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Potentiation of IL-19 expression in airway epithelia by IL-17A and IL-4/IL-13: Important implications in asthma  Fei Huang, PhD, Shinichiro Wachi, PhD,

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Presentation on theme: "Potentiation of IL-19 expression in airway epithelia by IL-17A and IL-4/IL-13: Important implications in asthma  Fei Huang, PhD, Shinichiro Wachi, PhD,"— Presentation transcript:

1 Potentiation of IL-19 expression in airway epithelia by IL-17A and IL-4/IL-13: Important implications in asthma  Fei Huang, PhD, Shinichiro Wachi, PhD, Philip Thai, MD, Artem Loukoianov, BS, Kin Hup Tan, BS, Rosanna Malbran Forteza, MD, Reen Wu, PhD  Journal of Allergy and Clinical Immunology  Volume 121, Issue 6, Pages e3 (June 2008) DOI: /j.jaci Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 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. Journal of Allergy and Clinical Immunology  , e3DOI: ( /j.jaci ) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 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. Journal of Allergy and Clinical Immunology  , e3DOI: ( /j.jaci ) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 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. Journal of Allergy and Clinical Immunology  , e3DOI: ( /j.jaci ) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 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. Journal of Allergy and Clinical Immunology  , e3DOI: ( /j.jaci ) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 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. Journal of Allergy and Clinical Immunology  , e3DOI: ( /j.jaci ) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 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. Journal of Allergy and Clinical Immunology  , e3DOI: ( /j.jaci ) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

8 NHBE cells treated with and without random oligomers (200 nmol/L) were compared for IL-19 expression. Results indicated IL-19 was induced equally in both control studies by IL-13, IL-17A, and their cotreatment, suggesting the siRNA effect in Fig 5 is specific. The experiments were repeated 3 times. ∗P < .01 for cytokine-treated samples and their non–cytokine-treated controls. Journal of Allergy and Clinical Immunology  , e3DOI: ( /j.jaci ) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

9 Isohelenin, an IκB-α degradation inhibitor, was shown to suppress IL-17A–induced IL-19 and HBD-2 expression. However, isohelenin had no effect on IL-13 or IL-13/IL-17A cotreatment–induced IL-19 expression. The data were from 3 experiments. ∗P < .05, as indicated by the line. CTL, Control. Journal of Allergy and Clinical Immunology  , e3DOI: ( /j.jaci ) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

10 In ChIP assays (Fig 6, C) the cycle threshold values of STAT6-III/IV real-time PCR were first normalized to their input controls, and then the normalized values for the negative IgG controls (left) were subtracted from the normalized values for the anti-STAT6 immunoprecipitation (middle), yielding the final value shown at right. ∗P < .05 for comparable non–cytokine-treated controls. Ab, Antibody. Journal of Allergy and Clinical Immunology  , e3DOI: ( /j.jaci ) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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