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Role of TWIK-related potassium channel-1 in chronic rhinosinusitis

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1 Role of TWIK-related potassium channel-1 in chronic rhinosinusitis
Ha Kyun Kim, MD, Jae Hyeong Kim, MD, Hyun Jung Kim, MD, Tae Hoon Kim, MD, Sang Hag Lee, MD, PhD  Journal of Allergy and Clinical Immunology  Volume 141, Issue 3, Pages e6 (March 2018) DOI: /j.jaci Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 The expression levels of TREK-1 mRNA (A) and protein (B) in normal sinus mucosa of control subjects (NE; n = 30) and inflammatory sinus mucosa of patients with CRSsNP (sP; n = 30) and patients with CRSwNP (cP; n = 30). Upper panels of Fig 1, A and B, show representative data for RT-PCR and Western blot, respectively. *Significant difference in the expression levels of TREK-1 between normal and inflammatory sinus mucosa (P < .05). Results are expressed as means ± SDs. C, Immunohistochemical localization of TREK-1 in normal and inflammatory sinus mucosa of patients with CRSsNP or CRSwNP. TREK-1 is distributed in the surface epithelium (arrowheads), submucosal glands (thick arrows), and vascular endothelium (thin arrows). Original magnification ×100. GAPDH, Glyceraldehyde-3-phosphate dehydrogenase. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 Real-time PCR (A and C) and Western blot analysis (B and D) of TREK-1 expression levels in cultured epithelial (Fig 2, A and B) and endothelial cells (Fig 2, C and D) after stimulation with IL-4, IL-5, IL-13, TNF-α, TGF-β1, and IFN-γ (30 ng/mL), respectively, for 24 hours. Lower Western blot panels (Fig 2, B and D) indicate representative images for the expression of TREK-1 in epithelial (Fig 2, B) and endothelial cells (Fig 2, D). Transepithelial permeability (E) and electrical resistance (F) were measured in cultured normal epithelial cells, epithelial cells isolated from CRSsNP or CRSwNP, normal epithelial cells treated with TREK-1 siRNA, stimulated with TREK-1 inhibitor (Fluox; fluoxetine; 5 μM), TREK-1 siRNA and TREK-1 activator (arachidonic acid; 10 μM), or TREK-1 inhibitor and TREK-1 activator. Cont indicates the epithelial permeability (Fig 2, E) and electrical resistance (Fig 2, F) measured in cultured normal epithelial cells without cytokine treatment. No Tx indicates various kinds of cultured epithelial cells without cytokine treatment. λ, λ λ, §, ф, ф ф, *, **, ***, #, ##, +, and ++ indicate significant difference between each group. Results are expressed as means ± SDs. Data are representative of 6 individual experiments. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig E1 Transendothelial pemeability (A), electrical resistance (B), and transendothelial leukocytes migration (C) were measured in cultured normal endothelial cells, endothelial cells isolated from CRSsNP or CRSwNP, normal endothelial cells treated with TREK-1 siRNA, stimulated with TREK-1 inhibitor, TREK-1 siRNA and TREK-1 activator, or TREK-1 inhibitor and TREK-1 activator. Cont indicates the endothelial cells permeability (A) and electrical resistance (B) measured in cultured normal endothelial cells without cytokine treatment. No Tx indicates the endothelial permeability and electrical resistance measured in various kinds of cultured endothelial cells without cytokine treatment. λSignificant difference between control cells and endothelial cells isolated from CRSwNP. λ λSignificant difference between control cells and normal endothelial cells treated with TREK-1 siRNA. §Significant difference between control cells and normal endothelial cells treated with TREK-1 inhibitor. фSignificant difference between normal endothelial cells treated with TREK-1 siRNA and normal endothelial cells treated with TREK-1 siRNA and arachidonic acid. ф фSignificant difference between normal endothelial cells treated with TREK-1 inhibitor and normal endothelial cells treated with TREK-1 inhibitor and arachidonic acid. *Significant difference between control cells and cytokine-treated normal endothelial cells. **Significant difference between cytokine-treated normal cells and cytokine-treated endothelial cells isolated from CRSsNP. ***Significant difference between cytokine-treated normal cells and cytokine-treated endothelial cells isolated from CRSwNP. #Significant difference between cytokine-treated normal cells and normal endothelial cells treated with TREK-1 siRNA and then followed by cytokine treatment. ##Significant difference between cytokine-treated normal endothelial cells and TREK-1 siRNA-treated normal endothelial cells followed by cytokine treatment. +Significant difference between TREK-1 siRNA-treated normal endothelial cells followed by cytokine treatment and normal endothelial cells treated by TREK-1 siRNA and arachidonic acid followed by cytokine treatment. ++Significant difference between TREK-1 inhibitor-treated normal endothelial cells followed by cytokine treatment and normal endothelial cells treated by TREK-1 inhibitor and arachidonic acid followed by cytokine treatment. Results are expressed as means ± SDs. Data are representative of 6 individual experiments using 6 wells per condition in each experiment. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig E2 Real-time PCR (A and C) and Western blot analysis (B and D) for the expression of junctional complex proteins (occludin, ZO-1, ZO-2, claudin-1, claudin-4) in epithelial cells treated with TREK-1 inhibitor (fluoxetine; 5 μM) (Fig E2, A and B) or silenced with TREK-1 siRNA (Fig E2, C and D), respectively, for 24 hours. Western blot panels on the right side (Fig E2, B and D) indicate representative patterns for the expression of occludin, ZO-1, ZO-2, claudin-1, and claudin-4 in epithelial cells. Results are expressed as means ± SDs. Data are representative of 6 individual experiments using 6 wells per condition in each experiment. *Significant difference between the nontreated control cells and treated cells (P < .05). E, Confocal microscopic findings of tight junctional proteins in cultured epithelial cells that were untreated (control; a, b, c, d, e), treated with TREK-1 inhibitor (fluoxetine; f, g, h, i, j), or treated with TREK-1 siRNA (k, l, m, n, o). Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig E3 Real-time PCR (A and C) and Western blot analysis (B and D) for the expression of vascular adhesion molecules (ICAM-1 and VCAM-1) in endothelia cells treated with TREK-1 inhibitor (5 μM) (Fig E3, A and B) or silenced with TREK-1 siRNA (Fig E3, C and D), respectively, for 24 hours. Western blot panels on the right side (Fig E3, B and D) indicate representative patterns for the expression of ICAM-1 and VCAM-1 in endothelial cells. Results are expressed as means ± SDs. Data are representative of 6 individual experiments using 6 wells per condition in each experiment. *Significant difference between the untreated control cells and treated cells (P < .05). E, Confocal microscopic findings of vascular adhesion molecules in nontreated control cells (a and d), in cells treated with TREK-1 inhibitor fluoxetine (b and e), and in cells treated with TREK-1 siRNA (c and f). Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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