Interleukin-13 (IL-13)/IL-13 receptor alpha1 (IL-13Ralpha1) signaling regulates intestinal epithelial cystic fibrosis transmembrane conductance regulator channel-dependent Cl- secretion

Abstract

Interleukin-13 (IL-13) has been linked to the pathogenesis of inflammatory diseases of the gastrointestinal tract. It is postulated that IL-13 drives inflammatory lesions through the modulation of both hematopoietic and nonhematopoietic cell function in the intestine. To delineate the relevant contribution of elevated levels of intestinal IL-13 to intestinal structure and function, we generated an intestinal IL-13 transgenic mouse (iIL-13Tg). We show that constitutive overexpression of IL-13 in the small bowel induces modification of intestinal epithelial architecture (villus blunting, goblet cell hyperplasia, and increased epithelial proliferation) and epithelial function (altered basolateral → apical Cl(-) ion conductance). Pharmacological analyses in vitro and in vivo determined that elevated Cl(-) conductance is mediated by altered cystic fibrosis transmembrane conductance regulator expression and activity. Generation of iIL-13Tg/Il13rα1(-/-), iIL-13Tg/Il13rα2(-/-), and iIL-13Tg/Stat6(-/-) mice revealed that IL-13-mediated dysregulation of epithelial architecture and Cl(-) conductance is dependent on IL-13Rα1 and STAT-6. These observations demonstrate a central role for the IL-13/IL-13Rα1 pathway in the regulation of intestinal epithelial cell Cl(-) secretion via up-regulation of cystic fibrosis transmembrane conductance regulator, suggesting an important role for this pathway in secretory diarrhea.

FIGURE 1.

Assessment of base-line serum IL-13, IL-13Rα2, and serum Ig in iIL-13Tg mice. A, diagrammatic representation of the rat intestinal fatty acid-binding protein (FABP) promoter (iFABPp)-mouse IL-13 (mIL-13) transgenic construct. B, intestinal IL-13. C, serum sIL-13Rα complexed to IL-13 (sIL-13Rα2-IL-13 complex) in wild type (WT) and iIL-13Tg mice. Values represent mean ± S.E.; n = 5–7 mice per group. Statistical significance is as follows: *, p < 0.05.

FIGURE 2.

Altered intestinal architecture and epithelial proliferation in iIL-13Tg mice. A, representative photomicrograph of H&E-stained jejunum from strain- and aged-matched WT and iIL-13Tg mice. B, villus length, crypt length, and villus/crypt ratio of jejunum of WT and iIL-13Tg mice. C, representative photomicrographs of PAS-stained jejunum and quantification of total PAS⁺ cells in the jejunum of WT and iIL-13Tg mice. D, representative photomicrographs of BrdU-stained jejunum and quantification of total number of BrdU⁺ cells in the jejunum of WT and iIL-13Tg mice. Values represent mean ± S.E.; n = 5–7 mice per group. Statistical significance is as follows: B–D, *, p < 0.01 versus WT.

FIGURE 3.

Altered transepithelial resistance and intestinal ion conductance in iIL-13Tg mice. Base-line TER and I_sc (A) and forskolin-induced I_sc (B) of isolated segments of jejunum from WT and iIL-13Tg. Values represent mean ± S.E.; A, n = 12–18 mice per group and B, n = 4–6 mice per group. Statistical significance is as follows: A, *, p < 0.01 versus WT; B, **, p < 0.01 versus WT; ***, p < 0.005 versus WT.

FIGURE 4.

IL-13-induced apical intestinal epithelial cell Cl⁻ conductance. TER (A) and I_sc (B) of a confluent CaCo2bbe cell monolayer 24 h following 0.1–10 units/ml IL-13 stimulation. Forskolin-induced (C) and acetylcholine-induced (D) I_sc of CaCo2bbe cells following 24 h of stimulation with 10 ng/ml IL-13. E, change in I_sc (ΔI_sc) of control- and IL-13-stimulated CaCo2bbe cells exposed basolaterally to amphotericin B (20 μm) and ouabain (1 mm) in the presence of basolateral to apical Cl⁻ concentration gradient (Baso → Ap [Cl⁻]). ΔI_sc of control- and IL-13-stimulated CaCo2bbe cells following basolateral addition of bumetanide (60 μm) (F) or following basolateral substitution of Cl⁻ with sodium gluconate (G). Values represent mean ± S.E.; n = 6–9 mice per group. Statistical significance is as follows: *, p < 0.05; **, p < 0.01; and ***, p < 0.005.

