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Inhibition of ABCC4 potentiates combination beta agonist and glucocorticoid responses in human airway epithelial cells  Ryan D. Huff, MSc, Christopher.

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Presentation on theme: "Inhibition of ABCC4 potentiates combination beta agonist and glucocorticoid responses in human airway epithelial cells  Ryan D. Huff, MSc, Christopher."— Presentation transcript:

1 Inhibition of ABCC4 potentiates combination beta agonist and glucocorticoid responses in human airway epithelial cells  Ryan D. Huff, MSc, Christopher F. Rider, PhD, Dong Yan, MSc, Robert Newton, PhD, Mark A. Giembycz, PhD, Chris Carlsten, MD, MPH, Jeremy A. Hirota, PhD  Journal of Allergy and Clinical Immunology  Volume 141, Issue 3, Pages e5 (March 2018) DOI: /j.jaci Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 ABCC4 expression is robust and restricted to human airway and submucosal epithelial cells in both healthy subjects and patients with asthma. Representative (n = 3 healthy subjects and n = 3 patients with asthma) immunohistochemical staining of ABCC4 (rust/brown) in proximal (A), distal (B), and submucosal gland epithelial cells (C) compared with isotype control antibody (D). For panels A-D, images were taken from a healthy subject. E, Immunoblot validation of anti-ABCC4 antibody with a single band of predicted molecular weight (159 kDa) in plasma membrane fractions of primary human airway epithelial cells, with EGFR as a control for membrane fraction and beta-actin as a loading control. F, ABCC4 transcript quantification in primary human airway epithelial cells from healthy control subjects and those with asthma under basal conditions (n = 7). G, ABCC4 transcript quantification in a human airway epithelial cell line (HBEC-6KT) following stimulation (n = 4). Results are shown as mean ± SD. EGFR, Epidermal growth factor receptor. Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 Inhibition of ABCC4 potentiates cAMP signaling and glucocorticoid responses in human airway epithelial cell lines (for details on cell lines, see this article's Online Repository at In human airway epithelial cells, ABCC4 inhibition with MK-571 attenuates extracellular transport of cAMP (*P < .05 compared with forskolin-stimulated cells) (A) and potentiates CRE-mediated luciferase expression as measured by area under the curve analysis (B and C), which is sensitive to the PKA inhibitor H89 (D) (&P < .05 compared with nonstimulated cells, *P < .05 compared with FSK + MK-571). ABCC4 inhibition with MK-571 potentiates GRE-mediated luciferase activity as measured by area under the curve analysis (E and F) (*P < .05 compared with dexamethasone, & P < .05 compared with dexamethasone and FSK), and RGS2 gene expression (G) (*P < .05 compared with FSK, & P < .05 compared with FSK and dexamethasone). MK-571 also potentiated budesonide- and formoterol-induced GRE-mediated luciferase activity (H and I) (*P < .05 compared with budesonide, & P < .05 compared with budesonide and formoterol) and RGS2 gene expression (J) (*P < .05 compared with formoterol, & P < .05 compared with formoterol and budesonide). Last, TNF-α–induced GM-CSF, but not IL-8, production was attenuated by combination budesonide, formoterol, and MK-571 (K and L) (*P < .05 compared with control, & P < .05 compared with TNF-α, and #P < .05 compared with TNF-α with budesonide and formoterol). All experiments (n = 5) with results shown as mean ± SD. CRE, cAMP response element; FSK, forskolin; GRE, glucocorticoid response element. Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig E1 Inflammatory cytokines do not regulate ABCC4 gene expression in the human airway epithelial cell line, HBEC-6KT. A, Quantitative PCR analysis of ABCC4 gene expression was performed following 24-hour treatment with combinations of recombinant human TNF-α, IL-6, and IFN-γ. B, Positive control of CCL5 gene expression following treatment with combinations of recombinant human TNF-α, IL-6, and IFN-γ. n = 5, *P < .05. Data expressed as mean ± SD. Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig E2 Inhibition of ABCC4 potentiates cAMP signaling and glucocorticoid responses in the BEAS-2B human airway epithelial cell line treated with budesonide and formoterol at 0.1 nM concentration. ABCC4 inhibition with MK-571 does not potentiate GRE-mediated luciferase activity as measured by area under the curve analysis (A and B) (*P < .05 compared with budesonide), but does potentiate RGS2 gene expression (C) (*P < .05 compared with formoterol, & P < .05 compared with formoterol and budesonide). Last, TNF-α–induced GM-CSF, but not IL-8, production was attenuated by combination budesonide, formoterol, and MK-571 (D and E) (*P < .05 compared with control, & P < .05 compared with TNF-α, and #P < .05 compared with TNF-α with budesonide and formoterol). All experiments (n = 5) with results shown as mean ± SD. Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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