IL-13 involvement in eosinophilic esophagitis: Transcriptome analysis and reversibility with glucocorticoids

IL-13 and IL-4 mRNA expression in biopsy samples from healthy (NL) subjects and patients with EE. The expression of IL-13 (A) and IL-4 (B) is shown. Each mRNA value is normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression from the same sample and is expressed as a fold increase. The black lines represent the mean value in each group. P values were calculated by using the Mann-Whitney U test (2 groups; n = 8-9 and 13-21 subjects for the healthy and EE groups, respectively).

Gene expression analysis in primary esophageal cells after IL-13 stimulation and comparison with the EE transcript signature. A, The 54,765 genes of the HG-U133 chip were subjected to fold-change filter in patients with EE versus healthy subjects and IL-13–stimulated primary cell cultures versus unstimulated cells. Spearman correlation and linear regression were calculated. B, The list displays 33 transcripts that were upregulated 5-fold or greater and 5 transcripts that were downregulated 4-fold or greater compared with unstimulated cells. C, The genes modified by 1.5-fold or greater on average in IL-13–stimulated cells (100 ng/mL) are presented in a heat diagram in 3 primary-culture patient biopsy specimens (1, 2, and 3), unstimulated and stimulated. Upregulated genes are shown in red, and downregulated genes are shown in blue. The magnitude of the gene changes is proportional to the darkness of the color. D, The fold increase of eotaxin-3 mRNA expression compared with the untreated value was quantified by means of real-time PCR. E, Eotaxin-3 released in the culture supernatant is expressed in nanograms per milliliter. Results are presented as means ± range and are representative of experiments performed in 5 different patients.

IL-13 receptor chain expression in esophageal cells and eotaxin-3 production by esophageal cell lines after IL-13 stimulation. A, The TE-1, TE-6, TE-7, and TE-13 esophageal cell lines were subjected to PCR analysis for IL-4Rα, IL-13Rα1, IL-13Rα2, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression. B, Flow cytometric analysis of IL-4R, IL-13Rα1, and IL-13Rα2 chain expression in the TE-7 cell line (dark lines). Controls were performed with an irrelevant IgG1 (gray lines). C, The TE-1, TE-6, TE-7, and TE-13 esophageal cell lines were stimulated for 24 hours with IL-13 (0, 1, 10, and 100 ng/mL). The fold increase of eotaxin-3 mRNA expression compared with that of the untreated cells is shown. D and E, TE-7 cells were cultured with IL-13 (0, 1, 10, and 100 ng/mL) for 1, 6, 12, 24, and 48 hours. Eotaxin-3 protein released in the culture supernatant was quantified by means of ELISA. Results are presented as means ± range and are representative of at least 3 experiments performed in triplicate.

Human eotaxin-3 promoter activity after IL-13 stimulation and the role of STAT6. A, TE-7 cells were transfected with pGL3 basic containing the eotaxin-3 promoter (P800) and phRLTK. Cells were stimulated with IL-13 (0, 1, 10, and 100 ng/mL) B, TE-7 cells were transfected with pGL3 basic containing different lengths of the eotaxin-3 promoter (P800, P500, and P100) and promoters containing mutations in the STAT6-responsive elements (MUT1, MUT2, and MUT1&2). C, The TE-7 cells were cotransfected with P800 and a dominant-negative form of STAT6 (DNSTAT6) or the empty vector (EV). D, TE-7 cells were cotransfected with P800 and the expression vector containing STAT6:ER. The cells were stimulated with 4-hydroxytamoxifene (4HT; 10 μmol/L). Results are presented as the ratio of the luciferase firefly/Renilla activities. E, Esophageal keratinocytes (TE-7) were pretreated with IL-13 (0 or 100 ng/mL) and actinomycin D (Actino; 0 or 10 μmol/L) for 0 to 48 hours. Results are presented as a percentage of eotaxin-3 mRNA compared with time 0 hours (100%; black and gray dashed lines for media and IL-13, respectively). Trend lines (black and gray lines for media and IL-13, respectively) were calculated.

Effect of glucocorticoids on the EE transcriptome and resistant genes. A and B, The expression of IL-13 (Fig 5, A) and eotaxin-3 (Fig 5, B) mRNA is shown in healthy subjects (NL), patients with EE, and patients with EE treated successfully with FP (n = 8-9, 13-19, and 6-8 subjects for the NL, EE, and FP groups, respectively). C, Total mRNA was subjected to microarray analysis. Upregulated genes are shown in red, and downregulated genes are shown in blue. Each column represents a separate patient (NL, EE, and FP Rx), and each line represents a gene. D, Genes that are resistant to glucocorticoid therapy are shown with their Affymetrix accession numbers and their fold change in patients with EE and in treated patients with EE. E, Expression of cadherin-26 (CDH26) was quantified by means of real-time PCR. Each data point corresponds to a separate individual (n = 9, 11, and 7 subjects for the NL, EE, and FP groups, respectively). P values were calculated using Kruskal-Wallis tests (3 groups).