FIGURE 5.

IL-13-induced Cl⁻ conductance in intestinal epithelial cells is CFTR-dependent. ΔI_sc of control- and IL-13-stimulated CaCo2bbe cells following basolateral addition of DIDS (20 μm) or the CFTR inhibitor (CFTR^Inh172). The figure is representative of n = 3 cultures per group from three separate experiments. Values represent mean ± S.E. Statistical significance is as follows: **, p < 0.05, and ***, p < 0.01.

FIGURE 6.

Increased Cl⁻ conductance in iIL-13Tg mice is CFTR-dependent. Forskolin-induced ΔI_sc of isolated segments of jejunum from WT and iIL-13Tg following basolateral exposure to DIDS (100 μm) or the CFTR inhibitor (CFTR^Inh172) (20 μm). The figure is representative of n = 3 isolated jejunal segments per group from two separate experiments. Values represent mean ± S.E. Statistical significance is as follows: *, p < 0.01 and ***, p < 0.005.

FIGURE 7.

IL-13-up-regulated CFTR expression in the CaCo2bbe intestinal epithelial cell line and jejunum of iIL-13Tg mice. Western blot analysis of protein lysates probing for CFTR and actin from control- and IL-13-stimulated intestinal epithelial cell CaCo2bbe cells (A) and jejunum segments from WT and iIL-13Tg mice (B). B, each lane represents an individual mouse. The figure is representative of duplicate experiments.

FIGURE 8.

IL-13-induced effects on intestinal epithelial morphology and epithelial barrier function are dependent on IL-13Rα1. Representative photomicrographs of H&E stained (A), PAS-stained (B), and BrdU-stained (C) jejunum from WT (BALB/c), iIL-13Tg, IL13ra1^−/−, IL13ra2^−/−, iIL-13Tg/IL13ra1^−/−, and iIL-13Tg/IL-13ra2^−/− mice. Endoplasmic reticulum (D) and forskolin-induced (E) ΔI_sc of jejunum WT, iIL-13, IL13ra1^−/−, IL13ra2^−/−, iIL-13Tg/IL13ra1^−/−, and iIL-13Tg/IL-13ra2^−/− mice are shown. Values represent mean ± S.E.; n = 5 mice per group from duplicate experiments. Statistical significance is as follows: D and E, *, p < 0.01 versus WT. **, p < 0.01 versus WT.

FIGURE 9.

IL-13-induced intestinal epithelial barrier dysfunction and CFTR expression are dependent on STAT-6. A, representative Western blot analyses probing for phosphorylated (pSTAT-6) and total STAT-6 (STAT-6) and actin in protein lysates from intestinal epithelial CaCo2bbe cells 0–30 min following 10 units/ml IL-13 stimulation. B, quantification of pSTAT-6/STAT-6/actin ratios in lysates from intestinal epithelial CaCo2bbe cells 0–30 min following 10 units/ml IL-13 stimulation. C, representative Western blot analyses probing for phosphorylated (pSTAT-6) and total STAT-6 (STAT-6) and actin in protein lysates from shRNA control- or shRNA-STAT-6-transduced CaCo2bbe cells following 15 min of stimulation with 10 ng/ml IL-13 (+) or control treatment (−). D, quantification of pSTAT6/actin and STAT6/actin ratios in protein lysates from shRNA-control- or shRNA-STAT-6-transduced CaCo2bbe cells following 15 min of stimulation with 10 ng/ml IL-13. I_sc (E), TER (F), forskolin-induced (G), and acetylcholine-induced (H) ΔI_sc of shRNA control- or shRNA-STAT-6-transduced CaCo2bbe cells following 24 h of stimulation with 10 ng/ml IL-13 is shown. Statistical significance is as follows: B, **, p < 0.01 versus control (0 min). D, *, p < 0.05. B and D, values represent mean ± S.E.; n = 3 separate experiments. E–H, values represent mean ± S.E.; n = 3–6 mice per group. Statistical significance is as follows: *, p < 0.05, and ***, p < 0.005.

FIGURE 10.

IL-13-induced effects on intestinal epithelial barrier function are dependent on STAT-6. TER (A), and I_sc (B), and , forskolin-induced (C) and carbachol-induced (D) ΔI_sc of jejunum WT, iIL-13Tg, Stat6^−/−, and iIL-13Tg/Stat6^−/− mice. Values represent mean ± S.E.; n = 4 mice per group from duplicate experiments. Statistical significance is as follows: A, **, p < 0.01 versus WT. ***, p < 0.01 versus WT.