for the effect of budesonide (Bud) addition. (D) The 370 genes induced (≥2-fold, P ≤ 0.05) at any time by budesonide in BEAS-2B cells were grouped according to the time of peak expression (log₂ fold) and presented as heat maps (red = induced, blue = repressed). (E) Expression of genes showing peak budesonide-induced expression at each time was summarized as in (B). For each time of peak budesonide-induced expression group, the genes were stratified according to the greatest effect (+1, 0, or −1) of formoterol occurring at any time: +1 = ≥2-fold, 0 = >0.5/<2-fold, and −1 = ≤0.5-fold produced by formoterol addition when compared with budesonide-induced expression. Line colors are as in (B). (F) Graphical representation showing the gene count for budesonide-induced genes meeting the criteria (+1, 0, or −1) in (E) for the effect of formoterol (Form) addition. (G) The 196 genes induced (≥2-fold, P ≤ 0.05) at any time by budesonide + formoterol, but not by either formoterol or budesonide alone in BEAS-2B cells, were grouped according to the time of peak expression and summarized as in (B). Line colors are as in (B).

Supra- and infra-additive effects of formoterol and budesonide on gene expression. (A) The 46 genes induced (≥2-fold, P ≤ 0.05) by both formoterol and budesonide at any time in BEAS-2B cells were grouped according to the time of peak expression, and expression was summarized using local polynomial regression (solid line) ± 95% confidence interval (CI; gray shading) of log₂ fold over time (hours) (left panel). For each group of genes, heat maps were generated to show fold gene expression for each treatment and time (center panel). Red = induced, blue = repressed, white = 1, and color intensity reflects fold change. For each gene (right-hand main panel), the effect (fold-1) of budesonide and formoterol was summed (B − 1 + F − 1 = B + F − 2) and displayed next to the effect of budesonide + formoterol (BF − 1) as a heat map using the same scale as the prior panel. Differences between B + F − 2 and BF − 1 were compared by one-way ANOVA followed by Tukey’s post hoc test (far right panel). P values presented as heat maps are shown in which purple color intensity corresponds to increasing −log₁₀(P value), whereas values below 1.3 (P < 0.05) are all white. (B) The 196 genes showing induced expression by budesonide + formoterol (2 ≥ fold, P ≤ 0.05), but not by budesonide or formoterol alone, were grouped, as in Fig. 2G, by time of peak expression. The effect of budesonide was summed with the effect of formoterol (B + F − 2) and displayed as a heat map next to the effect of budesonide + formoterol (BF − 1) (left panel) using the same scale as in (A). Similarly, significance was tested, and P values are presented as a heat map (right panel), as in (A). (C) For each time of budesonide + formoterol–induced peak expression, one gene showing budesonide+ formoterol, but not budesonide or formoterol alone, induced expression from (B) is shown. (D) Examples of formoterol-induced genes displaying significant supra- or infra-additivity (upper panels and lower panels, respectively) are shown for peak formoterol-induced expression at 1, 2, and 6 hours. Color coding is as in (C). (E) Examples of formoterol-induced genes displaying significant supra- or infra-additivity (upper panels and lower panels, respectively) are shown for peak formoterol-induced expression at 1, 2, and 6 hours. Color coding is as in (C). B, budesonide; BF, budesonide plus formoterol; F, formoterol.

Formoterol- and budesonide-repressed gene expression and effects of combination. (A) The 55 genes that were repressed (≤0.5-fold, P ≤ 0.05) at any time by formoterol in BEAS-2B cells were grouped according to the time of peak repression (log₂ fold) and presented as heat maps [red = induced, blue = repressed, white = log₂(1), and color intensity reflects increasing log₂ fold change]. (B) Expression of the 2, 25, 24, and 4 genes showing peak formoterol-induced repression at 1, 2, 6, or 18 hours was summarized using local polynomial regression (solid line) ± 95% confidence interval (CI; gray shading) of log₂ fold over time (hours). For each time of peak formoterol-induced repression group, the genes were further stratified according to the greatest effect (+1, 0, or −1) of budesonide occurring at any time: +1 = ≥2-fold, 0 = >0.5/<2-fold, and −1 = ≤0.5-fold produced by budesonide addition when compared with formoterol-repressed expression. Red line = formoterol, blue line = budesonide, and purple line = budesonide + formoterol. (C) Graphical representation showing the gene count for formoterol-repressed genes meeting the criteria (+1, 0, or −1) in (B) for the effect of budesonide (Bud) addition. (D) The 413 genes that were repressed (≤0.5-fold, P ≤ 0.05) at any time by budesonide in BEAS-2B cells were grouped according to the time of peak repression (log₂ fold) and presented as heat maps (red = induced, blue = repressed). (E) Expression of the 3, 17, 132, and 261 genes showing peak budesonide-induced repression at 1, 2, 6, or 18 hours was summarized as in (B). For each time of peak budesonide-induced repression group, the genes were further stratified according to the greatest effect (+1, 0, or −1) of formoterol occurring at any time: +1 = ≥2-fold, 0 = >0.5/<2-fold, and −1 = ≤0.5-fold produced by formoterol addition when compared with budesonide-repressed expression. Red line = formoterol, blue line = budesonide, and purple line = budesonide + formoterol. (F) Graphical representation showing the gene count for budesonide-repressed genes meeting the criteria (+1, 0, or −1) in (E) for the effect of formoterol (Form) addition. (G) The 198 genes, which were repressed (≤0.5-fold, P ≤ 0.05) at any time by budesonide + formoterol, but not but by either formoterol or budesonide alone, in BEAS-2B cells were grouped according to the time of peak repression. The 25, 77, and 96 genes with peak repression at 2, 6, or 18 hours were summarized as in (B). Red line = formoterol, blue line = budesonide, purple line = budesonide + formoterol. (H) The 117 genes that showed enhanced repression by budesonide plus formoterol when compared with both budesonide and formoterol alone were grouped according to the time of peak repression. There were 22, 76, and 31 genes showing peak repression at 2, 6, and 18 hours, respectively. Fold data for each group were expressed as log₂(fold) and are plotted as box and whiskers to show the effect of each treatment.

