Potentiation of IL-19 expression in airway epithelia by IL-17A and IL-4/IL-13: important implications in asthma

Abstract

Background: IL-17A and IL-19 are highly expressed in chronic inflammatory diseases, such as psoriasis and asthma. IL-19 plays a significant role in the enhancement of T(H)2 cytokine secretion in allergic diseases, but its cellular source in asthmatic patients remains unknown.

Objective: Our aims were to determine whether the epithelium is a major source of airway mucosal IL-19 and to elucidate the mechanism of gene expression regulation.

Methods: Immunofluorescent staining was used to determine IL-19 protein expression in tracheal tissue sections of various airway diseases. Well-differentiated primary human bronchial epithelial cultures and a corresponding cell line were used as in vitro models to study gene regulation.

Results: We found significantly higher IL-19 expression in airway epithelia of asthmatic patients than in epithelia of patients with other diseases. Using a cytokine panel, we demonstrated the upregulation of IL-19 expression in cultures by two T(H)2 cytokines, IL-4 and IL-13, in addition to the previously found T(H)17 cytokine IL-17A. Moreover, cotreatment of IL-17A and IL-4/IL-13 synergistically upregulated IL-19 expression. Using siRNA and chemical inhibitor approaches, we demonstrated a transcriptional regulation of IL-19 by nuclear factor kappaB and signal transducer and activator of transcription (STAT) 6. The addition of IL-13 to IL-17A stimulation triggers a shift from nuclear factor kappaB-dependent transcriptional regulation to one that is STAT6 based. Using chromatin immunoprecipitation assays, we demonstrated the presence of STAT6-binding elements in the IL-19 promoter region.

Conclusion: We propose that an IL-17A- and IL-13-induced synergism in IL-19 stimulation in airway epithelia occurs through a STAT6-dependent pathway.

FIG 1

Immunofluorescent staining on tracheal tissue sections from patients with no identifiable lung disease (A; normal), asthma (B), COPD (C), and CF (D). Deparaffinated sections were stained with anti-IL-19 antibody followed with Alexa Fluor 488–conjugated secondary antibody (green) and 4′-6-diamidino-2-phenylindole dihydrochloride (DAPI; blue, nucleus). Original images were at ×100 magnification by means of confocal microscopy. The results shown here are representative of at least 3 patients in each disease category.

FIG 2

Induction of IL-19 in NHBE cells: A, enhanced IL-19 message by IL-1β, IL-4, IL-13, and IL-17A; B, synergistic stimulation of IL-19 message by IL-17A and IL-4 or IL-13; C, bidirectional secretion of IL-19 in cultures. Data were from 3 independent experiments. *P < .01 compared with the control. #P < .05 for apical and basal secretion comparison. GAPDH, Glyceraldehyde-3-phosphate dehydrogenase.

FIG 3

Time- and concentration-dependent studies in NHBE cells: A, IL-19 induction by IL-13, IL-17A, or both in time course; B, various doses of IL-17A or IL-13 for IL-19 induction in the presence of a constant dose of IL-13 (25 ng/mL) or IL-17A (25 ng/mL), respectively. The experiments were repeated 3 times. *P < .05 compared with controls (no cytokine treatment). CTL, Control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

FIG 4

NHBE cells were treated with cytokines 24 hours before actinomycin D (Act; A) or cycloheximide (CHX; right) or vehicle (left; B) 30 minutes before cytokine treatments. RNA were harvested at indicated times. C, Nuclear translocation of STAT6, p65, and p50 in cytokine-treated HBE1 cells. The experiments were done 3 times. *P < .05 compared with controls. CTL, Control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

FIG 5

A and B, Inhibition of IL-17A–induced, but not IL-13– or IL-13/IL-17A–induced, IL-19 expression by p65 siRNA. IL-13 did not induce or potentiate IL-17A–induced p65-dependent HBD2 expression. C, Inhibition of IL-13– and IL-13/IL-17A–induced, but not IL-17A–induced, IL-19 expression by STAT6 siRNA. Data were from 3 independent experiments. *P < .05. RO, Random oligomer; CTL, control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

FIG 6

ChIP assays: A, identification of putative STAT6 sites in the 5′-flanking region of IL-19; B, PCR analysis of anti-STAT6 antibody–precipitated chromatins from HBE1 cells treated with cytokines; C, quantitative PCR analysis of anti-STAT6 ChIPs from preparations seen in Fig 6, B. Data were from 3 independent experiments. *P < .05. Ab, Antibody; IP, immunoprecipitation; CTL, control.