Functional aspects of formoterol-induced gene expression with effect of budesonide and overview GO analysis of genes modulated by formoterol, budesonide, and budesonide plus formoterol in BEAS-2B cells. (A) Functional annotation clustering of the 212 genes induced by formoterol resulted in 11 GO term clusters with enrichment scores ≥1.3 (Supplemental Fig. 3). (i) GO terms making up cluster 4 were depicted along with a bar graph showing the fraction of formoterol-induced genes that were further enhanced (≥2-fold) or repressed (≤0.5-fold) by budesonide. Expected fractions (enhanced = 0.22, repressed = 0.37) are indicated by dotted lines. Of the 21 genes in this cluster, three were further enhanced (≥2-fold) and 12 were repressed (≤0.5-fold) by budesonide cotreatment, making this cluster predominantly repressed by budesonide. (ii) Heat map showing effects of formoterol (Form), budesonide (Bud), and Bud + Form on the 21 genes within this cluster. Red = induced, blue = repressed, white = log₂(1), and color intensity reflects increasing change in log₂ fold. (B) Heap maps showing gene expression, as in (A), in response to formoterol, budesonide, and Bud + Form for all induced genes that were associated with the GO terms (i) “Pathways in cancer” and (ii) “cytokine activity” plus “Jak-STAT signaling pathway” with any treatment. (C) GO terms (biologic process, molecular function, KEGG) for the 267, 777, and 1165 genes modulated, either up (≥2-fold, P ≤ 0.05) or down (≤0.5-fold, P ≤ 0.05), by Form, Bud, or budesonide plus formoterol (B + F) in BEAS-2B cells were extracted using the DAVID. The number of individual genes within each functional grouping is indicated for each treatment. dev, development; diff, differentiation; prolif, proliferation.

Effects of formoterol and budesonide alone, or in combination, on gene expression in pHBECs. pHBECs from six normal individuals were either not stimulated (NS) or treated with formoterol (Form; 10 nM) and budesonide (Bud; 100 nM) or both together (Bud + Form or B + F) for 6 hours prior to RNA extraction and microarray analysis. Following robust multiarray averaging and normalization, data were expressed as fold relative to NS, and the genes showing ≥2-fold or ≤0.5 fold (P ≤ 0.05) were considered to be differentially expressed (Supplemental Table 5). (A) The number of differentially expressed genes showing up- or downregulation for each treatment is shown. Overall gene numbers of modulated (up- and downregulated) genes are shown below the graph. (B) Expression of genes showing different expression by formoterol was plotted against the effect of budesonide alone, and expression of the genes showing differential expression to budesonide was plotted against the effect of formoterol alone. Gene numbers in each corner quadrant are shown along with expected numbers in brackets. (C) The number of genes meeting the expression criteria for (i) induction (≥2-fold, P ≤ 0.05) or (ii) repression (≤0.5-fold, P ≤ 0.05) at any time with respect to each treatment is represented as Venn diagrams. Beside each Venn diagram, contingency tables assess the likelihood (χ² test) that the number of genes (induced or repressed) in common between formoterol and budesonide may have occurred by chance. ****P = 0.0001. (D) The 140 formoterol-induced genes were plotted to show the number of genes that were further induced (≥2-fold) or repressed (≤0.5-fold) with budesonide cotreatment, and the 94 budesonide-induced genes were plotted to show the number of genes that were further induced (≥2-fold) or repressed (≤0.5-fold) by formoterol cotreatment. (E). The 208 formoterol-repressed genes were plotted to show the number of genes that were induced (≥2-fold) or further repressed (≤0.5-fold) with budesonide cotreatment, and the 141 budesonide-repressed genes were plotted to show the number of genes that were induced (≥2-fold) or further repressed (≤0.5-fold) by formoterol cotreatment. E, expected, O, observed.